Biomedical subjects

M A Clark

Publications and source records attributed to M A Clark.

At least 19 recordsLinked to original sources

PubMed1992

Butyric acid-induced differentiation of HL-60 cells increases the expression of a single lysophospholipase.

Treatment of HL-60 cells with 0.5 mM-butyric acid resulted in morphological changes, including the formation of cytoplasmic granules, nuclear condensation and segmentation. These differentiated cells had an elevated phospholipase A2 activity and an increased capacity to synthesize a variety of eicosanoids, including both lipoxygenase and cyclooxygenase products. Phospholipase A2-mediated release of arachidonic acid is accompanied by an equimolar production of potentially cytotoxic lysophospholipid. In association with the differentiation process, there was a 2-3-fold increase in lysophospholipase activity. Subsequent studies were undertaken to identify and characterize the lysophospholipases in this cell system, with 1-[1-14C]palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine as substrate. Hydrophobic chromatography of both undifferentiated and differentiated cell extracts revealed three peaks of enzyme activity. Extracts of differentiated cells contained a dramatic increase in activity contained in peak 2. The increase in enzymic activity of peak 2 appeared to account for the increase in total lysophospholipase activity found in the differentiated cell homogenates. The lysophospholipases contained in peaks 2 and 3 were purified to homogeneity and were 20 and 22 kDa respectively, as determined by denaturing polyacrylamide-gel electrophoresis. Peaks 2 and 3 were similar on the basis of amino acid composition, but had distinctive C-terminal peptide amino acid sequences. Enzymic characterization of these proteins demonstrated that there was no detectable level of non-specific esterase, acyltransferase or transacylase activity associated with these proteins. We concluded that peak 2 lysophospholipase is regulated by differentiation in HL-60 cells and may play an important role in protecting these cells from the cytolytic effects of the lysophospholipids produced by the activation of phospholipase A2.

Amino Acid Sequence↗

PubMed1992

Isolation and characterization of three lysophospholipases from the murine macrophage cell line WEHI 265.1.

Anion exchange chromatography of WEHI 265.1 cell homogenates resolved the lysophospholipase activity into three peaks, when assayed using lysophosphatidylcholine as a substrate. Peaks 1 and 2 were purified by sequential hydrophobic interaction and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peaks 1 and 2 indicated homogeneous proteins with apparent masses of 28 and 27 kDa, respectively. Peak 3 lysophospholipases was partially purified by hydrophobic, hydroxyapatite and gel filtration chromatography. Peak 3 lysophospholipase also had calcium-dependent phospholipase A2 activity, which further co-purified with the lysophospholipase activity. The three lysophospholipases were characterized with respect to substrate specificity, additional enzymatic activities and the effects of lipids, metal ions and other compounds on enzymatic activity. Peaks 1, 2 and 3 hydrolyzed lysophosphatidylcholine most readily, but lysophosphatidylethanolamine also served as substrate for each enzyme. Furthermore, all three enzymes hydrolyzed platelet activating factor and acetylated lysophosphatidylcholine. Each lysophospholipase was inhibited by free fatty acids and by palmitoyl carnitine, although the relative sensitivities to these agents differed among the enzymes. The lysophospholipase activities of peaks 1 and 2, but not peak 3, were inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate and N-ethylmaleimide. Although they had similar masses, the amino acid compositions of peaks 1 and 2 differed, indicating that these are distinct proteins rather than posttranslational modifications of the same gene product.

Amino Acids↗

PubMed1992

High affinity histamine H3 receptors regulate ACTH release by AtT-20 cells.

The distribution of high affinity histamine H3 receptors in various tissues from guinea pig has been determined using [3H]N alpha-methylhistamine binding. In the course of those studies, it was observed that the pituitary gland contains H3 receptors. Using this radioligand, we have now identified and characterized H3 receptors on' the AtT-20 cell line from a murine anterior pituitary tumor. This line has approximately 5000 high affinity (KD = 0.7 nM) H3 binding sites per cell. Competition binding with standard H1, H2 and H3 agents has confirmed that these sites are, indeed, H3 receptors. The H3 receptor specific agonist, (R)-alpha-methylhistamine increased the release of adrenocorticotropic hormone (ACTH) from AtT-20 cells in a dose- and time-dependent manner, while histamine and the H2 agonist dimaprit were significantly less potent. Furthermore, this response was blocked by thioperamide, an H3 receptor specific antagonist, but not by the H1 and H3 antagonists, chlorpeniramine and cimetidine. These results identify, for the first time, a cell line expressing H3 receptors and indicate that the high affinity histamine H3 receptor regulates ACTH release from that cell.

Adrenocorticotropic Hormone↗

PubMed1992

IL-1 increases phospholipase A2 activity, expression of phospholipase A2-activating protein, and release of linoleic acid from the murine T helper cell line EL-4.

The early events in IL-1-mediated activation of T cells were investigated in the murine T cell line, EL-4. Treatment of EL-4 cells with human rIL-1 beta resulted in a rapid increase in phospholipase A2 (PLA2) activity. PLA2 activity increased approximately fivefold within 4 min after exposure to IL-1. Synthesis of the phospholipase A2- activating protein (PLAP) and its mRNA were also increased within 4 min of IL-1 treatment and preceded the increase in PLA2 enzyme activity. The increases in PLA2 activity and PLAP protein and mRNA levels were all transient and declined to baseline within 10 min after the addition of IL-1. The changes in levels of PLAP as a function of time after IL-1 treatment were consistent with PLAP playing an important role in the regulation of PLA2 activity in this system. The consequence of the elevated PLA2 activity was examined by analysis of the fatty acids released from IL-1-treated cells. There was a 20-fold increase in the release of radioactivity from [14C]-linoleic acid labeled cells whereas there was very little change in the release of radioactivity from [14C]-arachidonic acid labeled cells in response to the addition of IL-1. The radioactivity released from [14C]-linoleic acid labeled cells was analyzed by HPLC; no conversion of radiolabeled linoleic into arachidonic acid was observed. In EL-4 cells, IL-1 potentiates PMA-mediated release of IL-2 at suboptimal concentrations of PMA. Linoleic acid also augmented PMA-induced IL-2 release from the EL-4 cells. This fatty acid was more than 10 times more effective than arachidonic acid in this regard. Furthermore, the addition of exogenous PLAP to EL-4 cells could substitute for IL-1 in the stimulation of IL-2 release. These results suggest that the IL-1 effects on T cells may be mediated at least in part through increased PLA2 activity due to increased synthesis of PLAP. Furthermore, the release of the unsaturated fatty acid linoleic acid or its metabolites may be of functional importance in IL-1-mediated IL-2 production by EL-4 cells.

Animals↗

PubMed1992

Cardiac pathology in 2007 consecutive forensic autopsies.

The incidence of various types of cardiovascular disease was evaluated in 2007 consecutive forensic patients. Cardiovascular deaths accounted for 22.8% of the study patients and atherosclerotic coronary heart disease was the most common type of cardiac disease (18%). Among subjects dying of atherosclerotic coronary disease, sudden death was three times more frequent than acute myocardial infarction. Expected cardiac findings included the incidence of severe coronary atherosclerosis (21%), floppy mitral valves (5%), and congenital bicuspid aortic valves (1%). Major cardiac findings occurred in 32% and minor cardiac findings were found in 40%. Only 17% of hearts were anatomically normal. An unexpected cardiac necropsy finding included the high frequency of myocardial bridges (23%). Unexpected cardiac findings included the low incidence of acute myocarditis (0.6%) and common finding of tunneled epicardial coronary arteries ("myocardial bridges") (23%).

Adult↗

PubMed1992

Sequence analysis of an aphid endosymbiont DNA fragment containing rpoB (beta-subunit of RNA polymerase) and portions of rplL and rpoC.

The aphid Schizaphis graminum is dependent on an association with a prokaryotic endosymbiont (Buchnera aphidicola). The nucleotide (nt) sequence of a 5040 base pair (bp) DNA fragment of B. aphidicola, homologous to the rplL-rpoB-rpoC portion of the Escherichia coli beta operon, was determined. The DNA coded for the terminal 35 amino acids of RplL (large ribosomal subunit protein L7/L12), the complete RpoB (beta-subunit of RNA polymerase), and the first 209 amino acids of RpoC (beta'-subunit of RNA polymerase). The deduced sequences of B. aphidicola RplL, RpoB, and RpoC were 71, 84, and 91% identical, respectively, to the homologous proteins of E. coli. The sequences of two portions of the intergenic region between rplL and rpoB were nearly identical in both B. aphidicola and E. coli. One sequence constituted an inverted repeat that could be an RNase III-messenger RNA processing site; the other sequence preceded RpoB. A compilation of the codon usage for RpoB, RpoC, and other B. aphidicola proteins indicated a major preference for A or T in the first and third positions, a result consistent with the low guanine plus cytosine (G + C) content of the DNA of this organism.

Amino Acid Sequence↗

PubMed1992

Regulation of synovial cell growth: basic fibroblast growth factor synergizes with interleukin 1 beta stimulating phospholipase A2 enzyme activity, phospholipase A2 activating protein production and release of prostaglandin E2 by rheumatoid arthritis synovial cells in culture.

Cytokines have been implicated in the regulation of eicosanoid synthesis and synovial cell proliferation. To further define these mechanisms, we have compared the effects of basic fibroblast growth factor and platelet-derived growth factor on cell growth, prostaglandin E2 (PGE2) production and phospholipase A2 enzyme activity in long-term cultures of synovial cells from rheumatoid arthritis (RA) patients capable of proliferating in serum-free medium. Compared with serum-free medium alone, RA synovial cell growth was significantly enhanced by adding either basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF) to the culture medium. Growing RA synovial cells for 14 days in serum-free medium plus bFGF caused them to spontaneously release significant amounts of PGE2, an effect not seen if cells were grown in serum-free medium alone, or serum-free medium plus PDGF. Enhanced release of PGE2 occurred when arachidonic acid was added to bFGF but not PDGF-treated RA synovial cells, suggesting that bFGF increased cyclooxygenase enzyme activity in these cells. Moreover, phospholipase A2 (PLA2) enzyme activity was found to be significantly greater in RA synovial cells grown for 14 days in serum-free medium containing bFGF alone, or bFGF plus interleukin 1 beta (IL-1 beta) compared with cells grown in either serum-free medium alone, or serum-free medium plus PDGF. Similarly, bFGF plus IL-1 beta-stimulated release of PLA2 activating protein, a novel mammalian phospholipase stimulator found in high concentrations in RA synovial fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

Arthritis, Rheumatoid↗

PubMed1992

An autopsy case review of 142 nonpenetrating (blunt) injuries of the aorta.

This study retrospectively reviewed 142 fatal cases of aortic laceration. Data were collected from the autopsy reports (including toxicology results) and included the circumstances of the injury. In the case of automobile crashes this included the direction of impact, time of day, and vehicular damage. Seventy percent of the victims were men with a mean age of 37.5 years; 30% were women with a mean age of 46 years. Thirty-five percent of the 142 victims had elevated blood alcohol levels. Fifty-four percent of the lacerations were located at the classic site (the isthmus). The majority of the aortic lacerations (102) were sustained in automobile crashes. Of these, 42% were broadside collisions and 58% were head-on collisions. Seventy-three percent and 67% of the victims in broadside and head-on collisions, respectively, had aortic lacerations at the classic site. The results suggest that the pathogenesis of aortic rupture involves a lateral oblique compression impact to the chest, which causes thoracic mediastinal structures to shift and deflect the aortic arch, resulting in severe shearing and stretching at the isthmus. The use of seat belts and air bags may reduce the number of aortic injuries.

Accidents, Traffic↗

PubMed1992

Characterization of a digitonin-solubilized bovine brain H3 histamine receptor coupled to a guanine nucleotide-binding protein.

The H3 receptor is a high-affinity histamine receptor that inhibits release of several neurotransmitters, including histamine. We have characterized H3 receptor binding in bovine brain and developed conditions for its solubilization. Particulate [3H]histamine binding showed an apparently single class of sites (KD = 4.6 nM; Bmax = 78 fmol/mg of protein). Of the detergents tested, digitonin at a detergent/protein ratio of 1:1 (wt/wt) yielded the greatest amount of solubilized receptors, typically 15-30% of particulate binding. Neither equilibrium binding of [3H]histamine to receptors (KD = 6.1 nM; Bmax = 92 fmol/mg of protein) nor the inhibitor profile was substantially altered by digitonin solubilization. However, solubilization did increase the rate of [3H]histamine association with and dissociation from the receptor. Size-exclusion chromatography indicated an apparent molecular weight of 220,000 for the solubilized receptor, and peak binding from this column retained its guanine nucleotide sensitivity. These last two observations are consistent with the solubilized receptor occurring in complex with a guanine nucleotide-binding protein.

Animals↗

PubMed1992

Rat adrenal cortex is a source of a circulating ouabainlike compound.

To determine if the adrenal gland may be the source of plasma-borne ouabainlike compound (OLC) in rats, we 1) measured immunoreactivity expressed as OLC equivalents in extracts from a wide variety of central and peripheral tissues and, for adrenal cortex and medulla, chromatographed the extracts to determine to what extent immunoreactivity in the adrenal was OLC, and 2) measured OLC in the plasma of adrenalectomized and adrenal demedullectomized rats. The highest levels of immunoreactivity were found in adrenal cortex, adrenal medulla, atria, and the pituitary. Based on high-performance liquid chromatographic retention time, immunoreactivity in the adrenal cortex was almost exclusively immunoreactive OLC. Removal of this rich source of OLC from rats resulted in an approximate 50% decrease in circulating levels of OLC by 6 days after removal. Furthermore, although adrenal demedullectomy also caused a decrease in OLC 3 days after surgery, the decline was sustained only with total adrenalectomy, in that plasma levels of OLC in demedullectomized rats 6 days after surgery had returned to levels equal to those of sham controls. Taken together, these findings strongly suggest that the adrenal cortex is a major contributor to circulating OLC in the rat.

Adrenal Cortex↗

PubMed1992

Asphyxial deaths from the recreational use of nitrous oxide.

The recreational use of nitrous oxide is widespread. Nitrous oxide for recreational use is usually obtained from anesthesia tanks or whipped-cream machine chargers or cans. Twenty previously described deaths associated with recreational nitrous-oxide use describe anesthesia tanks and whipped-cream machine dispensors as a source. Five deaths associated with nitrous oxide use are presented; two involving whipped-cream cannisters as the source, two involving anesthesia tanks, and one involving a racing fuel tank as a source of nitrous oxide. Autopsy findings in our cases were subtle or negative, but usually suggestive of asphyxia. Through a laboratory simulation, we have confirmed that nitrous oxide displaces oxygen in a closed space, which probably leads to asphyxia. A review of the literature, neuropharmacology, and pathophysiology of nitrous oxide use is also presented.

Adolescent↗

PubMed1992

U.S. Navy and Marine Corps recruit training deaths in San Diego, California, 1973-1985; a review of 31 cases.

The deaths of military recruits associated with training activities nearly always fall under close scrutiny from relatives of the deceased recruit and the media. The literature contains isolated case reports of recruit deaths but no comprehensive reviews of all deaths at a single training facility. The purpose of this study is to describe the circumstances and causes of all recruit deaths occurring at the Naval Training Command and the Marine Corps Recruit Depot in San Diego, California, from 1973 through 1985. Thirty-one male recruits died in training during this period; eight died from medical conditions not detected by preenlistment questioning or examination. In five of these cases, the conditions were probably known to the recruit but were not listed on a medical history form. Seven recruits died in incidents related to training, and there were six cases of "sudden cardiac death," as well as eight deaths caused by infectious diseases.

Accidents↗

PubMed1991

Cloning of a phospholipase A2-activating protein.

Recently we have described the isolation and biochemical characterization of a phospholipase A2-activating protein (PLAP). We have cloned this protein and found it to be expressed as a 2.5-kilobase mRNA. The steady-state levels of PLAP mRNA are induced in smooth muscle and endothelial cells following treatment with leukotriene D4. The increased message levels coincide with increased amounts of PLAP. Synthetic antisense DNA was used to block the synthesis of PLAP and this treatment effectively blocked the activation of phospholipase A2 and the increased generation of prostanoids in smooth muscle and endothelial cells treated with leukotriene D4.

Amino Acid Sequence↗

PubMed1991

Specimens for teaching forensic pathology, odontology, and anthropology. I. Soft tissue.

A set of specimens has been collected, preserved, and organized specifically for the teaching of forensic pathology, odontology, and anthropology. Plastination of soft tissue, whole organs, bones, and teeth has proven valuable in preserving delicate, friable, and calcined specimens. The dry, odorless, biologically inert specimens are durable and resistant to damage caused by handling. Subtle features of soft tissue pathology are well preserved. Patterned injuries change due to shrinkage, but remain easily recognizable. Plastinated whole jaws are still readily identifiable from antemortem records. Radiographic density is essentially unchanged. Putrid and charred specimens become quite manageable.

Anthropology, Physical↗

PubMed1991

Specimens for teaching forensic pathology, odontology, and anthropology. II. Teeth and bone.

A set of specimens has been collected, preserved, and organized specifically for the teaching of forensic pathology, odontology, and anthropology. Plastination of soft tissue, whole organs, bones, and teeth has proven valuable in preserving delicate, friable, and calcined specimens. The dry, odorless, biologically inert specimens are durable and resistant to damage caused by handling. Subtle features of soft tissue pathology are well preserved. Patterned injuries change due to shrinkage, but remain easily recognizable. Plastinated whole jaws are still readily identifiable from antemortem records. Radiograph density is essentially unchanged. Putrid and charred specimens become quite manageable.

Anthropology↗

PubMed1991

Monoclonal antibodies differentiate between the haemagglutinating and the receptor-destroying activities of bovine coronavirus.

A relatively simple and sensitive method is described which enables the effect of monoclonal antibodies (MAbs) on the receptor-destroying enzyme (RDE) and the haemagglutination (HA) activity of bovine coronavirus (BCV) to be analysed in one assay. A lysate of HRT-18 cells infected with the L9 strain of BCV was found to have a higher RDE:HA ratio than purified virus. At 4 degrees C the lysate induced an HA pattern which completely disappeared upon raising of the temperature to 37 degrees C. This L9-infected cell lysate was used to determine the HA inhibition (HAI) titres of MAbs directed against the surface glycoproteins S and HE of BCV. Thereafter, the test plates were incubated at 37 degrees C to enable the ability of the MAbs to prevent elution of virus from BCV-erythrocyte complexes to be assessed. No inhibition of RDE was detectable with MAbs against glycoprotein S, which had HAI titres ranging from 1:16 to 1:128. On the other hand, MAbs directed against glycoprotein HE had similar HAI titres, but they inhibited elution of 8 HA units of BCV at titres of up to 1:65,000.

Animals↗

PubMed1991

Modification of the Bacillus sphaericus 51- and 42-kilodalton mosquitocidal proteins: effects of internal deletions, duplications, and formation of hybrid proteins.

The 51- and 42-kDa proteins which constitute the binary mosquitocidal toxin of Bacillus sphaericus 2362 have a low overall sequence similarity but share several regions of near identity (L. Baumann, A. H. Broadwell, and P. Baumann, J. Bacteriol. 170:2045-2050, 1988). By using site-directed mutagenesis, deletions of 6 to 16 amino acids in three of these regions of the 51- and 42-kDa proteins were made, and the modified proteins were expressed in Bacillus subtilis. Deletions in both of these proteins resulted in a loss of toxicity for mosquito larvae. Hybrid proteins containing exchanged fragments of the 51- and 42-kDa proteins were inactive when tested in a variety of combinations, thereby indicating that potentially analogous fragments of these two proteins were not functionally equivalent. An internal duplication of 73 amino acids in the 51-kDa protein and 72 amino acids in the 42-kDa protein resulted in a major reduction in toxicity. These results indicate that the conserved regions of the 51- and 42-kDa proteins are necessary for toxicity to larvae and that the 51- and 42-kDa proteins, despite their sequence similarity, are unique, differing from each other by at least one essential attribute.

Amino Acid Sequence↗

PubMed1991

Molecular characterization of nosA, a Pseudomonas stutzeri gene encoding an outer membrane protein required to make copper-containing N2O reductase.

A Pseudomonas stutzeri gene (nosA) encoding an outer membrane protein was cloned into the broad-host-range vector pRK290 and expressed in a mutant lacking the protein. Deletion analysis identified the approximate extent of the nosA region which was sequenced, and it was found to contain an open reading frame encoding 683 amino acids including a presumed signal sequence of 44 amino acids. The putative processed form had a molecular weight of 70,218, characteristics typical of outer membrane proteins, and considerable amino acid sequence homology with Escherichia coli BtuB. A short stretch of amino acids was homologous with the E. coli TonB-dependent outer membrane proteins, BtuB, IutA, FepA, and FhuA, suggesting a homologous function: interaction with a periplasmic protein or uptake of a specific substrate.

Amino Acid Sequence↗