bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of two germ-tube-specific cell wall antigens of Candida albicans
Authors: Ponton J , Jones J M
Published: January 2020

Outer cell wall layers of intact yeast- and mycelial-phase Candida albicans B311 were extracted with dithiothreitol. Antisera against mycelial-phase organisms were absorbed with yeast-phase organisms or yeast-phase extract and used to stain Western blots of sodium dodecyl sulfate-polyacrylamide gels loaded with yeast- and mycelial-phase extracts. Autoradiography of gels loaded with extracts from organisms surface labeled with 125I was used to detect surface antigens containing proteins. Antigen bands of interest identified in Western blots were cut from the blots and used to immunize rabbits. Two antigens were identified in the mycelial-phase extract which were not present in the yeast-phase extract. The first was a 19-kilodalton protein that was present in the cell walls of germ tubes but was not expressed on their surfaces. The second was a polysaccharide-rich high-molecular-weight antigen which was expressed on the surface of the germ tube. Treatment of mycelial-phase extract with protease and endo-beta-N-acetylglucosaminidase H demonstrated that this antigen was composed of polysaccharides linked through di-N-acetylchitobiose groups to proteins.
Granuloma formation and hemopoiesis induced by C36-48-mycolic acid-containing glycolipids from Nocardia rubra
Authors: Kaneda K , Sumi Y , Kurano F , Kato Y , Yano I
Published: January 2020

As previously reported (I. Yano, I. Tomiyasu, S. Kitabatake, and K. Kaneda, Acta Leprologica 2:341-349, 1984), Nocardia rubra, one of the nonpathogenic actinomycetes, possesses three classes of mycolic acid-containing glycolipid, i.e., glucose mycolate, trehalose dimycolate, and trehalose monomycolate. The carbon chain length of their mycolic acids is shorter (C36-48) than that in mycobacteria (longer than C70), and the glycolipid consists of only alpha-mycolic acid. One intravenous administration of 500 micrograms of each purified glycolipid to ICR mice in the form of water-in-oil-in-water emulsion without any protein antigens caused prominent granuloma formation in the lungs, spleen, and liver. The lung index in the treated mice was about 3.5 times larger than that in the control mice (given water-in-oil-in-water emulsion only) at 1 week after the injection and then rapidly declined, while spleen and liver indices peaked at 2 weeks after the injection and persisted longer. The granuloma consisted of macrophages, some of which phagocytized glycolipid micelles, lymphocytes, monocytes, and neutrophils. In addition, many small hemopoietic islands were observed in the liver sinusoids, where various immature blood cells were trapped by the prominent cytoplasmic projections of Kupffer cells. The granuloma formation and hemopoiesis observed here are considered to be the most characteristic morphological expression of macrophage activation in these organs. This is the first report to show that such histological changes can be induced by chemically defined and homogeneous mycolic acid-containing glycolipids other than those of mycobacteria.
Stability of expression of Neisseria gonorrhoeae lipooligosaccharides
Authors: Schneider H , Hammack C A , Shuman B A , Griffiss J M
Published: January 2020

We compared multiple lipooligosaccharide (LOS) extracts from individual strains of Neisseria gonorrhoeae. Each of the extracts was prepared from single mass cultures grown on solid media under similar conditions but separated by time. We found only subtle variations in the number, electrophoretic mobility, and concentration of components of the LOSs from individual strains. We found no variation in the expression of a 3.6-kilodalton LOS component that carries the L8 LOS epitope. A significant variation in the 3-deoxy-D-manno-octulosonic acid content was found among different extracts from the same strain, but this variation appeared to be unrelated to the other LOS characteristics studied.
Molecular basis for the pathological actions of Clostridium perfringens iota toxin
Authors: Simpson L L , Stiles B G , Zepeda H H , Wilkins T D
Published: January 2020

Clostridium perfringens type E iota toxin is composed of two separate and independent polypeptide chains that act synergistically in mouse lethal assays. The light chain is an enzyme that mono(ADP-ribosyl)ates certain amino acids. The enzyme displays substantial activity when homopoly-L-arginine is used as a substrate, but it shows little activity when polyasparagine, polylysine or polyglutamic acid are used. In keeping with the properties of an ADP-ribosylating enzyme, the toxin possesses the following characteristics. It produces incorporation of radioactivity into polyarginine when adenine-labeled NAD is used, but radioactivity is not incorporated when nicotinamide-labeled NAD is used. Irrespective of labeling, enzymatic activity is accompanied by the release of free nicotinamide. After incorporation of ADP-ribose groups into polyarginine, enzymatic and chemical techniques can be used to release the incorporated material. Snake venom phosphodiesterase releases mainly AMP; hydroxylamine releases AMP and ADP-ribose. The heavy chain of iota toxin has little or no enzyme activity, and it does not substantially affect the enzyme activity of the light chain. The heavy chain may be a binding component that directs the toxin to vulnerable cells. The data suggest that iota toxin is a representative of a novel class of ADP-ribosylating toxins.
Effects of oxygen on glucose-limited growth of Streptococcus mutans
Authors: Kodama T , Fukui K , Shimamoto T , Ohta H , Kokeguchi S , Kato K
Published: January 2020

Aerotolerance in the growth of Streptococcus mutans and related streptococci was examined under glucose-limited conditions. The growth rate of all strains tested was more or less retarded when they were transferred from anaerobic to aerobic conditions. As for growth yield, however, some strains (group 1) showed reduced values for the increment of cellular dry weight change (in grams per mole of glucose), whereas others showed either unaltered (group 2) or increased (group 3) yields. The characteristic feature of strains in groups 2 and 3 was their high activity of both glucose- and pyruvate-dependent oxygen uptake when strains were grown under aerobic conditions. By contrast, the activity of pyruvate-dependent oxygen uptake by group 1 strains was negligibly low, whether grown under aerobic or anaerobic conditions. Some strains (group 1a) consumed oxygen in the presence of glucose at a much faster rate than others did (group 1b). There seems to be a good correspondence of these aerotolerance groupings to those based on serotypes of S. mutans. Thus, the groups 1a, 1b, 2, and 3 correspond to serotypes g, a+d, c+f, and b, respectively.
Sialic acid of group B Neisseria meningitidis regulates alternative complement pathway activation
Authors: Jarvis G A , Vedros N A
Published: January 2020

The effect of meningococcal cell-associated sialic acid on activation of the human alternative complement pathway was examined by using a quantitative fluorescence immunoassay to assess alternative pathway-mediated C3 binding to a group B strain of Neisseria meningitidis from which graded amounts of sialic acid had been removed with neuraminidase. Using human serum absorbed with strain B16B6 (B:2a:L2,3) and chelated with 10 mM MgCl2 and 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, we found an increase in the amount of C3 bound by enzymatically desialylated B16B6 organisms over the amount bound by fully sialylated organisms. This increase was proportional to the amount of sialic acid cleaved from the bacteria. Enhanced C3 binding was accompanied by an increase in factor B deposition. A sialic acid-deficient mutant of strain B16B6, designated 2T4-1, bound C3 via the alternative pathway at a level equivalent to that bound by wild-type meningococci from which 88% of the sialic acid had been removed. Strain B16B6 was resistant to the alternative pathway-mediated bactericidal activity of both absorbed and hypogammaglobulinemic human sera, whereas noncapsular variant 2T4-1 was sensitive to these sera. The addition of purified immune immunoglobulin M (IgM) and IgG significantly increased the alternative pathway-mediated killing of strain B16B6 organisms. IgM mediated increased bactericidal activity without an increase in C3 or factor B deposition. In contrast, the IgG-mediated killing was associated with increased binding of C3 and factor B to the organisms. Absorption studies showed that the IgM bound to the sialic acid capsule, whereas the IgG bound to noncapsular surface antigens. We conclude from these results that the group B meningococcal sialic acid capsule inhibits activation of the alternative pathway in the nonimmune host and that both IgM and IgG, although specific for different surface antigens, are capable of augmenting the alternative pathway-mediated killing of group B meningococci.
Glycoprotein recognition mediates attachment of Plasmodium chabaudi to mouse erythrocytes
Authors: Rodriguez M H , Jungery M
Published: January 2020

The interaction between Plasmodium falciparum merozoites and human erythrocytes is mediated by specific parasite proteins and sialoglycoproteins (SGPs) on the surface of the host cell. To investigate whether a similar mechanism functions in rodent malaria, a series of experiments was performed to identify the proteins involved in the interaction of Plasmodium chabaudi parasites and mouse erythrocytes. Labeled parasite proteins incubated with purified mouse SGP bound specifically to glycoprotein 2.1. Two parasite proteins (72 and 126 kilodaltons [kDa]) were coprecipitated with antibody directed to mouse erythrocyte membrane proteins. The lower band (72 kDa) as well as a band of 105 kDa were also observed to bind to N-acetyl-D-galactosamine affinity columns, suggesting a carbohydrate component in the binding of these parasites to erythrocytes. These experiments indicate that P. chabaudi possesses specific proteins which recognized SGP on the surface of murine erythrocytes in a manner similar to that of the merozoites of P. falciparum. Thus P. chabaudi in mice may provide an in vivo model of the human parasite for testing ways to inhibit merozoite recognition and invasion of host cells.
Requirement of a properly acylated beta(1-6)-D-glucosamine disaccharide bisphosphate structure for efficient manifestation of full endotoxic and associated bioactivities of lipid A
Authors: Takahashi I , Kotani S , Takada H , Tsujimoto M , Ogawa T , Shiba T , Kusumoto S , Yamamoto M , Hasegawa A , Kiso M
Published: January 2020

Several synthetic acylated glucosamine monophosphates, with structures corresponding to the nonreducing or reducing moiety of the lipid A of the Escherichia coli or Salmonella minnesota type, and a synthetic compound corresponding to a biosynthetic disaccharide lipid A precursor (designated Ia or IVA) were examined for their endotoxic and related bioactivities in comparison with those of the synthetic and bacterial parent molecules, i.e., acylated beta(1-6)-D-glucosamine disaccharide bisphosphates. Some of the test monosaccharide compounds were definitely active in most of the in vitro assays. Their activities, except for complement activation, however, were weaker than those of the reference compounds, synthetic and bacterial acylated disaccharide bisphosphates. The differences between the test monosaccharide and disaccharide compounds were much more apparent in in vivo assays, in which the test acylated glucosamine monophosphates were scarcely active, though some test compounds exhibited weak lethal toxicity in galactosamine-loaded mice and were weakly active in pyrogenicity, immunoadjuvant activity, and possible tumor necrosis factor and alpha and beta interferon-inducing ability in Mycobacterium bovis BCG- and Propionibacterium acnes-primed mice, respectively. Mixture at an equimolar ratio of acyl glucosamine monophosphates, each of which has the structure of the reducing or nonreducing moiety of the reference disaccharide compound, did not restore the endotoxic or associated bioactivities of the corresponding parent molecules. No essential differences in bioactivity were noted between synthetic and bacterial monosaccharide compounds, i.e., lipid X, whose structure corresponds to the reducing moiety of E. coli-type lipid A.
Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa
Authors: Knutton S , Lloyd D R , McNeish A S
Published: January 2020

The adhesion of classic enteropathogenic Escherichia coli (EPEC) strains of human origin to isolated human small intestinal enterocytes and cultured small intestinal mucosa was investigated. An adhesion assay with isolated human enterocytes prepared from duodenal biopsy samples was developed and tested with EPEC strains known to cause diarrhea in healthy adult volunteers. In the assay a mean of 53 and 55% of enterocytes had brush border-adherent E. coli E2348 (O127;H6) and E851 (O142:H6), respectively, whereas the value for a nonpathogenic control strain and a plasmid-cured derivative of strain E2348 was 0%. A collection of 17 EPEC strains was also tested for the ability to colonize cultured human duodenal mucosa. Extensive colonization occurred with 13 strains, including serogroups O55, O86, O111, O114, O119, O127, O128, and O142; and in each case electron microscopic examination of colonized mucosa revealed the characteristic histopathological lesion reported by others in natural and experimental EPEC infections. EPEC strains were seen to adhere intimately to the enterocyte surface, causing localized destruction of microvilli. The plasmid-cured derivative of strain E2348, which colonized cultured mucosa much less efficiently than the parent strain, nevertheless produced an identical lesion, indicating that plasmid-encoded factors are not essential for adhesion and the brush border-damaging property of EPEC.
Primary murine Chlamydia trachomatis pneumonia in B-cell-deficient mice
Authors: Williams D M , Grubbs B , Schachter J
Published: January 2020

Mice were rendered deficient in B-cell activity by treatment with anti-mu antibody from birth. These animals were then infected intranasally with murine Chlamydia trachomatis (murine pneumonitis agent [MoPn]). They produced neither local nor systemic antibody to MoPn but had intact delayed-type hypersensitivity to MoPn. Anti-mu-treated mice were not significantly more susceptible to primary invasive infection with MoPn than were control mice, and unrestricted multiplication with MoPn did not occur. The dominant immune response controlling this type of infection is not likely to be antibody.
Characterization of an immunoprotective protein complex of Anaplasma marginale by cloning and expression of the gene coding for polypeptide Am105L
Authors: Barbet A F , Palmer G H , Myler P J , McGuire T C
Published: January 2020

Immunization with an Anaplasma marginale surface protein complex containing two polypeptides (Am105U and Am105L), each having a molecular weight of 105,000, protected cattle against challenge with virulent organisms. These polypeptides were immunoprecipitated together from detergent extracts of A. marginale by a neutralizing monoclonal antibody. After surface radioiodination of intact parasites, both Am105U and Am105L contained the radiolabel. To define the structural and antigenic relationships between Am105U and Am105L and to determine individual efficacies as protective immunogens, we cloned and expressed A. marginale DNA in Escherichia coli. We identified recombinant bacteria which expressed a novel protein of 105,000 molecular weight as a major cellular component. The recombinant protein was structurally and antigenically homologous to Am105L. There were multiple, partially homologous copies of the cloned DNA sequence in the rickettsial genome.
Susceptibility of pathogenic and nonpathogenic Naegleria spp. to complement-mediated lysis
Authors: Whiteman L Y , Marciano-Cabral F
Published: January 2020

The susceptibility of four species of Naegleria amoebae to complement-mediated lysis was determined. The amoebicidal activity of normal human serum (NHS) and normal guinea pig serum (NGPS) for Naegleria amoebae was measured by an in vitro cytotoxicity assay. Release of radioactivity from amoebae labeled with [3H]uridine and visual observation with a compound microscope were used as indices of lysis. Highly pathogenic mouse-passaged N. fowleri was less susceptible to the lytic effects of NHS and NGPS than the weakly pathogenic, axenically grown N. fowleri or N. australiensis and the nonpathogenic amoebae N. gruberi and N. lovaniensis. However, both pathogenic and nonpathogenic Naegleria spp. depleted complement as assessed by total hemolytic activity. Treatment of serum with EDTA, heat (56 degrees C, 30 min), cobra venom factor, or antibody to C3 or C9 complement components decreased the amoebicidal activity of NHS. The presence of specific agglutinating antibody to N. fowleri enhanced the amoebicidal activity of NGPS for N. fowleri.
Pregnancy and humoral immune response in mice chronically infected by Trypanosoma cruzi
Authors: Carlier Y , Rivera M T , Truyens C , Goldman M , Lambert P , Flament J , Bauwens D , Vray B
Published: January 2020

The effect of pregnancy on the humoral immune response induced by Trypanosoma cruzi was studied in groups of chronically infected and pregnant mice (IP) or chronically infected and nonpregnant mice (INP) of strain BALB/c. Groups of noninfected and nonpregnant mice (NINP) or noninfected and pregnant mice (NIP) served as controls. The pregnant mice were killed on day 17 of pregnancy. Anti-T. cruzi immunoglobulin G (IgG) and IgM antibodies, detected by immunofluorescence or enzyme-linked immunosorbent assay or both, underwent a pregnancy-associated decrease of 20 to 40%, whereas complement-mediated lytic antibodies were unaffected by pregnancy. Immunoblotting analysis indicated identical specificities of the anti-T. cruzi antibodies in IP and INP groups. The levels of all the immunoglobulin isotypes (particularly IgG2a and IgG3), circulating immune complexes, rheumatoid-like factor, and anti-DNA antibodies were considerably increased during chronic infection (NINP versus INP), which could be related to the high degree of polyclonal B-cell activation occurring in T. cruzi infection. However, pregnancy significantly decreased (by 20 to 60%) such parameters. IgG levels were particularly affected (by 40 to 60%), and the decreases could be ordered as follows: IgG3 greater than IgG2a greater than IgG1 greater than IgG2b for IP versus INP. Comparisons between the noninfected groups indicated differences only in IgG levels. These results indicate the following. (i) The specific humoral anti-T. cruzi immune response is weakly affected by pregnancy, which is not sufficient to modify the course of the mother's infection. (ii) Pregnancy does not modify the expression of the anti-T. cruzi antibody repertory. (iii) Pregnancy reduces the polyclonal B-cell activation, particularly the levels of the IgG isotypes undergoing the greatest activation.
Immunization of mice against tetanus with fragments of tetanus toxin synthesized in Escherichia coli
Authors: Fairweather N F , Lyness V A , Maskell D J
Published: January 2020

Two recombinant plasmids, pTet11 and pTet18, which express nontoxic protein fragments of tetanus toxin in Escherichia coli, were constructed. pTet11 protein (86 kilodaltons) is a fusion between part of the E. coli trpE protein and 441 amino acids of tetanus fragment C, and pTet18 (63 kilodaltons) consists of part of fragment B and all of fragment C of tetanus toxin. The synthesis of these proteins was induced in E. coli cultures, and the proteins were partially purified. Mice were immunized with these proteins, and dose-dependent titers of anti-tetanus toxoid antibodies were obtained. The proteins were able to induce neutralizing antibodies in mice, as demonstrated by the ability of mice immunized with 1 microgram or more of protein to survive challenge with 10 50% lethal doses of tetanus toxin.
In vivo colonization of the mouse large intestine and in vitro penetration of intestinal mucus by an avirulent smooth strain of Salmonella typhimurium and its lipopolysaccharide-deficient mutant
Authors: Nevola J J , Laux D C , Cohen P S
Published: January 2020

The relative abilities of an avirulent Salmonella typhimurium strain with wild-type lipopolysaccharide (LPS) character, SL5319, and a nearly isogenic LPS-deficient mutant, SL5325, to colonize the large intestines of streptomycin-treated CD-1 mice in vivo and to penetrate colonic mucus in vitro were studied. Previously it had been shown that, when fed simultaneously to streptomycin-treated mice (approximately 10(10) CFU each), the S. typhimurium strain with wild-type LPS colonized at 10(8) CFU/g of feces indefinitely, whereas the LPS-deficient mutant dropped within 3 days to a level of only 10(4) CFU/g of feces. In the present investigation, when SL5325 was allowed to colonize for 8 days before feeding mice SL5319 or when it was fed to mice simultaneously with an Escherichia coli strain of human fecal origin (10(10) CFU each), both strains colonized indefinitely at 10(7) CFU/g of feces. Moreover, when the wild-type and LPS-deficient mutant strains were fed to mice simultaneously in low numbers (approximately 10(5) CFU each) the strains survived equally well in the large intestines for 8 days, after which the LPS-deficient mutant was eliminated (less than 10(2) CFU/g of feces), whereas the wild-type colonized at a level of 10(7) CFU/g of feces. In addition although both strains were able to adhere to mucus and epithelial cell preparations in vitro, the wild-type strain was shown to have greater motility and chemotactic activity on CD-1 mouse colonic mucus in vitro and to more rapidly penetrate and form a stable association with immobilized colonic mucosal components in vitro. Based on these data, we suggest that the ability of an S. typhimurium strain to colonize the streptomycin-treated mouse large intestine may, in part, depend on its ability to penetrate deeply into the mucus layer on the intestinal wall and subsequently, through growth, colonize the mucosa.
A potassium thiocyanate extract vaccine prepared from Pasteurella multocida 3:A protects rabbits against homologous challenge
Authors: Lu Y S , Pakes S P , Massey L , Stefanu C
Published: January 2020

Potassium thiocyanate extracts of a virulent Pasteurella multocida 3:A rabbit isolate were prepared and used as a vaccine in rabbits. The extract contained protein, carbohydrate, hyaluronic acid, lipopolysaccharide, DNA, and RNA. The protein and lipopolysaccharide profiles of the extract were similar to those of the P. multocida cell membrane. Rabbits were vaccinated intranasally (i.n.) or intramuscularly (i.m.) four times at 1- or 3-week intervals and challenged i.n. with the homologous P. multocida 2 weeks after the last vaccination. Rabbits vaccinated with the extract by the i.n. route developed persisting serum immunoglobulin G (IgG) and nasal IgA antibodies, whereas rabbits immunized by the i.m. route produced persisting serum IgG and transient nasal IgA antibodies. The extract prevents the death of rabbits which were vaccinated by either route and challenged. Vaccination by the i.n. route in rabbits reduced the numbers of virulent P. multocida in nasal cavities and lungs and the prevalence and severity of rhinitis and pneumonia. These i.n.-vaccinated rabbits were also resistant to virulent P. multocida colonization in liver, spleen, uterus, and tympanic bullae. Similarly, i.m. vaccination in rabbits resulted in a reduction in the severity of rhinitis; the numbers of virulent P. multocida in lungs; and the prevalence of colonization in liver, spleen, uterus, and tympanic bullae. Vaccination by the i.n. route was superior to that by the i.m. route in that there was a significant reduction in the severity of pneumonia and numbers of virulent P. multocida in nasal cavities and lungs. Rabbits vaccinated with the extract without challenge showed no lesions.
Cross-reactivity of surface-exposed epitopes of outer membrane antigens of Haemophilus influenzae type b
Authors: Loeb M R , Woodin K A
Published: January 2020

The cross-reactivity of exposed surface epitopes of outer membrane proteins from a spectrum of Haemophilus influenzae type b isolates that varied in their evolutionary distance from each other and in their outer membrane protein composition was analyzed by using an immunoblot assay. The results for outer membrane proteins a, n, and b/c were as follows. (i) A total of 13 of 14 strains possessing a protein a with similar mobilities on gels (i.e., the same apparent molecular weight) as protein a of strain Eag absorbed antibodies to protein a of strain Eag. These strains represented a broad spectrum on a scale of evolutionary distance. (ii) In contrast, only one of seven strains possessing a protein a with different mobilities absorbed these antibodies. (iii) Of five isolates close to strain Eag on the evolutionary scale, the four with a protein n with the same mobility as protein n of strain Eag absorbed antibodies to protein n of strain Eag. (iv) In contrast, of five isolates distant from strain Eag on the evolutionary scale, none absorbed antibodies to protein n, including one strain that had a protein n of the same mobility as that of strain Eag. (v) All strains that absorbed antibodies to protein b/c also absorbed antibodies to lipopolysaccharide, and the reverse of this was also true. Evolutionary distance and mobility of protein b/c on gels were not factors. Control experiments indicated that this result was an artifact due to the strong association of lipopolysaccharide with protein b/c on the gel and subsequent blot. The important conclusions from these experiments, especially pertinent for consideration of these proteins in either whole or peptide vaccines, are that proteins with apparently identical molecular weights can possess different surface-exposed epitopes, that proteins with different molecular weights can possess cross-reactive surface-exposed epitopes, and that some surface-exposed epitopes have been conserved even though the bacterium has undergone evolutionary divergence. In addition, experiments were also performed to determine whether H. influenzae type b strains maintained their integrity during the absorption step, i.e., incubation in antiserum. Strain Eag, which was used as a prototype type b strain, released a small proportion of its membrane (0.13%), but this did not result in exposure of epitopes that were usually buried. In contrast, strain S2, an unencapsulated mutant of strain Eag, was quite unstable, releasing three times as much membrane and a large proportion of its periplasmic proteins.
Cloning and sequence analysis of cytadhesin P1 gene from Mycoplasma pneumoniae
Authors: Su C J , Tryon V V , Baseman J B
Published: January 2020

Mycoplasma pneumoniae cytadhesin P1 was purified by monoclonal antibody affinity chromatography followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal 18-amino-acid sequence of P1 was determined and used to design two synthetic oligonucleotides, a 14-mer corresponding to amino acids 1 to 5 and an 18-mer corresponding to amino acids 7 to 12. These oligonucleotides served as hybridization probes for the identification of the P1 gene by Southern blot analysis of M. pneumoniae DNA. The P1 gene was cloned into plasmid pUC19 and mapped by using appropriate restriction endonucleases. The DNA sequence of the entire P1 gene was determined by subcloning appropriate DNA fragments into bacteriophage M13 and sequencing the DNA by the dideoxy-chain-termination method. The P1 gene contains an open reading frame of 4,881 nucleotides coding for a protein of 1,627 amino acids with a calculated molecular weight of 176,288. Properties of the amino-terminal sequence suggest that protein P1 may be synthesized as a precursor with subsequent processing to a mature protein of a calculated molecular weight of 169,758. Potential antigenic sites were determined by hydrophilicity plots. A computer search revealed that part of the predicted P1 sequence is homologous to cytoskeletal keratin of mammalian species and human fibrinogen alpha chain precursor. These results demonstrate the uniqueness of P1 as a cytadhesin and virulence determinant.
Molecular analysis of the M protein of Streptococcus equi and cloning and expression of the M protein gene in Escherichia coli
Authors: Galán J E , Timoney J F
Published: January 2020

A Streptococcus equi gene bank was constructed in the bacteriophage lambda gt11 cloning vector, and hybrid phage plaques were screened with S. equi M protein antiserum. A hybrid phage expressing the S. equi M protein (lambda gt11/SEM7) was identified and lysogenized into Escherichia coli Y1089. The cloned M protein appeared in immunoblots as three polypeptides with relative molecular weights of 58,000, 53,000, and 50,000. When reacted with S. equi M protein antiserum in an agar double-diffusion assay, the cloned M protein formed a line of identity with a protein in an acid extract of S. equi. Furthermore, lambda gt11/SEM7 protein inhibited opsonization of S. equi by antiserum to S. equi M protein. In addition, the recombinant protein expressed determinants of the antigen in the immune complexes of purpura hemorrhagica. Native M protein obtained from S. equi and recombinant M protein showed very similar molecular weight distributions on immunoblots, appearing as multiple closely spaced bands with molecular weights ranging from 52,000 to 60,000. Antisera prepared separately against each of the acid-extracted polypeptides shown to be important in serum bactericidal responses (molecular weight, 29,000) and nasopharyngeal local antibody responses (molecular weights, 41,000 and 46,000) of the horse each reacted with all three polypeptides in an acid extract. Moreover, antisera against protoplasts and against recombinant M protein of S. equi also reacted with these polypeptides. These results suggest that the entire M protein molecule of S. equi is present in these preparations and that the fragments in acid extracts carry overlapping segments.
Ploidy and DNA content of Candida stellatoidea cells
Authors: Kwon-Chung K J , Wickes B L , Whelan W L
Published: January 2020

Three isolates of Candida stellatoidea contained approximately the same amount of DNA per blastospore (38.3 to 41.9 fg) as did a known diploid isolate of Candida albicans and about twice as much as did a haploid isolate of Saccharomyces cerevisiae. The diploidy of C. stellatoidea was supported by demonstration of mitotic segregation of an ade marker.
Cooperative action of complement component C3 and phagocytic effector cells in innate murine resistance to Trypanosoma lewisi
Authors: Desai B B , Albright J W , Albright J F
Published: January 2020

Mice display strong natural resistance to the rat-specific Trypanosoma lewisi. Intravenously injected intact T. lewisi parasites were eliminated by mice within 18 to 24 h. In comparison, "nude" T. lewisi organisms lacking the surface coat were rapidly and totally cleared from the bloodstream within 2 h postinoculation. Similarly, in vitro-cultivated trypanosomes were readily eliminated by mice with the conspicuous absence of a lag phase. Elimination of nude T. lewisi, like that of intact trypanosomes, required murine complement component C3. Splenectomy of mice did not affect their ability to eliminate T. lewisi. However, C3 depletion with cobra venom factor rendered splenectomized mice susceptible to this rat trypanosome; in these mice, T. lewisi established prolonged and frequently fatal infections. Beige mice were able to efficiently eliminate T. lewisi. But combined treatments of beige (bg/bg) and heterozygous (bg/+) mice with cobra venom factor and silica dust, or normal rat serum and silica dust, incapacitated the natural resistance of these mice to T. lewisi. Such combined treatments of beige and control mice resulted in fulminating parasitemias and death of the animals. Altogether, the results of the present studies indicate that T. lewisi elimination by mice requires the following: exposure of C3 acceptors on the surface of the parasites; activation of murine C3, probably via the alternative complement pathway; and destruction of the C3b-coated parasites by their interaction with C3b receptor-bearing, phagocytic effector cells that are abundant in the spleen and sensitive to silica dust.
Use of parasite antigens and interleukin-2 to enhance suppressed immune responses during Trypanosoma cruzi infection in mice
Authors: Choromanski L , Kuhn R E
Published: January 2020

Mice infected with Trypanosoma cruzi exhibit an early and profound suppression of parasite-specific and nonspecific immune responses. Earlier studies have shown that this suppression is due, at least in part, to suppressor macrophages, deficiency in production of interleukin-2 (IL-2), and reduced T helper (Th)-cell activity. In the present study, the effect of exogenously supplied IL-2 on enhancement of parasite-specific Th-cell activity, anti-parasite immunoglobulin G (IgG) and IgM antibody levels, parasitemia, and longevity was examined in infected mice. The results showed that administration of IL-2 with and without antigenic stimulation with trinitrophenylated T. cruzi significantly enhanced parasite-specific IgM and IgG levels. Injection of IL-2 and trinitrophenylated T. cruzi together significantly enhanced parasite-specific Th-cell activity and was more effective in enhancement of parasite-specific antibody levels. In addition, it was found that IL-2 alone had a rapid and lasting effect in reducing parasitemia. These results suggest that deficiency in IL-2 may play a major role in host susceptibility to T. cruzi.
Binding of Actinomyces naeslundii to glycosphingolipids
Authors: Brennan M J , Joralmon R A , Cisar J O , Sandberg A L
Published: January 2020

The type 2 fimbrial lectin of Actinomyces naeslundii WVU45 mediated the binding of this bacterium to glycosphingolipids chromatographed on thin-layer silica gel plates. Radioiodinated bacteria attached to GM1, GD1b, and globoside. After chromatograms were treated with sialidase, the bacteria also bound to GD1a and GT1b. The actinomyces lectin apparently recognized the Gal beta 3GalNAc termini of these gangliosides and the GalNAc beta 3Gal terminus of globoside, suggesting that glycolipids containing these sequences may serve as receptors for A. naeslundii on mammalian cells.
Production and characterization of serovar-specific monoclonal antibodies to serovars 4, 8, and 9 of Mycobacterium intracellulare
Authors: Nishimori K , Yugi H , Naiki M , Sugimura T , Tanaka Y , Nonomura I , Yokomizo Y , Kubo S
Published: January 2020

Serovar-specific monoclonal antibodies against Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex serovars 4, 8, and 9 were prepared. Nine, four, and one monoclonal antibodies, respectively, to the serovars were prepared by the usual cell fusion technique. All nine monoclonal antibodies to serovar 4 were monospecific for their homologous serovar and reacted with several native glycopeptidolipids (GPLs) and one major deacylated GPL from the homologous serovar. One of the four monoclonal antibodies to serovar 8 seemed to be monospecific for its homologous serovar, but the other cross-reacted with serovar 6 because serovar 6 organisms contain the same components as does the major deacylated GPL from serovar 8. One monoclonal antibody to serovar 9 was monospecific for its homologous serovar and reacted with one of the two major deacylated GPLs from this serovar. These antibody preparations proved useful for serovar identification.
Production, characterization, and antibody specificity of a mouse monoclonal antibody reactive with Cryptococcus neoformans capsular polysaccharide
Authors: Dromer F , Salamero J , Contrepois A , Carbon C , Yeni P
Published: January 2020

Two monoclonal immunoglobulin G1 antibodies reacting with Cryptococcus neoformans capsular polysaccharide (CNPS) were produced in mice by using a carefully defined procedure for immunization with unmodified CNPS purified from C. neoformans serotype A. Since the antibodies were found to have the same pattern of specificity, only one of them (E1) is described. This anti-CNPS monoclonal antibody reacted with the glucuronoxylomannan component of CNPS but not with the constituent monosaccharides or with the mannose alpha(1----3)-linked oligosaccharide structures present on CNPS. E1 appeared to be specific for C. neoformans serotype A by agglutination of whole cells; it was specific for soluble CNPS A by gel immunoprecipitation. However, indirect immunofluorescence and competitive-binding enzyme-linked immunosorbent assay experiments showed low levels of cross-reactivity with serotypes B and D but not with serotype C. Concentrations 10,000 times higher for serotypes B and D cells than for serotype A cells were required for a 50% inhibition of E1 anti-CNPS A activity as measured by enzyme-linked immunosorbent assay. Among the other yeasts tested, a cross-reaction was only detected with Trichosporon beigelii. The four serotypes of C. neoformans could be distinguished based on intensities and patterns of fluorescence in an indirect immunofluorescence assay using the monoclonal anti-CNPS A antibody. Monoclonal anti-CNPS A antibodies could be useful for fundamental studies on the glucuronoxylomannan structure, as well as for clinical applications such as serotyping and possibly the serological diagnosis of cryptococcosis.
Mechanism of activation of human basophils by Staphylococcus aureus Cowan 1
Authors: Marone G , Tamburini M , Giudizi M G , Biagiotti R , Almerigogna F , Romagnani S
Published: January 2020

We investigated the capacity of Staphylococcus aureus Cowan 1 and S. aureus Wood 46 to induce histamine release from human basophils in vitro. S. aureus Cowan 1 (10(5) to 10(7)/ml), which synthesizes protein A (Staph A), stimulated the release of histamine from basophils, whereas S. aureus Wood 46 (10(5) to 2 X 10(7)/ml), which does not synthesize Staph A, did not induce histamine secretion. Soluble Staph A (10(-3) to 10 micrograms/ml), but not staphylococcal enterotoxin A, induced histamine secretion from human basophils. Staph A binds through its classical site to the Fc region of human immunoglobulin G (IgG) and through its alternative site to the Fab portion of the different human immunoglobulins. Hyperiodination of Staph A, which destroys over 90% of the original Fc reactivity without altering the Fab-binding site, did not alter the ability of the protein to induce histamine release. The stimulating effect of Staph A was dose dependently inhibited by preincubation with human polyclonal IgG (0.3 to 100 micrograms/ml) and a human monoclonal IgM (0.3 to 100 micrograms/ml) which have F(ab')-Staph A reactivity. In contrast, rabbit IgG, which possesses only Fc-Staph A reactivity, and a Staph A-unreactive human monoclonal IgM did not inhibit Staph A activity. Similar results were obtained with intact S. aureus Cowan 1. Preincubation with either Staph A or anti-IgE (rabbit anti-Fc epsilon) resulted in complete desensitization to a subsequent challenge with the homologous stimulus. Staph A and anti-IgE induced partial cross-densensitization to the heterologous stimulus. Cells preincubated with anti-IgG (rabbit anti-Fc gamma) lost a small but significant part of their ability to release with Staph A but did not lose their response to anti-IgE. Basophils from which IgE had been dissociated by brief exposure to lactic acid no longer released histamine in response to anti-IgE and Staph A. When basophils from which IgE had been dissociated were incubated with human polyclonal IgE, they regained their ability to induce histamine in response to Staph A and anti-IgE. In contrast, two monoclonal IgEs which do not bind to Staph A did not restore the basophil responsiveness to Staph A. Furthermore, there was complete cross-desensitization between soluble Staph A and S. aureus Cowan 1, while cells desensitized to S. aureus Wood 46 released normally with Staph A and S. aureus Cowan 1.(ABSTRACT TRUNCATED AT 400 WORDS)
Protective immunity induced by outer membrane proteins of Salmonella typhimurium in mice
Authors: Udhayakumar V , Muthukkaruppan V R
Published: January 2020

Outer membrane proteins (OMP) extracted from both smooth (C5) and rough (Rb2) strains of Salmonella typhimurium were able to induce protective immunity to salmonellosis. The OMP-induced protection lasted for at least 6 months. The antibody level was estimated by passive hemagglutination. In the C5 OMP-immunized mice, antibodies to both proteins and lipopolysaccharide were detected. On the other hand, in the Rb2 OMP-immunized mice, antiprotein but not antilipopolysaccharide antibodies were detected. Delayed-type hypersensitivity appeared as early as the second week after immunization with OMP and persisted through the fourth week.
Characterization of antibody-reactive epitopes on the 65-kilodalton protein of Mycobacterium leprae
Authors: Buchanan T M , Nomaguchi H , Anderson D C , Young R A , Gillis T P , Britton W J , Ivanyi J , Kolk A H , Closs O , Bloom B R
Published: January 2020

Twenty-three monoclonal antibodies (MAbs) prepared in seven different laboratories were studied, all of which recognized the 65-kilodalton (kDa) protein of Mycobacterium leprae as determined by Western blotting or gel radioimmunoassay or both. Fourteen of the MAbs recognized different epitopes, as evaluated by cross-competition studies using radiolabeled MAb and unlabeled inhibitors; the species specificity of these epitopes was defined by nitrocellulose dot blot immunoassays with bacterial sonic extract antigen preparations from 23 species of mycobacteria. Each of the 14 distinct MAbs recognized a 65-kDa protein produced by a lysogenized Escherichia coli Y1089 host containing cloned rDNA which included the gene for the M. leprae 65-kDa protein. Of the 14 distinct MAbs, 1 recognized an epitope found only on M. leprae, and the others recognized epitopes present on as few as 8 or as many as all 23 of the mycobacterial species studied. Identification of these distinct 65-kDa protein epitopes and use of the MAbs which recognize them should assist future structural studies of this protein and characterization of the T-cell reactive and serodiagnostically useful portions of the molecule.
Stimulation of murine lymphocyte blastogenesis by mitogens in heat-killed Histoplasma capsulatum yeast cells
Authors: Nickerson D A , Friedrich L M
Published: January 2020

In vitro blastogenesis by normal murine splenocytes from several mouse strains has been detected after exposure to heat-killed Histoplasma capsulatum yeast cells. Maximal lymphocyte stimulation induced by 10(4) heat-killed cells resulted in 20- to 45-fold increases in [3H]thymidine uptake by splenocytes when compared with responses by normal unstimulated lymphocytes. The kinetics for this response to heat-killed H. capsulatum cells has shown peak mitogenesis 3 days after culture. Examination of the mitogenic potential of soluble antigen preparations from H. capsulatum has revealed stimulation of lymphocyte blastogenesis with yeast cell sonicates and autolysates but not substances from autoclaved yeast cells. The levels of lymphocyte blastogenesis induced by sonicates or autolysates were comparable to mitogen responses stimulated by heat-killed cells. Preliminary biochemical characterization of the mitogenic factor(s) associated with yeast cell sonicates show two peaks of activity, at 178,000 and less than 12,000 Mr, which have a protein or glycoprotein nature. Finally, analysis of lymphocyte blastogenesis in cultures enriched for selected lymphocyte subpopulations has shown that T lymphocytes are preferentially stimulated by yeast cell mitogens.
Helper T cells induced by a purified 28-kilodalton antigen of Schistosoma mansoni protect rats against infection
Authors: Auriault C , Balloul J M , Pierce R J , Damonneville M , Sondermeijer P , Capron A
Published: January 2020

Schistosoma mansoni adult worm 28-kilodalton (kDa) proteins were separated on polyacrylamide slab gels, recovered by electrophoretic elution, and used to immunize Fischer rats. After the second or third injection, inguinal lymph node T lymphocytes were propagated in vitro for 4 weeks in the presence of syngeneic antigen-presenting cells and adult worm antigens in medium containing interleukin-2. After this period of culture, 99% of the cells expressed the W3/13+ surface phenotype and 93% of the cells expressed the W3/25+ surface phenotype. These cells were then tested for their in vivo functional activity after transfer to Fischer rats that had been either infected with S. mansoni cercariae or immunized with the 28-kDa purified protein. In each case, an increase of S. mansoni-specific antibodies was observed. Whereas anti-28-kDa antibodies were only detectable at day 40 postinfection in controls injected with normal T lymphocytes, they appeared as early as day 13 postinfection when the animals received 28-kDa protein-specific T lymphocytes. This led to an effective protection of infected rats (45 to 85%) which correlated with the increase in S. mansoni-specific antibodies. These results therefore demonstrate that the 28-kDa protein possesses epitopes capable of activating helper T cells, which confer a strong protective immunity by enhancing the production of cytotoxic antibodies. The stimulation of the 28-kDa-specific T cells with recombinant proteins suggests that the major epitopes are located toward the carboxylic end of the molecule.
Structural requirements of muramylpeptides for induction of necrosis at sites primed with Mycobacterium tuberculosis in guinea pigs
Authors: Nagao S , Takada H , Yagawa K , Kutsukake H , Shiba T , Kusumoto S , Kawata S , Hasegawa A , Kiso M , Azuma I
Published: January 2020

Intracutaneous injection of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) in guinea pigs caused an extensive necrotic reaction in footpads prepared by injection of heat-killed Mycobacterium tuberculosis in water-in-mineral-oil emulsion. We examined a variety of analogs and derivatives of muramylpeptides for their ability to provoke this reaction. A maximum and a minimum structure responsible for the necrotic reaction were found to be N-acetylglycosaminyl-beta(1-4)-N-acetylmuramyl-tripeptide (GlcNAc-MurNAc-L-Ala-D-isoGln-meso-A2pm) and MDP, respectively. An unexpected finding was that GlcNAc-MurNAc-tetrapeptides having L-amino acids at their C termini, unlike comparable compounds having C-terminal D-amino acids, exhibited definite necrosis-inducing activity, probably due to their tendency to undergo in vivo degradation to GlcNAc-MurNAc-tripeptide. Introduction of some acyl groups, especially the stearoyl group, to the 6-O position of the muramic acid or the peptide moiety of muramylpeptides increased the necrosis-inducing activity of the parent molecules. However, this was not observed with 1-thio-muramic acid analogs of MDP. Modification of the alpha- or gamma-carboxyl groups of the glutamic acid residues of muramylpeptides tended to decrease their necrosis-inducing ability. Analogs and derivatives of muramylpeptides which are capable of inducing necrosis at a primed site, with few exceptions, exhibited powerful adjuvanticity against ovalbumin in guinea pigs. However, the reverse was not necessarily true.