U-2012: An improved Lowry protein assay, insensitive to sample color, offering reagent stability and enhanced sensitivity

Traditional colorimetric protein assays such as Biuret, Lowry, and modified Lowry (U-1988) are unsuitable for colored biological samples. Here we describe an improved Lowry protein assay (U-2012), which utilizes stable reagents and offers enhanced sensitivity over the U-1988 assay. U-2012 circumvents interference from colored pigments and other substances (for example sugars) bound to perchloric acid (PCA) precipitated proteins by hydrogen peroxide (H2O2) induced oxidation at 50°C. Unused hydrogen peroxide is neutralized with sodium pyruvate before protein estimation for a stable end color. The U-2012 assay is carried out on the PCA precipitated protein pellet after neutralization (with Na2CO3 plus NaOH), solubilization (in Triton-NaCl), decolorization (by H2O2) and pyruvate treatment. Protein contents in red wine and homogenates of beetroot and blueberry are calculated from standard curves established for various proteins and generated using a rectangular hyperbola with parameters estimated with Microsoft Excel's Solver add-in. The U-2012 protein assay represents an improvement over U-1988 and gives a more accurate estimation of protein content.     

Diversity and distribution of terricolous lichens as indicator of habitat heterogeneity and grazing induced trampling in a temperate-alpine shrub and meadow

Lichens are among the most sensitive biomonitors of ecosystem health and human induced disturbances. Terricolous lichens of Chopta–Tungnath (Garhwal, western Himalaya, India) were analysed for their ability to indicate habitat variability and disturbances induced by livestock grazing. Terricolous lichens were sampled from 12 sites, distributed across the three macrohabitats between 2,700 and 4,001 m, using 50 × 10 cm narrow frequency grids having five 10 × 10 cm sampling units. The terricolous lichen community of the area constituted, 20 species belonging to 10 genera, five families and four growth forms. Altitude and relative humidity were the major habitat factors found influencing the terricolous lichen community of the landscape. Fruticose and compound soil lichen growth forms were found indicative of habitat disturbance largely caused by grazing induced trampling. Terricolous lichen diversity of the area was delimited by grazing pressure at mid-altitudes (3,000–3,400 m) and by decreasing soil cover at higher altitudes (>3,400 m).     

Membrane—vanadium interaction: A toxicokinetic evaluation

Vanadium is an important trace metal widely distributed in environment. Interaction of vanadate with skeletal muscle sarcolemma and basement membrane has been focussed. Scatchard analysis indicated the presence of more than one binding site for vanadate. Vanadate inhibits sarcolemmal and intestinal brush border membrane enzymes in a non-competitive manner. Membrane-vanadium interaction may lead to several structural and functional changes. The binding of vanadium to basement membrane may have some protective role.     

Cannabinoid Modulation of Eukaryotic Initiation Factors (eIF2α and eIF2B1) and Behavioral Cross-Sensitization to Cocaine in Adolescent Rats

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Studies on the measurement of phospholipase A2 (PLA2) and PLA2 inhibitor activities in ram semen.

Extraction with Tris-citrate or Tris-NaCl-EGTA improved the yield of phospholipase A2 (PLA2) from ram semen by 40-50 fold over the previously recommended method of extraction by dilute (0.18 N) sulphuric acid. The enzyme activity in the citrate extract deteriorated more rapidly than in Tris-NaCl-EGTA. The semen PLA2 activity was optimum at pH 8.0, heat sensitive at 70 degrees C for 30 min, activated by Ca2+ (although approximately 60% activity was also found in the absence of calcium) and did not exist as a pro-enzyme. The semen PLA2 activity was equally distributed among the sperm and seminal plasma (SP) components of ram semen. However, the low levels of PLA2 activity in the SP of vasectomised rams tend to suggest that PLA2 in the SP fraction may have originated from testicular or epididymal secretions or leakage, from sperm. PLA, in sperm exists as a large molecular weight aggregate, whereas in SP it is present as a smaller aggregate. In addition to PLA2, semen also contained PLA2 inhibitor activities. Inhibition was observed against PLA2s from bee venom, pig pancreas and oviductal extracts. The inhibitory activity is presumed to be due to a large molecular weight protein as the inhibitor activity was not extracted in a chloroform:methanol (2:1; v/v) mixture, it was non-dialysable, precipitated by 10% trichloroacetic acid and destroyed by proteases. The inhibitor activity was distributed in various molecular weight fractions of sperm, SP and SP from vasectomised rams.     

In vitro regeneration of a mature leguminous liana (Bauhinia vahlii Wight & Arnott)

An in vitro propagation protocol has been developed from mature lianas of Bauhinia valii. Browning was the major obstacle in the establishment of cultures. Explants collected during the growing season (April–June) showed maximum browning; however, browning was minimal during the dormant phase. This problem was circumvented by soaking the sterilized explants in a solution of antioxidant (50 mg l^–1 ascorbic acid+75 mg l^–1 citric acid). The explants were thereafter transferred to culture room conditions after an initial incubation in the dark at 4 °C for 48 h. Shoot proliferation (58%), shoot number (4.5) and shoot length (35 mm) was best in Murashige and Skoog (MS) medium supplemented with 2.5 μM kinetin + 100 mg l^–1 adenine sulfate. Seasonal fluctuations significantly affected the proliferation potential of the explants. March– April was found to be the best season for shoot initiation. Microshoots were rooted on a half-strength, growth regulator-free, agar-gelled Murashige and Skoog medium after a dip in half-strength MS liquid medium containing 1-naph-thaleneacetic acid + indole-3-butyric acid (10 μM). Rooted plantlets were potted and acclimatized under culture room conditions for 4 weeks before transfer to a polyhouse. Received: 9 March 1998 / Revision received: 14 August 1998 / Accepted: 23 September 1998     

Bactericidal,quorum quenching and anti-biofilm nanofactories: a new niche for nanotechnologists

Despite several conventional potent antibacterial therapies, bacterial infections pose a significant threat to human health because they are emerging as the leading cause of death worldwide. Due to the development of antibiotic resistance in bacteria, there is a pressing demand to discover novel approaches for developing more effective therapies to treat multidrug-resistant bacterial strains and biofilm-associated infections. Therefore, attention has been especially devoted to a new and emerging branch of science “nanotechnology” to design non-conventional antimicrobial chemotherapies. A range of nanomaterials and nano-sized carriers for conventional antimicrobial agents have fully justified their potential to combat bacterial diseases by reducing cell viability, by attenuating quorum sensing, and by inhibiting/or eradicating biofilms. This communication summarizes emerging nano-antimicrobial therapies in treating bacterial infections, particularly using antibacterial, quorum quenching, and anti-biofilm nanomaterials as new approaches to tackle the current challenges in combating infectious diseases.     

Colorimetric estimation of inorganic phosphate in colored and/or turbid biological samples: assay of phosphohydrolases

A simple method of inorganic phosphate determination for colored and/or turbid biological samples is described. The procedure is mild, and so is suitable for routine phosphohydrolase assays. Following deproteinization by ice-cold trichloroacetic (or silicotungstic) acid, the sample was treated with acid-washed charcoal to remove interference due to color. The phosphate in the colorless supernatant was assayed either by measuring the phosphomolybdate spectrophotometrically at 310 nm, following its extraction in organic solvents or by a modified Fiske and Subbarow method. The turbidity interference in the latter case was eliminated either by centrifugation, by sodium dodecyl sulfate treatment, or by extraction of reduced phosphomolybdate blue color by cyclohexanone. Though deproteinization by silicotungstic acid eliminated the turbidity problem, its use in conjunction with charcoal treatment was not convenient.