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Biomedical subjects

A J Smith

Publications and source records attributed to A J Smith.

At least 19 recordsLinked to original sources
PubMed1992

Identification of topaquinone and its consensus sequence in copper amine oxidases.

The nature of the active site cofactor and the amino acid sequence flanking this structure have been determined in a range of copper amine oxidases. For enzymes from porcine plasma, porcine kidney, and pea seedlings, proteolytic digestion was performed on phenylhydrazone or p-nitrophenylhydrazone derivatives. Thermolysin treatment leads to relatively small active site peptides, which have been characterized by Edman degradation and by resonance Raman spectroscopy. Resonance Raman spectra of peptides show identical peak positions and intensities relative to each other and to a model p-nitrophenylhydrazone derivative of topaquinone hydantoin, establishing topaquinone as the cofactor in each instance. Edman degradation of peptides provides active site sequences for comparison to previous determinations with bovine serum and yeast amine oxidases. The available data establish a consensus sequence of Asn, Topa, Asp/Glu. Trypsin leads to significantly longer peptides, which reveal a high degree of sequence identity between plasma proteins from bovine and porcine sources (89%), with significantly decreased identity between the porcine serum and intracellular amine oxidases (56%). A lower degree of identity (45%) is observed between the pea seedling and mammalian enzymes. As an alternative to the isolation of active site peptides for topaquinone identification, visible spectra of intact proteins have been investigated. It is shown that p-nitrophenylhydrazone derivatives of native enzymes, active site-derived peptides, and a topaquinone model exhibit identical behavior, absorbing at 457-463 nm at neutral pH (pH 7.2) and at 575-587 nm in basic solution (1-2 M KOH). These spectral properties, which appear unique to topaquinone, provide a rapid and simple test for the presence of this cofactor in intact enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amine Oxidase (Copper-Containing)↗
PubMed1992

Proline-directed phosphorylation of human Tau protein.

The primary sequence of the microtubule-associated protein tau contains multiple repeats of the sequence -X-Ser/Thr-Pro-X-, the consensus sequence for the proline-directed protein kinase (p34cdc2/p58cyclin A). When phosphorylated by proline-directed protein kinase in vitro, tau was found to incorporate up to 4.4 mol of phosphate/mol of protein. Isoelectric focusing of the tryptic phosphopeptides demonstrated the presence of five distinct peptides with pI values of approximately 6.9, 6.5, 5.6-5.9, 4.7, and 3.6. Mapping of the tryptic phosphopeptides by high performance liquid chromatography techniques demonstrated three distinct peaks. Data from gas phase sequencing, amino acid analysis, and phosphoamino acid analysis suggest that proline-directed protein kinase phosphorylates tau at four sites. Each site demonstrates the presence of a proline residue on the carboxyl-terminal side of the phosphorylated residue. Two phosphorylation sites are located adjacent to the three-repeat microtubule-binding domain that has been found to be required for the in vivo co-localization of tau protein to microtubules. Two other putative phosphorylation sites are located within the identified epitope of the monoclonal antibody Tau-1. Phosphorylation of these sites altered the immunoreactivity of tau to Tau-1 antibody. Since the neuronal microtubule-associated protein tau is multiply phosphorylated in Alzheimer's disease, and Tau-1 immunoreactivity is similarly reduced in neurofibrillary tangles and enhanced after dephosphorylation, phosphorylation at one or more of these sites may correlate with abnormally phosphorylated sites in tau protein in Alzheimer's disease.

Amino Acid Sequence↗
PubMed1992

Bleomycin-induced unscheduled DNA synthesis in non-permeabilized human and rat hepatocytes is not paralleled by 8-oxo-7,8-dihydrodeoxyguanosine formation.

The genetic toxicity of the antitumour antibiotic bleomycin (BLM) is thought to involve the formation of a reactive oxygen intermediate. 8-Oxo-7,8-dihydrodeoxyguanosine (oxo8dG), an oxidation product of deoxyguanosine, is one of the major products formed when isolated DNA is exposed to oxygen radical generating systems. Gamma-irradiation (10-500 Gy 60Co; 10 Gy/min) or BLM and Fe2+ (37.5-150 U/L and 0.5 mM, respectively) treatment of isolated DNA (0.25 mg/mL) increased oxo8dG above background. In the latter case, the effect was greater than that with Fe2+ (0.5 mM) alone and was dependent on the dose of BLM. When DNA was irradiated with 500 Gy60Co, deoxyguanosine oxidation was inhibited by antioxidants (ethanol: 37.5 and 98% inhibition at 2 and 20 mM, respectively; mannitol: 20.5, 60 and 92% inhibition at 0.1, 1.0 and 10 mM, respectively). Similarly the BLM-induced production of oxo8dG was inhibited (64%) by mannitol (10 mM). BLM also caused production of base propenals on interaction with isolated DNA. In contrast, oxo8dG was not induced above background concentration (27 mol oxo8dG/10(6) mol dG) in permeabilized (37 degrees) and non-permeabilized (4 degrees and 37 degrees) rat hepatocytes treated with BLM (260 U/L). Despite this, there was extensive BLM-induced unscheduled DNA synthesis (10 and 100 U/L) in non-permeabilized rat and human hepatocytes in the absence of hydroxyurea. These findings, in accord with other observations, draw into question the role of .OH in BLM-induced DNA damage and the mimicry of ionizing radiation in cellular systems.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1992

Tyrosine codon corresponds to topa quinone at the active site of copper amine oxidases.

The recently discovered organic cofactor of bovine serum amine oxidase, topa quinone, is an uncommon amino acid residue in the polypeptide backbone (Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burlingame, A. L., and Klinman, J. P. (1990) Science 248, 981-987). The amine oxidase gene from the yeast Hansenula polymorpha has been cloned and sequenced (Bruinenberg, P. G., Evers, M., Waterham, H. R., Kuipers, J., Arnberg, A. C., and Geert, A. B. (1989) Biochim. Biophys. Acta 1008, 157-167). In order to understand the incorporation of topa quinone in eukaryotes, we have isolated yeast amine oxidase from H. polymorpha. Following protocols established with bovine serum amine oxidase, yeast amine oxidase was derivatized with [14C]phenylhydrazine, followed by thermolytic digestion and isolation of a dominant radiolabeled peptide by high pressure liquid chromatography. Comparison of resonance Raman spectra for this peptide to spectra of a model compound demonstrates that topa quinone is the cofactor. By alignment of a DNA-derived yeast amine oxidase sequence with the topa quinone-containing peptide sequence, it is found that the tyrosine codon, UAC, corresponds to topa quinone in the mature protein. In a similar manner, alignment of a tryptic peptide from bovine serum amine oxidase implicates tyrosine as the precursor to topa quinone in mammals.

Amine Oxidase (Copper-Containing)↗
PubMed1992

Characterization and dynamics of O-linked glycosylation of human cytokeratin 8 and 18.

The glycosylation of human cytokeratin (CK) 8 and 18 was studied after metabolic labeling of HT29 colonic cells with [3H]glucosamine. Labeling of CK8/18 was not inhibited by tunicamycin, suggesting that glycosylation was not N-linked. Acid hydrolysis of CK8 and CK18, purified from [3H]glucosamine-labeled cells, generated free glucosamine. In the presence of UDP-[3H]galactose, galactosyltransferase catalyzed the labeling of cytokeratin 8 and 18. beta-Elimination of the [3H]galactose- labeled CK8/18 generated the disaccharide N-acetyllactosaminitol, indicating that cytokeratin 8 and 18 contain single O-linked N-acetylglucosamine residues. Using chemical analysis, the stoichiometry of glycosylation was found to be 1.5 and 2 molecules of N-acetylglucosamine/protein molecule of CK8 and CK18, respectively. Peptide maps of [3H]glucosamine-labeled CK8/18 showed that multiple peptides were labeled with the amino sugar. The biosynthetic and degradation rates of the carbohydrate moiety were faster than the protein core as determined by metabolic radiolabeling or pulse-chase experiments, respectively. Our results show that CK8 and 18 are glycosylated at multiple sites with a single O-linked N-acetylglucosamine. Furthermore, CK8/18 glycosylation is a dynamic process which is likely to have functional relevance.

Amino Sugars↗
PubMed1992

Identification of the adipocyte acid phosphatase as a PAO-sensitive tyrosyl phosphatase.

We have partially purified an 18-kDa cytoplasmic protein from 3T3-L1 cells, which dephosphorylates pNPP and the phosphorylated adipocyte lipid binding protein (ALBP), and have identified it by virtue of kinetic and immunological criteria as an acid phosphatase (EC 3.1.3.2). The cytoplasmic acid phosphatase was inactivated by phenylarsine oxide (PAO) (Kinact = 10 microM), and the inactivation could be reversed by the dithiol, 2,3-dimercaptopropanol (Kreact = 23 microM), but not the monothiol, 2-mercaptoethanol. Cloning of the human adipocyte acid phosphatase revealed that two isoforms exist, termed HAAP alpha and HAAP beta (human adipocyte acid phosphatase), which are distinguished by a 34-amino acid isoform-specific domain. Sequence analysis shows HAAP alpha and HAAP beta share 74% and 90% identity with the bovine liver acid phosphatase, respectively, and 99% identity with both isoenzymes of the human red cell acid phosphatase but no sequence similarity to the protein tyrosine phosphatases (EC 3.1.3.48). HAAP beta has been cloned into Escherichia coli, expressed, and purified as a glutathione S-transferase fusion protein. Recombinant HAAP beta was shown to dephosphorylate pNPP and phosphoALBP and to be inactivated by PAO and inhibited by vanadate (Ki = 17 microM). These results describe the adipocyte acid phosphatase as a cytoplasmic enzyme containing conformationally vicinal cysteine residues with properties that suggest it may dephosphorylate tyrosyl phosphorylated cellular proteins.

3T3 Cells↗
PubMed1992

The effect of periodontal disease on sulphated glycosylaminoglycan distribution in the sheep periodontium.

The glycosylaminoglycan (GAG) distribution in the soft periodontal tissues of the sheep was investigated topographically in healthy tissue and the changes associated with periodontal disease determined and correlated with increasing disease severity. Total sulphated GAG content was determined spectrophotometrically and the proportions of individual GAGS measured after separation by cellulose acetate electrophoresis. In healthy tissue, total sulphated GAG distribution was found to mirror that described previously for collagen, being highest in the gum-pad region and adjacent to alveolar bone. This changed markedly in relation to periodontal disease. Early disease-related changes in GAGs were first detected adjacent to alveolar bone. In severely diseases tissue, an overall increase in sulphated GAG, particularly in the gum-pad region, was present. Increasing disease severity was accompanied by a decrease in dermatan sulphate and a concomitant increase in the proportion of chondroitin-4-sulphate. These changes may be due to the combined effects of chronic inflammation and alterations to the mechanical loading of the tissue owing to loss of attachment.

Animals↗
PubMed1992

State and trait differences in depressive self-perceptions.

A number of investigators have interpreted a tendency for depressed people to recall more negative than positive self-referent adjectives as evidence for a depressive self-schema made up of predominantly negative characterological information. We sought to confirm this account by eliciting the subjective self-perceptions of the depressed. Depressed patients and controls were required to rate whether or not a series of positive and negative adjectives applied to them during the previous week, at any time, and generally. The depressed distinguished clearly between these questions and, although describing themselves currently in largely negative terms, described their general state as equally composed of positive and negative elements.

Adult↗
PubMed1992

Effects of body condition and estradiol on luteinizing hormone secretion in post-partum beef cows.

An experiment was conducted to test the hypothesis that the effect of body fatness on LH pulsatility in post-partum cows is entirely independent of the negative feedback effects of ovarian steroids. Forty beef cows were fed in the last 100 d of gestation so that they achieved either a thin (mean score 1.97) or fat (mean score 2.79) body condition (0 to 5 scale) at calving and were fed after calving to maintain live weight and body condition. At 15 (sd 3.7) d post partum all cows were ovariectomised and half from each body condition score treatment group received a subcutaneous estradiol implant (+EST) while the remainder received no implant (-EST). At weeks 5 and 9 post-partum blood samples were collected via jugular catheter every 20 minutes for 10 hr on two consecutive d and on the third d cows were injected via the jugular vein with 2.5 micrograms GnRH. Blood samples were collected every 15 minutes for 1 hr before and 2 hr after GnRH injection. At 5 and 9 weeks the fatter cows had significantly higher mean LH concentrations, baseline LH concentrations, LH pulse amplitudes and pulse frequencies (P < 0.01). Implantation with estradiol in both fat and thin cows reduced mean LH concentrations, baseline LH concentrations, LH pulse amplitudes and pulse frequencies (P < 0.001). The lack of interaction between body condition and the presence or absence of estradiol implies that the effect of body condition on LH release is independent of ovarian steroid feedback mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

A computerized method for measurement and analysis of blood pressure in small animals.

A method is described for computerized measurement of blood pressure (BP) in small animals. As described, the system measures BP in up to four experimental animals at one time by sequential sampling but can be expanded to take data from up to 10. It requires an Apple II computer, and is run from a Grass pen-recording system. Pulse rate and systolic and diastolic BP are measured, and these variables are used to calculate mean arterial pressure (MAP) and the time integral of MAP minute by minute. The data are displayed in real time and stored hourly on floppy disc for further analysis.

Animals↗
PubMed1992

The effect of cocaine on sperm motility characteristics and bovine cervical mucus penetration.

OBJECTIVES: To determine the in vitro effects of cocaine on sperm motility and bovine mucus penetration because cocaine abuse is associated with decreased sperm motility, and related compounds, such as procaine, are known to decrease sperm motility. DESIGN: Human semen samples were exposed to a range of cocaine concentrations and the effects quantified using computer-assisted sperm analysis and the bovine mucus penetration test. SETTING: University research laboratory. PATIENTS, PARTICIPANTS: Samples were obtained from 18 healthy volunteers. INTERVENTIONS: Normal semen samples were exposed to concentrations of cocaine ranging from 10(-11) to 10(-4) M. Motility characteristics were evaluated after 2 hours, and bovine mucus penetration was evaluated after 30 minutes, 1 hour, and 2 hours. Mucus penetration by washed sperm was also evaluated. MAIN OUTCOME MEASURES: Motility characteristics were evaluated using computer-assisted sperm analysis, and functional sperm motility was evaluated using the bovine mucus penetration test. RESULTS: Cocaine exposure decreased the percentage of motile sperm in a concentration-dependent manner with a maximum decrease of 23% at 10(-4) M but had no effect on other motility characteristics. Cocaine decreased bovine mucus penetration by 12% at high cocaine concentrations (10(-4) M), but increased penetration by 69% at low concentrations (10(-9) M). Washing sperm before cocaine exposure attenuated the increased sperm penetration. CONCLUSION: The ability of cocaine to decrease the percentage of motile sperm at high concentrations may explain the decreased sperm motility associated with cocaine use. Cocaine's ability to augment sperm penetration at low concentrations suggests an interaction of cocaine with the sperm adrenergic system.

Analysis of Variance↗
PubMed1992

Effects of ibopamine on exercise-induced increase in norepinephrine in normal men.

The effects of 100 mg ibopamine, an orally active aselective dopamine (DA) agonist, on plasma catecholamines was evaluated in 8 healthy men during sympathetic stimulation by graded exercise in a single-blind, placebo-controlled cross-over study. The exercise consisted of progressive cycling activity less than or equal to 90% of the previously determined VO2max. Graded exercise resulted in an increase in systolic and mean blood pressure (SBP, MBP), heart rate, norepinephrine (NE) and epinephrine level, with a decrease in diastolic BP (DBP). The increase in NE was significantly blunted by ibopamine as compared with placebo. No differences for BP, heart rate (HR), or epinephrine between placebo- and ibopamine study day were noted. In previous studies, ibopamine decreased resting plasma NE in patients with congestive heart failure (CHF), whereas plasma NE was not altered by ibopamine in healthy volunteers. This different outcome in both categories might therefore be explained by the absence of substantial sympathetic stimulation in normal humans at rest. Because it is reasonable to assume that the effect of ibopamine on systemic and local hemodynamics is negligible as compared with the effect of exercise in the healthy volunteers, the plasma decrease caused by ibopamine is probably related to stimulation of DA2-receptors. In conclusion, ibopamine blunts the increase of plasma NE during graded exercise in healthy men.

Adult↗
PubMed1992

The origins of the human immunodeficiency viruses: an update.

In 1981 a new acquired immunodeficiency syndrome was first described. The disease has a 100% mortality rate and over 359,000 cases have been reported to the WHO from 162 countries. The WHO estimates that the cumulative global total of AIDS cases as of early 1991 is more than 1.5 million. The dental profession, in line with other health care professions, is involved with guiding rational efforts to stop transmission and with developing effective means of treatment and prevention. This paper reviews the nature of the virus, its possible origins, and the implications of such origins for treatment and prevention of the disease.

Acquired Immunodeficiency Syndrome↗