Biomedical subjects

E Rojas

Publications and source records attributed to E Rojas.

At least 73 records · Page 4Linked to original sources

PubMed1996

Genotoxicity of vanadium pentoxide evaluate by the single cell gel electrophoresis assay in human lymphocytes.

Vanadium compounds are extensively used in modern industry and occupational exposure to high doses of Vanadium is quite common. In this study, the genotoxicity of vanadium pentoxide (V2O5) was evaluated directly in whole blood leukocytes and in human lymphocyte cultures using the single-cell gel electrophoresis assay (Comet Assay) to detect DNA damage expressed as DNA strand breaks and alkali labile sites. This chemical produces a clear dose-response in DNA migration in whole blood leukocytes and a significative positive effect only with the highest tested concentration in human lymphocyte cultures. After different recovery times the level of DNA damage returned to the control values. These results indicate that V2O5 is capable to induce DNA single-strand breaks and/or alkali-labile damage.

Adult↗

PubMed1996

Zn2+ interaction with Alzheimer amyloid beta protein calcium channels.

The Alzheimer disease 40-residue amyloid beta protein (AbetaP[1-40]) forms cation-selective channels across acidic phospholipid bilayer membranes with spontaneous transitions over a wide range of conductances ranging from 40 to 4000 pS. Zn2+ has been reported to bind to AbetaP[1-40] with high affinity, and it has been implicated in the formation of amyloid plaques. We now report the functional consequences of such Zn2+ binding for the AbetaP[1-40] channel. Provided the AbetaP[1-40] channel is expressed in the low conductance (<400 pS) mode, Zn2+ blocks the open channel in a dose- dependent manner. For AbetaP[1-40] channels in the giant conductance mode (>400 pS), Zn2+ doses in the millimolar range were required to exert substantial blockade. The Zn2+ chelator o-phenanthroline reverses the blockade. We also found that Zn2+ modulates AbetaP[1-40] channel gating and conductance only from one side of the channel. These data are consistent with predictions of our recent molecular modeling studies on AbetaP[1-40] channels indicating asymmetric Zn(2+)-AbetaP[1-40] interactions at the entrance to the pore.

Amyloid beta-Peptides↗

PubMed1996

Induced mitotic death of HeLa cells by abnormal expression of c-H-ras.

When HeLa cells were selected for stable expression of a neo gene, linked either to mutated or wt c-H-ras genes, morphological examination of selected clones from several experiments revealed formation of giant multinucleated cells. These morphological alterations culminate in cell death, as a consequence of mitotic catastrophe (or mitotic death). Although clones expressing the mutated gene produced significantly larger numbers of these giant cells, those transfected with the normal allele were also found to produce significantly more giant multinucleated cells than non-transfected HeLa cells. Northern blot analysis of mRNA revealed overexpression of the normal H-ras gene in these clones. Chromatin structure analysis of these clones showed gross alterations, including the presence of micronuclei and heteroploid nuclei. Interestingly, odd numbers of nuclei were found in colonies of these giant cells. In addition, alterations in cell cycle parameters were observed, including the appearance of a subpopulation of cells with an abnormal content of DNA, probably representing dying cells. Our data support the notion that abnormal expression of H-ras contributes to mitotic catastrophe and death of a subpopulation of HeLa cells.

Blotting, Northern↗

PubMed1996

Reprotoxic and genotoxic studies of vanadium pentoxide in male mice.

Effects of vanadium pentoxide (V2O5) treatment on reproductive function and testicular DNA in male mice were investigated. These functions were evaluated with fertility rate, implants, resorptions, sperm counts, motility, and morphology. The DNA damage in individual testis cells was analyzed by single-cell gel electrophoresis technique (COMET assay). V2O5 treatment resulted in a decrease in fertility rate, implantations, live fetuses, and fetal weight, and an increase in the number of resorptions/dam. Sperm count, motility, and morphology were impaired with the advancement of treatment. Vanadium treatment induced DNA damage depending on the dose in the testis cells that was expressed and detected as DNA migration in the COMET assay. The distribution of DNA migration among cells, a function of dose, revealed that the majority of cells of treated animals expressed more DNA damage than cells from control animals. It is concluded that vanadium pentoxide was a reprotoxic and genotoxic agent in mice.

Animals↗

PubMed1996

Similarity in calcium channel activity of annexin V and matrix vesicles in planar lipid bilayers.

Matrix vesicles (MVs), structures that accumulate Ca2+ during the initiation of mineral formation in growing bone, are rich in annexin V. When MVs are fused with planar phospholipid bilayers, a multiconductance Ca2+ channel is formed, with activity essentially identical to that observed when annexin V is delivered to the bilayer with phosphatidylserine liposomes. Ca2+ currents through this channel, from either MV or annexin V liposomes, are blocked by Zn2+, as is Ca2+ uptake by MV incubated in synthetic cartilage lymph. Blockage by Zn2+ was most effective when applied to the side containing the MV or liposomes. ATP and GTP differentially modulated the activity of this channel: ATP increased the amplitude of the current and the number of conductance states; GTP dramatically reduced the number of events and conductance states, leading to well-defined Ca2+ channel activity from either MV or the annexin V liposomes. In the distinctive effects of ATP, GTP, and Zn2+ on the Ca2+ channel activity observed in both the MV and the liposome systems, the common factor was the presence of annexin V. From this we conclude that Ca2+ entry into MV results from the presence of annexin V in these membrane-enclosed structures.

Adenosine Triphosphate↗

PubMed1996

Genetic mapping using haplotype, association and linkage methods suggests a locus for severe bipolar disorder (BPI) at 18q22-q23.

Manic depressive illness, or bipolar disorder (BP), is characterized by episodes of elevated mood (mania) and depression. We designed a multistage study in the genetically isolated population of the Central Valley of Costa Rica to identify genes that promote susceptibility to severe BP (termed BPI), and screened the genome ot two Costa Rican BPI pedigrees (McInnes et al., submitted). We considered only individuals who fulfilled very stringent diagnostic criteria for BPI to be affected. The strongest evidence for a BPI locus was observed in 18q22-q23. We tested 16 additional markers in this region and seven yielded peak lod scores over 1.0. These suggestive lod scores were obtained over a far greater chromosomal length (about 40 cM) than in any other genome region. This localization is supported by marker haplotypes shared by 23 of 26 BPI affected individuals studied. Additionally, marker allele frequencies over portions of this region are significantly different in the patient sample from those of the general Costa Rican population. Finally, we performed an analysis which made use of both the evidence for linkage and for association in 18q23, and we observed significant lod scores for two markers in this region.

Alleles↗

PubMed1996

Genotoxic effects of Karwinskia humboldtiana toxin T-514 in peripheral blood lymphocytes.

Toxicity by Karwinskia humboldtiana, a Rhamnaceae plant, has been assessed in a number of studies. Four dimmeric anthracenones, named T-496, T-514, T-516 and T-544 for their molecular weight, have been isolated from this plant. T-514, in particular, has been shown to be toxic to liver and lung as well as to tumoral cell lines, preferentially to those from liver tumors. For this reason it has been suggested that the toxin could be used as an antineoplastic agent. The present study was performed to characterize the biological activity of T-514 as a potential cytostatic and genotoxic agent. Peripheral blood lymphocytes in culture were used as a test system, where chromosomal aberrations and sister chromatid exchanges were scored in order to evaluate genotoxicity, and mitotic index and cell proliferation kinetics were used as parameters for cytostatic and cytotoxic ability. Genotoxicity to lymphocytes was negative. However, proliferation was affected by the toxin, demonstrating a cytostatic activity independent of genotoxic damage.

Adult↗

PubMed1996

Immortalized GnRH neurons express large-conductance calcium-activated potassium channels.

The expression and function of large-conductance Ca2+ -activated K+ (BK) channels in the GT1-7 line of immortalized gonadotropin-releasing hormone (GnRH) neurons was investigated. Ionic currents were recorded using the patch-clamp technique, cytoplasmic free Ca2+ concentration ([Ca2+]i) was monitored using the fluorescent indicator, fura-2, and GnRH secretion was measured by radioimmunoassay. In cell-attached and inside-out patch-clamp recordings, K+ channels with a single-channel conductance of approximately 200 pS were detected. Depolarizing the patch increased the unitary current size and the open probability. In perforated-patch recordings, depolarizing pulses (50 ms) to potentials of -10 to +60 mV from a holding potential of -90 mV elicited outward current with early transient and sustained components. The transient current peaked 2-10 ms after the beginning of each pulse and increased in a voltage-dependent manner. This current was: (1) unaffected by the small-conductance Ca2+ -activated K+ channel blocker, apamin (100 nM); (2) reduced by the BK channel blocker, charybdotoxin (5 nM); (3) abolished by the Ca2+ channel blocker, CdCl2 (25 mu M), and (4) prolonged by the endoplasmic reticulum Ca2+ -ATPase inhibitor, thapsigargin (1 mu M). Based on the single-channel and whole-cell conductances, the number of channels per patch, the patch area, and the surface area of the cell, each GT1-7 cell contains 30-60 BK channels. The functional role of BK channels in GT1-7 cells was evaluated by measuring the effect of charybdotoxin (100 nM) on basal [Ca2+]i and GnRH secretion, as well as on the [Ca2+]i and GnRH secretory responses to gamma-aminobutyric acid (GABA, 100 mu M), an excitatory neurotransmitter in this system. Charybdotoxin had no effect on basal [Ca2+]i or GnRH secretion, or on the GABA-evoked [Ca2+]i and GnRH secretory responses. These results indicate that GT1-7 cells express BK channels; however, the physiological role of BK channels in GT1-7 cells remains elusive.

Animals↗

PubMed1996

[Presence of metacyclical promastigotes of Leishmania pifanoi in the hypopharynx of Lutzomyia youngi and their sugar consumption].

The presence of metacyclical promastigotes of Leishmania pifanoi in the hypopharyngeal duct of Lutzomyia youngi is reported. The insects were experimentally infected by engorgement on the tarsal lesions of hamsters. The metacyclics, whose morphology is illustrated, appeared in the hypopharynx 5 to 9 days after engorgement; they were more frequently found in the insects fed on unrefined commercial sugar. They role of amino derivates of glucose and galactose, as well as of amino acids in the development and migration of metacyclics, is investigated.

Animals↗

PubMed1996

Muscarinic receptor activated Ca2+ channels in non-excitable cells.

We have provided preliminary characterization of a single channel Ca2+ conductance in CHO cells. We have demonstrated that the channel conducts Ca2+, is regulated by m5 receptors, is voltage-independent, has an extremely low conductance, and is second messenger-independent. This channel may be the receptor-operated channel required for downstream activation of several signaling events. It is not known what other cell types express the channel or if it is one of a larger group of related channels. It seems likely that Ca2+ influx-dependent signaling pathways, activated by the muscarinic m5 receptor, would utilize a plasma membrane resident Ca2+ channel to provide a steady source of Ca2+ from outside the cell. The transient nature of IP3-activated increases in intracellular Ca2+ make it an unlikely source of the sustained Ca2+ rise required for phospholipase regulation. This is especially surprising, since levels of intracellular Ca2+ achieved from the release of intracellular Ca2+ stores can be at least one order of magnitude higher than those achieved from extracellular influx (Berridge, 1993). The phospholipase A2 and phospholipase D involved in muscarinic receptor-mediated signaling have not been purified or cloned. It is possible that receptor-activated and Ca2+ influx-dependent phospholipases are integral membrane proteins located adjacent to both receptors and channels. The phospholipases may also translocate to the membrane following activation where they would gain access to the continuous Ca2+ flow. Purification and cloning of this and other related channels should provide better insight into their role in cell signaling.

Animals↗

PubMed1995

Identification of full-length and truncated forms of Ehk-3, a novel member of the Eph receptor tyrosine kinase family.

Factors that bind and activate receptor tyrosine kinases are known to play key roles during development and in the adult. The Eph-related receptors constitute the largest known family of receptor tyrosine kinases. Members of the Eph family exhibit intriguing patterns of expression in the embryo, implicating them in a variety of developmental processes, and their expression is often restricted to particular subpopulations of postmitotic neurons in the adult. We describe the identification and characterization of a novel member of the Eph receptor family, which we have termed Ehk-3 for Eph Homologous Kinase 3. Ehk-3 displays all the major structural features shared by other members of the Eph family, including a cysteine-rich region and tandem fibronectin type-III domains in its extracellular portion. Ehk-3 is expressed in two forms in a developmentally-regulated fashion: a conventional full-length version containing the intracellular tyrosine kinase domain, as well as a truncated form that lacks this domain. Both forms of Ehk-3 are quite restricted to the nervous system in the adult, but Ehk-3 is more widely expressed in the embryo, suggesting that Ehk-3 mediates different functions during development and in the adult.

Age Factors↗

PubMed1995

Genomic organization and chromosomal localization of the human and mouse genes encoding the alpha receptor component for ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) has recently been found to share receptor components with, and to be structurally related to, a family of broadly acting cytokines, including interleukin-6, leukemia inhibitory factor, and oncostatin M. However, the CNTF receptor complex also includes a CNTF-specific component known as CNTF receptor alpha (CNTFR alpha). Here we describe the molecular cloning of the human and mouse genes encoding CNTFR. We report that the human and mouse genes have an identical intron-exon structure that correlates well with the domain structure of CNTFR alpha. That is, the signal peptide and the immunoglobulin-like domain are each encoded by single exons, the cytokine receptor-like domain is distributed among 4 exons, and the C-terminal glycosyl phosphatidylinositol recognition domain is encoded by the final coding exon. The position of the introns within the cytokine receptor-like domain corresponds to those found in other members of the cytokine receptor superfamily. Confirming a recent study using radiation hybrids, we have also mapped the human CNTFR gene to chromosome band 9p13 and the mouse gene to a syntenic region of chromosome 4.

Amino Acid Sequence↗

PubMed1995

Single-microelectrode voltage clamp measurements of pancreatic beta-cell membrane ionic currents in situ.

A conventional patch clamp amplifier was used to test the feasibility of measuring whole-cell ionic currents under voltage clamp conditions from beta-cells in intact mouse islets of Langerhans perifused with bicarbonate Krebs buffer at 37 degrees C. Cells impaled with a high resistance microelectrode (ca. 0.150 G omega) were identified as beta-cells by the characteristic burst pattern of electrical activity induced by 11 mM glucose. Voltage-dependent outward K+ currents were enhanced by glucose both in the presence and absence of physiological bicarbonate buffer and also by bicarbonate regardless of the presence or absence of glucose. For comparison with the usual patch clamp protocol, similar measurements were made from single rat beta-cells at room temperature; glucose did not enhance the outward currents in these cells. Voltage-dependent inward currents were recorded in the presence of tetraethylammonium (TEA), an effective blocker of the K+ channels known to be present in the beta-cell membrane. Inward currents exhibited a fast component with activation-inactivation kinetics and a delayed component with a rather slow inactivation; inward currents were dependent on Ca2+ in the extracellular solution. These results suggest the presence of either two types of voltage-gated Ca2+ channels or a single type with fast and slow inactivation. We conclude that it is feasible to use a single intracellular microelectrode to measure voltage-gated membrane currents in the beta-cell within the intact islet at 37 degrees C, under conditions that support normal glucose-induced insulin secretion and that glucose enhances an as yet unidentified voltage-dependent outward K+ current.

Animals↗

PubMed1995

Direct control of a large conductance K(+)-selective channel by G-proteins in adrenal chromaffin granule membranes.

We report here the presence of a Ca(2+)-independent K(+)-channel of large conductance in adrenal chromaffin cell secretory vesicle membranes which is controlled by inhibitory as well as stimulatory heterotrimeric GTP-binding proteins. Using antibodies against specific alpha subunits for immunoblot analysis, we were able to identify the presence of the inhibitory G(i)2 and G(i)3 subtypes, as well as the stimulatory G(o) and Gs subtypes, but not G(i)1 in adrenal chromaffin granules. Furthermore, functional analysis of the K(+)-channel incorporated into planar lipid bilayers showed that GDP beta S and GTP gamma S have opposite effects on channel activity inducing interconversions between a low and a high open-probability state. Consistent with these findings, the same antibodies antagonized the effects of the nonhydrolyzable analogues on the open probability of the K(+)-channel.

Adrenal Medulla↗

PubMed1995

Magnitude and modulation of pancreatic beta-cell gap junction electrical conductance in situ.

The parallel gap junction electrical conductance between a beta-cell and its nearest neighbors was measured by using an intracellular microelectrode to clamp the voltage of a beta-cell within a bursting islet of Langerhans. The holding current records consisted of bursts of inward current due to the synchronized oscillations in membrane potential of the surrounding cells. The membrane potential record of the impaled cell, obtained in current clamp mode, was used to estimate the behavior of the surrounding cells during voltage clamp, and the coupling conductance was calculated by dividing the magnitude of the current bursts by that of the voltage bursts. The histogram of coupling conductance magnitude from 26 cells was bimodal with peaks at 2.5 and 3.5 nS, indicating heterogeneity in extent of electrical communication within the islet of Langerhans. Gap junction conductance reversibly decreased when the temperature was lowered from 37 to 30 degrees C and when the extracellular calcium concentration was raised from 2.56 to 7.56 mM. The coupling conductance decreased slightly during the active phase of the burst. Activation of adenylate cyclase with forskolin (10 microM) resulted in an increase in cell-to-cell electrical coupling. We conclude that beta-cell gap junction conductance can be measured in situ under near physiological conditions. Furthermore, the magnitude and physiological regulation of beta-cell gap junction conductance suggest that intercellular electrical communication plays an important role in the function of the endocrine pancreas.

Animals↗

PubMed1995

Ion channel hypothesis for Alzheimer amyloid peptide neurotoxicity.

1. Alzheimer's disease (AD) is a chronic dementia and neurodegenerative disorder affecting the oldest portions of the population. Brains of AD patients accumulate large amount of the A beta P peptide in amyloid plaques. 2. The A beta P[1-40] peptide is derived by proteolytic processing from a much larger amyloid precursor protein (APP), and has been circumstantially identified as the toxic principle causing cell damage in the disease. 4. The A beta P[1-40] peptide is able to form quite characteristic calcium channels in planar lipid bilayers. These channels have conductances in the nS range, and can dissipate ion gradients quickly. The peptide can also cause equivalent cation conductances in cells. 5. We suggest that amyloid channel blocking agents might be therapeutically useful in Alzheimer's Disease, and have constructed molecular models of the channels to aid in the design of such compounds.

Alzheimer Disease↗

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

GnRH-induced cytosolic calcium oscillations in pituitary gonadotrophs: phase resetting by membrane depolarization.

Cultured rat pituitary gonadotrophs under whole-cell voltage clamp conditions respond to the hypothalamic hormone GnRH with synchronized oscillatory changes in both cytosolic Ca2+ concentration ([Ca2+]i) and [Ca2+]i-activated, apamin-sensitive K+ current (IK(Ca)). We found, and report here for the first time, that in GnRH-stimulated cells a brief depolarizing pulse can elicit a transient [Ca2+]i rise similar to the endogenous cycle. Furthermore, Ca2+ entry during a single depolarizing pulse was found to shift the phase of subsequent endogenous [Ca2+]i oscillations, which thereafter continue to occur at their previous frequency before the pulse. Application of two consecutive depolarizing pulses showed that the size of the [Ca2+]i rise evoked by the second pulse depended on the time lapsed between two consecutive pulses, indicating that each endogenous or evoked [Ca2+]i rise cycle leaves the Ca2+ release mechanism of the gonadotroph in a refractory state. Recovery from this condition can be described by an exponential function of the time lapsed between the pulses (time constant of ca. 1 s). We propose that the underlying mechanism in both refractoriness after endogenous cycles and phase resetting by a brief pulse of Ca2+ entry involves the InsP3 receptor-channel molecule presumed to be located on the cytosolic aspect of the endoplasmic reticulum membrane.

Animals↗