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Biomedical subjects

N Porter

Publications and source records attributed to N Porter.

18 recordsLinked to original sources
PubMed1995

Xenovulene A, a novel GABA-benzodiazepine receptor binding compound produced by Acremonium strictum.

Xenovulene A, a novel inhibitor of benzodiazepine binding to the GABA-benzodiazepine receptor is produced by submerged fermentation of Acremonium strictum. It was isolated from the mycelium by solvent extraction and purified by chromatography on Sephadex LH-20 and octadecyl silica. The structure of xenovulene A was determined to be a novel oxygenated sesquiterpene containing a humulene moiety by interpretation of various spectroscopic data, especially from 2D NMR experiments. Xenovulene A inhibited binding of the benzodiazepine, flunitrazepam, with an IC50 of 40 nM in an in vitro assay using bovine synaptosome membrane preparations.

Acremonium↗
PubMed1991

Observation of the light-triggered binding of pyrone to chymotrypsin by Laue x-ray crystallography.

Crystals of gamma-chymotrypsin inhibited with the photodissociable group trans-p-diethylamino-o-hydroxy-alpha-methylcinnamate were irradiated with a 1-msec flash from a high-energy xenon flashlamp in the presence of the mechanism-based inhibitor 3-benzyl-6-chloro-2-pyrone. The ensuing reaction was monitored by collection of sequential, single-exposure Laue x-ray diffraction patterns. The experiment was also performed in solution to verify the regeneration of catalytic activity and the subsequent inhibition of the enzyme by pyrone after photolysis. The resulting crystallographic structures show the presence of covalently bound cinnamate prior to photolysis, the generation of "free" enzyme after irradiation of the crystal, and the slow formation of a pyrone-inhibited complex several hours after photolysis. The structure of the free enzyme shows a significant proportion of the active sites in the crystal to contain a naturally occurring, noncovalently bound tetrapeptide inhibitor [Dixon, M.M. & Matthews, B.W. (1989) Biochemistry 28, 7033-7038], even after cinnamate acylation and photolysis. Data collected simultaneously with irradiation show the crystal to be slightly disordered during photolysis, leading to streaked x-ray photos. The resulting maps are suggestive of a bicyclic coumarin species produced by photolysis and deacylation; however, the electron density is difficult to model unambiguously by one unique chemical state. Nevertheless, Laue crystallography is shown to be capable of visualizing time-dependent chemical changes in the active site of an enzyme.

Binding Sites↗
PubMed1991

Lichen-forming fungi: potential sources of novel metabolites.

The challenge for today's pharmaceutical industry lies in the discovery and development of new, pharmacologically active molecules. Metabolites produced by microorganisms, and fungi in particular, are a resource for which the therapeutic potential has been recognized, but one that remains largely unexplored and unexploited. Approximately 20% of all known fungal species are obligate symbionts in lichens; this major group of fungi has been long neglected by mycologists, and overlooked by industry.

Ascomycota↗
PubMed1991

Positive selection of antibiotic-producing soil isolates.

Stepwise discriminant analysis was used to identify the most powerful selective substrates which could be used to formulate media capable of enriching for antibiotic-producing soil isolates. This was achieved by characterizing a collection of 74 soil bacteria, including eubacteria and actinomycetes, according to their ability to produce antibacterial antibiotics and their growth responses to 43 physiological and nutritional tests. The characters which were selective for actinomycetes relative to eubacteria included growth on proline (1%, w/v) and humic acid (0.1%) as sole sources of both carbon and nitrogen, growth on nitrate as a nitrogen source, and growth at pH 7.7-8.0. Growth on proline (1%) and humic acid (0.1%) as sole carbon/nitrogen sources, growth on asparagine as a nitrogen source, and growth in the presence of vitamins were among the characteristics which allowed antibiotic-producing actinomycetes to be differentiated from non-antibiotic-producing strains. Several simple isolation media which incorporated the selective substrates identified by discriminant analysis succeeded in increasing the proportion of actinomycetes isolated from soil samples. Furthermore, the percentage of isolates capable of antibiotic production was considerably increased.

Actinomycetales↗
PubMed1990

Photolysis and deacylation of inhibited chymotrypsin.

Inhibited chymotrypsin was reactivated through the photolysis of the covalently bound light-reversible cinnamates described in our previous paper [Stoddard, B.L., Bruhnke, J., Porter, N.A., Ringe, D., & Petsko, G. (1990) Biochemistry 29, 4871-4879]. The light-induced deacylation was accomplished both in solution and in protein crystals, with the release of inhibitor from the crystal monitored and confirmed by X-ray diffraction. The product of photolysis has been characterized as a 3-methylcoumarin, leading to a mechanism for light-driven deacylation of an internal lactonization that is dependent on the presence of an internal hydroxyl nucleophile. The acyl enzyme formed from cinnamate A is not suitable for photochemical studies, as the complex has a short half-life in solution and does not have a chromophore that is well separated from protein absorbance. Cinnamate B, with a p-diethylamino substituent, shows an enzyme deacylation rate enhancement of 10(9) for the cis photoisomer relative to the trans starting material. The half-life and deacylation rate of this compound in the E-I complex after photon absorption have been directly measured by subsecond UV absorption studies. X-ray diffraction studies of photoactivation using a flow cell show that the cinnamate B acyl enzyme complex is fully capable of light-induced isomerization and regeneration of native enzyme in the crystalline state. The E-I complex formed upon binding of cinnamate A, however, shows little if any effect from irradiation due to competitive absorbance by the highly concentrated protein at the shorter UV wavelengths. Photolysis of cinnamate B appears to occur on a time scale fast enough for applications in crystallographic studies of enzymatic intermediate-state structures.

Chymotrypsin↗
PubMed1990

Structure and activity of two photoreversible cinnamates bound to chymotrypsin.

The serine protease gamma-chymotrypsin was covalently inhibited with two different photoreversible cinnamate compounds, and the structures of the resulting complexes were determined to 1.9-A resolution. The inhibitors show different kinetics of binding, inhibition, and nonphotochemical deacylation relative to each other in solution activity assays. The crystal structures of the enzyme-cinnamate complexes show that both compounds acylate serine 195 and that the two molecules are bound in similar nonproductive conformations which have drastic effects on their ability to turn over. Substitution of a diethylamino group on the para position of the cinnamate ring causes a 1000-fold increase in the thermal stability of the inhibitor toward hydrolysis and deacylation.

Binding Sites↗
PubMed1987

The influence of hands-on laser training on usage of the CO2 laser.

A questionnaire was distributed to all participants in the Rochester General Hospital CO2 laser training workshops for surgeons (1982 to January 1985). The results of this study indicate that, while laser training is necessary prior to being able to use the laser, only a small fraction of trainees actually use the laser in their clinical practice. Surgeons who are using the laser do so in a minority of their cases. New incentives for laser usage must be investigated.

Education, Medical, Continuing↗
PubMed1985

Effect of stanozolol on factors VIII and IX and serum aminotransferases in haemophilia.

The treatment of haemophilia has been dramatically improved since the introduction of factor VIII and IX concentrates, however these concentrates have brought new problems such as hepatitis and A.I.D.S. An oral agent which could raise endogenous levels of factor VIII and IX would be of great benefit. Danazol, an anabolic steroid, has recently been shown to increase levels of factors VIII and IX in haemophilia. We therefore studied the effect of stanozolol, a closely related anabolic steroid, in 15 patients with haemophilia A or Christmas disease over a 2-4 week period. There was no consistent change in factor VIIIc or factor IX, and fibrinolysis was significantly enhanced. No effect was apparent on the incidence of spontaneous bleeds. However serum aminotransferases which were abnormal in 11 of the 15 patients at the start of the study fell significantly with stanozolol therapy. This raises the interesting possibility that anabolic steroids may be beneficial in patients with chronic liver diseases.

Blood Viscosity↗
PubMed1983

The effects of cyanide on the growth and respiration of Enterobacter aerogenes in continuous culture.

The effect of cyanide on the physiology of lactate- and oxygen-limited Enterobacter aerogenes NCTC 10336 was studied in chemostat culture (D = 0.1 h-1). In the absence of cyanide, the molar growth yield from oxygen (YO2) under oxygen limitation was 60% of the carbon-limited value. A similar decrease in yield was observed in a lactate-limited culture (excess oxygen) which was continuously fed low concentrations of potassium cyanide. The cultures with the lower growth yields possessed respiratory systems less sensitive to inhibition by cyanide. This was particularly marked in cultures grown in the presence of cyanide. Increased cyanide resistance was associated with an increase in the concentration of a cytochrome oxidase tentatively identified as a d-type and the appearance of additional cytochromes tentatively identified as b-type.

Cyanides↗
PubMed1982

Enzymatic properties of human glucuronyltransferase and a sensitive method for its assay in a stable B lymphocyte cell line.

Properties of lymphocyte glucuronyltransferase were studied in homogenates of SN1006 cells. A sensitive assay procedure for lymphocyte glucuronyltransferase was developed utilizing radioactive testosterone as the acceptor substrate and TLC for separation of the metabolite. The method is capable of detecting picomolar quantities of the product. The enzyme activity exhibited a broad pH optimum, and was subject to activation by the detergent Lubrol WX and Mn++ ions. The activity conformed to the Michaelis-Menten kinetics giving apparent Km values of 0.8 mM and 11 microM, for UDPGA and testosterone, respectively. 4-Methylumbelliferone, a-naphthol and p-nitrophenol behaved as competitive inhibitors of testosterone glucuronidation. The results indicate that the method could be used for genetic studies of human lymphocyte glucuronyltransferase, and that the enzyme is of consequence in detoxication of exogenous as well as endogenous substrates.

B-Lymphocytes↗
PubMed1981

Substrate specificity of human UDP-glucuronyltransferase in cultured lymphocytes.

1. This study establishes the presence of UDP-glucuronyltransferase activity for non-steroidal as well as steroidal substrates, in cultured human B-lymphocytes. Glucuronidation of alpha-naphthol and testosterone was demonstrated in homogenates of two cell lines, SN1006 and RPMI-1788, and that of phenolphthalein, 4-methylumbelliferone, p-nitrophenol and estradiol in the cell line with the higher glucuronyltransferase activity, SN1006. 2. Kinetic studies of testosterone glucuronidation in homogenates of both cell lines revealed a similarity in the behaviour of glucuronyltransferase of these cells. Thus, comparable apparent Km values for UDPGA (0.63 mM) and for testosterone (14 microgram) were observed, although apparent maximal velocities, Vmax, differed several-fold (3.0 versus 0.55 pmol/10(6) cells per min, in SN1006 and RPMI-1788 cells, respectively). 3. Kinetic studies of glucuronidation of testosterone, estradiol, phenolphthalein, alpha-naphthol, 4-methylumbelliferone, and p-nitrophenol yielded comparable apparent Km values for UDPGA (0.56-0.67 mM), suggesting that the same, or similar, glucuronyltransferase(s) catalyse(s) glucuronidation of this wide range of substrates in lymphocytes. This was reinforced by the observation of competitive inhibition of testosterone glucuronidation by alpha-naphthol (Ki 0.25mM), 4-methylumbelliferone (Ki 0.8mM) and p-nitrophenol (Ki 0.8 mM). Thus, lymphocyte glucuronyltransferase activity with a broad substrate specificity, for steroidal and non-steroidal aglycones, is indicated.

Cell Line↗
PubMed1980

Elevation of lysosomal enzymes in primary Lewis lung tumor correlated with the initiation of metastasis.

Lysosomal enzymes were elevated about two-fold in primary s.c. Lewis lung carcinoma as compared with metastatic nodules in the lung. In a time course experiment, a general two-fold elevation of acid phosphatase and several glycosidases was observed in the primary tumor between the 14th and 17th postimplant day following s.c. inoculation of Lewis lung carcinoma. This increase in hydrolytic enzyme activity was not due to necrosis in the primary tumor since a comparison of enzyme activities in the nonnecrotic and necrotic areas demonstrated much higher activities in the nonnecrotic areas. No increases in lysosomal enzyme activity were observed with time in Sarcoma 180, a tumor which does not metastasize. There was no change with time in primary Lewis lung tumor lactate dehydrogenase activity while a 7-fold increase in serum lactate dehydrogenase activity was observed in tumor-bearing mice. Mitochondrial succinate-2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium reductase levels fell in the primary Lewis lung tumor as the tumor size increased. A positive correlation was observed between the time of the elevations of tumor lysosomal enzymes in Lewis lung carcinoma and the appearance of micro- and macrometastatic lesions in the lungs. The mechanisms accounting for the increased intratumoral lysosomal enzymes are unknown, but they may be related to macrophage infiltration or other tumor-host interactions which may facilitate the dissemination of tumor cells.

Acid Phosphatase↗
PubMed1978

Platelet aggregation in patients with peripheral vascular disease.

Platelet aggregation has been studied in 28 control subjects and 46 patients with peripheral vascular disease. The platelets from the patients with peripheral vascular disease were significantly more sensitive to the aggregating effects of adenosine diphosphate and adrenaline than those from the control group (P less than 0.001). In addition, spontaneous platelet aggregation was observed in vitro in several of the vascular patients; this response was not apparent in the control group. Increased platelet aggregation could add to the vascular impairment in the unoperated subject and might also play a part in premature graft failure. Platelet function studies should perhaps be included as a routine in the assessment of patients with peripheral vascular disease.

Adenosine Diphosphate↗