Calorimetric characterization of microorganisms

A short survey is given on recent calorimetric studies of microbial growth, which raised the question whether calorimetrically obtained thermograms can be used for identification of different organisms. Advantages and the efficiency of the method are critically discussed.     

Specific substrates for spectrophotometric determination of penicillin acylase activity

Penicillin acylase substrates suitable for colorimetric determination of the enzyme activity have been tested in this study. The kinetic parameters (Km and kcat) have been elucidated for the following nine substrates: six phenylacetic acid derivatives (p-nitroanilide, p-nitrophenyl ester, p-nitro-m-carboxyanilide, p-nitro-o-carboxyanilide, p-nitro-o-hydroxyanilide, m-nitro-p-carboxyanilide), two D-phenylglycine derivatives (p-nitroanilide, p-nitro-m-carboxyanilide), and also p-nitrophenyl ester of acetic acid (p-nitrophenyl acetate). With the exception of p-nitrophenyl acetate, all the compounds studied are highly specific chromogenic substrates for penicillin acylase, but their reactivity is very variable and kcat/Km values are in a range from 0.8.10(4) to 5.10(6) M(-1).sec(-1).     

Fate and activity of microorganisms introduced into soil

Gastrointestinal (GI) hemorrhage during compromised liver function is known to precipitate portal-systemic encephalopathy (PSE). Hypothetically, the induced hyperammonemia depletes cerebral glutamate pools. To investigate this hypothesis, rats were studied 14 days after portacaval shunt (PCS) or sham surgery (SHAM). Rats received 3 mL bovine erythrocytes or saline at t = 0, 1, 2, and 3h via a previously placed gastrostomy catheter. At t = 0, 2, 4, 6 and 8h arterial blood and at t = 8h cerebral cortex were sampled for determination of ammonia and amino acids. Control rats (NORM) were sampled without previous surgery. Repeated intragastric blood administration increased the already elevated arterial ammonia levels in PCS rats further. This resulted in higher cerebral cortex ammonia and glutamine levels after blood administration. Despite the accumulation of ammonia and glutamine, cerebral cortex glutamate concentrations remained unaltered. Yet, PCS rats became more encephalopathic after blood gavages, suggesting that there is not a clear-cut relation between cerebral cortex glutamate concentrations and degree of PSE. Interestingly, cerebral cortex concentrations of GABA, tyrosine and phenylalanine were markedly increased. Whether these observations are pathogenetically related to PSE remains to be established. The present model of simulated GI hemorrhage in PCS rats seems to be a suitable, clinically valid model for future research regarding hepatic encephalopathy.     

Deoxyribonuclease activity associated with adenovirus 5 and 7

A DNase activity was found associated with isolated components of adenovirus types 5 and 7. The enzymatic activity was associated with the purified virus and viral components extracted from the soluble material of infected cells. The DNase activity of adenovirus type 5 was maximum at pH 5.5 and 7.0 whereas adenovirus 7 was active only at pH 7.0. Both DNases were shown to be located in the penton fraction.     

Endogenous 7-oxocholesterol is an enzymatic product: characterization of 7 alpha-hydroxycholesterol dehydrogenase activity of hamster liver microsomes

Previously, we described a new metabolite derived from endogenous cholesterol in the presence of hamster liver microsomal protein and NADPH (Song et al., 1991, Biochem. Pharmacol. 41, 1439-1447). Through gas chromatography/mass spectral analysis of the metabolite and its methoxime-3-dimethyl-t-butylsilyl ether derivative, this metabolite has been definitively identified as 7-oxocholesterol. Isotope incorporation experiments using molecular 18O2 demonstrated that no oxygen atoms from molecular oxygen were incorporated into the product, 7-oxocholesterol, when 7 alpha-hydroxycholesterol was used as substrate. In contrast, one atom of 18O was incorporated into cholesterol from 18O2 during its metabolism to form 7 alpha-hydroxycholesterol. Formation of 7-oxocholesterol was dependent upon the presence of NADP+, 7 alpha-hydroxycholesterol, and hamster liver microsomes. This enzyme appears to be a membrane-bound protein and its activity was most abundant in liver microsomal fractions and to a lesser extent in mitochondrial fractions; little or no activity was observed in nuclei or cytosol. The enzyme activity was present in highest content in the livers of hamsters and was also observed in human and bovine liver microsomes, but not those of mouse, rabbit, or rat. The reaction was inhibited by 2'-AMP, but not by anti-NADPH:cytochrome-P450 oxidoreductase globulin, carbon monoxide, metyrapone, nor miconazole. In contrast to the previously characterized 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase activity, NAD+ did not serve as an effective cofactor for 7-oxocholesterol formation. The ability of NADPH to partially serve as a cofactor in this reaction was shown to be due to a high NADPH-oxidase activity of hamster liver microsomes, thereby providing sufficient NADP+ to serve as the oxidizing pyridine nucleotide for the reaction. These results document the existence of a non-P450, NADP(+)-dependent 7 alpha-hydroxycholesterol dehydrogenase in liver microsomes which catalyzes this reaction. The product, 7-oxocholesterol, is produced enzymatically in the livers of hamsters and other mammals and may regulate bile acid metabolism or other processes due to its action as an oxysterol.     

Douglas-fir root-associated microorganisms with inhibitory activity towards fungal plant pathogens and human bacterial pathogens

A microbial culture collection composed of 1820 bacterial strains, including 298 actinomycete strains, was established from the roots of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings harvested from conifer nurseries and forest sites. Two hundred and thirty-four strains inhibited the growth of Fusarium, Cylindrocarpon, and (or) Pythium spp. in in vitro assays. A significantly greater proportion of bacterial strains from actinomycete genera exhibited antifungal properties compared with bacterial strains from nonactinomycete genera. Eighty-nine percent of identified inhibitory strains were Streptomyces, Streptoverticillium, Bacillus, Pseudomonas, or Burkholderia species. The actinomycete species were isolated almost exclusively from forest seedlings. Recovery of inhibitory strains representing 29 microbial species was enhanced using a variety of methods to isolate microorganisms from the roots of seedlings from nursery and forest sites. Bacterial strains (including actinomycete strains) with antifungal activity were tested for in vitro growth inhibition of six clinical human bacterial pathogens (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa). Forty-eight percent of the tested strains inhibited one or more human pathogens, Inhibitory activity towards fungal and bacterial pathogens was strain specific, not species specific, and many inhibitory strains exhibited broad-spectrum activity. Strains with antifungal activity against several conifer root pathogens were also more likely to inhibit multiple species of clinical bacterial pathogens.     

Isolation and characterization of 7-hydroxydiftalone beta-glucuronide

Metabolic studies on diftalone (I), a new non-steroidal antiinflammatory agent, demonstated that 7-hydroxydiftalone beta-glucuronide (V) is the main urinary metabolite. The isolation of (V) from the urine of a dog treated with 14C-diftalone was performed by liquid-liquid partition after precolation through Amberlite XAD-2 and IRC-50 (H+). All the steps were followed by radio-detection and by thin layer radio-chromatography. Compound (V) was characterized mainly by mass spectrometry after esterification with diazomethane and silylation with BSA.     

Two-step autocatalytic processing of the glutaryl 7-aminocephalosporanic acid acylase from Pseudomonas sp. strain GK16

The glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase of Pseudomonas sp. strain GK16 is an (alphabeta)2 heterotetramer of two nonidentical subunits. These subunits are derived from nascent polypeptides that are cleaved proteolytically between Gly198 and Ser199 after the nascent polypeptides have been translocated into the periplasm. The activation mechanism of the GL-7-ACA acylase has been analyzed by both in vivo and in vitro expression studies, site-directed mutagenesis, in vitro renaturation of inactive enzyme precursors, and enzyme reconstitution. An active enzyme complex was found in the cytoplasm when its translocation into the periplasm was suppressed. In addition, the in vitro-expressed GL-7-ACA acylase was processed into alpha and beta subunits, and the inactive enzyme aggregate of the precursor was also processed and became active during the renaturation step. Mutation of Ser199 to Cys199 and enzyme reconstitution allowed us to identify the secondary processing site that resides in the alpha subunit and to show that Ser199 of the beta subunit is essential for these two sequential processing steps. Mass spectrometry clearly indicated that the secondary processing occurs at Gly189-Asp190. All of the data suggest that the enzyme is activated through a two-step autocatalytic process upon folding: the first step is an intramolecular cleavage of the precursor between Gly198 and Ser199 for generation of the alpha subunit, containing the spacer peptide, and the beta subunit; the second is an intermolecular event, which is catalyzed by the N-terminal Ser (Ser199) of the beta subunit and results in a further cleavage and the removal of the spacer peptide (Asp190 to Gly198).     

FT-IR spectroscopy as an emerging method for rapid characterization of microorganisms

Effects of a novel 5-HT4 receptor agonist TKS159 on the cardiovascular system were assessed in comparison with cisapride using an in vivo canine model. TKS159 in doses of 0.1, 1.0, and 10 mg/kg (n = 6) or cisapride in 1/10 doses of 0.01, 0.1, and 1.0 mg/kg (n = 6) was cumulatively infused over 10 min with a pause of 20 min. The doses of the drugs were determined according to the previous knowledge of their pharmacokinetics. Clinically effective plasma concentrations as a gastrointestinal prokinetic drug were obtained after the infusion of 0.1 mg/kg of the respective drugs. In TKS159-administered animals, no significant change was induced in each cardiovascular parameter by an infusion of 0.1 mg/kg. The blood pressure was decreased, and the effective refractory period and repolarization phase of the ventricle were prolonged after 1.0 mg/kg. The heart rate was decreased, and the atrioventricular, as well as intraventricular, conduction were suppressed after 10 mg/kg, while no significant changes were observed in the cardiac output and the ventricular contraction and the relative refractory period of the ventricle during the study. Meanwhile, in cisapride-administered animals, the repolarization phase and the effective refractory period were prolonged after 0.01 mg/kg. The heart rate and the blood pressure were decreased after 0.1 mg/kg. The cardiac output, the ventricular contraction, and the atrioventricular conduction were suppressed, the relative refractory period was prolonged, and early afterdepolarization was detected after 1.0 mg/kg, while no significant change was observed in the intraventricular conduction during the study. Thus, TKS159 may have a safer cardiovascular profile than cisapride.     

Orientation degree characterization of diamond etched with Ce-7%Fe alloy

An increasing {110} orientation degree behavior was observed during etching of chemical vapor deposition (CVD) diamond films by partially melting Ce-7%Fe alloys. In order to accurately investigate this phenomenon, the X-ray diffraction method was used to identify the changes in the surface crystal orientation of the diamond films etched by Ce-7%Fe alloys, and evolution of orientation along the growth direction of the un-etched diamond film was analyzed by electron backscattering diffraction (EBSD), and then the morphology of etched diamond surface was observed by scanning electron microscopy (SEM). The results showed that the {110 } orientation degree of diamond surface increased due to the anisotropy in diamond etching with Ce-7%Fe, which was verified by the etching pit in SEM micrographs.     

Purification and properties of penicillin acylase of Bovista plumbea

Lipids from the insoluble material obtained by pulmonary lavage of 6 patients with alveolar proteinosis and from lamellar organelles of normal rabbit lungs were isolated and characterized. In both types of samples, dipalmitoylphosphatidylcholine was the predominant lipid. Phosphatidylethanolamine, phosphatidylglycerol, lysophosphatidylglycerol, and 2 glycolipids, GM3 and GL1 were also present in both types of preparations. Sphingomyelin, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidyl-N, N-dimethylethanolamine, phosphatidylserine, and lyso(bis)phosphatidic acid were found in the sedimented lavage material from humans but were not detected in lamellar organelles from rabbits. Significant quantities of neutral lipids were present in the lavage material, but only trace amounts, mainly as cholesterol and triglycerides, were detected in lamellar organelles. Phosphatidylcholine and the 2 glycolipids contained mostly saturated fatty acids and essentially no polyunsaturated fatty acids. Sphingomyelin, lysophosphatidycholine, and phosphatidyl-N, N-dimethylethanolamine, found only in the lavage, were also highly saturated. In addition to the fact that several phospholipids found in the lavage were not present in lamellar organelles, another striking difference between the lipids from these 2 sources was that phosphatidylglycerol of lamellar organelles contained predominantly palmitic acid, whereas the phosphatidylglycerol obtained by lavage of humans contained large amounts of stearic and oleic acids.     

Antimicrobial activity of newly synthesized isothiocyanate derivatives against pathogenic plant microorganisms

Fifteen reaction products of isothiocyanates with cysteine, seven reaction products of isothiocyanates with 2,3-dimercapto-1-propanol, and four reaction products of isothiocyanates with sulfanilamide were synthesized. Their antimicrobial activity against pathogenic plant microorganisms was investigated.     

In-vitro activity of purpuromycin and MDL 63,604 against microorganisms that cause vaginitis and vaginosis

Purpuromycin and its semi-synthetic derivative MDL 63,604 had in-vitro activity similar to that of amphotericin B against isolates of Candida albicans. MDL 63,604 had activity similar to that of metronidazole against Trichomonas vaginalis. Both compounds were very active against most species of Gram-positive and Gram-negative anaerobes and against Gardnerella vaginalis. MDL 63,604 had significantly lower MICs than purpuromycin against T. vaginalis and most of the bacteria, probably due to antagonism of purpuromycin's activity by medium supplements (blood or serum). Purpuromycin or related compounds may have a potential role in the topical treatment of vaginitis and vaginosis.     

Effect of amino acids on the production and activities of cephalosporin C acylase and penicillin V acylase from Aeromonas species ACY 95

In this article, we document flow disturbance due to internal thoracic artery spasm (ITA) in a patient undergoing minimally invasive coronary artery grafting. We used intraoperative duplex scanning. Application of systemic vasodilators resulted in rapid improvement of ITA flow, as demonstrated by serial duplex examinations.     

Immunologic characterization of CD7-deficient mice

Human CD7 is an Ig superfamily molecule that is expressed on mature T and NK lymphocytes. Although in vitro studies have suggested a role for CD7 in lymphoid development and function, the exact function of CD7 in vivo has remained elusive. One patient has been reported with SCID syndrome attributed to CD7 deficiency. To study in vivo functions of CD7, we have generated CD7-deficient mice and assessed their lymphoid development and function. CD7-deficient mice were viable, had normal peripheral blood and spleen lymphocyte numbers, and had normal specific Ab responses with Ag-driven Ig isotype switching. Thymocyte numbers were normal in 4-wk-old, 6-mo-old, and 1-yr-old CD7-deficient mice, but in 3-mo-old CD7-deficient mice, total thymocyte numbers were significantly increased by 60% (p < 0.02) compared with normal age-matched +/+ littermates. CD7-deficient splenocytes proliferated normally in response to various mitogens, including PHA, anti-CD3, Con A, and LPS. While NK cell numbers and cytolytic activity to YAC targets were normal, CD7-deficient mice had lower Ag-induced MHC class I-restricted CTL activity against OVA-transfected target cells than did +/+ control mice. Thus, CD7-deficient mice did not have a SCID syndrome, but rather had transient increases in thymocyte numbers at age 3 mo and altered splenocyte Ag-specific CTL effecter cell activity. These data suggest a role for CD7 in regulating intrathymic T cell development and in mediating CTL effecter function.     

Screening of Australian medicinal plants for antiviral activity

The nanoencapsulation of a model protein drug, bovine serum albumin (BSA), using gelatin as the matrix material is reported. Nanoencapsulation was conducted using a modified water-in-oil (w/o) emulsion method, which is emulsifier-free and simple. The nanoencapsulation product, BSA-containing gelatin nanoparticles, is characterized in terms of nanoparticle morphology, size and size distribution, water content, and in vitro protein release. The BSA-containing gelatin nanoparticles obtained from this nanoencapsulation process are nearly spherical and have a log-normal size distribution. The average diameter of the BSA-containing gelatin nanoparticles is approximately 840 nm. They can absorb 51-72% of water. In vitro release experiments demonstrate that BSA has been successfully encapsulated in, and can be released from the gelatin nanoparticles. The release of BSA from the gelatin nanoparticulate matrix follows a diffusion-controlled release mechanism. It is found that temperature affects both the water content and the BSA release rate of the gelatin nanoparticles.