Chlorzoxazone esters of some non-steroidal anti-inflammatory (NSAI) carboxylic acids as mutual prodrugs: Design,synthesis, pharmacological investigations and docking studies

The discovery of the inducible isoform of cyclooxygenase enzyme (COX-2) spurred the search for anti-inflammatory agents devoid of the undesirable effects associated with classical NSAIDs. New chlorzoxazone ester prodrugs (6–8) of some acidic NSAIDs (1–3) were designed, synthesized and evaluated as mutual prodrugs with the aim of improving the therapeutic potency and retard the adverse effects of gastrointestinal origin. The structure of the synthesized mutual ester prodrugs (6–8) were confirmed by IR, ¹H NMR, mass spectroscopy (MS) and their purity was ascertained by TLC and elemental analyses. In vitro chemical stability revealed that the synthesized ester prodrugs (6–8) are chemically stable in hydrochloric acid buffer pH 1.2 as a non-enzymatic simulated gastric fluid (SGF) and in phosphate buffer pH 7.4 as non-enzymatic simulated intestinal fluid (SIF). In 80% human plasma, the mutual prodrugs were found to be susceptible to enzymatic hydrolysis at relatively faster rate (t_1/2 ≈ 37 and 34 min for prodrugs 6 and 7, respectively). Mutual ester prodrugs (6–8) were evaluated for their anti-inflammatory and muscle relaxation activities. Scanning electromicrographs of the stomach showed that the ester prodrugs induced very little irritancy in the gastric mucosa of rats after oral administration for 4 days. In addition, docking of the mutual ester prodrugs (6–8) into COX-2 active site was conducted in order to predict the affinity and orientation of these prodrugs at the enzyme active site.     

GC-MS SPME profiling of rhizobacterial volatiles reveals prospective inducers of growth promotion and induced systemic resistance in plants

Chemical and plant growth studies of Bacilli strains GB03 and IN937a revealed that the volatile components 2,3-butanediol and acetoin trigger plant growth promotion in Arabidopsis. Differences in growth promotion when cytokinin-signaling mutants are exposed to GB03 versus IN937a volatiles suggest a divergence in chemical signaling for these two bacterial strains. To provide a comprehensive chemical profile of bacterial volatiles emitted from these biologically active strains, headspace solid phase microextraction (SPME) coupled with software extraction of overlapping GC-separated components was employed. Ten volatile metabolites already reported from GB03 and IN937a were identified as well as 28 compounds not previously characterized. Most of the newly identified compounds were branched-chain alcohols released from IN937a, at much higher levels than in GB03. Principal component analysis clearly separated GB03 from IN937a, with GB03 producing higher amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and butane-1-methoxy-3-methyl. The branched-chain alcohols share a similar functional motif to that of 2,3-butanediol and may afford alternative structural patterns for elicitors from bacterial sources.     

Subcellular distribution of protein kinase C in rat adrenal glomerulosa cells

With the aid of a synthetic nonapeptide which is a selective substrate for protein kinase C the activity of this enzyme was determined in the crude cytosolic and particulate fractions of rat adrenal glomerulosa cells. When the cells were sonicated in the presence of Ca2+ chelators 65 per cent of their total protein kinase C activity was found in the cytosolic extract. The treatment of cells with angiotensin II under conditions where the maximal stimulation of inositol-lipid hydrolysis was observed did not cause a statistically significant change in the apparent subcellular distribution of protein kinase C. However, when the cytosolic extract was prepared in the presence of Ca2+ the protein kinase C activity was recovered nearly exclusively from the particulate fraction.     

cAMP dependent inhibition of thromboxane A2, prostacyclin and PGF2 alpha synthesis in mouse hepatocytes

The role of cAMP dependent regulation in thromboxane A2, prostacyclin and PGF2 alpha synthesis (measured by radioimmunoassay) was investigated in isolated mouse hepatocytes and in microsomal membranes prepared from these cells. In isolated hepatocytes N6,O2-dibutyryl cAMP inhibited the formation of all the three derivatives, while calcium ionophore A 23187 stimulated their synthesis. Addition of the dissociated catalytic subunit of cAMP dependent protein kinase and ATP to microsomal membranes inhibited the production of TXA2, PGI2 and PGF2 alpha by about 50% and this inhibition was counteracted by the combined addition of heat stable inhibitor protein of cAMP dependent protein kinase. It is concluded that in parenchymal liver cells cAMP dependent phosphorylation is directly involved in the inhibition of prostanoid synthesis.     

Ethylene as a Mediator of Rubigan Biological Action in Cowpea Plants

Rubigan (a systemic fungicide) inhibited the growth of Rhizoctonia solani in vitro when used at concentrations of 6, 12 and 24 μg/ml respectively. Application of Rubigan as a soil drench significantly reduced the damping-off incidence on cowpea (Vigna sinensis) plants. Symptoms of growth retardation appearing on cowpea plants treated with Rubigan could be mimiced using an ethylene releasing compound (Ethrel). Ethrel at 10 and 20 μl/ml added to the soil around the seeds induced severe dwarfing as evidenced from measurements of plant length, dry weight and leaf area. Gas chromatographic analysis revealed that ethylene was released either from Rubigan mixed or not with the soil. Rates of ethylene production from soils drenched with Rubigan were relatively inferiorto those produced by authentic Rubigan solutions not added to the soil. Moreover, in all cases ethylene was released at rates proportional to the Rubigan concentrations applied. Factors involved in growth retardation of Rubigan-treated cowpea plants were discussed in the light of the possible interaction between ethylene and endogenous gibberellin levels.     

Cyclic AMP-dependent protein kinase stimulates the formation of polyphosphoinositides in lymphocyte plasma membrane

Inside-out vesicles from lymphocyte plasma membrane were phosphorylated in the presence of [gamma -32P]ATP. The dissociated catalytic subunit of cyclic AMP-dependent protein kinase stimulated both membrane protein and membrane lipid phosphorylation, indicating the presence of a phosphorylation cascade. The phosphorylated membrane lipids were analyzed by thin-layer chromatography. Increase of 32P-labelling stimulated by the cyclic AMP-dependent protein kinase was found exclusively in polyphosphoinositides.     

Statistical procedure for evaluating the sensitivity of Limulus amoebocyte lysate by using a reference lysate.

A study was designed to estimate variability of the Limulus amoebocyte lysate test by comparing a reference lysate against itself. Three technicians performed parallel tests, i.e., titrated side by side, the contents of two vials of reference lysate on 4 different days using 24 vials of the United States reference lysate and 12 vials of the United States reference endotoxin. Each parallel test was replicated three times. From the sensitivity endpoints, ratios were calculated for each parallel test. These ratios were converted to the logarithm for estimating variability among technicians and among vials of endotoxin. By using the overall variability of log ratios, a statistical procedure was developed to evaluate the sensitivity of each lot of licensed lysate submitted to the Bureau of Biologics for release.     

Temperature and salinity regulation of growth and gas exchange of Salicornia fruticosa (L.) L.

Summary Salicornia fruticosa was collected from a salt marsh on the Mediterranean sea coast in Libya. Growth and gas exchange of this C₃ species were monitered in plants pretreated at various NaCl concentrations (0, 171, 342, 513 and 855 mM). Maximum growth was at 171 mM NaCl under cool growth conditions (20/10° C) and at 342 mM NaCl under warm growth conditions (30/15° C) with minimum growth at 0 mM NaCl (control). Net photosynthesis (Pn) was greatest in plants grown in 171 mM NaCl with plants grown at 513 and 855 mM having lowest rates. Maximum Pn was at 20–25° C shoot temperatures with statistically significant reductions at 30° C in control plants while salt treated plants showed such reductions at 35° C. Salt treatments increased dark respiration over the control at 171 and 342 mM but reduced it at higher concentrations. Photorespiration was reduced by salt treatment and increased by increasing shoot temperature. Greatest transpiration was in 171 mM NaCl treated plants and increasing shoot temperature increased transpiration in all treatments. Stomatal resistance to CO₂ influx was influenced only moderately by temperature while increasing salinity resulted in increased stomatal resistance. In general both temperature and salinity increased the mesophyll resistance to CO₂ influx. The species seems adapted to the warm saline habitat along the Mediterranean sea coast, at least partially, by its ability to maintain relatively high Pn at moderate NaCl concentrations over a broad range of shoot temperatures.     

Low molecular weight cyclic AMP binding protein isolated from the extract of human tonsillar lymphocytes

A protein fraction of molecular weight 33,000-36,000 accounted for about 40% of the cyclic AMP binding capacity of the cytoplasmic extract of human tonsillar lymphocytes. This cyclic AMP binding fraction (designated as R' protein [10]) proved to be a proteolytic fragment of the regulatory subunit of the cyclic AMP-dependent protein kinase. The Scatchard plot of cyclic AMP binding by the isolated R' fraction indicated positive cooperativity. 50% saturation of the cyclic AMP binding sites was achieved at about 4 . 10(-9) M cyclic AMP. An upward concave curve was obtained in the Scatchard plot of cyclic GMP binding by the R' protein. These results strongly suggest that more than one molecule of cyclic nucleotide can be bound by one molecule of the R' protein. The R' protein could not be detected in the physiological salt extract of isolated nuclei in which type I cyclic AMP-dependent protein kinase was the dominating isoenzyme (according to the terminology used by Corbin, S.D., Keely, S.L. and Park, C.R. (1975) J. Biol. Chem. 250, 218-225). The cytoplasm of cells contained a higher amount of type II than type I regulatory subunit. In the cytoplasm the predominant part of RII was present in the dissociated state in all preparations, while when the RII was found in the nucleus it was mainly in the holoenzyme form. The R' protein presumably from the dissociated type II regulatory subunit.     

Differential vascular actions of ethanol in feline middle cerebral and mesenteric artery.

The vascular actions of ethanol on feline middle cerebral and mesenteric arteries were investigated in vitro. Ethanol (20-500 mM) caused potent contraction in cerebral arteries, but it contracted the mesenteric arteries only weakly. In the middle cerebral artery (but not in the mesenteric artery) ethanol (300 mM) potentiated the noradrenaline (5.10(-6) M) induced contractions. Antiserum for endothelin (in an appropriate concentration to inhibit endothelin-induced contraction; 0.02 mg/ml) did not inhibit the ethanol-induced contractions. Endothelium-dependent relaxations induced by acetylcholine and ATP were also affected by ethanol (300 mM); in the cerebral artery acetylcholine- but not ATP-induced relaxations, whereas in the mesenteric artery ATP- but not acetylcholine-induced relaxations were inhibited significantly. The results suggest that ethanol causes strong (endothelin-independent) contraction and facilitates the response to noradrenaline in the middle cerebral, but not in the mesenteric artery; and it selectively inhibits endothelium-dependent relaxation. These actions of ethanol may contribute to the development of vascular diseases.