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Biomedical subjects

E G Tuddenham

Publications and source records attributed to E G Tuddenham.

At least 109 records · Page 6Linked to original sources
PubMed1990

The regulation of tissue factor mRNA in human endothelial cells in response to endotoxin or phorbol ester.

Tissue factor (TF) is the membrane-bound glycoprotein whose cofactor activity with factor VIIa causes activation of the extrinsic pathway of coagulation. The transition of endothelium to a procoagulant state by agents such as bacterial lipopolysaccharide (LPS) is the result of TF expression by these cells. The mechanism of TF induction in human umbilical vein endothelial cells (HUVEC) was investigated in response to LPS and phorbol 12-myristate 13-O-acetate (PMA). Northern blot analysis of total RNA from HUVEC showed a rapid rise in TF mRNA levels which was maximal at 2 h and had fallen to low levels by 6 h following both LPS (10 micrograms/ml) and PMA (10 ng/ml) stimulation. Nuclear-run on experiments showed at most a 2-fold increase in transcription of the TF gene following LPS stimulation but a 10-fold increase following PMA stimulation. In addition 24-h pre-incubation with PMA desensitized HUVEC to further PMA exposure, but caused no alteration in the response to LPS. Cycloheximide (10 micrograms/ml) alone caused induction of TF mRNA. Treatment of cells previously exposed to LPS for 1 or 4 h with actinomycin D indicated a 12-fold difference in the TF mRNA half-life. Therefore the rapid accumulation of TF mRNA in HUVEC stimulated by LPS is largely a result of an increase in mRNA stability rather than an increased rate of transcription of the gene.

Cell Nucleus↗
PubMed1990

Purification and characterization of factor VIII 372-Cys: a hypofunctional cofactor from a patient with moderately severe hemophilia A.

We have purified factor VIII from a patient with moderately severe hemophilia A (FVIII, 4 U/dL; FVIII:Ag, 110 U/dL) and subjected the protein to Western blot analysis after time course activation with thrombin. The cross reacting material-positive (CRM+) FVIII has the normal distribution of heavy and light chains before thrombin activation, and, after incubation with the enzyme, appropriate cleavages are made at positions 740 and 1689. However, the normal thrombin cleavage at position 372 in the heavy chain of this molecule does not occur. This result is consistent with the demonstration in the patient's leukocyte DNA of a C to T transition in codon 372, leading to the substitution of a cysteine for an arginine residue at the heavy chain internal cleavage site. The severely impaired functional activity of this molecule confirms that the heavy chain of FVIII must be proteolysed in order to effect full cofactor activation in vivo. However, a threefold activation was detected when this protein was incubated with thrombin. No evidence of thrombin-mediated cleavage at position 336 in the heavy chain was detected, in contrast to the variant recombinant B domainless-molecule, FVIII 372-Ile, described by Pittman and Kaufman (Proc Natl Acad Sci USA 85:2429, 1988). Using gel permeation studies of the FVIII/von Willebrand factor (vWF) complex before and after thrombin activation, we have demonstrated that the 40 Kd A2 domain of wild type FVIII dissociates from vWF after cleavage by the enzyme. In contrast, incomplete dissociation was detected in the case of FVIII 372-Cys. We conclude that the functional defect in FVIII 372-Cys is a consequence of the resistance to proteolysis of the internal scissile bond in the heavy chain.

Amino Acid Sequence↗
PubMed1990

CRM+ haemophilia A due to a missense mutation (372----Cys) at the internal heavy chain thrombin cleavage site.

We have used the polymerase chain reaction (PCR) and differential oligonucleotide melting to screen for mutations in selected CpG dinucleotides in the factor VIII genes of haemophilia A patients. By this means we have identified and confirmed by sequencing a novel point mutation in codon 372 (CGC) of the factor VIII gene of a moderately severe CRM+ haemophiliac. The first C of this codon has been substituted by T resulting in the non-conservative substitution of cysteine for arginine at an essential thrombin cleavage site in factor VIII. Analysis of three intragenic restriction fragment length polymorphisms was uninformative in the patient's family. However, DNA analysis for the specific mutation shows one sister and the patient's mother to be carriers, and the other sister to be normal. This DNA analysis confirmed the results of phenotype analysis by factor VIII coagulant to von Willebrand factor antigen ratios for the females at risk. The two carrier females had low factor VIII coagulant activity and excess VIII antigen as predicted but the non-carrier sister also had anomalously high VIII antigen in her plasma. When feasible, mutation specific DNA analysis is able to resolve the difficulties posed by variable phenotype data and unknown level of mutation in sporadic haemophilia A.

Base Sequence↗
PubMed1989

Homozygous protein C deficiency with delayed onset of symptoms at 7 to 10 months.

We report an inbred family with two cases of homozygous protein C deficiency and review 11 other such cases. Both patients presented in the second half of their first year of life with recurrent rapidly disappearing ecchymotic skin lesions, disseminated intravascular coagulation, and venous thrombosis. Successful treatment has been achieved by frequent infusions of plasma or prothrombin complex then maintained with Warfarin. Homozygous recessive protein C deficiency usually presents in the neonatal period with purpura fulminans. Two cases have been described elsewhere which presented in the second decade of life with milder symptoms. The present cases appear to be intermediate in time of presentation and severity of symptoms. We also review the distinction that is now evident between recessive and dominant protein C deficiency.

Consanguinity↗
PubMed1989

Biopsy of human preimplantation embryos and sexing by DNA amplification.

A single cell was removed, through a hole made in the zona pellucida, from each of 30 human embryos at the 6-10 cell cleavage stage three days after in-vitro fertilisation. A normal proportion of the embryos (37%) developed to the blastocyst stage by day six in culture and 6 hatched from the zona. Each male embryo was sexed from the DNA by amplification of a repeated sequence specific for the Y chromosome. In 15 embryos with the normal two pronuclei the sex was determined also by in-situ hybridisation with a Y specific probe or fluorescent chromosome staining to detect metaphase Y chromosomes; the results of Y specific amplification were confirmed. This approach may be valuable for couples at risk of transmitting X-linked disease.

Biopsy↗
PubMed1989

von Willebrand factor and its disorders: an overview of recent molecular studies.

von Willebrand factor (vWF) is a plasma protein with multiple functions in haemostasis. The vWF gene, located on chromosome 12p2.1, encodes a primary gene product of 2813 amino acids. Post-translational modification, assembly and secretion of vWF are highly complex. The pro vWF promoter is covalently linked by intermolecular disulphide bonds to form a dimer of MW approximately 440 kDa. This then polymerises to form multimers ranging in MW from 1-20 x 10(6). Simultaneously the pro piece of vWF is cleaved, releasing a 741 amino acid peptide known as vW Ag II from the polymerised protomers. Two distinct secretion pathways are found in the endothelial cell, a regulated pathway with storage in Weibl-Palade bodies and a constitutive pathway. Platelets store vWF in their alpha-granules. Mature vWF participates in platelet adhesion, spreading and aggregation and is a carrier of factor VIII, protecting the latter from degradation. Disorders of vWF are highly diverse. At least 20 subtypes of von Willebrand's disease have been described to date, based on features of the vWF present in or absent from patients plasma and platelets. Some patients have reduced amounts of apparently normal vWF whilst others have clearly abnormal vWF with aberrant structure and function. Rare patients virtually or completely lack vWF. The genetic and structural basis of some of these abnormalities is just beginning to emerge. This article outlines the molecular biology and physiology of vWF, and reviews some recent progress on the molecular pathology and genetics of von Willebrand's disease.

Animals↗
PubMed1989

Response to desmopressin in type IID von Willebrand's disease.

Dominant transmission of a variant of von Willebrand's disease (vWD) with aberrant polymerization of von Willebrand factor (vWF) has been identified in a Scottish family. Multimer analysis of plasma vWF from the propositus and her father revealed an identical pattern to that previously reported in families designated as type IID vWD. There is loss of the larger multimers and presence of an intermediate subsidiary band not seen in normal subjects or other vWD variants. Platelet/vWF interaction induced by ristocetin is not enhanced in these cases and the platelet vWF shows the same aberrant multimer pattern as plasma vWF. DDAVP infusion in two affected members of the Scottish family and in one of the index cases produced a rise in plasma vWF antigen and factor VIII. Higher molecular weight vWF multimers appeared transiently after infusion of desmopressin (1-deamino-8-D-arginine vasopressin, abbreviated DDAVP) coincident with shortening of the bleeding time. The platelet counts did not change after the DDAVP infusions. DDAVP should be considered for management of bleeding in this variant of von Willebrand's disease.

Adult↗
PubMed1989

Purification and characterization of factor VIII 1,689-Cys: a nonfunctional cofactor occurring in a patient with severe hemophilia A.

We have purified the factor VIII from a CRM+ Hemophilia A plasma (90 U/dL VIII:Ag but 0 U/dL VIII:C) and analyzed the protein before and after thrombin activation by Western blotting with monoclonal antibodies (MoAbs). Normal or patient citrated plasma was ultracentrifuged, cryo-ethanol-precipitated and chromatographed on Sepharose 6B. The void volume fractions were reduced and subjected to ion exchange chromatography yielding material of specific activity approximately 1,000 U/mg protein (VIII:C or VIII:Ag). Factor VIII purified in this way from normal plasma is fully activatable by thrombin with proteolytic fragmentation as previously described by F. Rotblat et al (Biochemistry 24: 4294, 1985). Factor VIII 1,689-Cys has the normal distribution of factor VIII light and heavy chains prior to thrombin activation. After exposure to thrombin the heavy chain polypeptides were fully proteolysed but the light chain was totally resistant to cleavage. This is consistent with the demonstration in the patient's leucocyte DNA of a C to T transition in codon 1,689 converting Arg to Cys at the light chain thrombin cleavage site as previously described by J. Gitschier et al (Blood 72:1022, 1988). Uncleaved light chain of Factor VIII 1,689-Cys is not released from von Willebrand factor (vWF) by thrombin, but this is not the sole cause of the functional defect since the protein purified free of vWF has no coagulant activity. We conclude that the functional defect in factor VIII 1,689-Cys is a consequence of failure to release the acidic peptide from the light chain upon thrombin activation.

Antibodies, Monoclonal↗
PubMed1988

Characterization of a murine homeo box gene, Hox-2.6, related to the Drosophila Deformed gene.

The Hox-2 locus on chromosome 11 represents one of the major clusters of homeo-box-containing genes in the mouse. We have identified two new members (Hox-2.6 and Hox-2.7), which form part of this cluster of seven linked genes, and it appears that the Hox-2 locus is related by duplication and divergence to at least one other mouse homeo box cluster, Hox-1. The Hox-2.6 gene encodes a predicted protein of 250 amino acids, which displays extensive similarity in multiple regions to certain mouse, human, Xenopus, and zebra fish homeo domain proteins. The Drosophila Deformed (Dfd) gene also shares these same regions of similarity, and based on this sequence conservation, we suggest that Hox-2.6 forms part of a vertebrate 'Dfd-like' family. Hox-2.6 is expressed in fetal and adult tissues and is modulated during the differentiation of F9 teratocarcinoma stem cells. In situ hybridization analysis of mouse embryos shows that the Hox-2.6 is expressed in ectodermal derivatives: spinal cord, hindbrain, dorsal root ganglia, and the Xth cranial ganglia. In the central nervous system, expression is observed in the most posterior parts of the spinal cord, with the anterior limit residing in a region of the hindbrain and no expression in the mid- or forebrain. In mesodermal structures, Hox-2.6 is expressed in the kidney, the mesenchyme of the stomach and lung, and the longitudinal muscle layer of the gut. Expression has not been observed in derivatives of embryonic endoderm. The patterns of Hox-2.6 expression in both mesoderm and ectoderm are spatially restricted and may reflect a role for the gene in the response to or establishment of positional cues in the embryo.

Amino Acid Sequence↗
PubMed1988

Monoclonal antibodies to human factor VII: a one step immunoradiometric assay for VII:Ag.

Three mouse monoclonal antibodies (RFF-VII/1, RFF-VII/2, and RFF-VII/3) which bind specifically to different epitopes on human factor VII antigen were raised. Two of the antibodies, RFF-VII/1 and RFF-VII/2, bound strongly to factor VII antigen (VII:Ag), but only RFF-VII/1 and RFF-VII/3 were potent inhibitors of factor VII coagulation activity (VII:C). RFF-VII/1 and RFF-VII/2 were used in a one step, double monoclonal immunoradiometric assay for VII:Ag. This was highly reproducible and detected as little as 0.05U/dl VII:Ag. Values for VII:Ag obtained for plasma samples from normal subjects (n = 20), patients with liver disease (n = 20), patients treated with warfarin (n = 20), and those congenitally deficient in factor VII (n = 7) correlated very well (r = 0.96) with data obtained in a radioimmunoassay using polyclonal rabbit antiserum to factor VII. This simple and sensitive monoclonal antibody based assay offers a convenient method for the detection of VII:Ag in various disease states.

Animals↗
PubMed1988

Monoclonal antibodies to human factor VII: production of immunodepleted plasma for VII:C assays.

A high affinity monoclonal antibody to factor VII (RFF-VII/1), coupled to sepharose, was used to immunodeplete factor VII from normal plasma. The plasma could be used as a substrate in a one stage coagulation assay and performed as well as, or better than, commercially available factor VII deficient plasma or plasma from congenitally deficient factor VII patients. Plasma from normal donors (n = 20), patients with liver disease (n = 20), and patients receiving warfarin (n = 20), or congenitally factor VII deficient patients (n = 7) was assayed for VII:C concentration in a one stage coagulation assay. The concentration of VII:C detected with the immunodepleted plasma substrate was comparable in all cases with that seen with a commercially available substrate (r = 0.95).

Antibodies, Monoclonal↗
PubMed1988

Mutations of factor VIII cleavage sites in hemophilia A.

Hemophilia A is caused by a defect in coagulation factor VIII, a protein that undergoes extensive proteolysis during its activation and inactivation. To determine whether some cases of hemophilia are caused by mutations in important cleavage sites, we screened patient DNA samples for mutations in these sites by a two-step process. Regions of interest were amplified from genomic DNA by repeated rounds of primer-directed DNA synthesis. The amplified DNAs were then screened for mutations by discriminant hybridization using oligonucleotide probes. Two cleavage site mutations were found in a survey of 215 patients. A nonsense mutation in the activated protein C cleavage site at amino acid 336 was discovered in a patient with severe hemophilia. In another severely affected patient, a mis-sense mutation results in a substitution of cysteine for arginine in the thrombin activation site at amino acid 1689. This defect is associated with no detectable factor VIII activity, but with normal levels of factor VIII antigen. The severe hemophilia in this patient was sporadic; analysis of the mother suggested that the mutation originated in her gametes or during her embryogenesis. The results demonstrate that this approach can be used to identify factor VIII gene mutations in regions of the molecule known to be important for function.

Amino Acids↗
PubMed1987

Identification of six functional clotting factor VIII:C epitopes by analysis of cross-reactive public idiotypes in murine monoclonal VIII:C inhibitors.

Six factor VIII:C epitopes which can elicit clotting factor inhibitory antibodies were demonstrated by analysis of public idiotypes of murine monoclonal anti-VIII:C inhibitors. Anti-idiotypic immunoradiometric assays were developed with rabbit antibodies to murine VIII:C inhibitors: Synbiotics, Hybritech, C7F7 and RFF-VIIIC/8. Crossreactions among 10 murine monoclonal antibodies (MoAbs) to VIII:C were tested, showing 6 functional and immunospecific VIII:C epitopes. One epitope on the C-terminal, 80,000 dalton fragment of VIII:C was identified with cross-reaction among 3 MoAbs (Synbiotics, Chemicon, and IB3). Another unique site on this same fragment was recognized with C7F7. Two MoAbs (RFF-VIIIC/6 and RFF-VIIIC/8) defined another site with cross-reactive idiotypes on the N-terminal, 90,000 dalton fragment. Hybritech MoAb identified a fourth functionally distinct site to which no other cross-reacting MoAbs bound. A fifth functional locus was defined with RFF-VIIIC/2 which reacted with an N-terminal site (distinct from the RFF-VIIIC/6 X RFF-VIIIC/8-defined site). A sixth functional locus was recognized with RFF-VIIIC/5 which reacted with a C-terminal site (distinct from the Synbiotics/Chemicon/IB3-defined site but possibly near the C7F7-defined site). RFF-VIIIC/10 identified a non-functional locus on the middle region of VIII:C. These MoAb-based assays resolve six sites to which high-titered inhibitors are directed and offer a path to further study the immunoregulation of human anti-VIII:C inhibitors.

Animals↗
PubMed1987

Production of factor VIII deficient plasma by immunodepletion using three monoclonal antibodies.

Factor VIII deficient plasma was made from pooled, HIV antibody and hepatitis B antigen screened, normal human plasma by cryoprecipitation and immuno-depletion, using three different monoclonal antibodies bound to Sepharose columns, in series. These monoclonal antibodies are specific respectively for von Willebrand factor, factor VIII heavy chain and factor VIII light chain. The immunodepleted plasma contained less than 0.002 u/ml factor VIII coagulation activity (VIII:C) less than 0.0001 u/ml von Willebrand factor antigen and 1-2 g/l fibrinogen, while the levels of other clotting factors were unchanged. This immunodepleted plasma was compared with commercial factor VIII deficient plasma obtained from a severe haemophilia A patient as substrate in the one-stage factor VIII assay. Plasmas obtained from 20 normal subjects and 28 patients with von Willebrand's disease or haemophilia A were assayed for VIII:C using the two substrates. The results were very highly correlated (r = 0.96). The columns have high capacity and can be regenerated at least 10 times. Large-scale production of a substrate for factor VIII assays free of virus contamination is now feasible.

Antibodies, Monoclonal↗