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Biomedical subjects

G E Garcia

Publications and source records attributed to G E Garcia.

At least 19 recordsLinked to original sources
PubMed2001

2-methoxyestradiol blocks cell-cycle progression at G(2)/M phase and inhibits growth of human prostate cancer cells.

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17beta-estradiol, is present in human blood and urine. Here we show for the first time that 2-ME significantly inhibited the growth of normal prostate epithelial cells and androgen-dependent LNCaP and androgen-independent DU145 prostate cancer cells. This growth inhibition was accompanied by a twofold increase in the G(2)/M population, with a concomitant decrease in the G(1) population, as shown by cell-cycle analysis. 2-ME treatment affected the cell-cycle progression of prostate cancer cells specifically by blocking cells in the G(2) phase. Immunoblot analysis of the key cell-cycle regulatory proteins in the G(2)/M phase showed a 14-fold increase in the expression of p21 and an eightfold increase in the expression of p34 cell division cycle 2 (cdc2). We also found an accumulation of phosphorylated cdc2 after 2-ME treatment. Furthermore, Wee 1 kinase was detectable after 2-ME treatment. 2-ME treatment also led to an increase in the activity of caspase-3, followed by apoptosis, as shown by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick end-labeling and fluorescein isothiocyanate-poly(ADP-ribose) polymerase assay. Estrogen receptor levels did not change after treatment with 2-ME. Examination of the signaling pathways that mediate 2-ME-induced apoptosis showed reduction in the level of p53 expression and its DNA-binding activity. Given the fact that p53 mutations are common in patients with metastatic prostate cancer, our finding that 2-ME-mediated growth inhibition of human prostate cancer cells occurred in a p53-independent manner has considerable clinical significance. These findings, combined with the limited toxicity of 2-ME, may have significant implications for alternative treatment of advanced prostate cancer.

2-Methoxyestradiol↗
PubMed2000

NF-kappaB-dependent fractalkine induction in rat aortic endothelial cells stimulated by IL-1beta, TNF-alpha, and LPS.

Fractalkine is an endothelial cell-derived CX3C chemokine that is chemotactic mainly to mononuclear cells. Fractalkine was induced in rat aortic endothelial cells (RAEC) by interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and lipopolysaccharide (LPS) transcriptionally and translationally. This induction correlated with increased NF-kappaB DNA binding activity as determined by gel mobility shift assay. Supershift assays revealed that the NF-kappaB subunits p50 and p65 were responsible for kappaB binding. Accordingly, we examined the role of NF-kappaB in fractalkine induction in RAEC through the use of an adenovirus-mediated mutant IkappaB as a specific inhibitor. Delivery of a dominant-negative form of IkappaBalpha in RAEC dramatically reduced the induction of fractalkine by these stimuli, suggesting a role for NF-kappaB activation in fractalkine induction. The inhibition of fractalkine expression by two potent NF-kappaB inhibitors, sulfasalazine and sanguinarine, further supported the central role of NF-kappaB in fractalkine transcription regulation and suggested a novel therapeutic target aimed at modulating leukocyte endothelial cell interaction.

Animals↗
PubMed2000

Heparin-binding EGF-like growth factor contributes to reduced glomerular filtration rate during glomerulonephritis in rats.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, is expressed during inflammatory and pathological conditions. We have cloned the rat HB-EGF and followed the expression of HB-EGF in rat kidneys treated with anti- glomerular basement membrane (anti-GBM) antibody (Ab) to induce glomerulonephritis (GN). We observed glomerular HB-EGF mRNA and protein within 30 minutes of Ab administration and showed by in situ hybridization that glomerular HB-EGF mRNA expression was predominantly in mesangial and epithelial cells. Expression of HB-EGF correlated with the onset of decreased renal function in this model. To test the direct effect of HB-EGF on renal function, we infused the renal cortex with active rHB-EGF, prepared from transfected Drosophila melanogaster cells. This treatment induced a significant decrease in single nephron GFR (SNGFR), single nephron plasma flow, and glomerular ultrafiltration coefficient and an increase in the glomerular capillary hydrostatic pressure gradient. In addition, anti-HB-EGF Ab administered just before anti-GBM Ab blocked the fall in SNGFR and GFR at 90 minutes without any change in the glomerular histologic response. These studies suggest that HB-EGF expressed early in the anti-GBM Ab GN model contributes to the observed acute glomerular hemodynamic alterations.

Animals↗
PubMed1999

Buforin I, a natural peptide, inhibits botulinum neurotoxin B activity in vitro.

Botulinum neurotoxin B (BoNT/B) serotype specifically cleaves between the amino acids glutamine and phenylalanine (Q and F bond) in position 76-77 of synaptobrevin (VAMP2). We evaluated peptides that contain the QF cleavage site but are not identical in primary structure to the VAMP2 sequence surrounding the QF site for both inhibition of BoNT/B proteolytic activity and as substrates for BoNT/B. A reverse-phase high-performance liquid chromatography (RP-HPLC) method was used to measure digested peptides. A dose as high as 600 microM of substance P, and 11-amino acid peptide containing the QF bond, was neither a substrate nor inhibitor of BoNT/B in our assay, suggesting that more than the QF bond is required to be recognized by BoNT/B. Buforin I (B-I, QF site 24-25) is 39 amino acids in length, and sequence comparison of B-I and VAMP2 indicated a similarity of 18% for conserved amino acids around the QF site. Furthermore, computer-aided secondary structure computations predict alpha-helical structures flanking the QF site for VAMP2 and for the upstream sequence of B-I. Although predictions for the downstream sequence give nearly equal tendencies for alpha-helical and beta-sheet structures, Yi et al. showed that the downstream sequence is likely to be the alpha-helix based on their examination of buforin II (B-II, a 21-amino acid subset of B-I (16-36)), which includes the QF site and the downstream sequence of B-I. Buforin I was found not to be a substrate for BoNT/B; however, B-I dose dependently and competitively inhibited BoNT/B activity, yielding IC(50) = 1 x 10(-6) M. In contrast, B-II was not a substrate for BoNT/B and exhibited only 25% of the B-I inhibition of BoNT/B. Two additional B-I deletion peptides were tested for inhibition of BoNT/B proteolysis: peptide 36 (36 mer; containing B-I amino acids 1-36) and peptide 24 (24 mer; B-I amino acids 16-39). Peptide 24 had a similar inhibitory effect to B-II (ca. 25% of B-I) but peptide 36 was almost 50% as potent as B-I. These findings suggest that the buforin tertiary structure is important for the inhibitory activity of these peptides for BoNT/B.

Amino Acid Sequence↗
PubMed1999

Prevention of crescentic glomerulonephritis by immunoneutralization of the fractalkine receptor CX3CR1 rapid communication.

BACKGROUND: Fractalkine is a newly identified T-cell and monocyte/macrophage (Mphi) chemokine with a transmembrane domain and is a cell-surface protein on activated endothelium. It can mediate adhesion of cells expressing the fractalkine receptor CX3CR1. These unique features make fractalkine well suited for leukocyte recruitment in tissues with high blood flow as in the renal glomerulus. METHODS: Fractalkine expression in glomeruli and response of isolated glomerular inflammatory cells to fractalkine were studied in the Wistar-Kyoto (WKY) crescentic glomerulonephritis model. Antibody was used to confirm the proinflammatory role of fractalkine. RESULTS: Fractalkine was markedly induced in the endothelium of nephritic rat glomeruli, and inflammatory leukocytes infiltrating the glomeruli expressed increased levels of CX3CR1. Anti-CX3CR1 antibody treatment dramatically blocked leukocyte infiltration in the glomeruli, prevented crescent formation, and improved renal function. CONCLUSIONS: Fractalkine plays a central role in leukocyte trafficking at the endothelium in the high-flow glomerular circuit and, in turn, implicates CX3CR1 as a prime drug target for therapeutic intervention of endothelium-related inflammatory diseases.

Animals↗
PubMed1998

In vivo inhibition of CC and CX3C chemokine-induced leukocyte infiltration and attenuation of glomerulonephritis in Wistar-Kyoto (WKY) rats by vMIP-II.

Chemokines play a central role in immune and inflammatory responses. It has been observed recently that certain viruses have evolved molecular piracy and mimicry mechanisms by encoding and synthesizing proteins that interfere with the normal host defense response. One such viral protein, vMIP-II, encoded by human herpesvirus 8, has been identified with in vitro antagonistic activities against CC and CXC chemokine receptors. We report here that vMIP-II has additional antagonistic activity against CX3CR1, the receptor for fractalkine. To investigate the potential therapeutic effect of this broad-spectrum chemokine antagonist, we studied the antiinflammatory activity of vMIP-II in a rat model of experimental glomerulonephritis induced by an antiglomerular basement membrane antibody. vMIP-II potently inhibited monocyte chemoattractant protein 1-, macrophage inflammatory protein 1beta-, RANTES (regulated on activation, normal T cell expressed and secreted)-, and fractalkine-induced chemotaxis of activated leukocytes isolated from nephritic glomeruli, significantly reduced leukocyte infiltration to the glomeruli, and markedly attenuated proteinuria. These results suggest that molecules encoded by some viruses may serve as useful templates for the development of antiinflammatory compounds.

Animals↗
PubMed1998

Xanthurenic acid induces gametogenesis in Plasmodium, the malaria parasite.

A small, heat stable chromophore extracted from mosquitoes has recently been implicated as the signal that induces mating of Plasmodium, the malaria parasite. We have used high resolution electrospray mass spectrometry to determine that this gamete activation factor (GAF) has a m/z = 205.0450, suggesting a molecular species composition of C10H7NO4. Xanthurenic acid (XA), a product of tryptophan catabolism, was determined to have an elemental composition, ultraviolet absorbance maxima, and mass spectrum consistent with those characteristics of GAF. XA activated gametogenesis of Plasmodium gallinaceum and P. falciparum in vitro at concentrations lower than 0.5 microM in saline buffered to pH 7.4. A structural analog of XA, kynurenic acid (C10H6NO3), also activated gametogenesis but only at higher concentrations and with less effect. We propose that XA is GAF. This is the first evidence that XA has induction activity.

Animals↗
PubMed1997

Neuroprotective role of c-fos antisense oligonucleotide: in vitro and in vivo studies.

We investigated the dose-response and time-course of c-fos antisense oligodeoxynucleotide (ASO) treatment against excitatory amino acid (EAA)-induced neurotoxicity in rat hippocampal neurons. Glutamate (in vitro) or NMDA (in vivo) produced significant neuronal degeneration. Neuroprotection produced by 30 min or 4 h pretreatment with c-fos ASO in cultured hippocampal neurons was dose-dependent. In vivo, bilateral intrahippocampal injections of c-fos ASO (0.025 nmol/site) was neuroprotective when administered 30 min before or after NMDA treatment. However, 4 h pretreatment was ineffective. A higher dose (0.125 nmol) of c-fos ASO was neurotoxic and failed to afford neuroprotection regardless of the treatment schedule. Collectively, these results demonstrate a neuroprotective effect of c-fos ASO against EAA-induced neuronal injury supporting a causative role of c-fos expression in EAA neurotoxicity.

Animals↗
PubMed1997

Isolation of a substance from the mosquito that activates Plasmodium fertilization.

We have isolated a small, heat stabile, hydrophilic molecule from the gut lumen of unfed, female Anopheles stephensi that is a potent inducer of gametogenesis in Plasmodium falciparum and P. gallinaceum at a hydrogen ion concentration, pH 7.4, that normally suppresses activation. This gamete activation factor (GAF) was purified using reverse phase high performance liquid chromatography and determined to have a major ion m/z of 206.1 by low resolution electrospray mass spectrometry. The molecule, which was also found in the heads of both female and male A. stephensi, absorbed light in the ultraviolet region at three maxima (lambda(max) = 213, 245 and 350 nm); the 245/350 nm absorbance ratio was 7.0. The structure of the molecule and its normal function in the mosquito are not yet known, but in a sample of diverse insect species, extracts from those that feed on blood were bioactive. We propose that GAF is the previously observed malaria exflagellation factor (MEF).

Animals↗
PubMed1997

Effect of reduction of nitric oxide on plasma and kidney tissue angiotensin II levels.

Nitric oxide synthase (NOS) blockade increases blood pressure (BP) and modifies glomerular and tubular function. Angiotensin II (AII) blockade restores glomerular and tubular function but does not lower BP. We measured plasma renin activity (PRA), plasma (AIIp), and kidney tissue (AIIk) AII with radioimmunoassay to investigate the dissociation between renal and systemic effects of NOS blockade. Two period clearance studies followed by plasma and renal tissue harvesting were performed in seven groups of rats. Groups 1 and 1A served as controls. Groups 2 and 2A received NaCl-NaHCO3 during the first period and N(G)-monomethyl-L-arginine (L-NMMA, 0.5 mg/kg/min) during the second period. Group 3 was similar to group 2 but renal perfusion pressure (RPP) was maintained constant by using an aortic snare. Groups 4 and 4A received N(G)-nitro-L-arginine-methyl ester (L-NAME, 5 mg/100 mL of drinking water) for 2 weeks. NOS blockers decreased AIIp (group 1, 74 +/- 7 pg/mL; group 2, 22 +/- 1 pg/mL; group 3, 26 +/- 1 pg/mL; group 4, 19 +/- 3 pg/mL). The decrease in AIIp was a direct effect of L-NMMA independent of changes in perfusion pressure, as AIIp was similar in group 3 (normal RPP) and groups 2 and 4 (increased RPP). Measurements of PRA and AIIp demonstrated a similar reduction in PRA and AIIp in rats treated with NOS blocker. Although NOS blockers decreased AIIp, acute or chronic administration of NOS blockers did not modify AIIk (group 1, 1,192 +/- 51; group 2, 1,354 +/- 85; group 3, 1,348 +/- 180; group 4, 1,276 +/- 172 pg/kidney). Our findings demonstrate that NO blockers produce a dissociation between plasma and kidney AII levels. This dissociation can explain the beneficial effects of AII blockers on renal function and their lack of antihypertensive effects in anesthetized rats treated with NOS blockers.

Angiotensin II↗
PubMed1996

Management of ocular emergencies and urgent eye problems.

Evaluation of the patient with an acute eye problem begins with documentation of the level of vision in each eye, except in the case of a splash injury. In such cases, immediate copious irrigation is of critical importance. Subconjunctival hemorrhage is common and, typically, completely benign. Herpes simplex infection is painful and can lead to extensive damage. Herpes zoster infection is usually accompanied by skin lesions and can be effectively treated with oral acyclovir or famcyclovir. In patients with Bell's palsy, the eye must be carefully protected to prevent secondary injury. Corneal abrasions heal rapidly when antibiotics and patch protection are provided. Acute infections of the eyelids and conjunctivae usually respond well to topical antibiotics and warm compresses. Traumatic injuries require careful evaluation and, frequently, referral to an ophthalmologist.

Emergencies↗
PubMed1996

Effect of angiotensin II on the renal response to amino acid in rats.

Administration of the nitric oxide (NO) synthase blocker, N(G)-monomethyl L-arginine prevents the increase in glomerular filtration rate (GFR) normally observed with glycine, an effect that is restored by angiotensin II (AII) blockers. These findings suggest that changes in NO and AII dictate the presence or absence of renal vasodilation during amino acid (AA) infusion. We examined the effect of branched-chain (BCAA) and non-branched-chain (NBCAA) AA on GFR, NO, and AII to determine if abnormal NO or AII responses could explain the absence of vasodilation with BCAA. Our findings demonstrated that NBCAA increased GFR and NO and did not modify AII, either plasma (AIIp) or kidney (AIIk) AII. The response with BCAA was strikingly different. L-Valine increased GFR without modifying NO or AII. L-Leucine increased AIIk and NO but did not increase GFR. Administration of AII blockers (captopril or losartan) was associated with an increase in GFR during infusion of leucine. Single nephron studies demonstrated that increased AIIk with L-leucine was associated with decreased absolute proximal reabsorption and probably activation of the tubuloglomerular feedback. An AA-specific increase in AIIk is critical to inhibition of the normal renal response to AA infusion. NO generation is an important mediator but not the sole mechanism that determines the increase in GFR during amino acid infusion.

Amino Acids↗
PubMed1995

Anti-human immunodeficiency virus 1 (HIV-1) activities of 3-deazaadenosine analogs: increased potency against 3'-azido-3'-deoxythymidine-resistant HIV-1 strains.

3-Deazaadenosine (DZA), 3-deaza-(+/-)-aristeromycin (DZAri), and 3-deazaneplanocin A (DZNep) are powerful modulators of cellular processes. When tested against H9 cells infected acutely with two different strains of human immunodeficiency virus 1 (HIV-1) and in the chronically infected monocytoid cell lines U1 and THP-1, the 3-deazanucleosides caused a marked reduction in p24 antigen production. Similar reductions in p24 antigen were seen in phytohemagglutinin-stimulated peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Strikingly, in comparing the therapeutic indices between the paired pre- and post-3'-azido-3'-deoxythymidine (AZT) treatment HIV-1 isolates, DZNep and neplanocin A showed an increase of 3- to 18-fold in their potency against AZT-resistant HIV-1 isolates. In H9 cells treated with DZNep and DZAri, the formation of triphosphate nucleotides of DZNep and DZAri was observed. The mode of action of DZNep and DZAri appears complex, at least in part, at the level of infectivity as shown by decreases in syncytia formation in HIV-1-infected H9 cells and at the level of transcription as both drugs inhibited the expression of basal or tat-induced HIV-1 long terminal repeat chloramphenicol acetyltransferase activity in stably transfected cell lines. Since DZNep induced in H9 cells a rapid expression of nuclear binding factors that recognize the AP-1 transcription site, the anti-HIV-1 activity of the DZA analogs could partly be the induction of critical factors in the host cells. Thus, the 3-deazanucleoside drugs belong to an unusual class of anti-HIV-1 drugs, which may have therapeutic potential, in particular against AZT-resistant strains.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Involvement of reactive oxygen intermediates in cyclooxygenase-2 expression induced by interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide.

Reactive oxygen intermediates (ROIs) play an important role in inflammatory processes as mediators of injury and potentially in signal transduction leading to gene expression. Cyclooxygenase (COX) is a rate-limiting enzyme in prostanoid biosynthesis, and its recently cloned inducible form, COX-2, is induced by proinflammatory cytokines. This study linked ROIs to the signaling pathways that induce COX-2 expression. The hydroxyl radical scavengers DMSO (1%), as well as di- and tetramethylthiourea, inhibited IL-1-, TNF alpha-, and LPS-induced COX-2 expression in rat mesangial cells. The suppression of COX-2 mRNA expression correlated with the COX-2 protein level. In comparison with the prolonged induction of the inducible gene encoding protein-tyrosine phosphatase by hydrogen peroxide, the COX-2 gene was only transiently induced. Protein-tyrosine phosphatase is also induced by heat shock and chemical stress, whereas COX-2 is not. Superoxide was a more potent inducer for COX-2 than hydrogen peroxide. In addition, NADPH stimulated COX-2 expression, and an inhibitor of NADPH oxidase blocked COX-2 expression induced by TNF alpha. COX-2 and KC gene expression costimulated by IL-1 were inhibited differentially by the scavengers. These studies demonstrate that oxidant stress is a specific and important inducer of COX-2 gene expression. This induction may contribute to the deleterious amplification of prostanoids in inflammation and compound the direct effects of ROI production.

Animals↗
PubMed1995

Role of angiotensin in the regulation of renal response to proteins.

Intrarenal and extrarenal humoral factors have been proposed as mediators and modulators of the renal hyperemic response to amino acid infusion. Among the potential modulators, angiotensin II (AII) constitutes the most important candidate due to its critical role in the control of glomerular and tubular function. The modulatory effect of AII has been assessed by (1) measuring the changes in plasma renin activity (PRA)/AII during the normal hyperemic response, and (2) by assessing the levels of PRA/AII and the response to AII-suppressing agents in conditions with no vasodilatory response during amino acid infusion. Administration of a protein load in normal animals or humans does not modify PRA/AII. Absence of a vasodilatory response in various experimental conditions (nitric oxide blockade in normal rats, experimental models of hypertension, diabetes mellitus, chronic glomerulonephritis, cyclosporine administration) is characterized by a significant decrease in proximal tubular reabsorption during amino acid infusion. Converting enzyme inhibitors or AII receptor antagonist restore normal tubular function and the increase in glomerular filtration rate during amino acid infusion. Absence of a vasodilatory response is also associated with increases in kidney AII levels in some of these conditions. These results suggest that (1) AII modulates the amino acid-induced hyperemia through its inhibitory effect on proximal tubular reabsorption and activation of the tubuloglomerular feedback system, and (2) that the expression of the modulatory effect of AII may depend on the interaction between AII and other intrarenal systems like nitric oxide.

Amino Acids↗
PubMed1994

Does chromostatin influence catecholamine release or blood pressure in vivo?

Although the structure of chromogranin A (CgA) is now known, its ultimate physiological role remains elusive. Recently, an interior fragment of CgA [CgA(124-143)], also called chromostatin, was reported to suppress catecholamine release from chromaffin cells in vitro. We therefore explored chromostatin's biological actions when administered in vivo to anesthetized rodents with normal (Wistar-Kyoto rats) or elevated blood pressure (spontaneously hypertensive rats). Neither mean arterial pressure nor plasma epinephrine concentrations were significantly altered following either chromostatin or vehicle administration. Plasma norepinephrine, on the other hand, tended to rise throughout all studies, with the rise reaching statistical significance only in the SHR subgroup receiving chromostatin. We conclude that, unlike its actions in vitro, chromostatin does not appear to suppress catecholamine release or modulate blood pressure in vivo.

Animals↗
PubMed1992

Clostridium sticklandii glycine reductase selenoprotein A gene: cloning, sequencing, and expression in Escherichia coli.

Gene grdA, which encodes selenoprotein A of the glycine reductase complex from Clostridium sticklandii, was identified and characterized. This gene encodes a protein of 158 amino acids with a calculated M(r) of 17,142. The known sequence of 15 amino acids around the selenocysteine residue and the known carboxy terminus of the protein are correctly predicted by the nucleotide sequence. An opal termination codon (TGA) corresponding to the location of the single selenocysteine residue in the polypeptide was found in frame at position 130. The C. sticklandii grdA gene was inserted behind the tac promotor of an Escherichia coli expression vector. An E. coli strain transformed with this vector produced an 18-kDa polypeptide that was not detected in extracts of nontransformed cells. Affinity-purified anti-C. sticklandii selenoprotein A immunoglobulin G reacted specifically with this polypeptide, which was indistinguishable from authentic C. sticklandii selenoprotein A by immunological analysis. Addition of the purified expressed protein to glycine reductase protein components B and C reconstituted the active glycine reductase complex. Although synthesis of enzymically active protein A depended on the presence of selenium in the growth medium, formation of immunologically reactive protein did not. Moreover, synthesis of enzymically active protein in a transformed E. coli selD mutant strain indicated that there is a nonspecific mechanism of selenocysteine incorporation. These findings imply that mRNA secondary structures of C. sticklandii grdA are not functional for UGA-directed selenocysteine insertion in the E. coli expression system.

Amino Acid Oxidoreductases↗