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Extracellular proteins of Cryptococcus neoformans and host antibody response.

Proteins secreted by the fungal pathogen Cryptococcus neoformans may be involved in invasion and could be useful in vaccine design. Despite the medical importance of this fungus, little is known about its extracellular proteins or the immune response to these antigens. To study C. neoformans extracellular proteins, 12 strains were metabolically radiolabeled and protein supernatants were analyzed. Both strain- and growth condition-dependent differences were observed. Enzymatic assays of filtered culture supernatants revealed butyrate esterase and caprylate esterase lipase activity for 11 of 12 strains, as well as acid phosphatase, naphthol-AS-BI-phosphohydrolase, and beta-glucosidase activities in some strains. Serum from infected rodents immunoprecipitated several secreted proteins, consistent with in vivo expression and development of an antibody response. For strain 24067, two immunodominant species, of approximately 75 and 30 kDa, were recognized. The relative intensity of the autoradiographic bands depended on the route of infection for both rats and mice. In summary, our results indicate that (i) there are multiple proteins in C. neoformans culture supernatants, (ii) there are strain differences in supernatant protein profiles, (iii) there are differences in supernatant protein profile depending on the growth conditions, (iv) there are several new extracellular and/or cell-associated enzymatic activities, and (v) antibodies to several supernatant proteins are made in the course of infection.

Acid Phosphatase↗

Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila.

Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular pathogen of protozoa and macrophages. Previously, we had determined that the Legionella pilD gene is involved in type IV pilus biogenesis, type II protein secretion, intracellular infection, and virulence. Since the loss of pili and a protease do not account for the infection defect exhibited by a pilD-deficient strain, we sought to define other secreted proteins absent in the mutant. Based upon the release of p-nitrophenol (pNP) from p-nitrophenyl phosphate, acid phosphatase activity was detected in wild-type but not in pilD mutant supernatants. Mutant supernatants also did not release either pNP from p-nitrophenyl caprylate and palmitate or free fatty acid from 1-monopalmitoylglycerol, suggesting that they lack a lipase-like activity. However, since wild-type samples failed to release free fatty acids from 1,2-dipalmitoylglycerol or to cleave a triglyceride derivative, this secreted activity should be viewed as an esterase-monoacylglycerol lipase. The mutant supernatants were defective for both release of free fatty acids from phosphatidylcholine and degradation of RNA, indicating that PilD-negative bacteria lack a secreted phospholipase A (PLA) and nuclease. Finally, wild-type but not mutant supernatants liberated pNP from p-nitrophenylphosphorylcholine (pNPPC). Characterization of a new set of mutants defective for pNPPC-hydrolysis indicated that this wild-type activity is due to a novel enzyme, as opposed to a PLC or another known enzyme. Some, but not all, of these mutants were greatly impaired for intracellular infection, suggesting that a second regulator or processor of the pNPPC hydrolase is critical for L. pneumophila virulence.

Bacterial Proteins↗

Characterization of esterases produced by a ruminal bacterium identified as Butyrivibrio fibrisolvens.

An obligately anaerobic ruminal bacterial isolate was selected from 18 tributyrin-degrading isolates and identified as Butyrivibrio fibrisolvens strain 53. The culture in late exponential phase contained enzymes which could be released by sonic disruption. These enzymes degraded substrates at a rate in the order 1-naphthyl acetate (NA) > 1-naphthyl butyrate > 1-naphthyl propionate but did not degrade 1-naphthyl palmitate or 1-naphthyl phosphate. The enzymes on NA were neither stimulated nor inhibited by CoCl(2), MgCl(2), and MnCl (each varied from 10(-6) to 10(-4) M). CaCl at 10(-3) M stimulated esterase activity by 16%. Aliphatic substrates were hydrolyzed at a rate in the order triacetin > tributyrin > tripropionin, and ethyl acetate > ethyl formate. Similarly, aromatic fluorescein diesters were degraded at a rate in the order acetyl > propionyl > caproyl > butyryl > capryl > lauryl. Polyacrylamide gel electrophoretic zymograms indicated that the enzyme composite contained cathodally migrating bands. By column chromatography, these enzymes were separated into six NA-degrading fractions. Fraction V contained an esterase which had an optimal temperature of 39 C, a K(m) of 7.6 x 10(-4) on NA, and a molecular weight of about 66,000. This enzyme was inhibited by paraoxon (41%, 10(-4) M), eserine (17%, 10(-2) M), NaF (17%, 10(-2) M), and diisopropyl fluorophosphate (62%, 10(-4) M) but not by 1-naphthyl N-methyl carbamate at 8.4 x 10(-4) M.

Anaerobiosis↗

The apeE gene of Salmonella typhimurium encodes an outer membrane esterase not present in Escherichia coli.

Salmonella typhimurium apeR mutations lead to overproduction of an outer membrane-associated N-acetyl phenylalanine beta-naphthyl ester-cleaving esterase that is encoded by the apeE gene (P. Collin-Osdoby and C. G. Miller, Mol. Gen. Genet. 243:674-680, 1994). This paper reports the cloning and nucleotide sequencing of the S. typhimurium apeE gene as well as some properties of the esterase that it encodes. The predicted product of apeE is a 69.9-kDa protein which is processed to a 67-kDa species by removal of a signal peptide. The predicted amino acid sequence of ApeE indicates that it is a member of the GDSL family of serine esterases/lipases. It is most similar to a lipase excreted by the entomopathogenic bacterium Photorhabdus luminescens. The Salmonella esterase catalyzes the hydrolysis of a variety of fatty acid naphthyl esters and of C6 to C16 fatty acid p-nitrophenyl esters but will not hydrolyze peptide bonds. A rapid diagnostic test reported to be useful in distinguishing Salmonella spp. from related organisms makes use of the ability of Salmonella to hydrolyze the chromogenic ester substrate methyl umbelliferyl caprylate. We report that the apeE gene product is the enzyme in Salmonella uniquely responsible for the hydrolysis of this substrate. Southern blot analysis indicates that Escherichia coli K-12 does not contain a close analog of apeE, and it appears that the apeE gene is contained in a region of DNA present in Salmonella but not in E. coli.

Amino Acid Sequence↗

Carbon catabolism and synthesis of macromolecules during spore germination of Microsporum gypseum.

Either d- or l-leucine (10(-3)m) and unsaturated long-chain fatty acids such as oleic, linoleic, and arachidonic (10(-4)m) significantly stimulated macroconidia germination of Microsporum gypseum. Saturated long-chain fatty acids did not affect germination, whereas saturated short-chain fatty acids such as caprylic, hexanoic, and butyric were completely inhibitory. Germination was followed by an increase in endogenous respiration and a decrease in dry weight of approximately 5% at 4 hr. Endogenous fatty acids and soluble carbohydrates were utilized significantly during germination. Tritiated leucine, uridine, and thymidine were incorporated respectively into protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) fractions within the first 5 min of germination. Incorporation of oleic-1-C(14) into RNA and protein was significantly increased after germ tube development. Net synthesis of RNA and protein started prior to germ tube protrusion. Increase in DNA could be detected only later. A significant increase in RNA and protein during the 4th hr of germination was correlated with vegetative development. Inhibition of respiration and incorporation of leucine-H(3) and uridine-H(3) into corresponding macromolecules by dl-fluorophenylalanine and phenethyl alcohol started before germ tube appearance. Griseofulvin significantly inhibited incorporation of uridine-H(3) and thymidine-H(3), but not of leucine-H(3). This inhibition occurred only after initial vegetative development. In contrast to the two other inhibitors, which substantially inhibited germination, griseofulvin only slightly retarded the period of germination and did not affect respiration.

Alcohols↗

Enzymatic profile of Legionella pneumophilia.

The enzyme activities of four strains of Legionella pneumophilia were investigated by using the API ZYM system (API System S.A., F-38390 Montalieu Vercieu, France) and synthetic substrates. Aminopeptidases were detected specifically against L-alanine, L-arginine, L-aspartic acid, L-cystine, L-glutaminic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-tryptophan, L-tyrosine, and L-valine. Furthermore, the bacteria possesses esterase activity splitting propionate, butyrate, caproate, caprylate, and caprate, but not laurate, myristate, palmitate, and stearate, esters. The enzymes studies were inhibited partially by aprotinin. No inhibition of phosphatase (pH range, 5.4 to 8.5) or of phosphoamidase was observed. Activities of arylsulfatase, chymotrypsin, trypsin, and glycosidases could not be detected.

Aminopeptidases↗

Enzymatic activities of Legionella pneumophila and Legionella-like organisms.

The API ZYM system was used to investigate enzymatic activities of Legionella pneumophila and other Legionella-like organisms. Leucine aminopeptidase, alkaline and acid phosphatase, butyrate and caprylate esterase, and phosphoamidase activities were consistently detected in all strains tested. No evidence of myristate lipase, trypsin, chymotrypsin, or glycosidase activity was found.

Acid Phosphatase↗

Enzymatic characterization of Aeromonas hydrophila complex by the API ZYM system.

Enzymatic characterization of 48 Aeromonas hydrophila complex isolates from various sources was determined with the API ZYM system (Analytab Products, Plainview, N.Y.). All isolates lacked valine and cystine aminopeptidases, chymotrypsin, alpha-mannosidase, alpha-fucosidase, alpha-galactosidase, and beta-glucuronidase but possessed caprylate esterase-lipase, leucine aminopeptidase, acid phosphatase, phosphoamidase, and N-acetyl-beta-glucosidase. Variability was found in the presence of alkaline phosphatase, butyrate esterase, myristate lipase, trypsin, beta-galactosidase, alpha-glucosidase, and beta-glucosidase. No significant differences were evident among the enzymatic profiles of isolates from various sources.

Aeromonas↗

Enzymatic characterization of Pseudomonas cepacia by API ZYM profile.

The enzymatic activities of 53 strains of Pseudomonas cepacia were determined by using the API ZYM system. Strong alkaline phosphatase, acid phosphatase, butyrate esterase, caprylate esterase, myristate lipase, leucine arylamidase, and phosphoamidase activities were consistently detected in all strains. Weak activities were observed for valine arylamidase, beta-glucosidase, and N-acetyl-beta-glucosaminidase. No activities could be demonstrated for cystine arylamidase, trypsin, chymotrypsin, alpha-galactosidase, beta-galactosidase, beta-glucuronidase, alpha-glucosidase, alpha-mannosidase, and alpha-fucosidase. Enzymatic activities of pseudomonads may provide useful information about their pathogenesis and information for identification of Pseudomonas species.

Chromogenic Compounds↗

Development of snake antivenom antibodies in chickens and their purification from yolk.

Adult white leghorn hens hyperimmunised with Brazilian snake venoms of the genus Bothrops and/or Crotalus produced antibodies capable of recognising, combining with and neutralising the toxic and lethal components of the venoms. The antibodies were first detected by an enzyme-linked immunosorbent assay two weeks after starting the immunisation schedule, reached the highest titres by the third week and remained high for at least 24 weeks. These antibodies are transferred to the egg yolk from which they were isolated as enriched IgY preparations by a combination of methods using positive and negative precipitation with sodium sulphate and/or caprylic acid. The yolk-derived IgY preparations contained antibodies which blocked the phospholipase A2-dependent haemolytic activity of both venoms and the haemorrhagic activity of Bothrops venom, and neutralised the toxic lethal activities of the venoms with good efficacy. The median effective dose (ED50) of the IgY anti-Bothrops venom was 592.5 microliters/2LD50 and, 1.0 ml neutralised 0.0675 mg of venom. The ED50 of the IgY anti-Crotalus venom was 457.5 microliters/3LD50 and 1.0 ml neutralised 0.075 mg of venom.

Animals↗

Exoenzymes of Propionibacterium acnes.

Thirty strains of Propionibacterium acnes were grown in basal salt medium containing lecithin as a lipid substrate and in other media. The cultures were assayed for production of lipase (measured as fatty acid esterase) and other exoenzymes. Lipase was assayed spectrophotometrically; other enzymes were assayed using the API ZYM system (Analytab Products Inc., Plainview, NY). Substance for lipase were alpha- and beta-naphthol esters of propionic, butyric, valeric, caprylic, lauric, myristic, and oleic acids. All strains showed fatty acid esterase activity. Using the API ZYM system 19 enzymes were detected, 8 of which were found frequently and had high activity in most strains. Acid and alkaline phosphatases, phosphoamidase, ester lipase, trypsin-chymotrypsin-like proteases, beta-glucuronidase (80%), beta-galactosidase (80%), and N-acetyl-beta-glucosaminidase were found. Many enzymes of P. acnes appear to be adaptive, dependent on the culture substrate.

Culture Media↗

Phenotypic and genotypic characterization of esterase-producing Ureibacillus thermosphaericus isolated from an aerobic digestor of swine waste.

Eight closely related thermophilic strains were isolated from an aerobic and thermophilic treatment of swine wastes. The pleomorphic cells (short and long rods; cocci) showed peritrichous flagella, terminally swollen sporangium, and liberated spores exhibiting hairy appendages. The Gram reaction was negative for both young (4 h) and old (48 h) cultures. Several features, such as colonial morphology, growth between 35 degrees C and 65 degrees C, presence of catalase, presence of spores, and strictly aerobic metabolism (except for one strain), are similar to those found for the genus Bacillus. The inability of the strains to use sugars, except esculin, as source of carbon and energy and the whole cell fatty acid composition are similar to those found in Bacillus thermosphaericus DSM 10633. Sequence analysis of the 16S rRNA gene revealed 99.8%-99.9% identity for seven of the thermophilic strains with this species. A new genus, Ureibacillus, was recently proposed for type strain B. thermosphaericus DSM 10633 The last strain exhibits 97.8% and 97.3% identity with Ureibacillus terrenus DSM12654 and Bacillus sp. TP-84, respectively. Esterase activities were detected for all strains, and assays on p-nitrophenyl butyrate and p-nitrophenyl caprylate revealed that strains were more active on the shorter substrate.

Aerobiosis↗

Epitrochlearis muscle. II. Metabolic effects of contraction and catecholamines.

Effects of isometric contraction and catecholamines on glucose and glycogen metabolism in skeletal muscle were investigated with the in vitro rat epitrochlearis preparation. Mechanical performance and glycogenolysis exhibited two phases. During the initial 30 min, muscle work was 30% greater and glycogenolysis 8- to 10-fold faster than the steady-state values in the subsequent 3-4 h of contraction. Glucose uptake was increased by contraction and remained relatively constant during stimulation. Epinephrine (10(-9) to 10(-6) M) and norepinephrine (10(-7) to 10(-5) M) produced inotropic and glycogenolytic effects blocked by propranolol but not phentolamine. Chemical sympathectomy and propranolol blocked the initial glycogenolytic and inotropic effects produced by isometric contraction, suggesting that they were caused by the release of endogenous catecholamines. Net lactate production in resting muscles accounted for > 50% of total glucosyl units utilized. During contraction net lactate production accounted < 10-15% of total glycosyl flux indicating that rat fast-twitch pale muscle is capable of significant rates of aerobic glucose oxidation. Oleate and caprylate did not affect mechanical performance, glycogen, or glucose metabolism in resting or contracting muscles.

Animals↗

Beta-subunit-dependent modulation of hSlo BK current by arachidonic acid.

In this study, we examined the effect of arachidonic acid (AA) on the BK alpha-subunit with or without beta-subunits expressed in Xenopus oocytes. In excised patches, AA potentiated the hSlo-alpha current and slowed inactivation only when beta2/3 subunit was co-expressed. The beta2-subunit-dependent modulation by AA persisted in the presence of either superoxide dismutase or inhibitors of AA metabolism such as nordihydroguaiaretic acid and eicosatetraynoic acid, suggesting that AA acts directly rather than through its metabolites. Other cis unsaturated fatty acids (docosahexaenoic and oleic acid) also enhanced hSlo-alpha + beta2 currents and slowed inactivation, whereas saturated fatty acids (palmitic, stearic, and caprylic acid) were without effect. Pretreatment with trypsin to remove the cytosolic inactivation domain largely occluded AA action. Intracellularly applied free synthetic beta2-ball peptide induced inactivation of the hSlo-alpha current, and AA failed to enhance this current and slow the inactivation. These results suggest that AA removes inactivation by interacting, possibly through conformational changes, with beta2 to prevent the inactivation ball from reaching its receptor. Our data reveal a novel mechanism of beta-subunit-dependent modulation of BK channels by AA. In freshly dissociated mouse neocortical neurons, AA eliminated a transient component of whole cell K(+) currents. BK channel inactivation may be a specific mechanism by which AA and other unsaturated fatty acids influence neuronal death/survival in neuropathological conditions.

Animals↗

Evaluation of kidney viability during hypothermic perfusion or cold storage.

The effect of up to 6 days of hypothermic perfusion and up to 72 h of cold storage on tissue viability were studied from measurements of sodium potassium pump activity and adenosine nucleotide content. For the hypothermic perfusion two different albumin-based perfusates were used, one with, the other without addition of caprylic acid and amino acids. No difference could be found in the parameters studied between perfusion with these two perfusates. For both perfusates total adenine nucleotide content (TAN) decreased by approximately 60% during perfusion while energy charge potential (ECP) and the ATP/ADP ratio remained constant. During cold storage a decrease of both TAN and ECP and the ratio ATP/ADP was seen, the main decrease occurring within the 1st h. The transmembrane potassium influx as deduced from potassium content in incubated cortical slices during steady state conditions decreased during 6 days of hypothermic storage to 24.5 +/- 2.4 mumol/kg which was a significantly higher level than after 24 h of cold storage. Thus, addition of extra substrate to the perfusate does not seem to improve kidney cortex viability during hypothermic perfusion. Parenchymal viability is better preserved with continuous hypothermic perfusion, compared to simple cold storage in a solution of the same ionic composition as that used for perfusion.

Adenine Nucleotides↗

Oral heparin prevents neointimal hyperplasia after arterial injury: inhibitory potential depends on type of vascular injury.

BACKGROUND: In animal models, heparin delivered as a continuous intravenous infusion or via frequent (BID) subcutaneous dosing inhibits neointimal hyperplasia after balloon injury or stent implantation. However, human trials of subcutaneous heparin after percutaneous intervention have proven ineffective against restenosis. It may be that these failures are due to unfavorable heparin pharmacokinetics. Recently, the drug delivery agent sodium N-[8-(2-hydroxybenzoyl)amino] caprylate (SNAC) has been found to facilitate the gastric absorption of heparin. METHODS AND RESULTS: To investigate the effects of orally delivered heparin on neointimal hyperplasia after varying forms of arterial injury, 57 New Zealand White rabbits underwent iliac artery balloon dilatation. In half of the rabbits, endovascular stents were implanted and heparin was delivered through a variety of methods. Arteries were harvested at 14 days. Neointimal area was assessed with computer-aided morphometry. After balloon injury, both intravenous (0.3 mg/kg per hour) and oral heparin (90 mg/kg BID) effectively inhibited neointimal hyperplasia (0.11+/-0.02 and 0.09+/-0.07 mm(2), respectively, versus 0.16+/-0.06 mm(2) in control; P<0.05). After stent implantation, intravenous administration of heparin (0.3 mg/kg per hour) effectively inhibited neointimal growth (0.35+/-0.05 mm(2) versus 0.51+/-0.09 mm(2) in control; P<0.05), but oral heparin at 90 mg/kg BID and 180 mg/kg BID (0.48+/-0.04 and 0.49+/-0.08 mm(2), respectively; P=NS versus control) did not. A dose of 120 mg/kg TID, however, was effective (0.40+/-0.10 mm(2); P<0.05 versus control). CONCLUSIONS: These data suggest that oral heparin may be an effective therapy against restenosis after percutaneous intervention. Stented arteries required higher and more frequent dosing for efficacy. These data suggest that differences in the type of vascular injury must be considered in the design of drug delivery.

Administration, Oral↗

The influence of intravenous medium- and long-chain triglycerides and carnitine on the excretion of dicarboxylic acids.

Four groups of male Wistar rats were alimented parenterally for 3 days. Groups 1 and 2 received medium-chain triglycerides (MCT) and groups 3 and 4 long-chain triglycerides (LCT). Groups 2 and 4 were supplemented with 100 mg L-carnitine/kg/day. The MCT-alimented rats presented with a distinct excretion of the dicarboxylic acids: adipic acid (C6), suberic acid (C8), and sebacic acid (C10). The acids excreted corresponded to the infused pattern of monocarboxylic acids: caproic acid (C6), caprylic acid (C8), and capric acid (C10). Dicarboxylic acid excretion after MCT administration may reflect an insufficient capacity of beta-oxidation on one hand or a preferential omega-oxidation of medium-chain fatty acids on the other. Carnitine supplementation lead to a further increase of the dicarboxylic acids in the MCT-group. beta-OH-butyric acid excretion decreased after carnitine in the MCT as well as in the LCT group. An increased transport of fatty acid-carnitine compounds out of the mitochondria is discussed as an important effect of carnitine supplementation. Hereby medium-chain fatty acids may be more accessible for omega-oxidation.

Animals↗

Lipase and antibacterial activities of a recombinant protein from the accessory glands of female Phlebotomus papatasi (Diptera: Psychodidae).

The reproductive accessory glands of the adult female have a functional versatility in insects, contributing to reproduction in various ways. The major protein secreted by the accessory glands of female Phlebotomus papatasi (Diptera, Psychodidae) has already been characterised and named PhpaLIP (for P. papatasi lipase) because, in terms of its amino-acid sequence, it is very similar to a wide range of vertebrate lipases. The gene coding for PhpaLIP has now been cloned into a pQE30 vector and expressed in Escherichia coli. When the recombinant PhpaLIP was tested in vitro, it was found to have not only lipase-like activity (when p-nitrophenyl caprylate was used as the substrate) but also specific antibacterial activity against some Gram-positive and Gram-negative bacteria. The possible physiological roles of PhpaLIP in P. papatasi are discussed, in the light of these results.

Actinomycetales↗