Gene Literature Dashboard

11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) intracellularly regenerates active corticosterone from circulating inert 11-dehydrocorticosterone (11-DHC) in specific tissues. The hippocampus is a brain structure particularly vulnerable to glucocorticoid neurotoxicity with aging. In intact hippocampal cells in culture, 11beta-HSD-1 acts as a functional 11beta-reductase reactivating inert 11-DHC to corticosterone, thereby potentiating kainate neurotoxicity. We examined the functional significance of 11beta-HSD-1 in the central nervous system by using knockout mice. Aged wild-type mice developed elevated plasma corticosterone levels that correlated with learning deficits in the watermaze. In contrast, despite elevated plasma corticosterone levels throughout life, this glucocorticoid-associated learning deficit was ameliorated in aged 11beta-HSD-1 knockout mice, implicating lower intraneuronal corticosterone levels through lack of 11-DHC reactivation. Indeed, aged knockout mice showed significantly lower hippocampal tissue corticosterone levels than wild-type controls. These findings demonstrate that tissue corticosterone levels do not merely reflect plasma levels and appear to play a more important role in hippocampal functions than circulating blood levels. The data emphasize the crucial importance of local enzymes in determining intracellular glucocorticoid activity. Selective 11beta-HSD-1 inhibitors may protect against hippocampal function decline with age.

This article describes the first patient with a deficiency of transaldolase (TALDO1 [E.C.2.2.1.2]). Clinically, the patient presented with liver cirrhosis and hepatosplenomegaly during early infancy. In urine and plasma, elevated concentrations of ribitol, D-arabitol, and erythritol were found. By incubating the patient's lymphoblasts and erythrocytes with ribose-5-phosphate and subsequently analyzing phosphate sugar metabolites, we discovered a deficiency of transaldolase. Sequence analysis of the transaldolase gene from this patient showed a homozygous deletion of 3 bp. This deletion results in absence of serine at position 171 of the transaldolase protein. This amino acid is invariable between species and is located in a conserved region, indicating its importance for enzyme activity. The detection of this new inborn error of pentose metabolism has implications for the diagnostic workup of liver problems of unknown etiology.

By using sensitive homology-search and gene-finding programs, we have found that a genomic region from the tip of the short arm of human chromosome 16 (16p13.3) encodes a putative secreted protein consisting of a domain related to the whey acidic protein (WAP) domain, a domain homologous with follistatin modules of the Kazal-domain family (FS module), an immunoglobulin-related domain (Ig domain), two tandem domains related to Kunitz-type protease inhibitor modules (KU domains), and a domain belonging to the recently defined NTR-module family (NTR domain). The gene encoding these WAP, FS, Ig, KU, and NTR modules (hereafter referred to as the WFIKKN gene) is intron-depleted--its single 1,157-bp intron splits the WAP module. The validity of our gene prediction was confirmed by sequencing a WFIKKN cDNA cloned from a lung cDNA library. Studies on the tissue-expression pattern of the WFIKKN gene have shown that the gene is expressed primarily in pancreas, kidney, liver, placenta, and lung. As to the function of the WFIKKN protein, it is noteworthy that it contains FS, WAP, and KU modules, i.e., three different module types homologous with domains frequently involved in inhibition of serine proteases. The protein also contains an NTR module, a domain type implicated in inhibition of zinc metalloproteinases of the metzincin family. On the basis of its intriguing homologies, we suggest that the WFIKKN protein is a multivalent protease inhibitor that may control the action of multiple types of serine proteases as well as metalloproteinase(s).

The Drosophila mutant methuselah (mth) was identified from a screen for single gene mutations that extended average lifespan. Mth mutants have a 35% increase in average lifespan and increased resistance to several forms of stress, including heat, starvation, and oxidative damage. The protein affected by this mutation is related to G protein-coupled receptors of the secretin receptor family. Mth, like secretin receptor family members, has a large N-terminal ectodomain, which may constitute the ligand binding site. Here we report the 2.3-A resolution crystal structure of the Mth extracellular region, revealing a folding topology in which three primarily beta-structure-containing domains meet to form a shallow interdomain groove containing a solvent-exposed tryptophan that may represent a ligand binding site. The Mth structure is analyzed in relation to predicted Mth homologs and potential ligand binding features.

Huntington's Disease is an inherited neurodegenerative disease that affects the medium spiny neurons in the striatum. The disease is caused by the expansion of a polyglutamine sequence in the N terminus of Huntingtin (Htt), a widely expressed protein. Recently, we have found that Htt is an antiapoptotic protein in striatal cells and acts by preventing caspase-3 activity. Here we report that Htt overexpression in other CNS-derived cells can protect them from more than 20 days exposure to fatal stimuli. In particular, we found that cytochrome c continues to be released from mitochondria into the cytosol of cells that overexpress normal Htt. However, procaspase-9 is not processed, indicating that wild-type Htt (wtHtt) acts downstream of cytochrome c release. These data show that Htt inhibits neuronal cell death by interfering with the activity of the apoptosome complex.

Netrins are a family of chemotropic factors that guide axon outgrowth during development; however, their function in the adult CNS remains to be established. We examined the expression of the netrin receptors DCC and UNC5H2 in adult rat retinal ganglion cells (RGCs) after grafting a peripheral nerve (PN) to the transected optic nerve and following optic nerve transection alone. In situ hybridization revealed that both Dcc and Unc5h2 mRNAs are expressed by normal adult RGCs. In addition, netrin-1 was found to be constitutively expressed by RGCs. Quantitative analysis using in situ hybridization demonstrated that both Dcc and Unc5h2 were down-regulated by RGCs following axotomy. In the presence of an attached PN graft, Dcc and Unc5h2 were similarly down-regulated in surviving RGCs regardless of their success in regenerating an axon. Northern blot analysis demonstrated expression of netrin-1 in both optic and sciatic nerve, and Western blot analysis revealed the presence of netrin protein in both nerves. Immunohistochemical analysis indicated that netrin protein was closely associated with glial cells in the optic nerve. These results suggest that netrin-1, DCC, and UNC5H2 may contribute to regulating the regenerative capacity of adult RGCs.

Oocytes and early embryos of multiple (non-mammalian) species lack the somatic form of the linker histone H1. To the best of our knowledge, a mammalian oocyte-specific linker (H1) histone(s) has not, as yet, been reported. We have uncovered the cDNA in question in the course of a differential screening (suppression subtractive hybridization (SSH)) project. Elucidation of the full-length sequence of this novel 1.2 kb cDNA led to the identification of a 912 bp open reading frame. The latter encoded a novel 34 kDa linker histone protein comprised of 304 amino acids, tentatively named H1oo. Amino acid BLAST analysis revealed that H1oo displayed the highest sequence homology to the oocyte-specific B4 histone of the frog, the respective central globular (putative DNA binding) domains displaying 54% identity. Substantial homology to the cs-H1 protein of the sea urchin oocyte was also apparent. While most oocytic mRNAs corresponding to somatic linker histones are not polyadenylated (and remain untranslated), the mRNAs of (non-mammalian) oocyte-specific linker histones and of mammalian H1oo, are polyadenylated, a process driven by the consensus signal sequence, AAUAAA, detected in the 3'-untranslated region of the H1oo cDNA. Our data suggest that the mouse oocyte-specific linker histone H1oo (1) constitutes a novel mammalian homolog of the oocyte-specific linker histone B4 of the frog and of the cs-H1 linker histone of the sea urchin; (2) is expressed as early as the GV (PI) stage oocyte, persisting into the MII stage oocyte, the oocytic polar bodies, and the two-cell embryo, extinction becoming apparent at the four- to eight-cell embryonic stage; and (3) may play a key role in the control of gene expression during oogenesis and early embryogenesis, presumably through the perturbation of chromatin structure.

Caenorhabditis elegans has three POU homeobox genes, unc-86, ceh-6 and ceh-18. ceh-6 is the ortholog of vertebrate Brn1, Brn2, SCIP/Oct6 and Brn4 and fly Cf1a/drifter/ventral veinless. Comparison of C. elegans and C. briggsae CEH-6 shows that it is highly conserved. C. elegans has only three POU homeobox genes, while Drosophila has five that fall into four families. Immunofluorescent detection of the CEH-6 protein reveals that it is expressed in particular head and ventral cord neurons, as well as in rectal epithelial cells, and in the excretory cell, which is required for osmoregulation. A deletion of the ceh-6 locus causes 80% embryonic lethality. During morphogenesis, embryos extrude cells in the rectal region of the tail or rupture, indicative of a defect in the rectal epithelial cells that express ceh-6. Those embryos that hatch are sick and develop vacuoles, a phenotype similar to that caused by laser ablation of the excretory cell. A GFP reporter construct expressed in the excretory cell reveals inappropriate canal structures in the ceh-6 null mutant. Members of the POU-III family are expressed in tissues involved in osmoregulation and secretion in a number of species. We propose that one evolutionary conserved function of the POU-III transcription factor class could be the regulation of genes that mediate secretion/osmoregulation.

The apparent diversity of linker histone subtypes may be related to their specific roles in defining functional states of chromatin in vivo. We have developed a novel method to study constitutive peptides throughout the cell cycle and have demonstrated that an exogenous linker histone could be introduced into a living cell of the slime mold Physarum polycephalum. Here, we have used this method to assess the functional differences between three somatic linker histone subtypes in vivo, and to demonstrate the general applicability of this method. Exogenous linker histone proteins H1 degrees, H5 and H1 were directly absorbed into living cell segments of the naturally synchronous Physarum macroplasmodia at precise cell cycle stages. Fluorescence microscopy, native nucleoprotein gels and immunoblotting of nuclei and chromatin with subtype-specific antibodies revealed that exogenous linker histones were efficiently transported into nuclei and were integrated into chromatin. The immunoreactivity of a preparation of anti-H1 degrees antibodies that are blocked from binding to specific H1 degrees epitopes in native chromatin indicates that the exogenous linker histones were similarly associated into Physarum chromatin. Interestingly, linker histones were found to be less stably associated with Physarum chromatin during S-phase than during G(2)-phase. Furthermore, we show that exogenous linker histones incorporated in early G(2)-phase inhibited transcription and that the level of inhibition correlates with the apparent role of the linker histone subtype in regulating transcription in cells where it normally occurs.

The serotonin transporter (5-HTT) plays a key role in the regulation of serotonin (5-hydroxytryptamine, 5-HT) transmission in the pathophysiology and therapeutics of several psychiatric disorders. The mean spontaneous firing rate of midbrain dorsal raphe 5-HT neurons was recorded in chloral hydrate-anesthetized mice. The serotonin transporter (5-HTT), which plays a key role in the regulation of serotonin was significantly decreased in homozygous mice lacking the 5-HT transporter (5-HTT -/-) by 66% and in heterozygous (5-HTT +/-) mice by 36% compared with their normal littermates (5-HTT +/+). Systemic injection of the selective 5-HT(1A) receptor antagonist WAY 100635 enhanced 5-HT neuronal firing by 127% in 5-HT -/- mice, thus indicating an enhanced synaptic availability of 5-HT at inhibitory 5-HT(1A) receptors. Nevertheless, the cell body 5-HT(1A) autoreceptors were desensitized in both 5-HTT -/- and 5-HTT +/- mice. At the postsynaptic level, the recovery time (RT(50)) of the firing rate of hippocampus CA(3) pyramidal neurons following iontophoretic applications of 5-HT was significantly prolonged only in 5-HTT -/- mice. The selective 5-HT reuptake inhibitor paroxetine significantly prolonged the RT(50) in 5-HTT +/+ and 5-HTT +/- mice, without altering the maximal inhibitory effect of 5-HT. These neurons in 5-HTT -/- mice showed an attenuated response to the 5-HT(1A) agonist 8-hydroxy-2-diproplyaminotetralin, but not to 5-HT itself. These results establish that the lack of 5-HTT causes a prolonged recovery of firing activity following 5-HT applications. The genetic deletion of the 5-HTT plays a key role on 5-HT(1A) receptor adaptation: a desensitization at pre- and postsynaptic levels in 5-HTT -/- mice, but to a different extent, and only at the presynaptic level in the 5-HTT +/- group.

<h4>Background</h4>Nasal polyposis often coexists with asthma in airway inflammatory conditions characterised by the infiltration of a range of immune cells. A potentially important role for ovarian hormones has been implicated in airway inflammation but the cellular target for such action is not known.<h4>Methods</h4>Expression of oestrogen receptors (ER) and progesterone receptors (PR) was examined using immunohistochemistry in formalin fixed nasal polyp tissues from 47 subjects. The cells positive for ER or PR were confirmed by spatial location, dual immunolabelling, and histochemical staining.<h4>Results</h4>Consistent with the known features of nasal polyps, CD4+ (T helper/inducer), CD8+ (cytotoxic/suppressor), CD68+ (macrophages), mast cells, eosinophils and neutrophils were all clearly detected by their relevant monoclonal antibodies or appropriate histochemical staining, but only mast cells tested positive for ER/PR labelling with their polyclonal and monoclonal antibodies. The frequencies for expression were 61.7% for ER positive and 59.6% for PR positive cells. The expression of ER/PR was independent of patient sex and age but was highly correlated with the numbers of mast cells (r = 0.973, p<0.001 for ER; r = 0.955, p<0.001 for PR). Fewer than 5% of mast cells were found to be negative for ER/PR expression.<h4>Conclusions</h4>Mast cells alone, but not lymphocytes, macrophages, or other immune cells, express ER/PR in human upper airways. Numerous ER/PR positive mast cells exist in nasal polyps, indicating that this may be a major route for the involvement of sex hormones in airway inflammation when exposed to the higher and varying concentration of oestrogen and progesterone characteristic of females.

Hereditary hemochromatosis (HHC) is an inherited disorder of iron metabolism affecting approximately 1 in 200-300 individuals of Northern European descent. Over time, the continued deposition of iron in parenchymal cells of many organs can eventually lead to diabetes mellitus, cardiomyopathy, and hepatic cirrhosis, the last of which is frequently followed by hepatocellular carcinoma. Although the complications of HHC can be devastating, its clinical management is simple and effective if the disease is identified early in its progression. The recent elucidation of the HFE gene has provided insight into the pathogenesis of HHC and provided a means for the early identification of individuals in whom HHC may develop. Two mutations have been implicated in HHC: C282Y and H63D. The former occurs in a homozygous state seen in 75-100% of patients with HHC. The high correlation of HFE to HHC has caused it to be considered as a candidate gene for population-based genetic testing for diagnosis and detection of predisposition to HHC. In addition, mechanisms of iron transport and metabolism are unfolding and are providing clues to the enigma of iron homeostasis and the pathophysiology of iron overload.

An assay is described for high-throughput single nucleotide polymorphism (SNP) genotyping on a microfabricated capillary array electrophoresis (CAE) microchip. The assay targets the three common variants at the HFE locus associated with the genetic disease hereditary hemochromatosis (HHC). The assay employs allele-specific PCR (ASPCR) for the C282Y (845g->a), H63D (187c->g), and S65C (193a->t) variants using fluorescently-labeled energy-transfer (ET) allele-specific primers. Using a 96-channel radial CAE microplate, the labeled ASPCR products generated from 96 samples in a reference Caucasian population are simultaneously separated with single-base-pair resolution and genotyped in under 10 min. Detection is accomplished with a laser-excited rotary four-color fluorescence scanner. The allele-specific amplicons are differentiated on the basis of both their size and the color of the label emission. This study is the first demonstration of the combined use of ASPCR with ET primers and microfabricated radial CAE microplates to perform multiplex SNP analyses in a clinically relevant population.

Transgenic mice expressing the Simian virus 40 large T antigen under the control of the liver-specific human antithrombin-III promoter all develop well-differentiated hepatocellular carcinoma. During tumour development serum ceruloplasmin (Cp) increases gradually until it reaches 30 times control levels in all transgenic mice at 6 months of age. The accumulation of Cp in the serum is due to the increased transcription of the Cp gene as well as to the increase in Cp mRNA stability in the livers of the transgenic mice. One-half of the overproduced Cp is charged with copper and Cp-associated serum oxidase activity increases in parallel with the holo-Cp concentration. Through its ferroxidase activity Cp is involved prominently in iron metabolism. Analysis of copper and iron in serum and liver revealed increased copper levels in the serum of tumour-bearing animals and which increased in parallel with Cp concentration; the amounts of copper in the liver were unchanged. In contrast, serum iron remained constant during tumour development whereas the iron concentration in the livers of the transgenic mice decreased.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated disease initiated by antigen-specific CD4(+) T cells. Signaling through CD28 is a critical second signal for activation of T cells, and CD28 knockout (CD28KO) mice have been reported to be resistant to induction of EAE. We now report that CD28KO mice have no intrinsic defect in mediating disease, because they developed EAE after passive transfer of primed T cells. After immunization, peripheral T cells from CD28KO mice were primed and developed memory phenotype, but had decreased antigen-specific IFN-gamma production as compared with cells from wild-type (WT) animals. Reimmunization of CD28KO mice brought out clinical disease and increased IFN-gamma production in vitro. Pathologically, there were cellular infiltrates in the central nervous system, in contrast to single-immunized mice. We show furthermore that blocking B7-1 or CTLA4, but not B7-2, in CD28KO mice induces disease after a single immunization. Thus, EAE can be induced in animals lacking CD28-dependent costimulation, suggesting that alternative costimulatory pathways were used. Blocking the OX40-OX40L costimulatory pathway differentially affected disease induction in CD28KO mice as compared with WT controls. Our data show that EAE may develop in the absence of CD28 T-cell costimulation. These findings have implications for therapies aimed at blocking costimulatory signals in autoimmune diseases.

The deleterious role of fibrin deposition in arthritic joints prompted us to explore the effect of the thrombin inhibition on the course of collagen-induced arthritis (CIA) in the mouse. CIA was induced in male DBA/1J mice using native chicken type II collagen. The thrombin inhibitor polyethyleneglycol-hirudin (PEG-hirudin) was given for 16 days, starting 20 days after the first immunization (preventive treatment) or at the onset of clinical signs of arthritis (curative treatment). All the mice treated with PEG-hirudin had a significantly prolonged clotting time compared with control mice. PEG-hirudin, administered in a preventive way, led to significantly reduced incidence and severity of CIA during most of the treatment period, as assessed by clinical scoring. Accordingly, histological features showed a significant diminution of synovial hyperplasia in PEG-hirudin-treated mice compared with untreated mice. There was also a significant downmodulation of the synovial proinflammatory IL-1beta and IL-12p35 cytokine mRNAs in treated mice. Intra-articular fibrin, evaluated by immunohistochemistry, was significantly reduced in treated mice compared with control mice and correlated with both clinical and histological scorings. Most importantly, once arthritis was established, PEG-hirudin also showed a curative effect. In conclusion, PEG-hirudin can both prevent the onset of CIA in a dose-dependent manner and ameliorate established arthritis, suggesting that thrombin inhibition may offer a new therapeutic approach in arthritis.

Netrins stimulate and orient axon growth through a mechanism requiring receptors of the DCC family. It has been unclear, however, whether DCC proteins are involved directly in signaling or are mere accessory proteins in a receptor complex. Further, although netrins bind cells expressing DCC, direct binding to DCC has not been demonstrated. Here we show that netrin-1 binds DCC and that the DCC cytoplasmic domain fused to a heterologous receptor ectodomain can mediate guidance through a mechanism involving derepression of cytoplasmic domain multimerization. Activation of the adenosine A2B receptor, proposed to contribute to netrin effects on axons, is not required for rat commissural axon outgrowth or Xenopus spinal axon attraction to netrin-1. Thus, DCC plays a central role in netrin signaling of axon growth and guidance independent of A2B receptor activation.

We evaluated more than 450 patients with thrombophilia or iron overload for the presence of a factor V Leiden (R506Q), prothrombin G20210A, or HFE C282Y mutation using a standard method (polymerase chain reaction [PCR]-restriction fragment length polymorphism) and a comparative real-time PCR fluorescent resonance energy transfer (FRET) hybridization probe melting curve method. There was 100% concordance between the genotypes ascertained by the 2 methods (at each loci). In addition, phenotypic biochemical laboratory parameters measured on a subset of referred patients correlated with their respective genotypes. In the iron overload cohort, HFE C282Y homozygotes (n = 74) had significantly higher (P < .0001) transferrin saturation levels (74% +/- 25%) than did nonhomozygotes (n = 340; 51.4% +/- 28%), suggesting a genotype-dependent increase in body iron loads. In the thrombophilic cohort, the degree of activated protein C resistance (APCR), measured by a clotting time-based test, was associated significantly with the presence of 0 (n = 255; APCR = 2.59 +/- 0.26), 1 (n = 84; APCR = 1.61 +/- 0.13), or 2 (n = 5; APCR = 1.16 +/- 0.04) copies of the mutant factor V Leiden allele. As the fluorescent genotyping method required no postamplification manipulation, genotypes could be determined more quickly and with minimized risk of handling errors or amplicon contamination. In addition to these practical advantages, the FRET method is diagnostically accurate and clinically predictive of phenotypic, disease-associated manifestations.

After neuronal injury and in several neurodegenerative diseases, activated microglia secrete proinflammatory molecules that can contribute to the progressive neural damage. The recent demonstration of a protective role of estrogen in neurodegenerative disorders in humans and experimental animal models led us to investigate whether this hormone regulates the inflammatory response in the CNS. We here show that estrogen exerts an anti-inflammatory activity on primary cultures of rat microglia, as suggested by the blockage of the phenotypic conversion associated with activation and by the prevention of lipopolysaccharide-induced production of inflammatory mediators: inducible form of NO synthase (iNOS), prostaglandin-E(2) (PGE(2)), and metalloproteinase-9 (MMP-9). These effects are dose-dependent, maximal at 1 nm 17beta-estradiol, and can be blocked by the estrogen receptor (ER) antagonist ICI 182,780. The demonstration of ERalpha and ERbeta expression in microglia and macrophages and the observation of estrogen blockade of MMP-9 mRNA accumulation and MMP-9 promoter induction further support the hypothesis of a genomic activity of estrogen via intracellular receptors. This is the first report showing an anti-inflammatory activity of estrogen in microglia. Our study proposes a novel explanation for the protective effects of estrogen in neurodegenerative and inflammatory diseases and provides new molecular and cellular targets for the screening of ER ligands acting in the CNS.

Proteolysis of mutant huntingtin (htt) has been hypothesized to occur in Huntington's disease (HD) brains. Therefore, this in vivo study examined htt fragments in cortex and striatum of adult HD and control human brains by Western blots, using domain-specific anti-htt antibodies that recognize N- and C-terminal domains of htt (residues 181-810 and 2146-2541, respectively), as well as the 17 residues at the N terminus of htt. On the basis of the patterns of htt fragments observed, different "protease-susceptible domains" were identified for proteolysis of htt in cortex compared with striatum, suggesting that htt undergoes tissue-specific proteolysis. In cortex, htt proteolysis occurs within two different N-terminal domains, termed protease-susceptible domains "A" and "B." However, in striatum, a different pattern of fragments indicated that proteolysis of striatal htt occurred within a C-terminal domain termed "C," as well as within the N-terminal domain region designated "A". Importantly, striatum from HD brains showed elevated levels of 40-50 kDa N-terminal and 30-50 kDa C-terminal fragments compared with that of controls. Increased levels of these htt fragments may occur from a combination of enhanced production or retarded degradation of fragments. Results also demonstrated tissue-specific ubiquitination of certain htt N-terminal fragments in striatum compared with cortex. Moreover, expansions of the triplet-repeat domain of the IT15 gene encoding htt was confirmed for the HD tissue samples studied. Thus, regulated tissue-specific proteolysis and ubiquitination of htt occur in human HD brains. These results suggest that the role of huntingtin proteolysis should be explored in the pathogenic mechanisms of HD.

The consequences of the absence of 5-HT reuptake on the functional properties of 5-HT(1A) receptors were examined in the dorsal raphe nucleus and the hippocampus of knock-out mice lacking the serotonin transporter (5-HTT). Extracellular recordings showed that application of selective 5-HT reuptake inhibitors such as paroxetine and citalopram onto brainstem slices resulted in a concentration-dependent inhibition of 5-HT neuron firing in the dorsal raphe nucleus of wild-type 5-HTT+/+ mice, but not 5-HTT-/- mutants. By contrast, the 5-HT(1A) receptor agonists ipsapirone and 5-carboxamidotryptamine inhibited the discharge in both groups. However, the potency of these agonists was markedly decreased (by approximately 55- and approximately 6-fold, respectively) in 5-HTT-/- compared with 5-HTT+/+ animals. Similarly, intracellular recordings showed that the potency of 5-carboxamidotryptamine to hyperpolarize 5-HT neurons in the dorsal raphe nucleus was significantly lower in 5-HTT-/- than in 5-HTT+/+ animals. These data contrasted with those obtained with hippocampal slices in which 5-carboxamidotryptamine was equipotent to hyperpolarize CA1 pyramidal neurons in both mutant and wild-type mice. As expected from their mediation through 5-HT(1A) receptors, the effects of ipsapirone and 5-carboxamidotryptamine were competitively inhibited by the selective 5-HT(1A) antagonist WAY 100635 in both groups. These data showed that 5-HTT gene knock-out induced a marked desensitization of 5-HT(1A) autoreceptors in the dorsal raphe nucleus without altering postsynaptic 5-HT(1A) receptor functioning in the hippocampus. Similarities between these changes and those evoked by chronic treatment with 5-HT reuptake inhibitors emphasize the existence of regional differences in 5-HT(1A) receptor regulatory mechanisms.

The Sox gene family encodes an important group of transcription factors harboring the conserved high-mobility group (HMG) box originally identified in the mouse and human testis determining gene Sry. We have cloned and sequenced SOX6, a member of the human Sox gene family. SOX6 cDNAs isolated from a human myoblast cDNA library show 94.3% amino acid identity to mouse Sox6 throughout the gene, and 100% identity in the critical HMG box and coiled-coil domains. The human SOX6 gene was localized to chromosome 11p15.2-11p15.3 in a region of shared synteny with distal mouse chromosome 7. An analysis of the genomic structure of the human SOX6 gene revealed 16 exons. We identified three SOX6 cDNAs that are generated by alternative splicing. Northern blot analysis revealed that SOX6 is expressed in a wide variety of tissues, most abundantly in skeletal muscle, suggesting an important role for SOX6 in muscle. Mice homozygous for a null mutation of Sox6 (p(100H)) die suddenly within the first 2 weeks after birth, most likely from cardiac conduction defects (Hagiwara et al., 2000). Thus, there is a possibility that human SOX6 is similarly involved in an, as yet, unidentified human cardiac disorder.

No abstract available.

Type XVIII collagen is a homotrimeric basement membrane molecule of unknown function, whose COOH-terminal NC1 domain contains endostatin (ES), a potent antiangiogenic agent. The Caenorhabditis elegans collagen XVIII homologue, cle-1, encodes three developmentally regulated protein isoforms expressed predominantly in neurons. The CLE-1 protein is found in low amounts in all basement membranes but accumulates at high levels in the nervous system. Deletion of the cle-1 NC1 domain results in viable fertile animals that display multiple cell migration and axon guidance defects. Particular defects can be rescued by ectopic expression of the NC1 domain, which is shown to be capable of forming trimers. In contrast, expression of monomeric ES does not rescue but dominantly causes cell and axon migration defects that phenocopy the NC1 deletion, suggesting that ES inhibits the promigratory activity of the NC1 domain. These results indicate that the cle-1 NC1/ES domain regulates cell and axon migrations in C. elegans.

PTEN is a putative tumour suppressor gene located on chromosome band 10q23. Mutations in PTEN have been identified in numerous human malignancies, including cancers of the brain, endometrium, ovary, and prostate. In this study, we screened 80 Barrett's oesophagus-associated adenocarcinomas (BOAd) for loss of heterozygosity (LOH) at 10q23, using the microsatellite markers D10S541, D10S219, and D10S551. Tumours demonstrating LOH were then screened for the presence or absence of PTEN mutations. LOH at one or more loci was identified in 17/80 (21%) cases. In none of these cases did we detect mutations in PTEN. The presence of LOH did not correlate with patient age, tumour stage, degree of differentiation, presence of perineural or vascular invasion, or overall survival. We conclude that LOH at chromosome 10q23 is uncommon in BOAd, is not associated with mutations in the PTEN tumour suppressor gene, and does not correlate with the clinical or pathologic features of these tumours. It is possible that PTEN is inactivated through other mechanisms in BOAd.

<h4>Background</h4>Porphyria cutanea tarda (PCT) is associated in most cases with iron overload, which may participate in decreased activity of uroporphyrinogen decarboxylase in the liver. The aetiology of this iron overload remains unknown; however, it has been demonstrated that mutations of HFE, the genetic haemochromatosis gene, might be present in a significant proportion of Anglo-Saxon and Italian patients. Furthermore, transferrin receptor polymorphism may influence the affinity of this receptor to its ligand with a subsequent increase of cellular iron absorption and storage.<h4>Objectives</h4>To evaluate the incidence and spectrum of HFE mutations and the relative frequency of the two main alleles of transferrin receptor in patients with PCT originating from southern France, and to evaluate the relationship of these genetic data with iron status, and with hepatitis B and C and human immunodeficiency virus (HIV) infections.<h4>Methods</h4>Thirty-six consecutive patients with either sporadic or familial PCT were prospectively included between 1997 and 2000. Search for the presence of the three main mutations of the HFE gene and identification of the transferrin receptor alleles were performed using polymerase chain reaction followed by enzymatic digestion. Iron parameters and viral status for hepatitis B and C viruses and HIV were determined.<h4>Results</h4>Seven patients (19%) showed heterozygous C282Y mutation, but no C282Y homozygote was present; five patients (14%) carried homozygous H63D mutation, while eight (22%) were heterozygous for this mutation. One patient was heterozygous for the S65C mutation (3%). Iron parameters demonstrated overload in all patients, without a clear difference between patients with and without deleterious mutations of the HFE gene. Infection by hepatitis C virus was documented in 20 patients (56%), and was significantly less frequent in patients with deleterious HFE mutations. The profile of transferrin receptor alleles in PCT patients did not show significant variation compared with the general population.<h4>Conclusions</h4>This study confirms the high frequency of HFE mutations in patients with PCT and supports the hypothesis that HFE gene abnormalities might play a significant part in the PCT pathomechanism, probably through iron overload; by contrast, transferrin receptor polymorphisms do not appear to play a significant part in iron overload in PCT.

People suffering from hereditary hemochromatosis (HH) can not regulate the uptake of iron properly and gradually accumulate iron in their body over their lifetime. The protein involved in HH, HFE, has been recently identified as a class I major histocompatibility complex (MHC) homolog. The wild-type HFE associates and co-traffics with the transferrin receptor (TfR). The mutation responsible for 83% of HH (C260Y) results in the failure of HFE to form a critical disulfide bond, bind beta2 microglobulin, bind TfR, and traffic to the cell surface. In non-polarized cells, the partnership of HFE and TfR results in decreased iron uptake into cells. The mechanism whereby a class I MHC homolog modifies the function of a membrane receptor and how this dynamic complex of molecules regulates iron transport across intestinal epithelial cells is the subject of this review.

The synthesis and structure-activity relationships (SARs) of a series of novel 10-arylacetyl-1,8-dihydroxy-9(10H)-anthracenones are described. Acylation of anthralin with either the appropriate arylacetyl chlorides or arylacetic acids in the presence of pyridine or via the coupling agent dicyclohexylcarbodiimide (DCC), respectively, furnished this structural class of antipsoriatic agents. Potential antipsoriatic activity was evaluated in complementary assays specifically addressed to three important aspects of psoriasis. First, several compounds were identified which are equally potent as inhibitors of human keratinocyte growth as the antipsoriatic agent anthralin. Furthermore, improved ratio of antiproliferative activity to cytotoxicity is demonstrated by the reduced potential of the novel analogues to induce membrane damage, which is a benefit of their reduced ability to generate oxygen radicals as documented by deoxyribose degradation. Second, analogue 3o bearing a hydroxamate functional group was also a highly potent inhibitor of LTB(4) biosynthesis in addition to its excellent antiproliferative activity. SARs of these inhibitors of both keratinocyte growth and LTB(4) biosynthesis with respect to the nature of the para-substitution in the 10-phenylacetyl side chain are discussed. Third, the compounds were also evaluated for their ability to induce the formation of cornified envelope protein in keratinocytes. Cross-linking of cellular protein as a marker of terminal differentiation of keratinocytes was observed for many 10-arylacetyl analogues at concentrations required to arrest cell growth. This newly uncovered activity of the novel anthracenones suggests antipsoriatic potential with respect to disturbance of keratinocyte differentiation, in addition to hyperproliferative and inflammatory aspects of psoriasis.

Expanded polyglutamine repeats have been proposed to cause neuronal degeneration in Huntington's disease (HD) and related disorders, through abnormal interactions with other proteins containing short polyglutamine tracts such as the transcriptional coactivator CREB binding protein, CBP. We found that CBP was depleted from its normal nuclear location and was present in polyglutamine aggregates in HD cell culture models, HD transgenic mice, and human HD postmortem brain. Expanded polyglutamine repeats specifically interfere with CBP-activated gene transcription, and overexpression of CBP rescued polyglutamine-induced neuronal toxicity. Thus, polyglutamine-mediated interference with CBP-regulated gene transcription may constitute a genetic gain of function, underlying the pathogenesis of polyglutamine disorders.

No abstract available.

In chromosomal rearrangements of acute myeloid leukaemia patients the mixed lineage leukaemia (MLL) gene, a human homolog of the Drosophila gene trithorax, is frequently fused to AF10. Here we describe the identification and a functional characterization of the Drosophila homolog dAF10. We show that dAF10 functions in heterochromatin-dependent genomic silencing of position effect variegation, a phenomenon associated with chromosomal rearrangements that cause mosaic expression of euchromatic genes when relocated next to heterochromatin. We also demonstrate that dAF10 can associate with the heterochromatin protein 1 (HP1) in vitro and in vivo. The results indicate that dAF10 is an HP1-interacting component of the heterochromatin-dependent gene silencing pathway, which either contributes to the stability of the heterochromatin complex or to its function.

Sepsis and multisystem organ failure are often associated with disseminated intravascular coagulation and consumption of coagulation inhibitors such as antithrombin III (ATIII). The "sick euthyroid syndrome" is also seen in association with significant illnesses and consists of decreased levels of circulating triiodothyronine (T3). We evaluated whether T3 supplementation would affect ATIII levels in septic rats. Thirty Sprague-Dawley rats were divided into three groups: sham laparotomy (S) plus saline, cecal ligation and puncture (CLP) plus saline, and CLP plus T3 (3 ng/hour) via an osmotic minipump. Twenty-four hours after laparotomy blood was drawn, and T3 and ATIII levels were then compared with baseline values. T3 supplementation partially negated the sepsis-induced decrease in circulating T3 levels. The levels are expressed as percentage change from the levels before surgery (S, -12.9 +/- 3.1; CLP, -60.0 +/- 5.3; CLP + T3, -34.9 +/- 4.3; mean +/- standard error; P < 0.05). T3 supplementation also statistically changed the percentage difference in ATIII levels toward the control (S, 9.6 +/- 2.8; CLP, -37.9 +/- 5.4; CLP + T3, -16.0 +/- 4.5; mean +/- standard error; P < 0.01). T3 supplementation reduced the sepsis-induced decrease in ATIII levels. Whether this was accomplished by decreased consumption or increased production of ATIII via the direct anabolic effect of T3 on acute-phase protein synthesis in the liver is unknown and warrants further investigation.

Human synovial sarcoma has been shown to exclusively harbor the chromosomal translocation t(X;18) that produces the chimeric gene SYT-SSX. However, the role of SYT-SSX in cellular transformation remains unclear. In this study, we have established 3Y1 rat fibroblast cell lines that constitutively express SYT, SSX1, and SYT-SSX1 and found that SYT-SSX1 promoted growth rate in culture, anchorage-independent growth in soft agar, and tumor formation in nude mice. Deletion of the N-terminal 181 amino acids of SYT-SSX1 caused loss of its transforming activity. Furthermore, association of SYT-SSX1 with the chromatin remodeling factor hBRM/hSNF2 alpha, which regulates transcription, was demonstrated in both SYT-SSX1-expressing 3Y1 cells and in the human synovial sarcoma cell line HS-SY-II. The binding region between the two molecules was shown to reside within the N-terminal 181 amino acids stretch (aa 1--181) of SYT-SSX1 and 50 amino acids (aa 156--205) of hBRM/hSNF2 alpha and we found that the overexpression of this binding region of hBRM/hSNF2 alpha significantly suppressed the anchorage-independent growth of SYT-SSX1-expressing 3Y1 cells. To analyze the transcriptional regulation by SYT-SSX1, we established conditional expression system of SYT-SSX1 and examined the gene expression profiles. The down-regulation of potential tumor suppressor DCC was observed among 1,176 genes analyzed by microarray analysis, and semi-quantitative reverse transcription--PCR confirmed this finding. These data clearly demonstrate transforming activity of human oncogene SYT-SSX1 and also involvement of chromatin remodeling factor hBRM/hSNF2 alpha in human cancer.

Quantitation of estrogen and progesterone receptors (PR) represents the standard of care in the treatment of patients with breast cancer. Historically this was performed by cytosolic assay; current methods utilize immunohistochemical staining, which may be quantitated visually or by image cytometry. Formalin-fixed paraffin embedded sections from 95 breast carcinomas were immunostained with an avidin-biotin complex technique. steam antigen retrieval, and a monoclonal PR antibody (1/40 Biogenex). Nuclear immunostain was quantitated visually as the percentage of immunopositive nuclei, scored as 0 to 4. By image cytometry, the percentage of positively staining nuclear area (PPNA) was determined in 15 hpf using the CAS 200 Image Analyzer. Dextran-coated charcoal (DCC) ligand binding assay data were divided into negative (<10 fmol), low positive (10-50), or positive (>50). A statistically significant correlation was found between stage (P = 0.0001), the presence of nodal metastases (P = 0.0001), cytosolic assay (P = 0.036), image cytometry (P = 0.01), and disease-free survival. Only stage (P = 0.0001) and PR quantitation per cytosolic assay (P = 0.0001) correlated with overall survival. The method of choice for the assessment of PR hormone status in breast carcinomas is the DCC ligand binding assay. This method correlates with both survival and disease-free survival. Image cytometric quantitation of PR immunohistochemical staining correlates only with disease-free survival. The commonly used method of visual quantitation of PR immunostaining fails to relate either to survival or disease-free survival.

<h4>Purpose</h4>The recent discovery of the HFE gene and its association with hereditary hemochromatosis has renewed the attention directed to iron-overload diseases. Population screening for hereditary hemochromatosis is under debate, and population-based estimates of morbidity associated with hereditary hemochromatosis are needed. The purpose of this study is to estimate the number of hemochromatosis-associated hospitalizations in the United States using a population-based dataset.<h4>Methods</h4>National Hospital Discharge Survey and census data were used to estimate hemochromatosis-associated hospitalization rates for persons 18 years of age and over.<h4>Results</h4>From 1979 through 1997, the rate of hemochromatosis-associated hospitalizations was 2.3 per 100,000 persons in the United States. The rate among persons 60 years of age and over increased more than 60% during this time period.<h4>Conclusion</h4>The increase in the rate of hereditary hemochromatosis-associated hospitalizations among older persons is consistent with recent trends in mortality data and may reflect the rising awareness of iron-overload disorders in the United States.

Colorectal adenomas can be morphologically classified as exophytic or flat. Polypoid cancers and cancers arising de novo (ie., without any adenomatous component) might be the results of genetic progression from exophytic and flat adenomas, respectively. In this study, we examined 94 morphologically distinct neoplastic specimens for mutations in K-RAS and analyzed 10 microsatellite loci tightly linked to the tumor suppressor genes APC, p53, DCC/SMAD4, hMSH2, and hMLH1. K-RAS mutations were significantly associated with exophytic adenomas [11 of 21 (52%)] compared to flat adenomas [2 of 13(15%), P < 0.03] and polypoid cancers [17 of 25 (68%)] compared to cancers arising de novo [7 of 25 (28%), P < 0.01]. Two polypoid cancer cases demonstrated three and four different K-RAS mutations, respectively, suggesting multiple areas of clonal expansion. Cancers arising de novo were significantly associated with loss of heterozygosity (LOH) at chromosome 3p compared to pol ypoid cancers [6 of 18(33%) versus 1 of 20(5%), P < 0.03], whereas the prevalence of LOH at chromosomes 2p, 5q, 17p, and 18q and microsatellite instability were not different between the groups. For all cancers, LOH at chromosomes 17p and 18q occurred in 47 and 51%, respectively. However, LOH at 17p and 18q occurred in 0 and 16% of benign lesions, respectively, suggesting their role in malignant transformation. There was no difference in LOH at chromosomes 17p and 18q between exophytic and flat lesions. These findings suggest that (a) mutant K-RAS is associated with the exophytic growth of colonic neoplasms, and that (b) some colorectal cancers arising de novo lose chromosome 3p during their evolution, which is not seen in polypoid cancers. Half of all cancers lose chromosomes 17p and 18q at or near the malignant transition of benign lesions as reported previously, irrespective of morphology. There may be more than one genetic avenue for colorectal cancer formation, and this correlates with the morphological characteristics.

Huntington's disease (HD) is caused by the inheritance of the huntingtin gene with an expanded CAG repeat. The function of the normal or mutant form of the huntingtin protein remains to be determined. We used differential display to determine differences in steady-state mRNA levels between wild-type and the R6/2 transgenic mouse model of HD. Using this method, we determined that the steady-state mRNA levels of protein kinase C beta II (PKC beta II) subunit are decreased in symptomatic HD mice compared with age-matched wild-type controls. The decrease in PKC beta II mRNA levels occurred in both the striatum and cortex. Previously, it had been demonstrated that PKC beta II immunoreactivity is decreased in the caudate-putamen of patients with Huntington's disease. PKC has been implicated in the long-term potentiation model of brain plasticity and learning, and the loss of PKC may affect information storage in HD. The expression of htt-HD throughout the brain affects the transcription of specific genes in regions not associated with widespread neurodegeneration.

Neonatal platelets have been occasionally reported to show a reduced response to various agonists. The molecular mechanism(s) of such a depressed reactivity remains unclear. To further address this problem we studied neonatal platelet activation with thrombin, TRAP (thrombin receptor activating peptide, Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe) and ADP in 42 healthy 1-2 day old neonates using a whole peripheral blood flow cytometry. The neonates did not show an increased fraction of P-selectin-positive circulating platelets, whereas the expression of GPIb (glycoprotein Ib) in resting neonatal platelets was significantly lower compared to adults. Neonatal platelets were significantly less reactive than adult platelets to thrombin and TRAP, especially at lower agonist concentrations, but not to ADP or when incubated for 1 h at room temperature. Activation of neonatal platelets with agonists resulted in a marked alterations in the expression of P-selectin, whereas the internalization of GPIb was not affected. The reduced neonatal platelet sensitivity to thrombin and TRAP was accompanied by significantly reduced ATIII (antithrombin III) and increased prothrombin fragment F(1+2) in neonatal plasma. We conclude that various receptor systems potentially able to bind thrombin are relatively insensitive in neonatal platelets. The novelty of our work is that neonatal platelet hyposensitivity is not a generalized phenomenon, but concerns only selected agonists and selected receptor systems.

To obtain "exact" laboratory data which show or reflect the pathophysiological conditions of patients, preanalytical, analytical and postanalytical processes should be checked. However, it is difficult to know the preanalytical situation or out of laboratories. We realize preanalytical issue is one of the weak points in current laboratory medicine. I reported the results of analyses on the issues concerning abnormal laboratory data produced by inadequate handling of samples and sample collection. Five issues are described as follows: 1) The effect of detergents on fecal occult blood test. Immunological methods were affected markedly to moderately by various sanitizers and detergents tested. In collecting feces for immunological fecal occult blood test, contact of feces with toilet flush water dissolving sanitizers should be inhibited. 2) Blood storage and potassium(K) release. Causes of slight elevation of K in serum are sometimes unknown. Addition of injection drugs in vitro showed that various drugs induced K release from erythrocytes. Cold storage of blood is well-known to bring elevation of K level, however, the detailed mechanism is still unknown. We developed a simple method to measure K flux activity of erythrocytes and the decreased activity was shown in patients with chronic renal insufficiency and the aged as well as the aged erythrocytes. 3) Thawed serum and concentration difference. Frozen serum samples were thawed without mixing and various layers contained different concentrations in 60 items tested. Comparing the concentration before freezing, the upper layer contained 1/2 and the bottom 2 times more. T3 and FT4 showed exceptionally smaller difference, significance of which is unknown. 4) Two syringe method in blood collection. Blood samples drawn in the first half and the latter half were comparatively measured for APTT, PT and TT. Blood amount of 1 ml or more showed no differences. TT showed elevation in the first half in 0.5 ml-sample. The effects of local treatment by warming, tapping and congestion were examined. In some cases, tissue factor level elevated by such physical pretreatment. However, in usual blood collection in human, two syringe method is not necessarily employed. 5) Heat-inactivation and cold storage of serum. Preheating sera at 56 degrees C for 30 min reduced markedly the levels of ALT, ALP, CK, ChE, ATIII and alpha 2-PI. In seroimmunology, some complement components are markedly affected and rheumatoid factor of IgM classes was also affected. In about a half of HCV-Ab positive sera, CH50 levels were decreased by the cold storage(cold activation). We reported previously the non-participation of C1q in this phenomenon. In such sera, temperature management is important. Scientific data on the storage and collection of samples should be informed not only to laboratory staffs but to nurses and doctors in order to provide "exact" data.

The human serotonin transporter gene (5-HTT) demonstrates two polymorphisms with possible functional impact: a 44-bp insertion/deletion polymorphism of the promoter region and a 17-bp variable number of tandem repeat polymorphism (VNTR) in intron 2 (STin2). Such genetic polymorphisms in the serotoninergic system may increase the susceptibility to schizophrenia or may serve as predictors of therapeutic response. We therefore analyzed these polymorphisms as susceptibility factors for schizophrenia by comparison of 684 schizophrenic inpatients with 587 healthy controls. We furthermore compared the therapeutic outcome of schizophrenic patients differentiated by the 5-HTT genotypes. Schizo-affective patients were more frequently homozygous for the 44-bp insertion allele (Odds ratio, OR: 1.6, 95% confidence interval, CI: 1.1--2.3, P < 0.03) than were all other schizophrenic patients and controls. The 17-bp VNTR alleles found were: STin2.7, 9, 10, and 12. Sequence analysis revealed seven different sequence motifs with an invariable arrangement. Patients with schizo-paranoid schizophrenia were more frequently homozygous for the STin2.12 allele than were controls (OR: 1.4, CI: 1.1--1.8, P < 0.007) and all other schizophrenic patients (OR: 1.6, CI: 1.2--2.3). The STin2.9 allele represented a risk factor for the residual subtype of schizophrenia (OR: 6.4, CI: 2.5--16.2, P < 0.001). On the basis of global clinical impressions, as well as measurements with the positive and negative syndrome scale we found no association of the polymorphisms with therapeutic response. In conclusion, the 44-bp polymorphism may be associated with the schizo-affective and the 17-bp VNTR with the residual and schizo-paranoid subtype of schizophrenia, findings which require further biochemical and epidemiological confirmation.

In children and adolescents, hyperkinetic disorder (HD) with conduct disorder (CD) and without CD and attention-deficit/hyperactivity disorder (ADHD) is known to be comorbid with psychiatric disorders (anxiety, depression, aggression), some of which are related to disturbed serotonergic neurotransmission. The efficiency of serotonergic signalling relates to the concentration of the neurotransmitter in the synaptic cleft and is controlled by the serotonin transporter (5-HTT), which selectively removes serotonin out of the synaptic cleft.(1)The activity of serotonin transport itself has been shown to be also controlled by a 5-HTT-linked polymorphism in its promotor region with a L/L genotype yielding higher levels of 5-HTT function than do L/S or S/S genotypes.(2) Considering an association between 5-HTT polymorphism, serotonergic neurotransmission and HD +/- CD, we genotyped for 5-HTT polymorphism and compared patients with controls. In contrast to the distribution of L/L: L/S: S/S in controls (0.245: 0.509: 0.245), we found an enhanced expression of the L/L genotype in HD patients with CD (0.393: 0.304: 0.304; chi(2) = 7.603; P = 0.0211) and a significant overexpression of L/L in HD without CD (0.542: 0.333: 0.125; chi(2) = 9.127; P = 0.0092). To our knowledge, this is the first finding providing evidence for an association between the 5-HTT polymorphism and hyperkinetic disorder, implying that serotonergic neurotransmission might be affected in this desease. As a consequence, for a successful treatment of these patients one should now also consider drugs which specifically modulate serotonergic signalling such as selective serotonin reuptake inhibitors.

The glycine site-specific N-methyl-D-aspartate (NMDA) antagonist 5-nitro-6,7-dichloro-2,3-quinoxalinedione (ACEA 1021, 4x30 mg/kg, i.p.) given 30 min before dexfenfluramine (4x15 mg/kg, i.p., every 2 h) was unable to prevent dexfenfluramine-induced depletion of 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) content, and 5-HT transporter (5-HTT) density. Another glycine site-specific NMDA antagonist, R(+)-3-aminohydroxypyrrolidin-2-one [(R)-HA 966] (2x30 mg/kg, ip), given 30 min before dexfenfluramine (2x10 mg/kg, ip, 2 hourly) was also unable to prevent regional depletion of 5-HT, 5-HIAA, and 5-HTT density. However, ACEA 1021 (4x30 mg/kg, i.p.) given 30 min before (S)-3,4-methylenedioxymethamphetamine (MDMA, 4x10 mg/kg, 2 hourly, ip) attenuated the regional depletion of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), 5-HT, 5-HIAA content, and 5-HTT density. ACEA 1021 combined with (S)-MDMA also prevented (S)-MDMA-induced hyperthermia without causing hypothermia or preventing an (S)-MDMA-induced increase in locomotor activity.

A prospective randomized trial was used to study the incidence of cerebral dysfunction in patients undergoing cardiopulmonary bypass (CPB) with heparin-bonded vs non-heparin-bonded circuits. Although the etiology of postoperative cerebral dysfunction is controversial, activation of the systemic inflammatory response may play a role. After institutional approval and informed written consent, 39 elective coronary artery bypass (CABG) patients were studied. A battery of neuropsychometric tests (NPMTs) was performed preoperatively, and 5 days and 6 weeks postoperatively. Significant change in NPMT performance was defined as a 25% or greater decrease in postoperative performance, compared to baseline. The number of abnormal tests per patient was calculated. Analysis using the Mann-Whitney rank test was performed for the first follow-up. Patients randomized to heparin-bonded circuits had fewer abnormal NPMTs (>1 abnormal test) on postoperative day 5 (58 vs 70%, n=19 and 20) than patients randomized to non-heparin-bonded circuits. Patients exposed to heparin-bonded circuits had fewer abnormal tests (>1 abnormal test) at 6 weeks (36 vs 63%, n=14 and 16). Results suggested that the attenuation of systemic inflammation by heparin-bonded CPB circuits may lower the incidence of cerebral injury in cardiac surgical patients.

Heterozygosity for beta-thalassemia (minor) by itself does not lead into iron overload; however, when it is inherited together with a homozygous state for either the H63D or the C282Y mutations of the hereditary hemochromatosis gene (HFE gene), iron overload may ensue. We describe here a kindred in which the propositus, being heterozygote for beta-thalassemia and the H63D mutation of the HFE gene, developed severe iron overload and in turn, chronic liver failure with portal hypertension. Other members of the family with either beta-thalassemia or heterozygous for the H63D gene mutation did not develop iron overload. The interaction between beta-thalassemia and hereditary hemochromatosis is briefly discussed and speculations about other possible genetic mutations leading into familial iron loading are done.

<h4>Objective</h4>To test the hypothesis that the heterozygous state for HFE gene mutations involved in the pathogenesis of hemochromatosis, that may induce an increase of hepatic iron content, may aggravate the liver damage induced by prolonged and excessive use of ethanol.<h4>Patients and methods</h4>C282Y and H63D mutations of HFE gene were identified through polymerase chain reaction (PCR) on leukocyte DNA, in 125 consecutive patients diagnosed of advanced alcoholic liver disease (109 men, mean age 54 years, SD 11) and 181 healthy controls. All subjects were white Spaniards. RESULTS (CASES/CONTROLS): 1. Genotype distribution: a) mutation C282Y: no homozygotes, 10/23 heterozygotes, 115/158 normal (p = 0.60); b) mutation H63D: 9/5 homozygotes, 46/52 heterozygotes, 70/124 normal (Chi square 6.51, p = 0.039). 2. Allele frequencies: a) mutation C282Y: 240/339 normal, 10/23 mutated (p = 0.21); b) mutation H63D: 186/300 normal, 64/62 mutated (odds ratio 1.66, 95% CI 1.10-2.52, p = 0.01).<h4>Conclusions</h4>Our results suggest that H63D mutation of the HFE gene, but not the C282Y mutation, is associated to the risk of developing advanced liver alcoholic disease.

The possible role of iron in facilitating the development of liver cancer is still debated. The aims of this study were to define the prevalence of the mutations 845G --> A and 187C --> G (C282Y and H63D) in the HFE gene associated with hereditary hemochromatosis in Italian patients with hepatocellular carcinoma occurring in cirrhosis and to analyze the interaction between these mutations and other established risk factors for hepatocellular carcinoma. The HFE gene mutations, performed by polymerase chain reaction, were analyzed in 81 patients (63 males, 18 females) with hepatocellular carcinoma. None of the patients had a phenotype compatible with homozygous hereditary hemochromatosis. Interaction between HFE mutations and exogenous risk factors was analyzed by collecting information on alcohol consumption, hepatitis B and C virus infections, and iron status at the time of diagnosis of chronic liver disease. This analysis was performed only in males to rule out gender influence on patients' iron status by using the case-only approach specifically designed to estimate departure from multiplicative risk ratios under the assumption of independence between genotype and environmental exposure. The prevalence of the C282Y mutation was significantly higher in patients with hepatocellular carcinoma than in normal controls (8.6% vs 1.6%, P < 0.03). At univariate analysis, iron overload was significantly associated with both HFE mutations (P < 0.0001), whereas ongoing hepatitis B virus infection was associated with the C282Y mutation (P < 0.05). By multivariate analysis, a trend for an increased risk of being positive for hepatitis virus markers (OR 2.9, CI 95% 0.9-9.5) and of having been alcohol abusers (OR 3, CI 95% 0.7-14) was observed in patients heterozygous for the HFE mutations. These data indicate that the prevalence of the main mutation associated with hereditary hemochromatosis is significantly higher in cirrhotic Italian patients with hepatocellular carcinoma compared to a normal population and suggest that heterozygotes for HFE mutations exposed to hepatitis virus infections or who had been alcohol abusers could have an increased risk of developing cirrhosis and later liver cancer than people without the mutations exposed to the same risk factors.

The gene responsible for hemochromatosis (HFE) has been identified on the short arm of chromosome 6, 4.5 Mb telomeric to HLA-A. A major mutation C282Y is closely correlated with the disease, as it accounts for 68 to 100\% of the cases of hemochromatosis. Nevertheless, some C282Y homozygotes subjects have no clinical or biological expression of the disease. Moreover, in Northern European populations a large discrepancy is observed between the number of C282Y homozygotes and the number of diagnosed hemochromatosis patients, suggesting incomplete penetrance of the mutation. To localize and identify the modifying genes, we investigated eight families including C282Y homozygous relatives showing no clinical signs of the disease, in addition to the hemochromatosis patients. Genomic DNA from 20 C282Y homozygotes (10 patients and 10 siblings presenting no or minor biological abnormalities) were studied. Five polymorphisms from the HFE gene were determined by PCR restriction. Extended haplotypes of the 6p21.3 region were constructed with 10 microsatellite markers. All the C282Y homozygotes shared the same HFE polymorphism. The haplotypes presented no significant difference between the probands and their unaffected relatives. These studies suggest that neither HFE polymorphism nor genes surrounding HFE are able to modulate HFE expression.

We have identified five single nucleotide polymorphisms (SNPs) upstream (5') of the transferrin coding region. One polymorphism is in the 5' UTR at nt +49, and four are in the promoter region at nt -34, -551, -617, and -739, numbering from the start of transcription. The -34 and -617 SNPs are tightly but not completely linked. The -34 polymorphism lies between a conserved Sp1 site and the TATA box. The -617 polymorphism is within the DRII enhancer region. Five haplotypes have been defined from these SNPs by the identification of at least one homozygous individual, and two other haplotypes were deduced from heterozygous individuals. The total iron-binding capacity associated with each transferrin haplotype was haplotype 2 > 1 > 4 > 3. Transferrin promoter haplotype 2 had a significantly higher mean TIBC and haplotype 3 had a significantly lower mean TIBC than the more common haplotype 1. Persons with haplotype 4, which includes the -34T and -617A minor alleles, have a lower mean TIBC but the difference was not statistically significant. In normal individuals, the differences in the haplotypes were not found to be associated with differences in transferrin saturation and ferritin levels. There was no difference in the extent of increase in the mean TIBC levels in individuals with iron deficiency anemia in regard to their haplotype. Furthermore, there was no difference in the relative frequencies of the transferrin haplotypes in the iron-deficient population. In hemochromatosis patients who were homozygous for the C282Y HFE mutation, no particular haplotype was associated with a significant difference in transferrin saturation or ferritin levels. In White patients with Parkinson's disease, a disorder in which there is abnormal iron deposition in the brain, the presence of transferrin haplotype 3 was in slight excess over the normal White population.

<h4>Objective</h4>To provide genetic evidence for understanding the mechanism of oncogenesis of intrahepatic cholangiocarcinoma (ICC) by detecting the multiple genetic spectrums.<h4>Methods</h4>Twenty-two cases of paraffin-embedded ICC tissue sections were microdissected and genotyped by PCR-based DNA sequencing to detect the frequencies of loss of heterozygosity (LOH) of tumor suppressor genes APC, MCC, DCC, OGG1, p53, and RB1 and of point mutation of Ki-ras-2 oncogene.<h4>Results</h4>The genetic alteration rate was 68.8% for APC, 46.2% for DCC, 41.7% for OGG1, 37.5% for p53, 27.3% for Ki-ras-2, 22.2% for RB1, and 14.3% for MCC.<h4>Conclusion</h4>ICC-related genetic spectrum is basically composed of APC, DCC, OGG1, p53, and Ki-ras-2. Multiple genetic alteration plays an important role in the carcinogenesis of ICC.

No abstract available.