Identification and characterization of a histone binding site of the non-structural protein 3 of hepatitis C virus.

BACKGROUND: Chronic hepatitis resulting from the hepatitis C virus (HCV) infection leads to cirrhosis in at least half the infected patients and increases the risk of hepatocellular carcinoma. There are indications that this pathogenic effect may result from the disturbance of intracellular signal cascades caused by the interaction with viral antigens. Although a great amount of data has been accumulated about functional regions in HCV proteins, relatively little is known about their intracellular targets. Previously, we have demonstrated that the full-length non-structural protein 3 of HCV (NS3) (Borowski P, Heiland M, Feucht H, Laufs R. Characterisation of non-structural protein 3 of hepatitis C virus as modulator of protein phosphorylation mediated by PKA and PKC. Evidences for action on the level of substrate and enzyme. Arch Virol 1999a; 144) and its NH2- and COOH-terminal truncated form (Borowski P, Heiland M, Oehlmann K, Becker B, Kornetzky L, Feucht HH, Laufs R. Non-structural protein 3 of hepatitis C virus inhibits phosphorylation mediated by cAMP-dependent protein kinase. Eur J Biochem 1996;237:611-618) associate to stable complexes with core histones H2B and H4. The changes of the properties of histones as substrate for cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) were found as a direct consequence of the interaction. OBJECTIVE: In the present study we further these observations, localize the histone binding domain of NS3 and investigate the mechanisms by which NS3 affects the functions of the histones in vitro. STUDY DESIGN: HCV protein exhibiting the mentioned histone binding activity was produced in a bacterial expression system, purified and binding to histones was biochemically characterized. The region of NS3 involved in the interaction with histones was defined by proteolytic fragmentation, microsequencing and a specific histone binding assay. Furthermore, a functional test to quantify the interaction of histones with DNA was established and the binding of DNA to histone as a function of NS3 concentration was analysed by means of graphical methods. RESULTS: The investigated fragment of HCV polyprotein consisting of amino acid residues 1189-1525 (HCV-polyprotein-(1189-1525)) displayed significant histone binding activity. The binding occurred at a molar ratio 1:1 of histone to HCV-polyprotein-(1189-1525) and was mediated by a linear stretch of amino acids located between the residues 1343 and 1379 of the HCV polyprotein. To demonstrate that HCV-polyprotein-(1189-1525) affects the binding of DNA to histones we used two independent methods: overlay assay and binding assay on Sepharose beads. Graphic analysis of the binding kinetics revealed an uncompetitive type of inhibition. CONCLUSIONS: Our results provide the first evidence that NS3 binds and affects the functions of core histones. The mechanism by which the NS3 interferes with the histone functions involves conformational changes of histone molecule.     

MSI1 suppresses hyperactive RAS via the cAMP-dependent protein kinase and independently of chromatin assembly factor-1

RAS hyperactivation in the yeast Saccharomyces cerevisiae leads to multiple nutritional growth defects associated with overstimulation of the cAMP signaling pathway. Hyperactive RAS can be suppressed by overexpression of MSI1, a subunit of chromatin assembly factor-1 (yCAF-1). MSI1 overexpression suppresses phenotypes induced by increased cAMP content in multiple genetic backgrounds. However, MSI1 does not inhibit cAMP synthesis or total cellular cAMP-dependent protein kinase (PKA) activity, nor does MSI1 stimulate expression of several cAMP-repressible genes critical for the acquisition of thermotolerance in the stationary phase. Our analysis indicates that yCAF-1 is dispensable for inhibition of hyperactive RAS by MSI1. We demonstrate that in the presence of the PKA regulatory subunit, BCY1, MSI1 inhibits phenotypes of a mutationally activated PKA catalytic subunit. These observations indicate that MSI1 affects PKA function in a BCY1-dependent manner via mechanisms other than direct overall inhibition of PKA catalytic activity. MSI1 appears to provide two distinct roles – in chromatin modeling as a component of yCAF-1, and in the inhibition of RAS signaling by modulating PKA. Received: 23 August 1999 / Accepted: 7 April 2000     

Activation by GABAb,reduction of the intracellular concentration of Ca++ and inhibition of protein kinases are possible mechanisms of the long-term aosttetanic modification of the efficiency of inhibitory transmission in the new cortex

A hypothetical mechanism is proposed for the induction of long-term posttetanic potentiation of the efficiency of inhibitory synaptic transmission (LTPi). The data we have previously obtained have made it possible to hypothesize that modifiable inhibitory synapses are situated on the dendritic spines on which there are metabotropic GABAb receptors. It is hypothesized that modification of inhibitory transmission is determined precisely by these receptors, the activation of which leads to inactivation of protein kinases C and A (PKC and PKA) as a result of a decrease in the intracellular concentration of Ca⁺⁺ and the inhibition of cAMP. The hypothesis is confirmed by experiments in which it was demonstrated that an effect similar to LTPi took place as a result of the inactivation of PKC and PKA. It is hypothesized that eicanoid [sic] acids may be retrograde messengers during LTPi. A new hypothetical mechanism underlying long-term depression of excitatory transmission (LTDe) is proposed, according to which tetanized afferent fibers must simultaneously monosynaptically excite and disynaptically inhibit one and the same postsynaptic cell. LTDe may be induced only in those pathways which activate [are activated by — unclear from Russian text — Trans.] GABAb receptors. The proposed hypotheses make it possible to explain the results of certain experiments. Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Moscow. Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti imeni I.P. Pavlova, Vol. 45, No. 1, pp. 18–28, January–February, 1995.     

Mass spectrometric analysis reveals a functionally important PKA phosphorylation site in a Kir3 channel subunit

Phosphorylation of the Kir3 channel by cAMP-dependent protein kinase (PKA) potentiates activity and strengthens channel–PIP₂ interactions, whereas phosphorylation by protein kinase C (PKC) exerts the opposite effects (Keselman et al., Channels 1:113–123, 2007; Lopes et al., Channels 1:124–134, 2007). Unequivocal identification of phosphorylated residues in ion channel proteins has been difficult, but recent advances in mass spectrometry techniques have allowed precise identification of phosphorylation sites (Park et al., Science 313:976–979, 2006). In this study, we utilized mass spectrometry to identify phosphorylation sites within the Kir3.1 channel subunit. We focused on the Kir3.1 C-terminal cytosolic domain that has been reported to be regulated by several modulators. In vitro phosphorylation by PKA exhibited a convincing signal upon treatment with a phosphoprotein stain. The phosphorylated C terminus was subjected to mass spectrometric analysis using matrix-assisted lased desorption/ionization–time of flight mass spectroscopy (MS). Peptides whose mass underwent a shift corresponding to addition of a phosphate group were then subjected to tandem MS (MS/MS) in order to confirm the modification and determine its precise location. Using this approach, we identified S385 as an in vitro phosphorylation site. Mutation of this residue to alanine resulted in a reduced sensitivity of Kir3.1* currents to H89 and Forskolin, confirming an in vivo role for this novel site of the Kir3.1 channel subunit in its regulation by PKA.     

Regulators of sperm function: Involvement of protein kinases in platelet activating factor-induced acrosome reaction of human spermatozoa

The involvement of protein kinases in platelet activating factor(PAF)-induced acrosome reaction of human spermatozoa was investigatedusing specific inhibitors of protein kinase A (PKA), proteinkinase C (PKC) and protein tyrosine kinase (PTK). PAF (10^–9–10^–11M) treatment of spermatozoa enhanced the acrosome reaction ina dose-dependent manner (32 ± 4% at 10^–9 M, 28±4%at 10^–10 M and 24 ± 3% at 10^–11 M respectively).When spermatozoa were preincubated with PKA, PKC and PTK inhibitor(KT5720, calphostin C and genistein) for 15 min prior to additionof PAF, there was a significantly reduced acrosome reactioninduced by PAF, but complete inhibition was not observed. Onthe other hand, combined use of three inhibitors completelyinhibited PAF-induced acrosome reaction to levels of non-treatedsamples. These results suggest that the induction of the acrosomereaction by PAF treatment may involve the activation of PKA,PKC and PTK signalling pathways, and that interaction betweenthese pathways may regulate complex mechanisms of PAF-inducedacrosome reaction. acrosome reaction/human spermatozoa/platelet activating factor/protein kinase     

v-ras and protein kinase C dedifferentiate thyroid cells by down-regulating nuclear cAMP-dependent protein kinase A.

Ras proteins are membrane-associated transducers of eternal stimuli to unknown intracellular targets. The constitutively activated v-ras oncogene induces dedifferentiation in thyroid cells. v-Ras appears to act by stimulating protein kinase C (PKC), which inhibits the nuclear migration of the catalytic subunit of the cAMP-dependent protein kinase A (PKA). Nuclear tissue-specific and housekeeping trans-acting factors that are dependent on phosphorylation by PKA are thus inactivated. Exclusion of the PKA subunit from the nucleus could represent a general mechanism for the pleiotropic effects of Ras and PKC on cellular growth and differentiation.     

Phosphorylation is required for high-affinity binding of DBP, a yeast mitochondrial site-specific RNA binding protein

All yeast mitochondrial mRNAs terminate at their 3′ ends with a conserved dodecamer sequence, a site for high-affinity binding by DBP (dodecamer binding protein). Using purified DBP, we show that binding requires an intact dodecamer site and is enhanced by the presence in an oligonucleotide of the immediate 4–5 upstream nucleotides. Binding affinity varied from 0.25 to 0.85 nM towards a set of RNA oligonucleotides containing messenger specific upstream sequences in addition to the dodecamer site. Furthermore, we show that phosphatase treatment of DBP abolishes its specific binding, indicating the involvement of reversible phosphorylation in the regulation of its binding activities. This finding will further our understanding of the mechanism of DBP in the regulation of RNA metabolism in yeast mitochondria. Received: 25 June 1999 / 19 February 2000     

Differential modulation of unapposed connexin 43 hemichannel electrical conductance by protein kinase C isoforms

Opening of unapposed connexin 43 hemichannels (Cx43Hc) in the plasma membrane results in altered ionic homeostasis leading to cell damage. Although it is generally acknowledged that Cx43Hc function is regulated by protein kinase C (PKC), information regarding the functional role of PKC in the modulation of Cx43Hc electrical conductance is lacking. In this work, we used the patch-clamp technique to study the effect of phorbol 12-myristate 13-acetate (PMA), a general PKC activator, on the electrical conductance of exogenous Cx43Hc expressed in tsA201 cells. Subsequently, a matrix of synthetic PKC isoform-specific inhibitor peptides was used to dissect the functional role of individual PKC isoforms in Cx43Hc regulation. Superfusion with 10 nM PMA abolished Cx43Hc currents by 74%, an effect that was prevented by pretreatment with a general PKC inhibitor, GF109203X. It is interesting to note that intracellular diffusion of ɛV_1–2 (0.1 μM), an ɛPKC-specific inhibitor peptide, completely antagonized PMA-induced current inhibition. Cell dialysis with either β_II- or δPKC inhibitor peptides partially decreased PMA effect. Neither α- nor β_IPKC inhibition altered PMA-induced current reduction. This study shows for the first time that Cx43Hc electrical conductance is inhibited after PKC activation. Moreover, this inhibition is predominantly mediated by the “novel” ɛPKC isoform, whereas partial inhibition may be provided by the “conventional” β_IIPKC as well as the “novel” δPKC isoforms.     

Proliferating activity in columnar cell lesions of the breast

With the introduction of mammographic screening, columnar cell lesions (CCLs) are observed more and more frequently because they are often associated with microcalcifications. Until now, the proliferative activity of these lesions has not been previously evaluated. Ki67 index was performed by immunohistochemistry in CCLs without atypia [columnar cell change (CCC) n = 20 and columnar cell hyperplasia without atypia (CCH without atypia) n = 20], flat epithelial atypia (FEA DIN1A n = 20), low-grade intraductal carcinoma (DIN1C n = 20), high-grade intraductal carcinoma (DIN 2–3 n = 20). Adjacent terminal duct-lobular unit (TDLU) of normal breast tissue served as control. Ki-67 index is extremely low and close in CCLs without atypia (CCC mean 0.1% and CCH mean 0.76%) and paradoxically is lower than in normal TDLU (mean 2.4%) (p < 0.001). In the FEA, in comparison with normal TDLU and CCLs without atypia, the Ki67 is higher (mean 8.2%) (p < 0.001) but extremely close to those of DIN1C (mean 8.9%) (p = 0.6 NS). Lastly, the Ki67 index is higher in DIN 2–3 (mean 25.4%) than in CCLs without atypia and FEA (p < 0.001). CCLs are disparate lesions having in common cells with columnar configuration but different proliferative characteristics. These data represent findings of biological interest which could help us to better understand these controversial lesions.     

Evaluation of a Single-Sample Serological Technique for Diagnosing Pertussis in Unvaccinated Children

 This study was performed to evaluate the sensitivity of immunoglobulin (Ig)G and IgA antibodies to pertussis toxin and filamentous hemagglutinin in diagnosing pertussis from a single serum sample. The pertussis group was defined according to the World Health Organization pertussis case definition. The control group coughed for 21 days or more but had no microbiological or serological evidence of Bordetella infection. Both cohorts were divided into infants (<12 months of age), toddlers (1–4 years) and school children (5–10 years). There were 525 subjects in the pertussis group and 321 in the control group, with an even distribution of genders. IgG and IgA antibodies to pertussis toxin and filamentous hemagglutinin were measured in a standardized enzyme immunoassay. Antibody levels beyond the 95 percentile of the control cohort were regarded as indicative of recent contact, setting the specificity level at 0.95. Acute serum samples drawn between 1 week and 3 weeks after the onset of coughing showed a low sensitivity (2–19%) for diagnosing pertussis. In convalescent samples taken 5–10 weeks after the onset of symptoms, detection of IgG anti-pertussis toxin was the best single test, with a sensitivity of 61%, 65%, and 74% in infants, toddlers and school children, respectively. A combination of IgG anti-pertussis toxin and IgA anti-filamentous hemagglutinin using age-specific reference values had a sensitivity of 81–89% in diagnosing pertussis from a single serum sample taken 5–10 weeks after the beginning of symptoms.     

Glucose 6-phosphate dehydrogenase from larval Taenia crassiceps (cysticerci): purification and properties

Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) was purified to homogeneity from the soluble fraction of larval Taenia crassiceps (Eucestoda: Cyclophyllidea) by a three-step protocol. Specific activity of the pure enzyme was 33.8 ± 2.1 U mg⁻¹ at 25°C and pH 7.8 with d-glucose 6-phosphate and NADP⁺ as substrates. The activity increases to 67.6 ± 3.9 U mg⁻¹ at 39°C, a more physiological temperature in the intermediary host. Enzyme activity was maximal between pH 6.7 and 7.8. K _m values were 14 ± 1.7 μM and 1.3 ± 0.4 μM for glucose 6-phosphate and NADP⁺, respectively. The enzyme showed absolute specificity for its sugar substrate. NAD⁺ was also a substrate but with a low catalytic efficiency (207 M⁻¹ s⁻¹). No essential requirement for Mg⁺⁺ or Ca⁺⁺ was observed. Relative molecular mass of the native enzyme was 134,000 ± 17,200, while a value of 61,000 ± 1,700 was obtained for the enzyme subunit. Thus, glucose 6-phosphate dehydrogenase from T. crassiceps exists as a dimeric protein. The enzyme’s isoelectric point was 4.5. The enzyme’s activity dependence on temperature was complex, resulting in a biphasic Arrhenius plot. Activation energies of 9.91 ± 0.51 and 7.94 ± 0.45 kcal mol⁻¹ were obtained. Initial velocity patterns complemented with inhibition studies by product and substrate’s analogues support a random bi bi sequential mechanism in rapid equilibrium. The low K _i value of 1.95 μM found for NADPH suggests a potential regulatory role for this nucleotide.     

Inhibition of the L-type calcium channel by the five muscarinic receptors (m1–m5) expressed in NIH 3T3 cells

 Modulation of L-type calcium channels by the five cloned muscarinic receptors was studied by expression of the receptors in NIH 3T3 cells. Application of acetylcholine (ACh) to cells transfected with m1–m5 resulted in a reduction in the L-type calcium current amplitude. Elevations in intracellular cAMP concentrations induced by 8-bromo-cAMP or forskolin resulted in no discernible change in the L-type calcium current. In addition, treatment with Rp-adenosine 3′,5′-cyclic monophosphothioate triethylamine (Rp-cAMPS), a protein kinase A (PKA) inhibitor, had no effect on the L-type currents. Conversely, application of phorbol dibutyrate, an activator of protein kinase C (PKC) or 8-bromo-cGMP, an activator of cGMP-dependent protein kinase (PKG), reduced the calcium currents. Incubation of the cells with KT5823, an inhibitor of PKG, resulted in a reduction of the response to 8-bromo-cGMP. The ACh-induced depression of L-type calcium current amplitude was sensitive to pertussis toxin (PTX) in cells transfected with the m2 or m4 receptor subtype. The m2-muscarinic-receptor-induced inhibition of the L-type calcium current was attenuated by preincubation of the cells with 8-bromo-cAMP and was unaffected by KT5823 or by calphostin C. The m1-muscarinic-receptor-induced inhibition of the L-type calcium conductance was insensitive to PTX treatment. However, the m1-induced response was blocked by preincubation of the cells with calphostin C. The present data indicate that the m2 (and possibly also the m4) muscarinic receptors inhibit the L-type calcium conductance by a reduction in cAMP concentration and that the m1 (and possibly also the m3 and m5) muscarinic receptors inhibit the L-type calcium channel via activation of PKC. Received: 2 September 1996 / Received after revision and accepted: 15 October 1996     

Enhancement of sperm-zona pellucida (ZP) binding capacity by activation of protein kinase A and C pathways in certain infertile men with defective sperm-ZP binding

BACKGROUND: Defective sperm–zona pellucida (ZP) binding (DSZPB) isa common cause of failure of fertilization in vitro. This studywas to determine if DSZPB is caused by defective pathways upstreamof protein kinase A (PKA) and C (PKC), or reduced protein tyrosinephosphorylation (TP). METHODS: Infertile men with DSZPB and either normal sperm morphology(NSM) 14% (n = 15) or 5% (n = 15) were studied. Sperm–ZPbinding test was performed by incubation of motile sperm withoocytes for 2 h with or without dibutyryl cyclic AMP (dbcAMP,PKA activator) or phorbol myristate acetate (PMA, PKC activator).TP of capacitated sperm in medium was assessed by immunofluorescencewith an anti-phosphotyrosine monoclonal antibody. RESULTS: For normal sperm with normal sperm–ZP binding, both PMAand dbcAMP significantly enhanced sperm–ZP binding ina dose–response manner. Only dbcAMP, but not PMA, significantlyincreased TP of capacitated sperm. In DSZPB men with severeteratozoospermia (NSM 5%), neither PMA nor dbcAMP enhancedsperm–ZP binding, despite dbcAMP significantly increasingthe TP of capacitated sperm for all samples. In contrast, forDSZPB with NSM 14%, PMA caused significantly increased spermbinding up to normal levels (40 sperm bound/ZP) in five men,and dbcAMP had a similar result in two men. Again TP was significantlyenhanced only by dbcAMP, but not by PMA. CONCLUSIONS: There is defective signalling in pathways upstream of PKC andPKA in some men with DSZPB and normal semen analysis. Stimulationof TP by dbcAMP does not enhance sperm–ZP binding capacityin DSZPB men with low TP, regardless of sperm morphology.     

Procaterol Inhibits Lung Fibroblast Migration

Fibroblasts are important cells that are involved in modulation of fibrosis after injuries. In some uncontrollable inflammatory processes, excess fibroblasts migrate around the small airway. The pathogenesis of chronic obstructive pulmonary disease is related to fibrosis around the small airways. The aim of the current study was to investigate the effect of procaterol, a second-generation beta (2)-agonist, on migration of human fetal lung fibroblasts (HFL-1) induced by human plasma fibronectin (HFn). Using the blindwell chamber technique, 10⁻⁸ M procaterol inhibited migration of HFL-1 (control, 100%; 10⁻⁸ M, 73.2 ± 4.9%; n = 6, p < 0.05). The inhibitory effect of procaterol was concentration-dependent. Although a beta 2-receptor inhibitor, ICI 181551, blocked the inhibitory effect of procaterol, a beta 1-receptor inhibitor, atenolol, did not. Because a cyclic AMP-dependent protein kinase (PKA) inhibitor, KT5720, blocked the effect of procaterol, the cyclic AMP–PKA pathway may be involved in the migration inhibitory process. Procaterol, which is prescribed mainly for treatment of bronchial asthma, might be a useful drug for inhibiting lung fibrosis following injuries to the lung.     

Acute Ca-dependent desensitization of 5-HT1A receptors is mediated by activation of protein kinase A (PKA) in rat serotonergic neurons

This report investigates acute changes in the sensitivity of 5-HT_1A receptors in dorsal raphe (dr) neurons in response to elevated serotonin. DR neurons were isolated from adult rats and measurements of inhibition of Ca²⁺ current by 5-HT were obtained using the whole cell patch clamp technique. During a 10-min application of 5-HT (with normal [Ca²⁺]_i∼100 nM) a desensitization occurred. The response to 20 nM 5-HT decreased by 66% relative to control and remained depressed for about 30 min. When the internal [Ca²⁺] was buffered to <1 nM only a weak transient desensitization occurred that was surmountable with higher [5-HT]. Adenylyl cyclase activation with forskolin mimicked the desensitization and selective inhibition of protein kinase A (PKA), but not protein kinase C (PKC), partially antagonized the desensitization induced by 5-HT. To measure the activity of PKA and phosphatase enzymes, dr slices were incubated with the selective agonist dipropyl-5-carboxamidotryptamine (DP-5-CT, 1 μM) for 10 min and the phosphorylation of the PKA substrate Kemptide was followed using ATP-γ³²P. DP-5-CT inhibited the cAMP stimulated maximal activity of PKA but raised basal PKA activity, thus increasing the percentage of PKA in the active state (activity ratio), an effect that was prevented by the selective 5-HT_1A antagonist WAY100635. DP-5-CT also caused a significant inhibition of phosphatase activity. These data support a model in the dr where 5-HT_1A-receptor stimulation of PKA promotes phosphorylation of a target and phosphatase inhibition leading to heterologous desensitization. The effect would be expected to have physiological consequences for 5-HT-mediated inhibitory post synaptic potentials and the Ca²⁺ component of the action potentials of dr neurons.     

Substrate utilization in boys during exercise with [13C]-glucose ingestion

The influence of glucose ingestion on substrate utilization during prolonged exercise in children and adolescents is currently unknown. In the present study we determined the effect of intermittent exogenous glucose (GLU_exo) ingestion on substrate utilization during prolonged exercise, in adolescent boys ages 13–17 years. Healthy untrained volunteers performed four 30-min exercise bouts on a cycle ergometer, separated by 5-min rest periods (≅60% maximum O₂ consumption), on two occasions spaced 1–4 weeks apart. Two trials were performed, a control trial (CT), in which subjects ingested water intermittently during the exercise, and a glucose trial (GT), in which subjects ingested a ¹³C-enriched GLU_exo drink (≅3 g glucose · kg body mass⁻¹), also intermittently during the exercise. Total free fatty acids (FAT_total), glucose (GLU_total) and carbohydrate (CHO_total) oxidation was determined from indirect calorimetry, while GLU_exo oxidation was calculated from the ¹³C/¹²C ratio in expired air after 5–10 min and 25–30 min of exercise in each bout. Heart rate and rating of perceived exertion (RPE) were determined at the same time intervals. The oxidation of CHO_total was 169.1 (12.9) g · 120 min⁻¹ and 203.1 (15.9) g · 120 min⁻¹ (P < 0.01) and that of FAT_total was 31.0 (4.2) g · 120 min⁻¹ and 17.1 (2.5) g · 120 min⁻¹ (P < 0.01) in CT and GT, respectively. GLU_exo oxidation in GT was 57.8 (4.3) g · 120 min⁻¹, or 34.2 (2.2)% of that ingested. Endogenous glucose oxidation was 169.1 (12.9) g · 120 min⁻¹ and 145.3 (11.9) g · 120 min⁻¹ (P < 0.01) in CT and GT, respectively. Insulin and glucose concentrations were higher in GT than in CT by 226% and 37%, respectively (both P < 0.05). Free fatty acids and glycerol concentrations were lower in GT than in CT, by 27% and 79%, respectively (both P < 0.05). Heart rate was similar between trials, but RPE was lower in GT vs CT at both 115 and 135 min. Thus, under these experimental conditions, GLU_exo intake spares endogenous carbohydrate and fat by 16% and 45%, respectively, contributes to approximately 25% of the total energy demand of exercise, and lowers the RPE. Accepted: 21 May 2000     

Involvement of protein kinase C isoenzymes in Trypanosoma cruzi metacyclogenesis induced by oleic acid

Previously, we showed that oleic acid (OA) induces Trypanosoma cruzi metacyclogenesis through a signaling pathway involving de novo diacylglycerol biosynthesis and simultaneous protein kinase C (PKC) activation. Herein, we demonstrated that OA also triggers a transient Ca²⁺ signal in epimastigotes, necessary for parasite differentiation, that could account for PKC activation. In addition, we found that this free fatty acid (FFA) directly stimulated in vitro the activity of T. cruzi PKC in a dose–response way. We determined the presence of classical and novel PKC isoenzymes that were differentially expressed in the infective amastigotes (α and δ) and tripomastigotes (α, β, and γ) and in the non-infective epimastigotes (α, β, γ, and δ). We also demonstrated that OA induced in epimastigotes the translocation of PKC α, β, γ, and δ to the membrane, indicating a selective effect of this FFA. To establish a correlation between T. cruzi metacyclogenesis induced by OA and the activation of a particular PKC isoenzyme, the specific PKC inhibitors Ro 32-0432 and Rottlerin (9–30 nM and 5–35 μM, respectively) were employed. These compounds, even at the lowest concentrations assayed, abrogated both epimastigote differentiation and membrane translocation of PKC β, γ, and δ. These findings strongly support a key role for classical and novel PKC isoenzymes in the signaling pathways involved in T. cruzi metacyclogenesis induced by OA.     

Expression of immunoreactive forms of the hepatitis C NS5A protein in E. coli and their use for diagnostic assays

Summary.  In this study different forms of the hepatitis C virus (HCV) NS5A protein, including a nearly full-length, an amino-terminal and a carboxy-terminal truncated form were produced in E. coli as fusion proteins with the MBP or the GST protein. The chimeric proteins were tested for their reactivity with sera from HCV infected patients by immunoblot and ELISA assays. A panel of 110 sera specimens, including 39 HCV-positive sera, 27 sera from patients with non-HCV-associated liver disease and 44 healthy individuals were analyzed for the presence of antibodies to NS5A. Twenty four (61 %) out of the 39 HCV positive sera, showed reactivity against the nearly full length NS5A, 21 (54 %) against the amino-terminal part of NS5A and 20 (51 %) against the carboxy-terminal part of the NS5A protein in immunoblot assays, suggesting that immunoreactive epitopes are present both at the carboxy- and the amino- terminal part of the protein. None of the 71 HCV-negative serum samples showed any reactivity against the NS5A antigens. With the exception of one patient, similar data were obtained with an ELISA assay based on the use of the nearly full-length NS5A antigen. The data indicate that new forms of NS5A may be potentially valuable antigens for the development of serological assays for HCV. Received December 26, 2001; accepted April 15, 2002 Published online July 10, 2002     

The influence of either no fluid or carbohydrate-electrolyte fluid ingestion and the environment (thermoneutral versus hot and humid) on running economy after prolonged, high-intensity exercise

This study investigated the effects on running economy (RE) of ingesting either no fluid or an electrolyte solution with or without 6% carbohydrate (counterbalanced design) during 60-min running bouts at 80% maximal oxygen consumption (V˙O_2max). Tests were undertaken in either a thermoneutral (22–23°C; 56–62% relative humidity, RH) or a hot and humid natural environment (Singapore: 25–35°C; 66–77% RH). The subjects were 15 young adult male Singaporeans [V˙O_2max = 55.5 (4.4 SD) ml kg⁻¹ min⁻¹]. The RE was measured at 3 m s⁻¹ [65 (6)% V˙O_2max] before (RE1) and after each prolonged run (RE2). Fluids were administered every 2 min, at an individual rate determined from prior tests, to maintain body mass (group mean = 17.4 ml min⁻¹). The V˙O₂ during RE2 was higher (P < 0.05) than that during the RE1 test for all treatments, with no differences between treatments (ANOVA). The mean increase in V˙O₂ from RE1 to RE2 ranged from 3.4 to 4.7 ml kg⁻¹ min⁻¹ across treatments. In conclusion, the deterioration in RE at 3 m s⁻¹ (65% V˙O_2max) after 60 min of running at 80% V˙O_2max appears to occur independently of whether fluid is ingested and regardless of whether the fluid contains carbohydrates or electrolytes, in both a thermoneutral and in a hot, humid environment. Accepted: 30 October 1997     

Inhibition of protein kinases A and C demonstrates dual modes of response in human eosinophils stimulated with platelet-activating factor

BACKGROUND: Platelet-activating factor (PAF) is a potent stimulator of human eosinophils involved in the pathogenesis of allergic diseases. However, intracellular signaling mechanisms in eosinophils involving the PAF receptor are incompletely understood. OBJECTIVE: We sought to determine the roles of protein kinase C (PKC) and cyclic AMP-dependent protein kinase (protein kinase A [PKA]) in signaling pathways of human eosinophils stimulated with PAF. METHODS: After pretreatment with a PKC inhibitor, bisindolylmaleimide I, or a PKA inhibitor, H89, we investigated PAF-evoked functions, such as CD11b expression, cellular adhesion, superoxide anion generation, and degranulation in human eosinophils. RESULTS: Preincubation of eosinophils with bisindolylmaleimide I resulted in enhancement of upregulated CD11b expression and adhesion induced by PAF. H89 pretreatment also enhanced PAF-induced cellular adhesion. Superoxide anion generation and degranulation were suppressed by means of inhibition of either PKC or PKA. CONCLUSION: PKC and PKA negatively regulate PAF-induced CD11b upregulation and cellular adhesion but promote eosinophil effector functions, such as superoxide anion generation and degranulation. PKC and PKA modulate PAF-evoked intracellular signaling of the eosinophil function in distinct ways.