Evaluation of the Effects of Biological Preparations on Phytopathogenic Fungi Didymella applanata and Botrytis cinerea

Fungicidal and fungistatic effects of biological preparations involving bacteria of the genera Pseudomonas and Bacillusand fungi of the genusChaetomium on phytopathogenic fungi Didymella applanata and Botrytis cinereawere evaluated. All the biological preparations under study inhibited the growth of colonies of the fungi; however, the degree of the inhibition depended on the nature of each particular microorganism and the concentration of each particular preparation. A preparation containing Bacillus subtilis at a concentration of 0.2% effected maximum suppression ofB. cinerea (the diameter of the colonies decreased sevenfold). Preparations containing bacteria of the genus Pseudomonas and fungi of the genus Chaetomium were most efficient in suppressing D. applanata.Preparations containingB. subtilis and Chaetomium spp. showed promise as agents against simultaneous development of spur blight and Botrytis blight.     

Separation and some properties of the major proteins of the human erythrocyte membrane

A fractionation procedure is described which allows the isolation of three major human erythrocyte membrane proteins. Their isolation involves three sequential extraction procedures followed by gel filtration in 1% sodium dodecyl sulphate and preparative gel electrophoresis. All three proteins can be isolated from a single preparation. One of the proteins is the erythrocyte sialoglycoprotein, for which no C- or N-terminal residues were found. The other two proteins, which have not previously been isolated, have subunit molecular weights of 74000 and 93000 and contain 9 and 7% carbohydrate respectively. These glycoproteins have blocked N-terminal residues and show similarities in their chemical properties. Preparations derived from blood-group O erythrocytes contain no N-acetylgalactosamine, but similar preparations from blood-group A erythrocytes do contain this sugar. These three proteins cannot easily be solubilized by gentle aqueous procedures and represent about half of the erythrocyte ;ghost' protein. They carry a large proportion of the cell-surface carbohydrate.     

A cytolytic protein from the edible mushroom, Pleurotus ostreatus

Aqueous extracts of the edible mushroom, Pleurotus ostreatus, contain a substance that is lytic in vitro for mammalian erythrocytes. The hemolytic agent, pleurotolysin, was purified to homogeneity and found to be a protein lacking seven of the amino acids commonly found in proteins. In the presence of sodium dodecyl sulfate it exists a monomers of molecular weight 12 050 whereas under non-dissociating conditions it appears to exist as dimers. It is isoelectric at about pH 6.4. The sensitivity of erythrocytes from different animals correlates with sphingomyelin content of the erythrocyte membranes. Sheep erythrocyte membranes inhibit pleurotolysin-induced hemolysis and the inhibition is time and temperature dependent. Ability of membranes to inhibit hemolysis is abolished by prior treatment of membranes with specific phospholipases. Pleurotolysin-induced hemolysis is inhibited by liposomes prepared from cholesterol, dicetyl phosphate and sphingomyelin derived from sheep erythrocytes whereas a variety of other lipid preparations fail to inhibit. It is concluded that sphingomyelin plays a key role in the hemolytic reaction.     

A cytolytic protein from the edible mushroom, Pleurotus ostreatus

Aqueous extracts of the edible mushroom, Pleurotus ostreatus, contain a substance that is lytic in vitro for mammalian erthrocytes. The hemolytic agent, pleurotolysin, was purified to homogeneity and found to be a protein lacking seven of the amino acids commonly found in proteins. In the presence of sodium dodecyl sulfate it exists as monomers of molecular weight 12 050 whereas under non-dissociating conditions it appears to exist as dimers. It is isoelectric at about pH 6.4. The sensitivity of erythrocytes from different animals correlates with sphingomyelin content of the erythrocyte membranes. Sheep erythrocyte membranes inhibit pleurotolysin-induced hemolysis and the inhibition is time and temperature dependent. Ability of membranes to inhibit hemolysis is abolished by prior treatment of membranes with specific phospholipases. Pleurotolysin-induced hemolysis is inhibited by liposomes prepared from cholesterol, dicetyl phosphate adn sphingomyelin derived from sheep erythrocytes whereas a variety of other lipid preparations fail to inhibit. It is concluded that sphingomyelin plays a key role in the hemolytic reaction.     

Isolation of a tryptic fragment from Clostridium perfringens theta-toxin that contains sites for membrane binding and self-aggregation

Trypsin cleaves Clostridium perfringens theta-toxin (perfringolysin O or PFO) at a single site between residues 303 and 304 (Ohno-Iwashita, Y., Iwamoto, M., Mitsui, K., Kawasaki, H., and Ando, S. (1986) Biochemistry 25, 6048-6053; Tweten, R. K. (1988b) Infect. Immun. 56, 3228-3234) and yields an amino-terminal fragment of 30,208 Da (T1) and a carboxyl-terminal fragment of 22,268 Da (T2). Both peptides were purified by reverse phase chromatography of trypsin-nicked PFO. Neither peptide retained hemolytic activity. Peptide T1 had no apparent effect on the hemolytic activity of PFO, whereas T2 was found to inhibit the hemolytic activity of PFO and was analyzed further. The order of binding of T2 and PFO to membranes did not alter the inhibitory effect of T2 on PFO-induced hemolysis, indicating that competitive binding by T2 for PFO membrane binding sites was not the basis for the observed inhibition. Further analysis showed that T2 could inhibit membrane-dependent fluorescence energy transfer (FET) between PFO molecules labeled with fluorescein (fluorescent donor) or tetramethylrhodamine (fluorescent acceptor). This provided evidence that T2 could complex with PFO. T2 was also found to be incapable of self-aggregation (as opposed to PFO), since preincubation of T2 with either erythrocytes or erythrocyte ghost membranes did not affect the T2-dependent inhibition of hemolysis or FET. These data indicate that T2 inhibits PFO-dependent hemolysis by forming a complex with PFO, which inhibits aggregation and that the membrane binding site and a single aggregation site remain intact on T2.     

柑橘内生真菌的分离鉴定及其发酵产物对柑橘溃疡病菌的抑制活性

本研究从柑橘抗病品种的健康植株不同组织中分离纯化和鉴定内生真菌,并测定其发酵产物对柑橘溃疡病菌的抑制活性,以明确柑橘抗病品种中内生真菌的组成及其产抗柑橘溃疡病菌活性代谢产物的潜力,为柑橘溃疡病抗菌剂的开发奠定基础.该研究通过组织培养法分离内生真菌,采用形态学和分子生物学方法对其进行鉴定;基于前期的拮抗预试验结果,选取代...     

Membrane fusion activity of reconstituted vesicles of influenza virus hemagglutinin glycoproteins

Reconstituted vesicles of hemagglutinin glycoproteins into egg yolk phosphatidylcholine/spin-labeled phosphatidylcholine/cholesterol (molar ratio 1.6:0.4:1) were prepared by dialysis. Preparations at appropriate protein-to-lipid ratios (1:44 and 1:105 mol/mol) contained vesicles with a diameter of 100-300 nm and a high density of spikes on the surface. These vesicles showed low pH-induced membrane fusion activity. At pH 5.2 and 37 degrees C, fusion with erythrocyte membranes took place very rapidly within 1-2 min and reached a plateau at 63-66% fusion. The fusion was negligibly small at neutral pH and was induced to occur at pH values lower than 6.0. The reconstituted vesicles caused hemolysis and fusion of human erythrocyte cells in the same pH range as that of the fusion with erythrocyte membranes. The low pH-induced fusion activity of the reconstituted vesicles is essentially the same as that of the parent virus. These vesicles can be used to deliver some reagents or drugs into target cell cytoplasm via fusion at lysosomes.     

Comparative characteristics of acetylcholinesterase preparations from human erythrocytes with varying degrees of purity

Preparations of human erythrocyte acetyl cholinesterase (HEACE) that differed in their specific activities (0.7 to 4.1 U/mg) and the final purification method were examined for protein and isoenzymic spectrum (by polyacrylamide gel disc electrophoresis), catalytic properties in the reaction of acetytriocholine hydrolysis, sensitivity to the specific organophosphorus inhibitor Gd-42, concentrations of active centres of HEACE, and activity of one catalytic centre. The preparations showed a stable spectrum of 11-12 proteins: HEACE had molecular heterogeneity and was electrophoretically separated into 3 fractions that differed in their aggregation level. Preparations with varying specific activity contained different HEACE quantities. Regardless of the purification degree, HEACE displayed similar activity of the catalytic centre and enzymic properties in reactions with the substrate and inhibitor.     

微生物菌剂对秸秆降解及秸秆中微生物变化规律的影响

为明确不同微生物菌剂对秸秆降解率及秸秆降解过程中秸秆周围微生物变化规律的影响，在温室大棚内进行不同微生物菌剂降解秸秆试验。以玉米秸秆为基质，设玉米秸秆（对照组）、玉米秸秆+哈茨木霉（Trichoderma harzianum）（处理1）、玉米秸秆+哈茨木霉+地衣芽胞杆菌(Bacillus licheniformis)（处理2）三个处理。研究结果显示，单独用哈茨木霉处理可显著提高秸秆降解率，前期作用尤其明显；在秸秆降解的前30 d，秸秆降解率与秸秆中可培养真菌、细菌、放线菌呈显著正相关。不同微生物菌剂对秸秆中可培养微生物数量变化有显著影响，单独接种哈茨木霉后，秸秆中可培养真菌、细菌、放线菌数在前30 d显著高于对照组和混合菌剂处理，不同处理可培养活菌数均在90～120 d达到峰值，然后开始下降。研究结果表明，不同微生物菌剂对秸秆降解有显著影响，单独用哈茨木霉处理可显著提高秸秆降解率；不同处理对秸秆中可培养真菌、细菌、放线菌的影响有显著差异。可为秸秆降解与微生物相关性研究提供参考。     

The hemagglutinins of the human influenza viruses A and B recognize different receptor microdomains

A cryptically I-active sialylglycoprotein (glycoprotein 2) isolated from bovine erythrocyte membranes as Sendai virus receptor (Suzuki, Y., Suzuki, T. and Matsumoto, M. (1983) J. Biochem. 93, 1621-1633) contains N-glycolylneuraminic acid (NeuGc) as its predominate sialic acid and exhibits poor receptor activity for a variety of influenza viruses. Enzymatic modification of asialoglycoprotein-2 to contain N-acetylneuraminic acid (NeuAc) in the NeuAc alpha 2-3Gal and NeuAc alpha 2-6Gal sequences using specific sialyltransferase resulted in the appearance of receptor activity toward human influenza viruses A and B. The biological responsiveness chicken erythrocytes treated with sialidase and then reconstituted with derivatized glycoprotein 2 showed considerable recovery to influenza virus hemagglutinin-mediated agglutination, low-pH fusion and hemolysis. Specific hemagglutination inhibition activity of derivatized glycoprotein 2 was 5-16-times higher than that of human glycophorin. A/PR/8/34 (H1N1) virus preferentially recognized derivatized glycoprotein 2 containing NeuAc alpha 2-3Gal sequence over that containing NeuAc alpha 2-6Gal while the specificity of A/Aichi/2/68 (H3N2) for the sialyl linkages was reversed. B/Lee virus recognized both sequences almost equally. The biological responsiveness to the viruses of the erythrocytes labeled with the derivatized glycoprotein 2 containing NeuGc was considerably lower than that of derivatized glycoprotein 2 containing NeuAc. The results demonstrate that the hemagglutinins of human isolates of influenza viruses A and B differ in the recognition of microdomains (NeuAc, NeuGc) of the receptors for binding and fusion activities in viral penetration and the sequence to which sialic acid (SA) is attached (SA alpha 2-3Gal, SA alpha 2-6Gal). Inner I-active neolacto-series type II sugar chains may be important in revealing the receptor activity toward the hemagglutinin of both human influenza viruses A and B.     

Evaluation of two human plasma pools as candidate international standard preparations for syphilitic antibodies

A collaborative study was designed to asses two freeze-dried human plasma preparations containing anti-Treponema pallidum antibodies, 05/132 and 05/122, for their suitability as international reference reagents for syphilis serology. Both preparations are intended as replacements of the first international standard (IS) for syphilitic serum antibodies (HS). Samples were tested by eight laboratories using the T. pallidum passive particle agglutination assay (TPPA), the venereal disease research laboratory test (VDRL) and the rapid plasma reagin test (RPR). In addition a range of immunoassays was also used. The outcome of the collaborative study revealed that candidate standard 05/132 contains T. pallidum-specific IgG and IgM and is reactive in VDRL or RPR, and that 05/122 contains T. pallidum-specific IgG but is not reactive in either the VDRL or RPR test. Both 05/132 and 05/122 are reactive in the TPPA. On the basis of these results the Expert Committee on Biological Standardization of the World Health Organization designated 05/132 as the 1st IS for human syphilitic plasma IgG and IgM with a unitage of 3 IU per ampoule relative to HS and 05/122 as the 1st IS for human syphilitic plasma IgG with a unitage of 300 mIU per ampoule relative to 05/132.     

Characterization of fatty acid desaturase activity in rat lung microsomes.

Preparations of rat lung microsomes containing 0.030-0.050 nmole of cytochromes P-450 and b5 per mg microsomal protein have been observed to contain significant levels of fatty acid desaturase activity. Both stearoyl CoA and palmitoyl CoA are desaturated to their monounsaturated analogues, oleic acid and palmitoleic acid, respectively. Activity (per mg microsomal protein) of the lung preparations varied according to the diet of the animals prior to killing in the order: fat free diet greater than normal rat chow greater than starvation. All preparations exhibited approximately 50% inhibition when incubated in the presence of 0.10 mM CN-. Maximal activity was obtained with the 0.50 mM NADH less activity with equal amounts of NADPH, and there was no synergistic interaction of NADH and NADPH together. The rate of desaturation was linear with protein concentrations between 0.15-1.5 mg microsomal protein/incubation at incubation times up to 8 min. A pH optimum range of 7.0-7.4 was observed. For all variables of fatty acid desaturase activity which were examined, the rate of desaturation of stearoyl CoA was approximately twice that for palmitoyl CoA. These results indicate that the same fatty acid desaturation system which is functional in the liver is also present in significant amounts in mammalian lungs.     

THYMUS DERIVED INHIBITOR OF LYMPHOCYTE PROLIFERATION I. ISOLATION AND ASSESSMENT OF TISSUE SPECIFICITY

Preparations from bovine thymus tissue were analyzed for their inhibitory effects during in vitro lymphocyte proliferation. The results indicate that these preparations strongly inhibit DNA synthesis in stimulated human peripheral lymphocytes, bone marrow cells, thymocytes and lymphoblastoid cells. This inhibition was dose-dependent and not due to cytotoxicity of the preparations. No inhibition was found of the spontaneous proliferation of HeLa cells and human fibroblasts indicating that the inhibitory effect was specific for proliferating lymphocytes. Control preparations from bovine liver or kidney did not show any suppression in the test systems used.     

Purification of maize streak virus and its relationship to viruses associated with streak diseases of sugar cane and Panicum maximum

Maize streak virus (MSV) was purified by homogenising infected leaf tissue in 0·01 m pH 3·9 phosphate buffer and clarifying the extract with n-butanol (7 ml/100 ml extract). Purified preparations contained particles 20 nm in diameter, some occurring singly, but most occurring in pairs, forming structures of 30 × 20 nm. The sedimentation coefficients of single and paired particles were 54 and 76 S respectively. When centrifuged in sucrose density gradients preparations made by extracting leaves at pH 3·9 gave a single intense light-scattering zone containing paired particles. Preparations made at pH 5·9 or 7·9 gave one or two additional upper zones containing single particles and fragmented material. Preparations treated with 0·05 or 0·1 m ethylene diamine tetra-acetic acid, disodium salt, (EDTA) contained no paired particles, few single particles and much fragmented material. In immunoelectrophoresis, the major component in preparations without EDTA migrated to the cathode whereas that in EDTA-treated preparations migrated to the anode. Virus isolates from streak-diseased sugarcane and guinea grass (Panicum maximum) were serologically related to MSV and had similar particles with identical sedimentation coefficients. No such particles were seen in purified preparations of healthy maize, sugarcane, or guinea grass. The viruses from sugarcane and guinea grass are probably host-adapted and are referred to correctly as the sugarcane and guinea grass strains of MSV. MSV probably contains single-stranded RNA, and the cryptogram is (R)/1:*/*:S/S:S/Au.     

Experimental investigation of the rheological activation of blood platelets

In order to define various aspects of platelet rheological activation, samples of whole blood and platelet-rich plasma (PRP) from the same donors were subjected for 5 min to shear rates increasing from 10 to 10000 sec-1 (shear stresses from 10(-2) to 30 Pa approximatively) in a Couette type viscometer. The following parameters were measured: erythrocyte hemolysis; lactic dehydrogenase activity; plasma B-Thromboglobulin (B-TG); adenine nucleotides, and platelet photometric aggregation. The experimental results reveal that: In whole blood, hemolysis only reached at maximum 2% of the total hemolysis. Plasma LDH activity increased regularly beyond 500 sec-1, in close correlation with B-TG plasma concentration. In contrast, ADP and ATP levels remained stable up to 1000 sec-1 then increased slowly. In PRP, the LDH, ADP and ATP levels remain practically stable up to shear rates around 5000 sec-1. In contrast, B-TG appeared to be released in plasma at shear rate values of 3000 sec-1 and its progression is only correlated with the other parameters, when the platelet lysis occurred. Finally, a rapid and complete inhibition of platelet aggregation to ADP was observed from 5000 sec-1.     

Native Trichoderma strains isolated from Bangladesh with broad spectrum antifungal action against fungal phytopathogens

Nineteen Trichoderma isolates, collected from different locations in Bangladesh, were characterised through phenotypic, biochemical and molecular means. Besides, they were assessed for their antifungal action in vitro. The isolates were divided into three groups: T. asperellum, T. virens and T. harzianum. A dual culture assay and a culture filtrate assay against 6 phytopathogens revealed that 9 of the 19 isolates showed significant antifungal activities. The isolate T. harzianum TR05 showed the highest inhibition against Fusarium oxysporum, Rhizoctonia solani, Fusarium circinatum and Phomopsis vexans, followed by T. asperellum TR08 and T. virens TR06. TR08 had the highest inhibition against Sclerotium rolfsii and Pythium aphanidermatum, followed by TR05 and TR06. These findings were in agreement with their activities of extracellular hydrolytic enzymes, including chitinase, β-1,3-glucanase, and proteinase. Our results suggest that isolates TR05, TR06 and TR08 have the potential to be effective biocontrol agents against the phytopathogenic fungi.     

Diversity and pharmaceutical screening of fungi from benthic mats of Antarctic lakes

During the MICROMAT project, the fungal diversity of microbial mats growing in the benthic environment of Antarctic lakes was accessed for the discovery of novel antibiotics and anticancers. In all, 160 filamentous fungi belonging to fifteen different genera and 171 yeasts were isolated from 11 lakes, classified and cultivated in different media and at different temperatures. Filamentous fungi were then screened to discover novel antimicrobial and cytotoxic compounds. A total of 1422 extracts were prepared by solid phase extraction of the culture broths or by biomass solvent extraction. 47 (29%) filamentous fungi showed antimicrobial activity; most of them inhibited the growth of gram-positive Staphyloccus aureus (14%), gram-negative E. coli (10%), and of yeasts Candida albicans (11%) and Cryptococcus neoformans (8%). Less activity was detected against representatives of enterobacteria and filamentous fungi. The most productive in terms of bioactivities were cold-tolerant cosmopolitan hyphomycetes such as Penicillium, Aspergillus, Beauveria and Cladosporium. Two bioactive bis-anthraquinones (rugulosin and skyrin) were identified by LC–MS as the main products in a strain of Penicillium chrysogenum isolated from a saline lake in the Vestfold Hills. LC–MS fractionation of extracts from two diverse species of Aspergillus, that exhibited relatively potent antimicrobial activities, evidenced a chemical novelty that was further investigated. To our knowledge, this is the first report of new antibiotics produced by fungi from benthic microbial mats from Antarctic lakes. It can be concluded that these microbial assemblages represent an extremely rich source for the isolation of new strains producing novel bioactive metabolites with the potential to be developed as drugs.     

Antimicrobic and hemolytic activity of low-molecular metabolits of brown seaweed <Emphasis Type="Italic">Laminaria cichorioides</Emphasis> (Miyabe)

Antimicrobial and hemolytic activity of ethanol extract of brown seaweed Laminaria cichorioides (Miyabe), its lipophilic fractions, various classes of substances of lipophilic fraction, such as chlorophylls, fucoxanthin, monogalactosyldiacylglycerols, digalactosyldiacylglycerols, sulfoquinovosyldiacylglycerols, and fatty acids were investigated. The antimicrobial activity was studied by means of yeast cells Safale S-04 and Candida albicans KMM 455, fungi Aspergillus niger KMM 4634 and Fusarium oxysporum KMM 4639, bacteria Staphylococcus aureus ATCC 21027 (gram-positive) and Escherichia coli ATCC 15034 (gram-negative) which demonstrated selective sensitivity to the studied substances. Hemolytic activity was investigated at concentrations of substances in the range of 0.2–200 μg/ml at different pH of erythrocyte suspension. All investigated substances caused hemolysis. The dependence of hemolytic activity of substances on pH of media was determined.     

Studies on the role of goat heart galectin-1 as an erythrocyte membrane perturbing agent

Galectins are β-galactoside binding lectins with a potential hemolytic role on erythrocyte membrane integrity and permeability. In the present study, goat heart galectin-1 (GHG-1) was purified and investigated for its hemolytic actions on erythrocyte membrane. When exposed to various saccharides, lactose and sucrose provided maximum protection against hemolysis, while glucose and galactose provided lesser protection against hemolysis. GHG-1 agglutinated erythrocytes were found to be significantly hemolyzed in comparison with unagglutinated erythrocytes. A concentration dependent rise in the hemolysis of trypsinized rabbit erythrocytes was observed in the presence of GHG-1. Similarly, a temperature dependent gradual increase in percent hemolysis was observed in GHG-1 agglutinated erythrocytes as compared to negligible hemolysis in unagglutinated cells. The hemolysis of GHG-1 treated erythrocytes showed a sharp rise with the increasing pH up to 7.5 which became constant till pH 9.5. The extent of erythrocyte hemolysis increased with the increase in the incubation period, with maximum hemolysis after 5 h of incubation. The results of this study establish the ability of galectins as a potential hemolytic agent of erythrocyte membrane, which in turn opens an interesting avenue in the field of proteomics and glycobiology.     

Antagonistic and antimicrobial activities of some bacterial isolates collected from soil samples

Thirty seven bacterial cultures isolated from soil samples obtained from different locations were tested for their antagonistic activity against some fungal pathogens, viz., Sclerotium rolfsii, Fusarium oxysporum and Rhizoctonia solani, causal agents of collar rot of sunflower, wilts and root rots, respectively. Among them, 5 bacterial strains, viz., A1 6 (Bacillus sphaericus), K1 24 (Pseudomonas fluorescens), M1 42 (Bacillus circulans), M1 66 (Bacillus brevis) and T1 22 (Bacillus brevis) showed positive antagonistic activity. M1 66 was the most effective in inhibiting mycelial growth of S. rolfsii in vitro followed by M1 42, T1 22, K1 24 and A1 6. Only one bacterial strain i.e. M1 42 exhibited antagonistic activity against F. oxysporum, and none of the bacterial strains gave positive activity against R. solani. Furthermore, antimicrobial activities of all the 5 strains were checked against different test organisms. These strains showed their extensive inhibition effect particularly against gram-positive test bacteria (Staphylococcus aureus and Bacillus subtilis) and the test fungal strain (Candida albicans). On the other hand, B. brevis M1 66 and B. brevis T1 22 strains had an inhibitory effect against gram positive and gram-negative test bacteria (Escherichia coli and Proteus vulgaris) as well as the test fungal strain.