Gene Literature Dashboard

The mammalian testis determining factor SRY and its related Sox factors are critical developmental regulators. They share significant similarity in their high mobility group (HMG) domain and display discrete patterns of tissue-specific expression. Here we show that SRY and the Sox protein SOX6 colocalize with splicing factors in the nucleus and are dynamically redistributed following the blockage of splicing in living cells. Anti-SOX6 antibodies supershift the spliceosomal complex from assembled splicing reactions and inhibit splicing in vitro of multiple pre-mRNA substrates. Most importantly, SOX6-depleted nuclear extracts have impaired splicing activity, which is efficiently restored by addition of the recombinant SOX6 HMG domain and also by recombinant SRY and the SOX9 HMG domain. These results reveal an unexpected biological function of the SRY, SOX6, and SOX9 gene products and provide a functional link to the biochemical mechanisms operating in mammalian sex determination and in other developmental processes regulated by Sox genes.

Structural maintenance of chromosomes (SMC) proteins play central roles in higher-order chromosome dynamics from bacteria to humans. In eukaryotes, two different SMC protein complexes, condensin and cohesin, regulate chromosome condensation and sister chromatid cohesion, respectively. Each of the complexes consists of a heterodimeric pair of SMC subunits and two or three non-SMC subunits. Previous studies have shown that a bacterial SMC homodimer has a symmetrical structure in which two long coiled-coil arms are connected by a flexible hinge. A catalytic domain with DNA- and ATP-binding activities is located at the distal end of each arm. We report here the visualization of vertebrate condensin and cohesin by electron microscopy. Both complexes display the two-armed structure characteristic of SMC proteins, but their conformations are remarkably different. The hinge of condensin is closed and the coiled-coil arms are placed close together. In contrast, the hinge of cohesin is wide open and the coiled-coils are spread apart from each other. The non-SMC subunits of both condensin and cohesin form a globular complex bound to the catalytic domains of the SMC heterodimers. We propose that the "closed" conformation of condensin and the "open" conformation of cohesin are important structural properties that contribute to their specialized biochemical and physiological functions.

The non-covalent association of beta 2-microglobulin with MHC class I molecules and MHC class I-type molecules such as FcRn or the hemochromatosis protein (HFE) is of major importance for their function, i.e., antigen presentation, IgG transport, and regulation of iron uptake, respectively. In the human hemochorial placenta, the syncytiotrophoblast forms a continuous epithelial layer covering the villous trees, where it directly contacts maternal blood and, among many other functions, mediates uptake of maternal IgG and iron. The villous syncytiotrophoblast lacks MHC class I molecules but expresses FcRn and HFE. Since data on beta 2-microglobulin synthesis and localization in the term villous syncytiotrophoblast were contradictory, we investigated the subcellular localization of beta 2-microglobulin by immunoelectron microscopy. Synthesis in the trophoblast is demonstrated by colocalization of beta 2-microglobulin with protein disulfide isomerase, a marker protein of the endoplasmic reticulum. The presence of beta 2-microglobulin at the apical plasma membrane corresponds to the recently observed association of beta 2-microglobulin with HFE and FcRn. Localization of beta 2-microglobulin in late endosomes/lysosomes, labeled with antibodies to lysosome membrane antigen LAMP 2, suggests also a degradative route of beta 2-microglobulin internalized by fluid-phase from the maternal blood.

Huntington's disease is caused by the expansion of CAG repeats coding for a polyglutamine tract in the huntingtin protein. The major pathological feature found in Huntington's disease neurons is the presence of detergent-insoluble ubiquitinated inclusion bodies composed of the huntingtin protein. However, the mechanisms that underlie inclusion body formation, and the precise relationship between inclusion bodies and events that initiate toxicity, remain unclear. Here, we analyzed the effects of drugs or genetic mutations that disrupt the microtubule cytoskeleton in a Saccharomyces cerevisiae model of the aggregation of an amino-terminal polyglutamine-containing fragment of huntingtin exon 1 (HtEx1). Treatment of yeast with drugs that disrupt microtubules resulted in less than 2% of the detergent-insoluble HtEx1 observed in mock-treated cells and prevented the formation of large juxtanuclear inclusion bodies. Disruption of microtubules also unmasked a potent glutamine length-dependent toxicity of HtEx1 under conditions where HtEx1 exists in an entirely detergent-soluble nonaggregated form. Results from the yeast model paralleled those from neuronal pheochromocytoma cells, where disruption of microtubules eliminated the formation of juxtanuclear and intranuclear inclusion bodies by HtEx1. Our results suggest that active transport along microtubules may be required for inclusion body formation by HtEx1 and that inclusion body formation may have evolved as a cellular mechanism to promote the sequestration or clearance of soluble species of HtEx1 that are otherwise toxic to cells.

We have generated eight mAbs (MW1-8) that bind the epitopes polyglutamine (polyQ), polyproline (polyP), or the C terminus of exon 1 in huntingtin (htt) protein. In the brains of Huntington's disease (HD) mouse models, the anti-polyQ mAbs bind to various cytoplasmic compartments, whereas the anti-polyP and anti-C terminus mAbs bind nuclear inclusions containing htt. To use these mAbs as intracellular perturbation agents, we have cloned and expressed the antigen-binding domains of three of the mAbs as single-chain variable region fragment Abs (scFvs). In 293 cells cotransfected with htt exon 1 containing an expanded polyQ domain, MW1, MW2, and MW7 scFvs colocalize with htt exon 1. Moreover, these scFvs coimmunoprecipitate with htt exon 1 in cell extracts. In perturbation experiments, MW7 scFv, recognizing the polyP domains of htt, significantly inhibits aggregation as well as the cell death induced by mutant htt protein. In contrast, MW1 and MW2 scFvs, recognizing the polyQ stretch, stimulate htt aggregation and apoptosis. Therefore, these anti-htt scFvs can be used to investigate the role of the polyP and polyQ domains in HD pathogenesis, and antibody binding to the polyP domain has potential therapeutic value in HD.

BIG2 is a guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and adaptor protein (AP)-1 coat protein complexes. A fungal metabolite, brefeldin A (BFA), inhibits ARF-GEFs and leads to redistribution of coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). To investigate the function of BIG2, we examined the effects of BIG2-overexpression on the BFA-induced redistribution of ARF, coat proteins, and organelle markers. The BIG2 overexpression blocked BFA-induced redistribution from membranes of ARF1 and the AP-1 complex but not that of the COPI complex. These observations indicate that BIG2 is implicated in membrane association of AP-1, but not that of COPI, through activating ARF. Furthermore, not only BIG2 but also ARF1 and AP-1 were found as queues of spherical swellings along the BFA-induced membrane tubules emanating from the TGN. These observations indicate that BFA-induced AP-1 dissociation from TGN membranes and tubulation of TGN membranes are not coupled events and suggest that a BFA target other than ARF-GEFs exists in the cell.

Transglutaminase (TGase) activity is increased in affected regions of brains from patients with Huntington's disease (HD). TGase activity is particularly elevated in the nucleus compared with the cytoplasm from these brains. Gamma-glutaminyl-lysyl cross-links have been detected in nuclear inclusions in HD brain, indicating that TGase may play a prominent role in the aggregation of huntingtin (htt). Attempts to ameliorate experimental disease, via inhibition of TGase in transgenic models of HD in mice, are under investigation.

Tissue transglutaminase (tTG) likely plays a role in numerous processes in the nervous system. tTG posttranslationally modifies proteins by transamidation of specific polypeptide bound glutamines (Glns). This reaction results in the incorporation of polyamines into substrate proteins or the formation of protein crosslinks, modifications that likely have significant effects on neural function. Huntington's disease is a genetic disorder caused by an expansion of the polyglutamine domain in the huntingtin protein. Because a polypeptide bound Gln is the determining factor for a tTG substrate, and mutant huntingtin aggregates have been found in Huntington's disease brain, it has been hypothesized that tTG may contribute to the pathogenesis of Huntington's disease. In vitro, polyglutamine constructs and huntingtin are substrates of tTG. Further, the levels of tTG and TG activity are elevated in Huntington's disease brain and immunohistochemical studies have demonstrated that there is an increase in tTG reactivity in affected neurons in Huntington's disease. These findings suggest that tTG may play a role in Huntington's disease. However in situ, neither wild type nor mutant huntingtin is modified by tTG. Further, immunocytochemical analysis revealed that tTG is totally excluded from the huntingtin aggregates, and modulation of the expression level of tTG had no effect on the frequency of the aggregates in the cells. Therefore, tTG is not required for the formation of huntingtin aggregates, and likely does not play a role in this process in Huntington's disease brain. However, tTG interacts with truncated huntingtin, and selectively polyaminates proteins that are associated with mutant truncated huntingtin. Given the fact that the levels of polyamines in cells is in the millimolar range and the crosslinking and polyaminating reactions catalyzed by tTG are competing reactions, intracellularly polyamination is likely to be the predominant reaction. Polyamination of proteins is likely to effect their function, and therefore it can be hypothesized that tTG may play a role in the pathogenesis of Huntington's disease by modifying specific proteins and altering their function and/or localization. Further research is required to define the specific role of tTG in Huntington's disease.

The role of secretory epididymal factors on sperm survival and storage in bovine cauda epididymides is poorly understood. Thus, the effects of bovine epididymal epithelium fluid (BEEF) on frozen-thawed bovine sperm motility have been evaluated in vitro. Sperm motion parameters were assessed by computer-assisted sperm analysis. Compared with serum bovine proteins, BEEF efficiently sustained bovine sperm motility after a 6-h incubation period. The positive effect of BEEF on sperm motility was even more apparent using a fractionated BEEF extract (>10 kDa, 2 mg/ml). This beneficial effect was abolished when the BEEF active fraction was heat treated before incubation. A minimal 2-h BEEF preincubation period was necessary to maintain sperm motility activity and to protect sperm against oxidative injury caused by 150 microM hydrogen peroxide. The proteins from the BEEF >10-kDa fractions were biotinylated to identify the proteins that bind to the sperm surface. Five specific sperm-surface-binding proteins were revealed by Western blot analysis probed with avidin-horseradish peroxidase conjugate. These proteins were digested with trypsin for identification by matrix-assisted laser desorption ionization time-of-flight peptide mass spectrometric analyzer. Under reducing conditions, 5 bovine proteins were identified: the beta (36-kDa spot) and alpha (38-kDa spot) chains of clusterin, the beta-adrenergic receptor kinase 2 (48-kDa spot), and the antithrombin-III and the fibrinogen gamma-B chains, both corresponding to a doublet of about 50-52 kDa. These proteins are known to be present at the sperm surface in other species and could play a role in sperm protection in vivo. These results provide new insights to explain how secretory epididymal proteins sustain sperm motility during storage in vitro.

The tfd genes of Ralstonia eutropha JMP134 are the only well-characterized set of genes responsible for 2,4-dichlorophenoxyacetic acid (2,4-D) degradation among 2,4-D-degrading bacteria. A new family of 2,4-D degradation genes, cadRABKC, was cloned and characterized from Bradyrhizobium sp. strain HW13, a strain that was isolated from a buried Hawaiian soil that has never experienced anthropogenic chemicals. The cadR gene was inferred to encode an AraC/XylS type of transcriptional regulator from its deduced amino acid sequence. The cadABC genes were predicted to encode 2,4-D oxygenase subunits from their deduced amino acid sequences that showed 46, 44, and 37% identities with the TftA and TftB subunits of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) oxygenase of Burkholderia cepacia AC1100 and with a putative ferredoxin, ThcC, of Rhodococcus erythropolis NI86/21, respectively. They are thoroughly different from the 2,4-D dioxygenase gene, tfdA, of R. eutropha JMP134. The cadK gene was presumed to encode a 2,4-D transport protein from its deduced amino acid sequence that showed 60% identity with the 2,4-D transporter, TfdK, of strain JMP134. Sinorhizobium meliloti Rm1021 cells containing cadRABKC transformed several phenoxyacetic acids, including 2,4-D and 2,4,5-T, to corresponding phenol derivatives. Frameshift mutations indicated that each of the cadRABC genes was essential for 2,4-D conversion in strain Rm1021 but that cadK was not. Five 2,4-D degraders, including Bradyrhizobium and Sphingomonas strains, were found to have cadA gene homologs, suggesting that these 2,4-D degraders share 2,4-D degradation genes similar to those of strain HW13 cadABC.

The AF10 gene encodes a putative transcription factor containing an N-terminal LAP/PHD zinc finger motif, a functional nuclear localization signal, an AT-hook domain, and a leucine zipper toward the C-terminus. AF10 is involved in 2 distinct chromosomal translocations associated with hematologic malignancy. The chimeric fusion proteins MLL/AF10 and CALM/AF10, resulting from the t(10;11)(p12;q23) and the t(10;11)(p12;q14), respectively, consistently retain the leucine zipper motif of AF10. This part of the C-terminal region was used as bait in a yeast 2 hybrid screening of a testis complementary DNA library. The leucine zipper interacted with GAS41, a protein previously identified as the product of an amplified gene in a glioblastoma. GAS41 shows significant homology to the Saccharomyces cerevisiae protein ANC1 and to the human MLL fusion partners AF9 and ENL. The interaction was confirmed in vivo. Furthermore, the study showed by coimmunoprecipitation that GAS41 interacts with INI1 (Integrase Interactor 1) and that INI1 was present in the AF10 immunoprecipitate. INI1 is the human homologue of the yeast SNF5 protein, a component of the SWI/SNF complex, which acts to remodel chromatin and to modulate transcription. The retention of the leucine zipper in the MLL and CALM fusions suggests that a key feature of these chimeric proteins may be their ability to interfere in normal gene regulation through interaction with the adenosine triphosphate-dependent chromatinremodeling complexes.

Ret proto-oncogene rearrangements are common in papillary thyroid carcinoma. The relationship of hyalinizing trabecular tumors (HTT) to papillary thyroid carcinoma (PTC) was analyzed in a recent report in which the presence of RET rearrangements was detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry in several HTT. This commentary examines this report and related studies in the literature emphasizing the limitations of these approaches and the practical implications of these findings.

Despite advances in supportive care, sepsis and septic shock continue to be major causes of morbidity and mortality in critically ill patients. The lack of efficacy of anti-inflammatory drugs in patients with sepsis has shifted interest toward developing alternative treatments. The observation that clotting system activation may in part underlie the physiological derangements of sepsis has resulted in efforts to target the clotting cascade as a therapeutic strategy. Anticoagulants have been shown to ameliorate physiological derangements and improve survival in animal sepsis models. Three agents have undergone extensive study in humans: recombinant human activated protein C (rhAPC, drotrecogin-alpha), antithrombin III (ATIII) and tissue factor pathway inhibitor (TFPI). While a recent Phase III study of rhAPC suggests a survival benefit in patients with sepsis, major concerns about this trial include the manner in which the study was conducted, the potential toxicity of rhAPC and the questionable efficacy of this agent in patients with low mortality risk. Further clinical testing of rhAPC appears to be necessary to better define the target population most appropriate for its use. In contrast, a large Phase III study of high dose ATIII in patients with sepsis failed to show a treatment benefit with this agent. Finally, while TFPI has undergone extensive preclinical and Phase II testing, the results of Phase III studies have not been published. In summary, while coagulation inhibitors may ultimately have a therapeutic role in selected subgroups of patients with sepsis, the efficacy and safety of this class of agents remain to be proven.

<h4>Objective</h4>Genetic epidemiologic and clinical data suggest that comorbid panic disorder may define a subtype of bipolar disorder. Comorbid panic disorder might thereby influence the strength of association between bipolar disorder and genes that have been implicated in bipolar disorder on the basis of their function in monoamine neurotransmission and previously reported linkage results. Polymorphic markers at catechol O-methyltransferase (COMT), serotonin transporter (5-HTT), and tryptophan hydroxylase (TPH) genes were analyzed in a case-control association study of bipolar disorder patients with or without lifetime panic disorder.<h4>Method</h4>Unrelated subjects of Italian descent meeting DSM-III-R criteria for lifetime bipolar disorder (N=111), with (N=49) or without (N=62) comorbid lifetime panic disorder, were compared to 127 healthy subjects. DNA was extracted from blood leukocytes. The frequencies of COMT Val158Met, 5-HTTLPR, and TPH IVS7+218C>A polymorphisms were determined. Genotype and allele frequency comparisons between affected (bipolar disorder, bipolar disorder without panic disorder, or bipolar disorder with panic disorder) and unaffected individuals were carried out with chi-square tests or Fisher's exact tests.<h4>Results</h4>Relative to the comparison subjects, subjects with bipolar disorder without panic disorder, but not those with comorbid bipolar disorder and panic disorder, showed significantly higher frequencies of the COMT Met158 and the short 5-HTTLPR alleles and genotypes. The differences in the frequencies of the TPH IVS7+218A alleles and genotypes approached statistical significance.<h4>Conclusions</h4>The findings support the hypothesis that comorbid panic disorder identifies a genetic subtype of bipolar disorder and suggest a role for COMT and 5-HTT in vulnerability to these disorders.

A 1933 bp cDNA fragment, coding a truncated testis-specific novel nucleoporin, was isolated from a human testis lambdaZAPII cDNA library, designated as BS-63 and assigned GenBank accession number: U64675. By applying the methods of rapid amplification of cDNA ends (5' RACE) and PCR, a full-length BS-63 cDNA composed of 5475 bp was obtained. BS-63 cDNA contained an open reading frame consisting of 1765 codons and XFXFG or GLFG repetitive sequence motifs. These repetitive motifs are structural characteristic of nucleoporins. BS-63 cDNA has high homology with Nup358/Ran BP2. A 1599 bp fragment, corresponding to the C-terminus of BS-63 cDNA, was prepared and expressed in E. coli BL21(DE3). The recombinant product was purified by affinity chromatography and SDS-PAGE and polyclonal antibodies raised. In rat testis section, the BS-63 protein was localized at the sites of nuclear pores in spermatids by immuno-gold transmission electron microscopy and on the nuclear membrane of Triton X-treated sperm by colloidal silver immuno-gold scanning electron microscopy. The recombinant BS-63 protein can be phosphorylated in vitro with PKC and p34(cdc2). A yeast two-hybrid system was used to screen a mouse testis cDNA library to identify proteins capable of interacting with BS-63. Using the 1.6 kb cDNA fragment as bait, the following interacting proteins were identified: Ran, transportin (karyopherin beta2), two proteins related to the nucleocytoplasmic transporter and aF10 protein. The latter protein is a putative transcriptor containing a cysteine-rich N-terminus, a LAP/PHD finger, a leucine zipper domain and a glutamine-rich C-terminus. Also it is highly expressed in murine testis and is located in the cell nucleus and cytoplasm. The interaction of BS-63 with aF10 (696-1001aa) was validated by surface plasmon resonance and by affinity precipitation combined with Western blot. aF10 (696-1001aa) interacted in vitro with BS-63 extracted from rat testis germ cells. It is hypothesized that BS-63 is a testis-specific nucleoporin and possibly acts as a docking site and a cotransporter of Ran and transportin. The complex performs the task of a carrier system in transporting aF10 into the nucleus of germ cells during spermiogenesis.

CDO is a cell surface receptor-like protein that positively regulates myogenic differentiation. Reported here is the identification of BOC, which, with CDO, defines a newly recognized subfamily within the immunoglobulin superfamily. cdo and boc are co-expressed in muscle precursors in the developing mouse embryo. Like CDO, BOC accelerates differentiation of cultured myoblast cell lines and participates in a positive feedback loop with the myogenic transcription factor, MyoD. CDO and BOC form complexes in a cis fashion via association of both their ectodomains and their intracellular domains. A soluble fusion protein that contains the entire BOC ectodomain functions similarly to full-length BOC to promote myogenic differentiation, indicating that the intracellular region is dispensable for its activity in this system. Furthermore, a dominant-negative form of CDO inhibits the pro-myogenic effects of soluble BOC, suggesting that BOC is dependent on CDO for its activity. CDO and BOC are proposed to be components of a receptor complex that mediates some of the cell-cell interactions between muscle precursors that are required for myogenesis.

Neurotransmitters are released by synaptic vesicle fusion at the active zone. The active zone of a synapse mediates Ca2+-triggered neurotransmitter release, and integrates presynaptic signals in regulating this release. Much is known about the structure of active zones and synaptic vesicles, but the functional relation between their components is poorly understood. Here we show that RIM1alpha, an active zone protein that was identified as a putative effector for the synaptic vesicle protein Rab3A, interacts with several active zone molecules, including Munc13-1 (ref. 6) and alpha-liprins, to form a protein scaffold in the presynaptic nerve terminal. Abolishing the expression of RIM1alpha in mice shows that RIM1alpha is essential for maintaining normal probability of neurotransmitter release, and for regulating release during short-term synaptic plasticity. These data indicate that RIM1alpha has a central function in integrating active zone proteins and synaptic vesicles into a molecular scaffold that controls neurotransmitter release.

T cell lymphoma carrying Epstein Barr virus (EBV(+) TL) is very rare among Western countries while it is much more common among Japanese. Here we report an EBV(+) TL which has been maintained for years by the use of mice with severe combined immune deficiency (SCID) mice. Lymphoma was obtained from a 55-year-old male suffering from oculomotor nerve palsy and lymphadenopathy. A small piece of biopsied tumor was transplanted into SCID mice and the lymphoma has been maintained for over 3 years with passages every 2 - 3 weeks. The maintained lymphoma, termed as TMS24, and the original lymphoma cells showed identical phenotype and genotype, including diffuse medium-sized cell morphology lacking granules, suppressor / cytotoxic immunophenotype and identical T cell receptor beta-chain gene rearrangement mode. Further, both were shown to carry an identical EBV clone in terms of the number of terminal repeats and the latency II-type restricted gene expression profile. Cytogenetically, TMS24 retained two characteristic chromosomal translocations of t(1;18)(q32;q21) and t(6;12)(p21;q24). Since only one cell line with such characters has been reported previously, TMS24 should be useful for detailed analysis of EBV(+) TL.

Previous studies have provided conflicting evidence regarding the association of the serotonin transporter (5-HTT) gene with autism. Two polymorphisms have been identified in the human 5-HTT gene, a VNTR in intron 2 and a functional deletion/insertion in the promoter region (5-HTTLPR) with short and long variants. Positive associations of the 5-HTTLPR polymorphism with autism have been reported by two family-based studies, but one found preferential transmission of the short allele and the other of the long allele. Two subsequent studies failed to find evidence of transmission disequilibrium at the 5-HTTLPR locus. These conflicting results could be due to heterogeneity of clinical samples with regard to serotonin (5-HT) blood levels, which have been found to be elevated in some autistic subjects. Thus, we examined the association of the 5-HTTLPR and VNTR polymorphisms of the 5-HTT gene with autism, and we investigated the relationship between 5-HTT variants and whole-blood 5-HT. The transmission/disequilibrium test (TDT) revealed no linkage disequilibrium at either loci in a sample of 96 families comprising 43 trios and 53 sib pairs. Furthermore, no significant relationship between 5-HT blood levels and 5-HTT gene polymorphisms was found. Our results suggest that the 5-HTT gene is unlikely to play a major role as a susceptibility factor in autism.

Nonhuman primates offer unique opportunities to study the effects of genes, environments, and their interaction, on physiology and complex behavior. We examined genotype and early environment contributions to CNS function in a large sample of rhesus monkeys. In humans, length variation of the serotonin (5-HT) transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) that results in allelic variation in 5-HTT expression is associated with decreased serotonergic function and 5-HT-mediated psychopathology. We report that an analogous variation of the gene's regulatory region in monkeys interacts with early experience to affect central 5-HT functioning. Monkeys with deleterious early rearing experiences were differentiated by genotype in cerebrospinal fluid concentrations of the 5-HT metabolite, 5-hydroxyindoleacetic acid, while monkeys reared normally were not. These findings demonstrate an environment-dependent effect of the rh5-HTTLPR genotype on CNS 5-HT function and suggest nonhuman primates may provide an important avenue for investigating gene/environment interactions using candidate genes for physiological and behavioral traits.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder. In 1993 the mutation that causes HD was identified as an unstable expansion of CAG repeats in the IT15 gene. Since then one of the most important advances in HD research has been the generation of various mouse models that enable the exploration of early pathological, molecular and cellular abnormalities produced by the mutation. In addition, these models have made it possible to test different pharmacological approaches to delay the onset or slow the progression of HD. In this article, insights gained from mouse models towards the understanding of HD and the design of new therapeutic strategies are discussed.

<h4>Background/aims</h4>A body of evidence suggests that ceruloplasmin (Cp), the major serum copper-containing protein, acts in iron metabolism due to its ferroxidase activity which appears essential for iron movements and exchanges.<h4>Methods</h4>The present study investigated the serum levels of Cp and its ferroxidase activity in 53 C282Y homozygote genetic hemochromatosis (38 iron overloaded, 15 iron depleted) patients as compared to age and sex-matched healthy volunteers.<h4>Results</h4>Serum levels of Cp were significantly decreased in iron-overloaded male hemochromatotic patients vs. the control group (P=0.02). Furthermore, serum ferroxidase activity was strongly and significantly lower in iron-overloaded male hemochromatotic patients (P<0.001). In contrast, in iron-depleted male hemochromatotic patients, who were under maintenance therapy by regular phlebotomies, serum levels of Cp and ferroxidase activity were not statistically different from those observed in controls.<h4>Conclusions</h4>These data: (i) show that serum Cp and ferroxidase activity are decreased when C282Y homozygote men are iron overloaded and normal when iron depleted; (ii) suggest that iron may modulate the Cp gene expression; and (iii) raise the issue of the putative role of decreased serum ferroxidase activity in the phenotypic expression of HFE-1 hereditary hemochromatosis.

Neurons destined to form several precerebellar nuclei are generated in the dorsal neuroepithelium (rhombic lip) of caudal hindbrain. They form two ventrally directed migratory streams, which behave differently. While neurons in the superficial migration migrate in a subpial position and cross the midline to settle into the contralateral hindbrain, neurons in the olivary migration travel deeper in the parenchyma and stop ipsilaterally against the floor plate. In the present study, we compared the behavior of the two neuronal populations in an organotypic culture system that preserves several aspects of their in vivo environment. Both migrations occurred in mouse hindbrain explants dissected at E11.5 even when the floor plate was ablated at the onset of the culture period, indicating that they could rely on dorsoventral cues already distributed in the neural tube. Nevertheless, the local constraints necessary for the superficial migration were more specific than for the olivary migration. Distinct chemoattractive and chemorespulsive signal were found to operate on the migrations. The floor plate exhibited a strong chemoattractive influence on both migrations, which deviated from their normal path in the direction of ectopic floor plate fragments. It was also found to produce a short-range stop signal and to induce inferior olive aggregation. The ventral neural tube was also found to inhibit or slow down the migration of olivary neurons. Interestingly, while ectopic sources of netrin were found to influence both migrations, this effect was locally modulated and affected differentially the successive phases of migration. Consistent with this observation, while neurons in the superficial migration expressed the Dcc-netrin receptor, the migrating olivary neurons did not express Dcc before they reached the midline. Our observations provide a clearer picture of the hierarchy of environmental cues that influence the morphogenesis of these precerebellar nuclei.

Germline maintenance in the nematode C. elegans requires global repressive mechanisms that involve chromatin organization. During meiosis, the X chromosome in both sexes exhibits a striking reduction of histone modifications that correlate with transcriptional activation when compared with the genome as a whole. The histone modification spectrum on the X chromosome corresponds with a lack of transcriptional competence, as measured by reporter transgene arrays. The X chromosome in XO males is structurally analogous to the sex body in mammals, contains a histone modification associated with heterochromatin in other species and is inactivated throughout meiosis. The synapsed X chromosomes in hermaphrodites also appear to be silenced in early meiosis, but genes on the X chromosome are detectably expressed at later stages of oocyte meiosis. Silencing of the sex chromosome during early meiosis is a conserved feature throughout the nematode phylum, and is not limited to hermaphroditic species.

Hereditary hemochromatosis is a genetic disease characterized by exaggerated absorption of intestinal iron leading to its accumulation in some organs over the years. Its prevalence is estimated to be 3-5/1000 in Caucasians. A single mutation, C282Y in the HFE gene explains 80-90% of all diagnosed cases in populations of northwestern European ancestry. The importance of another frequent mutation in this gene, H63D, as well as of C282Y/H63D compound heterozygotes, is still a matter of debate. We estimated the prevalence of these mutations in newborns from a genetically well defined French-Canadian population, in Quebec City. We compared genotype and allele frequencies between neonates and referred patients for HFE molecular analysis. We genotyped anonymous-unlinked cord blood samples for C282Y (n = 881) and H63D (n = 870) mutations from neonates. Referred patients (n = 1084) were genotyped in two different laboratories and pooled after verifying the similarity of both groups. No C282Y homozygote was found in neonates (allele frequency = 0.043). However, we identified 163 C282Y homozygotes (15%) among 1084 referred patients leading to a, not surprising, 97-fold enrichment of this genotype. We found a similar proportion of genotypes homozygous for H63D in both groups suggesting a weak association with the disease. However, we found a 5-fold enrichment of compound heterozygotes in the referred group. Fewer C282Y homozygotes were observed in the French-Canadian population than in northwest Europe populations. However, the strong enrichment of homozygotes between the neonates and the referred patients is an argument in favour of screening for this lethal disease.

We previously characterized major components of mitotic chromosomes assembled in Xenopus laevis egg extracts and collectively referred to them as Xenopus chromosome-associated polypeptides (XCAPs). They included five subunits of the condensin complex essential for chromosome condensation. In an effort to identify novel proteins involved in this process, we have isolated XCAP-F and found it to be the Xenopus ortholog of ISWI, a chromatin remodeling ATPase. ISWI exists in two major complexes in Xenopus egg extracts. The first complex contains ACF1 and two low-molecular-weight subunits, most likely corresponding to Xenopus CHRAC. The second complex is a novel one that contains the Xenopus ortholog of the human Williams syndrome transcription factor (WSTF). In the absence of the ISWI complexes, the deposition of histones onto DNA is apparently normal, but the spacing of nucleosomes is greatly disturbed. Despite the poor spacing of nucleosomes, ISWI depletion has little effect on DNA replication, chromosome condensation or sister chromatid cohesion in the cell-free extracts. The association of ISWI with chromatin is cell cycle regulated and is under the control of the INCENP-aurora B kinase complex that phosphorylates histone H3 during mitosis. Apparently contradictory to the generally accepted model, we find that neither chromosome condensation nor chromosomal targeting of condensin is compromised when H3 phosphorylation is drastically reduced by depletion of INCENP-aurora B.

<h4>Background</h4>There has been much interest in screening populations for disease-associated mutations. A favoured candidate has been the HFE gene, mutations of which are the most common cause of haemochromatosis in the European population. About five people in 1000 are homozygotes for the 845G-->A mutation, but little is known of how many have mutation-caused clinical manifestations.<h4>Methods</h4>We screened 41038 individuals attending a health appraisal clinic in the USA for the 845G--> A and 187C-->G HFE mutations, and analysed laboratory data and data on signs and symptoms of haemochromatosis as elicited by questionnaire.<h4>Findings</h4>The most common symptoms of haemochromatosis, including poor general health, diabetes, arthropathies, arrhythmias, impotence, and skin pigmentation were no more prevalent among the 152 identified homozygotes than among the controls. The age distribution of homozygotes and compound heterozygotes did not differ significantly from that of controls: there was no measurable loss of such individuals from the population during ageing. However, there was a significantly increased prevalence of a history of hepatitis or "liver trouble" among homozygotes and in the proportion of homozygotes with increased concentrations of serum aspartate aminotransferase and collagen IV; these changes were not related to iron burden or to age. Only one of the 152 homozygotes had signs and symptoms that would suggest a diagnosis of haemochromatosis.<h4>Interpretation</h4>The normal age distribution of people with the haemochromatosis genotype, and the lack of symptoms in patients of all ages, indicate that the penetrance of hereditary haemochromatosis is much lower than generally thought. The clinical penetrance of a disorder is an essential consideration in screening for genetic disease; disorders with low penetrance are more expensive candidates for screening than disorders with high penetrance. Our best estimate is that less than 1% of homozygotes develop frank clinical haemochromatosis.

Netrins are a family of secreted proteins that function as tropic cues directing cell and axon migration during neural development. We show that the netrin-1 receptor, deleted in colorectal cancer (DCC), is present at filopodia tips in growth cones of embryonic rat spinal commissural neurons. To further investigate DCC function, we characterized the expression of netrins and netrin receptors in HEK293T cells and NG108-15 cells and found that they express netrin-1 but do not express DCC. Ectopic expression of DCC produced a netrin-1-dependent increase in the number of filopodia and in cell surface area. Coexpression of DCC and dominant negative Cdc42 or dominant negative Rac1 blocked the increase in filopodia number and cell surface area, respectively. Furthermore, addition of netrin-1 to cells expressing DCC caused a persistent activation of Cdc42 and Rac1. These findings suggest that netrin-1, via DCC, influences cellular motility by regulating actin-based membrane extension through the activation of Cdc42 and Rac1.

Hereditary hemochromatosis (HH) is a common inborn error of iron metabolism characterized by excess dietary iron absorption and iron deposition in several tissues. Clinical consequences include hepatic failure, hepatocellular carcinoma, diabetes, cardiac failure, impotence, and arthritis. Despite the discovery of the mutation underlying most cases of HH, considerable uncertainty exists in the mechanism by which the normal gene product, HFE, regulates iron homeostasis. Knockout of the HFE gene clearly confers the HH phenotype on mice. However, studies on HFE expressed in cultured cells have not yet clarified the mechanism by which HFE mutations lead to increased dietary iron absorption. Recent discoveries suggest other genes, including a second transferrin receptor and the circulating peptide hepcidin, participate in a shared pathway with HFE in regulation of iron absorption. This review summarizes our current understanding of the relationship between iron stores and absorption and presents models to explain the dysregulated iron homeostasis in HH.

The digital coincidence counting system developed by NPL and ANSTO is briefly described along with its benefits in the data collection and processing for the 4pi beta-gamma coincidence counting technique of radionuclide standardization. One of these benefits is the automatic detection of and correction for out-of-channel coincidences in the Computer Discrimination method. Where the criteria for the use of the Cox-Isham/Smith correction formulae for dead times and resolving times are not met, a generalized approximation based on the work of Campion is suggested.

We have cloned a novel hematopoietic granulocyte colony-stimulating factor (G-CSF)-induced olfactomedin-related glycoprotein, termed hGC-1 (human G-CSF-stimulated clone-1). mRNA differential display was used in conjunction with a modified two-phase liquid culture system. Cultures were enriched for early precursors of erythroid, myeloid, and megakaryocytic lineages, which were isolated after induction with erythropoietin, G-CSF, and thrombopoietin, respectively. RNA from the enriched cells was subjected to differential display analysis to identify lineage-specific expressed genes. One clone specifically induced by G-CSF, hGC-1, was characterized. The 2861 bp cDNA clone of hGC-1 contained an open reading frame of 1530 nucleotides, translating into a protein of 510 amino acids with a signal peptide and six N-linked glycosylation motifs. The protein sequence of hGC-1 showed it to be a glycoprotein of the olfactomedin family, which includes olfactomedin, TIGR, Noelin-2 and latrophilin-1. Olfactomedin-like genes show characteristic tissue-restricted patterns of expression; the specific tissues expressing these genes differ among the family members. hGC-1 was strongly expressed in the prostate, small intestine, and colon, moderately expressed in the bone marrow and stomach, and not detectable in other tissues. In vitro translation and ex vivo expression showed hGC-1 to be an N-linked glycoprotein. The hGC-1 gene locus mapped to chromosome 13q14.3. Together, our findings indicate that hGC-1 is primarily expressed as an extracellular olfactomedin-related glycoprotein during normal myeloid-specific lineage differentiation, suggesting the possibility of a matrix-related function for hGC-1 in differentiation.

The potential involvement of membrane estrogen binding sites in the induction of ERE-dependent transcriptional activity as well as in the regulation of intracellular estrogen receptor alpha (ER-alpha) level under estradiol (E2) stimulation was investigated. Our approach relied upon the use of two DCC-treated E2-BSA (bovine serum albumin) solutions (E2-6-BSA and E2-17-BSA). The absence of detectable free E2 in these solutions was established. Both E2-BSA conjugates led to a transient dose-dependent stimulation of the expression of ERE-luciferase (LUC) reporter gene in MVLN cells (MCF-7 cells stably transfected with a pVit-tk-LUC reporter plasmid), a property not recorded with free E2, which maintained enhanced transcriptional activity during the whole experiment. A very low concentration of E2 (10 pM) synergistically acted with E2-BSA conjugates. Hence, ERE-dependent transcriptional activity induced by these conjugates appeared to result from their known interactions with membrane estrogen binding sites. Anti-estrogens (AEs: 4-OH-TAM and RU 58,668), which antagonize genomic ER responses, abrogated the luciferase activity induced by E2-BSA conjugates, confirming a potential relationship between membrane-related signals and intracellular ER. Moreover, induction of luciferase was recorded when the cells were exposed to IBMX (3-isobutyl-1-methylxanthine) and cyclic nucleotides (cAMP/cGMP), suggesting the implication of the latter in the signal transduction pathway leading to the expression of the reporter gene. Growth factors (IGF-I, EGF and TGF-alpha) also slightly stimulated luciferase and synergistically acted with 10 pM E2, or 1 microM E2-BSA conjugates, in agreement with the concept of a cross-talk between steroids and peptides acting on the cell membrane. Remarkably, E2-BSA conjugates, IBMX and all investigated growth factors failed to down-regulate intracellular ER in MCF-7 cells, indicating the need for a direct intracellular interaction of the ligand with the receptor to regulate its level. ER elimination was, however, found in the presence of conditioned media (CMs) prepared from cells pre-exposed to E2-BSA conjugates, suggesting that they may produce (a) modulator(s) that may enhance receptor down-regulation when released within the medium.

<h4>Background</h4>Iron may promote coronary atherosclerotic disease (CAD) by increasing lipid peroxidation. Studies on biochemical or genetic markers of body iron stores as risk factors for CAD have yielded conflicting results.<h4>Methods</h4>We studied 849 individuals with a clear-cut definition of the CAD phenotype, i.e., with (CAD; n = 546) or without (CAD-free; n = 303) angiographically documented disease. We determined serum ferritin, as a biochemical estimate of iron stores, and the C282Y mutation in the HFE gene, i.e., the main cause of hemochromatosis in Caucasians. The relationships of ferritin with serum markers of either inflammation [C-reactive protein (CRP)] or lipid peroxidation (malondialdehyde) were also investigated.<h4>Results</h4>Mean ferritin concentrations were slightly higher in CAD vs CAD-free individuals, but this difference disappeared after adjusting for sex and CRP. Ferritin was significantly correlated with CRP (Spearman's test, rho = 0.129; P <0.001). Heterozygotes for Cys282Tyr were 4.8% among the CAD group and 6.6% among the CAD-free group (P = 0.26). The prevalence of high concentrations of stored iron, defined as ferritin concentrations above the sex-specific upper quintiles of the control distribution, was also similar in the two groups. There was a higher prevalence of "iron depletion" in CAD-free vs CAD females (20% vs 8.8%, respectively), but this difference disappeared after adjustment for age and other cardiovascular risk factors (odds ratio, 0.66; 95% confidence interval, 0.21-2.08). No differences in iron markers were found in CAD patients with or without myocardial infarction.<h4>Conclusions</h4>Our results do not support a role for biochemical or genetic markers of iron stores as predictors of the risk of CAD or its thrombotic complications.

A patient with clinically manifest hereditary hemochromatosis was found to be heterozygous for the c.845 A-->G (C282Y) mutation. As simple heterozygotes for this mutation do not develop the hemochromatosis phenotype, the coding region of the patient's HFE gene was sequenced and a previously undescribed nonsense mutation was identified at c.211 C-->T (R74X). The patient's brother who also had the hemochromatosis phenotype shared his HFE genotype. To determine how common such mutations might be, the coding and 5' region of the HFE genes of 11 subjects who had been found in a large population survey to be heterozygous for the C282Y mutation and had elevated ferritin levels were sequenced. No mutations were found. Sequencing of the HFE gene also revealed two polymorphisms that had not previously been noted, -467 C-->G and -970 T-->G. Neither of these mutations appear to cause an abnormality in iron metabolism.

The HFE gene contains two main missense mutations: C282Y and H63D. Individuals with these mutations carry a risk of developing hereditary haemochromatosis (HH). The common form of this disease is due to homozygosity for the C282Y mutation. Population studies have shown the variation of the prevalence of these mutations in different countries and ethnic groups. The purposes of this current study were to determine the prevalence of the C282Y and H63D mutations in the Balearic Islands and the genotypic characterization of patients diagnosed with HH, as well as those with iron overload and liver diseases. A total of 1330 Balearic chromosomes were analyzed. The results showed that the populations of the Balearic Islands were not homogeneous. No C282Y carriers were observed in a group of descendants of Majorcan Jews (Chuetas) and the frequency was very low in Minorca (1.2%) in comparison with the other islands of Majorca (4.7%) and Ibiza (6.5%). The carrier frequency of the H63D mutation was similar in the three islands and very high (43.1%) in the descendants of Majorcan Jews. The study of patients was carried out in 129 individuals. The homozygous C282Y genotype was the principal one involved in hereditary haemochromatosis (90%), whereas the other HH patients were C282Y/H63D compound heterozygous and H63D homozygous.

Netrin-1 is a bifunctional secreted protein that directs axon extension in various groups of developing axonal tracts. The transmembrane DCC (deleted in colorectal cancer) receptor is described as netrin-1 receptor and is involved in the attractive effects of netrin-1. In this study, we examined the spatio-temporal expression patterns of both netrin-1 and DCC in the rat olfactory system at different stages of development and during axonal regeneration following unilateral bulbectomy. High DCC expression was detected on the pioneer olfactory axons as they are extending toward the telencephalon. This expression was transient since from embryonic day 16 onwards, DCC was no longer detected along the olfactory nerve path. From embryonic day 14 until birth, DCC was also expressed within the mesenchyme surrounding the olfactory epithelium. During the same period, netrin-1 protein was detected along the trajectory of olfactory axons up to the olfactory bulb and its expression pattern in the nasal mesenchyme largely overlapped that of DCC. Moreover, netrin-1 continued to be present during the two first post-natal weeks, and a weak protein expression still persisted in the dorso-medial region of the olfactory epithelium in adult rats. While unilateral bulbectomy induced a transient up-regulation of netrin-1 in the lamina propria, particularly in the dorso-medial region of the neuroepithelium, no DCC expression was detected on the regenerating olfactory axons. In the developing olfactory bulb, the extension of mitral cell axons was associated with DCC presence while netrin-1 was absent along this axonal path. DCC was also highly expressed in the newly formed glomeruli after birth, and a weak DCC expression was still detected in the glomerular layer in adult rats. Taken together, these data support the notion that netrin-1, via DCC expressed on axons, may play a role in promoting outgrowth and/or guidance of pioneering olfactory axons toward the olfactory bulb primordium. Moreover, association of netrin-1 with mesenchymal DCC may provide a permissive environment to the growth of both pioneer and later-growing axons. The maintenance of netrin-1 expression in the nasal mesenchyme of adult rats as well as its regional up-regulation following unilateral bulbectomy infer that netrin-1, even in the absence of DCC, may be involved in the process of axonal growth of newly differentiated olfactory receptor neurons probably through the use of other receptors.

We adopted firstly the dextran-coated charcoal(DCC) and SP methods to detect estrogen receptor (ER) expression of bone tissue in ovariectomized(OVX) rats. The results demonstrate that in OVX rats, XW630 can significantly promote ER expression in bone tissue and increase the ER content. XW630 is superior to estrone in effectiveness. The results also reveal that the ER expression in OVX rat bone tissue decreases with the lapse of time, indicating that the expression of ER depends on the existence of estrogen.

Quercetin 3-acetyl-7,3',4'-trisulphate (ATS) and quercetin 3,7,3',4'-tetrasulphate (QTS) obtained from Flaveria bidentis (Asteraceae) were investigated in vitro for anticoagulant activity. Three different concentrations of each flavonoid were assayed at different incubation times, showing at 1 mM significant prolongation on the activated partial thromboplastin time (APTT), less on the prothrombin time (PT), and no effect on the thrombin time (TT). In order to define the action mechanism of the anticoagulant activity, all coagulation factors were evaluated and no important activity decrease was observed, indicating that another mechanism is involved. Thus, thrombin inhibition mediated by antithrombin III (ATIII) and heparin cofactor II (HCII) activation was investigated in comparison to the physiological activators, heparin and dermatan sulphate (DS), respectively. As a conclusion, no activation on ATIII for neither flavonoids was observed. On the contrary, QTS much more than ATS produced an activation on HCII comparable to the one of DS, indicating that these flavonoids act as agonists of this inhibitor. A plausible explanation of the effects of both flavonoids could be due to the different degree of sulphation of these molecules. According to the results obtained, and taking in account the high solubility of these natural products in aqueous media and the nontoxic nature of this family of compounds, further investigation on the antithrombotic effects of these flavonoids are merited.

In contrast with two previous western reports, a recent study on a Chinese population found an association for allele 12 of the variable number tandem repeat (VNTR) in the second intron of the serotonin transporter (5-HTT) gene and schizophrenic disorders. We have replicated this finding for a Chinese population in Taiwan (114 schizophrenic patients and 127 controls), demonstrating a modest but significant statistical association for the 5-HTT-VNTR allele 12 and schizophrenic patients (one-sided p = 0.043). This positive finding further supports the proposition that the 5-HTT-VNTR allele 12 is a risk factor for schizophrenic disorders in Chinese populations, although the effect is weak.

A 14-year-old girl demonstrated increased iron concentration and transferrin saturation, suggesting iron overload of unknown origin. Liver biopsy showed no fibrosis or hepatocytic atrophia. Nevertheless, Prussian blue reaction for histochemical detection of iron demonstrated very weak positive granules in a few hepatocytes on the periphery of hepatic lobules in close connection to bile capillaries. This very early stage of hemochromatosis was confirmed by TEM and EELS for iron accumulation inside hepatocytic lysosomes and residual bodies. Such siderosomes were scarce in number and iron content, compared to a case of manifested hemochromatosis and liver cirrhosis (Jonas L, Fulda G, Salemeh T, et al. Ultrastruct Pathol. 2001; 25: 111-118.). Liver iron concentration as measured by inductively coupled plasma-mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) yielded 2.005 mg/g tissue dry weight, which was considered not significantly increased. In the absence of known causes for secondary iron overload, the early diagnosis was evidenced by genotyping, revealed homozygosity for the HFE gene C282Y mutation, demonstrating the presence of hereditary hemochromatosis.

No abstract available.

The purpose of this study was to evaluate the relationship between attention deficit hyperactivity disorder (ADHD) and polymorphism of the two regions of the 5-HTT gene [variable number of tandem repeats (VNTR) and 5-HTTLRR] in a sample of Turkish children. Using the PCR technique, these polymorphisms were assessed in 71 patients with ADHD and 128 healthy controls. The 5-HTTLPR S/S genotype was significantly lower in the patients than in the controls (p = 0.018). Homozygous and heterozygous L variant predominated in the ADHD group. But the VNTR STin2.12/12 genotype was significantly less found in the patients than in the controls (p = 0.001). There was no significant difference between the frequency of the short (S), long, 10, and 12 alleles of both groups. The lack of an S/S variant of 5-HTTLPR polymorphism of the STin2.12/12 variant of VNTR polymorphism appears to be associated with an increased risk of ADHD.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in some areas of the world with an extremely poor prognosis. The major etiologic risk factors for HCC development include toxins (alcohol, aflatoxin B1), androgens and estrogens, hepatitis B virus (HBV) and hepatitis C virus (HCV) infection as well as various inherited metabolic disorders, such as alpha-1-antitrypsin deficiency and hemochromatosis. The molecular pathogenesis of HCC development is very complex and involves alterations in the structure or expression of several tumor suppressor genes, oncogenes and, possibly, mechanisms leading to a genetic instability due to mismatch repair deficiency or chromosomal instability and aneuploidy due to defective chromosomal segregation. Central to the molecular pathogenesis of HCCs are mutations of various genes and a genetic instability which in most cases result from chronic liver disease and the associated enhanced liver cell regeneration and mitotic activity. The prognosis of HCC patients is generally very poor. Most studies report a five year survival rate of less than 5% in symptomatic HCC patients. Furthermore, these tumors have been shown to be quite resistant to radio- or chemotherapy. Investigations of the natural history and clinical course of HCCs revealed long-term survival of patients only with small asymptomatic HCCs that could be treated surgically or by non-surgical interventions. Apart from exploring and refining new HCC treatment strategies, the implementation of existing and the development of novel measures to prevent HCC development are most important. Primary HCC prevention includes among others universal hepatitis B vaccination, antiviral therapy of patients with chronic hepatitis B or C, reduction of food contamination with aflatoxins, elimination of excessive alcohol etc. Also for some genetic diseases there is the potential for HCC prevention by identifying affected family members at risk, such as patients with precirrhotic hemochromatosis. Reduction of iron overload by phlebotomy has been shown to eliminate the progression hemochromatosis to liver cirrhosis and HCC. Preventive measures, therefore, should have a major impact on the incidence of HCCs in patients with acquired and inherited liver diseases. Further, the prevention of a local recurrence or the development of new HCC lesions in patients after successful surgical or non-surgical HCC treatment (secondary prevention) is of paramount importance and is expected to significantly improve disease-free and overall patient survival. Based on rapid scientific advances, molecular diagnosis, gene therapy and molecular prevention are becoming increasingly part of our patient management and will eventually complement and in part replace existing diagnostic, therapeutic and preventive strategies. Overall, this should result in a reduction of the incidence of HCCs, one of the most devastating malignancies worldwide.

Heat waves result in excess deaths, excess emergency department visits, and intensive care unit admissions for heat stroke. We describe the clinical features and 3-month outcome of a patient with near-fatal heat stroke, admitted to our intensive care unit in July, 2001. After heavily working for hours at a construction site during a heat wave, the 28-year-old male presented with 41.4 degrees C body temperature and multiorgan failure, consisting of neurological impairment, rhabdomyolysis, acute renal failure, disseminated intravascular coagulation, and acute respiratory distress syndrome (ARDS). In the first week there was no evidence of infection. Treatment included cooling, aggressive volume resuscitation, administration of antithrombin-III concentrates and steroids. The patient survived and recovered normal neurological, renal, respiratory and haematological function, and no disability persisted. This case illustrates survival and complete recovery after multiorgan failure in heat stroke with vigorous intensive care. Treatment with antithrombin and steroids and may well have contributed to the favourable outcome. Correction of reduced antithrombin III levels to supranormal by therapeutic administration of antithrombin III concentrate in disseminated intravascular coagulation of heat stroke was not associated with any bleeding complications.

Flibanserin has preferential affinity for serotonin 5-HT(1A), dopamine D(4k), and serotonin 5-HT(2A) receptors. In vitro and in microiontophoresis, flibanserin behaves as a 5-HT(1A) agonist, a very weak partial agonist on dopamine D(4) receptors, and a 5-HT(2A) antagonist. In vivo flibanserin binds equally to 5-HT(1A) and 5-HT(2A) receptors. However, under higher levels of brain 5-HT (i.e., under stress), flibanserin may occupy 5-HT(2A) receptors in higher proportion than 5-HT(1A) receptors. The effects of flibanserin on adenylyl cyclase are different from those of buspirone and 8-OH-DPAT, two other purported 5-HT(1A) receptor agonists. Flibanserin reduces neuronal firing rate in cells of the dorsal raphe, hippocampus, and cortex with the CA1 region being the most sensitive in the brain. Flibanserin-induced reduction in firing rate in the cortex seems to be mediated through stimulation of postsynaptic 5-HT(1A) receptors, whereas the reduction of the number of active cells seems to be mediated through dopamine D(4) receptor stimulation. Flibanserin quickly desensitizes somatic 5-HT autoreceptors in the dorsal raphe and enhances tonic activation of postsynaptic 5-HT(1A) receptors in the CA3 region. Flibanserin preferentially reduces synthesis and extracellular levels of 5-HT in the cortex, where it enhances extracellular levels of NE and DA. Flibanserin displays antidepressant-like activity in most animal models sensitive to antidepressants. Such activity, however, seems qualitatively different from that exerted by other antidepressants. Flibanserin seems to act via direct or indirect stimulation of 5-HT(1A), DA, and opioid receptors in those animal models. Flibanserin does not display consistent effects in animal models of anxiety and seems to exert potential antipsychotic effects. Flibanserin may induce some sedation but does not induce observable toxic effects at pharmacologically relevant doses.

Hereditary hemochromatosis (HH) is a common genetic disease with iron overload in certain organs, especially the liver. Most cases are homozygous for the C282Y mutation in the HFE gene; a few are C282Y heterozygous, compound C282Y/H63D heterozygous, or have no known mutation. A third mutation, S65C, has been associated with HH, but this finding is disputed. We have studied the clinical significance of various genotypes with the S65C mutation. In a population-based screening for HH in 65,238 persons, 613 had high serum transferrin saturation in two blood samples and were invited for HFE genotyping. In 556 persons with complete data sets, we studied the serum ferritin concentration and the risk of being diagnosed with phenotypic HH in the various genotypic groups. The phenotypic diagnosis was given without knowing the genotypic result. Except for the C282Y homozygotes, no differences in median serum ferritin concentrations were found between the various genotypic groups. However, the C282Y/S65C compound heterozygous group had a higher risk of being diagnosed with phenotypic HH than the wild-type group, as did the C282Y homozygous and the C282Y/H63D compound heterozygous groups. When combined with the C282Y mutation, the S65C mutation is associated with an increased risk of being diagnosed with phenotypic HH.

Autism is a biologically-heterogeneous disease. Distinct subgroups of autistic patients may be marked by intermediate phenotypes, such as elevated serotonin (5-HT) blood levels, potentially associated with different underlying disease mechanisms. This could lead to inconsistent genetic association results, such as those of prior studies on serotonin transporter (5-HTT) gene promoter variants and autistic disorder. Contributions of 5-HTT gene promoter alleles to 5-HT blood levels were thus investigated in 134 autistic patients and 291 first-degree relatives. Mean 5-HT blood levels are 11% higher in autistic patients carrying the L/L genotype, compared to patients with the S/S or S/L genotype; this trend is not observed in first-degree relatives. The probability of inheriting L or S alleles is significantly enhanced in patients with 5-HT blood levels above or below the mean, respectively (P < 0.05), but quantitative TDT analyses yield a non-significant trend (P = 0.10), as this polymorphism explains only 2.5% of the variance in 5-HT blood levels of autistic patients. In conclusion, 5-HTT gene promoter variants seemingly exert a small effect on 5-HT blood levels in autistic children, which largely does not account for hyperserotoninemia. Nonetheless, the inconsistent outcome of prior association studies could partly stem from a selection bias of hyper- or hypo-serotoninemic probands.

The well-replicated platelet hyperserotonemia of autism has stimulated interest in serotonin (5-HT) in autism. We have examined the effects of the serotonin transporter gene (5-HTT, locus SLC6A4) promoter polymorphism (5-HTTLPR) on platelet 5-HT physiology in autism. Platelet 5-HT uptake rates and affinities (V(max) and K(m)), uptake site densities (B(max)) and 5-HT levels were examined in 31 French individuals with autism genotyped with respect to the 5-HTTLPR. Platelet 5-HT uptake and 5-HT levels were measured using HPLC; uptake sites were determined by radioligand binding. A 1.5-fold increased rate (V(max)) of platelet 5-HT uptake was observed in ll genotype individuals compared to those with ls and ss genotypes (Mann- Whitney U-test, P = 0.022). However, no significant relationship was observed between genotype and uptake site density (U-test, P = 0.51). Although median levels of platelet 5-HT in platelet-rich plasma were higher in the ll group, only trend level significance was observed (U-test, P= 0.069); platelet 5-HT content measured in whole blood was similar across genotypes. Uptake rates were well correlated with B(max) values (r = 0.66, P = 0.002); correlations between uptake and platelet 5-HT levels and between B(max) values and 5-HT levels were somewhat lower. While 5-HTTLPR alleles had an appreciable effect on platelet 5-HT uptake rates, effects on 5-HT levels and uptake site density were smaller or absent. Based on these preliminary data and prior studies of allele frequencies, we conclude that the 5-HTTLPR is not a major determinant of the group mean platelet serotonin elevation seen in autism. However, a role for increased uptake in the hyperserotonemia of autism can not be ruled out. In addition, it appears that studies of platelet 5-HT measures in autism and other disorders should take account of the effects of 5-HTTLPR genotype on 5-HT uptake

<h4>Background</h4>Fondaparinux sodium is a novel antithrombotic agent, the first of a new class of selective factor Xa inhibitors. It has favourable pharmacokinetics including 100% bioavailability, low variability and a mean terminal half-life of 17 hours for young and 21 hours for elderly healthy volunteers, enabling once-daily administration. Studies on the prevention of venous thromboembolism (VTE) after orthopaedic surgery demonstrated significantly improved efficacy over the low-molecular-weight heparin enoxaparin, with a >50% reduced risk of VTE and a similar safety profile.<h4>Objective</h4>To investigate the in vitro binding of fondaparinux sodium to purified antithrombin III (ATIII) and other plasma proteins.<h4>Methods</h4>Fondaparinux sodium was incubated with human plasma, antithrombin-depleted plasma or purified human plasma proteins, including antithrombin, alpha1-acid glycoprotein, serum albumin and immunoglobulin. Non-protein-bound fondaparinux sodium was determined using a validated chromogenic assay method, enabling the calculation of the free fraction of fondaparinux sodium and its binding parameters.<h4>Results</h4>At steady state, fondaparinux sodium at therapeutic concentrations [i.e. those attainable in the prevention (0.14 to 0.50 mg/L) and treatment (up to approximately 2 mg/L) of VTE] was extensively bound (>97%) to plasma proteins and specifically bound (>94%) to purified ATIII. The specific binding parameters B(max) (binding capacity) and K(D )(dissociation constant) were similar for human plasma (B(max) = 2072 nmol/L, K(D) = 28 nmol/L) and purified ATIII (B(max) = 1627 nmol/L and K(D) = 32 nmol/L). There was no specific binding of fondaparinux sodium to other purified plasma proteins.<h4>Conclusion</h4>At clinically relevant concentrations, fondaparinux sodium is highly and specifically bound to ATIII in human plasma, suggesting that potential interaction with drugs via albumin or alpha1-acid glycoprotein displacement is unlikely.

Carcinoid tumor of the appendix is an endocrine tumor that is histologically similar to, but biologically less aggressive than carcinoids arising from other parts of the gastrointestinal tract. In this study, we examined E-cadherin, beta-catenin, DCC, p53 and Ki67 immunoexpression in cases of carcinoid of the appendix and made a comparison with non-appendiceal carcinoid tumors. Nine cases of appendiceal carcinoid and 11 biopsies of carcinoid of other parts of the gastrointestinal tract, five cases of the small intestine and six of the stomach were immunohistochemically evaluated for Ki67, p53, DCC, E-cadherin and beta-catenin. Two main patterns of beta-catenin staining were observed. The first pattern was characterized as membranous and cytoplasmic, and was seen mainly in the peripheral cells of the nests. The second pattern was diffuse, predominantly membranous. Most (five of seven) appendiceal carcinoids and only three of 11 non-appendiceal cases showed the first staining pattern (p < 0.05). Immunoexpression of E-cadherin and DCC was similar in both groups. p53 and Ki-67 immunostaining revealed stronger nuclear positivity in the non-appendiceal carcinoid tumors (statistically not significant). We found a pattern of beta-catenin immunostaining in typical carcinoid tumors of the appendix that was different from the pattern seen in non-appendiceal carcinoid tumors. This alteration suggests that carcinoid of the appendix may represent a different subtype of carcinoid tumors with different immunohistochemical and biological behavior.

The hypothetical multistep model of breast carcinogenesis suggests a transition from normal epithelium to invasive carcinoma via intraductal hyperplasia (without and with atypia) and in situ carcinoma. These presumptive precursor lesions are currently defined by their histological features, and their prognosis is imprecisely estimated from indirect epidemiological evidence. Cytogenetic and molecular-genetic analysis of these lesions give evidence for an accumulation of various genetic alterations during breast tumorigenesis. Using immuno-histochemistry overexpression of the c-erbB-2 oncogene was found in ductal carcinoma in situ (DCIS), but not in atypical intraductal hyperplasia (AIDH) and intraductal hyperplasia (IDH). An expression of mutant p53 tumor suppressor gene as well as expression of cyclin D1 was identified in DCIS. In IDH lesions loss of heterozygosity (LOH) at various loci could be identified, and comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) studies delivered evidence for DNA amplification on chromosomal region 20q13 in the early stage of IDH.However, little is currently known about genetic alterations in those premalignant lesions, and the chronology of genetic alterations and histopathological changes during carcinogenesis is mainly undiscovered.

The implantation of immuno-isolated recombinant cell lines secreting a therapeutic protein in alginate microcapsules presents an alternative approach to gene therapy. Its clinical efficacy has recently been demonstrated in treating several genetic diseases in murine models. However, its application to humans will depend on the long-term structural stability of the microcapsules. Based on previous implantations in canines, it appears that survival of alginate-poly-L-lysine-alginate microcapsules in such large animals is short-lived. This article reports on the biological factors that may have contributed to the degradation of these microcapsules after implantation in dogs. Alginate microcapsules coated with poly-L-lysine or poly-L-arginine were implanted in subcutaneous or intraperitoneal sites. The retrieved microcapsules showed a loss of mechanical stability, as measured by resistance to osmotic stress. The polyamino acid coats were rendered fragile and easily lost, particularly when poly-L-lysine was used for coating and the intraperitoneal site was used for implantation. Various plasma proteins were associated with the retrieved microcapsules and identified with western blotting to include Factor XI, Factor XII, prekallikrein, HMWK, fibrinogen, plasminogen, ATIII, transferrin, alpha-1-antitrypsin, fibronectin, IgG, alpha-2-macroglobulin, vitronectin, prothrombin, apolipoprotein A1, and particularly albumin, a major Ca-transporting plasma protein. Complement proteins (C3, Factor B, Factor H, Factor I) and C3 activation fragments were detected. Release of the amino acids from the microcapsule polyamino acid coats was observed after incubation with plasma. indicating the occurrence of proteolytic degradation. Hence, the loss of long-term stability of the polyamino acid-coated alginate microcapsules is associated with activation of the complement system, degradation of the polyamino acid coating, and destabilization of the alginate core matrix, probably through loss of calcium-mediated ionic cross-linking of the guluronic acid polymers in the alginate. These destructive forces may be slightly mitigated by using poly-L-arginine instead of poly-L-lysine for coating and by implanting in a subcutaneous instead of an intraperitoneal site. However, the long-term stability of such devices may require significant improvements in the microcapsule polymer chemistry to withstand such biological impediments.

A polymorphism in the serotonin (5-HT) transporter gene regulatory region (5-HTTLPR) is associated with measures of 5-HT transporter (5-HTT) expression and 5-HT-mediated behaviors in humans. An analogous length variation of the 5-HTTLPR has been reported in rhesus monkeys (rh5-HTTLPR). A retrospective association study was conducted on 115 rhesus macaque infants either homozygous for the long 5HTTLPR variant (l/l) or heterozygous for the short and long form (l/s). To assess contributions of genotype and early rearing environment, 36 mother-reared monkeys (l/l = 26, l/s = 10) and 79 nursery-reared monkeys (l/l = 54, l/s = 25) were assessed on days 7, 14, 21, and 30 of life on a standardized primate neurobehavioral test designed to measure orienting, motor maturity, reflex functioning, and temperament. Both mother-reared and nursery-reared heterozygote animals demonstrated increased affective responding relative to l/l homozygotes. Nursery-reared, but not mother-reared, l/s infants exhibited lower orientation scores than their l/l counterparts. These results demonstrate the contributions of rearing environment and genetic background, and their interaction, in a nonhuman primate model of behavioral development.

The serotonin transporter (5-HTT) is the site of primary action for the selective serotonin reuptake inhibitors (SSRIs). Previous Western reports have demonstrated that the lallele of the 5-HTT gene-linked polymorphic-region (5-HTTLPR) polymorphism is associated with better SSRI antidepressive effects than the s allele, however, another study of a Korean population has produced a contrasting finding. The present study tested the hypothesis that the 5-HTTLPR genetic polymorphism is associated with SSRI antidepressant response by evaluating total and cluster depressive symptoms for 121 Chinese patients diagnosed with major depression. Analysis of the results reveals that patients with the l/l genotype had a significantly better response to SSRI (fluoxetine) when compared with s allele carriers, as evaluated on the basis of total (P = 0.013), core (P = 0.011), and psychic-anxiety (P = 0.005) and somatic-anxiety (P = 0.002) Hamilton Depression Rating Scale-score percentage change. Our findings confirm reports that the l allele is associated with better SSRI response.

In contrast to adult ventricular cardiomyocytes the developmentally early stage cardiomyocytes show a suppression of the basal voltage-dependent calcium channels (DCC L-type Ca2+ channels, I(Ca)) by carbachol (CCh). This effect is mediated by the endothelial NO-synthase (NOS III). In contrast late stage and adult cardiomyocytes a direct coupling of the muscarinic receptor to the adenylyl cyclase. Thus, NO may function as an early signal transduction molecule during development. This review elucidates the role of beta1-integrins in mediating signal transduction between muscarinic receptors and coupled downstream target proteins such as ion channels. The key finding is that in embryonic stem cell-derived cardiomyocytes deficient of beta1-integrins, the modulation of L-type Ca2+ channels via the M2 receptor is absent. Experiments indicate that this selective signaling defect occurs at the G-protein level. This suggests a novel critical role for integrins in membrane delimited signal transduction processes.

Analysis of the common C282Y and H63D mutations in the HFE gene is widely used to diagnose hereditary hemochromatosis (HH). The aim of this study was to evaluate the efficiency with which different hospitals and general practitioners select patients for HH genotype and to determine the distribution of HFE mutations in such patients. Nine hundred unrelated patients from Danish hospitals and general practitioners (group A) and 69 consecutive patients from a specialized liver unit (group B) were examined for HFE substitutions using multiplex real-time polymerase chain reaction. In group A we found 13.0% (0%) C282Y homozygotes, 5.8% (2.6%) H63D/C282Y compound heterozygotes and 1.9% (3.1%) S65C heterozygotes. The values for 420 Danish blood donors are shown in parentheses. The distribution of genotypes in group B was similar to that of the blood donors. Serum ferritin, transferrin iron saturation and pathological data were collected from 38 randomly selected C282Y homozygotes, 36 H63D/C282Y compound heterozygotes, 19 H63D heterozygotes, 17 S65C heterozygotes and 144 wild-types. All of the C282Y homozygotes and 28% of the compound heterozygotes were diagnosed as HH patients. There was no evidence of HH in the H63D homozygotes or S65C heterozygotes. Moreover, 7 wild-type patients, 2 C282Y heterozygote patients and one H63D heterozygote patient fulfilled the criteria for HH. The significant enrichment of HH among associated genotype samples submitted for HFE testing indicates that the clinical selection is generally adequate. However, the study showed substantial deviation in the selection efficiency among the various hospitals and general practitioners.

Huntington's disease is the most prominent basal ganglion disease. Huntington's gene, IT15, in chromosome 4p16.3, has 67 axons with 10,366 bp coding space and unstable CAG sequence that codes glutamine on 5' terminal. The molecular-genetic analysis of disease determined expansion of nucleotide repeated CAG sequences. In large Bosnian family with Huntington's disease specific DNA diagnosis of IT15 gene mutation is performed, according the wishes of one female member with "high genetic risk", that voluntarily accessed to DNA test in order to make plans for her own family "without risk" of pathologic gene transmission. A mutation in IT15 gene (number of CAG tandem repeats 46, size of DNA fragment 165 bp and 245 bp) is detected in DNA of her clinically affected brother. But, results of PCR analysis of her DNA sample showed 23 CAG tandem repeats (fragment size 180 bp) that excluded presence of Huntington's disease. We accentuate importance of DNA test in persons with "genetic risk", that are not gene carriers. In that case there are able to create own future without fear of pathological gene transmission.

Hereditary hemochromatosis (HH) is one of the most common autosomal recessive disorders of iron metabolism among Caucasians, and it is associated with C282Y mutation of the HFE gene in populations of Celtic origins. A second mutation, H63D, shows a very high widespread frequency, although its role in iron metabolism is still inconclusive. There are no data on the frequencies of these two mutations in Sardinia, an island in the Mediterranean sea that has not been invaded by Celtic peoples. We examined 836 chromosomes from Sardinian subjects and tested for the mutation by restriction enzyme digestion of PCR products. Among the 836 analyzed chromosomes, we found a C282Y allele frequency of 0.0036 and an H63D allele frequency of 0.173. These data could explain the observed rarity of HH in Sardinia. The high allele frequency of H63D and the rarity of HH in Sardinia is suggestive that this mutation is not a major contributor to this disease.

In the present paper, we describe a patient who is a compound heterozygote for three mutations in the HFE gene: C282Y, H63D, and E168Q. The patient's mother carries two copies of H63D and one copy of E168Q; the patient's father is heterozygous for C282Y. The family study indicates that the patient, as well as his sister, a maternal uncle, and a first cousin, all have inherited a single HFE allele that contains two mutations H63D and E168Q. The clinical symptoms and laboratory findings of the patient and his relatives are consistent with the conclusion that the E168Q mutation by itself is unlikely to result in hemochromatosis.

Transcriptional profiling (TP) offers a powerful approach to identify genes activated during memory formation and, by inference, the molecular pathways involved. Trace eyeblink conditioning is well suited for the study of regional gene expression because it requires the hippocampus, whereas the highly parallel task, delay conditioning, does not. First, we determined when gene expression was most regulated during trace conditioning. Rats were exposed to 200 trials per day of paired and unpaired stimuli each day for 4 days. Changes in gene expression were most apparent 24 h after exposure to 200 trials. Therefore, we profiled gene expression in the hippocampus 24 h after 200 trials of trace eyeblink conditioning, on multiple arrays using additional animals. Of 1,186 genes on the filter array, seven genes met the statistical criteria and were also validated by real-time polymerase chain reaction. These genes were growth hormone (GH), c-kit receptor tyrosine kinase (c-kit), glutamate receptor, metabotropic 5 (mGluR5), nerve growth factor-beta (NGF-beta), Jun oncogene (c-Jun), transmembrane receptor Unc5H1 (UNC5H1), and transmembrane receptor Unc5H2 (UNC5H2). All these genes, except for GH, were downregulated in response to trace conditioning. GH was upregulated; therefore, we also validated the downregulation of the GH inhibitor, somatostatin (SST), even though it just failed to meet criteria on the arrays. By during situ hybridization, GH was expressed throughout the cell layers of the hippocampus in response to trace conditioning. None of the genes regulated in trace eyeblink conditioning were similarly affected by delay conditioning, a task that does not require the hippocampus. These findings demonstrate that transcriptional profiling can exhibit a repertoire of genes sensitive to the formation of hippocampal-dependent associative memories.

Rheumatoid arthritis is an inflammatory joint and systemic disease believed to be of autoimmune origin. Predisposing factors also include genetic factors, such as the presence of alleles HLA-DRB1 *04, (HLA-DRB1 *0401, *0404, *0405 and *0408) and, in other ethnic groups, of subtypes DRB1 *0101, *0102 and DRB1 *1001. These genetic factors are believed to raise the risk of developing the disease. In rheumatoid arthritis, as in other chronic inflammatory diseases, iron metabolism dysfunction has been observed and attributed to inflammation. In hereditary hemochromatosis, tissue sideropexia is associated with a peculiar form of arthropathy. C282Y is a point mutation involving the replacement of a cysteine with a tyrosine at position 282 of the HFE protein. When found in homozygosis, there is a close association with hereditary hemochromatosis, accounting for one of the causes of iron metabolism dysfunction observed in this disease. The aim of this study was to compare the frequency of C282Y in patients with rheumatoid arthritis with that in patients with different forms of spondylarthritis and to correlate these findings with iron metabolism parameters. In the group of patients with rheumatoid arthritis, 2/24 (8.34%) were found to be positive for the C282Y mutation in the case of heterozygosis compared with 3/24 (12.5%) of patients with spondylarthritis. In patients with the C282Y mutation, ferritin levels were significantly higher than those in controls; conversely, serum iron levels were higher in patients with spondylarthritis. Serum transferrin levels, although slightly higher in rheumatoid arthritis patients, showed no statistically significant differences.

No abstract available.

Three ecdysteroid 7,9(11)-dien-7-ones (dacryhainansterone, 25-hydroxydacryhainansterone and kaladasterone) were prepared by dehydration of the corresponding 11a-hydroxy ecdysteroids (ajugasterone C, turkesterone and muristerone A, respectively). The biological activities of the dienones in the Drosophila melanogaster B(II) cell bioassay, which reflect the affinity for the ecdysteroid receptor complex, showed that the dienones retain high biological activity. Irradiation at 350 nm of the ecdysteroid dienones (100 nM) with bacterially-expressed dipteran and lepidopteran ecdysteroid receptor proteins (DmEcR/DmUSP or CfEcR/CfUSP), followed by loading with [(3)H]ponasterone A revealed that irradiation of dacryhainansterone or kaladasterone resulted in blocking of >70% of the specific binding sites. Thus, ecdysteroid dienones show considerable potential as photoaffinity analogues for ecdysteroid binding proteins.

No abstract available.

No abstract available.