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Biomedical subjects

K B Thomas

Publications and source records attributed to K B Thomas.

At least 37 records · Page 2Linked to original sources
PubMed1990

Arylalkylamine (serotonin) N-acetyltransferase assay using high-performance liquid chromatography with fluorescence or electrochemical detection of N-acetyltryptamine.

A sensitive, rapid, and economical method has been developed for determination of serotonin N-acetyltransferase activity from a variety of enzyme sources. The assay is based upon separation and detection of N-acetyltryptamine formed from tryptamine and acetyl coenzyme A, by means of high-performance liquid chromatography with either electrochemical or fluorometric detection. The limit of sensitivity with both detection methods is less than 20 pmol of N-acetyltryptamine formed per sample. A method for synthesis of N-acetyltryptamine, used as an external standard in the assay, is described.

Acetyltransferases↗
PubMed1989

Changes in the cofactor binding domain of bovine striatal tyrosine hydroxylase at physiological pH upon cAMP-dependent phosphorylation mapped with tetrahydrobiopterin analogues.

The structure of the cofactor binding domain of tyrosine hydroxylase (TH) was examined at physiological pH by determining kinetic parameters of (R)-tetrahydrobiopterin [(R)-BH4] and a series of tetrahydropterin (PH4) derivatives (6-R1-6-R2-PH4: R1 = H and R2 = methyl, hydroxymethyl, ethyl, methoxymethyl, phenyl, and cyclohexyl; R1 = methyl and R2 = methyl, ethyl, propyl, phenyl, and benzyl). A minimally purified TH preparation that was not specifically phosphorylated (designated as "unphosphorylated") was compared with enzyme phosphorylated with cAMP-dependent protein kinase. The Km for tyrosine with most tetrahydropterin analogues ranged between 20 and 60 microM with little decrease upon phosphorylation. Two exceptions were an unusually low Km of 7 microM with 6-ethyl-PH4 and a high Km of 120 microM with 6-phenyl-6-methyl-PH4, both with phosphorylated TH. Tyrosine substrate inhibition was elicited only with (R)-BH4 and 6-hydroxymethyl-PH4. With unphosphorylated TH (with the exception of 6-benzyl-6-methyl-PH4, Km = 4 mM) an inverse correlation between cofactor Km and side-chain hydrophobicity was observed ranging from a high with (R)-BH4 (5 mM) to a low with 6-cyclohexyl-PH4 (0.3 mM). An 8-fold span of Vmax was seen overall. Phosphorylation caused a 0.6-4-fold increase in Vmax and a 35-2000-fold decrease in Km for cofactor, ranging from a high of 60 microM with 6-methyl-PH4 to a low of 0.6 microM with 6-cyclohexyl-PH4. A correlation of the size of the hydrocarbon component of the side chain with affinity is strongly evident with phosphorylated TH, but in contrast to unphosphorylated enzyme, the hydroxyl groups in hydroxymethyl-PH4 (20 microM) and (R)-BH4 (3 microM) decrease Km in comparison to that of 6-methyl-PH4. Although 6,6-disubstituted analogues were found with affinities near that of (R)-BH4 (e.g., 6-propyl-6-methyl-PH4, 4 microM), they were frequently more loosely associated with phosphorylated TH than their monosubstituted counterparts (6-phenyl-PH4, 0.8 microM; cf. 6-phenyl-6-methyl-PH4, 8 microM). A model of the cofactor side-chain binding domain is proposed in which a limited region of nonpolar protein residue(s) capable of van der Waals contact with the hydrocarbon backbone of the (R)-BH4 dihydroxypropyl group is opposite to a recognition site for hydroxyl(s). Although interaction with either the hydrophilic or hydrophobic regions of unphosphorylated tyrosine hydroxylase is possible, phosphorylation by cAMP-dependent protein kinase appears to optimize the simultaneous operation of both forces.

Animals↗
PubMed1987

General practice consultations: is there any point in being positive?

A group of 200 patients who presented in general practice with symptoms but no abnormal physical signs and in whom no definite diagnosis was made were randomly selected for one of four consultations: a consultation conducted in a "positive manner," with and without treatment, and a consultation conducted in a "non-positive manner," called a negative consultation, with and without treatment. Two weeks after consultation there was a significant difference in patient satisfaction between the positive and negative groups but not between the treated and untreated groups. Similarly, 64% of those receiving a positive consultation got better, compared with 39% of those who received a negative consultation (p = 0.001) and 53% of those treated got better compared with 50% of those not treated (p = 0.5).

Attitude of Health Personnel↗
PubMed1987

Analysis of platelet von Willebrand factor antigen.

Platelet lysates were obtained from suspensions of normal washed platelets by freeze-thawing or Triton X-100 lysis. The resultant platelet lysates contained 0.34 +/- 0.15 U/10(9) platelets (n = 8) of von Willebrand factor antigen (vWf:Ag) as determined by radioelectroimmunoassay using a monospecific antibody to vWf:Ag. The vWf:Ag level was higher in platelet lysates prepared from freshly drawn blood than from outdated platelet packs. Platelet lysates from patients with severe von Willebrand's disease type I (n = 2) did not contain detectable vWf:Ag. When normal platelet lysates were analyzed by radiocrossed immunoelectrophoresis in agarose using a monospecific polyclonal antibody to plasma vWf:Ag, two immunochemically identical precipitin peaks were seen. One of the platelet vWf:Ag peaks corresponded in its electrophoretic mobility to plasma vWf:Ag, while the other peak, i.e. platelet vWf:Ag-peak II, migrated to a more anodal position. The presence of the platelet vWf:Ag-peak II suggests structural differences between plasma and platelet vWf:Ag and illustrates previously unrecognized heterogeneity of platelet vWf:Ag.

Antigens↗
PubMed1985

Use of heat-treated clotting-factor concentrates in patients with haemophilia and a high exposure to HTLV-III.

In a group of 126 Australian patients with haemophilia, who were receiving lyophilized clotting-factor concentrates prepared from locally collected plasma, a high prevalence of antibody to human T-cell lymphotropic virus III (HTLV-III) was demonstrated in those with severe disease. Patients with moderate or mild disease had a much lower prevalence of HTLV-III antibody. After heat treatment of lyophilized factor VIII and factor IX concentrates (60 degrees C for 72 hours) to inactivate the virus, the losses of activity of an intermediate-purity and of a fibrinogen-poor factor VIII concentrate, and of the coagulant activity of a factor IX concentrate, were within acceptable limits. The solubility of the intermediate-purity factor VIII concentrate was markedly decreased; the fibrinogen-poor factor VIII concentrate and the factor IX concentrate were readily soluble. In-vivo recovery and survival of heated concentrates were equivalent to those of the unheated products, and they were effective in the treatment of spontaneous and traumatic haemorrhages.

Acquired Immunodeficiency Syndrome↗
PubMed1983

Fast migrating protein, immunochemically related to human factor VIII, studied by crossed immunoelectrophoresis in agarose.

A fast migrating protein (FMP) was detected by agarose radio-crossed immunoelectrophoresis, in addition to factor VIII antigen (VIII:RAg), using antiserum to human factor VIII (FVIII). FMP had partial immunochemical identity with FVIII, migrated as an alpha-protein, and was distinct from alpha-2 macroglobulin, fibronectin or IgM. FMP was precipitated by concanavalin A and was separable from the bulk of VIII:RAg by ammonium sulphate fractionation. A significant amount of FMP was seen in normal serum (n = 12), plasma from patients with: (a) disseminated intravascular coagulation (n = 12) and (b) severe haemophilia A (n = 6). Trace amounts of FMP were observed in plasma from normal donors (n = 12), but neither VIII:ARg nor FMP was detectable in the plasma or serum from patients with severe von Willebrand's disease (n = 3). Freshly prepared cryoprecipitate contained trace amounts of FMP, similar to normal plasma, but increased levels were observed in antihaemophilic factor concentrates prepared for patient use. Significant levels of FMP were also seen in cryoprecipitate after storage at 4 degrees C for 7 d and this generation of FMP was diminished by the addition of protease inhibitors. The presence of significant levels of FMP in situations where proteolytic enzymes may be activated and inhibition of its generation by protease inhibitors, suggest that this protein is produced by proteolytic action of enzyme(s) on the FVIII molecule.

Antigens↗
PubMed1982

Simplified immunoradiometric assay for factor VIII coagulant antigen.

A simplified, non-competitive, solid phase immunoradiometric assay has been developed for the quantitation of factor VIII coagulant antigen (VIII:CAg)--the antigenic counterpart of FVIII coagulant activity (VIII:C). Both homologous and heterologous antibodies to human factor VIII (FVIII) were used in this assay. Initially, FVIII in a test sample was attached to immobilized, human IgG obtained from a polytransfused haemophilia A patient with a high titre antibody to VIII:C. The bound FVIII was then detected using rabbit 125I-IgG specific for human FVIII. The concentration of VIII:CAg correlated well with VIII:C levels in the plasma from normal donors (r = 0.84, n - 15). Homozygote von Willebrand's disease patients had undetectable levels of VIII:CAg in their plasma. Patients with severe haemophilia A (VIII:C less than 0.01 u/ml) could be divided into groups on the basis of the VIII:CAg levels, i.e. those having undetectable VIII:CAg and other with measurable VIII:CAg. VIII:CAg detected in normal serum was less than 0.002 u/ml. In this assay the use of human antibody to FVIII is considerably decreased compared to other methods for VIII:CAg, and the time-consuming steps to immunopurify human anti-FVIII antibody are eliminated.

Antigens↗
PubMed1982

Variant von Willebrand's disease type B--revisited.

Results of investigations of the factor VIII (FVIII) of a patient with an unusual variant form of von Willebrand's disease (vWD) are presented. A two-peak crossed-immunoelectrophoresis (CIE) pattern was seen when fresh plasma was electrophoresed, but the CIE pattern became normal by incubating the plasma at 37 degree C for more than 72 hr. The two peaks on CIE were separated by cryoprecipitation: the slow-moving peak precipitating and the fast-moving forms of FVIII remaining in the cryosupernate. An additional protein band was seen on multimeric analysis of FVIII. The platelet-rich plasma (PRP) from this patient did not respond to ristocetin, but agglutinated normally in response to botrocetin. Multimeric and CIE analysis of the FVIII post agglutination and 125I-FVIII binding studies to normal formalin-fixed platelets indicated that this patient's FVIII interacted normally with botrocetin but failed to interact with ristocetin. These data strongly suggest that the sites on the FVIII molecule or the multimeric forms involved for ristocetin and botrocetin are different and that the ristocetin reaction is more closely aligned to the physiologic function of FVIII.

Animals↗
PubMed1979

Abnormal factor VIII in chronic renal failure.

Factor VIII procoagulant activity (VIII-C), related antigen (VIII-AG), and von Willebrand factor (VIII-vWf) were measured in 38 patients with chronic renal failure (CRF), in 19 patients with normal renal function from a general medical ward, and in 17 normal subjects. Wide variation in the levels of factor VIII (FVIII) functions were found in the group of CRF patients. There was no correlation between the levels of the three individual activities of FVIII in renal disease. The levels of the three functions were elevated in the patient controls and no significant differences were found between the two groups of patients, although both groups were significantly different from the normal subjects. Increased electrophoretic mobility (EM) was detected in 22 of the 38 patients with CRF (58%) compared to seven of the 19 patients controls (37%). These studies suggest caution in interpretation of changes in FVIII status in patients with other clinical disorders, such as liver disease.

Antigens↗
PubMed1978

The consultation and the therapeutic illusion.

At 45 general-practice surgery sessions 200 patients in whom no definite diagnosis could be made were randomly selected for one of two procedures. Either they were given a symptomatic diagnosis and medications, or they were told that they had no evidence of disease and therefore they required no treatment. No difference in outcome was found between these two methods as judged by the return or not of the patient within one month and his statement that he did or did not get better.

Attitude to Health↗
PubMed1978

United they fall, divided they stand.

The historical context of unification or separation of the academic bodies of the various branches of the profession in Great Britain and Ireland is examined.

General Surgery↗