Immunochemical Characterization of Cell Wall Protein Antigen Purified from the Cell Wall Autolysate of Clostridium botulinum Type A

The cell wall protein antigen was solubilized from the isolated cell walls of Clostridium botulinum type A by autolysis and purified by diethylaminoethyl-cellulose column chromatography followed by gel filtration on Sephadex G-150. The two fractions showed a high degree of the serological activity and produced a main fused precipitin line in immunodiffusion tests against the homologous antiserum. The fact that antigenic fractions contained various kinds of amino acids but no detectable amounts of amino sugars or carbohydrates suggests that the antigens were principally composed of proteins. The protein antigen possessed multiple antigenic components on immunoelectrophoresis. As serological activity, the antigen was heat-stable and resistant to tryptic digestion but sensitive to the actions of pronase, nagarse or pepsin. The protein antigen appeared to be responsible for the common antigenicity among the proteolytic strains of C. botulinum.     

Detection of ‘long-haired’ Saprolegnia (S. parasitica) isolates using monoclonal antibodies

The ability of five monoclonal antibodies (Mabs) raised against a pathogenic Saprolegnia parasitica isolate from brown trout to detect and differentiate between isolates with bundles of long hairs (S. parasitica) and other Saprolegnia species was determined by means of an indirect immunofluorescence assay. Four of the Mabs used recognized some of the long-haired S. parasitica isolates but also cross-reacted with other Saprolegnia species without bundles of hairs and with Achlya sp. The other Mab (named 18A6) was able to differentiate between the asexual and most of the sexual isolates in the group of long-haired S. parasitica isolates, but did not recognize Achlya sp. or the Saprolegnia species without bundles of hairs, with the exception of S. hypogyna. These results indicate that isolates with bundles of long hairs are closely related with other members of genus Saprolegnia and share several antigens. However, Mab 18A6 seems to recognize an epitope that is expressed mainly in the asexual isolates in the long-haired S. parasitica isolates.     

Evaluation of the antigen from chlamydospores of Candida albicans in the serodiagnosis of candidiasis

Antigens have been prepared from the chlamydospores and blastospores of Candida albicans and their precipitin patterns were analysed by two-dimensional immunoelectrophoresis using specific antisera.The two antigens were used in routine serological tests of patients suffering from candidiasis. On double-diffusion tests for the detection of circulating antibodies of Candida albicans, the antigen from chlamydospores displays precipitin lines that differ in number and intensity from those obtained with the antigen from blastospores. The results are briefly discussed in the framework of C. albicans antigen standardization.     

Immunochemical characterization of cell wall protein antigen purified from the cell wall autolysate of Clostridium botulinum type A.

The cell wall protein antigen was solubilized from the isolated cell walls of Clostridium botulinum type A by autolysis and purified by diethylaminoethyl-cellulose column chromatography followed by gel filtration on Sephadex G-150. The two fractions showed a high degree of the serological activity and produced a main fused precipitin line in immunodiffusion tests against the homologous antiserum. The fact that antigenic fractions contained various kinds of amino acids but no detectable amounts of amino sugars or carbohydrates suggests that the antigens were principally composed of proteins. The protein antigen possessed multiple antigenic components in immunoelectrophoresis. As serological activity, the antigen was heat-stable and resistant to tryptic digestion but sensitive to the actions of pronase, nagarse or pepsin. The protein antigen appeared to be responsible for the common antigenicity among the proteolytic strains of C. botulinum.     

Immunochemical investigations of antigens isolated fromBacteroides ovatus strain ATCC 8483

A saline extract (SE) and a phenol/water extract (WL) were prepared fromBacteroides ovatus strain ATCC 8483. A fraction CS was isolated from the culture supernatant. WL was further split by ultracentrifugation into lipopolysaccharide (LPS) and supernatant (L₁). Fractions SE, WL, LPS and L₁ reacted serologically with homologous antiserum but did not cross-react with antisera against heterologousBacteroides serotypes. Fraction CS was inactive in haemagglutination, haemagglutination inhibition and immunoelectrophoresis tests. SE, WL, LPS and L₁ proved to be serologically heterogeneous. A distinct serological specificity for SE was demonstrated. The serological reactivity in SE and WL was not altered after treatment with proteolytic enzymes yet completely destroyed in WL and partially in SE by sodium metaperiodate. SE, WL, LPS and L₁ contained the sugar components rhamnose, fucose, ribose, mannose, galactose, glucose and glucosamine in different molar ratios for each fraction. Galactosamine was found in WL and LPS, uronic acid in WL and L₁. Two unidentified aminohexoses were detected in WL, one of which was also detectable in L₁ and SE. 2-Keto-3-deoxyaldonic acid was demonstrated in LPS and L₁ after strong acid hydrolysis.     

The in vitro antifungal activity of 30 Chinese herb extracts to Saprolegnia sp.

Chinese traditional medicines have been used for several thousands of years in Asian countries, not only in humans but also in many animal species. These compounds prevent and control different types of diseases including internal diseases as well as some infectious diseases, where the aetiological agent is viral, bacterial, parasitic or mycotic. Rhizoma coptidis is believed to inhibit Shigella dysenteriae and that Radix isatidis can prevent flu caused by the influenza virus. It is thus hypothesized that some of these traditional herbal compounds will have anti‐fungal activity. Saprolegniosis is a disease common in fish and their eggs in both fresh and brackish water; a newer, safer medication against Saprolegnia is needed after the prohibition in many countries of the extremely effective fungicide, malachite green. In the present study an attempt is made to identify herbal compounds that have anti‐Saprolegnia activity. A strain of Saprolegnia, CCF1301, was isolated from the skin of infected grass carp (Ctenopharyngodon idella) and identified as Saprolegnia ferax by the 26S rDNA D1/D2 region and ITS region. This strain was used to evaluate the antifungal activity of thirty Chinese traditional herbal medicine extracts, and a modified dish dilution method was developed for the evaluation. Saprolegnia‐infected rapeseeds with visible hyphae were transplanted onto prepared PDA plates containing 2 g L^?1 herbal plant extracts and incubated at 20°C for 48 h. Each herbal plant species was tested in triplicate. Those herbal plant extracts that showed negative mycelium presence at 2 g L^?1 were further tested for minimum inhibitory concentration (MIC) evaluation. The results showed that Syzygium aromaticum, Magnolia officinalis, Melaphis chinensis, Euphorbia fischeriana Steud, and Sophora flavescentis exhibited enhanced growth inhibition at 2 g L^?1 and MIC values of 500, 62.5, 250, 62.5, 250 mg L^?1 concentrations, respectively. It was obvious that Magnolia officinalis and Euphorbia fischeriana Steud exhibited the best antifungal activity. Since there is a high natural toxicity in Euphorbia fischeriana Steud, its applicability as the main ingredient in an aquaculture therapeutic formulation requires further research. Thus, Magnolia officinalis would appear to be the more valuable antifungal herbal species with which to pursue further research.     

Allelic differences in hemolytic activity and protein concentration of BF molecules are found in association with particular HLA haplotypes

After separating the *F and *S alleles by electrophoresis the allele-specific hemolytic activity was detected by agarose overlay method using the programmable densitometer for scanning. The hemolytic activity of BF allotypes was analyzed from 81 individuals. In thirteen FS heterozygous serum samples BF F had lower hemolysis than BF S. Four FF homozygous samples also exhibited lower hemolysis than a homozygous control sample. The low hemolytic activity of F in FS heterozygotes was not due to decreased protein concentrations relative to S. On the contrary, BF F was associated with higher protein concentration than BF S. The relative quantitation of the allele specific BF protein was done by crossed immunoelectrophoresis. BF F with low hemolytic activity but with high protein concentration associated strongly with HLA B35 phenotype and the family material confirmed the association with the haplotypes A3, Cw4, B35, DR1, BFFB, C4A3BQO (or A2BQO, A3,2BQO). The results suggest that particular MHC haplotypes contain a factor B allele with encoding for poor hemolytic activity or that MHC haplotype specific regulatory elements affect pre- or post-translational activity levels.     

Aquatic fungi of Iraq: Species of Saprolegnia

Five species of Saprolegnia have been isolated from the water samples obtained monthly from Shatt Al-Arab estuary from September, 1976 to August, 1977. These are S. ferax, S. anisospora, S. diclina, S. terrestris and S. parasitica. Out of 81 isolates 43 reached sexual maturity while 38 remained sterile. Maximum abundance was found during winter months while minimum in summer months.     

Serological cross-reactivity between sporothrix schenckii and various unrelated fungi

The serological cross-reactivity ofSporothrix schenckii with various unrelated fungi was investigated by use of immunodiffusion tests. A rabbit antiS. schenckii serum was obtained, which reacted withCladosporium werneckii, C. carrionii, C. bantianum, Coccidioides immitis, Phialophora jeanselmei, P. gougerotii, P. dermatitidis, Fonsecaea pedrosoi, Aspergillus fumigatus, Histoplasma capsulatum andTrichophyton mentagrophytes, but not withSaccharomyces cerevisiae antigens. The serological determinants responsible for the cross-reactions were suggested to be D-galactosyl residues.     

Properties of spinach yellow mottle, a distinctive strain of tobacco rattle virus

A virus (isolate SYM) obtained from spinach plants in England with a severe yellow mottle disease induced symptoms resembling those of tobacco rattle virus (TRV) in several indicator species but caused systemic necrosis in Chenopodium amaranticolor and C. quinoa. It was transmitted to bait plants grown in soil containing the nematode Trichodorus primitivus. Purified virus preparations contained rod-shaped particles that were predominantly of four modal lengths: 188 nm (L particles), 101 nm (S particles), 57 nm and 48 nm (together called VS particles), containing RNA with mol. wts of 2.4, 1.5, 0.7 and 0.6 million, respectively. L particles (s°₂₀= 300 S) and S particles (230 S) greatly outnumbered VS particles (c. 150 S). All particles contained a single polypeptide species with estimated mol wt of 24 700, slightly larger than those previously reported for tobraviruses. Purified L particles were infective but both L and S particles were needed to induce the production of virus nucleoprotein particles. VS particles were not infective and apparently had no qualitative or quantitative effect on infection by L or by L plus S particles. S particles carried determinants for serological specificity and ability to invade C. amaranticolor systemically. Isolate SYM produced pseudo-recombinants with isolate PRN of TRV. Also, isolates CAM, OR and PRN of TRV, and isolate SYM, were found to be distantly related by three kinds of serological test. No relationship was detected between these isolates and pea early-browning virus in gel-diffusion precipitin tests or electron microscope serological tests, but a distant relationship between isolate SYM and pea early-browning virus was found by micro-precipitin tests. Isolate SYM therefore has closer affinities with TRV than with pea early-browning virus and is considered to be a distinctive strain of TRV.     

Expression of a streptokinase gene from Streptococcus equisimilis in Streptococcus sanguis

Summary Using recombinant DNA techniques, we introduced a previously cloned streptokinase gene from Streptococcus equisimilis into the Challis strain of S. sanguis (group H). The gene was expressed in the new host under the control of its own promoter and the gene product had biological properties identical to authentic streptokinase. However, the molecular weight of cloned streptokinase (42 K) as expressed by S. sanguis was substantially lower than that of authentic streptokinase (47 K). Since the cloned streptokinase gene encoded a 47 K mature protein, the lowered molecular weight of S. sanguis streptokinase may reflect posttranslational proteolytic cleavage, which leaves the biological activity of the gene product and its serological reactivity unimpaired.     

Proteolytic Activities of the Starch-Fermenting Ruminal Bacterium, Streptococcus bovis

The objective of this study was to characterize the extracellular proteolytic activity of Streptococcus bovis. Strains KEG, JB1, NCFB 2476, and K11.21.09.6C produced very similar large molecular weight (160–200 kDa) extracellular proteases that were specifically inhibited by PMSF, a serine protease inhibitor. Further experiments with S. bovis KEG indicated that cultures grown with casein as the sole added N source produced the greatest level of proteolytic activity, and the level of proteolytic activity was independent of growth rate. Clarified ruminal fluid (CRF) decreased proteolytic activity by 54% compared with cultures grown with casein alone, and addition of exogenous peptides and carbohydrates (CHO) to the CRF further reduced the level of proteolytic activity by 44% and 52%, respectively. These results suggested that the proteolytic activity of S. bovis KEG was modulated by available N source and that the proteolytic activity was present for reasons other than providing N for growth. The role of S. bovis in ruminal proteolysis requires further definition, but phenotypic similarity among some ruminal strains would suggest a common niche in ruminal proteolysis. The uniformity of proteolytic activities could make S. bovis a prime candidate for manipulation in ruminal proteolysis control strategies. Received: 12 January 1999 / Accepted: 19 May 1999     

Extracellular proteolytic activity ofCryptococcus neoformans

Eight strains ofCryptococcus neoformans var.neoformans isolated from AIDS patients in the Infectious Disease Institute, University of Turin, Italy, were examined for growth and extracellular proteolytic activity in culture with solid and liquid media. All of the strains grew well on Yeast Carbon Base (YCB) agar medium supplemented with both 0.1% (w/v) bovine serum albumin (BSA) and 0.01% (w/v) polypeptone (Pp), and produced a clear proteolytic zone around their colonies, whereas they exhibited less growth and proteolytic activity on YCB medium supplemented with BSA alone. Strain #8 with a strong proteolytic activity was cultured in three different liquid media. Its growth was limited in YCB medium supplemented with 0.1% BSA, but was moderate in that with 0.01% Pp. Enhanced growth was supported by the addition of both BSA and Pp to the YCB medium. The relative value of the final cellular yields obtained with the above YCB-0.1% BSA, YCB-0.01% Pp and YCB-0.1% BSA-0.01% Pp media was approximately 1:10:20. In the culture with YCB medium containing both BSA and Pp, a rapid decrease in the amount of BSA was demonstrated by a spectrophotometric assay and gel electrophoresis of the culture supernatant after the log-to-stationary phase. The proteolytic activity in the culture supernatant became detectable after the log phase when tested with skim milk agarose plates. These results allowed us to conclude thatCr. neoformans var.neoformans is able to secrete protease and to utilize protein as a source of nitrogen.     

Fibrinolytic activity of some fungi isolated from self-heated composted fertilizer

Mesophilic fungi isolated from organic fertilizer compost samples accounted for 70.94% of the total fungal count, while thermophilic and thermotolerant fungi constituted 29.05% of that count. Eight mesophilic fungal species, namelyAspergillus niger, Monilia sitophila, Paecilomyces divaricata, Penicillium chrysogenum, P. fellutanum, Scopulariopsis brevicaulis, S. brumptii andZygorhynchus japonicus; two thermophilic fungiHumicola grisea andOidiodendron flavum and three thermotolerant speciesAspergillus fumigatus, Thermomyces lanuginosus andZygorhynchus vuilleminii were isolated during the study. Most of the tested fungi showed a proteolytic activity and liquified gelatin in the test tube method and in cup plates. The thermophilic fungusO. flavum was the most potent proteolytic fungus. The comparative fibrinolytic assay revealed the following sequence in the ability of the tested fungi to hydrolyse fibrin:O. fiavum>S. brevicaulis>H. grisea>A. fumigatus>T. lanuginosus.     

26S proteasome exhibits endoribonuclease activity controlled by extra-cellular stimuli

26S proteasome is a large multi-subunit protein complex involved in proteolytic degradation of proteins. In addition to its canonical proteolytic activity, the proteasome is also associated with recently characterized endoribonuclease (endo-RNAse) activity. However, neither functional significance, nor the mechanisms of its regulation are currently known. In this report, we show that 26S proteasome is able to hydrolyze various cellular RNAs, including AU-rich mRNA of c-myc and c-fos. The endonucleolytic degradation of these mRNAs is exerted by one of the 26S proteasome subunits, PSMA5 (α5). The RNAse activity of 26S proteasome is differentially affected by various extra-cellular signals. Moreover, this activity contributes to the process of degradation of c-myc mRNA during induced differentiation of K562 cells, and may be controlled by phosphorylation of the adjacent subunits, PSMA1 (α6) and PSMA3 (α7). Collectively, the data presented in this report suggest a causal link between cell signalling pathways, endo-RNAse activity of the 26S proteasome complex and metabolism of cellular RNAs.     

Isolation and identification of mucinolytic actinomycetes

Biochemical and physiological tests, and 16S rRNA gene sequences, were used to classify nine Actinomycete strains isolated from soil and sand samples in Thailand. These strains were isolated based on their ability to readily degrade mucin glycoproteins. A turbidometric based mucinolytic assay was developed to confirm this. In addition all strains showed significant production of proteases. Phylogenetic analysis of the strains revealed that from the nine isolated Actinomycete strains eight were closely related to Streptomyces species and one was identified as belonging to the genus Kitasatospora. The biochemical and physiological tests performed identified two strain pairs that were similar (with only 3.9% difference observed) and this was in accordance with the phylogenetic results obtained. The remaining strains were distinct from each other, with the soil-isolated strains forming a separate clade to the sand-isolated strains in the inferred phylogenetic trees. The isolated mucinolytic Actinomycete strains will be the subject of further investigations into their proteolytic and glycosidic activity. Mucin degrading enzymes such as these are studied for their potential to be used for the development of a drug delivery system.     

Studies in aquatic fungi of varanasi. X. Additional new or interesting records of aquatic phycomycetes

Summary Four species of the genusSaprolegnia Nees, viz.,Saprolegnia diclina, S. delica, S. mixta, S. monoica var.glomerata, and three from the achlyoid group, viz.,Achlya colorata, A. racemosa, andA. americana were isolated on various substrata used as baits from the vicinity of University campus. ExcludingS. diclina andA. americana, all the above fungi are reported as occurring new from this country.     

Structural and immunoelectrophoretic comparison of soluble and particulate ribulose bisphosphate carboxylases from the cyanobacterium Chlorogloeopsis fritschii

The soluble and particulate (carboxysomal) forms of ribulose 1,5-bisphosphate (RuBP) carboxylase from the cyanobacterium Chlorogloeopsis fritschii have been purified separately. A molecular weight of 520,000 was found in each case. Large (L, 53,000) and small (S, 13,000) subunits were obtained after dissociation, indicating a L₈S₈ quaternary structure for the enzyme from both sources. The L and S subunits are identical in molecular weight to the major polypeptides present in isolated dissociated C. fritschii polyhedral bodies (carboxysomes). Occasionally an additional polypeptide (mol. wt. 45,000) was found after dissociation of the soluble enzyme only, although the possibility that this may be due to proteolysis is not discounted. Immunochemical identity between the purified soluble and carboxysomal RuBP carboxylases was indicated by tandem-crossed and rocket immunoelectrophoresis.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl-sulphate - RuBP D-ribulose 1,5-bisphosphate - TCA trichloroacetic acid - LTIB low Tris isolation buffer - HTIB high Tris isolation buffer - CIE crossed immunoelectrophoresis - TCIE tandem-crossed immunoelectrophoresis - RIE rocket immunoelectrophoresis     

Mitochondrial Dysfunction Is Involved in the Toxic Activity of Boric Acid against Saprolegnia

There has been a significant increase in the incidence of Saprolegnia infections over the past decades, especially after the banning of malachite green. Very often these infections are associated with high economic losses in salmonid farms and hatcheries. The use of boric acid to control the disease has been investigated recently both under in vitro and in vivo conditions, however its possible mode of action against fish pathogenic Saprolegnia is not known. In this study, we have explored the transformation in Saprolegnia spores/hyphae after exposure to boric acid (1 g/L) over a period 4–24 h post treatment. Using transmission electron microscopy (TEM), early changes in Saprolegnia spores were detected. Mitochondrial degeneration was the most obvious sign observed following 4 h treatment in about 20% of randomly selected spores. We also investigated the effect of the treatment on nuclear division, mitochondrial activity and function using confocal laser scanning microscopy (CLSM). Fluorescence microscopy was also used to test the effect of treatment on mitochondrial membrane potential and formation of reactive oxygen species. Additionally, the viability and proliferation of treated spores that correlated to mitochondrial enzymatic activity were tested using an MTS assay. All obtained data pointed towards changes in the mitochondrial structure, membrane potential and enzymatic activity following treatment. We have found that boric acid has no effect on the integrity of membranes of Saprolegnia spores at concentrations tested. It is therefore likely that mitochondrial dysfunction is involved in the toxic activity of boric acid against Saprolegnia spp.     

Fatty acid profile,proteolytic activity and survival of Octopus bimaculatus paralarvae fed with enriched Artemia franciscana

Octopus bimaculatus is an economically important food resource and a potential candidate for aquaculture diversification in the Pacific coast of Mexico; knowledge of its nutritional requirements is nil. For this reason, the composition of fatty acids of the newly hatched paralarvae was determined, as a criterion to define their requirements for essential fatty acids. We also evaluated the nutritional effect of live Artemia franciscana (8.2?±?1.20?mm total length) enriched with tuna orbit oil (the TOO group) on the proteolytic activity and survival of the O. bimaculatus paralarvae in one experiment. The experimental cultivation of paralarvae fed with the TOO group was ended 17 days after hatching (DAH) with a survival of 1.93% and an increase of suckers per arm from five at hatching to nine, while those fed with the non-enriched A. franciscana (the control group) finished with only six suckers at 11 DAH and a 1.77% survival. Acid and alkaline proteolytic activity was detected at hatching, which increased in accordance with the age of the paralarvae. The TOO group had a significant influence on acid proteolytic activity of the paralarvae only at eight DAH; however, the final survival rate obtained in this treatment was slightly higher compared with those fed with the control group.