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In stomach, Helicobacter pylori (Hp) adheres to gastric mucous epithelial cells (GMEC) and initiates several different signal transduction events. Alteration of intracellular Ca2+ concentration ([Ca2+]i) is an important signaling mechanism in numerous bacteria-host model systems. Changes in [Ca2+]i induced by Hp in normal human GMEC have not yet been described; therefore, we examined effects of Hp on [Ca2+]i in normal human GMEC and a nontransformed GMEC line (HFE-145). Cultured cells were grown on glass slides, porous filters, or 96-well plates and loaded with fura 2 or fluo 4. Hp wild-type strain 60190 and vacA-, cagA-, and picB-/cagE- isogenic mutants were incubated with cells. Changes in [Ca2+]i were recorded with a fluorimeter or fluorescence plate reader. Wild-type Hp produced dose-dependent biphasic transient [Ca2+]i peak and plateau changes in both cell lines. Hp vacA- isogenic mutant produced changes in [Ca2+]i similar to those produced by wild type. Compared with wild type, cagA- and picB-/cagE- isogenic mutants produced lower peak changes and did not generate a plateau change. Preloading cultures with intracellular Ca2+ chelator BAPTA blocked all Hp-induced [Ca2+]i changes. Thapsigargin pretreatment of cultures to release Ca2+ from internal stores reduced peak change. Extracellular Ca2+ removal reduced plateau response. Hp-induced peak response was sensitive to G proteins and PLC inhibitors. Hp-induced plateau change was sensitive to G protein inhibitors, src kinases, and PLA2. These findings are the first to show that H. pylori alters [Ca2+]i in normal GMEC through a Ca2+ release/influx mechanism that depends on expression of cagA and picB/cagE genes.

The condensin complex and topoisomerase II (topo II) have different biochemical activities in vitro, and both are required for mitotic chromosome condensation. We have used Xenopus egg extracts to investigate the functional interplay between condensin and topo II in chromosome condensation. When unreplicated chromatin is directly converted into chromosomes with single chromatids, the two proteins must function together, although they are independently targeted to chromosomes. In contrast, the requirement for topo II is temporarily separable from that of condensin when chromosome assembly is induced after DNA replication. This experimental setting allows us to find that, in the absence of condensin, topo II becomes enriched in an axial structure within uncondensed chromatin. Subsequent addition of condensin converts this structure into mitotic chromosomes in an ATP hydrolysis-dependent manner. Strikingly, preventing DNA replication by the addition of geminin or aphidicolin disturbs the formation of topo II-containing axes and alters the binding property of topo II with chromatin. Our results suggest that topo II plays an important role in an early stage of chromosome condensation, and that this function of topo II is tightly coupled with prior DNA replication.

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine (polyQ) expansion in the protein huntingtin (htt). Pathogenesis in HD seems to involve the formation of neuronal intranuclear inclusions and the abnormal regulation of transcription and signal transduction. To identify previously uncharacterized htt-interacting proteins in a simple model system, we used a yeast two-hybrid screen with a Caenorhabditis elegans activation domain library. We found a predicted SH3 domain protein (K08E3.3b) that interacts with N-terminal htt in two-hybrid tests. A human homolog of K08E3.3b is the Cdc42-interacting protein 4 (CIP4), a protein involved in Cdc42 and Wiskott-Aldrich syndrome protein-dependent signal transduction. CIP4 interacted in vitro with full-length htt from lymphoblastoid cells. Neuronal CIP4 immunoreactivity increased with neuropathological severity in the neostriatum of HD patients and partially colocalized to ubiquitin-positive aggregates. Marked CIP4 overexpression also was observed in Western blot from human HD brain striatum. The overexpression of CIP4 induced the death of striatal neurons. Our data suggest that CIP4 accumulation and cellular toxicity may have a role in HD pathogenesis.

Despite extensive progress in determining structures within heparin and heparan sulfate (Hp/HS) and the discovery of numerous proteinaceous binding partners for Hp/HS so far; the only detailed characterization of a specific protein-glycosaminoglycan interaction is antithrombin III (ATIII) binding to a Hp pentasaccharide containing a unique 3-O-sulfated glucosamine residue. Previously, it was reported from our laboratories that a 16 amino acid synthetic peptide derived from the C-terminus of human HIP/RPL29 (HIP peptide-1) enriched for ATIII-dependent anticoagulant activity, presumably by specifically binding the ATIII pentasaccharide. Herein, we demonstrate that HIP peptide-1 cannot enrich ATIII-dependent anticoagulant activity from a starting pool of porcine intestinal mucosa Hp through a bio-specific interaction. However, a HIP peptide-1 column can be used to enrich for anticoagulantly active Hp from a diverse pool of glycosaminoglycans known as Hp byproducts by a mechanism of nonspecific charge interactions. Thus, HIP peptide-1 cannot recognize Hp via bio-specific interactions but binds glycosaminoglycans by non-specific charge interactions.

Fast cholinergic neurotransmission between superior cervical ganglion neurons (SCGNs) in cell culture is initiated by N-type Ca(2+) currents through Ca(v)2.2 channels. To test the ability of different Ca(2+)-channel subtypes to initiate synaptic transmission in these cells, SCGNs were injected with cDNAs encoding Ca(v)1.2 channels, which conduct L-type currents, Ca(v)2.1 channels, which conduct P/Q-type Ca(2+) currents, and Ca(v)2.3 channels, which conduct R-type Ca(2+) currents. Exogenously expressed Ca(v)2.1 channels were localized in nerve terminals, as assessed by immunocytochemistry with subtype-specific antibodies, and these channels effectively initiated synaptic transmission. Injection with cDNA encoding Ca(v)2.3 channels yielded a lower level of presynaptic labeling and synaptic transmission, whereas injection with cDNA encoding Ca(v)1.2 channels resulted in no presynaptic labeling and no synaptic transmission. Our results show that exogenously expressed Ca(2+) channels can mediate synaptic transmission in SCGNs and that the specificity of reconstitution of neurotransmission (Ca(v)2.1 > Ca(v)2.3 >> Ca(v)1.2) follows the same order as in neurons in vivo. The specificity of reconstitution of neurotransmission parallels the specificity of trafficking of these Ca(v) channels to nerve terminals.

<h4>Rationale</h4>Although many studies have focused on the mechanisms underlying MDMA-induced neurotoxicity, little is known about the subsequent long-term response to psychostimulants following exposure to a neurotoxic dose of MDMA.<h4>Objectives</h4>We investigated the effect of pre-exposure to neurotoxic and non-neurotoxic doses of MDMA on the response of mice to the psychomotor stimulating effects of MDMA and cocaine.<h4>Methods</h4>To investigate MDMA-induced neurotoxicity, male Swiss Webster mice were subjected to three regimens of MDMA: i) 40 mg/kg x 2, ii) 30 mg/kg x 2, and iii) 15 mg/kg x 2 for 2 days. On day 5 following the last exposure to MDMA, the levels of dopaminergic and serotonergic markers were determined. For the behavioral experiments, mice received either a single injection of 10 mg/kg MDMA [MDMA(L)] or one of the following doses of MDMA: 30 mg/kg x 2 or 15 mg/kg x 2 for 2 days [MDMA (H)]. A third group received saline as a control. On day 5 after the last pretreatment injection, the first MDMA (10 mg/kg) challenge was given, and on day 12, cocaine (20 mg/kg) was administered. Subsequently, mice were re-challenged with MDMA on days 35, 50 and 80, after which locomotor activity was monitored by infrared beam-interrupts. On day 83, mice were killed to detect the levels of dopaminergic and serotonergic markers.<h4>Results</h4>MDMA-induced mortality and depletion of dopaminergic and serotonergic markers were dose-dependent. MDMA (H) mice endured a sensitized response to MDMA challenge from days 5 through 80, e.g. a persistent 3-fold increase in locomotor activity compared to the response of mice that were not pretreated with a neurotoxic dose of MDMA. The depletion of DAT and 5-HTT binding sites was sustained throughout this time period (64-68% of control). The MDMA (L) mice showed a sensitized response to MDMA only on day 5. Both MDMA (L) and MDMA (H) mice were sensitized to the cocaine challenge.<h4>Conclusions</h4>The induction of sensitization to the locomotor stimulating effects of MDMA and cocaine was independent of MDMA-induced neurotoxicity. However, the long-lasting maintenance of the sensitized response to MDMA may be related to the enduring neurotoxicity caused by MDMA.

In addition to neurological deficits, Huntington's disease (HD) patients and transgenic mice expressing mutant human huntingtin exhibit reduced levels of brain-derived neurotrophic factor, hyperglycemia, and tissue wasting. We show that the progression of neuropathological (formation of huntingtin inclusions and apoptotic protease activation), behavioral (motor dysfunction), and metabolic (glucose intolerance and tissue wasting) abnormalities in huntingtin mutant mice, an animal model of HD, are retarded when the mice are maintained on a dietary restriction (DR) feeding regimen resulting in an extension of their life span. DR increases levels of brain-derived neurotrophic factor and the protein chaperone heat-shock protein-70 in the striatum and cortex, which are depleted in HD mice fed a normal diet. The suppression of the pathogenic processes by DR in HD mice suggests that mutant huntingtin promotes neuronal degeneration by impairing cellular stress resistance, and that the body wasting in HD is driven by the neurodegenerative process. Our findings suggest a dietary intervention that may suppress the disease process and increase the life span of humans that carry the mutant huntingtin gene.

Biochemical iron overload (IO) is a frequent metabolic abnormality. It may be caused by several diseases, and data regarding the relative frequency of these are scant. A single diagnostic protocol including clinical, biochemical, and genetic data was used to diagnose the cause of biochemical IO in a group of 150 patients referred by general practitioners. Severe alterations of the HFE gene (42 patients, 28%), hepatitis C virus infection (33 patients, 22%), and dysmetabolic syndrome with iron overload (DSIO) (22 patients, 15%) emerged as the main causes, and other single causes were found in 20 patients (13%). In 19 patients (13%), multiple causes of IO were found, and in 14 patients no cause was found, 5 of whom had classical criteria of genetic hemochromatosis (GH) without HFE mutations. Transferrin saturation index (TSI) had a very low positive predictive value (0.16) for GH among patients with biochemical IO in this setting. In conclusion, 90% of patients with biochemical IO were diagnosed with a specific disorder. GH, hepatitis C infection, and DSIO were the major causes, and a large group of patients had multiple causes of IO. TSI is not a useful indicator of GH in patients referred by general practitioners.

Like other guanine nucleotide-exchange proteins (GEPs) that activate ADP-ribosylation factor (ARF) GTPases, brefeldin A-inhibited GEP2, BIG2, contains an approximately 200-aa Sec7 domain that is responsible for this catalytic activity and its inhibition by brefeldin A. The Sec7 domain is located near the center of the molecule and serves to accelerate replacement of GDP bound to ARF with GTP. To explore possible functions of the N-terminal region of BIG2 (1-832), we used three coding-region constructs as bait to screen a human heart cDNA library in a yeast two-hybrid system, retrieving two unique clones that encode a type I protein kinase A (PKA) regulatory subunit, RI alpha. Coimmunoprecipitation experiments confirmed interaction of in vitro translated BIG2 and RI alpha, as well as of the endogenous proteins in cytosol of cultured HepG2 cells. Using 28 deletion mutants, we found three regions of BIG2 that interacted with R subunits of PKA. Residues 27-48 (domain A) interacted with RI alpha and RI beta, 284-301 (domain B) interacted with RII alpha and RII beta, and 517-538 (domain C) interacted with RI alpha, RII alpha, and RII beta. Sequence analysis and helical wheel projection of amino acids in the three domains revealed potential amphipathic wheel structures characteristic for binding of PKA R subunits. Western blot analysis of subcellular fractions demonstrated translocation of BIG2 (and BIG1) from cytosol to the Golgi and other membrane structures after incubation of cells with 8-Br-cAMP or forskolin. All findings are consistent with a role for BIG2 as an A kinase-anchoring protein (or AKAP) that could coordinate cAMP and ARF regulatory pathways.

The germ cell-deficient (gcd) mouse mutation is a recessive, transgenic insertional mutation associated with the disruption of two Chr11 genes, Pog (proliferation of germ cells) and Vrk2 (vaccinia virus-related protein kinase 2). We have recently shown that like gcd/gcd mice, targeted Pog-/- males and females show virtually no spermatogenesis or oogenesis at 4-6 wk of age. Because Pog is deleted in gcd/gcd and Pog-/- mice, a comparison of the phenotypes of the two mouse models is appropriate. Here, we report that unlike in POG-deficient females, the germ cells in POG-deficient males eventually populate the seminiferous tubules at 9 wk, and fertility can be achieved by 12 wk. Homozygous gcd/gcd males did not show a similar degree of germ cell population, and most gcd/gcd males remained infertile at 16 and 22 wk of age. A comparison of the degree of germ cell deficiency at 13.5 days postcoitum and 1 day postpartum between Pog-/- and gcd/gcd males revealed that gcd/gcd males had far fewer germ cells than Pog-/- males at both time points. Our data suggest that Pog is essential for proper primordial germ cell proliferation in the embryonic stage but is not needed for spermatogonial proliferation after birth. Thus, the difference in the spermatogenetic potential in adult Pog-/- and gcd/gcd mice may result from the severity of germ cell deficiency rather than from the inability of gcd/gcd spermatogonia to proliferate efficiently. The greater deficiency of germ cells before the onset of spermatogenesis seen in gcd/gcd males compared to Pog-/- mice suggests either that the different background affects the outcome of Pog deletion or that Vrk2 has additional effects on germ cell development.

Recently, it has been shown that mammalian PEBPs are implicated in several signalling pathways controlling the cellular cycle. In particular, during brain development, the N-terminal part of mammalian PEBP is specifically cleaved and the resulting 11 amino acid peptide stimulates the growth and activity of acetylcholinergic neurons. The crystallographic structure of bovine and human PEBPs has revealed that their N- and C-terminal parts are accessible and exposed to the solvent suggesting that they may be involved in specific interactions with cellular partners. We have chemically synthetized the two peptides corresponding to these terminal parts and studied their structure in solution by circular dichroism and NMR spectroscopies: both of them are well-structured. The N-terminal peptide is composed of a series of turns, leading to a hook conformation. The C-terminal peptide displays a globally helical conformation similar to that observed in the whole protein; it is characterized by an amphipatic feature with a hydrophobic cluster located on one side. These structural features enlighten previous fluorescence and monolayer experiments and give new insights on the roles of both PEBP termini.

Densin-180 is a brain-specific synaptic protein of the o-sialoglycoprotein family. It functions in specific adhesion between presynaptic and postsynaptic membranes at glutamatergic synapses. We have cloned a novel splice variant of densin-180 from a fetal brain cDNA library and termed it LRRC7. It is located on 1p31 and consists of 8 exons. It encodes a putative protein of 216 amino acids which contains a nuclear localization signal and four leucine-rich repeats (LRRs). LRRs are 20-29 residue sequence motifs present in a number of proteins with diverse functions. Expression analysis of LRRC7 shows it is expressed ubiquitously while its splice variant densin-180 is brain-specific.

Nonsense-mediated mRNA decay (NMD) is a conserved proofreading mechanism that protects eukaryotic cells from the potentially deleterious effects of truncated proteins. Studies of Saccharomyces cerevisiae imply that NMD is a predominantly cytoplasmic decay pathway, while studies of mammalian systems suggest that decay of most substrate mRNAs may occur while they are still associated with the nucleus, possibly during a round of translation that occurs during their export to the cytoplasm. Complete entry of the latter mRNAs into the cytoplasm appears to render them immune to further NMD; i.e., they escape further susceptibility to this decay pathway. To determine if yeast cytoplasmic nonsense-containing mRNAs that evade decay are subsequently immune to NMD, we examined the consequences of placing each of the three UPF/NMD genes under the control of a galactose-inducible promoter. The decay kinetics of ADE2 and PGK1 nonsense-containing mRNAs were then analyzed when expression of UPF1, NMD2, or UPF3 was either repressed or subsequently induced. Results from these experiments demonstrated that activation of NMD caused rapid and immediate degradation of both substrate transcripts, with half-lives of both stable mRNA populations shortened to approximately 7 min. These findings make it unlikely that yeast nonsense-containing mRNAs can escape degradation by NMD and indicate that such mRNAs are available to this decay pathway at each round of translation.

Nonalcoholic fatty liver disease (NAFLD) has emerged as a ubiquitous liver disorder with occasional serious overtones. Although diabetes and obesity were initially held culpable, insulin resistance (IR) is now considered the fundamental operative mechanism. IR is probably the "first step" in nonalcoholic steatohepatitis (NASH). Oxidative stress may be the elusive "second" of possibly multiple steps in the progression of steatosis to fibrosing steatohepatitis. Because hepatic iron promotes oxidative stress, it was mooted as a contributory cofactor in NASH. This proposal was strengthened by an association with hepatic fibrosis. Subsequent studies have shown neither a significant increase in hepatic iron nor an association between hepatic iron and any of the histologic determinants in NASH. Likewise, the increased prevalence of hemochromatosis gene (HFE) mutations in some studies appears to be largely irrelevant to the development of hepatic fibrosis. Excess hepatic iron may occur in insulin resistance-associated iron overload (IRHIO), characterized by hyperferritinemia with normal to mild increases in transferrin saturation. Although patients with IRHIO have a high prevalence of IR-related metabolic disorders, the relationship of IRHIO to NASH is unclear. A recent study showed improvement in insulin sensitivity with the use of venesection in patients with NAFLD, but this approach cannot be implemented without extensive review.

Huntingtin is the protein involved in Huntington disease (HD), an inherited neurodegenerative disease. Research activities have focused on the abnormal functions of mutant huntingtin. However, recent results indicate that wild-type huntingtin has important activities in brain neurons, suggesting that loss of these activities may play a role in HD.

Two major risk factors for the development of porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). To develop an animal model, Hfe knockout mice were treated continuously with 10% ethanol in drinking water. By 4 months, uroporphyrin (URO) was detected in the urine. At 6 to 7 months, hepatic URO was increased and hepatic uroporphyrinogen decarboxylase (UROD) activity was decreased. Untreated Hfe(-/-) mice or wild-type mice treated with or without ethanol did not show any of these biochemical changes. Treatment with ethanol increased hepatic nonheme iron and hepatic 5-aminolevulinate synthase activity in Hfe(-/-) but not wild-type mice. The increases in nonheme iron in Hfe(-/-) mice were associated with diffuse increases in iron staining of parenchymal cells but without evidence of significant liver injury. In conclusion, the results of this study suggest that the uroporphyrinogenic effect of ethanol is mediated by its effects on hepatic iron metabolism. Ethanol-treated Hfe(-/-) mice seem to be an excellent model for studies of alcohol-mediated PCT.

<h4>Objective</h4>To determine the optimal means of identifying patients with undiagnosed haemochromatosis.<h4>Design</h4>Case-control study where cases are defined by the presence of specific clinical diagnoses or symptoms.<h4>Setting</h4>Primary care patients were recruited from three Oxfordshire practices and secondary care patients were recruited from those patients attending specialist clinics in Amiens University Hospital.<h4>Subjects</h4>A total of 569 patients recruited via hospital clinics and 60 primary care patients (recruited from 4022 consultations) presenting with the following haemochromatosis associated conditions, diabetes, arthralgia/chronic fatigue, osteoporosis or arthropathy were studied. The control group, a total of 991 healthy volunteers, were recruited through a Health Appraisal Centre. Patients and controls were included in the study if they or their family members had not previously been diagnosed with hereditary haemochromatosis.<h4>Main outcome measures</h4>Serum ferritin concentration, transferrin saturation (Tsat) and presence of HFE mutations, C282Y and H63D. The check-up in controls consisted of a questionnaire, clinical examination, biochemical tests and screening for the presence of the C282Y and H63D mutations.<h4>Results</h4>Patient groups presenting with unstable diabetes or chronic fatigue and arthralgia together with a raised serum ferritin concentration showed an enrichment in the haemochromatosis-associated genotype HH/YY, odds ratio (OR) = 40.1, confidence interval (CI) = 8.0-202.1 and OR = 103, CI = 22.9-469.7, respectively.<h4>Conclusion</h4>Patients presenting to hospital clinics with haemochromatosis associated conditions should be screened biochemically for iron overload. Only those with a serum ferritin >300 microg L-1 or Tsat >40% should subsequently go on to be genotyped for HFE mutations. The patients at greatest risk of having undiagnosed haemochromatosis are those presenting with unstable diabetes, or fatigue and/or arthralgia in the absence of any other explanation.

The car and ant operons originally isolated from Pseudomonas resinovorans strain CA10 contain the genes encoding the carbazole/dioxin-degrading enzymes and anthranilate 1,2-dioxygenase, respectively, and are located on the plasmid pCAR1. The entire nucleotide sequence of pCAR1 was determined to elucidate the mechanism by which the car operon may have been assembled and distributed in nature. pCAR1 is a 199,035-bp circular plasmid, and carries 190 open reading frames. Although the incompatibility group of pCAR1 is unclear, its potential origin for replication, OriP, and Rep and Par proteins appeared to be closely related to those of plasmid pL6.5 isolated from Pseudomonas fluorescens. The potential tellurite-resistance klaABC genes identified in the neighboring region of repA gene were also related to those in IncP plasmid originally identified from pseudomonads. On the other hand, we found genes encoding proteins that showed low but significant homology (20-45% identity) with Trh and Tra proteins from Enterobacteriaceae, which are potentially involved in conjugative transfer of plasmids or genomic island, suggesting that pCAR1 is also a conjugative plasmid. In pCAR1, we found tnpAcCST genes that encoded the proteins showing >70% length-wise identities with those are encoded by the toluene/xylene-degrading transposon Tn4651 of TOL plasmid pWW0. Both car and ant degradative operons were found within a 72.8-kb Tn4676 sequence defined by flanking tnpAcC and tnpST genes and bordered by a 46-bp inverted repeat (IR). Within Tn4676 and its flanking region, we found the remnants of numerous mobile genetic elements, such as the duplicated transposase genes that are highly homologous to tnpR of Tn4653 and the multiple candidates of IRs for Tn4676 and Tn4653-like element. We also found distinct regions with high and low G+C contents within Tn4676, which contain an ant operon and car operon, respectively. These results suggested that multiple step assembly could have taken place before the current structure of Tn4676 had been captured.

The growth of patents that include genetic sequences has been accompanied by concern about their impact on the ability of physicians to provide clinical genetic testing services and to perform research. Therefore, we conducted a survey of clinical laboratory directors that perform DNA-based genetic tests to examine potential effects. We performed a telephone survey between July and September in 2001 of all laboratory directors in the United States who were members of the Association for Molecular Pathology or who were listed on the GENETESTS:org website. One hundred thirty-two of 211 (63%) laboratory directors were interviewed. Ten of these were excluded because they did not conduct DNA-based genetic tests. Almost all performed genetic tests for clinical purposes. Half performed tests for research purposes as well. Twenty-five percent of respondents reported that they had stopped performing a clinical genetic test because of a patent or license. Fifty-three percent of respondents reported deciding not to develop a new clinical genetic test because of a patent or license. In total, respondents were prevented from performing 12 genetic tests, and all of these tests were among those performed by a large number of laboratories. We found 22 patents that were relevant to the performance of these 12 tests. Fifteen of the 22 patents (68%) are held by universities or research institutes, and 13 of the 22 patents (59%) were based on research funded by the United States Government. Overall, respondents reported that their perceptions of the effects of patents on the cost, access, and development of genetic tests, or data sharing among researchers, were negative. In contrast, most respondents felt that patents did not have an effect on the quality of testing. We conclude that patents and licenses have had a significant effect on the ability of clinical laboratories to develop and provide genetic tests. Furthermore, our findings suggest that clinical geneticists feel that their research is inhibited by patents. The effects of patents and licenses on patients' access to tests, and the costs and quality thereof, remains to be determined.

Minichromosomes assembled on the mouse mammary tumor virus (MMTV) promoter in vitro exhibit positioned nucleosomes, one of which covers the binding sites for progesterone receptor (PR) and nuclear factor 1 (NF1). Incorporation of histone H1 into MMTV minichromosomes improves the stability of this nucleosome and decreases basal transcription from the MMTV promoter, as well as its response to either PR or NF1. However, histone H1-containing minichromosomes display better PR binding and support a more efficient synergism between PR and NF1, leading to enhanced transcription initiation. A mutant MMTV promoter lacking positioned nucleosomes does not display enhanced transcriptional synergism in the presence of H1. Binding of PR leads to phosphorylation of H1, which leaves the promoter upon transcription initiation. Thus, H1 assumes a complex and dynamic role in the regulation of the MMTV promoter.

Somatic instability of expanded HD CAG repeats that encode the polyglutamine tract in mutant huntingtin has been implicated in the striatal selectivity of Huntington's disease (HD) pathology. Here in Hdh(Q111) mice, we have tested whether a genetic background deficient in Msh2, expected to eliminate the unstable behavior of the 109 CAG array inserted into the murine HD gene, would alter the timing or striatal specificity of a dominant disease phenotype that predicts late-onset neurodegeneration. Our analyses of Hdh(Q111/+):Msh2(+/+) and Hdh(Q111/+): Msh2(-/-) progeny revealed that, while inherited instability involved Msh2-dependent and -independent mechanisms, lack of Msh2 was sufficient to abrogate progressive HD CAG repeat expansion in striatum. The absence of Msh2 also eliminated striatal mutant huntingtin with somatically expanded glutamine tracts and caused an approximately 5 month delay in nuclear mutant protein accumulation, but did not alter the striatal specificity of this early phenotype. Thus, somatic HD CAG instability appears to be a consequence of a striatal-selective disease process that accelerates the timing of an early disease phenotype, via expansion of the glutamine tract in mutant huntingtin. Therefore Msh2, as a striking modifier of early disease onset in a precise genetic HD mouse model, provides a novel target for the development of pharmacological agents that aim to slow pathogenesis in man.

<h4>Background</h4>The aim of the present study was to identify the clinicopathological and molecular biological characteristics of early-onset colorectal cancers.<h4>Methods</h4>The clinicopathological and molecular biological parameters of 138 consecutive patients with colorectal cancer aged less than 40 years were compared with those of 339 patients aged 60 years or more.<h4>Results</h4>The younger patients with colorectal cancer had more mucin-producing (14.5 versus 4.7 per cent; P < 0.001) and poorly differentiated (7.2 versus 3.3 per cent; P = 0.015) tumours, a higher incidence of synchronous (5.8 versus 1.2 per cent; P = 0.007) and metachronous (4.0 versus 0.6 per cent; P = 0.023) colorectal cancers, and more advanced tumour stage (P < 0.001) than older patients. The operative mortality rate was lower (0.7 versus 5.0 per cent; P = 0.026), and cancer-specific survival was similar (in stage I, II and III disease; P > 0.05) or better (in stage IV disease; 95 per cent confidence interval 22.50 to 28.41 versus 12.61 to 17.05 months; P < 0.001). There was a higher percentage of normal p53 expression (61.1 versus 46.8 per cent; P = 0.023) and high-frequency microsatellite instability (MSI-H) (29.4 versus 6.3 per cent; P < 0.001), and a similar family history of cancer (17.5 versus 14.2 per cent; P > 0.05), compared with older patients.<h4>Conclusion</h4>Young patients with colorectal cancer have several distinct clinicopathological and molecular biological features. The mechanisms underlying the inconsistency between the presence of MSI-H and a family history of cancer in these early-onset colorectal cancers deserve further investigation.

<h4>Background & aims</h4>Mild to moderate hepatic iron loading is common in patients with chronic hepatitis C. We sought to determine whether mutations in the hemochromatosis gene, HFE, are associated with iron overload and acceleration of disease progression in hepatitis C patients.<h4>Methods</h4>A total of 316 patients with chronic hepatitis C were studied: 198 consecutive patients undergoing liver biopsy for compensated liver disease and 118 who underwent liver transplantation for end-stage liver disease. Serum iron studies, quantitative hepatic iron concentration, histologic activity index, and HFE genotype were determined.<h4>Results</h4>Among patients with compensated liver disease, the presence of HFE mutations was independently associated with elevations in serum iron level, serum transferrin-iron saturation, serum ferritin level, and hepatic iron index (P < 0.05). After adjustment for duration of infection with hepatitis C virus, HFE mutations were also independently associated with the presence of bridging fibrosis or cirrhosis (odds ratio, 18; 95% confidence interval, 1.7-193). HFE mutations were not independently associated with iron loading in patients with end-stage liver disease. There was no significant difference in the prevalence of HFE mutations between patients with compensated and end-stage liver disease (42% vs. 33%, respectively; P = 0.67).<h4>Conclusions</h4>The presence of HFE mutations is independently associated with iron loading and advanced fibrosis in patients with compensated liver disease from chronic hepatitis C, especially after controlling for duration of disease. These results suggest that HFE mutations accelerate hepatic fibrosis in hepatitis C but may not be responsible for progression to end-stage liver disease.

The mechanisms by which neurons die in CAG triplet repeat (polyglutamine) disorders, such as Huntington's disease, are uncertain; however, mitochondrial dysfunction and disordered calcium homeostasis have been implicated. We previously demonstrated abnormal mitochondrial calcium handling in Huntington's disease cell lines and transgenic mice. To examine whether these abnormalities might arise in part from direct effects of the expanded polyglutamine tract contained in mutant huntingtin, we have exposed normal rat liver and human lymphoblast mitochondria to glutathione S-transferase fusion proteins containing polyglutamine tracts of 0, 19, or 62 residues. Similar to bovine serum albumin, each of the protein constructs nonspecifically inhibited succinate-supported respiration, independent of polyglutamine tract length. There was a small but significant reduction of mitochondrial membrane potential (state 4) only in the presence of the pathological-length polyglutamine tract. With successive addition of small Ca(2+) aliquots, mitochondria exposed to pathological-length polyglutamine tracts (approximately 5 microM) depolarized much earlier and to a greater extent than those exposed to the other protein constructs. These results suggest that the mitochondrial calcium handling defects seen in Huntington's disease cell lines and transgenic mice may be due, in part, to direct, deleterious effects of mutant huntingtin on mitochondria.

Structural genetic alterations in cancer often involve gene loss or gene amplification. With the advent of microarray approaches for the analysis of the genome, as exemplified by array-CGH (Comparative Genomic Hybridization), scanning for gene-dosage alterations is limited only by issues of DNA microarray density. However, samples of interest to the pathologist often comprise small clusters of just a few hundred cells, which do not provide sufficient DNA for array-CGH analysis. We sought to develop a simple method that would permit amplification of the whole genome without the use of thermocycling or ligation of DNA adaptors, because such a method would lend itself to the automated processing of a large number of tissue samples. We describe a method that permits the isothermal amplification of genomic DNA with high fidelity and limited sequence representation bias. The method is based on strand displacement reactions that propagate by a hyperbranching mechanism, and generate hundreds, or even thousands, of copies of the genome in a few hours. Using whole genome isothermal amplification, in combination with comparative genomic hybridization on cDNA microarrays, we demonstrate the ability to detect gene losses in yeast and gene dosage imbalances in human breast tumor cell lines. Although sequence representation bias in the amplified DNA presents potential problems for CGH analysis, these problems have been overcome by using amplified DNA in both control and tester samples. Gene-dosage alterations of threefold or more can be observed with high reproducibility with as few as 1000 cells of starting material.

The inverse relationship between serum albumin concentration and its half-life suggested to early workers that albumin would be protected from a catabolic fate by a receptor-mediated mechanism much like that proposed for IgG. We show here that albumin binds FcRn in a pH dependent fashion, that the lifespan of albumin is shortened in FcRn-deficient mice, and that the plasma albumin concentration of FcRn-deficient mice is less than half that of wild-type mice. These results affirm the hypothesis that the major histocompatibility complex-related Fc receptor protects albumin from degradation just as it does IgG, prolonging the half-lives of both.

No abstract available.

Smad4 is a candidate tumour-suppressor gene identified recently on chromosome 18q21.1. Both alleles are inactivated in nearly one-half of pancreatic carcinomas, but its role in the tumorigenesis of other tumours is still unknown. The aim of this study was to investigate the potential involvement of the Smad4 locus in early-stage colorectal cancers (stages I-III) in tumour samples from a randomised multicentre trial. Of a large collection of DNA samples, 73 with a loss of one allele of the Smad4 gene were analysed for the presence of point mutations in the remaining gene. Patients, from whom biopsies were isolated, were part of a previous randomised multicentre study of the Swiss Group for Clinical Cancer Research on the benefit of adjuvant chemotherapy (SAKK study 40/81). Mutation analysis was restricted to the highly conserved C-terminal domain (exons 8, 9, 10 and 11) of Smad4, using PCR and single-strand conformational variant analysis. Two of the 73 patients (3%) with loss of one allele of Smad4 had a point mutation in the remaining allele. These results indicate that whereas Smad4 point mutations are prevalent in pancreatic carcinoma, they are infrequent in early stages (I-III) of colorectal cancer.

SR31747A is a recently described sigma receptor ligand that binds SR31747A-binding protein 1 (SR-BP) and emopamil-binding protein (EBP) (also called the sigma 1 receptor and the human sterol isomerase (HSI), respectively), and has immunoregulatory and antiproliferative activities. To further investigate its antitumour activity and focusing on cancers, which are sensitive to the molecule, we measured the proliferation of different human epithelial breast or prostate cancer cell lines following in vitro and in vivo SR31747A treatment. Firstly, in vitro, we found that nanomolar concentrations of SR31747A dramatically inhibited cell proliferation in both hormono-responsive and -unresponsive cancer cell lines. Secondly, tumour development was significantly decreased in mice treated with SR31747A. In an attempt to decipher the SR31747A mode of action, we found that the two binding sites may not fully account for this activity. Indeed, while competitive experiments indicated that EBP prevails in mediating SR31747A antiproliferative activity, an analysis of the expression of both receptors indicated that the cellular sensitivity to SR31747A is not correlated with either EBP or SR-BP expression. These data suggest that additional binding sites may exist. Preliminary binding studies demonstrated that SR31747A also binds to sigma 2, a protein that has not yet been cloned, but which is considered as a potential marker of the proliferative status of tumour cells. Altogether, our data demonstrate the antitumoural activity of SR31747A both in vitro and in vivo in two different cancer models, broaden the spectrum of its binding proteins and enhance the potential for further therapeutic development of the molecule.

<h4>Purpose</h4>Adjuvant therapy, either preoperatively or postoperatively, and modifications of surgery have been used to try to improve outcome of surgery for rectal cancer in regard to both local recurrence and survival. Assessment of prognosis in patients after resection is currently primarily based on clinicopathologic factors. These predict the subsequent behavior of the tumor only imperfectly. The aim of this study was to evaluate three potential molecular genetic markers of prognosis (p53, deleted in colorectal cancer gene, and thymidylate synthase) in Dukes Stage B and C low rectal tumors treated with adjuvant therapy and to determine whether they correlate with survival, local recurrence, or the pathologic response to adjuvant therapy (assessed by extent of tumor regression and tumor down-staging).<h4>Methods</h4>Sixty locally advanced low rectal tumors resected after preoperative chemoradiotherapy or radiotherapy alone were studied by immunohistochemical staining for p53, deleted in colorectal cancer gene, and thymidylate synthase. In addition, p53 gene mutations were sought by polymerase chain reaction-single-strand conformation polymorphism analysis. These results were correlated with survival, local recurrence, and pathologic response to adjuvant therapy.<h4>Results</h4>Lack of thymidylate synthase staining by immunohistochemistry was associated with tumor down-staging after preoperative chemoradiotherapy but not after radiotherapy or for these two combined groups. There was no correlation between p53, deleted in colorectal cancer gene, or thymidylate synthase immunohistochemical staining or between p53 polymerase chain reaction-single-strand conformation polymorphism and local recurrence or survival in locally advanced low rectal cancers treated with preoperative adjuvant therapies.<h4>Conclusion</h4>Prediction of prognosis in patients with locally advanced low rectal cancers treated with preoperative adjuvant therapies continues to be problematic. Thymidylate synthase immunohistochemistry appears to be the most promising factor of those assessed in predicting tumor down-staging after preoperative chemoradiotherapy for locally advanced low rectal cancers.

Female Fischer 344 (F344) rats have been shown to display increased serotonin transporter (5-HTT) gene expression in the dorsal raphe, compared to female Lewis (LEW) rats. Herein, we explored, by means of synaptosomal preparations and in vivo microdialysis, whether central, but also peripheral, 5-HTT protein expression/function differ between strains. Midbrain and hippocampal [3H]paroxetine binding at the 5-HTT and hippocampal [3H]serotonin (5-HT) reuptake were increased in male and female F344 rats, compared to their LEW counterparts, these strain differences being observed both in rats of commercial origin and in homebred rats. Moreover, in homebred rats, it was found that these strain differences extended to blood platelet 5-HTT protein expression and function. Saturation studies of midbrain and hippocampal [3H]paroxetine binding at the 5-HTT, and hippocampal and blood platelet [3H]5-HT reuptake, also revealed significant strain differences in Bmax and Vmax values. Although F344 and LEW rats differ in the activity of the hypothalamo-pituitary-adrenal (HPA) axis, manipulations of that axis revealed that the strain differences in hippocampal [3H]paroxetine binding at 5-HTTs and [3H]5-HT reuptake were not accounted for by corticosteroids. Hippocampal extracellular 5-HT levels were reduced in F344 rats, compared to LEW rats, with the relative, but not the absolute, increase in extracellular 5-HT elicited by the local administration of citalopram being larger in F344 rats. Because the aforementioned strain differences did not lie in the coding sequences of the 5-HTT gene, our results open the promising hypothesis that F344 and LEW strains model functional polymorphisms in the promoter region of the human 5-HTT gene.

We recently identified the Modifier of Min 2 (Mom2) locus. Mom2 is a new modifier of intestinal tumorigenesis that resulted from a spontaneous mutation in a B6 Apc(Min)/+ mouse. The presence of one resistant Mom2(R) allele results in a significant reduction in small intestinal polyp number and colon polyp incidence in Apc(Min)/+ mice. Through linkage analysis, we previously localized Mom2 to a 14-cM region on mouse Chromosome (Chr) 18, distal to the Apc gene. This region is syntenic with human Chr 18q, which frequently undergoes loss of heterozygosity (LOH) in several human cancers, including colorectal cancer. Residing in this region are the Madh2 and Madh4 genes, which have both been implicated in human colorectal cancer. Based on meiotic recombinations within the Mom2 region in the derivation of our congenic animals, we have narrowed the location of the Mom2 locus and excluded Madh2, Madh4, and Madh7, as well as Mbd1, Mbd2, Dcc, and Tcf4, as candidates for the Mom2 gene.

Several findings have revealed a likely role for DNA ligase IV, and interacting protein XRCC4, in the final steps of mammalian DNA double-strand break repair. Recent evidence suggests that the human DNA ligase IV protein plays a critical role in the maintenance of genomic stability. To identify protein-protein interactions that may shed further light on the molecular mechanisms of DSB repair and the biological roles of human DNA ligase IV, we have used the yeast two-hybrid system in conjunction with traditional biochemical methods. These efforts have resulted in the identification of a physical association between the DNA ligase IV polypeptide and the human condensin subunit known as hCAP-E. The hCAP-E polypeptide, a member of the Structural Maintenance of Chromosomes (SMC) super-family of proteins, coimmunoprecipitates from cell extracts with DNA ligase IV. Immunofluorescence studies reveal colocalization of DNA ligase IV and hCAP-E in the interphase nucleus, whereas mitotic cells display colocalization of both polypeptides on mitotic chromosomes. Strikingly, the XRCC4 protein is excluded from the area of mitotic chromosomes, suggesting the formation of specialized DNA ligase IV complexes subject to cell cycle regulation. We discuss our findings in light of known and hypothesized roles for ligase IV and the condensin complex.

<h4>Objective</h4>Hepatocellular carcinoma (HCC) is a late consequence of severe liver disease. Patients with genetic hemochromatosis may be at risk for HCC, but limited information is available on the relationship of HCC and heterozygosity for the HFE gene mutations.<h4>Methods</h4>HFE mutations (C282Y and H63D) were assessed in 162 consecutive patients (131 men/31 women) with HCC. A total of 159 patients had cirrhosis. The most common etiologies of cirrhosis were chronic viral hepatitis (hepatitis C 39%, hepatitis B 9%) and alcoholic liver disease (36%).<h4>Results</h4>Five patients were C282Y homozygotes, four C282Y/H63D compound heterozygotes, and three H63D homozygotes. The C282Y and H63D allele frequencies in HCC were 8.3 (95% confidence limit = 5.3-11.3) and 11.1 (7.8-14.6), respectively, and not different from previously published data in healthy subjects or patients with chronic hepatitis C in Austria. Furthermore, there was no difference in the age at diagnosis in patients with or without HFE gene mutations. C282Y homozygotes had a 19-fold increased risk to develop HCC. In contrast, all other HFE allele constellations were not associated with such a risk.<h4>Conclusions</h4>Except for C282Y homozygotes, HFE gene mutations do not increase the risk to develop HCC in patients with cirrhosis.

Localized mRNAs are thought to be transported in defined particles to their final destination. These particles represent large protein complexes that may be involved in recognizing, transporting, and anchoring localized messages. Few components of these ribonucleoparticles, however, have been identified yet. We chose the strategy to biochemically enrich native RNA-protein complexes involved in RNA transport to identify the associated RNAs and proteins. Because Staufen proteins were implicated in intracellular RNA transport, we chose mammalian Staufen proteins as markers for the purification of RNA transport particles. Here, we present evidence that Staufen proteins exist in two different complexes: (i) distinct large, ribosome- and endoplasmic reticulum-containing granules preferentially found in the membrane pellets during differential centrifugation and (ii) smaller particles in the S100 from rat brain homogenates. On gel filtration of the S100, we identified soluble 670-kDa Staufen1-containing and 440-kDa Staufen2-containing particles. They do not cofractionate with ribosomes and endoplasmic reticulum but rather coenrich with kinesin heavy chain. Furthermore, the fractions containing the Staufen1 particles show a 15-fold enrichment of mRNAs compared with control fractions. Most importantly, these fractions are highly enriched in BC1, and, to a lesser extent, in the alpha-subunit of the Ca(2+)/calmodulin-dependent kinase II, two dendritically localized RNAs. Finally, both RNAs colocalize with Staufen1-hemagglutinin in particles in dendrites of transfected hippocampal neurons. We therefore propose that these Staufen1-containing particles may represent RNA transport intermediates that are in transit to their final destination within neurons.

<h4>Background</h4>Hereditary hemochromatosis (HH) is characterized by excess iron deposition. Two mutations in the HFE gene are associated with HH. Heterozygous carriers of HFE mutations are at higher risk of developing type 2 diabetes mellitus (T2DM). The aims of our project were to identify the frequency of C282Y and H63D mutations in a population from the Małopolska region of south-eastern Poland, and to search for an association of HFE mutations with T2DM.<h4>Material/methods</h4>We included 391 individuals in this study: 222 T2DM patients and 169 controls. Genotypes were determined by electrophoresis of the DNA digestion products from SnaBI and DpnII, respectively. Differences in distributions between the groups were then analyzed by the chi-squared test.<h4>Results</h4>The frequency of wild/C282Y alleles was 98.2%/1.8% in T2DM patients and 96.7%/3.2% in controls (p=0.19). The frequency of wild/H63D alleles was 85.6%/14.4% and 88.8%/11.2% (p= 0.19), respectively. The distribution of genotypes was not statistically different. However, in stratified analyses based on age of T2DM onset and gender, we observed a higher prevalence of wild/H63D and H63D/H63D genotypes among T2DM patients diagnosed at > 49 years of age, the mean age for the entire group (p=0.018), and among male T2DM individuals (p=0.005) than in controls.<h4>Conclusions</h4>The frequency of HH-associated mutations in this population from south-eastern Poland is similar to other Caucasians. We found no evidence for the association of the C282Y mutation with T2DM. The results do suggest, however, that the H63D mutation may play a role in the pathogenesis of late onset T2DM and in males in this Polish population.

GST pi (GSTP) is a member of the glutathione S-transferase (EC 2.5.1.18; GST) family of enzymes that catalyse the conjugation of electrophilic species with reduced glutathione and thus play an important role in the detoxification of electrophilic metabolites. Deletion of GSTP in mice has previously been shown to lead to enhanced susceptibility to chemical-induced skin carcinoma, consistent with its known metabolic functions. A decreased susceptibility to paracetamol hepatotoxicity has also been observed, which has not been fully explained. One possibility is that deletion of the GSTP gene locus results in compensatory changes in other proteins involved in defence against chemical stress. We have therefore used complementary protein expression profiling techniques to perform a systematic comparison of the protein expression profiles of livers from GSTP null and wild-type mice. Analysis of liver proteins by two-dimensional electrophoresis confirmed the absence of GSTP in null mice whereas GSTP represented 3-5% of soluble protein in livers from wild-type animals. There was a high degree of quantitative and qualitative similarity in other liver proteins between GSTP null and wild-type mice. There was no evidence that the absence of GSTP in null animals resulted in enhanced expression of other GST isoforms in the null mice (GST alpha, 1.48%, GST mu, 1.68% of resolved proteins) compared with the wild-type animals (GST alpha, 1.50%, GST mu, 1.40%). In contrast, some members of the thiol specific antioxidant family of proteins, notably antioxidant protein 2 and thioredoxin peroxidases, were expressed at a higher level in the GSTP null mouse livers. These changes presumably reflect the recently described role of GSTP in cell signalling and may underlie the protection against paracetamol toxicity seen in these animals.

This article represents the proceedings of a symposium at the 2002 meeting of the Research Society on Alcoholism in San Francisco, CA. It was organized by Mary-Anne Enoch and David Goldman and chaired by David Goldman. The presentations were (1) Two functional polymorphisms and their intermediate phenotypes in complex behaviors: COMT/executive cognition and anxiety and HTT/anxiety, by David Goldman; (2) Role of the EEG in determining genetic risk for alcoholism and anxiety disorders, by Mary-Anne Enoch; (3) The response to alcohol as an intermediate phenotype for alcoholism, by Marc A. Schuckit; and (4) Pharmacogenomic approaches to alcoholism treatment: toward a hypothesis, by Bankole A. Johnson.

<h4>Background</h4>The mechanisms responsible for disturbed iron homoeostasis in hereditary haemochromatosis are poorly understood. However, results of some studies indicate a link between hepcidin, a liver-derived peptide, and intestinal iron absorption, suggesting that this molecule could play a part in hepatic iron overload. To investigate this possible association, we studied the hepatic expression of the gene for hepcidin (HAMP) and a gene important in iron transport (IREG1) in patients with haemochromatosis, in normal controls, and in Hfe-knockout mice.<h4>Methods</h4>We extracted total RNA from the liver tissue of 27 patients with HFE-associated haemochromatosis, seven transplant donors (controls), and Hfe-knockout mice. HAMP and IREG1 mRNA concentrations were examined by ribonuclease protection assays and expressed relative to the housekeeping gene GAPD.<h4>Findings</h4>There was a significant decrease in HAMP expression in untreated patients compared with controls (5.4-fold, 95% CI 3.3-7.5; p<0.0001) despite significantly increased iron loading. Similarly, we noted a decrease in Hamp expression in iron-loaded Hfe-knockout mice. Hepatic IREG1 expression was greatly upregulated in patients with haemochromatosis (1.8-fold, 95% CI 1.5-2.2; p=0.002). There was a significant correlation between hepatic iron concentration and expression of HAMP (r=0.59, p=0.02) and IREG1 (r=0.67, p=0.007) in untreated patients.<h4>Interpretation</h4>Lack of HAMP upregulation in HFE-associated haemochromatosis despite significant hepatic iron loading indicates that HFE plays an important part in the regulation of hepcidin expression in response to iron overload. Our results imply that the liver is important in the pathophysiology of HFE-associated haemochromatosis. Furthermore, the increase in hepatic IREG1 expression in haemochromatosis suggests that IREG1 could function to facilitate the removal of excess iron from the liver.

Therapeutic apheresis is a widely used treatment alternative for several diseases. In 29 patients with different diseases, we have monitored the PT, aPTT, thrombin time (TT), fibrinogen, D-dimer, factor VIII, IX, X, XI, XII, VWF, Protein C, S, Active Protein C Resistance (APCR) and Antithrombin-III during TPE. Patients were divided into four groups based on the replacement fluids used: 3% VARIHES or ISOHES + 4% albumin (1:1) (group 1), fresh frozen plasma (FFP) (group 2), 3% VARIHES or ISOHES (group 3) and 4% albumin (group 4). In our study, the fibrinogen level decreased to 83% of the base line level after the end of 48 h therapy. The APTT, PT, and TT increased during TPE. However no statistical difference was observed between the groups. We found a significant change in factor levels with time, only the difference in factors IX and XI between the groups was significant. In addition, factor levels measured at 48 h were close to the levels measured before aphereses. In our study, time the related change in AT-3 values was significant. Time-related changes of the Protein S and APCR were not statistical significant significant but on the other hand, we found a significant difference in AT-III and Protein C values between groups. The side effects of HES on coagulation factors and tests were comparable to those of other replacement fluids. Its low cost makes it favourable.

Transporter-assisted uptake of serotonin (5-HT) and dopamine (DA) has been accounted for activities in human behavior or mental status, because they are the sites of action of widely used antidepressant and psychoactive drugs. Both the human serotonin transporter (5-HTT) and human dopamine transporter (DAT1) genes are good candidates for etiological involvement in various psychiatric conditions. The serotonin transporter gene has two types of functional polymorphisms. One is serotonin transporter linked polymorphic region (5-HTTLPR) consisting of length variation of the repetitive sequence containing 20-23-bp-long repeat elements in the 5'-upstream region of the gene. Another polymorphism is that serotonin transporter variable number of tandem repeats (5-HTTVNTR) in its second intron. Both polymorphisms affect the transcription ratio of 5-HTT gene and may modify neuronal transmission by changing its protein expression. On the other hand, DAT1 gene has a variable number of tandem repeats type polymorphism (DAT1VNTR) in the 3'-untranslated region of the mRNA, which was also reported to change its gene expression. So polymorphic variations of transporters would change the behavioral and neuropathological tendency. Here, the feature of those two transporters and their relations to psychiatric disorders are described.

To evaluate the possible role of prolactin (PRL) in T-lymphocytes, we monitored gene induction in one cytotoxic T-lymphocyte (CTL) clone derived from a patient with hemochromatosis and in several T-helper clones generated from a normal donor and a patient with multiple sclerosis. The CTL clone expressed conventional PRL receptor (PRLR), and PRL induced the expression of suppressor of cytokine signaling-3 (SOCS-3) and increased the expression of SOCS-2 and cytokine-inducible src homology-2 containing protein (CIS, another member of the SOCS family). As is the case in granulocytes, expression of a conventional receptor for PRL could not be shown by polymerase chain reaction analysis on three helper clones. In addition, as in granulocytes, PRL modulated the expression of genes such as the interferon-regulatory factor-1, inducible nitric oxide synthase, CIS, and SOCS-2. These effects were also elicited with ovine PRL and could be prevented by anti-PRL antibodies. Thus, the use of clones allowed the detection of direct effects of PRL on T-cells, even when these have few or no detectable PRLR, confirming that human T-lymphocytes are targets for PRL.

The relationship between high dietary iron intake, mutations of the HFE gene, and iron status, and their effects on human health are reviewed. Prolonged high dietary intakes of iron are unlikely to result in iron overload in the general population. Homozygotes for the C282Y mutation of the HFE gene have elevated body iron levels. Heterozygotes have normal iron stores but some may be at increased risk for cardiovascular disease. There is no convincing evidence that elevated iron status increases the risk of coronary heart disease or type 2 diabetes, but high iron intakes may increase the risk of colorectal cancer. The dietary levels of iron associated with health risks in different HFE genotypes must be determined.

Four types of hereditary haemochromatosis have been identified. Type 1 is due to a point mutation in the HFE gene (C282Y) and leads via an increase in intestinal iron absorption to iron overload and organ damage. Type 2 is a juvenile form with manifestation before age 30; it affects both gender and is associated with severe cardiomyopathy and hypogonadism. The genetic defect of type 3 is located on chromosome 7q22 and affects the transferrin receptor 2. The consequences of type 3 are similar to those of type 1. The autosomal-dominant type 4 is located on chromosome 2q32 and affects the basolateral iron carrier ferroportin 1. In contrast to types 1 and 3 iron deposits in type 4 are seen predominantly in macrophages; in type 4 serum ferritin is significantly increased although transferrin saturation is only slightly abnormal. The prognosis of haemochromatosis is normal when phlebotomy therapy is started prior to manifestation of cirrhosis or diabetes. Screening strategies should be implemented to improve early detection.

The two-subunit meta-cleavage enzyme, 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase (CarBaBb), from the carbazole degrader Pseudomonas resinovorans strain CA10 was purified to homogeneity from an Escherichia coli strain carrying the expression vector pUCA503, in which two copies of the carBaBb genes are tandemly linked. SDS-PAGE and gel filtration showed that CarB was a alpha2beta2-heterotetrameric enzyme with subunit molecular masses of approximately 10,000 for CarBa and 29,000 for CarBb. The optimum pH for activity was 8.5 and that of temperature was 35 degrees C. The CarB enzyme had a Km of 14 microM and a kcat/Km of 0.25 microM(-1) s(-1) for 2'-aminobiphenyl-2,3-diol, and the catalytic activities for biphenyl-type catecholic substrates were higher than those for monoaromatic catechol derivatives. The enzyme was originally isolated as a meta-cleavage enzyme for 2'-aminobiphenyl-2,3-diol involved in carbazole degradation, but the enzyme was highly specific for 2,3-dihydroxybiphenyl.

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 hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, toxins (alcohol, aflatoxin B1) and various inherited metabolic liver diseases, such as hemochromatosis and alpha-1-antitrypsin deficiency. Central to the molecular pathogenesis of HCC are mutations of various genes and genetic/chromosomal instability that result from chronic liver disease and the associated enhanced liver cell regeneration and mitotic activity. Alterations in the structure or expression of several tumor suppressor genes and oncogenes have been described. In addition, mechanisms leading to genetic instability due to mismatch repair deficiency or chromosomal instability and aneuploidy due to defective chromosomal segregation appear to be involved. The prognosis of HCC patients is generally very poor. Most studies have shown a five-year survival rate of less than 5% in symptomatic patients. HCC has been found to be quite resistant to radio- or chemotherapy. Investigations of the natural history and clinical course of HCC revealed a long-term survival of patients only with small asymptomatic HCC that could be treated surgically or nonsurgically. For patients with advanced symptomatic HCC, novel therapeutic strategies such as gene therapy are urgently needed. Apart from exploring and refining new HCC treatment strategies, the implementation of the existing measures or the development of novel measures to prevent HCC is most important. Primary HCC prevention could have a major impact on the incidence of HCC. Further, secondary prevention of a local recurrence or of new HCC lesions in patients after successful surgical or nonsurgical HCC treatment is of paramount importance and is expected to significantly improve disease-free and overall survival rates of patients. Based on rapid scientific advances, molecular diagnosis, gene therapy and molecular prevention are becoming increasingly part of our patient management and will eventually complement or in part replace the existing diagnostic, therapeutic and preventive strategies. Overall, this should result in a reduced HCC incidence and an improved clinical outcome for patients with HCC, one of the most devastating malignancies worldwide.