Expression and purification of Dengue virus type 2 envelope protein as a fusion with hepatitis B surface antigen in Pichia pastoris

The methylotrophic yeast, Pichia pastoris, has been used as a host to express the envelope protein (Den2E) of dengue type 2 virus (NGC strain) as a chimera with hepatitis B surface antigen (HBsAg): a protein known to self assemble into virus-like particles (VLPs) and to be efficiently expressed in P. pastoris. The Den2E gene used in this study is a truncated version encoding the first 395 amino acid (aa) residues of the mature Den2E protein; the HBsAg gene encodes the full length 226 aa HBsAg protein. Two in-frame gene fusions were constructed for intracellular expression in P. pastoris. The first one contains the HBsAg gene as the 5' partner and the Den2E gene as the 3'partner (HBsAg-Den2E). In the second one, the relative positions of the two partners of the gene fusion were reversed to create the hybrid Den2E-HBsAg gene. These fusion genes were integrated into the genome of P. pastoris under the control of the methanol-inducible alcohol oxidase (AOX1) promoter. Of the two fusions, the Den2E-HBsAg gene was expressed at higher levels in P. pastoris based on Northern analysis. The hybrid protein ( approximately 68 kDa) expressed by this clone was purified to near homogeneity using a combination of acid precipitation, hydrophobic interaction, and immunoaffinity chromatographic steps. Final purification achieved was approximately 1400-fold with a yield of approximately 26%. The chimeric protein was found to possess the ability to assemble into high molecular weight aggregates (akin to HBsAg particles). The recombinant fusion protein eluted close to the void volume of a Sepharose CL-4B column indicating its macromolecular nature. On a CsCl density gradient the recombinant fusion protein sedimented to a position very similar to that of HBsAg VLPs. The hybrid protein is recognized by the two neutralizing monoclonals against the two components of the chimeric protein.     

DNA amounts and chromatin compactness in Vicia

2C DNA amounts and areas of chromatin were determined with a M 86 Vickers microdensitometer in 56 species of Vicia (x=5, 6, 7), exhibiting large differences in chromosome size. There were significant differences between the species both in DNA content and chromatin area. The nuclear DNA amounts range from 3.85 to 27.07 pg. DNA distribution appears discontinuous; species cluster into distinct groups and the average nuclear DNA amount separating each successive pair is approximately the same (2.23 pg). The compaction of DNA in interphase nuclei increases with increasing DNA amount, which is, at least partly, due to a disproportionate increase in the heterochromatin relative to the euchromatin component of DNA. Comparisons of DNA readings at various stages of the cell cycle show that the DNA amounts are underestimated by microdensitometry in nuclei with high DNA density. Estimation of relative DNA content and area of individual chromosomes were made in twelve species. The results show that changes in DNA content within chromosomes affect the degree of metaphase coiling in an orderly fashion.     

Associated chromosomal DNA changes in polyploids.

The 2C and 4C nuclear DNA amounts were estimated in eight diploid species, belonging to three diverse genera (Vicia, Tephrosia, and Phlox) and their corresponding colchitetraploids. In P. drummondii, T. purpurea, and T. oxygona tetraploids the deviation from the expectation was highly significant. The DNA in P. drummondii was further discarded in subsequent (C1, C2) generations, thus attaining an overall reduction of about 25%. The DNA content in the subsequent generations was the same as that of C2. It is concluded that rapid DNA loss in the first and subsequent generations was not only associated with the substantial increase (30-66%) in the seed set, but it also helped in the establishment and stabilization of the tetraploid. The possible relationship between such a nucleotypic change and success of polyploids is discussed. The DNA change from the expected value in the P. drummondii tetraploid was achieved by equal decrement to each chromosome independent of size, i.e., small chromosomes loose the same amount of DNA as the large chromosomes.     

Liposomal delivery of Mycobacterium leprae antigen(s) with murabutide and Trat peptide inhibits Fas-mediated apoptosis of peripheral blood mononuclear cells derived from leprosy patients

Protective immunity against intracellular pathogen Mycobacterium leprae is dependent on the activation of T cells. Repeated stimulation of T cells by M. leprae antigens MLCwA (M. leprae total cell wall antigen) and ManLAM (mannose capped lipoarabinomannan) may lead to apoptosis in leprosy patients. In the present study, inhibition of the Fas-induced apoptosis of peripheral blood mononuclear cells of leprosy patients was investigated using above M. leprae antigen(s), in combination with immunomodulators murabutide (MB) and a Trat peptide in particulate form (liposome). Incubation of the cells with particulate mode of antigen presentation led to both decreased percentage of propidium iodide (PI) positive cells and T cells expressing Fas-FasL, as well as decreased caspase-8/-3 activities in the lepromatous patients, thereby inhibiting apoptosis, while converse was true with stimulation with soluble antigen. Concurrently, there was an upregulation of antiapoptotic protein Bcl-X(L) in the lepromatous patients, thereby inhibiting apoptosis. Thus, the liposomal formulation of antigen promoted proliferation of anergized T cell by inhibiting apoptosis through decreased expression of death receptors and caspase activities and increased expression of anti-apoptotic protein Bcl-X(L) in these patients.     

Review Article Number 138: Constituents of the yew trees

Yew trees, taxonomically classified under the genus Taxus, are sources of a number of physiologically active compounds of different classes. Taxane derivatives with various carbon skeletons, lignans, flavonoids, steroids and sugar derivatives have been isolated from different Taxus species. Compounds isolated from the genus Taxus between 1908 and December 1997 have been comprehensively reviewed.     

Fungicidal management of wilt complex of scented geranium

Systemic fungicides, carbendazim + mancozeb and carbendazim alone completely inhibited the mycelia growth even at 100 and 200 ppm, whereas among non-systemic fungicides, thiram was found to be the most effective and gave 52.77, 62.77 and 85.00% inhibition at 500, 1000 and 1500 ppm concentrations, respectively. Thiram was found to be most effective fungicides in inhibiting the both fungi (68.84 and 58.69%) followed by the indofil M 45 (47.59 and 51.38%). In pot house condition, carbendazim + mancozeb (0.2%) and carbendazim (0.1%) were most effective fungicides in reducing cent percent disease incidence and severity, but it was at par with thiram (82.35; 87.50). In field condition, mulching with transparent polyethylene mulch (25 μm) resulted in highest survival rate (95.83%) and least mortality rate (4.16%) of rooted geranium cuttings after 30 days of germination. The application of carbendazim (Bavistin) at 0.1% when applied individually was also found almost complete control as is evident from low disease incidence (1.04%) and severity (0.52%), considered to be second best treatment. Apparent infection rate (0.0) was observed in carbendazim + mancozeb and carbendazim followed by copper oxychloride (0.006) and carboxin (0.009). Area under disease progress curve was less than 100 in cabendazim + mancozeb and carbendazim.