Effect of Sodium Chloride and pH on Enterotoxin C Production

Growth and production of enterotoxin C by Staphylococcus aureus strain 137 in 3% + 3% protein hydrolysate powder N-Z Amine NAK broths with 0 to 12% NaCl and an initial pH of 4.00 to 9.83 were studied during an 8-day incubation period at 37 C. Growth was initiated at pH values as low as 4.00 and as high as 9.83 at 0% salt level as long as the inoculum contained at least 10(8) cells per ml. Rate of growth decreased as the NaCl concentration was increased gradually to 12%. Enterotoxin C was produced in broths inoculated with 10(8) cells per ml and above and having initial pH ranges of 4.00 to 9.83, 4.40 to 9.43, 4.50 to 8.55 and respective NaCl concentrations of 0, 4, and 8%. In the presence of 10% NaCl, the pH range supporting enterotoxin C production was 5.45 to 7.30 for an inoculum level of 10(8) cells per ml and 6.38 to 7.30 for 3.6 x 10(6) cells per ml. In repeated experiments in which the inoculum contained 10(8) cells per ml, we failed to demonstrate enterotoxin C production in broths with 12% NaCl and a pH range of 4.50 to 8.55 and concentrated up to 14 times. The effect of NaCl on enterotoxin C production followed the same pattern as its effect on enterotoxin B production. As the concentration of NaCl increased from 0 to 10%, yields of enterotoxin B and C decreased to undetectable amounts.     

The novel flightless-I gene brings together two gene families, actin- binding proteins related to gelsolin and leucine-rich-repeat proteins involved in Ras signal transduction

The Drosophila melanogaster gene flightless-I, involved in gastrulation and muscle degeneration, has Caenorhabditis elegans and human homologues. In these highly conserved genes, two previously known gene families have been brought together, families encoding the actin- binding proteins related to gelsolin and the leucine-rich-repeat (LRR) group of proteins involved in protein-protein interactions. Both these gene families exhibit characteristics of molecular changes involving replication slippage and exon shuffling. Phylogenetic analyses of 19 amino acid sequences of 6 related protein types indicate that actin- associated proteins related to gelsolin are monophyletic to a common ancestor and include flightless proteins. Conversely, comparison of 24 amino acid sequences of LRR proteins including the flightless proteins indicates that flightless proteins are members of a structurally related subgroup. Included in the flightless cluster are human and mouse rsp-1 proteins involved in suppressing v-Ras transformation of cells and the membrane-associated yeast (Saccharomyces cerevisae) adenylate cyclase whose analogous LRRs are required for interaction with Ras proteins. There is a strong possibility that ligands for this group could be related and that flightless may have a similar role in Ras signal transduction. It is hypothesized that an ancestral monomeric gelsolin precursor protein has undergone at least four independent gene reorganization events to account for the structural diversity of the extant family of gelsolin-related proteins and that gene duplication and exon shuffling events occurred prior to or at the beginning of multicellular life, resulting in the evolution of some members of the family soon after the appearance of actin-type proteins.      

Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid

Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge.     

A novel approach for whey recycling in production of bacterial insecticides

A simplified approach was devised to recycle sweet whey in production of spore-δ-endotoxin complex from certain entomopathogenic varieties ofBacillus thuringiensis Berliner. The process suggested aimed at the protection of the environment through dual channels namely biological oxygen demand (BOD) reduction of the byproduct under investigation and its incorporation in a microbial fermentation for production of pollution-free biological insecticides. The sweet whey could be used successfully for endotoxin production as complete fermentation media both as such and with simple treatments. Supplementation of whey media with ground leguminous seeds and fodder yeast resulted in marked increase in the yields of endotoxin produced but the toxicity was not increased proportionnally. Standard biological assays revealed high efficiency of certain strains ofB.t. var.entomocidus, kurstaki andgalleriae in producing endotoxins highly active against 3rd instar larvae ofSpodoptera littoralis Boisduval,Spodoptera exigua Hübner andHeliothis armigera Hübner. The suggested approach and the findings obtained are discussed in view of their application feasibilities.

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Résumé Une méthode simplifiée a été mise au point pour recycler le petit lait dans la production du complexe spore-δ-endotoxine de certaines variétés entomopathogènes deBacillus thuringiensis Berliner. Le procédé proposé a pour but la protection de l'environnement par un double canal chimique, c'est-à-dire la réduction de la demande biologique en oxygène du sous produit étudié et son incorporation dans une fermentation microbienne pour la fabrication d'insecticides biologiques non polluants. Le petit lait peut être employé avec succès pour la production d'endotoxine à la fois en tant que milieux complets de fermentation et avec des traitements simples. L'addition aux milieux à base de petit lait de graines de légumineuses et de levure alimentaire aboutit à une augmentation sensible des rendements en endotoxine mais la toxicité n'est pas accrue en proportion. Des essais biologiques normalisés montrent une activité élevée de certaines souches deB.t. var.entomocidus, kurstaki etgalleriae qui produisent des endotoxines très actives à l'égard des larves de 3e stage deSpodoptera littoralis Boisduval,Spodoptera exigua Hübner etHeliothis armigera Hübner. La méthode proposée et les résultats obtenus sont discutés en vue de leur faisabilité d'application.

     

Production physiology and properties of a novel fungal fibrinolytic enzyme.

A potent extracellular fibrinolytic enzyme was obtained from cultures of the imperfect fungus Fusarium semitectum under certain growth conditions. Nitrate addition to cultures increased enzyme production. The enzyme showed a versatile proteolytic activity against several protein substrates including casein, gelatin, haemoglobin, bovine serum albumin, and fibrin from both buffalo and human sources. Optimal fibrinolysis occurred at pH values around 7.0. The fibrinolytic activity exhibited marked heat stability in enzyme samples heated at 60 degrees C, and retained more than 40% of its activity in samples heated to 100 degrees C for 10 min. Fibrinolysis proceeded optimally in the temperature range between 50--60 degrees C. Copper ions significantly activated the enzyme. Other biochemical properties are also reported.     

Physiology and characterization of a fungal milk-clotting enzyme from Aspergillus flavus.

Aspergillus flavus produced extracellularly an active rennin-like enzyme when grown aerobically in whey media. The enzyme was detected at early stages of growth reaching a maximum after three to four days at 25 degrees. The activity was destroyed by heating to temperatures higher than 50 degrees, whereas the presence of skim milk during heating preserved the enzyme activity, at least, up to 70 degrees. Calcium chloride significantly stimulated the milk-clotting activity up to 1% final concentration. The clotting time was inversely proportional to protein concentration in the range 0.2-0.6 mg/ml and the enzyme exhibited marked stability when stored at 37 degrees at pH 6.     

Management of the droopy tip: a comparison of three alar cartilage-modifying techniques

The droopy tip is a common nasal deformity in which the tip is inferiorly rotated. Five hundred consecutive rhinoplasty cases were studied to assess the incidence and causes of the droopy tip deformity and to evaluate the role of three alar cartilage-modifying techniques--lateral crural steal, lateral crural overlay, and tongue in groove--in correcting such a deformity. The external rhinoplasty approach was used in all cases. Only one of the three alar cartilage-modifying techniques was used in each case, and the degree of tip rotation and projection was measured both preoperatively and postoperatively. The incidence of droopy tip was 72 percent, and the use of an alar cartilage-modifying technique was required in 85 percent of these cases to achieve the desired degree of rotation. The main causes of droopy tip included inferiorly oriented alar cartilages (85 percent), overdeveloped scrolls of upper lateral cartilages (73 percent), high anterior septal angle (65 percent), and thick skin of the nasal lobule (56 percent). The lateral crural steal technique increased nasal tip rotation and projection, the lateral crural overlay technique increased tip rotation and decreased tip projection, and the tongue-in-groove technique increased tip rotation without significantly changing the amount of projection. The lateral crural overlay technique resulted in the highest degrees of rotation, followed by the lateral crural steal and finally the tongue-in-groove technique. According to these results, the lateral crural steal technique is best indicated in cases with droopy underprojected nasal tip, the lateral crural overlay technique in cases of droopy overprojected nasal tip, and the tongue-in-groove technique in cases where the droopy nasal tip is associated with an adequate amount of projection.     

Rates and patterns of mitochondrial DNA sequence evolution in fringilline finches (fringilla spp.) and the greenfinch (Carduelis chloris)

Rates and patterns of evolution in partial sequences of five mitochondrial genes (cytochrome b, ATPase 6, NADH dehydrogenase subunit 5, tRNA(Glu), and the control region) were compared among taxa in the passerine bird genera Fringilla and Carduelis. Rates of divergence do not vary significantly among genes, even in comparisons with the control region. Rate variation among lineages is significant only for the control region and NADH dehydrogenase subunit 5, and patterns of variation are consistent with the expectations of neutral theory. Base composition is biased in all genes but is stationary among lineages, and there is evidence for directional mutation pressure only in the control region. Despite these similarities, patterns of substitution differ among genes, consistent with alternative regimes of selective constraint. Rates of nonsynonymous substitution are higher in NADH dehydrogenase subunit 5 than in other protein-coding genes, and transitions exist in elevated proportions relative to transversions. Transitions appear to accumulate linearly with time in tRNA(Glu), and despite exhibiting the highest overall rate of divergence among species, there are no transversional changes in this gene. Finally, for resolving phylogenetic relationships among Fringilla taxa, the combined protein-coding data are broadly similar to those of the control region in terms of phylogenetic informativeness and statistical support.      

Factors affecting growth physiology of Bacillus thuringiensis

Summary Physiological studies on Bacillus thuringiensis var. entomocidus revealed the failure of the organism to survive or sporulate under low aeration levels, notably in the presence of high sugar concentrations. Cell counts, sporulation titers and potency of resulting endotoxin were found to vary with the level of aeration. The incremental feeding of glucose with continuous pH adjustment prevented cell injury and death which results from prolonged exposure to acidity liberated at the high sugar concentrations which occur when glucose is added batchwise. Increasing of dipotassium phosphate concentration in growth medium increased the potency of the resulting endotoxin.