Fermentation of Bacillus thuringiensis for exotoxin production: Process analysis study

The exotoxin produced by certain serotypes of Bacillus thuringiensis was used as a means of microbiological control of the larval development of flies. The optimal batch cultivation conditions with respect to pH, temperature, aeration, agitation, and initial concentration of growth-limiting substrate were determined. A dynamic model describing the process was designed and fitted to the experimental data. The application of a method for estimating exotoxin and bacterial concentrations from on-line measurable quantities such as oxygen consumption and heat production is presented.     

Tubulin synthesis and heat shock-induced cell synchrony in Tetrahymena

This paper offers the suggestion that heat shock inhibition of tubulin synthesis accounts for the molecular mechanism by which periodic heat shocks induce cell synchrony in Tetrahymena. Each heat shock (34 °C) represses tubulin synthesis and blocks the division cycle at the point when the oral structure, rich in microtubules, would normally begin to assemble. Recovery (at 28 °C) from each heat shock is characterized by parallel derepression of tubulin synthesis and of oral development. Changes in protein synthesis patterns are complex when the temperature is shifted up and down between 28 and 34 °C and further experimental support is required in support of the hypothesis here forwarded.     

Potentiation of the in vitro cytotoxic response to syngeneic lymphoma cells by soluble products of tuberculin-sensitive lymphoid cells stimulated with PPD

Spleen cells from rats immunized with the syngeneic (C58NT)D Gross virus induced lymphoma have previously been shown to differentiate into cytotoxic effector cells following restimulation with tumor cells in vitro. Previous work has also demonstrated that the addition of PPD-primed syngeneic spleen cells and PPD to cultures of (C58NT)D-primed spleen cells will potentiate the in vitro cytotoxic response to tumor antigens. In the studies presented here, the potentiating effect was found to be mediated by a soluble factor(s) released by nonadherent cells from BCG-primed rats. The release of this immunopotentiating factor(IPF) required the presence of PPD and varied with the concentration of PPD added. IPF was produced by BCG-primed spleen, lymph node, and thymus cells. Maximal production of IPF in PPD-stimulated cultures was obtained after 6–12 hr of incubation. Supernatants obtained after 30 hr of incubation lacked apparent IPF activity when tested initially, but activity was recovered after mild heat treatment. Recovery of IPF activity after heat exposure is best explained by the presence of a heat-labile inhibitor. IPF itself is stable to heat treatment to 56 °C for 40 min. IPF was also shown to be capable of enhancing immune responses to histocompatibility antigens in vitro.     

Role of Heme in Synthesis and Membrane Binding of Succinic Dehydrogenase in Bacillus subtilis

A 5-aminolevulinic acid-requiring mutant of Bacillus subtilis was isolated. When the mutant is shifted from medium containing 5-aminolevulinic acid to medium lacking this growth factor, the bacteria continued to grow at undiminished rate for about three generations. The membranes from these bacteria contained severely reduced amounts of cytochrome. The mutant was used to study the role of heme synthesis on synthesis and membrane binding of succinic dehydrogenase (SDH). The amount of SDH in whole-cell lysates in the soluble cytoplasmic fraction and in membranes was determined by one-dimensional (rocket) immunoelectrophoresis with an SDH-specific antiserum. After heme synthesis was blocked, the relative amount of SDH in the membrane decreased, whereas increasing amounts of SDH antigen were found in the cytoplasm. When heme synthesis was resumed on readdition of 5-aminolevulinic acid, the amount of membrane-bound SDH antigen increased at a much faster rate than net synthesis. During a 3-h growth period without 5-aminolevulinic acid, there was little change in the pattern of membrane proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioactively labeled membranes, as compared to membranes from control cultures. However, both the 65,000-dalton and the 28,000-dalton polypeptides of the SDH complex (L. Hederstedt, E. Holmgren, and L. Rutberg, J. Bacteriol. 138:370-376, 1979) were present in decreasing amounts in membranes from 5-aminolevulinic acid-starved bacteria. From these results we suggest that SDH in B. subtilis is synthesized as a soluble protein and becomes membrane bound only when it attaches to a site in the membrane, (part of) which is a cytochrome of b type.     

Occurrence and characterization of "avium-like" Mycobacteria isolated from animals in Sweden

431 cultures of "avium-like" mycobacteria (ALM) were isolated from wild and domestic animals during 1974--76 at the National Veterinary Institute, Stockholm. Of these, 50 isolates from pigs were studied by growth-chromogenicity, pathogenicity, and biochemical tests. Furthermore, thin-layer chromathography was performed, and on some isolates serotyping. All 50 isolates belonged to Runyon's group III and were pathogenic for chicken; none was capable of splitting oleic acid from Tween 80. 47 gave tellurite reduction within a period of three days; one was arylsulphatase-positive after three days and a further four after 14 days. The biological and biochemical tests permit assignation of the 50 isolates to the M. avium-intracellulare complex. The lipid patterns of the isolates examined were analysed by thin-layer chromatography. Thirty-five of the isolates showed a lipid pattern similar to that of A 2 of the fowl reference strain; three belonged to lipid type A 1 and four to A 3. Eight could not be typed. Of 22 isolates, 14 could be assigned to M. avium serotypes.     

Circadian rhythms of sensitivity to insecticides in Musca domestica (Diptera, Muscidae)

Circadian rhythms of LD₅₀ values to DDT, dieldrin and malathion, topically applied, were determined for houseflies reared under LD 14:10 with dawn at 06.00 hr. There was a marked increase in susceptibility at 05.00 hr in each case. With dawn at 18.00 hr., DDT LD₅₀ values were lowest at 17.00 hr indicating independence of the flies' biological clocks from clock time of day. Flies reared under LD 18:6 and 10:14 also had circadian rhythms of sensitivity to DDT. Mean daily LD₅₀ values were inversely related to photophase length. The ratios of mean daily LD₅₀ to pre-dawn values were greatest for the longer photophases. Flies reared under LD 14:10 until the pupal stage, then DD until testing showed a normal circadian rhythm. Flies reared in total darkness (DD) showed no diel variations in susceptibility. W.H.O. standard strain flies were used for all the experiments. A fully susceptible (Cooper) and a DDT resistant (DEH-DOV) strain also showed significant circadian rhythms of sensitivity to DDT.

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Zusammenfassung Circadianrhythmen der LD 50-Werte gegenüber DDT, Dieldrin und Malathion-topical angewandt wurden bei Stubenfliegen ermittelt, die bei 14:10 h-Tag mit Tagesanbruch um 6.00 Uhr gezüchtet wurden. In allen Fällen war die Empfindlichkeit um 5.00 Uhr wesentlich erhöht. Bei Tagesanbruch um 18.00 Uhr waren die niedrigsten LD 50-Werte um 17.00 Uhr. Dies weist auf die Unabhängigkeit der biologischen Uhr der Fliegen von der Tageszeit hin. Fliegen, die bei 18:6 oder bei 10:14 LD gezüchtet wurden, zeigten ebenfalls einen Circadianrhythmus hinsichtlich der Empfindlichkeit gegenüber DDT. Die mittleren LD 50-Werte waren umgekehrt proportional zur Länge der Photophase. Das Verhältnis der mittleren täglichen LD 50-Werte zu den Vortagesanbruchwerten war am grössten bei längerer Photophase. Fliegen, die bei 14:10 LD bis zum Puppenstadium und anschliessend bei DD bis zur Testung gehalten wurden, zeigten einen normalen circadianen Rhythmus. Bei Züchtung in völliger Dunkelheit zeigten sie keine Tagesschwankungen in der Empfindlichkeit. Für alle Versuche wurde ein WHO-Standardstamm benutzt. Zwei andere Stämme, einer voll empfindlich (Cooper), der andere resistent (DEH-DOV) zeigten ebenfalls signifikante Circadianrhythmen in der DDT-Empfindlichkeir.

     

Glutamine-dependent asparagine synthetase from Lupinus luteus

Asparagine synthetase (glutamine-hydrolyzing [l-aspartate: l-glutamine amido-ligase (AMP-forming), E.C. 6.3.5.4] was purified over 500-fold from cotyledon extracts of 1-week-old yellow lupin seedlings. The enzyme was labile and required protection by high levels of thiols; glycerol and the substrates also stabilized it. The reaction products were shown to be asparagine, AMP, PPi and glutamate. The limiting K_m values were for aspartate 1·3 mM, for MgATP 0·14 mM and for glutamine 0·16 mM. Positive homotropic cooperativity was observed for MgATP only, and gel filtration studies indicated that the substrate-free enzyme (MW 160 000) associated to a dimer (MW 320 000 in the presence of MgCl₂ and ATP. The purified enzyme, which had some glutaminase activity, catalyzed an aspartate- and glutamine-independent ATP-PPi exchange reaction at a rate 5–7-fold higher than the rate of asparagine synthesis. Initial velocity studies and exchange data indicated an overall ping-pong mechanism. Compared to similar enzymes isolated from mammalian tumor cells, the lupin enzyme appears to be unique with respect to MW, reaction mechanism and regulatory properties. The allosteric properties observed suggest an important role for this enzyme in the regulation of asparagine biosynthesis.     

Spectral imaging for precise surgical intervention in Hirschsprung's Disease

We used advanced spectral imaging for intrasurgical decision making in a preclinical study, on a mouse model of Hirschsprung's Disease. Our imaging device sampled areas from normal and abnormal (aganglionic) colon in these animals. Spectral segmentation and classification of the resulting images showed a clear distinction between the normal and aganglionic regions, as confirmed by pathological analysis and use of mutant mice. We developed a simple algorithm that could distinguish normal from aganglionic colon with high spatial resolution and reproducibility, and the following statistics: sensitivity = 97%, specificity = 94%, positive predictive value = 92%, negative predictive value = 98%. These studies showed translational proof of concept that spectral imaging could be used during operations, in real time, to help surgeons precisely distinguish normal from abnormal tissue without requiring traditional biopsy. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)