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Biomedical subjects

W W Lin

Publications and source records attributed to W W Lin.

At least 19 recordsLinked to original sources
PubMed2000

Basal cPLA(2) phosphorylation is sufficient for Ca(2+)-induced full activation of cPLA(2) in A549 epithelial cells.

The release of [(3)H] arachidonic acid (AA) and its connection with the triggering of the MAP kinase cascade were studied in the human A549 epithelial cell line upon stimulation with thapsigargin. Thapsigargin can increase AA release along with the increase of intracellular calcium concentration, phosphorylation, and activation of extracellular regulated kinase (ERK) and cytosolic phospholipase A(2) (cPLA(2)). Both ERK and cPLA(2) phosphorylation in response to thapsigargin were inhibited by PD 98059, a specific inhibitor of MAP kinase kinase of the ERK group (MEK), and EGTA. cPLA(2) phosphorylation was not affected by Ro 31-8220 (an inhibitor of all PKC isoforms) or LY 379196 (a PKCbeta selective inhibitor), while both of them indeed attenuated ERK activation. On the other hand, rottlerin (the selective PKCdelta inhibitor), SB 203580 (the selective p38 MAPK inhibitor), and wortmannin (the PI 3-kinase inhibitor) can affect neither cPLA(2) nor ERK phosphorylation. In A549 cells, PKC activator PMA cannot increase either the basal or thapsigargin-induced (3)H-AA release, while it can induce the phosphorylation of ERK and cPLA(2.) The PMA-induced ERK phosphorylation was inhibited by Ro 31-8220, LY 379196, rottlerin, and PD 98059, but unaffected by SB 203580 and wortmannin. Moreover, the phosphorylation by PMA was non-additive with that of thapsigargin. This implies that intracellular Ca(2+) level is the key factor for induction of cPLA(2) activity and thapsigargin-elicited ERK activation itself is substantially sufficient for cPLA(2) activation upon intracellular Ca(2+) increase.

Acetophenones↗
PubMed2000

Acrylamide-regulated neurofilament expression in rat pheochromocytoma cells.

Using the rat pheochromocytoma cell line (PC12), we present molecular evidence that the neurotoxicant acrylamide directly induces neurofilament gene expression, and the signaling pathways are initially distinctive from, but eventually merged into, that for nerve growth factor (NGF)-induced neurofilament expression. In PC12 cells, acrylamide increased neurofilament protein levels and synthesis. Acrylamide had no effect on the stability of neurofilament mRNAs suggesting that it directly increased neurofilament mRNA synthesis. K252a, a selective inhibitor for NGF receptor gp140trk, had no effect on acrylamide induction, but completely inhibited NGF-induced neurofilament protein synthesis. Therefore, the initial step for acrylamide signaling was distinctive from NGF. Dexamethasone reversed the effects of both NGF and acrylamide on neurofilament protein levels and synthesis indicated that there is a dexamethasone-sensitive signaling step upon which NGF and acrylamide merge, suggesting involvement of transcription-activating proteins like AP-1. These results, taken together with previous studies of transgenic mice that overexpress neurofilament genes, may partially explain the mechanisms of neurofilament accumulation in distal parts of large axons, a pathognomonic feature of acrylamide neurotoxicity in animals.

Acrylamide↗
PubMed2000

Cycloheximide-induced cPLA(2) activation is via the MKP-1 down-regulation and ERK activation.

Extracellular signal-regulated kinase (ERK)-dependent phosphorylation is an important regulator for cytosolic phospholipase A(2) (cPLA(2)). In this study, we found that the protein synthesis inhibitor cycloheximide can potentiate thapsigargin-induced arachidonic acid (AA) release concomitant with ERK phosphorylation from murine RAW 264.7 macrophages. The cycloheximide effect is not due to the activation of p38 mitogen-activated protein kinase (MAPK) nor c-Jun NH(2)-terminal kinase (JNK), because the activator of both MAPKs anisomycin does not elicit AA release. Cycloheximide effect is additive to the tyrosine phosphatase inhibitor orthovanadate since these two stimuli induced sustained ERK activation respectively through inhibition of the translation and activity of MAPK phosphatase-1 (MKP-1).

Animals↗
PubMed2000

Pyrimidinoceptor potentiation of macrophage PGE(2) release involved in the induction of nitric oxide synthase.

We have previously demonstrated that Ca(2+)/calmodulin-dependent protein kinase (CaMK) mediates pyrimidinoceptor potentiation of LPS-elicited inducible nitric oxide synthase (iNOS) induction in murine J774 macrophages. In the present paper, we have explored the role of cyclo-oxygenase (COX)-dependent prostaglandin E(2) (PGE(2)) formation in this event. In J774 macrophages predominantly expressing P2Y(6) receptors, the simultaneous addition of UTP and lipopolysaccharide (LPS) resulted in potentiated increase in PGE(2) release. UTP-induced increased PGE(2) release was demonstrated by a concomitant increase in COX-2 protein expression, and was decreased by inhibitors specific for phosphatidylinositide-phospholipase C (PI-PLC), CaMK, protein kinase C (PKC), nuclear factor-kappa B (NF-kappaB) or COX-2. NS-398 (a selective COX-2 inhibitor) reduced LPS plus UTP-elicited iNOS induction and nitrite accumulation, supporting for the positive regulation of iNOS gene expression by endogenous PGE(2). Moreover, the cyclic AMP/PKA-dependent up-regulation of iNOS expression mediated by PGE(2) was drawn from the inhibitory effects of 2',5'-dideoxyadenosine, KT5720 and H-89. Exogenous PGE(2) induced NF-kappaB activation and potentiated nitrite accumulation in response to LPS. In addition to COX-2 induction, arachidonic acid (AA) release and steady-state mRNA levels of type V secretory phospholipase A(2) (sPLA(2)) and Ca(2+)-independent PLA(2) (iPLA(2)) were also increased in the presence of LPS and UTP; the LPS-induced increase in iPLA(2) activity was also potentiated by UTP. Taken together, we conclude that UTP-mediated COX-2 and iPLA(2) potentiation and PGE(2) formation contribute to the iNOS induction, and that CaMK activation is the primary step in the UTP enhancement of COX-2 induction.

Animals↗
PubMed1999

Cold-induced ependymin expression in zebrafish and carp brain: implications for cold acclimation.

Cold acclimation has been suggested to be mediated by alternations in the gene expression pattern in the cold-adapted fish. To investigate the mechanism of cold acclimation in fish brain at the molecular level, relevant subsets of differentially expressed genes of interest were identified and cloned by the PCR-based subtraction suppression hybridization. Characterization of the selected cold-induced cDNA clones revealed one encoding ependymin. This gene was shown to be brain-specific. The expression of ependymin was induced by a temperature shift from 25 degrees C to 6 degrees C in Cyprinus carpio or 12 degrees C in Danio rerio. Activation of ependymin was detected 2 h after cold exposure and peaked at more than 10-fold at 12 h. This peak level remains unchanged until the temperature returns to 25 degrees C. Although the amount of soluble ependymin protein in brain was not changed by cold treatment, its level in the fibrous insoluble polymers increased 2-fold after exposure to low temperature. These findings indicate that the increase in ependymin expression is an early event that may play an important role in the cold acclimation of fish.

Acclimatization↗
PubMed1999

Enhanced mRNA expression of neurofilament subunits in the brain and spinal cord of diisopropyl phosphorofluoridate-treated hens.

Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester, and a single injection of this compound (1.7 mg/kg, s.c.) produces delayed neurotoxicity (OPIDN) in hens in 7-14 days. Clinically, the disease is marked by hindlimb ataxia followed by paralysis after some time. A characteristic feature of this neuropathy is axonal swelling in the initial stages and comparative dissolution of the accumulated material and degeneration of distal axons with disease progression. Axonal swelling consists of aggregated neurofilaments, microtubules, and proliferated smooth endoplasmic reticulum. We studied expression of neurofilament (NF) mRNAs in brain regions and spinal cord to elucidate their role in OPIDN. There was a 50-200% increase in NF transcripts in 24 hr after DFP administration. The NF-L mRNA level started falling after 1-5 days and came down to control level in susceptible brain regions (i.e. cerebellum and brainstem) and spinal cord, but not in cerebral cortex, which does not show degeneration of axons in OPIDN. Cerebral cortex exhibited elevated levels of both NF-L and NF-M transcripts in DFP-treated hens throughout the period of observation. The induction of NF messages is consistent with the previously reported effect on extension of neurites of human neuroblastoma cells in culture. The transient increase in NF messages in susceptible tissues either may be responsible for the delayed degeneration of axons in OPIDN or is the result of interruption of regulatory signal due to progressive degeneration of axons.

Animals↗
PubMed1999

Protein kinase C epsilon-dependent pathway of extracellular signal-regulated protein kinase activation by P2Y1 and P2Y2 purinoceptors that activate cytosolic phospholipase A2 in endothelial cells.

The aim of this study was to investigate the stimulating effects on arachidonic acid release of P2Y1 and P2Y2 receptor-selective agonists, 2-methylthio-ATP (2MeSATP) and UTP, respectively, in bovine pulmonary artery endothelial cells. Exposure of cells to 2MeSATP and UTP led to the release of arachidonic acid, a response which was abolished by the removal of extracellular Ca2+ and methyl arachidonyl fluorophosphonate. Phorbol 12-myristate 13-acetate (PMA) itself not only stimulated arachidonic acid release but also played a permissive role in the response to UTP. However, PMA failed to enhance the arachidonic acid response induced by 2MeSATP, probably due to greater attenuation of the [Ca2+]i increase caused by 2MeSATP than UTP. Inhibition of protein kinase C with Ro 31-8220 (1-[3-(amidinothio) propyl-1H-indoyl-3-yl]-3-(1-methyl-1H-indoyl-3-yl)-maleimide -methane sulphate) and staurosporine, but not with Go 6976 (12-(-2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-indolo(2, 3-a)pyrrolo(3,4-c)carbazole), reduced the arachidonic acid response of 2MeSATP, UTP and PMA. PMA-induced potentiation of the UTP response reached a maximum after a 1-h preincubation, then declined and eventually lost its effect when the preincubation lasted up to 8 h. Among the protein kinase C isoforms present in endothelial cells, betaI and epsilon could be down-regulated by treatment with PMA for 4-24 h. PD 098059 (2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) inhibited extracellular signal-regulated protein kinase activation, cytosolic phospholipase A2 phosphorylation and arachidonic acid release caused by 2MeSATP, UTP and PMA. Taken together, our results demonstrate that P2Y1 and P2Y2 purinoceptors mediate arachidonic acid release by activating cytosolic phospholipase A2 through an elevation of [Ca2+]i and protein kinase C epsilon-, extracellular signal-regulated protein kinase-dependent phosphorylation.

Adenosine Triphosphate↗
PubMed1999

Molecular enzymology of mammalian Delta1-pyrroline-5-carboxylate synthase. Alternative splice donor utilization generates isoforms with different sensitivity to ornithine inhibition.

Delta1-Pyrroline-5-carboxylate synthase (P5CS; EC not assigned), a mitochondrial inner membrane, ATP- and NADPH-dependent, bifunctional enzyme, catalyzes the reduction of glutamate to Delta1-pyrroline-5-carboxylate, a critical step in the de novo biosynthesis of proline and ornithine. We utilized published plant P5CS sequence to search the expressed sequence tag data base and cloned two full-length human P5CS cDNAs differing in length by 6 base pairs (bp) in the open reading frame. The short cDNA has a 2379-bp open reading frame encoding a protein of 793 residues; the long cDNA, generated by "exon sliding," a form of alternative splicing, contains an additional 6-bp insert following bp +711 of the short form resulting in inclusion of two additional amino acids in the region predicted to be the gamma-glutamyl kinase active site of P5CS. The long form predominates in all tissues examined except gut. We also isolated the corresponding long and short murine P5CS transcripts. To confirm the identity of the putative P5CS cDNAs, we expressed both human forms in gamma-glutamyl kinase- and gamma-glutamyl phosphate reductase-deficient strains of Saccharomyces cerevisiae and showed that they conferred the proline prototrophy. Additionally, we found expression of the murine putative P5CS cDNAs conferred proline prototrophy to P5CS-deficient Chinese hamster ovary cells (CHO-K1). We utilized stable CHO-K1 cell transformants to compare the biochemical characteristics of the long and short murine P5CS isoforms. We found that both confer P5CS activity and that the short isoform is inhibited by L-ornithine with a Ki of approximately 0.25 mM. Surprisingly, the long isoform is insensitive to ornithine inhibition. Thus, the two amino acid insert in the long isoform abolishes feedback inhibition of P5CS activity by L-ornithine.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1999

Modulation of inducible nitric oxide synthase induction by prostaglandin E2 in macrophages: distinct susceptibility in murine J774 and RAW 264.7 macrophages.

Prostaglandin E2 (PGE2) is the major cyclooxygenase metabolite in macrophages with complex proinflammatory and immunoregulatory properties. In the present study, we have compared the modulatory role of PGE2/cAMP-dependent signaling on induced nitric oxide (NO) production in two murine macrophages, J774 and RAW 264.7. With no effect on NO release by itself, PGE2 co-addition with lipopolysaccharide (LPS) resulted in a concentration-dependent enhancement in NO release and inducible NO synthase induction in J774, but not in RAW 264.7, macrophages. The potentiation effect of PGE2 in J774 cells was still seen when applied within 9 h after LPS treatment. Whereas RAW 264.7 macrophages release PGE2 with greater extent than J774 macrophages in response to LPS, indomethacin and NS-398, upon abolishing LPS-induced PGE2 release, caused a more obvious inhibition of NO release from J774 than RAW 264.7 cells. Thus, we suggest a higher positive modulatory role of PGE2--either endogenous or exogenous--on NO formation in J774 cells. Supporting these findings, exogenous PGE2 triggers cAMP formation in J774 cells with higher potency and efficacy. Of interest, dBcAMP also elicits higher sensitivity in potentiating NO release in J774 cells. We conclude that the opposite effect of PGE2/cAMP signaling on macrophage NO induction depends on its signaling efficacy and might be associated with the difference in endogenous PGE2 levels.

Animals↗
PubMed1999

The role of autosomal cell apoptosis regulator genes in human spermatogenesis.

Immunohistochemical analysis of the expression of the autosomal apoptosis regulator genes, BAX, bcl-2, p53, and cyclic-AMP responsive element modulator (CREM) in testis biopsies from infertile men demonstrated that BAX, bcl-2, and p53 immunoreactivity was absent irrespective of seminiferous tubule histology. In contrast, cell-specific CREM immunoreactivity in round spermatids, with complete absence of CREM in patient biopsies showing spermatocyte maturation arrest and Sertoli cell only, was evident, suggesting a possible role of autosomal genes in the regulation of apoptosis in human spermatogenesis regulation.

Adult↗
PubMed1999

Induction of cyclo-oxygenase-2 expression by methyl arachidonyl fluorophosphonate in murine J774 macrophages: roles of protein kinase C, ERKs and p38 MAPK.

1. Methyl arachidonyl fluorophosphonate (MAFP), an inhibitor of phospholipase A2 (PLA2), has been widely used to assess the roles of PLA2 in various cell functions. Here, we report on a novel action of this compound at concentrations similar to those used for PLA2 inhibition. 2. The murine macrophage J774 released a large amount of prostaglandin E2 (PGE2) by MAFP (1-30 microM), which was abolished by indomethacin and NS-398 but not by valeryl salicylate, and results from increased cyclo-oxygenase-2 (COX-2) protein levels and gene expression. 3. This PGE2 release was blocked by inhibitors of tyrosine kinase (genistein), protein kinase C (PKC) (Ro 31-8220, Go 6976 or LY 379196), mitogen-activated protein kinase kinase (MEK) (PD 098059) or p38 mitogen-activated protein kinase (MAPK) (SB 203580). 4. Consistent with these results, MAFP caused membrane translocation of PKCbetaI and betaII isoforms and activated extracellular signal-regulated kinase (ERK) and p38 MAPK. 5. In accordance with these effects of MAFP, PKC activator phorbol 12-myristate 13-acetate (PMA) increased PGE2 release and caused activation of PKCbeta, ERKs and p38 MAPK. 6. This is the first report that the PLA2 inhibitor, MAFP, can induce COX-2 gene expression and PGE2 synthesis via the PKC-, ERK- and p38 MAPK-dependent pathways. Thus, the use of MAFP as a PLA2 inhibitor should be treated with caution.

Animals↗
PubMed1999

PKCbetaI mediates the inhibition of P2Y receptor-induced inositol phosphate formation in endothelial cells.

1. Bovine pulmonary artery endothelium (CPAE) expresses phospholipase C (PLC)-linked P2Y1 and P2Y2 receptors, for them 2-methylthio-ATP (2MeSATP) and UTP are respective agonists. Here, we have investigated the particular protein kinase C (PKC) isoform(s) responsible for the inhibition of P2Y1 and P2Y2 receptor-evoked inositol phosphate (IP) formation by phorbol 12-myristate 13-acetate (PMA). 2. Although short-term (20 min) pretreatment of cells with PMA attenuated 2MeSATP- and UTP-induced phosphoinositide (PI) breakdown, this inhibition was lost after 15 h. Preincubation with PMA for 24 h, on the contrary, potentiated 2MeSATP and UTP responses. The IP formation stimulated by NaF was unaltered by PMA pretreatment. 3. Western blot analysis showed that treatment of CPAE with PMA resulted in a rapid translocation of PKC isoform betaI, epsilon and mu, but not lambda, from the cytosol to the membrane fraction. 4. Pretreatment of the selective PKC inhibitor Ro 31-8220 attenuated the inhibitory effect of PMA on IP formation. Go 6976 (an inhibitor of conventional PKCalpha, beta and gamma) and LY 379196 (a selective PKCbeta inhibitor) also dose-dependently inhibited the PMA-mediated desensitization. 5. Transfection of PKCbeta-specific antisense oligonucleotide reduced PKCbetaI protein level and inhibited PMA-mediated PI reduction. 6. RT - PCR analysis showed that PMA treatment for 4 - 24 h up-regulated P2Y1 and P2Y2 receptors at the mRNA levels. 7. These results suggest that PKCbetaI may exert a negative feedback regulation on endothelial P2Y1 and P2Y2 receptor-mediated PI turnover. The down-regulation of PKCbetaI and enhanced P2Y receptor expression together might contribute to the late PI enhancing effect of PMA.

Adenosine Triphosphate↗
PubMed1999

Roles of atypical protein kinase C in lysophosphatidic acid-induced type II adenylyl cyclase activation in RAW 264.7 macrophages.

1 Lysophosphatidic acid (LPA) has been widely studied as a naturally occurring and multifunctional phospholipid messenger in diverse tissue and cell types and shown to inhibit adenylyl cyclase (AC) by a G protein-mediated mechanism. 2 In type II AC-expressing mouse RAW 264.7 macrophages, we showed that LPA at 3-50 microM increased cyclic AMP formation in a concentration-dependent manner, the effect being additive with that of forskolin or cholera toxin, and synergistic with that of prostaglandin E1 (PGE1) or isoproterenol. 3 The potentiation effect of LPA was unaffected by the removal of serum or pertussis toxin treatment. 4 Both colchicine and cytochalasin B potentiated the cyclic AMP response to PGE1, the effect being additive to that of LPA. 5 On studying the regulation of type II AC by protein kinase C (PKC), phorbol 12-myristate-13 acetate (PMA) potentiated the PGE1-elicited cyclic AMP response, this effect being non-additive to that of LPA, suggesting that PKC activation was the common mechanism involved in AC potentiation by LPA and PMA. 6 PKC inhibitor Ro 31-8220, but not Go 6976, significantly inhibited the LPA-induced cyclic AMP potentiation. 7 The potentiation effect of LPA was unaffected by long-term treatment with PMA, which resulted in the down-regulation of PKCalpha, betaI, betaII and PKCdelta, but not PKCepsilon, mu, lambda and zeta. 8 By in situ kinase assay, we found a marked increase in atypical PKC activity after LPA treatment. 9 Taken together, we conclude that LPA can elicit a unique signalling cascade in RAW 264.7 macrophages and increase type II AC activity via the activation of atypical PKC.

Adenylate Cyclase Toxin↗
PubMed1999

Involvement of p38 mitogen-activated protein kinase in lipopolysaccharide-induced iNOS and COX-2 expression in J774 macrophages.

Both the nitrite and prostaglandin E2 (PGE2) release caused by lipopolysaccharide (LPS) in J774 macrophages are inhibited by SB 203580, a specific p38 mitogen-activated protein kinase (MAPK) inhibitor, in a concentration-dependent manner. The 50% inhibitory concentration (IC50) for nitrite and PGE2 responses was 1 microm and 0.5 microm, respectively. Inhibition was marked following simultaneous treatment with SB 203580 and LPS, and was much reduced when SB 203580 was added 6 hr after LPS treatment. In parallel, LPS induction of inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2) proteins and their steady-state levels of mRNA were reduced by SB 203580. LPS activation of nuclear factor-kappa B (NF-kappaB), activator protein-1 (AP-1) and p38 MAPK was also inhibited by SB 203580. These results suggest a crucial role of p38 MAPK in regulation of the transcriptional level of endotoxin LPS-induced iNOS and COX-2 protein expression.

Animals↗
PubMed1999

Germ-cell nondisjunction in testes biopsies of men with idiopathic infertility.

Intracytoplasmic sperm injection (ICSI) has been used in combination with testicular sperm extraction to achieve pregnancies in couples with severe male-factor infertility, yet many of the underlying genetic mechanisms remain largely unknown. To investigate nondisjunction in mitotic and meiotic germ cells, we performed three-color FISH to detect numeric chromosome aberrations in testicular tissue samples from infertile men confirmed to have impaired spermatogenesis of unknown cause. FISH was employed to determine the rate of sex-chromosome aneuploidy in germ cells. Nuclei were distinguished as haploid or diploid, respectively. The overall incidence of sex-chromosome aneuploidy in germ cells was found to be significantly higher (P<.00001) in all three abnormal histopathologic patterns (range 39.0%-43.5%) as compared with normal controls (29.1%). The relative ratio of normal to aneuploid nuclei in the diploid cells of patients with impaired spermatogenesis was approximately 1.0, a >300% decrease when compared with the 4.42 ratio detected in patients with normal spermatogenesis. These results provide direct evidence of an increased incidence of sex-chromosome aneuploidy observed in germ cells of men with severely impaired spermatogenesis who might be candidates for ICSI with sperm obtained directly from the testis. The incidence of aneuploidy was significantly greater among the diploid nuclei, which suggests that chromosome instability is a result of altered genetic control during mitotic cell division and proliferation during spermatogenesis.

Biopsy↗
PubMed1999

Spatiotemporal expression of alternatively spliced IGF-I mRNA in the rat costochondral growth plate.

IGF-I acts as a local proliferation and maturation factor for chondrocytes in the growth plate. However, the expression of different alternative IGF-I mRNA classes in the growth plate has not been characterized. Using quantitative reverse transcription PCR, the abundance of each alternative IGF-I mRNA class in resting, proliferative and hypertrophic chondrocytes was measured in rat costochondral growth plates. Class 1Ea mRNA was the most abundant IGF-I transcript overall and was highly expressed in proliferative chondrocytes at 2 and 4 weeks of age; by 6 weeks, the majority of 1Ea mRNA expression had shifted to hypertrophic chondrocytes. Class 1Eb mRNA was the second most abundant transcript and its distribution was uniform across all the cell types at 2 weeks of age. The expression pattern changed with increasing age such that at 6 weeks a gradient existed with hypertrophic chondrocytes expressing higher levels of 1Eb than resting chondrocytes. Class 2Ea mRNA was constitutively expressed at low levels across the growth plate at all ages, while class 2Eb mRNA expression was negligible. The distribution of total IGF-I mRNA also shifted across growth plate cell types as the animals aged from 2 to 6 weeks. These findings suggest that IGF-I class 1 mRNA plays the predominant role in the maturation of the growth plate.

Alternative Splicing↗