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Biomedical subjects

L Nuñez

Publications and source records attributed to L Nuñez.

At least 19 recordsLinked to original sources
PubMed2006

In vitro antibacterial activity of the peptide PsVP-10 against Streptococcus mutans and Streptococcus sobrinus with and without glycocalyx.

The antibacterial activity of the peptide PsVP-10 obtained from Pseudomonas sp. R10 against Streptococcus mutans and Streptococcus sobrinus was investigated. One hundred and twenty strains of S. mutans and 120 strains of S. sobrinus with and without glycocalyx were isolated from saliva samples in trypticase-yeast-cysteine-sucrose-bacitracin (TYCSB) agar. Bacterial identification was made by polymerase chain reaction. Glycocalyx production was observed in modified TYCSB agar and confirmed with a modified version of the microplate adherence assay. The minimum inhibitory concentration (MIC) of PsVP-10 bacteriocin was determined by means of the agar dilution method, and the time of bacterial death was calculated by means of colony-forming unit counts. The MIC of the bacteriocin PsVP-10 for both bacterial species with and without glycocalyx was < 2 mg/L and the time of bacterial death was less than 240 s for all the studied bacterial strains. Thus, bacteriocin PsVP-10 could be an interesting possibility to combat these cariogenic bacterial species.

Anti-Bacterial Agents↗
PubMed2004

Single nucleotide polymorphisms and outcome in docetaxel-cisplatin-treated advanced non-small-cell lung cancer.

BACKGROUND: Platinum-based doublets are the standard chemotherapy for advanced non-small-cell lung cancer (NSCLC). Excision-repair cross-complementing 1 (ERCC1), xeroderma pigmentosum group D (XPD) and ribonucleotide reductase subunit M1 (RRM1) are essential to the repair of cisplatin DNA adducts. Multidrug resistance 1 (MDR1) has been related to antimicrotubule resistance. We assessed whether single nucleotide polymorphisms (SNPs) in ERCC1, XPD, RRM1 and MDR1, and ERCC1 mRNA expression, predicted survival in docetaxel-cisplatin-treated stage IV NSCLC patients. PATIENTS AND METHODS: Using the TaqMan 5' nuclease assay, we examined ERCC1 118, XPD 751 and 312, RRM1 -37C/A, and MDR1 C3435T SNPs in peripheral blood lymphocytes (PBLs) obtained from 62 docetaxel-cisplatin-treated advanced NSCLC patients. ERCC1 expression was measured in RNA isolated from PBLs using real-time reverse transcriptase PCR. RESULTS: Overall median survival was 10.26 months. Median survival was 9.67 months for 34 patients with ERCC1 118 C/T, 9.74 months for 17 patients with T/T, and not reached for 11 patients with C/C (P=0.04). Similar significant differences in time to progression were observed according to ERCC1 118 genotype (P=0.03). No other significant differences were observed. CONCLUSIONS: Patients homozygous for the ERCC1 118 C allele demonstrated a significantly better survival. ERCC1 SNP assessment could be an important component of tailored chemotherapy trials.

Adult↗
PubMed2003

Normal prism adaptation but reduced after-effect in basal ganglia disorders using a throwing task.

Prism adaptation is a form of visuomotor learning in which the visual and motor systems need to be adjusted because a visual perturbation is produced by horizontally displacing prisms. Despite being known for over two centuries, the neuronal substrates of this phenomenon are not yet completely understood. In this article the possible role of the basal ganglia in this kind of learning was analysed through a study of Huntington's and Parkinson's disease patients. A throwing technique requiring the use of open loop feedback was used. The variables analysed were visuomotor performance, adaptation rate and magnitude, and the after-effect. The results clearly showed that both Huntington's and Parkinson's disease groups learned at the same rate as control subjects. In addition, despite having a disturbed visuomotor performance, both experimental groups showed the same adaptation magnitude as the control group. Finally, the after-effect, which is measured after removing the prisms, is reduced in both patients groups. This reduction leads to a disruption in the normal adaptation-after-effect correlation found in normal volunteers. These results suggest that basal ganglia are not involved in this type of open-looped visuomotor learning. The large number of patients studied as well as the similarity of the findings between both populations support this hypothesis. By contrast, there is an impairment in the after-effect on both basal ganglia patient populations. This impairment may be the result of the deterioration of the perceptual recalibration process involved in visuomotor learning.

Adaptation, Ocular↗
PubMed2003

Serum DNA as a tool for cancer patient management.

PURPOSE: Genetic analysis has shown that cell-free circulating DNA in plasma or serum of cancer patients shares similar genetic alterations to those described in the corresponding tumor. One of the most important alterations involved in carcinogenesis is aberrant promoter methylation. The interest in this field has grown due to the implementation of the methylation-specific PCR (MSP) assay. The main objective of this study is to analyze the methylation status of different genes in tumor and serum DNA obtained at the time of surgery in two different tumor models (glioblastoma [GBM] and non-small-cell lung cancer [NSCLC]) and their relationship to clinico-pathological characteristics and response to chemotherapy. MATERIAL AND METHODS: Using MSP assay, we assessed the methylation status of MGMT, RASSF1A, p16, DAPK, TMS-1 in tumor and serum DNA obtained at time of surgery or stereotactic biopsy from 28 GBM patients and from 51 NSCLC patients. RESULTS: In GBM patients, the prevalence of MGMT, p16, DAPK, and RASSF1A promoter methylation was 38.1%, 66.7%, 52.4%, 57.1%, respectively, in glioma tissue, and 39.3%, 53.6%, 34.3%, 50%, respectively, in serum. A high correlation between methylation in tumor and serum (Spearman test p = 0.0001) was observed. In NSCLC patients, RASSF1A, DAPK and TMS-1 were methylated in 34%, 45% and 35% tumors, respectively, and in 34%, 40% and 34% serum, respectively. A good correlation was found between alterations found in tumor and serum (Spearman test p = 0.0001). CONCLUSIONS: The study of serum or plasma DNA has opened new roads for translational research and new strategies for molecular diagnosis. Due to the similarities of alterations found in serum DNA and primary tumor, we can use this tool to calculate the risk of local or distant recurrence and its relationship with survival and its value in patient follow-up to evaluate response to therapy.

Adult↗
PubMed1999

Differentiation stage-specific inhibition of the Raf-MEK-ERK pathway by Akt.

Extracellular signals often result in simultaneous activation of both the Raf-MEK-ERK and PI3K-Akt pathways (where ERK is extracellular-regulated kinase, MEK is mitogen-activated protein kinase or ERK kinase, and PI3K is phosphatidylinositol 3-kinase). However, these two signaling pathways were shown to exert opposing effects on muscle cell hypertrophy. Furthermore, the PI3K-Akt pathway was shown to inhibit the Raf-MEK-ERK pathway; this cross-regulation depended on the differentiation state of the cell: Akt activation inhibited the Raf-MEK-ERK pathway in differentiated myotubes, but not in their myoblast precursors. The stage-specific inhibitory action of Akt correlated with its stage-specific ability to form a complex with Raf, suggesting the existence of differentially expressed mediators of an inhibitory Akt-Raf complex.

Animals↗
PubMed1999

Mechanisms of resistance to ampicillin, chloramphenicol and quinolones in multiresistant Salmonella typhimurium strains isolated from fish.

Mechanisms of antibiotic resistance and epidemiological relationships were investigated for five multiresistant strains of Salmonella typhimurium isolated from fish in India. Four strains showed resistance to nalidixic acid, chloramphenicol, tetracycline, co-trimoxazole, gentamicin and beta-lactam antibiotics. The remaining strain was susceptible to all beta-lactam antibiotics tested and to co-trimoxazole but resistant to the other antibiotics tested. Epidemiological analysis performed by REP-PCR showed that the five isolates belonged to the same clone. Resistance to nalidixic acid was related to a single mutation in the gyrA gene. Chloramphenicol resistance was related to the production of chloramphenicol acetyl-transferase. An OXA-1 beta-lactamase, located in an integron, was responsible for resistance to ampicillin. These results indicate the health hazard posed by the fact that S. typhimurium may acquire or develop several mechanisms of resistance to a variety of antibiotics, including quinolones, and can thus cause disease in humans which may be difficult to treat.

Ampicillin↗
PubMed1998

Episodic gonadotropin-releasing hormone gene expression revealed by dynamic monitoring of luciferase reporter activity in single, living neurons.

The existence of an intrinsic oscillator for pulsatile gonadotropin-releasing hormone (GnRH) secretion in normal and transformed GnRH neurons raises the question of whether the corresponding gene also is expressed in an episodic manner. To resolve this question, we used a modification of conventional luciferase technology, which enabled continuous monitoring of GnRH gene activity in single, living neurons. With this method, the relative rate of endogenous gene expression is estimated by quantification of photons emitted by individual neurons microinjected with a GnRH promoter-driven luciferase reporter construct. Immortalized GT1-1 neurons, which secrete the decapeptide GnRH in a pulsatile manner conceptually identical to that of their nontransformed counterparts in vivo, were chosen as the model for these studies. First, we injected individual cells with purified luciferase protein and established that the reporter half-life was sufficiently short (50 min) to enable detection of transient changes in gene expression. Next, we subjected transfected GT1-1 cells to continuous monitoring of reporter activity for 16 h and found that the majority of them exhibited spontaneous fluctuations of photonic activity over time. Finally, we established that photonic activity accurately reflected endogenous GnRH gene expression by treating transfected GT1-1 cells with phorbol 12-myristate 13 acetate (a consensus inhibitor of GnRH gene expression) and observing a dramatic suppression of photonic emissions from continuously monitored cells. Taken together, these results demonstrate the validity of our "real-time" strategy for dynamically monitoring GnRH gene activity in living neurons. Moreover, our findings indicate that GnRH gene expression as well as neuropeptide release can occur in an intermittent manner.

Cells, Cultured↗
PubMed1998

alpha-MSH potentiates the responsiveness of mammotropes by increasing Ca2+ entry.

It is well known that the suckling stimulus renders mammotropes considerably more responsive to prolactin (PRL)-releasing stimuli, and the neurointermediate lobe peptide alpha-melanocyte-stimulating hormone (alpha-MSH) has been proposed to play a pivotal role in this priming. The objectives of the present study were to determine whether alpha-MSH could act directly on pituitary cells to potentiate PRL release in response to two physiologically relevant PRL secretagogues, thyrotropin-releasing hormone (TRH) and ATP, and, if so, to identify the mechanism by which this priming phenomenon is manifested. To this end, we cultured anterior pituitary cells from lactating rats overnight and then subjected them to a reverse hemolytic plaque assay for PRL to evaluate their responses to various test agents. We found that alpha-MSH, which had no effect on PRL export when tested alone, augmented by more than threefold the secretory responses to TRH and ATP. Next, we utilized digital-imaging fluorescence microscopy of fura 2 to evaluate the role of intracellular Ca2+ in this process. We found that PRL export induced by pharmacological activation of L-type voltage-operated calcium channels was also potentiated by alpha-MSH, as was Ca2+ entry induced by TRH. Our results indicate that alpha-MSH acts as a mammotrope-priming agent on a subset of mammotropes by increasing Ca2+ entry induced by PRL secretagogues.

Adenosine Triphosphate↗
PubMed1997

Cytosolic phospholipase A2 is coupled to muscarinic receptors in the human astrocytoma cell line 1321N1: characterization of the transducing mechanism.

The cholinergic agonist carbachol induced the release of arachidonic acid in the 1321N1 astrocytoma cell line, and this was blocked by atropine, suggesting the involvement of muscarinic receptors. To assess the mechanisms of signalling involved in the response to carbachol, a set of compounds characterized by eliciting responses through different mechanisms was tested. A combination of 4beta-phorbol 12beta-myristate 13alpha-acetate and thapsigargin, an inhibitor of endomembrane Ca2+-ATPase that induces a prolonged elevation of cytosolic Ca2+ concentration, induced an optimal response, suggesting at first glance that both protein kinase C (PKC) and Ca2+ mobilization were involved in the response. This was consistent with the observation that carbachol elicited Ca2+ mobilization and PKC-dependent phosphorylation of cytosolic phospholipase A2 (cPLA2; phosphatide sn-2-acylhydrolase, EC 3.1.1.4) as measured by a decrease in electrophoretic mobility. Nevertheless, the release of arachidonate induced by carbachol was unaltered in media containing decreased concentrations of Ca2+ or in the presence of neomycin, a potent inhibitor of phospholipase C which blocks phosphoinositide turnover and Ca2+ mobilization. Guanosine 5'-[gamma-thio]triphosphate added to the cell-free homogenate induced both [3H]arachidonate release and cPLA2 translocation to the cell membrane fraction in the absence of Ca2+, thus suggesting the existence of an alternative mechanism of cPLA2 translocation dependent on G-proteins and independent of Ca2+ mobilization. From the combination of experiments utilizing biochemical and immunological tools the involvement of cPLA2 was ascertained. In summary, these data indicate the existence in the astrocytoma cell line 1321N1 of a pathway involving the cPLA2 which couples the release of arachidonate to the occupancy of receptors for a neurotransmitter, requires PKC activity and G-proteins and might operate in the absence of Ca2+ mobilization.

Arachidonic Acid↗
PubMed1997

Extracellular ATP as an autocrine/paracrine regulator of prolactin release.

Recent evidence demonstrates that ATP, costored with a number of hormones and neurotransmitters in secretory granules, is coreleased during exocytosis of these agents. Here, we explored the possibility that extracellular ATP subserves an autocrine and/or paracrine role in the regulation of prolactin (PRL) release by subjecting rat pituitary cells to various experimental manipulations aimed at evaluating putative interactions between ATP and mammotropes. Our results strongly support the view that ATP functions as a local regulator of PRL secretion. To be more specific, we observed that ATP is released in a predictable manner by physiologically relevant secretagogues that are reasonably targeted to mammotropes. Moreover, we found that ATP can act directly on pituitary cells to stimulate the release of PRL from most (if not all) mammotropes. Finally, we determined that antagonism or removal of ATP leads to a diminution of PRL export from pituitary cells cultured under basal or thyrotropin-releasing hormone-stimulated conditions. On the basis of these results, we propose that ATP acts locally to amplify and prolong the PRL secretory response elicited by a more traditional hypophysiotropic signal.

Adenosine Triphosphate↗
PubMed1996

HSV-1 vector mediated transfer of BDNF into cerebellar granule cells.

Cerebellar granule cells offer a useful model system to study the effects of neurotrophins during development. We have used a defective herpes simplex virus type 1 (HSV-1) vector containing brain-derived neurotrophic factor (BDNF) to express this neurotrophin in aggregate cultures of granule cells. Viral infection led to easily detectable BDNF expression and neurite outgrowth of granule cells, expressing the high affinity receptor TrkB. Neurite elongation mediated by the HSV-1 vector producing BDNF was similar to that found after exposure to purified BDNF. This study demonstrates the efficacy of HSV-1 vectors for delivery and expression of neurotrophins in cerebellar granule cells. The biological responses measured indicate the effectiveness of HSV-1 vectors as potential therapeutic tools.

Animals↗
PubMed1996

Modulation of intrahepatic cholesterol trafficking: evidence by in vivo antisense treatment for the involvement of sterol carrier protein-2 in newly synthesized cholesterol transport into rat bile.

Biliary cholesterol represents one of the two major excretory pathways for sterol elimination from the body and plays a central role in cholesterol gallstone formation. Biliary cholesterol originates from a precursor pool of preformed and newly synthesized free cholesterol. Although it has been suggested that newly synthesized and preformed biliary cholesterol are secreted by independent pathways, the specific cellular and molecular mechanisms are unknown. We used male Wistar rats to study the time-course of the appearance of newly synthesized cholesterol, phosphatidylcholine and protein into bile. The specific role of sterol carrier protein-2 (SCP-2) in the transport of newly synthesized biliary cholesterol was evaluated by an in vivo antisense oligonucleotide approach. In contrast to [14C]phosphatidylcholine and [35S]proteins, the time-course of [14C]cholesterol appearance into bile was rapid, and microtubule- and Golgi-independent. In vivo SCP-2 antisense treatment reduced and delayed the appearance of biliary [14C]cholesterol. Furthermore, hepatic SCP-2 expression increased more than 3-fold over control values in rats that had been treated with diosgenin to increase biliary secretion of newly synthesized cholesterol. These results suggest that SCP-2 is necessary for the rapid transport of newly synthesized cholesterol into bile and that hepatocytes can induce SCP-2 expression according to the rate of biliary secretion of newly synthesized cholesterol.

Animals↗
PubMed1996

Two different constituents of plasma increase cytosolic calcium selectively in neurons or glia of primary rat cerebellar cultures.

1. The ability of several serum fractions to increase the cytosolic calcium concentration ([Ca2+]i) was tested in rat cerebellar cells maintained in primary culture. 2. Serum filtered through an ultrafiltration membrane with 3000 Da molecular mass cut-off (filtered serum, FS) selectively stimulated neurons whereas dialysed serum (DS) selectively stimulated glia. 3. The effects of FS were due to glutamate as they were reproduced by N-methyl-D-aspartate (NMDA), blocked by NMDA receptor antagonists and prevented by enzymatic removal of glutamate. 4. The effects of DS on glia were not reproduced by platelet-activating factor, thrombin or bradykinin. They were not lost on heating or extraction with diethyl ether. They were reproduced by a methanol-chloroform-HCl extract from DS and by several commercial fraction V plasma albumins. 5. These [Ca2+]i-increasing factors present in blood could contribute to brain damage during ischaemia if they reached the brain interstitium on disruption of the blood-brain barrier.

Animals↗
PubMed1996

Mechanisms for synchronous calcium oscillations in cultured rat cerebellar neurons.

Removal of Mg2+ caused oscillations of the cytosolic Ca2+ concentration ([Ca2+]i) and the membrane potential in cultured cerebellar granule neurons. Oscillations of [Ca2+]i were synchronous in all the cells, and were restricted to the neurons (immunocytochemically identified) that responded to exogenous N-methyl-D-aspartate (NMDA). Oscillations were blocked by Ca2+ removal, nickel, NMDA receptor antagonists, omega-agatoxin IVA, tetrodotoxin, sodium removal and gamma-aminobutyric acid, but not by dihydropyridines, omega-conotoxin M VIIA or by emptying the intracellular Ca2+ stores with thapsigargin or ionomycin. The upstroke of the [Ca2+]i oscillations coincided in time with an increase in manganese permeability of the plasma membrane. Propagation of the [Ca2+]i wave followed more than one pathway and the spatiotemporal pattern changed with time. Membrane potential oscillations consisted of transient slow depolarizations of approximately 20 mV with faster phasic activity superimposed. We propose that the synchronous [Ca2+]i oscillations are the expression of irradiation of random excitation through a neuronal network requiring generation of action potentials and functional glutamatergic synapses. Oscillations of -Ca2+-i are due to cyclic Ca2+ entry through NMDA receptor channels activated by synaptic release of glutamate, which requires Ca2+ entry through P-type Ca2+ channels activated by action potentials at the presynaptic terminal.

Animals↗
PubMed1995

Receptor tyrosine kinase specific for the skeletal muscle lineage: expression in embryonic muscle, at the neuromuscular junction, and after injury.

While a number of growth factors have been described that are highly specific for particular cell lineages, neither a factor nor a receptor uniquely specific to the skeletal muscle lineage has previously been described. Here we identify a receptor tyrosine kinase (RTK) specific to skeletal muscle, which we term "MuSK" for muscle-specific kinase. MuSK is expressed at low levels in proliferating myoblasts and is induced upon differentiation and fusion. In the embryo, it is specifically expressed in early myotomes and developing muscle. MuSK is then dramatically down-regulated in mature muscle, where it remains prominent only at the neuromuscular junction; MuSK is thus the only known RTK that localizes to the neuromuscular junction. Strikingly, MuSK expression is dramatically induced throughout the adult myofiber after denervation, block of electrical activity, or physical immobilization. In humans, MuSK maps to chromosome 9q31.3-32, which overlaps with the region reported to contain the Fukuyama muscular dystrophy mutation. Identification of MuSK introduces a novel receptor-factor system that seems sure to play an important and selective role in many aspects of skeletal muscle development and function.

Amino Acid Sequence↗
PubMed1995

Differential Ca2+ responses of adrenergic and noradrenergic chromaffin cells to various secretagogues.

The effects of several physiological agonists on the cytosolic Ca2+ concentration ([Ca2+]i) of immunnocytochemically identified single adrenergic and noradrenergic bovine chromaffin cells were compared. No differences were observed in the responses to stimulation by high-K+ solutions with or without BAY K 8644, suggesting that the density and properties of voltage-dependent Ca2+ channels were similar in both cell types. The increase of [Ca2+]i induced by acetylcholine was greater in adrenergic cells, and this was due to differences in the response mediated through nicotinic receptors. The responses to bradykinin and to ATP were slightly greater in noradrenergic cells. Only a small fraction of the cells (18-28%) was responsive to ATP. The responses to angiotensin II and to histamine were much greater in adrenergic than in noradrenergic cells. Histamine was almost a selective stimulator of adrenergic cells. These differences suggest differential distribution of functional membrane receptors in both cell types and may be relevant to understanding the differential contribution of epinephrine- and norepinephrine-secreting cells during stressful conflicts in physiological or pathophysiological situations.

Angiotensin II↗
PubMed1995

Identification of mammalian noggin and its expression in the adult nervous system.

The multiple roles of noggin during dorsal fate specification in Xenopus embryos, together with noggin's ability to directly induce neural tissue, inspired an effort to determine whether a similar molecule exists in mammals. Here we describe the identification of human and rat noggin and explore their expression patterns; we also localize the human NOGGIN gene to chromosome 17q22, and the mouse gene to a syntenic region of chromosome 11. Mammalian noggin is remarkably similar in its sequence to Xenopus noggin, and is similarly active in induction assays performed on Xenopus embryo tissues. In the adult mammal, noggin is most notably expressed in particular regions of the nervous system, such as the tufted cells of the olfactory bulb, the piriform cortex of the brain, and the Purkinje cells of the cerebellum, suggesting that one of the earliest acting neural inducers also has important roles in the adult nervous system.

Amino Acid Sequence↗