Selective impairment of protein kinase C isotypes in murine macrophage by Leismania donovani

Leishmania donovani, an obligate intracellular parasite resides and multiplies within macrophage of the reticuloendothelial system. The intracellular signalling mechanism involved in the impaired oxidative response in leishmaniasis has not yet been clearly established. Generation of superoxide anion (O₂ ^–) is supposed to be the first line of host defence during microbial invasion. We found a substantial inhibition of superoxide anion generation in parasitized macrophages, which was just the reverse in case of macrophages challenged with Lipophosphoglycan (LPG) deficient attenuated leishmanial parasite UR-6. The generation of O₂ ^– essentially needs the prior activation of protein kinase C (PKC) mediated phosphorylation events. Our study proposed that phosphorylation of 67, 54, 47 and 36 kDa proteins was attenuated during infection. This was supported by PKC activity study, where Ca-dependent PKC activity was inhibited but, Ca-independent PKC activity was enhanced. This result was further confirmed by using isotype specific pseudosubstrate inhibitors of Ca-dependent PKC and Ca-independent PKC . Application of -pseudosubstrate could not alter the Ca-dependent PKC activity but -pseudosubstrate inhibited the Ca-independent PKC activity in infected macrophages. Our immunoblot analysis with specific antibody against PKC and PKC isotypes showed down regulation of PKC -II expression with concomitant induction of PKC . Such inhibition of Ca-dependent PKC was reversed in macrophages treated with UR-6. Taken together, our observations revealed that infection with L. donovani selectively attenuates both the expression and activity of Ca-dependent PKC .     

Expression of fungal desaturase genes in cultured mammalian cells

Long-chain polyunsaturated fatty acids (LC-PUFA) are important components of cellular structure and function. Most of LC-PUFA are derived from linoleic acid and a-linolenic acid. In plants and fungi, these two acids can be synthesized from oleic acid via the action of two enzymes, 12 and 15-desaturases. Due to lack of these enzymatic activities and the ability to synthesize these two essential fatty acids, animals must obtain them from the diet. In this report, we demonstrated the expression of a fungal 12-desaturase gene in mouse L cells incubated in serum-free medium. The results showed a significant increase in the amount of linoleic acid with a concomitant decrease of oleic acid in cellular lipids. Most of the newly formed linoleic acid was incorporated into cellular phospholipids, particularly phosphatidylcholine. The increase of linoleic acid provided the substrate for the endogenous synthesis of (n-6) LC-PUFA, such as eicosadienoic acid (EDA), dihomo--linoleic acid (DGLA) and arachidonic acid (AA). Prolonged incubation further increased the levels of linoleic acid derived from oleic acid by the action of 12-desaturase, and the levels of 20:2n-6 produced from linoleic acid by the action of the endogenous elongase. However, prolonged incubation suppressed significantly the formation of DGLA and AA. In a separate study, a fungal 6-desaturase gene has also been expressed in the mouse L cells incubated in serum-containing medium. The result shows a significant increase in levels of 20:3n-6 and 20:4n-6. These findings demonstrate that through genetic modification, it is possible to (1) generate cell lines which no longer require dietary 'essential' fatty acids and (2) alter the endogenous fatty acid metabolism to enhance the production of LC-PUFA and their derivatives.     

Discovery of novel,orally available benzimidazoles as melanin concentrating hormone receptor 1 (MCHR1) antagonists

Melanin concentrating hormone (MCH) is an important mediator of energy homeostasis and plays role in several disorders such as obesity, stress, depression and anxiety. The synthesis and biological evaluation of novel benzimidazole derivatives as MCHR1 antagonists are described. The in vivo proof of principle for weight loss with a lead compound from this series is exemplified.     

Signaling pathways initiated by beta-hydroxy-beta-methylbutyrate to attenuate the depression of protein synthesis in skeletal muscle in response to cachectic stimuli

To investigate the mechanism by which beta-hydroxy-beta-methylbutyrate (HMB) attenuates the depression of protein synthesis in the skeletal muscle of cachectic mice, a study has been carried out in murine myotubes in the presence of proteolysis-inducing factor (PIF). PIF inhibited protein synthesis by 50% within 4 h, and this was effectively attenuated by HMB (25-50 muM). HMB (50 muM) alone stimulated protein synthesis, and this was attenuated by rapamycin (27 nM), an inhibitor of mammalian target of rapamycin (mTOR). Further evidence for an involvement of this pathway was shown by an increased phosphorylation of mTOR, the 70-kDa ribosomal S6 kinase (p70(S6k)), and initiation factor 4E-binding protein (4E-BP1) and an increased association of eukaryotic initiation factor 2 (eIF4E) with eIF4G. PIF alone induced a transient (1-2 h) stimulation of phosphorylation of mTOR and p70(S6k). However, in the presence of HMB, phosphorylation of mTOR, p70(S6k), and 4E-BP1 was increased, and inactive 4E-BP1-eIF4E complex was reduced, whereas the active eIF4G.eIF4E complex was increased, suggesting continual stimulation of protein synthesis. HMB alone reduced phosphorylation of elongation factor 2, but this effect was not seen in the presence of PIF. PIF induced autophosphorylation of the double-strand RNA-dependent protein kinase (PKR), leading to phosphorylation of eIF2 on the alpha-subunit, which would inhibit protein synthesis. However, in the presence of HMB, phosphorylation of PKR and eIF2alpha was attenuated, and this was also observed in skeletal muscle of cachectic mice administered HMB (0.25 g/kg). These results suggest that HMB attenuates the depression of protein synthesis by PIF in myotubes through multiple mechanisms.     

Cytoprotective Agent in <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis> Extracts

Adenosine 5′-diphosphoribose (ADP-ribose) has been identified as a significant contributor to the anti-cytotoxic activity of Lactobacillus bulgaricus extracts. Although the biological activities associated with the administration of probiotic bacteria and components thereof are sometimes attributed to the peptidoglycans that comprise a substantial portion of the Gram-positive bacterial cell wall, we found that the beta-nicotine adenine dinucleotide (NAD) hydrolysis product ADP-ribose was a significant contributor to the observed anti-cytotoxicity in our L. bulgaricus extracts. The ADP-ribose was isolated, identified, and quantitated by high performance liquid chromatography (HPLC) and by nuclear magnetic resonance (NMR) spectroscopy. ADP-ribose levels as low as 5 mg/L exhibited a measurable inhibition of tumor necrosis factor alpha (TNF-α) mediated cytotoxicity in an in vitro cell assay, whereas the ADP-ribose content of the L. bulgaricus extracts often exceeded 5 mg/g dry weight.     

Urokinase separation from cell culture broth of a human kidney cell line

A single step ion-exchange chromatography on a sulfo-propyl (SP)- Sepharose column was performed to separate both the high molecular weight (HMW)- and low molecular weight (LMW)- forms of enzymatically active urokinase type plasminogen activator from human kidney (HT1080) cell culture media. The level of urokinase secreted by the cell line reached to about 145 Plough units/ml culture broth within 48 h of cultivation. The conditioned cell culture media was applied directly to the column without any prior concentration steps. Polyacrylamide gel electrophoresis of the column eluates in the presence of sodium dodecyl sulphate showed that the cell line secretes three forms of two-chain high molecular weight (HMW) urokinase of molecular weights (M(r)) 64,000, 60,900 and 55,000. In addition, two low molecular weight (LMW) forms of M(r) 22,000 and 20,000; proteolytic cleavage products of HMW, were also found. The HMW and LMW forms had intrinsic plasminogen dependent proteolytic activity as judged by zymographic analysis. The specific activity of the pooled peak fractions increased (approximately 93-fold) to values as high as 1481 Plough units/ mg protein. Both HMW as well as LMW forms were obtained in significantly high yields.