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Biomedical subjects

D Navarro

Publications and source records attributed to D Navarro.

At least 55 records · Page 3Linked to original sources
PubMed1993

Multiresidue pesticide analysis by ion-trap mass spectrometry.

The ion trap has been demonstrated to be able to detect and quantify 245 target pesticides extracted via the Luke method while providing concurrent confirmation of presence via full scan data at the sub-ppm level. The precision and accuracy of the analytical approach was determined to be no greater than 15% relative standard deviation. A comparison study of over 100 incurred residues analyzed by the ion trap and gas chromatography with an array of element-selective detectors has indicated that sample clean-up will probably be necessary before quantification is acceptable for all target compounds. The data obtained using a combination of gas chromatography and mass spectrometry and presented for 250 target pesticides constitutes the basic information required to duplicate and extend the methodology.

Food Contamination↗
PubMed1993

Transport and secretion of truncated derivatives of herpes simplex virus 1 glycoprotein B.

Herpes simplex virus 1 (HSV-1) glycoprotein B (gB) is one of several glycoproteins that compose the virion envelope. In infected cells, gB is a transmembrane glycoprotein that dimerizes and is glycosylated during its transport through the exocytic pathway to the cell surface. To understand the structural requirements for the transport of gB, we analyzed the processing, dimerization, and transport of mutated forms lacking the transmembrane region and the carboxy terminus of the molecule. Our studies showed that conversion of the membrane-anchored form of gB into soluble forms of different lengths resulted in slowed transport rates and incomplete processing. The longer derivatives, gB-(1-690) and gB-(1-690 delta 849), formed dimers but were incompletely transported and were retained within cells. Similar results were obtained with gB-(1-600), which failed to form dimers. In contrast, gB-(1-475), which also failed to form dimers, was transported and secreted from cells. These findings indicate that dimerization is not required for the transport of short, soluble forms of gB through the exocytic pathway provided that conformational regions contained within these shortened molecules are properly assembled.

Animals↗
PubMed1993

Glycoprotein B of human cytomegalovirus promotes virion penetration into cells, transmission of infection from cell to cell, and fusion of infected cells.

Human cytomegalovirus (HCMV) glycoprotein B (gB) is an abundant glycoprotein in the virion envelope that elicits neutralizing antibodies in human infection and in immunized animals. Using a panel of monoclonal antibodies to gB with neutralizing activity, we analyzed in detail the mechanisms by which these antibodies block HCMV infection. Twelve antibodies with complement-independent neutralizing activity were studied for their effect on the attachment of virions to the cell surface, virion penetration into cells, the transmission of infection from cell to cell, and fusion of infected cells. All of the complement-independent antibodies blocked penetration of virions into cells but had no effect on virion attachment to the cell surface. The most potent neutralizing antibodies also limited the spread of infection from cell to cell. Fusion of HCMV-infected U373 glioblastoma cells was blocked by all but two of the neutralizing antibodies to gB, suggesting that fusion of infected cells is similar but not identical to the fusion of the virion envelope with the cell membrane that occurs during virion entry. Our findings provide the first evidence that HCMV gB is a multifunctional glycoprotein that promotes virion penetration into cells, the transmission of infection from cell to cell, and fusion of infected U373 cells.

Antibodies, Monoclonal↗
PubMed1993

Diagnosis of systemic candidiasis by enzyme immunoassay detection of specific antibodies to mycelial phase cell wall and cytoplasmic candidal antigens.

Diagnosis of systemic Candida infections was attempted by the use of an enzyme-linked immunosorbent assay (EIA) to detect IgG antibodies towards cell wall-bound and cytoplasmic candidal antigens. Cell wall antigens were sequentially solubilized by treatment of germinated blastoconidia of Candida albicans (ATCC 26555 strain) with beta-mercaptoethanol (beta ME extract) and digestion with Zymolyase 20T, a beta-glucanase preparation (Zymolyase extract). Protoplasts obtained after treatment with Zymolyase were osmotically lysed (cytoplasmic antigens). Sera were obtained from patients with systemic (n = 28) and superficial (n = 46) candidiasis. Control sera were obtained from normal healthy individuals (n = 31) and from hospitalized patients at low (n = 36) and at high (n = 13) risk of developing systemic candidiasis yet showing no symptoms of candidal infection. Detection of antibodies in crude sera samples by EIA using all of these antigenic extracts was highly specific (98-100%), but sensitivity of the method was low (3.5-17.8%). However, adsorption of sera with latex microspheres coated with purified Candida mannan in order to selectively remove anti-mannan antibodies prior to EIA improved the diagnostic efficiency of this test. Improvement was particularly noticeable when the beta ME extract was used as antigenic preparation, yielding a sensitivity of 89.2% and a specificity of 98.6%.

Antibodies, Fungal↗
PubMed1992

Domains of herpes simplex virus I glycoprotein B that function in virus penetration, cell-to-cell spread, and cell fusion.

Herpes simplex virus 1 glycoprotein B (gB) is one of 10 glycoproteins in the virion envelope and in the membranes of infected cells. It is required for infection of cells in culture and functions in penetration of the cell by fusing the virion envelope with the plasma membrane. In studies to map the functional domains on HSV-1 gB, we reported that epitopes of potent neutralizing antibodies cluster in three major antigenic domains, D1, D2, and D5a. D1 contains continuous epitopes in the very amino terminus of gB. D2 comprises discontinuous epitopes that are assembled on gB derivatives 457 amino acids in length. D5a contains discontinuous epitopes that map between amino acids 600 and 690. We have now analyzed the function of these domains in virion infectivity by a detailed examination of the effects of 16 neutralizing antibodies on virion adsorption, penetration, plaque development, and cell fusion. Our results are as follows. (i) Ten antibodies with complement-independent neutralizing activity blocked penetration of virions into cells but not their adsorption to the cell surface. Treating cell-bound, neutralized virus with the fusogenic agent polyethylene glycol promoted their entry into cells. (ii) Ten antibodies with complement-dependent and -independent neutralizing activity interfered with plaque development by preventing spread of virus from infected to neighboring uninfected cells. (iii) Nine neutralizing antibodies, all complement-independent, prevented cell fusion induced by strain HFEM syn. We conclude that domains mapping in three regions of gB function in penetration of virions into cells, and that most neutralizing antibodies to these domains also block cell-to-cell spread of virus and cell fusion. The findings that three complement-independent neutralizing antibodies that blocked penetration did not inhibit plaque development, and that only one of these blocked cell fusion, indicate that the cell-to-cell spread of virus and cell fusion are related processes, but not identical to the penetration function.

Antibodies, Monoclonal↗
PubMed1992

Endometrial stromal sarcoma expression of estrogen receptors, progesterone receptors and estrogen-induced srp27 (24K) suggests hormone responsiveness.

The endometrial stroma plays a decisive role in sustaining the gland epithelium along the menstrual cycle, and in preparing the microenvironment that allows embryo implantation. The stroma undergoes important changes during the menstrual cycle that affects both the cell number and differentiation. These changes are regulated by both estrogen and progesterone. Stromal sarcomas are extremely rare, occurring much less than any other uterine tumor. Their origin and biology are poorly understood. The purpose of this work was to try to learn more about the stromal physiology, and also to ascertain whether the stromal sarcoma has characteristics of hormone dependence. We studied the presence of estrogen receptors (ER), progesterone receptors (PR) and the stress-responsive protein of 27K (srp27, a protein first described as an estrogen-induced 24K protein in MCF-7 cells) in both normal stroma and stromal sarcoma. The ER and PR were measured by exchange assays. The srp 27 was studied both by Western-blot and by IHC by means of specific monoclonal antibodies. The stromal sarcomas studied showed a high concentration of both ER (96 to 116 fmol/mg prot.) and PR (565 to 995 fmol/mg prot.). These amounts of ER and PR were higher than the mean found in normal endometrium during the proliferative phase (43 and 637 fmol/mg prot., respectively), and much higher than that of the secretory phase (17 and 229 fmol/mg prot., respectively). The srp27 characterized by Western-blot in both the normal stroma and stromal sarcoma was found to be similar to the srp27 of breast cancer. The IHC results showed a very low expression of srp27 in the stroma during the proliferative phase that increases when the endometrium enters the secretory phase. The low-malignancy grade stromal sarcomas showed abundant expression of srp27, but the high-malignancy grade sarcomas showed no expression of srp27. The obtained results prove the stroma capability to express the srp27. A negative correlation between malignancy of stromal tumors and srp27 expression was found. The presence of ER and PR in some stromal sarcomas proves that they have characteristics of hormone responsiveness. These findings suggest that ER and PR assays should be routinely performed in stromal sarcomas as well as in endometrial adenocarcinomas, and also that antiestrogenic drugs might be considered for the treatment of ER and PR positive stromal sarcomas.

Antineoplastic Agents↗
PubMed1992

The estradiol induction of the microsomal low-affinity glucocorticoid binding sites (LAGS) in the male rat liver is independent of the endocrine status.

The low-affinity glucocorticoid binding sites (LAGS) are entities present in the microsomal fraction of the rat liver, capable of binding several glucocorticoids and progesterone with low affinity. The present work focuses on the demonstration that estradiol exerts a powerful stimulatory effect on the LAGS concentration. For this purpose, we studied the effect of this hormone in immature, hypothyroid, and hypophysectomized rats, three experimental models which present a very low level of LAGS. In all of them, estradiol showed ability to significantly increase the level of LAGS. The positive results obtained in hypophysectomized rats point to a direct action of estradiol on the liver. In immature rats, the estradiol induction of the LAGS was shown to be especially slow, 3-4 days after estradiol administration being necessary to obtain a significant rise in the level of LAGS. Moreover, the dose of estradiol necessary to obtain the LAGS induction in these rats (0.5 mg/100 g body weight) was clearly supraphysiological. From these data we concluded that: (A) estradiol is a powerful stimulator of the LAGS concentration, its effect probably being exerted directly on the liver; and (B) to elicit its effect, estradiol does not need the participation of other hormones known to be implicated in the endocrine regulation of the LAGS.

Animals↗
PubMed1992

Epitopes of herpes simplex virus type 1 glycoprotein B that bind type-common neutralizing antibodies elicit type-specific antibody-dependent cellular cytotoxicity.

A panel of 45 well-characterized monoclonal antibodies (MAbs) reactive to glycoprotein B (gB) of herpes simplex virus (HSV) type 1 was tested by ELISA and in antiviral functional assays (that included virus neutralization, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent complement-mediated lysis), using type 1 or 2 virus strains. All MAbs were ELISA-reactive. Eleven of the MAbs mediated neutralization and 9 mediated ADCC. All of the ADCC epitopes were contained within the amino-terminal half of the extracellular portion of gB. The ADCC reactions were strictly type 1-specific, whereas 9 of 11 neutralizing MAbs exhibited type-common activity. There was some association between the ADCC and neutralization activities, since of 12 MAbs with functional activity, 8 were positive in both assays. These results suggest that differences in the presentation of gB, and perhaps HSV-1 gB versus HSV-2 gB, on free virus and virus-infected cells determine epitope availability.

Animals↗
PubMed1992

Assembly of conformation-dependent neutralizing domains on glycoprotein B of human cytomegalovirus.

We analysed the antigenic properties of human cytomegalovirus (CMV) glycoprotein B (gB) by constructing a set of deletion derivatives lacking different portions of the carboxy terminus and reacting them with a panel of monoclonal antibodies with neutralizing activity. We found that two novel antigenic domains that bind neutralizing antibodies were assembled on truncated forms of gB, one in the amino-terminal half and one that spans the midregion of the molecule. Assembly of the conformation-dependent epitopes occurred independently of residues in the carboxy-terminal half of the molecule and did not depend on proteolytic cleavage of the molecule between amino acids 460 and 461. Ten antibodies recognized a derivative with 447 amino-terminal residues; their failure to recognize a derivative 411 residues long suggested that the amino acids required for assembly of these epitopes either were incorrectly folded, or had been totally or partially deleted in this derivative. Epitopes for three antibodies with complement-independent neutralizing activity were assembled when amino acids from the midregion of gB between residues 447 and 476 were present. Two other antigenic domains were formed by the addition of residues 476 to 618 and 619 to 645 from the carboxy-terminal half of gB. Our results underscore the importance of conformation in the antigenic structure and functional properties of both the amino- and carboxy-terminal portions of gB.

Animals↗
PubMed1992

The amino terminus of human cytomegalovirus glycoprotein B contains epitopes that vary among strains.

We mapped three antigenic domains of continuous epitopes on human cytomegalovirus (CMV) glycoprotein B (gB) by reacting a panel of independently derived monoclonal antibodies with deletion mutants expressed transiently in COS-1 cells. One of these antigenic domains, DC2, maps in the last 75 amino acids of the carboxy terminus. These epitopes are conserved in strains Towne and AD169, as well as in 19 clinical CMV isolates. ELISAs of DC2-reactive antibodies with a set of overlapping synthetic oligopeptides from the carboxy terminus showed that the epitopes of antibodies CH405-1 and CH421-5 map between amino acids 833 and 852 and that the epitope of antibody CH28-2 maps between amino acids 878 and 898. These linear epitopes were grouped into domain DC3. The third antigenic domain, DC1v, maps at the amino-terminal end of CMV strain AD169 gB but is not contained in strain Towne or in 17 of 19 clinical isolates. Epitopes in this domain are likely to map between amino acids 28 and 67, an area where differences occur in the nucleotide sequence of the gB genes from AD169 and Towne. Analysis of CMV-infected cells by flow cytometry with antibodies to the amino- and carboxy-terminal domains revealed that the amino terminus of gB is extracellular and that the carboxy terminus is not exposed on the cell surface.

Amino Acid Sequence↗
PubMed1992

The fibroblast growth factor receptor is not required for herpes simplex virus type 1 infection.

The early events mediating herpes simplex virus type 1 (HSV-1) infection include virion attachment to cell surface heparan sulfates and subsequent penetration. Recent evidence has suggested that the high-affinity fibroblast growth factor (FGF) receptor mediates HSV-1 entry. This report presents three lines of experimental evidence showing that the high-affinity FGF receptor is not required for HSV-1 infection. First, rat L6 myoblasts lacking FGF receptors were as susceptible to HSV-1 infection as L6 cells genetically engineered to express the FGF receptor. Second, a soluble FGF receptor fragment that inhibited FGF binding and receptor activation did not inhibit HSV-1 infection. Finally, basic FGF (but not acidic FGF) inhibited HSV-1 infection in L6 cells lacking FGF receptors, presumably by blocking cell surface heparan sulfates also required for HSV-1 infection. These results show that the high-affinity FGF receptor is not required for HSV-1 infection but instead that specific low-affinity basic FGF binding sites are used for HSV-1 infection.

Animals↗
PubMed1991

Mutations in conformation-dependent domains of herpes simplex virus 1 glycoprotein B affect the antigenic properties, dimerization, and transport of the molecule.

Glycoprotein B (gB) is a component of the herpes simplex virus 1 envelope that is required for penetration of virions into cells. We constructed 11 mutants in the gB gene by deleting the carboxy terminus of the molecule, inserting linkers into the ectodomain and intracellular region, and creating point mutations in cysteine residues. To identify regions of the molecule that affect the formation of epitopes on gB, we cloned the mutated genes into a eukaryotic expression vector, transfected them in COS-1 cells, and reacted the gene products in immunofluorescence and immunoprecipitation tests with a panel of monoclonal antibodies. Our findings are as follows. (i) The ectodomain of gB between residues 600 and 690 is highly antigenic and contains residues that specify 8 continuous epitopes and affect the conformation of 12 discontinuous epitopes. Residues that form a novel neutralizing domain and affect the assembly of gB dimers are contained in this region. Dimerization of gB does not require the transmembrane region or the intracellular carboxy terminus. (ii) Transport of the insertion mutants was aberrant and depended on the site mutagenized. Insertions of linkers at residues 391, 413, and 479 of the ectodomain precluded the binding of neutralizing antibodies that recognize residues in four discontinuous-epitope domains; the latter mutant in intact gB was not translocated to the cell surface. In contrast, insertions at residue 600 of the ectodomain and 810 of the intracellular domain did not affect the conformation-dependent epitopes or gB transport. (iii) Substitution of serines for cysteine residues in a discontinuous-epitope domain in the midregion of gB altered the conformation of both proximal and distal sites. Seven epitopes were lost by mutagenesis of cysteine 382 and 4 epitopes by mutagenesis of cysteine 334. Together with previous findings, these results indicate that the ectodomain of gB contains three topographically distinct neutralizing regions, one of continuous and two of discontinuous epitopes. The continuous-epitope domains that map at the amino terminus are not altered by distal mutations. In contrast, the domains of discontinuous epitopes, assembled by juxtaposing residues on the surface of gB, are affected by proximal and distal mutations that alter the antigenic structure, processing, and surface transport of gB.

Amino Acid Sequence↗
PubMed1991

A mutation in the ectodomain of herpes simplex virus 1 glycoprotein B causes defective processing and retention in the endoplasmic reticulum.

Herpes simplex virus 1 (HSV-1) glycoprotein B (gB) is one of several envelope glycoproteins required for virion infectivity and is the only one known to oligomerize into homodimers. To study the conformational constraints for translocation of HSV-1 gB to the surface of eukaryotic cells, we analyzed the transport through the exocytic pathway of the wild-type glycoprotein and of mutant forms with insertions in the ectodomain and intracellular carboxy terminus. Transient expression of the glycoproteins in COS-1 cells showed that an insertion at position 479 in the amino-terminal ectodomain of gB, shown previously by reactions with monoclonal antibodies to have altered the conformation of the molecule, also had a drastic effect on transport, precluding exit of the mutant from the endoplasmic reticulum (ER) and transport to the Golgi and the plasma membrane. The fact that the mutant, gB-(Lk479), formed dimers suggests that local changes in assembled regions caused the transport defect. Mutants containing insertions at residues 600 of the ectodomain and 810 in the intracellular domain were slightly retarded in their rate of transport from the ER to the Golgi. The glucose-regulated proteins GRP78 and GRP94, which are resident proteins of the ER, associated with partially glycosylated, faster-migrating forms of gB but not with the fully processed, more slowly migrating product. GRP78 and GRP94 formed complexes with the mutant gB-(Lk479), which was degraded in the ER. Our results indicate that GRP78, and perhaps also GRP94, acts as a chaperone in the assembly of native gB oligomers and also binds to aberrant forms of the molecule, arresting their transport from the ER and possibly serving as markers for protein degradation in this compartment of the exocytic pathway.

Animals↗
PubMed1991

Transactivation of the grp78 promoter by malfolded proteins, glycosylation block, and calcium ionophore is mediated through a proximal region containing a CCAAT motif which interacts with CTF/NF-I.

The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum (ER) protein that can form stable associations with a variety of proteins retained in the ER because of underglycosylation or other conformational changes. In this study, we provide evidence at the transcriptional level that a conformationally abnormal protein, an altered herpes simplex virus type 1 envelope protein that is retained in the ER of a mammalian cell line, transactivates the grp78 promoter. In contrast, the normal viral envelope glycoprotein does not elevate grp78 promoter activity. Using a series of 5' deletions, linker-scanning, and internal deletion mutations spanning a 100-bp region from -179 to -80, we correlate the cis-acting regulatory elements mediating the activation of grp78 by malfolded proteins, glycosylation block, and the calcium ionophore A23187. We show that they all act through the same control elements, suggesting that they share a common signal. We report here that the highly conserved grp element, while important for basal level and induced grp78 expression, is functionally redundant. The single most important element, by linker-scanning analysis, is a 10-bp region that contains a CCAAT motif. It alone is not sufficient for promoter activity, but a 40-bp region (-129 to -90) that contains this motif is essential for mediating basal level and stress inducibility of the grp78 promoter. We show that the transcription factor CTF/NF-I is able to transactivate the grp78 promoter through interaction with this CCAAT motif.

Animals↗
PubMed1991

Thyroid hormones and glucocorticoids act synergistically in the regulation of the low affinity glucocorticoid binding sites in the male rat liver.

The low affinity glucocorticoid binding sites (LAGS) have been described and partially characterized in both the nuclei and microsomes of rat liver. The LAGS concentration is under endocrine regulation, as proved by their decrease after adrenalectomy and their almost complete disappearance after hypophysectomy. This article describes new data that also implicate the thyroid hormones in the endocrine regulation of LAGS. The LAGS were measured by [3H]dexamethasone exchange assay in crude microsome suspensions of rat liver. Propylthiouracil-induced hypothyroidism (TX) provoked a 90% reduction in the LAGS levels with respect to the control value. The administration of T3 to TX rats was able to completely restore the LAGS level. On the other hand, adrenalectomy (ADX) provoked a 50% decrease in LAGS levels, and this effect could be reverted by treatment with corticosterone acetate. TX rats that were also adrenalectomized (TX-ADX) showed a LAGS level similar to that of the TX rats. However, treatment of these rats with T3 was much less effective than in TX rats. A complete restoration of the LAGS level in TX-ADX rats could be achieved only with a combined treatment of corticosterone acetate plus T3. Similar results to those obtained in TX-ADX rats were also obtained in immature or hypophysectomized rats, two experimental models known to possess very low or undetectable levels of LAGS. From these findings we conclude that: 1) thyroid hormones, as well as glucocorticoids, play an important role in the regulation of the LAGS level; 2) glucocorticoids and thyroid hormones act synergistically in the endocrine regulation of LAGS; and 3) the results obtained in the hypophysectomized rats point to a direct action of glucocorticoids and T3 on the LAGS level of the rat liver.

Adrenalectomy↗
PubMed1989

Steroid induction of low-affinity glucocorticoid binding sites in rat liver microsomes.

Rat liver contains two glucocorticoid binding sites: the high-affinity or glucocorticoid receptor (GR) and the low-affinity glucocorticoid binding sites, or LAGS. The Kd of LAGS predicts that they can be half-saturated by plasma corticosteroids in some physiological circumstances and, therefore, that they can play relevant roles in the rat liver. [3H]dexamethasone was used as a ligand in exchange assays, to study the relative abundance of GR and LAGS in cell fractions of rat liver. GR were found in the cytosol, but not in the purified nuclei, the mitochondria, or the microsomes. LAGS were found in all the particulate fractions, being more abundant in the smooth-surfaced microsomes, but they were not found in the cytosol. The LAGS of microsomes and purified nuclei showed the same Kd and also the same broad range of steroid competition with [3H]dexamethasone (cortisol = progesterone greater than dexamethasone greater than or equal to corticosterone greater than R5020 greater than DHEA greater than testosterone = estradiol). LAGS were found in liver, placenta and kidney, but not in other GR-containing organs. This suggests that the LAGS could be involved in physiological functions related to the metabolism of steroid hormones. The liver microsome LAGS were undetectable at rat birth, and became present in the 25-day-old rat. The level of LAGS then increased progressively, reaching its maximum level in the 2-3-month-old rats (10 pmol/mg protein), and declining afterwards to reach the adulthood level (5 pmol/mg protein) in 6-month-old rats. LAGS are mainly controlled by the corticoadrenal steroids, which is shown by their dramatic decrease after adrenalectomy, and especially after hypophysectomy. Many steroid hormones, like estradiol, testosterone, and corticosterone (but not progesterone) induce LAGS, estradiol being the most effective. A combination of T4 and corticosterone was more effective in inducing LAGS than when the two hormones were injected separately. It is possible to conclude that rat liver LAGS are mainly microsomal proteins, whose concentration is regulated by a multihormone system under pituitary control.

Adrenalectomy↗