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<h4>Background</h4>The origin of the nuclear compartment has been extensively debated, leading to several alternative views on the evolution of the eukaryotic nucleus. Until recently, too little phylogenetic information was available to address this issue by using multiple characters for many lineages.<h4>Results</h4>We analyzed 65 proteins integral to or associated with the nuclear pore complex (NPC), including all the identified nucleoporins, the components of their anchoring system and some of their main partners. We used reconstruction of ancestral sequences of these proteins to expand the detection of homologs, and showed that the majority of them, present all over the nuclear pore structure, share homologs in all extant eukaryotic lineages. The anchoring system, by contrast, is analogous between the different eukaryotic lineages and is thus a relatively recent innovation. We also showed the existence of high heterogeneity of evolutionary rates between these proteins, as well as between and within lineages. We show that the ubiquitous genes of the nuclear pore structure are not strongly conserved at the sequence level, and that only their domains are relatively well preserved.<h4>Conclusion</h4>We propose that an NPC very similar to the extant one was already present in at least the last common ancestor of all extant eukaryotes and it would not have undergone major changes since its early origin. Importantly, we observe that sequences and structures obey two very different tempos of evolution. We suggest that, despite strong constraints that froze the structural evolution of the nuclear pore, the NPC is still highly adaptive, modern, and flexible at the sequence level.

Efforts to develop synthetic methodologies allowing the preparation of alpha,alpha- difluorophosphonothioates, alpha,alpha-difluorophosphonodithioates, alpha,alpha-difluorophosphono- trithioates, and alpha,alpha-difluorophosphinates are reviewed in the light of applications in the field of modified oligonucleotides and cyclitol phosphates. Two successful approaches have been developed, based either on the addition of phosphorus-centered radicals onto gem-difluoroalkenes or on a process involving the addition of lithiodifluorophosphono- thioates 91 onto a ketone and the subsequent deoxygenation reaction of the adduct. The radical route successfully developed a practical route to alpha,alpha-difluoro-H-phosphinates which proved to be useful intermediates to a variety of phosphate isosters. The ionic route led to the first preparation of phosphonodifluoromethyl analogues of nucleoside- 3'-phosphates.

A serotonin transporter gene linked polymorphic region (5-HTTLPR) has been investigated in several genetic association studies, including studies of bipolar disorder (BD) and suicidality. The current study was designed to examine whether the new long (A/G) variant polymorphism of the 5-HTT gene may be associated with the suicide attempts in 305 families with at least one member having BD. No association with history of suicide attempt was found either in the multiallelic HTTLPR (LRS=0.15, df=2, P=0.92), or with the intron 2 variable number tandem repeat (VNTR) polymorphism (LRS=0.87 df=2 P=0.64). When we performed a haplotype analysis, we found no association between suicide attempt and haplotype distribution (LRS=1.84 df=4 P=0.76). These findings suggest that this new polymorphism in the 5-HTT gene may not influence suicidal behaviour in patients with bipolar disorder.

CNS neurons are endowed with the ability to recover from cytotoxic insults associated with the accumulation of proteinaceous polyglutamine aggregates via a process that appears to involve capture and degradation of aggregates by autophagy. The ubiquitin-proteasome system protects cells against proteotoxicity by degrading soluble monomeric misfolded aggregation-prone proteins but is ineffective against, and impaired by, non-native protein oligomers. Here we show that autophagy is induced in response to impaired ubiquitin proteasome system activity. We show that ATG proteins, molecular determinants of autophagic vacuole formation, and lysosomes are recruited to pericentriolar cytoplasmic inclusion bodies by a process requiring an intact microtubule cytoskeleton and the cytoplasmic deacetylase HDAC6. These data suggest that HDAC6-dependent retrograde transport on microtubules is used by cells to increase the efficiency and selectivity of autophagic degradation.

<h4>Background/aims</h4>Hormone replacement therapy (HRT) is associated with an increased risk of thromboembolism dependent on the type of HRT; therefore, we compared therapy effects of intranasal with oral estrogens on coagulation and fibrinolysis markers in postmenopausal women.<h4>Methods</h4>A randomized study in which 29 healthy hysterectomized women received intranasal 17beta-estradiol or oral estrogens for 3 months.<h4>Results</h4>There were no significant differences in the baseline characteristics between groups. Those women receiving intranasal estradiol showed a mild increment in plasminogen activator inhibitor-1 (PAI-I) (from 6.8 +/- 3.5 to 9.6 +/- 3.9 U/ml, p < 0.01); however, fibrinogen, factor VII-tissue factor complex (VIIa-rTF), antithrombin III (ATIII), protein C (PC) activity, protein S (PS) activity, plasminogen (PLG), and tissue-type plasminogen activator antigen (t-PA) were unchanged. In contrast, oral unopposed estrogens elevated t-PA (from 4.9 +/- 2.9 to 9.6 +/- 5.1 ng/ml, p < 0.01) in parallel with a decrement in PAI-I (from 5.2 +/- 4.0 to 2.7 +/- 1.7 U/ml, p < 0.05) and VIIa-rTF (from 201.2 +/- 181.0 to 140.6 +/- 108.7 mU/ml, p < 0.05). Fibrinogen, ATIII, PC, PS, and PLG were unchanged.<h4>Conclusions</h4>Nasal 17beta-estradiol had no effect on the coagulation markers, except a moderate increment in PAI-1. In contrast, oral estrogens elicited a decrement in both VIIa-rTF and PAI-1; however, those changes did not surpass normal limits.

Antigens encoded by genes of the LAGE family, including LAGE-1 and NY-ESO-1, are of interest for cancer immunotherapy because they are tumor-specific and shared by tumors of different histological types. Several clinical trials are in progress with NY-ESO-1 peptides, protein, recombinant poxviruses, and dendritic cells pulsed with peptides. In this study, CD8 T lymphocytes from an individual without cancer were stimulated with dendritic cells infected with a recombinant avian poxvirus encoding a complete LAGE-1 protein. A CTL clone was isolated that recognized a new LAGE-1 peptide, ELVRRILSR, which corresponds to position 103-111 of the protein sequence. It is presented by HLA-A6801 molecules. When tumor cells expressing LAGE-1 were transfected with HLA-A68, they were lysed by the CTL clone, indicating that the peptide is processed in tumor cells. These results indicate that the LAGE-1.A68 peptide can be used for antitumoral vaccination. We observed also that specific T cells could be detected in a blood sample with a high sensitivity by using an A68/LAGE-1 fluorescent multimer.

<h4>Background</h4>Mucin glycoprotein's are major components of mucus and are considered an important class of tumor associated antigens. The objective of this study was to investigate the expression of human MUC genes (MUC1, MUC2, MUC5B, MUC5AC and MUC8) in human endometrium and cervix, and to compare and quantitate the expression of MUC genes in normal and cancerous tissues.<h4>Methods</h4>Slot blot techniques were used to study the MUC gene expression and quantitation.<h4>Results</h4>Of the five-mucin genes studied, MUC1, MUC5B and MUC8 showed high expression levels in the normal and cancerous endometrial and cervical tissues, MUC2 and MUC5AC showed considerably lower expression. Statistically, higher levels of MUC1, MUC5B and MUC8 were observed in endometrial adenocarcinomas compared to normal tissues. In contrast, only MUC1 levels increased with no significant changes in expression of MUC5B and MUC8 in cervical tumors over normal cervical tissues.<h4>Conclusion</h4>Endometrial tumors showed increased expression of MUC1, MUC5B and MUC8 over normal tissues. Only MUC1 appears to be increase, in cervical tumors. All the studied tissues showed high and consistent expression of MUC8 mRNA. Low to neglible levels of MUC2 and MUC5AC were observed in all studied endometrial and cervical tissues.

Saitohin is a gene unique to humans and their closest relatives, the function of which is not yet known. Saitohin contains a single polymorphism (Q7R), and its Q and R alleles belong to the H1 and H2 tau haplotype, respectively. The Saitohin Q allele confers susceptibility to several neurodegenerative diseases. To get a handle on Saitohin function, we used it as a bait in a yeast two-hybrid screen. By this assay and subsequent co-immunoprecipitation and glutathione S-transferase pull-down assays, we discovered and confirmed that Saitohin interacts with peroxiredoxin 6, a unique member of that family that is bifunctional and the levels of which increase in Pick disease. The strength of the interaction appeared to be allele-specific, giving the first distinction between the two forms of Saitohin.

Thrombospondins (THBSs) are secreted glycoproteins that have key roles in interactions between cells and the extracellular matrix. Here, we describe the 2.6-A-resolution crystal structure of the glycosylated signature domain of human THBS2, which includes three epidermal growth factor-like modules, 13 aspartate-rich repeats and a lectin-like module. These elements interact extensively to form three structural regions termed the stalk, wire and globe. The THBS2 signature domain is stabilized by these interactions and by a network of 30 bound Ca(2+) ions and 18 disulfide bonds. The structure suggests how genetic alterations of THBSs result in disease.

In mammalian cells, nonsense-mediated messenger RNA decay (NMD) targets newly synthesized nonsense-containing mRNA bound by the cap-binding-protein heterodimer CBP80-CBP20 and at least one exon-junction complex (EJC). An EJC includes the NMD factors Upf3 or Upf3X and Upf2, and Upf2 recruits Upf1. Once this pioneer translation initiation complex is remodeled so that CBP80-CBP20 is replaced by eukaryotic initiation factor 4E, the mRNA is no longer detectably targeted for NMD. Here, we provide evidence that CBP80 augments the efficiency of NMD but not of Staufen1 (Stau1)-mediated mRNA decay (SMD). SMD depends on the recruitment of Upf1 by the RNA-binding protein Stau1 but does not depend on the other Upf proteins. We find that CBP80 interacts with Upf1 and promotes the interaction of Upf1 with Upf2 but not with Stau1.

In postmortem Huntington's disease brains, mutant htt is present in both nuclear and cytoplasmic compartments. To dissect the impact of nuclear and extranuclear mutant htt on the initiation and progression of disease, we generated a series of transgenic mouse lines in which nuclear localization or nuclear export signal sequences have been placed N-terminal to the htt exon 1 protein carrying 144 glutamines. Our data indicate that the exon 1 mutant protein is present in the nucleus as part of an oligomeric or aggregation complex. Increasing the concentration of the mutant transprotein in the nucleus is sufficient for and dramatically accelerates the onset and progression of behavioral phenotypes. Furthermore, nuclear exon 1 mutant protein is sufficient to induce cytoplasmic neurodegeneration and transcriptional dysregulation. However, our data suggest that cytoplasmic mutant exon 1 htt, if present, contributes to disease progression.

Possible association between the C282Y and H63D mutations in the HFE gene and estrogen-dependent cancer risk was assessed. Genotyping was performed using PCR amplification followed by digestion of products with specific restrictases. In a population of 260 healthy women (permanent residents of the southwest European Russia), mutant allele frequencies at the C282Y and H63D sites were evaluated as 3.3 and 16.3%, respectively. In patients with breast, ovarian, and endometrial cancer, C282Y frequencies were also low (1.0, 1.3, and 3.8%, respectively), and no cancer risk associated with the C282Y mutation was found. Odds ratios for breast cancer risk associated with the H63D mutation increased significantly with age: 0.5 in women below 48 years old, 1.0 in a range of 48-57 years, and 4.4 in older women (P(trend)=0.002). The latter value was statistically significant (95% CI, 1.4-14.1), indicating that women bearing the H63D mutation may be at an increased breast cancer risk at an age above 57 years. Preliminary results obtained in patients with two other estrogen-dependent malignancies revealed the same tendency to OR increase with age in ovarian cancer patients (P(trend)=0.008), but no age-related OR differences in endometrial cancer patients.

Myelotoxic injury in the bone marrow (BM) as a consequence of total body irradiation (TBI) or granulocyte colony-stimulating factor (G-CSF) mobilization results in the deposition of iC3b on BM stroma (stroma-iC3b). In the present study, we have examined how stroma-iC3b interacts with hematopoietic progenitor cells (HPCs) and the role of complement (C) and complement receptor 3 (CR3) in BM injury/repair. We demonstrate here that stroma-iC3b tethers HPCs via the inserted (I) domain of HPC complement receptor 3 (CR3, CD11b/CD18, Mac-1). Following irradiation, stroma-iC3b was observed in the presence of purified IgM and normal mouse serum (NMS), but not serum from Rag-2(-/-) mice, implicating a role for antibody (Ab) and the classic pathway of C activation. Furthermore, a novel role for soluble yeast beta-glucan, a ligand for the CR3 lectin-like domain (LLD), in the priming of CR3(+) HPC is suggested. Soluble yeast beta-glucan could enhance the proliferation of tethered HPCs, promote leukocyte recovery following sublethal irradiation, and increase the survival of lethally irradiated animals following allogeneic HPC transplantation in a CR3-dependent manner. Taken together, these observations suggest a novel role for C, CR3, and beta-glucan in the restoration of hematopoiesis following injury.

<h4>Background and aims</h4>Although most cases of hereditary haemochromatosis are associated with homozygosity for the C282Y mutation of the HFE gene, clinical penetrance varies and other genes may modify disease expression. If so, relatives from clinically affected families, by inheriting such genes, may accumulate more iron. To seek evidence for this, we compared iron status and morbidity in unselected first degree relatives of two groups of index cases from South Wales, namely asymptomatic C282Y homozygotes identified by genetic screening of blood donors (n = 56) and C282Y homozygous haemochromatosis patients presenting clinically (n = 60).<h4>Methods</h4>All participating relatives had a structured interview, clinical assessment, and laboratory investigations. Health related quality of life was measured (SF-36 version 2).<h4>Results</h4>In total, 92% of 180 eligible first degree relatives were interviewed in the "screened" family group and 85% of 143 eligible relatives in the "patient" group. Of 59 relatives homozygous for C282Y, 76% of men and 32% of women had the "iron phenotype" (raised transferrin saturation and serum ferritin). Logistic regression modelling of the iron phenotype risk showed that 42% of the initial model deviance could be explained by homozygosity for C282Y, another 6% by lifestyle factors, and 6% by being male. Family group membership was not a significant risk factor. Morbidity and SF-36 scores did not differ significantly either between C282Y homozygotes and relatives lacking C282Y, or between C282Y homozygotes from the "screened" and "patient" groups. Serious morbidity (including cirrhosis) was low in both groups of relatives.<h4>Conclusions</h4>HFE C282Y homozygosity has a high penetrance for iron accumulation but a low clinical penetrance. Lack of excess morbidity among C282Y homozygous relatives of index cases who presented clinically suggests that residual unknown genetic or environmental factors do not greatly influence clinical outcome among C282Y homozygotes.

<h4>Background</h4>Our aim was to determinate bone mineral density (BMD), levels of biochemical markers and cytokines in children with chronic hepatitis B treated with interferon (IFN)-alpha and to investigate effect of IFN-alpha therapy on these variables. To the best of our knowledge, this is first study carried out about BMD and cytokine levels in pediatric patients with chronic hepatitis B treated with IFN-alpha.<h4>Methods</h4>BMD, levels of parathyroid hormone (PTH), osteocalcin, C-terminal cross-linking telopeptide of type I collagen (CTX), calcium, alkaline phosphates (ALP), cytokines as TNF-alpha, interleukin (IL)-1beta, IL-2r, IL-6, and IL-8 were studied in 54 children with chronic hepatitis B (4-15 years old) treated with interferon alone (n = 19) or in combination with lamivudine (n = 35) for six months and as controls in 50 age-matched healthy children.<h4>Results</h4>There was no significant difference in respect to serum IL-1beta, TNF-alpha and osteocalcin levels while serum IL-2r (p = 0.002), IL-6 (p = 0.001), IL-8 (p = 0.013), PTH (p = 0.029), and CTX (p = 0.021) levels were higher in children with chronic hepatitis B than in healthy controls. BMD of femur neck (p = 0.012) and trochanter (p = 0.046) in patients were higher than in healthy controls. There was a statistically significant correlation between serum IL-1beta and osteocalcin (r = -0.355, p < 0.01); between serum IL-8 and CTX levels (r = 0.372, p = 0.01), and ALP (r = 0.361, p = 0.01); between serum ALP and femur neck BMD (r = 0.303, p = 0.05), and trochanter BMD (r = 0.365, p = 0.01); between spine BMD and IL-2R (r = -0.330, p < 0.05).<h4>Conclusion</h4>In conclusion, our study suggest that BMD of femur, serum IL-2r, IL-6, IL-8, PTH, and CTX levels were higher in children with chronic hepatitis B treated with IFN-alpha alone or combination with lamivudine than in healthy children. High femur BMD measurements found in patients may suggest that IFN-alpha therapy in children with chronic hepatitis B could contribute indirectly to prevent from hip osteoporosis. Additionally, further investigations on effects of IFN-alpha for bone structure in children should be performed in the future.

Neuronal Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) interacts with several prominent dendritic spine proteins, which have been termed CaMKII-associated proteins. The NR2B subunit of N-methyl-d-aspartate (NMDA)-type glutamate receptor, densin-180, and alpha-actinin bind comparable, approximately stoichiometric amounts of Thr(286)-autophosphorylated CaMKIIalpha, forming a ternary complex (Robison, A. J., Bass, M. A., Jiao, Y., Macmillan, L. B., Carmody, L. C., Bartlett, R. K., and Colbran, R. J. (2005) J. Biol. Chem. 280, 35329-35336), but their impacts on CaMKII function are poorly understood. Here we show that these interactions are differentially regulated and exert distinct effects on CaMKII activity. Nonphosphorylated and Thr(286)-autophosphorylated CaMKII bind to alpha-actinin with similar efficacy, but autophosphorylation at Thr(305/306) or Ca(2+)/calmodulin binding significantly reduce this binding. Moreover, alpha-actinin antagonizes CaMKII activation by Ca(2+)/calmodulin, as assessed by autophosphorylation and phosphorylation of a peptide substrate. CaMKII binding to densin (1247-1542) is partially independent of Thr(286) autophosphorylation and is unaffected by Ca(2+)-independent autophosphorylation or Ca(2+)/calmodulin. In addition, the CaMKII binding domain of densin-180 has little effect on CaMKII activity. In contrast, the interaction of CaMKIIalpha with NR2B requires either Thr(286) autophosphorylation or the binding of both Ca(2+)/calmodulin and adenine nucleotides. NR2B inhibits both the Ca(2+)/calmodulin-dependent and autonomous activities of CaMKII by a mechanism that is competitive with autocamtide-2 substrate, non-competitive with syntide-2 substrate, and uncompetitive with respect to ATP. In combination, these data suggest that dynamically regulated interactions with CaMKII-associated proteins could play pleiotropic roles in finetuning CaMKII signaling in defined subcellular compartments.

<h4>Background/aims</h4>In hereditary hemochromatosis, iron-loading of hepatocytes is associated with increased iron uptake while little is known about iron release. This study aims to characterise iron release and ferroportin expression by Hfe knockout hepatocytes to determine if they contribute to iron overload in haemochromatosis.<h4>Methods</h4>Iron release by hepatocytes from Hfe knockout, non-iron-loaded and iron-loaded wild-type mice was measured after incubation with nontransferrin-bound iron as iron-citrate.<h4>Results</h4>Iron release and ferroportin expression by hepatocytes from Hfe knockout, non-iron-loaded and in vivo iron-loaded wild-type mice were similar although, nontransferrin-bound iron uptake was significantly increased in Hfe knockout hepatocytes and decreased in iron-loaded wild-type hepatocytes compared with non-iron-loaded wild-type cells. When expressed as a percentage of total iron uptake, iron release was decreased in Hfe knockout hepatocytes (4.6+/-0.7 versus 13.7+/-1.2%, P<0.0001) and increased in iron-loaded wild-type hepatocytes (29.5+/-0.5 versus 13.5+/-0.7%; P<0.0001) compared with wild-type hepatocytes. In contrast, in vitro iron-loading increased iron release and ferroportin expression by both Hfe knockout and wild-type hepatocytes.<h4>Conclusions</h4>Hfe knockout hepatocytes accumulate iron as a result of limited iron export and enhanced iron uptake. The correlation between iron release and ferroportin expression suggests that iron export in hepatocytes is mediated by ferroportin.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized behaviorally by chorea, incoordination, and shortened lifespan and neuropathologically by huntingtin inclusions and neuronal degeneration. In order to facilitate studies of pathogenesis and therapeutics, we have generated a new inducible mouse model of HD expressing full-length huntingtin (Htt) using a tetracycline-regulated promoter. In double transgenic mice Htt was expressed widely in the brain under the control of the tet-transactivator (tTA) driven by the prion promoter PrP (in the absence of doxycycline). Mice expressing full-length mutant Htt, but not full-length normal Htt, displayed a progressive behavioral phenotype, consisting of slowed and irregular voluntary movements, gait ataxia, tremor and jerky movements, incoordination, and weight loss, with a shortened lifespan. Neuropathology included prominent intranuclear inclusions in cortex and striatum as well as cytoplasmic aggregates. This phenotype is very similar to the phenotypes of previous transgenic mice expressing N-terminal fragments of mutant Htt. The current HD-transgenic mice had nuclear accumulation of Htt, particularly an approximately 60-kDa fragment, which appears to represent an N-terminal cleavage product. This fragment is smaller than calpain or caspase-derived cleavage products of Htt, but it is comparable to a product, termed cp-A, which accumulates in nuclei of cells in a previously described cell model. This new mouse model may be useful in the future for pathogenic and preclinical therapeutic studies related to HD. The data suggest that proteolytic processing could be a part of the pathogenesis of HD, potentially representing an attractive therapeutic target.

<h4>Background</h4>As the genetic basis of many human diseases is being discovered, there is increasing need for the detection of single-nucleotide polymorphisms/mutations in medical laboratories. We describe an innovative approach that combines PCR amplification directly on whole blood and real-time detection PCR technology (WB-RTD PCR).<h4>Methods</h4>We compared WB-RTD PCR with the method for extracted DNA-RTD PCR for the detection of mutations in the prothrombin (n = 94), factor V Leiden (n = 49), and hemochromatosis (n = 22) genes. Mutation detection on the Roche LightCycler was based on use of fluorescence resonance energy transfer (FRET) probes and melting curve analysis. We also compared the WB-RTD PCR on the LightCycler and the ABI Prismtrade mark 7700 sequence detection system with minor groove- binding nonfluorescent quencher probes.<h4>Results</h4>We obtained complete concordance between both methods in assigning genotypes. We also demonstrated that the WB-RTD PCR method can be performed on real-time PCR instruments from Applied Biosystems and the LightCycler. Omission of the need for DNA extraction and gel electrophoresis allowed substantial labor and cost savings with this method.<h4>Conclusion</h4>This approach has applications for testing other medically relevant single-nucleotide polymorphisms.

<h4>Rationale</h4>Many of the interstitial lung diseases represent a diagnostic and therapeutic challenge because their clinical and even histologic features are often nonspecific. Likewise, the transcriptional signatures of most of them are unknown.<h4>Objective</h4>To compare the gene expression patterns from patients with idiopathic pulmonary fibrosis (IPF) hypersensitivity pneumonitis (HP), and nonspecific interstitial pneumonia (NSIP) using custom oligonucleotide microarrays.<h4>Methods</h4>We profiled lung biopsies from 15 patients with IPF, 12 with HP, and eight with NSIP. Labeled complementary ribonucleic acid was hybridized to a custom Affymetrix oligonucleotide DNA microarray using standard Affymetrix protocols. The custom array, Hu03, contained 59,619 probe sets representing an estimated 46,000 gene clusters.<h4>Results</h4>We identified statistically significant gene expression signatures that characterize HP and IPF. The HP gene expression signature was enriched for genes that are functionally associated with inflammation, T-cell activation, and immune responses, whereas the IPF signature was characterized by the expression of tissue remodeling, epithelial, and myofibroblast genes. We then compared these gene expression signatures to classify NSIP, a histologic pattern that is often difficult to differentiate consistently from HP and IPF. Two cases exhibited an IPF-like gene expression, another one could be more properly classified as HP, whereas others did not resemble HP or IPF, suggesting that they may represent idiopathic NSIP.<h4>Conclusions</h4>Our results underscore the value of gene expression signatures to classify the interstitial lung diseases and to understand pathogenic mechanisms, and suggest new ways to improve the diagnosis and treatment of patients with these diseases.

Previous research has demonstrated that a polymorphism in the serotonin transporter gene (5-HTTLPR) and adverse psychosocial circumstances interact to predict depression. The purpose of the present study was to explore the extent to which sex modulates these effects. Eighty-one boys and 119 girls (16-19 years old) were interviewed about psychosocial background variables and genotyped for the 5-HTT promoter polymorphism. There were two main results. First, boys and girls carrying the short 5-HTTLPR allele react to different kinds of environmental factors. Whereas males were affected by living in public housing rather than in own owned homes and by living with separated parents, females were affected by traumatic conflicts within the family. Second, the responses of males and females carrying the short 5-HTTLPR allele to environmental stress factors go in opposite directions. Thus, whereas females tend to develop depressive symptoms, males seem to be protected from depression. The results suggest that both the molecular and the psychosocial mechanisms underlying depression may differ between boys and girls.

We have identified a novel interaction between huntingtin (htt) and N-type calcium channels, a channel key in coupling calcium influx with synaptic vesicle exocytosis. Htt is a widely expressed 350-kDa cytosolic protein bearing an N-terminal polyglutamine tract. Htt is proteolytically cleaved by calpains and caspases and the resultant htt N-terminal fragments have been proposed to be biologically active; however, the cellular function of htt and/or the htt fragments remains enigmatic. We show that N-terminal fragments of htt (consisting of exon1) and full-length htt associate with the synaptic protein interaction (synprint) region of the N-type calcium channel. Given that synprint has previously been shown to bind syntaxin 1A and that this association elicits inhibition of N-type calcium channels, we tested whether htt(exon1) affects the modulation of these channels. Our data indicate that htt(exon1) enhances calcium influx by blocking syntaxin 1A inhibition of N-type calcium channels and attributes a key role for htt N-terminal fragments in the fine tuning of neurotransmission.

<h4>Background</h4>Recent evidence has suggested that the short allele of the serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR of the human serotonin gene [SLC6A4]) is associated with increased risk of depressive disorder but only among individuals exposed to social adversity. We report an investigation designed to replicate this finding.<h4>Methods</h4>Data were available from a non-clinical sample of 4,175 adult men and women, ages 41-80 years, selected from participants in the European Prospective Investigation into Cancer and Nutrition in Norfolk (EPIC-Norfolk, United Kingdom) study. Evidence of past-year prevalent episodic major depressive disorder (MDD), defined by restricted DSM-IV diagnostic criteria, was assessed through questionnaire. Adverse experiences in childhood and in adulthood (during the five years preceding assessment) were also assessed through self-report. The 5-HTTLPR variant was genotyped according to published protocols.<h4>Results</h4>One-year prevalent MDD criteria were met by 298 study participants. The experience of social adversity (both in childhood and adulthood) was strongly associated with increased rates of past-year prevalent MDD. No gene by environment (GxE) interactions between the 5-HTTLPR genotype, social adversity, and MDD were observed.<h4>Conclusions</h4>This study has not replicated a previous finding of a GxE interaction between the 5-HTTLPR genotype, social adversity, and depression.

<h4>Background</h4>Serotonin 1A receptors (5-HT(1A)) are implicated in the pathophysiology of major depressive disorder (MDD) and in the action of selective serotonin reuptake inhibitors (SSRI). SSRI desensitize 5-HT(1A) and down-regulate 5-HT transporters (5-HTT) with the latter persisting for weeks after discontinuation of SSRI. MDD subjects are more likely to be homozygous for the functional 5-HT(1A) G(-1019) allele of the promoter polymorphism and are postulated to have higher 5-HT(1A) than healthy volunteers (controls). We measure 5-HT(1A) in MDD, assess the effects of antidepressant exposure (AE), and examine the role of the C(-1019)G polymorphism.<h4>Methods</h4>Genotyped and determined 5-HT(1A) binding potential (BP) by positron emission tomography (PET) using [carbonyl-C-11]-WAY-100635 in 28 medication-free MDD subjects during a current major depressive episode and 43 controls.<h4>Results</h4>No difference in BP between controls and MDD subjects (p = .235). There was a difference in BP comparing the controls, antidepressant naive (AN) MDD subjects, and subjects with AE across all regions (p = .013). Post hoc testing reveals higher BP in AN compared to controls (p = .008) and to AE (p = .007). The GG genotype is overrepresented in MDD subjects (p = .059), and BP appears higher with the G allele.<h4>Conclusions</h4>AN have higher 5-HT(1A) than controls and AE suggesting a model of depression characterized by an over expression of autoinhibitory somatodendritic 5-HT(1A) receptors, perhaps due to the higher expressing G allele, that may result in reduced terminal field 5-HT release. AE appears to have long-term effects on 5-HT(1A).

The mechanism of excessive iron storage in patients with hereditary hemochromatosis caused by mutations of the HFE gene seems to be a failure to up-regulate hepcidin in the face of increased body iron. Since the cytokines IL-1 and IL-6 stimulate hepcidin transcription in the absence of HFE, chronic inflammatory states might counteract the effect of HFE mutations. We measured the pre-phlebotomy plasma levels of C reactive protein (CRP) and of interleukin 6 (IL-6) in homozygotes for the C282Y mutation of HFE. There was no difference in these levels in subjects with high iron stores than in those with low iron stores, suggesting that the phenotypic differences between such homozygotes is not appreciably affected by ongoing chronic inflammation.

In obesity-related insulin resistance, pancreatic islets compensate for insulin resistance by increasing secretory capacity. Here, we report the identification of sex-determining region Y-box 6 (SOX6), a member of the high mobility group box superfamily of transcription factors, as a co-repressor for pancreatic-duodenal homeobox factor-1 (PDX1). SOX6 mRNA levels were profoundly reduced by both a long term high fat feeding protocol in normal mice and in genetically obese ob/ob mice on a normal chow diet. Interestingly, we show that SOX6 is expressed in adult pancreatic insulin-producing beta-cells and that overexpression of SOX6 decreased glucose-stimulated insulin secretion, which was accompanied by decreased ATP/ADP ratio, Ca(2+) mobilization, proinsulin content, and insulin gene expression. In a complementary fashion, depletion of SOX6 by small interfering RNAs augmented glucose-stimulated insulin secretion in insulinoma mouse MIN6 and rat INS-1E cells. These effects can be explained by our mechanistic studies that show SOX6 acts to suppress PDX1 stimulation of the insulin II promoter through a direct protein/protein interaction. Furthermore, SOX6 retroviral expression decreased acetylation of histones H3 and H4 in chromatin from the promoter for the insulin II gene, suggesting that SOX6 may decrease PDX1 stimulation through changes in chromatin structure at specific promoters. These results suggest that perturbations in transcriptional regulation that are coordinated through SOX6 and PDX1 in beta-cells may contribute to the beta-cell adaptation in obesity-related insulin resistance.

"Cancer-germline" genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in other normal tissues. They encode tumor specific antigens that are used in cancer immunotherapy trials. MAGE-4 antigens represent promising targets for cancer immunotherapy because gene MAGE-4 is expressed in more than 50% of carcinomas of the esophagus, lung, bladder, and head and neck. To identify new MAGE-4 antigenic peptides, we have folded HLA-A*2402 soluble molecules with candidate peptide NYKRCFPVI, which corresponds to amino acids 143 to151 of the MAGE-4 protein. A24/MAGE-4 multimers were used to isolate a cytolytic T cell clone that recognized the MAGE-4 peptide from the blood cells of a donor without cancer. This clone lysed specifically A24 carcinoma cells expressing MAGE-4. The antigenic peptide is processed more efficiently in tumor cells pre-treated with IFN-gamma. This MAGE-4 peptide could represent an interesting target for immunotherapy because it is presented by HLA-A24 molecules, which are widely expressed in different ethnic groups.

CNS neurons are endowed with the ability to recover from cytotoxic insults associated with the accumulation of proteinaceous aggregates in mouse models of polyglutamine disease, but the cellular mechanism underlying this phenomenon is unknown. Here, we show that autophagy is essential for the elimination of aggregated forms of mutant huntingtin and ataxin-1 from the cytoplasmic but not nuclear compartments. Human orthologs of yeast autophagy genes, molecular determinants of autophagic vacuole formation, are recruited to cytoplasmic but not nuclear inclusion bodies in vitro and in vivo. These data indicate that autophagy is a critical component of the cellular clearance of toxic protein aggregates and may help to explain why protein aggregates are more toxic when directed to the nucleus.

C. elegans spermatogenesis employs lysosome-related fibrous body-membranous organelles (FB-MOs) for transport of many cellular components. Previous work showed that spe-10 mutants contain FB-MOs that prematurely disassemble, resulting in defective transport of FB components into developing spermatids. Consequently, spe-10 spermatids are smaller than wild type and contain defective FB-MO derivatives. In this article, we show that spe-10 encodes a four-pass integral membrane protein that has a DHHC-CRD zinc-finger motif. The DHHC-CRD motif is found in a large, diverse family of proteins that have been implicated in palmitoyl transfer during protein lipidation. Seven spe-10 mutants were analyzed, including missense, nonsense, and deletion mutants. An antiserum to SPE-10 showed significant colocalization with a known marker for the FB-MOs during wild-type spermatogenesis. In contrast, the spe-10(ok1149) deletion mutant lacked detectable SPE-10 staining; this mutant lacks a spe-10 promoter and most coding sequence. The spe-10(eb64) missense mutation, which changes a conserved residue within the DHHC-CRD domain in all homologues, behaves as a null mutant. These results suggest that wild-type SPE-10 is required for the MO to properly deliver the FB to the C. elegans spermatid and the DHHC-CRD domain is essential for this function.

Because a temporal arrest in the G1-phase of the cell cycle is a prerequisite for cell differentiation, this study investigated the involvement of cell cycle factors in the differentiation of cultured mouse prechondrocyte cell line ATDC5. Among the G1 cell cycle factors examined, both protein and mRNA levels of cyclin-dependent kinase (Cdk6) were downregulated during the culture in a differentiation medium. The protein degradation of Cdk6 was not involved in this downregulation because proteasome inhibitors did not reverse the protein level. When inhibitors of p38 MAPK, ERK-1/2, and PI3K/Akt were added to the culture, only a p38 MAPK inhibitor SB203580 blocked the decrease in the Cdk6 protein level by the differentiation medium, indicating that the Cdk6 inhibition was mediated by p38 MAPK pathway. In fact, p38 MAPK was confirmed to be phosphorylated during differentiation of ATDC5 cells. Enforced expression of Cdk6 in ATDC5 cells blocked the chondrocyte differentiation and inhibited Sox5 and Sox6 expressions. However, the Cdk6 overexpression did not affect the proliferation or the cell cycle progression, suggesting that the inhibitory effect of Cdk6 on the differentiation was exerted by a mechanism largely independent of its cell cycle regulation. These results indicate that Cdk6 may be a regulator of chondrocyte differentiation and that its p38-mediated downregulation is involved in the efficient differentiation.

Aberrant methylation of CpG islands (CGI) not only plays a role in gene silencing, but is also a potential cancer biomarker. To identify more CGI aberrantly methylated in human breast cancers, we carried out a genome-wide search for aberrant methylation, using methylation-sensitive-representational difference analysis. CGI in 5' upstream regions of 20 genes, TSPAN-2, AK5, LOC284999, HOXD11, FLJ25161, XT3, PCDH10, PCDHGB6, SIM1, LOC346978, COE2, TDH (FLJ25033), LOC346419, FLJ33790, GJB2, AMN, LOC201164, DLX4, DCC and FOXA2, were found to be methylated in at least one of 8 breast cancer cell lines. Fifteen of the 20 genes were methylated in more than one of 21 primary breast cancers in Stages I or II, and especially, those of LOC346978, HOXD11, SIM1, PCDHGB6 and FLJ25161 were methylated in more than 10 cancers. All the breast cancers had some aberrant methylation. Among the 13 genes whose CGI were completely methylated in one or more cell lines, FOXA2 and XT3 were expressed in normal human mammary epithelial cells (HMEC) and were not expressed in cancer cell lines with complete methylation. The other 11 genes examined were barely expressed, or not expressed even in HMEC. Our results showed that breast cancer cells accumulate aberrant methylation of the CGI identified here. This may serve as markers for early-stage breast cancers and suggests that aberrant methylation targets transcriptionally inactive genes in vivo.

The serotonin transporter gene (5-HTT) encodes a transmembrane protein that plays an important role in regulating serotonergic neurotransmission and related aspects of mood and behaviour. The short allele of a 44 bp insertion/deletion polymorphism (S-allele) within the promoter region of the 5-HTT gene (5-HTTLPR) confers lower transcriptional activity relative to the long allele (L-allele) and may act to modify the risk of serotonin-mediated outcomes such as anxiety and substance use behaviours. The purpose of this study was to determine whether (or not) 5-HTTLPR genotypes moderate known associations between attachment style and adolescent anxiety and alcohol use outcomes. Participants were drawn from an eight-wave study of the mental and behavioural health of a cohort of young Australians followed from 14 to 24 years of age (Victorian Adolescent Health Cohort Study, 1992 - present). No association was observed within low-risk attachment settings. However, within risk settings for heightened anxiety (ie, insecurely attached young people), the odds of persisting ruminative anxiety (worry) decreased with each additional copy of the S-allele (approximately 30% per allele: OR 0.77, 95% CI 0.62-0.97, P=0.029). Within risk settings for binge drinking (ie, securely attached young people), the odds of reporting persisting high-dose alcohol consumption (bingeing) decreased with each additional copy of the S-allele (approximately 35% per allele: OR 0.74, 95% CI 0.64-0.86, P<0.001). Our data suggest that the S-allele is likely to be important in psychosocial development, particularly in those settings that increase risk of anxiety and alcohol use problems.

Iron uptake and storage are tightly regulated to guarantee sufficient iron for essential cellular processes and to prevent the production of damaging free radicals. The placenta is the entry site for iron, which is delivered to the developing embryo. Iron is taken up by syncytiotrophoblast cells and is transported unidirectionally from mother to fetus against a concentration gradient. Several iron transporters and regulators were recently characterized, including DMT1 and ferroportin/Ireg1 that transport iron through membranes, and HFE that regulates TfR-mediated iron uptake. In this study we demonstrate that in a differentiated choriocarcinoma cell line BeWo, HFE, and TfR are localized mainly in recycling endosomes and a small percentage of these complexes is observed in late endosomes with DMT1 while in trophoblast cells, the level of TfR is significantly lower and it is detected with HFE and DMT1 mainly in late endosomes. Most interestingly, TfR and HFE, as well as TfR and DMT1 interact in placental trophoblast cells. Based on previous and these data we suggest that the level of intracellular iron may regulate both TfR expression (on the post-transcriptional and the post-translational levels) and TfR trafficking/transcytosis in polarized cells.

Cytokines are integral to the development of anaemia of chronic inflammation. Cytokines modulate hepcidin expression and iron sequestration by the reticuloendothelial system but their direct effects on small bowel iron transport are not well characterized. The aim of the present study was to examine the local effects of TNFalpha (tumour necrosis factor alpha) on small bowel iron transport and on iron transporter expression in the absence of hepcidin. The effects of TNFalpha on iron transport were determined using radiolabelled iron in an established Caco-2 cell model. The effect of TNFalpha on the expression and localization of the enterocyte iron transporters DMT-1 (divalent metal transporter 1), IREG-1 (iron-regulated transporter 1) and ferritin was determined utilizing Caco-2 cells and in a human ex vivo small bowel culture system. TNFalpha mediated an early induction in both iron import and iron export, which were associated with increased DMT-1 and IREG-1 mRNA and protein expression (P<0.05). However, by 24 h, both iron import and iron export were significantly inhibited, coinciding with an induction of ferritin heavy chain (P<0.05) and a decrease in DMT-1 and IREG-1 to baseline levels. In addition, there was a relocalization of IREG-1 away from the basolateral cell border and increased iron deposition in villous enterocytes. In conclusion, TNFalpha has a direct effect on small bowel iron transporter expression and function, leading to an inhibition of iron transport.

<h4>Background</h4>Sepsis, iron loading, and aging cause independent increases in gut epithelial and splenic apoptosis. It is unknown how their combination will affect apoptosis and systemic cytokine levels.<h4>Materials and methods</h4>Hfe-/- mice (a murine homologue of hemochromatosis) abnormally accumulate iron in their tissues. Aged (24-26 months) or mature (16-18 months) Hfe-/- mice and wild type (WT) littermates were subjected to cecal ligation and puncture (CLP) or sham laparotomy. Intestine, spleen, and blood were harvested 24 h later and assessed for apoptosis and cytokine levels.<h4>Results</h4>Gut epithelial and splenic apoptosis were low in both aged septic and sham Hfe-/- mice, regardless of the amount of iron in their diet. Mature septic WT mice had increased apoptosis compared to age-matched sham WT mice. Mature septic Hfe-/- mice had similar levels of intestinal cell death to age-matched septic WT mice but higher levels of splenic apoptosis. Apoptosis was significantly lower in septic aged Hfe-/- mice than septic mature Hfe-/- animals. Interleukin-6 was elevated in septic aged Hfe-/- mice compared to sham mice.<h4>Conclusions</h4>Although sepsis, chronic iron dysregulation, and aging each increase gut and splenic apoptosis, their combination yields cell death levels similar to sham animals despite the fact that aged Hfe-/- mice are able to mount an inflammatory response following CLP and mature Hfe-/- mice have elevated sepsis-induced apoptosis. Combining sepsis with two risk factors that ordinarily increase cell death and increase mortality in CLP yields an apoptotic response that could not have been predicted based upon each element in isolation.

Huntington's disease (HD) is caused by a polyglutamine expansion in the protein huntingtin and is characterized by intraneuronal inclusions and widespread neuronal death at the late stage of the disease. In research, most of the emphasis has been on understanding the cell death and its mechanisms. Until recently, it was believed that the vast majority, if not all, of the symptoms in HD are a direct consequence of neurodegeneration. However, increasing evidence shows that subtle alterations in synaptic function could underlie the early symptoms. It is of particular interest to understand the nature of this neuronal dysfunction. Normal huntingtin interacts with various cytoskeletal and synaptic vesicle proteins that are essential for exocytosis and endocytosis. Altered interactions of mutant huntingtin with its associated partners could contribute to abnormal synaptic transmission in HD. This review describes recent advances in understanding synaptic dysfunction in HD.

Three microcolin A and B analogs have been synthesized. Their biological activity profiles were evaluated against several cell lines, revealing the existence of a structural determinant whose role in mediating the antiproliferative effect of the microcolins has heretofore gone unrecognized. While previously described as potent immunosuppressive natural products, we found that these microcolin analogs possessed no selective cytotoxicity when comparing immune cell versus nonimmune cell proliferation. In addition, the amino-terminus of microcolin A has been modified to incorporate a biotinylated polyethylene glycol linker. The biological activity of this biotinylated microcolin A analog was determined. This affinity reagent was shown to retain limited biological activity and thus can serve as a useful probe for elucidating the mechanism of action of microcolin A.

Huntington's disease can be used as a model to study neurodegenerative disorders caused by aggregation-prone proteins. It has been proposed that the entrapment of transcription factors in aggregates plays an important role in pathogenesis. We now report that the transcriptional activity of CBP is already repressed in the early time points by soluble mutant huntingtin, whereas the histone acetylase activity of CBP/p300 is gradually diminished over time. Mutant huntingtin bound much stronger to CBP than normal huntingtin, possibly contributing to repression. Especially at the later time points, CBP protein level was gradually reduced via the proteasome pathway. In sharp contrast, p300 was unaffected by mutant huntingtin. This selective degradation of CBP was absent in spinocerebellar ataxia 3. Thus, mutant huntingtin specifically affects CBP and not p300 both at the early and later time points, via multiple mechanisms. In addition to the reduction of CBP, also the altered ratio of these closely related histone acetyltransferases may affect chromatin structure and transcription and thus contribute to neurodegeneration.

Periventricular heterotopia (PH) is clinically diagnosed on the basis of the radiographic characteristics of heterotopic nodules composed of disorganized neurons along the lateral ventricles of the brain. Epilepsy is the main presenting symptom of patients with PH. Behaviorally, patients generally are of normal intelligence, although there have been associated findings of learning disabilities, namely, dyslexia. Two genes responsible for PH have been identified: FilaminA, which encodes for the protein filamin A, and ARFGEF2, which encodes for the vesical transport-regulating protein ARFGEF2. The much more common X-linked dominant form of this disorder is due to filamin A, affects females, and is typically lethal in males. A much rarer autosomal recessive form due to ARFGEF2 mutations leads to microcephaly and developmental delay in addition to PH. Cell motility, adhesion defects, and weakening along the neuroepithelial lining may result from defects in these genes during cortical development and contribute to PH, but the mechanisms are not clear yet. Treatment of PH is largely symptomatic, following basic principles for epilepsy management and genetic counseling.

Axon navigation relies on the competence of growth cones to sense and interpret attractive and repulsive guidance cues present along their trajectory. For most neurons, this process is mediated by a limited number of conserved families of ligand-receptor signaling systems, including Ephrin/Eph, Netrins/DCC-Unc5, Slits/Robo, and Semaphorins/Plexin-Neuropilin. Recent studies have demonstrated that some neurons respond also to well-known secreted signaling molecules, best known for their roles as morphogens, such as BMP7, SHH, FGF8, and Wnt. Thus, retina ganglion cell axon navigation is influenced by FGF, SHH, and possibly BMP signaling. Similarly, commissural neurons in the spinal cord respond sequentially to the activity of BMP, SHH, and Wnt to extend toward and away from their intermediate target, the floor plate. The data that support this conclusion will be summarized and how morphogens may signal at the growth cone will be discussed.

To identify the molecular changes that occur in non-small cell lung carcinoma (NSCLC), we compared the gene expression profile of the NCI-H292 (H292) NSCLC cell line with that of normal human tracheobronchial epithelial (NHTBE) cells. The NHTBE cells were grown in a three-dimensional organotypic culture system that permits maintenance of the normal pseudostratified mucociliary phenotype characteristic of bronchial epithelium in vivo. Microarray analysis using the Affymetrix oligonucleotide chip U95Av2 revealed that 1,683 genes showed a >1.5-fold change in expression in the H292 cell line relative to the NHTBE cells. Specifically, 418 genes were downregulated and 1,265 were upregulated in the H292 cells. The expression data for selected genes were validated in several different NSCLC cell lines using quantitative real-time PCR and Western analysis. Further analysis of the differentially expressed genes indicated that WNT responses, apoptosis, cell cycle regulation and cell proliferation were significantly altered in the H292 cells. Functional analysis using fluorescence-activated cell sorting confirmed concurrent changes in the activity of these pathways in the H292 line. These findings show that (1) NSCLC cells display deregulation of the WNT, apoptosis, proliferation and cell cycle pathways, as has been found in many other types of cancer cells, and (2) that organotypically cultured NHTBE cells can be used as a reference to identify genes and pathways that are differentially expressed in tumor cells derived from bronchogenic epithelium.

Hereditary hemochromatosis (HH) is a common cause of primary iron overload induced by genetic impairment of iron metabolism. More than 80% of HH patients in populations of European origin are homozygotes for a single mutation C282Y, or compound heterozygotes for C282Y and H63D mutations in the HFE gene. However, in the majority of Asian, African, Australasian, and Amerindian populations, frequencies of C282Y are close to zero. Data on the prevalence of HFE mutations in Russian population and in Russian patients with HH are very limited. In this work, we determined frequencies of C282Y and H63D in ethnical Russians living in the Central European region of Russia. Furthermore, we tested whether homozygocity for C282Y is the major cause of HH in Russians. We found that, in the Russian population, the frequency of C282Y mutation in the HFE gene is relatively high and corresponds to mean European levels. However, in contrast to the majority of European populations, homozygocity for C282Y is found only in a small proportion (5%) of patients with biochemical and clinical signs of HH. These data suggest that either the penetrance of C282Y in Russia is lower than in Western countries, or that a more frequent non-HFE dependent mechanism of primary iron overload dominates in Russian population.

<h4>Background</h4>Considerable evidence indicates that serotonergic mechanisms, particularly the serotonin transporter (5-HTT) may be involved in psychological alterations. Recent findings have demonstrated that depression and stress are influenced by polymorphism of the promoter region of 5-HTT (5-HTTLPR) and that the short allele (S) is associated with reduced transcriptional efficiency resulting in reduced serotonin expression and uptake. As psychological and genetic factors have been implicated in the pathogenesis of recurrent aphthous stomatitis (RAS), the purpose of the present study was to investigate 5-HTTLPR polymorphism in patients with RAS compared with control subjects.<h4>Methods</h4>Sixty-nine consecutive subjects affected by minor and major forms of RAS and 70 healthy volunteers were genotyped at 5-HTTLPR. The chi-square test was used for statistical analysis.<h4>Results</h4>A significant increase in the genotype of SS (P = 0.05) and of the allele S (P = 0.04) in the group of RAS were observed.<h4>Conclusion</h4>Our findings demonstrate that RAS patients have a tendency to show polymorphism associated with anxiety-related traits.

<h4>Background and aims</h4>Pancreatic cancer remains a devastating diagnosis with only limited therapeutic options. Specific inhibition of expression of target genes has become possible using small interfering (si) RNAs. We therefore investigated how far siRNA specific for bcl-2 may serve as a therapeutic option for pancreatic cancer in vitro and in vivo.<h4>Methods</h4>siRNAs targeting two different regions in the bcl-2 gene were transfected to YAP C and DAN G pancreatic carcinoma cells and human foreskin fibroblasts. Permutations were generated by changing 3' and 5' overhangs and varying the length of the paired RNA duplex. Transfection efficacy was determined using FITC labelled siRNAs and fluorescence microscopy. Cell survival and apoptosis were quantified at 24-120 hours. Pancreatic cancer xenografts in male nude mice were treated intraperitoneally with siRNAs daily for 24 days. siRNA pharmacokinetics in vivo were assessed using radioactively labelled siRNAs. Total protein and RNA were extracted for western Blot analysis and quantitative polymerase chain reaction.<h4>Results and conclusions</h4>Bcl-2 specific siRNAs specifically inhibited expression of the target gene in vitro and in vivo. Antiproliferative and proapoptotic effects were observed in tumour cells but not in fibroblasts or non-malignant tissues. siRNA permutations and diverse overhangs influenced gene silencing efficacy. siRNA was quickly distributed to all organs and excreted via the kidney and liver. Bcl-2 specific siRNA is a promising adjunctive treatment for pancreatic carcinoma.

<h4>Background and aims</h4>The aims of the present study were to determine: (i) whether alcohol consumption is greater in individuals with HFE mutations; and (ii) whether common HFE mutations modify the effects of alcohol on serum iron and liver biochemistry or morbidity.<h4>Methods</h4>The residents of the town of Busselton in Western Australia were subject to cross-sectional health surveys between 1966 and 1983. In 1994/1995 all surviving participants of the earlier surveys were invited to take part in a follow-up survey. Logistic, linear and Poisson log-linear regression analyses were performed in 1490 men and 1452 women from the 1994/1995 survey to assess the relationships between HFE mutations, alcohol, iron levels, liver biochemistry and morbidity.<h4>Results</h4>Heavy or moderate alcohol consumption was present in 7% or 36% of men and 0.5% or 12% of women, respectively. Alcohol consumption strongly influenced levels of serum ferritin and gamma glutamyl transpeptidase (GGT) and mean cell volume (MCV) in men and women but only alanine aminotransferase (ALT) levels in women. These effects were independent of HFE gene mutations. Hospital admission rates for respiratory disorders were higher in men with the C282Y mutation.<h4>Conclusions</h4>Alcohol consumption strongly influences serum ferritin and GGT levels and MCV in men and women but only ALT levels in women, and these effects are independent of HFE mutations. HFE gene mutations do not predispose to moderate or heavy alcohol consumption. The C282Y mutation is associated with increased respiratory admission rates in men.

<h4>Objective</h4>To evaluate the association between a serotonin (5-HT) transporter gene polymorphism and migraines in Koreans.<h4>Background</h4>The 5-HT metabolism is believed to play an important part in the pathophysiology of migraines, and a polymorphism of the serotonin transporter (5-HTT) promoter region might influence 5-HTT expression and serotonin uptake.<h4>Methods</h4>This study compared 52 patients with migraine and 170 healthy control subjects in order to determine the association between a polymorphism of the 5-HTT gene-linked polymorphic region (5-HTTLPR) and migraine. Using PCR techniques, the products of the 484-base pair (bp) fragments were denoted as being short alleles (s) and those of 528 bp as being long (l).<h4>Results</h4>The genotype distribution of the healthy controls was s/s (56.5%), s/l (38.2%), and l/l (5.3%) and that of the patients with migraine was 65.4%, 30.8%, and 3.8%, respectively. No significant differences were noted in the genotype and allele frequencies of 5-HTTLPR between the patients with migraine and the control subjects.<h4>Conclusions</h4>A 5-HTTLPR polymorphism does not appear to be involved in the genetic predisposition to migraines.

The serotonin transporter (5-HTT) regulates serotonin transmission and modulates behavioral effects of drug of abuse. A polymorphism in the promoter region of the serotonin transporter gene (5-HTTLPR) yielding a short (S) and long (L) allele has been associated with severity of substance abuse. The aims of the study were to investigate whether 5-HTTLPR genotypes differed in their response to treatment in cocaine- and alcohol-abusing patients. Polymerase chain reaction-based genotyping of a 44 base pair insertion/deletion polymorphism was performed in 141 African American cocaine-dependent patients with concurrent alcohol use who were entering a 12-week behaviorally oriented outpatient treatment program. In treatment, end of treatment and 6-month follow-up outcome measures included changes in Addiction Severity Index (ASI) scores, urine drug screens, days in treatment, individual/group sessions, dropout and completion rates. As expected, there was a reduction in substance abuse by the end of treatment and follow-up (F = 5.15, p = 0.000). However, there were no differences in the reduction in cocaine use across the LL, LS and SS genotypes. Interestingly, individuals with the S allele showed greater severity of alcohol use at admission (F = 4.84, p = 0.03), and the SS genotype showed less improvement in alcohol measures than the LL at follow-up (F = 3.68, p = 0.03), after controlling for baseline variables. While we found no association of the 5-HTTLPR variants with severity of cocaine abuse or any cocaine-related outcome measures, the data suggested that the 5-HTTLPR polymorphism may distinguish responders from non-responders to behavioral treatment in terms of alcohol use. Further investigations are required to determine the role of the 5-HTTLPR polymorphism in influencing treatment - outcome among substance abusers.

<h4>Background</h4>Hereditary hemochromatosis is an autosomal recessive disorder of iron metabolism that is characterized by excess accumulation of iron in various organs and often leads to diabetes mellitus (DM). To study whether mutations in the hemochromatosis gene (HFE) could be a risk factor for the development of gestational diabetes mellitus (GDM), the prevalence of HFE mutations in patients with GDM was compared to that of healthy pregnant controls.<h4>Methods</h4>GDM was diagnosed in 208 of 2,421 pregnant woman screened between the 24th and 28th week of gestation over a period of 18 months. Patients and 170 matched control subjects were screened for the HFE gene mutations C282Y and H63D.<h4>Results</h4>In North and Central European GDM patients, the allele frequency of the C282Y mutation (7.7%) was higher than in pregnant controls (2.9%; p = 0.04), while the frequency of the H63D mutation was not different (p = 0.45). Three patients with GDM were homozygous for H63D (3.1%), 1 patient was homozygous for C282Y (1.0%), 2 patients were compound heterozygous (2.0%) and 26 were heterozygous [11 C282Y (11.2%) and 15 H63D (15.3%)]. C282Y and H63D allele frequencies were not different between controls and GDM patients of Southern European or non-European origin. Irrespective of the HFE-mutation status, serum ferritin levels were increased in patients with GDM compared to healthy pregnant controls (p = 0.01), while transferrin saturation was similar in both groups.<h4>Conclusions</h4>In North and Central European patients with GDM, the C282Y allele frequency is higher than in healthy pregnant women, suggesting a genetic susceptibility to the development of GDM.

<h4>Purpose</h4>Birdshot retinochoroidopathy (BSCR) is a rare posterior uveitis characterized by distinctive, multiple, hypopigmented choroidal and retinal lesions. At least 96% of patients, if not all, share the major histocompatibility antigen HLA-A29. Although it was hypothesized earlier that more frequently the A*2902 subtype was closely associated with BSCR, new patients were found to share the A*2901 subtype and were further investigated. The present study was designated to check patients' HLA-A*2901 subtyping and the polymorphisms available in the HLA region in patients and control subjects sharing the A*2901 and A*2902 subtypes.<h4>Methods</h4>HLA-A29 was assessed and subtyped by molecular biology. cDNA from one patient (HLA-A*2901) was sequenced. A29.1 antigenic expression on peripheral blood lymphocytes was checked by microlymphocytotoxicity (MLCT). Four homozygous A29.2 and 4 heterozygous A29.2 patients, 3 homozygous A29.2 healthy subjects, 3 heterozygous A29.1 patients, and 11 heterozygous A29.1 healthy subjects were tested for the microsatellite alleles MOGa, -b, -c, and e (of the myelin oligodendrocyte glycoprotein [MOG]gene), D6S265, D6S510, RF, C5_4_5, D6S105, and D6S276 and the mutation H63D of the familial hemochromatosis gene (HFE).<h4>Results</h4>The patients' cDNA sequences and MLCT reactivities of HLA-A29.1 subtypes were found to be identical with published data from healthy individuals. Surprisingly, though A*2901 and A*2902 differed only by a single mutation (G376C/ D102H) two strong A*2901 and A*2902 complotypes were observed in patients and control subjects, the polymorphisms being identical at all loci near HLA-A, whereas more distant loci exhibited some diversity.<h4>Conclusions</h4>Susceptibility to BSCR thus appeared to be located between the left and right remote markers C5_4_5 and D6S276, if not relying on the HLA-A29 molecule itself.

Results from studies investigating the relationship between colorectal cancer survival and chromosome 18q allelic imbalance (AI)/loss of DCC expression (LOE) have been inconsistent. We have reviewed and pooled published studies to estimate the prognostic significance of chromosome 18q status more precisely. Data from 27 studies were eligible. Survival data were pooled using standard meta-analysis techniques. Considerable variation between assessment method, marker choice, and threshold for assigning AI/LOE was observed. Pooling data from a 2189 cases from 17 studies showed significantly worse overall survival in patients with AI/LOE (HR = 2.00, 95%CI: 1.49-2.69), maintained both in the adjuvant setting (HR = 1.69, 95%CI:1.13-2.54), and also by method (HR = 1.67, 95%CI: 1.19-2.36, genotyping microsatellites; HR = 3.00, 95%CI: 1.98-4.56, immunohistochemistry). There was however evidence of heterogeneity and publication bias. Cancers with chromosome 18q loss appear to have a poorer prognosis. Prospective studies using consistent methodology are needed to precisely quantify its effect and role in patients with stage II-III disease.

<h4>Aim</h4>To identify chromosomal translocations specific to gastric cancer (GC), spectral karyotyping (SKY) analysis was performed on established cell lines and cancerous ascitic fluids.<h4>Methods</h4>SKY analysis of 10 established cell lines and seven cancerous ascitic fluid samples identified recurrent chromosomal breakpoints and translocations in GC, several of which involved chromosomal loci of oncogenes or tumor suppressor genes.<h4>Results</h4>A total of 630 chromosomal breaks were identified. Chromosome no.8 was the most frequently involved in rearrangements (65 breaks), followed by chromosomes no.11 (53), no. 1 (49), no. 7 (46), no. 13 (37), no. 3 (36), no. 17 (33), and no. 20 (29). Frequent breakpoints were detected in 8q24.1 (30 breaks), 11q13 (29), 13q14 (16), 20q11.2 (14), 7q32 (13), 17q11.2 (13), 18q21 (12), 17q23 (9), 18q11.2 (9). SKY analysis identified a total of 242 chromosomal rearrangements including 190 reciprocal and non-reciprocal translocations. The recurrent combinations of chromosomal bands involved in translocations were 8q24.1 and 13q14 (3 cases), 8q24.1 and 11q13 (3), 11q13 and 17q11.2 (2), and 18q11.2 and 20q11.2 (2). Our study validated the ability of SKY to characterize in detail the chromosomal rearrangements in solid tumors and derived cell lines. Moreover, fluorescence in situ hybridization helped to identify the insertions, translocations, and homogeneously staining regions of MYC and CCND1 gene loci.<h4>Conclusion</h4>The non-random co-localization of certain cytogenetic bands suggests the importance of chromosomal translocations in gastric carcinogenesis, by serving as landmarks for the cloning of GC causing genes.

During mammalian sexual development, the SOX9 transcription factor up-regulates expression of the gene encoding anti-Müllerian hormone (AMH), but in chickens, Sox9 gene expression reportedly occurs after the onset of Amh expression. Here, we examined expression of the related gene Sox8 in chicken embryonic gonads during the sex-determining period. We found that cSox8 is expressed at similar levels in both sexes at embryonic day 6 and 7, and only at the anterior tip of the gonad, suggesting that SOX8 is not responsible for the sex-specifi c increase in cAmh gene expression at these stages. We also found that several other chicken Sox genes (cSox3, cSox4 and cSox11) are expressed in embryonic gonads, but at similar levels in both sexes. Our data suggest that the molecular mechanisms involved in the regulation of Amh genes of mouse and chicken are not conserved, despite similar patterns of Amh expression in both species.

Hereditary hemochromatosis (HH) is a genetic iron overload disease, in the majority of cases associated with homozygosity for the C282Y mutation of the HFE gene. In spite of this genetic homogeneity, there is a great clinical heterogeneity among HH patients. Low CD8(+) lymphocyte numbers have been associated with a more severe expression of iron overload in HH patients, and in experimental models of iron overload. HH patients present low serum transferrin levels. Transferrin is an indispensable resource for lymphopoiesis. Lymphocyte homeostasis follows general ecology rules of population dynamics that involve competition for limiting resources. In the present study, we questioned whether transferrin levels could be associated with the anomalies seen previously in lymphocyte subset numbers in HH patients. Transferrin levels, total and subset T lymphocyte counts were done in 426 apparently healthy subjects genotyped for HFE. All HFE C282Y carriers presented significantly lower serum transferrin levels than the wild type group, a difference that could not be explained solely by the degree of iron overload. Significant differences were also seen in transferrin levels between males and females, with females presenting higher average serum Transferrin levels. In the population of subjects with Transferrin levels lower than 248 mg/dl, a positive correlation was seen between the peripheral CD8(+) lymphocyte numbers and serum transferrin levels (R(2) = 2.41; r = 0.16; P = 0.018). To test the possible limiting resource effect of transferrin, the correlation between transferrin levels and CD8(+) lymphocyte numbers was scrutinized in 34 HH patients, homozygous for the C282Y mutation. In the homozygous males, where the lowest average transferrin levels were seen, another highly significant correlation was observed between Transferrin levels and CD8(+) numbers. This correlation points to a possible role of transferrin as a limiting resource for MHC class I dependent lymphocyte proliferation, an effect that was not observed in C282Y homozygous female patients.

African swine fever virus (ASFV) infection leads to rearrangement of vimentin into a cage surrounding virus factories. Vimentin rearrangement in cells generally involves phosphorylation of N-terminal domains of vimentin by cellular kinases to facilitate disassembly and transport of vimentin filaments on microtubules. Here, we demonstrate that the first stage in vimentin rearrangement during ASFV infection involves a microtubule-dependent concentration of vimentin into an "aster" within virus assembly sites located close to the microtubule organizing center. The aster may play a structural role early during the formation of the factory. Conversion of the aster into a cage required ASFV DNA replication. Interestingly, viral DNA replication also resulted in the activation of calcium calmodulin-dependent protein kinase II (CaM kinase II) and phosphorylation of the N-terminal domain of vimentin on serine 82. Immunostaining showed that vimentin within the cage was phosphorylated on serine 82. Significantly, both viral DNA replication and Ser 82 phosphorylation were blocked by KN93, an inhibitor of CaM kinase II, suggesting a link between CaM kinase II activation, DNA replication, and late gene expression. Phosphorylation of vimentin on serine 82 may be necessary for cage formation or may simply be a consequence of activation of CaM kinase II by ASFV. The vimentin cage may serve a cytoprotective function and prevent movement of viral components into the cytoplasm and at the same time concentrate late structural proteins at sites of virus assembly.

It is increasingly accepted that steroidal receptor co-regulators may also function in the cytoplasmic compartment. Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) is a novel coregulator that plays a role in both the genomic and extranuclear actions of estrogen receptors (ER) in hormonally responsive tissues. In this study using breast tumor arrays, we found that PELP1 was localized only in the cytoplasm in 58% of the PELP1-positive breast tumors. To help explain the significance of the cytoplasmic localization of PELP1 in human breast tumors, we created a mutant protein that was expressed only in the cytoplasm (PELP1-cyto) and then generated a model system wherein MCF-7 breast cancer cells were engineered to specifically express this mutant. We found that PELP1-cyto cells were hypersensitive to estrogen but resistant to tamoxifen. PELP1-cyto cells, but not parental MCF-7 cells, formed xenograft tumors in nude mice. In addition, PELP1-cyto cells exhibited increased association of PELP1 with Src, enhanced mitogen-activated protein kinase (MAPK) activation, and constitutive activation of AKT. The altered localization of PELP1 was sufficient to trigger the interaction of PELP1 with the p85 subunit of phosphatidylinositol-3-kinase (PI3K), leading to PI3K activation. In addition, PELP1 interacted with epidermal growth factor receptors and participated in growth factor-mediated ER transactivation functions. Our results suggest that the altered localization of PELP1 modulates sensitivity to antiestrogens, potentiates tumorigenicity, presumably via the stimulation of extranuclear estrogen responses, such as the activation of MAPK and AKT, and also enhance cross-regulation of ER transactivation activity by growth factors.

<h4>Background</h4>MDM2 acts as a principal regulator of the tumour suppressor p53 by targeting its destruction through the ubiquitin pathway. A polymorphism in the MDM2 promoter (SNP309) was recently identified. SNP309 was shown to result, via Sp1, in higher levels of MDM2 RNA and protein, and subsequent attenuation of the p53 pathway. Furthermore, SNP309 was proposed to be associated with accelerated soft tissue sarcoma formation in both hereditary (Li-Fraumeni) and sporadic cases in humans.<h4>Methods</h4>We evaluated the possible contribution of SNP309 to three tumour types known to be linked with the MDM2/p53 pathway, using genomic sequencing or restriction fragment length polymorphism as screening methods. Three separate Finnish tumour materials (population based sets of 68 patients with early onset uterine leiomyosarcomas and 1042 patients with colorectal cancer, and a series of 162 patients with squamous cell carcinoma of the head and neck) and a set of 185 healthy Finnish controls were analysed for SNP309.<h4>Results</h4>Frequencies of SNP309 were similar in all four cohorts. In the colorectal cancer series, SNP309 was somewhat more frequent in women and in patients with microsatellite stable tumours. Female SNP309 carriers were diagnosed with colorectal cancer approximately 2.7 years earlier than those carrying the wild type gene. However, no statistically significant association of SNP309 with patients' age at disease onset or to any other clinicopathological parameter was found in these three tumour materials.<h4>Conclusion</h4>SNP309 had no significant contribution to tumour formation in our materials. Possible associations of SNP309 with microsatellite stable colorectal cancer and with earlier disease onset in female carriers need to be examined in subsequent studies.

We compiled sequences of previously published aberrant 3' splice sites (3'ss) that were generated by mutations in human disease genes. Cryptic 3'ss, defined here as those resulting from a mutation of the 3'YAG consensus, were more frequent in exons than in introns. They clustered in approximately 20 nt region adjacent to authentic 3'ss, suggesting that their under-representation in introns is due to a depletion of AG dinucleotides in the polypyrimidine tract (PPT). In contrast, most aberrant 3'ss that were induced by mutations outside the 3'YAG consensus (designated 'de novo') were in introns. The activation of intronic de novo 3'ss was largely due to AG-creating mutations in the PPT. In contrast, exonic de novo 3'ss were more often induced by mutations improving the PPT, branchpoint sequence (BPS) or distant auxiliary signals, rather than by direct AG creation. The Shapiro-Senapathy matrix scores had a good prognostic value for cryptic, but not de novo 3'ss. Finally, AG-creating mutations in the PPT that produced aberrant 3'ss upstream of the predicted BPS in vivo shared a similar 'BPS-new AG' distance. Reduction of this distance and/or the strength of the new AG PPT in splicing reporter pre-mRNAs improved utilization of authentic 3'ss, suggesting that AG-creating mutations that are located closer to the BPS and are preceded by weaker PPT may result in less severe splicing defects.

Hepcidin is the principal iron-regulatory hormone. It acts by binding to the iron exporter ferroportin, inducing its internalization and degradation, thereby blocking cellular iron efflux. The bioactive 25 amino acid (aa) peptide has a hairpin structure stabilized by 4 disulfide bonds. We synthesized a series of hepcidin derivatives and determined their bioactivity in a cell line expressing ferroportin-GFP fusion protein, by measuring the degradation of ferroportin-GFP and the accumulation of ferritin after peptide treatment. Bioactivity was also assayed in mice by the induction of hypoferremia. Serial deletion of N-terminal amino acids caused progressive decrease in activity which was completely lost when 5 N-terminal aa's were deleted. Synthetic 3-aa and 6-aa N-terminal peptides alone, however, did not internalize ferroportin and did not interfere with ferroportin internalization by native hepcidin. Deletion of 2 C-terminal aa's did not affect peptide activity. Removal of individual disulfide bonds by pairwise substitution of cysteines with alanines also did not affect peptide activity in vitro. However, these peptides were less active in vivo, likely because of their decreased stability in circulation. G71D and K83R, substitutions previously described in humans, did not affect hepcidin activity. Apart from the essential nature of the N-terminus, hepcidin structure appears permissive for mutations.

Chondrocalcinosis can be associated with hyperparathyroidism, hemochromatosis, hypophosphatasia, and hypomagnesemia. Gitelman syndrome (GS), an inherited disorder due to loss of function mutations of the gene encoding the distal convoluted tubule Na-Cl cotransporter (NCCT), is characterized by hypokalemia metabolic alkalosis, hypomagnesemia, and hypocalciuria. A 53-year-old man, with history of recurrent joint effusions and pains affecting knees and wrists, had transient episodes of muscle pain, weakness, cramping, and fatigue over a one-year period. Laboratory tests showed hypokalemia, metabolic alkalosis, hypocalciuria, and hypomagnesemia related to genetically proven GS. Radiographs of affected joints revealed calcium pyrophosphate dihydrate deposition. This observation points out the necessity to look for Mg depletion (and especially GS) in the biological investigation of chondrocalcinosis. Additionally, the association between GS (NCCT inactivation) and high bone mineral density provides a new insight into the possible role of thiazides in osteoporosis management.

In this study, 61 drug-resistant Streptococcus pneumoniae strains were characterized by multilocus sequence typing (MLST). These strains were representatives of 26 major clones (defined using pulsed-field gel electrophoresis) accounting for 93% of the 1,285 drug-resistant Streptococcus pneumoniae isolates recovered from the nasopharynges of healthy children attending day-care centers in Lisbon during 2001 to 2003. Using MLST, 13 of the 26 clones were found to be identical or closely related to 11 Pneumococcal Molecular Epidemiology Network (PMEN) clones, 4 clones were found to be unique as there were no identical or highly related allelic profiles deposited in the MLST database, and the remaining 9 clones had sequence types that matched or differed at a single or double locus from allelic profiles available in the MLST database. These nine clones were of serotypes 33F, 10A, 19A, 19F, 6A, 20, 24F, and 3, one was nontypeable, and, by MLST, they were found to be identical or highly related to isolates from disease origin that were dispersed internationally. Since the majority of these clones had serotypes that are not included in the 7-valent conjugate pneumococcal vaccine, monitoring of these clones is important for surveying their possible spread in the future. We propose the inclusion of these novel international clones in the PMEN.

<h4>Objective</h4>To observe the effect of the antithrombin-III (AT-III) on cytokine levels in multiple organ dysfunction syndrome (MODS) in rats.<h4>Methods</h4>Seventy Wistar rats were randomly divided into three groups: normal control group, MODS group, and AT-III treatment group. The rats in MODS group and the treatment group were given lipopolysaccharide (LPS) two times to replicate systemic inflammatory response syndrome (SIRS)/MODS model. One hour after the injections of LPS, the rats in the treatment group were given AT-III (25 U/kg, 0.5 ml/100 g) intravenously. Four hours after treatment, blood samples were collected in rats. The tissue samples were collected and preserved in formalin. By use of radioimmunoassay and histopathology, the effect of the AT-III was observed in rats with SIRS/MODS.<h4>Results</h4>Endotoxin (ET), interleukin-1 (IL-1), IL-6 and tumor necrosis factor-alpha (TNF-alpha) levels were all increased significantly in MODS animals (all P<0.01), and they were decreased after AT-III treatment (all P<0.01), though still higher than those of the normal controls (all P<0.01). The pathologic changes in lung, kidney and liver in the treatment group were milder than in the MODS group.<h4>Conclusion</h4>These data suggest that AT-III could inhibit systemic inflammatory response and it might be used in the treatment of SIRS/MODS.

The anti-arrhythmic agent amiodarone (AD) is associated with numerous adverse effects, but serious liver disease is rare. The improved safety of administration of daily low doses of AD has already been established and this regimen is used for long-term medication. Nevertheless, asymptomatic continuous liver injury by AD may increase the risk of step-wise progression of non-alcoholic fatty liver disease. We present an autopsy case of AD-induced liver cirrhosis in a patient who had been treated with a low dose of AD (200 mg/d) daily for 84 mo. The patient was a 85-year-old male with a history of ischemic heart disease. Seven years after initiation of treatment with AD, he was admitted with cardiac congestion. The total dose of AD was 528 g. Mild elevation of serum aminotransferase and hepatomegaly were present. Liver biopsy specimens revealed cirrhosis, and under electron microscopy numerous lysosomes with electron-dense, whorled, lamellar inclusions characteristic of a secondary phospholipidosis were observed. Initially, withdrawal of AD led to a slight improvement of serum aminotransferase levels, but unfortunately his general condition deteriorated and he died from complications of pneumonia and renal failure. Long-term administration of daily low doses of AD carries the risk of progression to irreversible liver injury. Therefore, periodic examination of liver function and/or liver biopsy is required for the management of patients receiving long-term treatment with AD.

The hematopoietic system is an invaluable model both for understanding basic developmental biology and for developing clinically relevant cell therapies. Using highly purified cells and rigorous microarray analysis we have compared the expression pattern of three of the most primitive hematopoietic subpopulations in adult mouse bone marrow: long-term hematopoietic stem cells (HSC), short-term HSC, and multipotent progenitors. All three populations are capable of differentiating into a spectrum of mature blood cells, but differ in their self-renewal and proliferative capacity. We identified numerous novel potential regulators of HSC self-renewal and proliferation that were differentially expressed between these closely related cell populations. Many of the differentially expressed transcripts fit into pathways and protein complexes not previously identified in HSC, providing evidence for new HSC regulatory units. Extending these observations to the protein level, we demonstrate expression of several of the corresponding proteins, which provide novel surface markers for HSC. We discuss the implications of our findings for HSC biology. In particular, our data suggest that cell-cell and cell-matrix interactions are major regulators of long-term HSC, and that HSC themselves play important roles in regulating their immediate microenvironment.

Modification of proteins with hydrophilic polymers is an effective strategy for regulation of protein pharmacokinetics. However, conjugates of slowly or non-biodegradable materials, such as poly(ethylene glycol), are known to cause long-lasting cell vacuolization, in particular in renal epithelium. Conjugates of more degradable polymers, e.g., polysaccharides, have a significant risk of immunotoxicity. Polymers that combine complete degradability, long circulation in vivo, and low immuno and chemical toxicity would be most beneficial as protein conjugate components. This study explores new fully biodegradable hydrophilic polymers, hydrophilic polyals. They are nontoxic, stable at physiological conditions, and undergo proton-catalyzed hydrolysis at lysosomal pH. The model enzyme-polyal conjugates were prepared with 61-98% yield using conventional and novel conjugation techniques and retained 90-95% of specific activity. The model conjugates showed a significant prolongation of protein circulation in rodents, with a 5-fold reduction in the renal accumulation. The data suggests that hydrophilic polyals may be useful in designing protein conjugates with improved properties.

<h4>Background and objectives</h4>Iron homeostasis is tightly regulated in mammals according to the needs of erythropoiesis and the iron stores present. This regulation is disrupted in hereditary hemochromatosis (HH), a genetic disorder characterized by increased intestinal iron absorption, leading to iron overload. The genes coding for HFE, transferrin receptor 2 (TFR2), ferroportin (SLC40A1 or FPN1), hepcidin (HEPC) and hemojuvelin (HJV or RGMC) are responsible for different types of genetic iron overload. All these genes are highly expressed in the liver and their protein products are likely components of a single hepcidin-related pathway. In order to gain insights into the molecular relationship among the HH proteins we evaluated the hepatic expression of HH genes in conditions of iron restriction or overload.<h4>Design and methods</h4>Data were obtained after phlebotomy, to activate the erythroid regulators and following parenteral iron dextran loading, to activate the store regulators, in two mice strains (C57BL/6 and DBA/2). HH genes and proteins expression were analyzed by quantitative real time polymerase chain reaction and by Western blotting, respectively.<h4>Results</h4>Hepc RNA was reduced after phlebotomy and increased in iron overload. A statistically significant reduction of hepatic Fpn1 RNA expression was observed after phlebotomy; this effect was more evident in the DBA/2 strain. Fpn1 increased in C57BL/6 mice, but not in the DBA/2 ones in parenteral iron loading. Fpn1 protein did not change substantially in either condition. Hfe, Rgmc and Tfr2 expression was not influenced by phlebotomy. In parenteral iron overload, Tfr2 gene and protein expression decreased concomitant to the increase in Hepc, while Hfe RNA remained constant.<h4>Interpretation and conclusions</h4>Our results indicate that regulation of hepatic Fpn1 differs from that reported for duodenal Fpn1. Furthermore, taken the differences in gene expression in dietary overload (increased Hfe but not Tfr2), distinct roles are suggested for Hfe and Tfr2 in Hepc activation.

The molecular mechanisms controlling the differentiation of neural progenitors into distinct subtypes of neurons during neocortical development are unknown. Here, we report that Fezl is required for the specification of corticospinal motor neurons and other subcerebral projection neurons, which are absent from Fezl null mutant neocortex. There is neither an increase in cell death in Fezl(-/-) cortex nor abnormalities in migration, indicating that the absence of subcerebral projection neurons is due to a failure in fate specification. In striking contrast, other neuronal populations in the same and other cortical layers are born normally. Overexpression of Fezl results in excess production of subcerebral projection neurons and arrested migration of these neurons in the germinal zone. These data indicate that Fezl plays a central role in the specification of corticospinal motor neurons and other subcerebral projection neurons, controlling early decisions regarding lineage-specific differentiation from neural progenitors.

<h4>Unlabelled</h4>(11)C-N,N-Dimethyl-2-(2-amino-4-methylphenylthio)benzylamine ((11)C-MADAM) is a newly synthesized radioligand with high selectivity and specificity for the serotonin transporter (5-HTT) in a monkey model. The purpose of this study in humans was to examine the suitability and potential of (11)C-MADAM for quantitative PET studies of 5-HTT in applied clinical studies on the pathophysiology and treatment of neuropsychiatric disorders.<h4>Methods</h4>PET examination was performed on each of 9 male subjects after intravenous injection of (11)C-MADAM with high specific radioactivity. Radioactive metabolites in plasma were determined with high-performance liquid chromatography. A metabolite-corrected arterial input function was used in kinetic 2- and 3-compartment analyses. Cerebellum was used as the reference region in a cross-validation of 6 reference tissue approaches.<h4>Results</h4>The highest radioactivity concentration was detected in the raphe nuclei, followed consecutively by the striatum, hippocampal complex, cingulate cortex, neocortex, and cerebellum. The time-activity curve for the fraction of unchanged (11)C-MADAM in plasma was best described by a sigmoid function. After 50 min, the fraction was 40%. The labeled metabolites were more polar than the mother compound. The compartment model approaches converged, and could describe the time-activity curves in all regions. The total volume of distribution (V(t)) was similar to the regional distribution volumes obtained by the linear graphic analysis. The binding potentials (BPs) for 6 different approaches yielded similar values in all regions but the raphe nuclei, where the 2 equilibrium methods provided lower values.<h4>Conclusion</h4>The regional binding distribution of (11)C-MADAM is consistent with postmortem data acquired with (3)H-MADAM as well as with that of other reference ligands in vitro. The time-activity curves were well described by current major quantitative approaches. The suitability of the cerebellum as a reference region for nonspecific (11)C-MADAM binding could be confirmed, thus paving the way for experimentally less demanding approaches, such as the simplified reference tissue model, for applied clinical studies.

Long-range migrating progenitor cells generate hypaxial muscle, for instance the muscle of the limbs, hypoglossal cord, and diaphragm. We show here that migrating muscle progenitors express the chemokine receptor CXCR4. The corresponding ligand, SDF1, is expressed in limb and branchial arch mesenchyme; i.e., along the routes and at the targets of the migratory cells. Ectopic application of SDF1 in the chick limb attracts muscle progenitor cells. In CXCR4 mutant mice, the number of muscle progenitors that colonize the anlage of the tongue and the dorsal limb was reduced. Changes in the distribution of the muscle progenitor cells were accompanied by increased apoptosis, indicating that CXCR4 signals provide not only attractive cues but also control survival. Gab1 encodes an adaptor protein that transduces signals elicited by tyrosine kinase receptors, for instance the c-Met receptor, and plays a role in the migration of muscle progenitor cells. We found that CXCR4 and Gab1 interact genetically. For instance, muscle progenitors do not reach the anlage of the tongue in CXCR4;Gab1 double mutants; this target is colonized in either of the single mutants. Our analysis reveals a role of SDF1/CXCR4 signaling in the development of migrating muscle progenitors and shows that a threshold number of progenitor cells is required to generate muscle of appropriate size.

<h4>Objective</h4>To investigate the frequency of HFE gene variants in patients with hepatocellular carcinoma following chronic hepatitis B and to analyze their relationships.<h4>Methods</h4>56 patients with hepatocellular carcinoma following chronic hepatitis B (HCC group) and 60 healthy blood donors (control group) were studied for the amino acid dimorphism at codon 63 (His63Asp=H63D) and codon 282 (Cys282Tyr = C282Y) of the HFE gene. The codon 63 and 282 dimorphisms were defined by PCR amplification of genomic DNA samples and restriction enzyme digestion using RsaI for C282Y and BclI for H63D. The association between hepatocellular carcinoma following chronic hepatitis B and HFE mutations were analyzed by Chi-square test.<h4>Results</h4>The genotype frequency of C2/C2 in the HCC group was markedly higher than that in the normal control group (10.7% vs 0) and there was a significant correlation between them. At the same time, the allele frequency of C2 in the HCC group was markedly higher than that in the normal control group (16.1% vs 1.7%) and there was a significant correlation between them also.<h4>Conclusion</h4>The mutation of C282Y may be related with susceptibility to HCC after chronic hepatitis B. This outcome suggests that host HFE mutation may be an important factor related to the pathogenesis of HCC.

Macroautophagy is a key pathway for the clearance of aggregate-prone cytosolic proteins. Currently, the only suitable pharmacologic strategy for up-regulating autophagy in mammalian cells is to use rapamycin, which inhibits the mammalian target of rapamycin (mTOR), a negative regulator of autophagy. Here we describe a novel mTOR-independent pathway that regulates autophagy. We show that lithium induces autophagy, and thereby, enhances the clearance of autophagy substrates, like mutant huntingtin and alpha-synucleins. This effect is not mediated by glycogen synthase kinase 3beta inhibition. The autophagy-enhancing properties of lithium were mediated by inhibition of inositol monophosphatase and led to free inositol depletion. This, in turn, decreased myo-inositol-1,4,5-triphosphate (IP3) levels. Our data suggest that the autophagy effect is mediated at the level of (or downstream of) lowered IP3, because it was abrogated by pharmacologic treatments that increased IP3. This novel pharmacologic strategy for autophagy induction is independent of mTOR, and may help treatment of neurodegenerative diseases, like Huntington's disease, where the toxic protein is an autophagy substrate.

Iron misregulation promotes oxidative stress and abnormally high iron levels have been found in the spinal cords of patients with ALS. The authors investigated whether HFE gene polymorphisms, linked to hemochromatosis, are associated with ALS using two independent populations of patients with sporadic ALS and controls (totaling 379 patients and 400 controls). They found that the H63D polymorphism is overrepresented in individuals with sporadic ALS (odds ratio 1.85, CI: 1.35 to 2.54).

Among 27 preclinical carriers of the Huntington disease mutation (PMC), the authors found normal striatal values for MRI volumetry in 88% and for fluorodesoxyglucose PET metabolic index in 67%. Raclopride PET binding potential (RAC-BP) was decreased in 50% and correlated with increases in the product of age and CAG repeat length (p < 0.0005). Dopamine D2 receptor availability measured by RAC-BP seems the most sensitive indicator of early neuronal impairment in PMC.

Chondrogenesis is an essential process in vertebrates. It leads to the formation of cartilage growth plates, which drive body growth and have primary roles in endochondral ossification. It also leads to the formation of permanent cartilaginous tissues that provide major structural support in the articular joints and respiratory and auditory tracts throughout life. Defects in chondrogenesis cause chondrodysostoses and chondrodysplasias. These skeletal malformation diseases account for a significant proportion of birth defects in humans and can dramatically affect a person's expectancy and quality of life. Chondrogenesis occurs when pluripotent mesenchymal cells commit to the chondrocyte lineage, and through a series of differentiation steps build and eventually remodel cartilage. This review summarizes and discusses our current knowledge and lack of knowledge about the chondrocyte differentiation pathway, from mesenchymal cells to growth plate and articular chondrocytes, with a main focus on how it is controlled by tissue patterning and cell differentiation transcription factors, such as, but not limited to, Pax1 and Pax9, Nkx3.1 and Nkx3.2, Sox9, Sox5 and Sox6, Runx2 and Runx3, and c-Maf.

We accurately measured the conductance responsible for the afterhyperpolarization (medium AHP) that follows a single spike in spinal motoneurons of anesthetized cats. This was done by using the dynamic-clamp method. We injected an artificial current in the neurons that increased the AHP amplitude, and we made use of the fact that the intensity of the natural AHP current at the trough of the voltage trajectory was related linearly to the AHP amplitude. We determined at the same time the conductance and the reversal potential of the AHP current. This new method was validated by a simple theoretical model incorporating AHP and hyperpolarization-activated (Ih) currents and could be applied when the decay time constant of the AHP conductance was at least five times shorter than the estimated Ih activation time. This condition was fulfilled in 33 of 44 motoneurons. The AHP conductance varied from 0.3 to 1.4 microS in both slow- and fast-type motoneurons, which was approximately the same range as the input conductance of the entire population. However, AHP and input conductances were not correlated. The larger AHP in slow-type motoneurons was mainly attributable to their smaller input conductance compared with fast motoneurons. The likeness of the AHP conductance in both types of motoneurons is in sharp contrast to differences in AHP decay time and explains why slow- and fast-type motoneurons have similar gain.

<h4>Background/aims</h4>Liver and lymph node metastasis the major prognostic factor in patients with colorectal carcinoma. The aim of this work was to search for tumor parameters which can be employed to predict whether this has occurred.<h4>Methodology</h4>A total of 211 patients with a colorectal carcinoma (Dukes' B group, 83; Dukes' C, 94; Dukes' D, 34) were investigated for 10 clinicopathological variables, as well as apoptotic activity, expression of Ki-67, p21(WAF1/CIP1), p53, bcl-2 and DCC proteins, and the c-Ki-ras mutations. Data were analyzed by univariate and multivariate statistics.<h4>Results</h4>Lymph node metastasis-predictive models were developed using the venous involvement index (the number of vascular involvements per elastica van Gieson-stained slide; Odds ratio [OR], 2.38; 95% confidence interval [CI], 1.52-3.71; p=0.0001), tumor size (OR, 0.82; 95% CI, 0.70-0.97; p=0.0179), and p21(WAF1/CIP1) immunolabeled index (the percentages of positive tumor cells; OR, 0.76; 95% CI, 0.64-0.90; p=0.0011). Liver metastasis-predictive models were developed using the venous involvement index (OR, 2.40; 95% CI, 1.71-3.37; p=0.0000) and tumor location (rectum vs. colon; OR, 9.31; 95% CI, 2.41-36.01; p=0.0012).<h4>Conclusions</h4>Down-regulation of p21(WAF1/CIP1) as well as marked venous involvement, small tumor size and colonic tumor are associated with lymph node and/or liver metastasis. Criteria for assessment of metastasis risk provide a basis for additional treatment guidelines.

The results of genetic analysis of the wavy shoots character supported the hypothesis on the existence of two genes, Wsh1 and Wsh2, whose mutations caused wavy shoots in radish lines 16 and 26, respectively. As shown by analysis of joint inheritance with marker characters, wavy shoots were inherited independently of the biochemical markers (genes Lap1, Ep, Dia1, and Aat). Gene Wsh1 was shown to be linked to genes A, ac, and ar; the mode of inheritance depended on the cross combination. In hybrids with mutant line 16, plants with novel morphological defects, which were absent in the parental forms, were detected. Possible mechanisms of new morphological abnormalities and change of linkage in the combinations analyzed are discussed.

Remodelling of the skeleton is initiated by multinucleated osteoclasts resorbing either trabecular or cortical bone. Termination of the resorptive phase is followed by new bone formation by osteoblasts. Under normal conditions, remodeling is balanced and bone mass is preserved, with the amount of new bone formed equaling the amount that was initially resorbed. Skeletal remodeling is thought to be controlled by several hormones and many different cytokines. Recent studies have indicated that signaling molecules found in the nervous system may also play an important role in bone remodeling. One of these signaling molecules is thought to be serotonin (5-hydroxytryptamin; 5-HT). Osteoblasts and osteoclasts have receptors for serotonin and both cells express the membrane protein that is responsible for serotonin reuptake (5-HTT). Gene knockout studies in mice and use of selective serotonin reuptake inhibitors (SSRls) in mice and humans have suggested that inhibition of 5-HTT leads to reduced bone mass. These observations raise the possibility that patients treated with SSRIs may have an increased risk for secondary osteoporosis and skeletal fractures.

Ferroportin disease, autosomal-dominant reticuloendothelial iron overload, may be more prevalent than hemochromatosis in Japan. Hyperferritinemia of 822 ng/ml with 24.8% transferrin saturation of iron was incidentally noted in a 43-year-old man. His iron overload was selective in Kupffer cells of the liver. Subsequently, his father was found to have asymptomatic hyperferritinemia of 2,283 ng/ml with 62.1% saturation. These affected subjects were heterozygous for 1467A>C (R489S) in SLC40A1, and without other mutations of the hemochromatosis genes. Here, we report a Japanese family with ferroportin disease, characterized by hyperferritinemia with relatively low transferrin saturations of iron.

Acidic proteins play an important role during mineral formation in biological systems, but the mechanism of mineral formation is far from understood. In this paper, we report on the relationship between the structure of a protein and hydroxyapatite deposition under biomimetic conditions. Sericin, a type of silk protein, was adopted as a suitable protein for studying structural effect on hydroxyapatite deposition, since it forms a hydroxyapatite layer on its surface in a metastable calcium phosphate solution, and its structure has been reported. Sericin effectively induced hydroxyapatite nucleation when it has high molecular weight and a beta sheet structure. This indicates that the specific structure of a protein can effectively induce heterogeneous nucleation of hydroxyapatite in a biomimetic solution, i.e. a metastable calcium phosphate solution. This finding is useful in understanding biomineralization, as well as for the design of organic polymers that can effectively induce hydroxyapatite nucleation.