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Biomedical subjects

L Pang

Publications and source records attributed to L Pang.

At least 91 records · Page 5Linked to original sources
PubMed1996

Spread of murine cytomegalovirus to inner ocular structures following disruption of the blood-retina barrier in immunosuppressed BALB/c mice.

PURPOSE: The aims of this study were to determine whether disruption of the blood-retina barrier (BRB) increases spread of murine cytomegalovirus (MCMV) to the eye after intraperitoneal inoculation and whether systemic immunosuppression influences the location of MCMV in the ocular compartment. METHODS: The BRB of the left eye of normal and immunosuppressed mice was disrupted by supraciliary inoculation of tissue culture medium followed 2 hours later by intraperitoneal injection of MCMV. Plaque assay of homogenized ocular tissue was used to determine the frequency of virus-positive eyes and the titer of virus in the eyes. Beta-galactosidase staining of frozen sections was used to locate virus in the eyes. RESULTS: In nonimmunosuppressed mice, the frequency of virus isolation, as well as the titer of virus, were significantly higher in eyes in which the BRB had been disrupted. Although the frequency of virus isolation was the same in both eyes of immunosuppressed mice, the titer of virus was significantly higher in the eye in which the BRB had been disrupted. The most striking result was that the location of virus was different in the nondisrupted eyes of immunosuppressed mice than it was in the disrupted eyes of immunosuppressed mice. In the former, virus was seen only in the outer ocular structures (conjunctiva, sclera, lacrimal gland), whereas in the latter, virus was observed in the retina and anterior segment (iris, ciliary body) as well as the outer ocular structures. CONCLUSIONS: The results of these studies suggest that ocular damage followed by increased spread of virus to and within the eye during systemic infection with CMV may be one mechanism by which development of CMV retinitis is facilitated in patients with acquired immune deficiency syndrome.

Animals↗
PubMed1996

[Effects of different natural stimulants on the activity of the rabbits' carotid chemoreceptor].

The effects of different natural stimulants on the afferent unit discharge of rabbit carotid chemoreceptors were studied in the in vitro carotid body- sinus nerve preparation. A total of 32 chemoreceptive units were recorded. The results were as follows: (1) Of the 32 units, 10 (31%) showed chemosensory responses only to PO2 decrease; 9 (28%) to all stimulants (PO2 decrease, PCO2 increase and pH decrease); 9 (28%) to PO2 decrease and PCO2 increase; 3 (9%) to PO2 decrease and pH decrease; only one to pH decrease. (2) The potency of the three natural stimulants in eliciting the changes in intensity of discharge showed a decreasing order as follows: PO2 decrease > PCO2 increase > pH decrease. The above results suggest that the carotid body may contain only one or a combination of 2 or 3 kinds of chemoreceptors respectively sensitive to decrease PO2 to increase PCO2 or decrease pH.

Afferent Pathways↗
PubMed1995

A synthetic inhibitor of the mitogen-activated protein kinase cascade.

Treatment of cells with a variety of growth factors triggers a phosphorylation cascade that leads to activation of mitogen-activated protein kinases (MAPKs, also called extracellular signal-regulated kinases, or ERKs). We have identified a synthetic inhibitor of the MAPK pathway. PD 098059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one] selectively inhibited the MAPK-activating enzyme, MAPK/ERK kinase (MEK), without significant inhibitory activity of MAPK itself. Inhibition of MEK by PD 098059 prevented activation of MAPK and subsequent phosphorylation of MAPK substrates both in vitro and in intact cells. Moreover, PD 098059 inhibited stimulation of cell growth and reversed the phenotype of ras-transformed BALB 3T3 mouse fibroblasts and rat kidney cells. These results indicate that the MAPK pathway is essential for growth and maintenance of the ras-transformed phenotype. Further, PD 098059 is an invaluable tool that will help elucidate the role of the MAPK cascade in a variety of biological settings.

3T3 Cells↗
PubMed1995

Signal transduction pathways leading to insulin-induced early gene induction.

We examined the signal transduction pathway leading to insulin stimulation of two immediate early genes, c-fos, and the early growth response gene, Egr-1. In Rat 1 fibroblasts overexpressing normal human insulin receptors (HIRc-B), insulin and IGF-I rapidly and transiently induced the expression of both c-fos and Egr-1 mRNA with maximum accumulation at 30 min, declining to basal levels at 120 min. Insulin (100 ng/mL) increased c-fos and Egr-1 mRNA expression 10-fold (EC50 = 20 ng/mL), whereas IGF-I (100 ng/mL) and serum (20%) led to a 3- and 11.5-fold increase, respectively. Insulin-stimulated c-fos protein expression was maximal at 1 h postinduction and undetectable at 4 h. The effects of insulin and IGF-I on both c-fos mRNA and protein expression were absent in Rat 1 fibroblasts expressing tyrosine kinase-defective human insulin receptors (A/K1018). In cells expressing insulin receptors in which the two C-terminal tyrosines are mutated to phenylalanine (Y/F2 cells), the insulin stimulated increase in Egr-1 and c-fos mRNA was comparable to that of HIRc cells, whereas, in cells expressing C-terminal truncated receptors (delta CT cells), the insulin induced increase in Egr-1 mRNA was normal, but the c-fos mRNA response was severely blunted. As expected, the insulin effect to increase ras GTP formation and MAP kinase activity was negligible in A/K1018 cells but normal, or supernormal, in Y/F2 cells. Importantly, stimulation of ras GTP was increased in delta CT cells, whereas stimulation of MAP kinase activity was almost absent.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor.

The mitogen-activated protein kinase (MAP kinase) pathway is thought to play an important role in the actions of neurotrophins. A small molecule inhibitor of the upstream kinase activator of MAP kinase, MAP kinase kinase (MEK) was examined for its effect on the cellular action of nerve growth factor (NGF) in PC-12 pheochromocytoma cells. PD98059 selectively blocks the activity of MEK, inhibiting both the phosphorylation and activation of MAP kinases in vitro. Pretreatment of PC-12 cells with the compound completely blocked the 4-fold increase in MAP kinase activity produced by NGF. Half-maximal inhibition was observed at 2 microM PD98059, with maximal effects at 10-100 microM. The tyrosine phosphorylation of immunoprecipitated MAP kinase was also completely blocked by the compound. In contrast, the compound was without effect on NGF-dependent tyrosine phosphorylation of the pp140trk receptor or its substrate Shc and did not block NGF-dependent activation of phosphatidylinositol 3'-kinase. However, PD98059 completely blocked NGF-induced neurite formation in these cells without altering cell viability. These data indicate that the MAP kinase pathway is absolutely required for NGF-induced neuronal differentiation in PC-12 cells.

Animals↗
PubMed1995

Nerve growth factor stimulates a novel protein kinase in PC-12 cells that phosphorylates and activates mitogen-activated protein kinase kinase (MEK).

Activation of mitogen-activated protein kinase (MAP kinase) plays an important role in the cellular effects of nerve growth factor (NGF). Although the precise pathway by which NGF activates MAP kinase is not clear, several enzymes have been identified that may form a linear phosphorylation cascade, in which MAP kinase is activated by MAP kinase kinase (MEK). A key enzyme that links the ras-GTP complex to MEK is widely believed to be the raf kinase. However, immunoprecipitation experiments in PC-12 cells revealed that raf is not the major NGF-dependent MEK kinase [Zheng, Ohmichi, Saltiel and Guan (1994) Biochemistry 33, 5595-5599]. We have identified a protein kinase from PC-12 cells that catalyses both the phosphorylation and activation of MEK. This activity is stimulated 3-fold in cells treated with NGF. The partial purification on FPLC and characterization of this MEK kinase indicate that it is distinct from raf, MEK, MAP kinase and other previously described NGF-stimulated protein kinases. The activity of this enzyme is unaffected by direct addition to the assay of heparin, staurosporine, K252A and the heat-stable cyclic AMP-dependent kinase peptide inhibitor, but is slightly inhibited by NaF and calcium ions. Comparison of its behaviour on gel permeation and sucrose-density gradients indicates a molecular mass in the region of 50,000 Da. Moreover, isoelectric focusing of the enzyme revealed a pI of approx. 7.3. The kinase activity is specific for ATP as substrate with a Km of 11 microM, and requires Mg2+ as a cofactor. Analysis of the activation of this enzyme in PC-12 cells transfected with a dominant inhibitory mutant of p21ras suggests that this MEK kinase resides downstream of ras in the MAP kinase activation pathway. Moreover, site-directed mutation of the residues on MEK that are phosphorylated by raf does not completely abrogate phosphorylation by the MEK kinase, suggesting that this enzyme may share some phosphorylation sites with raf, but also phosphorylates MEK on other sites.

Amino Acid Sequence↗
PubMed1995

Effects of hypoxia on the intracellular K+ of clustered and isolated glomus cells of mice and rats.

Carotid bodies of rats and mice were used to measure the intracellular potassium activity, ai(K), of clustered and isolated glomus cells normally oxygenated (pO2 102-139 Torr), and during hypoxia (pO2 2-82 Torr) induced by Na-dithionite. ai(K) was measured with intracellular ion-selective microelectrodes, and the resting potential (EM) with KCl-filled micropipettes. Under normoxia, the ai(K) of clustered cells in both species was higher than that of isolated cells. This resulted in more negative potassium equilibrium potentials (EK's). There was no correlation between ai(K) and EM in clustered cells, but this correlation was significant in isolated cells. Hypoxia significantly decreased ai(K) in clustered and single mouse cells, and in clustered rat cells, although its effects on single rat cells were variable. ai(K) decreases were accompanied by cell depolarization and positive shifts in EK. During hypoxia, there were significant correlations between ai(K) and EM in all cells. It is suggested that ai(K) did not influence the EM of clustered cells under normoxia because of interference by K+ pumping mechanisms toward glomus cells from surrounding sustentacular processes. This hindrance is not present when glomus cells are isolated. During hypoxia K+ pumping from sustentacular cells is disrupted, allowing the EM of clustered glomus cells to follow their ai(K) and behave like isolated cells. The different effects of hypoxia on isolated rat and mouse cells may be due to activation of different types of glomus cells.

Animals↗
PubMed1995

Short report: hepatitis E infection in the Brazilian Amazon.

This is the first report of serologic evidence of hepatitis E infection in Brazil. During a community-based survey of healthy individuals, six of 97 gold miners in the Amazon region of Mato Grosso had antibody to the virus. The mining camps have poor sanitation with a great potential for fecal-oral transmission of disease. Since levels of hepatitis E antibodies may quickly wane, studies to directly measure the incidence of seroconversion are planned to determine the intensity of transmission in this area.

Adult↗
PubMed1994

Mutation of the two carboxyl-terminal tyrosines in the insulin receptor results in enhanced activation of mitogen-activated protein kinase.

Activation of mitogen-activated protein (MAP) kinase represents an important mechanism in hormonal regulation. To clarify the role of MAP kinase activation in insulin action, we compared the activation of the enzyme in Rat-1 cells transfected with wild-type (Hirc) and mutant insulin receptors in which the 2 carboxyl-terminal tyrosines were substituted with phenylalanine (Y/F2). Expression of the Y/F2 mutant receptor enhanced the responsiveness of MAP kinase to insulin. Moreover, the insulin responsiveness of the activator of this enzyme, MAP kinase kinase, was also increased in these cells. To explore the early signaling events that might account for this increase in responsiveness, we evaluated the tyrosine phosphorylation of the insulin receptor substrate, IRS-1, and its subsequent association with phosphatidylinositol (PI)-3 kinase. In both cell types, insulin led to a dose-dependent increase in the association of tyrosine phosphorylated IRS-1 with the SH2 domain of the p85 regulatory subunit of PI-3 kinase, and also increased the amount of PI kinase activity detected in anti-IRS-1 immunoprecipitates. The effect of insulin was significantly greater in Y/F2 cells, as determined in both assays. In previous studies, cells bearing this receptor mutant exhibited an identical metabolic response but enhanced mitogenic response to insulin when compared with wild-type receptor. These data provide further evidence for divergence of the mitogenic and metabolic signaling pathways at or near the insulin receptor.

Animals↗
PubMed1994

Stage- and region-dependent chondrogenesis and growth of chick wing-bud mesenchyme in serum-containing and defined tissue culture media.

During development, limb-bud mesenchymal cells carry out complex spatiotemporal-patterns of growth and differentiation. Tissue and organ culture facilitate analysis of environmental influences on these cell behaviors, allowing their partial dissection into exogenous and endogenous components. Two factors that complicate such in vitro analyses are the heterogeneity of the cultured cells and imprecise knowledge of culture medium composition. Limb mesenchyme comprises a heterogenous cell population with important regional differences in cell type. Dividing the limb into subregions helps limit the cellular heterogeneity and using chemically defined, serum-free medium alloys concerns about medium composition. In the present study, mesenchyme from different regions along the anteroposterior and proximodistal axes of stage 21-22 and stage 23-24 chick wing buds was grown in high-density microtiter cultures in chemically defined and in serum-containing medium. Four-day cultures of the various regions were compared in terms of culture morphology and the accumulation of Alcian blue-positive cartilage matrix and DNA. The results demonstrate stage- and region-dependent differences in the in vitro growth, differentiation, and responsiveness of these cells. For example, mesenchyme from the distal anterior region of the wing bud exhibited lower intrinsic chondrogenic capacity and greater responsiveness to serum than other regions. Patterns of in vitro chondrogenesis also suggest that, at the stages examined, distal wing-bud mesenchyme may be less homogeneous than has been believed. A case is made for the suitability of serum-free medium for future in vitro studies of chick limb-bud mesenchyme. The results are considered in relation to the process of limb development and regional expression of pattern-related genes.

Animals↗
PubMed1994

Localization of HIV RNA in mitochondria of infected cells: potential role in cytopathogenicity.

The intracellular distribution of HIV-1 RNA transcripts in infected cells was studied using in situ hybridization detected by electron microscopy and cellular fractionation. Although viral RNA and core protein could be detected throughout the cytoplasm and nucleus, viral RNA was found in significantly increased amounts in mitochondria relative to the cytoplasm and nucleus. In contrast, cellular poly(A) RNA or viral gag proteins were not increased in the mitochondria. A cell line containing an integrated latent genome that could be induced to express viral RNA after phorbol ester stimulation showed an increase in viral RNA accumulation in mitochondria parallel with the increase in HIV expression levels. Concomitant with HIV expression, there was a decrease in mitochondrial viability. Using immunofluorescent markers to detect probes to HIV RNA transcripts and antibodies to mitochondrial proteins simultaneously in single cells, there was an inverse relationship between the amount of viral RNA and mitochondrial integrity. High levels of viral RNA in mitochondria were found in acutely (but not chronically) infected cells. We propose that HIV RNA import into mitochondria can compromise mitochondrial function.

Cell Line↗
PubMed1993

The stimulation of pp42mapkinase by insulin does not correlate with its metabolic actions in cells overexpressing mutant insulin receptors.

Naturally occurring human insulin receptor mutants Ser1200 and Thr1134, and a site-directed mutant Arg1030 overexpressed in Chinese hamster ovary (CHO) cells, bind insulin with affinities identical to wildtype receptors but are apparently kinase deficient. Cells expressing the Ser1200 receptor exhibit insulin stimulation of glycogen synthesis similar to these bearing the wildtype receptor, but fail to mediate insulin-responsive DNA synthesis. In contrast, the Thr1134 and Arg1030 mutants exhibit no response to insulin. The activity of Mitogen Activated Protein (MAP) kinase in cells transfected with wildtype receptor is more responsive to insulin than that detected in untransfected parental cells, while cells bearing any of the mutant receptors are less responsive than the parental cells. These differences in the stimulation of MAP kinase activity are paralleled by differences in insulin-dependent phosphorylation of the enzyme. These results suggest that the p42 MAP kinase is not universally required for the metabolic effects of insulin.

Animals↗
PubMed1993

Hypoxia affects differently the intracellular pH of clustered and isolated glomus cells of the rat carotid body.

Clustered and isolated glomus cells, cultured from rat carotid bodies, were exposed to hypoxia (pO2 2-30 torr) induced by applications of sodium-dithionite (Na2S2O4). Hypoxia decreased or increased intracellular pH (pHi) of clustered cells about equally, but lowered it in most isolated cells. The levels of intracellular acidification were similar in both groups whereas alkalinization was more pronounced in the clusters. The H+ equilibrium potential (EH) and its changes during hypoxia (delta EH), were determined almost exclusively by pHi. Seventy-five percent of clustered cells became depolarized whereas 80% of isolated cells underwent hyperpolarization. In both groups, changes in the resting potential (delta EM) were directly and significantly correlated with delta EH, thus delta pHi. These observations support the view that clustered and isolated rat glomus cells behave differently. This difference may occur because of the presence (in the clusters) or absence (in isolated cells) of enveloping sustentacular cell processes.

Animals↗
PubMed1993

The tyrosine kinase inhibitor tyrphostin blocks the cellular actions of nerve growth factor.

A series of the synthetic protein kinase inhibitors known as tyrphostins were examined for their effects on the tyrosine autophosphorylation of the pp140c-trk, nerve growth factor (NGF) receptor. One of the tyrphostins, AG879, inhibited NGF-dependent pp140c-trk tyrosine phosphorylation, but did not affect tyrosine phosphorylation of epidermal growth factor or platelet-derived growth factor receptors. In addition, the tyrosine phosphorylation of the receptor-associated protein pp38 was also attenuated by the tyrphostin. This effect was time- and dose-dependent, although inhibition of pp38 phosphorylation occurred earlier and at lower concentrations of the compound. AG879 also inhibited NGF-induced PLC-gamma 1 phosphorylation, phosphatidylinositol-3 (PI3) kinase activation, the association of the tyrosine-phosphorylated proteins pp100 and pp110 with the p85 subunit of PI-3 kinase, mitogen activated protein and raf-1 kinases, and c-fos induction. In addition, AG879 inhibited NGF-induced neurite outgrowth in PC12 cells. These data indicate that tyrosine kinase activity of the pp140c-trk NGF receptor is essential for the cellular actions of this growth factor.

Animals↗
PubMed1993

Bombesin and epidermal growth factor stimulate the mitogen-activated protein kinase through different pathways in Swiss 3T3 cells.

Both bombesin and epidermal growth factor (EGF) are potent mitogens in Swiss 3T3 cells that nonetheless have dissimilar receptor structures. To explore possible common intracellular events involved in the stimulation of cellular growth by these two peptides, we have evaluated the regulation of the mitogen-activated protein (MAP) kinase. Exposure of Swiss 3T3 cells to bombesin, EGF or the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) causes the rapid and transient stimulation of the enzyme activity. Pretreatment of cells with the protein kinase inhibitor H-7, or down-regulation of cellular protein kinase C by prolonged exposure to PMA, causes a decrease of over 90% in the activation of MAP kinase by bombesin. In contrast, these treatments have no effect on the stimulation of MAP kinase by EGF. The stimulation of MAP kinase activity by bombesin is dose-dependent, occurring over a narrow concentration range of the peptide. Both EGF and bombesin stimulate the phosphorylation of an immunoprecipitable MAP kinase protein migrating at 42 kDa on SDS/PAGE. Phosphoamino acid analysis of this phosphorylated protein reveals that EGF and bombesin stimulate phosphorylation on tyrosine, threonine and serine residues. Tyrosine phosphorylation of the enzyme, as evaluated by antiphosphotyrosine blotting of the immunoprecipitated protein, reveals that the time course of phosphorylation by both mitogens correlates with stimulation of enzyme activity. These results provide further evidence for the convergence of discrete pathways emanating from tyrosine kinase and G-protein-linked receptors in the regulation of MAP kinase.

3T3 Cells↗
PubMed1993

Divergence of signaling pathways for insulin in PC-12 pheochromocytoma cells.

A PC-12 pheochromocytoma cell line is described with roughly equivalent levels of functional receptors for nerve growth factor (NGF), epidermal growth factor (EGF), and insulin. Each of these receptors undergoes autophosphorylation upon binding of their respective ligands, and causes the activation of phosphatidylinositol-3 kinase via a mechanism involving tyrosine phosphorylation. In the case of insulin, this activation is due to the tyrosine phosphorylation of its major cellular substrate, IRS-1. Despite the presence of functional receptors in these cells, insulin does not stimulate the activity of the mitogen-activated protein (MAP) kinase, despite a 5- to 8-fold activation observed with both NGF and EGF under the same conditions. This failure to activate MAP kinase was not due to the insulin-dependent dephosphorylation of the enzyme, but correlated with the lack of activation of the MAP kinase kinase, although this enzyme was also activated by NGF and EGF. Similarly, the activation of the raf and ras protooncogenes in these cells was not observed with insulin, whereas NGF and EGF produced marked activation. In addition, insulin-dependent induction of the c-fos protein was impaired, in comparison to NGF. In contrast to a lack of effect on the MAP kinase pathway, these PC-12 cells were metabolically responsive to insulin, exhibiting increases in glucose, lipid, and protein synthesis in response to the hormone. The differential responses of phosphorylation events to insulin, NGF, and EGF in these cells indicates that divergence of signaling pathways may occur at or near the insulin receptor.

Animals↗
PubMed1993

Active surveillance and risk factors for leptospirosis in Hawaii.

A clinic-hospital-based leptospirosis surveillance program was conducted to determine the morbidity and risk factors in nonepidemic settings. The study was conducted on two islands, Kauai and Hawaii (Big Island), in the state of Hawaii for one year during 1988 and 1989. An active, more comprehensive case detection system was used on the Big Island that enabled us to determine the incidence of clinical disease. Subjects from both islands were used to conduct a case-control study for risk factors. One hundred seventy-two subjects from the Big Island (who presented with any two of the following symptoms: fever, headache, myalgia, or nausea/vomiting) were enrolled in the study. Twenty cases were diagnosed by culture, serology, or fluorescent antibody tissue staining at autopsy. Six cases required hospitalization and two succumbed to fatal infections. We estimated that these cases represented an annual incidence rate of 128 per 100,000 person-years in our target population. For 33 cases, 77 controls were matched for island, age, sex, and time of onset of illness. Interviews were conducted retrospectively in a double-blinded fashion with cases and controls and evaluated approximately 30 risk factors. Factors that were associated most strongly with development of leptospirosis were household use of rainwater catchment systems (P = 0.003), presence of skin cuts during the incubation period (P = 0.008), contact with cattle or the urine of cattle (P = 0.05 and P = 0.03, respectively), and handling of animal tissues (P = 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗