PubMed Health⌕ Search

Biomedical subjects

N L Martin

Publications and source records attributed to N L Martin.

At least 19 recordsLinked to original sources
PubMed1995

Evidence that the pathway of disulfide bond formation in Escherichia coli involves interactions between the cysteines of DsbB and DsbA.

Disulfide bond formation is catalyzed in the periplasm of Escherichia coli. This process involves at least two proteins: DsbA and DsbB. Recent evidence suggests that DsbA, a soluble periplasmic protein directly catalyzes disulfide bond formation in proteins, whereas DsbB, an inner membrane protein, is involved in the reoxidation of DsbA. Here we present direct evidence of an interaction between DsbA and DsbB. (Kishigami et al. [Kishigami, S., Kanaya, E., Kikuchi, M. & Ito, K. (1995) J. Biol. Chem. 270, 17072-17074] have described similar findings.) We isolated a dominant negative mutant of dsbA, dsbAd, where Cys-33 of the DsbA active site is changed to tyrosine. Both DsbAd and DsbA are able to form a mixed disulfide with DsbB, which may be an intermediate in the reoxidation of DsbA. This complex is more stable with DsbAd. The dominance can be suppressed by increasing the production of DsbB. By using mutants of DsbB in which one or two cysteines have been changed to alanine, we show that only Cys-104 is important for complex formation. Therefore, we suggest that in vivo, reduced DsbA forms a complex with DsbB in which Cys-30 of DsbA is disulfide-bonded to Cys-104 of DsbB. Cys-104 is rapidly replaced by Cys-33 of DsbA to generate the oxidized form of this protein.

Bacterial Proteins↗
PubMed1995

Epitope mapping of the Pseudomonas aeruginosa major outer membrane porin protein OprF.

The Pseudomonas aeruginosa major outer membrane protein OprF has been proposed for use as a vaccine and as a target for immunotherapeutic and diagnostic monoclonal antibodies. The well-conserved epitopes for 10 surface-reactive, OprF-specific monoclonal antibodies were localized by both overlapping peptide analysis and immunodetection of OprF peptides generated by cyanogen bromide and the protease papain. Three of the monoclonal antibodies bound to specific overlapping octapeptides, which had been synthesized on 160 pins to cover the entire 326 amino acids of OprF. The highest reactivities were as follows: MA7-1 to the pin with attached peptide GTYETGNK (amino acids 55 to 62), MA7-2 to NLADFMKQ (amino acids 237 to 244), and MA5-8 to TAEGRAIN (amino acids 307 to 314). The other monoclonal antibodies showed no reactivity, indicating that they do not recognize linear epitopes. Two polyclonal sera were also tested and demonstrated weak reactivity with discrete regions of OprF, suggesting that the majority of antibodies produced might recognize conformational epitopes. Utilizing defined peptides generated with cyanogen bromide and papain, the conformational epitopes recognized by the seven monoclonal antibodies were localized to regions that were 42 to 90 amino acids long. These regions were located on two adjacent loops in the middle of an amended structural model of OprF.

Amino Acid Sequence↗
PubMed1995

Abdominal wall hernias: review of herniography and correlation with cross-sectional imaging.

Herniography is an accurate means of identifying groin hernias when the clinical diagnosis is uncertain. Its role in evaluation of other types of ventral hernias is less clear; however, with minor modifications in technique, herniography is also useful in these cases. This article reviews the technique of herniography, normal variations in anatomy, and interpretation of herniograms of the groin and anterior abdominal wall on the basis of 72 patients studied over 3 1/2 years. Herniography is also compared with cross-sectional imaging techniques. Inguinal and femoral hernias are best shown and classified with herniography. Ventral hernias are well demonstrated by both herniography and cross-sectional imaging; however, herniation of only fat and supine imaging may lead to misdiagnosis with computed tomography or ultrasound. Spigelian hernias are probably best depicted with cross-sectional imaging. Diagnostic pitfalls of herniography are reviewed, including the need for obtaining postexercise radiographs, oblique radiographs, and tangential radiographs.

Female↗
PubMed1994

Two cysteines in each periplasmic domain of the membrane protein DsbB are required for its function in protein disulfide bond formation.

DsbB is a protein component of the pathway that leads to disulfide bond formation in periplasmic proteins of Escherichia coli. Previous studies have led to the hypothesis that DsbB oxidizes the periplasmic protein DsbA, which in turn oxidizes the cysteines in other periplasmic proteins to make disulfide bonds. Gene fusion approaches were used to show that (i) DsbB is a membrane protein which spans the membrane four times and (ii) both the N- and C-termini of the protein are in the cytoplasm. Mutational analysis shows that of the six cysteines in DsbB, four are necessary for proper DsbB function in vivo. Each of the periplasmic domains of the protein has two essential cysteines. The two cysteines in the first periplasmic domain are in a Cys-X-Y-Cys configuration that is characteristic of the active site of other proteins involved in disulfide bond formation, including DsbA and protein disulfide isomerase.

Alkaline Phosphatase↗
PubMed1994

HIV type 1 Tat protein induces immunoglobulin and interleukin 6 synthesis by uninfected peripheral blood mononuclear cells.

Tat is a potent trans-activating protein encoded by the HIV genome. It is essential for viral replication, but has pleiotropic effects on host cells as well. We demonstrated that exogenous recombinant Tat increases immunoglobulin (Ig) and interleukin 6 (IL-6) production in vitro by normal uninfected peripheral blood mononuclear cells by 100-500%. The optimal Tat concentration was 100 ng/ml, but even a low concentration of 1 ng/ml induced a response in most subjects. The observed induction was inhibited by monoclonal anti-Tat antibodies and 2,3-dimercapto-1-propanol. Both anti-IL-6 antibodies and IL-6 antisense oligonucleotides inhibited Tat-induced IgG and IgA synthesis to some degree, whereas induction of IgM appeared to be independent of IL-6. We conclude that Tat can function in vitro in the absence of any other viral structures and induce Ig and IL-6 production; the clinical significance of these findings remains as yet undetermined.

Adult↗
PubMed1993

Conservation of surface epitopes in Pseudomonas aeruginosa outer membrane porin protein OprF.

The outer membrane proteins of several prominent bacterial pathogens demonstrate substantial variation in their surface antigenic epitopes. To determine if this was also true for Pseudomonas aeruginosa outer membrane protein OprF, gene sequencing of a serotype 5 isolate was performed to permit comparison with the published serotype 12 oprF gene sequence. Only 16 nucleotide substitutions in the 1053 nucleotide coding region were observed; none of these changed the amino acid sequence. A panel of 10 monoclonal antibodies (mAbs) reacted with each of 46 P. aeruginosa strains representing all 17 serotype strains, 12 clinical isolates, 15 environmental isolates and 2 laboratory isolates. Between two and eight of these mAbs also reacted with proteins from representatives of the rRNA homology group I of the Pseudomonadaceae. Nine of the ten mAbs recognized surface antigenic epitopes as determined by indirect immunofluorescence techniques and their ability to opsonize P. aeruginosa for phagocytosis. These epitopes were partially masked by lipopolysaccharide side chains as revealed using a side chain-deficient mutant. It is concluded that OprF is a highly conserved protein with several conserved surface antigenic epitopes.

Antigens, Bacterial↗
PubMed1993

Serum levels of soluble CD8, neopterin, beta 2-microglobulin and p24 antigen as indicators of disease progression in children with AIDS on zidovudine therapy.

OBJECTIVE: To test the hypothesis that serum levels of soluble markers in children change after initiation of zidovudine therapy and that the extent and pattern of these longitudinal changes correlates with clinical outcome. PATIENTS AND METHODS: We measured serum levels of soluble CD8, neopterin, beta 2-microglobulin (beta 2M), and p24 antigen, and CD4 cell counts, before the initiation of zidovudine therapy and at 12, 24 and 48 weeks of treatment in 24 HIV-1-infected children (Centers for Disease Control classification P2) and 15 controls. RESULTS: Soluble CD8 levels were elevated before therapy in 70% of the infected children; subsequent decreases were associated with lower risk of disease progression. The mean serum neopterin level before treatment was elevated in infected children; decreases in neopterin levels marginally reflected improved or stable clinical status. Serum beta 2M levels and CD4+ cell counts were not associated with clinical outcome. Only 10 out of the 24 patients had detectable levels of serum p24 antigen before treatment; again, the amount of decline after initiation of therapy did not predict clinical outcome. CONCLUSION: Decreasing levels of soluble CD8 and neopterin in HIV-1-infected children receiving zidovudine therapy might reflect a good response to treatment and a slowing of disease progression.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Low number of antibody producing cells in patients with sickle cell anemia.

B cell function is impaired in patients with sickle cell anemia. Although the number of surface IgM positive cells was similar in sickle cell patients and controls, in vitro spontaneous IgM, and PWM stimulated IgA, IgM, and IgG synthesis was significantly lower in the patients than in controls. The number of PWM induced and antigen specific immunoglobulin producing cells after immunization with Pneumovax, containing 21 serotypes of Streptococcus pneumoniae, was about 100-fold lower in the patients as compared with controls. Finally, the ability of the patients' peripheral blood mononuclear cells to proliferate in response to mitogens (PWM, SAC, PHA) was diminished. Because of the observed impairments in both nonspecific and antigen specific immunoglobulin synthesis and cell proliferation assays in the patients, we determined serum concentrations of IL-4 and IL-6, two cytokines associated with antibody production. IL-4 concentrations appeared low in sickle cell patients, and correlated with that of serum IgM. We hypothesize that B cell maturation in sickle cell patients is arrested at an IL-4 dependent stage.

Adult↗
PubMed1992

Analysis of the Pseudomonas aeruginosa major outer membrane protein OprF by use of truncated OprF derivatives and monoclonal antibodies.

TnphoA mutagenesis of the cloned oprF gene was utilized to generate 16 classes of fusions encoding differing lengths of the amino terminus of OprF fused to either alkaline phosphatase or to peptide tags of 1 to 20 amino acids, depending on the orientation and reading frame into which TnphoA was inserted. Representatives of each of the 16 classes were sequenced to determine the precise fusion joint. Four of these 16 representatives which produced in-frame fusions to alkaline phosphatase and another 8 with fusion joints in the amino-terminal half of OprF failed to react with a panel of 10 specific monoclonal antibodies. In contrast, OprF derivatives with predicted fusion joints at amino acids 180, 204, 289, and 299 reacted with one to five of the monoclonal antibodies. Four other immunoreactive OprF derivatives were created by subcloning and encoded amino acids 1 to 187, 188 to 326, 1 to 273 and 1 to 170 plus 301 to 326. On the basis of reactivity with the TnphoA-truncated derivatives and subclones of oprF, the epitopes for all 10 monoclonal antibodies were localized, in part, to specific regions of OprF. Nine of the 10 monoclonal antibodies, 8 of which recognize surface-exposed epitopes, mapped within the carboxy-terminal region of OprF that is homologous to the Escherichia coli outer membrane protein OmpA. Thus, we concluded that parts of the carboxy terminus of OprF are exposed on the external face of the outer membrane. In addition, a clone containing only the first two cysteine residues of OprF demonstrated reactivity with monoclonal antibodies MA4-4 and MA7-8 that was destroyed by 2-mercaptoethanol treatment, as was reactivity with intact OprF. Thus, we conclude that this first pair of cysteines at residues 176 and 185 of mature OprF form a disulfide bond.

Antibodies, Monoclonal↗
PubMed1992

Reevaluation, using intact cells, of the exclusion limit and role of porin OprF in Pseudomonas aeruginosa outer membrane permeability.

Earlier studies that used model membrane reconstitution methods have come to different conclusions regarding the exclusion limit of the outer membrane of Pseudomonas aeruginosa and whether OprF is the major channel-forming protein in the outer membrane. In this study, a 6.2-kbp SalI fragment, encoding only two cytoplasmic enzymes, alpha-galactosidase and sucrose hydrolase, and the inner membrane raffinose permease, was cloned behind the m-toluate-inducible tol promoter of vector pNM185 to create plasmid pFB71. P. aeruginosa strains harboring pFB71, when grown with inducer, produced both enzymes encoded by the insert and had acquired the ability to grow on the disaccharide melibiose and the trisaccharide raffinose. The rate of growth was dependent on the concentration and size of the saccharide and was decreased three- to fivefold by the absence of OprF, as examined by measuring the growth on melibiose and raffinose of an isogenic OprF-deficient omega insertion derivative, H636(pFB71). At high concentrations, di-, tri-, and tetrasaccharides could pass across the outer membrane to plasmolyze P. aeruginosa, as measured by light scattering and confirmed by electron microscopy. The initial rate kinetics of light-scattering changes were dependent on the size of the saccharide being used. Furthermore, the rates of change in light scattering due to raffinose and stachyose uptake across the outer membrane for strain H636 were fivefold or more lower than for its OprF-sufficient parent H103. These data are consistent with model membrane studies showing that OprF is the most predominant porin for compounds larger than disaccharides in P. aeruginosa and suggest that the exclusion limit for this porin and the outer membrane is greater than the size of a tetrasaccharide. In addition, these data confirmed the existence of other porins with a predominant function in monosaccharide uptake and a more minor function in the uptake of larger saccharides.

Bacterial Outer Membrane Proteins↗
PubMed1992

Computed radiography in musculoskeletal imaging: state of the art.

Computed radiography is a 2K x 2K x 10 bit digital radiographic system that replaces the film-screen combination with a photo-stimulable phosphor plate. The advantages of this relatively new technology include linear detector response, improved detector efficiency, and digital processing capabilities. Musculoskeletal applications benefit significantly from these attributes, which result clinically in the ability to reduce both radiation dose and number of exposures. Studies of observers' performance have shown no statistically significant difference in diagnostic accuracy between film-screen and computed radiographic musculoskeletal images. Computed radiography is particularly useful in the evaluation of the musculoskeletal system in traumatized patients with portable radiographs, spine radiographs, scoliosis studies, and depiction of soft-tissue abnormalities. Limitations include change in image format and size, high cost, decreased spatial resolution, restricted throughput, increased perception of noise, and new artifacts that must be recognized. Spatial resolution limitations of computed radiography in identification of fine detail information can be improved by using magnification techniques. Radiation dose reduction with an exposure decrease of 25-50% can be achieved without loss of diagnostic accuracy, although this depends on the examination and the abnormality. An interactive workstation is important in the use of a computed radiographic system with capabilities to adjust display parameters to best depict images and disease. We conclude that computed radiography is an alternative to film-screen radiography without significant differences in diagnostic quality in the evaluation of musculoskeletal images.

Humans↗
PubMed1992

A screening test for the detection of anti-HIV-1 IgA in young infants.

A simple dot blot screening test for anti-HIV-1 IgA in infant sera was developed using recombinant HIV proteins. Ten control infants, 19 uninfected infants of seropositive mothers and 12 HIV culture positive infants were studied at 3 month and 18 month time points. Prior to IgG depletion of the serum samples, 11/12 (92%) of the infected infants, 2/19 (11%) of the uninfected and none of the control infants were anti-HIV IgA positive at 3 months of age. After depletion, no anti-HIV IgA antibodies could be detected in the samples from uninfected infants, whereas the antibodies persisted in all 11 samples from the infected infants, resulting in sensitivity and specificity of 91.7% (95% confidence limits 59.8-99.6%) and 100% (79.1-100%) respectively. The assay might prove useful in the early diagnosis of HIV infection and can be performed at a fraction of the cost of commercially available western blot strips.

HIV Antibodies↗
PubMed1991

Detection of infection with human immunodeficiency virus (HIV) type 1 in infants by an anti-HIV immunoglobulin A assay using recombinant proteins.

To diagnose infection with the human immunodeficiency virus (HIV) soon after birth in infants born to HIV type 1-infected women, we developed antiviral IgA Western blot and dot blot assays with recombinant HIV-1 proteins. Thirty-three infants born to HIV-1-seropositive mothers and nine infants born to HIV-1-seronegative intravenous drug-abusing mothers were followed prospectively. Infection was documented by positive virus culture. Results with the polymerase chain reaction were used for comparison. Twelve infants were found infected with HIV-1; the earliest age at which cultures became positive ranged from birth to 31 weeks of age. Of the 12 culture-positive infants, 10 had anti-HIV IgA antibodies detectable initially between birth (cord blood) and 27 weeks of age. Anti-HIV IgA was not present in the uninfected infants or in the control subjects, either by Western blot or dot blot assays. Testing for anti-HIV IgA antibodies with recombinant HIV-1 proteins is an effective method for detecting viral infection in newborn and young infants.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Serum interleukin-6 concentrations are elevated and associated with elevated tumor necrosis factor-alpha and immunoglobulin G and A concentrations in children with HIV infection.

Hypergammaglobulinemia is one of the most consistent, and usually the first observable abnormality in infants vertically infected with HIV. We have analyzed serum interleukin (IL)-4, IL-6, tumor necrosis factor (TNF)-alpha, and immunoglobulin (Ig) concentrations in 23 HIV-infected and 21 uninfected children. IL-6 and TNF-alpha concentrations in HIV-infected children were significantly higher than those in uninfected children, and mutually correlated. No differences in serum IL-4 levels between infected and uninfected children were observed. There was a correlation between serum IL-6 and IgG and between IL-6 and IgA concentrations. Furthermore, during follow-up changes in IL-6 levels were usually accompanied by corresponding changes in IgG levels. Our data indicate an association between HIV, IL-6, TNF-alpha and hypergammaglobulinemia. Regardless of the source and initial stimulus, continued production of IL-6 and TNF-alpha may result in augmentation in an auto-feedback manner, accompanied by increases in Ig synthesis and, more importantly, HIV replication. Thus, elucidation of the mechanisms responsible for overproduction of these two cytokines in HIV-infected patients is not only interesting from a biologic point of view, but is likely to have important clinical implications as well.

B-Lymphocytes↗