Feulgen staining of rat liver fixed in different fixatives.

In the present study rat liver pieces fixed in 1) 10 per cent buffered neutral formalin, 2) 4 per cent glutaraldehyde, 3) Heidenhain's-Susa fixative and 4) Flemming's fluid, and following hydrolysis in 1-0 N HC1 at 60degreesC for varying time periods have been stained with the UV Feulgen procedure. The results of this study reveal that following hydrolysis for different time periods the tissue material fixed in formalin show the same staining pattern as those fixed in glutaraldehyde. The material fixed in Heidenhain's-Susa displays an intense Feulgen staining after two different times of hydrolysis, and that fixed in Flemming's fluid shows particular staining intensity for a prolonged time period thus indicating better preservation of DNA than in the materials fixed in the other three fixtatives.     

Intracellular distribution of enzymes of phospholipid metabolism in several gram-negative bacteria.

Cell-free extracts of Salmonella typhimurium, Serratia marcescens, Enterobacter aerogenes, and Micrococcus cerificans contained the following enzymatic activities related to phospholipid metabolism: cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diglyceride):l-serine O-phosphatidyltransferase (phosphatidylserine synthase), phosphatidylserine decarboxylase, CDP-diglyceride:sn-glycero-3-phosphate phosphatidyltransferase (phosphatidylglycerophosphate synthase), phosphatidylglycerophosphate phosphatase, and CDP-diglyceride hydrolase. The intracellular distribution of these enzymatic activities as determined by sucrose density gradient centrifugation of cell-free extracts was shown to be similar in each species investigated. The phosphatidylserine decarboxylase, phosphatidylglycerophosphate synthase, and CDP-diglyceride hydrolase activities were all associated with the cell envelope fraction, whereas the phosphatidylserine synthase activity was associated mainly with the ribosomal fraction. These enzymatic activities are comparable and have an intracellular distribution similar to those found in Escherichia coli cell-free extracts. Therefore, the pathways established for phospholipid biosynthesis in E. coli can also account for the synthesis of the major phospholipids (phosphatidylethanolamine and phosphatidylglycerol) in several other gram-negative organisms. In addition, the unusual ribosomal association of the phosphatidylserine synthase from E. coli (Raetz and Kennedy, J. Biol. Chem. 247:2008-2014, 1972) appears to be a general property for this activity in several other bacterial species.     

Genetic Structuring of Nine Indian Domestic Goat Breeds Based on SNPs Identified in IGF-1 Gene

The caprine Insulin like Growth Factor1 (IGF1) gene was analyzed for identification of single nucleotide polymorphisms (SNPs) and genetic structuring of Indian goat breeds. A panel of 80 samples belonging to nine Indian goat breeds (Capra hircus) including three large sized breeds (Jamunapari, Beetal and Jakhrana); three medium sized breeds (Sirohi, Barbari, and Osmanabadi) and three small sized breeds (Black Bengal, Changthangi, and Gaddi) were screened for SNP identification and diversity analysis. The comparative gene sequence analysis of all the nine goat breeds studied revealed a total of 18 SNPs in IGF1 gene. All the nucleotide changes were found to be synonymous. The mean observed heterozygosity was found to be maximum (0.074) in Sirohi, Beetal, Osmanabadi, and Gaddi breeds of goat, whereas it is found to be minimum (0.019) in Black Bengal breed of goat. The rest of the breeds were intermediate in terms of heterozygosity. The same has been confirmed by allele frequency distribution across the studied loci. Barbari and Gaddi were found to be more differentiated (0.0123), Changthangi and Jamunapari were least differentiated (0.00110) based on Nei's genetic distance.     

A co-transformation system to produce transgenic grapevines free of marker genes

A co-transformation system was developed to produce grapevines free of selectable marker genes. This was achieved by transforming Vitis vinifera L. ‘Thompson Seedless’ somatic embryos with a mixture of two Agrobacterium strains. The first strain contained a binary plasmid with an egfp gene of interest between the T-DNA borders. The second strain harbored the neomycin phosphotransferase (nptII) gene for positive selection and the cytosine deaminase (codA) gene for negative selection, linked together by a bi-directional dual promoter complex. Our technique included a short positive selection phase on medium containing 100 mg l⁻¹ kanamycin before subjecting cultures to prolonged negative selection on medium containing 250 mg l⁻¹ 5-fluorocytosine. We regenerated 25 stable EGFP expressing transgenic lines. PCR analysis confirmed 18 lines contained only the egfp gene, whereas the remaining contained both egfp and codA/nptII genes. Presumably, the 18 monogenic lines arose through cross protection by being in close proximity to cells that expressed nptII and thus detoxified kanamycin in the immediate vicinity. This is the first report for grapevine using a combination of positive and negative selection to produce transgenic plants that do not contain marker genes.     

RPE-secreted factors: influence differentiation in human retinal cell line in dose- and density-dependent manner

Retinal pigment epithelial (RPE) cells play an important role in normal functioning of retina and photoreceptors, and some retinal degenerations arise due to malfunctioning RPE. Retinal pigment epithelium transplantation is being explored as a strategy to rescue degenerating photoreceptors in diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Additionally, RPE-secreted factors could rescue degenerating photoreceptors by prolonging survival or by their ability to differentiate and give rise to photoreceptors by transdifferentiation. In this study, we have explored what role cell density could play in differentiation induced in a human retinal progenitor cell line, in response to RPE-secreted growth factors. Retinal progenitors plated at low (1 × 10⁴ cells/cm²), medium (2–4 × 10⁴ cells/cm²), and high (1 × 10⁵ cells/cm²) cell density were exposed to various dilutions of RPE-conditioned medium (secreted factors) under conditions of defined medium culture. Progenitor cell differentiation was monitored phenotypically (morphological, biochemical analysis, and immunophenotyping, and western blot analysis were performed). Our data show that differentiation in response to RPE-secreted factors is modulated by cell density and dilutions of conditioned medium. We conclude that before embarking on RPE transplantation as a modality for treatment of RP and AMD, one will have to determine the role that cell density and inhibitory and stimulatory neurotrophins secreted by RPE could play in the efficacy of survival of transplants. We report that RPE-conditioned medium enhances neuronal phenotype (photoreceptors, bipolars) at the lowest cell density in the absence of cell–cell contact. Eighty percent to 90% of progenitor cells differentiate into photoreceptors and bipolars at 50% concentration of conditioned medium, while exposure to 100% conditioned medium might increase multipolar neurons (ganglionic and amacrine phenotypes) to a small degree. However, no clear-cut pattern of differentiation in response to RPE-secreted factors is noted at higher cell densities.     

IL-2 Regulates SEB Induced Toxic Shock Syndrome in BALB/c Mice

Background

Toxic Shock Syndrome (TSS) is characterized by fever, rash, hypotension, constitutional symptoms, and multi-organ involvement and is caused by Staphylococcus aureus enterotoxins such as Staphylococcal Enterotoxin B (SEB). SEB binds to the MHC-IIα chain and is recognized by the TCRβ chain of the Vβ8 TCR⁺ T cells. The binding of SEB to Vβ chain results in rapid activation of T cells and production of inflammatory cytokines, such as Interleukin-2 (IL-2), Interferon-γ and Tumor Necrosis Factor-α which mediate TSS. Although IL2 was originally identified as the T cell growth factor and was proposed to contribute to T cell differentiation, its role in TSS remains unexplored.

Methodology/Principal Findings

Mice were injected with D-Gal (25 mg/mouse). One hour after D-Galactosamine (D-Gal) injection each mouse was injected with SEB (20 µg/mouse. Mice were then observed for 72 hrs and death was recorded at different times. We tested Interleukin-12, IFNγ, and IL-2 deficient mice (IL-2^−/−), but only the IL-2 deficient mice were resistant to SEB induced toxic shock syndrome. More importantly reconstitution of IL-2 in IL-2 deficient mice restored the shock. Interestingly, SEB induced IL-2 production from T cells was dependent on p38MAPK activation in macrophages as inhibition of it in macrophages significantly inhibited IL-2 production from T cells.

Conclusion

This study shows the importance of IL -2 in TSS which has not been previously explored and it also shows that regulating macrophages function can regulate T cells and TSS.     

Tsunami and tropical island ecosystems: a meta-analysis of studies in Andaman and Nicobar Islands

Tropical islands are special and sensitive ecosystems which are subjected to various disturbances imposed by human activities and natural disasters. A detailed study about the changing landscape scenarios of these fragile island systems induced by various driving factors could be used for setting up measurements in support of conservation and sustainable development projects. The current research is a meta-analysis of the studies carried out in Andaman and Nicobar islands which analyzed the impact of tsunami of 2004 using geospatial tools. Based on the analysis, it was observed that the Nicobar islands were more affected compared to the Andaman islands. The majority of the researchers used pre- and post-tsunami satellite imagery and adopted visual interpretation method to delineate the changed classes. The study infers uplift of land in Andaman (exposing) and subsidence in Nicobar islands (inundation) with severe damage to the coastal elements like mangroves, coral reefs, plantations and in few cases interior forest. The analysis showed there were no records of the damage for some small islands. Finally, it is concluded that utility of microwave satellite data for change analysis will prove better in regions like Andaman and Nicobar where it is difficult to get cloud free optical data because of the high monsoon periods in these islands. It is also suggested that future work utilizing suitable temporal satellite imagery should focus on the extent of recovery of vegetation and other coastal elements which suffer the impact of disaster.