bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenolic glycolipids of Mycobacterium bovis: new structures and synthesis of a corresponding seroreactive neoglycoprotein
Authors: Chatterjee D , Bozic C M , Knisley C , Cho S N , Brennan P J
Published: January 2020

The glycolipid that characterizes the majority of isolates of Mycobacterium bovis and that has come to be known as M. bovis-identifying lipid is the phenolic glycolipid mycoside B described in the literature by others. However, when mycoside B obtained from M. bovis BCG, field isolates, and infected tissues was examined in detail, it was shown to be different from that described in the literature in some important respects. In particular, the glycosyl substituent is 2-O-methyl-alpha-L-rhamnopyranose rather than 2-O-methyl-beta-D-rhamnopyranose. With this information, a seroreactive neoglycoprotein (neoantigen) containing the 2-O-methyl-alpha-L-rhamnopyranosyl substituent suitable for the serodiagnosis of bovine tuberculosis was synthesized. M. bovis also contains other minor seroreactive phenolic glycolipids, one of which is a deacylated form of mycoside B and another of which contains an alpha-L-rhamnopyranosyl unit rather than 2-O-methyl-alpha-L-rhamnopyranose.
Surface characteristics of Pseudomonas aeruginosa grown in a chamber implant model in mice and rats
Authors: Kelly N M , Bell A , Hancock R E
Published: January 2020

Pseudomonas aeruginosa PAO1 was grown in vivo in chambers implanted into the peritoneums of mice and rats. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracts of bacterial cells taken from the chambers and washed to remove loosely bound host proteins revealed the presence of the major outer membrane proteins D2, E, F, G, and H2. Western immunoblotting with specific antisera confirmed the presence of porin protein F and lipoprotein H2. However, there was no apparent induction of the phosphate starvation-inducible porin P or the divalent cation starvation-inducible protein H1. Small amounts of proteins with molecular weights similar to those of the iron-regulated outer membrane proteins were found in cells grown in vivo; however, their presence could not be confirmed immunologically. The presence of pili and flagella on the cells grown in vivo was demonstrated by electron microscopy and Western immunoblotting. A consistent alteration in the lipopolysaccharide banding pattern was observed after growth in vivo. Compared with cells of strain PAO1 grown in vitro, cells grown in vivo appeared to lack a series of high-molecular-weight O-antigen-containing lipopolysaccharide bands and gained a new series of lower-molecular-weight lipopolysaccharide bands. This alteration in the lipopolysaccharide after growth in vivo did not affect the O-antigen serotype or the resistance of the bacteria to serum.
Systemic injection of group A streptococcal peptidoglycan-polysaccharide complexes elicits persistent neutrophilia and monocytosis associated with polyarthritis in rats
Authors: Wells A F , Hightower J A , Parks C , Kufoy E , Fox A
Published: January 2020

The perpetuation of inflammatory changes within joints elicited by persisting, poorly biodegradable group A streptococcal cell walls (peptidoglycan-polysaccharide complexes [PG-PS]) is well documented. Chronic changes in the bloodstream induced by PG-PS have not been described previously. We demonstrated that leukocytosis occurs within 3 days after intraperitoneal injection of PG-PS and remains elevated 20 weeks later. Chronic neutrophilia, monocytosis, and lymphocytosis were observed in all experiments. Chronic changes in platelet, erythrocyte, and reticulocyte counts were not seen. The newly documented leukocytosis, lasting for months after PG-PS administration, provided a circulating pool of leukocytes that may participate in chronic inflammatory events in the joint. Although the central role of the macrophage in PG-PS-mediated inflammation has been emphasized (F. G. Dalldorf, W. J. Cromartie, S. K. Anderle, R. L. Clark, and J. H. Schwab, Am. J. Pathol. 100:383-402, 1980), the polymorphonuclear cell may be involved in periods of exacerbation of streptococcal cell wall-mediated polyarthritis. This was supported by our observations that neutrophilia and monocytosis correlate well with the degree of chronic joint inflammation.
Cloning and expression of the phospholipase C gene from Clostridium perfringens and Clostridium bifermentans
Authors: Tso J Y , Siebel C
Published: January 2020

The phospholipase C gene from Clostridium perfringens was isolated, and its sequence was determined. It was found that the structural gene codes for a protein of 399 amino acid residues. The NH2-terminal residues have the typical features of a signal peptide and are probably cleaved after secretion. Escherichia coli cells harboring the phospholipase C gene-containing plasmid expressed high levels of this protein in the periplasmic space. Phospholipase C purified from E. coli transformants was enzymatically active, hemolytic to erythrocytes, and toxic to animals when injected intravenously. The phospholipase C gene from a related organism, Clostridium bifermentans, was also isolated. The two phospholipase C genes were found to be 64% homologous in coding sequence. The C. bifermentans protein, however, was 50-fold less active enzymatically than the C. perfringens enzyme.
Heterogeneity of the streptokinase gene in group A streptococci
Authors: Huang T T , Malke H , Ferretti J J
Published: January 2020

A molecular epidemiological study was conducted to determine the distribution of the streptokinase gene in group A streptococcal strains of different M types and in other streptococcal species. Plasmid pNC1, containing only the internal coding sequence of the streptokinase gene from group C streptococcal strain H46A, was used as a DNA probe in colony and Southern hybridization experiments. Only the pathogenic group A, C, and G streptococci contained a streptokinase gene; 12 other Lancefield group strains did not. A total of 134 group A strains, including 61 M types and 6 T types, were tested. Although only 62% (83 of 134) of the strains tested showed positive streptokinase activity by the casein-plasminogen overlay assay, all strains contained the streptokinase gene as evidenced by strong hybridization with the pNC1 probe. Southern blot DNA hybridizations were carried out with 101 strains of group A streptococci. The restriction enzymes HindIII and HaeIII were used to digest the genomic DNA. Six hybridization patterns were observed after HindIII digestion. Double hybridization bands appeared in all of the patterns, which indicated the existence of a highly conserved HindIII site. More complex hybridization results were obtained after HaeIII digestion. Twelve hybridization patterns were observed; three were characterized by a single hybridization band, and nine were characterized by double bands. Variations in hybridization patterns were observed in strains of both the same and different serotypes. The overall results at the gene level indicate that there is considerable heterogeneity among the streptokinases of group A streptococci, consistent with previous findings of immunological and chemical differences among streptokinases of group A streptococci.
Ceruloplasmin can substitute for rabbit serum in stimulating the growth of Treponema denticola
Authors: Suzuki M , Loesche W J
Published: January 2020

The growth of Treponema denticola in a complex medium was stimulated by human ceruloplasmin and, to a lesser extent, albumin, alpha-1-antitrypsin, alpha-1-acid glycoprotein, and alpha-2-macroglobulin. Human ceruloplasmin could stimulate growth of T. denticola as well as or better than 5% rabbit serum, with a dose-response relationship of between 1 and 5 mg of ceruloplasmin per ml of culture medium. These results suggest that ceruloplasmin could substitute for rabbit serum in stimulating the growth of T. denticola.
Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O7 lipopolysaccharide antigen of a human invasive strain of E. coli O7:K1
Authors: Valvano M A , Crosa J H
Published: January 2020

We have cloned and studied the expression in Escherichia coli K-12 of chromosomal rfb genes determining the biosynthesis of the O7 lipopolysaccharide (LPS) antigen from E. coli K1 strain VW187. Two E. coli K-12 strains carrying recombinant cosmids gave positive coagglutination reactions with protein A-rich staphylococcal particles bearing an O7-specific rabbit polyclonal antiserum. Silver-stained polyacrylamide gels of total membranes extracted with hot phenol showed O side chain material which had O7 specificity as determined by immunoblotting experiments. However, the amount of O7 LPS expressed in E. coli K-12 was considerably lower than that produced by the wild-type strain VW187. Deletion and transposition experiments identified a region of about 17 kilobase pairs which is essential for the expression of O7 LPS. The existence of homologies between the O7 LPS genes and other E. coli O side chain genes was investigated by Southern blot hybridization experiments. An O7-specific probe fragment of 15 kilobase pairs did not hybridize to genomic DNA digests of E. coli strains belonging to several different O types, demonstrating that the O7 LPS genes are unique.
Oral administration of a streptococcal antigen coupled to cholera toxin B subunit evokes strong antibody responses in salivary glands and extramucosal tissues
Authors: Czerkinsky C , Russell M W , Lycke N , Lindblad M , Holmgren J
Published: January 2020

Generation of local and systemic immune responses by the oral administration of antigens is frequently inefficient, requiring large quantities of immunogens and yielding only modest antibody responses. In this study, we have demonstrated that oral administration of microgram amounts of Streptococcus mutans protein antigen I/II covalently coupled to the B subunit of cholera toxin elicits vigorous mucosal as well as extramucosal immunoglobulin A and G antistreptococcal antibody responses in mice. These responses were manifested by the presence of large numbers of antibody-secreting cells in salivary glands, mesenteric lymph nodes, and spleens and by the development of high levels of circulating antibodies. This novel immunization strategy may find broad application in the construction of oral vaccines for the control of infectious diseases caused by pathogens encountered at mucosal and extramucosal sites.
Bordetella parapertussis invasion of HeLa 229 cells and human respiratory epithelial cells in primary culture
Authors: Ewanowich C A , Sherburne R K , Man S F , Peppler M S
Published: January 2020

Bordetella parapertussis, a respiratory tract pathogen commonly regarded as noninvasive, was found to invade HeLa 229 cell monolayers. Following treatment of the monolayers with gentamicin, numbers of viable B. parapertussis recovered were comparable to those of invasive Salmonella and Shigella isolates. Invasion occurs through a cytochalasin-sensitive process which appears to be distinct from receptor-mediated endocytosis. Hyperimmune antisera raised against filaments hemagglutinin, a major adhesion of B. pertussis, did not inhibit invasion by B. parapertussis, suggesting that alternate adhesin(s) are required for invasion. In addition, B. parapertussis was found to invade human respiratory epithelial cells in primary culture, as demonstrated in ultrathin sections viewed by transmission electron microscopy. Although viable intracellular B. parapertussis persist within HeLa cells, they do not multiply there and the monolayers remain intact, suggesting a possible mechanism of carriage for these organisms.
DNA sequence of mip, a Legionella pneumophila gene associated with macrophage infectivity
Authors: Engleberg N C , Carter C , Weber D R , Cianciotto N P , Eisenstein B I
Published: January 2020

In a previous study, a 24-kilodalton (kDa) protein surface antigen of Legionella pneumophila was cloned into Escherichia coli and found to be expressed on the host cell surface. Subsequently, a site-directed mutation in this gene (designated mip) in L. pneumophila was found to impair the capacity of this bacterium to initiate intracellular infection in human macrophages. The work presented here indicates that the antigenic gene product is distinct from the 24- to 29-kDa major outer membrane protein of L. pneumophila. In addition, the antigen was identified as a highly basic protein on two-dimensional nonequilibrium polyacrylamide gels and on two-dimensional monoclonal antibody immunoblots. When the DNA fragment encoding this protein was sequenced, a long open reading frame of 699 base pairs was identified within a region to which antigen expression was previously mapped. mip mRNA isolated from both L. pneumophila and transformed E. coli had the same 5' end, as determined by primer extension analysis, indicating that the same promoter sequences are used in both species. A likely factor-independent transcriptional terminator was found 20 residues downstream of the stop codon, suggesting that mip is encoded on a monocistronic message. The inferred polypeptide began with a possible 20- to 24-residue signal sequence, and, as predicted by two-dimensional electrophoresis, had a molecular weight of 24,868 and was a potent polycation with an estimated pI of 9.8.
Defects in sera from acquired immunodeficiency syndrome (AIDS) patients and from non-AIDS patients with Mycobacterium avium infection which decrease macrophage resistance to M. avium
Authors: Crowle A J , Cohn D L , Poche P
Published: January 2020

Some characteristics of the sera and macrophages (MP) of human immunodeficiency virus (HIV)-infected patients which might contribute to their unusual susceptibility to Mycobacterium avium infection were studied. Cultures of patient peripheral blood MP in medium supplemented with their sera or normal subject sera were infected with M. avium and compared with similar cultures of normal MP. Intracellular mycobacterial replication was measured in the infected MP by CFU counts of the bacteria made from lysed samples of the MP at 0, 4, and 7 days after MP infection. Sera from patients with chronic granulomatous infection with M. avium, but no HIV infection, also were studied. The sera from all of the patients with chronic granulomatous infection and from several HIV-infected patients were deficient or lacking in an inhibitor that in normal serum acts within normal MP to suppress intracellular growth of M. avium. Most of the HIV-infected patients also had MP that were abnormally permissive for M. avium because they responded poorly to the serum inhibitor. Elucidation of these associated defects in native defenses against M. avium may result in better prevention and therapy of M. avium infections.
Stimulation of macrophage phagocytic but not bactericidal activity by colony-stimulating factor 1
Authors: Cheers C , Hill M , Haigh A M , Stanley E R
Published: January 2020

The ability of mouse peritoneal cells to phagocytose and lyse Listeria monocytogenes was measured after the cells were incubated with purified murine macrophage-specific colony-stimulating factor (CSF-1). Activation of combined phagocytic and bacteriolytic ability required 24 h, with an optimal dose of 1,000 U of CSF-1 per ml. No activation was achieved with a shorter period of incubation, known to be sufficient for GM-CSF to stimulate phagocytosis by granulocytes, and there was no advantage in longer exposure. After 24 h in 1,000 U of CSF-1, macrophages showed visibly increased spreading on the plastic petri dish. Activated cells examined microscopically showed an increase in the number of phagocytic cells and in the numbers of bacteria per phagocytic cell. This increased phagocytic ability was evident also in the increase in the amount of radioactivity associated with the cells following a 30-min incubation with radiolabeled bacteria. When these cells were carefully washed, the percentage of this initial uptake released during the next 2 h was not increased by pretreatment of the cells with CSF-1, showing that the effect of this growth factor was on phagocytosis of the bacteria not on the killing mechanisms per se.
Human immune response to meningococcal outer membrane protein epitopes after natural infection or vaccination
Authors: Mandrell R E , Zollinger W D
Published: January 2020

Antibody levels in 41 sets of human acute- and convalescent-phase meningococcal sera were compared with those in 23 sets of human prevaccination and 2-week postvaccination sera. We used a modification of a solid-phase radioimmunoassay (SPRIA) technique to test each of the human serum samples as inhibitors of monoclonal antibodies (MAbs) that bind (HIMSPRIA) to the outer membrane complex from a 2a:P1.2:P5.1 strain. We used three murine MAbs specific for the 2a, P1.2, and P5.1 epitopes on meningococcal class 1, 2, and 5 proteins, respectively, to detect antibodies with similar specificities in human sera. Each of 40 available matching strains from patients were also screened with the three MAbs in a nitrocellulose spot blot assay. A total of 37 (92%) were positive for the 2a epitope, 36 (90%) were positive for the P1.2 epitope, and 16 (40%) were positive for the P5.1 epitope. Of 38 available convalescent-phase sera, 27 (71%) matched with these strains and had detectable inhibiting antibody for each of the MAb-defined protein epitopes of the infecting strain. Three convalescent-phase sera had no HIMSPRIA activity for MAb-defined epitopes that were present on the infecting strain; others had activity for one or two of the epitopes. The results were similar for pre- and postvaccination sera. The average level of HIMSPRIA activity for the P1.2 epitope was greater than fivefold higher in postvaccination sera compared with that in convalescent-phase sera. Sera with distinct patterns of HIMSPRIA activity also were tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis and showed a correlation between the HIMSPRIA activity for particular epitopes and the level of antibody binding to the immunoblotted proteins possessing those epitopes. A comparison of the HIMSPRIA and the bactericidal activity of selected postvaccination sera indicated a possible correlation between HIMSPRIA and bactericidal activity, but it also suggested the presence of bactericidal antibodies with specificities other than those defined by the MAbs.
Distribution of the P-associated-pilus (pap) region among Escherichia coli from natural sources: evidence for horizontal gene transfer
Authors: Plos K , Hull S I , Hull R A , Levin B R , Orskov I , Orskov F , Svanborg-Edén C
Published: January 2020

Variation in chromosomal DNA in Escherichia coli was studied with probes specific for the P-associated-pilus (pap) region. The presence of DNA homologous to pap was determined by dot blots. Variation in the number of copies of pap and in the organization of internal and flanking sequences was determined by Southern blot hybridization. The 229 strains studied were also classified by O:K:H serotyping and multilocus enzyme electrophoresis. There was considerable heterogeneity in the presence of pap and distribution of pap-homologous DNA in these E. coli strains from natural sources. In general, there was less variation in pap among strains of the same specific O:K:H serotype and enzyme electrophoretic type than among random isolates. There were, however, E. coli strains identified as members of the same clone by O:K:H serotyping and enzyme electrophoresis that were pap positive and pap negative or had different Southern blot patterns for the pap probes (pap type). There were also isolates of the same pap type that differed in two of three O:K:H serotype antigens and the majority of enzymes that determined their enzyme electrophoretic type. These latter two observations were interpreted as evidence for the horizontal (infectious) transfer of the pap-homologous sequences among clones of E. coli.
Cathepsin D-like activity in neutrophils and monocytes
Authors: Levy J , Kolski G B , Douglas S D
Published: January 2020

Monocytes-macrophages and polymorphonuclear leukocytes contain an acid proteolytic enzyme that cleaves tritiated hemoglobin. The monocyte-macrophage-derived enzymatic activity was completely inhibited by pepstatin A, a property of cathepsin D. Monocyte-derived macrophages developed detectable cathepsin D-like activity after 5 days in culture, and this activity coincided with the appearance of other known indicators of macrophage maturation. The cathepsin D activity further increased significantly with time after day 5 of culture. The proteinase activity extracted from neutrophils was only partially inhibitable by pepstatin A, which indicates that this activity is contributed by more than one proteolytic enzyme, including cathepsin D. Cathepsin D activity demonstrated in neutrophils and macrophages may be an important marker of phagocyte function.
Dependence of proliferation of Bacteroides forsythus on exogenous N-acetylmuramic acid
Authors: Wyss C
Published: January 2020

Bacteroides forsythus is the first wild-type or mutant bacterium found to require exogenous N-acetylmuramic acid for proliferation and maintenance of cell shape. This implies so far unknown pathways for peptidoglycan synthesis and a strict dependence of B. forsythus on other bacteria in its oral habitat. Addition of N-acetylmuramic acid to conventional bacteriological media allows routine cultivation of this fastidious organism.
Accumulation of chlamydial lipopolysaccharide antigen in the plasma membranes of infected cells
Authors: Karimi S T , Schloemer R H , Wilde C E
Published: January 2020

The presence of a chlamydia-specified antigen associated with the plasma membrane of infected cell lines was demonstrated by indirect immunofluorescence staining with a monoclonal antibody, designated 47A2, specific for the chlamydial genus-specific lipopolysaccharide (LPS) antigen. Staining of HeLa, L-929, and McCoy cells infected with the L2 or F serovar of Chlamydia trachomatis was observed either without fixation or following aldehyde fixation and brief drying. The 47A2-reactive antigen appeared to be present on the plasma membrane, on bleb-like structures on the host cell surface, and on proximal processes of neighboring uninfected cells. Antibodies to chlamydial protein antigens such as the major outer membrane protein produced no surface staining under similar conditions. Membrane vesicles elaborated from infected cells were enriched for the 47A2-reactive antigen. Superinfection of chlamydia-infected cells with vesicular stomatitis virus, an enveloped virus which buds from the plasma membrane, allowed purification of progeny virions that were enriched with chlamydial LPS. These results are consistent with the presence of chlamydial LPS in the plasma membranes of infected host cells.
Relatedness of structures of a major immunogen in Trichomonas vaginalis isolates
Authors: Alderete J F , Neale K A
Published: January 2020

Solubilization of live Trichomonas vaginalis organisms with detergent caused the release of cysteine proteinases in the detergent extract which were inhibitable with N-alpha-p-tosyl-L-lysine chloromethyl ketone. The detergent extracts of all isolates tested possessed similar cysteine proteinase activities. These parasite proteinases rapidly degraded a prominent immunogen whose surface disposition undergoes phenotypic variation in some isolates. The relatedness of the forms of this immunogen among all isolates tested was confirmed by identical immunoblot patterns of autolysed immunogen, and data suggest the presence of repeating units or at least equidistant sites for proteinase cleavage within the immunogen molecule.
Group A streptococcal pyrogenic exotoxin (scarlet fever toxin) type A and blastogen A are the same protein
Authors: Schlievert P M , Gray E D
Published: January 2020

Group A streptococcal pyrogenic exotoxins A, B, and C (also known as scarlet fever toxins and erythrogenic toxins) were evaluated for relatedness to another streptococcus-derived lymphocyte mitogen, blastogen A. Streptococcal pyrogenic exotoxin A and blastogen A were immunologically cross-reactive and shared the same molecular weight, N-terminal amino acid sequence, and capacity to stimulate rabbit splenocyte proliferation nonspecifically.
Activation and binding of opsonic fragments of C3 on encapsulated Cryptococcus neoformans by using an alternative complement pathway reconstituted from six isolated proteins
Authors: Kozel T R , Wilson M A , Pfrommer G S , Schlageter A M
Published: January 2020

Encapsulated Cryptococcus neoformans yeast cells are potent activators of the complement system. We examined the interaction of the yeast cells with an alternative complement pathway reconstituted from isolated factor D, factor B, factor H, factor I, C3, and properdin. Incubation of encapsulated cryptococci with the reconstituted pathway led to activation and binding of C3 fragments to the yeast cells that was quantitatively and qualitatively identical to that observed with normal human serum. Incubation with either normal serum or a mixture of isolated proteins led to binding of 4 x 10(7) to 5 x 10(7) C3 molecules to the yeast cells. The kinetics for activation and binding of C3 were identical, with maximum binding observed after a 20-min incubation. Immunoglobulin G was not needed for optimal activation kinetics. C3 fragments eluted from the yeast cells by treatment with hydroxylamine and subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the presence primarily of iC3b on yeast cells incubated with either normal serum or the reconstituted pathway. Ultrastructural examination of the opsonized yeast cells showed that the cryptococcal capsule was the site for binding of C3 activated from normal serum or the reconstituted pathway, with a dense accumulation of C3 at the periphery of the capsule. Thus, incubation of encapsulated cryptococci in the reconstituted pathway led to deposition of opsonic complement fragments at a site that was appropriate for interaction with phagocyte receptors. Cryptococci opsonized with the reconstituted pathway showed a markedly enhanced interaction with cultured human monocytes compared with unopsonized yeast cells, indicating that the alternative pathway alone is opsonic for yeast cells. However, the results indicate that additional serum factors are needed for optimal opsonization of yeast cells because a 35% reduction in the number of cryptococci bound to macrophages was observed with cryptococci opsonized with the reconstituted pathway compared with that observed when yeast cells were opsonized with normal serum.
Sequence of the structural gene (rmpM) for the class 4 outer membrane protein of Neisseria meningitidis, homology of the protein to gonococcal protein III and Escherichia coli OmpA, and construction of meningococcal strains that lack class 4 protein
Authors: Klugman K P , Gotschlich E C , Blake M S
Published: January 2020

The structural gene (rmpM) of the class 4 outer membrane protein of Neisseria meningitidis has been cloned and sequenced. The derived amino acid sequence reveals a 218-amino-acid protein following a 22-amino-acid signal peptide. The protein shows 94.2% homology with protein III of Neisseria gonorrhoeae and shares its two potential disulfide loops. The protein also shares limited homology with Escherichia coli OmpA. N. gonorrhoeae protein III has been shown to elicit blocking antibodies that prevent the killing of serum-resistant strains by immune sera (P. A. Rice, H. E. Vayo, M. R. Tam, and M. S. Blake, J. Exp. Med. 164:1735-1748, 1986). The very close homology of meningococcal class 4 protein with gonococcal protein III suggests that meningococcal outer membrane preparations containing class 4 protein may similarly stimulate blocking antibodies. In order to investigate the role of the class 4 protein in the pathogenesis of meningococcal infection, we have used an erythromycin resistance gene in developing two meningococcal strains that lack class 4 protein.
Characterization of integral membrane proteins of Leishmania major by Triton X-114 fractionation and analysis of vaccination effects in mice
Authors: Murray P J , Spithill T W , Handman E
Published: January 2020

The total integral membrane proteins of promastigotes of Leishmania major were extracted by using the Triton X-114 phase separation technique and were characterized by immunoprecipitation, Western blotting (immunoblotting), and lectin chromatography. Of the 40 or more proteins which partitioned into the detergent phase, only about 10 proteins could be surface radioiodinated on live promastigotes, suggesting their surface orientation. The abundance of the gp58-63 antigen varied markedly between two strains of L. major. Sera from patients with visceral leishmaniasis caused by Leishmania donovani chagasi recognized the gp58-63 complex and an additional Mr-42,000 polypeptide shared between L. major and L. donovani chagasi. A subpopulation of six surface proteins, including the abundant gp58-63 antigen and a group of proteins of Mr 81,000 to 105,000, were glycoproteins recognized by antiserum to wheat germ agglutinin- or concanavalin A-binding proteins. The membrane proteins of the LRC-L119 isolate of L. major could successfully vaccinate genetically susceptible mice, thus opening the way for a molecularly defined subunit vaccine composed of glycolipid and membrane protein antigens.
Biological activities and chemical composition of purified tracheal cytotoxin of Bordetella pertussis
Authors: Cookson B T , Cho H L , Herwaldt L A , Goldman W E
Published: January 2020

Specific destruction of ciliated epithelial cells lining the large airways is the primary respiratory tract cytopathology associated with human Bordetella pertussis infections. We have purified a single low-molecular-weight glycopeptide, tracheal cytotoxin (TCT), that appears to cause this pathology. By using a combination of solid-phase extraction and reversed-phase high-pressure liquid chromatography, about 700 nmol of biologically active peptide can be isolated from 1 liter of B. pertussis culture supernatant (approximately 60% yield). TCT at concentrations of 1 microM destroyed the ciliated cell population when incubated with respiratory epithelium in vitro. This concentration of TCT is similar to the concentrations found in the culture supernatant of growing B. pertussis. Purified TCT also inhibited DNA synthesis of hamster trachea epithelial cells in a quantitative, dose-dependent fashion. Endotoxin was not detected in the purified material, and neither B. pertussis nor Escherichia coli endotoxin could duplicate the biological activities of TCT. Amino acid and amino sugar analyses of purified TCT revealed the presence of glucosamine, muramic acid, alanine, glutamic acid, and diaminopimelic acid in molar ratios of 1:1:2:1:1. This suggests that TCT, the released ciliostatic principle of B. pertussis, is a disaccharide tetrapeptide subunit of peptidoglycan.
In vitro model of Treponema pallidum invasiveness
Authors: Riviere G R , Thomas D D , Cobb C M
Published: January 2020

The purpose of this investigation was to develop an in vitro model with which invasion of tissues by pathogenic Treponema pallidum could be studied. Double-sided culture chambers were created by mounting abdominal walls excised from mice between two halves of small dialysis cells. The integrity of tissue barriers was confirmed by dye exclusion. T. pallidum subsp. pallidum, including intrinsically radiolabeled organisms, was introduced into one side of each chamber, and fractions from the other side were evaluated over time by dark-field microscopy and scintillation counting. Tissues were evaluated by scanning electron microscopy and immunologic staining. Motile T. pallidum, but not nonpathogenic, host-indigenous Treponema phagedenis biotype Reiter, was able to pass from one side of the chamber to the other side within 10 h. Up to 12% of the inoculum crossed the chamber within 24 h. Spirochetes were found within tissue in the greatest numbers between 6 and 8 h postinoculation. The murine abdominal wall has epithelium only on the peritoneum side, and results showed that T. pallidum required an epithelial surface on the entry side of the double-chambered cell in order to traverse the tissue barrier. This new in vitro technique may be of value in studying spirochete virulence and host resistance.
Transcriptional regulation of the protective antigen gene of Bacillus anthracis
Authors: Bartkus J M , Leppla S H
Published: January 2020

Bicarbonate is required for production of the major virulence factors, the toxins and capsule, of Bacillus anthracis. In this study we examined the basis for stimulation of production of protective antigen (PA), a central component of the two anthrax toxins encoded by plasmid pXO1. RNA prepared from B. anthracis grown in media with and without added bicarbonate was probed for PA mRNA. Data showed that bicarbonate was required for increased transcription of the PA gene (pag) in minimal medium. Transcription of pag was low in rich medium and could not be stimulated by the addition of bicarbonate. To characterize further the factors required for transcriptional regulation of pag, the promoter region of pag was fused to the chloramphenicol acetyltransferase gene (cat-86) of vector pPL703 and transformed by electroporation into pXO1+ (Tox+) and pXO1- (Tox-) strains of B. anthracis. Analysis of chloramphenicol acetyltransferase produced by the pag-cat-86 fusion in each of these backgrounds confirmed the results obtained by hybridization. Data obtained with this fusion also revealed that the large toxin plasmid, pXO1, found in virulent strains of B. anthracis, was required for stimulation of transcription of pag by bicarbonate. This result suggests the existence of a trans-acting factor that is involved in the activation of pag transcription.
Modulation of colonization by black-pigmented Bacteroides species in squirrel monkeys by immunization with Bacteroides gingivalis
Authors: McArthur W P , Magnusson I , Marks R G , Clark W B
Published: January 2020

Periodontal diseases are inflammatory responses thought to be triggered by specific microorganisms colonizing in the gingival crevice. Theoretically, periodontal diseases could be prevented if the etiologic organisms were not allowed to colonize the subgingival area. The humoral immune response is one mechanism which may modulate bacterial colonization in the gingival crevice. To test the effect of systemic humoral immunity on subgingival colonization by bacteria, squirrel monkeys (Saimiri sciureus) were immunized with Bacteroides gingivalis, a black-pigmented Bacteroides sp. and putative periodontal pathogen. Immunized and sham-immunized monkeys were orally inoculated with 10(10) viable B. gingivalis during ligation of five teeth in one quadrant with bacterium-soaked suture material and distribution over the entire dentogingival margin. Immunization resulted in an increased level of immunoglobulin G anti-B. gingivalis in serum and was associated with a strong trend toward a statistically significant reduction in colonization of the gingival crevice by black-pigmented bacteroides.
Recombinant murine gamma interferon induces enhanced resistance to Listeria monocytogenes infection in neonatal mice
Authors: Chen Y , Nakane A , Minagawa T
Published: January 2020

Neonatal mice within 24 h of birth were highly susceptible to intraperitoneal infection with Listeria monocytogenes. The 50% lethal dose of bacterial cells was 6.3 X 10(1) CFU for neonates and 3.2 X 10(6) CFU for adult mice. A single injection of recombinant murine gamma interferon (rMuIFN-gamma) protected neonatal mice from simultaneous challenge with a lethal dose of L. monocytogenes cells. The rMuIFN-gamma effect was dose dependent: protection was consistently observed in mice treated with rMuIFN-gamma at doses of more than 4 X 10(2) U (0.1 microgram of protein) per mouse. Bacterial growth in the spleens and livers of rMuIFN-gamma-treated neonates was significantly suppressed in comparison with that in the nontreated controls. The infected neonatal mice showed acquired antilisterial resistance against secondary intravenous infection after 4 weeks of age, and this resistance was significantly augmented in mice that had been treated with rMuIFN-gamma.
Production and characterization of monoclonal antibodies against specific serotypes of Mycobacterium avium and the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex
Authors: Kolk A H , Evers R , Groothuis D G , Gilis H , Kuijper S
Published: January 2020

Serotype-specific and Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex (MAIS complex)-specific monoclonal antibodies (MAbs) were prepared. A series of MAbs were obtained, five specific for serotype 2, three specific for serotype 4, eight against a strain of serotype 19, two specific for the MAIS complex, and two against a common glycolipid shared by all the mycobacteria tested so far. The serotype-specific and the MAIS complex-specific MAbs reacted in immunofluorescence with intact mycobacteria and in enzyme-linked immunosorbent assay and immuno-thin-layer chromatography with lipid extracts of mycobacteria. The two MAbs against a common mycobacterial glycolipid reacted only in lipid enzyme-linked immunosorbent assay and immuno-thin-layer chromatography. All MAbs were directed against glycopeptidolipids (GPLs), except for four MAbs against proteins of serotype 19. The serotype- and MAIS complex-specific epitopes on GPLs are exposed on the mycobacterial cell wall, in contrast with the common mycobacterial glycolipid, which is probably located inside the cell wall. The serotype-specific MAbs reacted with native as well as deacetylated GPLs, in contrast with the MAIS complex-specific MAbs, which reacted only with native GPLs. The MAbs will be useful for the identification of MAIS complex and M. avium serotypes 2 and 4 and a strain of serotype 19, GPL analyses with immuno-thin-layer chromatography, and the localization of GPL epitopes in mycobacteria.
Phorbol myristate acetate modulates calcium ion-dependent superoxide anion generation induced by a monoclonal antibody raised against polymorphonuclear leukocytes
Authors: Ichinose Y , Hara N , Ohta M , Motohiro A , Kuda T , Aso H , Yagawa K
Published: January 2020

We used a monoclonal antibody, YI 51, raised against human polymorphonuclear leukocytes (PMN) to induce superoxide anion (O2-) generation in cells. Although YI 51 alone played only a small part in inducing O2- generation in PMN, the amount of O2- generation induced in 5 X 10(5) PMN was 3.7 to 5.5 nmol/min when F(ab')2 fragments of rabbit anti-mouse immunoglobulin antibody were added as a cross-linking agent. This O2- -inducing activity was high compared with that of wheat germ agglutinin (WGA), insoluble immunoglobulin G immune complexes (IC), or phorbol myristate acetate (PMA). The binding of YI 51 and soluble immunoglobulin G IC to PMN was not reciprocally inhibitory, indicating that YI 51 does not interfere with ligand binding to the Fc receptor-binding site. In the absence of calcium ion (Ca2+), O2- generation induced by YI 51 decreased to 10 to 20% of that in the presence of Ca2+. In contrast, O2- generation in response to WGA, IC, or PMA under Ca2+-free conditions was not affected. When PMN were pretreated with low concentrations of PMA (10(-10) to 10(-9) M), the amount of O2- generation by the cells in response to YI 51 in Ca2+-free buffer was enhanced in a concentration-dependent manner. It also equaled the O2- generated by the cells in buffer containing Ca2+. In cells pretreated with PMA, the amount of O2- induced by WGA was enhanced two- to threefold over that in untreated cells. In contrast, there was no augmentation over untreated cells with stimulation by IC. These results suggest that YI 51, IC, and WGA induce O2- generation in human PMN in different manners.
Immunological characterization of papain-induced fragments of Clostridium botulinum type A neurotoxin and interaction of the fragments with brain synaptosomes
Authors: Kozaki S , Miki A , Kamata Y , Ogasawara J , Sakaguchi G
Published: January 2020

After treatment of Clostridium botulinum type A neurotoxin with papain, three fragments (Mrs, 101,000, 45,000, and 43,000) were purified by hydrophobic and ion-exchange chromatography with a high-performance liquid chromatographic system. Immunoblotting analyses with monoclonal antibodies showed that the 101,000-dalton fragment consisted of the light chain and a part of the heavy chain (H-1 fragment) linked together by a disulfide bond, and the other two fragments were correlated to the remaining portion of the heavy chain (H-2 fragment). The 45,000- and 43,000-dalton fragments effectively competed for binding of the 125I-labeled neurotoxin to synaptosomes, while no inhibition was observed with the 101,000-dalton fragment. The results indicate that the H-2 fragment interacts with the binding site on the neural membrane. The binding of the neurotoxin was impaired by treatment of synaptosomes with neuraminidase. Incorporation of gangliosides into neuraminidase-treated synaptosomes resulted in the restoration of binding. The results suggest that gangliosides are one of the components of the toxin-binding site.
Characterization of native and recombinant 75-kilodalton immunogens from Chlamydia trachomatis serovar L2
Authors: Birkelund S , Lundemose A G , Christiansen G
Published: January 2020

A 75-kilodalton (kDa) immunogen from Chlamydia trachomatis serovar L2 was characterized. The 75-kDa protein was localized in the cytoplasm of chlamydiae and was shown to be a protein synthesized early in the developmental cycle of chlamydiae. A gene library was made by the recombinant DNA technique, using the expression vectors pEX1, pEX2, and pEX3. From this library one clone was found which reacted with a monoclonal antibody against the 75-kDa immunogen of C. trachomatis. The 75-kDa protein produced by the recombinant Escherichia coli was expressed independently of the promoter for the hybrid protein cro-betagalactosidase. Thus it is not produced as a fusion protein. Epitope mapping of the 75-kDa protein from C. trachomatis L2 and from the recombinant E. coli performed by Staphylococcus aureus V8 protease digestion showed that the two proteins are identical. Furthermore, patient sera reacted with both proteins.
Somnogenic activity of O-acetylated and dimeric muramyl peptides
Authors: Johannsen L , Rosenthal R S , Martin S A , Cady A B , Obal F , Guinand M , Krueger J M
Published: January 2020

Slow-wave sleep-promoting factors in brain and urine were identified as muramyl peptides (MPs), the building blocks of bacterial cell wall peptidoglycan. In this study, structural variations of MPs that occur naturally in bacterial peptidoglycan were investigated for somnogenic activity. Monomeric and dimeric MPs were isolated and purified from Neisseria gonorrhoeae and Actinomadura sp. strain R39. The structures of these MPs were verified by fast atom bombardment mass spectroscopy and tandem mass spectroscopy. After intracerebroventricular administration of MPs, electroencephalograms and brain temperatures of rabbits were recorded for 6 h and were analyzed to determine durations of slow-wave sleep, rapid-eye-movement sleep, and wakefulness. The 6-O acetylation of muramic acid enhanced the somnogenic effects of certain monomeric MPs relative to their non-O-acetylated (but otherwise identical) counterparts. Two monomeric MPs containing an unsubstituted amide (i.e., Iso-Gln) were inactive, thus confirming previous results showing that amidation of a variety of MPs can block somnogenic activity. Two peptide-cross-linked MP dimers tested had no effect on slow-wave sleep, although a third peptide-cross-linked MP containing a 1,6-anhydro muramyl end on one of its monomeric subunits, a structure that enhances somnogenic potency of un-cross-linked monomers, was somnogenic. Two dimers connected by glycosidic bonds and containing an Iso-Gln moiety were inactive. Two other glycosidically linked dimers that also contained an Iso-Gln moiety, but were of lower molecular weight, were somnogenic. In summary, 6-O acetylation of muramic acid in somnogenic MPs enhances activity, and as a class, peptide-linked dimeric MPs tend to be less active than their constituent monomers.
Clonal polymorphisms of outer membrane protein OspB of Borrelia burgdorferi
Authors: Bundoc V G , Barbour A G
Published: January 2020

The outer membrane protein OspB of Borrelia burgdorferi, the Lyme borreliosis agent, differs in relative molecular weight (Mr) among strains. To determine whether antigenic variation occurs in B. burgdorferi, a cell population of the human isolate HB19 was cloned first by being diluted in broth and then by being plated on agar medium. Several clones were obtained and characterized by polyacrylamide gel electrophoresis, in situ protease treatment, and Western (immunoblot), Southern, and Northern (RNA) blot analyses. Variants featuring OspB proteins that differed in Mrs and in reactivities with monoclonal antibodies were found. One variant made increased amounts of a 21,000-molecular-weight protein (21K protein) in addition to normal amounts of a 33K OspB protein. Another variant did not produce an OspB protein at all but did express an 18.5K protein. Both the 18.5K and 21K proteins were susceptible in situ to trypsin and were bound by a monoclonal antibody directed against the OspB of strain HB19. There were no differences in the Southern and Northern blot analyses of the different variants. The results led to the following conclusions. (i) Clonal polymorphisms in the surface protein OspB occurred in B. burgdorferi. (ii) Hitherto uncharacterized 18.5K and 21K proteins were protease susceptible, antigenically related to OspB, and apparently produced in greater amounts when an OspB either was not produced or was altered in structure. (iii) The OspB variations, including its absence from cells, were not accounted for by major DNA rearrangements or failure of transcription of the ospB gene.
Acylation of the 47-kilodalton major membrane immunogen of Treponema pallidum determines its hydrophobicity
Authors: Chamberlain N R , DeOgny L , Slaughter C , Radolf J D , Norgard M V
Published: January 2020

The 47-kilodalton (kDa) major integral membrane immunogen of Treponema pallidum was recently found to be a proteolipid. Similar two-dimensional electrophoretic mobilities and common hydrophobic properties displayed by the native (T. pallidum) and recombinant (Escherichia coli) 47-kDa antigens suggested that the recombinant antigen also possesses covalently bound lipid. Both intact E. coli and E. coli minicells acylated the 47-kDa antigen; immunoprecipitation with a monoclonal antibody specific for the 47-kDa immunogen supported the contention that the acylated product of E. coli corresponds to the cloned T. pallidum antigen. Triton X-114 phase partitioning was used to compare the relative hydrophobicities of 47-kDa molecules synthesized by in vitro and in vivo protein translation systems. The products synthesized by T. pallidum, intact E. coli, or E. coli minicells were hydrophobic, while the protein synthesized in an E. coli cell-free translation system was hydrophilic. Processing experiments with E. coli suggested that the primary gene translation product of the protein is not synthesized in a precursor form, unlike other bacterial proteolipids. These results indicate that the hydrophobicity of the 47-kDa integral membrane protein is conferred substantially by the covalently attached lipid(s). The biochemical similarities between the native and recombinant 47-kDa proteolipids will provide a foundation for future investigations into the structure and immunogenicity of this integral membrane protein of T. pallidum.
Selective binding of Trypanosoma cruzi to host cell membrane polypeptides
Authors: Davis C D , Kuhn R E
Published: January 2020

An adaptation of the immunoblotting technique was used to investigate binding interactions between Trypanosoma cruzi and mammalian host cells at the molecular level. A specific binding interaction was observed between T. cruzi and two host cell membrane polypeptides with molecular masses of approximately 32 and 34 kilodaltons. This molecular interaction was observed with antigen extracts of T. cruzi and with live, infective trypomastigote stages of the parasite, suggesting that the observed phenomenon may have relevance to the initial attachment of the parasite to the host cell membrane before invasion.