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Biomedical subjects

A M Rodriguez

Publications and source records attributed to A M Rodriguez.

At least 19 recordsLinked to original sources
PubMed2002

Opposite actions of testosterone and progesterone on UCP1 mRNA expression in cultured brown adipocytes.

The brown adipose tissue (BAT) thermogenic response to diet-induced obesity and cold has been found to be gender dependent. In the present work, we aimed to investigate the effects of the main physiological male and female sex hormones, i.e. testosterone, progesterone and 17-beta-estradiol, on the expression of uncoupling protein I (UCP1)--the main mediator of BAT thermogenesis--and on UCP2 and lipid accumulation in rodent brown adipocytes differentiated in culture. Testosterone-treated cells showed fewer and smaller lipid droplets than control cells and a dose-dependent inhibition of UCP1 mRNA expression, under adrenergic stimulation by norepinephrine (NE). These effects were reverted by the androgen receptor antagonist flutamide, suggesting they are dependent, at least in part, on the androgen receptor. Progesterone- and 17-beta-estradiol-treated cells showed more and larger lipid droplets and progesterone stimulated NE-induced UCP1 mRNA expression at the lower concentration tested, but not at higher concentrations, suggesting that for brown adipocytes, this hormone is dose dependent. 17-beta-Estradiol did not have any remarkable effect either on UCP1 or UCP2 mRNA expression. Interestingly, the specific progesterone receptor antagonist RU486 induced UCP1 and UCP2 mRNAs, including UCP1 mRNA expression in non-NE-treated brown adipocytes, suggesting a profound effect of this antiprogestagen on brown adipocyte thermogenic capacity. Thus, are conclude that testosterone, 17-beta-estradiol, progesterone and RU486 have distinct actions on brown adipocytes, thus modulating UCP1 and UCP2 mRNA expression and/or lipid accumulation, and that sex hormones are factors that may explain in part the gender-dependent BAT thermogenic response.

Adipose Tissue, Brown↗
PubMed2001

Manganese superoxide dismutase signals matrix metalloproteinase expression via H2O2-dependent ERK1/2 activation.

Manganese-superoxide dismutase (Sod2) removes mitochondrially derived superoxide (O(2)) at near-diffusion limiting rates and is the only antioxidant enzyme whose expression is regulated by numerous stimuli. Here it is shown that Sod2 also serves as a source of the intracellular signaling molecule H(2)O(2). Sod2-dependent increases in the steady-state levels of H(2)O(2) led to ERK1/2 activation and subsequent downstream transcriptional increases in matrix metalloproteinase-1 (MMP-1) expression, which were reversed by expression of the H(2)O(2)-detoxifying enzyme, catalase. In addition, a single nucleotide polymorphism has recently been identified (1G/2G) at base pair--1607 that creates an Ets site adjacent to an AP-1 site at base pair --1602 and has been shown to dramatically enhance transcription of the MMP-1 promoter. Luciferase promoter constructs containing either the 1G or 2G variation were 25- or 1000-fold more active when transiently transfected into Sod2-overexpressing cell lines, respectively. The levels of MMP-2, -3, and -7 were also increased in the Sod2-overexpressing cell lines, suggesting that Sod2 may function as a "global" redox regulator of MMP expression. In addition, Sod2(-/+) mouse embryonic fibroblasts failed to respond to the cytokine-mediated induction of the murine functional analog of MMP-1, MMP-13. This study provides evidence that the modulation of Sod2 activity by a wide array of pathogenic and inflammatory stimuli may be utilized by the cell as a primary signaling mechanism leading to matrix metalloproteinase expression.

Animals↗
PubMed2001

Flank transparency: transparent filters seen in dynamic two-color displays.

Flank transparency is the perception of a colored transparent filter evoked by apparent-motion displays containing as few as two colors. Displays of flank transparency contain a random array of line segments placed on a uniform background. Small flanks are added to the line segments if the segments fall in the interior of a moving virtual shape, such as a virtual disk. This leads to the perception of a colored transparent disk with well-defined boundaries moving over the array of lines. Current qualitative and quantitative models of luminance and color conditions for perceptual transparency do not account for flank transparency as they require displays containing at least three different colors.

Color Perception↗
PubMed2001

Potential mechanisms for the inhibition of tumor cell growth by manganese superoxide dismutase.

Studies from many laboratories have shown that overexpression of manganese superoxide dismutase (MnSOD) inhibits the growth of numerous tumor cell types. The inhibition of tumor cell growth can be attributed to the increase in the steady-state levels of H2O2 as a result of the increased dismuting activity of MnSOD. Here we demonstrate that overexpression of MnSOD enhances the activity of the superoxide (O2*-)-sensitive enzyme aconitase, decreases the intracellular GSH/GSSG ratio, and dose-dependently inhibits pyruvate carboxylase activity. Thus, alterations in the steady-state concentrations of mitochondrial O2*- and H2O2 as a result of MnSOD overexpression can alter the metabolic capacity of the cell leading to inhibition of cell growth. Furthermore, we propose that MnSOD overexpression can modulate the activity of nitric oxide (*NO) by preventing its reaction with O2*-. This hypothesis suggests that the redox environment of the mitochondria can be altered to favor the activity of *NO rather than peroxynitrite (ONOO-) and may explain the enhanced toxicity of *NO-generating compounds toward MnSOD-overexpressing cell lines. These findings indicate that therapeutic strategies targeted at overexpressing MnSOD in tumor tissue may be more effective when used in combination with agents that deplete the oxidant-buffering and enhance the *NO-generating capacity of the tumor and host, respectively.

Aconitate Hydratase↗
PubMed2001

Benzylsuccinate synthase of Azoarcus sp. strain T: cloning, sequencing, transcriptional organization, and its role in anaerobic toluene and m-xylene mineralization.

Biochemical studies in Azoarcus sp. strain T have demonstrated that anaerobic oxidation of both toluene and m-xylene is initiated by addition of the aromatic hydrocarbon to fumarate, forming benzylsuccinate and 3-methyl benzylsuccinate, respectively. Partially purified benzylsuccinate synthase was previously shown to catalyze both of these addition reactions. In this study, we identified and sequenced the genes encoding benzylsuccinate synthase from Azoarcus sp. strain T and examined the role of this enzyme in both anaerobic toluene and m-xylene mineralization. Based on reverse transcription-PCR experiments and transcriptional start site mapping, we found that the structural genes encoding benzylsuccinate synthase, bssCAB, together with two additional genes, bssD and bssE, were organized in an operon in the order bssDCABE. bssD is believed to encode an activating enzyme, similar in function to pyruvate formate-lyase activase. bssE shows homology to tutH from Thauera aromatica strain T1, whose function is currently unknown. A second operon that is upstream of bssDCABE and divergently transcribed contains two genes, tdiS and tdiR. The predicted amino acid sequences show similarity to sensor kinase and response regulator proteins of prokaryotic two-component regulatory systems. A chromosomal null bssA mutant was constructed (the bssA gene encodes the alpha-subunit of benzylsuccinate synthase). This bssA null mutant strain was unable to grow under denitrifying conditions on either toluene or m-xylene, while growth on benzoate was unaffected. The growth phenotype of the DeltabssA mutant could be rescued by reintroducing bssA in trans. These results demonstrate that benzylsuccinate synthase catalyzes the first step in anaerobic mineralization of both toluene and m-xylene.

Anaerobiosis↗
PubMed2001

Cloning of the mouse sodium iodide symporter and its expression in the mammary gland and other tissues.

Iodide concentration in milk by mammals is a necessary step for thyroid hormone synthesis by the newborn. With the purpose of using the mouse as an animal model to analyse the role of the sodium iodide symporter (NIS) in iodide transport and its regulation in the mammary gland, mouse NIS (mNIS) cDNA was isolated from lactating mice. The cloned sequence shows an open reading frame of 1854 nucleotides encoding a protein of 618 amino acids highly homologous to the rat and human NIS (95% and 81% identity respectively). Expression of mNIS in cultured mammalian cells induced cellular iodide accumulation. This iodide uptake process is sodium dependent and inhibited by thiocyanate and perchlorate. Tissue distribution analysis revealed that mNIS mRNAs are predominantly expressed in thyroid, stomach and in the lactating mammary gland and are present to a lower extent in several other tissues. Our data show for the first time that the level of mNIS mRNA is upregulated in the mammary gland during lactation.

Amino Acid Sequence↗
PubMed2000

Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing.

During hypoxia, hypoxia-inducible factor-1alpha (HIF-1alpha) is required for induction of a variety of genes including erythropoietin and vascular endothelial growth factor. Hypoxia increases mitochondrial reactive oxygen species (ROS) generation at Complex III, which causes accumulation of HIF-1alpha protein responsible for initiating expression of a luciferase reporter construct under the control of a hypoxic response element. This response is lost in cells depleted of mitochondrial DNA (rho(0) cells). Overexpression of catalase abolishes hypoxic response element-luciferase expression during hypoxia. Exogenous H(2)O(2) stabilizes HIF-1alpha protein during normoxia and activates luciferase expression in wild-type and rho(0) cells. Isolated mitochondria increase ROS generation during hypoxia, as does the bacterium Paracoccus denitrificans. These findings reveal that mitochondria-derived ROS are both required and sufficient to initiate HIF-1alpha stabilization during hypoxia.

Androstadienes↗
PubMed2000

Interaction of Leishmania mexicana promastigotes with the plasminogen-plasmin system.

The binding of human plasminogen and plasmin to the promastigote form of Leishmania mexicana was investigated. L. mexicana was capable to bind both molecules, the binding being inhibited by epsilon-aminocaproic acid. Scatchard plot analysis revealed a dissociation constant (Kd) value of 2.4+/-0.8 microM and 0.9+/-0.1 x 10(4) binding sites per cell for plasminogen and a Kd value of 1.2+/-0.4 microM and 1.6+/-0.2 x 10(5) binding sites per cell for plasmin. C-terminal lysine residues are involved in plasminogen binding to cells, since carboxypeptidase B treatment reduced this binding by 34%. Ligand blotting analysis showed a group of proteins, with molecular masses between 105 and 115 kDa, capable to interact with plasminogen. Zymogram analysis showed that the protease activity acquired by L. mexicana, due to the interaction with either plasminogen or plasmin, comprises an important fraction of the total protease activity at pH 7.7. Plasminogen activation by tissue-type plasminogen activator (t-PA) was enhanced by the presence of L. mexicana promastigotes. These results raise the question whether the interaction of L. mexicana with components of the fibrinolytic system is involved in the virulence of the parasite.

Animals↗
PubMed2000

Survival of various implant-supported prosthesis designs following 36 months of clinical function.

BACKGROUND: The use of endosseous dental implants to replace natural teeth lost to trauma, dental caries, or periodontal disease has become a predictable form of prosthetic treatment since gaining popularity in the early 1980s. While numerous clinical studies have focused on the survival of implants, few address the survival of different prosthesis designs. METHODS: Beginning in 1991, 882 prostheses supported by more than 2,900 implants (687 patients) were placed by the Department of Veterans Affairs Dental Implant Clinical Research Group (DICRG). These prostheses were divided into five research strata based on arch location. The recommended design for each stratum was: bar-supported overdenture (maxillary completely edentulous); screw-retained hybrid denture (mandibular completely edentulous); screw-retained fixed partial denture (mandibular and maxillary posterior partially edentulous); and cemented single crown (maxillary anterior single tooth). Alternative overdenture designs were utilized in the edentulous arches when the recommended prosthesis could not be fabricated. Prosthesis success rates for the research strata were calculated for an observation time of up to 36 months following prosthesis placement. RESULTS: Success rates for the maxillary edentulous stratum ranged from 94.6% for the bar-retained overdenture supported by five to six fixtures to 81.8% for the cap-retained overdenture. The mandibular edentulous strata produced success rates of 98.1% for the fixed hybrid prosthesis to 91.7% for the cap-retained prosthesis. Success rates for maxillary and mandibular posterior fixed partial dentures were 94.3% and 92.6%, respectively, while the maxillary anterior single-tooth prosthesis yielded a success rate of 98.1% for the 36-month observation period. CONCLUSIONS: The recommended prosthesis designs investigated in this study proved to be reliable, with encouraging success rates for an observation period of 36 months following placement.

Alloys↗
PubMed1999

Overexpression of catalase in cytosolic or mitochondrial compartment protects HepG2 cells against oxidative injury.

HepG2 cells were transfected with vectors containing human catalase cDNA and catalase cDNA with a mitochondrial leader sequence to allow comparison of the effectiveness of catalase overexpressed in the cytosolic or mitochondrial compartments to protect against oxidant-induced injury. Overexpression of catalase in cytosol and in mitochondria was confirmed by Western blot, and activity measurement and stable cell lines were established. The intracellular level of H(2)O(2) induced by exogenously added H(2)O(2) or antimycin A was lower in C33 cell lines overexpressing catalase in the cytosol and mC5 cell lines overexpressing catalase in the mitochondria as compared with Hp cell lines transfected with empty vector. Cell death caused by H(2)O(2), antimycin A, and menadione was considerably suppressed in both the mC5 and C33 cell lines. C33 and mC5 cells were also more resistant to apoptosis induced by H(2)O(2) and to the loss of mitochondrial membrane potential induced by H(2)O(2) and antimycin A. In view of the comparable protection by catalase overexpressed in the cytosol versus the mitochondria, catalase produced in both cellular compartments might act as a sink to decompose H(2)O(2) and move diffusable H(2)O(2) down its concentration gradient. The present study suggests that catalase in cytosol and catalase in mitochondria are capable of protecting HepG2 cells against cytotoxicity or apoptosis induced by oxidative stress.

Antimycin A↗
PubMed1999

cDNA and protein characterization of human MAGE-10.

MAGE genes are frequently expressed in several types of human malignancy and code for antigens recognized by cytotoxic T lymphocytes. We have previously described a monoclonal antibody (MAb), named 6C1, that recognizes the MAGE-1 protein and cross-reacts with a 72-kDa protein present in lysates of melanoma cells such as MZ2-MEL. To identify this protein, we have screened an expression library prepared from MZ2-MEL cells. Several clones that encoded a protein recognized by antibody 6C1 contained a sequence identical to that of MAGE-10, another member of the MAGE-A gene family. Full-length MAGE-10 cDNA clones, obtained after screening additional cDNA melanoma libraries, were found to be approximately 2.5 kb in length. In vitro translation and transient transfection experiments indicated that MAGE-10 codes for a protein of approximately 72 kDa. This product was recognized by MAb 6C1 as well as by a polyclonal serum raised against a MAGE-10 peptide, thus demonstrating its identity with MAGE-10. Analysis of MAGE-10 mRNA by RT-PCR confirmed its presence in testis and placenta but not in other normal tissues. Expression of MAGE-10 in melanoma tumors was found to parallel that of MAGE-1. Western blot analysis with the polyclonal anti-MAGE-10 antibody showed the presence of MAGE-10 in lysates of purified trophoblast cells. Immuno-cytochemistry of cultured melanoma cells indicated that MAGE-10 is a nuclear protein.

Antibodies, Monoclonal↗
PubMed1999

Brown adipose tissue response to cafeteria diet-feeding involves induction of the UCP2 gene and is impaired in female rats as compared to males.

Noradrenaline-dependent brown adipose tissue (BAT) thermogenesis is activated by the cold and excess energy intake, largely depends on the activity of the uncoupling protein 1 (UCP1), and is mediated mainly through the beta3-adrenoceptor (beta3-AR). We investigated the expression of ucp2, a gene that encodes a putative UCP1-like uncoupling protein, along with that of ucp1 and beta3-ar, in the interscapular BAT (IBAT) of male and female rats chronically fed a cafeteria diet. After 3 months on this diet, male rats attained a 34% excess body mass and showed IBAT hypertrophy and increased IBAT thermogenic potential, in terms of both UCP1 and UCP2 mRNA expression (both by 1.6-fold), UCP1 protein expression (by 1.75-fold) and GDP binding to IBAT mitochondria (by 2.2-fold); female rats attained a larger excess body weight (50%) and their IBAT, although hypertrophied, showed no signs of increased thermogenic potential per gram of tissue. Interestingly, the IBAT of female rats was already activated compared to males. Treatment of mouse brown adipocytes in primary culture with noradrenaline also triggered a dose-dependent increase of the levels of UCP1 mRNA and UCP2 mRNA. Retroregulatory down-regulation of the beta3-AR mRNA levels was found in the two models used. The results support a physiological role for UCP2, along with UCP1, in rodent BAT thermogenesis.

Adipose Tissue, Brown↗
PubMed1999

Genetic and molecular analysis of cglB, a gene essential for single-cell gliding in Myxococcus xanthus.

Gliding movements of individual isolated Myxococcus xanthus cells depend on the genes of the A-motility system (agl and cgl genes). Mutants carrying defects in those genes are unable to translocate as isolated cells on solid surfaces. The motility defect of cgl mutants can be transiently restored to wild type by extracellular complementation upon mixing mutant cells with wild-type or other motility mutant cells. To develop a molecular understanding of the function of a Cgl protein in gliding motility, we cloned the cglB wild-type allele by genetic complementation of the mutant phenotype. The nucleotide sequence of a 2.85-kb fragment was determined and shown to encode two complete open reading frames. The CglB protein was determined to be a 416-amino-acid putative lipoprotein with an unusually high cysteine content. The CglB antigen localized to the membrane fraction. The swarming and gliding defects of a constructed DeltacglB mutant were fully restored upon complementation with the cglB wild-type allele. Experiments with a cglB allele encoding a CglB protein with a polyhistidine tag at the C terminus showed that this allele also promoted wild-type levels of swarming and single-cell gliding, but was unable to stimulate DeltacglB cells to move. Possible functions of CglB as a mechanical component or as a signal protein in single cell gliding are discussed.

Amino Acid Sequence↗
PubMed1999

Nitric oxide enhances the manganese superoxide dismutase-dependent suppression of proliferation in HT-1080 fibrosarcoma cells.

The overexpression of manganese superoxide dismutase (MnSOD), an enzyme that catalyzes the removal of superoxide (O2*-) from the mitochondria, has been shown to be closely associated with tumor regression in vivo and loss of the malignant phenotype in vitro. To investigate the mechanism by which MnSOD overexpression mediates this reversal, we have established 29 independent, clonal MnSOD-overexpressing HT-1080 fibrosarcoma cells. MnSOD activity is inversely correlated with cell proliferation in our cell lines. Incubating cells in 3% oxygen can prevent the inhibition of cellular proliferation mediated by MnSOD, suggesting that oxygen is a prerequisite component of the MnSOD-dependent proliferative inhibition. Confocal laser microscopy was used in combination with the oxidant-sensitive fluorescent dyes dihydrorhodamine-123, dihydroethidium, and 2',7'-dichlorodihydrofluorescein diacetate to determine the oxidizing capacity of the MnSOD-overexpressing cells. When compared with parental or control cell lines, there was a significant decrease in the rate of oxidation of the fluorophores in the MnSOD-overexpressing cell lines. Thus, an increase in the oxidizing capacity of the cells does not appear to mediate the inhibition of proliferation associated with MnSOD overexpression. Superoxide dismutase has also been shown to enhance the cytotoxic activity of NO* toward tumor cells. In this study, we have shown that MnSOD overexpression enhances the cytostatic action of the NO* donors, sodium nitroprusside, 3-morpholinosydnonomine, and (Z)-1-[2-aminethyl)-N-(2-ammonioethyl)amino]diazen-1-+ ++ium-1,2-diolate in a dose-dependent manner. In addition, the NO* toxicity is blocked by oxyhemoglobin, a NO* scavenger. Our findings suggest that NO* may play a role in the reversal of tumorigenicity associated with MnSOD overexpression.

Cell Division↗
PubMed1998

Absence of imprinting of HLA class Ia genes leads to co-expression of biparental alleles on term human trophoblast cells upon IFN-gamma induction.

Human trophoblast cells have developed various efficient regulatory mechanisms to prevent cell surface expression of the classical HLA-A, -B, and (but not always) -C class I molecules. This allows them to escape maternal alloimmune attack during pregnancy. However, recent results have demonstrated that such a lack of expression could be reversed in villous cytotrophoblast cells purified from term placenta by in vitro IFN-gamma treatment. In this context, we investigated whether both maternal and paternal HLA class Ia antigens were co-dominantly expressed in such trophoblast cells. Using polymerase chain reaction sequence-specific primers for HLA-A and HLA-C alleles, we detected transcripts of both paternal and maternal origins, showing that these genes were not affected by genomic imprinting, at least in term placenta. After in vitro IFN-gamma treatment, the polymorphic HLA-A and HLA-B antigens of both parental origins become detectable at the cell surface, as assessed by flow cytometry and/or complement-dependent microtoxicity test. Appearance of paternal antigens on trophoblast cells upon IFN-gamma induction raises the question of the in vivo biological consequences of this phenomena, in term of materno-fetal tolerance and in particular of a potential allogeneic cytotoxic immune response.

Alleles↗
PubMed1998

Analysis of a Streptomyces antibioticus chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring.

A 6-kb region from the chromosome of Streptomyces antibioticus, an oleandomycin producer, was cloned and sequenced. This region was located between the 3' end of the gene encoding the third subunit of the oleandomycin type I polyketide synthase and the oleP and oleB genes, which encode a cytochrome P450 monooxygenase and an oleandomycin resistance gene, respectively. Analysis of the nucleotide sequence revealed the presence of five genes encoding a cytochrome P450-like protein (oleP1), two glycosyltransferases (oleG1 and oleG2) involved in the transfer of the two 6-deoxysugars (L-oleandrose and D-desosamine) to the oleandomycin macrolactone ring, a methyltransferase (oleM1), and a gene (oleY) of unknown function. Insertional inactivation of this region by gene disruption generated an oleandomycin non-producing mutant which accumulated a compound that, according to mass spectrometry analysis, could correspond to the oleandomycin macrolactone ring (oleandolide), suggesting that the mutation affects oleandrosyl glycosyltransferase.

Amino Acid Sequence↗
PubMed1997

Identification, localization, and expression of two novel human genes similar to deoxyribonuclease I.

Two novel cDNAs, DNAS1L2 and DNAS1L3, are predicted to encode proteins of 299 and 305 amino acids with 56 and 46% residue identity (71 and 63% similarity), respectively, to deoxyribonuclease I (DNase I). DNAS1L2 is located on a 16p13.3 cosmid, while DNAS1L3 maps to 3p14.3-p21.1 by fluorescence in situ hybridization and by PCR analysis of a radiation hybrid panel. Northern analysis revealed DNAS1L3 expression nearly exclusively in liver, while DNAS1L2 expression was detected in brain by RT-PCR. The previously defined DNL1L or DNAS1L1 is expressed highest in heart and skeletal muscle, while DNase I is expressed in the pancreas, parotid gland, and kidney. Thus, to date, four DNase I-like genes that show different tissue expression patterns are known. A comparison of DNAS1L1, DNAS1L2, and DNAS1L3 with the well-characterized DNase I suggests that the DNAS1L proteins are unlikely to be glycosylated or bind actin; however, catalytic and calcium- and DNA-binding residues are conserved, and potentially cleavable signal peptides are present among all these proteins. This analysis also identifies regions of high conservation among these proteins with no currently assigned function.

Amino Acid Sequence↗