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C Redman

Publications and source records attributed to C Redman.

At least 37 records · Page 2Linked to original sources
PubMed1996

Fibrinogen assembly and secretion. Role of intrachain disulfide loops.

Human fibrinogen is a homodimer composed of three different (Aalpha, Bbeta, gamma) polypeptide chains. The chains are linked by 29 inter- and intrachain disulfide bonds. Each half-molecule has 6 intrachain disulfide bonds, which form loops in the carboxyl-terminal region of each of the chains. Aalpha chain has one disufide loop (Cys442-Cys472), Bbeta has three (Cys201-Cys286, Cys211-Cys240, and Cys394-Cys407), and gamma has two loops (Cys153-Cys182 and Cys326-Cys339). The intrachain loops are conserved in fibrinogens of different species. We changed, by site-directed mutagenesis, the cysteines, which form the intrachain loops, to serine or alanine. Fibrinogen chain assembly and secretion was determined in transiently transfected COS cells expressing two normal and a mutant fibrinogen chain. In the Bbeta and gamma chains, disruption of the disulfide loops closest to the "coiled-coil" region (CysBbeta211-Cys240, CysBbeta201-Cys286, and Cysgamma153-Cys182) abolished chain assembly and secretion, indicating that the disulfide loops closest to the coiled-coil region are essential for chain assembly. By contrast, preventing formation of the disulfide loops, which are toward the carboxyl termini of each of the chains, had different effects. Disruption of the single Aalpha disulfide loop had no effect, as did disruption of BbetaCys394-Cys407. However, disruption of Cysgamma326-Cys339, which is similar in size and location to CysBbeta394-Cys407, allowed chain assembly to occur, but the assembled chains were not secreted.

Animals↗
PubMed1996

In vitro assembly of the component chains of fibrinogen requires endoplasmic reticulum factors.

Human fibrinogen (340 kDa) is a dimer, with each identical half-molecule composed of three different polypeptides (Aalpha, 66 kDa; Bbeta, 55 kDa; and gamma, 48 kDa). To understand the mechanisms of chain assembly, a coupled in vitro transcription translation system capable of assembling fibrinogen chains was developed. Fibrinogen chain assembly was assayed in an expression system coupled to rabbit reticulocyte lysate in the presence or absence of dog pancreas microsomal membranes. Fibrinogen chain assembly required microsomal membranes and oxidized glutathione. Co-expression of two of the chains, Bbeta and gamma or Aalpha and gamma, yielded free chains and two-chain complexes. Unlike combinations of Aalpha with gamma and Bbeta with gamma, co-expression of Aalpha and Bbeta did not form a single two-chain complex but produced a mixture of two-chain complexes. Co-expression of all three chains yielded free chains, two-chain complexes, and higher molecular weight complexes that corresponded to a half-molecule and to fully formed fibrinogen. Upon treatment of this mixture with thrombin and factor XIIIa, a gamma.gamma dimer, similar to that obtained from cross-linked human fibrin, was produced, indicating that properly folded fibrinogen was formed in vitro. Molecular chaperones may participate in fibrinogen assembly, since antibodies to resident proteins of the endoplasmic reticulum (BiP, Hsp90, protein disulfide isomerase, and calnexin) co-precipitated the chaperones together with nascent fibrinogen chains and complexes.

Animals↗
PubMed1996

Epithelial ovarian cancer: influence of polymorphism at the glutathione S-transferase GSTM1 and GSTT1 loci on p53 expression.

The importance of polymorphism in the glutathione S-transferase GSTM1, GSTT1 and, cytochrome P450, CYP2D6 loci in the pathogenesis of epithelial ovarian cancer has been assessed in two studies; firstly, a case-control study designed to determine the influence of these genes on susceptibility to this cancer, and secondly, the putative role of these genes in the protection of host cell DNA has been studied by comparing p53 expression in patients with different GSTM1, GSTT1 and CYP2D6 genotypes. The frequencies of GSTM1, GSTT1 and CYP2D6 genotypes in 84 cases and 325 controls were not different. Immunohistochemistry was used to detect p53 expression in 63 of these tumours. Expression was found in 23 tumours. Of the patients demonstrating immunopositivity, 20 (87%) were GSTM1 null. The frequency distributions of GSTM1 genotypes in p53-positive and -negative samples were significantly different (P = 0.002) and those for GSTT1 genotypes approached significance (exact P = 0.057). The proportion of patients with both GSTM1 null and GSTT1 null was also significantly greater in the immunopositive (4/22) than in the immunonegative group (1/40) (P = 0.0493). Single-strand conformational polymorphism (SSCP) analysis was used to detect mutations in the 23 tumour samples demonstrating p53 positivity. A shift in electrophoretic mobility of amplified fragments was found in 11 patients (exons 5, 6, 7 and 8) and these exons were sequenced. In eight samples a mutation was found. No SCCP variants were identified in the other 12 immunopositive patients. Sequencing of exons 4-9 of p53 from these tumours resulted in the detection of mutations in two patients (exons 5 and 7). Thus, in 23 patients who demonstrated immunopositivity, p53 mutations were found in nine patients with GSTM1 null (90.0%). In the 13 patients in whom no mutations were identified, 11 were GSTM1 null (84.6%). The data show that overexpression of p53 is associated with the GSTM1 null genotype. We propose the data are compatible with the view that GSTM1 and GSTT1 are critical in the detoxification of the products of oxidative stress produced during the repair of the ovarian epithelium. Thus, failure to detoxify products of this stress may result in damage to various genes in the host cell, including to p53, resulting in persistent expression of mutant protein. In other patients, oxidative stress effects damage to various genes, but not including p53, resulting in overexpression of wild-type p53.

Case-Control Studies↗
PubMed1996

Point mutations characterize KEL10, the KEL3, KEL4, and KEL21 alleles, and the KEL17 and KEL11 alleles.

BACKGROUND: The Kell blood group system is complex, consisting of five sets of alleles and expressing high- and low-incidence antigens and at least 11 other independently expressed antigens. The molecular basis of two sets of alleles: KEL1 (K) and KEL2 (k) and KEL6 (Jsa) and KEL7 (Jsb) have been elucidated as single-base mutations leading to amino acid changes. The molecular basis for the KEL3 (Kpa), KEL4 (Kpb), and KEL21 (Kpc) alleles, the KEL11(Cote) and KEL17(Wka) alleles, and for KEL10 (UIa) is now reported. STUDY DESIGN AND METHODS: Genomic DNA from unrelated individuals with KEL:3,-4,-21 [Kp(a+b-c-)], KEL:-3,-4,21 [Kp(a-b-c+)], KEL:17,-11, and KEL:10 (UIa) phenotypes was amplified by polymerase chain reaction (PCR) with primers for the 19 exons of KEL. The PCR products were sequenced and compared to the DNA sequences of a common Kell system phenotype, KEL:-3,4,-21,-17,-10. Base mutations found were confirmed by restriction fragment length polymorphism analysis in which DNA of unrelated persons with similar red cell phenotypes was used. RESULTS: In all cases, single-base mutations were responsible for the expression of the various antigens. In KEL3 (Kpa), KEL4 (Kpb), and KEL21 (Kpc), point mutations at the same codon in exon 8, encoding amino acid residue 281, distinguish the three genes. KEL4 has the CGG codon for arginine, KEL3 has the TGG codon for tryptophan, and KEL21 has the CAG codon for glutamine. KEL17 has a T1025C mutation in exon 8, encoding a valine-to-alanine amino acid change at residue 302. KEL10 has an A1601T mutation in exon 13, encoding a glutamic acid-to-valine change at residue 494. In all cases, the point mutations created restriction enzyme sites, and PCR-based restriction fragment length polymorphisms confirmed that these point mutations occurred in unrelated persons with the same red cell phenotype. CONCLUSION: Single-base substitutions characterize the KEL3, KEL21, KEL17, and KEL10 genes. The allelic relationship of KEL3, KEL4, and KEL21 was confirmed because the mutations occur in the same codon, expressing different amino acids. PCR-based restriction fragment length polymorphisms can be used to distinguish genotypes.

Alanine↗
PubMed1995

Inhibition of diacylglycerol kinase by the antitumor agent calphostin C. Evidence for similarity between the active site of diacylglycerol kinase and the regulatory site of protein kinase C.

Calphostin C is an anti-tumor agent that binds to the regulatory domain of protein kinase C and inhibits the binding of phorbol dibutyrate. Recent studies suggest that there may be structural similarities between protein kinase C (PKC) and diacylglycerol kinase (DGK). Both enzymes bind diacylglycerol and phosphatidylserine, and sequencing of the 80 kDa diacylglycerol kinase shows that it contains zinc finger-like sequences, similar to those occurring in PKC. Similarities in some enzymatic properties of PKC and DGK led us to examine whether regulatory-site inhibitors of PKC also might inhibit DGK. For these studies, the membrane-bound DGK was partially purified from porcine testis membranes. Calphostin C inhibited DGK with an IC50 in the micromolar range. The inhibition of DGK by calphostin C was competitive with respect to diacylglycerol and was not affected by the presence or absence of phosphatidylserine. Other inhibitors of protein kinase C were without effect, with the exception of Adriamycin, which inhibited at millimolar concentrations. Staurosporine, which binds to the catalytic domain of protein kinase C, did not inhibit DGK. The results suggest that there are functional similarities between the substrate binding site of DGK and the regulatory site of protein kinase C.

Alkaloids↗
PubMed1995

Organization of the gene encoding the human Kell blood group protein.

Kell is one of the major blood group systems in human erythrocytes. It is a complex system containing a large number of different antigens. Previously we cloned the Kell cDNA, which was predicted to encode an integral membrane protein with 731 amino acids. Now we have isolated overlapping genomic clones and determined the exon-intron structure of the KEL gene; it spans approximately 21.5 kb with its coding sequence being organized in 19 exons that range in size from 63 bp to 288 bp. The size of introns ranges from 93 bp to approximately 6 kb. The donor and acceptor splice sites all conform to the consensus splicing sequences. Exon 1 encodes only the initiation amino acid, methionine, and contains a consensus Sp1 binding site. The single membrane spanning region of Kell protein is encoded in exon 3 and the putative zinc endopeptidase active site is in exon 16. The amino acids encoded by the 19 exons are identical to those of a person with a common Kell phenotype, as determined by RNA polymerase chain reaction of peripheral blood. Amplification of cDNA 5' ends, derived from human fetal liver, indicated three transcription initiation sites located 30, 81, and 120 bp upstream of the initiation codon. The 5' flanking region of KEL from -176 does not contain a TATA sequence, but has possible GATA-1 binding sites and has significant promoter activity when determined by chloramphenicol acetyltransferase activity in K562 cells.

Amino Acid Sequence↗
PubMed1995

Molecular basis of the Kell (K1) phenotype.

K1 (K, Kell) is a strong immunogen; its antibodies can cause severe reactions if incompatible blood is transfused and may cause hemolytic disease of the newborn in sensitized mothers. K1 is a member of the Kell blood group system, which is complex, containing over 20 different antigens. Some of the antigens are organized in allelic pairs of high and low prevalence whereas others are independently expressed. K1, which is present in 9% of the population, is antithetical to the high-prevalence K2 (k) antigen. We have determined the molecular basis of the K1/K2 polymorphism by sequencing the 19 exons of the Kell gene (KEL) of a K1/K1 person. Polymerase chain reaction was performed on genomic DNA isolated from peripheral blood and the amplified products were either directly sequenced or subcloned and sequenced. Comparisons of K1/K1 and K2/K2 DNA showed a C to T base substitution in exon 6 that predicts a threonine to methionine change at amino acid residue 193. This amino acid substitution occurs at a consensus N-glycosylation site (Asn. X. Thr) and probably prevents N-glycosylation, leading to a change in phenotype. The C to T substitution creates a Bsm I restriction enzyme site, which was tested in 42 different samples to confirm that this base change identifies the K1/K1 genotype. This test differentiates genotypes, K1/K1, K2/K2, and the K1/K2 heterozygote and should prove useful in the prenatal diagnosis of K1-related hemolytic disease of the newborn.

Amino Acid Sequence↗
PubMed1995

Long-term follow-up of patients with advanced ovarian cancer treated in randomised clinical trials.

The data from two prospective randomised phase III trials that were initiated by the West Midlands Ovarian Cancer Study Group (WMOCSG) in 1981 and 1986, recruiting 167 and 195 patients respectively, have been pooled and the survival patterns of the 362 patients treated for advanced epithelial ovarian cancer within clinical trials in the West Midlands over the 10 year period (1981-91) have been explored. All patients had histologically proven epithelial ovarian cancer and all had residual disease after primary surgery, with the majority having stage III/IV disease. The primary treatment for all patients was debulking surgery followed by platinum-based chemotherapy. Eligible patients were further randomised to undergo a second debulking operation. The main end point, survival, was assessed using Kaplan-Meier curves and the log-rank test. A Cox proportional hazards model identified performance status (P = 0.002), residual disease (P = 0.005) and albumin level (P = 0.04) as independent prognostic factors. A multivariate model to predict survival curves for patients with the best and worst prognoses was developed with predicted 5 year survival of 30% and 3% for those in the best and worst prognostic groups respectively. The identification of clinical interventions to improve outcome is an urgent matter since the prognosis for patients with advanced ovarian cancer remains poor.

Animals↗
PubMed1995

Molecular basis of the K:6,-7 [Js(a+b-)] phenotype in the Kell blood group system.

BACKGROUND: The Kell blood group system consists of at least 21 antigens. KEL6(Jsa) is a low-incidence antigen that has an antithetical relationship with the high-incidence KEL7(Jsb) antigen. The molecular basis of KEL6 that appears in less than 1.0 percent of the general population, but in up to 19.5 percent of African Americans, was unknown. STUDY DESIGN AND METHODS: Nineteen exons of the Kell gene (KEL) were amplified by polymerase chain reaction (PCR) assays of genomic DNA obtained from individuals with K:6,-7 [Js(a+b-)] phenotype. The PCR products were sequenced. A comparison was made of the sequence of the PCR products and the sequence of K:-6,7, the common phenotype. RESULTS: KEL from individuals with the K:6,-7 phenotype had two base substitutions in exon 17. One was a missense mutation (T-to-C base substitution) at nucleotide (nt) 1910, which predicts an amino acid change from leucine to proline; the other was a silent substitution (A-to-C) at nt 2019. The T-to-C substitution eliminated a restriction site for Mnl I, whereas the A-to-G substitution eliminated a Dde I site. Analyses of exon 17 in seven unrelated persons with K:6,-7 phenotype by Mnl I and Dde I enzymes showed the expected presence of restriction fragment length polymorphisms. CONCLUSION: The base substitutions T-to-C at nt 1910 and A-to-G at nt 2019 are unique to KEL6. The predicted Leu-->Pro change may disrupt the alpha-helical structure and thus form the epitope for KEL6.

Alleles↗
PubMed1995

Maternal 3;13 chromosome insertion, with severe pre-eclampsia.

A previously healthy young primigravida suffered very severe pre-eclampsia and was delivered at 32 weeks gestation. The baby was growth retarded with dysmorphic features, and died aged 4 days. Chromosome analysis of the baby revealed partial trisomy 13 resulting from recombination within a maternal insertion of part of 13q into 3p. To date, the maternal insertion has been identified in a further three members of the family and may have contributed to a number of spontaneous abortions, stillbirths and neonatal deaths in other family members. The various possibilities for recombination and malsegregation are discussed. An association between pre-eclampsia and trisomy 13 has been reported previously. To our knowledge this present paper constitutes the first report of partial trisomy for 13q occurring with pre-eclampsia.

Adult↗
PubMed1995

Flow cytometric detection of activated platelets in pregnant women prior to the development of pre-eclampsia.

Pre-eclampsia is a common complication of pregnancy, in which platelets may have an early pathogenetic role. In this prospective study a whole blood flow cytometric method has been used to detect circulating activated platelets in pregnant women prior to the development of pre-eclampsia. Activated platelets were identified by bound fibrinogen or by CD63 antigen expression. Of 121 healthy primiparous women studied at 28 weeks of pregnancy, 18 (15%) developed clinical pre-eclampsia six to thirteen weeks later. The platelets of these women showed increased fibrinogen binding ex vivo (5.1% platelets positive, compared with 3.4% in those who completed a normal pregnancy, p < 0.02), and increased CD63 antigen expression (0.73% positive compared to 0.45%, p = 0.01). In contrast, no differences between the women with different outcomes were detected at 28 weeks in platelet counts, or plasma beta-thromboglobulin levels. These findings confirm that whole blood flow cytometry is a sensitive technique for investigating platelet activation in a clinical setting and support the hypothesis that platelets have a critical role in the pathogenesis of pre-eclampsia.

Antigens, CD↗
PubMed1995

Serum squamous cell carcinoma antigen: a potential marker for benign vulval disease?

Benign vulval disease comprises a variety of disorders and can affect women of all ages. To date, the optimal management of these conditions has been uncertain and not subjected to a systemic prospective approach. It is recognized that benign vulval disease has a potential premalignant potential. Squamous cell carcinoma antigen (SCCA) has shown in different studies to be an effective means of monitoring the course of the disease in cervical carcinoma. Elevated levels of SCCA have been found in the skin. In addition, raised SCCA levels have been found in non-carcinomatous inflammatory dermatoses, and the levels observed correlated with the extent of the disease and the response to therapy. It was thought that SCCA might prove to be a useful marker for benign vulval disease, and in our pilot study the objectives were to determine if levels of SCCA are elevated in patients with that disease and to assess whether there is an association between SCCA and clinical response to treatment.

Antigens, Neoplasm↗
PubMed1994

Overexpression of any fibrinogen chain by Hep G2 cells specifically elevates the expression of the other two chains.

Earlier studies showed that overexpression of B beta fibrinogen chains, by transfection of Hep G2 cells with B beta cDNA, specifically enhanced the synthesis of all three fibrinogen chains (Roy, S. N., Mukhopadhyay, G., and Redman, C. M. (1990) J. Biol. Chem. 265, 6389-6393). To determine whether overexpression of any of the three component chains of fibrinogen affects the synthesis of the other two chains, we developed stable Hep G2 cell lines transfected with individual fibrinogen chain cDNAs. As a control, cells were also transfected with expression vector, which did not contain fibrinogen cDNA. Transfection with any fibrinogen cDNA increased the synthesis of all three fibrinogen chains but not of other plasma proteins. Hep G2 cells transfected with B beta cDNA produced 3-4-fold more fibrinogen than control cells, and cells transfected with A alpha or gamma cDNA made about 2-fold more fibrinogen. Northern blot analyses showed that levels of all 3 fibrinogen mRNAs were increased and were highest in Hep G2-B beta cells. Nuclear run-on transcription assays demonstrated that increased expression of the chains was due to increased transcriptional activity. These studies show that transcription of the three fibrinogen chains is tightly linked, and increased expression of any chain specifically leads to increased synthesis of the other two chains.

Blood Proteins↗
PubMed1994

Iron(II)/reductant(DH2)-induced activation of dioxygen for the hydroxylation and ketonization of hydrocarbons; mimics for the cytochrome P-450 hydroxylase/reductase system.

Several metal complexes [(FeII(DPAH)2 (DPAH2 = 2,6-dicarboxyl pyridine), FeII(PA)2 (PAH = picolinic acid), FeII(bpy)2(2+), FeII(OPPh3)4(2+), (Cl8TPP)FeIIIX (X = Cl, OH, SCH2Ph) [Cl8TPP = tetrakis (2,6-dichlorophenyl)porphyrin], (TPP) FeIIICl (TPP = tetraphenylporphyrin), and CuI(tpy)2+ (typ = 2,2'-6,2"-terpyridine)] in combination with one of several reductants [DH2; PhNHNHPh (mimic of dihydroflavin), PhNHNH2, ascorbic acid (H2asc), and PhCH2SH (model ligand for cysteine residue)] catalytically activate O2 (1 atm) for the hydroxylation of saturated hydrocarbons (e.g. c-C6H12-->c-C6H11OH). This chemistry closely parallels that of cytochrome P-450 proteins, and both appear to involve a Fenton-like reactive intermediate), [LxFeOOH(DH)]. With cyclohexane as the substrate the dominant product is its ketone (as well as significant amounts of its hydroperoxide). 1,4-Cyclohexadiene (with two double-allylic carbon centers) undergoes dehydrogenation to give benzene, but also yields substantial amounts of phenol via ketonization of an allylic carbon. The 1:1 FeII(bpy)2(2+)/(PhNHNH2 or H2asc), FeII(PA)2/H2asc, and (Cl8TPP)FeIIICl/PhNHNH2 combinations initiate the autoxidation of 1,4-cyclohexadiene with turnover numbers (moles of product per mole of reductant) from 71 to 26, respectively (alpha-tocophenol reduces the turnover numbers by 20-80%). With respect to aerobic biology, the present results indicate that dysfunctional transition metals (degradation products of metalloproteins) in combination with biological reductants activate O2 for reaction with organic substrates. The level of activation is similar to that for Fenton reagents and cytochrome P-450 hydroxylases. Hence, dysfunctional transition metals, reductants, and O2 are a hazardous combination within a biological matrix.

Cytochrome P-450 Enzyme System↗