Flocculation of Bacillus thuringiensis var. israelensis suspension and its efficacy against mosquito larvae.

Bacillus thuringiensis ssp. israelensis (Bti) is increasingly used as an ecologically friendly anti-mosquito agent. The bacterium cells undergo fermentation in dilute suspensions; before practical use, therefore it is necessary to concentrate the suspensions. Aggregation by polymers is a powerful tool with which to regulate the stability of suspensions. Typically, polymers at low concentrations destabilize and at high concentrations stabilize colloidal systems. Bti suspensions can be flocculated efficiently by either cationic or anionic polyelectrolytes. Cationic polyelectolytes were found to be the most efficient flocculants for bacterial suspensions. It was shown that the degree of toxicity of the flocculated Bti suspensions for biting mosquito larvae was in the same range than in non-flocculated suspension.

Stress related changes of cell surface hydrophilicity in Bacillus subtilis.

The changes of cell surface hydrophilicity in Bacillus subtilis were analyzed in response to oxygen-limitation, heat shock, salt stress, pH-shock, phosphate- and carbon-limitation. Although cell surface hydrophilicity varied during growth phases, an increase of surface hydrophilicity was observed under several of these stress conditions. An observed drop in intracellular GTP and/or ATP may be an element of the signal transduction pathway leading to an increase in surface hydrophilicity in response to environmental stresses. Attachment of cells to soil particles under salt stress conditions is strongly influenced by the degS/degU two-component system, which thereby provides a mechanism for the bacteria to escape from the hostile environment.

Changes in activity of extracellular enzymes in dual cultures of Lentinula edodes and mycoparasitic Trichoderma strains.

AIMS: The main problem that arises during the cultivation of Lentinula edodes, the Asian Shiitake mushroom, is that the logs on which the cultivation is performed are contaminated by competing micro-organisms, especially Trichoderma spp. The aim of this study was to examine the changes in activity of extracellular enzymes in dual cultures of Trichoderma spp. and L. edodes. METHODS AND RESULTS: Extracellular enzyme activities were determined spectrophotometrically. Trichoderma enzymes important for the degradation of fungal cell walls (N-acetyl-beta-glucosaminidase and laminarinase) were shown to be induced by inactive L. edodes mycelia in liquid culture. The changes that occurred in the extracellular enzyme activities of L. edodes and mycoparasitic Trichoderma spp. (T. aureoviride, T. harzianum and T. viride) were examined during antagonistic interactions on solid medium. The extracellular enzyme patterns of both partners proved to be altered. Trichoderma spp. were induced to produce N-acetyl-beta-glucosaminidase and laminarinase in the presence of active L. edodes mycelia, similarly as observed in liquid culture. The activities of both laccase and manganese peroxidase of L. edodes decreased after physical contact with active Trichoderma mycelia, possibly in consequence of the beginning of degradation of L. edodes by the Trichoderma enzymes. However, besides a decrease in manganese peroxidase activity, an enhancement of L. edodes laccase activity was observed on solid media containing crude culture fluids from Trichoderma liquid cultures. The metabolites responsible for these effects proved to be heat stable. CONCLUSIONS: Induction and inhibition of several extracellular enzymes of both partners were shown in dual cultures of L. edodes and Trichoderma strains, indicating the important role of these enzymes in the antagonistic interaction between the two species. SIGNIFICANCE AND IMPACT OF THE STUDY: As the main problem during the large-scale cultivation of L. edodes is the contamination of the growth substrate by Trichoderma mycelia, the particular knowledge of the mechanism of this competition might be relevant.

pH-dependent activation of the alternative transcriptional factor sigmaB in Bacillus subtilis.

The expression of the sigB gene of Bacillus subtilis was analysed in response to a mild acid shock. This gene is subject to sigmaB-dependent regulation. It has been found that the expression of sigB is induced as part of the acid-tolerant response. In that respect sigB is similar to the previously described gene gsiB which is also a member of the sigmaB regulon. Through this induction, the sigmaB regulon provides protection against acid shock. Besides its protective role against acid shock, no other general function could be directly associated with the sigmaB regulon. An acidification of the cytoplasmic environment induces synthesis of general stress proteins in B. subtilis.

Interactions between human and porcine interferons.

To investigate the possible interactions between human and porcine interferons (IFNs) in vitro, human transformed (FL) and nontransformed (HEF) cells were treated with either HuIFN alpha and/or gamma and porcine alpha and/or gamma. In both cases the antiproliferative activity was measured to determine the effects of different combinations between human and porcine IFNs on cell proliferation. Combinations of human and porcine IFNs acted mostly antagonistically with exception of IFN combination Hu-alpha/Po-gamma which showed a synergic cooperativity in therms of antiproliferative activity on human transformed cells.

Effect of storage and 60Co irradiation on the antiviral activity and subtype pattern of native human interferon-alpha preparations.

The stability of native human leukocyte IFN-alpha was investigated after one year storage at different temperatures, or after sterilization with 60Co-irradiation by determining the total antiviral activity of samples. Inactivation of VSV and Aujeszky virions by 60Co-irradiation was directly related to the radiolysis of samples, indicating the uselessness of this procedure for sterilization of IFN-alpha preparations. The presence and proportion of subtypes in the stored or irradiated preparations (with 50 and 25-70% inactivation, respectively) was analysed by chromatofocusing, comparing their patterns with that of the untreated controls. A logarithmic correlation was found between the pI values and temperature/irradiation sensitivity of subtypes.

Glucocorticoid-induced low molecular mass anti-inflammatory factors which do not inhibit phospholipase A2.

The peritoneal lavage fluid of dexamethasone (DXM)-pretreated rats was filtered through an Amicon YM-10 membrane (cutsize 10 kD). The retentate inhibited dextran oedema. Its 40 kD fraction (lipocortin) obtained by molecular sieving on Sephadex (SG)-75, suppressed carrageenin-induced foot swelling and phospholipase A2 (PLA2) activity, but it had no effect on the dextran response. The Amicon filtrate was chromatographed on SG-25 gel and yielded 6 and 2 kD fractions suppressing dextran and serotonin (5-HT) oedema but not carrageenin inflammation and PLA2 activity. These fractions may be active fragments of vasocortin, a glucocorticoid-induced mediator regulating vascular permeability.

Effect of amino acids on the expression of antiviral activity of different types of human interferon. I. Effect of single amino acids.

It has been observed that certain amino acids may influence the antiviral activity of different types of human interferon (IFN). The effect may be enhancing or inhibitory. A qualitative survey is provided on all three types of human IFN using 23 different amino acids. A more detailed study was carried out with 10 amino acids which proved to be active towards HuIFN-alpha. Their influences on the synergistic antiviral effects of HuIFN-alpha and HuIFN-gamma were also investigated. The dose-dependence curves of their effects on HuIFN-alpha were established.

Effect of amino acids on the expression of antiviral activity of different types of human interferon. II. Effects of various amino acid pairs.

Seven amino acids which influence the antiviral activity of IFN-alpha were tested in various pairs on human leukocyte and immune IFN and on their synergistic antiviral effect. Different kinds of interactions were observed between amino acids, ranging from extinction through competition to synergism. In some cases, different types of interactions could be observed in relation to HuIFN-alpha and HuIFN-gamma. It has been reported that certain amino acids influence the antiviral activity of human interferons (IFNs). Some of them are even capable of affecting synergistic cooperation between HuIFN-alpha and HuIFN-gamma. In our experiments we tested whether these effects can be modified further by applying two amino acids simultaneously. We hoped to shed light on the problem of their mode of action by analysing their interactions.

Direct evidence for the anti-inflammatory effect of human interferon-alpha in CFLP mice. Brief report.

In CFLP mice, intravenously administered partially purified interferon-alpha (IFN-alpha 2 X 10(6) mu/mg protein), prepared from human leukocytes, reduced carrageenan-induced paw swelling and produced slight irritation when injected into the footpad. Anti-human IFN-alpha serum abolished the anti-inflammatory effect but did not influence local phlogogenic activity. Highly purified human IFN-alpha (1.2 X 10(8) mu/mg protein) was also found to be anti-inflammatory after intravenous administration, and devoid of irritative effect at the injection site. These results suggest that human IFN-alpha possesses a direct inhibitory effect on acute inflammation in mice, and the irritation appearing at the site of its application might be due to some impurities being present in the partially purified preparations.

Semi-crude human leukocyte interferon production in a simple medium.

Semi-crude human leukocyte interferon (specific activity at least 10(5) international units per mg protein) was produced by replacement of priming medium with protein poor medium before the production period. A simpler medium than Eagle's medium was suitable to obtain substantial quantities of interferon.

Purification of human leukocyte interferon alpha by carboxymethyl controlled pore glass bead chromatography.

Human leukocyte-derived alpha interferon [HuIFN-alpha(Le)] has been purified and/or concentrated on Carboxymethyl derivatized Controlled Pore Glass (CML-CPG240) beads. These glass beads adsorb HuIFN-alpha(Le) efficiently at acid pH and at physiological ionic strengths. Elution of HuIFN-alpha(Le) may be accomplished by several methods. Using buffers at relatively high ionic strengths (approximately 0.6 M) and pH values ranging from 2.6 to 6.9 for elution, preparations with specific activities of 10(5)-10(6) IU/mg were obtained with approximately 90 percent recoveries. Alternatively, using elution buffers at the same high ionic strength and at pH values ranging from 7.0 to 8.0, five-fold or better concentration and complete recovery of crude HuIFN-alpha(Le) were achieved. The resulting preparations were suitable for direct application to an antibody affinity chromatography column.

Mouse monoclonal antibody with specificity for human interferon gamma.

We developed a monoclonal antibody (MAb) specific for human interferon gamma (HuIFN-gamma) by hybridizing cells from the NS-1 myeloma cell line with spleen lymphocytes from BALB/c mice immunized with partially purified HuIFN-gamma. Hybridoma culture supernatants were screened for neutralization of antiviral activity of HuIFN-gamma by the method determining the inhibition of nucleic acid synthesis assay (INAS), employing human fibroblasts infected with encephalomyocarditis virus (EMC). Clones exhibiting neutralization of antiviral activity of HuIFN-gamma were recloned, retested and an MAb with maximum neutralization activity was selected. This MAb was of IgM subclass and was specific for HuIFN-gamma. Antiviral activities either of human leukocyte-derived (HuIFN-alpha) or human fibroblast-derived interferon (HuIFN-beta) were not affected by this monoclonal antibody as determined by the INAS test. The specificity of the MAb for HuIFN-gamma was further confirmed by an indirect immunoprecipitation method, where monoclonal antibody-HuIFN-gamma complexes were immunoprecipitated with rabbit anti-mouse immunoglobulin and remaining IFN activity in the supernatants was determined by virus yield reduction assay. Ammonium sulfate precipitated preparations of this MAb were able to significantly increase (range of 230- to 1300-fold) the virus yield when compared with that obtained in the presence of IFN-gamma. SDS-PAGE analysis revealed that the MAb immunoprecipitates a molecule of Mr = 47 kD under nonreducing conditions. Under reducing conditions, two additional bands of Mr = 26 kD (major band) and Mr = 21 kD (minor band) were observed. A sepharose affinity column was constructed using this MAb and was able to retain approximately 60% of the partially purified HuIFN-gamma preparation applied. Significant amounts of HuIFN-gamma were eluted by increasing the ionic strength and decreasing the pH. HuIFN-alpha and HuIFN-beta were not retained by this column.

In vivo hyporesponsiveness induced by Sendai virus in CFLP mice.

Sendai and Semliki Forest viruses (SFV) raised the interferon (IFN) level in blood and suppressed the acute inflammatory response induced by carrageenan in CFLP mice. After Sendai virus had been inoculated, unresponsiveness developed to repeated challenge either with the same virus or with SFV. The hyporeactive state culminated 48 hr after first virus inoculation. It was characterized (1) by absence of IFN induction and (2) by disappearance of the virus-induced anti-inflammatory effect. In contrast, the anti-inflammatory effect of indomethacin and dexamethasone remained unchanged. In addition, peripheral white blood cells were counted upon Sendai virus inoculation either in normal or in hyporesponsive mice. Six hr after inoculation, Sendai virus induced a marked granulocytosis with lymphopenia. In hyporesponsive mice leukocytosis was observed. Repeated Sendai virus injection was followed by a less pronounced granulocytosis, while the decreased number of mononuclear cells remained unchanged. These alterations in mice inoculated with Sendai virus offers a model of hyporesponsiveness established in vivo.

On the anti-inflammatory effect of sendai virus inoculation.

Sendai virus given intravenously into CFLP mice produced dose-related suppression of the acute inflammation produced by carrageenan and 5-HT. The inhibition correlated with interferon levels in the blood, suggesting that interferon may account for the virus-induced anti-inflammatory effect.

Antiviral effect of 3-[bis-(2-hydroxyethyl)-amino]-acetophenone-[4,5-diphenyl-oxyazolyl-(2)]hydrazone (IMET 98/69) in mice.

The potential in vivo antiviral activity of 3-[bis-(2-hydroxyethyl)-amino]-acetophenone-[4,5-diphenyl-oxazolyl-(2)]-hydrazone (IMET 98/69) was evaluated on model infections in mice. Animals treated subcutaneously (s.c.) with 1 mmole of the drug per kg body weight once daily for five days were significantly protected against a lethal infection with cardioviruses, Semliki forest virus and vaccinia virus. In influenza A and B virus models no antiviral activity was observed either after s.c. or oral treatment.

Enhancement of interferon induction in mice by polycationic modified polypeptides.

Combined intraperitoneal treatment of mice with poly I: C and a polycationic modified polypeptide (poly-DMAE-glutamine) was investigated. It was established that the presence of appropriate amounts of poly-DMAE-glutamine produced markedly enhanced serum interferon levels as compared to those produced by poly I: C alone. Similar combinations injected into mice produced full protection against lethal doses of mouse-virulent Semliki forest virus.

Regulation of resistance to various toxicants by PCN (pregnenolone-16alpha-carbonitrile) and thyroxine.

In rats, PCN (the most potent catatoxic steroid known to date) at the usual dose level powerfully inhibited the toxicity of antazoline, carbamazepine, cocaine, guanethidine, ibuprofen, ketamine, LSD, nembutal and reserpine, whereas (except in the case of nembutal) thyroxine sensitized the animals to intoxication with these same compounds. Even much lower doses of PCN or thyroxine exerted similar but weaker effects. PCN-induced resistance to the various substrates was generally not altered by concurrent administration of thyroxine but in a few cases its protective action was partially or totally inhibited, depending upon the respective dose levels of both compounds.