Biomedical subjects

C Malone

Publications and source records attributed to C Malone.

10 recordsLinked to original sources

PubMed1992

Differential mapping of Fc gamma-binding and monoclonal antibody-reactive epitopes on gE, the Fc gamma-binding glycoprotein of herpes simplex virus type 1.

The entire 396 residue extracellular sequence of gE the HSV-1 Fc gamma-binding glycoprotein has been studied to determine epitopes binding to two mAb II-481 and 88S previously demonstrated to react with gE at or near the Fc gamma-binding regions. Overlapping 7-mers constructed from the established sequence were tested with mAb II-481 and 88S along with their Fab fragments. Control mAb of the same IgG 2b subclass as well as whole rabbit and human IgG and Fc were also tested for binding to overlapping linear sequences using the ELISA pin assay to map Fc gamma-binding regions. Six sequences PKTSWRRVS, GLYTLSV, QVASVVLVVQP, PAPPRSWP, CLYHPQLP, and ASTWTSRL were found that constituted major regions binding to the two different mAb of the same specificity. Glycine substitution for each residue within these sequences indicated that arginine 29, tryptophane 70, valine 144, valine 157, arginine 208, histidine 283, and arginine 305 constituted important portions of the II 481 mAb-reactive epitope. Many of the same regions along with one other, GPLHPSW, appeared to be involved in Fc gamma binding. Substitution of glycine for each residue indicated that histidine 67, tryptophane 70, valine 71, valine 157, valine 158, valine 160, valine 161, tryptophane 210, serine 279, cysteine 280, leucine 281, tyrosine 282, histidine 283, proline 284, glutamine 285, proline 287, tryptophane 302, and arginine 305 were important for Fc gamma-binding. Inhibition by gE peptides of rosetting of E sensitized with rabbit IgG antibody around HSV-1-infected cells, as well as inhibition of rosetting using F(ab)2 fragments of rabbit antibodies to these peptides was used to assay relative contributions of all seven regions to Fc gamma-binding activity. Our results provide a tentative map of mAb binding and Fc gamma-reactive sites on gE. mAb and Fc gamma binding of a limited number of individual antigenic amino acids widely distributed among the separate reactive regions suggest that many of the same separate residues contribute both to antigenicity as well as to Fc gamma-binding activity.

Amino Acid Sequence↗

PubMed1992

Specific sequences and a hairpin structure in the template strand are required for N4 virion RNA polymerase promoter recognition.

Coliphage N4 virion-encapsidated, DNA-dependent RNA polymerase (vRNAP) is inactive on double-stranded N4 DNA; however, denatured promoter-containing templates are accurately transcribed. We report that all determinants of vRNAP promoter recognition exist in the template strand, indicating that this enzyme is a site-specific, single-stranded DNA-binding protein. We show that conserved sequences and the integrity of inverted repeats present at the promoters are essential for activity, suggesting the necessity for specific secondary structure. Evidence for such a structure is presented. We propose a model for in vivo utilization of vRNAP promoters in which template negative supercoiling yields single-strandedness at the promoter to reveal the determinants of vRNAP binding. This structure is stabilized by the binding of E. coli single-stranded DNA-binding protein to yield an "activated promoter."

Affinity Labels↗

PubMed1992

Human anti-F(ab')2 antibodies show preferential reactivity for F(ab')2 molecules bearing lambda light chains.

In order to evaluate binding specificities of anti-F(ab')2 antibodies from patients with systemic lupus erythematosus (SLE) and from normal healthy controls, F(ab')2 fragments were prepared from 24 IgG myelomas with defined isoelectric points, DNA-associated idiotypes, and kappa/lambda light chain types. Using ELISA and hemagglutination assays, anti-F(ab')2 antibodies from 12 healthy controls and 29 SLE patients were observed to exhibit preferential binding (lambda > kappa) to myeloma F(ab')2 fragments composed of lambda light chains (P < 0.0001). No correlation of anti-F(ab')2 binding and presence of cationic, neutral, or anionic isoelectric points or for DNA-associated idiotypes on monoclonal F(ab')2 was detected. Anti-F(ab')2 antibodies, often elevated in SLE during remission, show preferential specificity for F(ab')2 fragments bearing lambda light chains.

Adult↗

PubMed1992

Escherichia coli single-stranded DNA-binding protein is a supercoiled template-dependent transcriptional activator of N4 virion RNA polymerase.

Coliphage N4 is a double-stranded DNA virus that requires the sequential activity of three different RNA polymerases during infection. The N4 virion RNA polymerase, which is carried in the virion and is injected with the DNA at the start of infection, is responsible for the synthesis of N4 early RNAs. In vitro, the virion RNA polymerase can transcribe double-stranded N4 DNA accurately and efficiently but only when the DNA is denatured. We have shown previously that the activity of DNA gyrase is required for in vivo early N4 transcription. We report here that Escherichia coli single-stranded DNA-binding protein (SSB) is also required for N4 early transcription. In vitro, linear or relaxed templates cannot be activated by SSB; however, supercoiled template and SSB allow the virion polymerase to recognize its promoters on duplex DNA and activate transcription. The effects of supercoiling are limited to transcript initiation and are not required for transcript elongation. The activation is specific for SSB; no other single-stranded DNA-binding proteins can substitute. Therefore, SSB is one of a small number of proteins that function to stimulate both replication and transcription. The basis for the specificity of SSB, the mechanism of transcriptional activation by SSB and template supercoiling, and their role in the N4 transcriptional program during development are discussed.

Base Sequence↗

PubMed1991

Rheumatoid factors react with Fab fragments of monoclonal antibodies to herpes simplex virus types 1 and 2 Fc gamma-binding proteins.

Human polyclonal IgM rheumatoid factors (RF) were tested in an enzyme-linked immunosorbent assay with monoclonal antibodies (MAb) (II-481 and B10/A8) to glycoprotein E (gE), the Fc gamma-binding protein of herpes simplex virus type 1 (HSV-1), as well as with MAb 88-S to gE of HSV-2. Most of the RF reacted with II-481 and 88-S. Positive reactions were recorded for RF reacting with whole MAb II-481 and 88-S, as well as with their Fab, but not their Fc, fragments. Human monoclonal IgM RF isolated from mixed cryoglobulins showed a similar profile, with reactivity for both whole MAb II-481 and 88-S and for their Fab fragments. Reactivity with MAb to gE was observed regardless of the Gm specificity of the polyclonal RF and the cross-reactive idiotypes (6B6, 17.109, or G6) of the monoclonal RF. No positive reactions were noted between protein A and Fab fragments of any of the anti-gE MAb. These findings indicate that many RF may bear the internal image of the Fc gamma-binding regions of 2 different herpesviruses: HSV-1 and HSV-2.

Antibodies, Monoclonal↗

PubMed1991

Parallelism of serum anti-F(ab')2 and anti-cationic IgG reactivities in patients with systemic lupus erythematosus.

Cationic anti-DNA antibodies may be related to glomerular injury in murine lupus nephritis or in patients with systemic lupus erythematosus (SLE). Therefore, anti-cationic antibodies in SLE could include antibodies with regulatory function on such pathogenic cationic molecules. Since anti-F(ab')2 antibodies may be involved in the idiotype control of anti-DNA antibodies in some patients with inactive SLE, the present study was aimed to determine if SLE patients with significant serum levels of anti-F(ab')2 produce antibodies reacting with cationic IgG molecules. Three SLE sera with high titers of anti-F(ab')2 antibodies were individually adsorbed by sequential affinity chromatography on three Sepharose columns coupling normal IgG from Cohn Fraction II, pooled cationic IgG myeloma paraproteins displaying idiotypic anti-DNA markers (F4 and 8.12), and F(ab')2 fragment from allogeneic IgG, respectively. Eluates obtained from cationic IgG adsorption showed predominant anti-F(ab')2 reactivity. A similar profile was also detected in a serum from a normal control donor with high levels of anti-F(ab')2. Biotinylation of anti-cationic eluates showed that such antibodies were significantly more reactive with cationic than anionic or neutral IgG, confirming their apparent affinity for positively charged antigens on IgG molecules. Since anti-cationic absorptions were able to remove the anti-F(ab')2 activities in the SLE sera studied, it is possible that anti-cationic antibodies could function as immunoregulatory antibodies in the idiotypic control of some SLE autoreactive phenomena, including glomerular anti-DNA deposition.

Adult↗

PubMed1991

Monoclonal antibodies against human anti-F(ab')2 antibodies react with light chain epitopes.

Anti-F(ab')2 antibodies affinity isolated from sera of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or normal SLE relatives were used to produce monoclonal antibodies (mAbs) in Balb/c and NZB mice. Four of five mAbs showed only primary light chain specificity. Only one mAb produced in an NZB mouse against anti-F(ab')2 from a single SLE patient showed anti-mu-chain specificity. Parallel identical control immunizations with IgG or a single human IgG kappa myeloma produced mAbs with a predominant gamma-chain/Fc fragment specificity. Anti-light chain specificity of mAbs was demonstrated to involve epitopes requiring tertiary structure of the entire light chain instead of antigens confined to Ckappa/lambda or Vkappa/lambda fragments. Anti-kappa specificity of three mAbs was extremely similar but not identical to that defined by anti-Km1 allotyping systems. No evidence was obtained with any of the mAbs produced for antigens unique to SLE or RA anti-F(ab')2 antibodies. The light chain antigenic prominence of many anti-F(ab')2 antibodies may reflect structural features shared by this group of immunoglobulins somehow important for their biologic function.

Animals↗

PubMed1990

Synthesis and transport of storage proteins by testes in Heliothis virescens.

The synthesis of two storage protein subunits, 76,000-Mr and 82,000-Mr polypeptides, by the testes sheath has been studied in Heliothis virescens. Like fat body, which is the primary site of synthesis for the large extratesticular pool, cells of the testes sheath secrete glycosylated storage proteins assembled into hexamers. The testis sheath differed from fat body in several important respects, including the failure to synthesize an abundant (in the hemolymph) 74,000-Mr storage protein, its relatively reduced expression of the 76,000-Mr polypeptide, and the absence of resorption of storage proteins from the lumen of the testis during pupal development. Cyst cells were also shown to import actively the 82,000-Mr storage protein by pinocytosis of testicular fluid and transfer it to the developing spermatids. Unlike other cell types that sequester storage proteins in the form of cytoplasmic granules, their localization within spermatids was exclusively mitochondrial. These observations suggest that expression of the storage protein genes is regulated tissue specifically and reveal novel pathways for their transport and, perhaps, utilization and function during development.

Animals↗

PubMed1976

Pediatric-psychiatric liaison as a model for teaching pediatric residents.

The pediatrician's function as a comprehensive family health practitioner has frequently been ignored. A major problem is that pediatric residents are not trained or are inadequately trained to assess emotional, behavioral, and family problems that may be an integral part of the patient's symptoms. At the Children's Hospital of Philadelphia, a pediatric-psychiatric liaison program has evolved in which pediatric residents learn the principles and skills necessary to manage these problems. Ultimately, the liaison program will enable the staff pediatricians who use these principles to assume appropriate responsibility for educating the pediatric trainees.

Child Psychiatry↗

PubMedDate not supplied

Intensive pressures.

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Clinical Protocols↗