Modulation of cellular processes by H7, a non-selective inhibitor of protein kinases.

H7 has been described as a potent inhibitor of protein kinase C (PKC) and has been widely used to investigate the regulatory role of this enzyme in intact cell systems. In this comparative study between H7 and the microbial alkaloid, staurosporine, we found that the former inhibited rat brain PKC and cAMP dependent protein kinase with IC50 values of 18 and 16 microM respectively whereas the latter was a much more potent inhibitor of both kinases with IC50 values of 9.5 nM and 42 nM respectively. H7, at concentrations up to 100 microM, failed to block cellular events induced by phorbol esters, agents which specifically stimulate PKC, yet was a potent inhibitor of IL-2 induced T cell proliferation with an IC50 value of 19 microM. In contrast, staurosporine was a potent inhibitor of both phorbol ester induced p47 phosphorylation in platelet (I50 value = 540 nM) and also CD3 and CD4 down-regulation in T cells (I50 values 200 nM and 50 nM respectively). Staurosporine was also a potent inhibitor of IL-2 induced T cell proliferation I50 value = 9 nM). These results provide a strong argument against the use of H7 to probe for PKC involvement in cellular processes.

Biologic properties of romazarit (Ro 31-3948), a potential disease-modifying antirheumatic drug.

The biologic effects of a new potential disease-modifying antirheumatic drug, romazarit (Ro 31-3948, 2-[[2-(4-chlorophenyl)-4-methyl-5-oxazolyl]methoxy]-2-methylpropio nic acid), have been investigated. In a 5-day adjuvant arthritis model, romazarit inhibited the development of hindpaw inflammation with a minimum effective dose of 30 mg kg-1. Plasma levels of the acute phase reactants seromucoid and haptoglobulin were also significantly reduced. Romazarit was equally effective in adrenalectomized animals, indicating that the compound is not acting via stimulation of the pituitary/adrenal axis. When the developing adjuvant arthritis was extended to 15 days romazarit showed dose-related improvements of all the symptoms of arthritis with a minimum effective dose of 25 mg kg-1. Romazarit caused a dose-dependent (range 20-250 mg kg-1) reduction in both the inflammatory and bony changes occurring during collagen arthritis in the rat, without any significant effect on anticollagen antibody titers except at the highest dose. Collagenase and prostaglandin E2 production in cultures of talus bones taken from rats with collagen arthritis were reduced by romazarit. In vitro romazarit was an extremely weak inhibitor of prostaglandin synthetase activity in both sheep seminal vesicle (IC50 6500 microM) and rat renal medulla (IC50 greater than 300 microM) cell-free preparations. Romazarit showed little or no activity in models of acute inflammation such as rabbit skin edema, carrageenan pleurisy or UV-induced erythema. In both acute and chronic tests romazarit displayed no ulcerogenic potential. In comparison with the structurally similar compound clobuzarit, hepatic changes such as increases in catalase and peroxisome proliferation-associated 80,000 mol.wt. protein were markedly less with romazarit. Clinical studies with romazarit are currently in progress.

Potent selective inhibitors of protein kinase C.

A series of potent, selective inhibitors of protein kinase C has been derived from the structural lead provided by the microbial broth products, staurosporine and K252a. Our inhibitors block PCK in intact cells (platelets and T cells), and prevent the proliferation of mononuclear cells in response to interleukin 2 (IL2).

Effect of recombinant cytokines on glycolysis and fructose 2,6-bisphosphate in rheumatoid synovial cells in vitro.

Recombinant-derived human interleukin 1 (IL1) alpha and beta and interferon gamma (IFN-gamma) each produced similar increases in rheumatoid synovial cell (RSC) glycolysis, as judged by increased values for glucose uptake, lactate production and cellular fructose 2,6-bisphosphate [Fru(2,6)P2]. Measurement of Fru(2,6)P2 proved to be the most sensitive parameter for an assessment of glycolysis: IL1 alpha, IL1 beta and IFN-gamma all produced a 3-6-fold increase in this metabolite whereas tumour necrosis factor (TNF alpha) was far less effective. Prostaglandin E production was stimulated predominantly by IL1 alpha and IL1 beta rather than by IFN-gamma or TNF alpha. When combinations of cytokines were examined the addition of IFN-gamma with either IL1 alpha, IL1 beta or murine IL1 produced a synergistic increase in cellular Fru(2,6)P2. The three forms of IL1 increased Fru(2,6)P2 via the same pathway, whereas IFN-gamma acted via a different mechanism. The increase in Fru(2,6)P2 in subcultured RSC produced by addition of medium from a primary culture exceeded the maximal effects of any of the single cytokines studied, suggesting the presence of a mixture of cytokines in the primary RSC culture medium.

Anti-inflammatory effects of synthetic retinoids may be related to their immunomodulatory action.

The effects of retinoids have been studied in a model of delayed-type hypersensitivity using the T-cell-dependent antigen, methylated bovine serum albumin to elicit inflammation in the hind paws of mice. A number of synthetic retinoids, including etretinate and arotinoids, showed a marked anti-inflammatory action in this model. Using differential dosing schedules, the anti-inflammatory effect of retinoids was clearly distinguished from conventional cyclooxygenase inhibitors. By screening a number of synthetic retinoids, structure-activity relationships for this effect can be deduced.

9-alkyl anthracyclines. Absence of cross-resistance to adriamycin in human and murine cell cultures.

Four cell lines of human (CCRF CEM and U266BL) or murine (L1210 and P388D1) origin, resistant to the anthracycline antibiotic Adriamycin (doxorubicin) were selected in vitro from cultured cells by serial passage in the presence of Adriamycin. The resistant sublines were also cross-resistant to Mitoxantrone, 4'-epi Adriamycin and a number of novel anthracyclines including 4'-deoxy and 4'-methoxy analogues. However, a series of 9-alkyl substituted 4-demethoxyanthracyclines retained full activity against all the resistant sublines as did Aclacinomycin A. These results suggest that 9-alkyl substitution of 4-demethoxy-anthracyclines is an important determinant of activity against Adriamycin-resistant cell lines in vitro.

Synergistic antibacterial activity between L-norvalyl-L-1-aminoethylphosphonic acid and nocardicin A.

The phosphonopeptide L- norvalyl -L-1- aminoethylphosphonic acid [ Nva -Ala(P)] has been studied in combination with 12 beta-lactam antibiotics for activity against Pseudomonas aeruginosa. Nocardicin A was found to give the most potent synergistic combination with Nva -Ala(P). This interaction was widely observed in clinical isolates of P. aeruginosa in vitro and in a mouse septicemia model. Synergy was also observed in vitro and in vivo in several other species, including Proteus mirabilis, indole-positive Proteus spp., and Serratia marcescens. The interaction between Nva -Ala(P) and nocardicin A involved a strongly bacteriolytic mechanism. In addition, the individual components were complementary to one another in their action against organisms not showing synergy. These properties resulted in a broad spectrum of activity of the combination Nva -Ala(P) plus nocardicin A when used to treat experimental gram-negative bacterial infections.

Phosphonopeptides as substrates for peptide transport systems and peptidases of Escherichia coli.

Peptide transport and peptidase susceptibility of the antibacterial agent alafosfalin and other phosphonopeptides have been characterized in Escherichia coli. Phosphonodipeptides were accumulated by a process which appeared to involve multiple permeases; saturation was not achieved even at concentrations of 128 microM. Competition studies showed that these compounds had only a low affinity for the system transporting phosphonooligopeptides and were rapidly taken up by and were inhibitory to E. coli mutants unable to transport the toxic peptide triornithine. Phosphonodipeptides containing D-residues were not appreciably transported. By contrast, phosphonooligopeptides were generally transported by a distinct saturable permease system for which they had a high affinity. This system was identical to that utilized by triornithine. Phosphonooligopeptides with simple monoalkyl substituents at the amino terminus were also transported except in the case of a t-butyl substituent. The oligopeptide permease was also able to transport certain derivatives which contained some residues having D rather than L stereochemistry. Intracellular metabolism of phosphonooligopeptides was initiated almost exclusively by hydrolysis from the N terminus by an L-specific peptidase. This initial hydrolytic activity was unaffected by the aminopeptidase inhibitor bestatin, unlike the final hydrolysis step which yields L-1-aminoethylphosphonic acid from the phosphonodipeptide intermediate.

The fractional inhibitory concentration (FIC) index as a measure of synergy.

The value of the FIC index as a predictor of synergy has been investigated using the antibacterial agents alafosfalin and cephalexin combined together with themselves in fully blind experiments. Under the conditions used, even weak interaction (FIC index 0.5-0.99) proved to be statistically highly significant. The use of such fully controlled blind studies would greatly enhance the credibility of many of the claims of synergy published in the literature. The representation of results as average isobolograms is only of value with combinations which show moderate to strong interaction.

Antibacterial activity and mechanism of action of phosphonopeptides based on aminomethylphosphonic acid.

Phosphonopeptides based on aminomethylphosphonic acid as the C-terminal residue linked to L-amino acids possessed antibacterial activity in vitro and in vivo. Analogs in this series were generally less potent than corresponding compounds based on L-1-aminoethylphosphonic acid such as alafosfalin (L-alanyl-L-1-aminoethylphosphonic acid). Significant differences in antibacterial spectra were observed. The mechanism of action involved active transport of the peptide mimetics into the bacterial cells, followed by intracellular release of high concentrations of aminomethylphosphonic acid which inhibited bacterial cell wall biosynthesis. Aminomethylphosphonic acid behaved as a mimetic of both D- and L-alanine and inhibited D-Ala-D-Ala synthetase (EC 6.3.2.4.), alanine racemase (EC 5.1.1.1.), and UDP-N-acetylmuramyl-L-alanine synthetase (EC 6.3.2.8.). The minimal inhibitory concentration of L-norvalyl-aminomethylphosphonic acid was essentially unaffected by the presence of D-alanine, whereas the activity of the corresponding L-norvalyl derivative of L-1-aminoethylphosphonic acid was markedly decreased. Substantial differences in the inhibitory and lytic activity of the L-norvalyl derivatives of aminomethylphosphonic and L-1-aminoethylphosphonic acids were also observed when these agents were combined with other inhibitors of bacterial cell wall biosynthesis.

Antibacterial properties of alafosfalin combined with cephalexin.

The phosphonopeptide alafosfalin (L-alanyl-L-1-aminoethylphosphonic acid) exhibited synergy in vitro and in animal studies against a range of bacterial genera when combined with cephalexin. Alafosfalin also showed synergy with mecillinam and, to a much lesser extent, with ampicillin. Synergy with cephalexin was more pronounced when the bacteria were relatively insensitive to the beta-lactam component. The action of this combination involved both an inhibitory and a bacteriolytic mechanism which was abolished by concurrent treatment with the aminopeptidase inhibitor, bestatin. Regrowth of subpopulation resistant to either component was markedly reduced by the combination. The potential of alafosfalin combined with cephalexin for use in therapy is discussed.

Effects of lysozyme on Bacillus cereus 569: rupture of chains of bacteria and enhancement of sensitivity to autolysins.

Bacillus cereus 569 is known to be resistant to lysis by lysozyme because of the presence of deacetylated glucosamine residues in its peptidoglycan, and cultures continued to grow even in the presence of lysozyme at 200 microgram ml-1. However, lysozyme caused rupture of the chains of bacteria and promoted the rate of autolysis in a non-growing cell suspension, causing a doubling of the rate of release of radioactively labelled wall material. Heat-inactivated cells did not autolyse and were not lysed by lysozyme unless they were supplemented by unheated cells or cell-free autolysate. Enhancement of autolysin activity could also be effected by pre-treatment of heated cells with lysozyme. The action of lysozyme on isolated cell walls released some free reducing groups, indicating limited breakage of the polysaccharide chains of peptidoglycan, and it was concluded that lysozyme modified the peptidoglycan and made it more susceptible to autolysin(s). Lysozyme also enhanced the rate of septum separation and the probable significance of the results in relation to the control of cell separation is discussed.

The effect of nalidixic acid on the cell cycle of synchronous Rhodopseudomonas palustris cultures.

The influence of the DNA synthesis inhibitor, nalidixic acid, on the properties of synchronous cultures of selected Rhodopseudomonas palustris swarmer cells was examined. There was little alteration in the changes in morphology, extinction, volume distribution and leucine incorporation up to bud development, and photosynthetic membrane lamellae were still synthesized de novo in the bud. However, there was no subsequent division, or flagellum or holdfast synthesis. Instead cells elongated by continued outgrowth of the abortive bud. Since DNA synthesis was also inhibited, this suggested a dependence of cell division, and flagellum and holdfast synthesis, on the completion of chromosome replication. By addition or removal of nalidixic acid at various times in the cell cycle, periods were demonstrated when the organism was insensitive to the antibiotic indicating that there was a pre-synthetic and post-synthetic gap in the pattern of DNA synthesis in R. palustris swarmers.

Synchronous growth of Rhodopseudomonas palustris from the swarmer phase.

Rhodopseudomonas palustris was chosen as a model organism for studying bacterial differentiation. Synchronous populations selected by sucrose gradient centrifugation yielded more than 95% swarmer cells. The appearance and disappearance of cell morphological groupings and the doubling of cell numbers in cultures of such swarmer populations were very well defined. Cells were only motile for the first half of the division cycle, but motility was regained before division. Development gave rise to a distinct and characteristic pattern of extinction increase and particle volume distribution. The development of swarmers into mother cells and the dimorphic division of R. palustris are discussed as simple examples of differentiation.