Phorbol Myristoyl Acetate Research Articles

Identification of a novel CHN1 p.(Phe213Val) variant in a large Han Chinese family with congenital Duane retraction syndrome

Duane retraction syndrome (DRS) is a neuromuscular dysfunction of the eyes. Although many causative genes of DRS have been identified in Europe and the United States, few reports have been published in regard to Chinese DRS. The aim of the present study was to explore the genetic defect of DRS in a Chinese family. Exome sequencing was used to identify the disease-causing gene for the two affected family members. Ophthalmic and physical examinations, as well as genetic screenings for variants in chimerin 1 (CHN1), were performed for all family members. Functional analyses of a CHN1 variant in 293T cells included a Rac-GTP activation assay, α2-chimaerin translocation assay, and co-immunoprecipitation assay. Genetic analysis revealed a NM_001822.7: c.637T > G variant in the CHN1 gene, which resulted in the substitution of a highly conserved C1 domain with valine at codon 213 (NP_001813.1: p.(Phe213Val)) (ClinVar Accession Number: SCV001335305). In-silico analysis revealed that the p.(Phe213Val) substitution affected the protein stability and connections among the amino acids of CHN1 in terms of its tertiary protein structure. Functional studies indicated that the p.(Phe213Val) substitution reduced Rac-GTP activity and enhanced membrane translocation in response to phorbol-myristoyl acetate (PMA). Together with previous studies, our present findings demonstrate that CHN1 may be an important causative gene for different ethnicities with DRS.

Cytokine release by Jurkat T‐lymphocytes is modulated following treatment with polyphenols or Lactobacillus rhamnosus probiotic extracts (1044.10)

Quercetin and (‐)‐epigallocatechingallate (EGCG) are dietary polyphenols claimed to exert anti‐inflammatory effects. Jurkat T‐lymphocytes were incubated with 5μM quercetin or ECGC for 48h, with or without 25ng/ml phorbol myristoyl acetate and 5μg/ml phytohaemagglutinin (PMA/PHA) at 24h to induce cytokine release. Inflammatory cytokines interleukin 2 (IL2), 8 (IL8) and tumour necrosis factor α (TNFα) were measured. Unexpectedly, polyphenols had no significant anti‐inflammatory effects and EGCG increased IL8 release from stimulated cells by 174±18%. Polyphenols are digested to phenolic acids by the colonic microflora, thus we hypothesised anti‐inflammatory effects may be mediated by these metabolites. Quercetin and EGCG were fermented with normal gut bacteria (E. coli) or probiotic strain (L. rhamnosus) for 6h and phenolic acids were then isolated by solid phase extraction. Jurkat cells were incubated with the extracts for 48h with or without PMA/PHA stimulation at 24h.E. coli extract increased TNFα release from stimulated cells by 268±51% while L. rhamnosus extract reduced IL8 release from unstimulated cells by 34±2%. EGCG fermented with L. rhamnosus increased IL8 release from stimulated cells by 128±16%, consistent with effects of unfermented EGCG. The findings showed anaerobic bacterial fermentation did not modify polyphenol activity, but supported anti‐inflammatory effects of L. rhamnosus.Grant Funding Source: Supported by BBSRC DRINC

Low-dose diet supplement of a natural flavonoid, luteolin, ameliorates diet-induced obesity and insulin resistance in mice

Mast cells play important roles in diet-induced obesity and diabetes, and some synthetic mast cell stabilizers can improve related metabolic disturbances in mice. Luteolin (LU) is a potent natural mast cell stabilizer. However, a direct correlation between LU and these common metabolic diseases is not established. Male C57BL/6 mice were fed low-fat diet, high-fat diet (HFD), HFD with 0.002 and 0.01% LU for 12 wk, respectively. Dietary LU suppressed HFD-induced body weight gain, fat deposition, and adipocyte hypertrophy. Meanwhile, glucose intolerance and insulin sensitivity was also improved. Interestingly, dietary LU ameliorated angiogenesis and associated cell apoptosis and cathepsin activity in epididymis adipose tissues, which is a critical mechanism that mast cells are involved in diet-induced obesity and diabetes. Further, we showed dietary LU reduced mast cell and macrophage infiltrations and inflammatory cytokine levels in epididymis adipose tissues. Finally, LU inhibited mast cell-derived IL-6 expression, which is a key cytokine that contributes to mast cell-associated metabolic derangements, and protein kinase C activator phorbol myristoyl acetate reversed the inhibitory effects. As a natural flavonoid, low-dose diet supplement of LU ameliorates diet-induced obesity and insulin resistance in mice, suggesting a new therapeutic and interventional approach for these diseases.

Calmodulin kinase II and protein kinase C mediate the effect of increased intracellular calcium to augment late sodium current in rabbit ventricular myocytes

An increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) augments late sodium current (I(Na.L)) in cardiomyocytes. This study tests the hypothesis that both Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) mediate the effect of increased [Ca(2+)](i) to increase I(Na.L). Whole cell and open cell-attached patch clamp techniques were used to record I(Na.L) in rabbit ventricular myocytes dialyzed with solutions containing various concentrations of [Ca(2+)](i). Dialysis of cells with [Ca(2+)](i) from 0.1 to 0.3, 0.6, and 1.0 μM increased I(Na.L) in a concentration-dependent manner from 0.221 ± 0.038 to 0.554 ± 0.045 pA/pF (n = 10, P < 0.01) and was associated with an increase in mean Na(+) channel open probability and prolongation of channel mean open-time (n = 7, P < 0.01). In the presence of 0.6 μM [Ca(2+)](i), KN-93 (10 μM) and bisindolylmaleimide (BIM, 2 μM) decreased I(Na.L) by 45.2 and 54.8%, respectively. The effects of KN-93 and autocamtide-2-related inhibitory peptide II (2 μM) were not different. A combination of KN-93 and BIM completely reversed the increase in I(Na.L) as well as the Ca(2+)-induced changes in Na(+) channel mean open probability and mean open-time induced by 0.6 μM [Ca(2+)](i). Phorbol myristoyl acetate increased I(Na.L) in myocytes dialyzed with 0.1 μM [Ca(2+)](i); the effect was abolished by Gö-6976. In summary, both CaMKII and PKC are involved in [Ca(2+)](i)-mediated augmentation of I(Na.L) in ventricular myocytes. Inhibition of CaMKII and/or PKC pathways may be a therapeutic target to reduce myocardial dysfunction and cardiac arrhythmias caused by calcium overload.

Oxytocin and Interferon-Tau Regulation of Prostaglandin Transporter (PGT) and Multidrug Resistant Associated Protein 4 (MRP4) in the Bovine Endometrium.

Prostaglandins (PG) are involved in many female reproductive processes and are regulated at the levels of biosynthesis, secretion and catabolism. Transmembrane transport of PGs is also an important site of regulation and we have shown the requirement of PG transporter (PGT/SLCO21) for functional luteolysis in the bovine. A comprehensive screening of the endometrial expression of different PGTs indicated that MRP4/ABCC4 of ATP Binding Cassette (ABC) family was regulated during the bovine estrous cycle apart from the previously studied PGT/SLCO21 of Solute Carrier Organic Anion family. Accordingly, we have confirmed the expression of these carriers in our recently developed endometrial epithelial and stromal cell lines bEEL and CSC. Using these models, we have studied the effect of oxytocin (OT) and/or interferon tau (IFNt) and phorbol myristoyl acetate (PMA) on the regulation of PGT and MRP4 in relation with PGs production. Downregulation of MRP4 expression using siRNA induced a significant increase of PGF2α accumulation relative to PGE2 in the culture medium of CSC. Treatment of bEEL cells with OT stimulated COX2 expression and PGF2αproduction. These responses correlated well with upregulation of MRP4/ABCC4 but not PGT/SLCO2A1. In contrast, IFNt at a concentration that stimulated COX-2 without a corresponding increase in PG accumulation stimulated the expression of PGT/SLCO2A1 but not that of MRP4/ABCC4. Inhibition of OT induced PG production by the COX2 inhibitior NS-398 did not affect the induction of ABCC2 or COX2 expression in bEEL cells. Since OT at the origin of luteolytic PGF2α regulates MRP4 and IFNt, the embryonic antiluteolytic signal, regulates PGT, it is possible that these modulators are involved in selective or preferential transport of terminal PGs like PGE2 and PGF2α. (poster)

Regulation of the Cardiac Na+/Ca2+ Exchanger by Calcineurin and Protein Kinase C

Na+/Ca2+ exchanger (NCX) activity is markedly inhibited in hypertrophic neonatal rat cardiomyocytes subjected to chronic phenylephrine treatment. This inhibition is reversed partially and independently by acute inhibition of calcineurin and protein kinase C (PKC) activities. Similar NCX inhibition occurs in CCL39 cells expressing cloned wild-type NCX1, when they are infected with adenoviral vectors carrying activated calcineurin A and then treated acutely with phorbol myristoyl acetate or protein phosphatase-1 inhibitors. The data obtained with these cells suggest that calcineurin activity, PKCalpha-mediated NCX1 phosphorylation, and the central loop of NCX1 (possibly its beta1 repeat) are required for the observed NCX inhibition. We observe partial inhibition of NCX activity independent of NCX1 phosphorylation when CCL39 cells are infected with activated calcineurin A but not further treated with phorbol myristoyl acetate or phosphatase inhibitors. Calcineurin thus appears to downregulate NCX activity via two independent mechanisms, one involving NCX1 phosphorylation and the other not involving NCX1 phosphorylation. These data indicate the existence of a novel regulatory mechanism for NCX1 involving calcineurin and PKC, which may be important in cardiac pathology.

Inhibiting Long-Chain Fatty Acyl CoA Synthetase Does Not Increase Agonist-Induced Release of Arachidonate Metabolites from Human Endothelial Cells

Background: Triacsin C, a fatty acid analog, inhibits endothelial nitric oxide synthetase (eNOS) palmitoylation, increases nitric oxide synthesis and enhances methacholine-induced relaxation of vascular rings. The experiments presented here tested the hypothesis that triacsin C increases the synthesis of PGI₂ and/or endothelial-derived hyperpolarizing factor. Methods: Long-chain fatty acyl CoA synthetase activity (LCFACoAS), agonist-induced prostacyclin synthesis and agonist-induced release of radioactivity in endothelial cells labeled with [³H]arachidonic acid were measured in the presence and absence of triacsin C. Results: Inhibition by triacsin C of palmitoyl CoA formation was significantly greater than inhibition of arachidonoyl CoA formation in solubilized endothelial cell preparations. While 24-hour triacsin C treatment significantly reduced basal 6-keto synthesis, it had no effect on agonist-stimulated synthesis. The release of arachidonic acid metabolites was examined in [³H]arachidonate-labeled cells. Triacsin C treatment had no effect on basal or vasopressin-, angiotensin-II-, bradykinin- or ionomycin-induced release of radioactivity, but significantly reduced release in response to isoproterenol or phenylephrine. Expression of neither immunoreactive eNOS nor immunoreactive inducible nitric oxide synthetase (iNOS) was changed by triacsin C treatment, but the fraction of immunoreactive eNOS in the cytoplasm of treated cells was significantly greater as compared to vehicle control cells. Phorbol myristoyl acetate or fenofibrate significantly increased in vitro LCFACoAS activity, and significantly decreased the nitrite/eNOS ratio. Conclusions: These data indicate that, while triacsin C can inhibit arachidonoyl CoA synthetase in endothelial cells, it does not increase the availability of endogenous substrate for basal or agonist-induced PGI₂ synthesis, nor does it enhance release of arachidonic acid or its metabolites.

Role of protein kinase C alpha in calcium induced keratinocyte differentiation: defective regulation in squamous cell carcinoma.

Calcium induces both involucrin and transglutaminase-K in normal keratinocytes (NHK) but not in squamous carcinoma cell lines (SCC). The protein kinase C (PKC) agonist phorbol myristoyl acetate potentiates and the PKC antagonist Ro31-8220 blocks the ability of calcium to stimulate the involucrin promoter in normal human keratinocytes but not in SCC4. We thus examined the ability of calcium to regulate the levels of five PKC isozymes in NHK and two SCC. In the normal keratinocytes, the levels of PKC [alpha], PKC [delta], PKC [eta], and PKC [zeta] increased over the first one to two weeks in a calcium-and time-dependent manner. PKC [epsilon] decreased in a time-and calcium-dependent fashion over the three-week period. All five isozymes showed little change during culture in SCC4 at any calcium concentration. Calcium and time of culture had partial effects on SCC12B2, a carcinoma that shows partial differentiation characteristics. Since PKC [alpha] is the only calcium responsive PKC isozyme in keratinocytes and most likely to be directly involved in calcium induced differentiation, we evaluated the effect of inhibiting its production with antisense oligonucleotides on calcium-regulated markers of differentiation. We found that the PKC [alpha] specific antisense oligonucleotide blocked calcium stimulated involucrin promoter activity as well as PKC [alpha], involucrin, and transglutaminase protein production, whereas the sense oligonucleotide control did not. We conclude that although a number of PKC isozymes are regulated during calcium-induced differentiation, PKC [alpha] plays a necessary role in mediating calcium-induced differentiation. Failure to regulate PKC [alpha] in SCC4 may underlie at least part of the failure of calcium to promote differentiation in these cells.

Induction of matrix metalloproteinase‐9 (MMP‐9) in lipopolysaccharide‐stimulated primary astrocytes is mediated by extracellular signal‐regulated protein kinase 1/2 (Erk1/2)

In the present study, we investigated whether the activation of protein kinase C (PKC) and extracellular signal-regulated kinase 1/2 (Erk1/2) are involved in the induction of MMP-9 in lipopolysaccharide (LPS)-stimulated primary astrocytes. The expression of MMP-9 but not MMP-2 was increased by LPS. LPS treatment induced activation of Erk1/2 within 30 min, which was dose-dependently inhibited by PD98059, a specific inhibitor of the Erk kinase (MEK). In this condition, PD98059 blocked the increase in MMP-9 protein and mRNA level as well as gelatin-digesting activity. Inhibition of PKC activity blocked the LPS-induced activation of Erk1/2 as well as MMP-9 expression. In addition, activation of PKC by phorbol myristoyl acetate (PMA) activated Erk1/2 with concomitant increase in MMP-9 production. Moreover, treatment of PD98059 dose-dependently decreased the PMA-induced MMP-9 expression. The results from the present study suggest that induction of MMP-9 by LPS in rat primary astrocytes is mediated, at least in part, by the sequential activation of PKC and Erk1/2. The Erk1/2-mediated MMP-9 induction may provide insights into the regulation of MMP-9 production in CNS, which may occur in vivo in pathological situations such as CNS inflammation.

Mixed lineage kinase 3 inhibits phorbol myristoyl acetate-induced DNA synthesis but not osteopontin expression in rat mesangial cells.

Mixed lineage kinase 3 (MLK 3) (also called SPRK or PTK-1) is a recently described member of the family of the mixed lineage kinase subfamily of Ser/Thr protein kinases that interacts with mitogen-activated protein kinase pathways. In order to test the biological relevance and potential interaction of MLK 3 with protein kinase C-mediated signaling pathways, human MLK 3 was stably expressed in rat glomerular mesangial cells using a retroviral vector (LXSN) and the effects of phorbol myristoyl acetate (PMA) on DNA synthesis and osteopontin mRNA expression were examined. In control (vector-transfected) mesangial cells PMA increased [3H]-thymidine incorporation in a concentration-dependent manner. In mesangial cells stably expressing MLK 3, the PMA-induced increase in [3H]-thymidine incorporation was significantly reduced (> 50%). However, the PMA-induced increase in osteopontin mRNA was not affected by MLK 3 expression. To determine the mechanisms of these effects, activation of ERK2, JNK1 and p38 in response to PMA was examined in both vector and MLK 3 transfected cells. ERK2 activation was increased several fold by PMA in control cells but was attenuated significantly in MLK 3 expressing cells, suggesting that MLK 3 expression in mesangial cells can negatively regulate the ERK pathway. PMA had no significant effect on JNK and P38 activation, in either vector- or MLK 3-expressing cells. PD98059, a MEK inhibitor blocked PMA-induced DNA synthesis without affecting osteopontin expression. These results suggest that while protein kinase C activation increases cellular proliferation and osteopontin mRNA expression, over-expression of MLK 3 affects only the PKC-induced DNA synthesis, probably through inhibition of ERK. These results also indicate a novel mechanism of growth regulation by a member of the mixed-lineage kinase family that might have significant therapeutic implications in proliferative glomerulonephritis.

Controls of EGF-induced morphological transformation of human bronchial epithelial cells.

Human bronchial epithelial cells, both normal primary (NHBE) and the BEAS-2B line, respond to epidermal growth factor (EGF) by extruding lengthy filaments, or filapodia. The morphological transformation of BEAS-2B cells maximized at 48 h using 1-10 nM EGF. EGF-induced filapodia extension was inhibited by co-exposure to transforming growth factor beta, which did not affect tyrosine phosphorylation of the EGF receptor (EGFR). Inhibition was also effected by phorbol myristoyl acetate (PMA), which reduced the rate of EGFR tyrosine phosphorylation. Dibutyryl-cAMP had no effect, whereas the protein kinase inhibitor H-89 stimulated the EGF response. The ability to regulate cellular responses to EGF by hormonal and chemical approaches has implications for current investigations into the roles of EGF in lung growth, differentiation, and wound repair.

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O2- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O2- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O2- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O2- production and MPO release from stimulated neutrophils.

Expression of a peptide binding to receptor for activated C-kinase (RACK1) inhibits phorbol myristoyl acetate-stimulated phospholipase D activity in C3H/10T1/2 cells: dissociation of phospholipase D-mediated phosphatidylcholine breakdown from its synthesis

The C3H/10T1/2 Cl8 HAβC2-1 cells used in this study express a peptide with a sequence shown to bind receptor for activated C-kinase (RACK1) and inhibit cPKC-mediated cell functions. Phorbol myristoyl acetate (PMA) strongly stimulated phosphatidylcholine (PtdCho)-specific phospholipase D (PLD) activity in the C3H/10T1/2 Cl8 parental cell line, but not in Cl8 HAβC2-1 cells, indicating that full PLD activity in PMA-treated Cl8 cells is dependent on a functional interaction of α/βPKC with RACK1. In contrast, the PMA-stimulated uptake of choline and its subsequent incorporation into PtdCho, were not inhibited in Cl8 HAβC2-1 cells as compared to Cl8 cells, indicating a RACK1-independent but PKC-mediated process. Increased incorporation of labelled choline into PtdCho upon PMA treatment was not associated with changes of either CDP-choline: 1,2-diacylglycerol cholinephosphotransferase activity or the CTP:phosphocholine cytidylyltransferase distribution between cytosol and membrane fractions in Cl8 and Cl8 HAβC2-1 cells. The major effect of PMA on the PtdCho synthesis in C3H/10T1/2 fibroblasts was to increase the cellular uptake of choline. As a supporting experiment, we inhibited PMA-stimulated PtdH formation by PLD, and also putatively PtdH-derived DAG, in Cl8 cells with 1-butanol. Butanol did not influence the incorporation of [ 14C]choline into PtdCho. The present study shows: (1) PMA-stimulated PLD activity is dependent on a functional interaction between α/βPKC and RACK1 in C3H/10T1/2 Cl8 fibroblasts; and (2) inhibition of PLD activity and PtdH formation did not reduce the cellular uptake and incorporation of labelled choline into PtdCho, indicating that these processes are not directly regulated by PtdCho-PLD activity in PMA-treated C3H/10T1/2 Cl8 fibroblasts.

Intracellular survival of Burkholderia cepacia complex isolates in the presence of macrophage cell activation.

Strains of the Burkholderia cepacia complex have emerged as a serious threat to patients with cystic fibrosis due to their ability to infect the lung and cause, in some patients, a necrotizing pneumonia that is often lethal. It has recently been shown that several strains of the B. cepacia complex can escape intracellular killing by free-living amoebae following phagocytosis. In this work, the ability of two B. cepacia complex strains to resist killing by macrophages was explored. Using fluorescence microscopy, electron microscopy and a modified version of the gentamicin-protection assay, we demonstrate that B. cepacia CEP021 (genomovar VI), and Burkholderia vietnamiensis (previously B. cepacia genomovar V) CEP040 can survive in PU5-1.8 murine macrophages for a period of at least 5 d without significant bacterial replication. Furthermore, bacterial entry into macrophages stimulated production of tumour necrosis factor and primed them to release toxic oxygen radicals following treatment with phorbol myristoyl acetate. These effects were probably caused by bacterial LPS, as they were blocked by polymyxin B. Infected macrophages primed with interferon gamma produced less nitric oxide than interferon-gamma-primed uninfected cells. We propose that the ability of B. cepacia to resist intracellular killing by phagocytic cells may play a role in the pathogenesis of cystic fibrosis lung infection. Our data are consistent with a model where repeated cycles of phagocytosis and cellular activation without bacterial killing may promote a deleterious inflammatory response causing tissue destruction and decay of lung function.

THE NEUROPEPTIDE CALCITONIN GENE-RELATED PEPTIDE INHIBITS TNF-α BUT POORLY INDUCES IL-6 PRODUCTION BY FETAL RAT OSTEOBLASTS

Tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6) are potent inflammatory cytokines produced by osteoblasts and whose contribution to bone loss occurring in oestrogen deficiency is well documented. Calcitonin gene-related peptide (CGRP) is a neuropeptide abundantly concentrated in sensory nerve endings innervating bone metaphyses and periosteum suggesting that it controls bone homeostasis locally. Since CGRP was shown to inhibit TNF-α production by T cells and stimulate IL-6 expression by fibroblasts, this study was designed to investigate whether CGRP regulated TNF-α and IL-6 production by osteoblasts. We show that CGRP inhibits the production of TNF-α by both lipopolysaccharide (LPS)- and IL-1-stimulated fetal rat osteoblasts. Like CGRP, the cAMP agonists prostaglandin E 2(PGE 2), dibutyryl cAMP (Bt 2cAMP) and forskolin inhibit TNF-α production by osteoblasts. Exposure of osteoblasts to a high dose of phorbol myristoyl acetate (PMA) to deplete PKC activity abolished CGRP-mediated TNF-α suppression. In contrast with its potent inhibition of TNF-α production, we show that CGRP is a weak inducer of IL-6 when compared to PGE 2, Bt 2cAMP and forskolin. However, in presence of isobutylmethylxanthine (IBMX) CGRP stimulates the production of IL-6. Collectively, these data suggest that the inhibition of TNF-α CGRP is cAMP dependent and PMA sensitive and that the concentration of intracellular cAMP may be a regulatory mechanism for IL-6 expression in osteoblasts.

Activation of protein kinase C enhances the infection of endothelial cells by human cytomegalovirus

The infection of cultured endothelial cells with human cytomegalovirus (HCMV) is generally limited to less than 10% of the cells in contrast to HCMV infection of fibroblasts, where essentially all cells can be infected. It is known that HCMV infection influences a number of signal transduction pathways of infected cells. We therefore questioned whether, conversely, the infectivity of human umbilical vein endothelial cells could be influenced by the deliberate activation of these pathways. When endothelial cells were treated prior to infection with phorbol myristoyl acetate, an activator of protein kinase C, the number of HCMV-positive cells increased two to three times. On the other hand, pretreatment of the cells with RO 31-8220, a specific protein kinase C inhibitor, or with staurosporine, a general protein kinase inhibitor, resulted in a decreased infection level and in abolishment of the PMA-induced effect. Pretreatment with the protein phosphatase inhibitor, okadaic acid, caused a slight increase in infectivity, whereas pretreatment with the protein tyrosine kinase inhibitor, genistein, was without effect. Furthermore, neither forskolin and ilomedine, compounds known to activate the endothelial adenylate cyclase, nor the calcium ionophore A23187 were able to influence HCMV infectivity. It is concluded that: (a) the HCMV infection level of unstimulated endothelial cells is influenced by the basal level of protein kinase C; and (b) stimulation of protein kinase C prior to infection results in an increase of infection by HCMV.

Changes in prostaglandin synthesis and metabolism associated with labour, and the influence of dexamethasone, RU 486 and progesterone

The objective was to compare the changes in prostaglandin synthesis and metabolism occurring within the fetal membranes that are associated with the onset of parturition and to study the effect of steroid hormones on prostaglandin metabolism. A tissue explant study was made of discs of amnion and chorion obtained from 24 pregnant women at 37-42 weeks' gestation following spontaneous labour and delivery (12 women) and elective caesarean section (12 women). Significantly more prostaglandin E2 (PGE2) and PGF2 alpha were synthesized by amnion obtained following spontaneous labour than elective caesarean section. Arachidonic acid stimulated both PGE2 and PGF2 alpha synthesis by amnion in both groups. Phorbol myristoyl acetate stimulated PGE2 synthesis in both groups. There was no difference between the groups in the capacity of the chorion to metabolize prostaglandins. Mifepristone (RU 486) reduced the metabolism of added PGE2 following spontaneous labour, while dexamethasone and progesterone had no effect on prostaglandin metabolism. In conclusion, the increase in concentration of PGE2 and PGF2 alpha associated with the onset of spontaneous labour is the result of an increase in synthesis rather than a reduction in metabolism. There was no decrease in metabolism to account for the increase in prostaglandin concentrations and, with the exception of mifepristone, metabolism was not altered by the addition of steroid hormones.

Hierarchical analysis of the nerve growth factor‐dependent and nerve growth factor‐independent differentiation signaling pathways in PC12 cells with protein kinase inhibitors

The effects of a series of protein kinase inhibitors on nerve growth factor (NGF)-dependent and NGF-independent neurite outgrowth in PC12 cells have established an ordered relationship among those protein kinases sensitive to down regulation by bryostatin, stimulation by staurosporine, inhibition by sphingosine, or inhibition by 6-thioguanine (6-TG). Quantitation of the biphasic staurosporine effects on NGF-induced neurite outgrowth (Hashimoto and Hagino: J Neurochem 53:1675-1685, 1989) gave an IC50 of 2-4 nM for inhibition and an EC50 of 15-20 nM for induction of neurite extension. Both sphingosine and 6-TG inhibited neurite outgrowth induced by staurosporine and basic fibroblast derived growth factor (bFGF), as well as by NGF; therefore, sphingosine- and 6-TG-sensitive protein kinase steps occur after the convergence of the NGF, bFGF, and staurosporine signal pathways. Down regulation of protein kinase C by bryostatin chronic treatment, which inhibits NGF- and bFGF-induced neuritogenesis (Singh et al.: Biochemistry 33:542-551, 1994), did not inhibit the staurosporine-induced neurite outgrowth. Thus, the bryostatin-sensitive protein kinase C must occur subsequent to the convergence of the bFGF and NGF pathways, but before (or parallel to) staurosporine initiation of neurite outgrowth. In contrast, low concentrations of phorbol myristoyl acetate (PMA) or bryostatin, which activate protein kinase C activity, enhanced the staurosporine- or NGF-induced neurite extension. These data indicate that stimulation of one or more protein kinase C isozymes can synergistically interact with the signaling pathway to increase the rate of neuritogenesis. Inhibition by 5-7.5 nM staurosporine acted rapidly to arrest and decrease development of neurites up to 24 hr after NGF treatment, as did K252a and NGF polyclonal antibody addition. Our cellular data support the concept that staurosporine acts to inhibit the NGF receptor Trk (Nye et al.: Mol Biol Cell 3:677-686, 1992), but that downstream steps can be activated by the higher concentration of staurosporine to bypass Trk and lead to neurite generation. Effects of staurosporine, 6-TG, and sphingosine on c-fos gene induction with or without NGF were not correlated with the generation of neurites. The sequence of protein kinases sensitive to these effectors appears to be in the order (but not consecutive) bryostatin, staurosporine, sphingosine, and 6-TG.

Identification and characterization of rhesus macaque interleukin-8

To establish a direct link between IL-8 and inflammation in vivo, we first isolated the gene encoding rhesus macaque IL-8. The open reading frame directs the translation of a 101 amino acid (aa) precursor, which is 94% identical to human IL-8. Rhesus IL-8 was expressed in bacteria and purified to homogeneity with ion-exchange chromatography. Pure rhesus IL-8 was biologically active as measured by its ability to bind specifically to either rhesus (Kd = 0.5 nM) or human (Kd = 2 nM) IL-8 receptors and to promote in vitro chemotaxis of rhesus (EC50 = 2 nM) or human neutrophils (EC50 = 4 nM). Moreover, a mouse monoclonal antibody, DM/C7, which neutralizes human IL-8 activity, also recognized and neutralized (IC50 = 0.5-3.0 microgram/ml) rhesus IL-8 in vitro. Systemic administration of DM/C7 completely inhibited the dermal inflammation of rhesus ears induced by the external application of phorbol myristoyl acetate. These observations reveal that rhesus IL-8 is structurally and functionally similar to human IL-8 and suggests that IL-8 plays a prominent role in a primate model of inflammation.

In vitro reverse cholesterol transport from THP-1-derived macrophage-like cells with synthetic HDL particles consisting of proapolipoprotein A1 or apolipoprotein A1 and phosphatidylcholine.

The human monocytic leukaemia cell line THP-1 was induced to differentiate to macrophage-like cells by the addition of phorbol myristoyl acetate (PMA). Subsequently, the cells were enriched in cholesterol and these cholesterol laden cells were used to study the capability of reconstituted discoidal complexes (RDCs), consisting of either human apolipoprotein A1 (apo A1) or recombinant human proapolipoprotein A1 (proapo A1) and phosphatidylcholine (PC), to promote cholesterol efflux. RDCs containing apo A1 and proapo A1 were both effective in the mobilization of intracellular cholesterol, whether this was measured by intracellular cholesterol mass or by the appearance of radiolabelled cholesterol in the supernatant. Using the radiolabelling technique, the activity was saturable and followed Michaelis-Menten kinetics. For both types of complexes and for native HDL the maximum rate of cholesterol removed was approximately 0.5 nmol h-1 per 10(6) cells. For RDCs of proapo A1 and apo A1 and for native HDL the Km values were 3.7, 2.9 and 64.8 micrograms ml-1 respectively. A significant in vitro cholesterol efflux could only be achieved with protein-lipid complexes; no significant export was observed with either free proapo A1 or multilamellar PC liposomes without apolipoprotein. Both RDCs were found to be more active in the mobilization of intracellular cholesterol than HDL isolated from human plasma. The combined results demonstrate that synthetic complexes consisting either of apo A1 or proapo A1 and PC are both active in the in vitro reverse transport of cholesterol.