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A new carbazole (CAR)-degrading bacterium, called strain OM1, was isolated from activated sludge obtained from sewage disposal plants in Fukuoka Prefecture, and it was identified as Pseudomonas stutzeri. Anthranilic acid (AN), 2'-aminobiphenyl-2,3-diol and its meta-cleavage product, 2-hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4-dienoic acid, were identified as metabolic intermediates of CAR in the ethyl acetate extract of the culture broth. Therefore, the CAR catabolic pathway to AN in strain OM1 was indicated to be identical to those found in the Pseudomonas sp. strains CA06 and CA10. The strain OM1 degraded catechol (CAT) via a meta-cleavage pathway in contrast to strains CA06 and CA10, which transform catechol into cis, cis-munonic acid. Clones containing a 6.9-kb EcoRI fragment and a 3-kb PstI-SphI fragment were isolated from colonies, forming a clear zone of CAR and a yellow ring-cleavage product from CAT, respectively. Recombinant E. coli carrying the 6.9-kb fragment degraded CAR in the L-broth and produced AN. Cell-free extract from the clone carrying a 3-kb PstI-SphI fragment had high meta-ring-cleavage dioxygenase activity for CAT. The nucleotide sequences of these fragments were determined. The 6.9-kb fragment showed a very high degree of homology with the CAR catabolic genes of strain CA10. The amino acid and nucleotide sequences of the 3-kb fragment were found to exhibit significant homology with the genes for the CAT-catabolic enzymes of TOL plasmid pWW0, plasmid NAH7, and plasmid pVI150.

The serotonin transporter (HTT) is a candidate gene for obsessive-compulsive disorder (OCD) that has been associated with anxiety-related traits. The long (l) and short (s) variants of the HTT promoter have different transcriptional efficiencies. HTT promoter genotype and blood 5-HT concentration were examined in 70 subjects from 20 families ascertained through children and adolescents with a DSM-III-R diagnosis of OCD. The HTT promoter variant had a significant effect on blood 5-HT content. Subjects with the l/l and l/s genotypes had significantly higher blood 5-HT levels than did those with the s/s genotype. There was a significant interaction between HTT promoter genotype and seasonal variation in blood 5-HT content, with significant seasonal differences in 5-HT occurring only in the subjects with the l/l genotype. Further studies of the regulation of HTT gene expression are indicated.

Cdk7 has been shown previously to be able to phosphorylate and activate many different Cdks in vitro. However, conclusive evidence that Cdk7 acts as a Cdk-activating kinase (CAK) in vivo has remained elusive. Adding to the controversy is the fact that in the budding yeast Saccharomyces cerevisiae, CAK activity is provided by the CAK1/Civ1 protein, which is unrelated to Cdk7. Furthermore Kin28, the budding yeast Cdk7 homolog, functions not as a CAK but as the catalytic subunit of TFIIH. Vertebrate Cdk7 is also known to be part of TFIIH. Therefore, in the absence of better genetic evidence, it was proposed that the CAK activity of Cdk7 may be an in vitro artifact. In an attempt to resolve this issue, we cloned the Drosophila cdk7 homolog and created null and temperature-sensitive mutations. Here we demonstrate that cdk7 is necessary for CAK activity in vivo in a multicellular organism. We show that cdk7 activity is required for the activation of both Cdc2/Cyclin A and Cdc2/Cyclin B complexes, and for cell division. These results suggest that there may be a fundamental difference in the way metazoans and budding yeast effect a key modification of Cdks.

The estrogen receptor is capable of binding a diverse set of ligands that are broadly categorized as agonists or antagonists, depending on their abilities to induce or interfere with transcriptional responsiveness. We show, using a fusion protein assay for ligand-binding which does not rely on transcriptional responsiveness, that agonists and antagonists differently position the C-terminus of the ligand-binding domain (helix 12) and the F domain. Upon antagonist binding, the F domain interferes with the fusion protein activity. Mutational disruption of helix 12 alters the position of the F domain, imposing interference after agonist or antagonist binding. Genetically selected inversion mutations where only agonists, but not antagonists, induce interference are similarly reliant on helix 12 and F domain positioning. Our results demonstrate that agonists and antagonists differently position helix 12 and implicate the F domain in mechanisms of antagonist action.

<h4>Background & aims</h4>The identification of a candidate gene for hereditary hemochromatosis in 69%-100% of patients with hemochromatosis has resulted in a diagnostic genotypic test (C282Y). The aim of this study was to reassess the phenotypic diagnostic criteria for hemochromatosis in patients homozygous for the C282Y mutation of the HFE gene.<h4>Methods</h4>Transferrin saturation, ferritin, hepatic iron index, and iron removed by venesection were studied in C282Y++ homozygotes and C282Y-- putative homozygotes.<h4>Results</h4>Patients were homozygous for the C282Y mutation in 122 of 128 cases (95%). In C282Y homozygotes, the results were as follows: hepatic iron index, >1.9 in 91.3%; transferrin saturation, >55% in 90%; serum ferritin, >300 microg/L in 96% of men and >200 microg/L in 97% of women; and iron removed, >5 g in 70% of men and 73% of women. There were four homozygotes for C282Y with no biochemical evidence of iron overload.<h4>Conclusions</h4>The sensitivity of the phenotypic tests in decreasing order was as follows: serum ferritin, hepatic iron index, transferrin saturation, and iron removed by venesection. Although the genetic test is useful in the diagnostic algorithm, this study has shown both iron-loaded patients without the mutation and homozygous patients without iron overload.

Huntingtin is the protein product of the gene for Huntington's disease (HD) and carries a polyglutamine repeat that is expanded in HD (>36 units). Huntingtin-associated protein (HAP1) is a neuronal protein and binds to huntingtin in association with the polyglutamine repeat. Like huntingtin, HAP1 has been found to be a cytoplasmic protein associated with membranous organelles, suggesting the existence of a protein complex including HAP1, huntingtin, and other proteins. Using the yeast two-hybrid system, we found that HAP1 also binds to dynactin P150(Glued) (P150), an accessory protein for cytoplasmic dynein that participates in microtubule-dependent retrograde transport of membranous organelles. An in vitro binding assay showed that both huntingtin and P150 selectively bound to a glutathione transferase (GST)-HAP1 fusion protein. An immunoprecipitation assay demonstrated that P150 and huntingtin coprecipitated with HAP1 from rat brain cytosol. Western blot analysis revealed that HAP1 was enriched in rat brain microtubules and comigrated with P150 and huntingtin in sucrose gradients. Immunofluorescence showed that transfected HAP1 colocalized with P150 and huntingtin in human embryonic kidney (HEK) 293 cells. We propose that HAP1, P150, and huntingtin are present in a protein complex that may participate in dynein-dynactin-associated intracellular transport.

A method is described for measuring the diversity of combinatorial oligonucleotide libraries that entails extrapolating the base composition of a co-synthesized model library (dNC, N = A, C, G, T) to that of a multibase library template. The base composition of dNC was measured by HPLC. The ability of dNC to predict the base composition of a multibase library template was corroborated by measuring the composition of a 12 base combinatorial library. The base composition of the 12 base library was determined by several template dependent incorporation assays: measurement of restriction fragment specific activities from polymerase incorporation/restriction enzyme digests, template directed radionucleotide primer extension and quantitative dideoxynucleotide sequencing. Additionally, a convention for describing oligomeric combinatorial library (OCL) diversity is proposed. The convention uses a quantity termed the diversity quotient (Qd) to describe library breadth and the mole fraction of the least represented monomeric unit of the OCL to calculate minimum library quantity requirements. Similar methods/conventions could presumably be developed/adopted for non-nucleic acid libraries.

A 37-year-old untransfused, non-drinking man with Hemoglobin H-CS disease presented with insulin-dependent diabetes mellitus, markedly elevated serum ferritin level, and marked iron deposition in hepatocytes. He did not carry either of the two common mutations of the HLA-H gene for hereditary hemochromatosis, namely, Cys282Tyr and His68Asp, nor did he have the associated HLA marker (HLA-A3, B7 nor B-14) for the disease. Patient with HbH disease should be monitored for iron overload.

It is unclear how polyglutamine expansion is associated with the pathogenesis of Huntington disease (HD). Here, we provide evidence that polyglutamine expansion leads to the formation of large intracellular aggregates in vitro and in vivo. In vitro these huntingtin-containing aggregates disrupt normal cellular architecture and increase in frequency with polyglutamine length. Huntingtin truncated at nucleotide 1955, close to the caspase-3 cleavage site, forms perinuclear aggregates more readily than full-length huntingtin and increases the susceptibility of cells to death following apoptotic stimuli. Further truncation of huntingtin to nucleotide 436 results in both intranuclear and perinuclear aggregates. For a given protein size, increasing polyglutamine length is associated with increased cellular toxicity. Asymptomatic transgenic mice expressing full-length huntingtin with 138 polyglutamines form exclusively perinuclear aggregates in neurons. These data support the hypothesis that proteolytic cleavage of mutant huntingtin leads to the development of aggregates which compromise cell viability, and that their localization is influenced by protein length.

We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis called HFE. The gene product, a member of the major histocompatibility complex class I-like family, was found to have a mutation, Cys-282 --> Tyr (C282Y), in 85% of patient chromosomes. This mutation eliminates the ability of HFE to associate with beta2-microglobulin (beta2m) and prevents cell-surface expression. A second mutation that has no effect on beta2m association, H63D, was found in eight out of nine patients heterozygous for the C282Y mutant. In this report, we demonstrate in cultured 293 cells overexpressing wild-type or mutant HFE proteins that both the wild-type and H63D HFE proteins form stable complexes with the transferrin receptor (TfR). The C282Y mutation nearly completely prevents the association of the mutant HFE protein with the TfR. Studies on cell-associated transferrin at 37 degrees C suggest that the overexpressed wild-type HFE protein decreases the affinity of the TfR for transferrin. The overexpressed H63D protein does not have this effect, providing the first direct evidence for a functional consequence of the H63D mutation. Addition of soluble wild-type HFE/beta2m heterodimers to cultured cells also decreased the apparent affinity of the TfR for its ligand under steady-state conditions, both in 293 cells and in HeLa cells. Furthermore, at 4 degrees C, the added soluble complex of HFE/beta2m inhibited binding of transferrin to HeLa cell TfR in a concentration-dependent manner. Scatchard plots of these data indicate that the added heterodimer substantially reduced the affinity of TfR for transferrin. These results establish a molecular link between HFE and a key protein involved in iron transport, the TfR, and raise the possibility that alterations in this regulatory mechanism may play a role in the pathogenesis of hereditary hemochromatosis.

We describe a new class of fluorescence polarization immunoassays based on the luminescence from a Re(I) metal-ligand complex. Re(I) complexes are extremely photostable and possess useful photophysical properties including long lifetimes, high quantum yields, and high emission polarization in the absence of rotational diffusion. In the present study, a conjugatable, highly luminescent Re(I) metal-ligand complex, [Re(bcp)(CO)3(4-COOHPy)](ClO4), where bcp is 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline and 4-COOHPy is isonicotinic acid, has been evaluated for use in fluorescence polarization immunoassays (FPIs) with high-molecular-weight antigens. This Re(I) complex (Re) displays highly polarized emission (with a maximum anisotropy near 0.3) in the absence of rotational diffusion and a long average lifetime (2.7 microseconds) when bound to human serum albumin (HSA) in oxygenated aqueous solution. The emission polarization of the Re-HSA conjugate is sensitive to the binding of anti-HSA, resulting in a significant increase in anisotropy. The labeled HSA was also used in a competition immunoassay where unlabeled HSA was also used as an antigen. These experimental results, combined with theoretical predictions, demonstrate the potential of this Re(I) metal-ligand complex as a luminescence probe in FPIs of high-molecular-weight analytes (10(5)-10(8) Da).

<h4>Background</h4>The cause of polycythemia vera, which originates from a multipotent hematopoietic progenitor cell, is unknown. Thrombopoietin is a hematopoietic growth factor that regulates the production of multipotent hematopoietic progenitor cells and platelets. To evaluate the possibility that an abnormality in thrombopoietin-mediated signal transduction might be involved in the pathogenesis of polycythemia vera, we examined thrombopoietin-induced tyrosine phosphorylation of proteins and the expression of the thrombopoietin receptor in platelets from patients with the disease.<h4>Methods</h4>Platelets were isolated from the blood of patients with polycythemia vera or other chronic myeloproliferative disorders and control subjects. The platelets were exposed to either thrombopoietin or thrombin and then lysed for analysis of tyrosine phosphorylation of platelet proteins and the expression of the proteins by means of immunoblotting. Expression of the thrombopoietin receptor (Mpl) by platelets and megakaryocytes was also assessed.<h4>Results</h4>Thrombopoietin-mediated tyrosine phosphorylation of proteins was impaired in platelets from 20 patients with polycythemia vera and 3 with idiopathic myelofibrosis, but not in 4 patients with essential thrombocytosis, 3 with chronic myelogenous leukemia, 6 with secondary erythrocytosis, 2 with iron-deficiency anemia, 4 with hemochromatosis, or 5 normal subjects. Thrombin-mediated tyrosine phosphorylation of proteins was intact in platelets from patients with polycythemia vera, and the tyrosine kinases and substrates involved in the process were present in normal amounts. However, expression of the platelet thrombopoietin receptor MpI was markedly reduced or absent in 34 of 34 patients with polycythemia vera and in 13 of 14 patients with idiopathic myelofibrosis. Impaired thrombopoietin-induced tyrosine phosphorylation of proteins in patients with these two diseases was uniformly associated with markedly reduced expression of MpI or the lack of its expression. In patients with polycythemia vera, reduced expression of MpI by platelets was associated with reduced expression of MpI by megakaryocytes.<h4>Conclusions</h4>Reduced expression of the thrombopoietin receptor MpI is characteristic of polycythemia vera and idiopathic myelofibrosis. The abnormality appears to distinguish polycythemia vera from other-forms of erythrocytosis.

Protein expression of the putative tumour-suppressor gene DCC on chromosome 18q was evaluated in a panel of 16 matched colorectal cancer and normal colonic tissue samples together with DCC mRNA expression and allelic deletions (loss of heterozygosity, LOH). Determined by a polymerase chain reaction (PCR)-LOH assay, 12 of the 16 (75%) cases were informative with LOH occurring in 2 of the 12 cases. For DCC mRNA, transcripts could be detected in all analysed normal tissues (eight out of eight) by RT-PCR, whereas 6 of the 15 tumours were negative. DCC protein expression, investigated by immunohistochemistry using the monoclonal antibody 15041 A directed against the intracellular domain, was homogeneously positive in all normal tissue samples. In tumour tissues, no DCC protein was seen in 11 out of 16 samples (69%). For the DCC codon 201, we found a loss of a wild-type codon sequence caused by mutation or LOH in at least 8 out of 15 cases (53%) compared with the corresponding normal tissue. DCC protein expression was undetectable in eight of the nine tumours missing both wild-type codons. Only one of the five tumours with retained DCC protein expression had no detectable wild-type codon 201. In addition, 9 out of 15 normal tissue specimens were mutated in codon 201. In two out of three cases with homozygous wild-type codons in peripheral blood lymphocyte (PBL) DNA, mutations were already observed in the tumour adjacent normal colonic mucosa. We conclude that DCC immunostaining should be introduced in the clinicopathological routine because of its strong correlation with the known prognostic markers 18q LOH and mutation of codon 201.

The human major histocompatibility complex (MHC) on chromosome 6 encodes three classical class I genes: human leukocyte antigen-A (HLA-A), HLA-B, and HLA-C. These polymorphic genes encode a 43- to 45-kDa cell surface glycoprotein that, in association with the 12-kDa beta 2-microglobulin molecule, functions in the presentation of nine amino acid peptides to the T cell receptor of CD8-bearing T lymphocytes and killer inhibitory receptors on natural killer cells. In addition to these ubiquitously expressed polymorphic proteins, the human genome also encodes a number of nonclassical MHC class I-like, or class Ib, genes that in general encode nonpolymorphic molecules involved in a variety of specific immunologic functions. Many of these genes, including CD1, the neonatal Fc receptor for immunoglobulin G, HLA-G, the MHC class I chain-related gene A, and Hfe, are prominently displayed on epithelial cells, suggesting an important role in epithelial cell biology.

Stainable iron in the liver (hemosiderosis) is most commonly seen in individuals with homozygous genetic hemochromatosis, prior transfusion, hemolysis, porphyria cutanea tarda, and chronic alcohol-induced liver disease. In chronic viral hepatitis, however, significant hepatocellular hemosiderosis is uncommon. This report describes unusual foci of hepatocellular hemosiderosis ("iron-rich foci" or IRF) in liver biopsy specimens from three patients with chronic hepatitis with or without cirrhosis (two hepatitis C-related, one hepatitis B-related). IRF present within the lobular parenchyma or cirrhotic nodules contrasted sharply with the immediately adjacent hemosiderin-negative liver tissue. Serum iron indices were abnormal in all three patients, but homozygous hemochromatosis was ruled out based on the hepatic iron concentration and hepatic iron index for each case. These cases highlight the potential for irregular iron storage in chronic viral liver disease and possible confusion with genetic hemochromatosis. The possible pathogenesis of IRF and the relationship of iron storage to the outcome of interferon therapy in chronic viral hepatitis are discussed.

Carcinoma is an important complication of ulcerative colitis (UC) and develops from dysplastic precursor lesions. Genetic changes involved in the malignant transformation have not been fully characterized. We studied 19 cases of UC with high-grade dysplasia (HGD) and eight samples of associated carcinoma (CA). Microdissection of normal epithelium, epithelium at the site of chronic inflammation, HGD, and CA was performed. Polymerase chain reaction (PCR) amplification for loss of heterozygosity (LOH) of the following polymorphic microsatellites of putative tumor suppressor gene loci was done: APC (5q), DCC (18q), p16 (9p), p53 (17p), and 8p12. To compare genetic alterations, 22 typical adenomas of the colon were studied with the markers for APC and pl6 gene loci. The results indicated that LOH of p16 and p53 were present in nondysplastic epithelium, HGD, and CA. However, the LOH in nondysplastic epithelium was detected in some associated HGD, but not all. Whereas LOH of p16 was present in 7 of 14 cases of HGD (50%), it was noted in only 1 of 22 adenomas (5.0%). LOH in the APC and DCC gene loci in UC was noted in HGD with associated CA, but LOH of APC was not present either in cases of nondysplastic epithelium or in HGD alone. Conversely, LOH in APC was present in 4 of 19 colonic adenomas. We conclude that LOH of p53 and p16 in nondysplastic epithelium may be associated with chronic reparative processes. These changes may lead to susceptibility to further genetic damage involving the APC and DCC gene loci in the development of dysplasia and progression of CA in UC. The low frequency of LOH in the p16 gene (9p) in adenomas compared with dysplasia in UC combined with infrequent LOH in APC gene loci in cases of pure dysplasia in UC may support this combination of markers as a clinical test for the differentiation of polypoid dysplasia from adenomas in UC.

No abstract available.

The usefulness of gene information was studied when used in conjunction with a morphological diagnosis of either dysplasia or carcinoma that later develops into ulcerative colitis (UC). The cases investigated consisted of those operated on for UC with carcinoma complications and those operated on for UC over 7 years previously without carcinoma complications. Ras and DCC were examined for the presence of any point mutations in codon 12 and polymorphism in codon 201 using the PCR-RFLP method, while p53 was also studied immunohistologically. A mutation in ras was found in 25% of the UC-IV cases and also in 17% of the UC-III cases, while no mutation at all was found in the UC-I and UC-II cases. p53 showed a high rate of positivity in the UC-IV and UC-III cases with carcinoma complications, while it was negative in all cases in the control group cases. Gly in DCC codon 201 was also found in many cases including the control group. This study demonstrated that a gene aberration can thus influence the pathophysiology and cancerization of UC and therefore the p53 findings were thus considered to be useful in the morphological diagnosis of dysplasia and carcinoma.

The objective of this article is to evaluate the plasma levels of coagulation and fibrinolysis parameters in the third trimester of gestation and 72 hr postdelivery. Antithrombin III (ATIII), thrombin-antithrombin III complexes (TAT), heparin cofactor II (HCII), protein C (PC), protein S (PS), tissue plasminogen activator (t-PA), D-dimer, and plasminogen activator inhibitors (PAI-1 and PAI-2) levels in uncomplicated pregnancies and in pregnancies complicated by intrauterine growth retardation (IUGR) have been determined. Normal pregnant women (n = 63) and women whose was complicated by IUGR (n = 10) formed the study population. Coagulation and fibrinolysis parameters were estimated using commercial tests. There were no differences in ATIII, HCII, and PS levels between normal and IUGR pregnancies. TAT, t-PA, and D-dimer levels were higher in IUGR pregnancy than in the uncomplicated pregnancy group. PAI-1 and PAI-2 were found depressed in IUGR pregnancy when compared with normal pregnancy. Changes in coagulation and fibrinolytic systems occur in plasma of women with pregnancies complicated by IUGR. The results suggest an activation of the coagulation system in pregnancies complicated by IUGR. Reduced PAI-2 and high TAT levels correlate with birth weight. In IUGR pregnancies a hypercoagulative state with hyperfibrinolytic compensatory mechanisms is suggested.

In a population-based association study, we tested the hypothesis that allelic variants of the human serotonin transporter (5-HTT) gene confer susceptibility to mood disorders. Both a biallelic repeat polymorphism in the 5' promotor region that differentially modulates gene expression and a second intron variable-number-tandem-repeat (VNTR) marker were genotyped in 294 controls and 115 patients with mood disorders. Subjects were of West European descent and included 36 patients with major depressive disorder (MDD) and 79 patients with bipolar I disorder (BD). No significant differences in genotype or allele frequencies were found at either locus between controls and combined patients, nor between controls and MDD or BD patients separately. Thus, our data do not support the association between depressive disorder and a nine-repeat allelic variant of the 5-HTT VNTR marker recently reported by Ogilvie et al. (Lancet 347:731-733, 1996). More importantly, no association between alleles conveying functional differences in 5-HTT gene expression and MDD or BD could be found. Taken together, our data suggest that the 5-HTT gene is not commonly involved in the susceptibility to mood disorders.

Interactions with antidepressants, as well as other biochemical evidence, implicate the serotonin transporter 5-HTT in the etiology of affective disorders. However, genetic studies have produced conflicting results concerning an association of 5-HTT with bipolar disorder. We examined a variable number tandem repeat in the regulatory region of this gene to investigate the possible contribution of 5-HTT to bipolar disorder susceptibility in a 22-pedigree series. By affected-sib-pair analysis and the transmission/disequilibrium test, we found no significant linkage or association of 5-HTT to bipolar disorder. During the course of this study, we adapted a PCR technique designed to amplify long templates to replicating long GC stretches with complex structure. We also refined the location of 5-HTT by radiation hybrid mapping, placing the locus between D17S1294 and SHGC11022 on 17q11.2.

Recombinant glycosaminoglycan-modified urinary thrombomodulin (GAG-UTM), which partially improved the amino acid sequence of human urinary thrombomodulin (UTM), was expressed in C127 cells. GAG-UTM accelerates protein C activation by thrombin and also thrombin inhibition by antithrombin III (ATIII) in the buffer system. Both accelerating activities of GAG-UTM are more potent than those of unmodified recombinant UTM (r-UTM) without a GAG chain. As ATIII in plasma also inhibits protein C activation by a thrombin-thrombomodulin complex, we studied whether GAG-UTM accelerates protein C activation in plasma. GAG-UTM suppressed the generation of thrombin in activating plasma protein C stronger than r-UTM. By Western blot analysis using anti-protein C antibody, activated protein C was generated by GAG-UTM more than by r-UTM. From these results, the acceleration of activated protein C formation by GAG-UTM was confirmed in plasma too.

<h4>Objective</h4>To determine the impact of mutations in the HFE gene (human leukocyte antigen H) on predisposition to coronary artery disease (CAD) in patients not diagnosed with hereditary hemochromatosis.<h4>Background</h4>Elevated iron stores can predispose to acute myocardial infarction. Two mutations (C282Y and H63D) in the novel major histocompatibility complex (MHC) class 1 gene HFE were found in most patients with hereditary hemochromatosis causing high iron stores. The effect of these mutations on predisposition to CAD has not been investigated previously.<h4>Methods</h4>Three hundred patients with a history of myocardial infarction or angina pectoris and angiographically documented CAD were studied. Patients were divided into two groups: group 1 (150 patients), manifesting early onset CAD and presenting with these findings under age 50 years; and group 2 (150 patients), presenting for the first time over age 65 years. Prevalence of the C282Y and H63D mutations was assessed by molecular analysis, and plasma ferritin was measured immunochemically.<h4>Results</h4>There was no difference in the prevalence of homozygous, heterozygous or compound heterozygous (C282Y/H63D) states between the groups. Males in group 1 had higher plasma ferritin than those in group 2 (234 +/- 174 micrograms/L versus 136 +/- 103 micrograms/L, P < 0.0001), but this was not significantly different in females (75 +/- 54 micrograms/L versus 92 +/- 73 micrograms/L, P = 0.26). Ferritin remained higher in group 1 than in group 2 males after exclusion of mutation carriers (195 +/- 121 micrograms/L versus 109 +/- 76 micrograms/L, respectively, P < 0.0001), but did not change in females.<h4>Conclusions</h4>Higher iron stores were found in males with early onset CAD. This association was not related to the C282Y or H63D mutation in HFE. It is suggested that association of the MHC locus with delayed onset CAD is probably unrelated to HFE in these patients, and that HFE mutations are not a major risk factor in the development of high iron stores in early onset CAD.

Decorsin is a 39-residue RGD-protein crosslinked by three disulfide bridges isolated from the leech Macrobdella decora belonging to the family of GPIIb-IIIa antagonists and acting as a potent inhibitor of platelet aggregation. Here we report the solid-phase synthesis of decorsin using the Fmoc strategy. The crude polypeptide was purified by reverse-phase HPLC in its reduced form and allowed to refold in the presence of glutathione. The homogeneity of the synthetic oxidized decorsin was established by reverse-phase HPLC and capillary zone electrophoresis. The results of amino acid analysis after acid hydrolysis of the synthetic protein, NH2-terminal sequencing and mass determination (4,377 Da) by electrospray mass spectrometry were in full agreement with this theory. The correct pairing of the three disulfide bridges in synthetic decorsin was determined by a combined approach of both peptide mapping using proteolytic enzymes and analysis of the disulfide chirality by CD spectroscopy in the near-UV region. Synthetic decorsin inhibited human platelet aggregation with an IC50 of approximately 0.1 microM, a figure quite similar to that determined utilizing decorsin from natural source. In particular, the synthetic protein was 2,000-fold more potent than a model RGD-peptide (e.g., Arg-Gly-Asp-Ser) in inhibiting platelet aggregation. Thermal denaturation experiments of synthetic decorsin, monitored by CD spectroscopy, revealed its high thermal stability (Tm approximately 74 degrees C). The features of the oxidative refolding process of reduced decorsin, as well as the thermal stability of the oxidized species, were compared with those previously determined for the NH2-terminal core domain fragment 1-41 or 1-43 from hirudin. This fragment shows similarity in size, pairing of the three disulfides and three-dimensional structure with those of decorsin, even if very low sequence similarity. It is suggested that the less efficient oxidative folding and the enhanced thermal stability of decorsin in respect to those of hirudin core domain likely can be ascribed to the presence of the six Pro residues in the decorsin chain, whereas none is present in the hirudin domain. The results of this study indicate that decorsin can be obtained by solid-phase methodology in purity and quantities suitable for structural and functional studies and thus open the way to prepare by chemical methods novel decorsin derivatives containing unusual amino acids or even non-peptidic moieties.

The MHC fold, with its well-characterized peptide-binding groove, can perform other functions in addition to presentation of antigenic peptides to T cells. Homologs of MHC molecules have diverse roles that include presentation of lipid antigens (by CD1), transport of immunoglobulins (by the neonatal Fc receptor), regulation of iron metabolism (by the hemochromatosis gene product, HFE), and deception of the host immune system (by viral homologs). Recent crystal structures of two of these non-standard MHC-like molecules have allowed comparison of the recognition properties of classical. MHC molecules with those of their unusual homologs.

Although iron is an essential dietary requirement, the amount absorbed by the body is well regulated and depends on body iron stores and on dietary iron availability. There is very little iron excreted under normal conditions. Iron deficiency is a worldwide problem but iron overload, as seen in the inherited disease, hemochromatosis, is a major cause of morbidity in some Caucasian populations. This is a problem particularly where there is an adequate dietary iron intake and especially in males. A mutation has recently been described in an MHC Class l-like gene (HFE) that encodes for a protein (HFE) of 343 amino acids. The molecule contains a signal sequence peptide-binding region, alpha, and alpha(2) domains, and an immunoglobulinlike alpha(3) domain, in addition to a transmembrane region and a small cytoplasmic tail. It is a candidate gene for hemochromatosis. Several possibilities as to the function of this gene and the corresponding protein have been suggested but none has yet been confirmed. The mutation has been detected by several different groups in 80%-100% of subjects with the disease. However, in one study, 18%-20% of patients with the mutation did not exhibit significant iron overload. The discovery of this gene has important implications for both clinical studies and the elucidation of the pathways of iron metabolism.

Patterns of allele loss (loss of heterozygosity, LOH) have been studied in order to investigate the genetic pathways involved in the pathogenesis of three types of colorectal cancer (CRC): sporadic CRC without replication errors (RER-) (32 cases); sporadic RER+ CRC (23 cases); and ulcerative colitis-associated CRC (UCACRC) (16 cases). Each tumour was assessed for allele loss at ten microsatellite markers which map close to known or putative tumour-suppressor genes: APC (5q21-q22); DCC (18q21.1); 1p35-p36; p16 (9p21); 22q; 8p; E-cadherin (16q22.1); beta-catenin (3p22-p21.3); RB1 (13q14.1-q14.2); and HLA. Overall, high frequencies of allele loss (> 30 per cent) were found near DCC (42 per cent), p16 (38 per cent), 22q (37 per cent), 1p35-p36 (34 per cent) and APC (31 per cent), and low frequencies (< 20 per cent) near RB1 (16 per cent) and E-cadherin (13 per cent). LOH near beta-catenin, HLA, and on 8p occurred at frequencies between 20 and 30 per cent. The overall frequency of allele loss did not differ among the three tumour groups, but some variation was seen at individual loci. There was a significantly higher frequency of LOH at 1p35-36 in RER+ tumours compared to RER- tumours. Allele loss at this site was also associated with a more advanced Dukes' stage at presentation. In addition, RER- tumours showed a higher frequency of allele loss at p16 than RER+ tumours. No significant difference existed at any locus between the frequency of LOH in sporadic CRC and in UCACRC. Pairwise analysis showed a negative association between LOH at APC and DCC, and between LOH at chromosome 22p and p53 overexpression. Thus, there may be specific differences between the mutation spectra of RER+ and RER- CRCs, but there are large degrees of overlap among the underlying genetic pathways of these cancers and UCACRCs.

<h4>Aim</h4>To assess allelic imbalance and microsatellite instability in the region of the "deleted in colorectal cancer" (DCC) gene on chromosome 18q using fluorescent DNA technology in colorectal cancer in patients under the age of 35.<h4>Methods</h4>Thirty two cases of colorectal cancer in patients under the age of 35 and with no family history of colon cancer were retrieved. DNA was extracted by standard methods, polymerase chain reaction (PCR) was performed using Cy5 labelled primers to microsatellite markers (D18S21, D18S34, and D18S58) in the DCC gene. The results were analysed using software attached to an automated DNA sequencer.<h4>Results</h4>The patients ranged in age from 17 to 35 years. Nineteen were women, all had left sided tumours (tumours distal to the splenic flexure). Twenty eight cases were either stage C or D (using the Astler Coller system). The informativity of the three markers were as follows: D18S21, 25 of 32 (78.1%); D18S34, 18 of 32 (56.25%); D18S58, 24 of 32 (75%). Allelic imbalance for the markers, after excluding homozygous and microsatellite instability cases, was: D18S21, 31.8%; D18S34, 11.7%; and D18S58, 0%. Nine cases showed allelic imbalance for both D18S21 and D18S34, yielding a combined allelic imbalance frequency of 39.1%. Ten cases showed microsatellite instability in at least one marker, with microsatellite instability seen most commonly for D18S58. Three cases showed microsatellite instability for all three markers.<h4>Conclusions</h4>Approximately 39% of cases showed allelic imbalance for D18S21 and D18S34 markers, while microsatellite instability was found in 31.25% of cases. This figure is higher than that encountered in sporadic colorectal cancer over the age of 50, suggesting a role for the DNA repair genes in the pathogenesis of these cancers occurring under the age of 35.

A new human myeloid leukemia cell line (OIH-1), with alterations in chromosome 18 and the deleted in the colorectal carcinoma (DCC) gene and its product, was established from the peripheral blood (PB) of a patient with acute myeloblastic leukemia (AML) after myelodysplastic syndrome (MDS). Serial cytogenetics showed the presence of two clones, one with i(18)(q11) and another with trisomy 18. Southern blot analysis of OIH-1 cells with i(18)(q11) showed an extremely reduced intensity of 20- and 14-kb EcoRI fragments, suggesting the allelic loss of the DCC gene. Immunoprecipitation (IP) analysis by the murine monoclonal antibody (MoAb) AF5, specific for the DCC extracellular domain, failed to detect normal 180-kDa DCC protein, however extra 85-kDa protein was detected. However, Southern blot analysis of the latter clone of OIH-1 with trisomy 18 showed normal structure of the DCC gene. IP analysis with AF5 or G92-13 (specific for the extracellular domain) did not detect the DCC protein, but a 150-kDa protein other than the DCC-specific 180-kDa protein was detected with G97-449, specific for the cytoplasmic domain of the DCC protein. RT-PCR analysis showed the expression of the DCC mRNA in OIH-1 cells carrying each type of chromosome 18 abnormalities. These alterations in the DCC gene and protein may contribute to progression of malignancy for OIH-1 cells. The OIH-1 cell line may be useful for studying the role of the DCC gene in leukemogenesis of MDS or AML.