Evaluation of immunostimulatory and growth promoting effect of seed fractions of Achyranthes aspera in common carp Cyprinus carpio and identification of active constituents

Immunostimulatory and growth promoting properties of Achyranthes aspera seeds were studied with larvae of common carp Cyprinus carpio. Four experimental diets were prepared using raw (D1) and alcohol (D2), petroleum ether (D3) and 50% aqueous alcohol (D4) extracts of A. aspera seeds. Diet without seed served as control (D5). Fish were fed with test/control diet for 30 days and then immunized with 10 μl of c-RBC. Blood samples were collected 7 days after immunization. Survival (93 ± 3%) of fish was significantly (P < 0.05) higher in D1 diet fed group compared to others. Highest specific growth rate was found in fish fed with diet D2. Significantly (P < 0.05) higher levels of serum protein and albumin were found in D1 and D3 compared to others. Highest serum globulin level was found in D1, which was followed by D3, D2, D4 and D5. Hemagglutination titer level was 5-18 folds higher in diet D3 fed fish compared to others. SGOT and SGPT levels were significantly (P < 0.05) higher in control group compared to the treated groups. Myeloperoxidase activity was significantly (P < 0.05) higher in D1 (2.513 ± 0.27 λ 450 nm) and D3 (2.38 ± 0.07 λ 450 nm) diets fed groups compared to others. The best performance of fish was found in raw A. aspera seeds incorporated diet fed group and the active constituents were identified as ecdysterone and two essential fatty acids linolenic acid and oleic acid.     

Can contact potentials reliably predict stability of proteins?

The simplest approximation of interaction potential between amino acid residues in proteins is the contact potential, which defines the effective free energy of a protein conformation by a set of amino acid contacts formed in this conformation. Finding a contact potential capable of predicting free energies of protein states across a variety of protein families will aid protein folding and engineering in silico on a computationally tractable time-scale. We test the ability of contact potentials to accurately and transferably (across various protein families) predict stability changes of proteins upon mutations. We develop a new methodology to determine the contact potentials in proteins from experimental measurements of changes in protein's thermodynamic stabilities (DeltaDeltaG) upon mutations. We apply our methodology to derive sets of contact interaction parameters for a hierarchy of interaction models including solvation and multi-body contact parameters. We test how well our models reproduce experimental measurements by statistical tests. We evaluate the maximum accuracy of predictions obtained by using contact potentials and the correlation between parameters derived from different data-sets of experimental (DeltaDeltaG) values. We argue that it is impossible to reach experimental accuracy and derive fully transferable contact parameters using the contact models of potentials. However, contact parameters may yield reliable predictions of DeltaDeltaG for datasets of mutations confined to the same amino acid positions in the sequence of a single protein.     

The proteome of dissimilatory metal-reducing microorganism Geobacter sulfurreducens under various growth conditions

The proteome of Geobacter sulfurreducens, a model for the Geobacter species that predominate in many Fe(III)-reducing subsurface environments, was characterized with ultra high-pressure liquid chromatography and mass spectrometry using accurate mass and time (AMT) tags as well as with more traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Cells were grown under six different growth conditions in order to enhance the potential that a wide range of genes would be expressed. The AMT tag approach was able to identify a much greater number of proteins than could be detected with the 2-D PAGE approach. With the AMT approach over 3,000 gene products were identified, representing about 90% of the total predicted gene products in the genome. A high proportion of predicted proteins in most protein role categories were detected; the highest number of proteins was identified in the hypothetical protein role category. Furthermore, 91 c-type cytochromes of 111 predicted genes in the G. sulfurreducens genome were identified. Differences in the abundance of cytochromes and other proteins under different growth conditions provided information for future functional analysis of these proteins. These results demonstrate that a high percentage of the predicted proteins in the G. sulfurreducens genome are produced and that the AMT tag approach provides a rapid method for comparing differential expression of proteins under different growth conditions in this organism.     

Investigations on feasibility of in situ development of amphotericin B liposomes for industrial applications

Amphotericin B (AmB) liposome formulations are very successful in the treatment of fungal infections and leishmaniasis. But higher cost limits its widespread use among people in developing countries. Therefore, we have developed a modified ethanol-injection method for the preparation of AmB liposomes. Two liposomal formulations were developed with dimyristoyl phosphatidylcholine [F-1a] and soya phosphatidylcholine [F-2a], along with egg phosphatidyl glycerol and cholesterol. AmB was dissolved in acidified dimethyl acetamide and mixed with ethanolic lipid solution and rapidly injected in 5% dextrose to prepare liposomes. Liposomes were characterized on the basis of size (~100?nm), zeta (-43.3?±?2.8 mV) and percent entrapment efficiency (>95%). The in vitro release study showed an insignificant difference (P?≥?0.05) for 24-hour release between marketed AmB liposomes (AmBisome) and F-1a and F-2a. Proliposome concentrate, used for the preparation of in situ liposomes, was physically stable for more than 3 months at experimental conditions. Similarly, AmB showed no sign of degradation in reconstituted liposomes stored at 2-8°C for more than 3 months. IC(50) value of Ambisome (0.18 μg/mL) was comparatively similar to F-1a (0.17 μg/mL) and F-2a (0.16 μg/mL) against intramacrophagic amastigotes. Under experimental conditions, a novel modified method for AmB liposomes is a great success and generates interest for development as a platform technology for many therapeutic drug products.     

Inhibitory effect of antibodies against human chorionic gonadotropin on the growth of colorectal tumour cells

Human chorionic gonadotropin (hCG) was initially believed to be secreted exclusively by the embryo with its primary function being "rescue" of the corpus luteum. However, recently it has been found that the hormone (or its individual subunits) is also secreted by many cancers and that in many cases secretion is associated with poor patient prognosis. In this study, we assessed the presence of hCG in colorectal cancer cells (CCL-253) and evaluated the anti-tumour effects of anti-hCG antibodies in vitro and in vivo. Anti-hCG antibodies were reactive with CCL-253, as revealed by confocal immunoflourescence microscopy; both cell surface and intracellular expression were observed. Western blot analysis showed that antibodies appeared to interact with several moieties, indicating a level of cross-reactivity. Anti-hCG antiserum specifically reduced the viability of tumor cells and the addition of complement increased in vitro anti-tumor effects. In nude mice implanted with CCL-253 cells, administration of anti-hCG antiserum caused a significant reduction in tumor volume; all treated animals survived, while mortality was observed in control animals. Results suggest that anti-hCG antibodies can mediate significant anti-tumor activity both in vitro and in vivo and lend support to the rationale of anti-hCG immunization in the therapy of gonadotropin- sensitive cancers.     

Latitude dependent arrhythmicity in the circadian oviposition rhythm of Drosophila ananassae

Latitude dependent arrhythmicity in the circadian rhythm of oviposition of Drosophila ananassae strains originating from 8.1°N to 32.7°N was studied by inbreeding them in cycles of 12 h of light at 20 lux and 12 h of darkness. The number of inbreeding generations required to initiate arrhythmicity in oviposition rhythm was dependent on the origin of latitude of the strain. The strains from the lower latitudes became arrhythmic after notably more numbers of generations than those from the higher latitudes. This might be attributed to the higher inherent degree of oviposition rhythmicity in the F₁ generation, and enhanced photic sensitivity of the circadian pacemaker mediating entrainment of oviposition rhythm of the strains from lower latitudes as compared to those from the higher latitudes.     

Modulation of gene expression in autoimmune disease and aging by food restriction and dietary lipids

Several recent observations carried out by many investigators have offered some clues in understanding the mechanism of how food restriction (FR) acts in the prolongation of life-span, but the precise mechanisms involved in modulating the immune system have not been clearly understood. Our own ongoing studies indicate that FR may act at the molecular level and may extend the life-span by modulating functional activities of several genes in various target tissues. For instance, while cytochrome P-450 IIB1 and IIB2 expression is known to decline with age in ad libitum-fed rats, FR prevented the loss of (drug-inducible) P-450 enzymes in liver tissues. In addition, both alpha 2u-globulin and senescence marker protein 2 expressions, which are regulated by hormones, were also modulated during aging by FR in Fischer 344 male rats. In short-lived autoimmune-prone mice, both FR and omega-3 (n-3) fatty acids diet lowered the severity of autoimmune disease both in lupus-prone (NZB x NZW)F1 mice and in mice prone to develop lymphoproliferative and renal diseases, whereas saturated (n-9) and polyunsaturated (n-6) dietary lipids not only exacerbated autoimmune disease, but also significantly enhanced expression of several oncogenes in lymphoid tissues. FR and omega-3 fatty acids decreased the expression of certain oncogenes. Both FR and omega-3 fatty acids may modulate the aging and autoimmune disease processes by not only altering the fatty acid composition, membrane fluidity, and signal transduction, but also by modulating the lymphokine hormone receptors and their functions and thereby modulating expression of several genes in various tissues during the aging process.     

One-step purification of glucoamylase by affinity precipitation with alginate.

It was found that alginate binds to glucoamylase, presumably through the recognition of starch binding domain of the latter. The present work exploits this for purification of glucoamylases from commercial preparation of Aspergillus niger and crude culture filtrate of Bacillus amyloliquefaciens by affinity precipitation technique in a single-step protocol. Glucoamylase is selectively precipitated using alginate as macroaffinity ligand and later eluted with 1.0 M maltose. In the case of A. niger, 81% activity is recovered with 28-fold purification. The purified glucoamylase gave a single band on SDS-PAGE corresponding to 78 kDa molecular weight. The developed affinity precipitation process also works efficiently for purification of Bacillus amyloliquefaciens glucoamylase from its crude culture filtrate, giving 78% recovery with 38-fold purification. The purified preparation showed a major band corresponding to 62 kDa and a faint band about 50 kDa on SDS-PAGE. The latter corresponds to the molecular weight for alpha-amylase of Bacillus amyloliquefaciens.     

A pregnane ester diglycoside from Sarcostemma brevistigma

A pregnane ester diglycoside, brevinine, has been isolated from the dried twigs of Sarcostemma brevistigma. Its chemical and spectroscopic data are consistent with the structure 11-O-benzoyl-sarcogenin-3-O-α-L-diginopyranosyl-(1 → 4)-α-L-diginopyranoside.     

Deoxycholic acid differentially regulates focal adhesion kinase phosphorylation: role of tyrosine phosphatase ShP2

Environmental factors, including dietary fats, are implicated in colonic carcinogenesis. Dietary fats modulate secondary bile acids including deoxycholic acid (DCA) concentrations in the colon, which are thought to contribute to the nutritional-related component of colon cancer risk. Here we demonstrate, for the first time, that DCA differentially regulated the site-specific phosphorylation of focal adhesion kinase (FAK). DCA decreased adhesion of HCA-7 cells to the substratum and induced dephosphorylation of FAK at tyrosine-576/577 (Tyr-576/577) and Tyr-925. Tyrosine phosphorylation of FAK at Tyr-397 remained unaffected by DCA stimulation. Interestingly, we found that c-Src was constitutively associated with FAK and DCA actually activated Src, despite no change in FAK-397 and an inhibition of FAK-576 phosphorylation. DCA concomitantly and significantly increased association of tyrosine phosphatase ShP2 with FAK. Incubation of immunoprecipitated FAK, in vitro, with glutathione-S-transferase-ShP2 fusion protein resulted in tyrosine dephosphorylation of FAK in a concentration-dependent manner. Antisense oligodeoxynucleotides directed against ShP2 decreased ShP2 protein levels and attenuated DCA-induced FAK dephosphorylation. Inhibition of FAK by adenoviral-mediated overexpression of FAK-related nonkinase and gene silencing of Shp2 both abolished DCA's effect on cell adhesion, thus providing a possible mechanism for inside-out signaling by DCA in colon cancer cells. Our results suggest that DCA differentially regulates focal adhesion complexes and that tyrosine phosphatase ShP2 has a role in DCA signaling.