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R C Judd

Publications and source records attributed to R C Judd.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of naturally elaborated blebs from serum-susceptible and serum-resistant strains of Neisseria gonorrhoeae.

Outer-membrane blebs from two serum-susceptible and two serum-resistant strains of Neisseria gonorrhoeae were characterized. In general, bleb surfaces resembled cell surfaces, but there were qualitative and quantitative protein differences in blebs released by serum-susceptible and serum-resistant strains. Relative to blebs from serum-resistant strains, blebs from serum-susceptible strains expressed reduced amounts of major outer-membrane proteins I and III, and little if any 68,000 Dalton outer-membrane protein.

Bacterial Outer Membrane Proteins↗
PubMed1992

The interaction of naturally elaborated blebs from serum-susceptible and serum-resistant strains of Neisseria gonorrhoeae with normal human serum.

We studied the interaction of normal human serum immunoglobulins with outer-membrane bleb antigens of Neisseria gonorrhoeae. Gonococcal 68,000 Dalton and Lip (H.8 antigen) outer-membrane proteins were recognized by normal human serum immunoglobulins in blebs from serum-resistant strains, but not in blebs from serum-susceptible strains. The addition of blebs from a serum-resistant strain to bactericidal assays resulted in significantly greater inhibition of serum killing than the addition of blebs from a serum-susceptible strain. Our results indicate that blebs from two serum-resistant gonococcal strains have an enhanced ability to bind and remove cell-targeted bactericidal factors, and that outer-membrane blebbing may contribute to serum resistance.

Antibodies, Bacterial↗
PubMed1991

Identification and characterization of a conserved outer-membrane protein of Neisseria gonorrhoeae.

A previous study in our laboratory identified a surface-exposed peptidoglycan-associated protein of Neisseria gonorrhoeae which had an apparent molecular mass of 44,000 daltons (44kDa) (Hill and Judd, 1989). This paper reports results which confirm that the 44kDa protein is surface-exposed, and that the protein is expressed in, and is structurally invariant among, 14 strains of N. gonorrhoeae. The fact that the 44kDa outer-membrane protein is found in a conserved form in all gonococci examined strongly suggests that it is crucial to the bacterium's survival. Moreover, it appears that this protein is a penicillin-binding protein (PBP3) (Shafer and Judd, 1991). This invariant, surface-exposed, peptidoglycan-associated outer-membrane protein deserves further investigation to elucidate its role in the immunobiology of N. gonorrhoeae, and its possible use as an immunoprophylactic reagent.

Antigens, Surface↗
PubMed1991

Gonococcal penicillin-binding protein 3 and the surface-exposed 44kDa peptidoglycan-binding protein appear to be the same molecule.

The outer membrane of Neisseria gonorrhoeae contains a 44,000 dalton (44kDa) surface-exposed protein which has the reported ability to form covalent interactions with peptidoglycan (PG). This PG-binding outer-membrane protein (OMP) appears to be highly conserved since it has been detected in all isolates examined. It also appears to be invariant since its primary structure among strains gives evidence of being identical (Judd et al., 1991). While studying the interaction of gonococcal penicillin-binding proteins (PBPs) with human lysosomal cathepsin G, we noticed that the 44kDa PG-binding OMP exhibited certain properties similar to PBP3. In this study we sought to obtain biochemical evidence to ascertain whether these proteins were the same. We found that both proteins fractionated with other sarkosyl-insoluble OMPs and that they exhibited similar susceptibility to cleavage in situ by enzymatically active cathepsin G. Moreover, a purified preparation of the 44kDa OMP was found to covalently bind radiolabelled benzylpenicillin in vitro. Thus, the data presented herein suggest that the 44kDa PG-binding OMP and PBP3 are the same OMP.

Bacterial Outer Membrane Proteins↗
PubMed1990

Comparison of two serum bactericidal assays for Neisseria gonorrhoeae.

Pooled normal human serum killing of 14 strains of Neisseria gonorrhoeae was assessed by dilution plate and microtiter methods. In both assays, the strains presented a spectrum of sensitivity to the serum. In the dilution plate assay, results with two different concentrations of human serum were similar for most, but not all of the strains tested. When data for all of the strains were compared, no correlation was found between the dilution plate and microtiter bactericidal assays. Finally, we found that the bactericidal capacities of intact and complement-depleted human sera were very similar when assessed by microtiter methods, suggesting a non-complement-mediated serum killing mechanism.

Analysis of Variance↗
PubMed1990

Characterization of reverse transcriptase from feline immunodeficiency virus.

Reverse transcriptase has been purified from feline immunodeficiency virus (FIV) by DEAE-cellulose and phosphocellulose chromatography. The purified enzyme consists of a single protein with a Mr of 67,000. When proteolysis is not minimized during purification, a fragment of Mr 54,000 is also observed. This is similar to the reverse transcriptase from human immunodeficiency virus type 1 (HIV), which consists of a polypeptide of Mr 66,000; when proteolysis is not minimized during purification, a fragment of Mr 51,000 is also observed. In direct comparisons, the FIV reverse transcriptase is very similar to the HIV reverse transcriptase in template specificity and requirements for Mg2+. In contrast to these similarities, the FIV and HIV reverse transcriptases are substantially different in primary sequence, as determined by peptide mapping.

Animals↗
PubMed1990

Peptide mapping.

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Autoradiography↗
PubMed1990

Characterization of fourteen strains of Neisseria gonorrhoeae: structural analyses and serum reactivities.

Resistance to normal human serum (NHS) killing in Neisseria gonorrhoeae has been associated with particular types of Protein I (PI) and lipopolysaccharide (LPS), but many exceptions exist, and the role of these structures in determining serum reactivities remains controversial. In reality, the response of the gonococcus to NHS is probably governed by several parameters involving a number of outer-membrane (OM) components. We surveyed the serum reactivities of 14 strains of N. gonorrhoeae and characterized each of their major OM components. The strains presented a spectrum of sensitivity to pooled NHS. As assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, immunoblotting, and peptide mapping, the strains were also quite heterogeneous in terms of PI, H.8 antigen, and LPS type, and the presence of the 2-1-L8 epitope. Five of the strains had identical PIAs in varying LPS and H.8 backgrounds, and four had identical PIBs in varying LPS and H.8 backgrounds. As assessed by electrophoretic migration and monoclonal antibody binding, Protein III and the 44,000 Dalton protein were identical in these strains. We found no association between PI subclass and serum sensitivity, while H.8 and LPS variation appeared to be related to bactericidal responses. The diversity and close interaction of gonococcal components in the OM are undoubtedly involved in differential abilities to survive NHS killing.

Antigens, Bacterial↗
PubMed1989

Complement binding on serum-sensitive and serum-resistant transformants of Neisseria gonorrhoeae: effect of presensitization with a non-bactericidal monoclonal antibody.

The binding of C3 and C9 on serum sensitive (FA635) and serum resistant (FA638) transformants of serum sensitive Neisseria gonorrhoeae strain F62 was examined. Previous studies showed that these transformants have Protein IAs which are minimally different by proteinase K cleavage and primary structural and peptide mapping and bear LPS which vary slightly on SDS-PAGE. Binding of C3 and C9 on FA635 exceeded binding on FA638 in NHS and in adsorbed NHS. Monoclonal antibody 4G5, which binds to PI on FA638 but not FA635, increases C9 binding on FA638 to levels 3-3.5 fold greater than on FA635 but does not result in killing. The majority of additional 125IC9 deposited on FA638 following presensitization with 4G5 is released from the bacterial surface by trypsin. These results extend our earlier results with N. gonorrhoeae by showing that, although PI monoclonals can lead to substantial deposition of non-bactericidal C5b-9, this C5b-9 is not fully inserted into the gonococcal outer membrane.

Antibodies, Bacterial↗
PubMed1989

Topographical alterations in proteins I of Neisseria gonorrhoeae correlated with lipooligosaccharide variation.

Four transformant strains of Neisseria gonorrhoeae were generated, two of which (WS3 and WS5) had protein I subclass A (P.IA) and two which (WS2 and WS4) had protein I subclass B (P.IB). Analysis of the strains demonstrated that the two P.IA-bearing strains differed in lipooligosaccharide (LOS) and H.8 antigen, as assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The WS5 strain had slow-migrating LOS and H.8 antigen, and the WS3 strain had fast-migrating LOS and H.8 antigen. The P.IB-bearing strains also had either slow-migrating LOS and H.8 antigen (WS4) or fast-migrating LOS and H.8 antigen (WS2). Structural and exposure analysis revealed that although the P.IAs were identical in the WS3 and WS5 strains, there was a slight alteration of the exposure of the proteins which correlated with altered LOS and/or H.8 antigen. The P.IBs were also shown to be structurally identical, but the LOS and/or H.8 antigen variation in these strains correlated with a more pronounced alteration in the exposure of the P.IB molecules. The differences in protein I (P.I) exposure were generally found in highly negatively charged regions of the molecule, suggesting that the immunogenicity and/or antigenicity of the P.I molecules may vary as a result of LOS and/or H.8 antigen alterations.

Antigenic Variation↗
PubMed1989

Identification and characterization of peptidoglycan-associated proteins in Neisseria gonorrhoeae.

The principal proteins associated with Neisseria gonorrhoeae peptidoglycan (PG), as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, are the following: two proteins at approximately 90 kilodaltons (kDa), single major species at both 60 and 44 kDa, a 34- to 36-kDa protein, and three proteins between 28 and 32 kDa. A protein analogous to Escherichia coli Braun lipoprotein was not detected with gonococcal cell wall preparations. The identity of the PG-associated proteins was confirmed immunologically with antibody generated against purified cell walls. Two types of protein species, dithiothreitol extractable (the majority) and alkylation dependent (primarily the 34- to 36-kDa protein), appeared to be associated with the N. gonorrhoeae cell wall fraction. It was found that a crucial step in the extraction of the proteins from the PG fraction was the inclusion of an acetone-water wash of the purified PG pellet. Studies with cell wall preparations obtained from N. gonorrhoeae intrinsically labeled with 32P revealed that the acetone wash was removing phospholipid from the cell wall fraction and thus facilitating protein extraction. Autoradiographic analysis with PG material derived from 125I-surface-labeled cells indicated that the 44-kDa protein is exposed on the surface of the organism even when associated with the PG layer. Radioimmunoprecipitation with anti-PG antibody confirmed these findings. Lectin analysis (wheat germ agglutinin conjugated to horseradish peroxidase) suggested that the 34- to 36-kDa protein is covalently attached to the PG layer.

Acetone↗
PubMed1989

Export and intercellular transfer of DNA via membrane blebs of Neisseria gonorrhoeae.

Naturally elaborated membrane bleb material is frequently observed in cultures of Neisseria gonorrhoeae. This material was purified and analyzed for protein, lipopolysaccharide, and nucleic acid content. The electrophoretic protein profiles of two bleb-rich fractions, called BI and BII, were distinct, with only BII containing lipopolysaccharide and outer membrane proteins I and III. Both fractions contained RNA, circular DNA, and linear DNA. Exogenous pancreatic DNase I appeared to hydrolyze all bleb-associated DNA in fraction BI and the linear DNA in fraction BII. The circular DNA molecules associated with fraction BII resisted digestion. Electron microscopy of the bleb fractions verified their DNA content. Fixing blebs with glutaraldehyde before mounting them for microscopy prevented release of internal DNA. Such fixation produced little change in the micrographs of BI; however, only traces of DNA were observed in fixed BII preparations. Incubation of wild-type gonococci in mixtures of DNase and blebs purified from antibiotic-resistant strains resulted in efficient exchange of penicillinase-specifying R plasmids. Recipients incorporated plasmids independently of endogenous and exogenous chromosomal streptomycin resistance markers. These in vitro results suggest that bleb formation by N. gonorrhoeae may serve to transfer plasmids intercellularly in vivo, perhaps constituting a previously unexplored genetic exchange mechanism in these bacteria.

Bacterial Outer Membrane Proteins↗
PubMed1988

Purification of outer membrane proteins of the gram-negative bacterium Neisseria gonorrhoeae.

A system of protein purification, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting, that results in purified outer membrane proteins of the gram-negative bacterium Neisseria gonorrhoeae is described. The proteins, which ranged in apparent molecular mass from approximately 31,000 to approximately 92,000 Da, were located by naphthol blue black staining, eluted from nitrocellulose membranes using 88% formic acid, and precipitated by the addition of concentrated ammonium hydroxide. Up to 65% of the original protein present was recovered by this procedure. The resultant purified protein could then be resuspended in aqueous buffer by brief sonication, making it available for further structural and in vivo immunological analyses. Proteins purified in this manner retain their original antigenicity when probed with polyclonal and monoclonal antibodies, and are structurally unaltered by the purification process. This procedure makes it possible to acquire easily usable quantities of highly insoluble outer membrane proteins of gram-negative bacteria.

Bacterial Outer Membrane Proteins↗
PubMed1987

Radioiodination and 125I-labeled peptide mapping of proteins on nitrocellulose membranes.

A rapid procedure for generating dozens of 125I-labeled peptide maps from a protein band excised from a single lane of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel has been developed. Proteins, which can be rapidly purified by 2 X SDS-PAGE separation, are electroblotted onto nitrocellulose paper (NCP) and located by aqueous naphthol blue-black staining. All subsequent steps of radioiodination, and enzyme or chemical cleavage, are carried out on the NCP making it possible to test a variety of cleavage reagents on the same protein sample. The resultant peptidic residues, which can be separated by thin-layer electrophoresis-thin-layer chromatography (2D TLE-TLC), SDS-PAGE, or HPLC, can be used in comparative studies or they can be recovered for further structural and immunological analyses.

Bacterial Proteins↗
PubMed1987

Characterization of protein I from serum-sensitive and serum-resistant transformants of Neisseria gonorrhoeae.

The protein IAs of serum-sensitive (FA635) and serum-resistant (FA638) transformants of Neisseria gonorrhoeae, which have identical pedigrees, have been shown to be different by the use of a monoclonal antibody and were also shown to be different by proteinase K cleavage and primary structural and surface peptide mapping. The difference in structure is within the surface-exposed region of the molecule. The only other difference observed between the two strains was a very slight difference in lipooligosaccharide silver staining in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that protein I alone or in combination with lipooligosaccharide may significantly contribute to serum resistance.

Antigens, Bacterial↗