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Chronic liver disorders represent a serious health problem, considering that 300 million people worldwide are hepatitis B virus carriers, and 8,000-10,000 patients per year, in the U.S. alone, die as a result of liver failure caused by hepatitis C infection. Nitric oxide synthase (NOS) regulates hepatic vasculature; however, the patterns of expression and activity of NOS proteins in healthy and diseased human livers are unknown. Sections of diseased (n = 42) and control livers (n = 14) were collected during orthotopic liver transplants and partial hepatectomy. The diseased sections included alcoholic cirrhosis, viral hepatitis, cholestasis, acute necrosis, and uncommon pathologies including alpha(1)-anti-trypsin disorder. The endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) were studied by using the citrulline assay, Western immunoblot, immunohistochemistry, and in situ hybridization. The systemic generation of plasma NO metabolites was measured by HPLC. In control livers, Ca(2+)-dependent and -independent NOS activities were identified by Western analysis as eNOS and iNOS, respectively. The eNOS was uniformly distributed in the hepatocytes and also detected in the endothelium of hepatic arteries, terminal hepatic venules, sinusoids, and in biliary epithelium. The iNOS was detected in hepatocytes and localized mainly in the periportal zone of the liver acinus. This pattern of distribution of eNOS and iNOS in normal liver was confirmed by in situ hybridization. In diseased livers, there was a significant increase in Ca(2+)-independent NOS with the corresponding strong appearance of iNOS in the cirrhotic areas. The eNOS was translocated to hepatocyte nuclei. Thus, eNOS and iNOS proteins are differentially expressed in healthy human liver, and this expression is significantly altered in cirrhotic liver disorders.

SFT, a stimulator of iron (Fe) transport, has been described as a transmembrane protein that facilitates the uptake of ferrous and ferric iron in mammalian cells. This study was initiated to investigate the 5' regulatory region of SFT and its role in the etiology of hereditary hemochromatosis. Sequence analyses of the putative 5' regulatory region revealed that the SFT cDNA sequence corresponds to intron 6/exon 7 of UbcH5A, a member of E2 ubiquitin-conjugating enzymes, which is involved in the iron-dependent ubiquitination of the hypoxia-inducible factor (HIF) by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. Further mRNA expression studies using a sequence-specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay showed that UbcH5A is significantly up-regulated in the liver of iron-overloaded patients with hereditary hemochromatosis, as previously published for SFT. However, in vitro studies on HepG2 cells failed to demonstrate any significant UbcH5A regulation in response to iron loading or iron chelation. In conclusion, in vivo mRNA expression data previously obtained for SFT might be attributed to UbcH5A. The role of UbcH5A and the ubiquitination pathway in the etiology of hereditary hemochromatosis remains to be elucidated further.

Hirschsprung disease (HSCR) is a common congenital disorder characterized by aganglionosis of the gut. The seemingly unrelated multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant disorder characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism. Yet, germline mutations in the RET proto-oncogene are associated with both MEN 2 and HSCR. In the former, gain-of-function mutations in a limited set of codons is found, whereas, in the latter, loss-of-function mutations are found. However, germline RET mutation is associated with only 3% of a population-based series of isolated HSCR, and little is known about susceptibility to sporadic MTC. We have found previously that specific haplotypes comprising RET coding single-nucleotide polymorphisms (SNPs) comprising exon 2 SNP A45A were strongly associated with HSCR, whereas haplotypes associated with exon 14 SNP S836S were associated with MTC. In this study, we describe three novel intron 1 SNPs, and, together with the coding SNP haplotypes, the data suggest the presence of distinct ancestral haplotypes for HSCR and sporadic MTC in linkage disequilibrium with a putative founding susceptibility locus/loci. The data are consistent with the presence of a very ancient, low-penetrance founder locus approximately 20-30 kb upstream of SNP A45A, but the failure of the SNPs to span the locus presents challenges in modeling mode of transmission or ancestry. We postulate that this founding locus is germane to both isolated HSCR and MTC but also that different mutations in this locus would predispose to one or the other.

Hereditary hemochromatosis is a common iron-loading disorder found in populations of European descent. It has been proposed that mutations causing loss of function of HFE gene result in reduced iron incorporation into immature duodenal crypt cells. These cells then overexpress genes for iron absorption, leading to inappropriate cellular iron balance, a persistent iron deficiency of the duodenal mucosa, and increased iron absorption. The objective was to measure duodenal iron content in Hfe knock-out mice to test whether the mutation causes a persistent decrease in enterocyte iron concentration. In both normal and Hfe knock-out mice, duodenal nonheme iron content was found to correlate with liver iron stores (P <.001, r = 0.643 and 0.551, respectively), and this effect did not depend on dietary iron levels. However, duodenal iron content was reduced in Hfe knock-out mice for any given content of liver iron stores (P <.001).

<h4>Background</h4>The incidence of deep vein thrombosis and pulmonary embolism following laparoscopic surgery is unknown and studies on alterations of hemostasis after laparoscopy are inconclusive.<h4>Methods</h4>In this study we prospectively evaluated changes in prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen (Fg), antithrombin III (ATIII), prothrombin fragment F 1 + 2, beta-thromboglobulin (betaTG) and D-dimer (D-D), preoperatively and 24 h after laparoscopic surgery in 16 patients.<h4>Results</h4>Comparing pre- and postoperative values, no statistical differences were observed in aPTT, F1 + 2, and ATIII measurements. Postoperative PT values increased slightly (p approximately 0.05) after surgery. Conversely, Fg, betaTG, and D-D values were statistically higher in the 24-h evaluation (p = 0.008, 0.01, and 0.045, respectively).<h4>Conclusions</h4>These data suggest that laparoscopic surgery induces activation of coagulation and fibrinolytic pathways and, additionaly, betaTG elevation, which has never been reported and might account for postoperative platelet activation and a greater risk of thrombogenicity. Therefore, routine thromboembolic prophylaxis in patients undergoing laparoscopic surgery is recommended.

Alterations in the genetic content of a cell are the underlying cause of many human diseases, including cancers. We have developed a method, called digital karyotyping, that provides quantitative analysis of DNA copy number at high resolution. This approach involves the isolation and enumeration of short sequence tags from specific genomic loci. Analysis of human cancer cells by using this method identified gross chromosomal changes as well as amplifications and deletions, including regions not previously known to be altered. Foreign DNA sequences not present in the normal human genome could also be readily identified. Digital karyotyping provides a broadly applicable means for systematic detection of DNA copy number changes on a genomic scale.

Overexpression studies have suggested that Siah1 proteins may act as effectors of p53-mediated cellular responses and as regulators of mitotic progression. We have tested these hypotheses using Siah gene knockout mice. Siah1a and Siah1b were not induced by activation of endogenous p53 in tissues, primary murine embryonic fibroblasts (MEFs) or thymocytes. Furthermore, primary MEFs lacking Siah1a, Siah1b, Siah2, or both Siah2 and Siah1a displayed normal cell cycle progression, proliferation, p53-mediated senescence, and G(1) phase cell cycle arrest. Primary thymocytes deficient for Siah1a, Siah2, or both Siah2 and Siah1a, E1A-transformed MEFs lacking Siah1a, Siah1b, or Siah2, and Siah1b-null ES cells all underwent normal p53-mediated apoptosis. Finally, inhibition of Siah1b expression in Siah2 Siah1a double-mutant cells failed to inhibit cell division, p53-mediated induction of p21 expression, or cell cycle arrest. Our loss-of-function experiments do not support a general role for Siah genes in p53-mediated responses or mitosis.

<h4>Background</h4>Mechanical prosthetic heart valve thrombosis is a serious complication with an incidence of 1-6%. The reduction in active vitamin-K dependent protein C and S levels caused by warfarin treatment also results in a prothrombotic state. This study was conducted to investigate the connection between protein C (PC), protein S (PS), antithrombin III (ATIII) deficiency and prosthetic mechanical valve thrombosis.<h4>Methods</h4>Twenty-nine of the 283 patients who underwent valve replacement with St. Jude medical prosthesis had mechanical valve thrombosis (group 2). The rest were considered as group 1. Twelve of the 29 patients (41.4%) had isolated aortic valve replacement, 12 had isolated mitral valve replacement (41.4%) and 5 patients had double valve replacement (17.2%). Most of the patients had rheumatic valve disease at their 1st operation. The mean time of occurrence for mechanical valve occlusion was 4.1+/-1.0 years following surgery.<h4>Results</h4>The values of PC, PS and ATIII were obtained when the mechanical valves stuck or at routine follow-up. PC, PS and ATIII levels were significantly lower in the mechanical valve thrombosis group. PC levels were 75.4+/-37.6% and 49.9+/-32.2% in group 1 and 2, respectively (p=0.001). PC, PS and ATIII values were mostly lower in the 2nd group but this difference only became significant after at least 2 years of warfarin usage.<h4>Conclusions</h4>Natural anticoagulant levels can be low during the use of warfarin. In which case the dose can be increased in order to hold the international normalized ratio (INR) at 3-3.5. However, more frequent follow-up is required and patients should be investigated for hypercoagulation states or deficiency in anticoagulant proteins. Patients referred to hospital with any mechanical valve thrombosis or recurrent thromboembolism should be evaluated for hypercoagulant proteins.

Conventional DNA hybridization assay kinetics depends solely on the diffusion of target to surface-bound probes, causing long hybridization times. In this study, we examined the possibilities of accelerating the hybridization process by using microfluidic channels ("biochannels") made of polycarbonate, optionally with an integrated pump. We produced two different devices to study these effects: first, hybridization kinetics was investigated by using an eSensor electrochemical DNA detection platform allowing kinetic measurements in homogenous solution. We fabricated an integrated cartridge for the chip comprising the channel network and a micropump for the oscillation of the hybridization mixture to further overcome diffusion limitations. As a model assay, we used an assay for the detection of single-nucleotide polymorphisms in the HFE-H gene. Second, based on the biochannel approach, we constructed a plastic microfluidic chip with a network of channels for optical detection of fluorescent-labeled targets. An assay for the simultaneous detection of four pathogenic bacteria surrogate strains from multiple samples was developed for this device. We observed high initial hybridization velocities and a fast attainment of equilibrium for the biochannel with integrated pump. Experimental results were compared with predictions generated by computer simulations.

Anti-beta -glycoprotein I antibody (abetaGPI) has been recognized in raising the risk of cerebral ischemia in patients with antiphospholipid antibody syndrome (APS), especially by protein C (PC) axis perturbation. Although a high potential is also seen in non-APS patients, the mechanism is substantially unknown. In the present study, we examined the effect of abetaGPI on PC and antithrombin-III (AT-III) activity in non-APS patients with non-cardiac cerebral ischemia (NCCI). A total of 111 NCCI patients and 30 healthy controls were enrolled. They were free of APS manifestation, and their anticardiolipin antibody and lupus anticoagulant tests were within normal range. There were 14.4% patients found to have an abnormal increase of blood abetaGPI. The PC, AT-III, albumin, aminotransferases, creatinine, prothrombin time and activated partial thromboplastin time did not differ between our patients and controls, or patients with or without increased abetaGPI. However, a marked decrease of the PC/AT-III ratio was found in patients with increased abetaGPI. The correlation between PC and AT-III activity was highly significant in patients with an increase of abetaGPI (P = 0.001), only marginal in controls (P = 0.042), and was insignificant in patients with a normal abetaGPI (P = 0.277). The abetaGPI did not correlate to PC or AT-III activity in either patients or controls. These findings suggest that high PC/AT-III coupling may relate to NCCI in non-APS patients associated with an increase of abetaGPI. This coupling effect seems not to be caused by abetaGPI directly.

<h4>Background</h4>There is increasing evidence for an interaction between iron and copper metabolism.<h4>Methods</h4>Iron indices (ferritin, transferrin saturation [TS], serum iron), liver parameters, the prevalence and significance of C282Y and H63D HFE mutations were studied in 40 unrelated, Caucasian patients with Wilson's disease and 295 healthy controls. Due to specific treatment Wilson's disease was well controlled in all but one patient.<h4>Results</h4>The allele frequencies for the C282Y (11.3% vs. 6.2%) and the H63D (18.8% vs. 16.4%) mutation did not differ between patients with Wilson's disease and healthy controls. One patient with C282Y homozygous HH and Wilson's disease was identified showing progressive liver disease despite reasonable venesection and copper chelation therapy. No differences in iron indices and liver values were seen between HFE heterozygous and HFE wildtype patients with Wilson's disease. Higher serum ferritin levels were noticed in patients with Wilson's disease compared to healthy controls (149 +/- 26 microg/l vs. 87 +/- 8 microg/l; P < 0.03).<h4>Conclusions</h4>It appears reasonable to assess iron indices in patients with Wilson's disease in order to detect iron overload. HFE mutations other than C282Y homozygosity seem to have no impact on iron indices and liver parameters as long as Wilson's disease is controlled.

<h4>Background/aims</h4>Several abnormalities in the immune status of hereditary hemochromatosis patients have been reported. We evaluated the peripheral blood lymphocytes phenotype and cytokine profile of CD8(+) and CD4(+) T cells in C282Y homozygous hereditary hemochromatosis patients compared to control subjects.<h4>Methods</h4>Peripheral blood lymphocytes from 17 asymptomatic patients and 14 control subjects were analyzed. We determined the distribution of lymphocyte subsets and investigated at single-cell level by flow-cytometry the potential of cytokines production. The frequency of cytokine (interferon gamma, tumor necrosis factor alpha, interleukin 2 (IL-2), IL-4, IL-5, IL-10 and IL-13) producing cells was assessed in total T-lymphocytes, CD3(+)CD8(+) and CD3(+)CD4(+) subsets.<h4>Results</h4>The patients studied showed a significant decrease of total lymphocyte count, T CD4(+)CD3(+), CD28(+), CD8(+)CD28(+) lymphocytes and natural killer (NK) CD56(+)CD16(+)CD3(-) cells. The reduction of CD28(+) and CD8(+)CD28(+) lymphocyte count was inversely related to transferrin saturation index. An increase in the ability of T-cells to produce all the cytokines studied and a major increase in IL-4 and IL-10 production in the CD3(+)CD8(+) subset was found. Our results demonstrate that activated Th1 and Th2 lymphocytes coexist in the peripheral blood of hereditary hemochromatosis patients and that T-cytotoxic (Tc) 2 subset is more expanded than in control population.<h4>Conclusions</h4>The association of a decreased number of T CD8(+) cytotoxic lymphocytes and NK cells, and the development of Tc2 cells in asymptomatic C282Y homozygous patients represents an imbalance in their immune function that might contribute to the high incidence of hepatocarcinoma.

<h4>Background/aims</h4>Recent evidence implicates iron as a comorbid factor for development of non-hemochromatotic liver diseases. Mutations or polymorphisms in the HFE gene or the TfR1 gene may influence the accumulation of iron in the liver or other tissues or may influence chronic viral hepatitis apart from effects on iron homeostasis. The aim of this study was to assess the role of hepatic iron, HFE and TfR1 variations on development and progression of chronic hepatitis C infection.<h4>Methods</h4>We studied 119 consecutive patients with chronic hepatitis C, correlating clinical, laboratory, histopathological, and genetic data. Frequencies of genetic variations were compared with local and national controls.<h4>Results</h4>HFE mutations were more common in patients than controls (48% vs. 38%, P=0.04), and advanced degrees of fibrosis developed at younger ages in subjects with the C282Y mutation (38.6 vs. 46.5 years, P=0.03). Patients carrying C282Y had higher mean hepatic iron concentrations (P=0.02), hepatic iron indices (P<=0.0001), and hepatic fibrosis scores (P=0.01). Hepatic fibrosis was correlated with hepatic iron concentration (P=0.03). TfR1 polymorphisms bore no detectable relation to disease severity or response to therapy.<h4>Conclusions</h4>Hepatic iron and HFE mutations are comorbid factors that increase development and progression of chronic hepatitis C.

<h4>Background</h4>There is a marked difference in the degree of expression of the homozygous C282Y HFE genotype that is associated with hereditary hemochromatosis. It has been reported that individuals with the haptoglobin 2-2 type manifest increased iron concentrations, including serum iron, transferrin saturation, and ferritin.<h4>Methods</h4>We studied 232 patients, 115 homozygous for the c.845G-->A (C282Y) mutation and 117 matched controls with the wild-type HFE genotype, for haptoglobin phenotypes. Haptoglobin types were determined by electrophoresis of the denatured protein. The HFE genotype was determined by allele-specific oligonucleotide hybridization. Ferritin and transferrin saturation were measured by standard methods.<h4>Results</h4>There was no relationship between haptoglobin type and ferritin concentration or transferrin saturation.<h4>Conclusions</h4>The effect of haptoglobin type on iron homeostasis cannot account for the marked phenotypic variation that is seen in patients homozygous for the HFE C282Y mutation.

This study examined whether serotonin transporter (5-HTT) gene knockout influences adrenomedullary, sympathoneural, or hypothalamo-pituitary-adrenal responses to acute immobilization. In conscious, cannulated mice, arterial plasma concentrations of catecholamines, ACTH, and corticosterone were measured at baseline and after 15 min of immobilization. Tissue levels of serotonin (5-HT), catecholamines, and hormones were also measured in pituitary and adrenal glands. At baseline, adrenal and pituitary 5-HT concentrations in knockout (5-HTT(-/-)) mice were markedly lower than those in littermate control (5-HTT(+/+)) mice, whereas the groups did not differ in levels of catecholamines or hormones in plasma or tissue. Immobilization increased plasma levels of catecholamines, ACTH, and corticosterone in all genotypes. 5-HTT(-/-) mice had exaggerated responses of plasma epinephrine to immobilization and significant reductions in adrenal epinephrine, norepinephrine, and 5-HT contents compared with values in littermate controls. Pituitary ACTH was significantly reduced after immobilization in 5-HTT(-/-) mice only, but increases in plasma ACTH and corticosterone levels did not differ between genotypes. The results suggest that one 5-HTT function is to restrain adrenomedullary activation in response to immobilization. Exaggerated adrenomedullary responses seem to be an autonomic correlate of the anxiety-like behaviors in 5-HTT knockout mice.

The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 consists of terminal oxygenase (CarAa), ferredoxin (CarAc), and ferredoxin reductase (CarAd). Each component of CARDO was expressed in Escherichia coli strain BL21(DE3) as a native form (CarAa) or a His-tagged form (CarAc and CarAd) and was purified to apparent homogeneity. CarAa was found to be trimeric and to have one Rieske type [2Fe-2S] cluster and one mononuclear iron center in each monomer. Both His-tagged proteins were found to be monomeric and to contain the prosthetic groups predicted from the deduced amino acid sequence (His-tagged CarAd, one FAD and one [2Fe-2S] cluster per monomer protein; His-tagged CarAc, one Rieske type [2Fe-2S] cluster per monomer protein). Both NADH and NADPH were effective as electron donors for His-tagged CarAd. However, since the k(cat)/K(m) for NADH is 22.3-fold higher than that for NADPH in the 2,6-dichlorophenolindophenol reductase assay, NADH was supposed to be the physiological electron donor of CarAd. In the presence of NADH, His-tagged CarAc was reduced by His-tagged CarAd. Similarly, CarAa was reduced by His-tagged CarAc, His-tagged CarAd, and NADH. The three purified proteins could reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc seemed to be indispensable for electron transport, while His-tagged CarAd could be replaced by some unrelated reductases.

Migrating axons require the correct presentation of guidance molecules, often at multiple choice points, to find their target. Netrin 1, a bifunctional cue involved in both attracting and repelling axons, is involved in many cell migration and axon pathfinding processes in the CNS. The netrin 1 receptor DCC and its Caenorhabditis elegans homolog UNC-40 have been implicated in directing the guidance of axons toward netrin sources, whereas the C. elegans UNC-6 receptor, UNC-5 is necessary for migrations away from UNC-6. However, a role of vertebrate UNC-5 homologs in axonal migration has not been demonstrated. We demonstrate that the Unc5h3 gene product, shown previously to regulate cerebellar granule cell migrations, also controls the guidance of the corticospinal tract, the major tract responsible for coordination of limb movements. Furthermore, we show that corticospinal tract fibers respond differently to loss of UNC5H3. In addition, we observe corticospinal tract defects in mice homozygous for a spontaneous mutation that truncates the Dcc transcript. Postnatal day 0 netrin 1 mutant mice also demonstrate corticospinal tract abnormalities. Last, interactions between the Dcc and Unc5h3 mutations were observed in gene dosage experiments. This is the first evidence of an involvement in axon guidance for any member of the vertebrate unc-5 family and confirms that both the cellular and axonal guidance functions of C. elegans unc-5 have been conserved in vertebrates.

Hereditary haemochromatosis is the prototype disease for primary iron overload. The disorder is very common, especially amongst subjects of Northern European extraction. It is characterized by an autosomal recessive mode of inheritance, and most cases are homozygous for the C282Y mutation in the HFE gene. Haemochromatosis is now recognized to be a complex genetic disease with probable significant environmental and genetic modifying factors. The early diagnosis of individuals at risk for the development of haemochromatosis is important, because survival and morbidity are improved if phlebotomy therapy is instituted before the development of cirrhosis. The cost-effectiveness and utility of large-scale screening for haemochromatosis have been questioned given that many individuals with the homozygous C282Y mutation do not have iron overload or end-organ damage. However, the use of phenotypic tests, such as serum transferrin-iron saturation, for initial screening avoids the problem of the identification of non-expressing homozygotes. Liver biopsy remains important in management to determine the presence or absence of cirrhosis, particularly amongst patients with serum ferritin levels greater than 1000 ng/mL or elevated liver enzymes. Those with non-HFE haemochromatosis who cannot be identified on genotypic testing should have a liver biopsy to establish diagnosis. Patients with end-stage liver disease may develop liver failure or primary liver cancer, and liver transplantation may be required. Liver transplantation for haemochromatosis is associated with a poorer outcome compared with other indications because of infections and cardiac complications.

Endocrine-disrupting organochlorines, such as the pesticide dichlorodiphenyltrichloroethane (DDT), bind to and activate estrogen receptors (ERs), thereby eliciting estrogen-like effects. Although ERs function predominantly through activation of transcription via estrogen-responsive elements, both ERs, alpha and ss, can interact with various transcription factors such as activator protein-1 (AP-1). Additionally, estrogens may regulate early signaling events, suggesting that the biological effects of environmental estrogens may not be mediated through classic ER (alpha and ss) activity alone. We hypothesized that known environmental estrogens, such as DDT and its metabolites, activate AP-1-mediated gene transactivation through both ER-dependent and ER-independent means. Using two Ishikawa human endometrial adenocarcinoma cell line variants that we confirmed to be estrogen responsive [Ishikawa(+)] and estrogen unresponsive [Ishikawa(-)], we generated stably transfected AP-1 luciferase cell lines to identify the role of an estrogen-responsive mechanism in AP-1-mediated gene expression by various stimuli. Our results demonstrate that DDT and dichlorodiphenyldichloroethane (DDD) were the most potent activators of AP-1 activity; 2,2-bis(p-chlorophenyl) acetic acid failed to activate. Although stimulated in both Ishikawa(+) and Ishikawa(-) cells by DDT and its congeners, AP-1 activation was more pronounced in the estrogen-unresponsive Ishikawa(-) cells. In addition, DDT, DDD, and dichlorodiphenyldichloroethylene (DDE) could also stimulate AP-1 activity in the estrogen-unresponsive human embryonic kidney 293 cells using a different promoter context. Thus, our data demonstrate that DDT and its metabolites activate the AP-1 transcription factor independent of ER (alpha or ss) status.

We analyzed a prostate cancer xenograft derived from a locally advanced tumor using combined cytogenetic, array-based comparative genomic hybridization and expression analyses. This analysis revealed that genes in the 20q13 chromosomal region, CSE1L, ZNF217, MYBL2, and STK15, were significantly overexpressed in this tumor. The expression pattern of these genes was then confirmed in two large human prostate cancer microarray databases. Furthermore, the MYBL2 and STK15 have been significantly overexpressed in prostate metastases, allowing a clear distinction between localized tumors and metastases. Our data suggest these genes to be involved in advanced stages of prostate tumorigenesis and as such, they may serve as markers for tumor progression.

Because of the major clinical impact of bronchial cancer worldwide, the possibility that human papillomavirus (HPV) contributes to its pathogenesis as a co-carcinogen is an intriguing one. Bronchial squamous cell carcinoma develops through well defined precursor lesions, often at the sites of squamous metaplasia. Benign squamous cell papillomas are rare but HPV DNA has been found in almost half of those studied, implicating a causal association. In invasive bronchial cancer, morphological changes seen in HPV lesions elsewhere are often seen. HPV DNA has been detected in 21.7% of the 2,468 bronchial carcinomas analysed to date and the same high risk types implicated in other squamous cell cancers have been identified. Clearly, more effort should be focused on assessing the role of HPV in bronchial carcinogenesis, by analysing the synergistic effects of carcinogenic agents (cigarette smoke, radiation, asbestos, etc) and HPV in different experimental settings.

As a resident of early endosomal phagosomes, Mycobacterium tuberculosis is connected to the iron uptake system of the host macrophage. beta-2-microglobulin (beta2m) knockout (KO) mice are more susceptible to tuberculosis than wild-type mice, which is generally taken as a proof for the role of major histocompatibility complex class I (MHC-I)-restricted CD8 T cells in protection against M. tuberculosis. However, beta2m associates with a number of MHC-I-like proteins, including HFE. This protein regulates transferrin receptor mediated iron uptake and mutations in its gene cause hereditary iron overload (hemochromatosis). Accordingly, beta2m-deficient mice suffer from tissue iron overload. Here, we show that modulating the extracellular iron pool in beta2m-KO mice by lactoferrin treatment significantly reduces the burden of M. tuberculosis to numbers comparable to those observed in MHC class I-KO mice. In parallel, the generation of nitric oxide impaired in beta2m-KO mice was rescued. Conversely, iron overload in the immunocompetent host exacerbated disease. Consistent with this, iron deprivation in infected resting macrophages was detrimental for intracellular mycobacteria. Our data establish: (a) defective iron metabolism explains the increased susceptibility of beta2m-KO mice over MHC-I-KO mice, and (b) iron overload represents an exacerbating cofactor for tuberculosis.

Inhibition of the serotonin transporter (5-HTT) is a principal initial target of many antidepressants. However, the contribution of the 5-HTT to their therapeutic efficacy is incompletely understood. We utilized a targeted gene mutation approach to examine the role of the 5-HTT in the behavioral actions of antidepressants. The 5-HTT mutation was bred onto two separate genetic backgrounds, C57BL/6J and 129S6. On a preliminary screen for gross physical, neurological and behavioral functions, all measures were normal with the exception that 5-HTT -/- mice on the C57BL/6J background showed increased body weight and poor rotarod performance, and 5-HTT -/- mice on the 129S6 background showed reduced neuromuscular strength. On the tail suspension test, 5-HTT -/- mice on the 129S6 background showed a baseline antidepressant-like reduction in immobility. In contrast, the same mice showed increased immobility in the forced swim test, possibly due to compromised neuromuscular strength. 5-HTT -/- mice on the C57BL/6J background showed no baseline antidepressant-related phenotype on either test. The behavioral effects of three antidepressants were tested in 5-HTT mutant mice (C57BL/6J background) in the tail suspension test. The anti-immobility effects of the serotonin reuptake inhibitor, fluoxetine (30 mg/kg), were abolished in 5-HTT -/- mice, confirming that the 5-HTT gene is required for the behavioral effects of fluoxetine. In contrast, 5-HTT-/- mice retained sensitivity to the anti-immobility effects of the norepinephrine reuptake inhibitor, desipramine (20 mg/kg), and the mixed serotonin/norepinephrine reuptake inhibitor, imipramine (25 mg/kg). 5-HTT knockout mice provide a valuable tool for delineating the neuropsychopharmacological actions of antidepressants.

No abstract available.

The unique property of stem cells to self-renew suggests specific mechanisms that regulate their cell-cycle progression. In the present study, we identify a novel protein, nucleostemin, found in the nucleoli of CNS stem cells, embryonic stem cells, and several cancer cell lines and preferentially expressed by other stem cell-enriched populations. It contains an N-terminal basic domain and two GTP-binding motifs. When stem cells differentiate, nucleostemin expression decreases rapidly prior to cell-cycle exit both in vitro and in vivo. Depletion or overexpression of nucleostemin reduces cell proliferation in CNS stem cells and transformed cells. Mutation analysis indicates that excessive nucleostemin, particularly mutants that lack the GTP-regulatory domain, prevents cells from entering mitosis and causes apoptosis in a p53-dependent manner. The N-terminal basic domain specifies nucleolar localization, the p53 interaction, and is required for the cell death caused by overexpression. This work describes a novel nucleolar mechanism that controls the cell-cycle progression in CNS stem cells and cancer cells.

The establishment of metaphase chromosomes is an essential prerequisite of sister chromatid separation in anaphase. It involves the coordinated action of cohesin and condensin, protein complexes that mediate cohesion and condensation, respectively. In metazoans, most cohesin dissociates from chromatin at prophase, coincident with association of condensin. Whether loosening of cohesion at the onset of mitosis facilitates the compaction process, resolution of the sister chromatids, or both, remains unknown. We have found that the prophase release of cohesin is completely blocked when two mitotic kinases, aurora B and polo-like kinase (Plx1), are simultaneously depleted from Xenopus egg extracts. Condensin loading onto chromatin is not affected under this condition, and rod-shaped chromosomes are produced that show an apparently normal level of compaction. However, the resolution of sister chromatids within these chromosomes is severely compromised. This is not because of inhibition of topoisomerase II activity that is also required for the resolution process. We propose that aurora B and Plx1 cooperate to destabilize the sister chromatid linkage through distinct mechanisms that may involve phosphorylation of histone H3 and cohesin, respectively. More importantly, our results strongly suggest that cohesin release at the onset of mitosis is essential for sister chromatid resolution but not for condensin-mediated compaction.

Mutations in NPHS2, encoding podocin, have been identified in childhood onset focal and segmental glomerulosclerosis (FSGS). The role of NPHS2 in adult disease is less well defined. We studied 30 families with FSGS and apparent autosomal recessive inheritance and 91 individuals with primary FSGS. We screened family members for NPHS2 mutations. NPHS2 mutations appeared to be responsible for disease in nine of these families. In six families, the affected individuals were compound heterozygotes for a nonconservative R229Q amino acid substitution. This R229Q variant has an allele frequency of 3.6% in a control population. In these families, R229Q was the only mutation identified on one of the two disease-associated NPHS2 alleles. We used in vitro-translated podocin and purified nephrin to investigate the effect of R229Q on their interaction and found decreased nephrin binding to the R229Q podocin. These data suggest that this common polymorphism contributes to the development of FSGS. Chromosomes bearing the R229Q mutation share a common haplotype defining an approximately 0.2-Mb region. R229Q appears to enhance susceptibility to FSGS in association with a second mutant NPHS2 allele. Identification of R229Q mutations may be of clinical importance, as NPHS2-associated disease appears to define a subgroup of FSGS patients unresponsive to corticosteroids.

1. Increasing the lipophilicity is a strategy often used to improve a compound's cellular uptake and retention but this may also convert it into a substrate for an ATP-dependent transporter such as P-glycoprotein or the multidrug resistance-associated protein (MRP1), which are involved in cellular efflux of drugs. Tris-Lipidation of compounds is a convenient way of modifying drug lipophilicity and generating an array of derivatives with diverse properties. 2. To determine the effect of Tris-Lipidation on a drug's cytoxicity in multidrug resistant cells, various glycyl-Tris-mono- (GTP1), di- (GTP2) and tri-palmitate (GTP3) derivatives were prepared of the cancer chemotherapeutic drugs chlorambucil and methotrexate, and of the anti-HIV drug AZT. The cytotoxicity of these derivatives and their parent compounds was determined in the CEM/VLB(100) cells with increased P-glycoprotein expression, the CEM/E1000 cells that overexpress MRP1 and the parent, drug-sensitive CCRF-CEM cells. 3. Increasing the lipophilicity of AZT increased its cytotoxicity in the sensitive CCRF-CEM parental cell line while decreased cytotoxicity was observed for the methotrexate derivatives. For the chlorambucil derivatives, both increased (GTP1) and decreased (GTP2) cytotoxicity occurred in the CCRF-CEM cells. With the exception of AZT-GTP1, all GTP1 and GTP2 derivatives of chlorambucil, methotrexate and AZT had decreased cytotoxicity in the P-glycoprotein-expressing CEM/VLB(100) cells while chlorambucil-GTP1, methotrexate-GTP2 and methotrexate-GTP3 were the only compounds with decreased cytotoxicity in the MRP1-overexpressing CEM/E1000 cells. 4. The number of palmitate residues, the position of derivatisation and the type of linkage all may affect the P-glycoprotein and MRP1 substrate properties. 5. Tris-Lipidation may therefore provide a useful way of manipulating the pharmacokinetic properties of drugs.

Evidence is provided that some condensed linker histone-containing chromatin structures are highly flexible in solutions containing 2 mM Mg2+. Chromatin assembled in vitro +/- histone H5 on a 6.3 kb linear DNA fragment in 90 mM NaCl using the polyglutamic acid method sedimented fairly homogeneously. The H5-containing sample had s(20, w) values that were 58-69% greater than the sample lacking H5. Chromatin assembled on linear pUC19 plasmid DNA was treated with T4 DNA ligase in solutions containing 2 mM Mg2+ over a range of DNA concentrations. It was found that the intramolecular DNA ends of the chromatin could be joined together more efficiently than the intramolecular ends of the naked DNA at the higher DNA concentrations. This result could not be attributed to the effective reduction in DNA length by nucleosome formation. The chromatin structures formed did not have naked DNA tails extending from the ends as assessed by exonuclease III digestion. Chromatin assembled on DNA shortened by up to 420 bp gave very similar results, suggesting that the structure was a flexible one, rather than a rigid one having DNA ends that were fortuitously juxtaposed.

Expansion of a CAG tract within the huntingtin gene, leading to the production of a protein with an expanded polyglutamine tract, is responsible for Huntington's disease. We show here that the 5' untranslated region (UTR) of the huntingtin gene plays an important role in controlling the synthesis of huntingtin. In particular, the 5' UTR contains an upstream open reading frame (uORF) encoding a 21 amino acid peptide. We demonstrate that the presence of this uORF negatively influences expression from the huntingtin mRNA. Our results suggest a role for the uORF in limiting ribosomal access to downstream initiation sites. Mechanisms involving the post-transcriptional regulation of huntingtin are not well understood, and this may be an important way of regulating huntingtin protein levels.

The anion [Fe(2)(S(2)C(3)H(6))(CN)(CO)(4)(PMe(3))](-) (2(-)) is protonated by sulfuric or toluenesulfonic acid to give HFe(2)(S(2)C(3)H(6))(CN)(CO)(4)(PMe(3)) (2H), the structure of which has the hydride bridging the Fe atoms with the PMe(3) and CN(-) trans to the same sulfur atom. (1)H, (13)C, and (31)P NMR spectroscopy revealed that HFe(2)(S(2)C(3)H(6))(CN)(CO)(4)(PMe(3)) is stereochemically rigid on the NMR time scale with four inequivalent carbonyl ligands. Treatment of 2(-) with (Me(3)O)BF(4) gave Fe(2)(S(2)C(3)H(6))(CNMe)(CO)(4)(PMe(3)) (2Me). The Et(4)NCN-induced reaction of Fe(2)(S(2)C(3)H(6))(CO)(6) with P(OMe)(3) gave [Fe(2)(S(2)C(3)H(6))(CN)(CO)(4)[P(OMe)(3)]](-) (4). Spectroscopic and electrochemical measurements indicate that 2H can be further protonated at nitrogen to give [HFe(2)(S(2)C(3)H(6))(CNH)(CO)(4)(PMe(3))](+) (2H(2)(+)). Electrochemical and analytical data show that reduction of 2H(2)(+) gives H(2) and 2(-). Parallel electrochemical studies on [HFe(2)(S(2)C(3)H(6))(CO)(4)(PMe(3))(2)](+) (3H(+)) in acidic solutions led also to catalytic proton reduction. The 3H(+)/3H couple is reversible, whereas the 2H(2)(+)/2H(2) couple is not, because of the efficiency of the latter as a proton reduction catalyst. Proton reduction is proposed to involve protonation of reduced diiron hydrides. DFT calculations establish that the regiochemistry of protonation is subtly dependent on the coligands but is more favorable to occur at the Fe-Fe bond for [Fe(2)(S(2)C(3)H(6))(CN)(CO)(4)(PMe(3))](-) than for [Fe(2)(S(2)C(3)H(6))(CN)(CO)(4)(PH(3))](-) or [Fe(2)(S(2)C(3)H(6))(CN)(CO)(4)[P(OMe)(3)]](-). The Fe(2)H unit stabilizes the conformer with eclipsed CN and PMe(3) because of an attractive electrostatic interaction between these ligands.

Huntington's disease (HD) and spinocerebellar ataxia type 7 (SCA7) belong to a group of progressive neurodegenerative diseases caused by polyglutamine (polyQ) expansions. SCA7 is the only one to display degeneration in the retina, a tissue usually spared in HD. We previously described a SCA7 transgenic retinal model expressing mutant full length ataxin-7 in rod photoreceptors. These mice develop a severe and characteristic retinopathy. We show here that R6 transgenic mice, which reproduce many features of HD, express mutant huntingtin in the retina leading to strong vision deficiencies and retinal dystrophy. These two different polyQ mouse models exhibit comparable early and progressive retinal degeneration and dysfunction. These abnormalities are reminiscent of other retinal degeneration phenotypes (in particular rd7/rd7 mice) where photoreceptor cell loss occurs. Retinopathy in R6 and R7E models can be monitored in living mice by ERG and fundus examination, which can facilitate in vivo evaluation of therapeutic agents in polyQ disorders.

<h4>Background</h4>The increased thromboembolic risk associated with total hip arthroplasty is multifactorial. We assessed whether the prevalence of abnormalities shown by newer genetic screening tests for thrombophilia and hypofibrinolysis was higher in patients in whom pulmonary embolism had developed after total hip arthroplasty than it was in matched control patients.<h4>Methods</h4>Fourteen patients with documented pulmonary embolism after total hip arthroplasty and fourteen matched control patients who had undergone total hip arthroplasty without any clinical indication of thromboembolism were evaluated for risks of thrombophilia and hypofibrinolysis. Functional tests of hemostasis included evaluations of prothrombin time; activated partial thromboplastin time; levels of fibrinogen, serum homocysteine, protein C and S, and antithrombin III; activated protein-C resistance; and dilute Russell viper venom time. Molecular genetic testing was performed for factor-V Leiden, prothrombin promoter G20210A, methylenetetrahydrofolate reductase C677T, plasminogen activator inhibitor-1 4G/4G, and platelet glycoprotein IIb/IIIa A1/A2 or A2/A2 mutations.<h4>Results</h4>The total number of genetic thrombophilic abnormalities identified was higher in the pulmonary embolism group (twenty-four abnormalities) than in the control group (fifteen abnormalities). Only patients with pulmonary embolism were found to have heterozygosity or homozygosity for the prothrombin G20210A mutation (four of fourteen patients; p = 0.05 compared with the control group) and a decreased antithrombin-III level (three of thirteen patients; p = 0.10 compared with the control group). Patients with pulmonary embolism were much more likely than control patients to have at least one thrombophilic abnormality: seven of fourteen patients with pulmonary embolism had a low antithrombin-III level or the prothrombin G20210A gene mutation compared with none of the fourteen in the control group (Fisher exact test, p < 0.01). The presence of the prothrombin G20210A gene mutation was significantly correlated with pulmonary embolism (r = 0.41, p = 0.03), as was the presence of least one abnormality (a low antithrombin-III level or the presence of the prothrombin G20210A gene mutation) (r = 0.58, p = 0.001).<h4>Conclusions</h4>Genetic thrombophilia and hypofibrinolysis were more frequent in patients who had had pulmonary embolism after total hip arthroplasty than in those who had not. The presence of multiple genetic thrombophilic polymorphisms, particularly prothrombin G20210A and antithrombin III, rather than any single genetic prothrombotic abnormality, appears to signal an increased thromboembolic risk in patients undergoing total hip arthroplasty. Future refinements and availability of these tests will likely allow preoperative identification of patients with an increased genetic predisposition for thromboembolism.

No abstract available.

Recent studies suggest that molecules important for guiding neuronal migration and axon path-finding also play a role in modulating leukocyte chemotaxis. Neuronal migration and leukocyte chemotaxis may share some common regulatory mechanisms. Intracellular signal transduction mechanisms guiding neuronal migration and leukocyte chemotaxis are beginning to be elucidated. Studying molecular mechanisms modulating cell migration may provide new insights into understanding of endogenous inhibitors of inflammation.

To characterize the metabolic role of peroxisomes in yeast cells under physiological conditions, we performed a comprehensive meta-analysis of published microarray data. Previous studies of yeast peroxisomes have mainly been focused on the function of peroxisomes under extreme conditions, such as growth on oleate or methanol as the sole carbon source, and may therefore not be representative of the normal physiological role of yeast peroxisomes. Surprisingly, our analysis of the microarray data reveals that the only pathway responding to peroxisome deficiency in mid-log phase is lysine biosynthesis, whereas classical peroxisomal pathways such as beta-oxidation are unaffected. We show that the upregulation of lysine biosynthesis genes in peroxisome-deficient yeasts shares many characteristics with the physiological response to lysine starvation. We provide data that suggest that this is the result of a "pathological" stimulation of the Lys14p transcriptional activator by the pathway intermediate aminoadipate semialdehyde. Mistargeting of the peroxisomal lysine pathway to the cytosol increases the active concentration of aminoadipate semialdehyde, which is no longer contained in the peroxisome and can now activate Lys14p at much lower levels than in wild-type yeasts. This is the first well-documented example of pathway misregulation in response to peroxisome deficiency and will be useful in understanding the phenotypic details of human peroxisome-deficient patients (Zellweger syndrome).

No abstract available.

The high-mobility-group (HMG) domain containing transcription factor Sox10 is an important regulator of various processes including the development of neural crest cells and glial cells. Target gene promoters contain multiple Sox10-binding sites, which either support monomeric or cooperative, dimeric binding. The latter is unusual for Sox proteins and might contribute to functional specificity of Sox10. We find that specific amino acid residues in a conserved region immediately preceding the HMG domain of Sox10 are required for cooperative binding. These residues cooperate with the HMG domain during dimeric binding in a manner dependent on specific determinants within the first two alpha-helices of the HMG domain. Cooperativity of DNA binding is surprisingly refractory to changes in the overall conformation of the DNA-bound dimer. Whereas maintenance of cooperativity is essential for full activation of the promoter of the myelin protein zero target gene, dimer-dependent conformational changes such as the exact bending angle introduced into the promoter appear to be less important, shedding new light on the architectural function of Sox proteins.

<h4>Aims</h4>Heparin releases tissue factor pathway inhibitor (TFPI) from the endothelium and this release may decrease after repeated high dose heparin administration. The primary aim was to investigate and quantify this phenomenon during a short low dose heparin infusion. Also, the effects of heparin on tissue plasminogen activator (t-PA) were studied.<h4>Methods</h4>Nine healthy, nonsmoking, male volunteers (range 19-23 years) received a continuous heparin infusion (2000 IU) over 40 min. The endothelial TFPI release rate was estimated from the total TFPI concentration profile using a pharmacokinetic model.<h4>Results</h4>Mean +/- s.d. total and free TFPI increased from 62.9 +/- 9.4/8.3 +/- 2.1 ng ml-1 at baseline to 237.2 +/- 40.9/111.0 +/- 19.9 ng ml-1 after 40 min infusion. The relationship between heparin concentration (anti-IIa activity) and TFPI concentration followed a maximum effect model and a clockwise loop (proteresis) was observed. The TFPI release rate rapidly increased to maximum of 200 +/- 45 micro g min-1 after 17.5 min heparin infusion but did not increase further although heparin concentrations further doubled. In contrast to TFPI, t-PA antigen decreased from 5.6 +/- 1.0 at baseline to 4.5 +/- 1.0 ng ml-1 at the end of infusion (t = 40 min) (difference of 1.1 ng ml-1 (95% confidence interval; 0.9, 1.3).<h4>Conclusions</h4>Our application of concentration-effect models and pharmacokinetic principles to these haemostatic variables showed that endothelial TFPI release has a maximum that is already reached at low heparin dose, corresponding with an anti-IIa activity of 0.08 IU ml-1. The relationship between anti-IIa activity and TFPI release rate showed signs of acute tolerance (clockwise loop) indicating exhaustion of endothelial TFPI pools. These findings may be of importance for the heparin dose used in conditions such as unstable angina, in which the favourable effects of heparin have been ascribed to its ability to release TFPI.

Recent years have witnessed important advances in the understanding of the pathophysiology of primary pulmonary hypertension (PPH). Both genetic and mechanistic studies have succeeded in identifying new molecular pathways relevant to the process of pulmonary vascular remodelling, which underlies PPH. Mutations in the type II bone morphogenetic protein (BMP) receptor (BMPR)-II are now considered to be the genetic basis for familial PPH and approximately 30% of cases of sporadic PPH. The identification of the relevance of the BMP pathway to the aetiology of PPH now raises many questions about the link between the BMPR-II mutant genotype and the PPH phenotype. As PPH does not develop in all subjects with BMPR-II mutations, environmental or associated genetic factors may play a crucial role. Among these, the finding of an association between PPH and the L-allelic variant of the serotonin transporter (5-HTT) gene indicates that 5-HTT, which controls smooth muscle hyperplasia, probably contributes to susceptibility to PPH or is an important modifier of the PPH phenotype. Recognition of these molecular pathways should provide insight into the pathogenesis not only of primary pulmonary hypertension, but also of secondary forms of pulmonary hypertension. This should soon lead to the development of new and more selective therapeutic approaches to pulmonary hypertension.

We have identified and cloned GLUT14, a novel member of the glucose transporter family. GLUT14 (SLC2A14) maps to chromosome 12p13.3 (17.1M), about 10 Mb upstream of GLUT3, with which it shares remarkable identity. Until now GLUT14 was thought to be a pseudogene. It consists of 11 exons with a genomic organization similar to that of GLUT3 and likely resulted from a duplication of GLUT3. GLUT14 has two alternatively spliced forms; the shorter form of GLUT14 (GLUT14-S) consists of 10 exons and produces a 497-amino-acid protein that is 94.5% identical to GLUT3. The long form (GLUT14-L) has an additional exon and codes for a protein with 520 amino acids that differs from GLUT14-S only at the N-terminus. GLUT14-S/L contain 12 putative membrane-spanning helices along with sugar-transporter signature motifs that have previously been shown to be essential for sugar transport activity. The putative glycosylation sites of GLUT14-S/L are present in loop 1. In contrast to the expression of GLUT3 in many tissues, both isoforms of GLUT14 are specifically expressed in testis. The mRNA level of GLUT14 in testis is about four times higher than that of GLUT3. Interestingly, the ortholog of GLUT14 is not found in mice. The multiple duplications of GLUT genes suggest that the GLUT family probably emerged by gene duplications and mutations during evolution in different lineages.

Primary hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. However, the viral-chemical etiology as well as molecular mechanisms of HCC pathogenesis remains largely unknown. Recent studies in our laboratory have identified several potential factors that may contribute to the pathogenesis of HCC. Oxidative stress and chronic inflammation have been linked to an increased risk of liver cancer. For example, oxyradical overload diseases such as Wilson disease and hemochromatosis result in the generation of oxygen/nitrogen species that can cause mutations in the p53 tumor suppressor gene. The Hepatitis B virus X gene (HBx), a viral transactivator with oncogenic potentials, has been shown to bind to and inactivate p53-mediated apoptosis. HBx mutants derived from HCC have a diminished ability to act as a transactivator. However, they still retain the ability to bind to and abrogate p53-mediated apoptosis. The comparison of gene expression profiles between HBx-expressing primary human hepatocytes and HBV-infected liver samples by cDNA microarrays indicate a unique alteration of a subset of oncogenes and tumor suppressor genes including p53. Our studies implicate both viral and endogenous chemical processes in the etiology of HCC, and p53 may be a common target for the inactivation during liver carcinogenesis.

<h4>Background</h4>The actual prevalence and the clinical relevance of gene mutations of HFE (which are linked to hemochromatosis) have not yet been established in patients on chronic dialysis. On the basis of theoretical premises, it could be hypothesized that these genetic determinants might influence the response to iron intake and the susceptibility for iron overload in patients in parenteral iron therapy. Furthermore, carriers for these mutations might be prone to develop sporadic porphyria cutanea tarda and cardiovascular events.<h4>Methods</h4>C282Y/H63D mutations of HFE gene were evaluated in 132 patients (34 in peritoneal dialysis, 98 in HD) and correlated with biochemical parameters of iron status (ferritin (FER) concentration and transferrin saturation (TSAT)), red cell parameters (red cell size and hemoglobin content), erythropoietin (EPO) dosage, major cardiovascular events and C-reactive protein as marker of chronic inflammation, in patients without iron therapy and after i.v. iron supplementation (< or = 60 mg/week) and with the presence of biopsy-proven porphyria.<h4>Results</h4>C282Y heterozygous mutation was found in 8/132 (6.6%); H63D homozygous and heterozygous mutations were found in 3/132 (2.3%) and 22/132 (16%) patients, respectively. Two patients (1.5%) showed double heterozygosis. No differences in baseline serum FER and TSAT and the other biochemical and clinical parameters were found in patients bearing mutations alleles nor after continuous iron therapy at low dosages. However, the prevalence of patients capable of maintaining normal hemoglobin (Hb) level without EPO therapy is increased in the C282Y-mutated patients. Only 1 patient out of the 4 with biopsy-proven porphyria cutanea tarda was bearing gene mutations (H63D heterozygosis).<h4>Conclusion</h4>C282Y/H63D HFE gene mutations do not seem to be related to major abnormalities in biochemical parameters of iron status in dialysis patients without iron therapy or after i.v. iron supplementation, granted that low dosages are employed. Obviously, as our patients were exposed to a relatively uniform iron regimen in our clinical center (< or = 60 mg/week), it is unclear if other dosing regimens will unmask clinically significant differences between the heterozygotes and normals. The fact that the C282Y-mutated patients more frequently maintain high Hb values without EPO is interesting as could suggest a better use of available iron for erythopoiesis, but needs to be confirmed in larger samples. No clear association is demonstrated with porphyria cutanea tarda and major cardiovascular events.

Biochemical markers of bone metabolism are divided into two groups: formation and resorption markers. Bone turnover is a dynamic process, which increases in postmenopausal period. Hormone replacement therapy (HRT) can diminish this increased bone turnover. Biochemical markers reflect acute changes in bone metabolism. Therefore, they may be very useful for the prediction of subsequent bone mineral density changes after HRT in menopausal women. Both oral and transdermal routes of HRT are efficacious in decreasing the levels of biochemical markers. However, markers do not replace bone mineral density measurement. Collagen type I cross linked N-telopeptide, collagen type I cross linked C-telopeptide, and osteocalcin are the most promising markers.

Huntington's disease is a dominantly inherited neurological disorder where specific neurodegeneration is caused by an extended polyglutamine stretch in the huntingtin protein. Proteins with expanded polyglutamine regions have the ability to self-aggregate and previous work in our laboratory, and by others, revealed sparse amyloid-like deposits in the Huntington's disease brain, supporting the hypothesis that the polyglutamine stretches may fold into regular beta-sheet structures. This process of folding has similarities to other neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and the prion diseases which all exhibit beta-sheet protein accumulation. We were therefore interested in testing the hypothesis that TATA-binding protein may play a role in Huntington's disease as it contains an elongated polymorphic polyglutamine stretch that ranges in size from 26 to 42 amino acids in normal individuals. A proportion of TBP alleles fall within the range of glutamine length that causes neurodegeneration when located in the huntingtin protein. In this study the distribution and cellular localisation of TATA-binding protein was compared to the distribution and cellular localisation of the huntingtin protein in the middle frontal gyrus of Huntington's disease and neurologically normal subjects. Seven different morphological forms of TATA-binding protein-positive structures were detected in Huntington's disease but not in control brain. TATA-binding protein labelling was relatively more abundant than huntingtin labelling and increased with the grade of the disease. At least a proportion of this accumulated TBP exists as insoluble protein. This suggests that TBP may play a role in the disease process.

To help understand the biological functions of carbonic anhydrase-related proteins (CA-RPs VIII, X, and XI), we obtained cDNA clones of murine CA-RPs X and XI and studied the tissue distribution of all three CA-RPs and their developmental expression in the murine embryonic brain. The amino acid sequences of murine CA-RPs are highly conserved with their human homologues, indicating a fundamental biological role of CA-RPs. Among a panel of vital organs, the strongest mRNA expression of all three CA-RPs was consistently observed in the brain. Immunohistochemical analysis revealed the expression of all three CA-RPs in the neural cell body and neurites. CA-RP X was also observed in the myelin sheath, as demonstrated using the shiverer demyelinated mouse. In murine embryos, CA-RP VIII and X messages first appeared in the middle of the gestation period, while the CA-RP XI message was seen at an early gestational stage and expressed to a lesser extent as gestation progressed. All CA-RPs were expressed in the neuroprogenitor cells in the subventricular area and subsequently detected in the neural cells migrating to the cortex. Although the exact function of CA-RPs is still undefined, these findings suggest an important role of CA-RPs in the CNS.

Hereditary hemochromatosis (HH) is an iron-overload disease common in populations of Northern European origin. Patients display increased iron absorption leading to excessive iron deposition and potential multiorgan failure. Using polymerase chain reaction sequence-specific primer (PCR-SSP) technology, we have developed an HH diagnosis assay capable of detecting 19 non-synonymous HFE mutations (including a previously unreported mutation, V295A) and several TFR2, SLC11A3 and H ferritin alleles implicated in HH. As part of the validation process, 159 UK renal donors were genotyped to determine HH allele frequencies in the UK population. The alleles nominally identified as HFE*01 (C282Y), HFE*02 (H63D) and HFE*03 (S65C) were found at frequencies of 0.085, 0.173 and 0.009, respectively. All other potential HH-associated alleles were absent, confirming their low prevalence in this population. This assay enables comprehensive routine HH genotyping, producing rapid, accurate and reproducible results at low cost.

In a transgenic mouse model of the neurodegenerative disorder Huntington's disease (HD), age-dependent neurologic defects are accompanied by progressive alterations in glucose tolerance that culminate in the development of diabetes mellitus and insulin deficiency. Pancreatic islets from HD transgenic mice express reduced levels of the pancreatic islet hormones insulin, somatostatin, and glucagon and exhibit intrinsic defects in insulin production. Intranuclear inclusions accumulate with aging in transgenic pancreatic islets, concomitant with the decline in glucose tolerance. HD transgenic mice develop an age-dependent reduction of insulin mRNA expression and diminished expression of key regulators of insulin gene transcription, including the pancreatic homeoprotein PDX-1, E2A proteins, and the coactivators CBP and p300. Disrupted expression of a subset of transcription factors in pancreatic beta cells by a polyglutamine expansion tract in the huntingtin protein selectively impairs insulin gene expression to result in insulin deficiency and diabetes. Selective dysregulation of gene expression in triplet repeat disorders provides a mechanism for pleiotropic cellular dysfunction that restricts the toxicity of ubiquitously expressed proteins to highly specialized subpopulations of cells.

The human cell cycle was synchronized and the chromosomes were isolated by a centrifugation method using two representative solutions for chromosome isolation (a polyamine buffer, PAB and citric acid solution, CAS) and fixatives. The centrifugation method yielded sufficient amounts of human metaphase chromosomes. Observation of the isolated chromosomes by scanning electron microscopy (SEM) revealed two types of surface structure which have been repeatedly reported to date: the human chromosomes in the PAB were relatively smooth but covered irregularly with scaly structures, while the surface of the chromosomes in the CAS exhibited a dense fibrous structure with a uniform diameter of 50-70 nm. Comparison of proteins extracted from chromosomes isolated with the PAB and CAS clearly indicated the removal of linker histones, H1, from chromosomes isolated with the CAS. These findings imply that the two different images of human chromosomes frequently observed by SEM are due to the removal of peripheral chromosomal materials including linker histones and/or the depletion of linker histones which prevent the surface chromatin fibers from scattering.

<h4>Objective</h4>To determine whether patients previously diagnosed as chronic fatigue syndrome (CFS) actually have primary haemochomatosis (PH).<h4>Methods</h4>The setting was a Dutch referral centre. Transferrin saturation (TS) was retrospectively evaluated in banked blood samples of 88 patients diagnosed as CFS. Patients with elevated TS values were asked to provide a new overnight fasting blood sample for a second determination of TS and measurement of serum ferritin. The DNA was investigated for mutations in the HFE gene when one of these iron parameters was elevated.<h4>Results</h4>For 19 out of 88 patients with CFS an elevated TS was found. A new blood sample was obtained from 11 of these 19: six had increased TS and two had elevated serum ferritin values. These eight patients were neither C282Y homozygotes nor compound C282Y-H63D heterozygotes. In the eight cases where no new blood samples could be obtained, the TS was > 50% for two of the five men and < 45% for the three female patients.<h4>Conclusion</h4>In a group of 88 CFS patients we could exclude PH in all but two of them (prevalence 2.3%; 95% confidence interval 0-5.5%). In our population of CFS patients PH is not more common than in a control population of northern European descent (prevalence 0.25-0.50%).

Erythrocyte uroporphyrinogen decarboxylase (UROD) activity was measured to classify 118 Spanish patients with porphyria cutanea tarda (PCT) into three subtypes: sporadic-, familial- and type III-PCT. Seventy-four patients (63%) had eythrocyte UROD activity within the normal range (74% to 126% of the mean activity of 43 healthy controls) and were classified as sporadic-PCT (47%) or as type III-PCT (16%) whenever a family history of PCT was documented. Forty-four patients (37%) had decreased UROD activity and were classified as familial-PCT. The frequency of both familial-PCT and type III-PCT was higher than reported in other countries. The clinical expression of PCT was associated with the coexistence of two or more risk factors in 80% of the sporadic-PCT patients and in 89% of the familial-PCT patients. Hepatitis C virus and alcohol abuse were risk factors frequently found in these patients, being unrelated to age of onset of skin lesions. A heavy alcohol intake was the main risk factor for type III-PCT. Estrogens appeared as a precipitating factor for women with familial-PCT. The H63D mutation in the hemochromatosis type 1 gene was more frequently found than the C282Y mutation. Both mutations appeared to play a role as precipitating factors in sporadic-PCT when associated with hepatitis C virus infection and alcohol abuse.

A number of factors, including increased iron stores and alcohol consumption, are known to be associated with the development of porphyria cutanea tarda (PCT) in susceptible individuals. Recent reports have described a significant association between inheritance of the C282Y and H63D mutations in the HFE gene, associated with genetic hemochromatosis (GH) and PCT. A strong association between hepatitis C virus infection and PCT has also been demonstrated, while case reports record a link between human immunodeficiency virus (HIV) and PCT. We have investigated the frequency of these factors in a racially-mixed population of patients with PCT in Cape Town, South Africa. 57 patients with PCT drawn from three ethnic groups were screened for the presence of the C282Y and H63D mutations linked to GH, and the prevalences were compared with corresponding healthy control populations. The seroprevalence of markers for HCV, hepatitis B (HBV) and HIV infection were examined in 28 of these. In the control populations, we found that both the C282Y and H63D mutations are highly prevalent in South Africans of European origin. In a population of mixed or Asian origin, the C282Y mutation is very rare whereas the H63D mutation is common. Neither mutation was encountered in any African subject. Both mutations are associated with PCT, but the association is dependent on the ethnic origins of the population to which the patient belongs. In contrast to other studies, HCV infection is numerically unimportant in PCT in our patients. HIV infection is increasingly encountered in our patients with PCT, but the strength of the association cannot be determined in view of the high background prevalence of HIV infection in some sectors of the South African population. The contribution of specific risk factors may be heavily dependent on the population from which patients are drawn, and care should be taken in extrapolating from observations in one racial or geographic population to any other.

There is much evidence for the involvement of central monoaminergic systems, the key targets of stress, in the regulation of mood. Animal and human findings indicate that genetics play a role in the etiology of mood disorders, and so we selected divergent inbred rat strains according to their anxiety-related behaviors on exposure to novel environments. We compared these strains for psychoneuroendocrine response to stressors and/or antidepressants. Molecular genetic studies were also performed to localize the genomic regions associated with these strain-dependent anxiety profiles. We then examined human results indicating that allelic variations in the serotonin transporter (5-HTT) may play a role in the etiology of neuroticism and depression. Thus, we compared inbred rat strains for the 5-HTT, with regard to central and peripheral (platelet) protein expression and function, and the consequences of local application of a selective serotonin reuptake inhibitor (SSRI) on extracellular serotonin (5-HT) levels. Our results indicate that spontaneously hypertensive rats and Lewis rats (LEW) selectively diverge in terms of anxiety-related behaviors and that this divergence is located on chromosome 4. The use of social defeat in LEW and the analysis of its psychoneuroendocrine consequences strongly suggest that such a paradigm, which is sensitive to repeated SSRI treatment, models posttraumatic stress disorder. The Wistar-Kyoto rat may be an adequate model to study the consequences of a genetically driven hypersensitivity to stress and noradrenergic antidepressants. Our most recent findings show that the Fischer 344 and LEW strains differ in protein expression and function of hippocampal and platelet 5-HTT; the divergence in protein expression is not due to allelic variations in the gene-coding sequences and leads to marked differences in extracellular 5-HT levels under basal conditions or SSRI. These examples illustrate how the use of inbred rat strains may complement our knowledge on the genetics of behavior, in the same way as the use of transgenic mice.

No abstract available.