Henning Juhl

Acyclovir in herpes zoster.

Abstract In a double-blind randomised trial patients with acute herpes zoster received either 5 mg/kg acyclovir (27) or placebo (29) intravenously three times daily. Acyclovir significantly improved the rate of healing of the skin lesions and shortened the period of pain in the acute phase of zoster. Particularly responsive patients were those above 67 years, those with fever, and those with less than four days of pain before treatment. No adverse effects were observed. Acyclovir seems to be active against varicella/zoster virus.

Ca2+ and phorbol ester activation of protein kinase C at intracellular Ca2+ concentrations and the effect of TMB-8

A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from human polymorphonuclear leukocytes and shown to be identical to bovine protein kinase C. The Ca2+ activation of the enzyme was studied and the Ca2+ concentrations required to activate the enzyme were compared to free cytosolic Ca2+ concentrations in resting and activated polymorphonuclear leukocytes. The free calcium concentrations in the cytosol and in the enzyme assay mixture were determined using the calcium indicator quin 2. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range, but was not activated by phosphatidylserine at Ca2+ concentrations corresponding to the intracellular free Ca2+ concentration under resting conditions. However, at similar Ca2+ concentrations (less than 2.5 X 10(-7) M) the enzyme was highly activated by phorbol 12-myristate 13-acetate (PMA) or diolein in the presence of phosphatidylserine. It was demonstrated that PMA stimulation of human polymorphonuclear leukocytes did not induce any increase in the level of the intracellular free calcium concentration. It was concluded that PMA activation of protein kinase C occurred independently of a rise in the intracellular Ca2+ concentration. K0.5 (half-maximal activation) for the PMA activation of purified protein kinase C was shown to be equivalent to the K0.5 for PMA stimulation of superoxide (O-2) production in human polymorphonuclear leukocytes, suggesting that protein kinase C is involved in activation of the NADPH oxidase. The presumed intracellular Ca2+ antagonist TMB-8 inhibited the PMA-induced superoxide production, but neither by an intracellular Ca2+ antagonism nor by a direct inhibition of protein kinase C activity.

Purification and properties of protein kinase C from bovine polymorphonuclear leucocytes

A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from bovine polymorphonuclear leucocytes. The purified enzyme had a specific activity of 10 000 U/mg protein and on SDS gelelectrophoresis the Mr was 88 000. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range. At lower concentrations of calcium (less than 1 X 10(-7) M) the enzyme was only activated by the simultaneous presence of phosphatidylserine and diolein. Phorbol 12-myristate 13-acetate mimicked the effect of diolein and partially activated the enzyme. Protein kinase C activity and the phorbolester binding protein co-purified throughout all the purification steps.

Activation of the glycogenolytic cascade in human polymorphonuclear leucocytes by different phagocytic stimuli

Abstract. Human polymorphonuclear leucocytes were found to respond to activation by immunoglobulin opsonized latex particles and to complement opsonized zymosan particles with a rapid transient increase in cAMP concentration, dissociation of the cAMP dependent protein kinase, activation of gly‐cogen phosphorylase and glycogen break down. However, since phosphorylase kinase was not activated, the activation of phosphorylase is believed to be secondary to non‐covalent activation of phosphorylase kinase by Ca2+.

Purification and properties of cAMP dependent and independent histone kinases from human leukocytes

SummaryHistone kinase activity was purified from human polymorphonuclear leukocytes by ammonium sulphate precipitation of a 180 000 × g supernatant, followed by DEAF-cellulose chromatography and gelfiltration. On DEAE-cellulose cAMP dependent kinase activity eluted in two peaks, I and III, at 1.2 mmho and 6.5 mmho, respectively. Catalytic subunit (C) from both peaks had Mr 33 000, 3.0S. Regulatory subunit (R) from peak I and III both had Mr 33 000 upon gelfiltration, but sedimented at 2.8–3.0S and 3.0–3.2S, respectively. R2 and R4 subunits were identified. The R-C dimer from peak I and III sedimented at 4.8S and (4.8)–5.1S, respectively. The holoenzyme from peak I had Mr 165 000, 6.7S, which suggest a R2C2 structure, while that of peak III sedimented at 6.7S, but eluted at Mr 330 000 (2R2C2) by gelfiltration.The Kmappfor peak I and III enzymes were, respectively: histone IIA 0.5 mg/ml (both forms), ATP 18 μm and 23 μm, and cAMP 5 × 10−8m and 6.3 × 10−8m. Both enzymes had pH optimum 6.7–6.9 and were equally sensitive to Ca2+ temperature and protein kinase inhibitor. The substrate specificity was histone VS ≫ histone IIA = histone VIS ≫ casein > phosvitin. Peak I enzyme, but not peak III enzyme, was dissociated by histone and high ionic strength and reassociation of R and C subunits were facilitated by ATP-Mg. It is concluded that peak I and III enzymes represent type I and II cAMP dependent protein kinases, respectively. Type I comprises 20–30% of cAMP dependent protein kinase activity and is absent from the 180 000 × g supernatant of gently disrupted cells.Purified catalytic subunit had Kmapp(ATP) 20 μm with rabbit muscle glycogen synthase I as substrates. Synthase I from rabbit muscle and human leukocytes were phosphorylated by catalytic subunit to synthase D (ratio of independence less than 0.07).cAMP independent histone kinase activity eluted in one peak (Peak II) at 3 mmho. The enzymatic activity sedimented at 3.4S and eluted from gelfiltration with Mr 78 000. Kmappfor ATP was 78 μm and for histone IIA 0.5 mg/ml. The enzyme was sensitive to temperature, but less sensitive than cAMP dependent protein kinase to Ca2+, and insensitive to protein kinase inhibitor. The substrate specificity was histone IIA > histone VS = histone VIS, while casein and phosvitin were poor substrates. Glycogen synthase I was not phosphorylated. The cAMP independent histone kinase activity comprised 15% of the total histone kinase activity in a crude homogenate of leukocytes. Its physiological substrate is unknown.

Membrane-associated protein kinases in phorbol ester-activated human polymorphonuclear leukocytes

Membrane-associated protein kinases in human polymorphonuclear leukocytes were studied. In unstimulated polymorphonuclear leukocytes the protein kinase C was predominantly present in the cytosol but in phorbol 12-myristate 13-acetate- (PMA-) activated cells a time and dose-dependent translocation of the kinase to the particulate fraction occurred. Two new protein kinase activities also appeared in the particulate fraction upon PMA activation. The one had a Mr of 40,000 and its activity was independent of phospholipids. The other (Mr 90,000) as partially activated by phospholipids, but separated from protein kinase C on DEAE-cellulose chromatography.

Membrane-associated protein kinase C activity in superoxide-producing human polymorphonuclear leukocytes.

Protein kinase C activity was studied in superoxide‐producing human polymorphonuclear leukocytes. Using equivalent cell concentrations, superoxide production and particulate fraction‐associated protein kinase C activity increased in parallel in phorbol 12‐myristate 13‐acetate (PMA), oleoyl‐acetyl‐glycerol (OAG), opsonized zymosan, and A23187‐activated leukocytes. Also, an increase in particulate fraction‐associated phospholipid‐independent kinase activity was observed upon stimulation with these activators. In contrast, in formyl‐methionyl‐leucine‐phenylalanine (FMLP)‐activated cells the increase in superoxide production was only accompanied by an increase in particulate fraction‐associated protein kinase C activity if the cells were pretreated with cytochalasin B. Purified protein kinase C activity was stimulated by OAG and PMA, whereas no stimulation was observed using A23187 or opsonized zymosan. It is suggested that the activation induced in human neutrophils by PMA, OAG, opsonized zymosan, and A23187 involves a thight membrane association of phospholipid‐dependent and ‐independent protein kinase activity. This contrasts to FMLP‐activated neutrophils, in which a membrane‐bound form is only observed after pretreatment with cytochalasin B.

Phosphorylation of glycogen synthase I from human polymorphonuclear leukocytes

SummaryGlycogen synthase I from human polymorphonuclear leukocytes was phosphorylated with cAMP dependent protein kinase, synthase kinase or phosvitin kinase prepared from these cells. Limited tryptic hydrolysis released four phosphopeptides (t-A, t-B, t-C, t-D). Subsequent α-chymotryptic hydrolysis of the trypsin resistant core released three phosphopeptides. (c-A, c-B, c-C). The kinetic changes of glycogen synthase were compared with the phosphorylation of the peptides. Equivalent kinetic changes (Kc=0.2–0.3 mM Glc-6-P) were obtained when 1 Pi/subunit was introduced by cAMP dependent protein kinase, 0.5 Pi/subunit by synthase kinase and 0.8 Pi/subunit by both kinases. Initially, cAMP dependent protein kinase phosphorylated peptides c-A and t-C in parallel and somewhat later also t-B, whereas synthase kinase initially phosphorylated only c-A. The ultimate effect of the two kinases on c-A was additive. It was concluded that the initial kinetic changes were dependent on phosphorylation of c-A, which contained two sites, one for each kinase. The same kinetic changes were induced by phosphorylation on each of the sites.In the subsequent phosphorylation the kinases, separately or together, phosphorylated peptide c-C indicating one non-specific phosphorylatable site in this peptide. The cAMP dependent protein kinase alone phosphorylated t-C maximally, whereas both kinases were required for an equal phosphorylation of t-A and t-B. It is suggested that the cAMP dependent protein kinase phosphorylated t-A and t-C, whereas the data did not allow a similar suggestion for t-B. The kinetic changes occurring during the later stages of phosphorylation were an increase in Kc for Glc. 6-P to 4–5 mM at 1.85 Pi/subunit and to 20 mM at 3.3 Pi/subunit, but the changes could not be assigned to phosphorylation of any specific peptide. Phosphorylation of the peptides t-D and c-B were insignificant, but c-B may be phosphorylated under other experimental conditions (25).The phosvitin kinase phosphorylated glycogen synthase extremely slowly to an extent of 0.8 Pi/subunit, mainly in peptide c-C. Glycogen synthase would appear without physiological importance as substrate for this kinase. Phosphorylase kinase from rabbit skeletal muscle incorporated 0.7 Pi/subunit, mainly in peptide c-A causing a decrease in RI to 0.3, which upon further incubation remained constant.The rate of decrease in RI to 0.5 was unaffected by several synthase modifiers, including Glc-6-P, but was inhibited by ADP and Pi. The rate of phosphorylation by cAMP dependent protein kinase and synthase kinase was diversely affected in different buffers, however, without affecting the ultimate phosphorylation pattern.

Effect of glycogenelytic agents on glycogen synthase activity in polymorphonuclear leukocytes: Evidence for a Ca2+-mediated regulation of glycogen synthase activity

Abstract Human polymorphonuclear leukocytes were found to respond to the β-receptor activators, adrenalin and isoproterenol, with a rapid transient increase in cyclic AMP, activation of cyclic AMP-dependent protein kinase, phosphosrylase kinase, deactivation of glycogen synthase and glycogen breakdown. This response was unaffected by the presence of 10 mM EGTA. Incubation of leukocytes with phorbol myristate acetate, which stimulates the hexose monophosphate shunt by a Ca2+ mediated mechanism, resulted in activation of phophorylase without affecting cyclic AMP-dependent protein kinase or phosphorylase kinase activity, thus indicating a Ca2+-mediated activation of phosphorylase. This was, however, unaffected by EGTA. Prolonged incubation with phorbol myristate acetate was found to result in a parallel activation of phosphorylase and glycogen synthase secondary to a pronouned depletion of cellular glycogen. Addition of glucose to polymorphonuclear leukocytes resulted in total conversion of phosphorylase a to the b form and activation of glycogen synthase, however, when EGTA was included, the response to glucose was greatly amplified, thus indicating the synthase conversion is regulated by Ca2+ sensitive mechanisms which do not involve phosphorylse kinase. Addition of adrenalin to cells previously activated by glucose resulted in an increase in the concentration of cyclic AMP and activation of cyclic AMP-dependent protein kinase but deactivation of synthase was not effectuated under these conditions.

Phosphorylation of rabbit skeletal muscle glycogen synthase 1 by the cAMP dependent protein kinase, the cAMP independent synthase kinase and the phosvitin kinase from human polymorphonuclear leukocytes

SummarycAMP dependent protein kinase and cAMP independent synthase kinase incorporated up to two Pi/subunit in rabbit skeletal muscle glycogen synthase I. The first Pi/subunit was incorporated much faster than the second. After incorporation of one Pi/subunit by the CAMP dependent protein kinase, the ratio of independence (RI) was 0.20 and the dissociation constant Kc for Glc-6-P was 0.3 mm, and quite different from the RI of 0.02 and Kc (Glc-6-P) of 1 mM, obtained when one Pi/subunit was incorporated by the cAMP independent synthase kinase. Within the first Pi/subunit, the cAMP dependent protein kinase predominantly phosphorylated in the trypsin sensitive region (60–70%), corresponding to two trichloro-acetic acid soluble tryptic phosphopeptides, termed site-1 and site-2. Site-2 was found to be phosphorylated prior to site-1. CNBr degradation resolved the phosphorylated regions in two phosphopeptides with Mr 28,000 and 10,000.The larger CNBr phosphopeptides were derived from the trypsin sensitive region. Within the first Pi/subunit, synthase kinase almost exclusively phosphorylated in the trypsin insensitive region (80%) corresponding to the smaller CNBr phosphopeptide. However, when two Pi/subunit were incorporated by either the cAMP dependent protein kinase or the synthase kinase the phosphates were almost equally distributed between the trypsin sensitive and insensitive regions and Kc (Glc-6-P) increased to 2 mm, Maximum phosphorylation (2.8–3.3 Pi/subunit and Kc (Glc-6-P) 9–11 mm) was only obtainable when both the cAMP dependent protein kinase and the synthase kinase were present.The phosvitin kinase very slowly incorporated one Pi/subunit.We suggest that within the first P1subunit phosphorylation in the trypsin insensitive region determine the affinity for the allosteric activator, glucose-6-phosphate. Thereafter phosphorylation in the trypsin sensitive region is the major determinant. Purified glycogen-free rabbit skeletal muscle glycogen synthase binds glycogen with lower affinity than polymorphonuclear leukocyte glycogen synthase. Glycogen was found to increase the initial rate of phosphorylation and facilitate the phosphorylation of site-1.