Biomedical subjects
Publications and source records attributed to K Takumi.
A nonfimbrial hemagglutinin (HA) of Yersinia pseudotuberculosis was observed by immunoelectron microscopy using monospecific HA antiserum and protein A-gold conjugate. The HA, an amorphous but morphologically identifiable entity, was located in a region distal to or detached from the outer edge of bacteria.
Yersinia pseudotuberculosis which were screened out depending on auto-agglutination and Ca2+ dependency, were examined for their production of hemagglutinin (HA), and its purification and characterization were performed. The HA with a broad reactivity with various mammalian erythrocytes was recovered from the culture supernatant of these strains grown at 37 C but not 25 C. HAs from two strains, R148R and T1040, were purified by salt precipitation, gel filtration and anion-exchange chromatography by HPLC. Both purified HAs were cysteine-deficient acidic protein with an apparent molecular weight in the range of 15,000 to 16,000. N-terminal amino acid sequences of the first 25 residues were found to share 12% identity with that of afimbrial adhesin from enterotoxigenic Escherichia coli 2230. Immunoelectron microscopy and immunodiffusion test with polyclonal antiserum raised against the purified R148RHA demonstrated that the HA was associated with the amorphous aggregates which were detached from bacteria. These results suggest that the HA of Y. pseudotuberculosis belongs to a third type of HA produced by the yersinial species.
S layer of Clostridium botulinum type E Saroma and its subunits were isolated and characterized for their chemical and morphological properties. The S layer was composed of a number of subunits with apparent molecular weights ranging from about 10 to 150 kDa. The isolated S layer subunits possess the ability to assemble into recrystallized flat sheets in the absence of any supporting layer and to reattach to the cell wall from which they have been removed. Immunoblot analysis using an antiserum against whole cells of the organism showed that 60 kDa and 90 kDa subunits of the S layer were major somatic antigens of the organism. Immunogold-labeling using monospecific antiserum raised to the individual 60 and 90 kDa proteins revealed that both subunits were exposed evenly over the entire cell surface. The amino acid compositions of both subunits showed that aspartate and glutamate were predominant whereas cystine and methionine were poor. The amino acid composition and acidic property of the two subunits of the S layer agree well with the results obtained from the S layers of other bacterial species as well as other pathogenic clostridia.
Regularly arrayed surface component (S layer) of Clostridium difficile strain GAI 0714 was isolated with 4 M guanidine hydrochloride from the cell wall of the organism, and examined for self-assembly in vitro. The S layer was composed of two different protein subunits with molecular weights of 32 kDa and 45 kDa. Optical diffraction analysis revealed that the morphological units of both native and self-assembled S layer were essentially identical and composed of a rhombus possessing each side of 8.1 nm and interior angle of 88 degrees. The self-assembly of S layer subunits were induced in the presence of divalent cations such as Ca2+ or Zn2+, but Ba2+ or monovalent cations including K+, Na+ and Li+ failed to induce self-assembly. These results suggest that Ca+2 or Zn+2 may act as bridges to link negatively charged surface subunits.
The S layer of Clostridium difficile GAI0714 was shown to be composed of two proteins, of 32 kDa and 45 kDa, as determined by SDS-PAGE. The two proteins were extracted with 8 M-urea (pH 8.3) from a cell wall preparation and purified by DEAE-Sepharose CL-6B chromatography followed by HPLC gel filtration. When solubilized in 0.1 M-urea, both proteins appeared to exhibit dimeric forms, with respective molecular masses of about 61 kDa and 99 kDa, upon HPLC. Although the amino acid compositions of the two proteins differed from each other, both proteins had a high content of acidic amino acids, very low contents of histidine and methionine, and no cysteine. The 32 kDa protein exhibited multiple isoelectric forms (pI 3.7-3.9), whereas the 45 kDa protein had a single form (pI 3.3). Radioiodination and immunogold labelling revealed that both proteins were exposed evenly over the entire cell surface. Based on immunodiffusion analysis using monospecific antiserum raised to the individual proteins, there was no antigenic relationship between the two proteins. Furthermore, immunoblot analysis showed that the antigenicity of the 32 kDa protein appeared to be strain specific, whereas that of the 45 kDa protein appeared to be group specific.
Two different cell wall antigens, carbohydrate (CHO) and protein (P), from Clostridium botulinum type E Saroma were extracted with sodium dodecyl sulfate (SDS) and purified by chromatography on DEAE-Sepharose CL-6B and Sephadex G-75 or G-100. The CHO antigen was composed of glucose, galactose, glucosamine, galactosamine, alanine and phosphorus with a molar ratio of 1.5:1.5:0.25:0.25:1:1. The P antigen was an acidic protein with a molecular weight of 60 kDa, in which the major amino acids were aspartate, glutamate and serine, while the minor ones were cysteine and methionine. Thin sections of the intact or SDS-extracted cells of the organism demonstrated that the cell wall was composed of a two-layered structure, an inner layer about 20 nm thick and an outer layer about 10 nm, and by the extraction with SDS, the outer layer disappeared from the cell surface, leaving the inner layer. Immunogel diffusion tests demonstrated that either CHO antigen or P antigen was common among the nonproteolytic strains of C. botulinum.
An autolysis-deficient mutant was isolated from Clostridium botulinum type A 190L by treatment with ethyl methanesulfonate. The cell wall prepared from the mutant autolyzed at much slower rate than that from the parent strain, accompanying with much less liberation of both amino terminals and reducing groups. Electron microscopic observation revealed that the mutant strain was converted to short rod or curved spherical form with thickened cell walls when the growth temperature was shifted from 37 to 45 C. The mutant had a significantly larger amount of non-peptidoglycan-carbohydrate complexes than did the parent strain and became markedly resistant to the autolysin partially purified from the parent, compared with the parent strain. Furthermore, the mutant was fairly tolerant to killing by penicillin. These results suggest that the autolysis deficiency of the mutant was due not only to the deficient production of autolysin but also to the excess accumulation of carbohydrate in the cell wall.
A soluble hemagglutinin (HA) produced by Yersinia pseudotuberculosis strain Inoue, serotype 5b, was purified by ammonium sulfate precipitation, gel filtration on Sepharose CL-6B and high performance liquid chromatography on a DEAE-5PW anion-exchange column. The purified HA was a 14.5 kDa protein with an isoelectric point of 4.5. Amino acid analysis indicated that the HA consisted of 133 residues, corresponding to the molecular weight of 14,100. The amino acid sequence of N-terminal 38 amino acid residues showed no homology with that of several fimbrial proteins from Escherichia coli.
S layer protein of Clostridium tetani strain AO 174, a nontoxigenic derivative of strain Harvard A 47, was prepared from the cell walls by 4 M urea extraction and purified by DEAE-Sepharose CL-6B chromatography followed by a combination of anion-exchange chromatography and reverse-phase chromatography using an HPLC system. The molecular weight of the S layer protein was estimated to be 140 kilodaltons (kDa) by SDS-PAGE. The amino acid composition of the 140 kDa protein was very similar to those of S layer proteins from the other bacterial species: it was rich in acidic amino acid and lacked cysteine. Also, the protein was unique in its extremely low content of proline (0.02 to 0.03 mol%). Multiple isoelectric forms ranging from pH 4.0 to 4.5 were observed in the purified preparation. Immunodiffusion analysis showed that the 140 kDa protein was a common antigen to the three strains of C. tetani tested.
The in vivo distribution of lectin receptors in the photoreceptors and interphotoreceptor matrix (IPM) was studied by histochemistry and light microscopy. Horseradish peroxidase-conjugated lectins (peanut agglutinin, PNA; Ricinus communis agglutinin-1, RCA-1; or wheat germ agglutinin, WGA) were administered into the subretinal space of living rats, and 1 and 6 hours later eyeballs were enucleated to examine retinal tissues with a light microscope. The surfaces of cone photoreceptor outer segments and inner segments, and/or cone-associated IPM were labeled preferentially with PNA at 1 and 6 hours after its subretinal administration. One hour after subretinal injection, RCA-1 labeling appeared in the distal surface of the outer segments, the basal region of the inner segments, and the surface of the retinal pigment epithelium (RPE). At 6 hours the basal portion of the outer segments showed RCA-1 labeling as well. The distribution of labeling by WGA was uniform throughout the outer segments and/or the IPM domain except for the most distal portion of the outer segments. These observations conform for the most part to previous in vitro experiments, and indicate, together with previously not explicitly described observations, that a spatial heterogeneity in the vertical and horizontal directions exists in the IPM domain.
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Using peroxidase-antiperoxidase immunocytochemistry, we demonstrated a dense accumulation of substance P-like (SP) immunoreactive terminals in the internal layer of the pregeniculate nucleus and in the pretectal olivary nucleus of the Japanese monkey. These distributions of immunostaining were similar to those for retinofugal terminals in the same nuclei. Bilateral eye enucleation markedly decreased SP immunoreactivity in the nuclei. The reduced immunoreactivity probably reflected the loss of SP-containing retinal ganglion cells that sent axons to the pregeniculate and pretectal olivary nuclei.
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A wall-surface protein antigen, designated 32K antigen, was extracted from whole cells of Clostridium difficile strain ATCC 11011 with phosphate buffered saline and purified by ion-exchange chromatography, gel filtration, and chromatofocusing. The 32K antigen preparation was determined to be highly homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of the antigen was characteristic in the predominance of the acidic amino acids, the very low contents of methionine and histidine, and the lack of cysteine. A monomeric molecular weight of the 32K antigen was estimated to be 32,000 by SDS-PAGE and 30,200 by sedimentation equilibrium. The antigen exhibited two isoelectric forms (IP, 4.12 and 3.96). Neither carbohydrate nor phosphorus was detectable in the antigen. The antigen was relatively resistant to trypsin but sensitive to pepsin. Immunoblot analysis of the wall proteins isolated from other strains of C. difficile probed with monospecific antiserum against the antigen from ATCC 11011 showed that the antigenicity of 32K wall protein was common among some of the strains containing 32K wall proteins.
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