Germination of concentrated suspensions of spores from Aspergillus niger

Summary Germination of condiospores fromA.niger in very concentrated suspension was required to inoculate solid fermentation media, but a germination self-inhibitor was detected in spores. It was found that the inhibitory effect depended on the composition of the medium and was reduced when glucose was used as a carbon source. The effect of the self-inhibitor was eliminated by washing the spores and using glucose and a protein nitrogen source in the germination medium. By this method it was possible to increase about 100 times (10⁶ to 10⁸) spore concentration, keeping more than 90% germination.     

Functional alpha 2-adrenoceptors in rat adipocytes: inhibition of cyclic AMP accumulation

Alpha 2-adrenoceptor activation inhibits cyclic AMP accumulation in fat cells from many species. However, the presence of alpha 2-adrenoceptors in rat adipocytes has been difficult to demonstrate. We observed that alpha 2-adrenergic activation inhibits forskolin-stimulated cyclic AMP accumulation both in rat and hamster adipocytes; UK 14304, p-amino clonidine and clonidine were the agents with higher efficacy. The effect of UK 14304 was blocked by yohimbine but not by prazosin demonstrating the involvement of alpha 2-adrenoceptors. Pertussis toxin blocked the alpha 2-adrenergic effect. Our results demonstrate the presence in rat fat cells of alpha 2-adrenoceptors coupled to adenylate cyclase via "Gi".     

Spin label and microcalorimetric studies of the interaction of DNA with unilamellar phosphatidylcholine liposomes

High-molecular DNA from chicken erythrocytes interacts with 1,2-dipalmitoylphosphatidylcholine in unilamellar liposomes, both in the presence and absence of Mg2+ ions. This interaction results in a phase separation in liposome membranes. The new phase induced by DNA and Mg2+ has a higher gel-liquid crystal phase transition temperature as measured by microcalorimetry. In the liquid crystalline state, the 16- and 5-doxyl stearic acid spin labels indicate changed local bilayer properties at the label position in the new phase.     

Susceptibility of autologous target cells to lysis by lymphokine-activated effectors from interferon-α-treated chronic myelogenous leukaemia patients

Summary Chronic myelogenous leukemia (CML) patients in chronic phase display compromised lymphokine-activated killer (LAK) cell induction, which is partly restored after therapy with interferon . However, the relative resistance of the leukemic cells from these patients to autologous or allogeneic LAK lysis is not affected by this treatment. In an attempt to render CML cells more susceptible to lysis or cytostasis, they were precultured in serum-free medium with or without recombinant growth factors. In eight patients studied, interleukin-3 (IL-3) significantly enhanced the spontaneous short-term (6-day) proliferation of CML cells, with retention of ability to form colonies in methylcellulose. Culture in either medium alone or IL-3 led to a significant enrichment of CD14⁺ and CD33⁺ cells but to a reduction in CD34⁺ cells. In contrast, culture of the same cells in IL-2 (to generate autologous LAK activity) resulted in a loss of CD14⁺ and CD33⁺ as well as CD34⁺ cells but in a significant increase in CD3⁺ and CD56⁺ cells. Despite similarities in their phenotypes, IL-3 cultured cells but not those cultured in medium alone acquired susceptibility to lysis by the IL-2-cultured autologous LAK cells. These results may have significance for the design of novel combination immunotherapy in CML.This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 120)     

Insulin-stimulated alpha-(methyl)aminoisobutyric acid uptake in skeletal muscle. Evidence for a short-term activation of uptake independent of Na+ electrochemical gradient and protein synthesis.

1. The present study was designed to explore the mechanisms by which insulin stimulates system A of amino acid transport in extensor digitorum longus (EDL) muscles, by using a system A analogue, alpha-(methyl)aminoisobutyric acid (MeAIB). 2. Insulin stimulation of MeAIB uptake was noted after only 30 min of incubation and was maximal at 60 min. Kinetics of the insulin effect on MeAIB uptake were characterized by an increased Vmax. without modification of Km for MeAIB. 3. Incubation of EDL muscles with cycloheximide for 90 min did not modify MeAIB uptake in either the presence or the absence of insulin, indicating the independence of insulin action from protein synthesis de novo. Incubations for 180 min with cycloheximide caused a decrease in basal MeAIB uptake; however, the percentage stimulation of amino acid transport by insulin was unaltered. Basal MeAIB uptake was increased by incubation for 180 min, but under these conditions no change in the percentage effect of insulin was found. 4. Ouabain, gramicidin D, or both, markedly decreased basal MeAIB uptake by EDL muscle, but the percentage effect of insulin was unaltered. 5. We conclude that insulin action on amino acid transport through system A in muscle is rapid, is characterized by an increased Vmax., and is independent of protein synthesis de novo and the Na+ electrochemical gradient. Our data are compatible with insulin acting directly on the system A transporter.     

Effect of pectin on pectinases in autolysis of Botrytis cinerea

Pectic activity in autolyzed cultures of Botrytis cinerea in a medium with and without pectin was similar, but in the medium with pectin maximal activities occurred in younger cultures. The pectic activities found were polygalacturonase, polymethylgalacturonase, endo activity (pectin as substrate) and pectin lyase. The molecular weights of polygalacturonase, polymethylgalacturonase and endo activity (pectin as substrate) were 36000, 33000 and 30200 daltons respectively, and the molecular weight of pectin lyase was 18200 daltons. By gel electrophoresis four different pectic activities were detected, three in the top of the gel and one in the bottom. Two enzymes were characterized, the polygalacturonase activity (first band in the top) inhibited by Ca⁺⁺ and the pectin lyase activity (in the bottom) which was not inhibited by Ca⁺⁺. These enzymes are not induced by the presence of pectin in the medium during degradation of Botrytis cinerea.     

Asymmetric incorporation of [14C]cyanate and of fluorescein isothiocyanate in mamillary body of conditioned rats

A marked decrease in overall learning capacity has been observed in rats injected with cyanate. Therefore it was of interest to test whether learning influenced carbamylation of brain proteins. Incorporation of [¹⁴C]cyanate into proteins of the mamillary body was selectively modified following operant conditioning of the rat, so that trained rats showed an asymmetric image with higher levels of incorporation in the right side than in the left side, as compared to control rats. These results were confirmed using fluorescein isothiocyanate. The asymmetry persisted once the learning had been well established.     

Characterization of the cellulase complex from Cellulomonas grown on bagasse pith

Summary The effect of physico-chemical parameters on the cellulolytic activity of Cellulomonas sp. IIbc grown on sugarcane bagasse pith was investigated, and the optimum ranges for enzyme activity were established. The cellulases were more stable when incubated at the optimum growth temperature (32°C) than under optimum activity conditions (45°C for -glucosidases and 50°C for CMC- and FP-cellulases). The -glucosidases were the thermostability-limiting enzymes of the complex. Two types of endoglucanases could be recognized according to their adsorption properties on bagasse: one weakly-bound and one tightly-bound type, the latter constituting approximately 73% of the extracellular endoglucanases at exponential growth phase. Four forms active on filter paper and three active on CMC were obtained by HPLC separation of the extracellular fraction of the culture at stationary phase.Abbreviations CMC carboxymethylcellulose - FP filter paper     

Evidence suggesting a role for sperm metalloendoprotease activity in penetration of zona-free hamster eggs by human sperm

It has been reported that metalloendoprotease (MEP) activity is involved in somatic cell membrane fusion events and in the sea urchin sperm acrosome reaction (AR). MEP activity also has been demonstrated in human and other mammalian sperm. The present study was concerned with investigating whether a human sperm MEP is important in membrane events necessary for sperm egg fusion. Ejaculated human sperm were washed, capacitated in vitro, and preincubated with the competitive MEP inhibitors phosphoramidon (50 microM) or CBZ-L-phenylalanine (1 mM), with 100 microM diethylenetriaminepentaacetic acid (DTPA), a heavy metal chelator, or as controls, with the appropriate solvents. The AR was initiated in vitro with preovulatory human follicular fluid and the sperm washed to dilute inhibitors and then coincubated with zona-free golden hamster eggs (zonae and cumuli removed with trypsin and hyaluronidase, respectively). Eggs were washed after 0.5 h, and the number of sperm remaining bound was counted. After 2.5 h further incubation, the eggs were stained with acetolacmoid or acetoorcein and penetration was assayed by counting the number of decondensed sperm heads per egg (penetration index) and the percent of penetrated eggs. The inhibitor treatments did not decrease the percentage of penetrated eggs (range 80-90%), but a significant reduction in the penetration index was observed. Phosphoramidon reduced the penetration index by 45%, CBZ-L-phenylalanine by 57%, and DTPA by 56%. None of the inhibitors decreased the penetration index or the percentage of penetrated eggs when added directly to suspensions of acrosome-reacted sperm and zona-free eggs at the diluted levels that would have been present after washing inhibitor-treated sperm.(ABSTRACT TRUNCATED AT 250 WORDS)