Antibacterial Natural Peptide Fractions from Indian <Emphasis Type="Italic">Ganoderma lucidum</Emphasis>

Natural peptides are emerging as a leading alternative to conventional drugs and antibiotics, owing to their remarkable potency, better stability and less toxicity. Such peptides encompass numerous healing properties such as antimicrobial, anti-inflammatory, immunomodulatory, etc. Though plant- derived peptides have been widely studied for their therapeutic benefits, however, fungal peptides are still lesser explored. Ganoderma lucidum, a highly medicinal oriental mushroom comprises a vast array of phytoconstituents, namely flavonoids, phenolics, terpenoids, polysaccharides, proteins, glycolipids, etc and hence, is being used since several decades in traditional Chinese medicine (TCM) for its various ameliorative effects e.g. anti-inflammatory, antimicrobial, anti-proliferative and antioxidant properties. This study presents the isolation and characterization of antibacterial peptide fractions from fruiting body (GLF) and mycelium (GLM) of Indian G. lucidum. Representative amide bonds were identified in the fractions using established standard techniques. Peptide mass fingerprinting and HPLC confirmed the presence of cationic and hydrophobic amino acids in the peptide fractions which are known to be major structural features of antimicrobial peptides. Secondary structure prediction showed abundance of α-helices and random coils in GLF and GLM fractions respectively. The fractions exhibited appreciable antioxidant potential. Besides, these also possessed substantial antibacterial activity against Escherichia coli and Salmonella typhi wherein it was observed that generation of reactive oxygen species and induction of intracellular protein leakage within the bacterial cells were the possible mechanisms of inhibitory action.     

Dopamine antagonist binding: a significant decrease with morphine dependence in the rat striatum

(³H)-Spiroperidol specific binding was determined in striatal tissue of rats which received a single dose of, or made dependent on morphine. Acute morphine (30 mg/kg i.p.) did not alter (³H)-spiroperidol specific binding. However, morphine-dependent rats with two 50 mg pellets when withdrawn for 24 or 48 hours, significantly decreased the binding and increased K_d. Binding sites were reduced with a decrease in K_d in rats implanted with four-50 mg pellets or receiving high doses of morphine. These results indicate that binding characteristics of (³H)-spiroperidol depend on the relative dose of morphine used to induce dependence. Low dose dependence (2 pellets) results in a decrease in binding affinity while high dose dependence (4 pellets or chronic injection) results in an increase of (³H)-spiroperidol affinity in the presence of fewer binding sites.     

IL-2-PE40 prevents the development of tumors in mice injected with IL-2 receptor expressing EL4 transfectant tumor cells

A number of different immunotherapeutic reagents are currently being developed to target IL-2R for the treatment of leukemia, graft rejection, and certain autoimmune diseases. Previously, we have shown that IL-2-PE40, a chimeric protein composed of human IL-2 linked to the N-terminus of a truncated form of Pseudomonas exotoxin (PE), could effectively kill a variety of cell lines in vitro expressing either low, intermediate, or high affinity IL-2R. Here, we demonstrate that IL-2-PE40 can successfully retard or prevent the growth of a lethal ascites tumor or a solid tumor composed of EL4J murine thymoma cells transfected with the p55 murine IL-2R. The transfected line, EL4J-3.4, expresses 1,000 to 3,000 high affinity IL-2R. Survival extension in the ascites model was achieved by initiating treatment either after 4 to 6 h or within 5 days post-tumor injection in both athymic nude and C57BL/6 mice. Similarly, the growth of an aggressive s.c. solid tumor could also be inhibited. Extension of survival was not achieved either by using the truncated toxin alone not attached to IL-2 or by using an IL-2-PE40Asp553 mutant lacking a functional toxin. Survival extension was not caused by IL-2 activated NK or other host effector mechanisms as IL-2-PE40 was unable to prevent the receptor-negative EL4J parental line from forming a lethal ascites or a solid tumor. Thus, IL-2-PE40 is a potent, specific cytolytic reagent that may prove useful in the arsenal of anti-IL-2R immunotherapeutics.     

Optimization of medium and process parameters for the production of inulinase from a newly isolated Kluyveromyces marxianus YS-1

A newly isolated strain of Kluyveromyces marxianus YS-1 was used for the production of extra cellular inulinase in a medium containing inulin, meat extract, CaCl2 and sodium dodecyl sulphate (SDS). Fermentation medium pH 6.5, cultivation temperature 30 degrees C and 5% (v/v) inoculum of 12 h-old culture were optimal for enzyme production (30.8 IU/ml) with a fermentation time of 72 h at shake flask level. Raw inulin (2%, w/v) extracted from dahlia tubers by processing at 15 kg/cm2 for 10 min was optimum for bioreactor studies. Maximum enzyme production (55.4 IU/ml) was obtained at an agitation rate of 200 rpm and aeration of 0.75 vvm in a stirred tank reactor with a fermentation time of 60 h.     

Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi

The acid alpha-mannosidase of Trypanosoma cruzi is a broad-specificity hydrolase involved in the catabolism of glycoconjugates, presumably in the digestive vacuole. We have cloned the alpha-mannosidase gene from a T.cruzi epimastigote genomic library. The alpha-mannosidase gene was determined to be single copy by Southern analysis, and similar sequences were not detected in genomic digests of either Trypanosoma brucei or Leishmania donovani. The coding region was subcloned into the Pichia pastoris expression vector pPICZ, and alpha-mannosidase activity was detected in the medium of induced cultures. The recombinant alpha- mannosidase demonstrated a pH optimum, inhibition by swainsonine, Km, and substrate specificity consistent with the characteristics of the alpha-mannosidase previously purified from T.cruzi epimastigotes. The recombinant enzyme was purified 103-fold from the culture medium of Pichia pastoris and had a native molecular mass of 359 kDa by gel filtration. A combination of SDS-PAGE, deglycosylation with endo H, and NH2-terminal sequencing indicates that the enzyme is originally synthesized as a homodimeric polypeptide that is subsequently cleaved to form a heterotetramer composed of 57 and 46 kDa subunits. A polyclonal antibody raised to the recombinant enzyme was shown to immunoprecipitate the alpha-mannosidase from T.cruzi cell extracts and will be used in future immunolocalization studies.      

Enhancement of morphine analgesia after acute and chronic haloperidol.

In the male rats haloperidol pretreatment caused an enhancement of morphine analgesia. After chronic haloperidol the analgesia enhancing effect of haloperidol was observed up to 10 days after its discontinuation. No analgesia was present in the haloperidol treated rats not given morphine.