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The variable clinical manifestations of cystic fibrosis (CF) suggest the influence of modifier genes. For example, meconium ileus is present in approximately 10-15% of neonates with cystic fibrosis; however, the genetic and, or environmental factors that determine whether an individual will develop this complication have not been determined. We propose the HFE gene as a candidate modifier locus for CF based on (1) the suggestion of an association between the HLA loci and CF phenotypes; (2) the location of the HFE gene near the HLA loci and; (3) the similarity between the gastrointestinal manifestations of hereditary hemochromatosis and CF. We have determined the frequency of the C282Y and H63D mutations in a group of 89 CF patients who were homozygous for delta F508 and for whom meconium ileus status was known. The carrier frequency of C282Y among the CF patients with meconium ileus was significantly different from that of our unaffected control group (19.4% versus 7.7%). However, the difference between the meconium ileus and the nonmeconium ileus groups was not significant (19.4% versus 10.3%). There was no difference in the frequency of the H63D among the three groups that were studied. These data are suggestive of a relationship between the development of meconium ileus or other gastrointestinal diseases in CF and the HFE gene. Further study of a larger group of patients is warranted.

Alterations in various hemostatic factor have been identified as risk factors for survival after acute myocardial infarction (AMI). However, these clinical data are primarily limited to observations made during the postinfarct stage. We assessed the effects of 50 minutes of left anterior descending artery occlusion on several hemostatic factors and analyzed their changes with outcome in 18 Yorkshire swine. Blood samples were obtained from the systemic circulation at base-line and at 25 and 50 minutes of occlusion. Platelet aggregability and plasma antithrombin-III protein C, protein S, fibronectin, endothelin-1, as well as the metabolites of thromboxane and prostacyclin were measured. Of the 18 swine, 7 survived the infarct and 11 animals developed fatal ventricular fibrillation. Both groups demonstrated significant decreases in platelet aggregation, and a decline in plasma protein S when compared with baseline. Thromboxane, prostacyclin, and ondothelin-1 plasma concentrations were also markedly reduced at the end of occlusion. There were significant changes in antithrombin-III, protein C, and fibronectin levels between surviving animals and those that died of ventricular fibrillation. Acute coronary artery occlusion is associated with substantial changes in the hemostatic factors in swine. Plasma levels of fibronectin, antithrombin-111, and protein C differed between survivors and nonsurvivors and thus might serve as predictors of mortality due to fatal ventricular fibrillation during AMI. The mechanisms of these changes during the acute phase of AMI are unclear. Immediately AMI prognosis may be related to hemostatic changes not only after thrombolysis or spontaneous reperfusion, but also during the occlusion phase as well.

Background: Little is known about the relationship between myocardial infarction and the coagulation inhibitors protein C, protein S, and antithrombin III (ATIII). Methods: We explored the hypothesis. that partial deficiencies in protein C, protein S, and ATIII may be associated with acute myocardial infarction by comparing the baseline levels of these anti coagulants in a sample of participants in the Thrombolysis in Myocardial Infarction Phase II Trial (TIMI II) to the levels in a sample of Red Cross donors. We also examined the changes in concentrations of hemostatic and inhibitory factors during thrombolytic therapy, the correlations between the inhibitors and plasminogen and fibrinogen, and the association of baseline inhibitor levels with the degree of plasmin generation during treatment. Levels of protein C, free and total protein S, ATIII, fibrinogen, tissue-type plasminogen activator, (t-PA), plasminogen, and alpha-2 antiplasmin were measured in plasma samples obtained at baseline before treatment with thrombolytic therapy and at 50 minutes, 5 hours, and 8 hours after the start of therapy (n=92). Protein C, free and total protein S, and ATIII were also measured in plasma samples from comparable Red Cross donors (n=92). Results: Baseline protein C and ATIII levels were higher in the TIMI II subjects than in controls (3.7 µ/ml vs 3.3 for protein C, P=0.002; 118% vs 111% for ATIII, P=0.006). Free and total protein S levels were lower in the TIMI II subjects than in controls (4.3 µ/ml vs 5.2 for free protein S, and 15.3 µ/ml vs 17.2 for total protein S, P=0.001). There were no major changes in protein C or protein S levels after initiation of thrombolytic therapy, but there was a 13% decrease in ATIII. Significant correlations were found between baseline protein C, Protein S, ATIII, and plasminogen; none of these factors were correlated with baseline fibrinogen concentration. The pretreatment levels of the inhibitors were correlated with markers of the amount of plasmin generated during therapy. Conclusions: It is not likely that partial protein C or ATIII deficiency is commonly associated with myocardial infarction. It is more likely that at the time of infarction, these inhibitors are somewhat higher possibly due to a homeostatic response to previous plaque rupture and thrombosis. Partial protein S deficiency may play a role in coronary thrombosis. The levels of these inhibitors at the time of infarction may be important in the efficacy of thrombolytic therapy.

The pathogenesis of polycythemia vera (PV), a disease involving a multipotent hematopoietic progenitor cell, is unknown. Thrombopoietin (TPO) is a newly characterized hematopoietic growth factor which regulates the production of multipotent hematopoietic progenitor cells as well as platelets. To evaluate the possibility that an abnormality in TPO-mediated signal transduction might be involved in the pathogenesis of PV, we examined TPO-induced protein tyrosine phosphorylation using platelets as a surrogate model system. Platelets were isolated from the blood of patients with PV as well as from patients with other chronic myeloproliferative disorders and control subjects. Impaired TPO-mediated platelet protein tyrosine phosphorylation was a consistent observation in patients with PV as well as those with idiopathic myelofibrosis (IMF), in contrast to patients with essential thrombocytosis, chronic myelogenous leukemia, secondary erythrocytosis, iron deficiency anemia, hemochromatosis, or normal volunteers. Thrombin-mediated platelet protein tyrosine phosphorylation was intact in PV platelets as was expression of the appropriate tyrosine kinases and their cognate substrates. However, expression of the platelet TPO receptor, Mpl, as determined by immunoblotting, chemical crosslinking or flow cytometry was markedly reduced or absent in 34 of 34 PV patients and also in 13 of 14 IMF patients. Impaired TPO-induced protein tyrosine phosphorylation in PV and IMF platelets was uniformly associated with markedly reduced or absent expression of Mpl. We conclude that reduced expression of Mpl is a phenotypic characteristic of platelets from patients with PV and IMF. The abnormality appears to distinguish PV from other forms of erythrocytosis and may be involved in the platelet function defect associated with PV.

Neoplastic transformation in the normal human brain occurs as a result of the accumulation of a series of genetic alterations. These genetic alterations include the loss, gain or amplification of different chromosomes which lead to altered expression of proteins that play important roles in the regulation of cell proliferation. Several common genetic alterations at the chromosomal level (loss of 17p, 13q, 9p, 19, 10, 22q, 18q and amplification of 7 and 12q) have been observed. These alterations lead to changes in the expression of several genes; protein 53 (p53), retinoblastoma (RB), interferon (INF) alpha/beta, cyclic AMP dependent kinase number 2 (CDKN2), mutated in multiple advanced cancers 1 (MMAC1), deleted-in-colon carcinoma (DCC), epidermal growth factor receptor (EGFR), platelet derived growth factor (PDGF), platelet derived growth factor receptor (PDGFR), MDM2, GL1, CDK4 and SAS during the genesis and progression of human gliomas. Recent studies suggest that altered expression of several other genes [MET; MYC; transforming growth factor beta (TGF beta); CD44; vascular endothelial growth factor (VEGF); human neurological-related cell adhesion molecule (hNr-CAM); neuroglial cell adhesion molecule (NCAM L1); p21waf1/Cip1; TRKA; mismatch repair genes (MMR); C4-2; D2-2] and proteins [e.g., cathepsins, tenascin, matrix metalloproteases, tissue inhibitors of metalloproteases, nitric oxide synthase, integrins, interleukin-13 receptor (IL-13R), Connexin43, urokinase-type plasminogen activator receptors (uPARs), extracellular matrix proteins and heat shock proteins] are associated with the genesis of human gliomas. Taken together, these findings point to the accumulation of multiple genetic mutations coupled with extensive changes in gene expression in the etiology of human gliomas.

We studied the regulation of spontaneous activity in the embryonic (day 10-11) chick spinal cord. After bath application of either an excitatory amino acid (AP-5 or CNQX) and a nicotinic cholinergic (DHbetaE or mecamylamine) antagonist, or glycine and GABA receptor (bicuculline, 2-hydroxysaclofen, and strychnine) antagonists, spontaneous activity was blocked for a period (30-90 min) but then reappeared in the presence of the drugs. The efficacy of the antagonists was assessed by their continued ability to block spinal reflex pathways during the reappearance of spontaneous activity. Spontaneous activity ceased over the 4-5 hour monitoring period when both sets of antagonists were applied together. After application of glycine and GABA receptor antagonists, the frequency of occurrence of spontaneous episodes slowed and became highly variable. By contrast, during glutamatergic and nicotinic cholinergic blockade, the frequency of occurrence of spontaneous episodes initially slowed and then recovered to stabilize near the predrug level of activity. Whole-cell recordings made from ventral spinal neurons revealed that this recovery was accompanied by an increase in the amplitude of spontaneously occurring synaptic events. We also measured changes in the apparent equilibrium potential of the rhythmic, synaptic drive of ventral spinal neurons using voltage or discontinuous current clamp. After excitatory blockade, the apparent equilibrium potential of the rhythmic synaptic drive shifted approximately 10 mV more negative to approximately -30 mV. In the presence of bicuculline, the apparent equilibrium potential of the synaptic drive shifted toward the glutamate equilibrium potential. Considered with other evidence, these findings suggest that spontaneous rhythmic output is a general property of developing spinal networks, and that GABA and glycinergic networks alter their function to compensate for the blockade of excitatory transmission.

The present study sought to determine whether doses of methamphetamine in the range of those used recreationally by humans produce brain dopamine (DA) neurotoxicity in baboons and to ascertain whether positron emission tomography (PET) imaging with the DA transporter (DAT) ligand [11C]WIN-35,428 ([11C]2beta-carbomethoxy-3beta-(4-fluorophenyl)-tropane) could be used to detect methamphetamine-induced DAT loss in living primates. Baboons were treated with saline (n = 3) or one of three doses of methamphetamine [0.5 mg/kg (n = 2); 1 mg/kg (n = 2); and 2 mg/kg (n = 3)], each of which was given intramuscularly four times at 2 hr intervals. PET studies were performed before and 2-3 weeks after methamphetamine treatment. After the final PET studies, animals were killed for direct neurochemical determination of brain DA axonal markers. PET-derived binding potential values, used to index striatal DAT density, were significantly decreased after methamphetamine, with larger decreases occurring after higher methamphetamine doses. Reductions in striatal DAT documented by PET were associated with decreases in DA, dihydroxyphenylacetic acid, and specific [3H]WIN-35,428 and [3H]DTBZ binding determined in vitro. Decreases in DAT detected with PET were highly correlated with decreases in specific [3H]WIN-35,428 binding determined in vitro in the caudate of the same animal (r = 0.77; p = 0.042). These results indicate that methamphetamine, at doses used by some humans, produces long-term reductions in brain DA axonal markers in baboons, and that it is possible to detect methamphetamine-induced DAT loss in living nonhuman primates by means of PET.

Both human and murine cytomegaloviruses (HCMV and MCMV) down-regulate expression of conventional class I major histocompatibility complex (MHC) molecules at the surfaces of infected cells. This allows the infected cells to evade recognition by cytotoxic T cells but leaves them susceptible to natural killer cells, which lyse cells that lack class I molecules. Both HCMV and MCMV encode class I MHC heavy-chain homologs that may function in immune response evasion. We previously showed that a soluble form of the HCMV class I homolog (U(L)18) expressed in Chinese hamster ovary cells binds the class I MHC light-chain beta2-microglobulin and a mixture of endogenous peptides (M. L. Fahnestock, J. L. Johnson, R. M. R. Feldman, J. M. Neveu, W. S. Lane, and P. J. Bjorkman, Immunity 3:583-590, 1995). Consistent with this observation, sequence comparisons suggest that U(L)18 contains the well-characterized groove that serves as the binding site in MHC molecules for peptides derived from endogenous and foreign proteins. By contrast, the MCMV homolog (m144) contains a substantial deletion within the counterpart of its alpha2 domain and might not be expected to contain a groove capable of binding peptides. We have now expressed a soluble version of m144 and verified that it forms a heavy chain-beta2-microglobulin complex. By contrast to U(L)18 and classical class I MHC molecules, m144 does not associate with endogenous peptides yet is thermally stable. These results suggest that U(L)18 and m144 differ structurally and might therefore serve different functions for their respective viruses.

Zwitterionic, net neutral oligonucleotides containing alternating negatively charged N3'-->P5' phosphoramidate monoester and positively charged phosphoramidate diester groups were synthesized. The ability of zwitterionic phosphoramidates to form complexes with complementary DNA and RNA was evaluated. Stoichiometry and salt dependency of these complexes were determined as a function of the nature of the heterocyclic bases of the zwitterionic compounds. Unlike the melting temperatures of the natural phosphodiester-containing oligomers, the T m of the duplexes formed with the zwitterionic oligothymidylates was salt concentration independent. The thermal stability of these duplexes was much higher with Delta T m values of 20-35 degrees C relatively to phosphodiester counterparts at low salt concentrations. The zwitterionic oligoadenylate formed only 2Py:1Pu triplexes with complementary poly(U) or poly(dT) strands. The thermal stability of these complexes was dependent on salt concentration. Also, the T m values of the complexes formed by the zwitterionic oligoadenylate with poly(U) were 6-41 degrees C higher than for the natural phosphodiester counterpart. Triplexes of this compound with poly(dT) were also more stable with a Delta T m value of 22 degrees C at low salt concentrations. Complexes formed by the zwitterionic oligonucleotides with complementary RNAs were not substrates for RNase H. Surprisingly, the duplex formed by the all anionic alternating N3'-->P5'phosphoramidate-phosphodiester oligothymidylate and poly(A) was a good substrate for RNase H.

In an attempt to improve physico-chemical and biological properties of peptide nucleic acids (PNAs), particularly water solubility and cellular uptake, the synthesis of chimeric oligomers consisted of PNA and phosphono-PNA analogues (pPNAs) bearing the four natural nucleobases has been accomplished. To produce these chimeras, pPNA monomers of two types containing N-(2-hydroxyethyl)phosphonoglycine, or N-(2-aminoethyl)phosphonoglycine backbone, were used in conjunction with PNA monomers representing derivatives of N-(2-aminoethyl)glycine, or N-(2-hydroxyethyl)glycine. The oligomers obtained were composed of either PNA and pPNA stretches or alternating PNA and pPNA monomers. The examination of hybridization properties of PNA-pPNA chimeras to DNA and RNA complementary strands in comparison with pure PNAs, and pPNAs as well as DNA-pPNA hybrids and DNA fragments confirmed that these chimeras form stable complexes with complementary DNA and RNA fragments. They were found to be resistant to degradation by nucleases. All these properties together with good solubility in water make PNA-pPNA hybrids promising for further evaluation as potential therapeutic agents.

Sporadic porphyria cutanea tarda (PCT) is caused by a reduced activity of uroporphyrinogen decarboxylase (URO-D) in the liver. Mild to moderate iron overload is common in PCT, as iron is one of the factors which trigger the clinical manifestations of the disease through the inactivation of URO-D. A role for genetic hemochromatosis in the development of iron overload in sporadic PCT has been hypothesized in the past. The aim of this work was to investigate whether mutations of HFE, which is a candidate gene for hemochromatosis, play the role of genetic susceptibility factors for PCT in Italian patients, who have a high prevalence of acquired triggering factors, such as hepatitis C virus (HCV) chronic infection and alcohol. We determined HFE genotypes of 68 male patients with PCT. Our data do not confirm an association of PCT with the Cys282Tyr HFE mutation, strongly associated with hemochromatosis in Northern European countries. A second mutation of HFE, His63Asp, however, had a significantly increased frequency as it was present in half of the patients. Surprisingly, the presence of the His63Asp mutation was not related to the iron status of patients, suggesting that a subtle abnormality of iron metabolism induced by this mutation could escape detection by the standard parameters of iron status. In PCT patients with liver disease, the presence of the mutation could contribute to the inactivation of URO-D, either directly or through a synergistic action with other factors that cause liver damage.

Integration of human papillomavirus (HPV) DNA into the host cell genome is an important step in cervical carcinogenesis. In tumour cells with integrated HPV DNA, transcription of viral oncogenes E6 and E7 continues into the flanking cellular sequences thereby producing viral-cellular fusion transcripts. Analysis of cellular sequences flanking the integrated HPV68 DNA in the cervical carcinoma cell line ME180 revealed homozygosity of the mutant allele in ME180 cells. We speculated that this could indicate the existence of a cellular tumour suppressor gene in the integration region. We report here the identification of a novel human gene, named APM-1, which is co-transcribed with the HPV68 E6 and E7 genes and is present in the 3'-cellular part of the ME180 viral-cellular fusion transcripts. The APM-1 gene encodes a protein with a BTB/POZ domain and four zinc fingers, and is located at chromosome 18q21. APM-1 transcripts are detected in normal cervical keratinocytes, but not in the majority of cervical carcinoma cell lines analysed. The APM-1 gene caused a reduction of clonal cell growth in vitro of HeLa and CaSki tumour cells. These characteristics make APM-1, the first novel human gene identified in a HPV integration region, a likely candidate for the postulated tumour suppressor gene.

The transcription factor HNF3 and linker histones H1 and H5 possess winged-helix DNA-binding domains, yet HNF3 and other fork head-related proteins activate genes during development whereas linker histones compact DNA in chromatin and repress gene expression. We compared how the two classes of factors interact with chromatin templates and found that HNF3 binds DNA at the side of nucleosome cores, similarly to what has been reported for linker histone. A nucleosome structural binding site for HNF3 is occupied at the albumin transcriptional enhancer in active and potentially active chromatin, but not in inactive chromatin in vivo. While wild-type HNF3 protein does not compact DNA extending from the nucleosome, as does linker histone, site-directed mutants of HNF3 can compact nucleosomal DNA if they contain basic amino acids at positions previously shown to be essential for nucleosomal DNA compaction by linker histones. The results illustrate how transcription factors can possess special nucleosome-binding activities that are not predicted from studies of factor interactions with free DNA.

The v-Myb DNA-binding domain differs from that of c-Myb mainly by deletion of the first of three repeats. This truncation correlates with efficient oncogenic transformation and a decrease in DNA-binding activity. Here we demonstrate that the D-type cyclins, cyclin D1 and D2 in particular, specifically inhibit transcription when activated through the v-Myb DNA-binding domain, but not the c-Myb DNA-binding domain. Analysis of a cyclin D1 mutant and a dominant-negative CDK4 mutant implied that this repression is independent of complex formation with a CDK partner. Association of cyclin D1 and D2 with the Myb DNA-binding domain could be demonstrated. Increased levels of cyclin D1 and D2 resulted in a stabilization of the Myb proteins, but not in an alteration in binding of the Myb proteins to DNA. These results highlight an unexpected role for cyclin D as a CDK-independent repressor of transcriptional activation by v-Myb but not c-Myb. This differential effect of D-type cyclins on v-Myb and c-Myb might help to explain the mechanism underlying the oncogenic activity of v-Myb, which appears to be a stronger transcriptional activator following the TPA-induced differentiation of transformed monoblasts when cyclin D1 and D2 are down-regulated.

Hemichrome formation resulting from oxidation of heme proteins has been proposed as sensitive indicator of iron-induced oxidative stress. The Heme Protein Spectra Analysis Program was used to quantitate oxidized heme proteins (OHP) in chronic hepatic iron overload in vivo. Rats were fed a diet containing carbonyl iron for 14 months with or without vitamin E supplementation. A 30-fold increase in hepatic iron concentration was observed in rats fed the iron-containing diet. At baseline, total OHP and TBARS were significantly elevated in iron-loaded livers but no increase in hemichrome was seen. During 3 h of spontaneous oxidation, hemichrome formation increased significantly in iron-loaded livers compared to controls. Although supplemental vitamin E was associated with lower levels of OHP and TBARS in iron-loaded livers at baseline, it did not significantly inhibit hemichrome formation during in vitro oxidation. In conclusion, an increase in hemichrome formation in iron-loaded livers was observed only during oxidation in vitro. These results suggest that total OHP is more sensitive than hemichrome formation as an indicator of oxidative stress in this in vivo model of iron overload.

A cDNA encoding a beta-1,4-galactosyltransferase named beta-1,4-GalT II was cloned from a cDNA library of the human breast tumor cell line, MRK-nu-1. Initially, a 860-bp PCR fragment was obtained from MRK-nu-1 mRNA by 3'-rapid amplification of cDNA ends by using two nested degenerate oligonucleotide primers based on a highly conserved amino acid sequence found in the catalytic domain of mammalian beta-1,4-galactosyltransferases and Lymnaea stagnalis beta-1,4-N-acetylglucosaminyltransferase (beta-1,4-GlcNAcT), both of which utilize the same sugar acceptor. This subsequently was used as a probe to isolate a 4.7-kb cDNA that contained an ORF of 1,164 bp predicting a polypeptide of 388 aa. Its deduced amino acid sequence shows an identity of 37% with that of the previously characterized human beta-1,4-galactosyltransferase (referred to as beta-1,4-GalT I) and of 28% with that of L. stagnalis beta-1,4-GlcNAcT. Study of the properties of the beta-1,4-GalT II fused to protein A expressed as a soluble form in COS-7 cells revealed that it is a genuine beta-1,4-GalT but has no lactose synthetase activity in the presence of alpha-lactalbumin. Northern blot analysis of 24 human tissues showed that they all express the beta-1,4-GalT II transcript, although the levels varied. These results indicate that human cells contain another beta-1,4-GalT.

Although HLA-linked hemochromatosis greatly increases the risk for hepatocellular carcinoma in people of European ancestry, iron overload in Africa is not thought to be etiologically related to this malignancy. To determine if African iron overload may be associated with hepatocellular carcinoma, we reviewed 320 consecutive diagnostic liver biopsies processed at the University of Zimbabwe from 1992 to 1994 and we selected for analysis 215 biopsies from adults that were suitable for the histological assessment of hepatocellular iron. Subjects were stratified according to hepatocellular iron grades of 0-2+ (normal levels to mild siderosis; n = 183) and grades of 3+ and 4+ (distinctly elevated levels consistent with iron overload; n = 32). Thirty-six subjects had hepatocellular carcinoma. Logistic regression modeling revealed a significant association between iron overload and hepatocellular carcinoma after adjustment for age, sex and and the presence of portal fibrosis or cirrhosis (p = 0.041). The odds of hepatocellular carcinoma in subjects with iron overload was 3.1 (95% confidence interval of 1.05-9.4) times that of subjects without iron overload. While we could not test for exposure to viral hepatitis or to aflatoxins in this study, our findings suggest that iron overload may be a risk factor for hepatocellular carcinoma in Africa.

We evaluated the effect of r-hirudin on an experimental model of disseminated intravascular coagulation (DIC) in rabbits, through the continuous infusion of 100 microg/kg/hr of Escherichia coli endotoxin for a period of 6 hours. r-Hirudin (0.05, 0.3, and 0.6 mg/kg/hr) as treatment, or saline solution as placebo, were administered simultaneously with endotoxin. Severe DIC in the endotoxin control group was shown by impairment in hemostatic parameters, kidney fibrin deposition, and a high mortality rate. Medium and high doses of r-hirudin led to an improvement in such DIC-related parameters as platelet numbers and fibrinogen and protein C concentrations. High-dose r-hirudin also reduced consumption of antithrombin III (ATIII). All doses of r-hirudin prevented decreases in tissue plasminogen activator (t-PA) and reduced the increase in plasminogen activator inhibitor-1 (PAI-1) activity observed at 2 hours after endotoxin administration. A significant reduction in kidney fibrin deposition was seen in medium- and high-dose r-hirudin groups. Additionally, the mortality rate in rabbits receiving medium- and high-dose r-hirudin was 10%, and that in rabbits receiving low-dose r-hirudin was 20%, as compared with a mortality rate of 70% in the control group. Protein C activity was significantly lower (p < 0.001) in nonsurviving rabbits. Moreover, there was a strong positive correlation (r = 0.68, p < 0.001) between protein C consumption and kidney fibrin deposition. We conclude that r-hirudin can be a useful drug in the clinical treatment of DIC.

(3S,4R)-4-(4-Fluorophenyl)-3-[[3,4-(methylenedioxy)phenoxy]methyl] piperidine [(3S,9R)-3, paroxetine] is a selective serotonin reuptake inhibitor (SSRI) used as an antidepressant in humans. In previous studies, we reported that certain (1R)-3 beta-(substituted phenyl)nortropane-2 beta-carboxylic acid methyl esters (2a) exhibited high affinity and reasonable selectivity for the serotonin transporter (5-HTT). The major structural differences between 2a and (3S,4R)-3 are that 2a possesses a different absolute stereochemistry and has an ethylene bridge not present in 3. In addition, 2a possesses a carbomethoxy substituent adjacent to the aryl ring, whereas (3S,4R)-3 contains a [3,4-(methylenedioxy)phenoxy]methyl group. In this study, we present the synthesis and biological evaluations of six of the possible eight isomers of 3-(4-fluorophenyl)-2-[[3,4-(methylenedioxy)phenoxy]methyl]nortropane+ ++ (4). The data for inhibition of [3H]paroxetine binding show that (1R)-2 beta, 3 alpha-4c, which has the same stereochemistry as paroxetine, has the highest affinity at the 5-HTT. Strikingly, the most potent compounds for inhibition of [3H]WIN-35,428 binding were not the (1R)-2 beta, 3 beta-isomers but rather (1R)-2 beta, 3 alpha-4c and (1S)-2 beta, 3 alpha-4f. Conformational analyses show that these isomers exist in a flattened boat conformation with pseudoequatorial substituents. Thus, the binding data show that this conformation is recognized by the DAT-associated binding site and also suggest that this conformation of paroxetine is recognized by the 5-HTT-associated binding site.

It has been reported that the rho genes, which consist of a ras-related small GTPase protein family, regulate cytoskeletal structures and have the potential to transform cultured cells. To investigate the biological relevance of the rho genes in pancreatic carcinogenesis, we examined expressions of the rhoA, B and C genes by polymerase chain reaction after reverse transcription (RT-PCR) in 33 cases of ductal adenocarcinoma of the pancreas. In addition, mutations of the K-ras, rhoA, B and C genes were studied in the same series of tumour tissues to correlate with rho gene expressions. The expression levels of the rhoC gene were significantly higher in tumours than in non-malignant portions (P < 0.001). Metastatic lesions overexpressed the rhoC gene compared with primary tumours (P < 0.05). Carcinoma tissues with perineural invasion and lymph node metastasis exhibited significantly higher expressions of the rhoC gene than tumours without these manifestations (P < 0.001 and P < 0.05 respectively). Overexpression of the rhoC gene significantly correlated with poorer prognosis of patients with pancreatic adenocarcinoma (P < 0.05). In contrast, the expression levels of the rhoA and B genes showed no significant relationship with clinicopathological findings. Mutation was not found either in the rhoA, B or C gene sequences examined. K-ras gene mutation, detected in 27 out of 33 (81.8%) cases, did not affect the expression levels in any of the rho genes. These suggest that elevated expression of the rhoC gene may be involved in the progression of pancreatic carcinoma independent of K-ras gene activation.

The human leukocyte antigen (HLA)-linked iron-loading gene (HFE) associated with the autosomal recessive disorder known as hereditary hemochromatosis occurs in about 10% of subjects of European descent, most of whom are unaffected heterozygotes. In contrast, the 3 to 5 per 1,000 who are homozygotes are at risk of developing severe and potentially lethal iron overload, with damage to a number of organs, including the liver, pancreas, heart, joints, and the endocrine glands. Although the removal of the excess iron by repeated venesections is simple, effective, and safe therapy, much of the organ damage, once it has occurred, is irreversible. Because symptoms are often nonspecific, it is important for physicians in the relevant specialties to develop a high index of suspicion and to apply widely the appropriate screening tests, including transferrin saturation and serum ferritin concentration. Equally important is the detection of affected family members, who are usually siblings, before they have developed significant iron overload. In addition, screening of populations in which the prevalence of hereditary hemochromatosis is high has become an attractive and cost-effective option, especially now that the molecular structure of the HFE gene has been defined. Using this approach it is now possible to detect individuals homozygous or heterozygous for the gene using a simple polymerase chain reaction-based test. The application of this exciting new tool promises to provide fresh insights into the range of phenotypic expression in hereditary hemochromatosis. A challenge for the future will be to define the genetic or environmental factors responsible for iron overload in up to 20% of patients with clinical hemochromatosis who do not have the HFE gene.

One of the loci for neuroblastoma suppressor genes is chromosome 18q21 where the DPC4 tumor suppressor gene, as well as the DCC and MADR2 genes, is located. DPC4 is a molecule of the TGF-beta signal which regulates differentiation of the neural crest precursor cells from which neuroblastoma originates. During the search for the significance of DPC4 as a candidate neuroblastoma suppressor gene, we found that there are at least two variant forms of the DPC4 transcripts by using the reverse-transcriptase-PCR procedure. The subsequent sequencing analysis has revealed that one is missing exons 5 and 6 and the other is missing exons 4-6. Both splice variants were frequently observed in neuroblastomas and at low levels in normal tissues. Though the functional role of the DPC4 splice variants is unknown, they might be important in regulating the TGF-beta signaling not only in neuroblastomas but also in other tumors and normal tissues.

Using a bacterial fusion protein, a deleted colorectal carcinoma (DCC)-specific monoclonal antibody (MAb) 127-22 was established. Although MAb 127-22 reacted with almost all normal tissues, it did not react or only weakly reacted with many cancer cell lines, including colonic cancer lines, in flow cytometry. In Western immunoblots, the MAb reacted with a single 190-kDa molecule in a myeloma line Ara-10 extract. This component was scarcely detected in colonic cancer cell lines. Immunoblots of samples from 25 pairs of colonic cancers and adjacent normal tissues and from five adenoma tissues revealed that all normal colonic and adenoma tissues significantly expressed the DCC protein, whereas colonic cancer tissues showed poor expression. These results indicate not only deletion of and lowered mRNA expression of the DCC gene, but also marked reduction of DCC protein occurred in colonic cancer tissues. In addition, colonic cancer patients with liver metastasis expressed significantly lower levels of DCC than those without, suggesting the prognostic value of DCC expression.

To understand the mechanisms that guide migrating cells, we have been studying the embryonic migrations of the C. elegans canal-associated neurons (CANs). Here, we describe two screens used to identify genes involved in CAN migration. First, we screened for mutants that died as clear larvae (Clr) or had withered tails (Wit), phenotypes displayed by animals lacking normal CAN function. Second, we screened directly for mutants with missing or misplaced CANs. We isolated and characterized 30 mutants that defined 14 genes necessary for CAN migration. We found that one of the genes, ceh-10, specifies CAN fate. ceh-10 had been defined molecularly as encoding a homeodomain protein expressed in the CANs. Mutations that reduce ceh-10 function result in Wit animals with CANs that are partially defective in their migrations. Mutations that eliminate ceh-10 function result in Clr animals with CANs that fail to migrate or express CEH-23, a CAN differentiation marker. Null mutants also fail to express CEH-10, suggesting that CEH-10 regulates its own expression. Finally, we found that ceh-10 is necessary for the differentiation of AIY and RMED, two additional cells that express CEH-10.

Specification of the R7 photoreceptor cell in the developing Drosophila eye requires the seven in absentia (sina) gene. We demonstrate that ectopic expression of sina in all cells behind the morphogenetic furrow disrupts normal eye development during pupation, resulting in a severely disorganized adult eye. Earlier events of cell fate specification appear unaffected. A genetic screen for dominant enhancers and suppressors of this phenotype identified mutations in a number of genes required for normal eye development, including UbcD1, which encodes a ubiquitin conjugating enzyme; SR3-4a, a gene previously implicated in signaling downstream of Ras1; and a Drosophila homolog of the Sin3A transcriptional repressor.

Patients with systemic lupus erythematosus (SLE) are at an increased risk of developing osteonecrosis (ON). Twenty-six patients with SLE were studied. Fifteen of these had ON and 11 did not. The latter were used as control subjects. Various coagulation analytes including antithrombin III (ATIII) activity, protein C activity, protein S activity, alpha 2-antiplasmin activity, anticardiolipin antibodies (aCL), plasminogen activator inhibitor (PAI-1) activity and tissue type plasminogen activator (tPA) antigen were measured using citrated plasma samples from the patients. A significant proportion (80%) of patients had at least one laboratory abnormality that has been associated with a thrombotic predisposition. ON was significantly associated with elevated levels of PAI-1 activity; it was also associated with elevated PAI-1/tPA ratio. There was no association between ON of SLE and abnormalities of the other measured coagulation analytes. These results suggest that defective fibrinolysis seems to be operative in the pathogenesis of ON associated with SLE. The defect appears to involve an imbalance between tPA and its inhibitor, PAI-1. This imbalance could represent an important risk factor in the pathogenesis of ON.

To investigate the frequency of estrogen receptor (ER) gene mutation in metastatic or recurrent breast cancer, metastatic lymph nodes or recurrent breast cancer tissue from 35 patients with ER-positive primary tumors were screened for mutations in the hormone-binding domain of the ER gene by sequence analysis. Four missense mutations, Val316Ile, Gly344Val, Ala430Val and Gly494Val, were identified in these lesions. Second, to clarify whether there is any disparity in hormone receptor status between primary and metastatic or recurrent tumors, we immunohistochemically studied 117 specimens including the above 35 specimens obtained from metastatic or recurrent breast cancer patients using monoclonal anti-ER and progesterone receptor (PgR) antibodies. Although hormone receptor status, especially ER, was highly maintained through disease progression, negative change in PgR expression at relapse (33%) was identified more frequently than in metastatic lymph nodes (6.7%). Therefore, it was suggested that development of PgR-negative phenotype might correlate with disease progression in some breast cancer patients. These results suggest that ER mutations in metastatic or recurrent breast cancer may be more frequent than in primary lesions, irrespective of high maintenance of ER protein expression through disease progression.

The aim of this study was to clarify whether or not the status of gene alteration is heterogeneous in intramucosal carcinoma and homogeneous within invasive carcinoma. We selected 10 colorectal carcinoma cases (1 mucosal, 5 submucosal and 4 advanced carcinomas including 2 cases with lymph node metastasis) and analyzed the p53 gene sequence. Six colorectal cancers in this study showed heterogeneity in p53 mutations in cells from the intramucosal part. In the invasive part of a carcinoma, p53 mutation status was homogeneous intratumorally in all cases. These data indicate that, in regard to p53 gene alterations, colorectal cancers can be composed of various subclones when limited to the mucosa, but clonal selection occurs when one of these subclones commences invasion to the submucosa, generating a monoclonal invasive carcinoma.

<h4>Background</h4>Unrelated individuals (n = 242) were interviewed directly for the presence of migraine, anxiety disorders, and major depression.<h4>Materials and methods</h4>The data described in this study are derived from a clinical genetic relational database that was developed initially for the genetic analysis of migraine. Genotyping of the DRD2 NcoI C to T polymorphism located in exon 6 (His313His) was performed using previously described primers.<h4>Results</h4>A significantly increased incidence of migraine with aura (MWA), major depression, generalized anxiety disorder (GAD), panic attacks, and phobia was observed in individuals with the DRD2 NcoI C/C genotype compared with individuals with an DRD2 NcoI T allele. Specifically, 69% (91/131) of DRD2 NcoI C/C individuals in the present study met criteria for at least one of these neuropsychiatric disorders versus only 22% (4/18) of the DRD2 NcoI T/T individuals (Chi-square = 15.29; p < 0.00005). The DRD2 NcoI C allele frequency is significantly higher (Chi-square = 17.13; p < 0.00002) in individuals with MWA, anxiety disorders, and/or major depression (C allele frequency = 0.80) than in individuals who have none of these disorders (C allele frequency = 0.67).<h4>Conclusions</h4>These data indicate that MWA, anxiety disorders, and major depression can be components of a distinct clinical syndrome associated with allelic variations within the DRD2 gene. Clinical recognition of this genetically based syndrome has significant diagnostic and therapeutic implications.

Genetic testing for hereditary haemochromatosis is likely to be a significant workload for diagnostic laboratories. The C282Y and H63D mutations in the HFE gene associated with hereditary haemochromatosis have previously been detected using a number of methods including alterations in the restriction digest pattern of polymerase chain reaction (PCR) amplified products. An amplification refractory mutation system (ARMS) has been developed that will simultaneously detect both hereditary haemochromatosis mutations. Comparison of the results obtained from the analysis of 46 samples referred for hereditary haemochromatosis testing showed no discrepancies between ARMS and restriction enzyme digestion. Furthermore, consistent results were obtained by ARMS from both blood and buccal mouthwash samples. The ARMS test is quicker and less expensive in terms of consumables and scientist time than restriction enzyme analysis, and is therefore suited to the routine diagnostic analysis of hereditary haemochromatosis.

Flow-cytometric DNA analysis was performed retrospectively from paraffin-embedded blocks in 158 consecutive ductal infiltrative breast carcinoma patients grades I-III. Normal breast tissue was used as control. Tumor proliferative activity, cell ploidy, and DNA index (DI) were related to age of patients, histological grade of tumor, tumor size, axillary lymph node status, estrogen and progesterone receptor status, menopausal status,TNM clinical classification, and survival. There was a significant association between DNA aneuploidy and a high cellular proliferative activity, increased DI, poor differentiation of tumor, primary tumor size, number of positive lymph nodes, and postmenopausal state. Increased proportion of cells in S-phase was associated with positive lymph node status and higher number of positive lymph nodes. The cell cycle parameters had no prognostic value either for overall survival of disease-free survival of the patients.

Severely burned patients often present a hypercoagulability situation. However, its magnitude, time course, and relationship with organ failure and outcome remains to be established. Forty-three patients were studied on the first and seventh day after burn for hypercoagulability and fibrinolysis parameters. A hypercoagulability and hyperfibrinolysis state was found the first day after burn demonstrated by high levels of activated factor VII (VIIa, p<0.01), thrombin-antithrombin III complex (TAT, p<0.01), tissue plasminogen activator (t-PA, p<0.001) and D dimer (DD, p<0.01) and low levels of antithrombin III (ATIII, p<0.01), protein C (PC, p<0.01), plasminogen (PG, p<0.001) and alpha2 antiplasmin (AP, p<0.001). A paradoxical coexisting hypofibrinolysis was found as suggested by a low global fibrinolytic activity in the euglobulin plasma fraction fibrin plate assay (FA, p<0.01) and high levels of tissue plasminogen activator inhibitor type 1 (PAI-1, p<0.01). On day 7, a less marked hypercoagulability situation was found, with low ATIII (p<0.01) and PC (p<0.01), persisting the hypofibrinolytic situation observed on the first day. Non-survivors (NS) showed higher levels of VIIa (p<0.01), TAT (p<0.05) and t-PA (p<0.05), and lower levels of ATIII (p<0.05), PC (p<0.05) and AP (p<0.001) than survivors (S) on the first day. Also, there was a positive correlation of Marshall organ failure score with ATIII, (r2=0.49, p<0.001), PC, (r2=0.14, p<0.045) and PG levels, (r2=0.41, p<0.0003). Severely burned patients show a state of transient disseminated intravascular coagulation, related to the development of organ failure and outcome.

Von Hippel-Lindau (VHL) disease is an inherited autosomal dominant neoplastic disorder causing central nervous system haemangioblastomas. The VHL gene (3p25-3p26) is known to be a tumour suppressor gene, with its inactivation being responsible for a predisposition to tumour development. As far as we know, the present report of VHL disease manifestation in identical twins is unique. Genetic inquiry into the family background did not reveal this disease among their progenitors. For presymptomatic diagnosis of 17 presently unaffected family members, constitutional DNA of the twins was screened for VHL germline mutations, using loss of heterozygosity studies and exon-specific DNA sequencing. To determine the influence of somatic mutations of the VHL gene in tumourigenesis, DNA of five surgically removed intracerebral haemangioblastomas of the identical twins was analyzed in comparison with their constitutional DNA by DNA sequencing of the complete VHL coding region. However, no allelic losses were found for the VHL gene or for various other tumour suppressor genes (p53, BRCA1, BRCA2, DCC, and MCC). Furthermore, no mutations were found in the constitutional DNA of either twin sister or in the DNA of all five tumour lesions. Based on our observations, we conclude that in certain VHL families, presymptomatic molecular diagnosis of the disease is not feasible and requires close clinical surveillance of all individuals at risk.

Microsatellite instability (MSI) has been reported to occur in various types of malignant neoplasms. We performed a polymerase-chain-reaction-based assay for MSI between the initial and the most recently available ("latest") samples from 23 patients with myelodysplastic syndrome (MDS). Of these patients, 15 were informative at more than three microsatellite loci. Seven patients showed an increase in leukemic cells while 8 patients did not during the interval between the two analyses. Only 1 of the patients, who had refractory anemia with excess blasts, which changed to acute myelogenous leukemia, showed microsatellite alteration at the analysis times. Among all 23 patients, two alterations were detected in the 42 informative paired samples that showed an increase in leukemic cells (4.8%), while none was detected in the 59 paired samples without such an increase. In total, therefore only two alterations were detected among 101 informative paired samples (2%). This indicates that MSI is rare in the clinical course of MDS irrespective of disease status, and is consequently not a critical genetic event for disease progression in most MDS patients.

A possible dysregulation of serotonergic neurotransmission has been implicated in the aetiology of schizophrenic psychoses. In the present study we analysed allelic and genotypic variations of a recently described functional polymorphic region in the promoter of the human serotonin transporter gene (5-HTTLPR) and a variable tandem repeat (VNTR) in intron 2 of the 5-HTT gene. We investigated 413 unrelated individuals, 180 schizophrenic patients and 233 blood donors as controls. With regard to the 5-HTTLPR, both the schizophrenic and the control group did not significantly differ between genotype frequencies (chi2, p = 0.920) and allele frequencies (chi2, p = 0.836). The odds ratio for subjects with schizophrenia who were homozygous for the short allele was 1.04 (95% CI 0.59-1.84). No evidence of allelic association to specific schizophrenia subtypes was found. The 5-HTT associated VNTR also showed no significant differences between either the allelic or the genotypic distributions. Haplotype analysis revealed a significant overall linkage disequilibrium at a level of p = 0.00004. Our findings indicate that both polymorphisms are unlikely to play a substantial role in the genetic predisposition to schizophrenic disorders.

Mutation analysis of the HLA-H gene in French hemochromatosis patients and genetic counseling in families: Genotype analysis of 61 hemochromatosis patients living in France and 126 controls confirms that the disease is strongly associated with homozygosity for the mutation C282Y of the HLA-H gene: in our sample 67.2% patients, and none of the controls, carry two copies of the C282Y mutation; the frequency of the C282Y allele in a control population is 3.97%. A second variant (H63D) at the HLA-H gene is somehow more enriched (8.2%) in patient chromosomes that do not carry the C282Y mutation. The C282Y mutation was used in 10 hemochromatosis families for genetic counseling.

<h4>Background and objectives</h4>The purpose of this study was to establish a rapid method suitable for large-scale population screening, including blood donors, for the detection of two genetic mutations at codons 63 and 282 on the HFE gene that are associated with haemochromatosis.<h4>Materials and methods</h4>A method using the polymerase chain reaction with sequence-specific primers (PCR-SSP) was designed and tested using a panel of 185 individuals previously typed for HFE mutations by PCR-RFLP.<h4>Results</h4>The PCR-SSP method detected the two mutations showing complete agreement with the genotypes obtained by PCR-RFLP. Three HFE alleles, termed HFE-1, -2, and -3, were identified.<h4>Conclusions</h4>This PCR-SSP method allows efficient HFE genotyping for large numbers of individuals.

A case control study was conducted among cases with schizophrenia (DSM IV criteria) and screened adult controls from three cohorts. Bi-allelic polymorphisms in the promoter region of the serotonin transporter gene (5-HTT) were examined in conjunction with those of the serotonin 5-HT2a receptor (HTR2). No significant association with 5-HTT was detected among US Caucasians (n = 207), African-Americans (n = 84) or Caucasians from Sweden (n = 221). However, survival analysis suggested an association with the age at onset among the Swedish cases. The association should be considered tentative as it was not evident in the smaller US samples. The following exploratory analyses among the US samples were also not significant: associations with subgroups of patients based on familiality or response to medications, or altered risk due to the joint effects of 5-HTT and HTR2 genotypes.

Hemochromatosis (GH) is an inborn error of iron metabolism, characterized by progressive iron loading that, if untreated, causes high morbidity and death. The gene responsible for the disease (HFE), located 4.5 megabases telomeric to the HLA-A locus, encodes a protein homologous to class I MHC molecules. A main mutation, C282Y, has been identified within the gene. Although hemochromatosis is considered as the most frequent inherited disease in the populations of Northern European origin, its prevalence in Brittany had not been evaluated yet. In this issue we report the C282Y mutation frequency in a cohort of 1000 newborns from maternity hospitals of the four breton départements. The homozygote frequency was 5/1000 and heterozygote frequency was 12%; such high frequencies raise the question of the penetrance of the disease and the relevance of systematic genotypic screening for hemochromatosis.

Many of the complications of high-dose therapy with hematopoietic stem cells are caused by or lead to the multiple-organ dysfunction syndrome (MODS). In hematopoietic stem cell transplantation (HSCT), acquired antithrombin III (ATIII) deficiency is independently associated with MODS to the exclusion of transplant type, preparative regimen, and bacteremia. In experimental settings, replacement of ATIII can ameliorate the severity of MODS that develops in response to a variety of pathologic stimuli, suggesting that ATIII supplementation might improve the clinical course of MODS in patients undergoing HSCT. We performed a study to determine if ATIII can improve the morbidity of MODS in HSCT. Forty-nine patients undergoing HSCT, who developed pulmonary dysfunction (oxygen saturation of <90%), central nervous system dysfunction (drop of >4 points in the mini-mental status exam), or hepatic dysfunction (bilirubin >34 micromol/L [2.0 mg%], weight gain of >5% over baseline, and abdominal pain, possibly of hepatic origin) with a concomitant ATIII activity of <84% were double-blind randomized to receive ATIII concentrate, 70 units/kg within 24 hours of recognition of initial organ dysfunction followed by 50 units/kg 8, 16, 48, and 72 hours later, or albumin placebo. The group randomized to ATIII had a lower severity-of-illness score (15.7 +/- 19.2 vs. 28.6 +/- 25.2, p = 0.03), shorter duration of hospitalization (14.9 +/- 16.7 vs. 25.7 /- 17.9 days, p = 0.03), and lower hospital charges ($138,700 +/- $23,500 vs. $206,400 +/- $34,000). ATIII concentrate was associated with improved morbidity of MODS in patients undergoing HSCT when given early in the evolution of the syndrome.

No abstract available.

No abstract available.