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Biomedical subjects

R Herrera

Publications and source records attributed to R Herrera.

At least 19 recordsLinked to original sources
PubMed1996

Adhesion through the interaction of lymphocyte function-associated antigen-1 with intracellular adhesion molecule-1 induces tyrosine phosphorylation of p130cas and its association with c-CrkII.

The B-lymphoblastoid cell line JY undergoes homotypic aggregation in a lymphocyte function-associated antigen-1 (LFA-1)-mediated, intracellular adhesion molecule-1 (ICAM-1)-dependent manner when stimulated with phorbol 12-myristate 13-acetate or anti-LFA-1 antibodies. Under conditions that lead to cell aggregation, we observed rapid tyrosine phosphorylation of p130cas, a protein previously identified to be phosphorylated on tyrosine in both v-src- and v-crk-transformed cells. Phosphorylation of p130cas was dependent on binding of LFA-1 to its ligand, ICAM-1, as demonstrated by the use of anti-ICAM-1 antibodies. Several observations suggest that this event may be an important step in the signaling pathway initiated by LFA-1. p130cas phosphorylation was rapidly reversible upon disengagement of the LFA-1-ICAM-1 complex and required cell adhesion since binding of phorbol 12-myristate 13-acetate-stimulated JY cells to purified ICAM-1 or cross-linking of either LFA-1 or ICAM-1 was not sufficient to induce phosphorylation of p130cas. The integrin-stimulated phosphorylation of p130cas created binding sites that were recognized in vitro by the SH2 domain of c-CrkII, a key adaptor protein involved in cell differentiation and transformation. Moreover, we also showed that the LFA-1-stimulated tyrosine phosphorylation of p130cas induces the formation of a p130cas.CrkII and p130cas.CrkL complex in intact cells. This observation suggests that adhesion mediated by the interaction of LFA-1 and ICAM-1 initiates a signaling cascade that involves the activation of protein tyrosine kinases and leads to the regulation of protein-protein interaction via SH2 domains, a key process shared with growth factor signaling pathways.

Antibodies↗
PubMed1996

The product of the cbl oncogene forms stable complexes in vivo with endogenous Crk in a tyrosine phosphorylation-dependent manner.

The cellular homologs of the v-Crk oncogene product are composed exclusively of Src homology region 2 (SH2) and SH3 domains. v-Crk overexpression in fibroblasts causes cell transformation and elevated tyrosine phosphorylation of specific cellular proteins. Among these proteins is a 130-kDa protein, identified as p130cas, that forms a stable complex in vivo with v-Crk. We have explored the role of endogenous Crk proteins in Bcr-Abl-transformed cells. In the K562 human chronic myelogenous leukemia cell line, p130cas is not tyrosine phosphorylated or bound to Crk. Instead, Crk proteins predominantly associate with the tyrosine-phosphorylated proto-oncogene product of Cbl. In vitro analysis showed that this interaction is mediated by the SH2 domain of Crk and can be inhibited with a phosphopeptide containing the Crk-SH2 binding motif. In NIH 3T3 cells transformed by Bcr-Abl, c-Cbl becomes strongly tyrosine phosphorylated and associates with c-Crk. The complex between c-Crk and c-Cbl is also seen upon T-cell receptor cross-linking or with the transforming, tyrosine-phosphorylated c-Cbl. These results indicate that Crk binds to c-Cbl in a tyrosine phosphorylation-dependent manner, suggesting a physiological role for the Crk-c-Cbl complex in Bcr-Abl tyrosine phosphorylation-mediated transformation.

3T3 Cells↗
PubMed1995

The effects of high altitude on hypothalamic-pituitary secretory dynamics in men.

OBJECTIVE: Individuals adapted to high altitude (HA) have abnormalities in endocrine function and specifically in the pituitary-thyroid axis and aldosterone regulation. In this study we assessed hypothalamic-pituitary function in men adapted to high altitude living using exogenous administration of synthetic hypothalamic hormones. DESIGN: Growth hormone releasing hormone (Geref 1-29) 1 microgram/kg, TRH 500 micrograms, GHRH 100 micrograms and ovine corticotrophin releasing hormone (oCRH) 1 microgram/kg were simultaneously administered intravenously to two groups of men: 12 born and raised in the city of Pasto, Colombia, South America, located at an altitude of 2600 m in the southern Colombian Andes (HA group) and 10 men living at sea level (SL) in Tampa, Florida. MEASUREMENTS: The following hormones were measured: GH, IGF-I, TSH, T4, free T4, free T3, PRL, ACTH, beta-endorphin and cortisol. IGF binding protein-3 (IGFBP-3) and cortisol binding globulin (CBG) were also measured. RESULTS: GH response to GHRH in HA men was exaggerated compared to SL men, and IGF-I concentration was also significantly increased in the presence of normal levels of IGFBP-3. No differences in TSH or PRL responses were found following TRH. HA men had lower basal total T4 levels, but higher free T4 and free T3 concentrations. The basal ACTH concentrations in the HA men were significantly lower than SL, although the response to oCRH was still present. beta-Endorphin basal levels were similar at HA and SL but the response to oCRH at HA was blunted. At HA, basal cortisol levels as well as CBG were elevated compared to SL and, in contrast to SL, did not increase significantly after endogenous ACTH secretion. CONCLUSIONS: This study is the first description of exaggerated GH response to the administration of GHRH in HA men and also of a significant increase in IGF-I concentration in the same subjects in the presence of normal levels of IGFBP-3. An altered hypothalamic-pituitary response was found in HA men after administration of oCRH characterized by a significantly lower basal ACTH concentration at HA, although the response to oCRH was present but the beta-endorphin response to oCRH was blunted. At HA, basal cortisol levels, as well as CBG, were elevated and the cortisol levels did not significantly increase after endogenous ACTH secretion. We have characterized the differences in hypothalamic-pituitary dynamics after the administration of TRH, GnRH and oCRH in HA men comparing their response to age/sex matched SL men.

Adaptation, Physiological↗
PubMed1995

Glycosylation sites encoded by exon 2 of the human insulin receptor gene are not required for the oligomerization, ligand binding, or kinase activity of the insulin receptor.

Asparagine-linked glycosylation of the insulin receptor is required for complete biosynthesis and acquisition of function. However, the relative role of each individual glycosylation site has not been elucidated. Previously, it has been shown that removal, by site-directed mutagenesis, of the four amino terminal glycosylation sites (N16,N25,N78, and N111) results in a mutant insulin receptor that remained in the endoplasmic reticulum as an unprocessed proreceptor (Collier E., Carpentier J.-L., Beitz L., Caro L. H. P., Taylor S. I., and Gorden P. [1993] Biochemistry 32, 7818-7823). In the present study, the contribution of these independent glycosylation sites to dimerization and insulin binding has been evaluated. Chinese hamster ovary cells were transfected with the wild-type human insulin receptor cDNA, or cDNA that had Q substituted for N at one, two, or all four of these glycosylation sites. Electrophoretic characterization of the proteins immunoprecipitated from 35S-labeled cells showed that both the wild-type and the quadruple mutant receptor had similar profiles, indicating that the mutant receptor is capable of undergoing dimerization. Analysis of the biochemical properties of this mutant showed that this receptor binds insulin, but ligand binding does not result in kinase stimulation. We demonstrated that the absence of kinase activation is not a property of the mutated receptor since the wild-type proreceptor behaves in a similar manner. Only partial glycosylation in this region of the receptor is required for its targeting to the cell membrane since single and double glycosylation mutants were found processed to their alpha and beta subunits on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Activators of protein kinase C stimulate association of Shc and the PEST tyrosine phosphatase.

Using the yeast two-hybrid system, complementary DNA clones were isolated from a HeLa cell library encoding proteins that interacted with p52shc. One of these clones encoded the non-catalytic, COOH-terminal half of the cytosolic protein tyrosine phosphatase PTP-PEST. Expression of truncated forms of p52shc in the two-hybrid system revealed that the amino-terminal half of p52shc was sufficient for interaction with PTP-PEST. The p52 and p66 forms of Shc, but not the p46 form, bound to a glutathione S-transferase fusion protein containing the region of PTP-PEST isolated from the two-hybrid screen. Similarly, when HeLa cell lysates were immunoprecipitated with PTP-PEST antiserum, p52shc and p66shc proteins, but not p46shc, co-precipitated. Shc-PTP-PEST complex formation was stimulated 6-8-fold by the protein kinase C activator phorbol 12-myristate 13-acetate, while epidermal growth factor and serum had no effect. Phorbol 12-myristate 13-acetate also stimulated phosphorylation of p52shc and p66shc. The muscarinic agonist carbachol (also an activator of protein kinase C) stimulated complex formation 3-5-fold in SH-SY5Y neuroblastoma cells. These results suggest a role for PTP-PEST in G protein receptor signaling and in cross-talk between G protein receptor and tyrosine kinase receptor pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Expression of alpha 1-chimaerin (rac-1 GAP) alters the cytoskeletal and adhesive properties of fibroblasts.

The small GTP-binding protein rac-1, a member of the ras gene superfamily of GTPases, is thought to be a key component of a signal transduction pathway that mediates cell membrane ruffling and actin stress fiber formation induced by growth factors. rac-1 protein is regulated by the interplay of several activities: proteins that enhance GDP dissociation (GDP Dissociation Stimulator, GDS), inhibit nucleotide exchange (GDP Dissociation Inhibitor, GDI), or accelerate GTP hydrolysis (GTPase Activating Protein, GAP). We have assessed the relative contribution of the rac-1/GAP interactions to the overall activity of rac-1 by expressing alpha 1-chimaerin, a rac-1-specific GAP, in fibroblasts. NIH 3T3 cells were transfected with alpha 1-chimaerin cDNA-containing expression vector and stable clones were established. Extracts prepared from alpha 1-chimaerin-expressing cells showed rac-1 GAP activity that was regulated by phosphatidylserine and phorbol ester. The cells expressing alpha 1-chimaerin showed a distinct phenotype. They had altered adhesive properties as measured by their ability to bind to a fibronectin-coated glass surface, suggesting that the expression of a rac-1 GAP alters the assembly of integrin receptors, actin and cytoskeletal proteins such as vinculin and talin. Direct demonstration of this phenomenon was achieved by studying the organization of actin stress fiber and formation of focal adhesions in the alpha 1-chimaerin expressing cells following stimulation by growth factors. Mock transfected cells, upon serum or lysophosphatidic acid stimulation, organize actin as a dense array of parallel fibers running the length of the cell. This process did not take place in the cells expressing rac-1 GAP. Similarly, the formation of focal adhesions as measured by the appearance of vinculin clusters was impaired in the alpha 1-chimaerin expressing cells. These results demonstrate that expression of a GAP for rac-1 in fibroblasts produces profound changes in the cytoskeletal organization and suggest that GAP activity negatively regulates rac-1 function.

3T3 Cells↗
PubMed1994

Surgical intervention for the complications of meningococcal-induced purpura fulminans.

Purpura fulminans is an infrequent but sometimes catastrophic illness that usually complicates a viral, rickettsial or bacterial infection. This communication presents a retrospective review of 152 patients with meningococcemia hospitalized at Children's Medical Center of Dallas from January, 1983, through December, 1993. Eighteen (11.9%) of the 152 patients developed purpura fulminans. Thirteen (72%) of the 18 patients with purpura fulminans needed one or more surgeries including skin grafts, local debridement, microvascular flaps or amputations. Five patients (28%) died.

Amputation, Surgical↗
PubMed1992

[Ovarian cancer (experience with 186 patients)].

An experience with 186 ovarian carcinomas seen at the Oncology Service of The General Hospital of Mexico, between 1981 to 1990 is reported. Fifty patients, (26.9%) were in stage I; 2, (1.0%) were in stage II; 113 in stage III, (60.7%) and 21, (11.2%) were in stage IV. Epidemiological and clinical aspects are reviewed as well as results of treatment. In 137 cases, (73.6%) we have follow up and it was without evidence of disease between 12 and 60 months with a mean of 18 months in 32, (23.3%). This number includes 22 of 26 patients in stage I, (84.6%); 1 of 2 in stage II; 9 of 89, (10.1%) in stage III and 0 of 20 in stage IV. There were not significant differences in prognosis for stage I with the schemes of treatment employed: only surgery, postoperative radiotherapy and postoperative chemotherapy. Patients in stage III evolutioned without evidence of disease when the surgery left residual disease of 2 cms or less for unit: There were 1 of 59 patients treated only with surgery (1.6%); 3 of 15, (20%) treated with radiotherapy to whole abdomen plus over dosage to pelvis and 5 of 15, (33.3%) treated with two agents of chemotherapy. (cyclophosphamide plus adriamycin of cyclophosphamide plus cisplatin). In 3 of 11 stage III patients, (27.2%) with second look surgeries, we found macroscopic residual tumor.

Adult↗
PubMed1990

A direct role for c-fos in AP-1-dependent gene transcription.

Transcription factor activator protein 1 (AP-1) is a protein fraction that contains c-fos, c-jun, and several other related proteins. Although this protein fraction can stimulate transcription in vitro, the relative contributions of c-fos and c-jun to the transcriptional effect of AP-1 are not clear. In order to approach this question, we have overexpressed both proteins using a baculovirus-mediated expression system and defined their DNA-binding and transcriptional enhancement activities in vitro. Gel mobility-shift and DNase 1 footprinting assays showed that c-jun protein specifically binds to DNA through an AP-1 binding site. Under the same conditions, no detectable binding of c-fos protein was observed. However, when the DNA binding assays were performed in the presence of both c-jun and c-fos, a marked increase in the affinity of c-jun for the AP-1 site was observed. An AP-1-dependent transcription assay was used to test the capability of both proteins to stimulate correctly initiated RNA synthesis in vitro. Under our conditions, c-jun protein was capable of stimulating specific RNA transcription in an AP-1 site-dependent manner. In contrast, c-fos protein showed no detectable transcriptional activation by itself. However, a transcription assay carried out in the presence of both c-fos and c-jun proteins showed that the c-fos/c-jun complex was more active as a transcriptional regulator than c-jun protein alone. These experimental results indicate that c-fos and c-jun proteins are required to reconstitute full AP-1-dependent transcriptional activation and directly demonstrate that c-fos is a regulator of gene expression.

Animals↗
PubMed1989

A direct role for C/EBP and the AP-I-binding site in gene expression linked to adipocyte differentiation.

Adipocyte differentiation is accompanied by the transcriptional activation of many new genes, including the gene encoding adipocyte P2 (aP2), an intracellular lipid-binding protein. Using specific deletions and point mutations, we have shown that at least two distinct sequence elements in the aP2 promoter contribute to the expression of the chloramphenicol acetyltransferase gene in chimeric constructions transfected into adipose cells. An AP-I site at -120, shown earlier to bind Jun- and Fos-like proteins, serves as a positive regulator of chloramphenicol acetyltransferase gene expression in adipocytes but is specifically silenced by adjacent upstream sequences in preadipocytes. Sequences upstream of the AP-I site at -140 (termed AE-1) can function as an enhancer in both cell types when linked to a viral promoter but can stimulate expression only in fat cells in the intact aP2 promoter. The AE-1 sequence binds an adipocyte protein identical or very closely related to an enhancer-binding protein (C/EBP) that has been previously implicated in the regulation of several liver-specific genes. A functional role for C/EBP in the regulation of the aP2 gene is indicated by the facts that C/EBP mRNA is induced during adipocyte differentiation and the aP2 promoter is transactivated by cotransfection of a C/EBP expression vector into preadipose cells. These results indicate that sequences that bind C/EBP and the Fos-Jun complex play major roles in the expression of the aP2 gene during adipocyte differentiation and demonstrate that C/EBP can directly regulate cellular gene expression.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

[Pre-existing diseases as risk factors and prognosis of genito-urinary infection in pregnancy].

With the intention of reducing the abortion, preterm delivery and perinatal mortality caused by the genito urinary infection (GUI) we attempt to identify the risk factors and with those make a diagnostic test, that permits detection and prediction of GUI during gestation period. Morbidity records that suggests alterations of immune response were studied in a group of 165 pregnant women that presented GUI and were compared with 86 pregnant women without GUI. As risk antecedents, we found: spontaneous abortions of second trimester 42.4% (study group) and 6.9% (control group) (p < 0.01), repeated vaginitis 31.5% and 2.3% (p < 0.01) urinary tract infection (UTI) 29.7% and 6.92% (p < 0.01), preterm deliveries 17.6% and 3.52% (p < 0.01), puerperal infection 9.12% and 0.0% (p < 0.05), familiar diabetes 21.2% and 8.12% (p < 0.05). Psychiatric pathology 12.72 and 3.5% (p < 0.05), typhoid fever 12.1% and 3.5% (p < 0.05) and surgical infections 16.4% and 8.12% (p < 0.1). A score was elaborated assigning 1/2 point to: puerperal infections, familiar diabetes, psychiatric pathology, typhoid fever and surgical infections; 1 point to: abortion, vaginitis, UTI and preterm delivery and 2 points to: repeated abortion, UTI and preterm delivery. Patients were divided in multiparous and primiparas. In multiparous with 1 or more points we found: sensitivity 75.3%, specificity 81%, positive predictive value 89.3%, negative predictive value 60.7% and accuracy 77.1%. In primiparas with 1/2 or more points we found: sensitivity 61.8%, specificity 70.5%, positive predictive value 78.3%, negative predictive value 51.7% and accuracy 65%.

Abortion, Spontaneous↗
PubMed1988

Synthesis, purification, and characterization of the cytoplasmic domain of the human insulin receptor using a baculovirus expression system.

The cytoplasmic domain of the beta subunit of the human insulin receptor has been overexpressed in insect cells using the baculovirus expression system. A recombinant baculovirus (BIR-2) was constructed by inserting the human insulin proreceptor cDNA fragment that encodes the cytoplasmic domain of the receptor into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Synthesis of the protein (baculovirus insulin receptor kinase (BIRK), Mr 48,000) in BIR-2-infected Spodoptera frugiperda (Sf9) cells was detected 24 h after infection and maximal accumulation (2-3% of the cytosolic protein) was achieved 48-72 h post-infection. The expressed protein is active as a soluble protein tyrosine kinase, both in Sf9 cells and in vitro. Rapid purification to near homogeneity was accomplished by sequential chromatography on Fast-Q-Sepharose and phenyl-Superose with an overall yield of 35% and a specific activity with histone as substrate of 20 nmol/min/mg protein. Autophosphorylation activated the intrinsic kinase activity of BIRK and decreased its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using a combination of tryptic digestion and immunoprecipitation with specific antipeptide antisera, it was ascertained that 30-40% of the 32P incorporated into BIRK by autophosphorylation is in the carboxyl-terminal domain (that includes tyrosyl residues 1316 and 1322 of the human proreceptor). Of the remaining radioactivity, 75% is in the amino-terminal domain (that includes tyrosyl residues 953, 960, 972, 999, and 1075) and 25% is in the conserved autophosphorylation domain (including tyrosyl residues 1146, 1150, and 1151). Limited digestion of BIRK with trypsin yielded a fragment, Mr 38,000, that lacks the carboxyl-terminal domain. This fragment exhibits protein tyrosine kinase activity that is stimulated by autophosphorylation. The properties of the soluble, monomeric BIRK are similar to those of the intact, activated, oligomeric insulin receptor kinase with respect to specificity, immunoreactivity, divalent cation requirements, and specific activity. These observations coupled with the ease of producing 0.4 mg of purified enzyme from 100 ml of suspension culture suggest that BIRK will be useful for biochemical and biophysical analysis of the insulin receptor protein tyrosine kinase.

Chromatography↗
PubMed1987

Denture plaque and palatal mucosa in denture stomatitis: scanning electron microscopic and microbiologic study.

Denture plaque is an etiologic factor in denture stomatitis. In this study, denture and mucosa samples from nine patients were examined in the SEM and processed for microbiologic cultures. Denture plaque in patients with denture stomatitis showed a considerable thickness containing cocci, filaments, rods, yeasts, and desquamated epithelial cells. Some microorganisms were revealed in the connective tissue in one patient and phagocytizing polymorphonuclear leukocytes were found in the palatal mucosa of the patients with denture stomatitis.

Bacteria↗