Protein estimation in tissues containing high levels of lipid: modifications to Lowry's method of protein determination

A method to estimate protein in detergent-solubilized homogenates of lipid-rich biological samples (e.g., adipose tissue, myelin-enriched fractions of sheep brain) is described. The method is also suitable for samples in which protein is present as a protein-detergent complex. The method involves homogenization of tissue in the presence of a suitable detergent and KCl. Protein is then estimated in an aliquot of this homogenate by Lowry's method in the presence of excess sodium dodecyl sulfate, the solutions being clarified by extraction with ethyl acetate. Protein solubilization by Triton X-100 from adipose tissue was biphasic, extracting two to three times more protein under optimum conditions [1.7 +/- 0.1% (v/v) Triton X-100 and 0.75 M KCl], compared with homogenization without salt and detergent. Unlike adipose tissue, protein solubilization from myelin-enriched fractions of sheep brain peaked at 1% (v/v) Triton X-100, resulting in the extraction of approximately three times more protein than homogenization in the absence of detergent and salt.     

Effectiveness of action in India to reduce exposure of Gyps vultures to the toxic veterinary drug diclofenac

Contamination of their carrion food supply with the non-steroidal anti-inflammatory drug diclofenac has caused rapid population declines across the Indian subcontinent of three species of Gyps vultures endemic to South Asia. The governments of India, Pakistan and Nepal took action in 2006 to prevent the veterinary use of diclofenac on domesticated livestock, the route by which contamination occurs. We analyse data from three surveys of the prevalence and concentration of diclofenac residues in carcasses of domesticated ungulates in India, carried out before and after the implementation of a ban on veterinary use. There was little change in the prevalence and concentration of diclofenac between a survey before the ban and one conducted soon after its implementation, with the percentage of carcasses containing diclofenac in these surveys estimated at 10.8 and 10.7%, respectively. However, both the prevalence and concentration of diclofenac had fallen markedly 7-31 months after the implementation of the ban, with the true prevalence in this third survey estimated at 6.5%. Modelling of the impact of this reduction in diclofenac on the expected rate of decline of the oriental white-backed vulture (Gyps bengalensis) in India indicates that the decline rate has decreased to 40% of the rate before the ban, but is still likely to be rapid (about 18% year(-1)). Hence, further efforts to remove diclofenac from vulture food are still needed if the future recovery or successful reintroduction of vultures is to be feasible.     

Chemical Composition and Biological Activities of Trans-Himalayan Alga Spirogyra porticalis (Muell.) Cleve

The freshwater alga Spirogyra porticalis (Muell.) Cleve, a filamentous charophyte, collected from the Indian trans-Himalayan cold desert, was identified on the basis of morpho-anatomical characters. Extracts of this alga were made using solvents of varying polarity viz. n-hexane, acetonitrile, methanol and water. The antioxidant capacities and phenolic profile of the extracts were estimated. The methanol extract showing highest antioxidant capacity and rich phenolic attributes was further investigated and phytochemical profiling was conducted by gas chromatography-mass spectrometry (GC/MS) hyphenated technique. The cytotoxic activity of methanol extract was evaluated on human hepatocellular carcinoma HepG2 and colon carcinoma RKO cell lines. The anti-hypoxic effect of methanol extract of the alga was tested on in vivo animal system to confirm its potential to ameliorate oxidative stress. The antioxidant assays viz. ferric reducing antioxidant power (FRAP), 2,2''-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and nitric oxide (NO) radical scavenging capacities, β-carotene-linoleic acid bleaching property and lipid peroxidation exhibited analogous results, wherein the algal extracts showed significantly high antioxidant potential. The extracts were also found to possess high content of total proanthocyanidin, flavonoid and polyphenol. GC/MS analysis revealed the presence of thirteen chemotypes in the methanol extract representing different phytochemical groups like fatty acid esters, sterols, unsaturated alcohols, alkynes etc. with substantial phyto-pharmaceutical importance. The methanol extract was observed to possess anticancer activity as revealed from studies on HepG2 and RKO cell lines. In the present study, S. porticalis methanol extract also provided protection from hypoxia-induced oxidative stress and accelerated the onset of adaptative changes in rats during exposure to hypobaric hypoxia. The bioactive phytochemicals present in this trans-Himalayan alga are of enormous interest and can be utilized sustainably for discovery of novel drugs against oxidative stress.     

Gβγ and the C terminus of SNAP-25 are necessary for long-term depression of transmitter release

Background

Short-term presynaptic inhibition mediated by G protein-coupled receptors involves a direct interaction between G proteins and the vesicle release machinery. Recent studies implicate the C terminus of the vesicle-associated protein SNAP-25 as a molecular binding target of Gβγ that transiently reduces vesicular release. However, it is not known whether SNAP-25 is a target for molecular modifications expressing long-term changes in transmitter release probability.

Methodology/Principal Findings

This study utilized two-photon laser scanning microscopy for real-time imaging of action potential-evoked [Ca²⁺] increases, in single Schaffer collateral presynaptic release sites in in vitro hippocampal slices, plus simultaneous recording of Schaffer collateral-evoked synaptic potentials. We used electroporation to infuse small peptides through CA3 cell bodies into presynaptic Schaffer collateral terminals to selectively study the presynaptic effect of scavenging the G-protein Gβγ. We demonstrate here that the C terminus of SNAP-25 is necessary for expression of LTD, but not long-term potentiation (LTP), of synaptic strength. Using type A botulinum toxin (BoNT/A) to enzymatically cleave the 9 amino acid C-terminus of SNAP-25 eliminated the ability of low frequency synaptic stimulation to induce LTD, but not LTP, even if release probability was restored to pre-BoNT/A levels by elevating extracellular [Ca²⁺]. Presynaptic electroporation infusion of the 14-amino acid C-terminus of SNAP-25 (Ct-SNAP-25), to scavenge Gβγ, reduced both the transient presynaptic inhibition produced by the group II metabotropic glutamate receptor stimulation, and LTD. Furthermore, presynaptic infusion of mSIRK, a second, structurally distinct Gβγ scavenging peptide, also blocked the induction of LTD. While Gβγ binds directly to and inhibit voltage-dependent Ca²⁺ channels, imaging of presynaptic [Ca²⁺] with Mg-Green revealed that low-frequency stimulation only transiently reduced presynaptic Ca²⁺ influx, an effect not altered by infusion of Ct-SNAP-25.

Conclusions/Significance

The C-terminus of SNAP-25, which links synaptotagmin I to the SNARE complex, is a binding target for Gβγ necessary for both transient transmitter-mediated presynaptic inhibition, and the induction of presynaptic LTD.     

Synthesis and anticancer activity of sclerophytin-inspired hydroisobenzofurans

Three structurally related sets of hydroisobenzofuran analogs of sclerophytin A were prepared in three or four steps from (S)-(+)-carvone via an aldol-cycloaldol sequence. The most potent members of each set of analogs exhibited IC₅₀’s of 1–3 μM in growth inhibitory assays against KB3 cells. The NCI 60-cell line 5-dose assay for analog 6h revealed a GI₅₀ = 0.148 μM and LC₅₀ = 9.36 μM for the RPMI-8226 leukemia cell line, and a GI₅₀ = 0.552 μM and LC₅₀ = 26.8 μM for the HOP-92 non-small cell lung cancer cell line.     

Effect of chronic choline availability on lung and lymph nodes of rat

Male albino rats were given intraperitoneal injections of choline chloride (0. 1,0.33 or 0.5 × lethal dose 50) for a total period of one month and then killed at the end of 30,90 and 240 days for the study of pathotoxicokinetics of choline. Chronic choline administration in rats caused a decrease in growth rate, a dose dependent modulating effect on the somatic tissue indices of lung and lymph nodes, as well as cellularity of lymph nodes. In another experiment, the effect of choline on mica induced pulmonary lesions was studied. The combined effect of choline and mica caused adenocarcinoma of bronchiolar epithelium and marked lymphadenopathy with abnormal cells in the lymph nodes at the termination of experiment (330 days). The results of the present investigation suggest that excess choline availability not only produces pulmonary pathological lesions by itself but it also further enhances the lung lesions in altered pulmonary conditions     

Attempted sexing of bovine spermatozoa by fractionation on a Percoll density gradient

Bovine spermatozoa were fractionated on Percoll density gradients into two major subpopulations of motile spermatozoa and a minor fraction containing mostly nonmotile spermatozoa with abnormal morphology. Fractionation required the addition of bovine serum albumin and a continuous Percoll gradient buffered with sodium bicarbonate. It is postulated that, under suitable ionic conditions, the binding of bovine serum albumin to spermatozoa amplifies subtle differences between subpopulations. These studies were directed toward separating Y- and X-bearing spermatozoa. However, when the subpopulations were evaluated by flow cytometry, their Y:X ratios were similar to that of an unfractionated control.     

Micropropagation ofBauhinia vahlii Wight & Arnott — a leguminous liana

Anin vitro propagation protocol for a leguminous liana,Bauhinia vahlii, has been established. In the first experiment, cotyledonary nodes fromin-vitro-germinated seedlings were cultured on various basic media (Murashige and Skoog medium, Woody Plant medium, B5, and 1/2 Murashige and Skoog medium) containing 1.0M thidiazuron. Shoot proliferation (96.20%) and multiplication (5.55 shoots/explant) was best when cultured on Murashige and Skoog medium. The second experiment compared responses to benzylaminopurine, kinetin, zeatin and thidiazuron. Murashige and Skoog medium supplemented with 1.0M thidiazuron proved most effective for both shoot proliferation and shoot multiplication. The effect of cytokinin type and concentration and their interaction was found to be significant (P<0.001) for explant proliferation, shoot number and length. Subsequent rooting (55.14%) of the regenerated shoots was achieved on half-strength Murashige and Skoog medium supplemented with IM 1- naphthaleneacetic acid. Successful transfer of regenerants to soil has been accomplished, and efforts are being made to gradually transfer them to field conditions.     

A step towards developing the expertise to control hunger and satiety: Regulatory role of satiomem—A membrane proteoglycan

Regulation of hunger and satiety is a complex process thought to be controlled by a complex interplay of neurotransmitters in the hypothalamic region of the brain. Reduced food intake or anorexia has also been observed under various disease or disorder conditions including AIDS and cancer. On the other hand, increased appetite because of some impairment of central mechanisms regulating the food intake could also cause obesity. A large number of substances including neuropeptides, hormones, drugs, and synthetic peptides have been implicated in the regulation of appetite and food intake behavior in normal as well as disease or disorder conditions. Most of these substances are not directly involved in the regulation of normal hunger and satiety but exert their effect indirectly via other media. Some of them are involved under certain pathologic conditions and during the course they become involved directly or indirectly in the triggering of hunger and satiety regulatory mechanism. Recently, we have been able to isolate and purify an endogenous proteoglycan from membranes of animal and plant sources. This membrane anchored proteoglycan termed as Satiomem reduces food intake without any rebound effects and has no apparent toxicity. It also fulfils all the criteria of a true satiety or anorexigenic substance. The release of satiomem from the cell surface could be mediated by a specific phospholipase-C. Satiomem seems to be involved in transducing activating signals and may also act as a source of second messenger for the regulatory mechanism of appetite. This article summarizes the regulatory aspects of hunger and satiety mechanisms controlled by endogenous substances with the emphasis on our present knowledge about satiomem.Dedicated to Dr. Sydney Ochs.