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Biomedical subjects

M A Clark

Publications and source records attributed to M A Clark.

At least 199 records · Page 11Linked to original sources

Splenectomy for immune thrombocytopenic purpura: Auckland experience 1979-87.

Of 148 elective splenectomies performed in Auckland during 1979-87, 48 were performed as therapy for immune thrombocytopenic purpura (ITP). In all cases medical therapy had either failed to maintain their platelet count (36 patients), or side-effects of medical treatment had made continuation unacceptable (12 patients). The mean age of the group was 31 years and there were 15 males and 33 females. Thirty-four of 48 patients (71%) had a favourable response to splenectomy: platelet count was maintained above 150 x 10(9)/l and medical therapy was no longer required. There was no operative or inhospital mortality. Seven patients experienced postoperative complications (mainly infections) of which none were life-threatening. There was no difference in response between males and females (67% and 73% response rate, respectively), and the response rates of Europeans and non-Europeans were similar (71% and 69%, respectively). By contrast, the average age of the responders [29 years) was almost 10 years younger than the non-responders (38 years). It is concluded that the use of splenectomy as therapy for patients with ITP, who have either failed to respond to medical management or for various reasons cannot tolerate the complications associated with steroid therapy, is a safe procedure which in the present series was associated with no operative mortality and an acceptably low rate of morbidity. Younger ITP patients (less than 30 years) have a better chance of responding favourably to splenectomy than do older patients (greater than 40 years).

Adolescent↗

Vasopressin induces V1 receptors to activate phosphatidylinositol- and phosphatidylcholine-specific phospholipase C and stimulates the release of arachidonic acid by at least two pathways in the smooth muscle cell line, A-10.

The rat thoracic aortic smooth muscle cell line, A-10, expresses vasopressin receptors of the V1 subtype. Vasopressin treatment of these cells stimulated the release of arachidonic acid and the formation of diacylglycerol and phosphocholine. These responses to vasopressin were inhibited by the V1-specific antagonist SK&F 100273, indicating that these were receptor-mediated phenomena. The mechanisms by which V1 receptors mediate arachidonic acid release appeared to be unaffected by cycloheximide or actinomycin D, suggesting that the release is independent of protein and RNA synthesis. The V1 receptors also appeared to be coupled to a phospholipase C which can hydrolyze phosphatidylcholine, a possible source of the released arachidonic acid. Phosphocholine and diacylglycerol were also generated. The release of arachidonic acid, phosphocholine, or diacylglycerol was not affected by prior treatment of the cells with pertussis toxin (islet-activating protein). Thus, the release of these second messengers is not mediated by the guanine nucleotide-binding protein Gi or other pertussis toxin-sensitive substrates. We conclude that V1 receptors induce the release of arachidonic acid and the formation of diacylglycerol and phosphocholine via the activation of both a phosphatidylinositol- and phosphatidylcholine-specific phospholipase C.

Animals↗

Tumour necrosis factor (cachectin) induces phospholipase A2 activity and synthesis of a phospholipase A2-activating protein in endothelial cells.

Tumour necrosis factor (TNF) is an important mediator of endotoxin-induced vascular collapse and other inflammatory reactions. Eicosanoids have been implicated in the pathogeensis of these responses. In order to explore further the potential interactions between TNF and eicosanoid metabolism in eliciting vascular responses, we studied the effects of TNF on the bovine endothelial cell line CPAE. TNF induced cellular retraction observed by light microscope. This morphological change was monitored by the passage of iodinated protein A between adjacent cells and by release of [3H]arachidonic acid metabolites from cells. Both the morphological and functional responses were abrogated by inhibition of eicosanoid synthesis with BW755c. The release of [3H]arachidonic acid metabolites appeared to be mediated by a transient increase in phospholipase A2 activity. Phospholipase C activity was not affected by TNF. The maximal increase in phospholipase A2 activity occurred at 5 min following the addition of TNF. Phospholipase A2 activation, [3H]arachidonic acid-metabolite synthesis and passage of iodinated protein A, required both RNA and protein synthesis and were associated with an increase in the synthesis of a recently described phospholipase A2-activating protein. The Bordetella pertussis toxin, islet-activating protein, also inhibited the increase in phospholipase A2 activity, the release of [3H]arachidonic acid metabolites and the passage of iodinated protein A, suggesting that the TNF receptor-ligand interaction resulting in cellular retraction, phospholipase A2 activation and eicosanoid synthesis, is coupled through the Ni guanine nucleotide regulatory protein in these cells.

Animals↗

Selective purification of cardiac myosin by a high-performance salicylate affinity column.

The cardiac muscle proteins, myosin and actin, were purified in one step using a salicylate-silica affinity column. The affinity columns were prepared by coupling sodium salicylate via its hydroxyl group to an Altex Ultraffinity-EP column. Crude detergent extracts from guinea pig hearts were passed through the column and the myosin-actin complex was then eluted with excess free salicylate or high salt. The affinity of cardiac myosin for immobilized salicylate was unique as myosin heavy chain from guinea pig leg muscle detergent extracts could not be purified by this procedure. Commercially purified rabbit leg muscle myosin also appeared to have no interaction with the salicylate affinity column, suggesting that the column is specific for cardiac myosin.

Amino Acids↗

The role of fatty acid metabolites in the differentiation of the human monocyte-like cell line U937.

The human monocyte cell line, U937, can be induced to terminally differentiate into macrophage-like cells when treated with gamma-interferon. However, if these cells were treated with gamma-interferon and esculetin, an inhibitor of the lipoxygenase pathway, or BW755C, an inhibitor of both the lipoxygenase and the cyclooxygenase pathways, a marked inhibition in cellular differentiation occurred. In contrast, inhibitors of only the cyclooxygenase pathway had no effect on differentiation. These studies suggest a role for lipoxygenase products of arachidonic acid in the differentiation of the human U937 cell line. Arachidonic acid utilized in the production of eicosanoids is derived from phospholipids by the action of phospholipase A2 and phospholipase C. When U937 cells were cultured in medium supplemented with gamma-interferon, there was a striking increase in the level of phosphatidylcholine and phosphatidylethanolamine-specific phospholipase A2 activities and phosphatidylinositol-specific phospholipase C activity as compared to control cells. More ever, although there was not a significant difference in the incorporation of labeled arachidonic acid or linoleic acid into the major phospholipids of differentiated U937 cells as compared to undifferentiated control cells, there was a marked increase in the relative amount of the labeled arachidonic acid released from the differentiated cells as lipoxygenase products compared to cyclooxygenase products. These data suggest that lipoxygenase products may be essential in the differentiation process of U937 cells and that enhanced phospholipase enzyme activities that occur during differentiation help explain how arachidonic acid becomes available to form lipoxygenase products.

Cell Differentiation↗

Sutureless renal repair after low-velocity ballistic trauma.

The tissue adhesive butyl cyanoacrylate was evaluated in the repair of injured porcine kidneys following low-velocity ballistic trauma. This technique was compared to the classic renal repair using chromic suture and a patch of perirenal fat. The repair with butyl cyanoacrylate was less time consuming (four minutes vs. 28 minutes for the controls) and decreased blood loss (67 cc vs. 180 cc for the controls). Ten days post injury the kidneys repaired with tissue adhesive showed minimal intraparenchymal hemorrhage or perirenal bleeding, while two of the four (50%) suture-repaired kidneys showed intrarenal microhemorrhages and one (25%) developed a perirenal hematoma. Perirenal adhesions were noted in both groups. Vital signs, IVP's, creatinine clearance, and serum and urine chemistries were nearly identical in the two groups. The results show that low-velocity ballistic injuries of renal tissue can be repaired effectively and more efficiently with the use of butyl cyanoacrylate when compared to conventional suturing methods.

Animals↗

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators.

Amino Acid Sequence↗

Medical evaluation of the victims of the 1986 Lake Nyos disaster.

A cloud of carbon dioxide gas, with an estimated volume of 1 km3 was released from Lake Nyos, a volcanic crater lake in Cameroon, Africa, causing 1700 to 2000 human fatalities as well as killing thousands of livestock and wild animals. At the request of the Cameroonian Government, the Office of Foreign Disaster Assistance of the U.S. Department of State sent a multidisciplinary team which included 2 forensic pathologists to assist the Government of Cameroon in investigating this natural disaster. The medical evaluation was concentrated in 3 areas: the autopsy of human and animal fatalities, examination and interview of survivors, and examination of the scene of the disaster. Toxicologic specimens were obtained at autopsy, and numerous samples of lake water were collected. The autopsy findings were consistent with asphyxia. The results of chemical analyses excluded many volatiles but not carbon dioxide as the toxic agent. The exact source of this gas continues to be a subject of a heated geologic debate, but fermentation of organic materials in the lake water has been eliminated on the basis of C14 isotope studies. This investigation underlines the value of forensic pathologists in epidemiological studies and in the examination of living persons.

Animals↗

The shielding capacity of the standard military flak jacket against ballistic injury to the kidney.

An animal model was designed to evaluate the ability of the standard military flak jacket to protect the kidneys from direct ballistic trauma. Yorkshire swine were anesthetized and their flanks were protected by flak jackets. Ballistic insults ranging from a low-velocity handgun to a high-velocity rifle were evaluated. The results demonstrate that a flak jacket provides adequate protection against low- and mid-velocity projectiles since none of the animals from this group had evidence of internal trauma and because only minor superficial surgical attention would have been required. Survivability in each case was considered to be excellent. The flak jacket failed to provide any protection against several types of high-velocity bullets. These weapons caused severe internal injury to the genitourinary system as well as to other major organ systems. Survival would have been unlikely.

Animals↗

The effect of sn-2 fatty acid substitution on phospholipase C enzyme activities.

The human monocyte cell line U937 expresses phospholipase A2 and phospholipase C activities and produces eicosanoids. The phospholipase C (PLC) activity exhibits substrate preference for phosphatidyl-choline (PC), rather than phosphatidylinositol or phosphatidylethanolamine. In order to characterize the PLC activity found in these cells, the effects of substitution of the sn-2 fatty acid on this activity were examined. PC substrates with palmitic acid (PC-2P), oleic acid (PC-2O), arachidonic acid (PC-2A) and linoleic acid (PC-2L) at the sn-2 position were used. The sn-1 fatty acid was palmitic acid. PC-2L and PC-2A with the longer-chain less-saturated fatty acids linoleic acid and arachidonic acid esterified at sn-2 were found to be better substrates for PLC activity than PC-2P or PC-2O in these cells. This preference was maintained even when substrate phospholipid was solubilized in non-ionic, anionic, cationic and zwitterionic amphiphiles. Furthermore, when a 500-fold excess of 1,2-diolein or 1,2-dipalmitin was added to the reaction, the specificity of the PLC activity for PC-2A and PC-2L remained unchanged. When similar experiments were performed with phosphatidylinositol as a substrate, we did not observe any effect when the sn-2 position was altered. These data show that the fatty acid constituent at the sn-2 position affects the observed PLC activity when phosphatidylcholine, but not phosphatidylinositol, is used as a substrate by these cells.

Binding Sites↗

Identification and isolation of a mammalian protein which is antigenically and functionally related to the phospholipase A2 stimulatory peptide melittin.

Antibodies prepared against the phospholipase A2 stimulatory peptide melittin were used to identify and isolate a novel mammalian protein with similar functional and antigenic properties. The mammalian protein of Mr 28,000 was isolated from cell sonicates by high performance immunoaffinity chromatography and size exclusion chromatography. This stimulatory protein was stable for several months when frozen at -70 degrees C. The purified protein selectively stimulated phospholipase A2 when phosphatidylcholine was used as a substrate but had no effect on phospholipase A2 activity when phosphatidylethanolamine was used as a substrate. Furthermore, this protein had no effect on phospholipase C activity or on pancreatic or snake venom phospholipase A2. The stimulatory activity was unaffected by RNase or DNase treatment. However, boiling or trypsin digestion inactivated the phospholipase stimulatory activity. The mechanism of phospholipase A2 stimulation appeared to result from an increase in the apparent Vmax of the enzyme.

Animals↗

Stimulus-specific induction of phospholipid and arachidonic acid metabolism in human neutrophils.

Phospholipid remodeling resulting in arachidonic acid (AA) release and metabolism in human neutrophils stimulated by calcium ionophore A23187 has been extensively studied, while data obtained using physiologically relevant stimuli is limited. Opsonized zymosan and immune complexes induced stimulus-specific alterations in lipid metabolism that were different from those induced by A23187. [3H]AA release correlated with activation of phospholipase A2 (PLA2) but not with cellular activation as indicated by superoxide generation. The latter correlated more with calcium-dependent phospholipase C (PLC) activation and elevation of cellular diacylglycerol (DAG) levels. When cells that had been allowed to incorporate [3H]AA were stimulated with A23187, large amounts of labeled AA was released, most of which was metabolized to 5-HETE and leukotriene B4. Stimulation with immune complexes also resulted in the release of [3H]AA but this released radiolabeled AA was not metabolized. In contrast, stimulation with opsonized zymosan induced no detectable release of [3H]AA. Analysis of [3H]AA-labeled lipids in resting cells indicated that the greatest amount of label was incorporated into the phosphatidylinositol (PI) pool, followed closely by phosphatidylcholine and phosphatidylserine, while little [3H]AA was detected in the phosphatidylethanolamine pool. During stimulation with A23187, a significant decrease in labeled PI occurred and labeled free fatty acid in the pellet increased. With immune complexes, only a small decrease was seen in labeled PI while the free fatty acid in the pellets was unchanged. In contrast, opsonized zymosan decreased labeled PI, and increased labeled DAG. Phospholipase activity in homogenates from human neutrophils was also assayed. A23187 and immune complexes, but not zymosan, significantly enhanced PLA2 activity in the cell homogenates. On the other hand, PLC activity was enhanced by zymosan and immune complexes. Stimulated increases in PLC activity correlated with enhanced superoxide generation induced by the stimulus.

Adult↗

A Pseudomonas stutzeri outer membrane protein inserts copper into N2O reductase.

Among a set of frameshift mutagen (ICR-191; Polysciences, Inc.)-induced mutations that confer inability to grow anaerobically with N2O as the sole electron acceptor, one class was found that produced an inactive N2O reductase which lacked copper. All of these mutant strains failed to produce a 61,000-Mr protein located in the outer membrane. This protein, termed NosA, seems not to be responsible for bringing copper into the cell because the mutant strains and their parent were similarly sensitive to the copper content of the growth medium and no intermediate copper concentration in the medium permitted the mutant strains (nosA) to grow anaerobically with N2O as the sole electron acceptor. We conclude that NosA is necessary to insert copper into N2O reductase or to maintain it there.

Bacterial Outer Membrane Proteins↗

The phs gene and hydrogen sulfide production by Salmonella typhimurium.

Salmonella typhimurium produces H2S from thiosulfate or sulfite. The respective pathways for the two reductions must be distinct as mutants carrying motations in phs, chlA, and menB reduced sulfite, but not thiosulfate, to H2S, and glucose repressed the production of H2S from thiosulfate while it stimulated its production from sulfite. The phs and chlA mutants also lacked a methyl viologen-linked thiosulfate reductase activity present in anaerobically grown wild-type cultures. A number of hydroxylamine, transposon Tn10 insertion, and Mu d1(Apr lac) operon fusion mutants defective in phs were characterized. One of the hydroxylamine mutants was an amber mutant, as indicated by suppression of its mutation in a supD background. The temperature-sensitive phs mutants produced H2S and methyl viologen-linked thiosulfate reductase at 30 degrees C but not at 42 degrees C. The reductases in all such mutants grown at 30 degrees C were as thermostable as the wild-type enzyme and did not differ in electrophoretic relative mobility, suggesting that phs is not the structural gene for thiosulfate reductase. Expression of beta-galactosidase in phs::Mu d1(Apr lac) mutants was dependent on anaerobiosis and the presence of reduced sulfur. It was also strongly influenced by carbon source and growth stage. The results are consistent with a model in which the phs gene encodes a regulatory protein essential for the reduction of thiosulfate to hydrogen sulfide.

Genes, Bacterial↗

Effect of auranofin and other gold complexes on the activity of phospholipase C.

Auranofin (AF) is an orally active chrysotherapeutic agent used for the treatment of rheumatoid arthritis, a self-perpetuating inflammatory disease. Because of reports suggesting that AF and other gold complexes can, under certain circumstances, exacerbate rheumatoid inflammatory lesions in humans and adjuvant arthritic rats and that phospholipase C (PLC) and phospholipase A2 activities are increased in rheumatoid patients, the effects of AF and a related gold complex on in situ mammalian and purified Bacillus cereus PLC were examined. Results of our studies show that 1) AF and triethylphosphine gold chloride (TEPG), an AF analog, stimulated PLC activity in the sonicate of RAW 264.7 macrophages; 2) AF and TEPG stimulated B. cereus PLC activity in a concentration-dependent manner, but the pattern of stimulation and concentrations of drugs required to stimulate the purified enzyme differ from those seen with the macrophage PLC; 3) metals (cobalt and zinc) and sulfhydryl reagents (N-ethylmaleimide, iodoacetic acid, and glutathione), tested at the same concentrations of AF that enhanced PLC activity, had no effect on the enzyme. These data suggest that stimulation of PLC may be a generic phenomenon since two divergent PLCs are affected by gold complexes. Additionally, these studies may provide one potential explanation for rheumatoid lesion flares seen in patients and animals on chrysotherapy.

Animals↗

A fatal wound from an unusual military projectile: potential dangers of live military ordnance to the autopsy pathologist.

During ground maintenance on an F-14 aircraft, a worker was removing the Mark 124 cartridge activated devices (CADS) from the aircraft when a second worker entered the cockpit of the aircraft and energized the electrical system, causing the four CADS to detonate. One of the four CADS became an airborne projectile. It struck the first worker in front of the right arm, passed through his chest, and became embedded in his thoracic spine. An immediate concern at autopsy was whether or not the device retained any explosive potential. Recommendations for autopsy procedures in cases involving military ordnance are discussed.

Accidents, Occupational↗

Inhibition of phosphoinositide-specific phospholipase C by manoalide.

Manoalide is a novel sesterterpenoid which has previously been shown to be a potent inhibitor of venom phospholipases A2. To determine whether manoalide inhibited other phospholipases, the sensitivity of phosphoinsitide-specific phospholipase C (PI-PLC) to inactivation by manoalide was examined using crude cytosolic PI-PLC and a PI-PLC purified to homogeneity from guinea pig uterus cytosol (PI-PLC I). Manoalide inhibited both cytosolic and purified PI-PLC I in a concentration-dependent fashion, exhibiting an IC50 of 3-6 microM. Inactivation of PI-PLC I was calcium- and pH-dependent, with greater inactivation occurring at alkaline pH. Manoalide inhibited hydrolysis of all three phosphoinositides by purified PI-PLC I. The substrate kinetics of PI-PLC I suggest that manoalide does not inhibit purified PI-PLC I by simple competitive or noncompetitive inhibition. Enzyme activity was not recovered after dialysis of manoalide-treated PI-PLC I, indicating that inactivation of PI-PLC I was irreversible. To determine whether manoalide inhibited PI-PLC in cells, the effects of manoalide on norepinephrine (NE)-stimulated phosphoinositide hydrolysis and calcium mobilization were investigated in a smooth muscle-like cell line, DDT1MF-2. Manoalide inhibited NE-induced inositol 1,4,5-trisphosphate and inositol 1-phosphate formation in a concentration-dependent manner. The IC50 for inhibition of inositol 1-phosphate formation was 1.5 microM. Manoalide also inhibited NE-induced calcium transients in DDT1MF-2 cells, exhibiting an IC50 of 2 microM. These data suggest that inhibition of PI-PLC may account, in part, for the anti-inflammatory actions of manoalide.

Animals↗