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Biomedical subjects

C A West

Publications and source records attributed to C A West.

At least 37 records · Page 2Linked to original sources
PubMed1994

Dietary copper deficiency increases the mast cell population of the rat.

Mast cell-released histamine has been implicated in the enhanced acute inflammatory response of copper-deficient rats. The present study examined possible changes in the copper-deficient mast cell which may account for increased macromolecular leakage and edema formation. Mast cell populations were determined in the cremaster muscle of copper-adequate and copper-deficient rats. Total histamine content, unstimulated histamine release and concentration-dependent histamine release with the mast cell secretagogue compound 48/80 were also determined in isolated peritoneal mast cells. A significantly higher number of mast cells were found in the cremaster muscle of the copper-deficient rats (78 +/- 7 cells/5 microns section) than in the copper-adequate controls (51 +/- 4). Total histamine content per cell as well as unstimulated and stimulated release of the inflammatory mediator per cell were not different between the groups. The results suggest that dietary copper deficiency increases the mast cell population but does not alter the mast cell histamine content or sensitivity to degranulation in the rat. This increase in the number of mast cells may be a mechanism by which acute inflammation is enhanced in copper deficiency.

Animals↗
PubMed1993

The neurotoxicity of sulfur-containing amino acids in energy-deprived rat hippocampal slices.

The rat hippocampal slice preparation and its electrophysiology were used to assess the toxicity of two sulfur-containing amino acids, L-cysteate (CA) and L-cysteine (CYS). Both compounds were innocuous under normal conditions but became toxic in energy-deprived (lack of oxygen or glucose) slices. CA and CYS toxicity was apparent as both reduced the number of slices that normally recover their neuronal function (evoked CA1 population spike) after a standardized period of hypoxia or glucose deprivation (GD). The competitive N-methyl-D-aspartate (NMDA) antagonist DL-2-amino-5-phosphonovalerate blocked the toxicity of both CA and CYS in hypoxic slices, but it was effective only against CYS toxicity in glucose-deprived slices. The glycine antagonist 7-chlorokynurenate blocked CA and CYS toxicity in hypoxic slices but was unable to block their toxicity in glucose-deprived tissue. Perfusing slices with medium containing a high magnesium concentration blocked the toxicity of CA in both hypoxic and glucose-deprived slices, a treatment that was ineffective against CYS toxicity under either condition. Calcium depletion from the perfusion medium completely blocked the damaging effect of both amino acids in hypoxic slices, but it only partially blocked the toxicity of CA and did not block that of CYS in glucose-deprived slices. These results suggest that CA and CYS activate different NMDA receptor subsets and other glutamate receptor subtypes. Moreover, the results indicate a possible difference between the mechanism that lead to hypoxic neuronal damage and the one that lead to hypoglycemic neuronal damage.

Amino Acids, Sulfur↗
PubMed1992

The excitotoxicity of heterocyclic dicarboxylic acids in rat hippocampal slices: structure-activity relationships.

The structural resemblance of certain heterocyclic dicarboxylates to aspartate and glutamate led investigators to study their potency as agonists and antagonists of the N-methyl-D-aspartate (NMDA) receptor. The sensitivity of hypoxic rat hippocampal slices to NMDA ligands is several fold greater than that of normoxic slices. In the present study, the excitotoxic potency of heterocyclic dicarboxylates was assessed electrophysiologically by measuring their ability to enhance hypoxic and hypoglycemic neuronal damage in the rat hippocampal slice preparation. Four compounds were tested: quinolinate (QUIN), 4,5-imidazole-dicarboxylate (IZDA), 1,2,3-triazole-4,5-dicarboxylate (TZDA), and 2,3-pyrazinedicarboxylate (PZDA). QUIN was the most toxic drug in enhancing both hypoxic and hypoglycemic neuronal damage. IZDA and TZDA were slightly less toxic than QUIN, while PZDA was innocuous. The effect of the 3 active drugs was blocked by the NMDA competitive antagonist DL-2-amino-5-phosphonovalerate. The sequence -N-CH(COOH)-CH(COOH)- appears to be a prerequisite for a heterocyclic dicarboxylate to exert NMDA-type agonistic properties. A 5-membered ring heterocyclic compound which contains more than one nitrogen atom in its ring retains its NMDA-type toxicity while a 6-membered ring with more than one nitrogen atom (PZDA) does not.

Animals↗
PubMed1992

Biosynthesis of rice phytoalexins: identification of putative diterpene hydrocarbon precursors.

A procedure for the preparation of a cell-free enzyme solution from rice leaves capable of catalyzing the biosynthesis of diterpene hydrocarbons from geranylgeranyl pyrophosphate or copalyl pyrophosphate as added substrates has been developed. The rates of synthesis of a group of "pimaradiene-like" diterpene hydrocarbons are about 75-fold higher with geranylgeranyl pyrophosphate as substrate and about 8-fold higher with copalyl pyrophosphate as substrate in comparison with extracts from untreated control leaves. The maximum rate of diterpene hydrocarbon biosynthesis is seen in extracts prepared at 40 h after uv irradiation. Five diterpene hydrocarbons (compounds A-E) were present in the hydrocarbon fraction biosynthesized from [3H]geranylgeranyl pyrophosphate in large-scale incubation mixtures prepared from uv-treated rice leaves. Three of these diterpenes were identified as ent-kaur-16-ene (B), ent-sandaracopimara-8(14), 15-diene (D), and 9 beta H-pimara-7,15-diene (E) from GC retention times and GC-MS spectral characteristics in comparison with those of authentic reference compounds. Compound C has spectral characteristics analogous to those of a pimaradiene, but a specific structural assignment from the data available was not possible. Similar incubations with [3H]copalyl pyrophosphate as the substrate and enzyme prepared from uv-treated rice leaves produced ent-kaurene (B), ent-sandaracopimara-8(14),15-diene (D), and compound C, but not 9 beta H-pimara-7,15-diene (E). These results are consistent with a proposed biosynthetic scheme in which 9 beta H-pimara-7,15-diene serves as a precursor of the momilactone family, and ent-sandaracopimara-8(14),15-diene serves as a precursor of the oryzalexin family of rice phytoalexins. ent-Kaurene was the only diterpene detected in incubation mixtures containing enzyme extract from untreated rice leaves and [3H]copalyl pyrophosphate as the substrate. It is suggested that kaurene biosynthesis in rice leaves is probably associated with gibberellin biosynthesis and the regulation of vegetative growth rather than stress metabolism. The diterpene cyclization enzymes in extracts of uv-treated rice leaves show only a relatively modest inhibition by the plant growth retardants AMO-1618 and Phosfon D. No evidence was obtained for the subcellular localization of these cyclization enzymes in organellar preparations; it is tentatively concluded that the enzymes are present predominantly in the extraorganellar cytoplasm of rice leaves.

Alkyl and Aryl Transferases↗
PubMed1992

Functional expression in Escherichia coli of proteins B and C from soluble methane monooxygenase of Methylococcus capsulatus (Bath).

Methylococcus capsulatus (Bath) uses a soluble methane monooxygenase (sMMO) to catalyse the oxidation of methane to methanol. sMMO is comprised of three components; A, B and C. Protein C (the reductase) transfers electrons from NADH to protein A (the hydroxylase) which contains the active site, and protein B regulates this electron flow. The five genes encoding the sMMO proteins and their subunits are clustered and have been cloned in Escherichia coli. A DNA fragment containing mmoB, the gene encoding protein B, was subcloned into pT7-5, a plasmid of the T7 RNA polymerase promoter expression system. Upon induction, E. coli expressed protein B which was fully functional after purification. The gene encoding protein C, mmoC, was amplified with unique restriction sites at each end using the polymerase chain reaction and then subcloned into pT7-7 (a plasmid similar to pT7-5 but containing its own ribosome-binding site and ATG start codon). Protein C expressed in E. coli was also found to be functional. This is the first report of the functional expression of methanotroph methane monooxygenase genes in a heterologous host and represents a significant step forward in our analysis of the assembly and catalysis of sMMO.

Base Sequence↗
PubMed1992

Elicitation of Diterpene Biosynthesis in Rice (Oryza sativa L.) by Chitin.

Cell-free extracts of UV-irradiated rice (Oryza sativa L.) leaves have a much greater capacity for the synthesis from geranylgeranyl pyrophosphate of diterpene hydrocarbons, including the putative precursors of rice phytoalexins, than extracts of unstressed leaves (KA Wickham, CA West [1992] Arch Biochem Biophys 293: 320-332). An elicitor bioassay was developed on the basis of these observations in which 6-day-old rice cell suspension cultures were incubated for 40 hours with the substance to be tested, and an enzyme extract of the treated cells was assayed for its diterpene hydrocarbon synthesis activity as a measure of the response to elicitor. Four types of cell wall polysaccharides and oligosaccharide fragments that have elicitor activity for other plants were tested. Of these, polymeric chitin was the most active; a suspension concentration of approximately 7 micrograms per milliliter gave 50% of the maximum response in the bioassay. Chitosan and a branched beta-1,3-glucan fraction from Phytophthora megasperma f. sp. glycinea cell walls were only weakly active, and a mixture of oligogalacturonides was only slightly active. A crude mycelial cell wall preparation from the rice pathogen, Fusarium moniliforme, gave a response comparable to that of chitin, and this activity was sensitive to predigestion of the cell wall material with chitinase before the elicitor assay. N-Acetylglucosamine, chitobiose, chitotriose, and chitotetrose were inactive as elicitors, whereas a mixture of chitin fragments solubilized from insoluble chitin by partial acid hydrolysis was highly active. Constitutive chitinase activity was detected in the culture filtrate and enzyme extract of cells from a 6-day-old rice cell culture; the amount of chitinase activity increased markedly in both the culture filtrate and cell extracts after treatment of the culture with chitin. We propose on the basis of these results that soluble chitin fragments released from fungal cell walls through the action of constitutive rice chitinases serve as biotic elicitors of defense-related responses in rice.

Journal Article↗
PubMed1991

Neurotoxicity of quinolinic acid and its derivatives in hypoxic rat hippocampal slices.

The excitotoxicity of quinolinic acid (2,3-pyridinedicarboxylic acid), a potent endogenous N-methyl-D-aspartate (NMDA)-type agonist, was characterized in the hypoxic hippocampal slice preparation. A series of other pyridinedicarboxylic acids was also tested in this preparation in order to obtain information about the structural requirements for the interaction between the NMDA receptor and its agonists. Of the 7 pyridinedicarboxylic acids tested, only quinolinic acid and its anhydride exerted their excitotoxicity by enhancing hypoxic neuronal damage in rat hippocampal slices at a relatively low concentration (100 microM). Much higher concentration (1 mM) of 3,4-pyridinedicarboxylic acid was required to exhibit any enhancement of hypoxic neuronal damage. The rest of the derivatives were innocuous. The effect of quinolinic acid was blocked by DL-2-amino-5-phosphonovaleric acid, by elevated magnesium levels in the incubation medium or by perfusion with a medium depleted of calcium. Aglycemic damage was also enhanced by quinolinic acid. It appears from the present study that two adjacent carboxylic groups on the pyridine ring, preferably at positions 2 and 3, are a prerequisite for an interaction between the NMDA receptor and its agonist. However, other factors may have great influence on that interaction as was evident from the total impotency of 6-methyl-quinolinic acid. The hypoxic hippocampal slice preparation and its neuronal function is an inexpensive model system, sensitized to the neurotoxins, and thus, allows the easy screening and evaluation of potential ligands of the glutamate receptor and its subtypes.

2-Amino-5-phosphonovalerate↗
PubMed1990

Characterization of the adenosine deaminase-adenosine deaminase complexing protein binding reaction.

Glutaraldehyde-fixed membranes from rabbit kidney cortex were used to characterize binding of monomeric adenosine deaminase to the adenosine deaminase complexing protein. With the use of bovine adenosine deaminase it was shown that enzyme binding is a saturable, high affinity process. The K value for binding of the bovine enzyme was 11 nM. Maximum enzyme binding and rate of binding to a constant amount of membrane did not vary significantly from pH 5.0 to 9.5. Metal ions, with the exception of Hg2+, sulfhydryl reagents, and other proteins had little or a slightly stimulatory effect on maximum binding. Mercuric ion inhibited binding. Using biotinylated bovine adenosine deaminase it was shown that purified rabbit, human, and monkey enzymes compete for binding sites on fixed membranes. The K values for the rabbit and human enzymes were 9 and 6 nM, respectively. Mouse or guinea pig adenosine deaminase did not bind to the membranes or compete with the biotinylated bovine enzyme for binding sites. The retention of characteristics required for binding by enzymes from rabbit, human, monkey, and calf tissues argues for biologic significance of the adenosine deaminase-complexing protein interaction. The basis for the apparent failure of rodent adenosine deaminase to bind to complexing protein remains to be determined.

Adenosine Deaminase↗
PubMed1990

Regulation of expression of the casbene synthetase gene during elicitation of castor bean seedlings with pectic fragments.

A castor bean cDNA library has been constructed in the expression vector lambda gt11. Screening of 30,000 plaques with antibodies against casbene synthetase yielded six positive clones, from which three purified clones were obtained after sequential screening. The cDNA inserts in these clones ranged in size from 200 to 500 base pairs and were shown to share homologous sequences. One of the clones (pCS4) was examined in hybrid-selected and hybrid-arrested translation experiments and shown to contain a partial sequence of the casbene synthetase coding region. Northern analysis using the pCS4 clone as a probe revealed that the total hybridizable casbene synthetase mRNA amount increased to a maximum at 6 h after treatment of seedlings with pectic fragment elicitor and then decreased steadily with almost the same kinetics as observed previously for the changes in the translatable casbene synthetase mRNA activity under these same conditions. Run-on transcription experiments were performed with nuclei isolated from castor bean seedlings at various time periods after treatment with pectic fragment elicitors. Transcription of the casbene synthetase gene was first detected at 2 h after elicitation, rose to a maximum at 5 h, and fell rapidly thereafter. These results suggest that the accumulation of casbene synthetase, and hence casbene, is governed primarily by the rate of transcription of the casbene synthetase gene during elicitation by pectic fragments.

Ricinus communis↗
PubMed1990

Determinants of survival following repair of interrupted aortic arch in infancy.

Between January 1971 and March 1987, surgery was performed in 26 infants with interrupted aortic arch. At operation the 14 boys and 12 girls weighted between 1.71 and 4.23 kg (mean +/- SD = 3.1 +/- 0.63 kg) and ranged in age from 2 to 90 days (13 +/- 18 days). The interruption was distal to the left subclavian artery in 4 (15%), between the left carotid and subclavian arteries in 20 (77%) and between the brachiocephalic (innominate) and left carotid arteries in 2 (8%). Associated complex cardiac lesions in 8 patients included complete transposition (2), common arterial trunk (2), aortopulmonary window (2), double inlet left ventricle (1) and tricuspid atresia (1). The remaining patients had an isolated ventricular septal defect. The arch was reconstructed with a prosthetic conduit in 14 patients; by a direct anastomosis in 6; using the subclavian artery in 3; and with the pulmonary trunk and the arterial duct in 2. Twenty patients (77%) underwent palliative surgery as the first stage of management, and banding of the pulmonary trunk was also performed in 16 of these. Five patients (19%) underwent primary complete repair of the interruption and intracardiac anomalies. One patient (4%) died soon after thoracotomy for palliative surgery. Of the 15 (57%, 70% confidence limits CL = 46-69%) early deaths, 7 occurred in patients with complex associated defects and 4 occurred when single stage repair was attempted. Survival following first-stage palliative surgery for arch interruption with isolated ventricular septal defect was 64% (9/14) [70% CL = 47-79%]. All of these patients subsequently underwent complete repair. Chi-squared and t-tests showed the year of operation and the type of operation (two-stage repair) to be associated with improved survival. It is concluded that a two-stage repair of interrupted aortic arch offers a reasonable alternative to primary complete correction and will lead to satisfactory subsequent repair in most cases.

Anastomosis, Surgical↗
PubMed1990

Localization of adenosine deaminase and adenosine deaminase complexing protein in rabbit heart. Implications for adenosine metabolism.

The distribution of adenosine deaminase and adenosine deaminase complexing protein in rabbit heart has been compared using immunohistochemical staining procedures. Sections (4-5 microns) of tissue fixed in Clarke's solution or paraformaldehyde and embedded in paraffin were stained by the peroxidase anti-peroxidase method for adenosine deaminase or complexing protein, using affinity purified antibodies. Staining for adenosine deaminase and complexing protein was observed in the central myocardium of all heart chambers. Adenosine deaminase was detected in endothelial cells of blood vessels and adjacent pericytes. The nuclei of arteries stained heavily for adenosine deaminase, whereas those of venules and small veins, although positive, stained much more lightly. The cytoplasm of blood vessel endothelial cells and smooth muscle cells of the tunica media were also weakly positive for adenosine deaminase. Endothelial cells of the endocardium and epicardium did not stain. Randomly distributed mononuclear inflammatory cells and interstitial connective tissue fibroblasts were also negative for adenosine deaminase. These results raise the possibility that endothelial cells containing adenosine deaminase could serve as a metabolic barrier preventing the free exchange of plasma and interstitial adenosine. Positive staining for complexing protein was restricted to blood vessel endothelial cells, especially cytoplasmic processes. Colocalization experiments carried out with biotinylated primary antibodies indicate that some vessels are positive for both adenosine deaminase and complexing protein. This is the first experimental evidence of possible in situ association of adenosine deaminase and complexing protein.

Adenosine↗
PubMed1989

Electrophysiology of energy metabolism and neuronal function in the hippocampal slice preparation.

The brain slice preparation offers a unique opportunity to study synaptic function in vitro. Employing electrophysiological methods to measure synaptic activity, we manipulated the extracellular environment of the rat hippocampal slice preparation: (1) by exposing it to different degrees of hypoxia, (2) by changing the levels of glucose, (3) lactate, and (4) H+, separately and in combination with each other. The lower the oxygen level during hypoxia and the longer its duration were, the lower was the recovery rate of synaptic function in the slice upon restoration of oxygenation. Reduction or complete depletion of glucose from the perfusion medium had similar effects, although synaptic function could recover after longer periods of glucose lack as compared with oxygen lack. Reduction in the levels of both oxygen and glucose had an additive effect on the recovery rate of synaptic function when compared with the effect of each of them alone. 'Hyperglycemic' concentration of glucose prolonged the hypoxic period slices could tolerate. Acidosis, induced either by lactic acid or HCl, had no adverse effect on hypoxic slices when the pH was held at or above 6.0 or when lactic acid concentration was below 20 mM. At 10 mM, lactic acid appeared to have a beneficial effect on hypoxic slices. Consequently, it was found that lactate can replace glucose as the sole aerobic energy substrate to support synaptic function in cerebral tissue in vitro.

Action Potentials↗
PubMed1989

Protection against cerebral hypoxia by local anesthetics: a study using brain slices.

The ability of the local anesthetics lidocaine, 2-chloroprocaine and cocaine to protect neuronal tissue against hypoxic damage was evaluated. Rat hippocampal slices were incubated with non-depressive doses of these agents 60 min prior to their exposure to 15 min hypoxia. The rate of recovery of synaptic function (evoked field potentials) following the hypoxic episode was used as an index of hypoxic damage. Slices treated with 0.1 mM of any of the three local anesthetics exhibited a significant increase in the recovery rate of synaptic function from hypoxia as compared to control, untreated slices. These results indicate that local anesthetics, by reducing neuronal sodium influx (and possibly its concomitant calcium influx) which occurs upon hypoxic depolarization, are able to prolong the hypoxic insult a cerebral tissue could tolerate.

Action Potentials↗
PubMed1989

Homologues of catalytic domains of Cellulomonas glucanases found in fungal and Bacillus glycosidases.

We demonstrate homology between the catalytic domains of exoglucanase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) from Cellulomonas fimi and those of endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8) from Bacillus sp. strain C-125 and the fungus Cryptococcus albidus; and between the catalytic domains of endoglucanase (1,4-(1,3:1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) from Cellulomonas fimi and exoglucanase II from Trichoderma reesei. These five enzymes apparently evolved by reshuffling of two catalytic domains and several substrate-binding domains.

Actinomycetales↗
PubMed1989

Elicitation of lignin biosynthesis and isoperoxidase activity by pectic fragments in suspension cultures of castor bean.

Suspension cultures of castor bean (Ricinus communis L.) which have been treated with pectic fragment elicitor rapidly accumulate lignin as measured by derivatization with thioglycolic acid. The responsiveness of cultured cells to elicitor is dependent on the stage of culture growth. In 6-day (maximally responsive) cultures, increases in lignin are first evident 3 hours after addition of pectic fragment elicitor with maximal rates of lignin synthesis between 4 and 10 hours. The abundance of lignin in cultures after 12 hours of elicitor treatment is 10- to 20-fold higher than in untreated control cultures and can thereby account for as much as 2% of the dry cell weight. Only intermediate sizes of pectic oligomer are active as elicitors of lignin. Half-maximal accumulation of lignin occurs at 250 to 300 micrograms per milliliter of an optimal elicitor preparation with an average degree of polymerization of seven. We consider the synthesis of lignin in elicited cultures to be a mechanism of plant disease resistance which is induced by the elicitor. Plant peroxidases have been proposed to catalyze the last enzymatic steps in the biosynthesis of both lignin and hydrogen peroxide. Six extracellular isoenzymes of peroxidase (two anionic, designated A1 and A2, and four cationic, designated C2, C3, C4, and C7) are detectable in healthy suspension cultures of castor bean by native gel electrophoresis. Treatment of cultures with elicitor causes substantial changes in the activity of four of these species (A1, C2, C3, and C7). Elicitor treatment also results in the appearance of three new peroxidase isoenzymes that are not readily detectable in healthy cultures (C1, C5, and C6). Increases in the activities of these isoenzymes are concurrent with or slightly precede the accumulation of lignin in elicited 6-day cultures. By 12 hours after addition of elicitor, C1 becomes the most abundant extracellular isoperoxidase. The differential regulation of expression of peroxidase isoenzymes following elicitor treatment suggests that individual isoenzymes of peroxidase may have specific functional roles in the biosynthesis of disease-lignin.

Journal Article↗
PubMed1988

Lactate-supported synaptic function in the rat hippocampal slice preparation.

The present study was undertaken to examine the possibility that cerebral energy metabolism can be fueled by lactate. As a sole energy substrate, lactate supported normal synaptic function in rat hippocampal slices for hours without any sign of deterioration. Slices that were synaptically silent as a result of glucose depletion could be reactivated with lactate to show normal synaptic function. When slices were exposed to the glycolytic inhibitor iodoacetic acid, lactate-supported synaptic function was unaffected, whereas that supported by glucose was completely abolished. This indicated that lactate was metabolized directly via pyruvate to enter the tricarboxylic acid cycle. Thus, under conditions that lead to lactate accumulation (cerebral ischemia) this "end product" may be a useful alternative as a substrate for energy metabolism.

Animals↗
PubMed1988

Lactic acidosis and recovery of neuronal function following cerebral hypoxia in vitro.

The rat hippocampal slice preparation was used to study the combined effects of hypoxia and lactic acidosis on neuronal function. Control slices were exposed to a standard hypoxic insult while being perfused with normal artificial cerebrospinal fluid (ACSF). Experimental slices were perfused with ACSF containing 1.0, 2.0, 10.0 or 20.0 mM lactic acid, 30 min before and during the same standard hypoxic insult. Following at 30-min recovery period the ability of these slices to respond to orthodromic stimulation by displaying a population spike (synaptic function) was tested. No significant decreases in the recovery rate of synaptic function were found between control and experimental groups, excluding the combination of 20 mM lactic acid and 10 min hypoxia, where such a decrease was found. The combination of 10 mM lactic acid and 12 min hypoxia brought about an increase in the recovery rate of synaptic function. Thus, the adverse effects attributed to lactic acid in vivo were not seen in the present in vitro study. Neuronal tissue appears to be able to handle excess lactic acid by yet, unknown mechanism (high intracellular buffer capacity?). The suggested in vivo damage due to lactic acidosis could originate in the cerebrovascular system. On the other hand, the possibility that lactic acidosis is harmless under hypoxic conditions should also be considered.

Acidosis, Lactic↗