Viggo Esmann

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.

Prednisolone does not prevent post-herpetic neuralgia.

In a randomised, double-blind, controlled study of the effect of prednisolone on the development of post-herpetic neuralgia 78 patients with herpes zoster whose pain and exanthema had been present for less than 96 h were given 800 mg acyclovir five times daily for 7 days and prednisolone in a total dose of 575 mg, starting with 40 mg daily in the first week and tapering off over the next 2 weeks. 18 (23%) of the patients had post-herpetic neuralgia at 6 months after the acute zoster, 9 (24.3%) having received prednisolone and 9 (22.5%) placebo. The 95% CI for the difference between the placebo and prednisolone groups in the proportion of patients having pain at 6 months was minus 17% to plus 20%. Prednisolone, however, relieved pain for the first 3 days. The 1-2 week interval between admission and reappearance of pain and development of triggered pain seems to be the time needed to establish neuralgia. Once established, the type and intensity of pain remained largely unaltered.

A new assay of phosphorylase based on the filter paper technique

A rapid method for the assay of phosphorylase has been developed, similar in technique to the method described by Thomas et al. (8) for glycogen synthetase. It utilizes the ability of phosphorylase to incorporate 14C-labeled glucose from 14C-glucose 1-phosphate into glycogen, which is then precipitated on filter paper squares by immersion in ice cold 66% ethanol. After removal of surplus 14C-glucose 1-phosphate, the papers are counted directly in a vial containing scintillation fluid. Several buffers commonly used in phosphorylase assays were compared and it was found that measuring in a 100 mM maleate buffer gave approximately 15% lower activity than assaying in any of the other seven buffer systems tested.

Quantitation of superoxide production in human polymorphonuclear leukocytes from normals and 3 types of chronic granulomatous disease.

Abstract Conditions for quantitating the superoxide producing capacity of polymorphonuclear leukocytes are given. It is shown that the production is partly dependent on glucose but independent on Mg 2+ , Ca 2+ and extracellular pH in the range 6.6–8.2. The capacity of normal cells to produce superoxide is estimated to 13.4±0.13 SEM (n=9) femtomoles per min per cell, whereas superoxide production is absent in all cases of chronic granulomatous disease, irrespective of the type. It is suggested that at least 3 enzymes cooperate intimately in the superoxide producing system of the cell. This supports the theory of an electron transport chain of significance for oxygen consumption.

A study on the role of protein kinase C and intracellular calcium in the activation of superoxide generation

Accumulating evidence indicates that protein kinase C plays an essential role in the activation of NADPH oxidase. In the present study, the correlation between superoxide generation, intracellular calcium, activation of purified protein kinase C and stabilized membrane-bound protein kinase C was studied. Phorbol 12-myristate 13-acetate (PMA) and 1-deacyl-2-acetyl-rac-glycerol (OAG) were found to induce equal activation of purified protein kinase C and translocation of protein kinase C to the membrane fraction, but differed significantly in their ability to induce superoxide generation. Intracellular calcium was varied using calcium ionophores and increasing the intracellular calcium concentration to more than 1 microM was found to induce increased superoxide generation in maximally OAG-stimulated cells; this contrasted to maximally PMA-stimulated leukocytes. Ionomycin and A23187 were both found to induce a translocation of protein kinase C to the membrane fraction. This translocation was highly dependent upon extracellular calcium. In contrast, PMA- and OAG-induced translocation of protein kinase C was not dependent upon extracellular calcium. In conclusion, our results indicate that although PMA, OAG and calcium ionophores seem to activate protein kinase C in human polymorphonuclear leukocytes these activators differ in their ability to induce superoxide generation.

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.

Glycogen metabolism: the integrated cellular response to a bi-directional metabolic stimulus.

Abstract The control exerted by phosphorylase a over the I-conversion of glycogen synthase (1) does not apply to glycogen synthesis, as judged from experiments with intact human leucocytes. 1. In incubated leucocytes conversion of glycogen synthase (GS) (E.C.2.4.1.11) to GS-I is preceeded by inactivation of glycogen phosphorylase a (GPh-a). 2. By the addition of latex particles a flash-activation of GPh-a can be elicited within 10–30 sec. in the intact cells. 3. GPh is inactivated when a glucose load is given. 4. When, in glycogen depleted cells, glucose is given immediately after latex the I-conversion of GS is completely abolished, and GS-I remains low for 20 min although GPh is rapidly inactivated after the glucose is given. 5. However, glycogen synthesis as compared with a control in which only glucose is given, and in which a large I-conversion occurs, is not the least depressed. 6. This apparent contradiction is completely resolved taking into account a newly discovered, intermediate form of GS (2) (GS-R). 7. An important physiological role can be proposed for GS-R.

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.

Folate deficiency in malnutrition, malabsorption, and during phenytoin treatment diagnosed by determination of serine synthesis in lymphocytes.

Abstract A new method for the detection of folate deficiency by measuring the rate of incorporation of 14C‐formate into serine in isolated lymphocytes has been applied to a group of 24 control subjects and 23 patients. 5 of the patients suffered from malnutrition, 14 had malabsorption and 4 were under treatment with phenytoin.