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.     

Growth patterns of early versus late successional multipurpose trees of the western Himalaya

In the mid-western Himalaya (altitude 1350 m, rainfall 1100 mm), multipurpose trees found as escapees in agricultural fields or naturally growing in the forests, play an important role in providing fuel, fooder and small timber to the farmers. Shoot elogation, and tree architecture of 4 year old trees of Grewia optiva, Robinia pseudoacacia and Celtis australis (early successionals), and Quercus leucotrichophora, Q. glauca and Ilex odorata (late successionals), were analyzed. All the late successional species showed a proleptic type of bud and branch production, while the early successional trees made growth through syllepsis. The shoot elongation differed significantly (P <0.05) with the crown position, and ranged from 11 to 30 cm in different species. Early successional species tended to maintain a comparatively narrow crown and showed a significantly (P <0.05) higher ramification ratio, and multilayered canopy. The late successionals, in contrast, showed a wide crown with monolayered canopy, adapted to the weak light intensity. There was only one flush of leaves in Q. leucotrichophora and Q. glauca while in the rest of the species there were two distinct flush periods. The results are important for the management of agroforestry trees.     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.     

Mapping of rDNA on the chromosomes of Eleusine species by fluorescence in situ hybridization

Mapping of rDNA sites on the chromosomes of four diploid and two tetraploid species of Eleusine has provided valuable information on genome relationship between the species. Presence of 18S-5.8S-26S rDNA on the largest pair of the chromosomes, location of 5S rDNA at four sites on two pairs of chromosomes and presence of 18S-5.8S-26S and 5S rDNA at same location on one pair of chromosomes have clearly differentiated E. multiflora from rest of the species of Eleusine. The two tetraploid species, E. coracana and E. africana have the same number of 18S-5.8S-26S and 5S rDNA sites and located at similar position on the chromosomes. Diploid species, E. indica, E. floccifolia and E. tristachya have the same 18S-5.8S-26S sites and location on the chromosomes which also resembled with the two pairs of 18S-5.8S-26S rDNA locations in tetraploid species, E. coracana and E. africana. The 5S rDNA sites on chromosomes of E. indica and E. floccifolia were also comparable to the 5S rDNA sites of E. africana and E. coracana. The similarity of the rDNA sites and their location on chromosomes in the three diploid and two polyploid species also supports the view that genome donors to tetraploid species may be from these diploid 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.     

Chemical fingerprinting and antibacterial activity of Saussurea lappa clarke

Costunolide and dehydrocostus lactone of Saussurea lappa are the active compounds having various biological activities used in medicine. HPLC and HPTLC were used for chemical profiling of S. lappa samples collected from different geographical regions of Uttarakhand, India. Costunolide was found to be 0.19 to 0.39% and 0.25 to 0.56% while dehydrocostus lactone was reported in the ranges of 0.27 to 0.70 and 0.50 to 1.06% by HPLC and HPTLC, respectively. Antibacterial activity of methanol extracts of S. lappa was evaluated in order to compare the effects of active constituents against gram positive and negative bacteria. It was determined by well diffusion method. S. lappa exhibited inhibitory effect on bacterial strains with the MICs ranging from 3.12 to 12.50 μg/μL for Escherichia coli and Citrobacter freundii, from 6.25 to 25.00 μg/μL for Enterococcus faecalis and from 6.25 to 50.00 μg/μL for Staphylococcus aureus. It was observed that the samples of S. lappa containing the higher contents of costunolide and dehydrocostus lactone showed the higher antibacterial activity. The results demonstrated that concentrations of both active constituents depended on altitude at which the collected plants were located.     

Interaction studies of novel cell selective antimicrobial peptides with model membranes and E. coli ATCC 11775

Cationic antimicrobial peptides (CAMPs) are novel candidates for drug development. Here we describe design of six short and potent CAMPs (SA-1 to SA-6) based on a minimalist template of 12 residues H+HHG+HH+HH+NH2 (where H: hydrophobic amino acid and +: charged hydrophilic amino acid). Designed peptides exhibit good antibacterial activity in micro molar concentration range (1-32 μg/ml) and rapid clearance of Gram-positive and Gram-negative bacterial strains at concentrations higher than MIC. For elucidating mode of action of designed peptides various biophysical studies including CD and Trp fluorescence were performed using model membranes. Further based on activity, selectivity and membrane bound structure; modes of action of Trp rich peptide SA-3 and template based peptide SA-4 were compared. Calcein dye leakage and transmission electron microscopic studies with model membranes exhibited selective membrane active mode of action for peptide SA-3 and SA-4. Extending our work from model membranes to intact E. coli ATCC 11775 in scanning electron micrographs we could visualize different patterns of surface perturbation caused by peptide SA-3 and SA-4. Further at low concentration rapid translocation of FITC-tagged peptide SA-3 into the cytoplasm of E. coli cells without concomitant membrane perturbation indicates involvement of intracellular targeting mechanism as an alternate mode of action as was also evidenced in DNA retardation assay. For peptide SA-4 concentration dependent translocation into the bacterial cytoplasm along with membrane perturbation was observed. Establishment of a non specific membrane lytic mode of action of these peptides makes them suitable candidates for drug development.     

Genome-scale metabolic reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> for strain improvement

Background  

Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.