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Using differential display analysis, we compared the expression of RNA in v-mos-transformed cells and their flat revertant and isolated a novel gene, drm (down-regulated in mos-transformed cells), whose expression is down-regulated in parental v-mos-transformed cells but which is expressed at a high level in the revertant and normal rat fibroblasts (REF-1 cells). Analysis of different oncogene-transformed cells revealed that drm gene expression was also suppressed in REF-1 cells transformed by v-ras, v-src, v-raf, and v-fos. The drm cDNA contains a 184-amino-acid-protein-encoding open reading frame which shows no significant homologies to known genes in DNA databases. Polyclonal antibodies raised against drm peptide detect a protein with the predicted size of 20.7 kDa in normal cells and under nonpermissive conditions in cells conditionally transformed by v-mos but not in parental v-mos-transformed cells. Northern analysis of normal adult tissues shows that drm is expressed as a 4.4-kb message in a tissue-specific manner, with high expression in the brain, spleen, kidney, and testis and little or no expression in the heart, liver, and skeletal muscle. In situ hybridization analysis in adult rat tissue reveals good correlation with this pattern and indicates that drm mRNA is most highly expressed in nondividing and terminally differentiated cells, such as neurons, type 1 lung cells, and goblet cells. Transfection of a drug-selectable drm expression vector dramatically reduced the efficiency of colony formation in REF-1 and CHO cells, and the drm-transfected REF-1 survivors expressed low or nondetectable levels of exogenous drm mRNA. The toxic effects of drm can be overcome by cotransfection with constructs expressing oncogenic ras; furthermore, cells expressing high levels of drm and conditionally transformed with mos-expressing Moloney murine sarcoma virus rapidly undergo apoptosis when shifted to the nonpermissive temperature. Taken together, our data suggest that cells expressing high levels of drm undergo apoptotic death in the absence of oncogene-induced transformation and that drm represents a novel gene with potential roles in cell growth control or viability and tissue-specific differentiation.

The heparin binding site of the anticoagulant protein antithrombin III (ATIII) has been defined at high resolution by alanine scanning mutagenesis of 17 basic residues previously thought to interact with the cofactor based on chemical modification experiments, analysis of naturally occurring dysfunctional antithrombins, and proximity to helix D. The baculovirus expression system employed for this study produces antithrombin which is highly similar to plasma ATIII in its inhibition of thrombin and factor Xa and which resembles the naturally occurring beta-ATIII isoform in its interactions with high affinity heparin and pentasaccharide (Ersdal-Badju, E., Lu, A., Peng, X., Picard, V., Zendehrouh, P., Turk, B., Björk, I., Olson, S. T., and Bock, S. C. (1995) Biochem. J. 310, 323-330). Relative heparin affinities of basic-to-Ala substitution mutants were determined by NaCl gradient elution from heparin columns. The data show that only a subset of the previously implicated basic residues are critical for binding to heparin. The key heparin binding residues, Lys-11, Arg-13, Arg-24, Arg-47, Lys-125, Arg-129, and Arg-145, line a 50-A long channel on the surface of ATIII. Comparisons of binding residue positions in the structure of P14-inserted ATIII and models of native antithrombin, derived from the structures of native ovalbumin and native antichymotrypsin, suggest that heparin may activate antithrombin by breaking salt bridges that stabilize its native conformation. Specifically, heparin release of intramolecular helix D-sheet B salt bridges may facilitate s123AhDEF movement and generation of an activated species that is conformationally primed for reactive loop uptake by central beta-sheet A and for inhibitory complex formation. In addition to providing a structural explanation for the conformational change observed upon heparin binding to antithrombin III, differences in the affinities of native, heparin-bound, complexed, and cleaved ATIII molecules for heparin can be explained based on the identified binding site and suggest why heparin functions catalytically and is released from antithrombin upon inhibitory complex formation.

The extraction of motion information from time varying retinal images is a fundamental task of visual systems. Accordingly, neurons that selectively respond to visual motion are found in almost all species investigated so far. Despite its general importance, the cellular mechanisms underlying direction selectivity are not yet understood in most systems. Blocking inhibitory input to fly visual interneurons by picrotoxinin (PTX), we demonstrate that their direction selectivity arises largely from interactions between postsynaptic signals elicited by excitatory and inhibitory input elements, which are themselves only weakly tuned to opposite directions of motion. Their joint activation by preferred as well as null direction motion leads to a mixed reversal potential at which the postsynaptic response settles for large field stimuli. Assuming the activation ratio of these opponent inputs to be a function of pattern velocity can explain how the postsynaptic membrane potential saturates with increasing pattern size at different levels for different pattern velocities ("gain control"). Accordingly, we find that after blocking the inhibitory input by PTX, gain control is abolished.

During humoral immune responses, B-lymphocyte activation is followed by differentiation along either the plasma cell pathway or the memory B-cell pathway. Recent studies suggest that CD40-CD40 ligand, OX-OX40 ligand, a group of cytokines and intracellular transcriptional factors may all contribute to B-lymphocyte differentiation control.

The DNA fragment encoding meta-cleavage enzymes and the meta-cleavage compound hydrolase, involved in carbazole degradation, was cloned from the carbazole-utilizing bacterium Pseudomonas sp. strain CA10. DNA sequence analysis of this 2.6-kb SmaI-SphI fragment revealed that there were three open reading frames (ORF1, ORF2, and ORF3, in this gene order). ORF1 and ORF2 were indispensable for meta-cleavage activity for 2'-aminobiphenyl-2,3-diol and its easily available analog, 2,3-dihydroxybiphenyl, and were designated carBa and carBb, respectively. The alignment of CarBb with other meta-cleavage enzymes indicated that CarBb may have a non-heme iron cofactor coordinating site. On the basis of the phylogenetic tree, CarBb was classified as a member of the protocatechuate 4,5-dioxygenase family. This unique extradiol dioxygenase, CarB, had significantly higher affinity and about 20-times-higher meta-cleavage activity for 2,3-dihydroxybiphenyl than for catechol derivatives. The putative polypeptide encoded by ORF3 was homologous with meta-cleavage compound hydrolases in other bacteria, and ORF3 was designated carC. The hydrolase activity of CarC for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the meta-cleavage compound of 2,3-dihydroxybiphenyl, was 40 times higher than that for 2-hydroxy-6-oxohepta-2,4-dienoic acid, the meta-cleavage compound of 3-methylcatechol. Alignment analysis and the phylogenetic tree indicate that CarC has greatest homologies with hydrolases involved in the monoaromatic compound degradation pathway. These results suggest the possibility that CarC is a novel type of hydrolase.

Nucleotide sequence analysis of the flanking regions of the carBC genes of Pseudomonas sp. strain CA10 revealed that there were two open reading frames (ORFs) ORF4 and ORF5, in the upstream region of carBC. Similarly, three ORFs, ORF6 to ORF8, were found in the downstream region of carBC. The deduced amino acid sequences of ORF6 and ORF8 showed homologies with ferredoxin and ferredoxin reductase components of bacterial multicomponent dioxygenase systems, respectively. ORF4 and ORF5 had the same sequence and were tandemly linked. Their deduced amino acid sequences showed about 30% homology with large (alpha) subunits of other terminal oxygenase components. Functional analysis using resting cells harboring the deleted plasmids revealed that the products of ORF4 and -5, ORF6, and ORF8 were terminal dioxygenase, ferredoxin, and ferredoxin reductase, respectively, of carbazole 1,9a-dioxygenase (CARDO), which attacks the angular position adjacent to the nitrogen atom of carbazole, and that the product of ORF7 is not indispensable for CARDO activity. Based on the results, ORF4, ORF5, ORF6, and ORF8 were designated carAa, carAa, carAc, and carAd, respectively. The products of carAa, carAd, and ORF7 were shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be polypeptides with molecular masses of 43, 36, and 11 kDa, respectively. However, the product of carAc was not detected in Escherichia coli. CARDO has the ability to oxidize a wide variety of polyaromatic compounds, including dibenzo-p-dioxin, dibenzofuran, biphenyl, and polycyclic aromatic hydrocarbons such as naphthalene and phenanthrene. Since 2,2',3-trihydroxydiphenyl ether and 2,2',3-trihydroxybiphenyl were identified as metabolites of dibenzo-p-dioxin and dibenzofuran, respectively, it was considered that CARDO attacked at the angular position adjacent to the oxygen atom of dibenzo-p-dioxin and dibenzofuran as in the case with carbazole.

The effect of the Arg506 --> Gln mutation in factor VLEIDEN on thrombin generation was evaluated in a reconstituted system using the purified components of the tissue factor (TF) pathway to thrombin and the components of the protein C pathway. Recombinant full-length tissue factor pathway inhibitor (RTFPI) was included in the system because of a previously observed synergistic inhibitory effect of TFPI and the protein C pathway on TF-initiated thrombin generation. Thrombin generation initiated by 1.25 pM factor VIIa.TF in the absence of the protein C pathway components occurs following an initiation phase, after which prothrombin is quantitatively converted to 1.4 microM thrombin. The factor VLEIDEN mutation did not influence thrombin generation in the reconstituted model in the absence of the protein C pathway. In the presence of 2.5 nM TFPI, 65 nM protein C, and 10 nM recombinant soluble thrombomodulin (Tm), thrombin generation catalyzed by normal factor V was abolished after the initial formation of 25 nM thrombin. In contrast, persistent thrombin generation was observed in the presence of factor VLEIDEN in the same system, although the rate of thrombin generation was slower compared with the reaction without protein C and Tm. The rate of thrombin generation with factor VLEIDEN increased with time and ultimately resulted in quantitative prothrombin activation. When the TFPI concentration was reduced to 1.25 nM, thrombin generation is still curtailed in the presence of normal factor V. In contrast, under similar conditions using factor VLEIDEN, the protein C pathway totally failed to down-regulate thrombin generation. The dramatic effect of a 50% reduction in TFPI concentration on the inhibitory potential of the protein C pathway on thrombin generation catalyzed by factor VLEIDEN suggests that the observed synergy between TFPI and the protein C pathway is directly governed by the TFPI concentration and by cleavage of the factor Va heavy chain at Arg506. This cleavage appears to have a dramatic regulatory effect in the presence of low concentrations of TFPI. Markedly increased thrombin generation in the presence of both 1.25 nM TFPI and factor VLEIDEN was also observed when antithrombin-III was added to the system to complete the natural set of coagulation inhibitors. Protein S (300 nM) had a minimal effect in the model on the inhibition of thrombin generation by protein C, Tm, and TFPI, with either normal factor V or factor VLEIDEN. Protein S also failed to significantly potentiate the action of the protein C pathway in the presence of antithrombin-III in reactions employing normal factor V or factor VLEIDEN. The absence of an effect of protein S in the model, which employs saturating concentrations of phospholipid, suggests that the reported interactions of protein S with coagulation factors are not decisive in the reaction. Altogether the data predict that TFPI levels in the lower range of normal values are a risk factor for thrombosis when combined with the Arg506 --> Gln mutation in factor VLEIDEN.

CTLA-4 plays a critical role in regulating the immune response. It is mainly located in cytoplasmic vesicles and is expressed only transiently on the surface after T cell activation. In this study, we demonstrate that CTLA-4 is associated with AP50, the medium chain of the clathrin-associated coated pit adaptor protein complex AP2. In a yeast two-hybrid screen, three individual cDNA clones that encode mouse AP50 were isolated, all of which can interact specifically with the cytoplasmic domain of mouse CTLA-4, but not with the cytoplasmic domain of mouse CD28. We have shown that CTLA-4 can bind specifically to AP50 when CTLA-4 and AP50 are cotransfected into human 293T cells. A Y201 to F201 mutation in the YVKM intracellular localization motif of the CTLA-4 cytoplasmic domain significantly diminished its binding to AP50. We also found that AP50 bound to a CTLA-4 peptide containing unphosphorylated Y201 but not to a peptide containing phosphorylated Y201. Conversely, the p85 subunit of phosphatidylinositol 3-kinase and, to a lesser extent, protein tyrosine phosphatase SYP (SHP-2) and SHP (SHP-1) bind only to the CTLA-4 peptide containing phosphorylated Y201. Therefore, the phosphorylation status of Y201 in the CTLA-4 cytoplasmic domain determines the binding specificity of CTLA-4. These results suggest that AP50 and the coated pit adaptor complex AP2 may play an important role in regulating the intracellular trafficking and function of CTLA-4.

Thiazole-4-carboxamide adenine dinucleotide (TAD) analogue 7 containing a fluorine atom at the C2' arabino configuration of the adenine nucleoside moiety was found to be a potent inducer of differentiation of K562 erythroid leukemia cells. This finding prompted us to synthesize its hydrolysis-resistant methylenebis(phosphonate) and difluoromethylenebis(phosphonate) analogues 8 and 9, respectively. Since both TAD and benzamide adenine dinucleotide (BAD) are potent inhibitors of inosine monophosphate dehydrogenase (IMPDH), the corresponding fluorine-substituted methylenebis(phosphonate) analogue 12 of BAD was also synthesized. Thus, 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine (13) was converted in five steps into the corresponding methylenebis(phosphonate) analogue 18. Dehydration of 18 with DCC led to the formation of the bicyclic trisanhydride intermediate 19a, which upon reaction with 2',3'-O-isopropylidenetiazofurin (20) or -benzamide riboside (21) followed by hydrolysis and deprotection afforded the desired methylene-bridged dinucleotides 8 and 12, respectively. The similar displacement of the 5'-mesyl function of 2',3'-O-isopropylidene-5'-O-mesyltiazofurin (24) with the difluoromethylenebis(phosphonic acid) derivative gave the phosphonate 25 which was coupled with 13 to afford 26. The desired difluoromethylenebis(phosphonate) analogue 9 was obtained by deprotection with Dowex 50/H+. This compound as well as beta-CF2-TAD (4) showed improved differentiation-inducing activity over beta-CH2-TAD (3), whereas analogues containing the -CH2-linkage (8 and 12) were inactive.

In filamentous fungi, the ability to form a productive heterokaryon with a genetically dissimilar individual is controlled by specific loci termed het loci. Only strains homozygous for all het loci can establish a heterokaryon. In Neurospora crassa, 11 loci, including the mating-type locus, regulate the capacity to form heterokaryons. An allele of the het-c locus (het-cOR) of N. crassa has been previously characterized and encodes a nonessential 966 amino acid glycine-rich protein. Herein, we describe the genetic and molecular characterization of two hei-c alleles, het-cPA and het-cOR, that have a different specificity from that of het-cOR, showing that vegetative incompatibility is mediated by multiple alleles at het-c. By constructing chimeric alleles, we show that het-c specificity is determined by a highly variable domain of 34-48 amino acids in length. In this regard, het-c is similar to loci that regulate recognition in other species, such as the (S) self-incompatibility locus in plants, the sexual compatibility locus in basidiomycetes and the major histocompatibility complex (MHC) genes in vertebrates.

<h4>Objectives</h4>To establish the prevalence of increased hepatic iron content in patients with hepatitis C virus-related chronic hepatitis and to assess the accuracy of serum iron and ferritin in detecting tissue iron overload.<h4>Methods</h4>Serum iron, serum ferritin, and hepatic iron content were determined in 81 consecutive patients undergoing liver biopsy for chronic ALT elevation and hepatitis C virus infection. Moreover, in a subgroup of 28 patients, outcome of a 6-month course of interferon (IFN) treatment (6 million U of recombinant IFN, three times weekly) was determined after a mean follow-up of 24 +/- 6 months and the outcome was compared with the pretreatment values of hepatic iron content.<h4>Results</h4>Elevated serum iron or ferritin levels were detected in approximately 40% of patients, but elevated hepatic iron content was observed in only eight patients (10%). One of these patients had a hepatic iron index > 1.9, indicating hemochromatosis. Liver iron content and serum iron levels were not correlated. No differences in hepatic iron content were observed among patients with a sustained response to IFN (seven patients), short-term responders (seven patients), or nonresponders (14 patients).<h4>Conclusions</h4>Ten percent of patients with chronic hepatitis C have elevated hepatic iron content. These patients cannot be identified using serum markers of iron status. The relationship between liver iron and response to IFN treatment requires further prospective investigations.

Neogenin is a member of the N-CAM family of cell adhesion molecules and is closely related to the DCC tumor suppressor gene product. Recently, it has been demonstrated that the DCC/Neogenin subfamily plays a key role in axonal guidance within the embryonic nervous system, however little is known about the function of DCC or Neogenin in non-neuronal tissues in vertebrates. To gain an understanding of Neogenin function outside of the nervous system we have cloned and sequenced the mouse homologue of Neogenin. We describe three alternatively spliced exons within the extracellular domain of Neogenin and a fourth alternatively spliced exon within the cytoplasmic domain. We further demonstrate that three of these alternatively spliced exons are developmentally regulated. Analysis of Neogenin mRNA expression showed that two distinct Neogenin transcripts are expressed at significant levels in a broad spectrum of adult mouse tissues and throughout the mid to late stages of embryogenesis. In situ hybridization studies on day 15.5 mouse embryos revealed that Neogenin is expressed widely throughout the developing mouse embryo, in both neuronal and non-neuronal tissues. These observations suggests that Neogenin may play an integral role in regulating differentiation programmes and/or cell migration events within many embryonic and adult tissues.

Molecular biology is making a tremendous impact on the diagnosis and treatment of liver diseases. Methods such as the polymerase chain reaction are changing the way physicians diagnose and monitor patients with viral hepatitis. Assays based on recombinant protein antigens allow for detection of specific autoantibodies in diseases such as primary biliary cirrhosis. The diagnosis of inherited metabolic diseases, such as hemochromatosis and Wilson disease, is being revolutionized by discovery of the defective genes involved and the development of methods to rapidly sequence DNA and identify mutations. Treatments and preventive measures are now possible with use of drugs and vaccines produced by recombinant DNA technology. Gene therapy and nucleic acid-based therapeutics are also realistic future treatment options for individuals with liver diseases.

Chronic mild liver enzyme abnormalities are attributable to hereditary hemochromatosis in at least 3% of cases. Hemochromatosis formerly was diagnosed late with diabetes and hepatic and cardiac failure. Only recently have the autosomal recessive inheritance and subtle early presentations been understood. However, patients still wait many years and see many physicians before receiving a correct diagnosis. Increased serum transferrin saturation is currently the best test for detection of those likely to accumulate iron. Serum ferritin identifies those requiring treatment. When liver biopsy (controversial in asymptomatic individuals) is indicated, chemical measurement of liver iron content is helpful and therapeutic phlebotomy is the only effective treatment. Caucasian-type hemochromatosis (prevalence of 0.005) is associated with genetic abnormalities in HLA-H but also occurs in other ethnic groups. Those of African descent may have a different but also heritable iron-loading disease. Caucasian-type and to a lesser extent African iron loading are detectable early by laboratory testing. Early treatment restores normal expectations of length and quality of life in the Caucasian disease. Long-term treatment data are not yet available in African iron loading. Laboratory-initiated screening programs using unsaturated iron-binding capacity can eliminate symptomatic hemochromatosis.

Huntington's disease (HD) is one of an increasing number of human neurodegenerative disorders caused by a CAG/polyglutamine-repeat expansion. The mutation occurs in a gene of unknown function that is expressed in a wide range of tissues. The molecular mechanism responsible for the delayed onset, selective pattern of neuropathology, and cell death observed in HD has not been described. We have observed that mice transgenic for exon 1 of the human HD gene carrying (CAG)115 to (CAG)156 repeat expansions develop pronounced neuronal intranuclear inclusions, containing the proteins huntingtin and ubiquitin, prior to developing a neurological phenotype. The appearance in transgenic mice of these inclusions, followed by characteristic morphological change within neuronal nuclei, is strikingly similar to nuclear abnormalities observed in biopsy material from HD patients.

The mechanism by which an elongated polyglutamine sequence causes neurodegeneration in Huntington's disease (HD) is unknown. In this study, we show that the proteolytic cleavage of a GST-huntingtin fusion protein leads to the formation of insoluble high molecular weight protein aggregates only when the polyglutamine expansion is in the pathogenic range. Electron micrographs of these aggregates revealed a fibrillar or ribbon-like morphology, reminiscent of scrapie prions and beta-amyloid fibrils in Alzheimer's disease. Subcellular fractionation and ultrastructural techniques showed the in vivo presence of these structures in the brains of mice transgenic for the HD mutation. Our in vitro model will aid in an eventual understanding of the molecular pathology of HD and the development of preventative strategies.

Diallelic polymorphisms have been identified in the HLA-H gene and the ZNF192 gene located about 2 megabases centromeric to HLA-H. The three polymorphic sites in HLA-H together with the two hemochromatosis mutations in this gene give rise to 8 different haplotypes. The three polymorphic sites in ZNF192 give rise to 4 different haplotypes. The haplotypes in HLA-H are in complete linkage disequilibrium with the two common mutations in that gene, 845A (C282Y) and 187G (H63D). The 845A mutation is in weak linkage disequilibrium with the ZNF192 polymorphisms and the 187G mutation appears to be in equilibrium with this polymorphism. The 187G mutation therefore appears to be the older of the two HLA-H mutations.

Recent understanding of the mechanism of immunoglobulin G (IgG) catabolism has yielded new insight into antibody-mediated diseases. We proposed that beta2-microglobulin (beta2m)-deficient mice have been protected from systemic lupus erythematosis (SLE)-like syndromes because they lack the beta2m-associated IgG protection receptor (FcRn) and therefore catabolize IgG, including pathogenic IgG autoantibodies, considerably more rapidly than normal mice. Such an hypothesis would predict that beta2m-deficient mice would also be resistant to experimental bullous pemphigoid, a disease with a pathogenesis thought to be much simpler than SLE, being the result of antibody directed toward a pathogenic epitope on the epidermal hemidesmosome that anchors basal keratinocytes to the basement membrane. To test this hypothesis, we administered pathogenic rabbit antibody directed toward the hemidesmosome to beta2m-deficient mice and to normal control mice, both intraperitoneally and intradermally, and assessed the mice clinically, histologically, and immunologically for manifestations of skin disease. We found that the beta2m-deficient mice were protected when the antibody was given intraperitoneally whereas intradermal administration resulted in blisters only slightly less severe than those seen in normal mice. These data would indicate that autoantibody-mediated inflammation might be prevented or controlled by appropriate modulation of FcRn function.

A Drosophila cDNA encoding a structural homologue of the mammalian coated vesicle component alpha-adaptin (AP2 adaptor complex) has been cloned and sequenced. The mammalian and invertebrate sequences are highly conserved, especially within the amino terminal region, a domain that mediates interactions with other components within the AP2 complex and with specific receptors tails. Mammalian alpha-adaptins are encoded by two genes; however, Drosophila alpha-adaptin has a single gene locus, within polytene bands 21C2-C3 on the left arm of the chromosome 2, closely adjacent to the paired homeobox gene aristaless. There seem to be at least two Drosophila alpha-adaptin transcripts expressed, plausibly by alternative splicing. One of the transcripts is more abundant during early embryogenesis and may be of maternal origin. We have studied the distribution of the alpha-adaptin protein throughout embryogenesis and at the neuromuscular junction of the third instar larva. During cellularization of the blastoderm embryo, the protein is seen between and ahead of the elongating nuclei, and then redistributes to the cell surface during gastrulation. These observations suggest a role for endocytosis in cellularization and are consistent with the finding that dynamin (the shibire gene product), another component of the endocytic mechanism, is required for cellularization. At later stages of embryogenesis, alpha-adaptin is expressed in complex and dynamic patterns. It is strongly induced in elements of the central and peripheral nervous system (e.g., in neuroblasts, the presumptive stomatogastric nervous system, and the lateral chordotonal sense organs), in the Garland cells, the adult midgut precursors, the antenno-maxillary complex, the endoderm, the fat bodies, and the visceral mesoderm. In the larva, alpha-adaptin is localized at the plasma membrane in the synaptic boutons of the neuromuscular junctions. The cells expressing high levels of alpha-adaptin are known or expected to support high levels of endocytosis; thus, this coated vesicle protein seems to be an excellent marker for endocytic activity. The expression patterns of dynamin, detected in the embryo by in situ hybridization methods, are very similar to those reported here for alpha-adaptin reflecting the likely coordinated expression of endocytic components. Taken together with previous evidence, our results suggest that endosomal vesicle trafficking, membrane recycling, and the regulation of endocytosis play critical roles in the wide range of developmental processes.

Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by dramatic episodes of fever and serosal inflammation. This report describes the cloning of the gene likely to cause FMF from a 115-kb candidate interval on chromosome 16p. Three different missense mutations were identified in affected individuals, but not in normals. Haplotype and mutational analyses disclosed ancestral relationships among carrier chromosomes in populations that have been separated for centuries. The novel gene encodes a 3.7-kb transcript that is almost exclusively expressed in granulocytes. The predicted protein, pyrin, is a member of a family of nuclear factors homologous to the Ro52 autoantigen. The cloning of the FMF gene promises to shed light on the regulation of acute inflammatory responses.

Microsatellite instability (MSI) is intrinsic to most colorectal carcinomas (CRC) from patients with hereditary nonpolyposis colorectal cancer (HNPCC), reflecting germline mutations in the mismatch-repair (MMR) genes. Its occurrence and chronological sequence of development in sporadic CRC appears less well defined. To explore the time sequence in acquisition of MSI, and the role it plays during tumor progression in sporadic CRC, we compared the incidence of MSI in tissue samples from 40 Dukes'-B and 30 Dukes'-D CRC patients with liver metastases, at 4 different microsatellite loci, representing sites on the APC, DCC and p53 genes respectively as well as the D2S123 site. Among the 30 patients with hepatic metastases, MSI was found in 9 (30%) of the primary, and 13 (43.3%) of the metastatic tumors. In comparison, among the 40 Dukes'-B CRC, MSI was found in only 8 cases (20%). CRC with MSI were more frequently located in the right colon, less frequently on the left side, and seldom in the rectum. Tumor ploidy analysis shows that 46.2% of Dukes'-D primary tumors with MSI are diploid (chi2 = 4.46, p = 0.035). With a mean follow-up time of 4.2 years for the Dukes'-B CRC, there were no recurrences in the 8 patients with MSI, whilst 6 (18.8%) relapses occurred amongst the 32 patients without MSI, average time to recurrence being 15 months. In Dukes'-D CRC, mean survival time for patients with MSI was 37 months (95% CI, 24 to 51 months), for those without MSI 26 months (95% CI, 18 to 35 months), although this was not statistically significant. Our data suggest that tumor progression may involve increased genetic instability.

Controversy currently exists regarding the use of diltiazem in the treatment of acute myocardial infarction (AMI). due to conflicting results from clinical trials and animal studies. The purpose of this project was to evaluate the changes in the hemostatic profile during AMI following low dose intracoronary diltiazem infusion. Fourteen Yorkshire swine underwent thoracotomy and 50 min LAD occlusion, followed by 3 h of reperfusion. The first group (n = 8) received 2.5 mg of diltiazem intracoronary at a rate of 5.6 micrograms kg min-1 at the onset of reperfusion. The second group (n = 6) received 0.9% saline intracoronary at the onset of reperfusion and served as the control. The dynamics of plasma antithrombin-III (AT-III), Protein C, total Protein S, fibronectin, endothelin-1 (ET-1), and the stable metabolites of thromboxane (TxB2) and prostacyclin (6-keto-PGF1a) were determined at baseline, then twice during occlusion and finally three times during reperfusion. Diltiazem infusion resulted in diminished ET-1 (34.5%), fibronectin (23.2%), and TxB2 (35.6%); and elevated Protein C (29.3%) when compared with controls. We conclude that intracoronary diltiazem favorable influences hemostasis during AMI in swine. The cardioprotective effects of diltiazem during AMI may be related to the improved hemostatic profile and the reduced incidence of thrombotic complications in such patients.

<h4>Objective</h4>The objective of this study was to ascertain the exact relation between specific oncogenes and long- and short-term survival rates in patients with esophageal cancer.<h4>Summary background data</h4>Recent developments in molecular biology have shown that several oncogenes and suppressor genes are involved in the development of esophageal cancer. However, the role of these genes still is unknown.<h4>Methods</h4>The clinical outcome of 84 cases of R0-resected esophageal carcinomas (from 1986-1993) and the molecular and biologic characteristics of these tumors were studied. The patients studied were divided into three groups, which were designated as follows: shortest term survivors (up to 6 months), short-term survivors (7-12 months), and long-term survivors (>5 years). These groups included 23, 17, and 44 subjects, respectively. For the genomic analysis, CyclinD1, int-2, murine double minute 2 (MDM2), retinoblastoma, p53, adenomatous polyposis coli (APC), deleted in colorectal carcinoma (DCC), and human papillomavirus were studied in these patients. The regrowth capability of primary cultures and the clinicopathologic characteristics of these patients also were analyzed.<h4>Results</h4>The CyclinD1 and int-2 genes, which are located in the 11q13 chromosome, and the MDM2 gene were related to short survival. However, the p53 mutation and human papillomavirus infection were not related to short-term survival. The average ratio of genomic abnormalities to genes examined was higher in the shortest and short-term survival groups than in the long-term survival group. Regrowth capability in primary cultures also was related to short-term survival. Among the long-term survival patients, 7 (16%) of 44 cases suffered further cancer after esophagectomy.<h4>Conclusions</h4>These results suggest that the 11q13 amplicon and MDM2 may play an important role in the progression of esophageal cancer, and an accumulation of genomic abnormalities may result in poor prognosis. Careful follow-up testing for double cancer is needed in long-term survivors of esophageal cancer.

Two mutations have been described on the gene considered to be responsible for genetic hemochromatosis, the HLA-H or HFE gene. The C282Y mutation is a disease-causing mutation in most cases of genetic hemochromatosis, but involvment of the H63D substitution in the pathogenesis of the disease is unclear. Compound heterozygotes for both substitutions could help to determine whether or not the second mutation is a worsening factor when associate in trans with the C282Y mutant. We found twenty nine compound heterozygotes during DNA analysis of patients referred to our laboratory for the screening of those mutations. Clinical and biological data were obtainable for 23 of them. Compound heterozygotes could be divided into two groups: subjects with or without iron overload. Five (22%) individuals had normal ferritin levels, whereas 18 had elevated ferritin concentrations (78%). Among those 18 patients, 7 (30% of the total) had clinical and biological criteria of genetic hemochromatosis. Eleven had iron overload without all the criteria of genetic hemochromatosis. Such a high proportion of genetic hemochromatosis is not found in heterozygotes for the C282Y mutation alone neither in our series nor in the literature. Compound heterozygotes for the C282Y and the H63D mutations may have a higher risk of iron overload or genetic hemochromatosis than single heterozygotes for the C282Y mutation. We propose a schematic theoretical representation that could explain this fact at the protein level. Further fundamental studies on the protein, and clinical follow up of compound heterozygotes could help to ascertain this hypothesis.

The DNA of 287 healthy white elderly volunteers in the New Mexico Aging Process Study, between 63 and 91 years of age, was examined for mutations of the HLA-H gene at nt 845 and nt 187. None were found to be homozygous for the 845A mutation and there were no gender differences in the percentage of the various mutations. The frequency of the 845A mutation was 0.061 resulting in a carrier frequency of 12.2%. The frequency of the 187G mutation was 0.136 resulting in a carrier frequency of 19.9% for a single mutation; 2.4% were compound heterozygous, 834A/187G and 2.4% were homozygous for the 187G mutation. After excluding 5 men and 4 women with microcytic or macrocytic anemia, mean percent transferrin saturation (PSAT) and iron stores, as estimated from serum ferritin concentrations, were calculated for each mutation. Estimated iron stores were normally distributed (range approximately 50 to 1,550 mg) with men (n=111) having significantly higher mean estimated iron stores than women (n=167), 826 +/- 318 and 753 +/- 287 mg, respectively. More men, 15 of 28, (54%) with estimated iron stores in the upper quartile, >/= 1,050 mg, had a HLA-H mutation compared to 25 of 83 (30%) who had a mutation and whose estimated iron stores were < ,050 mg, P<0.05. Seven were heterozygous for the 845A mutation with mean estimated iron stores of 1,300 +/- 127 mg, 7 were heterozygous for the 187G mutation with mean estimated iron stores of 1,439 mg. Similar differences were not noted in women. Even though the potential role of the 187G mutation in the phenotypic expression of HH is less certain than the 845A mutation, the increase in PSAT seen in men with the 187G mutation and the equal distribution of 845A and 187G mutations seen with iron stores >/= 1,050 mg lends support for the involvement of the 187G mutation, or a linked mutation, in iron metabolism. We concluded that men having either a single chromosomal 845A and/or 187G mutation results in higher PSAT's and estimated iron stores than if no HLA-H mutation were present.

We have examined normal individuals and all the patients currently being treated for hemochromatosis at the Norfolk and Norwich hospital for mutations in the HLA-H gene. We found a gene frequency in 200 normal subjects for teh 845A (C282Y) allele of 0.085, corresponding to a carrier frequency of 17% which is among the highest reported anywhere in the world. The frequency for the less penetrant 187G (H63D) allele was 0.16 among 58 of the normal subjects, which corresponds to a carrier frequency of 32%. All 18 hemochromatosis patients were homozygous for the 845A allele which is not significantly different from other reports in our subset of 12 unrelated patients. These findings present a snapshot of a relatively stable population containing a predicted 3,500 individuals homozygous for the 845A allele but not diagnosed with hemochromatosis. This population will be an excellent model for studies on the penetrance of the 845A homozygous genotype and population screening.

This report assesses the degree of iron overload in a cohort of patients in relationship to the presence or absence of the recently described 845 G-->A (C282Y) and 187 C-->G (H63D) mutations in the HFE (HLA-H) gene. Sixty-one patients with hereditary hemochromatosis diagnosed either with liver biopsy or on clinical grounds were included in this analysis. Forty-one patients were homozygous for C282Y, the genotype considered to be characteristic of hereditary hemochromatosis. At the time of this analysis, 37 of these 41 patients had achieved a state of iron depletion and mobilizable iron was calculated: 19 had less than 4 grams. Twenty-five of these 41 patients had liver biopsies; 4 of these patients had a hepatic iron index less than 1.9. Of the 4 patients with a normal hepatic iron index, 3 had a quantitative hepatic iron of greater than 50 micromol/g dry weight, and one had an inadequate biopsy sample. These findings support our suspicion that individuals may have hereditary hemochromatosis and homozygous C282Y despite relatively low body iron stores. Five patients were compound heterozygotes for C282Y and H63D. Four of these patients underwent liver biopsy; two had a hepatic iron index greater than 1.9. a third patient had a hepatic iron index of 1.3 but a quantitative hepatic iron of 90.6 micromol/g dry weight. All patients were phlebotomized to a state of iron depletion and only one of these patients had a mobilizable iron greater than 4 grams. Three patients were homozygous for H63D; these patients had either a hepatic iron index >1.9 or greater than 4 grams of mobilizable iron. Patients with homozygous H63D and significant iron overload are not well described. Seven patients were heterozygous for either C282Y or H63D; 4 had significant iron overload but three did not. Five patients had no HFE mutations; one of these patients unequivocally has iron overload with a hepatic iron index of 4.4 We conclude that: (1) Identification of HFE mutations will be clinically useful in identifying patients with hereditary hemochromatosis, (2) Patient genotyping will help confirm a diagnosis of hereditary hemochromatosis in some patients with relatively low body iron stores, (3) Significant iron loading can occur in the absence of homozygous C282Y, adding to the evidence that genes other than HFE may be involved in iron loading, and (4) Homozygous H63D can be associated with significant iron overload.