Accumulation and turnover of the classical Folch-Lees proteolipid proteins in developing and adult rat brain.

The turnover of classical Folch-Lees proteolipid proteins was studied after administration of [2,3-3H]tryptophan to both developing and adult rat brain. The animals were killed from 2h to 250 days after subcutaneous injections of [3H]tryptophan. The measured specific radioactivity in developing brain attained maximum value 24h after the administration of label, whereas the total radioactivity per brain reached a maximum 21 days after injection. The half-life of proteolipid protein from the measured specific radioactivity was 7-20 days, depending on the time-points used for the calculation, whereas calculation from total radioactivity between 28-77 and 91-257 days gave half-lives of 35-40 and 188 days respectively. In contrast, in animals injected at 40 days of age, the half-life from the whole-brain-radioactivity data was 188 days. The problem of the recycling of radioactivity for the synthesis of myelin proteins from either a general or a discrete amino acid pool is discussed.     

Effective Protocol for in Vitro Shoot Production Through Nodal Explants of Simmondsia Chinensis

Nodal explants excised from 18 to 20-year-old female plants of Simmondsia chinensis if cultured on Murashige and Skoog's medium supplemented with 20 M N⁶-benzyladenine (BA) differentiated an average of 2.7 ± 0.4 shoots in 11.5% explants. The percentage of nodal explants inducing multiple shoots enhanced significantly if in vitro raised shoots were used as source of explants. Nearly 100% cultures differentiated an average of 4.7 ± 2.0 shoots per explant on the same medium. Nearly 85% of the shoots induced roots when a pulse treatment of 50 M indole-3-butyric acid (IBA) was given prior to their transfer to semi-solid MS medium containing 10 M IBA + 0.5% activated charcoal + 1 M BA. Plantlets were gradually hardened in Soilrite and acclimatized to soil.     

Proteome analysis of differentially displayed proteins as a tool for investigating ozone stress in rice (Oryza sativa L.) seedlings

Employing classical two-dimensional electrophoresis (2-DE), amino acid sequencing and immunoblot analysis, we examine for the first time the effect of ozone, a highly notorious environmental pollutant, on rice seedling proteins. Drastic visible necrotic damage to leaf by ozone and consequent increase in ascorbate peroxidase protein(s) was accompanied by rapid changes in the 2-DE protein profiles, over controls. Out of a total of 56 proteins investigated, which were reproducible in repeated experiments, 52 protein spots were visually identified as differentially expressed over controls. Six proteins were N-terminally blocked, and the sequence of 14 proteins could not be determined, whereas 36 proteins were N-terminally and one was internally sequenced. Ozone caused drastic reductions in the major leaf photosynthetic proteins, including the abundantly present ribulose-1, 5-bisphosphate carboxylase/oxygenase, and induction of various defense/stress related proteins. Most prominent change in leaves, within 24 h post-treatment with ozone, was the induced accumulation of a pathogenesis related (PR) class 5 protein, three PR 10 class proteins, ascorbate peroxidase(s), superoxide dismutase, calcium-binding protein, calreticulin, a novel ATP-dependent CLP protease, and an unknown protein. Present results demonstrate the highly damaging effect of ozone on rice seedlings at the level of the proteome.     

Octadecanoid signaling component "burst" in rice (Oryza sativa L.) seedling leaves upon wounding by cut and treatment with fungal elicitor chitosan

Octadecanoid pathway components, 12-oxo-phytodieonic acid (OPDA) and jasmonic acid (JA), are key biologically active regulators of plant self-defense response(s). However, to date these compounds have been studied mostly in dicots, and used large (1-10 g fresh weight, FW) samples for quantification, even when examined in mature rice plants, which is a drawback considering their rapid responsiveness to stress. Focusing on rice--a monocot cereal crop research model--this work describes an efficient and simultaneous quantification of both OPDA and JA using a minimum amount of 200mg FW seedling leaf tissue upon wounding (by cut) and treatment with fungal elicitor, chitosan (CT) by high-pressure liquid chromatography-turboionspray tandem mass spectrometry. Transient OPDA/JA "burst" was consistently and reproducibly detected within 3 min in wounded and CT treated leaves. OPDA peaked dramatically around 5 min and returned to its basal level within 15 min, whereas JA induction upon wounding and CT treatment were in parallel to OPDA production, peaking at 30 and 60 min, respectively. Present results mark a major advance in our understanding of key inducible octadecanoid pathway components in rice, and strongly suggest a role for the octadecanoid pathway downstream of perception of at least these two fundamentally different extracellular stimuli.     

Production of xylanase by Emericella nidulans NK-62 on low-value lignocellulosic substrates

Thermotolerant Emericella nidulans NK-62 was isolated from bird nesting material and was tested for its ability to produce xylanase. The fungus when grown on a medium containing wheat bran (2% w/v) supplemented with Czapek's mineral salt solution at 45 °C for 7 days produced 362 IU/ml of xylanase (EC 3.2.1.8). The specific activity of E. nidulans NK-62 xylanase was found to be 275 IU/mg of total protein. The enzyme was found to be active over a broad temperature and pH range with 60 °C as optimum temperature for enzyme activity. The enzyme was stable at 50 °C and its half-life at 55 °C was 45 min. -xylosidase (EC 3.2.1.37) and carboxymethylcellulase (EC 3.2.1.4) activities, 0.018 and 0.21 IU/ml respectively, were also noticed. The fungus was screened for its ability to produce xylanase on four different lignocellulosic substrates. It produced 318.9 IU/ml of cellulase-free xylanase on corn cobs. The fungus could also utilize lentil bran (seed husk of Lens esculentus) and meal of groundnut shells to produce 84.8 and 17.3 IU/ml xylanase respectively.     

In vitro activation and differentiation of naïve CD4 and CD8 T cells into HCV Core- and NS3-specific armed effector cells: A new role for CD4 T cells

Viral clearance in hepatitis C virus (HCV) infection has been correlated with strong, multi-specific and sustained T cell responses. The number of functionally active effector T cells determines the outcome of infection. Only a small number of antigen-specific naïve T cells are originally present. Upon infection, they undergo activation, clonal expansion and differentiation to become effector cells. In this study, we determined the ability of dendritic cells (DCs) to prime T cells in vitro to become effector cells upon stimulation with various TLR ligands or IFNα. T cell priming and activation was determined by proliferation and production of effector molecules, IFN-γ and Granzyme B (GrB). HCV Core-specific T cells showed significant increase in proliferation, and the number of HCV Core-specific CD4⁺ and CD8⁺ T cells producing IFN-γ and GrB was higher than control or NS3-specific T cells. These in vitro-primed CD4⁺ and CD8⁺ T cells exhibit the phenotype of just-activated and/or armed effector lymphocytes confirming the transition of naïve T cells to effector cells. This is the first study demonstrating the activation of GrB⁺CD4⁺ T cells against antigen(s) derived from HCV. Our study suggests a novel role of CD4⁺ T cells in immunity against HCV.     

Glutathione and citric acid modulates lead- and arsenic-induced phytotoxicity and genotoxicity responses in two cultivars of <Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.

This study was conducted through the pot experiments to understand the mechanism of lead (Pb) and arsenic (As)-induced phytotoxicity and their possible alleviation by glutathione (GSH) and citric acid (CA) in two cultivars of Solanum lycopersicum L., i.e., Pusa ruby (PR) and Arka vikas (S22). Therefore, tomato seedlings were germinated in soil-rite supplemented with seven treatments, i.e., control, 10 µM Pb, 10 µM As, 10 µM Pb + 250 µM GSH, 10 µM As + 250 µM GSH, 10 µM Pb + 250 µM CA and 10 µM As + 250 µM CA for 7 days and examined for growth parameters, lipid peroxidation, photosynthetic pigments and antioxidative mechanism. Results of our study showed that Pb and As alone decrease seed germination, growth parameter, chlorophylls and increase anthocyanins and lipid peroxidation in both the cultivars. Pb- and As-induced oxidative stress resulted into significant changes in the plant responses that attributed by increased activity of antioxidative enzymes and non-enzymatic antioxidants. GSH and CA showed potential to alleviate Pb- or As-induced phytotoxicity and strengthen the plant antioxidative machinery and structural integrity. Cultivar PR showed better response than cv. S22. Pb and As treatment caused significant damages to the DNA molecules and structural integrity of the cv. PR roots. These findings can be useful for understanding the Pb- and As-induced phytotoxic biomarkers along with GSH- and CA-mediated alleviation mechanisms, which will provide new insight in developing better system for phytoremediation technology.     

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.     

Computational tool for the early screening of monoclonal antibodies for their viscosities

Highly concentrated antibody solutions often exhibit high viscosities, which present a number of challenges for antibody-drug development, manufacturing and administration. The antibody sequence is a key determinant for high viscosity of highly concentrated solutions; therefore, a sequence- or structure-based tool that can identify highly viscous antibodies from their sequence would be effective in ensuring that only antibodies with low viscosity progress to the development phase. Here, we present a spatial charge map (SCM) tool that can accurately identify highly viscous antibodies from their sequence alone (using homology modeling to determine the 3-dimensional structures). The SCM tool has been extensively validated at 3 different organizations, and has proved successful in correctly identifying highly viscous antibodies. As a quantitative tool, SCM is amenable to high-throughput automated analysis, and can be effectively implemented during the antibody screening or engineering phase for the selection of low-viscosity antibodies.     

Vitamin D Supplementation Reduces Induction of Epithelial-Mesenchymal Transition in Allergen Sensitized and Challenged Mice

Asthma is a chronic disease of the lung associated with airway hyperresponsiveness (AHR), airway obstruction and airway remodeling. Airway remodeling involves differentiation of airway epithelial cells into myofibroblasts via epithelial-mesenchymal transition (EMT) to intensify the degree of subepithelial fibrosis. EMT involves loss in E-cadherin with an increase in mesenchymal markers, including vimentin and N-cadherin. There is growing evidence that vitamin D has immunomodulatory and anti-inflammatory properties. However, the underlying molecular mechanisms of these effects are still unclear. In this study, we examined the contribution of vitamin D on the AHR, airway inflammation and expression of EMT markers in the airways of mice sensitized and challenged with a combination of clinically relevant allergens, house dust mite, ragweed, and Alternaria (HRA). Female Balb/c mice were fed with vitamin D-sufficient (2000 IU/kg) or vitamin D-supplemented (10,000 IU/kg) diet followed by sensitization with HRA. The density of inflammatory cells in the bronchoalveolar lavage fluid (BALF), lung histology, and expression of EMT markers by immunofluorescence were examined. Vitamin D-supplementation decreased AHR, airway inflammation in the BALF and the features of airway remodeling compared to vitamin D-sufficiency in HRA-sensitized and -challenged mice. This was accompanied with increased expression of E-cadherin and decreased vimentin and N-cadherin expression in the airways. These results indicate that vitamin D may be a beneficial adjunct in the treatment regime in allergic asthma.