Summary of the 22nd United States-Japan Joint Cholera Conference.
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Bacterial Toxins↗
Biomedical subjects
N Ohtomo
Publications and source records attributed to N Ohtomo.
Increase in intradermal vascular permeability caused by pertussis toxin from Bordetella pertussis.
Rabbits that were injected intradermally with pertussis toxin (PT), produced from Bordetella pertussis, showed slight edema and erythema at the injection sites, but not hemorrhage nor necrosis. The edema lesions were stained blue by the intravenous injection of Pontamine Sky Blue 6B dye, suggesting that PT caused increased vascular permeability, similarly to the permeability factor (PF) of cholera toxin. The reaction of the PF of PT could be determined by measuring the diameter of the blue area. The diameter of the blue area bore a good linear relationship to the logarithm of the dose of PT. The activity of the PF was neutralized by anti-PT rabbit serum. Detoxification of PT with formalin did not increase the vascular permeability, but reverted pertussis toxoid showed a PF reaction in proportion to the reverted leukocytosis-promoting and histamine-sensitizing activities of PT. The supernate of a Bordetella pertussis culture also induced a PF reaction and the reaction could be made clear by heating the supernate at 56 C for 30 min, but the supernate of Bordetella bronchiseptica did not induce the reaction at all.
Animals↗
Localization of mono-L-aspartyl chlorin e6 (NPe6) in mouse tissues.
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Animals↗
Expression of hepatitis B virus middle and large surface antigen genes in Saccharomyces cerevisiae.
The hepatitis B virus genome carries the surface antigen (SAg) gene and an open reading frame that encodes two SAg-related polypeptides: SAg with a 55-amino-acid N-terminal extension polypeptide and SAg with a 174-amino-acid N-terminal extension polypeptide. These are termed middle S and large S, respectively. These polypeptides or their glycosylated derivatives have been detected in Dane particles, but their chemical and biological properties have remained largely unknown because of their limited availability. We attempted to produce these proteins in Saccharomyces cerevisiae by placing the coding regions under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Yeast cells carrying middle S and large S coding sequences produced 33,000- and 42,000-dalton products, respectively, each of which reacted with anti-S antibody and bound to polymerized human serum albumin, in accordance with the known properties of pre-S proteins from particles in human sera (K. H. Heermann, U. Goldmann, W. Schwartz, T. Seyffarth, H. Baumgarten, and W. H. Gerlich, J. Virol. 52:396-402, 1984; A. Machida, S. Kishimoto, H. Ohnuma, K. Baba, Y. Ito, H. Miyamoto, G. Funatsu, K. Oda, S. Usuda, S. Togami, T. Nakamura, Y. Miyakawa, and M. Mayumi, Gastroenterology 86:910-918, 1984). The middle S polypeptide is glycosylated and can be assembled into particles whose size and density are similar to those of SAg. However, this polypeptide was highly susceptible to proteolytic degradation into 29,000- and 26,000-dalton polypeptides, of which only the former retained the binding activity to polymerized albumin. The large S polypeptides are nonglycosylated, relatively stable, and do not seem to assemble into particles by themselves.
Cloning, Molecular↗
Immunogenicity of purified glycoprotein gB of herpes simplex virus.
The efficacy of a herpes simplex virus (HSV) component vaccine consisting of viral glycoprotein gB was examined in a mouse system. Immunization of mice with HSV type 1 (HSV-1) gB emulsified in Freund's complete adjuvant or with HSV-1 gB adsorbed to aluminum gel was fully protective against subsequent challenge with HSV-1 or HSV type 2. Latent infection in the trigeminal ganglion was also prevented by immunization with gB.
Animals↗
Expression of hepatitis B virus core antigen gene in Saccharomyces cerevisiae: synthesis of two polypeptides translated from different initiation codons.
Two recombinant plasmids were constructed that allow expression of the hepatitis B core (HBc) antigen gene in the yeast Saccharomyces cerevisiae under the control of the repressible acid phosphatase promoter. One plasmid was designed to produce polypeptide I, which consists of 183 amino acids, and the other plasmid was designed to produce polypeptide II, which has an additional 29-amino-acid sequence at the amino terminus of polypeptide I. The viral genome may code for either one or both of these two polypeptides, depending upon the selection of initiation codons. Both polypeptides produced in yeast cells reacted with anti-HBc antibody and were assembled into spherical particles approximately 27 nm in diameter. Particles made of polypeptide I were stable, whereas those made of polypeptide II readily dissociated when exposed to high salt levels. The antigenicity of the HBc (as defined by its reactivity to anti-HBc antibody in the reversed passive hemagglutination assay) disappeared as the particle dissociated, leaving materials that sedimented slowly and that reacted to anti-hepatitis B e antibody. These observations strongly suggest that native viral cores are mostly (if not all) made of polypeptide I, because it is reasonably stable, and that the N-terminal portion of this polypeptide has some, but not a profound, influence on the assembly of polypeptides into particles.
Capsid↗
Expression of herpes simplex virus glycoprotein B gene in yeast.
The DNA sequence coding for herpes simplex virus type 1 glycoprotein B was placed under control of the acid phosphatase promoter of the yeast Saccharomyces cerevisiae in a plasmid capable of replicating in both yeast and Escherichia coli. Yeast transformed by the plasmid synthesized immunologically active glycoprotein B polypeptide.
Animals↗
Two mammalian cell systems for propagation of the hepatitis B virus genome in extrachromosomal and chromosomally integrated states: production of the surface and e antigens.
A recombinant plasmid consisting of (i) the entire genome of hepatitis B virus (HBV) DNA, (ii) the replication origin of SV40 virus, and (iii) a deletion derivative of pBR322 was introduced either into COS cells of monkey origin which constitutively express SV40 large T antigen, or into thymidine kinase(TK)-deficient mouse L cells together with the TK DNA of Herpes simplex virus. In the COS cell system, the transfecting recombinant DNA replicates via SV40 origin and is maintained in an autonomously replicating state. The cells carrying these extrachromosomal elements express the hepatitis B surface antigen gene at moderate rate, and release the products into the culture medium. However, neither core antigen nor e antigen expression was detected in this system. In the L cell system, the transformed L cells carry the recombinant DNA in a chromosomally integrated state. Such cells express the surface antigen gene at high rate, and release the products into the culture medium. This system also excretes the e antigen into the culture medium. The core antigen was not detected.
Animals↗
Passive immunization of mice with monoclonal antibodies to glycoprotein gB of herpes simplex virus.
To investigate the protective ability of monoclonal antibodies (MCAs) to viral glycoprotein in herpes simplex virus (HSV) infection, athymic nude mice were inoculated intracutaneously with HSV type-1 (HSV-1) in the midflank. Three hours after inoculation, one group of mice was passively immunized with one of a series of MCAs to glycoprotein gB of HSV-1, and a control group of mice was given phosphate buffered saline alone. The control mice died within 16 days after infection, whereas the mice passively immunized with any of the MCA showed suppressed development of skin lesions. Three of six mice given MCA failed to develop any visible lesions and no HSV could be isolated from the lumbar dorsal root ganglia of these mice 60 days after the challenge. BALB/c mice were also protected from infection with HSV type 2 by passive immunization with MCA to HSV-1 gB.
Animals↗
An improved method for preparation of C1-depleted serum and its application to hemolytic assay of C1.
Serum depleted of the first component of complement (C1D) was prepared by treating fresh human serum with Sepharose-IgG in the presence of triethylenetetramine-N,N,N',N'',N''',N'''-hexa-acetic acid and di-isopropyl fluorophosphate at acidic pH (5.2). The total hemolytic activity of this C1D could be increased to approximately 75% of that of the original serum by the addition of excess purified C1. A linear relation was obtained on determination of C1 hemolytic activity using C1D, and the C1 titers of sera from patients measured by this simple method showed a good correlation with those measured using intermediate cells.
Animals↗
[Increase of intradermal vascular permeability by pertussis toxin].
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Animals↗
[Bacterial infection and immunoglobulin].
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Agammaglobulinemia↗
Distributions and recoveries of complement components during cold ethanol fractionation of human plasma.
The distributions and recoveries of the hemolytic activities and quantities of protein of complement components during cold ethanol fractionation were studied. Most of the components were recovered in Cohn's fraction III, with slight activities in fractions I and IV. The hemolytic activities of C1 and C5 were recovered in high yields in fraction III. The recoveries of activities of late components of complement (C6-C9) in fraction II + III were also high. These results suggest that many complement components can be isolated from Cohn's ethanol fractions.
Cold Temperature↗
Cloning and structural analyses of hepatitis B virus DNAs, subtype adr.
Entire genomes of hepatitis B virus (subtype adr) have been cloned. The nucleotide sequence data were compared with other sequences of HBV genome including: adw [Valenzuela et al. (1981) in Animal Virus Genetics. Fields et al. eds. Academic Press, Inc., NY. pp. 57-70], ayw [Galibert et al. (1979) Nature, 281, 646-650], and adyw [Pasek et al. (1979) Nature 282, 575-579]. Four open coding frames for polypeptides larger than 6,000 dalton were found to be conserved and were highly compressed by overlapping with each other in one strand (L-strand). Sites of initiation of the S gene and termination of the P gene were not conserved. No conserved coding frame was found on the opposite strand (S strand). Amino acid sequences of six surface antigen (HBsAg) peptides, including subtypes adr, adw, and ayw, are deduced from the DNA sequences, and the substitution of amino acid residues which are consistent with the change of subtypes are demonstrated.
Amino Acid Sequence↗
Degradation of choleragen bound to cultured human fibroblasts and mouse neuroblastoma cells.
125I-choleragen bound to human fibroblasts was degraded slowly with a t1/2 of 2-3 days; the radiolabel in bound 125I-choleragen was present in both the A and B subunits. During degradation, radiolabel was lost more rapidly from the 125I-A1 (t1/2 approximately 2 days) than from the 125I-B peptides (t1/2 greater than 5 days). 125I-Choleragen bound to neuroblastoma cells showed a considerably shorter t1/2 for both the 125I-A1 and 125I-B peptides; as with the fibroblasts, radiolabel was lost more rapidly from the 125I-A1 than from the 125I-B peptides. The continued presence of choleragen in the fibroblasts and neuroblastoma cells was associated with a prolonged activation of adenylate cyclase. In addition, fibroblasts, previously exposed to toxin and then washed free of unbound choleragen, only slowly recovered their ability to bind 125I-choleragen with a t1/2 of 7 days. Fibroblasts exposed to choleragen also showed evidence of persistent toxin on the surface based on the ability of the cells to bind antitoxin, antisubunit A or antisubunit B antibodies followed by 3H-protein A. It appears that choleragen remains persistently bound to fibroblasts, is degraded at a slow rate, and may prevent the binding of new toxin molecules to the fibroblast. The relatively slow degradation of toxin by fibroblasts may explain the prolonged activation of adenylate cyclase by toxin. The loss of 125I-toxin binding capacity following incubation with toxin may result from continued presence of toxin subunits on the cell surface.
Adenylyl Cyclases↗
Expression of hepatitis B surface antigen gene in yeast.
The DNA sequence coding for hepatitis B virus surface antigen (HBsAg) was placed under control of the repressible acid phosphatase promoter of the yeast Saccharomyces cerevisiae in a plasmid capable of autonomous replication in both yeast and Escherichia coli. Yeast transformed by this plasmid synthesized up to 5 X 10(5) molecules per cell of immunologically active HBsAg polypeptide in phosphate-free medium. The HBsAg polypeptides produced in the yeast cells were assembled into 20- to 22-nm spherical or oval particles and were immunogenic.
Acid Phosphatase↗
Entrance of cholera enterotoxin subunits into cells.
Quantitative analysis of the staining of cholera enterotoxin on the surface of cells with specific antibodies against each subunit of cholera toxin, using a Fluorescence-Activated Cell Sorter, showed that not only subunit A but also subunit B penetrates the cell membrane. The detection of each subunit inside the cell was facilitated by the use of saponin, an agent which increases membrane permeability.
Animals↗
Mechanism of antibody-mediated protection against herpes simplex virus infection in athymic nude mice: requirement of Fc portion of antibody.
Experiments were performed to investigate the resistance of the host due to antibody-mediated mechanisms to herpes simplex virus (HSV) infection. Transfer of hyperimmune anti-HSV mouse serum inhibited the development of skin lesions and prolonged the survival of lethally HSV-infected nude mice. Relatively high concentrations of antibody were required to achieve this protection. Antisera prepared in heterologous animals were also effective, while administration of anti-cowpox virus serum or interferon provided no protection. This type of protection is therefore due to specific antibody and cannot be attributed to interferon. In order to delineate the requirement for antibody in antibody-mediated protection, human gamma globulin preparations were transferred to lethally HSV-infected nude mice. Transfer of intact human gamma globulin (GG) was effective in controlling infection. S-sulfonation of GG did not diminish the protective ability. However, purified F(ab')2 did not have any protective action even when it was administered frequently to maintain serum neutralizing antibody titer. GG was effective in C5-deficient mice lethally infected with HSV. These results indicate that in vivo antibody-mediated protection to HSV infection requires the Fc region of the intact IgG molecule and suggest that antibody-dependent cell-mediated cytotoxicity may be operative in vivo.
Animals↗