Phenology of plants in relation to ambient environment in a subalpine forest of Uttarakhand,western Himalaya

Observations on phenology of some representative trees, shrubs, under-shrubs and herbs in a subalpine forest of Uttarakhand, western Himalaya were recorded. With the commencement of favorable growth season in April, occurrence of leaf fall was indicatory growth phenomenon in Quercus semecarpifolia, Q. floribunda and Abies spectabilis. However, active vegetative growth in herbaceous species starts onward April and fruit maturation and seed dehiscence are completed from mid of September to October. In general, vegetative growth and reproductive stages in majority of the studied species seems to be dependent on adequate moisture content and also flowering and fruiting in subalpine plants correlate ambient temperature.     

Synaptic mutation-driven male sterility in Panax sikkimensis Ban. (Araliaceae) from Eastern Himalaya, India

In higher plants, synaptic mutation-associated gametic abnormalities are reported mostly in crop plants, but studies have rarely focused on the natural plant populations. This is particularly so in threatened herbaceous perennials, some of which are known to suffer from loss of sexual reproduction driven by the genetic mutations. Cytological investigations of Panax species, viz. P. sikkimensis, P. sokpayensis and P. bipinnatifidus, revealed that all the species were diploid with 2n = 24 chromosomes. Natural occurrence of synaptic mutation was recorded in Panax sikkimensis in the Kalep population of North Sikkim, India. We recorded that 86.03% of pollen mother cells (PMCs) lacked bivalent formation and had 24 distinct univalents at prophase I in the mutant plants of P. sikkimensis. We found a significantly lower mean number of chiasmata per cell (0.31 ± 0.91; t test = 38.24, P < 0.001) in the mutant plants as compared to the normal plants (21.04 ± 4.56). The chromosomal associations in the PMCs of the synaptic mutants ranged from 25% bivalents and 75% univalents to 100% univalents at diplotene/diakinesis. The unequal distribution of chromosomes at anaphase I and II resulted in the formation of microspores and microcytes of differing sizes. The pollen stainability test in the mutant population of P. sikkimensis revealed very low (0.12%) pollen fertility reflecting the consequences of synaptic mutation. Synaptic mutation in the herbaceous perennial P. sikkimensis was considered to be responsible for the male sterility in the species.     

In Vitro Culture of Functionally Active Buffalo Hepatocytes Isolated by Using a Simplified Manual Perfusion Method

Background

In farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes.

Results

Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3±0.66)×10⁷ cells per gram of liver tissue with a viability of 82.3±3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies.

Conclusion

We developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active hepatocytes for studying gene expression, regulation, hepatic genomics and proteomics in farm animals.     

Mountain Aspect Influences the Genetic Clustering of Psychrotolerant Phosphate Solubilizing <Emphasis Type="Italic">Pseudomonads</Emphasis> in the Uttarakhand Himalayas

Fourteen cold tolerant phosphate solubilizing bacteria isolated from high altitude representative locations of the two major mountain aspects of the Uttarakhand Himalayas (cooler north and warmer south facing slopes) were selected for this study. The tricalcium phosphate (TCP) solubilizing abilities of the isolates were estimated at three different incubation temperatures viz., 4, 15, and 30°C under in vitro conditions. Irrespective of their geographical origin, all the isolates recorded maximum P release values at 30°C. At 4°C, the isolates from the north facing slope were found to release significantly higher levels of P, as compared to the isolates from the south facing slopes. Alternatively at 15°C, the isolates from the south facing slope were found to release significantly higher levels of P. Initial confirmation of their genus level identity as Pseudomonads was arrived by amplification of a 990 bp fragment of the 16S rRNA gene using genus specific primers. Further putative species level identification was arrived by sequencing of the 16S rRNA gene. The diversity among the isolates was determined by rep-PCR using the primers BOX, ERIC, and ERIC2. A composite dendrogram constructed using the rep-PCR profiles revealed that the isolates from the north and south mountain aspects formed separate major clusters. The extent of diversity was greater among the isolates from the south mountain aspect. This study reveals the potential of rep-PCR in determining the genetic diversity among Pseudomonads selected for a single functional trait, but varying in their geographical origin.     

Evolution,Expression Differentiation and Interaction Specificity of Heterotrimeric G-Protein Subunit Gene Family in the Mesohexaploid Brassica rapa

Heterotrimeric G-proteins, comprising of Gα, Gβ, and Gγ subunits, are important signal transducers which regulate many aspects of fundamental growth and developmental processes in all eukaryotes. Initial studies in model plants Arabidopsis and rice suggest that the repertoire of plant G-protein is much simpler than that observed in metazoans. In order to assess the consequence of whole genome triplication events within Brassicaceae family, we investigated the multiplicity of G-protein subunit genes in mesohexaploid Brassica rapa, a globally important vegetable and oilseed crop. We identified one Gα (BraA.Gα1), three Gβ (BraA.Gβ1, BraA.Gβ2, and BraA.Gβ3), and five Gγ (BraA.Gγ1, BraA.Gγ2, BraA.Gγ3, BraA.Gγ4, and BraA.Gγ5) genes from B. rapa, with a possibility of 15 Gαβγ heterotrimer combinations. Our analysis suggested that the process of genome triplication coupled with gene-loss (gene-fractionation) phenomenon have shaped the quantitative and sequence diversity of G-protein subunit genes in the extant B. rapa genome. Detailed expression analysis using qRT-PCR assays revealed that the G-protein genes have retained ubiquitous but distinct expression profiles across plant development. The expression of multiple G-protein genes was differentially regulated during seed-maturation and germination stages, and in response to various phytohormone treatments and stress conditions. Yeast-based interaction analysis showed that G-protein subunits interacted in most of the possible combinations, with some degree of subunit-specific interaction specificity, to control the functional selectivity of G-protein heterotrimer in different cell and tissue-types or in response to different environmental conditions. Taken together, this research identifies a highly diverse G-protein signaling network known to date from B. rapa, and provides a clue about the possible complexity of G-protein signaling networks present across globally important Brassica species.     

The Jak2 Small Molecule Inhibitor,G6, Reduces the Tumorigenic Potential of T98G Glioblastoma Cells In Vitro and In Vivo

Glioblastoma multiforme (GBM) is the most common and the most aggressive form of primary brain tumor. Jak2 is a non-receptor tyrosine kinase that is involved in proliferative signaling through its association with various cell surface receptors. Hyperactive Jak2 signaling has been implicated in numerous hematological disorders as well as in various solid tumors including GBM. Our lab has developed a Jak2 small molecule inhibitor known as G6. It exhibits potent efficacy in vitro and in several in vivo models of Jak2-mediated hematological disease. Here, we hypothesized that G6 would inhibit the pathogenic growth of GBM cells expressing hyperactive Jak2. To test this, we screened several GBM cell lines and found that T98G cells express readily detectable levels of active Jak2. We found that G6 treatment of these cells reduced the phosphorylation of Jak2 and STAT3, in a dose-dependent manner. In addition, G6 treatment reduced the migratory potential, invasive potential, clonogenic growth potential, and overall viability of these cells. The effect of G6 was due to its direct suppression of Jak2 function and not via off-target kinases, as these effects were recapitulated in T98G cells that received Jak2 specific shRNA. G6 also significantly increased the levels of caspase-dependent apoptosis in T98G cells, when compared to cells that were treated with vehicle control. Lastly, when T98G cells were injected into nude mice, G6 treatment significantly reduced tumor volume and this was concomitant with significantly decreased levels of phospho-Jak2 and phospho-STAT3 within the tumors themselves. Furthermore, tumors harvested from mice that received G6 had significantly less vimentin protein levels when compared to tumors from mice that received vehicle control solution. Overall, these combined in vitro and in vivo results indicate that G6 may be a viable therapeutic option against GBM exhibiting hyperactivation of Jak2.