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Biomedical subjects

D J Perry

Publications and source records attributed to D J Perry.

At least 55 records · Page 3Linked to original sources
PubMed1995

Factor V and antithrombin gene mutations in patients with idiopathic central retinal vein occlusion.

A number of anticoagulants are found in plasma, helping to maintain the balance between thrombosis and haemorrhage. Two of the most important are antithrombin and protein C, which inactivates factors V and VIII. Deficiencies are well-recognised predisposing factors for systemic thrombosis. To establish whether the factor V or Cambridge II antithrombin mutations were present with an increased frequency in patients with idiopathic central retinal vein occlusion (CRVO) we screened 50 such patients. DNA was isolated and the regions of the gene encoding for factor V and antithrombin were amplified by means of the polymerase chain reaction. Following digestion with restriction enzymes the products were electrophoresed in agarose gels. We identified a single patient with the factor V mutation and none with the antithrombin mutation. These findings suggest that resistance to activated protein C and antithrombin mutations does not play a major role in CRVO.

Aged↗
PubMed1995

Two novel antithrombin variants, Asn187Asp and Asn187Lys, indicate a functional role for asparagine 187.

Three unrelated families have been identified with mutations involving asparagine 187. Two of these families are asymptomatic and were identified during the screening of random blood donors, whilst the third has a history of recurrent thromboembolic disease. In two families the mutation (6460 AAC-->GAC) results in an asparagine to aspartate substitution and is associated with normal immunological levels of antithrombin but a reduction in functional activity. In the third family the mutation (6462 AAC-->AAA) results in an asparagine to lysine substitution at residue 187 and is associated with a parallel reduction in both immunological and functional antithrombin levels. Asparagine 187 is located in the middle of the F helix of antithrombin and forms the major link between the F helix and strand 3 of the A sheet. The F helix is seen to overlie the A sheet of the molecule and moves with strands 2 and 3 of this sheet as they open to allow entry of the reactive site loop to form strand 4. Substitutions of asparagine 187 are, therefore, likely to disrupt this sliding movement leading to a loss of inhibitory activity.

Adult↗
PubMed1995

Ectopic transcript analysis in human antithrombin deficiency.

Reverse transcription and amplification of total cellular RNA isolated from peripheral blood mononuclear cells has been used to identify correctly spliced transcripts for human antithrombin. We have explored the potential of this technique for identifying the underlying mutation in five individuals with a Type I deficiency and one individual with a Type II deficiency. In each case correctly spliced transcripts of the correct size were obtained, direct sequencing of which clearly demonstrated the mutation. This method simplifies the detection of mutations in cases of antithrombin deficiency and further extends the use of ectopic transcript analysis.

Antithrombin III↗
PubMed1995

Gunshot wounds to the knee.

Between 1989 and 1993, 67 fractures of the knee secondary to gunshot wounds were treated at the King/Drew Medical Center in Los Angeles, California. Based on this experience, a treatment protocol was devised. The protocol addresses the evaluation and management of neurovascular injuries, soft-tissue wounds, and fractures. These concepts are summarized in an algorithm.

Adult↗
PubMed1994

Antithrombin and its inherited deficiencies.

Human antithrombin is the major inhibitor of the coagulation serine proteases accounting for approximately 80% of the thrombin inhibitory activity of plasma. It is a member of the serpin family of serine protease inhibitors and in common with some other members of this family it undergoes a dramatic increase in its inhibitory activity in the presence of heparin and other sulphated glycosaminoglycans. Two functional domains in antithrombin are recognised, the reactive site domain which interacts with the active site serine residue of the protease and the heparin binding domain. The gene for antithrombin has been cloned and its entire nucleotide sequence determined. A deficiency or functional abnormality of antithrombin may result in an increased risk of thromboembolic disease. Such deficiencies are estimated to affect as many as 1:300 of the general population and 3 to 5% of patients with thrombotic disease. On the basis of functional and immunological antithrombin assays, antithrombin deficiency may be subdivided into Types I and II. Type I disease is due to a wide variety of heterogeneous DNA mutations whilst in Type II disease missense mutations leading to single amino acid substitutions have been identified in all cases. Clinically, Type I antithrombin deficiency is associated with recurrent thromboembolic disease whereas in Type II deficiency the risk of thrombosis is closely related to the position of the mutation within the protein. Thus, heterozygotes with mutations within the heparin binding domain of antithrombin have a relatively low risk of thrombosis compared to those with mutations at or close to the reactive site of the molecule.

Amino Acid Sequence↗
PubMed1994

Uptake of BRL 41897A, a C(7) alpha-formamido substituted cephalosporin, via the ferri-pyochelin transport system of Pseudomonas aeruginosa.

Several catechol-substituted cephalosporin antibiotics are transported into Gram-negative organisms via components of the iron uptake system. We provide evidence that the C(7) alpha-formamido substituted cephalosporin BRL 41897A enters Pseudomonas aeruginosa via the high molecular weight pyochelin transport system. No MIC could be recorded when cells were grown under iron-replete conditions, but when grown in iron-depleted succinate medium strain PAO1 the MIC was 4 mg/L. A derivative of PAO1 that is deficient in the biosynthesis of pyochelin and pyoverdin, IA1, was more resistant, having MIC of 16 mg/L. This was reduced to 4 mg/L by growing the cells in the presence of pyochelin, which specifically induces the pyochelin uptake system. Growth in the presence of pyochelin also increased the rate and extent of uptake of a radiolabelled iron-antibiotic complex. Growth in the presence of 1/8 of the MIC resulted in significant reduction in expression of the ferripyochelin receptor in the outer membrane, coupled with decreased sensitivity to the agent.

Bacterial Outer Membrane Proteins↗
PubMed1994

Prevalence of antithrombin deficiency in the healthy population.

In a cohort of 9669 blood donors we have identified 16 cases of congenital AT deficiency (1 in 600) by way of family studies and AT gene analysis. Two donors had type I AT deficiency (prevalence 0.21 per 1000; 95% CI = 0.03/1000 to 0.75/1000), their families displaying a symptomatic phenotype. 14 donors had a type II deficiency (prevalence 1.45 per 1000; 95% CI = 0.79/1000 to 2.43/1000): one recurring and three unique mutations. None of these type II deficiencies appeared to confer a high thrombotic risk despite many of the affected individuals having experienced potentially prothrombotic challenges. The high frequency of these relatively asymptomatic variants may reflect a selection bias in the study population. However, their existence should not only add to our understanding of structure-function relationships of AT but may also influence our management of asymptomatic deficient individuals identified in epidemiological or presurgical screening programmes.

Adult↗
PubMed1994

Thromboembolic disease due to thermolabile conformational changes of antithrombin Rouen-VI (187 Asn-->Asp)

A new variant of antithrombin (Rouen-VI, 187 Asn-->Asp) with increased heparin affinity was shown to have normal inhibitory activity which decreased slowly at 4 degrees C and rapidly at 41 degrees C. On electrophoresis the freshly isolated variant had an anodal shift relative to native antithrombin due to the mutation. A further anodal transition occurred after either prolonged storage at 4 degrees C or incubation at 41 degrees C due to the formation of a new inactive uncleaved component with properties characteristic of L-form (latent) antithrombin. At the same time, polymerization also occurred with a predominance of di-, tri-, and tetra-mers. These findings fit with the observed mutation of the conserved asparagine (187) in the F-helix destabilizing the underlying A-sheet of the molecule. Evidence of A-sheet perturbation is provided by the increased rate of peptide insertion into the A-sheet and by the decreased vulnerability of the reactive loop to proteolysis. The spontaneous formation of both L-antithrombin and polymers is consistent with our crystal structure of intact antithrombin where L-form and active antithrombin are linked together as dimers. The nature of this linkage favors a mechanism of polymerization whereby the opening of the A-sheet, to give incorporation of the reactive center loop, is accompanied by the bonding of the loop of one molecule to the C-sheet of the next. The accelerated lability of antithrombin Rouen-VI at 41 versus 37 degrees C provides an explanation for the clinical observation that episodes of thrombosis were preceded by unrelated pyrexias.

Adult↗
PubMed1994

Models of fast-electron penetration.

We introduce multiple scattering models of charged-particle penetration which are based on the previous analyses of Yang and Perry (Radiat. Res. 115, 26-43, 1988). Our development removes the main limitations of the Fermi-Eyges approach while retaining its considerable potential as a theory which is useful for applied work. We illustrate key predictions with sample calculations that are of particular interest in therapeutic applications, 5-20 MeV electrons incident on water.

Electrons↗
PubMed1992

Insertions/deletions in the antithrombin gene: 3 mutations associated with non-expression.

We have investigated the molecular basis of antithrombin deficiency in 3 individuals, 2 of whom had a proven family history of thromboembolic disease. An approximate 50% reduction in functional and immunologic levels of antithrombin was detected in plasma from the propositi indicating an allelic deficiency of antithrombin. In each case direct sequencing of amplified DNA revealed a novel mutation involving single bases: two being insertions, of a T in codon 48 and an A in codon 208, and the third being the deletion of an A in codon 370. The three mutations, which were confirmed by cloning and sequencing the normal and variant alleles, all caused frameshifts leading to premature termination of protein translation. In no case could a truncated antithrombin be detected in plasma from the propositus suggesting either that it fails to be secreted, or is rapidly degraded.

Adult↗
PubMed1992

Hydrolink gels: a rapid and simple approach to the detection of DNA mutations in thromboembolic disease.

AIMS: To develop a simple and rapid technique for detecting DNA mutations based on the polymerase chain reaction, followed by electrophoresis, in a novel polymer--Hydrolink D5000--specifically designed to separate double stranded DNA fragments. METHODS: Eleven subjects with previously characterised mutations within the antithrombin gene (including single base pair mutations and insertions) and three normal controls were studied. DNA was amplified and one sixth of the PCR product electrophoresed in a 20 cm x 20 cm x 1 mm 100% Hydrolink D5000 gel for two to six hours, followed by staining in ethidium bromide for 20 minutes. The gel was then visualised under ultraviolet light. RESULTS: After amplification and electrophoresis a single additional band was observed in five out of nine variants in which the mutations involved a single base pair substitution, while two additional bands were seen in four out of nine mutants which arose as a result of a single base pair insertion. No abnormality was detected in two known variants. CONCLUSION: This method provides a simple and rapid approach to the screening and detection of mutations at the DNA level which does not involve the use of either toxic reagents or radioisotopes. It may also provide evidence about the type of mutation.

Antithrombins↗
PubMed1992

PET evaluation of soft-tissue masses with fluorine-18 fluoro-2-deoxy-D-glucose.

Positron emission tomography (PET) with fluorine-18 fluoro-2-deoxy-D-glucose (FDG) was performed in 19 patients referred for clinical evaluation of soft-tissue masses. These patients had 20 different lesions and had been evaluated previously with computed tomography (CT) and/or magnetic resonance (MR) imaging. The diagnoses were subsequently confirmed with open biopsy or excision (19 lesions) or by clinical and radiographic follow-up (one lesion). Semiquantitative assessment of FDG accumulation (differential uptake ratio) within the suspected tumor helped correctly separate the 10 malignant tumors from the 10 benign lesions. In contrast, a simple ratio of FDG uptake within the suspected tumor to that within comparable normal soft tissue was less successful in helping make this distinction, with overlap in 12 of the 20 cases. Careful comparison with findings from other available imaging studies is essential for accurate interpretation of PET studies of soft-tissue masses, but in many cases, PET may be a useful adjunct in the preoperative evaluation of suspected soft-tissue tumors, yielding valuable information that is not provided with CT or MR imaging.

Deoxyglucose↗
PubMed1991

Antithrombin III: a database of mutations.

Elucidation of the molecular defects responsible for antithrombin III deficiency is proceeding rapidly. In order that a record is kept of the new and duplicated mutations that are found, we have compiled a database that we plan to update annually. In this, the first report of the database, we list 6 antithrombin III locus sequence polymorphisms and 94 recorded mutations causing functional deficiency of the protein, 38 of which are novel. As is the case with mutations affecting other protein genes, most mutations of antithrombin III involve a CG to TG or CA change.

Antithrombin III↗
PubMed1991

Antithrombin Cambridge II, 384 Ala to Ser. Further evidence of the role of the reactive centre loop in the inhibitory function of the serpins.

Four unrelated individuals have been identified with an identical antithrombin variant, associated in one of them with episodes of recurrent venous thromboses. In each case, the plasma antithrombin concentration was normal and the only function abnormality was a minor but consistent decrease in the heparin-induced thrombin inhibition suggesting a mutation at or near the reactive centre of the molecule. Amplification and direct sequencing of exon 6 showed a G----T mutation at nucleotide 1246, which corresponds to a substitution of a serine for an alanine at residue 384. This is one of a series of conserved alanines that form the stalk to the reactive centre loop. The observed changes in this variant are compatible with recent structural studies that infer that mobility of this stalk with partial re-entry into the A-sheet of the molecule is necessary for optimal inhibitory activity.

Aged↗
PubMed1991

Chemotherapy with 5-fluorouracil (5-FU) and cisplatin or 5-FU, cisplatin, and vinblastine for advanced non-small cell lung cancer. A randomized phase II study of the cancer and leukemia group B.

Two hundred forty-seven patients with previously untreated nonresectable non-small cell lung cancer (NSCLC) were entered in a prospective, randomized Phase II trial. Response assessment was possible in 232 patients, and 237 patients were evaluable for survival. Thirteen partial responses (11%) and 5 regressions (4%) of evaluable disease were obtained for the 116 patients treated with 5-fluorouracil (5-FU) and cisplatin (C) (95% confidence interval [CI], 8.5% to 21.5%). The median time to progression was 2.2 months and the median survival time was 4.6 months for 5-FU plus C. Twenty-three partial responses (20%) and 4 regressions (3%) of evaluable disease were obtained for the 116 patients treated with 5-FU, C, and vinblastine (V) (95% CI, 15.3% to 30.7%). The median time to progression was 2.8 months and the median survival time was 5.6 months for 5-FU, C, and V. The 5-FU and C doses were equivalent in the two treatment regimens. Sixteen of 85 patients (19%) with a performance status of 0 and 18 of 103 patients (17%) with a performance status of 1 responded, whereas only 2 of 44 patients (5%) with a performance status of 2 or greater responded (P = 0.009). Patients who had received locoregional radiation therapy had a lower overall response rate then those in the no prior radiation therapy group (P = 0.028). The median survival time for patients with a performance status of 0 or 1 was 6.3 months compared with 1.9 months for patients with a performance status of 2 or greater (P less than 0.001). Performance status also appeared to be a significant factor for time to progression. More frequent and severe leukopenia, fever, genitourinary (GU) toxicity, and pulmonary toxicity was reported with 5-FU, C, and V. There were three treatment-related deaths with 5-FU, C, and V and one treatment-related death with 5-FU plus C. Grade III/VI myelotoxicity was not influenced by prior radiation therapy or performance status. Neither regimen is active enough to be considered as standard therapy for advanced NSCLC.

Adenocarcinoma↗
PubMed1991

Investigation of copper-PTSM as a PET tracer for tumor blood flow.

Copper-PTSM has been shown in previous studies to act as a fluid microsphere and to be useful in quantitating blood flow in brain, myocardium, and kidneys. In this study we have evaluated this agent as a PET tumor blood flow agent. 64Cu- or 67Cu-labeled Cu-PTSM was administered (i.v.) to Golden Syrian hamsters with colorectal carcinoma cell implants (GW39). One minute prior to sacrifice (10-60 min after Cu-PTSM was administered) 125I-iodoantipyrine (125I-IAP), an agent known to measure tumor blood flow, was administered intravenously by a 3-stage, 1 min ramp infusion. Following sacrifice, samples of tumor and brain were removed (within 40s) and the tumor and brain levels of Cu-PTSM and iodoantipyrine determined. Since the brain uptake of both Cu-PTSM and IAP is perfusion rate limited, the brain was used as a reference organ to normalize tumor levels of the two tracers. The plot of Cu-PTSM versus 125I-IAP tumor/brain ratios showed a good linear correlation (r value of 0.97), suggesting that Cu-PTSM could be used to quantify tumor blood flow. Since the mechanism of Cu-PTSM trapping is likely to be due to glutathione levels in the tissue, and because tumor tissue glutathione levels might vary, the temporal uptake of Cu-PTSM was investigated by PET imaging both the tumor-bearing hamsters and approximately 300 g Copenhagen rats bearing R3227 prostate tumors. The tumors were clearly visualized and the retained copper radioactivity in the tumor was constant over the 30 min imaging period.

Animals↗
PubMed1991

The incidence of dysfunctional antithrombin variants: four cases in 210 patients with thromboembolic disease.

210 patients, with a history of venous thrombosis, have undergone prothrombotic investigations. In nine cases a consistent deficiency of antithrombin was identified. In five there was a reduction in the plasma antigenic concentration of antithrombin and in a further four cases deficiency was due to the presence of a dysfunctional antithrombin variant. The variants have all been characterized by DNA analysis and in three the mutations have been confirmed by peptide sequencing (antithrombin Basel (41 Pro to Leu). Hamilton (382 Ala to Thr). Cambridge I (384 Ala to Pro) and Cambridge II (384 Ala to Ser). The incidence of antithrombin deficiency in patients with a history of venous thrombosis has previously been quoted at between 2% and 3%: there is no published data available on the incidence of antithrombin variants. In our series 5% of patients who presented before the age of 40 years had antithrombin deficiency, and 2% of the total number of patients investigated had a dysfunctional variant. Our figures indicate that a significant number of cases of antithrombin deficiency are due to dysfunctional variants and that the true incidence of antithrombin deficiency in patients with a history of venous thrombosis is in the order of 5%.

Adult↗