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Biomedical subjects

M H Brown

Publications and source records attributed to M H Brown.

At least 19 recordsLinked to original sources
PubMed1995

Genetic rearrangements in the rfb regions of Vibrio cholerae O1 and O139.

The recent emergence of a pathogenic new non-O1 serotype (O139) of Vibrio cholerae has led to numerous studies in an attempt to identify the origins of this new strain. Our studies indicate that O139 strains have clear differences in the surface polysaccharides when compared with O1 strains: the lipopolysaccharide can be described as semi-rough. Southern hybridization with the O1 rfb region demonstrates that O139 strains no longer contain any of the rfb genes required for the synthesis of the O1 O-antigen or its modification and also lack at least 6 kb of additional contiguous DNA. However, O139 strains have retained rfaD and have a single open reading frame closely related to three small open reading frames of the O1 rfb region. This region is closely related to the H-repeat of Escherichia coli and to the transposases of a number of insertion sequence elements and has all the features of an insertion sequence element that has been designated VcIS1. Transposon insertion mutants defective in O139 O-antigen (and capsule) biosynthesis map to the same fragment as VcIS1. Preliminary sequence data of complementing clones indicate that this DNA encodes a galactosyl-transferase and other enzymes for the utilization of galactose in polysaccharide biosynthesis. We propose a mechanism by which both the Ogawa serotype of O1 strains and the O139 serotype strains may have evolved.

Amino Acid Sequence↗
PubMed1995

CD79a: a novel marker for B-cell neoplasms in routinely processed tissue samples.

The CD79 molecule, comprising two polypeptide chains, mb-1 (CD79a) and B29 (CD79b), is physically associated in the B-cell membrane with immunoglobulin. It transmits a signal after antigen binding and may, therefore, be considered the B cell equivalent of CD3. It appears before the pre-B-cell stage, and the mb-1 (CD79a) chain can still be present at the plasma cell stage. In this report, we describe a new anti-CD79a monoclonal antibody, JCB117, which reacts with human B cells in paraffin embedded tissue sections, including decalcified bone marrow trephines. When tested on a total of 454 paraffin embedded tissue biopsies, gathered from a number of different institutions, it reacted with the great majority (97%) of B-cell neoplasms, covering the full range of B-cell maturation, including 10 of 20 cases of myeloma/plasmacytoma. It is of interest that the antibody labels precursor B-cell acute lymphoblastic leukemia samples, making it the most reliable B-cell marker detectable in paraffin-embedded specimens in this disorder. All neoplasms of T cell or nonlymphoid origin were negative, indicating that antibody JCB117 may be of value to diagnostic histopathologists for the identification of B-cell neoplasms of all maturation stages.

Antibodies, Monoclonal↗
PubMed1995

Variables affecting symmetry of the nipple-areola complex.

Currently, little is known about which variables are most critical to the subjective appearance of symmetry of the nipple-areola complex. Clinical photographs were transferred onto a compact disc and then imported into a computer photoshop program. Alterations to the nipple-areola complex were then performed, creating 1 symmetrical image and 19 asymmetrical images. The asymmetries were produced by altering position, diameter, or pigmentation. The images were converted to 35-mm slides and shown to four separate panels of three members each. A "symmetry score" was given for each slide. Results showed that any deviation from symmetrical was reliably detectable by the observers. Changes in areola diameter were detected most easily, and the specific direction of positional change was not found to be a significant factor. Differences were seen between individual panel assessments. This study has shown an application for CD-ROM technology in analyzing well-identified variables and has helped in delineating which factors are most critical to the subjective impression of symmetry of the nipple-areola complex.

CD-ROM↗
PubMed1995

Mortality of iron foundry workers: IV. Analysis of a subcohort exposed to formaldehyde.

In the final phase of the mortality study of workers at an automotive iron foundry, a subset (N = 3929) of the original cohort of 8147 men, consisting of those exposed to formaldehyde during the period from January 1960 through May 1987, was analyzed. In addition to the external US population, an internal population (N = 2032), consisting of men who had worked in the same foundry during the same time period but not in formaldehyde-exposed jobs, was also used as a referent. Follow-up continued through December 31, 1989. Smoking status was ascertained for 65.4% of the exposed and for 55.1% of the unexposed cohorts. Detailed work histories and evaluation of occupational exposures by an industrial hygienist enabled us to categorize cumulative formaldehyde and silica exposures. Standardized mortality ratios were used to compare the mortality experience of the exposed cohort with the US population and, because of concerns about the healthy worker effect, with an occupational referent population. Relative risks for race, formaldehyde exposure status, smoking status, and silica exposure level were estimated by fitting a Poisson regression model to four causes of death: cancers of the buccal cavity and pharynx, lung cancer, diseases of the respiratory system, and emphysema. No association between formaldehyde exposure and deaths from malignant or nonmalignant diseases of the respiratory system was found. Cigarette smoking and silica exposure were found to be significantly associated with deaths attributed to lung cancer and disease of the respiratory system.

Adolescent↗
PubMed1995

Molecular characterization of the staphylococcal multidrug resistance export protein QacC.

The QacC polypeptide is a member of a family of small membrane proteins which confer resistance to toxic compounds. The staphylococcal qacC gene confers resistance to toxic organic cations via proton-dependent export. The membrane topology of the QacC polypeptide was investigated by constructing and analyzing a series of qacC-phoA and qacC-lacZ fusions. From these analyses, most of the predicted features of the QacC protein were verified, although data regarding the possible orientation of the COOH region were not conclusive. The role of the sole cysteine residue, Cys-42, in QacC was studied by using the sulfhydryl reagent N-ethylmaleimide and site-directed mutagenesis. N-Ethylmaleimide was shown to inhibit qacC-mediated ethidium export. Multiple amino acid substitutions were made for Cys-42, and mutations at this location had various effects on resistance specificity. This suggests that the Cys-42 residue may be located near a region of QacC that is involved in substrate recognition. Mutagenesis of conserved residues in QacC indicated that Tyr-59 and Trp-62 also play an essential structural or functional role in QacC.

Alkaline Phosphatase↗
PubMed1994

Color dissociation artifacts in double Maddox rod cyclodeviation testing.

BACKGROUND: The double Maddox rod test, based on a red Maddox rod in front of one eye and a clear Maddox rod in front of the other, is used to measure cyclodeviation, typically in patients with superior oblique muscle pareses. Discrepant results between the double Maddox rod test and other torsion measures, and reports of "paradoxic" cyclodeviation in the normal eye of some patients with superior oblique paresis, suggest the two-color format of the double Maddox rod test may produce artifactual torsion measures. METHODS: Forty patients with superior oblique paresis were tested twice using the double Maddox rod test, reversing the red and white Maddox rods between eyes for the second test, and 18 were tested further with same-color red or clear Maddox rods in front of both eyes. RESULTS: With the standard double Maddox rod test, 33 (83%) of 40 patients localized their cyclodeviation to the eye viewing through the red Maddox rod, irrespective of laterality of the paresis or fixation preference. In all 33 patients, laterality of the perceived torsion changed between eyes when testing was repeated with red and white Maddox rods interchanged between eyes. With same-color Maddox rods before both eyes, 17 (94%) of 18 patients localized extorsion to the paretic eye. There was 7.6:1 ratio of luminance transmission and a 1.6:1 ratio of grating spatial frequency bandpass in the plano meridian between the clear and red Maddox rods, which appear to be responsible for the double Maddox rod test artifact. CONCLUSION: The traditional double Maddox rod test may produce artifactual cyclodeviation measurements. An alternative version of the test, based on same-color Maddox rods in front of both eyes, is proposed. The relatively high spatial frequency bandpass characteristics of the plano meridian of the Maddox rod (as high as 20/25 Snellen equivalent resolution through the clear Maddox rod) also suggests double Maddox rod testing should be conducted in a dark room to avoid biases from visual environment cues.

Artifacts↗
PubMed1994

Expression of immunoglobulin and scavenger receptor superfamily domains as chimeric proteins with domains 3 and 4 of CD4 for ligand analysis.

One approach to the analysis of leucocyte cell surface proteins is to express their domains with part of another protein as a carrier. We report the use of two immunoglobulin superfamily (IgSF) domains from rat CD4 (CD4d3 + 4) in producing domains from various superfamilies as chimeric proteins in Chinese hamster ovary cell lines. Four types of construct were successfully expressed containing: (i) the two IgSF domains of CD48; (ii) the IgSF domain of mb-1 which is part of the B cell antigen recognition complex; (iii) a T cell receptor V domain; and (iv) the N-terminal domain of CD5 which belongs to the scavenger receptor superfamily. This CD5 chimeric protein was antigenic for a panel of CD5 mAbs showing that mAbs with functional effects reacted with the N-terminal domain of CD5. The CD48 chimeric protein has been used both as multivalent complexes produced by cross-linking with mAbs recognizing CD4 and in a monomeric form to analyse the kinetics of the interaction between CD48 and CD2 [van der Merwe et al. (1993) EMBO J., 12, 4945-4954].

Animals↗
PubMed1994

Improvement of outer membrane-permeabilizing and lipopolysaccharide-binding activities of an antimicrobial cationic peptide by C-terminal modification.

Antimicrobial cationic peptides have been discovered in many different organisms and often possess a broad range of activity. In this study, we investigated the mechanisms of actions of melittin and two synthetic peptides, CEME (a cecropin-melittin hybrid) and CEMA, against gram-negative bacteria. CEMA was produced by recombinant DNA procedures and is an analog of CEME with a modified C terminus resulting in two additional positive charges. All three peptides showed good antimicrobial activity against four different gram-negative bacteria, but only CEMA was able to somewhat augment the activity of some conventional antibiotics in synergy studies. Studies using the bacteria Pseudomonas aeruginosa and Enterobacter cloacae showed that the peptides all possessed the ability to permeabilize bacterial outer membranes to the hydrophobic fluorophor 1-N-phenylnaphthylamine and the protein lysozyme, with CEMA being the most active. CEMA also had the strongest relative binding affinity for bacterial endotoxin (lipopolysaccharide). These data collectively indicated that these peptides all cross the outer membrane by the self-promoted uptake pathway and that CEMA is the peptide most effective at accessing this pathway.

Amino Acid Sequence↗
PubMed1994

Mortality of iron foundry workers. III. Lung cancer case-control study.

A nested case-control study was undertaken to identify the determinants of lung cancer mortality in a cohort of 8147 foundry men among whom an excess of lung cancer deaths was previously observed. The present study consisted of all lung cancer deaths (N = 220) that occurred within this cohort between 1950 and 1989. both living and dead controls, matched on race and attained age, were selected in the ratio of 10:1 (N = 2200) by means of the incidence density sampling procedure. All cases and two controls per case, randomly selected from each case's 10 controls, were included in a smoking history survey. Basic smoking history information was obtained for about 71% of these study subjects. For the purpose of this study, formaldehyde exposure levels were categorized as high, medium, low, and none. Airborne silica exposure was categorized only as high, medium, and low levels, because all foundry workers were known to be exposed to silica. Conditional logistic regression analyses indicated that cigarette smoking was a strong predictor of lung cancer mortality in this cohort. Neither exposure to formaldehyde nor silica exposure level, nor employment in any of the six major work areas within the foundry, showed an association with lung cancer.

Aged↗
PubMed1993

Affinity and kinetic analysis of the interaction of the cell adhesion molecules rat CD2 and CD48.

CD2 is a plasma membrane glycoprotein present on T lymphocytes that functions as a cell adhesion molecule (CAM). The CD2 counter-receptor in rodents is the structurally-related CAM CD48. Intercellular adhesion involves the formation of multiple CAM complexes between adhering cells and de-adhesion requires disruption of these complexes. To gain an insight into the initiation and termination of intercellular adhesion, the kinetics and affinity of the rat CD2-CD48 interaction was analysed using a BIAcore instrument, which enables the monitoring of protein binding in real time. A soluble chimeric protein, comprising the extracellular portion of rat CD48 and domains 3 and 4 of rat CD4 (sCD48-CD4), bound to immobilized soluble CD2 (sCD2) with a KD of 90 microM. The affinity was also determined in the reverse orientation and sCD2 was shown to bind immobilized sCD48-CD4 with a comparable KD of 60 microM. sCD48-CD4 bound to immobilized deglycosylated sCD2 with a KD of 125 microM, indicating that glycosylation of sCD2 has little effect on the affinity of the interaction. The low affinity was the result of an extremely rapid off-rate constant (K(off) > or = 6 s-1), whereas the on-rate constant was unremarkable (K(on) > or = 10(5) M-1s-1). The kinetic analysis revealed that small amounts of multimeric aggregates of sCD48-CD4 formed in concentrated preparations. Our experience suggests that even low concentrations (< 2%) of these aggregates may be a cause of artifactually high affinity values when analysing low-affinity protein interactions. In conclusion, this study provides the first detailed analysis of the kinetics and affinity of monomeric CAM interactions and suggests that binding between CAMs may be weaker than anticipated.

Animals↗
PubMed1993

Recombinant DNA procedures for producing small antimicrobial cationic peptides in bacteria.

Natural polycationic antibiotic peptides have been found in many different species of animals and insects and shown to have broad antimicrobial activity. To permit further studies on these peptides, bacterial expression systems were developed. Attempts to produce these peptides with an N-terminal signal sequence were unsuccessful due to the lability of the basic peptides. Therefore, a number of different fusion protein systems were tested, including fusions to glutathione-S-transferase (GST) (on plasmid pGEX-KP), Pseudomonas aeruginosa outer membrane protein OprF (on plasmid pRW5), Staphylococcus aureus protein A (on plasmid pRIT5), and the duplicated IgG-binding domains of protein A (on plasmid pEZZ18). In the first three cases, stable fusion proteins with the defensin, human neutrophil peptide 1 (HNP-1), and/or a synthetic cecropin/melittin hybrid (CEME) were obtained. In the course of these studies, we developed a novel method of purifying inclusion bodies, using the detergent octyl-polyoxyethylene (octyl-POE), as well as establishing methods for preventing fusion protein proteolytic breakdown. Cationic peptides could be successfully released from the carrier protein with high efficiency by chemical means (CNBr cleavage) and with low efficiency by enzymatic cleavage (using factor Xa protease). Fusions of protein A to cationic peptides were secreted into the culture supernatant of S. aureus clones and after affinity purification, CNBr digestion and column chromatography, pure cationic peptide was obtained. CEME produced by this procedure had the same amino acid (aa) content, aa sequence, gel electrophoretic mobility and antibacterial activity as CEME produced by protein chemical procedures.

Amino Acid Sequence↗
PubMed1993

The NH2-terminal domain of rat CD2 binds rat CD48 with a low affinity and binding does not require glycosylation of CD2.

CD2, CD48 and CD58 are structurally similar cell adhesion-molecules forming a subset of the immunoglobulin superfamily (IgSF). In humans CD58 is a ligand for CD2 while in mice CD2 binds CD48. We constructed a soluble chimeric molecule comprising the extracellular portion of rat CD48 and domains 3 and 4 of rat CD4 (sCD48-CD4) and used it to examine whether CD2 is a ligand for CD48 in rats. sCD48-CD4-coated polystyrene Dynabeads formed rosettes on rat CD2-transfected COS-7 cells, and this rosetting was blocked by anti-CD2 (OX34) and anti-CD48 (OX45) monoclonal antibodies. We used sucrose-gradient ultracentrifugation to show that sCD48-CD4 binds, in solution, to soluble forms of rat CD2 including the single NH2-terminal IgSF domain of rat CD2 expressed in bacteria. The upper limit of the affinity of the rat CD48-CD2 interaction is 4 x 10(5) M-1, lower than the published affinity of human CD2 for CD58. These results show that rat CD48 binds CD2 on its NH2-terminal IgSF domain with a low affinity and that binding is independent of glycosylation.

Animals↗
PubMed1992

CD3 and CD2 antigen-mediated CD3 gamma-chain phosphorylation in permeabilized human T cells. Regulation by cytosolic phosphatases.

The role of cytosolic and membrane-associated phosphatases in regulating dephosphorylation of the CD3 antigen gamma-chain has been investigated using streptolysin-O-permeabilized T lymphoblasts and Jurkat T leukaemia cells. Permeabilization of T cells caused a rapid extrusion of cytosolic type 2A phosphatases, but a membrane-associated phosphorylase phosphatase activity remained inside the cells. This activity had the properties characteristic of type 2A phosphatases, being resistant to inhibition by type 1 phosphatase inhibitors, though it was inhibited in a time-dependent manner by ATP or by non-hydrolysable ATP analogues, but not by GTP, CTP, ITP or PPi. The membrane-associated type 2A phosphatase in permeabilized cells did not dephosphorylate the CD3 antigen gamma-chain, suggesting that cytosolic phosphatases dephosphorylate the gamma-chain in situ. Cross-linking the CD2 and CD3 antigens with a bivalent monoclonal antibody in the absence of cytosolic phosphatases induced marked phosphorylation of the CD3 gamma-chain, immunoprecipitated using a novel gamma-chain peptide analogue directed antiserum (TG1). Phosphorylation was inhibited by a protein kinase C (PKC) pseudosubstrate inhibitor, indicating that CD2/CD3-induced gamma-chain phosphorylation is a PKC-mediated event. Activation of T cells either with phorbol 12,13-dibutyrate or by CD2-CD3 cross-linking caused [32P]Pi incorporation into the same gamma-chain Ser residues. The site-mapping data suggested that PKC in situ may incorporate phosphate at the CD3 gamma-chain Ser-123 and Ser-126 residues, but that phosphate is rapidly lost from Ser-123 by cytosolic phosphatase action. Our findings underline the importance of the dual actions of kinases and phosphatases as potential regulators of T cell antigen-receptor complex function.

Adenosine Triphosphate↗
PubMed1992

Differential thymus dependence of rat CD8 isoform expression.

Expression of the rat CD8 molecule was studied using five novel monoclonal antibodies (mAb), four of which are specific for the V-like domain of CD8 alpha, whereas one reacts either with the beta chain or with a determinant only expressed on the CD8 alpha/beta heterodimer. mAb to both chains effectively blocked purified lymph node CD8 T cells in mixed lymphocyte reaction and in cell-mediated cytotoxicity. Flow cytometric analysis showed that CD8 T cells from lymph nodes or spleen of normal rats almost exclusively express the alpha/beta isoform, regardless of the T cell receptor isotype (alpha/beta or gamma/delta). In contrast, natural killer (NK) cells carry only CD8 alpha chains. This CD8 alpha + beta - phenotype was also prominent among CD8 T cells from athymic rats and from intestinal epithelium of normal rats. CD8 alpha homodimers can also be expressed as a result of activation, as shown by analysis of CD4 CD8 double-positive T cells obtained from highly purified lymph node CD4 T cells by in vitrok stimulation. Such CD4+CD8 alpha + beta - cells also represent a major subset among adult intestinal intraepithelial lymphocytes (IEL), suggesting local activation. Taken together, the difference in CD8 isoform expression among T cells from athymic rats, NK cells, and gut IEL versus CD8 T cells from peripheral lymphatic organs of euthymic animals suggests that like in mice, expression of the CD8 heterodimer is more dependent on intrathymic maturation than that of the homodimer. Since the more stringent thymus dependence of CD8 alpha + beta + T cells may be due to a requirement for thymic selection on self major histocompatibility complex class I antigens, the virtually exclusive CD8 alpha + beta + phenotype of peripheral rat gamma/delta T cells could mean that antigen recognition by this subset is also restricted by MHC class I molecules.

Animals↗
PubMed1992

Air-conditioned environments do not prevent deterioration of human semen quality during the summer.

OBJECTIVE: To determine if air conditioning might mitigate summer reductions in semen quality. DESIGN: Prospective study of semen quality in summer and winter. SETTING: Normal human volunteers were studied in the setting of a fertility laboratory. PATIENTS, PARTICIPANTS: Two groups of volunteers were selected from the vicinity of New Orleans: 64 men who worked indoors during the summer in air-conditioned environments and 76 others who worked outdoors. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Parameters of manual semen analysis were examined for seasonal and group differences. RESULTS: Remarkably similar reductions in semen quality during summer as compared with winter were observed in both indoor and outdoor workers, respectively, with regard to the following parameters of semen quality: 19% and 19% in sperm concentration, 25% and 27% in total sperm per ejaculate, 17% and 20% in motile sperm concentration, 13% and 15% in percent sperm with normal morphology, and 23% and 23% in concentration of morphologically normal motile sperm. CONCLUSIONS: These findings do not support the hypothesis that the heat of the summer is detrimental to male reproductive capacity. The available evidence suggests instead a possible role of photoperiod in causing the seasonal changes in semen quality.

Adolescent↗
PubMed1992

The hinge region of the CD8 alpha chain: structure, antigenicity, and utility in expression of immunoglobulin superfamily domains.

The lymphocyte surface CD8 antigen is a heterodimer with each chain containing a single Ig-related domain, a hinge-like sequence, a transmembrane segment, and a short cytoplasmic sequence. A soluble form of the rat CD8 alpha chain was produced by introducing a stop codon into the cDNA at the end of the region encoding the extracellular sequence and expressed in Chinese hamster ovary cells. sCD8 alpha was produced at 20 mg/l, and consisted of monomers, dimers, and higher aggregates. The latter could be minimized, but not eliminated, by removal of one of the two cysteine residues in the hinge region by mutation and by growth in serum-free medium. The positions of the N- and O-linked glycosylation sites and the disulphide bond in the Ig-like domain were determined. The MRC OX-8 antibody was shown to react with a region from the CD8 alpha hinge containing 24 amino acids and the antigenic determinant was sensitive to neuraminidase digestion. A construct encoding the Ig-like domain of rat CD8 alpha without the hinge was not expressed in CHO cells, indicating the importance of the hinge region for expression. It seemed possible that the CD8 alpha hinge might facilitate expression of other Ig-related domains and such expression could be detected using the MRC OX-8 antibody. To test the system cDNA constructs were made with the rat CD8 alpha hinge spliced to the V-like domain of mouse CD8 alpha, to the V alpha and V beta domains of a T lymphocyte antigen receptor, and to one or both of the Ig-like domains of the MRC OX-47 membrane antigen. All these forms were expressed as soluble proteins that were detected with the MRC OX-8 antibody. This method may prove useful for the expression of Ig superfamily domains for raising antibodies and other studies.

Aged↗
PubMed1992

Immunohistological detection of human cytotoxic/suppressor T cells using antibodies to a CD8 peptide sequence.

AIMS: To evaluate whether cytotoxic/suppressor T cells can be detected in paraffin wax embedded human tissue samples using antibodies to a synthetic CD8 peptide sequence. METHODS: Polyclonal and monoclonal antibodies were raised against a 13 amino acid peptide sequence from the cytoplasmic portion of the alpha chain of the human CD8 molecule. RESULTS: These antibodies specifically detected the native form of the CD8 polypeptide when tested by immunoprecipitation with radiolabelled T cells, and gave the expected staining pattern for cytotoxic/suppressor T cells in cryostat sections. Being raised in rabbits, the polyclonal antibodies were also useful for double labelling for CD8 in conjunction with monoclonal antibodies. CD8 positive cells could also be detected in paraffin wax embedded tissues. This was achieved without prior treatment of the sections if the tissue had been fixed in Bouin's fixative. When tissues had been exposed to conventional formalin fixation, preliminary microwave treatment was required. CONCLUSIONS: These findings provide further evidence that antibodies against leucocyte associated antigens, capable of reacting on paraffin wax embedded tissue, can be produced by immunisation with synthetic peptide sequences.

Amino Acid Sequence↗