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The mechanism for heparin activation of antithrombin III has been postulated to involve disruption of interactions between its reactive loop P1 residue and Glu(255) on the underlying protein surface. To test this hypothesis, the potential P1-constraining Arg(393)-Glu(255) hydrogen bond and ionic interactions were eliminated by converting Glu(255) to alanine. E255A and wild-type ATIIIs have identical reactive loop sequences (including the P1 and P14 residues), but differ in that Glu(255)-mediated, P1-constraining interactions with the underlying surface cannot form in the mutant. Relative to its wild-type parent, E255A had a 5-fold higher affinity for heparin and pentasaccharide. In the absence of cofactor, E255A exhibited a 5-fold activation of thrombin inhibition but no activation of factor Xa inhibition. Pentasaccharide addition elicited no further activation of thrombin inhibition but increased the factor Xa inhibition rate 100-fold. E255A heparin-dependent thrombin and factor Xa inhibition rates were 1000- and 2-fold faster, respectively, than pentasaccharide-catalyzed rates. Although "approximation" is the predominant factor in heparin activation of ATIII thrombin inhibition, and removal of the P1 constraint plays a distinct but minor role, the primary determinant for activation of factor Xa inhibition is the pentasaccharide-induced conformational change, with approximation making a further minor contribution, and removal of the P1 constraint playing no role at all.

A-kinase (or PKA)-anchoring protein AKAP95 is a zinc-finger protein implicated in mitotic chromosome condensation by acting as a targeting molecule for the condensin complex. We have identified determinants of chromatin-binding, condensin-targeting and chromosome-condensation activities of AKAP95. Binding of AKAP95 to chromatin is conferred by residues 387-450 and requires zinc finger ZF1. Residues 525-569 are essential for condensation of AKAP95-free chromatin and condensin recruitment to chromosomes. Mutation of either zinc finger of AKAP95 abolishes condensation. However, ZF1 is dispensable for condensin targeting, whereas the C-terminal ZF2 is required. AKAP95 interacts with Xenopus XCAP-H condensin subunit in vitro and in vivo but not with the human hCAP-D2 subunit. The data illustrate the involvement of overlapping, but distinct, domains of AKAP95 for condensin recruitment and chromosome condensation and argue for a key role of ZF1 in chromosome condensation and ZF2 in condensin targeting. Moreover, condensin recruitment to chromatin is not sufficient to promote condensation.

A variety of RNA molecules have been found over the last 20 years to have a remarkable range of functions beyond the well-known roles of messenger, ribosomal and transfer RNAs. Here, we present a general categorization of all non-coding RNAs and briefly discuss the ones that affect transcription, translation and protein function.

Reading disability (RD), or dyslexia, is a common heterogeneous syndrome with a large genetic component. Several studies have consistently found evidence for a quantitative-trait locus (QTL) within the 17 Mb (14.9 cM) that span D6S109 and D6S291 on chromosome 6p21.3-22. To characterize further linkage to the QTL, to define more accurately the location and the effect size, and to identify a peak of association, we performed Haseman-Elston and DeFries-Fulker linkage analyses, as well as transmission/disequilibrium, total-association, and variance-components analyses, on 11 quantitative reading and language phenotypes. One hundred four families with RD were genotyped with a new panel of 29 markers that spans 9 Mb of this region. Linkage results varied widely in degree of statistical significance for the different linkage tests, but multipoint analysis suggested a peak near D6S461. The average 6p QTL heritability for the 11 reading and language phenotypes was 0.27, with a maximum of 0.66 for orthographic choice. Consistent with the region of linkage described by these studies and others, there was a peak of transmission disequilibrium with a QTL centered at JA04 (chi2=9.48; empirical P=.0033; orthographic choice), and there was strong evidence for total association at this same marker (chi2=11.49; P=.0007; orthographic choice). Although the boundaries of the peak could not be precisely defined, the most likely location of the QTL is within a 4-Mb region surrounding JA04.

Hereditary hemochromatosis (HH) is a disorder of iron metabolism in which enhanced iron absorption of dietary iron causes increased iron accumulation in the liver, heart, and pancreas. Most individuals with HH are homozygous for a C282Y mutation in the HFE gene. The function of HFE protein is unknown, but it is hypothesized that it acts in association with beta(2)-microglobulin and transferrin receptor 1 to regulate iron uptake from plasma transferrin by the duodenum, the proposed mechanism by which body iron levels are sensed. The aim of this study was to test this hypothesis by comparing clearance of transferrin-bound iron in Hfe knockout (KO) mice with that observed in C57BL/6 control mice. The mice were fed either an iron-deficient, control, or iron-loaded diet for 6 weeks to alter body iron status. The mice then were injected i.v. with (59)Fe-transferrin, and blood samples were taken over 2 h to determine the plasma (59)Fe turnover. After 2 h, the mice were killed and the amount of radioactivity in the duodenum, liver, and kidney was measured. In both Hfe KO and C57BL/6 mice, plasma iron turnover and iron uptake from plasma transferrin by the duodenum, liver, and kidney correlated positively with plasma iron concentration. However, duodenal iron uptake from plasma transferrin was decreased in the Hfe KO mice compared with the control mice. Despite this difference in duodenal uptake, the Hfe KO mice showed no decrease in iron uptake by the liver and kidney or alteration in the plasma iron turnover when compared with C57BL/6 mice. These data support the hypothesis that HFE regulates duodenal uptake of transferrin-bound iron from plasma, and that this mechanism of sensing body iron status, as reflected in plasma iron levels, is impaired in HH.

During recent years, piggyback liver transplantation (pOLT) with preservation of the retrohepatic vena cava has been introduced in adults. The objective of this study was to evaluate hemostatic changes associated with this transplantation technique. Fifty-seven patients undergoing elective pOLT for endstage liver disease were studied. Most significant changes were observed after graft reperfusion, when PT showed a 49% decrease and activated partial thromboplastin time (aPTT) as well as TT a 2- to 3-fold prolongation. At the same time, factors of the extrinsic coagulation pathway (II, V, VII) revealed an overall 50% decline. Similar changes were observed for antithrombin III (ATIII) and fibrinogen plasma levels. However, only 42% of all patients required intraoperative substitution with coagulation components. There was an association between preoperative fibrinogen (<1.7 g/dl) and ATIII (<50%) plasma levels and the substitution requirement. Multiple linear regression showed a significant correlation between preoperative ATIII activity and intraoperative blood loss. Despite a marked impairment of hemostasis, pOLT can frequently be performed with minimized substitution therapy.

Tobacco ( Nicotiana tabacum L.) has two major H1 variants (H1A and H1B), which account for over 80% of chromatin linker histones, and four minor variants: H1C, H1D, H1E and H1F. We have shown previously [M. Prymakowska-Bosak et al. (1999) Plant Cell 11:2317-2329] that reversal of the natural proportion of major to minor H1 variants in transgenic tobacco plants results in a characteristic male-sterility phenotype identical to that occurring in many plant species subjected to water deficit at the time of male meiosis. It has been proposed by others that the drought-induced arrest of male gametophyte development is linked to decreased sugar delivery to reproductive tissues. Within the family of angiosperm H1s there is a well-defined class of minor H1 variants named "drought inducible" because some of its members have been shown to be induced by water deficit. We have identified and cloned the tobacco H1C gene, which, based on sequence similarity, represents a "drought-inducible" minor H1 variant. Analysis of the un-translated mRNA and promoter regions of H1C suggests a regulation by sucrose concentration. Antisense silencing of H1C and its close homologue H1D in plants that do not express H1A and H1B does not affect the characteristic H1A(-)/ H1B(-) male-sterility phenotype. Silencing of H1C and H1D also has no effect on growth and development of plants. Our findings demonstrate that H1C and H1D are dispensable for normal growth and development of tobacco, and that the compensatory up-regulation of "drought-inducible" H1s observed in H1A(-)/ H1B(-) plants is not the direct cause of male sterility linked to alterations in H1 variants.

A variety of valuable therapeutic proteins are expressed in mammalian cells. Currently, rate-limiting for secretion of recombinant glycoproteins are activities in the secretory pathway of eukaryotic cells, i.e., folding and glycosylation of the naked polypeptide chain. In this paper we provide evidence that elevation of expression level alone is sufficient to cause intracellular aggregation of a structurally relatively simple glycoprotein, antithrombin III (ATIII). Elevation of expression level by selection for increased drug resistance in Chinese hamster ovary cells stably expressing ATIII resulted in formation of disulfide-bonded aggregates of ATIII. Aggregated ATIII displayed incomplete sialylation and Endo H-sensitivity and located to the endoplasmic reticulum and the cis-Golgi compartment in subcellular fractionations. To explore possible causes for aggregation of ATIII at elevated expression levels we investigated the influence of the two major energy sources of cultured mammalian cells, D-glucose and L-glutamine, on the ATIII-yield. We found that utilization of D-glucose was not limiting for synthesis of ATIII at elevated expression levels. However, the amount of ATIII-synthesized per L-glutamine consumed did not seem to increase steadily with expression level for ATIII, indicating that secretion of ATIII may be limited by the capacity of the cell to utilize L-glutamine.

The mammalian Siah genes encode highly conserved proteins containing a RING domain. As components of E3 ubiquitin ligase complexes, Siah proteins facilitate the ubiquitination and degradation of diverse protein partners including beta-catenin, N-CoR, and DCC. We used gene targeting in mice to analyze the function of Siah1a during mammalian development and reveal novel roles in growth, viability, and fertility. Mutant animals have normal weights at term but are postnatally growth retarded, despite normal levels of pituitary growth hormone. Embryonic fibroblasts isolated from mutant animals grow normally. Most animals die before weaning, and few survive beyond 3 months. Serum gonadotropin levels are normal in Siah1a mutant mice; however, females are subfertile and males are sterile due to a block in spermatogenesis. Although spermatocytes in mutant mice display normal meiotic prophase and meiosis I spindle formation, they accumulate at metaphase to telophase of meiosis I and subsequently undergo apoptosis. The requirement of Siah1a for normal progression beyond metaphase I suggests that Siah1a may be part of a novel E3 complex acting late in the first meiotic division.

Hereditary hemochromatosis (HHC) is one of the most frequent genetic disorders in humans. In healthy individuals, absorption of iron in the intestine is tightly regulated by cells with the highest iron demand, in particular erythroid precursors. Cloning of intestinal iron transporter proteins provided new insight into mechanisms and regulation of intestinal iron absorption. The aim of this study was to assess whether, in humans, the two transporters are regulated in an iron-dependent manner and whether this regulation is disturbed in HHC. Using quantitative PCR, we measured mRNA expression of divalent cation transporter 1 (DCT1), iron-regulated gene 1 (IREG1), and hephaestin in duodenal biopsy samples of individuals with normal iron levels, iron-deficiency anemia, or iron overload. In controls, we found inverse relationships between the DCT1 splice form containing an iron-responsive element (IRE) and blood hemoglobin, serum transferrin saturation, or ferritin. Subjects with iron-deficiency anemia showed a significant increase in expression of the spliced form, DCT1(IRE) mRNA. Similarly, in subjects homozygous for the C282Y HFE mutation, DCT1(IRE) expression levels remained high despite high serum iron saturation. Furthermore, a significantly increased IREG1 expression was observed. Hephaestin did not exhibit a similar iron-dependent regulation. Our data show that expression levels of human DCT1 mRNA, and to a lesser extent IREG1 mRNA, are regulated in an iron-dependent manner, whereas mRNA of hephaestin is not affected. The lack of appropriate downregulation of apical and basolateral iron transporters in duodenum likely leads to excessive iron absorption in persons with HHC.

<h4>Background & aims</h4>Increased body iron, genetic hemochromatosis (GH) mutations, and nonalcoholic fatty liver disease (NAFLD) tend to cluster in carbohydrate-intolerant patients. In an attempt to further clarify the interrelationships among these conditions, we studied 42 carbohydrate-intolerant patients who were free of the common GH mutations C282Y and H63D, and had a serum iron saturation lower than 50%.<h4>Methods</h4>We measured body iron stores, and induced iron depletion to a level of near-iron deficiency (NID) by quantitative phlebotomy.<h4>Results</h4>In the 17 patients with clinical evidence of NAFLD, we could not demonstrate supranormal levels of body iron (1.6 +/- 0.2 vs. 1.4 +/- 0.2 g; P = 0.06). However, at NID, there was a 40%-55% improvement (P = 0.05-0.0001) of both fasting and glucose-stimulated plasma insulin concentrations, and near-normalization of serum alanine aminotransferase activity (from 61 +/- 5 to 32 +/- 2 IU/L; P < 0.001).<h4>Conclusions</h4>These results reflect the insulin-sparing effect of iron depletion and indicate a key role of iron and hyperinsulinemia in the pathogenesis of NAFLD.

Diffuse astrocytomas have a median survival of 6-8 years. However, in a minority of cases that are histologically low grade, progression is rapid, leading to death within 2 years. Loss of p16, retinoblastoma protein, and deleted-in-colon-carcinoma protein expression, and monosomy of chromosome 10 have been shown to occur in malignant astrocytic tumors. We have investigated the prognostic value of expression of these markers, using techniques applicable in many histopathology laboratories, in diffuse astrocytomas that are histologically low grade. Paraffin sections from 71 diffuse, supratentorial, low-grade astrocytomas, from patients with at least 8-year survival data, were immunostained with antibodies to p16, deleted-in-colon-carcinoma protein, p53, Ki67, and retinoblastoma protein. In situ hybridization with a digoxigenin-labeled probe to chromosome 10 was used to assess chromosomal loss. In most cases there was immunostaining of virtually all tumor cell nuclei with antibodies to p16 and retinoblastoma protein. Three of the 68 tumors in which assessment of p16 was possible included discrete foci with lack of detectable immunoreactivity in tumor cells. The three patients concerned had a significantly shortened median survival (1.1 years vs 4.4 years in those without loss of p16; p <0.01). In six of the 61 cases where assessment of retinoblastoma protein was possible, <70% of tumor cell nuclei showed immunoreactivity. These six patients had a shorter survival (4.0 years) than had the remaining patients (5.4 years), although this difference was not statistically significant. The tumor from one of these patients included areas where only 36% of tumor cells showed retinoblastoma protein immunoreactivity, and this patient survived only 1.5 years. Tumors showing loss of both p16 and retinoblastoma were not seen. p53 and deleted-in-colon-carcinoma protein expression was highly variable and did not correlate with survival. Tumors with monosomy for chromosome 10 were not identified. Both polyploidy and the Ki67 labeling index were significantly associated with the p53 labeling index but not with survival. Focal loss of p16 or retinoblastoma protein is demonstrable in approximately 5% and 10% of diffuse low-grade diffuse astrocytomas, respectively. Tumors with focal loss of immunoreactivity for these proteins are associated with shorter survival than those without, suggesting that immunohistochemistry for p16 and retinoblastoma protein may be a useful adjunct to other methods for assessing the prognosis of astrocytomas.

Resistance to the antiestrogen tamoxifen is the main stumbling block for the success of breast cancer therapy. We focused our study on cellular alterations induced by a prolonged treatment with the active tamoxifen metabolite hydroxytamoxifen (OHT). We show that a prolonged OHT treatment (for up to 7 days) led to a progressive increase in the level of phosphorylated p44/42 mitogen activated kinase (MAP kinase) induced by 10(-7) M TPA stimulation, without any significant change in the protein level. This effect was also observed in MCF-7 cells grown first in medium containing dextran-coated charcoal-treated FCS (DCC medium) for 20 days prior to OHT treatment, indicating a specific effect of the antiestrogen and not an effect of estrogen deprivation. It was prevented by cotreatment with estradiol and not observed in the estrogen receptor negative HeLa cell line, suggesting that it was mediated by the estrogen receptor. TPA induced phosphorylation of MEK1/2 was also raised by OHT treatment, without any change in their protein level or Raf-1 and H-Ras levels. When the MCF-7R OHT resistant cell line was grown in antiestrogen containing medium, the level of phosphorylated p44/42 MAP kinase was also high but reversed when the antiestrogen was removed. The 2 other MAP kinase, JNK and P38 pathways were not affected in the same way by OHT treatment. In conclusion, our data reveal that a prolonged OHT treatment, by increasing p44/42 MAPK activity, affects a key step in the growth control of MCF-7 cells, although not sufficiently to overcome the growth inhibitory effect of the drug.

Dysfunctions of the central serotonin (5-hydroxytryptamine, 5-HT) system seem to be associated with psychiatric disorders such as schizophrenia or depression. Previous studies suggested that a 44-bp insertion/deletion polymorphism of the 5-HT transporter (5-HTT) promoter region might influence the transcriptional activity of the 5-HTT gene, and the insertion variant resulted in increased 5-HTT expression and 5-HT uptake. Moreover, a 17-bp variable number of tandem repeat (VNTR) polymorphism of the second intron may act as a transcriptional regulator with allele dependent differential enhancer-like properties. Since the 5-HTT of human platelets shares many properties with the transporter of neural tissue, platelets are widely used as a surrogate tissue source, possibly reflecting central 5-HT metabolism. Therefore, we investigated the impact of the 44-bp polymorphism and the 17-bp VNTR for 5-HT uptake in platelets of 50 male subjects. We found no significant effect of the 44-bp polymorphism and of the 17-bp VNTR on maximum rate (Vmax) of 5-HT uptake. However, individuals homozygous for the 5-HTT intron 2 allele with 12 repeats (STin2.12) of the 17-bp VNTR appeared to have lower affinity of 5-HT uptake than individuals heterozygous for the STin2.10/STin2.9 allele. This was also observed for the combined analysis of both polymorphisms. In conclusion, we found no association between the different genotypes of the 44-bp polymorphism and the 17-bp VNTR and maximum rate of 5-HT uptake into platelets.

Five candidate genes, the receptors DRD2, DRD3, HTR2A and GABA(A)gamma2, and the serotonin transporter (5-HTT) were analyzed for association with heroin abuse. We examined three polymorphisms (promoter - 141DeltaC, Ser311Cys, and TaqI) in the DRD2 gene, one polymorphism (Ser9Gly) in the DRD3 gene, two polymorphisms (promoter - 1438G/A and T102C) in the HTR2A gene, two polymorphisms (VNTR and Del/Ins) in 5-HTT gene, and one polymorphism (G3145A) in GABA(A)gamma2 gene in 121 Chinese heroin addicts and 194 controls. None of the polymorphisms differed significantly for allele, genotype, or haplotype frequencies, except for the DRD2 promoter polymorphism - 141DeltaC (genotype-wise and allele-wise, P = 0.05, uncorrected). An additional 344 subjects with heroin abuse and 104 controls were investigated for the - 141DeltaC polymorphism. In the second sample, there were no significant difference of genotype or allele frequencies between subjects with heroin abuse and normal controls. When we divided the sample by route of administration into nasal inhalers and IM or IV injectors, however, it produced a significant difference between inhalers of heroin and controls (genotype-wise, P = 0.006, allele-wise, P = 0.016) but not for injectors of heroin (genotype-wise, P = 0.81, allele-wise, P = 0.69). We also found that LD between all polymorphisms we examined in the gene was weak, possibly explaining why we see association of this polymorphism with heroin abuse but not with other markers in the gene. Overall our results indicates that the HTR2A, 5-HTT, DRD3 and GABA(A)gamma2 genes are not likely to be a major genetic risk factor for heroin abuse in this population, with the exception of possible association between nasal inhalation and DRD2 promoter - 141DeltaC polymorphism.

To elucidate mechanisms underlying mitochondrial dysfunctions induced by glutamate, we have examined the effects of in vivo treatment with the ionotropic glutamate receptor agonist kainate on localization of the transcription factor activator protein-1 (AP-1) in mitochondria as well as nuclei of murine brain. A systemic administration of kainate dramatically enhanced AP-1 DNA binding in both mitochondrial and nuclear extracts of mouse cerebral cortex and hippocampus 1 hr to 3 d later. Unlabeled AP-1 probe selectively competed for AP-1 DNA binding in mitochondrial extracts of cortex and hippocampus obtained from mice injected with kainate. Supershift and immunoblotting analyses revealed participation of c-Fos, Fos-B, and Jun-B proteins in potentiation by kainate of mitochondrial AP-1 DNA binding in cortex and hippocampus. An immunohistochemical study demonstrated marked expression by kainate of c-Fos protein in the pyramidal and dentate granular layers, whereas an immunoelectron microscopic analysis showed localization of c-Fos protein within mitochondria, as well as nuclei, of the CA1 pyramidal and dentate granular cells in hippocampus obtained 2 hr after the administration of kainate. Mitochondrial AP-1 DNA binding was inhibited by particular unlabeled oligonucleotides containing sequences similar to the AP-1 site found in the noncoding region of mitochondrial DNA. Kainate markedly potentiated binding of radiolabeled oligonucleotide probes containing sequences effective in competing for AP-1 DNA binding in hippocampal mitochondrial extracts. These results suggest that kainate may facilitate expression of the AP-1 complex and subsequent translocation into mitochondria to participate in mechanisms associated with transcriptional regulation of mitochondrial DNA in murine hippocampus.

Multiple types of voltage-activated Ca(2+) channels (T, L, N, P, Q, R type) coordinate Ca(2+)-dependent processes in neurons and neuroendocrine cells. Expressional and functional data have suggested a role for Ca(v)2.3 Ca(2+) channels in endocrine processes. To verify its role in vivo, Ca(v)2.3(-/-) mutant mice were generated, thus deficient in alpha 1E/R-type Ca(2+) channel. Intraperitoneal injection of D-glucose showed that glucose tolerance was markedly reduced, and insulin release into plasma was impaired in Ca(v)2.3-deficient mice. In isolated islets of Langerhans from these animals, no glucose-induced insulin release was detected. Further, in stressed Ca(v)2.3-deficient mice, the rate of glucose release into the blood was only 29% of that observed for wild-type animals. Thus, the deletion of Ca(v)2.3 causes deficits not only in insulin release but also in stress-induced hyperglycemia. The complex phenotype of Ca(v)2.3-deficient mice has dual components related to endocrine and neurological defects. The present findings provide direct evidence of a functional role for the Ca(v)2.3 subunit in hormone secretion and glucose homeostasis.

We recently reported the hemochromatosis-like phenotype observed in our Usf2 knockout mice. In these mice, as in murine models of hemochromatosis and patients with hereditary hemochromatosis, iron accumulates in parenchymal cells (in particular, liver and pancreas), whereas the reticuloendothelial system is spared from this iron loading. We suggested that this phenotypic trait could be attributed to the absence, in the Usf2 knockout mice, of a secreted liver-specific peptide, hepcidin. We conjectured that the reverse situation, namely overexpression of hepcidin, might result in phenotypic traits of iron deficiency. This question was addressed by generating transgenic mice expressing hepcidin under the control of the liver-specific transthyretin promoter. We found that the majority of the transgenic mice were born with a pale skin and died within a few hours after birth. These transgenic animals had decreased body iron levels and presented severe microcytic hypochromic anemia. So far, three mosaic transgenic animals have survived. They were unequivocally identified by physical features, including reduced body size, pallor, hairless and crumpled skin. These pleiotropic effects were found to be associated with erythrocyte abnormalities, with marked anisocytosis, poikylocytosis and hypochromia, which are features characteristic of iron-deficiency anemia. These results strongly support the proposed role of hepcidin as a putative iron-regulatory hormone. The animal models devoid of hepcidin (the Usf2 knockout mice) or overexpressing the peptide (the transgenic mice presented in this paper) represent valuable tools for investigating iron homeostasis in vivo and for deciphering the molecular mechanisms of hepcidin action.

HFE gene is a major histocompatibility complex class I-like gene, which was identified as a candidate gene for hemochromatosis in 1996. The proposed role for HFE is its part in the regulation of the interaction of the transferrin receptor with transferrin. Hemochromatosis, the common autosomal recessive disease of iron overload, affects at least 1 in 300 Caucasians. The identification of the C282Y mutation in the HFE gene has led to population screening studies. Much of this work has also included the analysis of a second mutation, H63D, which appears to have a low penetrability. HFE protein was recently found to coprecipitate with the transferrin receptor and to affect the reaction between transferrin and the transferrin receptor. Functional data suggest that the mutation C282Y abolishes the association of the HFE protein with beta 2-microglobulin (beta 2M), making the complex unable to reach the cell surface. Clearly, if the mutation protein is unable to reach the cell surface, this regulatory feature is missing. The role of a second mutation in the HFE gene, H63D, is less clear. Current data suggest that this mutation protein can associate with beta 2-microglobulin and does reach the cell surface and that the defect lies in a failure to modify the affinity of the transferrin receptor for transferrin. This does not explain the low degree of penetrability associated with this mutation.

<h4>Background/aims</h4>HFE-related haemochromatosis is a common disorder of iron metabolism. Most affected individuals are homozygous for the C282Y mutation of HFE. Some are compound heterozygotes for C282Y/H63D. A small proportion have neither of these genotypes. We have investigated the phenotype of compound heterozygotes for C282Y and another missense mutation S65C.<h4>Methods</h4>Genotype for the S65C mutation was determined in 309 subjects heterozygous for C282Y and negative for H63D, referred because of increased serum iron indices or family screening. A control sample comprising 315 individuals was also studied.<h4>Results</h4>Twelve individuals were compound heterozygotes for C282Y and S65C. Seven, referred for family screening, had normal serum iron indices. Five subjects had elevated serum iron indices; three of these had elevated hepatic iron and have received treatment for iron overload. Transferrin saturation was significantly elevated in C282Y/S65C compound heterozygotes compared with simple C282Y heterozygotes.<h4>Conclusions</h4>Some C282Y/S65C compound heterozygotes have elevated serum iron indices and iron overload. The penetrance of this genotype is low and other genetic and environmental factors may influence the expression of iron loading. Screening for S65C may be useful in individuals with iron overload who are not homozygous for C282Y or compound heterozygous for C282Y/H63D.

Members of the ATP-dependent family of chromatin remodeling enzymes play key roles in the regulation of transcription, development, DNA repair and cell cycle. Each of these enzymes are multi-subunit assemblies that hydrolyze thousands of molecules of ATP in order to change nucleosome positions, disrupt DNA-histone interactions and perhaps destabilize chromatin folding. Here I review recent studies that suggest these potent machines can be 'tamed' by one of several mechanisms: targeting their activity to localized regions, blocking their chromatin binding activity or inhibiting their remodeling activity.

We examined whether JTV-803, a specific activated factor X inhibitor independent of antithrombin III (ATIII), is effective against disseminated intravascular coagulation (DIC) in rat models induced by tissue factor (TF) or lipopolysaccharides (LPS). In male Wistar rats, DIC was induced by a 4 h infusion of thromboplastin (3.75 U/kg) or LPS (50 mg/kg). The rats were given JTV-803 (0.3 or 3 mg/kg, bolus intravenously) (JTV-803 groups) or low molecular weight heparin (LMWH groups) (200 U/kg, bolus intravenously) prior to an injection of TF or LPS. The results showed that JTV-803 was dose-dependently effective against DIC in both TF-induced and LPS-induced rat models. This anti-DIC effect of JTV-803 at higher doses was almost equivalent to that of LMWH in both types of DIC. Plasma ATIII activity was more prominent in the group treated with JTV-803 than in that treated with LMWH. None of rats died in the TF-induced DIC model with or without drug administration. On the contrary, seven of 22 rats died (mortality rate, 31.8%) in the LPS-induced DIC model without drug administration. Although the mortality rate of rats induced with LPS and treated with LMWH was quite high (6/16, 37.5%), none of the LPS-induced rats treated with JTV-803 died. These findings suggested that JTV-803 can treat both TF-induced and LPS-induced DIC models, and that this drug has greater potential in preserving ATIII and in improving the prognosis of DIC.

This review focuses on genes other than the high penetrance genes BRCA1 and BRCA2 that are involved in breast cancer susceptibility. The goal of this review is the discovery of polymorphisms that are either associated with breast cancer or that are in strong linkage disequilibrium with breast cancer causing variants. An association with breast cancer at a 5% significance level was found for 13 polymorphisms in 10 genes described in more than one breast cancer study. Our data will help focus on the further analysis of genetic polymorphisms in populations of appropriate size, and especially on the combinations of such polymorphisms. This will facilitate determination of population attributable risks, understanding of gene-gene interactions, and improving estimates of genetic cancer risks.

Although most colorectal cancer develops based on the adenoma-adenocarcinoma sequence, morphologically, colorectal cancer is not a homogeneous disease entity. Generally, there are two distinct morphological types: polypoid and ulcerative colorectal tumours. Previous studies have demonstrated that K-ras codon 12 mutations are preferentially associated with polypoid growth of colorectal cancer; however, little is known about the molecular mechanism that determines ulcerative growth of colorectal cancer. beta-catenin complex plays a critical role both in tumorigenesis and morphogenesis. We examined the differential expression of beta-catenin and its related factors among different types of colorectal cancer in order to determine any relationship with gross tumour morphology. Immunohistochemical staining of beta-catenin, E-cadherin and MMP-7 was performed on 51 tumours, including 26 polypoid tumours and 25 ulcerative tumours. Protein truncation tests and single-strand conformational polymorphism for mutation of the adenomatous polyposis coli tumour suppressor gene, as well as single-strand conformational polymorphism for the mutation of beta-catenin exon 3 were also done. Nuclear expression of beta-catenin was observed in 18 out of 25 (72%) cases of ulcerative colorectal cancer and seven out of 26 (26.9%) cases of polypoid colorectal cancer. A significant relationship of nuclear beta-catenin expression with ulcerative colorectal cancer was found (P<0.001). However, this finding was independent of adenomatous polyposis coli tumour suppressor gene mutation and E-cadherin expression. Together with previous data, we propose that different combinations of genetic alterations may underlie different morphological types of colorectal cancer. These findings should be taken into consideration whenever developing a new genetic diagnosis or therapy for colorectal cancer.

To know the function of the Ca2+ channel containing alpha(1)2.3 (alpha1E) subunit (Ca(v)2.3 channel) in spermatozoa, we analyzed Ca2+ transients and sperm motility using a mouse strain lacking Ca(v)2.3 channel. The averaged rising rates of Ca2+ transients induced by alpha-D-mannose-bovine serum albumin in the head region of Ca(v)2.3-/- sperm were significantly lower than those of Ca(v)2.3+/+ sperm. A computer-assisted sperm motility assay revealed that straight-line velocity and linearity were greater in Ca(v)2.3-/- sperm than those in Ca(v)2.3+/+ sperm. These results suggest that the Ca(v)2.3 channel plays some roles in Ca2+ transients and the control of flagellar movement.

A study of papers on amyloid fibers suggested to us that cylindrical beta-sheets are the only structures consistent with some of the x-ray and electron microscope data. We then found that our own 7-year-old and hitherto enigmatic x-ray diagram of poly-L-glutamine fits a cylindrical sheet of 31 A diameter made of beta-strands with 20 residues per helical turn. Successive turns are linked by hydrogen bonds between both the main chain and side chain amides, and side chains point alternately into and out of the cylinder. Fibers of the exon-1 peptide of huntingtin and of the glutamine- and asparagine-rich region of the yeast prion Sup35 give the same underlying x-ray diagrams, which show that they have the same structure. Electron micrographs show that the 100-A-thick fibers of the Sup35 peptide are ropes made of three protofibrils a little over 30 A thick. They have a measured mass of 1,450 Da/A, compared with 1,426 Da/A for a calculated mass of three protofibrils each with 20 residues per helical turn wound around each other with a helical pitch of 510 A. Published x-ray diagrams and electron micrographs show that fibers of synuclein, the protein that forms the aggregates of Parkinson disease, consist of single cylindrical beta-sheets. Fibers of Alzheimer A beta fragments and variants are probably made of either two or three concentric cylindrical beta-sheets. Our structure of poly-L-glutamine fibers may explain why, in all but one of the neurodegenerative diseases resulting from extension of glutamine repeats, disease occurs when the number of repeats exceeds 37-40. A single helical turn with 20 residues would be unstable, because there is nothing to hold it in place, but two turns with 40 residues are stabilized by the hydrogen bonds between their amides and can act as nuclei for further helical growth. The A beta peptide of Alzheimer's disease contains 42 residues, the best number for nucleating further growth. All these structures are very stable; the best hope for therapies lies in preventing their growth.

A pathological hallmark of polyglutamine diseases is the presence of inclusions or aggregates of the expanded polyglutamine protein. Polyglutamine inclusions are present in the neuronal nucleus in a number of inherited neurodegenerative disorders, including Huntington disease (HD). Recent studies suggest that polyglutamine inclusions may sequester polyglutamine-containing transcription factors and deplete their concentration in the nucleus, leading to altered gene expression. To test this hypothesis, we examined the expression and localization of the polyglutamine-containing or glutamine-rich transcription factors TBP, CBP and Sp1 in HD mouse models. All three transcription factors were diffusely distributed in the nucleus, despite the presence of abundant intranuclear inclusions. There were no differences in the nuclear staining of these transcription factors between HD and wild-type mouse brains. Although some CBP staining appeared as dots in the selective brain regions (e.g. hypothalamus and amygdala), double labeling showed that most CBP was not co-localized with huntingtin nuclear inclusions. Electron microscopy confirmed that CBP was diffusely distributed in the nucleus. Western blots showed that these transcription factors were not trapped in huntingtin inclusions. In the striatum of HD mice, which suffers a significant reduction in the expression of a number of genes, mutant huntingtin was present in both an aggregated and a diffuse form. These findings suggest that altered gene expression may result from the interactions of soluble mutant huntingtin with nuclear transcription factors, rather than from the depletion of transcription factors by nuclear inclusions.

HAP-1 is a huntingtin-associated protein that is enriched in the brain. To gain insight into the normal physiological role of HAP-1, mice were generated with homozygous disruption at the Hap1 locus. Loss of HAP-1 expression did not alter the gross brain expression levels of its interacting partners, huntingtin and p150glued. Newborn Hap1(-/-) animals are observed at the expected Mendelian frequency suggesting a non-essential role of HAP-1 during embryogenesis. Postnatally, Hap1(-/-) pups show decreased feeding behavior that ultimately leads to malnutrition, dehydration and premature death. Seventy percent of Hap1(-/-) pups fail to survive past the second postnatal day (P2) and 100% of Hap1(-/-) pups fail to survive past P9. From P2 until death, Hap1(-/-) pups show markedly decreased amounts of ingested milk. Hap1(-/-) pups that survive to P8 show signs of starvation including greatly decreased serum leptin levels, decreased brain weight and atrophy of the brain cortical mantel. HAP-1 is particularly enriched in the hypothalamus, which is well documented to regulate feeding behavior. Our results demonstrate that HAP-1 plays an essential role in regulating postnatal feeding.

We have expressed, purified and crystallized recombinant human phosphatidylcholine transfer protein (PC-TP) and selenomethionyl PC-TP bound to dilinoleoyl phosphatidylcholine. The biochemical properties of native and selenomethionyl PC-TP were indistinguishable, and the two proteins crystallized under similar conditions. Both native and selenomethionyl PC-TP crystallized in two distinct space groups and diffracted X-rays to 2.4 A resolution.

<h4>Objective</h4>The increased risk of developing hepatocellular carcinoma in hereditary hemochromatosis has been associated with cirrhosis and hepatic iron overload. The aim of this study was to investigate the association between HFE gene mutations (C282Y, H63D) and hepatocellular carcinoma in patients with alcoholic and virus-related cirrhosis.<h4>Methods</h4>Serum markers of iron status and HFE mutations were determined in 179 patients with alcoholic cirrhosis and 98 patients with hepatitis B and/or hepatitis C virus-related cirrhosis. A total of 43 patients with alcoholic cirrhosis and 34 patients with virus-related cirrhosis had hepatocellular carcinoma. The control group consisted of 159 healthy bone marrow donors.<h4>Results</h4>No differences were found in the frequencies of mutations among patients with alcoholic cirrhosis, those with virus-related cirrhosis, and the control subjects. However, nine (20.9%) of the 43 patients with alcoholic cirrhosis and hepatocellular carcinoma were heterozygous for the C282Y mutation, compared with six (4.4%) of the 136 patients without tumor (p = 0.002). This difference was not found in patients with virus-related cirrhosis, with or without hepatocellular carcinoma, or the H63D mutation. The transferrin saturation was the only serum iron marker the value of which was significantly higher among C282Y heterozygotes with alcoholic cirrhosis compared to those without mutation.<h4>Conclusions</h4>The high frequency of heterozygosity for the C282Y mutation in patients with alcoholic cirrhosis plus hepatocellular carcinoma suggests that the presence of this mutation could be associated with an increased risk of developing hepatocellular carcinoma in these patients.

The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) may be involved in neuronal disease and in programmed cell death. Recent investigations indicate an in vitro physical association between GAPDH and huntingtin, the mutated protein in Huntington's disease (HD). Previous studies reveal the functional diversity of GAPDH as a membrane, cytoplasmic and nuclear protein. These activities are independent of its classical glycolytic function. Thus, huntingtin-GAPDH interactions could affect not only energy production but also result in pleiotropic effects involving various biochemical pathways in HD cells. We now report the identification of a nuclear high molecular weight (HMW) GAPDH species in Huntington's disease cells. In contrast, nuclei from age-matched control normal human cells did not contain the HMW GAPDH species. Further, this GAPDH structure was not observed in HD whole cell sonicates which are characterized by normal GAPDH activity. The disruption of intracellular structure is implicit in the preparation of whole cell sonicates. Therefore, these results suggest that the dissociation of the GAPDH protein from its high molecular weight structure results in the recovery of its function. These findings reveal a singular, new subcellular phenotype in HD cells. As such, they indicate an interrelationship between nuclear GAPDH function and huntingtin localization in this CAG expansion neuronal disease.

Investigations on the effects of serotonin (5-HT) and the serotonin transporter (5-HTT) on the pulmonary circulation are of special interest because of the reported increased risk of primary pulmonary hypertension (PPH) in patients who used some appetite suppressants that interfere with 5-HT. In addition to its vasoactive effects, 5-HT exerts mitogenic and comitogenic effects on pulmonary artery smooth muscle cells (PASMCs). These mitogenic and comitogenic effects require 5-HT internalization by the high-affinity 5-HTT, which can be competitively inhibited by specific drugs such as fluoxetine and paroxetine. In a recent study, we showed that hypoxia increases the rate of 5-HTT gene transcription in PASMCs and potentiates the growth-promoting effect of 5-HT on these cells. An increase in the levels of 5-HTT messenger ribonucleic acid was observed in smooth-muscle cells from remodeled pulmonary arteries in rats subjected to long-term hypoxia. Two series of especially relevant data further support the idea that 5-HT plays a key role in PASMC proliferation in vivo: (1) treatments that increase plasma 5-HT levels aggravate pulmonary hypertension in rats subjected to long-term hypoxia, and this effect can be prevented by combined simultaneous treatment with 5-HTT inhibitors; and (2) knockout mice with disruption of the 5-HTT gene exhibit lesser degree of hypoxic pulmonary hypertension and pulmonary vascular remodeling than control mice despite increased hypoxic pulmonary vasoconstriction. These observations indicate that 5-HTT expression, activity, or both in PASMCs contribute to pulmonary vascular remodeling and that the inducing effects of some appetite suppressants on pulmonary hypertension may be related to possible effects of these drugs on 5-HTT expression, activity, or both.

The aims of the study were to standardise an immunohistochemical (IHC) method for the detection of progesterone receptors (PR) on formalin-fixed, paraffin wax-embedded tissue sections of feline mammary gland tumours and dysplasias, comparing the results with those obtained using the radiolabelled ligand dextran coated charcoal (DCC) assay applied to frozen tissue samples from the same cases. Also, to define the immunohistochemical distribution of PR in the different cellular compartments of the lesions and to compare the oestrogen receptor (ER) and PR status of the feline mammary lesions. Proliferative mammary lesions collected from 34 cats were studied; 25 malignant tumours and 9 benign tumours and dysplasias. PR protein was present at a concentration of 5 fmol mg(-1) (positivity threshold) in 37.5 per cent of malignant tumours and 66.7 per cent of benign tumours and dysplasias while immunoreative products to PR antibody were found in the nuclei of tumour cells in 38.5 per cent and 66.7 per cent of the cases, respectively. Concordance between DCC-PR and IHC-PR was 88.5 per cent (P<0.001). The specificity (true negatives) and sensitivity (true positives) of the IHC method were 89.4 per cent and 87.5 per cent respectively. The presence of PR was linked to the absence of ovariectomy (P<0.02). Oestrogen receptors, detected by either method, were also detected in half the cases in which PR had been detected. In malignant tumours, the most prevalent groups were the ER + PR + and ER-PR + groups.

Pseudomonas resinovorans strain CA10 assimilates catechol, which is an intermediate of carbazole degradation, by ortho cleavage pathway enzymes encoded by the catR, catBCA operon. Cat proteins of strain CA10 were very similar to those of P. putida, although the relatedness in non-coding regions was not high. It was found that catBCA genes were induced in carbazole-grown cells as a single transcriptional unit.

Iron and copper are essential nutrients that must be meticulously regulated to exploit their usefulness in biological reactions while protecting against their tendency to promote formation of toxic free-radicals. This review summarizes recently described steps in the transport of these metals, and explores how defects in these steps lead to human diseases including hemochromatosis, Menkes disease and Wilson disease.

Previous comparative mapping suggested that at least five pig chromosomes (Sscr4, 6, 9, 10 and 14) share homology with human chromosome 1 (Hsap1). A significant quantitative trait loci (QTL) for fat deposition has been identified on Sscr4 that appears to be near the junction region between Sscr4 and Sscr9 relative to Hsap1. It is of interest to define the boundaries of conserved synteny between pig chromosomes and Hsap1 to use human map information to identify putative comparative positional candidates for this QTL. Eleven genes, including Janus kinase 1 (JAK1), Prostaglandin E receptor3 (PTGER3), urate oxidase (UOX), coagulation factor 3 (F3), vascular cell adhesion molecule 1 (VCAM1), ribosomal protein L5 (RPL5), POU domain, class 2, transcription factor 1 (POU2F1), coagulation factor 5 (F5), Prostaglandin endoperoxide synthase-2 (PTGS2), myosin binding protein H (MYBPH) and Antithrombin III (SERPINC1), were selected to refine the boundaries of the blocks of conserved synteny between Hsap1 and pig chromosomes. Pig sequence tagged sites (STSs) were developed and used to physically map these 11 genes using a somatic cell hybrid panel. Eight loci have been mapped by using fluorescent in situ hybridization (FISH) to improve map resolution. Heterologous FISH was used to refine the location of VCAM1 on human chromosomes. In addition, human yeast artificial chromosomes (YACs) were mapped by heterologous FISH on pig metaphases to refine the boundaries of the regions of homology between Sscr4 and Sscr9 on Hsap1. Results from this study suggest the precise break in conserved synteny on Hsap1 corresponding to the Sscr4/6 and Sscr4/9 transitions are most likely on the Hsap1p22 and Hsap1q24-25 regions, respectively. Further, our data predict that Hsap1q21-24 is a candidate region for the backfat QTL localized to Sscr4.

A 52-year-old man presented to his primary care physician with blisters and sores on the backs of his hands. Laboratory studies supported a diagnosis of porphyria cutanea tarda, complicated by the presence of both the C282Y and H63D mutations in the HFE gene, with susequent iron over-load. This case illustrates the need to understand the pathogenesis of porphyria cutanea tarda, particularly the role of excess iron in the overproduction of uroporphyrin. Iron, by catalyzing the formation of reactive oxygen species, can enhance uroporphyrin formation by increasing the rate at which uroporphyrinogen is oxidized to urophophyrin. Iron may also act indirectly to inhibit uroporphyrinogen decarboxylase activity by enhancing the formation of non-porphyrin products of porphyrinogen oxidation that are themselves direct inhibitors of the enzyme. Finally, iron can act to increase urophorphyrin production by inducing delta-aminolevulinic acid synthase, thus increasing the amount of delta-aminolevulinic acid, the precursor to uroporphyrinogen, present in the cell. After the diagnosis, the patient underwent an aggressive series of therapeutic phlebotomies to reduce iron levels, and gradually the cutaneous manifestations of porphyria cutanea tarda improved.

Hereditary hemochromatosis (HH) comprises several inherited disorders of iron homeostasis characterized by increased gastrointestinal iron absorption and secondary tissue iron deposition. The most common form of this disorder is called HFE-related HH and is caused by homozygosity for the C282Y mutation in the HFE gene. Recently, other less common hereditary forms of iron overload have been recognized and are designated as non-HFE-related HH. The identification and cloning of HFE and other genes involved in iron metabolism has greatly expanded our understanding of many aspects of HH. The introduction of a commercially available genetic test for the C282Y and H63D mutations of HFE allows presymptomatic diagnosis, and adds precision to studies of the population genetics of HFE-related HH. It is now recognized that a substantial proportion of C282Y homozygotes does not develop clinically significant iron overload, and modifier genes may be involved in this phenomenon. Mouse models of HH and cell culture studies have increased our understanding of the normal physiology and pathophysiology of iron homeostasis. Future investigations will refine our knowledge of the mechanisms of action of HFE protein, the phenotypic variability observed in persons homozygous for the C282Y mutation, and the mechanisms responsible for non-HFE-related HH.

The presence of an expanded polyglutamine produces a toxic gain of function in huntingtin. Protein aggregation resulting from this gain of function is likely to be the cause of neuronal death. Two main mechanisms of aggregation have been proposed: hydrogen bonding by polar-zipper formation and covalent bonding by transglutaminase-catalyzed cross-linking. In cell culture models of Huntington's disease, aggregates are mostly stabilized by hydrogen bonds, but covalent bonds are also likely to occur. Nothing is known about the nature of the bonds that stabilize the aggregates in the brain of patients with Huntington's disease. It seems that the nature of the bond stabilizing the aggregates is one of the most important questions, as the answer would condition the therapeutic approach to Huntington's disease.

<h4>Objectives</h4>Thrombolytic therapy in patients with acute myocardial infarction is hampered by bleeding complications and procoagulant effects favoring early reocclusion. TNK-tPA was shown in vitro to have considerable fibrin specificity. We investigated the effects of tenecteplase (TNK-tPA) and alteplase (rt-PA) on the haemostasis and fibrinolytic system.<h4>Methods and results</h4>We enrolled 30 patients with AMI into the study. Twenty patients received front-loaded rt-PA up to 100 mg; 10 patients were given TNK-tPA in a single bolus up to 50 mg. All patients received aspirin and intravenous heparin. During the first 2 days, the following parameters were repetitively determined: thrombin-antithrombin III complexes (TAT), antithrombin III (ATIII), prothrombin fragment F 1 + 2 (F 1 + 2), kallikrein-like activity (KK), activated factor XII (FXIIa), plasmin alpha 2-antiplasmin complexes (PAP), fibrinogen, D-dimers (DD), tissue-type plasminogen activator (t-PA). A total of 75 healthy persons served as control group. TAT increased significantly after rt-PA but not after TNK-tPA (3 h: 38.1 +/- 29.4 versus 10.5 +/- 4.2 microg/l; p < 0.01), indicating paradoxical thrombin activation. F 1 + 2 increased transiently after rt-PA but not after TNK-tPA. Fibrinogen was significantly lower after rt-PA versus TNK-tPA (3 h: 163 +/- 27 versus 380 +/- 54 mg/dl; p < 0.05). KK activities in the rt-PA group were significantly (p < 0.01) increased over 48 h versus TNK-tPA. PAP and D-dimers were lower over the time course of 48 h in the tenecteplase group versus rt-PA.<h4>Conclusions</h4>This study indicates that tenecteplase has higher fibrin specificity not only in vitro but also in vivo versus alteplase. TNK-tPA consecutively has no paradoxical systemic procoagulant effect due to the lower extent of activation of the kallikrein-factor XII system than alteplase.

Hereditary hemochromatosis is a disorder of iron metabolism, which is currently the most prevalent autosomal recessive disorder in the world, with an expression of the homozygous form occurring in approximately 1 in 200 individuals of European descent. Approximately one third of patients with hemochromatosis die of iron-induced cardiac complications. Although the exact mechanism is not known, it is believed that the toxicity of excess iron in biological systems is due to its ability to catalyze the generation of harmful reactive oxygen free radical species (ROS), which can damage proteins, lipids, and DNA. There is preliminary evidence to suggest that non-transferrin-bound iron uptake in the myocardium may occur through voltage-dependent L-type calcium channels, and that calcium channel blockers (CCBs) may possess antioxidant properties. Accordingly, the authors hypothesized that the administration of amlodipine besylate would (1) decrease iron uptake in the myocardium and (2) decrease oxygen free radical production as measured by cytotoxic aldehyde-derived peroxidation products in a murine model of iron overload cardiomyopathy. The findings show that the CCB amlodipine is partially effective in limiting iron uptake in the heart and significantly inhibits the production of ROS in chronically iron-loaded mice. These are important preliminary findings because they suggest that CCBs may have significance in the clinical management of genetic disorders of iron metabolism.