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<h4>Background and aims</h4>In rectal cancer altered expression of p53 or DCC may be indicative of poor patient prognosis. This study determined by immunohistochemistry the tumour status of p53 protein and DCC protein in patients with rectal cancer who had a curative resection and were followed-up prospectively and examined the correlation to clinical and pathology variables.<h4>Patients and methods</h4>The study included 171 who had a curative resection for rectal cancer: 88 at Concord Hospital (CH) followed up for a mean of 11 years and 83 at Royal Prince Alfred Hospital (RPAH) followed up prospectively for a mean of 4.3 years. Specimens were assessed by immunohistochemical assay of p53 expression ( n=170) and of DCC expression ( n=168).<h4>Results</h4>p53 over-expression was demonstrated in 58% of CH tumours and 59% of RPAH tumours. Absence of normal DCC expression was demonstrated in 66% of CH tumours and 52% of RPAH tumours. On both separate and combined analysis of these groups there were no significant associations by univariate analysis between p53 expression or DCC expression or combinations of p53 and DCC expression and the pathology variables: extent of penetration through bowel wall, lymph node involvement, presence of venous invasion, and tumour differentiation.<h4>Conclusion</h4>The immunohistochemical p53 and DCC status of rectal tumours was not associated with other clinical or pathology variables, nor predictive of outcome.

Cellular iron uptake in most tissues occurs via endocytosis of diferric transferrin (Tf) bound to the transferrin receptor (TfR). Recently, a second transferrin receptor, transferrin receptor 2 (TfR2), has been identified and shown to play a critical role in iron metabolism. TfR2 is capable of Tf-mediated iron uptake and mutations in this gene result in a rare form of hereditary hemochromatosis unrelated to the hereditary hemochromatosis protein, HFE. Unlike TfR, TfR2 expression is not controlled by cellular iron concentrations and little information is currently available regarding the role of TfR2 in cellular iron homeostasis. To investigate the relationship between TfR and TfR2, we performed a series of in vivo and in vitro experiments using antibodies generated to each receptor. Western blots demonstrate that TfR2 protein is expressed strongest in erythroid/myeloid cell lines. Metabolic labeling studies indicate that TfR2 protein levels are approximately 20-fold lower than TfR in these cells. TfR and TfR2 have similar cellular localizations in K562 cells and coimmunoprecipitate to only a very limited extent. Western analysis of the receptors under nonreducing conditions reveals that they can form heterodimers.

Mycobacterium tuberculosis multiplies within the macrophage phagosome and requires iron for growth. We examined the route(s) by which intracellular M. tuberculosis acquires iron. During intracellular growth of the virulent Erdman M. tuberculosis strain in human monocyte-derived macrophages (MDM), M. tuberculosis acquisition of (59)Fe from transferrin (TF) provided extracellularly (exogenous source) was compared with acquisition when MDM were loaded with (59)Fe from TF prior to M. tuberculosis infection (endogenous sources). M. tuberculosis (59)Fe acquisition required viable bacteria and was similar from exogenous and endogenous sources at 24 h and greater from exogenous iron at 48 h. Interferon-gamma treatment of MDM reduced (59)Fe uptake from TF 51% and TF receptor expression by 34%. Despite this, intraphagosomal M. tuberculosis iron acquisition in IFN-gamma-treated cells was decreased by only 30%. Macrophages from hereditary hemochromatosis patients have altered iron metabolism. Intracellular M. tuberculosis acquired markedly less iron in MDM from these individuals than in MDM from healthy donors, regardless of the iron source (exogenous and endogenous): 36 +/- 3.8% and 17 +/- 9.6% of control, respectively. Thus, intraphagosomal M. tuberculosis can acquire iron from both extracellular TF and endogenous macrophage sources. Acquisition of iron from macrophage cytoplasmic iron pools may be critical for the intracellular growth of M. tuberculosis. This acquisition is altered by IFN-gamma treatment to a small extent, but is markedly reduced in macrophages from hemochromatosis patients.

<h4>Background</h4>G-protein-coupled receptors (GPCRs) are the largest and most diverse family of transmembrane receptors. They respond to a wide range of stimuli, including small peptides, lipid analogs, amino-acid derivatives, and sensory stimuli such as light, taste and odor, and transmit signals to the interior of the cell through interaction with heterotrimeric G proteins. A large number of putative GPCRs have no identified natural ligand. We hypothesized that a more complete knowledge of the phylogenetic relationship of these orphan receptors to receptors with known ligands could facilitate ligand identification, as related receptors often have ligands with similar structural features.<h4>Results</h4>A database search excluding olfactory and gustatory receptors was used to compile a list of accession numbers and synonyms of 81 orphan and 196 human GPCRs with known ligands. Of these, 241 sequences belonging to the rhodopsin receptor-like family A were aligned and a tentative phylogenetic tree constructed by neighbor joining. This tree and local alignment tools were used to define 19 subgroups of family A small enough for more accurate maximum-likelihood analyses. The secretin receptor-like family B and metabotropic glutamate receptor-like family C were directly subjected to these methods.<h4>Conclusions</h4>Our trees show the overall relationship of 277 GPCRs with emphasis on orphan receptors. Support values are given for each branch. This approach may prove valuable for identification of the natural ligands of orphan receptors as their relation to receptors with known ligands becomes more evident.

<h4>Background</h4>Huntington's disease (HD) pathogenesis is due to an expanded polyglutamine tract in huntingtin, but the specificity of neuronal loss compared with other polyglutamine disorders also implies a role for the protein's unknown inherent function. Huntingtin is moderately conserved, with 10 HEAT repeats reported in its amino-terminal half. HD orthologues are evident in vertebrates and Drosophila, but not in Saccharomyces cerevisiae, Caenorhabditis elegans or Arabidopsis thaliana, a phylogenetic profile similar to the NF-kB/Rel/dorsal family transcription factors, suggesting a potential functional relationship.<h4>Results</h4>We initially tested the potential for a relationship between huntingtin and dorsal by overexpression experiments in Drosophila S2 cells. Drosophila huntingtin complexes via its carboxyl-terminal region with dorsal, and the two enter the nucleus concomitantly, partly in a lipopolysaccharide (LPS)- and Nup88-dependent manner. Similarly, in HeLa cell extracts, human huntingtin co-immunoprecipitates with NF-kB p50 but not with p105. By cross-species comparative analysis, we find that the carboxyl-terminal segment of huntingtin that mediates the association with dorsal possesses numerous HEAT-like sequences related to those in the amino-terminal segment. Thus, Drosophila and vertebrate huntingtins are composed predominantly of 28 to 36 degenerate HEAT-like repeats that span the entire protein.<h4>Conclusion</h4>Like other HEAT-repeat filled proteins, huntingtin is made up largely of degenerate HEAT-like sequences, suggesting that it may play a scaffolding role in the formation of particular protein-protein complexes. While many proteins have been implicated in complexes with the amino-terminal region of huntingtin, the NF-kB/Rel/dorsal family transcription factors merit further examination as direct or indirect interactors with huntingtin's carboxyl-terminal segment.

<h4>Background</h4>We wanted to quantify HLA-A and -B allele and haplotype frequencies in Alabama hemochromatosis probands with HFE C282Y homozygosity and controls, and to compare results to those in other populations.<h4>Methods</h4>Alleles were detected using DNA-based typing (probands) and microlymphocytotoxicity (controls).<h4>Results</h4>Alleles were determined in 139 probands (1,321 controls) and haplotypes in 118 probands (605 controls). In probands, A*03 positivity was 0.7482 (0.2739 controls; p = or < 0.0001; odds ratio (OR) 7.9); positivity for B*07, B*14, and B*56 was also increased. In probands, haplotypes A*03-B*07 and A*03-B*14 were more frequent (p < 0.0001, respectively; OR = 12.3 and 11.1, respectively). The haplotypes A*01-B*60, A*02-B*39, A*02-B*62, A*03-B*13, A*03-B*15, A*03-B*27, A*03-B*35, A*03-B*44, A*03-B*47, and A*03-B*57 were also significantly more frequent in probands. 37.3% of probands were HLA-haploidentical with other proband(s).<h4>Conclusions</h4>A*03 and A*03-B*07 frequencies are increased in Alabama probands, as in other hemochromatosis cohorts. Increased absolute frequencies of A*03-B*35 have been reported only in the present Alabama probands and in hemochromatosis patients in Italy. Increased absolute frequencies of A*01-B*60, A*02-B*39, A*02-B*62, A*03-B*13, A*03-B*15, A*03-B*27, A*03-B*44, A*03-B*47, and A*03-B*57 in hemochromatosis cohorts have not been reported previously.

Some members of the major histocompatibility complex (MHC) class I gene family are encoded outside the MHC. Here we describe a family of mouse class I-like genes mapping to the vicinity of the leukocyte receptor complex (LRC) on chromosome 7. This family, which we call Mill (MHC class I-like located near the LRC), has two members designated Mill1 and Mill2. Both genes are predicted to encode membrane glycoproteins with domain organization essentially similar to that of MHC class I heavy chains. The following features of Mill are noteworthy. (i) The deduced MILL proteins lack most of the residues known to be involved in the docking of peptides in classical MHC class I molecules. (ii) Among the known members of the class I gene family, MILL1 and MILL2 are related most closely to MICA/MICB encoded in the human MHC. (iii) Unlike all other known members of the class I gene family, Mill1 and Mill2 have an exon between those coding for the signal peptide and the alpha1 domain. (iv) Mill1 has a more restricted expression profile than Mill2. (v) The gene orthologous to Mill1 or Mill2 apparently is absent in the human. (vi) Mill1 and Mill2 show a limited degree of polymorphism in laboratory mice. The observation that the Mill family is related most closely to the MIC family, together with its apparent absence in the human, suggests its involvement in innate immunity.

We developed an effective strategy to restrict the amino acid usage in a relatively large protein to a reduced set with conservation of its in vivo function. The 213-residue Escherichia coli orotate phosphoribosyltransferase was subjected to 22 cycles of segment-wise combinatorial mutagenesis followed by 6 cycles of site-directed random mutagenesis, both coupled with a growth-related phenotype selection. The enzyme eventually tolerated 73 amino acid substitutions: In the final variant, 9 amino acid types (A, D, G, L, P, R, T, V, and Y) occupied 188 positions (88%), and none of 7 amino acid types (C, H, I, M, N, Q, and W) appeared. Therefore, the catalytic function associated with a relatively large protein may be achieved with a subset of the 20 amino acid. The converged sequence also implies simpler constituents for proteins in the early stage of evolution.

The survival of endothelial cells is dependent on interactions between the matrix and integrins mediated through focal adhesions. Focal adhesion kinase (FAK) is thought to play a key role in maintaining focal adhesion function and cell survival, whereas caspase-mediated FAK proteolysis is implicated in focal adhesion disassembly during apoptosis. We examined the relationship between changes in FAK phosphorylation and proteolysis during apoptosis of primary porcine aortic endothelial cells (PAEC) induced by staurosporine, a widely used apoptogenic agent in diverse cell types. Staurosporine-induced PAEC apoptosis was detected after 1 h and was preceded by disruption and loss of FAK localization to focal adhesions within a few minutes, whereas staurosporine-induced cleavage of FAK occurred only after 8-24 h. Staurosporine induced a very rapid dephosphorylation of FAK at Tyr(861) and Tyr(397) and caused dissociation of phosphorylated FAK from focal adhesions as early as 30 s. The effect of staurosporine was very potent with striking inhibition of Tyr(861) and Tyr(397) phosphorylation and focal adhesion disruption occurring in the range 10-100 nM. Selective inhibition of a known target of staurosporine, protein kinase C, using GF109203X, and of phosphoinositide 3'-kinase using wortmannin, did not reduce FAK tyrosine phosphorylation at Tyr(861) and Tyr(397), or cause disruption of focal adhesions. Cycloheximide, the protein synthesis inhibitor, induced PAEC apoptosis more slowly than staurosporine, but did not induce FAK dephosphorylation or rapid focal adhesion disruption, and instead caused a slower loss of focal adhesions and a marked increase in FAK proteolysis. These studies show that FAK dephosphorylation and focal adhesion disassembly are very early events mediating the onset of staurosporine-induced endothelial cell apoptosis and are dissociated from FAK proteolysis. Cycloheximide induces apoptosis through a pathway involving FAK proteolysis without dephosphorylation.

In the present study, we used 22 microsatellite markers flanking to or within 13 known or candidate tumor suppressor genes (TSGs) to detect loss of heterozygosity (LOH) in these chromosomal regions among 41 cases of non-small cell lung cancer, including 28 squamous cell carcinoma (SCC) and 13 adenocarcinoma (ADC). The studied TSGs comprised FHIT, VHL, APC, PRLTS, p16, IFNA, PTEN, p57, ATM, p53, BRCA1, DPC4 and DCC. Our data demonstrated frequent allelic losses of FHIT, p53, IFNA, VHL and p16 in both SCC and ADC. PTEN and ATM showed the least frequency of LOH, while no deletion of BRCA1 was detected in all tumor samples. LOH analysis of PRLTS was extended to 26 cases of ADC, which demonstrated significantly higher frequency of LOH than SCC. Our data indicated a possible correlation between specific TSG(s) and either histological type of lung cancer, and more attention should be paid to the PRLTS gene, which might play an important role in the development of ADC.

To investigate genetic alterations in primary cutaneous B-cell lymphomas (PCBCLs), we have analyzed 29 cases of PCBCL. Comparative genomic hybridization showed chromosome imbalances (CIs) in 12 cases (41%). The mean number of CIs per sample was 2.05 +/- 2.97, with gains (1.48 +/- 2.38) more frequent than losses (0.56 +/- 1.40). The common regions of gains were 18/18q (50%), 7/7p (42%), 3/3q (33%), 20 (33%), 1p (25%), 12/12q (25%), and 13/13q (25%), whereas loss of 6q was frequent (42%). Among the different subsets of PCBCLs, CI was seen in 50% of diffuse large-cell lymphomas (DLCLs), 33% of marginal zone lymphomas, and 8% of follicle center cell lymphomas and unclassified lymphomas. A similar pattern of CI was observed in these lymphomas, but loss of 6q and gains of 3/3q were present only in DLCLs. Microarray-based genomic analysis of four DLCL cases identified oncogene gains of SAS/CDK4 (12q13.3) in three cases and MYCL1 (1p34.3), MYC (8q24), FGFR2 (10q26), BCL2 (18q21.3), CSE1L (20q13), and PDGFB (22q12-13) in two cases, whereas losses of AKT1 (14q32.3), IGFR1 (15q25-26), and JUNB (19p13.2) were identified in three cases, and losses of FGR (1p36), ESR (6q25.1), ABL1 (9q34.1), TOP2A (17q21-22), ERBB2 (17q21.2), CCNE1 (19q13.1), and BCR (22q11) were each identified in two cases. In addition, real-time-polymerase chain reaction detected amplification of BCL2 in 5 of 29 cases. These findings suggest that there are complex but consistent genetic alterations associated with the pathogenesis of PCBCLs.

We investigated the association between serotonergic polymorphisms and incidence of nausea, which is the most frequent side-effect of selective serotonin reuptake inhibiters (SSRIs), in 66 patients treated with fluvoxamine in a protocolized-dosing method. We focused on three polymorphisms of serotonin (5-HT) neuronal systems such as 5-HT transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR), a variable number of tandem repeat (VNTR) polymorphism in the second intron of the 5-HTT gene (STin2) and tryptophan hydroxylase (TPH) gene polymorphism in intron 7 (TPH-A218C), which have been reported to possess positive association with treatment response to SSRIs. In addition to this, the relationship between development of nausea and treatment response was also analyzed. Results suggested that these three polymorphisms did not affect the development of fluvoxamine-induced nausea, and that incidence of nausea was not a phenomenon that predicts the treatment response to fluvoxamine.

The RING domain protein Sina, together with Phyllopod and the F-box protein Ebi, forms a Ras-regulated E3 ubiquitin ligase complex that activates photoreceptor cell differentiation in the eye of Drosophila melanogaster. The expression of Phyllopod is induced upon Ras activation, allowing the complex to degrade the transcription repressor Tramtrack and removing its block of neuronal development in photoreceptor precursors. We show that Phyllopod functions as an adaptor in the complex, physically linking Sina with Tramtrack via separate binding domains. One 19-amino-acid domain in Phyllopod interacts with a region of Sina's SBD domain. Another domain in Phyllopod interacts with a C-terminal helix in the POZ domain of Tramtrack. This interaction is specific to the Tramtrack POZ domain and not to other POZ domain proteins present in photoreceptor precursors. Degradation of Tramtrack is dependent upon association of Sina with its cognate binding site in Phyllopod. These results illustrate how Ras signaling can modulate an E3 ligase activity not by the phosphorylation of substrate proteins but by regulating the expression of specific E3 adaptors.

The 4-chloro- and 2,4-dichlorophenol-degrading strain Rhodococcus opacus 1CP has previously been shown to acquire, during prolonged adaptation, the ability to mineralize 2-chlorophenol. In addition, homogeneous chlorocatechol 1,2-dioxygenase from 2-chlorophenol-grown biomass has shown relatively high activity towards 3-chlorocatechol. Based on sequences of the N terminus and tryptic peptides of this enzyme, degenerate PCR primers were now designed and used for cloning of the respective gene from genomic DNA of strain 1CP. A 9.5-kb fragment containing nine open reading frames was obtained on pROP1. Besides other genes, a gene cluster consisting of four chlorocatechol catabolic genes was identified. As judged by sequence similarity and correspondence of predicted N termini with those of purified enzymes, the open reading frames correspond to genes for a second chlorocatechol 1,2-dioxygenase (ClcA2), a second chloromuconate cycloisomerase (ClcB2), a second dienelactone hydrolase (ClcD2), and a muconolactone isomerase-related enzyme (ClcF). All enzymes of this new cluster are only distantly related to the known chlorocatechol enzymes and appear to represent new evolutionary lines of these activities. UV overlay spectra as well as high-pressure liquid chromatography analyses confirmed that 2-chloro-cis,cis-muconate is transformed by ClcB2 to 5-chloromuconolactone, which during turnover by ClcF gives cis-dienelactone as the sole product. cis-Dienelactone was further hydrolyzed by ClcD2 to maleylacetate. ClcF, despite its sequence similarity to muconolactone isomerases, no longer showed muconolactone-isomerizing activity and thus represents an enzyme dedicated to its new function as a 5-chloromuconolactone dehalogenase. Thus, during 3-chlorocatechol degradation by R. opacus 1CP, dechlorination is catalyzed by a muconolactone isomerase-related enzyme rather than by a specialized chloromuconate cycloisomerase.

Several methods have been used to develop rodent models with the hepatic manifestations of porphyria cutanea tarda (PCT). Acute iron administration or mutations of the hemochromatosis gene (Hfe) have been used to generate hepatic siderosis, a nearly uniform finding in PCT. Heterozygosity for a null mutation at the uroporphyrinogen decarboxylase (Uro-D+/-) locus has been developed to mimic familial PCT in humans. This study examines the interplay of these 2 genetic risk factors and their influence, alone and combined with polychlorinated-biphenyl exposure. Neither an Hfe-null mutation nor iron-dextran administration alone or in combination with polychlorinated biphenyl exposure was porphyrinogenic in a 3-week model using mice wild-type at the Uro-D locus. Homozygosity for an Hfe-null mutation significantly elevated hepatic iron but not to the extent seen with parenteral iron-dextran administration. Homozygosity for an Hfe-null mutation but not iron-dextran administration was porphyrinogenic in animals heterozygous for the Uro-D mutation. Polychlorinated biphenyls were also porphyrinogenic in these animals. Uroporphyria in Uro-D+/- animals was exacerbated by combinations of the homozygous Hfe-null mutation and polychlorinated biphenyls and iron-dextran and polychlorinated biphenyls. In all cases in which uroporphyria developed, a greater degree of experimental uroporphyria was seen in female animals. All elevated hepatic uroporphyrin concentrations were accompanied by depressed uroporphyrinogen decarboxylase activity and the presence of a factor in cytosol that inhibits recombinant human uroporphyrinogen decarboxylase. In conclusion, the expression of the uroporphyric phenotype, dependent on the susceptibility imparted by a genetic mutation, provides a uniquely facile model for dissecting the molecular pathogenesis of the disease.

Netrin-1 and its receptors deleted in colorectal cancer (DCC) and neogenin are expressed within the developing visual system and have been implicated in mediating axon guidance and cell migration. However, the expression of the netrin-1 receptor UNC-5 has not yet been addressed. Here we clone Xenopus UNC-5 (XUNC-5) and describe its expression within the developing visual system. Xunc-5 transcripts are expressed within the developing optic vesicles and later become restricted to the dorsal ciliary marginal zone, a site of retinoblast proliferation and differentiation. This highly restricted expression pattern could make XUNC-5 an excellent marker of dorsal retinal precursor cells.

Caenorhabditis elegans UNC-40, Drosophila Frazzled, and vertebrate Neogenin and DCC constitute a subgroup of the immunoglobulin superfamily (IgSF). They possess four immunoglobulin-like domains and six fibronectin-type III repeats at the extracellular region, a single transmembrane region, and a approximately 300 amino-acid intracellular region. UNC-40, Frazzled and DCC can function in axon guidance as the receptor of Netrin (Cell Mol. Life Sci. 56 (1999) 62; Curr. Opin. Cell Biol. 10 (1998) 609). Neogenin binds to Netrin-1 with the same affinity as DCC in vitro (Cell 87 (1996) 175), and is expressed by neurons as they project axons (J. Cell Biol. 127 (1994) 2009), suggesting that it is also a DCC-like Netrin receptor. A zebrafish homologue of DCC (zDCC) is reported recently (Mech. Dev. 109 (2001) 105), but so far there is no report of zebrafish Neogenin. To elucidate a possible neural function of vertebrate Neogenin, we cloned and characterized a zebrafish homologue of neogenin, zneo1, and identified four alternative splice sites within it. In the adult, despite broad tissue distribution, our reverse transcription polymerase chain reaction and Northern analyses demonstrated the dominant expression of zneo1 mRNA in brain. We detected zneo1 mRNA in the embryos from 10 hpf onward and revealed its spatiotemporally regulated expression pattern in both neuronal and non-neuronal tissues by in situ hybridization. Our data showed that during early brain development, zneo1 mRNA was not only present in the proliferative ventricular zones but also in the domains of several first postmitotic neuron clusters when they extended axons. Alternative splicing generates several isoforms of zneo1. Most of them are developmentally regulated, showing distinct distribution in brain and other tissues.

In the rodent primary somatosensory cortex, the thalamocortical axons (TCAs) are organized into clusters that correspond to functional units in the periphery. Around these axons, neurons in layer IV aggregate as barrels. To understand how this organization emerges, we analyzed TCA development in mice that do not form barrels, the monoamine oxidase A knock-out (MAOA-KO), and in MAOA/5-HT(1B) receptor double-KO mice, which have a restored barrel field. We show that TCAs already attain cortical layer IV on the day of birth. They are uniformly distributed in this layer from postnatal day 0 (P0) to P2 and secondarily coalesce into barrel domains in layer IV, over a 3 d period (P3-P5), with no prepatterning in the deeper layers. In MAOA-KO mice, the uniform distribution of the TC projection is maintained, and no axon clusters emerge. Individual TCA arbors were traced after carbocyanine injections. At P1, TCAs were poorly branched and covered variable tangential widths, encompassing one to two prospective barrels. At P7 the number of TCA branches increased 10-fold in layer IV and became restricted to one barrel. In MAOA-KO mice, there was a 50% reduction of the TCA terminal branches in layer IV, with a 40% increase in their tangential extent. These defects were corrected in the MAOA/5-HT(1B) double knock-out mice, indicating an effect of the presynaptic 5-HT(1B) receptor on axon branching. Our results indicate that the barrel-deficient phenotype of MAOA-KO mice results from an altered refinement of the TCA arbors in their target layer IV, involving branch elaboration and collateral retraction during early postnatal life.

The first several months of life are a critical period for neuronal plasticity in the visual cortex during which anatomic and physiological development depends on visual experience. In cats, electrophysiologically assessed neuronal plasticity is minimal until approximately 3 weeks, peaks at 5 weeks, gradually declines to low levels at 20 weeks, and disappears at approximately 1 year of age (Daw, 1994). Rearing in darkness slows the entire time course of this critical period, such that at 5 weeks of age, normal cats are more plastic than dark-reared cats, whereas at 20 weeks, dark-reared cats are more plastic (Mower, 1991; Beaver et al., 2001). Thus, a stringent criterion is that genes that are important for plasticity in visual cortex will show differences in expression between normal rearing and dark rearing that are of opposite direction in young versus older animals. The present study reports the identification by differential display PCR of Munc13-3, a mammalian homolog of the Caenorhabditis elegans "uncoordinated" gene (unc-13), as a candidate gene for critical-period neuronal plasticity, the expression of which is regulated according to this criterion specifically in visual cortex and not in frontal cortex. Other members of the Munc13 family (Munc13-1 and Munc13-2) do not meet this criterion in visual cortex, indicating that Munc13-3 is the only family member that is regulated by age and dark rearing in the same manner as physiological plasticity during the visual cortical critical period.

Huntington's disease (HD) is caused by a pathological expansion of a CAG repeat in the first exon of the gene coding for huntingtin, resulting in an abnormally long polyglutamine stretch. Despite its widespread expression, mutant huntingtin leads to selective neuronal loss in the striatum and cortex. Here we report that the neurospecific phosphoprotein PACSIN 1, which has been implicated as playing a central role in synaptic vesicle recycling, interacts with huntingtin via its C-terminal SH3 domain. Moreover, two other isoforms, PACSIN 2 and 3, which show a wider tissue distribution including the brain, do not interact with huntingtin despite a highly conserved SH3 domain. Furthermore, this interaction is repeat-length-dependent and is enhanced with mutant huntingtin, possibly causing the sequestration of PACSIN 1. Normally, PACSIN 1 is located along neurites and within synaptic boutons, but in HD patient neurons, there is a progressive loss of PACSIN 1 immunostaining in synaptic varicosities, beginning in presymptomatic and early-stage HD. Further, PACSIN 1 immunostaining of HD patient tissue reveals a more cytoplasmic distribution of the protein, with particular concentration in the perinuclear region coincident with mutant huntingtin. Thus, the specific interaction of huntingtin with the neuronal PACSIN isoform, PACSIN 1, and its altered intracellular distribution in pathological tissue, together with the observed differences in the binding behavior, suggest a role for PACSIN 1 during early stages of the selective neuropathology of HD.

T cell activation and cytokine secretion are important mediators of inflammation in allergic asthma. The costimulatory pathway CD28/CD80/CD86 has been shown to play an important role in T cell activation in allergic asthma, but less is known about the effect of other costimulatory molecules in allergy. The costimulatory molecule OX40 ligand (OX40L), a member of the tumor necrosis factor superfamily, has been shown to be important in T cell priming and cytokine production. We investigated the role of OX40L in a murine model of allergic inflammation using OX40L(-/-) mice. In this model, following OVA sensitization and challenge, mice develop features of allergic inflammation including elevated levels of total serum IgE, pulmonary eosinophils, cytokines, and pulmonary inflammation. In the absence of OX40L, total serum IgE, pulmonary eosinophils, cytokines, and pulmonary inflammation were all significantly reduced compared to wild-type controls. Levels of eotaxin mRNA, an eosinophil-specific chemoattractant, were also markedly reduced, paralleling the significant reduction in pulmonary eosinophils. Levels of allergen-induced Th1 as well as Th2 cytokines were also significantly reduced. Together, the data support a critical role for OX40L signals in allergic responses.

The serpin superfamily of serine proteinase inhibitors has a central role in controlling proteinases in many biological pathways in a wide range of species. The inhibitory function of the serpins involves a marked conformational transition, but this inherent molecular flexibility also renders the serpins susceptible to point mutations that result in aberrant intermolecular linkage and polymer formation. The effects of such protein aggregation are cumulative, with a progressive loss of cellular function that results in diseases as diverse as cirrhosis and emphysema. The recent recognition that mutations in a serpin can also result in late-onset dementia provides insights into changes that underlie other conformational diseases, such as the amyloidoses, the prion encephalopathies and Huntington and Alzheimer diseases.

An in vitro model using co-culture of bovine in vitro-produced (IVP) embryos and bovine oviduct epithelial cells (bOECs) was established to study embryo-maternal interactions in the oviductal environment. In vitro conditions maintaining differentiated growth of oviductal cells were determined by evaluating several media supplemented with different sera at various concentrations. Morphological features were used as indicators of physiological growth, and it became obvious that synthetic oviduct fluid (SOF) supplemented with either oestrous cow serum (OCS) or dextran-coated charcoal-treated fetal calf serum (DCC-FCS) helped to prevent dedifferentiation of bOECs (Expt 1). RT-real-time-PCR analysis revealed an increased mRNA content of the oviduct-specific glycoprotein GP 85-97 when using lower serum concentrations (2 and 5% compared with 10%; Expt 2). In subsequent experiments in which cell-free cultured controls and co-cultured embryos were compared, co-cultured embryos showed an increased rate of cleavage (P < 0.05) after 3 days. Successive cell-free culture until day 8 resulted in a lower rate of blastocyst development (P < 0.05) and reduced ATP content (P < 0.05) of co-cultured versus control embryos (Expt 3). Long-term co-culture (8 days) in SOF with 5% OCS increased the expression of developmentally relevant genes (glucose transporter 1 (Glut-1) and heat shock protein (HSP 70)) in co-cultured versus control embryos (Expt 4). Higher embryonic Glut-1 mRNA expression after co-culture was obvious when using 10% DCC-FCS, but was not significant when culture medium was supplemented with 10% rather than 5% OCS (Expt 5). In conclusion, SOF with 5% OCS supports differentiated growth of bOECs. Co-culture under these conditions improves early cleavage rate, but not blastocyst development, and increases the expression of developmentally relevant genes influenced by type of serum and serum concentration.

<h4>Introduction</h4>The pathogenesis of iron overload in alcoholic individuals is not fully elucidated. The frequency of mutations in hereditary hemochromatosis (HFE) is high in this population. Heterozygotes show data of iron overload similar to those in alcoholic individuals.<h4>Objective</h4>To analyze whether iron excess among alcoholic individuals is associated with mutations in C282Y, H63D or S65C in the HFE gene. Patients and methods. Thirty-two active alcoholic individuals (29 males and 3 females, age range 30-67 years) with data of iron overload (increased serum ferritin with or without saturation of increased transferrin) were studied. In all individuals, mutations C282Y, H63D and S65C were investigated. From 16 cases, liver histology was available. Data on iron overload were compared between patients with and without mutations.<h4>Results</h4>Twenty-two patients (68.8%) did not show any mutation, one (3.1%) was heterozygous for C282Y, three (9.4%) were homozygous for H63D, four (12.5%) were heterozygous for H63D, and two patients (6.3%) were heterozygous for C282Y/H63D. None of the patients was homozygous for C282Y or presented mutation in S65C. Transferrin saturation, serum ferritin and liver iron index were similar among with and without mutations. Three patients (9.3%) were diagnosed of hemochromatosis. One of them was homozygous for H63D, other patient was heterozygous for the combined C282Y/H63D, and in the remaining patient none of the mutations was found.<h4>Conclusions</h4>In our setting, iron overload among alcoholic individuals seems to be independent of the presence of mutations C282Y, H63D and S65C in the HFE gene.

The absence of bile in the gastrointestinal tract stimulates bacterial overgrowth and bacterial translocation. In the response to endotoxin and LPS-induced endotoxemia which may be prevented by antithrombin-III (AT-III); endothelial cells; and various cells release cytokines, nitric oxide (NO) and other mediators. The purpose of this study was to examine blood NO levels and renal inducible NO synthase (iNOS) expression and determine whether AT-III has an inhibiting effect on renal injury and iNOS expression in obstructive jaundice (OJ). Forty rats were randomized into four groups: group A (Sham), group B (Sham+AT-III, 250 IU kg(-1)), group C (OJ), group D (OJ+AT-III, 250 IU kg(-1)). All animals were sacrificed on the 10th day and blood samples were taken for bilirubin and NO level determination. In addition, iNOS expression of the renal tissues was evaluated immunohistochemically. Blood NO levels were found to be 32.99 micromol l(-1) in group A, 32.26 micromol l(-1) in group B, 46.33 micromol l(-1) in group C, and 34.71 micromol l(-1) in group D. The intensity of iNOS staining in the OJ+AT-III group was less than the intensity of iNOS staining in the OJ group in the renal tissue. This study shows that OJ causes increased production of NO in blood and increased iNOS expression in the kidney. AT-III inhibits iNOS expression and reduces the level of blood NO. Thus, our findings indicate that under conditions of OJ, AT-III limits renal cellular injury by inhibiting LPS-induced iNOS expression.

Generalised lipodystrophy of the Berardinelli-Seip type (BSCL) is a rare autosomal recessive human disorder with severe adverse metabolic consequences. A gene on chromosome 9 (BSCL1) has recently been identified, predominantly in African-American families. More recently, mutations in a previously undescribed gene of unknown function (BSCL2) on chromosome 11, termed seipin, have been found to be responsible for this disorder in a number of European and Middle Eastern families. We have studied the genotype/phenotype relationships in 70 affected subjects from 44 apparently unrelated pedigrees of diverse ethnic origin. In all subjects, hepatic dysfunction, hyperlipidaemia, diabetes mellitus, and hypertrophic cardiomyopathy were significant contributors to morbidity with no clear differences in their prevalence between subjects with BSCL1 or BSCL2 and those with evidence against cosegregation with either chromosome 9 or 11 (designated BSCLX). BSCL2 appears to be a more severe disorder than BSCL1 with a higher incidence of premature death and a lower prevalence of partial and/or delayed onset of lipodystrophy. Notably, subjects with BSCL2 had a significantly higher prevalence of intellectual impairment than those with BSCL1 or BSCLX (p<0.0001, OR 17.0, CI 3.6 to 79.0). The higher prevalence of intellectual impairment and the increased risk of premature death in BSCL2 compared to BSCL1 emphasise the importance of molecular diagnosis of this syndrome and have clear implications for genetic counselling.

We have investigated the role of two vasoactive substances, nitric oxide (NO)and endothelin (ET), in the pathophysiology of disseminated intravascular coagulation (DIC), using two types of DIC models. Experimental DIC was induced by sustained infusion of 0.1, 1, 10, or 50 mg/kg lipopolysaccharide (LPS), or 3.75 U/kg thromboplastin (TF), for 4 h via the rat tail vein. Plasma levels of both NOX (metabolites of NO) and ET were significantly increased following infusion of 0.1 mg/kg or greater of LPS in the LPS-induced DIC rat model. In contrast, although a marked increase in the plasma levels of NOX was observed, only a slight increase in plasma ET levels was seen in the TF-induced DIC rat model. No significant differences in the plasma levels of platelets or thrombin-ATIII complex were observed among the TF-induced and LPS (50 mg/dl)-induced DIC models. However, plasma NOX levels rose significantly higher in the TF-induced model, relative to the LPS-induced model (p <0.01). Conversely, plasma ET levels were significantly greater after LPS-induction, compared to TF-induction, of DIC (p <0.01). Vasoconstriction, as well as depressed fibrinolytic activity, may be additional factors leading to severe organ dysfunction in the LPS-induced DIC rat model. Moreover, vasodilatation, as well as enhanced fibrinolytic activity, may help to prevent rats from severe organ dysfunction in the TF-induced DIC model. Our results suggest that modulator of vasoactive substances should be examined in the treatment of DIC.

<h4>Background and purpose</h4>Increased serum iron is found to be a risk factor for stroke. Carriers of HFE C282Y and H63D mutations have elevated serum iron levels and may have an increased risk for stroke. We studied the association between HFE gene mutations, carotid atherosclerosis, and stroke.<h4>Methods</h4>We compared the frequency of the HFE C282Y and H63D gene mutations in 202 prevalent and incident cases of stroke with that of 2730 controls from a population-based study, the Rotterdam Study. The influence of HFE mutations on the relationship between hypertension, smoking, and stroke was studied by use of a logistic regression model. In the analyses of hypertension, we used noncarriers and nonhypertensives as reference; in the analysis of smoking, we used noncarriers and those who never smoked as the reference group. Furthermore, we studied the mean intima-media thickness of the common carotid artery in relation to hypertension, smoking, and the HFE genotype in subjects without stroke.<h4>Results</h4>The percentage of both C282Y and H63D carriers in cases (43.7%, n=87) did not differ significantly (P=0.09) from that of controls (37.6%, n=986). The odds ratio for stroke for HFE carriers who also suffered from hypertension was 3.0 (95% CI, 1.9 to 4.6), and for HFE carriers who were also smokers, the odds ratio for stroke was 2.6 (95% CI, 1.4 to 5.0). The mean+/-SD intima-media thickness of the carotid artery was 0.77+/-0.14 mm for noncarriers without a history of hypertension or smoking compared with 0.81+/-0.17 mm for HFE carriers who smoked (P<0.004) and 0.84+/-0.20 mm for HFE carriers who were hypertensive (P<0.001).<h4>Conclusions</h4>Mutations in the HFE gene were not significantly related to stroke or atherosclerosis in the carotid artery. The HFE gene may modify the relationship between smoking and stroke.

Both Helicobacter pylori (HP) and Epstein-Barr virus (EBV) have been implicated in carcinogenesis of the stomach. Fifty-seven gastric carcinomas were tested for microsatellite instability and allelic loss at several tumor suppressor loci using 21 polymorphic microsatellite markers. Furthermore, immunohistochemistry for p53 and DPC4/SMAD4 was performed. Results were analyzed according to HP and EBV status of the tumors, as assessed by immunohistochemistry and RNA in situ hybridization, respectively. Fractional allelic loss was lower in EBV-positive carcinomas (n = 15) when compared to EBV-negative carcinomas (P < 0.001). EBV positivity was inversely associated with allelic loss at specific markers on chromosomal arms 5q (APC), 17p (TP53), and 18q (DPC4/SMAD4). Allelic loss at the TP53 locus was not encountered in EBV-positive carcinomas, but occurred in 51% of EBV-negative carcinomas (P < 0.005). Moreover, none of the EBV-positive carcinomas showed unequivocal p53 immunopositivity in contrast to 39% of the EBV-negative carcinomas (P < 0.01). EBV-status was not related to microsatellite instability. There was no correlation between HP-status and any of the molecular alterations tested. In conclusion, EBV-positive gastric carcinomas follow a distinct pathogenesis at the molecular level, in which p53 is not, or differently inactivated.

The present study was aimed at determining whether hepcidin, a recently identified peptide involved in iron metabolism, plays a role in conditions associated with both iron overload and iron deficiency. Hepcidin mRNA levels were assessed in two models of anemia, acute hemolysis provoked by phenylhydrazine and bleeding provoked by repeated phlebotomies. Hepcidin response to hypoxia was also studied, both ex vivo, in human hepatoma cells, and in vivo. Anemia and hypoxia were associated with a dramatic decrease in liver hepcidin gene expression, which may account for the increase in iron release from reticuloendothelial cells and increase in iron absorption frequently observed in these situations. A single injection of turpentine for 16 hours induced a sixfold increase in liver hepcidin mRNA levels and a twofold decrease in serum iron. The hyposideremic effect of turpentine was completely blunted in hepcidin-deficient mice, revealing hepcidin participation in anemia of inflammatory states. These modifications of hepcidin gene expression further suggest a key role for hepcidin in iron homeostasis under various pathophysiological conditions, which may support the pharmaceutical use of hepcidin agonists and antagonists in various iron homeostasis disorders.

The interaction between OX40 and OX40 ligand (OX40L) is suggested to provide T cells with an effective costimulatory signals during T cell-APC interaction. To examine the in vivo effect of constitutive OX40/OX40L interaction during immune regulation, we report the establishment of OX40L-transgenic (OX40L-Tg) mice that constitutively express OX40L on T cells. Markedly elevated numbers of effector memory CD4(+) T cells, but not CD8(+) T cells, were observed in the secondary lymphoid organs of OX40L-Tg mice. Upon immunization with keyhole limpet hemocyanin in the absence of adjuvant, profound T cell proliferative responses and cytokine productions were seen in the OX40L-Tg mice as compared with wild-type mice. Furthermore, in OX40L-Tg mice administrated with superantigen, this constitutive OX40/OX40L interaction on CD4(+) T cells completely prevented normal in vivo clonal T cell deletion. Interestingly, OX40L-Tg mice on the C57BL/6 background spontaneously developed interstitial pneumonia and inflammatory bowel disease that was accompanied with a significant production of anti-DNA Ab in the sera. Surprisingly, these diseases were not evident on the OX40L-Tg mice on the BALB/c strain. However, such inflammatory diseases were successfully reproducible in recombination-activating gene (RAG)2-deficient mice upon transfer of OX40L-Tg CD4(+) T cells. Blockade of OX40/OX40L interaction in the recipient RAG2-deficient mice completely prevented disease development. The present results orchestrated in this study indicate that OX40/OX40L interaction may be a vital link in our understanding of T cell-mediated organ-specific autoimmunity.

The purpose of this study was to examine the methylation profile of various oral squamous cell carcinomas and to correlate the methylation of particular chromosomal loci with the clinicopathological features of the tumors. A semiquantitative analysis of the methylation status of 12 loci in 96 primary tumors and 13 cell lines was carried out. Methylation frequency was calculated as the percentage of methylated alleles detected by bisulfate-PCR. Of the 12 loci examined, 9 (p16INK4A, p15INK4B, p14ARF, DCC, DAP kinase, MINT1, MINT2, MINT27, and MINT31) exhibited aberrant methylation at various frequencies, whereas 3 (hMLH1, HRK, and CACNA1G) showed no methylation. Dense methylation of the 5' CpG island of DAP kinase and MINT1 was well correlated with loss of gene expression. In addition, methylation of DCC was correlated with bone invasion by gingival tumors (P = 0.036), with aggressive invasiveness of tumors of the tongue (P = 0.046), and with reduced survival (P = 0.050). Methylation of MINT1 and MINT31 also correlated with poor prognoses (P = 0.058 and 0.041), whereas methylation of p14ARF correlated with a good prognosis (P = 0.021). Cox regression analysis showed methylation of MINT31 to be an independent predictor of outcome (hazard ratio, 3.79; 95% confidence interval, 1.58-9.10) and to be associated with the T4 disease group (hazard ratio, 5.71; 95% confidence interval, 1.25-26.07). Analysis of DNA methylation is a useful approach to evaluation of the biological characteristics of oral cancers and may be a useful diagnostic indicator of patient prognosis.

Stimulation of p21-activated kinase-1 (Pak1) induces cytoskeleton reorganization and signaling pathways in mammary cancer cells. Here, we show that inhibition of Pak1 kinase activity by a dominant-negative fragment or by short interference RNA markedly reduced the estrogen receptor-alpha (ER) transactivation functions. To understand the role of Pak1 in mammary glands, we developed a murine model expressing constitutively active Thr423 glutamic acid Pak1 driven by the beta-lactoglobulin promoter. We show that mammary glands from these mice developed widespread hyperplasia associated with apocrine metaplasia and lobuloalveolar hyperdevelopment during lactation. Mammary tissues with active Pak1 also exhibited an increased activation of mitogen-activated protein kinase and stimulated transactivation functions of the ER and expression of endogenous ER target genes. Furthermore, Pak1 directly phosphorylated the activation function-2 domain of the ER at the N-terminal residue Ser305, and its mutation to Ala (S305A) abolished the Pak1-mediated phosphorylation and transactivation functions of the ER, while its mutation to glutamic acid (S305E) promoted transactivation activity of ER. These findings reveal a novel role for the Pak1-ER pathway in promoting hyperplasia in mammary epithelium.

The central role of sequential accumulation of genetic alterations during the development of cancer has been firmly established since the pioneering cytogenetic studies successfully defined recurrent chromosome changes in specific types of tumor. In the course of carcinogenesis, cells experience several genetic alterations that are associated with the transition from a preneoplastic lesion to an invasive tumor and finally to the metastatic state. Tumor progression is characterized by stepwise accumulation of genetic alterations. So does the dominant metastatic clone. Modern molecular genetic analyses have clarified that genomic changes accumulate during the development and progression of cancers. In comparison with the corresponding primary tumor, additional events of chromosomal aberrations (including gains or allelic losses) are frequently found in metastases, and the incidence of combined chromosomal alterations in the primary tumor, plus the occurrence of additional aberrations in the distant metastases, correlated significantly with decreased postmetastatic survival. The deletions at 3p, 4p, 6q, 8p, 10q, 11p, 11q, 12p, 13q, 16q, 17p, 18q, 21q, and 22q, as well as the over-representations at 1q, 8q, 9q, 14q and 15q, have been found to associate preferentially with the metastatic phenotype of human cancers. Among of them, the deletions on chromosomes 8p, 17p, 11p and 13p seem to be more significant, and more detail fine regions of them, including 8p11, 8p21-12, 8p22, 8p23, 17p13.3, 11p15.5, and 13q12-13 have been suggested harboring metastasis-suppressor genes. During the past decade, several human chromosomes have been functionally tested through the use of microcell-mediated chromosome transfer (MMCT), and metastasis-suppressor activities have been reported on chromosomes 1, 6, 7, 8, 10, 11, 12, 16, and 17. However, it is not actually known at what stage of the metastatic cascade these alterations have occurred. There is still controversial with the association between the chromosomal aberrations and the metastatic phenotype of cancer. As the progression of human genome project and the establishment of more and more new techniques, it is hopeful to make clear the genetic mechanisms involved in the tumor metastasis in a not very long future, and provide new clues to predicting and controlling the metastasis.

<h4>Aim</h4>To evaluate the role of APC mutation in gastric carcinogenesis and to correlate APC mutation with microsatellite instability (MSI) in gastric carcinomas.<h4>Methods</h4>APC mutation was measured with multiplex PCR, denaturing gradient gel electrophoresis and DNA sequencing; and MSI was analyzed by PCR-based methods.<h4>Results</h4>Sixty-eight cases of sporadic gastric carcinoma were studied for APC mutation at exon 15 and MSI. APC mutations were detected in 15(22.1 %) gastric cancers. Frequence of APC mutation (33.3 %) in intestinal type of gastric cancer was significantly higher than that in diffuse type (13.1 %, P<0.05). On the contrary, no association was observed between APC mutation and tumor size, differentiation, depth of invasion, metastasis or clinical stages. Using five microsatellite markers, MSI in at least one locus was detected in 17 of 68 (25 %) of the tumors analyzed. APC mutations were all detected in MSI-L (only one locus, n=9) or MSS(tumor lacking MSI or stable, n=51), but no mutation was found in MSI-H (> or =2 loci, n=8).<h4>Conclusion</h4>APC mutation is involved in carcinogenesis of intestinal type of gastric cancer and is independent of MSI phenotype but related to the LOH pathway in gastric cancer.

<h4>Aim</h4>To study the role of lamivudine in improving the efficiency of interferon for the treatment of mutant type chronic hepatitis B.<h4>Methods</h4>Fifteen patients with mutant type chronic hepatitis B were prospectively studied. All patients had liver histology and serology to prove the diagnosis of chronic hepatitis B. Each patient received 4.5 million units of interferon alpha-2a thrice weekly and 100 mg of oral lamivudine daily for 24 weeks. Patients were observed and tested for blood chemistry every week for the initial 4 weeks and every 2 weeks thereafter during the treatment until 24 weeks. After the end of treatment, patients were followed up at 4-week intervals for an additional 6 months. Serum HBV DNA levels were tested using the liquid phase molecular hybridization assay. Those with non-detectable HBV DNA were also tested using the real-time polymerase chain reaction. One patient, who did not finish treatment due to depression, was excluded.<h4>Results</h4>At the end of treatment, 7 (50 %) patients had serum ALT levels within normal limits; 12 (86 %) patients had serum HBV DNA levels <5 pg/mL using the liquid phase molecular hybridization assay, but only 8 (67%) were <20 copies/dL using the real-time polymerase chain reaction. Six months after treatment, only two (14 %) patients had a sustained complete response to the combination therapy with serum ALT level <35 iu/L and undetectable serum HBV DNA levels.<h4>Conclusion</h4>These pilot data showed that lamivudine did not increase the efficacy of interferon in the treatment of mutant type chronic hepatitis B. The liquid phase molecular hybridization assay was not sensitive enough to detect the low HBV DNA levels during combined interferon and lamivudine therapy.

<h4>Unlabelled</h4>We previously reported the correlation of placenta grade with low antithrombin III (ATIII) in pregnant women at term. In this report, the clinical relevance of grade III placenta in the assessment of low plasma ATIII was compared with other factors that raise the risk of blood hypercoagulability.<h4>Methods</h4>We investigated the correlation of low ATIII with ultrasonographic placental grade as well as with age, parity, body mass index (BMI), blood pressure, platelet counts and hematocrit in 164 healthy pregnant women at term. We calculated the odds ratio for each variable (clinical factor) to predict low ATIII activity of less than 80% using a multiple logistic regression model.<h4>Results</h4>Low ATIII activity was associated with BMI>/=28, systolic blood pressure>/=136 mmHg, diastolic blood pressure>/=84 mmHg and the presence of grade III placenta. The odds ratio of low ATIII activity was 3.2 for women with BMI>/=28 (95%Confidential Interval (CI) : 1.1-9.7); 2.7 (95% CI: 0.7-10.3) for women with systolic blood pressure>/=136 mmHg; 1.9 (95% CI: 0.3-11.9) for those with diastolic blood pressure>/=84 mmHg; and 2.4 (95% CI: 0.8-6.9) for those with placental grade III.<h4>Conclusion</h4>We can assess low placenta ATIII activity by using placental grade with similar odds ratios to BMI and blood pressure in pregnant women at term.

Hereditary hemochromatosis (hh, type 1 hemochromatosis) is an autosomal recessive trait characterized by hyperabsorption of dietary iron. The disease trait occurs in approximately five per thousand Caucasians of northern European descent. The causative gene, designated HFE, was isolated and characterized in 1996; most individuals with hh are homozygous for a mutation resulting in a change from cysteine to tyrosine at residue 282 of the HFE protein (C282Y). Wild-type HFE protein binds to the transferrin receptor, and by an undefined mechanism the enterocyte is "programmed" to absorb an amount of dietary iron precisely matched to the body's needs. The C282Y mutant protein is not expressed on the cell surface and does not bind to the transferrin receptor; the result is an enterocyte programmed to absorb slightly more iron than required. Most individuals with hh display a common laboratory phenotype, an elevated transferrin saturation. Iron stores in excess of normal eventually occur in most men and some women. The prevalence of organ damage due to iron overload, however, remains a controversial issue. Published estimates range from less than 1% to "nearly all." The main reason for this discrepancy has been ascertainment bias. Retrospective studies have been biased in favor of individuals with morbid complications of hh, whereas screening studies of groups such as blood donors generally include only healthy subjects. We focus here on a review of studies that have attempted to avoid ascertainment bias. If biopsy-proven hepatic fibrosis and/or cirrhosis is employed as the single criterion for disease-related morbidity, clinical penetrance of hh occurs in 4% to 25% of homozygotes. This range, although narrower than in biased studies, is still wide and requires clarification. A large-scale population-based study has been sponsored by the National Institutes of Health to address this issue. Until results become available, the pragmatic approach is to continue to screen for hemochromatosis in the primary care setting and to maintain serum ferritin values at approximately 100 micro g/L or lower with phlebotomy therapy.

Iron is a vitally important element in mammalian metabolism because of its unsurpassed versatility as a biologic catalyst. However, when not appropriately shielded or when present in excess, iron plays a key role in the formation of extremely toxic oxygen radicals, which ultimately cause peroxidative damage to vital cell structures. Organisms are equipped with specific proteins designed for iron acquisition, export, transport, and storage as well as with sophisticated mechanisms that maintain the intracellular labile iron pool at an appropriate level. These systems normally tightly control iron homeostasis but their failure can lead to iron deficiency or iron overload and their clinical consequences. This review describes several rare iron loading conditions caused by genetic defects in some of the proteins involved in iron metabolism. A dramatic decrease in the synthesis of the plasma iron transport protein, transferrin, leads to a massive accumulation of iron in nonhematopoietic tissues but virtually no iron is available for erythropoiesis. Humans and mice with hypotransferrinemia have a remarkably similar phenotype. Homozygous defects in a recently identified gene encoding transferrin receptor 2 lead to iron overload (hemochromatosis type 3) with symptoms similar to those seen in patients with HFE-associated hereditary hemochromatosis (hemochromatosis type 1). Transferrin receptor 2 is primarily expressed in the liver but it is unclear how mutant forms cause iron overload. Mutations in the gene encoding the iron exporter, ferroportin 1, cause iron overload characterized by iron accumulation in macrophages yet normal plasma iron levels. Plasma iron, together with dominant inheritance, discriminates iron overload due to ferroportin mutations (hemochromatosis type 4) from hemochromatosis type 1. Heme oxygenase 1 is essential for the catabolism of heme and in the recycling of hemoglobin iron in macrophages. Homozygous heme oxygenase 1 deletion in mice leads to a paradoxical accumulation of nonheme iron in macrophages, hepatocytes, and many other cells and is associated with low plasma iron levels, anemia, endothelial cell damage, and decreased resistance to oxidative stress. A similar phenotype occurred in a child with severe heme oxygenase 1 deficiency. Recently, a mutation in the L-subunit of ferritin has been described that causes the formation of aberrant L-ferritin with an altered C-terminus. Individuals with this mutation in one allele of L-ferritin have abnormal aggregates of ferritin and iron in the brain, primarily in the globus pallidus. Patients with this dominantly inherited late-onset disease present with symptoms of extrapyramidal dysfunction. Mice with a targeted disruption of a gene for iron regulatory protein 2 (IRP2), a translational repressor of ferritin, misregulate iron metabolism in the intestinal mucosa and the central nervous system. Significant amounts of ferritin and iron accumulate in white matter tracts and nuclei, and adult IRP2-deficient mice develop a movement disorder consisting of ataxia, bradykinesia, and tremor. Mutations in the frataxin gene are responsible for Friedreich ataxia, the most common of the inherited ataxias. Frataxin appears to regulate mitochondrial iron (or iron-sulfur cluster) export and the neurologic and cardiac manifestations of Friedreich ataxia are due to iron-mediated mitochondrial toxicity. Finally, patients with Hallervorden-Spatz syndrome, an autosomal recessive, progressive neurodegenerative disorder, have mutations in a novel pantothenate kinase gene (PANK2). The cardinal feature of this extrapyramidal disease is pathologic iron accumulation in the globus pallidus. The defect in PANK2 is predicted to cause the accumulation of cysteine, which binds iron and causes oxidative stress in the iron-rich globus pallidus.

Iron overload is common in rural sub-Saharan African populations that have the custom of drinking a traditional fermented beverage with high iron content. As with both excessive alcohol exposure and HFE hemochromatosis, hepatic portal fibrosis and micronodular cirrhosis are prominent sequelae of African iron overload. Two observations are therefore important in characterizing iron overload in Africa. First, the hepatic iron concentrations associated with African iron overload often far exceed those seen in alcoholic liver disease and histologic changes of alcohol effect are almost always absent. Second, the pattern of iron accumulation in African dietary iron is prominent in both macrophages and hepatic parenchymal cells; this pattern is in contrast to HFE homochromatosis, which is marked by predominantly parenchymal iron-loading. For a long time, it was thought that African iron overload was purely dietary in nature, that increased iron and alcohol in the diet could fully explain markedly elevated tissue iron levels sometimes seen with this condition. Recent studies of pedigrees suggest that, in addition to high dietary iron content, a genetic defect may also be implicated in iron overload in Africans. These studies indicate that the possible defect is different from mutations in the HFE gene frequently found in Caucasians with iron overload, but the putative gene has not been identified. Recent studies also indicate that non-HFE iron overload occurs in African-Americans, but the prevalence and possible genetic basis is yet to be determined.

The aim of this study was to establish the prevalence of HFE gene mutations in the population of the Czech Republic. Altogether, 257 randomly selected newborn screening cards (Guthrie cards) were analyzed for the C282Y and H63D mutations within the HFE gene. Complete results were obtained from 254 cards for the C282Y mutation and 257 cards for the H63D mutation. No sample was identified as homozygous for C282Y, and two (0.78%) were homozygous for H63D. Twenty (7.87%) samples were C282Y-heterozygous, and 69 (26.84%) were H63D-heterozygous. Of these, two (0.79%) carried one copy of each mutation, i.e., were compound heterozygous. Allele frequencies for C282Y and H63D were 0.039 and 0.142, respectively. The observed genotype frequencies for both C282Y and H63D mutations in the Czech Republic are in the range of values reported for other Central and Western European populations.

No abstract available.

The precise cause of neuronal death in Huntington's disease (HD) is unknown. Proteolytic products of the huntingtin protein can contribute to toxic cellular aggregates that may be formed in part by tissue transglutaminase (Tgase). Tgase activity is increased in HD brain. Treatment in R6/2 transgenic HD mice, using the transglutaminase inhibitor cystamine, significantly extended survival, improved body weight and motor performance, and delayed the neuropathological sequela. Tgase activity and N(Sigma)-(gamma-L-glutamyl)-L-lysine (GGEL) levels were significantly altered in HD mice. Free GGEL, a specific biochemical marker of Tgase activity, was markedly elevated in the neocortex and caudate nucleus in HD patients. Both Tgase and GGEL immunoreactivities colocalized to huntingtin aggregates. Cystamine treatment normalized transglutaminase and GGEL levels in R6/2 mice. These findings are consistent with the hypothesis that transglutaminase activity may play a role in the pathogenesis of HD, and they identify cystamine as a potential therapeutic strategy for treating HD patients.

Pharmacogenetic research dedicated to the investigation of inherited factors that influence drug response has produced exciting results over the past decade. Adding to the knowledge that genetic variation in metabolic enzymes may cause drug-related adverse reactions, recent studies indicate that variation in neurotransmitter receptors can also be the cause of treatment failure. In addition, recent studies have attempted to use genetic information for the prediction of treatment outcome. The aim of this review is to summarize the most significant findings in pharmacogenetic research in relation to CNS drugs and to outline how these studies could lead to the individualization of drug treatment.

Pulmonary hypertension is one of the major causes of morbidity and mortality of patients with sickle cell hemoglobinopathy (SCH). Although a clinically recognized complication of sickle cell disease (SCD), there are few published pathologic studies of pulmonary findings in these patients. The aim of this study was to define the pulmonary pathologic changes and to investigate correlation between the pathologic changes, the antemortem diagnosis of pulmonary hypertension, and the severity of SCH. Cases of SCH were identified from the autopsy database using Snomed codes. Clinical and echocardiograph data were collected for correlation with the pathologic data. A total of 20 adult patients (12 males and 8 females) were identified. Hemoglobin electrophoresis results were available for 16 patients, with hemoglobin S fraction percentages ranging from 23% to 97.8%. Eleven patients had SCD, 5 patients had sickle cell trait (SCT), and the remaining 4 patients without hemoglobin electrophoresis were included in the SCT group. The mean age of the SCT group was higher than that of the SCD group (P = 0.03). Histologically, all 20 patients demonstrated changes in pulmonary vasculature considered diagnostic of pulmonary hypertension grade I to grade IV, associated with plexiform lesions in 60% of patients. Medial hypertrophy and intimal hyperplasia/fibrosis, considered potentially reversible lesions, were seen in all patients. A weak association was found between SCD and plexiform lesions. Fibroelastic degeneration of small arteries, arterioles, and venules was identified in almost all (95%) cases. Clinically, tricuspid regurgitation was detected by echocardiogram in 10 of 20 (50%) patients; 6 of these 10 had significant regurgitation to allow estimation of systolic pressure. Sudden death occurred in 8 patients, with males having a significantly higher incidence. Cardiomegaly was present in 95% of patients, however, autosplenectomy and hepatic cirrhosis/hemochromatosis were observed almost exclusively in patients with SCD. Cirrhosis was found to have a strong positive association with SCD. This study demonstrates pulmonary hypertensive changes in all 20 autopsied patients who had SCH but died from various causes. We conclude that a high prevalence of pulmonary hypertension is associated with SCH with consequent high mortality. Therefore, patients with SCH would benefit from a regular periodic assessment for pulmonary hypertension regardless of age, sex, and severity of hemoglobinopathy.

We used unpublished reports, published manuscripts, and communication with investigators to identify and summarize 49 anthrax-related epidemiologic field investigations conducted by the Centers for Disease Control and Prevention from 1950 to August 2001. Of 41 investigations in which Bacillus anthracis caused human or animal disease, 24 were in agricultural settings, 11 in textile mills, and 6 in other settings. Among the other investigations, two focused on building decontamination, one was a response to bioterrorism threats, and five involved other causes. Knowledge gained in these investigations helped guide the public health response to the October 2001 intentional release of B. anthracis, especially by addressing the management of anthrax threats, prevention of occupational anthrax, use of antibiotic prophylaxis in exposed persons, use of vaccination, spread of B. anthracis spores in aerosols, clinical diagnostic and laboratory confirmation methods, techniques for environmental sampling of exposed surfaces, and methods for decontaminating buildings.

There is growing evidence that Ca(v)2.3 (alpha1E, E-type) transcripts may encode the ion-conducting subunit of a subclass of R-type Ca(2+) channels, a heterogeneous group of channels by definition resistant to blockers of L-, N-, and P/Q-type Ca(2+) channels. To understand whether splice variation of Ca(v)2.3 contributes to the divergence of R-type channels, individual variants of Ca(v)2.3 were constructed and expressed in HEK-293 cells. With Ba(2+) as charge carrier, the tested biophysical properties were similar. In Ca(2+), the inactivation time course was slower and the recovery from short-term inactivation was faster; however, this occurred only in variants containing a 19-amino-acid-long insertion, which is typical for neuronal Ca(v)2.3 Ca(2+) channel subunits. This different Ca(2+) sensitivity is not responsible for the major differences between various R-type channels, and future studies might clarify its importance for in vivo synaptic or dendritic integration and the reasons for its loss in endocrine Ca(v)2.3 splice variants.

Iron and copper are essential transition metals that permit the facile transfer of electrons in a series of critical biochemical pathways. Recent work has identified the specific proteins involved in the absorption, transport, utilization, and storage of iron and copper. Remarkable progress is being made in understanding the molecular basis of disorders of human iron and copper metabolism. This review describes these proteins and examines the clinical consequences of new insights into the pathophysiology of genetic abnormalities affecting iron and copper metabolisms. Hereditary hemochromatosis is the most common genetic disorder of iron metabolism caused by mutations in the HFE gene. Aceruloplasminemia is a rare iron metabolic disorder that results from deficiency of ceruloplasmin ferroxidase activity as a consequence of mutations in the ceruloplasmin gene. Menkes disease and Wilson's disease are inherited disorders of copper metabolism resulting from the absence or dysfunction of homologous copper-transporting ATPases.

Hereditary hemochromatosis (HH) is a disorder of iron metabolism in which enhanced absorption of dietary iron causes increased iron accumulation in the liver, heart, and pancreas. Most individuals with HH are homozygous for a point mutation in the HFE gene, leading to a C282Y substitution in the HFE protein. The function of HFE protein is unknown, but the available evidence suggests that it acts in association with beta2-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 identification of HFE has established the foundation for a better understanding of the molecular and cellular biology of iron homeostasis and its altered regulation in HH. Additionally, the ability to accurately diagnose iron overload disorders has been strengthened, family screening has been improved, and evaluation of patients with other forms of liver disease complicated by moderate-to-severe iron overload is now possible. However, the role of HFE testing in generalized population screening for HH is still controversial. Recently, other forms of HH have been described that are not related to HFE but are due to mutations in genes coding iron transport proteins.

Previously, we demonstrated that Ath1 is a quantitative trait locus for aortic fatty streak formation, located on Chromosome (chr) 1, with susceptibility in C57BL/6J mice and resistance in C3H/HeJ and BALB/cJ mice fed an atherogenic diet. In this study, we find an atherosclerosis susceptibility locus in the same region of Chr 1 by constructing two congenic strains with the resistance phenotype transferred from different resistant strains, PERA/EiJ or SPRETUS/EiJ. By backcrossing one congenic strain to C57BL/6J and testing recombinant animals, we reduced the distance of the atherosclerosis susceptibility region to 2.3 cM between D1Mit14 and D1Mit10. Further testing of nine recombinant animals showed that eight of the nine were consistent with a further narrowing between D1Mit159 and D1Mit398 a distance of 0.66 cM. This region encompasses a number of potential candidate genes including the thiol-specific antioxidant gene Aop2, also known as peroxiredoxin 5 (Prdx5). AOP2 is capable of reducing hydroperoxides and lipid peroxides in the cell. To investigate Aop2 as a potential candidate, we mapped Aop2 in our backcross and localized it to the atherosclerosis susceptibility interval. We determined that Aop2 is highly expressed in atherosclerosis-related tissues including liver and heart. We also found an inverse correlation between Aop2 mRNA in liver and atherosclerosis phenotype for strains C57BL/6 and the resistant congenic derived from SPRETUS/EiJ. Since LDL oxidation has been implicated in the pathogenesis of this disease, and AOP2 possesses antioxidant activity, we suggest the role of Aop2 in atherosclerosis susceptibility needs to be further explored.

Genetic haemochromatosis is an autosomal recessive inherited disorder of iron metabolism due to mutation of the HFE gene. In homozygotes (1 in 300 of the UK population), this results in excessive iron absorption from the gut and its deposition in major body organs. This may give rise to fatigue, arthritis, cardiac failure, diabetes mellitus, hepatic cirrhosis or skin pigmentation, occurring predominantly in males over 50 years of age. Identification uses measurement of serum iron, iron-binding capacity (or transferrin) and ferritin, together with initial or confirmatory genetic DNA studies.

<h4>Purpose</h4>Mutations of the HFE gene that cause hereditary hemochromatosis may be associated with an increased risk of cardiovascular disease. We examined the relation between two HFE mutations (C282Y and H63D), indicators of iron homeostasis, and the prevalence of coronary heart disease in a large population of white adults.<h4>Subjects and methods</h4>We conducted a cross-sectional study of 30,916 white adults aged 25 to 98 years who attended a health appraisal center and underwent screening for HFE mutations. Coronary heart disease and cardiovascular risk factors were ascertained by questionnaire and medical records.<h4>Results</h4>Overall, 12% (1798/15,362) of men and 7% (1074/15,554) of women had a history of coronary heart disease. Of 10 HFE genotypes tested (five genotypes by sex), only men with the C282Y/H63D genotype (compound heterozygotes) had a significantly higher prevalence of coronary heart disease compared with men with no HFE mutations (odds ratio = 1.6; 95% confidence interval: 1.1 to 2.4; P = 0.01) after adjusting for cardiovascular risk factors. Elevated serum ferritin levels (>250 ng/mL) were associated with a lower prevalence of coronary heart disease in men (10% [255/2209] vs. 12% [1515/12,461] in controls, P = 0.008), which was not significant after adjusting for use of aspirin and anticoagulants. There were no significant associations between elevated transferrin saturation in either men or women, or between elevated serum ferritin levels or HFE mutations in women, and the prevalence of coronary heart disease.<h4>Conclusion</h4>The results do not support a consistent association between HFE mutations or serum iron indicators and the prevalence of coronary heart disease.

Amino-terminal fragments of huntingtin, which contain the expanded polyglutamine repeat, have been proposed to contribute to the pathology of Huntington's disease (HD). Data supporting this claim have been generated from patients with HD in which truncated amino-terminal fragments forming intranuclear inclusions have been observed, and from animal and cell-based models of HD where it has been demonstrated that truncated polyglutamine-containing fragments of htt are more toxic than full-length huntingtin. We report here the identification of a region within huntingtin, spanning from amino acids 63 to 111, that is cleaved in cultured cells to generate a fragment of similar size to those observed in patients with HD. Importantly, proteolytic cleavage within this region appears dependent upon the length of the polyglutamine repeat within huntingtin, with pathological polyglutamine repeat-containing huntingtin being more efficiently cleaved than huntingtin containing polyglutamine repeats of nonpathological size.

Huntington's disease, with its dominant loss of striatal neurons, is triggered by an expanded glutamine tract in huntingtin. To investigate a proposed role for increased activation of the apoptotic cascade in mutant huntingtin's trigger mechanism, we examined huntingtin cleavage and lesion severity after mild ischemic injury in Hdh(Q92) mice. We found activation of calpain and caspase proteases and proteolysis of huntingtin in lesioned striatum. However, huntingtin fragments resembled products of calpain I, not caspase-3, cleavage and turnover was accompanied by augmented levels of full-length normal and mutant protein. By contrast, the number of apoptotic cells, total and striatal infarct size, and degree of neurologic deficit were similar in Hdh(Q92) and wild-type mice, indicating that the disease process neither strongly protected nor sensitized striatal neurons to apoptotic death. Thus, our findings do not support a role for increased apoptosis or caspase-3 cleavage in the mechanism by which mutant huntingtin triggers disease. However, they suggest that calpain activation and huntingtin regulation merit investigation as modifiers of disease progression in neurons injured by the harmful consequences of full-length mutant huntingtin.

A 2-Keto-L-gulonic acid (2-KLG) production process using stationary Pantoea citrea cells and a Corynebacterium 2,5-diketo-D-gluconic acid (2,5-DKG) reductase enzyme has been developed which may represent an improved method of vitamin C biosynthesis. Experimental data was collected using the F22Y/A272G 2,5-DKG reductase mutant and NADP(H) as a cofactor. An extensive kinetic analysis was performed and a kinetic rate equation model for this process was developed. A recent protein engineering effort has resulted in several 2,5-DKG reductase mutants exhibiting improved activity with NADH as a cofactor. The use of NAD(H) in the bioreactor may be preferable due to its increased stability and lower cost. The kinetic parameters in the rate equation model have been replaced in order to predict 2-KLG production with NAD(H) as a cofactor. The model was also extended to predict 2-KLG production in the presence of a range of combined cofactor concentrations. This analysis suggests that the use of the F22Y/K232G/R238H/A272G 2,5-DKG reductase mutant with NAD(H) combined with a small amount of NADP(H) could provide a significant cost benefit for in vitro enzymatic 2-KLG production.

Cellular signaling by proteases of the blood coagulation cascade through members of the protease-activated receptor (PAR) family can profoundly impact on the inflammatory balance in sepsis. The coagulation initiation reaction on tissue factor expressing cells signals through PAR1 and PAR2, leading to enhanced inflammation. The anticoagulant protein C pathway has potent anti-inflammatory effects, and activated protein C signals through PAR1 upon binding to the endothelial protein C receptor. Activation of the coagulation cascade and the downstream endothelial cell localized anticoagulant pathway thus have opposing effects on systemic inflammation. This dichotomy is of relevance for the interpretation of preclinical and clinical data that document nonuniform responses to anticoagulant strategies in sepsis therapy.