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Biomedical subjects

T W Young

Publications and source records attributed to T W Young.

At least 19 recordsLinked to original sources
PubMed2001

The "open" and "closed" structures of the type-C inorganic pyrophosphatases from Bacillus subtilis and Streptococcus gordonii.

Recently, a new class of soluble inorganic pyrophosphatase (type-C PPase) has been described that is not homologous in amino acid sequence or kinetic properties to the well-studied PPases (types A and B) found in many organisms from bacteria to humans and thought to be essential to the cell. Structural studies of the type-C PPases from Streptococcus gordonii and Bacillus subtilis reveal a homodimeric structure, with each polypeptide folding into two domains joined by a flexible hinge. The active site, formed at the interface between the N and C-terminal domains, binds two manganese ions approximately 3.6 A apart in a conformation resembling binuclear metal centres found in other hydrolytic enzymes. An activated water molecule bridging the two metal ions is likely poised for nucleophilic attack of the substrate. Importantly, the S. gordonii and B. subtilis enzymes have crystallised in strikingly different conformations. In both subunits of the S. gordonii crystal structure (1.5 A resolution) the C-terminal domain is positioned such that the active site is occluded, with a sulphate ion bound in the active site. In contrast, in the B. subtilis structure (3.0 A resolution) the C-terminal domain is rotated by about 90 degrees, leaving the active site wide open and accessible for substrate binding.

Amino Acid Sequence↗
PubMed2000

Methanococcus jannaschii ORF mj0608 codes for a class C inorganic pyrophosphatase protected by Co(2+) or Mn(2+) ions against fluoride inhibition.

Openreading frame mj0608 of the Methanococcus jannaschii genome, recognized by its sequence similarity to that of the gene coding for class C inorganic pyrophosphatase in Bacillus subtilis, was cloned and over-expressed in Escherichia coli. The protein was purified and characterized by SDS-PAGE, M(r), and N-terminal sequence. Under suitable conditions it catalyzed the specific hydrolysis of PPi at about 600 micromol x min(-1) x mg(-1) at 25 degrees C, and at 8000 micromol x min(-1) x mg(-1) at 85 degrees C. Therefore this protein is a specific inorganic pyrophosphatase. The activities of Mg(2+), Mn(2+), Co(2+), and Zn(2+) ions as cofactors for hydrolysis of PPi were compared at pH 7.5 and 9.0. Unlike the class C pyrophosphatase of B. subtilis, this enzyme required no prior activation by low concentrations of Mn(2+) or Co(2+) ions. However, prior exposure to these ions afforded striking protection against inhibition by sodium fluoride, to which the enzyme was otherwise very sensitive.

Amino Acid Sequence↗
PubMed2000

Reduced hepatic content of dehydroepiandrosterone sulphotransferase in chronic liver diseases.

AIMS/BACKGROUND: Dehydroepiandrosterone sulphotransferase (DHEA ST) is the enzyme responsible for sulphation of lithocholic acid and other potentially hepatotoxic steroids. We have previously shown that DHEA ST activity is reduced in cytosol of liver from miscellaneous patients with chronic liver disease. The aim of this study was to investigate the cause of diminished sulphotransferase activity in order to further our understanding of whether a reduction in the ability to sulphate potentially hepatotoxic bile acids might play a role in the aetiology of primary cholestatic liver disease. METHODS: We quantified DHEA ST in human liver cytosol from groups of patients with chronic liver diseases and normal subjects using a semiquantitative sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE)/ immunoblotting method, and an enzyme-linked immunosorbent assay (ELISA). We determined DHEA ST enzyme activity and correlated it with its immunoreactive concentration in 87 samples of human liver tissue. RESULTS: DHEA ST activity and concentration were significantly reduced in primary biliary cirrhosis, primary sclerosing cholangitis, chronic active hepatitis and alcoholic cirrhosis but not in cryptogenic cirrhosis when compared to normal liver. There were no significant differences among disease groups. In all groups enzyme activity and cellular concentration correlated, suggesting that no aberrant non-functional enzyme was produced. CONCLUSION: These results confirm that DHEA ST activity is diminished in liver disease and that the reduction is due to diminished enzyme presence. Further studies are required to show whether the reduction has any pathogenetic significance or is merely a consequence of disease.

Adult↗
PubMed1998

Secretion and pH-dependent self-processing of the pro-form of the Yarrowia lipolytica acid extracellular protease.

The secretion and maturation of the acid extracellular protease (AXP) of the yeast Yarrowia lipolytica have been characterized using antiserum raised against this enzyme. A 42 kDa pro-enzyme form of AXP was identified from lysates of radiolabelled Y. lipolytica cells and found to contain no N-linked carbohydrate moieties. Using pulse-chase immune precipitation it was demonstrated that the AXP precursor was secreted into the extracellular medium where, under conditions of low pH, it underwent autocatalytic activation forming the mature enzyme. Conversion of the AXP pro-form in the presence of the protease inhibitor pepstatin indicated that an intramolecularly-catalysed reaction mechanism was involved in AXP maturation. Further evidence supporting the role of autocatalytic processing came from the side-chain specificity of mature AXP towards the oxidized B-chain of insulin.

Amino Acid Sequence↗
PubMed1996

The extracellular acid protease gene of Yarrowia lipolytica: sequence and pH-regulated transcription.

The gene encoding an acid extracellular protease (AXP) from Yarrowia lipolytica (Candida olea) 148 was cloned and the complete nucleotide sequence was determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature AXP consists of 353 amino acids with an M, of 37427. The gene also encodes a putative 17 amino acid hydrophobic prepeptide and a 27 amino acid propeptide containing no potential N-glycosylation sites. The mature extracellular enzyme is produced by cleavage between Phe and Ala. AXP is a member of the aspartyl family of proteases. AXP shows homology to proteases of several fungal genera and to human progastricin. The coding sequence is preceded by a potential regulatory region of 1982 bp. Transcription of both AXP and alkaline extracellular protease genes of Y. lipolytica 148 is regulated by the pH of culture.

Amino Acid Sequence↗
PubMed1995

Purification of human hepatic arginase and its manganese (II)-dependent and pH-dependent interconversion between active and inactive forms: a possible pH-sensing function of the enzyme on the ornithine cycle.

Purification of human liver arginase by chromatography on DEAE-Sepharose, CM-Sepharose, hydroxylapatite, and MonoS yielded protein of greater than 95% purity by sodium dodecyl sulfate-gel electrophoresis. Detailed kinetic studies of the interconversion of active and inactive forms of arginase showed the effects of metal ion addition and withdrawal, metal ion type, time, temperature, and pH. At pH 7 and 37 degrees C, removal of Mn2+ caused a first-order deactivation with half-life of 1 h. Reactivation was completed within 0.5 min (1 mM Mn2+) or 90 min (ca. 6 nM Mn2+). Activation by Mn2+ showed a hyperbolic response, with Kd for Mn2+ of about 36 nM. Mn2+ apparently displaced about 2 H+, resulting in sigmoid dependence upon concentration of OH-. Both the maximal velocity of catalysis and the Km toward arginine were markedly pH-dependent in the physiological range. The findings lead to a model where Mn2+ allosterically activates arginase by a sequential, and pH-sensitive, mechanism. The combined pH sensitivities of activation, Vmax, and Km are likely to give arginase a role in mediating the demonstrated pH control of the ornithine cycle and hence in the regulation of body pH.

Arginase↗
PubMed1993

Misclassification of deaths caused by cocaine. An assessment by survey.

The use of U.S. vital statistics for surveillance of drug-related mortality may be limited by the way in which certifiers complete death certificates and by the constraints of the International classification of diseases, Ninth Revision (ICD-9). ICD-9 is the system used by the National Center for Health Statistics (NCHS) to compile national, cause-specific mortality data from information reported on death certificates. To investigate the extent of variability in certification practices among medical examiners (MEs), we conducted a mailout survey in which we asked a national sample of 49 MEs to review summaries of 28 death scenarios and, for each death, assign the cause and manner of death. Cocaine use was the unequivocal cause of death for 17 of the 28 deaths. We then asked a nosologist at NCHS to code the verbatim survey responses in accordance with the rules and rubrics of the ICD-9 system. Of the 20 MEs who responded, 14 provided complete cause and manner determinations. For the cocaine-caused deaths, the 14 respondents provided 238 cause-of-death statements; 220 (92.4%) explicitly mentioned cocaine. However, only 45 of the 238 responses (18.9%) led to a cocaine-specific ICD-9 code for the underlying cause of death. Our findings illustrate how death certification practices, coupled with the ambiguities of the ICD-9 system, may lead to substantial loss of detail about cocaine-caused deaths and misclassification of these deaths in official compilations of mortality statistics.

Cause of Death↗
PubMed1992

Pain during sexual response due to bilateral Bartholin's gland adenomas. A case report.

Benign adenomas of the major vestibular gland are very rare. Presented is a case of bilateral vestibular gland adenomas in a woman whose chief complaint was vulvar pain occurring with sexual arousal and orgasm. Surgical removal of the adenomas relieved the symptoms. The development of pain during sexual response in this case appears to be mediated by vascular engorgement.

Adenoma↗
PubMed1992

Reye's syndrome. A diagnosis occasionally first made at medicolegal autopsy.

Reye's syndrome, a condition characterized pathologically by cerebral edema and fatty change of the liver, has been described extensively in the medical literature as a disease manifested clinically by encephalopathy and coma. This is a report of five cases of Reye's syndrome occurring as sudden, unexpected deaths outside of the hospital. In each of these cases, there is a vague history of a previous viral illness. A history of aspirin intake is inconstant. Each child either had no significant past illnesses or there was a history of repeated upper respiratory infections. The classic progression of signs and symptoms usually described for Reye's syndrome, where vomiting usually precedes encephalopathy and coma, was not present in any of the cases. Results of autopsies showed the characteristic findings for Reye's syndrome, and additional tests showed no other explanation for the deaths. This manifestation of the disease is seldom described in medical literature, but it may be encountered occasionally by the medical examiner.

Autopsy↗
PubMed1987

Extracellular acid and alkaline proteases from Candida olea.

Candida olea 148 secreted a single acid protease when cultured at acidic pH. In unbuffered medium, the culture pH eventually became alkaline and a single alkaline protease was produced. This was the only proteolytic enzyme produced when the organism was grown in buffered medium at alkaline pH. Both proteolytic enzymes were purified to homogeneity (as assessed by SDS-PAGE). The Mr of the acid protease was 30900, the isoelectric point 4.5; optimum activity against haemoglobin was at 42 degrees C and pH 3.3. This enzyme was inactivated at temperatures above 46 degrees C and was inhibited by either pepstatin and diazoacetyl-norleucine methyl ester but was insensitive to inhibition by either 1,2-epoxy-3-(p-nitrophenoxy)-propane or compounds known to inhibit serine, thiol or metallo proteases. The acid protease contained 11% carbohydrate. The alkaline protease had an Mr of 23400 and isoelectric point of 5.4. The activity of this enzyme using azocoll as substrate above 42 degrees C and was inhibited by phenylmethyl-sulphonyl fluoride and irreversible inactivated by EDTA. The enzyme was also partially inhibited by DTT but was insensitive to either pepstatin or p-chloromercuribenzoic acid.

Aspartic Acid Endopeptidases↗
PubMed1984

Red cell fragmentation in human disease (a light and scanning electron microscope study).

Although the mechanism of schizocyte formation has been amply documented in animal experiments and in in-vitro models, the fragmentation encounter between flowing red cells and fibrin strands has not previously been successfully demonstrated in human, microangiopathic disease. If blood flow is restored briefly in vitro immediately prior to tissue fixation, the instant of red cell fragmentation can be captured. Examination of tissue specimens fixed in this manner shows a pathophysiologic process that amplifies the findings previously described in other studies. In the patient under study, the microangiopathic process was widespread in all specimens of pulmonary and renal tissue that had been fixed after brief restoration of blood flow. Small arteries as well as the true microcirculation of both organs were involved. The microangiopathic process in the small arteries and arterioles presented as a partially occlusive thrombus of characteristic histology. The pulmonary capillaries contained linear fibrin microclots festooned with distorted and partially fragmented red cells. The microcirculation of the kidney showed the same findings as well as amorphous, sludged, occlusive red cell masses, particularly in the renal medulla.

Adenocarcinoma↗
PubMed1979

Ultrastructure of the asexual apparatus in Mycotypha (Mucorales).

The development of the asexual apparatus of Mycotypha africana and Mycotypha poitrasii observed by means of scanning electron microscopy is reported. Using ultrathin sections and freeze-fracture replicas, sporangiole structure is described. Sporangiospore structure is compared with that of other members of the Thamnidiaceae.

Cell Membrane↗
PubMed1978

A comparison of the killer character in different yeasts and its classification.

The interactions between 20 killer yeasts of various genera and species were examined. Ten distinct groups were recognised with respect to killer activity and 10 distinct groups with respect to resistance to killer action. Using both killing and resistance phenotypes, 13 classes of killer yeast were found. With the exception of Torulopsis glabrata NCYC 388, non-Saccharomyces strains of yeast were not killed by a member of the genus Saccharomyes. The killer character of the 3 killing groups of Saccharomyces identified could be cured by treatment with cycloheximide or incubation at elevated temperature and the effectiveness of these procedures was indicative of the category of killer yeast examined. Killer yeasts not belonging to the genus Saccharomyces could not be cured of their activity. Double-stranded ribonucleic acids were extracted only from Saccharomyces spp. and the molecular weights of the species present were a function of the killer class to which a strain belonged. By an analysis of the effects of proteolytic enzymes, temperature and pH on killer activity and by gel chromatography of crude preparations of killer factors, the toxins of different killer classes were shown to be biochemically distinct. However all toxins had certain properties in common consistent with there being a protein component essential to killer action.

Cycloheximide↗