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<h4>Background</h4>It is generally assumed that linker histones contribute to condensation of chromatin and to the repression of genetic activity by binding to chromatin with increased affinity. The aim of this study is to investigate a possible correlation between linker histone binding to chromatin in situ and chromatin condensation using a cytochemical approach that largely preserves nuclear structure.<h4>Methods</h4>Ionically bound H1 was extracted with increasing concentrations of sodium chloride. Thereafter, the cells were fixed in formaldehyde and stained with 50 nM 4',6-diamidino-2-phenylindole (DAPI) and fluorescence intensity was measured by image cytofluorometry.<h4>Results</h4>The association between linker histones and chromatin was stronger in cultured human fibroblasts and rat smooth muscle cells than in both human T lymphocytes and granulocytes, and in particular, frog erythrocytes, which exhibited highly condensed chromatin. Our data indicate a lower affinity of linker histones for metaphase chromosomes than for interphase chromatin, and we observed a reduction in linker histone affinity during terminal differentiation of frog erythrocytes in vivo.<h4>Conclusions</h4>Taken together, our findings imply that the affinity of linker histones for chromatin in situ is unrelated or inversely related to chromatin condensation.

Myelinating glia express high levels of a unique set of genes which code for structural proteins of the myelin sheath. Few transcription factors have so far been implicated in the regulation of any myelin gene. Here we show that the protein zero (P(0)) gene, a myelin gene exclusively expressed in the Schwann cell lineage of the peripheral nervous system, is controlled in its expression by the high-mobility-group domain protein Sox10 both in tissue culture and in vivo. Induction of wild-type Sox10, but not of other transcription factors or Sox10 mutants, strongly increased endogenous P(0) expression in tissue culture. This activation was mediated by the P(0) promoter, which was stimulated by Sox10 in transient transfections. Detailed analyses revealed the involvement of a proximal and a distal promoter region. The distal region functioned only in conjunction with the proximal one and contained a single Sox consensus binding site, which accounted for most of its activity. In contrast, the proximal region mediated Sox10 responsiveness on its own. It contained multiple binding sites for Sox proteins, with two high-affinity sites being the most significant. P(0) expression also depended on Sox10 in vivo, as evident from the analysis of Schwann cell precursors in mouse embryos with Sox10 mutation at day 12.5 of embryogenesis. To our knowledge this is the most conclusive link to date between a glial transcription factor and cell-specific activation of myelin gene expression.

<h4>Background/aims</h4>Inhibition of programmed cell death (apoptosis) is associated with increased tumour aggressiveness, and expression of Survivin, an antiapoptosis gene, in colorectal carcinomas may provide important prognostic information.<h4>Patients/methods</h4>Expression of Survivin messenger RNA was evaluated by reverse transcription-polymerase chain reaction in 144 colorectal carcinomas and 86 adjacent histologically normal mucosa samples from patients for whom long term follow up data were available.<h4>Results</h4>Survivin transcripts were detected in a significantly greater proportion of carcinomas (63.5%) than normal mucosa samples (29.1%; p<0.001). The prevalence of Survivin expression was independent of advancing pathological stage. Death due to recurrent cancer following curative resection was predicted independently by tumour expression of Survivin (hazard ratio (HR) 2.60; 95% confidence interval (95% CI) 1. 17-5.75) and lymph node metastases (HR 2.38; 95% CI 1.21-4.70). On stage wise analysis, the predictive value of Survivin expression was limited to patients with stage II colorectal carcinomas; those with Survivin negative tumours had a five year survival rate of 94.4% compared with 44.8% for patients with Survivin positive tumours (p=0. 004, log rank test).<h4>Conclusion</h4>In patients with stage II colorectal carcinomas, Survivin expression provides prognostic information that may have important therapeutic implications.

<h4>Background</h4>In the UK approximately 1 in 140 people are homozygous for the C282Y mutation of the HFE gene and are at risk from iron overload caused by genetic haemochromatosis (GH). Early detection can prevent organ damage secondary to iron deposition and increase life expectancy.<h4>Aim</h4>To screen for GH in all blood samples sent to the laboratory for routine liver function tests in which raised serum alanine aminotransferase (ALT) activity was detected.<h4>Methods</h4>ALT was measured in sera sent to the laboratory for routine liver function tests. In those samples found to have raised activity, transferrin saturation and ferritin were measured followed by genetic testing when transferrin saturation was increased.<h4>Results</h4>Of the 35 069 serum samples assayed for routine liver function tests, 1490 (4.2%) had raised ALT levels (>50 u/l). Transferrin saturation and serum ferritin concentrations were measured in these patient samples, and in 56 transferrin saturation was >60%. Further blood samples were requested from these patients for genetic testing: 33 samples were obtained. There were nine patients homozygous for the C282Y mutation of the HFE gene and three compound heterozygotes (heterozygous for both C282Y and H63D mutations).<h4>Conclusions</h4>The association of raised ALT activity and transferrin saturation of >60% could provide a simple, cost effective method for detecting individuals with clinical haemochromatosis. Although many patients with GH may have been missed, this study suggests that the clinical penetrance of the disorder may be much lower than is generally supposed and that genetic screening will identify many people who may never develop clinical haemochromatosis.

Seventy malignant, premalignant and histologically normal biopsies from 7 oesophagogastrectomy specimens of adenocarcinomas of the lower oesophagus and gastroesophageal junction were analysed for loss of heterozygosity (LOH) at 9 known or putative gene loci. LOH was detected in 20 of 27 (74%) malignant biopsies, 4 of 7 (57%) biopsies of dysplasia, 2 of 12 (25%) biopsies of histologically normal oesophagus adjacent to adenocarcinoma, and in 2 of 14 (14%) biopsies of histologically normal stomach adjacent to adenocarcinoma. LOH at the VHL, APC, CDKN2 and DCC tumour suppressor and MSH3 mismatch repair gene loci can be detected in histologically normal tissue and in adjacent adenocarcinoma, and are potential markers of early neoplastic progression.

Alcohol is a major cause of liver cirrhosis in the Western world and accounts for the majority of cases of liver cirrhosis seen in district general hospitals in the UK. The three most widely recognised forms of alcoholic liver disease are alcoholic fatty liver (steatosis), acute alcoholic hepatitis, and alcoholic cirrhosis. The exact pathogenesis of alcoholic liver injury is still not clear but immune mediated and free radical hepatic injury are thought to be important. There is increasing interest in genetic factors predisposing to hepatic injury in susceptible individuals. Diagnosis is based on accurate history, raised serum markers such as gamma-glutamyltransferase, mean corpuscular volume, and IgA and liver histology when obtainable. Abstinence is the most important aspect of treatment. Newer drugs such as acamprosate and naltrexone are used to reduce alcohol craving. Vitamin supplements and nutrition are vital while corticosteroids have a role in acute alcoholic hepatitis where there is no evidence of gastrointestinal haemorrhage or sepsis. Liver transplantation has excellent results in abstinent patients with end stage liver disease but there are concerns about recidivism after transplant.

Foamy viruses (FVs) are highly fusogenic, and their replication induces massive syncytium formation in infected cell cultures which is believed to be mediated by expression of the envelope (Env) protein. The FV Env is essential for virus particle egress. The unusually long putative membrane-spanning domain (MSD) of the transmembrane subunit carries dispersed charged amino acids and has an important function for particle envelopment. To better understand the capsid-envelope interaction and Env-mediated cell fusion, we generated a variety of FV MSD mutations. C-terminal deletions revealed the cytoplasmic domain to be dispensable but the full-length MSD to be required for fusogenic activity. The N-terminal 15 amino acids of the MSD were found to be sufficient for membrane anchorage and promotion of FV particle release. Expression of wild-type Env protein rarely induced syncytia due to intracellular retention. Coexpression with FV Gag-Pol resulted in particle export and a dramatic increase in fusion activity. A nonconservative mutation of K(959) in the middle of the putative MSD resulted in increased fusogenic activity of Env in the absence of Gag-Pol due to enhanced cell surface expression as well as structural changes in the mutant proteins. Coexpression with Gag-Pol resulted in a further increase in the fusion activity of mutant FV Env proteins. Our results suggest that an interaction between the viral capsid and Env is required for FV-induced giant-cell formation and that the positive charge in the MSD is an important determinant controlling intracellular transport and fusogenic activity of the FV Env protein.

The C-peptide of proinsulin is important for the biosynthesis of insulin but has for a long time been considered to be biologically inert. Data now indicate that C-peptide in the nanomolar concentration range binds specifically to cell surfaces, probably to a G protein-coupled surface receptor, with subsequent activation of Ca(2+)-dependent intracellular signaling pathways. The association rate constant, K(ass), for C-peptide binding to endothelial cells, renal tubular cells, and fibroblasts is approximately 3. 10(9) M(-1). The binding is stereospecific, and no cross-reaction is seen with insulin, proinsulin, insulin growth factors I and II, or neuropeptide Y. C-peptide stimulates Na(+)-K(+)-ATPase and endothelial nitric oxide synthase activities. Data also indicate that C-peptide administration is accompanied by augmented blood flow in skeletal muscle and skin, diminished glomerular hyperfiltration, reduced urinary albumin excretion, and improved nerve function, all in patients with type 1 diabetes who lack C-peptide, but not in healthy subjects. The possibility exists that C-peptide replacement, together with insulin administration, may prevent the development or retard the progression of long-term complications in type 1 diabetes.

Association of the condensin multiprotein complex with chromatin is required for chromosome condensation at mitosis. What regulates condensin targeting to chromatin is largely unknown. We previously showed that the nuclear A kinase-anchoring protein, AKAP95, is implicated in chromosome condensation. We demonstrate here that AKAP95 acts as a targeting protein for human chromosome-associated protein (hCAP)-D2/Eg7, a component of the human condensin complex, to chromosomes. In HeLa cell mitotic extract, AKAP95 redistributes from the nuclear matrix to chromatin. When association of AKAP95 with chromatin is prevented, the chromatin does not condense. Condensation is rescued by a recombinant AKAP95 peptide containing the 306 COOH-terminal amino acids of AKAP95. Recombinant AKAP95 binds chromatin and elicits recruitment of Eg7 to chromosomes in a concentration-dependent manner. Amount of Eg7 recruited correlates with extent of chromosome condensation: resolution into distinct chromosomes is obtained only when near-endogenous levels of Eg7 are recruited. Eg7 and AKAP95 immunofluorescently colocalize to the central region of methanol-fixed metaphase chromosomes. GST pull-down data also suggest that AKAP95 recruits several condensin subunits. The results implicate AKAP95 as a receptor that assists condensin targeting to chromosomes.

Hereditary hemochromatosis (HH) is a prevalent human disease caused by a mutation in HFE, which encodes an atypical HLA class I protein involved in regulation of intestinal iron absorption. To gain insight into the pathogenesis of hemochromatosis, we have bred Hfe knockout mice to strains carrying other mutations that impair normal iron metabolism. Compound mutant mice lacking both Hfe and its interacting protein, beta-2 microglobulin (B2m), deposit more tissue iron than mice lacking Hfe only, suggesting that another B2m-interacting protein may be involved in iron regulation. Hfe knockout mice carrying mutations in the iron transporter DMT1 fail to load iron, indicating that hemochromatosis involves iron flux through DMT1. Similarly, compound mutants deficient in both Hfe and hephaestin (Heph) show less iron loading than do Hfe knockout mice, indicating that iron absorption in hemochromatosis involves the function of Heph as well. Finally, compound mutants lacking Hfe and the transferrin receptor accumulate more tissue iron than do mice lacking Hfe alone, consistent with the idea that interaction between these two proteins contributes to the control of normal iron absorption. In addition to providing insight into the pathogenesis of HH, our results suggest that each of these genes might be a candidate modifier of the human hemochromatosis phenotype.

Obsessive-compulsive disorder (OCD) is a severe and disabling anxiety disorder with a marked genetic contribution. Pharmacological data indicated involvement of the serotonergic and dopaminergic systems. We studied the association between OCD and six candidate genes encoding important components of the serotonergic and dopaminergic pathways in 75 biologically unrelated patients and 172 ethnically matched controls (Ashkenazi and non-Ashkenazi Jews). Polymorphisms in the following genes were studied: tryptophan hydroxylase (TPH), serotonin 2A receptor (HTR2A), serotonin 2C receptor (HTR2C), serotonin transporter (5-HTT), dopamine receptor D4 (DRD4), and dopamine transporter (DAT1). The genotypic and allelic distribution of all polymorphisms tested did not show statistically significant differences between patients and controls. Our results suggest that these polymorphisms do not play a major role in the genetic predisposition to OCD, although a minor contribution cannot be ruled out.

Early diagnosis and treatment of hemochromatosis is essential to prevent organ damage. Screening strategies to detect early hemochromatosis include testing for iron overload and/or genetic testing. Voluntary blood donors numbering 5,211 were screened with unbound iron-binding capacity (UIBC), transferrin saturation (TS), and genetic testing for the C282Y mutation of the HFE gene. The study found 16 C282Y homozygotes (1 in 327), 69 compound heterozygotes, 371 simple heterozygotes, and 4,755 normals. There were 5 men and 11 women homozygotes with a mean age of 42, range 28 to 57. Mean UIBC (24 +/- 7 microL) and TS (48% +/- 17%) in homozygotes were significantly different from compound heterozygotes, simple heterozygotes, and normals (ANOVA). Only 3 homozygotes had an elevated serum ferritin. Family studies found an additional 4 iron-loaded homozygotes. Optimal thresholds were < or =28 micromol/L for UIBC and > or =46% for TS. Receiver operating characteristic (ROC) curve analysis showed an area under the curve for UIBC of 0.93 (0. 85-1.0, 95% confidence interval), and for TS of 0.83 (0.7-0.95). Screening with UIBC to preselect those for genotyping is a cost-efficient strategy for population screening for hemochromatosis.

<h4>Background</h4>Although normal prostatic development is androgen-dependent, the prostate continues to grow in the neonate despite castration. However, the manner in which neonatal growth of the prostate occurs, in the absence of the testis, remains largely unknown. The purpose of this study was to examine the differentiation of prostatic epithelial cells after neonatal castration.<h4>Methods</h4>Immunohistochemistry was utilized to detect the expression of differentiation products: basal-cell cytokeratin (CK 5), luminal-cell cytokeratin (CK 18), and prostatic steroid-binding protein (PBP), a ventral prostate-specific marker indicative of secretory function in luminal cells. The reverse transcription-polymerase chain reaction was used to detect transcription products of the three polypeptide subunits of PBP, designated C1, C2, and C3. Rats were castrated on day 5 after birth, and ventral prostates were collected on day 14. Dihydrotestosterone was injected (100 microg/animal every 2 days) in castrated animals to determine if PBP expression could be initiated by androgen.<h4>Results</h4>Although no major effects of castration were detected on the differentiation of stromal or basal cells (which differentiate prior to day 5), castration had a pronounced effect on luminal-cell differentiation. Castration inhibited PBP protein expression, but did not affect the expression of luminal-cell cytokeratin (CK 18) protein. Furthermore, castration reduced C1, C2, and C3 transcription. Androgen replacement to castrated animals allowed for the initiation of PBP expression, although its onset was delayed.<h4>Conclusions</h4>These observations indicate that the testis is not necessary for prostatic luminal-cell differentiation, but is necessary for full expression of luminal-cell secretory phenotype. Furthermore, our study suggests that factors of testicular origin, in addition to androgen, are needed for proper timing of PBP expression. This investigation establishes that the cytological and the physiological differentiation of the rat prostate are differentially regulated.

Netrins are soluble neurite-outgrowth-promoting proteins related to the laminin B2 chain. Since these proteins and their receptor DCC (the "deleted in colorectal carcinoma" gene product) bind heparin, glycosaminoglycans may modulate their biological actions in a similar fashion as described for several other ligand-receptor systems. Here we show that a polypeptide encompassing the C-terminal cluster of basic amino acids of netrin-1 (i) adopts an alpha-helical conformation in water-trifluoroethanol mixtures according to circular dichroism experiments and (ii) binds electrostatically to heparin with high affinity under physiological ionic conditions (K(D) = 15 nM for the binding to immobilized heparin according to surface plasmon resonance, K(D) = 50 nM in solution as determined with isothermal titration calorimetry). These data indicate that the cluster of basic amino acids at the C-terminus of netrin-1 forms an alpha-helical structural element which can contribute to the glycosaminoglycan-binding activity of this neurotrophic guidance molecule.

alpha(1) subunit of the voltage-dependent Ca(2+) channel is essential for channel function and determines the functional specificity of various channel types. alpha(1E) subunit was originally identified as a neuron-specific one, but the physiological function of the Ca(2+) channel containing this subunit (alpha(1E) Ca(2+) channel) was not clear compared with other types of Ca(2+) channels because of the limited availability of specific blockers. To clarify the physiological roles of the alpha(1E) Ca(2+) channel, we have generated alpha(1E) mutant (alpha(1E)-/-) mice by gene targeting. The lacZ gene was inserted in-frame and used as a marker for alpha(1E) subunit expression. alpha(1E)-/- mice showed reduced spontaneous locomotor activities and signs of timidness, but other general behaviors were apparently normal. As involvement of alpha(1E) in pain transmission was suggested by localization analyses with 5-bromo-4-chloro-3-indolyl beta-d-galactopyranoside staining, we conducted several pain-related behavioral tests using the mutant mice. Although alpha(1E)+/- and alpha(1E)-/- mice exhibited normal pain behaviors against acute mechanical, thermal, and chemical stimuli, they both showed reduced responses to somatic inflammatory pain. alpha(1E)+/- mice showed reduced response to visceral inflammatory pain, whereas alpha(1E)-/- mice showed apparently normal response compared with that of wild-type mice. Furthermore, alpha(1E)-/- mice that had been presensitized with a visceral noxious conditioning stimulus showed increased responses to a somatic inflammatory pain, in marked contrast with the wild-type mice in which long-lasting effects of descending antinociceptive pathway were predominant. These results suggest that the alpha(1E) Ca(2 +) channel controls pain behaviors by both spinal and supraspinal mechanisms.

Haemochromatosis is a common recessive disorder characterized by progressive iron overload, which may lead to severe clinical complications. Most patients are homozygous for the C282Y mutation in HFE on 6p (refs 1-5). A locus for juvenile haemochromatosis (HFE2) maps to 1q (ref. 7). Here we report a new locus (HFE3) on 7q22 and show that a homozygous nonsense mutation in the gene encoding transferrin receptor-2 (TFR2) is found in people with haemochromatosis that maps to HFE3.

Expansion of a polyglutamine sequence in the N terminus of huntingtin is the gain-of-function event that causes Huntington's disease. This mutation affects primarily the medium-size spiny neurons of the striatum. Huntingtin is expressed in many neuronal and non-neuronal cell types, implying a more general function for the wild-type protein. Here we report that wild-type huntingtin acts by protecting CNS cells from a variety of apoptotic stimuli, including serum withdrawal, death receptors, and pro-apoptotic Bcl-2 homologs. This protection may take place at the level of caspase-9 activation. The full-length protein also modulates the toxicity of the poly-Q expansion. Cells expressing full-length mutant protein are susceptible to fewer death stimuli than cells expressing truncated mutant huntingtin.

We have characterized five genes encoding condensin components in Saccharomyces cerevisiae. All genes are essential for cell viability and encode proteins that form a complex in vivo. We characterized new mutant alleles of the genes encoding the core subunits of this complex, smc2-8 and smc4-1. Both SMC2 and SMC4 are essential for chromosome transmission in anaphase. Mutations in these genes cause defects in establishing condensation of unique (chromosome VIII arm) and repetitive (rDNA) regions of the genome but do not impair sister chromatid cohesion. In vivo localization of Smc4p fused to green fluorescent protein showed that, unexpectedly, in S. cerevisiae the condensin complex concentrates in the rDNA region at the G2/M phase of the cell cycle. rDNA segregation in mitosis is delayed and/or stalled in smc2 and smc4 mutants, compared with separation of pericentromeric and distal arm regions. Mitotic transmission of chromosome III carrying the rDNA translocation is impaired in smc2 and smc4 mutants. Thus, the condensin complex in S. cerevisiae has a specialized function in mitotic segregation of the rDNA locus. Chromatin immunoprecipitation (ChIP) analysis revealed that condensin is physically associated with rDNA in vivo. Thus, the rDNA array is the first identified set of DNA sequences specifically bound by condensin in vivo. The biological role of higher-order chromosome structure in S. cerevisiae is discussed.

<h4>Background and purpose</h4>MR imaging sheds new light on CNS involvement in the course of acquired chronic liver disease; however, the exact pathogenetic mechanisms of hepatic encephalopathy and associated MR abnormalities remain unclear. Our purpose was to relate MR signal intensity abnormalities of the CNS to clinical, biochemical, and pathologic features of childhood-onset chronic liver disease.<h4>Methods</h4>Twenty-one patients (12 male and nine female patients) were included in the study; two had Crigler-Najjar disease type 2, 17 had chronic liver disease of different causes, and two had idiopathic copper toxicosis. Twelve patients had histologically proved liver cirrhosis, with a median disease duration of 175 months at the time of MR study. None had clinical symptoms of hepatic encephalopathy. MR imaging was performed using spin-echo T1- and T2-weighted sequences.<h4>Results</h4>Eleven patients had abnormal MR imaging findings of the brain revealed by T1-weighted MR sequences; two of the 11 had idiopathic copper toxicosis. The affected sites were the hypothalamus and globus pallidus, presenting symmetrical and bilateral high signal intensities, or the pituitary gland, which appeared homogeneously hyperintense, or both findings. Eight of the 12 patients with cirrhosis had abnormal MR signals of the brain. In these, the median cirrhosis duration was shorter (169 months) than in the remaining four patients with normal MR signals (177 months). A significant correlation was found between abnormal MR signals of the brain and cirrhosis (P = .008) and factor V activity (P = .008).<h4>Conclusion</h4>MR imaging confirms the presence of abnormal brain signals in the globus pallidus, hypothalamus, and pituitary gland in patients with childhood-onset liver disease in the absence of clinical symptoms of encephalopathy. Signal intensity abnormalities are likely caused by an as yet unidentified metabolic process partially correlated with the severity of liver disease.

<h4>Background</h4>Hereditary hemochromatosis (HH) is a common hereditary disorder of iron metabolism causing iron overload, organ failure, and malignancy. Preclinical diagnosis using HFE gene analysis followed by prophylactic phlebotomy can completely prevent the disease.<h4>Methods</h4>We conducted a mail survey of all registered primary care physicians, gastroenterologists, and hematologists in Arkansas (n = 860) to determine utilization of HFE mutation analysis in clinical medicine a year after the new molecular test first became available.<h4>Results</h4>Of 346 responding physicians (40%), 71 (21%) were aware of the test, 36 (10%) knew that the test was available in Arkansas, and 10 (3%) had used the test. One physician had used the test to screen first-degree relatives of a homozygous HH proband.<h4>Conclusions</h4>Because of poor utilization of the test, the discovery of the role of HFE mutations in HH has had minimal impact on clinical care in Arkansas.

<h4>Background/aims</h4>Histological and biochemical iron overload has been reported in non-tumoral liver of most patients presenting an hepatocellular carcinoma (HCC) developed in non-cirrhotic liver (NCL). The aim of our study was to investigate HFE mutations in patients with HCC in NCL.<h4>Methods</h4>Thirty-five patients with HCC in NCL were included either retrospectively or prospectively. Clinical data, iron and viral status, and HFE gene mutations were compared between groups with (I+, n = 19) or without histological iron overload (I-, n = 16).<h4>Results</h4>Twenty per cent of patients were HBV or HCV positive. Fifty-four per cent had hepatocytic iron overload at histology. Mean hepatic iron concentration was 100.2 +/- 14.6 micromol/g in I+ versus 23.2 +/- 2.1 micromol/g in I- (p<0.001). Among the 19 I+ patients, eight mutations were found: two C282Y/C282Y, three C282Y/WT, two C282Y/H63D and one H63D/H63D. None of these mutations was found in the I- group. There was no significant difference concerning the H63D heterozygous mutation between I+ or I- patients.<h4>Conclusions</h4>In patients with HCC in NCL, HBV and HCV markers are rare (20%), and mild iron overload is frequent (54%). In patients with HCC in NCL and iron overload, C282Y mutations are frequent (36.8% of cases) and significantly increased (p<0.009) compared to HCC in NCL without iron overload; these mutations are mostly heterozygous. H63D heterozygosity is not associated with liver iron overload. Because of the small size of the series, HFE C282Y mutation should be investigated on a larger scale in patients with HCC in NCL with iron overload in order to confirm this association.

The identification of mutations in the haemochromatosis gene (HFE) (C282Y and H63D) provides the unique opportunity to test whether genetic variants that are associated with tissue iron accumulation may influence the risk of coronary atherosclerosis. To this aim the prevalence of C282Y and H63D mutations was determined in 174 patients with angiographically documented CAD (>50% stenosis) and history of MI, 187 healthy free-living individuals and 142 blood donors. C282Y and H63D mutations were not found to be more frequent in coronary patients as compared to controls. Moreover, these HFE variants were unrelated to the severity of coronary atherosclerosis. These findings did not provide evidence of an association between HFE mutations and the presence of coronary atherosclerosis or its major ischaemic complications, thus indicating that HFE mutations are poor genetic markers of coronary risk.

A 30 degrees reverse Trendelenburg position and the pneumoperitoneum performed in patients undergoing at Laparoscopic Cholecystectomy (LC) cause haemodynamic modifications in inferior cava vein and in femoral veins producing a venous stasis in lower limbs. So that activation of coagulation with hypercoagulability occurs. The aim of this study is to value the modification of the haemocoagulative and fibrinolytic factors during LC and OC. 18 patients with symptomatic and non complicated lithiasis were examined. They were divided in two groups, nine patients for each group. LC was performed in first group and OC in the other group. Prothrombin time (PT), plasma-activated partial thromboplastin time (PTT), Antithrombin III (AT III) fibrinogen degradation products (FDP) were valued before and at 6, 12, 24, 48 hours after operation. For the statistical analysis Student "t" for average comparison was used. There were no relevant alterations of PT, PTT and ATIII after operation in the two groups. FP was increased after surgery, especially at 24th-48th hours in the group in which OC (p < 0.05) was performed. FDP were increased in both groups but only at 24 and 48 hour after OC (p < 0.05) the result was significant. Earlier mobilization and reduced invasivity of laparoscopy could contrast thrombotic effects of the blood stasis in the lower limbs when 30 degrees-45 degrees reverse Trendelenburg and pneumoperitoneum are performed.

It is not known how the efficiency of intestinal iron absorption is regulated. One hypothesis suggests that an interaction between the transferrin receptor (TfR) and the haemochromatosis protein (HFE) regulates the level of iron loading in crypt cells. The hypothesis goes on to suggest that this determines the amount of transport protein, expressed in villus enterocytes, that is involved in iron absorption. Mice with haploinsufficiency for TfR are iron deficient and this is thought to be caused by reduced iron absorption. This suggests that TfR may play a role in the regulation and/or mechanism of iron absorption. We investigated TfR function and distribution by measuring iron uptake from plasma transferrin and by immunohistochemistry. The uptake of transferrin-bound iron (Tf-Fe2) into mucosal cells subsequently separated along the crypt-villus axis was compared to the presence of TfR, determined by immunohistochemistry using frozen and wax sections. Frozen sections showed TfR staining in crypt and villus epithelial cells. In wax sections TfR was only identified in a supranuclear region commencing in enterocytes at the crypt-villus junction and attaining greatest levels at the villus tip. This indicates that the processing of wax tissue exposes a TfR epitope that otherwise remains undetectable when studied in frozen sections. This appearance in paraffin sections was inversely related to the uptake of Tf-Fe2. Supranuclear TfR was not associated with lysosomes, since there was no difference in the uptake of normal Tf-Fe2 and that of the non-digestible cellobiose Tf-Fe2, and Western blot analysis revealed similar amounts of TfR in crypt and villus cells. Also the uptake of Tf-Fe2 increased linearly with time, albeit less in villus than crypt cells, suggesting that maturation of an efflux system in villus cells is not responsible for this difference. We hypothesise that TfR in the supranuclear region of villus enterocytes may play a role in iron absorption.

Thanks to the discovery of the HFE gene and of its mutations, it is now established that the most frequent form of hemochromatosis is related to homozygosity for the mutation C282Y, and that other types of hemochromatosis, unrelated to HFE mutations, do exist such as the juvenile hemochromatosis. From a pathophysiological standpoint, the C282Y mutation impairs HFE protein expression at the surface of the membrane and disturbs the cellular entry of iron (carried by circulating transferrin) into the cryptic duodenal cell. This, in turn, is likely to lead to an aberrant programmation of the degree of iron influx from the digestive lumen into the apical duodenal cells. The resulting hyperabsorption, which forms the basis of iron overload in hemochromatosis, is likely to implicate an overexpression of the transmembrane iron carrier DMT1. It is remarkable to observe that these major improvements in the knowledge of hemochomatosis have been accompanied by similar improvements in the understanding of normal iron metabolism.

Genetic hemochromatosis is an autosomal recessive disease, characterized by an increased iron absorption, leading to progressive iron overload. The fully expressed phenotype comprises fatigue, skin pigmentation, liver disease with hepatomegaly, cirrhosis and hepatocellular carcinoma, and diabetes. Arthralgias are frequent, cardiopathy or impotence may occur. This presentation is now unfrequent with earlier diagnosis, and patients are often asymptomatic--with only biochemical expression--or pauci-symptomatic (mild fatigue, arthralgias or increased transaminases). Transferrin saturation is always increased. Serum ferritin is proportional to iron burden. Diagnosis is now easy, since most patients are homozygote for the C282Y mutation of the HFE gene. Liver biopsy can be useful to quantify iron overload and assess liver fibrosis. The disease can be lethal due to liver disease, carcinoma or heart disease, but life expectancy goes to normal if patients are treated before the occurrence of cirrhosis. Treatment relies on regular venesections. Familial screening is essential.

<h4>Rationale</h4>A 44-base-pair insertion/deletion polymorphism in the promoter region of the human serotonin (5-HT) transporter (5-HTT) gene gives rise to a bi-allelic polymorphism designated long (l) and short (s). The s variant is associated with a lower expression of 5-HTT sites and a reduced efficiency of 5-HT reuptake.<h4>Objective</h4>The aim of the present study was to determine whether the increase in brain 5-HT function produced by acute 5-HT reuptake blockade is influenced by the 5-HTT promoter l/s polymorphism.<h4>Methods</h4>We measured the increase in plasma prolactin that follows acute administration of the tricyclic antidepressant clomipramine as an index of 5-HT neurotransmission in 14 healthy female subjects (7 with ss genotype and 7 with ll genotype) using a placebo-controlled crossover design.<h4>Results</h4>Clomipramine-induced prolactin release was significantly greater in subjects with the ll genotype.<h4>Conclusion</h4>Our findings suggest that acute 5-HT reuptake blockade produces a greater increase in 5-HT neurotransmission in subjects with the ll genotype than in those with an ss genotype. These results are consistent with clinical data indicating that subjects with an ss genotype may have a poorer therapeutic response to selective serotonin reuptake inhibitor (SSRI) monotherapy.

<h4>Purpose</h4>The extremely low clearance and small distribution volume of UCN-01 in humans could be partly due to the high degree of binding to hAGP. The quantitative effects of hAGP on the pharmacokinetics of UCN-01 at several levels of hAGP and UCN-01 were estimated in rats given an infusion of hAGP to mimic the clinical situation and a physiological model for analysis was developed.<h4>Methods</h4>The plasma concentrations of UCN-01 (72.5-7250 nmol/kg i.v.) in rats given an infusion of hAGP, 15 or 150 nmol/h/kg, were measured by HPLC. Pharmacokinetic analysis under conditions assuming rapid equilibrium of protein binding and incorporating the dissociation rate was conducted.<h4>Results</h4>The Vdss and CLtot of UCN-01 (725 nmol/kg i.v.) in rats given an infusion of hAGP, 150 nmol/h/kg, fell to about 1/250 and 1/ 700 that in control rats. The Vdss and CLtot following 72.5-7250 nmol/kg UCN-01 to rats given 150 nmol/h/kg hAGP were 63.9-688 ml/kg and 3.18-32.9 ml/h/kg, respectively, indicating non-linearity due to saturation of UCN-01 binding. The CLtot estimated by the physiological model assuming rapid equilibrium of UCN-01 binding to hAGP, was six times higher than the observed value while the CLtot estimated by the model incorporating k(off), measured using DCC, was comparable with the observed value.<h4>Conclusions</h4>These results suggest that the slow dissociation of UCN-01 from hAGP limits its disposition and elimination.

The present study evaluated the singular and interactive effects of a functional polymorphism (variation) in the serotonin transporter (5-HTT) gene and a psychological trait (anxiety sensitivity [AS], i.e., fear of arousal symptoms) in predicting subjective and physiological responses to a 35% carbon dioxide (CO2) challenge in a community sample (N = 72). Genotypes were divided into 2 groups in accord with prior research. Findings were partially supportive of the hypothesized risk model. These indicated that the Group L genotype (homozygous for the 1 allele), compared with the Group S genotype (homozygous for the s allele plus heterozygous individuals), predicted greater fearful response to the biological challenge. There was also an AS x Genotype interaction predicting heart rate variability (HRV) in response to the CO2, suggesting that high AS plus Group L status predicts decreased HRV.

Familial dilated cardiomyopathy (DCM) should be an "evidence-based" diagnosis derived from clinical and echocardiographic screening of informed and consenting relatives of index patients, and on the examination of clinical reports for deceased relatives. Most familial dilated cardiomyopathy pedigrees show an autosomal pattern of inheritance. Very few of them are X-linked and matrilinear. Autosomal recessive inheritance is difficult to be assessed in an evidence-based setting. By linkage analysis, several loci, but no disease gene, have been identified. At present, few cases of familial dilated cardiomyopathy can benefit of a molecular diagnosis. The diagnosis of dystrophin defect-related dilated cardiomyopathy is important for patients and families, especially for carrier detection. These patients present X-linked inheritance, dominant cardiac involvement and raised levels of serum creatine phosphokinase. Defects of the glycoprotein complex associated to dystrophin (DAG) are rare skeletal muscle diseases with possible cardiac involvement. Mitochondrial diseases, both pure cardiomyopathies and multiorgan syndromes involving the heart, are associated to defects of mitochondrial DNA genes or of nuclear genes coding for mitochondrial proteins. Barth's syndrome develops in male children with granulocytopenia, dilated cardiomyopathy, and methylglutaconic aciduria. Cardiomyopathies with atrioventricular block are observed in hemochromatosis, Emery-Dreifuss syndrome, desmin storage disease, and in isolated familial dilated cardiomyopathy. Actin defects were recently identified in 2 unrelated patients with familial dilated cardiomyopathy. Desmin defects were also recently identified in 1 familial dilated cardiomyopathy. The overall knowledge, although in progression, is still limited. Clinical family screening identifies familial forms, preclinical cases, and inheritance pattern. By candidate gene screening, the molecular diagnosis can be provided for dystrophin, DAG, mitochondrial DNA, actin and desmin gene defects.

We have curated a reference set of cancer- related genes and reanalyzed their sequences in the light of molecular information and resources that have become available since they were first cloned. Homology studies were carried out for human oncogenes and tumor suppressors, compared with the complete proteome of the nematode, Caenorhabditis elegans, and partial proteomes of mouse and rat and the fruit fly, Drosophila melanogaster. Our results demonstrate that simple, semi-automated bioinformatics approaches to identifying putative functionally equivalent gene products in different organisms may often be misleading. An electronic supplement to this article provides an integrated view of our comparative genomics analysis as well as mapping data, physical cDNA resources and links to published literature and reviews, thus creating a "window" into the genomes of humans and other organisms for cancer biology.

Inactivation of the gene "deleted in colon cancer" (DCC) on chromosome 18 is known to be associated with the tumorigenesis and metastasis of colorectal cancer. In the present study, we investigated the expression of DCC, P53 and HER-2/neu product in surgical specimens from 79 patients with invasive breast cancer by immunohistochemistry staining and found the expression of DCC to be decreased in 42 tumors (52%). Overexpression of HER-2/neu and P53 was detected in 29 (36.8%) and 26 (32.9%) of this 79 breast cancer specimens, respectively. To evaluate the outcomes of the 79 breast cancer patients, we followed up the patients during the period from May 1990 to August 1998. The average length of follow-up was 52 months (ranging from 4 to 94 months). Patients with tumors having a combination of DCC-negative and HER-2/neu overexpression showed a marginal influence on survival time of breast cancer (P = 0.06). However, patients with tumors having a combination of DCC-negative and P53 overexpression showed no influence of these on survival time of breast cancer (P = 0.36). These findings suggest that a decreased DCC expression and HER-2/neu overexpression may influence the prognosis of breast cancer.

Since autism was first described by Leo Kanner the view on its etiology and pathogenesis has been changing. Recently there are more data on genetic and neurobiological background of autism. At the beginning it was noticed that autism appeared more frequently among boys, in population studies it was found that autism appeared more frequently among siblings, mostly among monozygotic twins. Many disorders like Tourett syndrome and tuberous sclerosis were reported in connection with autism. Recently research is focused mostly on chromosome abnormalities: chromosome 15 (locus 15q11-13), chromosome 7 (locus 7q), chromosome 16 (locus 16p) and gens of particular receptors (GABRB3, UBE3A/E6-AP, 5-HTT). These abnormalities may also be one of the causes of autism.

Iron, either in the form of heme or non-heme compounds, is essential to life, but it can also pose serious health risks. The liver is a principal target for iron toxicity because it is chiefly responsible for taking up and storing excessive amounts of iron. The major hepatic toxicities of iron overload include damage to multiple cell types (hepatocytes, Kupffer cells, hepatic stellate cells) and to multiple subcellular organelles (mitochondria, lysosomes, and smooth endoplasmic reticulum). Heavy iron overload, as occurs in primary (hereditary) or secondary forms of hemochromatosis, may cause cirrhosis, liver failure, and hepatocellular carcinoma. In addition, iron has been shown to be a contributory factor in the development or progression of alcoholic liver disease, nonalcoholic liver steatohepatitis, chronic viral hepatitis, prophyria cutanea tarda, and, perhaps, in alpha 1-antitrypsin deficiency and end-stage liver disease, regardless of cause.