Augmentation of an antitumor CTL response In vivo by inhibition of suppressor macrophage nitric oxide

Evidence is provided that inhibition of macrophage NO production can augment in vivo CTL responses. Specifically, administration of NG-monomethyl-l -arginine (NGMMA) via osmotic pumps increases the tumor-specific CTL response against the P815 mastocytoma in the peritoneal cavity of preimmunized mice. Both the magnitude and duration of the CTL response were increased. That the augmented CTL response resulted from inhibition of the NO synthase pathway is supported by the finding that macrophage NO production from NGMMA-treated mice was reduced. Also, in vitro inhibition of NO production by peritoneal exudate cells from P815 tumor-challenged mice augmented the secondary CTL response observed. Cell proliferation was augmented by NGMMA in these cultures, suggesting that macrophage NO may suppress CTL by inhibiting clonal expansion. NO-mediated inhibition was observed in vivo in this experimental system, even though the CTL response is not suppressed, in that tumor rejection occurs. Therefore, the present results are consistent with the conclusion that macrophage NO-mediated inhibition of the CTL response is a side effect of activating macrophages rather than resulting from the action of a distinct subset of what have long been termed suppressor macrophages. Most important, the results indicate that NO-mediated suppressor macrophage activity can be an important CTL immunoregulatory element in vivo.     

Dietary phytochemicals as rumen modifiers: a review of the effects on microbial populations

In the recent years, the exploration of bioactive phytochemicals as natural feed additives has been of great interest among nutritionists and rumen microbiologists to modify the rumen fermentation favorably such as defaunation, inhibition of methanogenesis, improvement in protein metabolism, and increasing conjugated linoleic acid content in ruminant derived foods. Many phytochemicals such as saponins, essential oils, tannins and flavonoids from a wide range of plants have been identified, which have potential values for rumen manipulation and enhancing animal productivity as alternatives to chemical feed additives. However, their effectiveness in ruminant production has not been proved to be consistent and conclusive. This review discusses the effects of phytochemicals such as saponins, tannins and essential oils on the rumen microbial populations, i.e., bacteria, protozoa, fungi and archaea with highlighting molecular diversity of microbial community in the rumen. There are contrasting reports of the effects of these phytoadditives on the rumen fermentation and rumen microbes probably depending upon the interactions among the chemical structures and levels of phytochemicals used, nutrient composition of diets and microbial components in the rumen. The study of chemical structure–activity relationships is required to exploit the phytochemicals for obtaining target responses without adversely affecting beneficial microbial populations. A greater understanding of the modulatory effects of phytochemicals on the rumen microbial populations together with fermentation will allow a better management of the rumen ecosystem and a practical application of this feed additive technology in livestock production.     

Reversal of boswellic acid analog BA145 induced caspase dependent apoptosis by PI3K inhibitor LY294002 and MEK inhibitor PD98059

PI3K/Akt and ERK pathways are important for growth and proliferation of many types of cancers. Therefore, PI3K inhibitor LY294002 (LY) and MEK1/2 inhibitor PD98059 (PD) are used to sensitize many types of cancer cell lines to chemotherapeutic agents, where AKT and ERK pathways are over activated. However, in this study, we show for the first time that PD could protect the leukemia cells independent of ERK pathway inhibition, besides, we also report a detailed mechanism for antiapoptotic effect of LY in HL-60 cells against the cytotoxicity induced by a boswellic acid analog BA145. Apoptosis induced by BA145 is accompanied by downregulation of PI3K/Akt and ERK pathways in human myelogenous leukemia HL-60 cells, having activating N-Ras mutation. Both LY and PD protected the cells against mitochondrial stress caused by BA145, and reduced the release of cytochrome c and consequent activation of caspase-9. LY and PD also diminished the activation of caspase-8 without affecting the death receptors. Besides, LY and PD also reversed the caspase dependent DNA damage induced by BA145. Further studies revealed that LY and PD significantly reversed the inhibitory effect of BA145 on cell cycle regulatory proteins by upregulating hyperphosphorylated retinoblastoma, pRB (S795) and downregulating p21 and cyclin E. More importantly, all these events were reversed by caspase inhibition by Z-VAD-fmk, suggesting that both LY and PD act at the level of caspases to diminish the apoptosis induced by BA145. These results indicate that inhibitors of PI3K/Akt and ERK pathways can play dual role and act against chemotherapeutic agents.     

Developing procedures for the large-scale purification of human serum butyrylcholinesterase

Human serum butyrylcholinesterase (Hu BChE) is the most viable candidate for the prophylactic treatment of organophosphate poisoning. A dose of 200 mg/70 kg is predicted to protect humans against 2× LD₅₀ of soman. Therefore, the aim of this study was to develop procedures for the purification of gram quantities of this enzyme from outdated human plasma or Cohn Fraction IV-4. The purification of Hu BChE was accomplished by batch adsorption on procainamide-Sepharose-CL-4B affinity gel followed by ion-exchange chromatography on a DEAE-Sepharose column. For the purification of enzyme from Cohn Fraction IV-4, it was resuspended in 25 mM sodium phosphate buffer, pH 8.0, and fat was removed by decantation, prior to batch adsorption on procainamide-Sepharose gel. In both cases, the procainamide gel was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0, containing 0.05 M NaCl, and the enzyme was eluted with the same buffer containing 0.1 M procainamide. The enzyme was dialyzed and the pH was adjusted to 4.0 before loading on the DEAE column equilibrated in sodium acetate buffer, pH 4.0. The column was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0 containing 0.05 M NaCl before elution with a gradient of 0.05–0.2 M NaCl in the same buffer. The purity of the enzyme following these steps ranged from 20% to 40%. The purity of the enzyme increased to >90% by chromatography on an analytical procainamide affinity column. Results show that Cohn Fraction IV-4 is a much better source than plasma for the large-scale isolation of purified Hu BChE.     

Characterization of cross-linked immobilized lipase from thermophilic mould Thermomyces lanuginosa using glutaraldehyde

Cross-linked enzyme aggregates (CLEAs) have emerged as an interesting biocatalyst design for immobilization. Using this approach, a 1,3 regiospecific, alkaline and thermostable lipase from Thermomyces lanuginosa was immobilized. Efficient cross-linking was observed when ammonium sulphate was used as precipitant along with a two fold increase in activity in presence of SDS. The TEM and SEM microphotographs of the CLEAs formed reveal that the enzyme aggregates are larger in size as compared to the free lipase due to the cross-linking of enzyme aggregates with glutaraldehyde. The stability and reusability of the CLEA with respect to olive oil hydrolysis was evaluated. The CLEA showed more than 90% residual activity even after 10 cycles of repeated use.     

In vitro cytotoxic potential of Polyalthia longifolia on human cancer cell lines and induction of apoptosis through mitochondrial-dependent pathway in HL-60 cells

Polyalthia longifolia is a lofty evergreen tree found in India and Sri Lanka. We are reporting first time the anticancer potential of P. longifolia leaves extract (A001) and its chloroform fraction (F002). Both inhibited cell proliferation of various human cancer cell lines in which colon cancer cells SW-620 showed maximum inhibition with IC(50) value 6.1 microg/ml. Furthermore, F002 induce apoptosis in human leukemia HL-60 cells as measured by several biological end points. F002 induce apoptotic bodies formation, DNA ladder, enhanced annexin-V-FITC binding of the cells, increased sub-G(0) DNA fraction, loss of mitochondrial membrane potential (DeltaPsi(mt)), release of cytochrome c, activation of caspase-9, caspase-3, and cleavage of poly ADP ribose polymerase (PARP) in HL-60 cells. All the above parameters revealed that F002-induced apoptosis through the mitochondrial-dependent pathway in HL-60 cells.     

Acetylsalicylic acid enhances and synchronizes thidiazuron-induced somatic embryogenesis in geranium (Pelargonium x hortorum Bailey) tissue cultures

Summary Thidiazuron (TDZ) effectively induced somatic embryogenesis in cultured hypocotyl explants of geranium (Pelargonium x hortorum Bailey) during only a 3-day period of induction. The presence of acetylsalicylic acid (ASA) during this period caused a two-fold increase in the number of somatic embryos and enhanced synchronization of embryo development compared to the TDZ treatment alone. Salicylic acid was ineffective in modulating similar embryogenic responses as ASA. The ASA-induced enhancement and synchronization of somatic embryogenesis could possibly be used as an experimental system to study the interplay of growth regulators in somatic embryogenesis.Abbreviations ASA acetylsalicylic acid - SA salicylic acid     

Differences in amino acid residues in the binding pockets dictate substrate specificities of mouse senescence marker protein-30, human paraoxonase1, and squid diisopropylfluorophosphatase

Senescence marker protein-30 (SMP-30) is a candidate enzyme that can function as a catalytic bioscavenger of organophosphorus (OP) nerve agents. We purified SMP-30 from mouse (Mo) liver and compared its hydrolytic activity towards various esters, lactones, and G-type nerve agents with that of human paraoxonase1 (Hu PON1) and squid diisopropylfluorophosphatase (DFPase). All three enzymes contain one or two metal ions in their active sites and fold into six-bladed β-propeller structures. While Hu PON1 hydrolyzed a variety of lactones, the only lactone that was a substrate for Mo SMP-30 was d-(+)-gluconic acid δ-lactone. Squid DFPase was much more efficient at hydrolyzing DFP and G-type nerve agents as compared to Mo SMP-30 or Hu PON1. The K(m) values for DFP were in the following order: Mo SMP-30>Hu PON1>squid DFPase, suggesting that the efficiency of DFP hydrolysis may be related to its binding in the active sites of these enzymes. Thus, homology modeling and docking were used to simulate the binding of DFP and selected δ-lactones in the active sites of Hu SMP-30, Hu PON1, and squid DFPase. Results from molecular modeling studies suggest that differences in metal-ligand coordinations, the hydrophobicity of the binding pockets, and limited space in the binding pocket due to the presence of a loop, are responsible for substrate specificities of these enzymes.