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Biomedical subjects

R Perez-Polo

Publications and source records attributed to R Perez-Polo.

12 recordsLinked to original sources
PubMed2000

Insulin-like growth factor-I cDNA gene transfer in vitro and in vivo.

Our hypothesis is that gene transfer of an IGF-I CMV-cDNA with cholesterol containing cationic liposomes is an efficient tool for transient transfection of growth factors in vitro and in vivo. In vitro, we transiently cotransfected IGF-I cDNA with a CMV construct and a Lac Z beta-galactosidase cDNA/CMV construct using cholesterol containing cationic liposomes and measured beta-galactosidase and IGF-I mRNA and protein. In vivo, we subcutaneously injected 3-month-old male Sprague-Dawley rats with IGF-I cDNA and beta-galactosidase cDNA into rat skin. After IGF-I and beta-galactosidase were cotransfected into PC12 cells, Northern blot analysis showed that the peak time of IGF-I expression was 2 days for mRNA and 5 days for protein. In vivo, a cDNA/liposome ratio of 1:2 was most effective. IGF-I protein expression in IGF-I-transfected skin resulted in significant transfection from day 5 to day 7. In situ determination of beta-galactosidase activity confirmed that transfections resulted in a restricted expression area.

Animals↗
PubMed2000

One path to cell death in the nervous system.

Both acute and chronic insults to the nervous system can result in changes in homeostasis that result in cell death or recovery processes that alter function. The signaling mechanisms for this broad spectrum of events that impair neurological function span the gamut from abrupt injury to the slow onset of neurodegenerative diseases in extreme aging. A common element in all of these events is the triggering of signal cascades that determine cellular commitment to apoptosis as a ameliorative alternative to inflammatory necrosis. Key in these cascades is the activation of the caspase and Bcl-family of proteins by the NF-kappaB transcription factor. Here we consider aspects of specificity of activation as a result of the differential expression of NF-kappaB proteins and their regulation of selective genes as a result of binding to select DNA consensus sequences out of the 64 different combinations that constitute the NF-kappaB DNA binding consensus sequence.

Aging↗
PubMed1999

NGF levels decrease in the spinal cord and dorsal root ganglion after spinal hemisection.

To examine changes in nerve growth factor (NGF) levels in spinal cord and dorsal root ganglia (DRG) after spinal injury, male Sprague-Dawley rats weighing 150-175 g were given spinal hemisections. NGF content was measured at various post-surgical times and compared with naive controls (n = 4 per time point) in the spinal cord, DRG and blood serum by ELISA techniques (Promega). Levels of NGF in the blood serum were significantly increased 8-fold at 48h but were significantly decreased in the spinal cord and DRG by 2- to 4-fold until 7 days postsurgery (ANOVA, p < 0.05). Contrary to accepted dogma, spinal injury results in decreased levels of NGF in the spinal cord and DRG following spinal injury.

Animals↗
PubMed1998

Brain-derived neurotrophic factor (BDNF) protects cultured rat cerebellar granule neurons against glucose deprivation-induced apoptosis.

In the present study, cell death induced by glucose deprivation in primary cultures of cerebellar granule neurons was examined. Glucose deprivation-induced apoptotic cell death was demonstrated using the terminal transferase-mediated (TdT) deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL) method and DNA fragmentation assays. When the effects of different neurotrophins on the survival of cerebellar granule neurons after glucose deprivation were assessed, BDNF, but not NT-3 or NGF, was found to protect cerebellar granule neurons against glucose deprivation-induced cell death. In addition, BDNF treatment increased c-Fos immunoreactivity in the cerebellar granule neurons. These results are consistent with the hypothesis that neuronal death due to glucose deprivation has a significant apoptotic component and that neurotrophins can protect against hypoglycemic damage.

Animals↗
PubMed1997

Regulation of antioxidant enzyme expression by NGF.

The rapid decreases in viability seen in H2O2-treated PC12 cells reflect enhanced susceptibility of neural cell types to oxidant injury. The dose-response relationship between NGF concentration and survival after H2O2 treatment resembles that for NGF effects on PC12 survival in serumless medium. Previously we have shown that NGF treatment enhances the activity of GSH-Px and catalase which catalyze the degradation of H2O2. Here in order to ascertain whether NGF stimulates transcription, affects mRNA stability, or acts post-transcriptionally, we measured catalase and GSH-Px mRNA half-lives. While both catalase and GSH-Px transcripts are stable with a relatively long half life and a gradual decay in mRNA levels, NGF had different effects on their stability. NGF had marked effects on catalase mRNA stability. The catalase gene has a 3' flanking region with T-rich clusters and CA repeats known to be susceptible to regulation by destabilization or ubiquination. NGF maintained catalase mRNA levels of actinomycin D (ACT-D) treated PC12 cells at twice that of cells exposed to ACT-D alone, delaying the rate of decay for catalase mRNA for 24 h. The NGF induction of GSH-Px and catalase mRNA was inhibited by cycloheximide (CHX) treatment with a slight decrease in their mRNA levels due to prolonged exposure to CHX. When the CHX treatment was delayed relative to the NGF treatment there was no effect on NGF effects on catalase and GSH-Px. The GSH-Px gene has conserved sequences in the open reading frame and 3' untranslated region which forms a stem-loop structure necessary for the incorporation of Se into this selenoprotein. While Se is important in stabilizing GSH-Px transcripts, it did not affect transcription rates or mRNA stability. These results are consistent with the hypothesis that NGF regulates catalase and GSH-Px expression via a primary effect on transcription factor pathways.

Animals↗
PubMed1996

Increased uptake of L-cysteine and L-cystine by nerve growth factor in rat pheochromocytoma cells.

Nerve growth factor is a neurotrophic factor which promotes cell survival and differentiation in the central and peripheral nervous system. The rat pheochromocytoma (PC12) cell has been frequently used to study the actions of nerve growth factor (NGF). Our previous studies demonstrate that pretreatment with NGF for 24 h protects PC12 cells from oxidative stress by increasing glutathione (GSH) concentrations and the activity of gamma-glutamylcysteine synthetase, which is a rate-limiting enzyme in GSH synthesis. The synthesis of intracellular GSH is dependent on the availability of the precursor amino acid, L-cysteine. Cells take up L-cystine from the extracellular fluid and convert it to L-cysteine intracellularly. L-Cysteine is then released from cells to maintain extracellular L-cysteine. Here we report that NGF increased the uptake of L-cysteine or L-cystine. The increased concentrations of L-cysteine or L-cystine by NGF was responsible for the enhanced intracellular GSH concentrations. The increased GSH and L-cysteine concentrations by NGF also served as intracellular antioxidants. The protection of PC12 cells by NGF from oxidative stress was due to the stimulated increased levels of intracellular glutathione and L-cysteine or L-cystine.

Animals↗
PubMed1996

Regulation of gamma-glutamylcysteine synthetase activity by nerve growth factor.

Nerve growth factor is a neurotrophic factor that regulates neuronal cell development, maintenance, and injury responses in the peripheral and central nervous system. Nerve growth factor reduces injury owing to oxidative stress in rat pheochromocytoma (PC12) cells by increasing intracellular glutathione, in part owing to its stimulation of the activity of gamma-glutamylcysteine synthetase, which is the rate-limiting enzyme in the synthesis of glutathione. Here we show that nerve growth factor did not increase the activity of gamma-glutamylcysteine synthetase in PC12 cells at the transcriptional level. Rather, nerve growth factor enhanced the stability of gamma-glutamylcysteine synthetase mRNA in PC12 cells. These results suggest that, during oxidative stress, nerve growth factor extended the half-life of gamma-glutamylcysteine synthetase mRNA, thus increasing gamma-glutamylcysteine synthetase mRNA levels compared to nerve growth factor-deprived PC12 cells.

Animals↗
PubMed1994

Regional CNS uptake of blood-borne nerve growth factor.

Nerve growth factor (NGF), in addition to being a neurotrophic substance, has effects on the endocrine and immune systems. For example, intravenous injection of NGF results in a cascade of events leading to an increase in glucocorticoid secretion. While this response appears to be mediated centrally, there has been no evidence that circulating NGF has access to the CNS. Using intravenous injections of 125I-NGF, we find specific uptake at 1 hr but none at 6 hr, into homogenates of the basal forebrain, cerebellum, frontal cortex, hippocampus, and olfactory bulb. By autoradiography, uptake is localized to circumventricular organs, deep layers of the cerebellum, and all layers of the hippocampal region CA1, but not the dentate gyrus. Thus, uptake of blood-borne NGF could affect the hypothalamic-pituitary-adrenal axis via binding to NGF receptors present in the hippocampus. However, the sources of endogenous NGF, the mechanism of access through the blood-brain barrier, the eventual fate of NGF entering from the blood, and the physiological significance of this uptake remain to be elucidated.

Animals↗
PubMed1993

Role of nerve growth factor in oxidant homeostasis: glutathione metabolism.

Free radicals are generated in the CNS by ongoing oxygen metabolism and biological events associated with injury and inflammation. Increased free radical levels may also persist in some chronic neurological diseases and in the aged. Nerve growth factor (NGF) is a member of the neurotrophin family of proteins that can regulate neuronal development, maintenance, and recovery from injury. NGF protected rat pheochromocytoma PC12 cells, an adrenal chromaffin-like NGF-responsive cell line, from the oxidant stress accompanying hydrogen peroxide treatment by stimulating GSH levels and enzymes in the GSH metabolism cycle and in the GSH/GSH peroxidase antioxidant redox system, a ubiquitous cellular antioxidant system. Specifically, NGF increased gamma-glutamylcysteine synthetase (GCS) activity, the rate-limiting enzyme for GSH synthesis, by 50% after 9 h and GSH levels by 100% after 24 h of treatment. NGF stimulated GSH peroxidase by 30% after 3 days and glucose 6-phosphate dehydrogenase by 50% after 2 days. Treatment with NGF and cycloheximide, or actinomycin D, which inhibit protein and RNA synthesis, respectively, blocked the NGF stimulation of GCS and glucose 6-phosphate dehydrogenase. Increased GSH levels due to NGF treatment were responsible for the significant protection of PC12 cells from hydrogen peroxide-induced stress. Pretreatment of PC12 cells with NGF for 24 h rescued cells from the toxic effects of the extracellular hydrogen peroxide generated by the glucose/glucose oxidase system but did not rescue cells that were subjected to GSH deprivation due to treatment with 10 microM L-buthionine-(S,R)-sulfoximine, an inhibitor of GCS.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Nerve growth factor binding in aged rat central nervous system: effect of acetyl-L-carnitine.

The nerve growth factor protein (NGF) has been demonstrated to affect neuronal development and maintenance of the differentiated state in certain neurons of the peripheral and central nervous system (CNS) of mammals. In the CNS, NGF has sparing effects on cholinergic neurons of the rodent basal forebrain (BF) following lesions where it selectively induces choline acetyltransferase (ChAT). NGF also induces ChAT in the areas to which BF provides afferents. In aged rats, there is a reduction in the NGF-binding capacity of sympathetic ganglia. Here, we wish to report that there is a decrease in the NGF-binding capacity of the hippocampus and basal forebrain of aged (26-month-old) rats as compared to 4-month-old controls but no change in NGF binding in cerebellum. In all instances, equilibrium binding dissociation constants did not differ significantly. Treatment of rats with acetyl-L-carnitine, reported to improve cognitive performance of aged rats, ameliorates these age-related deficits.

Acetylcarnitine↗