Role of protein subunits in Proteus rettgeri penicillin G acylase.

Penicillin G acylase from Proteus rettgeri is an 80,000- to 90,000-dalton enzyme composed of two nonidentical subunits. Both subunits were required for enzymatic activity. The 65,000-dalton beta subunit contained a phenylmethylsulfonyl fluoride-sensitive residue required for enzymatic activity, and the 24,500-dalton alpha subunit contained the domain that imparts specificity for the penicillin side chain.     

Production of pectinases byAspergillus sp using differently pretreated lemon peel as the carbon source

Summary Aspergillus sp strains from decaying lemons were tested for extracellular pectinase production, testing differently pretreated lemon peel as the carbon source instead of pectin. It was found that the production of extracellular polygalacturonase was about the same and that of pectinesterase substantially higher when unwashed fresh lemon peel was used instead of pectin. The culture filtrate obtained showed a clarifying capacity similar to that of a commercial pectinase preparation, but the vitamin C of the juice was less affected by the treatment.     

Biological ensiling of sugarcane tops

The following lactic acid bacteria were isolated from sugarcane tops silage:Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus brevis, Lactobacillus delbrueckii, Pediococcus cerevisiae, Leuconostoc mesenteroides andStreptococcus lactis. The isolates were grown in a synthetic medium and the final pH, sugar uptake and the effect of adding CaCO₃ and L1, L2 and L3 factors was observed. The importance of the buffering capacity of the ensiled material for the silage process with a view to the occurrence of lactic acid bacteria is discussed.     

Catabolite repression of the synthesis of inducible polygalacturonase and pectinesterase byAspergillus niger sp.

Several recent reports have described large numbers of monoclonal antibodies that cross-react with toxins A and B ofClostridium difficile; this suggests that the toxins share major epitopes. Our results show that monoclonal antibodies (MAb) against other antigens bind nonspecifically to both toxins. Therefore, we believe that the cross-reacting MAb bind by this manner and not by a true immune reaction.     

Batch culture of Candida utilis in a medium deprived of a phosphorous source

Summary Candida utilis var. major NRRL-Y-1084 was grown in a defined medium without a phosphorous (P) source. During the exponential phase, cells divided according to a specific growth rate of 0.32 h^-1, which is lower than the usual rate for a balanced medium (0.4–0.6 h^-1). The relative P content of the biomass decreased from 2.70% to 0.75% over a period of 6 h, including 2 h of cell division arrest. At the end of this period there was another interruption of cell division. After that, multiplication restarted at a considerably lower rate and it deviated slightly from the exponential pattern. The stationary phase began when biomass P content reached 0.4%–0.5%, slowly decreasing afterwards to 0.25–0.20%. Biomass synthesis was less affected than cell division by the relative decrease of endogenous P, the two processes differing partially in their kinetics. Cell lysis started shortly before the stationary phase and affected about 20% of the population by the end of the assay. RNA and P content of the resulting biomass were 2.4% and 0.25% respecitvely, P being mainly incorporated to RNA.The relationship of biomass production to glucose uptake was very low, probably because the marked P deficiency called for an increase in energy consumption for growth and specially for maintenance. Compared with yeasts grown in a balanced medium, 40% increase in glycogen was observed, whereas no mean changes in the content of cell wall carbohydrates (glucan and mannan) and that of true protein were found.Member of the Scientific Researcher's Career of the Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET). Agrentina     

Aflatoxin — induced alteration in the levels of membrane chemicals of subcellular organelles isolated from excised,incubated Glycine max,cv. ‘Essex’ roots

Isolates of aflatoxin-producing strains of Aspergillus grow on autoclaved and field-grown (lesser extent) Glycine max beans. Both mixed and aflatoxin B₁ inhibit G. max, cv. Essex bean germination and elongation of either attached or excised cultured roots. Because B₁ impairs the latter roots' ability to intracellularize [¹⁴C]-leucine, it may alter plasmalemma structure and/or function. To determine whether incubation of excised roots for 18 hours in toxin-containing medium could affect cellular membrane chemical content, organelles were isolated by differential centrifugation (1 000, 40 000, and 80 000 xg) of homogenates and characterized chemically. Statistically significant differences between treated and untreated roots in acid insoluble protein but not either sterol or lipid phosphorus levels were observed for both 40,000 and 80,000 xg pellets. Protein and sterol recoveries were 81 (treated) and 84 (untreated) % for the former and 77 (treated) and 79 (untreated) % for the latter. Lipid phosphorus recoveries were 87.3 (treated) and 136 (untreated) % with and 96 (treated) and 83 (untreated) without membrane stabilization. Protein:sterol:lipid phosphorus were 35.74.51 (1 000 xg), 18.93.61 (40000 xg), 26.34.61 (80 000 xg) and 1,010291 (80 000 xg supernatant) for untreated and 36.93.31 (1,000 xg), 23.13.81 (40 000 xg), 36.24.81 (80 000 xg) and 1,05321.71 (80 000 xg supernatant) for treated roots. Significant differences in RNA content between treated and untreated roots were found for both 1 000 and 40 000 xg pellets but not for the 80 000 xg pellet and its supernatant. Whereas a significant increase in the 1 000 xg pellet occurred upon treatment, a decrease was noted for the 40 000 xg pellet but not for the 80 000 xg pellet and its supernatant. Similar pH 6 (plasmalemma marker enzyme) and 9 (mitochondrial marker enzyme) K⁺-stimulated ATPase activities were demonstrated for 40 000 and 80 000 xg pellets. The 1 000 xg pellet contained greater than 50% of the NADH-cytochrome c-reductase activity (endoplasmic reticulum marker enzyme) recovered from fractions examined for this activity which was absent from the 40 000 xg pellet. Both the 80 000 xg pellet and its supernatant possessed equivalent reductase activities. Inosine diphosphatase activity (dictyosome marker enzyme) was not present in 1 000 xg pellets obtained from either treated or untreated roots but was in both 40 000 and 80 000 xg pellets. Based on these results, a tentative assignment of organelles to each fraction (xg force) is reported.Abbreviations used AFB₁ aflatoxin B₁ - AFB₂ aflatoxin, B₂ - AFG₁ aflatoxin G₁ - AFG₂ aflatoxin G₂ - ATPase adenosine triphosphatase - IDPase inosine diphosphatase - NADH reduced nicotinamide-adenine dinucleotide - PCA perchloric acid - TCA trichloroacetic acid - 2, 4-D 2,4-dichlorophenoxyacetic acid Aided by grant IN-127 from the American Cancer Society to WVD and funds from the Departments of Biology, West Virginia University and Virginia Commonwealth University as well as a Sigma Xi award to JMD.     

Biological ensiling of sugarcane tops

When a mixture of sugarcane tops, alfalfa (20%) and milled malt (0.5%) was ensiled, a pronounced decrease of pH was observed during the first week corresponding to a rapid production of laotic and acetic acids. After that pH and acid production detached. The number of microorganisms increased during the first days of silage. Then aerobes rapidly decreased and were the fewer after 50 days of silage. Anaerobes also decreased but not so substantially. Lactic aoid producers rose very quickly during the first 4–5 days to became more or less constant after that. Filamentous fungi were predominant at the beginning among the fungal population, but later on almost disappeared and yeasts were found instead. During the silage process the amount of total reducing hydrolyzate increased and readily utilizable sugars were found after the microbial activity had ceased.     

In vitro hemolysis of autologous erythrocytes caused by immune murine spleen cells and spherules of the fungus Coccidioides immitis.

This communication describes an in vitro system wherein mouse erythrocytes are lysed in the presence of spherules of the fungus Coccidioides immitis and spleen cells from syngeneic mice immunized with a variety of antigens. The antigens include: tobacco mosaic virus in complete Freund's adjuvant (CFA), CFA alone, separate components of CFA, sheep erythrocytes, and allogeneic tumor. Spleen cells from mice sublethally infected with C. immitis are also capable of participating in this response. The lytic phenomenon, which does not require complement, is dependent upon the number of spleen cells per culture, the number of spherules per culture, the time of culture incubation, the amount of antigen injected into the animal and the time after immunization at which spleen cells are recovered. Live spherules or spherules killed with heat, with dimethylsulfoxide, or with formalin were effective participants, together with immune spleen cells, in the lytic reaction.     

HIV Populations Are Large and Accumulate High Genetic Diversity in a Nonlinear Fashion

HIV infection is characterized by rapid and error-prone viral replication resulting in genetically diverse virus populations. The rate of accumulation of diversity and the mechanisms involved are under intense study to provide useful information to understand immune evasion and the development of drug resistance. To characterize the development of viral diversity after infection, we carried out an in-depth analysis of single genome sequences of HIV pro-pol to assess diversity and divergence and to estimate replicating population sizes in a group of treatment-naive HIV-infected individuals sampled at single (n = 22) or multiple, longitudinal (n = 11) time points. Analysis of single genome sequences revealed nonlinear accumulation of sequence diversity during the course of infection. Diversity accumulated in recently infected individuals at rates 30-fold higher than in patients with chronic infection. Accumulation of synonymous changes accounted for most of the diversity during chronic infection. Accumulation of diversity resulted in population shifts, but the rates of change were low relative to estimated replication cycle times, consistent with relatively large population sizes. Analysis of changes in allele frequencies revealed effective population sizes that are substantially higher than previous estimates of approximately 1,000 infectious particles/infected individual. Taken together, these observations indicate that HIV populations are large, diverse, and slow to change in chronic infection and that the emergence of new mutations, including drug resistance mutations, is governed by both selection forces and drift.