Immunochemical and Biochemical Studies of Fungi

Polysaccharides were separated from mycelia and culture filtrates of the filamentous fungi Aspergillus niger and Penicillium chrysogenum, and purified by column chromatography on Sephadex G-50, DEAE- and CM-cellulose, successively. No nitrogen and phosphorus were detected in the polymer, and the sugar components were observed to be galactose and mannose. The polysaccharides were confirmed to be galactomannans which were easily hydrolyzed by weak acid, liberating galactofuranose and oligosugar in dialyzable fractions.     

On the Immunochemical and Biochemical Studies of Fungi

The nature of the cross reaction of the mycelial mannan of Trichophyton rubrum and galactomannan isolated from the culture medium of Aspergillus fumigatus with antisera of Saccharomyces cerevisiae and Candida albicans is described. Cross-reactivity of polysaccharides of both T. rubrum and A. fumigatus was weak with antisera of C. albicans and S. cerevisiae, but the galactomannan of A. fumigatus showed slightly stronger activity than the mannan of T. rubrum which possesses more closely related chemical structure of the mannans of S. cerevisiae and C. albicans.     

Studies on Trypanosomatid Actin I. Immunochemical and Biochemical Identification

In this study, the presence of actin in cultured trypanosomatids was investigated using polyclonal antibodies to heterologous actin. Polyclonal antisera to rabbit muscle actin and a monospecific anti-actin antibody react with a 43-kDa polypeptide in extracts of Trypanosoma cruzi, Herpetomonas samuelpessoai and Leishmania mexicana amazonensis on protein immunoblots. The 43-kDa polypeptide co-migrates with skeletal muscle actin and is retained within trypanosomatid cytoskeletons. Attempts to isolate H. samuelpessoai actin through DNase I affinity chromatography showed that the 43-kDa polypeptide did not bind to the column. Instead, low yields of a 47-kDa polypeptide were obtained indicating that the trypanosomatid actin displays unusual DNase I binding behavior when compared to actins from higher eukaryotes. Immunofluorescence studies confirmed that cytoskeletons retain the actin-like protein. In H. samuelpessoai , actin is localized in the region close to the flagellum, whereas in T. cruzi it is more homogeneously distributed. The data presented here show that trypanosomatid actin displays biochemical characteristics similar to actins of other protozoa.     

Biochemical and Immunochemical Studies on the GABAergic System in the Rat Fallopian Tube and Ovary

gamma-Aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) activities were measured in the ovary and the Fallopian tube of rats and compared with brain values. GABA levels in the Fallopian tube were about twice as high as in the brain, while in the ovary they represented only about 5% of the amino acid content of the CNS. In vitro decarboxylation of glutamate, measured via CO2 formation, occurred both in the Fallopian tube and in the ovary. These two organs contained, respectively, 10% and 1% of brain GAD activity. However, the actual formation of GABA from glutamate in a high-speed supernatant was detectable only in the Fallopian tube, where it represented about 5% of brain GAD activity. In contrast with the enzyme present in ovary, liver, anterior pituitary, and kidney, that in the Fallopian tube was quantitatively precipitated by a specific antiserum directed against rat neuronal GAD. Moreover, subcutaneous transplantation resulted in a quantitative decrease of both GABA levels and GAD activity in the Fallopian tube while no change occurred in the ovary, and vagus nerve section induced a 50% decrease of GAD activity in the Fallopian tube, although GABA levels were not significantly altered. The findings suggest an extrinsic GABAergic innervation in the rat Fallopian tube but not in the ovary.     

Immunochemical Studies of the Muscarinic Acetylcholine Receptor

Abstract

Muscerinic receptors have been purified from calf forebrain plasma cell membranes by affinity chromatography on a dexetimide-agarose gel. SDS-PAGE analysis showed a single 70kDa band. Monoclonal antibodies have been prepared against these affinity purified 70kDa protein(s). One antibody, M-35, Immunoprecipitated up to 80% of digitonin-solubilized muscarinic receptors. M-35 had agonist-like effects on guinea-pig myometrium: it increased the intracellular cyclic GMP content, decreased prostaglandin-induced cyclic AMP accumulation and caused muscle contractions. The two first affects were inhibited by atropine. M-35 was used to visualize muscarinic receptors at the surface of human fibroblastic cells. In the particular cell line used, the receptors have a low affinty for pirenzepine, were negatively coupled to adenylate cyclase and mediated increase in the phosphatidyl-insitol breakdown.     

Immunochemical Studies on Bacterial Blood Group Substances

The terminal α-galactosyl linkage was hydrolyzed by an enzyme preparation from either Clostridium maebashi or coffee beans. Reduction of blood group B activity of Salmonella milwaukee and Escherichia coli O₈₆ lipopolysaccharides and of some oligosaccharides occurred consequently. The degraded lipopolysaccharides showed significant blood group O (H) activity indicating the presence of terminal α-fucosyl residues in them and the same activity was demonstrated with oligosaccharide A-3 after the enzyme treatment. α-Fucosidase derived from Bacillus fulminans caused liberation of fucose from the α-galactosidasc-treated, materials and abolishment of O (H) activity. The results of quantitative analysis, borohydride reduction, Morgan-Elson reaction and treatments with several kinds of enzyme preparations on a series of oligosaccharides indicated that the structure of O-somatic side chain of E. coli O₈₈ is probably; It was evident that there is a similarity in the terminal structure of lipopolysaccharides of E. coli O₈₆, S. milwaukee and human B blood group substance.     

Immunochemical Studies on Bacterial Blood Group Substances

A-decomposing enzyme preparation (α-N-acetylgalactosaminidase) from hog liver, liberates from the lipopolysaccharide of Salmonella riogrande, which contains O antigen 40, 15.5% of its original amount of N-acetylgalactosamine, destroying its ability of inhibiting the agglutination of human A red ceils by anti-S. riogrande rabbit serum. From the structures of blood group A-active oligosaccharides, obtained from the lipopolysaccharide by mild acid-hydrolysis, it was assumed that the antigenic determinant of A specificity might have the following structure, which indicated the presence of N-acetylgalactosaminyl residue as the non-reducing end; α-N-acetylgalactosaminyl-(1→3)-mannosyl-glucosyl-(1→3)-N-acetylgalactosaminyl-glucose.     

Immunochemical Studies on Bacterial Blood Group Substances

Blood group H-active polysaccharide has been prepared from “smooth” strain Escherichia coli 2B-V by Freeman's method. a-Fucosidase derived from Bacillus fulminans caused the liberation of fucose from this polysaccharide, together with concomitant loss of blood group H activity. The results of quantitative microanalysis, borohydride reduction, the Morgan-Elson reaction and enzymic hydrolysis with β-galactosidase using isolated oligosaccharides obtained by partial acid hydrolysis indicated that the O-specific side chain of the polysaccharide has a pentasaccharide unit which is β-d-Gal-(1→3)-d-GalNAc-(1→3)-d-GalNAc-Fuc with a D -glucose residue bound at some undetermined point on this structure. It was considered that terminal non-reducing fucose of the polysaccharide was liberated by partial acid hydrolysis.     

Immunochemical Studies on Bacterial Blood Group Substances

Two oligosaccharides were isolated from a partial hydrolysis of the lipopolysaccharide from Shigella dysenteriae which had a common antigen with human red blood cells (RBC). A disaccharide sh-2 had a structure of O-β-D-galactosyl-(l→2)-D-galactose, and was active in the hemagglutination inhibition test between human O RBC and anti-S. dysenteriae chicken serum. A tetrasaccharide sh-12 was composed of galactose and rhamnose and was a more effective inhibitor than sh-2. Both oligosaccharides were assumed to be derived from the determinant structure of a common antigen. Serological specificity of the hemagglutinin in chicken serum was examined using these oligosaccharides and human milk oligosaccharides, and the 1→2 linked galactosyl residue was supposed to be important for determination of the heterophile RBC antigenicity.     

虫生真菌抗逆的生化分子基础及利用

虫生真菌在生活史各个阶段均面临多种环境因子的危害。为了避开这些不利的因素,虫生真菌进化出多种抗逆的机制。近年来,以球孢白僵菌(Beauveria bassiana)和金龟子绿僵菌(Metarhizium anisopliae)为代表的虫生真菌抗逆的生化分子基础的研究取得了一定的进展,明确了某些生化成份和抗逆基因与菌株抗逆性能的相关性。就环境胁迫因子、虫生真菌抗逆的生化分子基础及其在改良菌株抗逆性能方面的应用进行综述,这将为环境适应性强的虫生真菌制剂的研发与应用提供理论依据。     

Immunochemical Studies on Lipopolysaccharide from Agglutinable and Non-Agglutinable Vibrios

Lipopolysaccharide (LPS) prepared from Vibrio cholerae and a non-agglutinable (NAG; not agglutinable with O-group I serum according to Gardner and Venkatraman [13]) vibrio strain, isolated from a patient with cholera-like clinical symptoms, have been compared with respect to their chemical composition and immunological behavior. In addition to a significant difference in the chemical composition between the two lipopolysaccharides, the LPS from V. cholerae, unlike that from the NAG vibrio, requires prior treatment with alkali for it to be an effective antigen in the indirect hemagglutination test with sheep cells. It has been suggested that the alkali acts by removing excess O-acetyl group from LPS of agglutinable vibrios. LPS from the NAG vibrio consistently showed a lower antibody response in rabbits in terms of agglutinin and vibriocidal titer. Also, the class of agglutinin antibody elicited by LPS of the NAG vibrio was predominantly immunoglobin M, and that from V. cholerae was immunoglobulin G under comparable conditions.     

真菌HSP30的研究概况

热休克蛋白30是小分子热休克蛋白(small heat shock proteins,sHSPs)中的一种,也是真菌中研究最广泛的小分子热休克蛋白。多种真菌编码热休克蛋白的基因序列已经被克隆和检测,HSP30的研究主要集中在应激水平下的表达和转录水平的调控,HSP30在应激反应中的合成机制仍不是很清楚,综述了它的研究概况以及应用前景。     

Immunochemical Studies of Bovine and Human Choline-O-Acetyltransferase Using Monoclonal Antibodie

Abstract: Immunochemical properties of bovine and human choline acetyltransferase (ChAT, EC 2.3.1.6, acetyl-CoA:choline- O -acetyltransferase) were studied using six monoclonal antibodies (AB1, AB5, AB6, AB7, AB8, and AB9) reactive with the enzyme. All antibodies except AB1 bound specifically to two proteins of 68,000 and 70,000 MW on "Western" blots of sodium dodecyl sulfate-polyacrylamide gels containing human or bovine ChAT. The enzyme was specifically absorbed to immobilized antibody and could not be eluted by low pH and/or high salt concentrations, although the enzyme retained activity on the immunoabsorbent. Pure bovine enzyme consisting of the same two proteins as seen in the Western blotting studies was eluted from immobilized AB1 in the presence of sodium dodecyl sulfate. Although active enzyme could not be eluted from immobilized antibodies by standard conditions, various combinations of free and immobilized antibodies were effective in competing off bound enzyme. Free antibody AB1 quantitatively eluted the active enzyme from immobilized AB1. The different capacities of the antibodies to elute enzyme from various immunoabsorbents reflect interesting properties of both the enzyme and the antibodies.     

Biochemical Studies on Yeast

The present authors obtained direct proof of the occurrence of glucose dehydrogenase in yeast. Optimum pH of the glucose dehydrogenation system in yeast was about 7.0. After dialysis of the salting out preparation, the dialysate revealed only a trace of activity. The addition of DPN or TPN restored the activity. The majority of the yeast glucose dehydrogenase precipitated below about 0.70 ammonium sulfate saturation, and there was no marked activity in 0.30 ammonium sulfate saturation. The yeast glucose dehydrogenase was observed to be highly specific for β-d-glucose.     

Biochemical Studies on Yeast

The growth of Zygosaccharomyces soja was strongly inhibited by the addition of 10^?2 m-monoiodoacetic acid and 10^?3 m-potassium cyanide. The formation of a considerable amount of riboflavin was recognized after 5 days cultivation of this organism in presence of the aforementioned two inhibitors, and its growth was considerably improved after the formation and accumulation of riboflavin. Furthermore, the effect of the aforementioned two inhibitors upon the growth of this organism in young stage was compensated with the addition of riboflavin.     

Biochemical Studies on Cycasin

Purification of β-glucosidase from the seeds of Japanese cycad, and properties of the purified preparation are described. The enzyme activity was determined by colorimetry using ONPG as substrate. Crude preparation was obtained easily by adsorption on fibrous CMC pulp. It was further purified by chromatography on CMC powder, and a preparation which showed an activity of 135-folds of the original extract was obtained. Influences of pH, temperature, and substrate concentration upon the enzyme activity were examined. Michaelis constant of the enzyme for ONPG was 3.3×10^–3M.