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Biomedical subjects

D Hernandez

Publications and source records attributed to D Hernandez.

At least 55 records · Page 3Linked to original sources
PubMed1994

Molecular basis of drug phototoxicity: photosensitized cell damage by the major photoproduct of tiaprofenic acid.

Tiaprofenic acid is a photosensitizing nonsteroidal anti-inflammatory drug, whose major photoproduct (decarboxytiaprofenic acid) is also a potent photosensitizer. Because of the lack of the carboxylate moiety, this photoproduct is more lipophilic and might bind more efficiently to cell membranes, thereby causing phototoxic damage. To verify the feasibility of this hypothesis, we have prepared the 3H-labeled analogs of tiaprofenic acid and its photoproduct and examined the binding, persistence and phototoxicity of the photoproduct using poorly metabolizing (fibroblasts) and actively metabolizing cells (hepatocytes). The photoproduct of tiaprofenic acid accumulates in both cell types as it is formed. Upon removal of the photoproduct from the culture medium, it rapidly disappears from hepatocytes but not from fibroblasts. Consequently, irradiation of fibroblasts previously incubated with the photoproduct and kept in culture in the dark for 20 h results in generalized cell damage while this effect is not observed in hepatocytes. Because of its long persistence in poorly metabolizing skin cells and its reluctance to photobleaching, the formation of this photoproduct in skin may be of relevance to explain the in vivo phototoxicity of tiaprofenic acid.

Binding Sites↗
PubMed1993

Identification of Ser143 as the site of modification in the active site of histidine ammonia-lyase.

Histidine ammonia-lyase (histidase) from Pseudomonas putida was irreversibly inactivated by L-cysteine at pH 10.5 in the presence of oxygen. Inactivation was accompanied by the formation of a new uv-absorbing species centered around 340 nm. L-[35S]cysteine labeling experiments revealed that 4 mol of L-cysteine was bound per mole of enzyme tetramer upon complete modification. However, the radiolabel was dissociated from the protein under denaturing conditions without loss of the 340-nm absorbance. Prior inactivation of histidase by cyanide, borohydride, or bisulfite precluded the formation of the 340-nm species in subsequent L-cysteine modification experiments. This suggests a common target site for modification of histidase by all of these reagents. Based on its strong absorbance at 340 nm an octapeptide was isolated from L-cysteine-inactivated histidase following trypsin and staphylococcal V8 protease digestion. Electrospray MS/MS revealed that this peptide (Gly138-SerValGlyAlaSerGlyAsp145) contained an unidentified modification of mass 184 Da located on Ser143. This peptide and the serine residue are conserved in all histidases and phenylalanine ammonia-lyases for which the amino acid sequence is available. Ser143 represents the binding site for an electrophilic cofactor required for histidase activity.

Amino Acid Sequence↗
PubMed1993

The order and orientation of a cluster of metalloproteinase genes, stromelysin 2, collagenase, and stromelysin, together with D11S385, on chromosome 11q22-q23.

A cluster of metalloproteinase genes, stromelysin, fibroblast collagenase, and stromelysin 2 together with the anonymous DNA marker D11S385, was mapped using pulsed-field gel electrophoresis to a 135-kb region of chromosome 11q22-q23. The physical proximity of these markers was subsequently confirmed using two YAC clones, and their relative order was established as stromelysin 2-collagenase-stromelysin-D11S385. The pattern of marker representation in a panel of radiation-reduced chromosome 11 hybrids suggests that the metalloproteinase gene/D11S385 cluster is orientated with STMY2 closest to the centromere.

Animals↗
PubMed1993

Purification and characterization of Pseudomonas putida histidine ammonia-lyase expressed in Escherichia coli.

Histidine ammonia-lyase (HAL) from Pseudomonas putida PRS1 contains a catalytically important electrophilic center reported to be dehydroalanine. Little is known about the origin of this group or its linkage to the protein. To initiate structural studies on this enzyme, P. putida HAL was purified from an Escherichia coli high-expression clone in which the HAL gene (hutH) was under the control of the lambda PL promoter on a plasmid vector. In this clone from 6 to 10% of the soluble cell protein after heat induction was HAL and approximately 200 mg of 95% pure HAL could be obtained from 120 g wet weight of cells in a 40 to 60% yield. The overexpressed protein was identical to P. putida HAL in native molecular weight (220 kDa), subunit composition (four identical subunits of 53 kDa each), affinity for substrate (L-histidine Km of 5.3 mM at pH 9.0), and its sensitivity to inactivation by cyanide and bisulfite. The N-terminal amino acid sequence was in agreement with the DNA-predicted sequence, indicating proper translational initiation. These features make this enzyme an appropriate candidate for protein structure investigations regarding the nature of the electrophilic center and its association with the protein.

Amino Acid Sequence↗
PubMed1993

A family showing no evidence of linkage between the ataxia telangiectasia gene and chromosome 11q22-23.

We have studied an inbred family in which two cousins presented with the same clinical features of ataxia telangiectasia (AT). Both patients are still ambulatory at ages 25 and 20. Cellular features of both patients are typical of AT and include increased radiosensitivity and an increased level of spontaneously occurring chromosome aberrations in peripheral blood lymphocytes. Linkage studies and haplotype analysis show no clear evidence that the gene for AT in this family is on chromosome 11q22-23. As previously reported AT families from complementation groups AB, C, and D have all shown linkage to this region of 11q22-23. Our study is of importance in suggesting additional locus heterogeneity.

Adult↗
PubMed1993

Amoebological study of the atmosphere of San Luis Potosi, SLP, Mexico.

A one year round survey was conducted for isolation of free living amoebae (FLA) in the city of San Luis Potosi, (SLP), Mexico, which is placed in a desert environment. Samples were taken by modified impinger method and cultivated in laboratory conditions for FLA isolation following a week period of rehidration. 57 strains were isolated, 39% belonged to Acanthamoeba genus (which is important because it bears opportunistic pathogens that produce amoebic keratitis and granulomatous amoebic encephalitis in humans), 16% to Hartmannella, 9% to Vahlkampfia and the other proportion was divided among 6 other genera. The isolations were more abundant during dry season and the main genera were present in all four stands. The difference among them was the species variety which is discused as connected with abundance of organic wastes and lack of urbanization near the stations.

Air Microbiology↗
PubMed1992

Etretinate increases carcinoembryonic antigen in palmar scrapings.

BACKGROUND: Retinoids can affect epithelial structure and function. Patients who take etretinate can develop stickiness of their palms and soles. OBJECTIVE: We measured carcinoembryonic antigen, a representative glycoprotein in palmar scrapings, to see whether levels were increased in patients taking etretinate when compared with a variety of controls. METHODS: Carcinoembryonic antigen was measured by a standard immunoassay in scrapings taken from the palm. RESULTS: Carcinoembryonic antigen is increased in palmar scrapings in patients taking etretinate. CONCLUSION: Etretinate usage may affect sweat gland function or possibly adherence of carcinoembryonic antigen to surface keratinocytes. The presence of increased amounts of glycoprotein on the surface of the skin may explain the stickiness often noted by patients taking etretinate.

Carcinoembryonic Antigen↗
PubMed1992

Brucella abortus stimulates human T cells from uninfected and HIV-infected individuals to secrete IFN gamma: implications for use of Brucella abortus as a carrier in development of human vaccines.

Brucella abortus has been characterized as a T-independent type 1 antigen/carrier in human and murine antibody responses. In this report it is shown that BA can activate human CD3+ T cells to secrete interferon-gamma (IFN gamma). Unlike mitogens, such as phytohemagglutinin, this stimulation was associated with minimal T-cell proliferation or upregulation of interleukin-2 (IL-2) receptor. Monocytes inhibited BA-mediated IFN gamma secretion since their removal resulted in increased responses, whereas adding monocytes back to cultures caused inhibition. BA elicited IFN gamma from CD4+ and CD8+ T cells, although CD4+ T cells secrete significantly more (p less than 0.05) IFN gamma than CD8+ T cells. The ability of BA to elicit IFN gamma from human T cells was inhibited in the presence of anti-Tac, suggesting that BA also induces IL-2 secretion and that IL-2 is involved in BA-mediated IFN gamma secretion. Detectable IL-2 secretion was induced by BA in the presence of anti-Tac. Exogenous IL-2 acted synergistically with BA to enhance IFN gamma secretion, suggesting that the amount of IL-2 released by BA alone was insufficient for optimal IFN gamma release. Furthermore, addition of IL-2 to T cells from individuals with poor or absent responses to BA, including individuals infected with HIV-1, restored their ability to secrete IFN gamma in response to BA. These data indicate that BA is capable not only of activating human B cells but can also induce T cells, probably of the TH1 phenotype, to secrete IFN gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Brucella abortus↗
PubMed1991

A polymer containing a repeating peptide sequence can stimulate T-cell-independent IgG antibody production in vivo.

The immunogenicity of a CD4 peptide sequence 303-315 (V4 domain) was determined for three forms of the peptide: (a) a polymer consisting of repeating peptide units, (b) a peptide linked to a large protein carrier, chicken serum albumin, and (c) unmodified free peptide. Two in vivo systems were used: a T-cell-competent BALB/c mouse and a T-cell-deficient nude mouse. In BALB/c mice the IgG antibody responses to the peptide-CSA conjugate were at least 100-fold greater than the response to the polymer, and the free peptide gave no response. However, in contrast to the normal BALB/c mice, in the nude mice the polymer produced the only significant response. Nude mice immunized with the polymer produced a predominantly IgG response with almost equal amounts of IgG1 and IgG 2a + 2b. This IgG subclass distribution was very similar to that obtained in the BALB/c for the peptide-CSA conjugate. Positive sera from both groups were able to react with immobilized CD4 molecule on Western blots, confirming the specificity of both the T-cell-independent as well as T-cell-dependent response. These results suggest that repeating epitopes on the same peptide polymer molecule may cause cross-linking of antigen-specific immunoglobin receptors on B-cells, and thus behave as a relatively T-cell-independent immunogen which can induce isotype switching.

Amino Acid Sequence↗
PubMed1991

Immunogenicity of lipopolysaccharide derived from Brucella abortus: potential as a carrier in development of vaccines for AIDS.

In view of its unique ability to stimulate human B cells, we have considered using Brucella abortus (BA) as a carrier for human vaccines. Recently we showed that HIV-1 coupled to BA, but not unconjugated HIV-1, was able to stimulate murine responses even in the relative absence of CD4+ T cells. This result suggested that HIV-BA may be useful in boosting the immunity of individuals infected with HIV-1 and who have impaired CD4+ T cell function. In order to refine this carrier we purified lipopolysaccharide (LPS) from BA and examined its effects on immune responses. Similar to LPS from E. coli (LPS-EC), LPS-BA was capable of stimulating mouse B cells to proliferate. In addition, LPS-BA could activate mouse spleen cells to secrete antibodies in vitro. Isotype analysis revealed that IgM and all the IgG subclasses were elicited. When comparing these responses to those of LPS-EC, LPS-BA induced a greater percentage of IgG2a and LPS-EC evoked more IgG3. IgG2a is probably important in protection against murine viral infection. LPS-BA was haptenated with trinitrophenol TNP-LPS (BA) and tested for carrier effect. Similar to TNP-BA and TNP-LPS (EC), TNP-LPS (BA) triggered anti-TNP antibody of the IgM and all IgG subclasses. In contrast, TNP-ficoll induced mainly IgM and only small amounts of IgG3. These results suggest that LPS-BA, like intact BA, behaves as a T-independent type 1 carrier, and as such may be advantageous as a carrier for human vaccines development.

AIDS Vaccines↗
PubMed1991

Nitrate transport and its regulation by O2 in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an obligate respirer which can utilize nitrate as a terminal electron acceptor under anaerobic conditions (denitrification). Immediate, transient regulation of nitrate respiration is mediated by oxygen through the inhibition of nitrate uptake. In order to gain an understanding of the bioenergetics of nitrate transport and its regulation by oxygen, the effects of various metabolic inhibitors on the uptake process and on oxygen regulation were investigated. Nitrate uptake was stimulated by the protonophores carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol, indicating that nitrate uptake is not strictly energized by, but may be affected by the proton motive force. Oxygen regulation of nitrate uptake might in part be through redox-sensitive thiol groups since N-ethylmaleimide at high concentrations decreased the rate of nitrate transport. Cells grown with tungstate (deficient in nitrate reductase activity) and azide-treated cells transported nitrate at significantly lower rates than untreated cells, indicating that physiological rates of nitrate transport are dependent on nitrate reduction. Furthermore, tungstate grown cells transported nitrate only in the presence of nitrite, lending support to the nitrate/nitrite antiport model for transport. Oxygen regulation of nitrate transport was relieved (10% that of typical anaerobic rates) by the cytochrome oxygen reductase inhibitors carbon monoxide and cyanide.

2,4-Dinitrophenol↗
PubMed1991

Production of a novel antigen by conjugation of HIV-1 to Brucella abortus: studies of immunogenicity, isotype analysis, T-cell dependency, and syncytia inhibition.

In the present study inactivated human immunodeficiency virus type 1 (HIV-1) was conjugated to Brucella abortus and tested for immunogenicity in normal and anti-L3T4-treated BALB/c mice. HIV-BA was more immunogenic than uncoupled HIV in normal mice, since 6-fold less virus in HIV-BA preparations elicited higher titer responses than HIV-1 alone. Furthermore, the HIV-BA antibody response reached higher levels before the HIV-1 response. Immunoblot analysis showed that most of the HIV-1 antigens were recognized by antibodies induced by either HIV-1 or HIV-BA. Isotype analysis revealed that HIV-1 induced similar levels of IgG1 and IgG2a antibodies, whereas the IgG2a responses to HIV-BA were more pronounced than the IgG1 response. These different IgG subclass patterns suggest that conjugation of HIV-1 to BA changed the immunogenic nature of HIV-1. The requirement for helper T cells was examined by immunizing mice that were depleted of CD4+ T cells by in vivo anti-L3T4 treatment. Under these conditions the IgG responses to HIV-1 were completely eliminated. Although HIV-BA antibody responses were markedly reduced in anti-L3T4-treated mice, anti-HIV-1 antibodies, mainly of the IgG2a isotype, were produced. The antibodies generated by HIV-1 and HIV-BA immunization were also tested for their ability to inhibit syncytia formed by infecting CD4 + CEM cells with gp160 vaccinia. Sera from normal mice, immunized with either HIV-1 or HIV-BA were capable of inhibiting syncytia. In contrast, following anti-L3T4 treatment, only mice immunized with HIV-BA, but not HIV-1, produced antibodies capable of inhibiting syncytia.

Animals↗
PubMed1991

Effect of thiazides on colonic NaCl absorption: role of carbonic anhydrase.

The mechanisms by which the benzothiadiazide class of diuretics inhibit electroneutral NaCl absorption are not fully understood. We studied the mechanisms of thiazide action in perfused loops of distal colon in anesthetized male Sprague-Dawley rats. Hydroflumethiazide (1 mM) reversibly inhibited greater than 40% of Na, Cl, and water absorption. Prior exposure of the colon to the carbonic anhydrase inhibitor methazolamide (0.1 mM) prevented the effects of hydroflumethiazide and metolazone, a thiazide-like drug, on colonic absorption. In Ussing flux chambers, addition of hydroflumethiazide to both the mucosal and serosal bathing solutions (but not to the mucosal solution alone) caused marked decreases in Na and Cl absorption. Such inhibition only occurred at concentrations of hydroflumethiazide (0.1 and 1.0 mM) that inhibited greater than 90% of carbonic anhydrase activity in homogenized colonic mucosa. We conclude that an important mechanism by which thiazides inhibit NaCl absorption in the rat distal colon is by inhibition of mucosal carbonic anhydrase. In tissues containing this enzyme, this mechanism of thiazide effect on ion flux must be considered.

Acetazolamide↗