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<h4>Purpose</h4>This study utilized both cDNA microarray and 2D protein gel electrophoresis technology to investigate the multiple interactions of the genes and proteins involved in the pathophysiology of uterine leiomyomas. Also, Gene Ontology (GO) analysis was used to systematically characterize the global expression profiles, which were found to correlate with the leiomyosarcomas.<h4>Materials and methods</h4>The uterine leiomyoma biopsies were obtained from patients in the Department of Obstetrics and Gynecology, The Catholic University of Korea. Differentially expressed transcriptome and proteome, in 6 paired leiomyoma and normal myometrium, were profiled. The total RNAs from the leiomyoma and normal myometrium were labeled with Cy5 and Cy3. All specimens were punch-biopsy-obtained, and frozen in liquid nitrogen.<h4>Results</h4>Screening of up to 17,000 genes identified 71 that were either up-regulated or down-regulated (21 and 50, respectively). The gene expression profiles were classified into 420 mutually dependent functional sets, resulting in 611 cellular processes, according to the gene ontology. Also, the protein analysis, using 2D gel electrophoresis, identified 33 proteins (17 up-regulated and 16 down-regulated) with more than 500 total spots, which were classified into 302 cellular processes. O f these functional profilings, transcriptomes and proteoms down-regulations were shown in the cell adhesion, cell motility, organogenesis, enzyme regulator, structural molecule activity and responses to external stimulus functional activities, which are supposed to play important roles in the pathophysiology. In contrast, up-regulation was only shown in the nucleic acid binding activity. The CDKN2A, ADH1A, DCX, IGF2, CRABP2 and KIF5C were found to increase the reliability of this study, and correlate with the leiomyosarcomas.<h4>Conclusion</h4>Potentially significant pathogenetic cellular processes showed that down-regulated functional profiling has an important impact on the discovery of the pathogenic pathways in leiomyomas and leiomyosarcomas. GO analysis can also overcome the complexity of the expression profiles of cDNA microarrays and 2D protein analyses, via a cellular process level approach. Thereby, a valuable prognostic candidate gene, with real relevance to disease-specific pathogenesis, can be found at cellular process levels.

<h4>Purpose</h4>Although androgens are the main steroids controlling the growth of prostate glands, estrogens are also important in the regulation of its growth. Prostate cancer cells, like other cancer cells, maintain high levels of polyamines. In LNCaP cells, apoptosis is induced by mifepristone. During the process of cell death, the regulation of ROS production, caspase-3 activation and poly (ADP-ribose) polymerase cleavage were investigated in the presence of estrogen and polyamines to identify their possible roles.<h4>Materials and methods</h4>The cell growth was assessed using the MTT assay, and the intracellular ROS production by the DCFH-DA assay. The p53 protein expression, activation of caspase-3 and PARP cleavage were checked by Western blotting, with specific antibodies to each.<h4>Results</h4>The growth and viability of the cells were significantly inhibited, in a dose- and time-dependent manners, by mifepristone (MIF) treatment. The production of ROS were dependent on the MIF dosage. The activation of caspase-3 and cleavage of PARPalso increased with the duration of MIF treatment. The expression of p53 protein also increased with increases in the MIF incubation time. E(2) severely inhibited the ROS production, caspase-3 activation and PARP cleavage. However, polyamines only inhibited the ROS production, without influencing the caspase-3 activation or PARP cleavage.<h4>Conclusion</h4>In LNCaP cells, MIF induces apoptosis through ROS production. The expression of p53 protein, caspase-3 activation and PARP cleavage accompanied the process of apoptosis. The apoptotic processes were inhibited by E(2), but polyamines only inhibited the ROS production, implying the multifunctional role of E(2), in addition to its role as a free radical scavenger.

Paraventricular (PVN) and supraoptic nuclei of the hypothalamus maintain homeostasis by modulating pituitary hormonal output. PVN and supraoptic nuclei contain five major cell types: oxytocin-, vasopressin-, CRH-, somatostatin-, and TRH-secreting neurons. Sim1, Arnt2, and Otp genes are essential for terminal differentiation of these neurons. One of their common downstream genes, Brn2, is necessary for oxytocin, vasopressin, and CRH cell differentiation. Here we show that Sim2, a paralog of Sim1, contributes to the expression of Trh and Ss genes in the dorsal preoptic area, anterior-periventricular nucleus, and PVN. Sim2 expression overlaps with Trh- and Ss-expressing cells, and Sim2 mutants contain reduced numbers of Trh and Ss cells. Genetically, Sim1 acts upstream of Sim2 and partially compensates for the loss of Sim2. Comparative expression studies at the anterior hypothalamus at early stages reveal that there are separate pools of Trh cells with distinctive molecular codes defined by Sim1 and Sim2 expression. Together with previous reports, our results demonstrate that Sim1 and Otp utilize two common downstream genes, Brn2 and Sim2, to mediate distinctive sets of neuroendocrine hormone gene expression.

Juvenile hemochromatosis is an early-onset form of iron storage disease characterized by hypogonadotrophic hypogonadism and cardiomyopathy. Recently, the putative causative gene (LOC148738) encoding a protein designated hemojuvelin was cloned. The previously proposed designation of this gene as HFE2 is contrary to established convention, because it is not a member of the HFE family. We suggest that it be designated HJV. We sequenced this gene in members of 2 previously reported kinships that manifest typical juvenile hemochromatosis. In one kinship, 2 previously undescribed mutations of HJV were identified, c.238T>C (C80R) and c.302T>C (L101P). In the second kinship, 2 previously identified mutations, G320V and I222N, were found. These studies confirm that mutations in HJV cause juvenile hemochromatosis.

<h4>Background</h4>Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths in the Western world, and despite the fact that metastases are usually the ultimate cause of deaths, the knowledge of the genetics of advanced stages of this disease is limited. In order to identify potential genetic abnormalities underlying the development of local and distant metastases in CRC patients, we have, by comparative genomic hybridization, compared the DNA copy number profiles of 10 primary carcinomas, 14 local recurrences, 7 peritoneal carcinomatoses, and 42 liver metastases from 61 CRC patients.<h4>Results</h4>The median number of aberrations among the primary carcinomas, local recurrences, carcinomatoses, and liver metastases was 10, 6, 13, and 14, respectively. Several genetic imbalances, such as gains of 7, 8q, 13q, and 20, and losses of 4q, 8p, 17p, and 18, were common in all groups. In contrast, gains of 5p and 12p were more common in the carcinomatoses than in other stages of the disease. With hierarchical cluster analysis, liver metastases could be divided into two main subgroups according to clusters of chromosome changes.<h4>Conclusions</h4>Each stage of CRC progression is characterized by a particular genetic profile, and both carcinomatoses and liver metastases are more genetically complex than local recurrences and primary carcinomas. This is the first genome profiling of local recurrences and carcinomatoses, and gains of 5p and 12p seem to be particularly important for the spread of the CRC cells within the peritoneal cavity.

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine (polyQ) tract expansion near the N terminus of huntingtin (Htt). Proteolytic processing of mutant Htt and abnormal calcium signaling may play a critical role in disease progression and pathogenesis. Recent work indicates that calpains may participate in the increased and/or altered patterns of Htt proteolysis leading to the selective toxicity observed in HD striatum. Here, we identify two calpain cleavage sites in Htt and show that mutation of these sites renders the polyQ expanded Htt less susceptible to proteolysis and aggregation, resulting in decreased toxicity in an in vitro cell culture model. In addition, we found that calpain- and caspase-derived Htt fragments preferentially accumulate in the nucleus without the requirement of further cleavage into smaller fragments. Calpain family members, calpain-1, -5, -7, and -10, have increased levels or are activated in HD tissue culture and transgenic mouse models, suggesting they may play a key role in Htt proteolysis and disease pathology. Interestingly, calpain-1, -5, -7, and -10 localize to the cytoplasm and the nucleus, whereas the activated forms of calpain-7 and -10 are found only in the nucleus. These results support the role of calpain-derived Htt fragmentation in HD and suggest that aberrant activation of calpains may play a role in HD pathogenesis.

In response to retinoic acid, embryonic stem and carcinoma cells undergo differentiation to embryonic primitive endoderm cells, accompanied by a reduction in cell proliferation. Differentiation does not reduce the activation of cellular MAPK/Erk, but does uncouple mitogen-activated protein kinase (MAPK) activation from phosphorylation/activation of Elk-1 and results in inhibition of c-Fos expression, whereas phosphorylation of the cytoplasmic substrate p90RSK remains unaltered. Cell fractionation and confocal immunofluorescence microscopy demonstrated that activated MAPK is restricted to the cytoplasmic compartment after differentiation. An intact actin and microtubule cytoskeleton appears to be required for the restriction of MAPK nuclear entry induced by retinoic acid treatment because the cytoskeletal disrupting agents nocodazole, colchicine, and cytochalasin D are able to revert the suppression of c-Fos expression. Thus, suppression of cell proliferation after retinoic acid-induced endoderm differentiation of embryonic stem and carcinoma cells is achieved by restricting nuclear entry of activated MAPK, and an intact cytoskeleton is required for the restraint.

<h4>Background</h4>Gene expression is regulated mainly by transcription factors (TFs) that interact with regulatory cis-elements on DNA sequences. To identify functional regulatory elements, computer searching can predict TF binding sites (TFBS) using position weight matrices (PWMs) that represent positional base frequencies of collected experimentally determined TFBS. A disadvantage of this approach is the large output of results for genomic DNA. One strategy to identify genuine TFBS is to utilize local concentrations of predicted TFBS. It is unclear whether there is a general tendency for TFBS to cluster at promoter regions, although this is the case for certain TFBS. Also unclear is the identification of TFs that have TFBS concentrated in promoters and to what level this occurs. This study hopes to answer some of these questions.<h4>Results</h4>We developed the cluster score measure to evaluate the correlation between predicted TFBS clusters and promoter sequences for each PWM. Non-promoter sequences were used as a control. Using the cluster score, we identified a PWM group called PWM-PCP, in which TFBS clusters positively correlate with promoters, and another PWM group called PWM-NCP, in which TFBS clusters negatively correlate with promoters. The PWM-PCP group comprises 47% of the 199 vertebrate PWMs, while the PWM-NCP group occupied 11 percent. After reducing the effect of CpG islands (CGI) against the clusters using partial correlation coefficients among three properties (promoter, CGI and predicted TFBS cluster), we identified two PWM groups including those strongly correlated with CGI and those not correlated with CGI.<h4>Conclusion</h4>Not all PWMs predict TFBS correlated with human promoter sequences. Two main PWM groups were identified: (1) those that show TFBS clustered in promoters associated with CGI, and (2) those that show TFBS clustered in promoters independent of CGI. Assessment of PWM matches will allow more positive interpretation of TFBS in regulatory regions.

Huntington's disease is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in the huntingtin protein that results in intracellular aggregate formation and neurodegeneration. Pathways leading from polyglutamine tract expansion to disease pathogenesis remain obscure. To elucidate how polyglutamine expansion causes neuronal dysfunction, we generated Drosophila transgenic strains expressing human huntingtin cDNAs encoding pathogenic (Htt-Q128) or nonpathogenic proteins (Htt-Q0). Whereas expression of Htt-Q0 has no discernible effect on behavior, lifespan, or neuronal morphology, pan-neuronal expression of Htt-Q128 leads to progressive loss of motor coordination, decreased lifespan, and time-dependent formation of huntingtin aggregates specifically in the cytoplasm and neurites. Huntingtin aggregates sequester other expanded polyglutamine proteins in the cytoplasm and lead to disruption of axonal transport and accumulation of aggregates at synapses. In contrast, Drosophila expressing an expanded polyglutamine tract alone, or an expanded polyglutamine tract in the context of the spinocerebellar ataxia type 3 protein, display only nuclear aggregates and do not disrupt axonal trafficking. Our findings indicate that nonnuclear events induced by cytoplasmic huntingtin aggregation play a central role in the progressive neurodegeneration observed in Huntington's disease.

Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) is an approximately 200-kDa brefeldin A-inhibited guanine nucleotide-exchange protein that preferentially activates ADP-ribosylation factor 1 (ARF1) and ARF3. BIG1 was found in cytosol in a multiprotein complex with a similar ARF-activating protein, BIG2, which is also an A kinase-anchoring protein. In HepG2 cells growing with serum, BIG1 was primarily cytosolic and Golgi-associated. After incubation overnight without serum, a large fraction of endogenous BIG1 was in the nuclei. By confocal immunofluorescence microscopy, BIG1 was localized with nucleoporin p62 at the nuclear envelope (probably during nucleocytoplasmic transport) and also in nucleoli, clearly visible against the less concentrated overall matrix staining. BIG1 was also identified by Western blot analyses in purified subnuclear fractions (e.g., nucleoli and nuclear matrix). Antibodies against BIG1, nucleoporin, or nucleolin coimmunoprecipitated the other two proteins from purified nuclei. In contrast, BIG2 was not associated with nuclear BIG1. Also of note, ARF was never detected among proteins precipitated from purified nuclei by anti-BIG1 antibodies, although microscopically the two proteins do appear sometimes to be colocalized in the nucleus. These data are consistent with independent intracellular movements and actions of BIG1 and BIG2, and they are also evidence of the participation of BIG1 in both Golgi and nuclear functions.

Extensive striatal neuronal loss occurs in Huntington's disease (HD), which is caused by an expanded polyglutamine tract in huntingtin (htt). Evidence suggests that mutant htt directly or indirectly compromises mitochondrial function, contributing to the neuronal loss. To determine the role of compromised mitochondrial function in the neuronal cell death in HD, immortalized striatal cells established from Hdh(Q7) (wild-type) and Hdh(Q111) (mutant) mouse knock-in embryos were treated with 3-nitropropionic acid (3-NP), a mitochondrial complex II toxin. 3-NP treatment caused significantly greater cell death in mutant striatal cells compared with wild-type cells. In contrast, the extent of cell death induced by rotenone, a complex I inhibitor, was similar in both cell lines. Although evidence of apoptosis was present in 3-NP-treated wild-type striatal cells, it was absent in 3-NP-treated mutant cells. 3-NP treatment caused a greater loss of mitochondrial membrane potential (deltapsim) in mutant striatal cells compared with wild-type cells. Cyclosporine A, an inhibitor of mitochondrial permeability transition pore (PTP), and ruthenium red, an inhibitor of the mitochondrial calcium uniporter, both rescued mutant striatal cells from 3-NP-induced cell death and prevented the loss of deltapsim. These data show that mutant htt specifically increases cell vulnerability to mitochondrial complex II inhibition and further switched the type of cell death induced by complex II inhibition from apoptosis to a non-apoptotic form, caused by mitochondrial membrane depolarization, probably initiated by mitochondrial calcium overload and subsequent PTP opening. These findings suggest that impaired mitochondrial complex II function in HD may contribute to non-apoptotic neuronal cell death.

<h4>Background</h4>We sought to compare frequencies of HFE C282Y and H63D alleles and associated odds ratios (OR) in 100 consecutive unrelated white adults with malignancy to those in 318 controls.<h4>Methods</h4>Data from patients with more than one malignancy were analyzed according to each primary malignancy. For the present study, OR > or =2.0 or < or =0.5 was defined to be increased or decreased, respectively.<h4>Results</h4>There were 110 primary malignancies (52 hematologic neoplasms, 58 carcinomas) in the 100 adult patients. Allele frequencies were similar in patients and controls (C282Y: 0.0850 vs. 0.0896, respectively (OR = 0.9); H63D: 0.1400 vs. 0.1447, respectively (OR = 0.9)). Two patients had hemochromatosis and C282Y homozygosity. With C282Y, increased OR occurred in non-Hodgkin lymphoma, myeloproliferative disorders, and adenocarcinoma of prostate (2.0, 2.8, and 3.4, respectively); OR was decreased in myelodysplasia (0.4). With H63D, increased OR occurred in myeloproliferative disorders and adenocarcinomas of breast and prostate (2.4, 2.0, and 2.0, respectively); OR was decreased in non-Hodgkin lymphoma and B-chronic lymphocytic leukemia (0.5 and 0.4, respectively).<h4>Conclusions</h4>In 100 consecutive adults with malignancy evaluated in a community medical oncology practice, frequencies of HFE C282Y or H63D were similar to those in the general population. This suggests that C282Y or H63D is not associated with an overall increase in cancer risk. However, odds ratios computed in the present study suggest that increased (or decreased) risk for developing specific types of malignancy may be associated with the inheritance of HFE C282Y or H63D. Study of more patients with these specific types of malignancies is needed to determine if trends described herein would remain and yield significant differences.

<h4>Introduction</h4>PROWESS (Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis) was a phase III, randomized, double blind, placebo controlled, multicenter trial conducted in patients with severe sepsis from 164 medical centers. Here we report data collected at study entry for 1690 patients and over the following 7 days for the 840 patients who received placebo (in addition to usual standard of care).<h4>Methods</h4>Nineteen biomarkers of coagulation activation, anticoagulation, fibrinolysis, endothelial injury, and inflammation were analyzed to determine the relationships between baseline values and their change over time, with 28-day survival, and type of infecting causative micro-organism.<h4>Results</h4>Levels of 13 of the 19 biomarkers at baseline correlated with Acute Physiology and Chronic Health Evaluation II scores, and nearly all patients exhibited coagulopathy, endothelial injury, and inflammation at baseline. At study entry, elevated D-dimer, thrombin-antithrombin complexes, IL-6, and prolonged prothrombin time were present in 99.7%, 95.5%, 98.5%, and 93.4% of patients, respectively. Markers of endothelial injury (soluble thrombomodulin) and deficient protein C, protein S, and antithrombin were apparent in 72%, 87.6%, 77.8%, and 81.7%, respectively. Impaired fibrinolysis (elevated plasminogen activator inhibitor-1) was observed in 44% of patients. During the first 7 days, increased prothrombin time (which is readily measurable in most clinical settings) was highly evident among patients who were not alive at 28 days.<h4>Conclusion</h4>Abnormalities in biomarkers of inflammation and coagulation were related to disease severity and mortality outcome in patients with severe sepsis. Coagulopathy and inflammation were universal host responses to infection in patients with severe sepsis, which were similar across causative micro-organism groups.

Cancer cells are more susceptible to chemotherapeutic agent-induced apoptosis than their normal counterparts. Although it has been demonstrated that the increased sensitivity results from deregulation of oncoproteins during cancer development (Evan, G. I., and Vousden, K. H. (2001) Nature 411, 342-348; Green, D. R., and Evan, G. I. (2002) Cancer Cell 1, 19-30), little is known about the signaling pathways leading to changes in the apoptotic threshold in cancer cells. Here we show that low RKIP expression levels in tumorigenic human prostate and breast cancer cells are rapidly induced upon chemotherapeutic drug treatment, sensitizing the cells to apoptosis. We show that the maximal RKIP expression correlates perfectly with the onset of apoptosis. In cancer cells resistant to DNA-damaging agents, treatment with the drugs does not up-regulate RKIP expression. However, ectopic expression of RKIP resensitizes DNA-damaging agent-resistant cells to undergo apoptosis. This sensitization can be reversed by up-regulation of survival pathways. Down-regulation of endogenous RKIP by expression of antisense and small interfering RNA (siRNA) confers resistance on sensitive cancer cells to anticancer drug-induced apoptosis. Our studies suggest that RKIP may represent a novel effector of signal transduction pathways leading to apoptosis and a prognostic marker of the pathogenesis of human cancer cells and tumors after treatment with clinically relevant chemotherapeutic drugs.

We present a method for creating a network of gene co-occurrences from the literature and partitioning it into communities of related genes. The way in which our method identifies communities makes it likely that the component genes of each community will be related by their function. The method processes a large database of article abstracts, synthesizing information from many sources to shed light on groups of genes that have been shown to interact. It is a tool to be used by researchers in the biomedical sciences to swiftly search for known interactions and to provide insight into unexplored connections. The partitioning procedure is designed to be particularly applicable to large networks in which individual nodes may play a role in more than one community. In this paper, we explain the details of the method, in particular the partitioning process. We also apply the method to produce communities of genes related to colon cancer and show that the results are useful.

<h4>Background</h4>Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA surveillance mechanism that detects and degrades mRNAs with premature termination codons (PTC+ mRNAs). In mammals, a termination codon is recognized as premature if it lies more than about 50 nucleotides upstream of the final intron position. More than a third of reliably inferred alternative splicing events in humans have been shown to result in PTC+ mRNA isoforms. As the mechanistic details of NMD have only recently been elucidated, we hypothesized that many PTC+ isoforms may have been cloned, characterized and deposited in the public databases, even though they would be targeted for degradation in vivo.<h4>Results</h4>We analyzed the human alternative protein isoforms described in the SWISS-PROT database and found that 144 (5.8% of 2,483) isoform sequences amenable to analysis, from 107 (7.9% of 1,363) SWISS-PROT entries, derive from PTC+ mRNA.<h4>Conclusions</h4>For several of the PTC+ isoforms we identified, existing experimental evidence can be reinterpreted and is consistent with the action of NMD to degrade the transcripts. Several genes with mRNA isoforms that we identified as PTC+--calpain-10, the CDC-like kinases (CLKs) and LARD--show how previous experimental results may be understood in light of NMD.

Atomic co-ordinates and structure factors for the T67R/S92D metMbCN mutant have been deposited with the Protein Data Bank, under accession codes 1h1x and r1h1xsf, respectively. Protein engineering and cofactor replacement have been employed as tools to introduce/modulate peroxidase activity in sperm whale Mb (myoglobin). Based on the rationale that haem peroxidase active sites are characterized by specific charged residues, the Mb haem crevice has been modified to host a haem-distalpropionate Arg residue and a proximal Asp, yielding the T67R/S92D Mb mutant. To code extra conformational mobility around the haem, and to increase the peroxidase catalytic efficiency, the T67R/S92D Mb mutant has been subsequently reconstituted with protohaem-L-histidine methyl ester, yielding a stable derivative, T67R/S92D Mb-H. The crystal structure of T67R/S92D cyano-metMb (1.4 A resolution; R factor, 0.12) highlights a regular haem-cyanide binding mode, and the role for the mutated residues in affecting the haem propionates as well as the neighbouring water structure. The conformational disorder of the haem propionate-7 is evidenced by the NMR spectrum of the mutant. Ligand-binding studies show that the iron(III) centres of T67R/S92D Mb, and especially of T67R/S92D Mb-H, exhibit higher affinity for azide and imidazole than wild-type Mb. In addition, both protein derivatives react faster than wild-type Mb with hydrogen peroxide, showing higher peroxidase-like activity towards phenolic substrates. The catalytic efficiency of T67R/S92D Mb-H in these reactions is the highest so far reported for Mb derivatives. A model for the protein-substrate interaction is deduced based on the crystal structure and on the NMR spectra of protein-phenol complexes.

To identify tumor antigens for glioma, a human testis cDNA library was screened by serological identification of antigens by recombinant expression cloning with sera from glioma patients. In this screening, the most frequently isolated antigen was SOX6, an Sry-related high-mobility group (HMG) box-containing gene. SOX6 is a transcriptional factor that is specifically expressed in the developing central nervous system and in the early stages of chondrogenesis in mouse embryos. IgG antibodies against SOX6 were detected in sera from 12 of 36 glioma patients (33.3%), 0 of 14 patients with other brain disease (0%), and one of 54 other cancer patients (1.9%). In sera from 37 healthy individuals, no IgG responses against SOX6 were detected, except in an elderly female. Furthermore, Western blot and ELISA analyses with sera from glioma patients revealed that the DNA-binding domain, the HMG box of SOX6, might be a dominant epitope of IgGs against SOX6. RT-PCR and Northern blot analysis revealed that the SOX6 gene was more highly expressed in glioma tissues than in normal adult tissues, except testis. Western blot analysis with an anti-SOX6 antibody demonstrated that the SOX6 protein was expressed in glioma tissues, but not in normal adult brain tissue. Immunohistochemical analysis with the anti-SOX6 antibody showed that all the glioma tissues analysed expressed SOX6 in tumor cells, but only a few SOX6-positive cells were detected in non-neoplastic tissues from the cerebral cortex. In summary, these results indicate that the developmentally regulated transcription factor SOX6 is aberrantly expressed in glioma and specifically recognized by IgGs from glioma patients' sera.

Histamine is a neurotransmitter with actions throughout the nervous system of vertebrates and invertebrates. Nevertheless, the actions of only a few identified histamine-containing neurons have been characterized. Here, we present the actions of a histaminergic projection neuron on the rhythmically active pyloric and gastric mill circuits within the stomatogastric ganglion (STG) of the crab Cancer borealis. An antiserum generated against histamine labeled profiles throughout the C. borealis stomatogastric nervous system. Labeling occurred in several somata and neuropil within the paired commissural ganglia as well as in neuropil within the STG and at the junction of the superior oesophageal and stomatogastric nerves. The source of all histamine-like immunolabeling in the STG neuropil was one pair of neuronal somata, the previously identified inferior ventricular (IV) neurons, located in the supraoesophageal ganglion. These neurons also exhibited FLRFamide-like immunoreactivity. Activation of the IV neurons in the crab inhibited some pyloric and gastric mill neurons and, with inputs from the commissural ganglia eliminated, terminated both rhythms. Focal application of histamine had comparable effects. The actions of both applied histamine and IV neuron stimulation were blocked, reversibly, by the histamine type-2 receptor antagonist cimetidine. With the commissural ganglia connected to the STG, IV neuron stimulation elicited a longer-latency activation of commissural projection neurons which in turn modified the pyloric rhythm and activated the gastric mill rhythm. These results support the hypothesis that the histaminergic/peptidergic IV neurons are projection neurons with direct and indirect actions on the STG circuits of the crab C. borealis.

<h4>Objective</h4>Serotonin reuptake inhibitors (SRIs) are the most efficient pharmacological treatment of obsessive-compulsive disorders (OCD). Previous studies have suggested that some peripheral serotonergic parameters can be used to predict the clinical outcome of the treatment of OCD patients with SRIs. We tried to identify further peripheral serotonergic parameters that could help predict the clinical outcome of SRI treatment in a sample of patients with OCD.<h4>Methods</h4>We compared 19 OCD patients before and after 8 weeks of SRI treatment with 19 sex-matched and age-matched controls. We assessed clinical improvement and whole-blood serotonin (5-HT) concentration, platelet 5-HT transporter (5-HTT) and 5-HT2A receptor binding characteristics and platelet IP3 content.<h4>Results</h4>Before treatment, OCD patients had higher platelet IP3 content and fewer 5-HTT binding sites than the controls. Treatment with SRIs further lowered the number of 5-HTT binding sites, normalized platelet IP3 contents, and lowered the number of platelet 5-HT2A binding sites and whole-blood 5-HT concentrations below control values. The patients who improved most following SRI treatment had higher whole-blood 5-HT concentrations before treatment.<h4>Conclusion</h4>Our results confirm that whole-blood 5-HT concentration is a predictor for clinical improvement and indicate that abnormal intracellular mechanisms may be involved in OCD patients, in particular, the overstimulation of the phosphoinositide signaling system.

Hereditary hemochromatosis is a common autosomal- recessive disorder of iron overload usually occurring in individuals who are homozygous for a C282Y mutation in the hemochromatosis (HFE) gene. Current screening methods can detect affected individuals early in disease pathogenesis, enabling early institution of effective treatment that can restore normal life expectancy. Phenotypic screening of adults using transferrin saturation and serum ferritin levels identifies the majority of individuals who develop iron overload. HFE genotyping, when combined with serum biochemical measurements, has reduced reliance on liver biopsy as a diagnostic tool and is the preferred initial screening modality for families with an affected individual. Genetic testing has altered previously held views regarding the high level of penetrance of the disease. Although the majority of C282Y homozygotes develop increased body iron stores, end-organ damage occurs much less frequently than previously thought. Screening is recommended in high-risk groups and in those with a high index of clinical suspicion. Opportunistic screening during routine health assessments may also be recommended. However, large-scale screening of the average-risk population is not recommended on the basis of current evidence.

H1 linker histones (H1s) are key regulators of chromatin structure and function. The functions of different H1s during early embryogenesis, and mechanisms regulating their associations with chromatin are largely unknown. The developmental transitions of H1s during oocyte growth and maturation, fertilization and early embryogenesis, and in cloned embryos were examined. Oocyte-specific H1FOO, but not somatic H1s, associated with chromatin in oocytes (growing, GV-stage, and MII-arrested), pronuclei, and polar bodies. H1FOO associated with sperm or somatic cell chromatin within 5 min of intracytoplasmic sperm injection (ICSI) or somatic cell nuclear transfer (SCNT), and completely replaced somatic H1s by 60 min. The switching from somatic H1s to H1FOO following SCNT was developmentally regulated. H1FOO was replaced by somatic H1s during the late two- and four-cell stages. H1FOO association with chromatin can occur in the presence of a nuclear envelope and independently of pronucleus formation, is regulated by factors associated with the spindle, and is likely an active process. All SCNT constructs recapitulated the normal sequence of H1 transitions, indicating that this alone does not signify a high developmental potential. A paucity of all known H1s in two-cell embryos may contribute to precocious gene transcription in fertilized embryos, and the elaboration of somatic cell characteristics in cloned embryos.

The most distinctive feature of oocyte-specific linker histones is the specific timing of their expression during embryonic development. In Xenopus nuclear transfer, somatic linker histones in the donor nucleus are replaced with oocyte-specific linker histone B4, leading to the involvement of oocyte-specific linker histones in nuclear reprogramming. We recently have discovered a mouse oocyte-specific linker histone, named H1foo, and demonstrated its expression pattern in normal preimplantation embryos. The present study was undertaken to determine whether the replacement of somatic linker histones with H1foo occurs during the process of mouse nuclear transfer. H1foo was detected in the donor nucleus soon after transplantation. Thereafter, H1foo was restricted to the chromatin in up to two-cell stage embryos. After fusion of an oocyte with a cell expressing GFP (green fluorescent protein)-tagged somatic linker histone H1c, immediate release of H1c in the donor nucleus was observed. In addition, we used fluorescence recovery after photobleaching (FRAP), and found that H1foo is more mobile than H1c in living cells. The greater mobility of H1foo may contribute to its rapid replacement and decreased stability of the embryonic chromatin structure. These results suggest that rapid replacement of H1c with H1foo may play an important role in nuclear remodeling.

<h4>Objective</h4>To determine the rate and correlates of weight change in a large, well characterised sample of patients with Huntington's disease followed at 44 sites by the Huntington Study Group.<h4>Participants and methods</h4>Weight change was assessed in 927 adults with a definite diagnosis of Huntington's disease who were followed prospectively for (mean (SD)) 3.4 (1.4) years. The unified Huntington's disease rating scale was used to assess weight, motor dysfunction (including chorea and dystonia), depressive symptoms, and functional decline.<h4>Results</h4>Random effects modelling determined that patients gained an average of 0.11 (1.7) kg/year and their chorea scores increased by 0.36 (0.78) points/year. There were significant but weak relations between weight loss and increasingly severe chorea (r = -0.13), worse baseline motor performance (r = -0.12), less severe baseline depressed mood (r = 0.14), and poorer baseline independence ratings (r = 0.07). Patients who were within 0 to 2 years of symptom onset at the time of the baseline visit gained more weight than those with longer disease duration.<h4>Conclusions</h4>Weight loss following symptom onset is not a consistent feature of Huntington's disease. The mechanisms contributing to weight change in this condition are unclear and probably multifactorial. Future studies examining asymptomatic carriers of the mutation could be helpful in identifying incipience of low body weight and may be better suited for identifying clinical correlates of weight loss than studies in symptomatic patients.

Mutations in the BRAF gene have recently been detected in a wide range of neoplastic lesions with a particularly high prevalence in melanoma and papillary thyroid carcinoma (PTC). The hot-spot mutation BRAF(V599E) is frequently detected in PTC (36-69%), in contrast to its absence in other benign or malignant thyroid lesions. In order to unravel whether there is any association between the occurrence of the BRAF mutation and the histological pattern of PTC, in this study a previous series of 50 PTCs was extended to 134 cases, including ten cases of PTC-related entities-hyalinizing trabecular tumour (HTT) and mucoepidermoid carcinoma (MEC). Using PCR/SSCP and sequencing, the BRAF(V599E) mutation was detected in 45 of the 124 PTCs (36%). No mutations were detected in any case of HTT and MEC. BRAF(V599E) was present in 75% of Warthin-like PTCs and 53% of conventional PTCs, whereas no BRAF(V599E) mutations were detected in any of the 32 cases of the follicular variant of PTC. BRAF(V599E) was also detected in 6 of 11 cases of the oncocytic variant of PTC that displayed a papillary or mixed follicular-papillary growth pattern and in none of the four oncocytic PTCs with a follicular growth pattern. A distinct mutation in BRAF (codon K600E) was detected in three cases of the follicular variant of PTC. This study has confirmed the high prevalence of BRAF(V599E) in PTC and has shown that the mutation is almost exclusively seen in PTC with a papillary or mixed follicular-papillary growth pattern, regardless of the cytological features of the neoplastic cells. The results support the existence of an oncocytic variant of PTC that should be separated from the oncocytic variant of follicular carcinoma and suggest that the follicular variant of PTC may be genetically different from conventional PTC.

<h4>Introduction</h4>For patients with hepatocellular carcinoma (HCC) in particular, living donor liver transplant (LDLT) improves access to transplant. We report our results in 36 patients with HCC who underwent LDLT with a median follow-up >1 year. METHODS Underlying diagnoses included: hepatitis C (24), hepatitis B (9), cryptogenic cirrhosis (1), hemochromatosis (1), and primary biliary cirrhosis (1). Patients with tumors >or= 5 cm received IV doxorubicin intraoperatively and 6 cycles of doxorubicin at 3-week intervals. Patients were followed with CT scan and alpha-fetoprotein levels every 3 months for 2 years posttransplant. Mean waiting time, pretransplant treatment, tumor variables, and survival were analyzed. Univariate and multivariate analysis were done to analyze tumor variables; Kaplan-Meier and log rank were used to compare survivals. P < 0.05 was considered significant. RESULTS Mean wait for LDLT was 62 days, compared with 459 days in 50 patients with HCC transplanted with cadaveric organs during the same time period (P = 0.0001). At median follow-up of 450 days, there have been 10 deaths due to non-tumor-related causes and 3 deaths from recurrence; recurrence has also been observed in 3 other patients. On univariate and multivariate analysis, bilobar distribution was the only significant tumor variable (P = 0.03, log rank = 0.02). Fifty-three percent of patients exceeded UNOS priority criteria. One- and two-year patient survivals were 75% and 60%, respectively. Freedom from recurrence at 365 and 730 days was 82% and 74%, respectively. Overall and in patients with HCC > 5 cm (n = 12), there were no statistically significant differences in survival or in freedom from recurrence between recipients of living donor and cadaveric grafts. CONCLUSION Although one third of patients had tumors > 5 cm, the incidence of recurrence as well as patient survival and freedom from recurrence are comparable to results after cadaveric transplant. LDLT allows timely transplantation in patients with early or with large HCC.

Temperature-sensitive (ts) mutants of vaccinia virus defective in the B1 kinase demonstrate a conditionally lethal defect in DNA synthesis. B1 is the prototypic member of a new family of protein kinases (vaccinia virus-related kinases, or VRK) that possess distinctive B1-like sequence features within their catalytic motifs (R. J. Nichols and P. Traktman, J. Biol. Chem., in press). Given the striking sequence similarity between B1 and the VRK enzymes, we proposed that they might share overlapping substrate specificity. We therefore sought to determine whether the human and mouse VRK1 enzymes (hVRK1 and mVRK1, respectively) could complement a B1 deficiency in vivo. Recombinant ts2 viruses expressing hVRK1, mVRK1, or wild-type B1 were able to synthesize viral DNA at high temperature, but those expressing the more distantly related human casein kinase 1 alpha 2 could not. Complementation required the enzymatic activity of hVRK1, since a catalytically inactive allele of hVRK1 was unable to confer a temperature-insensitive phenotype. Interestingly, rescue of viral DNA synthesis was not coupled to the ability to phosphorylate H5, the only virus-encoded protein shown to be a B1 substrate in vivo. Expression of hVRK1 during nonpermissive ts2 infections restored virus production and plaque formation, whereas expression of mVRK1 resulted in an intermediate level of rescue. Taken together, these observations indicate that enzymatically active cellular VRK1 kinases can perform the function(s) of B1 required for genome replication, most likely due to overlapping specificity for cellular and/or viral substrates.

<h4>Object</h4>Few studies have been conducted to investigate the genomic survey of oncogene amplification in medulloblastoma. Low frequency of N-myc, C-myc, and epidermal grow factor receptor (EGFR) gene amplification (< 10%) has been reported in medulloblastoma. Previous comparative genomic hybridization (CGH) study of primary medulloblastomas has revealed chromosomal amplification on 2p21, 3p, 5p15.3, 7q, 8q24, 11q22.3, and 17q. The aim of this study was to detect common oncogenes involved in medulloblastoma tumorigenesis.<h4>Methods</h4>The authors studied a series of 14 samples by performing CGH and array-based CGH. The CGH analysis detected nonrandom losses on 8p, 17p, 16q, 8q, and 1p, whereas gains were found on 17q, 12q, 7q, and 1p. Array-based CGH was conducted to investigate amplification of 58 oncogenes throughout the genome of these samples. Gene amplifications identified for the first time included PGY1 at 7q21.1, MDM2 at 12q14.3-q15, and ERBB2 at 17q21.2. The highest frequencies of oncogene gain were detected in D17S1670 (61.5%), PIK3CA (46.2%), PGY1 (38.5%), MET (38.5%), ERBB2 (38.5%), and CSE1L (38.5%). The gain in gene copy numbers was confirmed in 34 additional archival medulloblastoma cases by using fluorescence in situ hybridization analysis.<h4>Conclusions</h4>This is the first genome-wide survey of multiple oncogene amplifications involved in the development of medulloblastoma. Gains of several candidate oncogenes such as D17S1670, ERBB2, PIK3CA, PGY1, MET, and CSE1L were frequently detected. These genes may be used as molecular markers and therapeutic targets of medulloblastomas.

We previously performed a global analysis of the gene expression of gastric cancer cell lines established from metastases to the peritoneal cavity with the cDNA microarray method, which made it possible to analyse the expression of approximately 21168 genes for the identification of novel markers for the detection of micrometastases in the peritoneal cavity. One of the upregulated genes is dopa decarboxylase (DDC), which is responsible for the synthesis of the key neurotransmitters dopamine and serotonine. We have examined its potential as a novel marker for the detection of peritoneal micrometastases of gastric cancer.DDC mRNA in the peritoneal wash from 112 gastric cancer patients was quantified for comparison of carcinoembryonic antigen (CEA) mRNA by means of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) with a fluorescently labelled probe to predict peritoneal recurrence. The quantity of DDC and CEA correlated with wall penetration. Real-time RT-PCR could quantitate 10-10(6) DDC-expressing gastric cancer cells per 10(7) mesothelial cells. The cutoff value was set at the upper limit of the quantitative value for noncancer patients, and those above this cutoff value constituted the micrometastasis (MM+) group. Of 15 cases with peritoneal dissemination, 13 were MM+DDC (87% sensitivity), and one of 48 t1 cases was MM+ (98% specificity). DDC levels in peritoneal washes from patients with synchronous peritoneal metastases were more than 50 times higher than in those from patients without metastasis (P<0.01). For 15 cases of peritoneal dissemination (seven cases were cytologically positive), DDC was positive in 13 cases (87% sensitivity), but CEA failed to detect micrometastases in four cases (73% sensitivity), indicating that DDC is in some cases superior to CEA for the detection of peritoneal micrometastases of gastric cancer in terms of sensitivity as well as specificity, especially for poorly differentiated adenocarcinomas. A combination of CEA and DDC improved the accuracy of diagnosis up to 94%. These results suggest that DDC is potentially a novel marker for peritoneal dissemination of gastric cancer and that quantitative RT-PCR of DDC is reliable and efficient for the selection of patients for adjuvant intraperitoneal chemotherapy to prevent peritoneal recurrence.

High-altitude pulmonary oedema (HAPE) is a potentially fatal condition affecting fit and previously well individuals at altitudes in excess of 3000 m. This article discusses the mechanisms of HAPE, considers the contribution of hypoxic pulmonary vasoconstriction and alterations in sodium transport to the pathological process. It discusses the various biochemical mediators such as nitric oxide (NO), endothelin-1 (ET-1), and the renin-angiotensin-aldosterone system (RAS) that may be involved and considers possible oxygen-sensing mechanisms involved in hypoxic adaptation such as hypoxia-inducible factor-1 (HIF-1). Those who have had HAPE once run an unpredictable but significant risk of recurrence; therefore, there may be a constitutional or genetic component in its aetiology. This paper considers the possible involvement of genes that may be involved in physiological adaptation to hypoxia (e.g., angiotensin-1 [AT(1)]-converting enzyme [ACE], tyrosine hydroxylase, serotonin transporter [5-HTT], and endothelial NO synthase [eNOS] genes). As yet, no formal association has been identified between an identified genetic polymorphism and HAPE, but genetic variation provides a possible mechanism to explain interindividual variation in response to hypoxia and enhanced or reduced performance at altitude.

Serpins are unique among the various types of active site proteinase inhibitors because they covalently trap their targets by undergoing an irreversible conformational rearrangement. Members of the serpin superfamily are present in the three major domains of life (Bacteria, Archaea and Eukarya) as well as several eukaryotic viruses. The human genome encodes for at least 35 members that segregate evolutionarily into nine (A-I) distinct clades. Most of the human serpins are secreted and circulate in the bloodstream where they reside at critical checkpoints intersecting self-perpetuating proteolytic cascades such as those of the clotting, thrombolytic and complement systems. Unlike these circulating serpins, the clade B serpins (ov-serpins) lack signal peptides and reside primarily within cells. Most of the human clade B serpins inhibit serine and/or papain-like cysteine proteinases and protect cells from exogenous and endogenous proteinase-mediated injury. Moreover, as sequencing projects expand to the genomes of other species, it has become apparent that intracellular serpins belonging to distinct phylogenic clades are also present in the three major domains of life. As some of these serpins also guard cells against the deleterious effects of promiscuous proteolytic activity, we propose that this cytoprotective function, along with similarities in structure are common features of a cohort of intracellular serpin clades from a wide variety of species.

<h4>Context</h4>Type 2 diabetes is a common manifestation of hemochromatosis, a disease of iron overload. However, it is not clear whether higher iron stores predict the development of type 2 diabetes in a healthy population.<h4>Objective</h4>To examine plasma ferritin concentration and the ratio of the concentrations of transferrin receptors to ferritin in relation to risk of type 2 diabetes.<h4>Design, setting, and participants</h4>Prospective nested case-control study within the Nurses' Health Study cohort. Of the 32 826 women who provided blood samples during 1989-1990 and were free of diagnosed diabetes, cardiovascular disease, and cancer, 698 developed diabetes during 10 years of follow-up. The controls (n = 716) were matched to cases on age, race, and fasting status; and on body mass index (BMI) for cases in the top BMI decile.<h4>Main outcome measure</h4>Incident cases of type 2 diabetes.<h4>Results</h4>Among cases, the mean (SD) concentration of ferritin was significantly higher (109 [105] vs 71.5 [68.7] ng/mL for controls; P<.001 for difference) and the mean (SD) ratio of transferrin receptors to ferritin was significantly lower (102 [205] vs 141 [340], respectively; P =.01). In conditional logistic regression stratified on the matching factors and controlled for BMI and other diabetes risk factors, the multivariate relative risks [RRs] of incident type 2 diabetes across increasing quintiles of ferritin were 1.00, 1.09 (95% confidence interval [CI], 0.70-1.70), 1.26 (95% CI, 0.82-1.95), 1.30 (95% CI, 0.83-2.04), and 2.68 (95% CI, 1.75-4.11) (P<.001 for trend). The RRs across increasing quintiles of transferrin receptors to ferritin ratio were 2.44 (95% CI, 1.61-3.71), 1.00 (95% CI, 0.64-1.56), 1.13 (95% CI, 0.73-1.74), 0.99 (95% CI, 0.64-1.53), and 1.00 (P =.01 for trend). Further adjustment for an inflammatory marker (C-reactive protein) did not change the results appreciably. The associations persisted within strata defined by levels of BMI, menopausal status, alcohol consumption, and C-reactive protein.<h4>Conclusion</h4>Higher iron stores (reflected by an elevated ferritin concentration and a lower ratio of transferrin receptors to ferritin) are associated with an increased risk of type 2 diabetes in healthy women independent of known diabetes risk factors.

The subventricular zone (SVZ) contains undifferentiated cells, which proliferate and generate olfactory bulb (OB) interneurons. Throughout life, these cells leave the SVZ and migrate along the rostral migratory stream (RMS) to the OB where they differentiate. In vitro, the septum and the choroid plexus (CP) secrete repulsive factors that could orient the migration of OB precursors. Slit1 and Slit2, two known chemorepellents for developing axons, can mimic this effect. We show here that the Slit receptors Robo2 and Robo3/Rig-1 are expressed in the SVZ and the RMS and that Slit1 and Slit2 are still present in the adult septum. Using Slit1/2-deficient mice, we found that Slit1 and Slit2 are responsible for both the septum and the CP repulsive activity in vitro. In adult mice lacking Slit1, small chains of SVZ-derived cells migrate caudally into the corpus callosum, supporting a role for Slits in orienting the migration of SVZ cells. Surprisingly, in adult mice, Slit1 was also expressed by type A and type C cells in the SVZ and RMS, suggesting that Slit1 could act cell autonomously. This hypothesis was tested using cultures of SVZ explants or isolated neurospheres from Slit1-/- or Slit1+/- mice. In both types of cultures, the migration of SVZ cells was altered in the absence of Slit1. This suggests that the regulation of the migration of OB precursors by Slit proteins is complex and not limited to repulsion.

We present the first application of a multiplex multicolour assay for the simultaneous detection of three of the most frequent mutations related to hereditary haemochromatosis (C282Y, H63D and S65C), using fluorescent detection and capillary electrophoresis. We describe two methods: the first is based on a single base extension assay, resulting in a single base difference of the extended products; and the second is a competitive allele-specific polymerase chain reaction (PCR), based on competition between allele-specific primers. Specificity of the latter primers is enhanced with a mismatch at the antepenultimate nucleotide. Primers are designed to amplify products of different sizes and with different fluorescent dyes in order to accurately distinguish all possible combinations of genotypes (homozygous and heterozygous for each mutation) in a multiplex PCR analysis. An advantage of the present approach is that capillary electrophoresis analysis of the amplified products enables easy, rapid, unambiguous and high resolution discrimination between wild-type and mutant alleles, although different mutations may be present in the multiplex analysis. This will facilitate automated genotyping for routine molecular diagnostics and large-scale genetic studies.

Studies of the association between polymorphisms of the serotonin transporter gene (5-HTT) and trait anxiety have produced inconsistent results, raising questions about the strength of the relationship and the methodological conditions under which the relationship holds. We conducted a meta-analysis of existing studies to provide formal statistical measures of the strength of the linked polymorphic region of the serotonin transporter gene (5-HTTLPR)-anxiety relationship. For the entire collection of 26 studies, results provided no support for a relationship between anxiety and the presence of the short form of the 5-HTTLPR polymorphism. There was strong evidence of the presence of moderating variables, however, and subsequent analysis revealed that choice of the measure of trait anxiety was significant. Studies using the Neuroticism scale of Costa and McCrae were found to produce a small positive effect (d=0.23). Other potential moderators (country of study origin, type of subject) did not have a meaningful impact on d statistics. These findings indicate that 5-HTTLPR may in fact have a small but reliable influence on personality, particularly in the manifestation of trait anxiety when measured with a neuroticism scale based on the five-factor model of personality. Our results suggest that the success of future personality genetics research will be maximized by the use of personality measures from both the psychobiological and five-factor models.

<h4>Aim</h4>Pituitary tumor transforming gene (PTTG1) is overexpressed in a variety of tumors, including carcinomas of the lung, breast, colon, as well as in leukemia, lymphoma and pituitary adenomas. However, there is little information on its expression in gastric carcinoma. We sought to investigate the expression of PTTG1 in gastric carcinoma and to explore the relationship between its expression and clinicopathological factors.<h4>Methods</h4>We studied 75 primary human gastric adenocarcinomas, including 17 mucosal carcinomas, 21 submucosal infiltrative carcinomas, 12 carcinomas invading proprial muscle layers, 6 carcinomas reaching the subserosa, and 19 carcinomas penetrating the serosal surface. Immunohistochemical analysis was performed using paraffin-embedded sections of gastric adenocarcinomas.<h4>Results</h4>PTTG1 was expressed heterogeneously in carcinomas. Positive PTTG1 staining was observed in 65.3% of the carcinomas (49 of 75). Its expression did not correlate significantly with either the histological type or the depth of infiltration of the gastric carcinomas. However, a statistical analysis showed significant differences between the primary adenocarcinomas and the associated metastatic lymph nodes.<h4>Conclusion</h4>The results of this study demonstrate that PTTG1 expression is enhanced in metastatic lymph nodes in comparison to that in primary carcinomas. We suggest that PTTG1 may contribute to lymph node metastases in gastric carcinoma.

<h4>Aim</h4>This study was designed to compare the levels of v5 and v6 splice variants of CD44 evaluated using ELISA test in the serum of patients with colorectal cancer in different stages of progression of the disease estimated in pT stage according to WHO score, histopathological grade of malignancy and some clinicopathological features.<h4>Methods</h4>The serum obtained from 114 persons with colorectal adenocarcinomas was examined using ELISA method. pT stage and grade of malignancy of the tumour were examined in formalin fixed and paraffin embedded materials obtained during operation.<h4>Results</h4>Only the level of CD44 v5 in the serum of patients before operation with G2 pT4 tumour was lower than that in other probes and the difference was statistically significant. We did not find any other correlations between the level of v5 and v6 CD44 variants and other evaluated parameters.<h4>Conclusion</h4>The level of CD44 v5 and v6 estimated by ELISA test in the serum can not be used as a prognostic factor in colorectal cancer.

<h4>Unlabelled</h4>This study investigated the role of transcription factor AP-2alpha in chondrocyte differentiation in vitro. AP-2alpha mRNA declined during differentiation, and overexpression of AP-2alpha inhibited differentiation. The results demonstrated that AP-2alpha plays a negative role in chondrocyte differentiation.<h4>Introduction</h4>Transcription factor AP-2alpha has been detected in growth plate and articular chondrocytes and has been shown to regulate cartilage matrix gene expression in vitro. However, the precise functional role of AP-2alpha in chondrocyte differentiation is not known. In this study, we assessed the expression and the function of AP-2alpha in chondrocyte differentiation of ATDC5 cells.<h4>Materials and methods</h4>Chondrocyte differentiation of ATDC5 cells was induced with insulin or transforming growth factor beta (TGF-beta). Proteoglycan production was assessed by alcian blue staining, and expression levels of chondrocyte marker genes and AP-2 gene family were determined by quantitative real time reverse transcriptase-polymerase chain reaction (RT-PCR). Overexpression of AP-2alpha in ATDC5 cells was accomplished by retroviral infection. Infected cells were selected for G418 resistance and pooled for further analysis.<h4>Results and conclusions</h4>Quantitative real time RT-PCR analysis showed that among the four members of the AP-2 gene family, AP-2alpha mRNA was the most abundant. AP-2alpha mRNA levels progressively declined during the differentiation induced by either insulin or TGF-beta treatment. Retroviral expression of AP-2alpha in ATDC5 cells prevented the formation of cartilage nodules, suppressed the proteoglycan production, and inhibited the expression of type II collagen, aggrecan, and type X collagen. Expression profile analysis of key transcription factors involved in chondrogenesis showed that overexpression of AP-2alpha maintained the expression of Sox9 but suppressed the expression of SoxS and Sox6. Taken together, we provide, for the first time, molecular and cellular evidence suggesting that AP-2alpha is a negative regulator of chondrocyte differentiation.

Mutations in the Drosophila gene greatwall cause improper chromosome condensation and delay cell cycle progression in larval neuroblasts. Chromosomes are highly undercondensed, particularly in the euchromatin, but nevertheless contain phosphorylated histone H3, condensin, and topoisomerase II. Cells take much longer to transit the period of chromosome condensation from late G2 through nuclear envelope breakdown. Mutant cells are also subsequently delayed at metaphase, due to spindle checkpoint activity. These mutant phenotypes are not caused by spindle aberrations, by global defects in chromosome replication, or by activation of a caffeine-sensitive checkpoint. The Greatwall proteins in insects and vertebrates are located in the nucleus and belong to the AGC family of serine/threonine protein kinases; the kinase domain of Greatwall is interrupted by a long stretch of unrelated amino acids.

To achieve X-chromosome dosage compensation, organisms must distinguish X chromosomes from autosomes. We identified multiple, cis-acting regions that recruit the Caenorhabditis elegans dosage compensation complex (DCC) through a search for regions of X that bind the complex when detached from X. The DCC normally assembles along the entire X chromosome, but not all detached regions recruit the complex, despite having genes known to be dosage compensated on the native X. Thus, the DCC binds first to recruitment sites, then spreads to neighboring X regions to accomplish chromosome-wide gene repression. From a large chromosomal domain, we defined a 793-base pair fragment that functions in vivo as an X-recognition element to recruit the DCC.

Significantly increased plasma total homocysteine levels (t-Hcys) appeared in treated Huntington disease (HD) patients compared to controls and untreated HD subjects. Because the protein Huntingtin interacts with the homocysteine metabolism modulating enzyme cystathionine beta-synthase, we hypothesize that homocysteine promotes neurodegeneration in HD.

<h4>Objective</h4>We searched for genes whose alleles cause obesity and novel pathways correlated with obesity.<h4>Research methods and procedures</h4>BSB mice are a model of complex obesity due to interactions among genes from C57BL/6J (B) and Mus spretus (SPRET) in (B x SPRET) x B backcross mice. Stringent criteria identified 50 genes differentially expressed in epididymal adipose tissue from 7 pairs of lean vs. obese BSB mice. Quantitative reverse transcription-polymerase chain reaction of adipose tissue RNA from 48 BSB mice with a range of obesity was assayed. Leptin was evaluated in inbred (SPRET/Ei) and outbred (SPRET/Pt) BSB mice.<h4>Results</h4>Leptin (Lep) and adipsin expressions had the greatest fold differences between obese and lean mice. Four genes involved in iron homeostasis were included in the 50 differentially expressed genes [hemochromatosis (Hfe), diaphorase 1, transferrin receptor (Trfr) 2, and protoporphyrinogen oxidase] and two additional iron-related genes did not quite meet the stringent criteria for differential expression (Trfr and lactotransferrin). Hfe and Trfr mRNA levels and liver iron were negatively correlated with fat mass. Variation in obesity phenotypes explained 49%, 40%, and 37%, respectively, of the variance in Hfe, Lep, and Trfr mRNA levels. Leptin differed by haplotype at the Lep locus in outbred BSB. The quantitative trait locus identified in the outbred cross did not occur in inbred BSB.<h4>Discussion</h4>Our results suggest that iron homeostasis in BSB mice is coordinately regulated in vivo in adipose depots in response to obesity. Lep alleles derived from outbred, but not inbred, SPRET are a positional candidate for the chromosome 6 quantitative trait locus in BSB mice.

No abstract available.

Recent studies have shown that human myeloid leukaemia cells can differentiate into dendritic cell (DC)-like cells (leukaemia-DCs) when cultured with a combination of cytokines. In the present study, we examined whether the transduction of leukaemia-DCs with OX40 ligand (OX40L), a member of the tumour necrosis factor (TNF) family, resulted in augmentation of their antigen presenting activity. Bicistronic retroviral vectors expressing both human OX40L and enhanced green fluorescent protein (EGFP) or EGFP alone were generated and used for transduction. Fresh leukaemic cells from five patients with acute myeloid leukaemia (AML) were isolated and retrovirally transduced with OX40L during the culture with a combination of cytokines from stem cell factor, fms-like tyrosine kinase (Flt)-3 ligand, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and TNF-alpha. After 7 d, the majority of cells showed DC-like morphology, and expressed higher levels of CD80, CD86 and HLA-DR than fresh leukaemic cells. The transduction efficiency was 8.5-27.2%. Leukaemia-DCs transduced with OX40L elicited higher proliferative response of allogeneic CD4+ T cells than fresh leukaemic cells, non-transduced, or mock-transduced leukaemia-DCs. Co-culture of allogeneic CD4+ T cells with OX40L-transduced leukaemia-DCs was superior in the generation of interferon (IFN)-gamma producing CD4+ T cells and in production of IFN-gamma. Furthermore, OX40L-transduced leukaemia-DCs could elicit significant proliferative response of human leucocyte antigen-matched T cells from the donor in allogeneic stem cell transplantation. These results indicate that retroviral transduction of leukaemia-DCs with OX40L augments their antigen presenting cell activity and thus renders them more suitable for tumour vaccines or ex vivo stimulation of leukaemia-specific T cells.

Nine neurodegenerative diseases including Huntington's disease (HD) and spinocerebellar ataxia type 7 (SCA7) are caused by an expansion of a polyglutamine (polyQ) stretch in the respective proteins. Aggregation of expanded polyQ-containing proteins into the nucleus is a hallmark of these diseases. Recent evidence indicates that transcriptional dysregulation may contribute to the molecular pathogenesis of these diseases. Using SCA7 and HD mouse models in which we recently described a retinal phenotype, we investigated whether altered gene expression underlies photoreceptor dysfunction. In both models, rhodopsin promoter activity was early and dramatically repressed, suggesting that downregulation of photoreceptor-specific genes plays a major role in polyQ-induced retinal dysfunction. Because the rhodopsin promoter drives mutant ataxin-7 expression in our SCA7 mice, we also assessed whether downregulation of mutant SCA7 transgene would reverse retinopathy progression and aggregate formation. Although residual expression of mutant ataxin-7 was found negligible from 9 weeks of age, SCA7 transgenic mice showed a progressive decline of photoreceptor activity leading to a complete loss of electroretinographic responses from 1 year of age. At this age, aggregates were cleared in only half of the photoreceptors, indicating that their formation is not fully reversible in this model. We demonstrate here that abolishing full-length mutant ataxin-7 expression did not reverse retinopathy progression in SCA7 mice, raising the possibility that some polyQ-induced pathological events might be irreversible.

Striatal and cortical intranuclear inclusions and cytoplasmic aggregates of mutant huntingtin are prominent neuropathological hallmarks of Huntington's disease (HD). We demonstrated previously that transglutaminase 2 cross-links mutant huntingtin in cells in culture and demonstrated the presence of transglutaminase-catalyzed cross-links in the HD cortex that colocalize with transglutaminase 2 and huntingtin. Because calmodulin regulates transglutaminase activity in erythrocytes, platelets, and the gizzard, we hypothesized that calmodulin increases cross-linking of huntingtin in the HD brain. We found that calmodulin colocalizes at the confocal level with transglutaminase 2 and with huntingtin in HD intranuclear inclusions. Calmodulin coimmunoprecipitates with transglutaminase 2 and huntingtin in cells transfected with myc-tagged N-terminal huntingtin fragments containing 148 polyglutamine repeats (htt-N63-148Q-myc) and transglutaminase 2 but not in cells transfected with myc-tagged N-terminal huntingtin fragments containing 18 polyglutamine repeats. Our previous studies demonstrated that transfection with both htt-N63-148Q-myc and transglutaminase 2 resulted in cross-linking of mutant huntingtin protein fragments and the formation of insoluble high-molecular-weight aggregates of huntingtin protein fragments. Transfection with transglutaminase 2 and htt-N63-148Q-myc followed by treatment of cells with N-(6-aminohexyl)-1-naphthalenesulfonamide, a calmodulin inhibitor, resulted in a decrease in cross-linked huntingtin. Inhibiting the interaction of calmodulin with transglutaminase and huntingtin protein could decrease cross-linking and diminish huntingtin aggregate formation in the HD brain.

The Sox6 gene is a member of the Sox gene family that encodes transcription factors. Previous studies have suggested that Sox6 plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia and fibroblast-like cells. In this report, we have shown that Sox6 mRNA increased rapidly in P19 cells during RA induction and then decreased during the differentiation of P19 into neuronal cells. To explore the possible roles of Sox6 during this process, stably Sox6-overexpressing P19 cell lines (P19[Sox6]) were established. These P19[Sox6] had acquired both characteristics of the wild-type P19 induced by RA. First, P19[Sox6] cells showed a marked cellular aggregation in the absence of RA. Second, P19[Sox6] could differentiate into microtubule-associated protein 2 (MAP2)-expressing neuronal cells in the absence of RA. Sox6 expression could cause the activation of endogenous genes including the neuronal transcription factor Mash-1, the neuronal development-related gene Wnt-1, the neuron-specific cell adhesion molecule N-cadherin, and the neuron-specific protein MAP2, resulting in neurogenesis. Moreover, E-cadherin, a major cell adhesion molecule of wild-type P19, was strongly induced by Sox6, resulting in cellular aggregation without RA. Thus Sox6 may play a critical role in cellular aggregation and neuronal differentiation of P19 cells.

Calcium pyrophosphate dihydrate (CPPD) crystal induced arthropathy (CPPD-CA; systemic articular chondrocalcinosis) characterized by deposition of CPPD-crystals in fibro- and hyaline cartilage, joint capsule and periarticular tissues is associated with a variable clinical spectrum of inflammatory degenerative and occasionally destructive joint and vertebral manifestations including neurologic complications and rarely tophaceous-tumoral tissue calcifications. Microcrystal formation in the extracellular matrix of cartilage and tendons is based on genetic and acquired dysregulation of chondrocyte pyrophosphate metabolism and presumably linked to gene mutants on the short arm of chromosome-5 (gene locus 5p15.1 and ANKH gene). Idiopathic disease occurs rarely in hereditary-familial and frequently in sporadic manifestation with increasing prevalence due to aging; a secondary form is significantly related to endocrine and metabolic disorders (as to hyperparathyroidism, hemochromatosis etc.). The impact of the clinical syndrome in practice is important in differential diagnosis of age related conditions like acute mono- or oligoarthritis, systemic osteoarthritis and polymyalgia rheumatica. Actual treatment ignores established prevention of systemic cartilage calcification and is oriented to symptomatic relief.

Axons from the retina traverse different molecular territories as they navigate to the tectum. A single territory might span only a few cell diameters and harbour multiple guidance cues, many of which are beginning to be characterized. Also present in the pathway are 'modulators' that influence a growth cone's response to a coincident signal but do not guide growth directly. An emerging principle is that the growth cone, itself, changes molecularly as it journeys through the visual pathway. Growing retinal axons contain mRNAs, ubiquitinating and apoptotic enzymes, translation and degradation machinery. Guidance cues can trigger rapid and local synthesis, degradation and endocytosis of proteins, providing a fast and flexible way for growth cones to respond to cues in their microenvironment and to alter their responsiveness. The data raise the idea that the localized synthesis and downregulation of proteins might help to steer retinal axon growth and, further, might contribute to the changing character of a growth cone as it ages.

A large number of data derived from molecular analyses support the hypothesis that human cancer is a genetic disease and a distinct subset of genes have been found to be genetically changed in most tumors. Molecular alterations in pancreatic cancer include: (1) oncogenes such as K-ras, c-myc, c-fos, and c-erbB-2; (2) tumor suppressor genes such as p53, p16, DPC4/SMAD4, and DCC; and (3) growth factors such as EGF, FGF, HGF, PDGF, VEGF, TGF-beta. Genetic alterations of K-ras and p53 are common in human pancreatic cancer, but the occurrence of pancreatic cancer is a multi-step phenomenon in which the accumulation of genetic changes is extremely important.

<h4>Objective</h4>To study the effects of coagulation dysfunctions on patients with the development of systemic inflammatory response syndrome (SIRS).<h4>Methods</h4>All the patients studied were divided into 2 groups: SIRS and non-SIRS according to the criteria for selection of SIRS. The SIRS group included 100 patients and the non-SIRS group 50 patients. A control group included 50 healthy adults. SIRS group was further divided into two subgroups: survivor and non-survivor. Levels of platelet (PLT), D-dimer, protein C (PC), protein S (PS), antithrombin III (ATIII), thrombomodulin (TM), tissue plasminogen activator (TPA), plasminogen-activator inhibitor (PAI-1), prothrombin test (PT), thrombin time (TT), activated partial thromboplastin time (APTT) and fibrinogen (FBG) were measured in all the patients and healthy persons.<h4>Results</h4>(1) Levels of TT, APTT, PT, D-dimer, TM, PAI-1 were significantly higher in SIRS than those in non-SIRS and the control group (P < 0.05). Levels of PC, PS, TPA, PLT, FBG were significantly lower in SIRS group than those in non-SIRS and the control group (P < 0.05). (2) Levels of ATIII were not different among SIRS, non-SIRS and the control group (P > 0.05). (3) Survivors levels of D-dimer, APTT and PAI-1 in SIRS group were significantly higher than there in non-survivors (P < 0.05); they were both negatively correlated with the prognosis of SIRS (P < 0.01).<h4>Conclusions</h4>(1) There are dysfunctions of coagulation in patients with systemic inflammatory response syndrome. (2) Fibrinolytic system plays an important role in the development of SIRS. (3) Levels of PAI-1 and D-dimer are both negatively correlated with the prognosis of SIRS.

<h4>Objective</h4>To analysis and define the clinical phenotype and related mutation of hereditary hemorrhagic telangiectasia. (HHT).<h4>Methods</h4>The proband of a Chinese HHT family, female, aged 48, and 2 of her family members: her father, aged 74, and her brother, aged 44 underwent nasal cavity endoscopy and automatic photochonography to observe the capillaries in nasal mucosa. Samples of peripheral blood were collected. The exons 3, 7, and 8 of the activin receptor-like kinase 1 (ALK1) gene of the proband and her family members and 2 healthy people as contrds were amplified by polymerase chain reaction, and the PCR products were sequenced. The levels of plasma thrombomodulin (TM) of the proband and her family members were detected by Western blotting combined with density screening.<h4>Results</h4>Except the mother and sister-in-law, other 4 of the family members had epistaxis or bleeding in other sites. The proband and her father had obvious telangiectasis of nasal mucosa or finger skin respectively. ALK1 gene analysis confirmed that a C1231T mutation (CGG-->TGG) in the exon 8 existed in the proband, her brother, and her father, no mutation was found in her sister in law, her nephew, and the healthy persons. Six bands were shown in the expression of plasma thrombomodulin. Density screening was conducted for the 2 bands with differential expression: those with the molecular masses of 56,000 and 28,000. The result of density screening showed that the average value of screening at the 56,000 band was 218.3 in the normal controls, significantly higher than that of the HTT patients (145.1); and the average value of screening at the 28,000 band was 222.0 in the normal controls, significantly higher than that in the HTT patients (145.1).<h4>Conclusion</h4>ALK1 gene mutation, a C1231T variation on exon 8, exists in Chinese type 2 HHT individuals. The levels of plasma thrombomodulin levels at the 56,000 and 28,000 fragments of the HTT patients are lower than of normal subjects whose significance and mechanism remain to be elucidated.

<h4>Aim</h4>It is not clear whether the mutations in hemochromatosis (HFE) gene and hepatitis C virus (HCV) infection act independently in the pathogenesis of porphyria cutanea tarda (PCT). The prevalence of both risk factors varies greatly in different parts of the world. PCT patients from Hungary were evaluated to assess both factors.<h4>Methods</h4>The prevalence of C282Y and H63D mutations in the HFE gene was determined in 50 PCT patients and compared with the reported control frequencies. Furthermore, the presence of HCV infection was determined and related to the patients' HFE gene status.<h4>Results</h4>The C282Y mutation was found in 8/50 cases (three homozygotes and five heterozygotes), with an 11% allele frequency (vs. 3.8% control) (P<0.05). Seventeen patients were heterozygous, one was homozygous for the H63D mutation, allele frequency 19%, which did not differ significantly from the reported control prevalence of 12.3%. Twenty-two patients (44%) were HCV-RNA positive; six out of them were heterozygous for H63D mutation, one only for the C282Y mutation and one was compound heterozygous for both mutations.<h4>Conclusion</h4>HCV infection and HFE C282Y mutation may probably be independent predisposing factors for development of PCT in Hungarian patients.

Wilson's disease (WD) and hereditary hemochromatosis (HH) are two inherited disorders with potentially devastating and life-threatening complications. Their eminent treatability makes diagnosis in adolescence or young adulthood critical. WD is the result of abnormal copper homeostasis, causing copper overload and end-organ damage. Chelation therapy can be highly efficacious in preventing manifestations of WD. HH is caused by inappropriate absorption of dietary iron, typically as the result of a specific mutation, C282Y, in the HFE gene. End-organ disease from iron accumulation is protean and includes progressive damage of the liver, pancreas, skin, heart, and pituitary. It is important to permit therapeutic phlebotomy to commence before the onset of complications.

Dyslipoproteinaemia is a cardinal feature of the metabolic syndrome that accelerates atherosclerosis. It is usually characterised by high plasma concentrations of triglyceride-rich and apolipoprotein (apo) B-containing lipoproteins, with depressed concentrations of high-density lipoprotein (HDL). Dysregulation of lipoprotein metabolism in these subjects may be due to a combination of overproduction of very-low-density lipoprotein (VLDL) apoB-100, decreased catabolism of apoB-containing particles, and increased catabolism of HDL apoA-I particles. These abnormalities may be consequent on a global metabolic effect of insulin resistance that increases the flux of fatty acids from adipose tissue to the liver, the accumulation of fat in the liver, the increased hepatic secretion of VLDL-triglycerides and the remodelling of both low-density lipoprotein (LDL) and HDL particles in the circulation; perturbations in lipolytic enzymes and lipid transfer proteins contribute to the dyslipidaemia. Our in vivo understanding of the kinetic defects in lipoprotein metabolism in the metabolic syndrome has been chiefly achieved by ongoing developments in the use of stable isotope tracers and mathematical modelling. Knowledge of the pathophysiology of lipoprotein metabolism in the metabolic syndrome is well complemented by extensive cell biological data. Nutritional modifications and increased physical exercise may favourably alter lipoprotein transport in the metabolic syndrome by collectively decreasing the hepatic secretion of VLDL-apoB and the catabolism of HDL apoA-I, as well as by increasing the clearance of LDL-apoB. Pharmacological treatments, such as statins, fibrates or fish oils, can also correct the dyslipidaemia by several mechanisms of action including decreased secretion and increased catabolism of apoB, as well as increased secretion and decreased catabolism of apoA-I. The complementary mechanisms of action of lifestyle and drug therapies support the use of combination regimens to treat dyslipidaemia in the metabolic syndrome.