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Biomedical subjects

H Rodriguez

Publications and source records attributed to H Rodriguez.

At least 37 records · Page 2Linked to original sources
PubMed2000

Estrogen and progesterone modulation of eosinophilic infiltration of the rat uterine cervix.

Ripening of the rat cervix involves widespread collagenolysis that follows an eosinophilic leukocyte infiltration. The hormonal control of these events is not well understood. The aims of this study were to investigate the mechanism through which progesterone (P) and 17beta-estradiol (E(2)) modulate eosinophilic invasion and to determine if this event is protein synthesis mediated. Cervical eosinophilic invasion was measured in intact rats during the second half of pregnancy and compared with values from ovariectomized (O) pseudopregnant (PSP) rats treated with P and E(2) in doses that mimicked the levels of pregnancy. Other O-PSP rats were treated with an E(2) antagonist (tamoxifen) and the antiprogestin RU-486. To study the role of protein synthesis in eosinophilic invasion of the cervix, rats were treated with actinomycin-D (an inhibitor of mRNA synthesis), and animals were sacrificed on D21 or D22 to evaluate eosinophilic invasion. Rats treated with E(2) showed high levels of infiltration and tamoxifen blocked this E(2) effect. On the other hand, P antagonized the stimulatory effects of E(2) on eosinophilic invasion, however when the P and E(2) treated rats were injected with RU-486 the inhibitory effect of P was reversed. In intact pregnant rats a sharp rise in eosinophilic infiltration was detected on D23, 20 h after the fall of serum P. Finally, E(2) treated rats injected with actinomycin-D had no invasion of eosinophils. In conclusion, the estrogen-triggered eosinophil invasion is affected by the classic estrogen receptor antagonist tamoxifen and by the mRNA synthesis blocker actinomycin-D suggesting a genomic action of E(2). Furthermore, the estrogen effect is blocked by P and this inhibition is reversed by RU-486.

Animals↗
PubMed2000

Isolated torsion of the fallopian tube in an adolescent: a case report.

Isolated torsion of the fallopian tube is an uncommon event. It is a difficult condition to evaluate clinically and surgery is often necessary to establish the diagnosis. This report focuses on a 15-year-old female who presented with acute pelvic pain, nausea, and vomiting. Pelvic ultrasound showed an adnexal mass. A diagnostic laparoscopy was performed which confirmed the diagnosis of isolated tubal torsion. Based on this experience as well as other similar reported cases, isolated torsion of the fallopian tube should be considered in the differential diagnosis of acute lower abdominal/pelvic pain in the female patient. Prompt surgical intervention may allow for preservation of the tube.

Abdominal Pain↗
PubMed2000

Localization of concealed norplant implants and implant fragments by a modified mammography technique.

A modified mammography technique for localization of concealed Norplant implants and Norplant fragments was applied to four patients, ages 18, 35, 22, and 25. A dedicated mammography unit and mammography film screen system affording high resolution and high contrast was applied under the same conditions to each patient. 26 kilovoltage peak (KVp) was used, ranging from 60 to 150 milliamperes. Using both the automatic exposure control and manual techniques, specific milliampere range depended on the specific case. The technique proved successful, as concealed Norplant capsules were exquisitely visualized by films obtained with a Siemens Mammomat 3000. Applying the same modified technique, precise location of the Norplant implants was determined using a fenestrated compression plate with an alphanumeric grid in a manner similar to hookwire localization of breast lesions under mammographic guidance. The capsules were then safely retrieved. We conclude that this modified technique may provide practitioners with valuable assistance in the exact localization of the Norplant capsule(s) or fragments, thereby facilitating their removal. Application of a modified mammography technique reveals precise localization of concealed Norplant capsules facilitating their removal.

Adolescent↗
PubMed2000

Mapping oxidative DNA damage and mechanisms of repair.

We developed a method to map oxidative-induced DNA damage at the nucleotide level using ligation-mediated polymerase chain reaction (LMPCR) technology. In vivo and in vitro DNA base modification patterns inflicted by reactive oxygen species (ROS) in the human P53 and PGK1 gene were nearly identical in vitro and in vivo. In human male fibroblasts, these patterns are independent of the transition metal used (Cu (II), Fe(II), or Cr(VI). Therefore, local probability of H2O2-mediated DNA base damage is determined primarily by DNA sequence. Moreover, in cells undergoing severe oxidative stress, extranuclear sites contribute metals that enhance nuclear DNA damage. The role of the base excision repair pathway in human cells responsible for the repair of the majority of ROS base damage is also discussed.

Ascorbic Acid↗
PubMed2000

Enzymatic activities of P450c17 stably expressed in fibroblasts from patients with the polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting approximately 5-10% of women of reproductive age. The clinical features of PCOS include oligo/anovulation, hyperandrogenemia, and hyperinsulinemia. Because P450c17 is the single enzyme catalyzing both 17alpha-hydroxylase and 17,20-lyase activities in the ovary and adrenal, some have suggested that defects in P450c17 may cause the hyperandrogenism of PCOS. Previous studies have shown that serine hyperphosphorylation of P450c17 increases the enzyme's 17,20-lyase activity, thereby favoring androgen production, and that serine phosphorylation of the insulin receptor beta-chain (IR-beta) inhibits IR-beta tyrosine phosphorylation, causing insulin resistance in vitro. We previously suggested that a gain of function mutation in a single serine kinase might cause the hyperandrogenism and insulin resistance observed in PCOS patients by excessive phosphorylation of both P450c17 and IR-beta. To test this hypothesis, we obtained fibroblasts from nine previously studied patients: three controls, three PCOS patients with normal levels of IR-beta serine phosphorylation, and three PCOS patients with increased levels of IR-beta serine phosphorylation. Initial studies showed that such skin fibroblasts could not be transfected effectively by calcium phosphate, diethylaminoethyl-dextran, lipofection or adenovirus procedures. Therefore, we employed a retroviral infection system to stably express human P450c17 in the primary cultures of fibroblast cells from the PCOS patients and controls and measured the resulting 17alpha-hydroxylase and 17,20-lyase activity. The cells were analyzed in a blinded fashion until the study was complete. The 17alpha-hydroxylase and 17,20-lyase activities in each cell line correlated well with the amount of P450c17 protein expressed, but there was no correlation between either enzymatic activity (or their ratio) with the clinical phenotype of the cells' donors even when results were corrected for the number of P450c17 complementary DNA inserts per cell line. Overnight incubation with 1 micromol/L insulin also did not affect enzymatic activity. Thus, we were unable to find evidence for the hypothesis that in PCOS a single abnormal kinase hyperphosphorylates both IR-beta, causing insulin resistance, and P450c17, causing hyperandrogenism. However, because fibroblasts do not normally express either P450c17 or the accessory proteins needed for its optimal activity, these results cannot exclude a role for serine phosphorylation in the hyperandrogenism and insulin resistance of PCOS.

Adolescent↗
PubMed2000

Association of a polymorphism in the TNFR2 gene with low bone mineral density.

Previous genetic linkage data suggested that a gene on chromosome 1p36.2-36.3 might be linked to low bone mineral density (BMD). Here, we examine the gene for tumor necrosis factor receptor 2 (TNFR2), a candidate gene within that interval, for association with low BMD in a group of 159 unrelated individuals. We assess two polymorphic sites within the gene, a microsatellite repeat within intron 4, and a three-nucleotide variation in the 3' untranslated region (UTR) of the gene. The latter has five alleles of which the rarest allele is associated with low spinal BMD Z score (p = 0.008). Lowest mean spinal BMD Z scores were observed for individuals having genotypes that were heterozygous for the rarest allele. No homozygotes for the rarest allele were observed. Preliminary analysis suggests that there is a difference in the genotype frequency distribution between the group with low BMD and a control group.

3' Untranslated Regions↗
PubMed2000

An in vitro model to evaluate the effect of an organophosphoric agropesticide on cell proliferation in mouse seminiferous tubules.

Recently, there has been public concern about the toxic effects of organophosphoric pesticides (OP) upon human and animal populations. Since the seminiferous epithelium is an actively proliferating tissue, it was of interest to study germ cell proliferation in the isolated seminiferous tubules of mice that were cultured in the presence of Parathion or paraoxon, its metabolite. Eighteen 3-month-old CF-1 male mice were used. Paraoxon (PO) and Parathion (PT) were added to cultures of seminiferous tubules in the following groups: (1) Ham F-10 medium pH 7.4 (M) (control); groups 2 to 5, M + decreasing doses of either PO or PT (0.8; 0.4; 0.04; 0.004; 0.0004 mM). Each group consisted of six mice. Incubation of the tubules was carried out for 5 h at 35 degrees C, 5% CO2, 95% air. One hour before the end of incubation 5 microCi of 3H-thymidine was added to the cultures. DNA uptake was measured by scintillation counting. PO and PT at concentrations of 0.8 mM elicited a sharp decrease in testicular DNA synthesis. Recuperation at concentrations under 0.4 mM was different. With PO it was rapid, possibly due to the high detoxificative ability of the testis, which contains high quantities of the enzyme 'type A esterases' that hydrolyses PO. At concentrations lower than PO, PT has an inhibitory effect upon germ cell proliferation, which deserves further research.

Animals↗
PubMed1999

Mapping of peroxyl radical induced damage on genomic DNA.

We have examined the DNA damage produced by reaction of peroxyl radicals with human fibroblast DNA. DNA damage consisted of both strand breaks and base modifications. The extent of strand breaks and base modifications induced as a function of peroxyl radical concentration was determined by quantitation of fragment size distributions using denaturing glyoxal-agarose gel electrophoresis. Both strand breaks and base modifications increased in a log linear fashion with respect to peroxyl radical concentration. Oxidative base modifications were observed to occur to a greater extent than strand breaks at every concentration measured. The sequence-specific distribution of peroxyl radical induced base damage was mapped for 803 nucleotide positions using the method of ligation mediated PCR. A total of 87% of all guanine positions in the examined sequences was found to be significantly oxidized. The order of reactivity of DNA bases toward oxidation by peroxyl radicals was found to be G >> C > T. Adenine is essentially unreactive. The yield of oxidative base modifications at guanines and cytosines by peroxyl radicals depends on the exact specification of 5' and 3' flanking bases in a polarity dependent manner. Every guanine in the 5'XGC3' motif was found to be oxidized, where X is any 5' neighbor. In contrast, 5' and 3' purine flanks drastically reduced the extent of peroxyl radical G oxidation. The pattern of base modification and the influence of nearest neighbors differs substantially from that previously reported for hydrogen peroxide damage mediated by low valent transition metal ions for the identical DNA sequences.

3' Untranslated Regions↗
PubMed1999

Molecular characterization of two light-induced, gamete-specific genes from Chlamydomonas reinhardtii that encode hydroxyproline-rich proteins.

Gametic differentiation in Chlamydomonas reinhardtii is a two-step process, which is controlled by the sequential action of the two extrinsic signals, nitrogen starvation and blue light. The gamete-specific genes GAS28 and GAS29 are expressed in the late phase of gametogenesis. Their light-induced expression is restricted to cells that have completed the first, nitrogen starvation-activated, phase of differentiation. A comparison of the two genes revealed striking similarities as well as differences. Their most prominent shared feature is an extended sequence homology of over 90% in their 5'-untranslated regions, suggesting a role in translational regulation. GAS28 and GAS29 both encode hydroxyproline-rich proteins (HRGPs) of very similar sizes that exhibit typical features of volvocalean cell wall constituents. GAS28 shows a high degree of homology with the Volvox pherophorin gene family, suggesting a relationship between these genes.

Amino Acid Sequence↗
PubMed1998

Mutagenesis by peroxy radical is dominated by transversions at deoxyguanosine: evidence for the lack of involvement of 8-oxo-dG1 and/or abasic site formation.

Oxidative damage of DNA by endogenously generated oxygen radicals contributes to the mutagenic process. Hydroxy, alkoxy, and peroxy radicals all have the potential to react with DNA, giving rise to strand breaks and potentially mutagenic oxidative base damage. Although reactions of the hydroxy radical with DNA have been well studied, far less is known about the reactivities of these other radicals with DNA and their mutation-inducing potential. Frequencies of DNA base modifications and strand break densities caused by peroxy radical (ROO*) oxidation were measured by glyoxal gel electrophoretic analysis. We report the spectrum of mutations induced in Escherichia coli upon transfection with peroxy radical treated DNA carrying the lacZ alpha gene as a reporter. Transfection of DNA exposed to micromolar amounts of peroxy radical resulted in a 30-fold increase in mutation frequency in non-SOS-inducible cells. Sequencing analysis of DNA isolated from mutants showed that among base substitution mutants 88% consisted of transversions at G, with a nearly equal number of G --> C and G --> T mutants. Transition mutations were rarely detected, in contrast to control experiments. Electrophoretic analysis of peroxy radical treated DNA exposed to NaOH, Nth, and Fpg proteins demonstrated that abasic sites are not formed to any detectable degree. The oxidative G lesions are sensitive to digestion by the Fpg protein. We were unable to detect the formation of 8-oxo-dG by HPLC/electrochemical analysis of peroxy radical oxidation of dG, suggesting that the G --> T transversions were not caused by this base lesion.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1998

Antigenic homology of HIV-1 GP41 and human platelet glycoprotein GPIIIa (integrin beta3).

Fifty-eight of 89 serum samples (65.17%) from HIV-1-infected individuals at various disease stages contain antibodies that react with a platelet peptide located in the cytoplasmic domain of integrin beta3, glycoprotein GPIIIa (aa749-761; sequence DRKEFAKFEEERA). Rabbit polyclonal antibodies raised against the synthetic platelet peptide also react with the structurally homologous HIV-1 gp41-derived peptide (EKNEQELLELDKW(A)) and bind to a Western blot band with molecular weight corresponding to HIV-1 gp41. These findings point to molecular mimicry between HIV-1 and a human membrane protein found in platelets and other cells that could be of pathologic consequence.

Amino Acid Sequence↗
PubMed1998

Large scale isolation of genes as DNA fragment lengths by continuous elution electrophoresis through an agarose matrix.

A simple procedure for the isolation of genes as DNA fragment lengths is described. By using a commercial continuous elution protein electrophoresis apparatus and incorporating an agarose matrix, preparative scale amounts (300 microg) of DNA can be purified by fragment lengths from a mixture of genomic fragment lengths with high recovery yields. Fractions corresponding to unique fragment length ranges are screened for individual genes by dot-blot analysis. Using this technique, we have isolated two genes: PGK1, a single copy housekeeping gene; and p53 (exons 3-11), a tumor suppressor gene, whose DNA fragment lengths elute at 4 and 7.5 kbp, respectively, from a single preparative run. As an example of the utility of the technique, we applied it to improving the sensitivity of the ligation-mediated polymerase chain reaction (LMPCR)--a nucleic acid amplification technique used for the detection and mapping of DNA damage along genes. By eliminating excess nontargeted genomic DNA, the agarose matrix continuous elution electrophoresis (CEE) procedure provided a 24-fold increase in signal strength attributable to base damage caused by exposing DNA to reactive oxygen species. Genomic DNA fragment length purification by agarose matrix CEE should also prove useful in other research areas requiring gene isolation, such as genomics and molecular biology.

DNA↗
PubMed1998

Mapping oxidative DNA damage at nucleotide level.

DNA damage induced by reactive oxygen species (ROS) is considered an important intermediate in the pathogenesis of human conditions such as cancer and aging. By developing an oxidative-induced DNA damage mapping version of the Ligation-mediated polymerase chain reaction (LMPCR) technique, we investigated the il vivo and in vitro frequencies of DNA base modifications caused by ROS in the human p53 and PGK1 gene. Intact human male fibroblasts were exposed to 50mM H2O2, or purified genomic DNA was treated with 5 mM H2O2, 100 microM Ascorbate, and 50 microM, 100 microM, or 100 microM of Cu(II), Fe(II), or Cr(VI) respectively. The damage pattern generated in vivo was nearly identical to the in vitro Cu(II) or Fe(III) damage patterns; damage was non-random with guanine bases heavily damaged. Cr(VI) generated an in vitro damage pattern similar to the other metal ions, although several unique thymine positions were damaged. Also, extra nuclear sites are a major contributor of metal ions (or metal-like ligands). These data show that the local probability of H2O2-mediated DNA damage is determined by the primary DNA sequence, with chromatin structure having a limited effect. The data suggest a model in which DNA-metal ion binding domains can accommodate different metalions. LMPCR's unique aspect is a blunt-end ligation of an asymmetric double-stranded linker, permitting exponential PCR amplification. An important factor limiting the sensitivity of LMPCR is the representation of target gene DNA relative to non-targeted genes; therefore, we recently developed a method to eliminate excess non-targeted genomic DNA. Restriction enzyme-digested genomic DNA is size fractionated by Continuous Elution Electrophoresis (CEE), capturing the target sequence of interest. The amount of target DNA in the starting material for LMPCR is enriched, resulting in a stronger amplification signal. CEE provided a 24-fold increase in the signal strength attributable to strand breaks plus modified bases created by ROS in the human p53 and PGK1 genes, detected by LMPCR. We are currently taking advantage of the enhanced sensitivity of target gene-enriched LMPCR to map DNA damage induced in human breast epithelial cells exposed to non-cytotoxic concentrations of H2O2.

Ascorbic Acid↗
PubMed1997

Metal ion-dependent hydrogen peroxide-induced DNA damage is more sequence specific than metal specific.

The frequency of oxidative base damage along the human p53 and PGK1 genes was determined at nucleotide resolution by cleaving DNA at oxidized bases with endonuclease III and formamidopyrimidine DNA glycosylase and then using the ligation-mediated PCR technique to map induced break frequency. Damage was induced either in vivo by exposing cultured human male fibroblasts to H2O2 or in vitro by exposing purified genomic DNA to H2O2 plus ascorbate in the presence of Cu(II), Fe(III), or Cr(VI) metal ions. All four base damage patterns from either in vivo or in vitro treatments were nearly identical in both regions of the genome. The frequency of base damage varied along the DNA, with guanine being the most commonly damaged base. In the Fe(III)-mediated in vitro reactions, single-stranded breaks were almost completely suppressed by addition of sucrose, which facilitated mapping of base damage. The in vitro base damage pattern generated by Cr(VI), ascorbate, and H2O2 was similar to that of the other metal ions, with the exception of several unique positions; these were heavily damaged only in the presence of Cr(VI). Isolated nuclei suffered little oxidative base damage in the presence of ascorbate and H2O2, and we conclude that during H2O2 in vivo treatment of cells, metal ions (or metal-like ligands) are freed from the cytoplasm to migrate into the nucleus and supply the redox cycling ligands necessary for oxidative base damage. These data simplify the complexity of H2O2-induced oxidative damage and mutagenesis studies by demonstrating the commonality of damage catalyzed by different transition metal ions and by showing that the pattern of H2O2-mediated oxidative base damage is determined almost entirely by the primary DNA sequence, with chromatin structure having a limited effect. Our data suggest a model for base damage in which DNA-metal ion binding domains can equally accommodate a variety of different metal ions and thus are a key factor in determining the local probability of DNA damage.

Ascorbic Acid↗
PubMed1997

Stimulated human leukocytes cause activating mutations in the K-ras protooncogene.

Human tissues which are chronically infiltrated with inflammatory leukocytes are more likely to develop malignancies than non-inflamed tissues, however the mechanism(s) by which leukocytes contribute to carcinogenesis is unknown. Stimulated human leukocytes release superoxide anion and hydrogen peroxide which, in the presence of iron, can be converted into the potent oxidant, hydroxyl radical (.OH). Previous studies have shown that leukocyte-derived .OH (or a .OH-like species) can cause DNA damage, however a relationship between leukocyte-induced DNA damage and carcinogenesis has not been established. The present report demonstrates that leukocyte-derived .OH-induced DNA damage can cause K-ras oncogene activation, and suggests that there may be a characteristic pattern of .OH-induced K-ras oncogene activation. Since activation of the K-ras oncogene is believed to play a crucial role in the pathogenesis of many human malignancies, .OH-induced K-ras oncogene activation could be an important mechanism by which human leukocytes contribute to carcinogenesis.

3T3 Cells↗
PubMed1997

A hot spot for hydrogen peroxide-induced damage in the human hypoxia-inducible factor 1 binding site of the PGK 1 gene.

Using ligation-mediated polymerase chain reaction to separately map the distribution of induced oxidized bases and strand breaks along the human PGK1 promoter at nucleotide resolution, we previously described the pattern of oxidative DNA damage induced in vitro by Cu(II)/ascorbate/H2O2 [J. Biol. Chem. 270, 17633-17640 (1995)]. Here we report that the pattern of in vivo base damage caused by H2O2 is almost identical to that of the previously used in vitro system with the exception of transcription factor-associated footprints. An unusually strong positive footprint for both strand breaks and oxidized bases is associated with binding of the hypoxia-inducible transcription factor-1. Base damage at this footprint was 52-91% repaired in 24 h, which was similar to the global base damage repair rate. However, strand breaks at this footprint were only 39-55% repaired in 24 h or approximately 100-fold slower than the global strand break repair rate.

Ascorbic Acid↗