Adenoviruses as vectors for delivering vaccines to mucosal surfaces

Immunization of mucosal surfaces has become an attractive route of vaccine delivery because of its ability to induce mucosal immunity. Although various methods of inducing mucosal immunity are being developed, our laboratory has focused on developing adenoviruses as replication-competent and replication-incompetent vectors. The present report will summarize our progress in sequencing the entire bovine adenovirus-3 genome and identifying regions which can be deleted and subsequently used as insertion sites for foreign genes in developing recombinant viral vaccines. Using these recombinant viruses, we demonstrated the 'proof-of-principle' in developing mucosal immunity and, more importantly, inducing protection against bovine herpes virus in a natural host-cattle. Finally, we demonstrated that immunity and protection occurred even in animals that had pre-existing antibodies to the vector.     

SARS-CoV 9b protein diffuses into nucleus, undergoes active Crm1 mediated nucleocytoplasmic export and triggers apoptosis when retained in the nucleus

BACKGROUND: 9b is an accessory protein of the SARS-CoV. It is a small protein of 98 amino acids and its structure has been solved recently. 9b is known to localize in the extra-nuclear region and has been postulated to possess a nuclear export signal (NES), however the role of NES in 9b functioning is not well understood. PRINCIPAL FINDINGS/METHODOLOGY: In this report, we demonstrate that 9b in the absence of any nuclear localization signal (NLS) enters the nucleus by passive transport. Using various cell cycle inhibitors, we have shown that the nuclear entry of 9b is independent of the cell cycle. Further, we found that 9b interacts with the cellular protein Crm1 and gets exported out of the nucleus using an active NES. We have also revealed that this NES activity influences the half-life of 9b and affects host cell death. We found that an export signal deficient SARS-CoV 9b protein induces apoptosis in transiently transfected cells and showed elevated caspase-3 activity. CONCLUSION/SIGNIFICANCE: Here, we showed that nuclear shuttling of 9b and its interaction with Crm1 are essential for the proper degradation of 9b and blocking the nuclear export of this protein induces apoptosis. This phenomenon may be critical in providing a novel role to the 9b accessory protein of SARS-CoV.     

1,2,4-Oxadiazoles: a new class of anti-prostate cancer agents

Sulfide and sulfonyl derivatives of 1,2,4-oxadiazoles were synthesized and screened by MTT assay on the prostate cancer cells, DU-145. Six compounds were identified as potential anti-prostate cancer agents with IC(50) values ranging from 0.5 to 5.1μM. These compounds exhibited good activity on the androgen independent cells PC-3, while the results were moderate on androgen dependent LNCaP cells, suggesting the possibility of a mechanism of action different from that of the bioisosteric bicalutamide. Also a very low cytotoxicity was observed on non-cancerous cells MCF-10A.     

An intra-specific consensus genetic map of pigeonpea [Cajanus cajan (L.) Millspaugh] derived from six mapping populations

Pigeonpea (Cajanus cajan L.) is an important food legume crop of rainfed agriculture. Owing to exposure of the crop to a number of biotic and abiotic stresses, the crop productivity has remained stagnant for almost last five decades at ca. 750?kg/ha. The availability of a cytoplasmic male sterility (CMS) system has facilitated the development and release of hybrids which are expected to enhance the productivity of pigeonpea. Recent advances in genomics and molecular breeding such as marker-assisted selection (MAS) offer the possibility to accelerate hybrid breeding. Molecular markers and genetic maps are pre-requisites for deploying MAS in breeding. However, in the case of pigeonpea, only one inter- and two intra-specific genetic maps are available so far. Here, four new intra-specific genetic maps comprising 59-140 simple sequence repeat (SSR) loci with map lengths ranging from 586.9 to 881.6?cM have been constructed. Using these four genetic maps together with two recently published intra-specific genetic maps, a consensus map was constructed, comprising of 339 SSR loci spanning a distance of 1,059?cM. Furthermore, quantitative trait loci (QTL) analysis for fertility restoration (Rf) conducted in three mapping populations identified four major QTLs explaining phenotypic variances up to 24?%. To the best of our knowledge, this is the first report on construction of a consensus genetic map in pigeonpea and on the identification of QTLs for fertility restoration. The developed consensus genetic map should serve as a reference for developing new genetic maps as well as correlating with the physical map in pigeonpea to be developed in near future. The availability of more informative markers in the bins harbouring QTLs for sterility mosaic disease (SMD) and Rf will facilitate the selection of the most suitable markers for genetic analysis and molecular breeding applications in pigeonpea.     

Effect of coenzymes on the quaternary structure conformation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Kinetics of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle have been studied under different pH conditions in the absence and presence of various concentrations of NAD+ and NADH. The data have been discussed with respect to the effect of the coenzymes on the quaternary structure symmetry of the two enzymes and their binding isotherms. Both the (homo-tetrameric) apo-enzymes exhibit biphasic kinetics of thermal inactivation, characteristic of C2 symmetry, at lower pH values and a single exponential decay of enzyme activity, characteristic of D2 symmetry, at higher pHs. In each case, NAD+ has no effect on the biphasic kinetic pattern of thermal inactivation at lower pH values, but NADH brings about a change to single exponential decay. At higher pH values, NADH does not affect the kinetic pattern (single exponential decay) of any enzyme, but NAD+ alters it to biphasic kinetics in each case. The data suggest that NAD+ and NADH have higher affinity for the C2 and D2 symmetry conformation, respectively. With mung beans enzyme, the effect of NAD+ on the two rate constants of biphasic inactivation at pH 7.3 is consistent with a Kdiss equal to 110 microM. The NAD(+)-dependent changes in the kinetic pattern of thermal inactivation of this enzyme at pH 8.6 suggest a positive cooperativity in the coenzyme binding (nH = 3.0). In the binding of NADH to the mung beans enzyme, a weak positive cooperativity is observed at pH 7.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine adenovirus‐3 protein VIII associates with eukaryotic initiation factor‐6 during infection

Adenovirus protein VIII appears to connect core with the inner surface of the adenovirus capsid. Because protein–protein interactions are central to virus replication, identification of proteins interacting with protein VIII may help in understanding their role in adenovirus infection. Our yeast 2‐hybrid assay indicated that protein VIII interacts with eukaryotic initiation factor 6 (eIF6). These findings were confirmed by Glutathione S‐transferase‐pull down assay, bimolecular fluorescent complementation assay, and coimmunoprecipitation assay in plasmid DNA transfected and bovine adenovirus‐3 (BAdV‐3) infected cells. The C‐terminus amino acids 147 to 174 of protein VIII and N‐terminus amino acids 44 to 97 of eIF6 are involved in these interactions. Polysome analysis demonstrated increased level of 60S ribosomal subunit and decreased level of 80S complex in protein VIII expressing cells or BAdV‐3 infected cells. Our results suggest that formation of functional 80S ribosome appears impaired in the presence of protein VIII at late times post infection. We speculate that this impaired ribosome assembly may be responsible for the inhibition of cellular mRNA translation observed late in adenovirus infected cells. Moreover, analysis of recombinant BAdV‐3 expressing mutant protein VIII (deletion of eIF6 interacting domain) suggests that interaction of protein VIII and eIF6 may help in preferential translation of adenovirus genes during late phase of adenovirus infection.     

Incidence of parasites ofLymantria obfuscata [Lymantriidae: Lepidoptera] in Kashmir

Incidence of parasites ofLymantria obfuscata Walker [Lymantriidae: Lepidoptera] was studied in Kashmir during 1983 and 1984. The only egg parasite reared wasAnastatus kashmirensis Mathur parasitising between 4.49 to 11.92 percent of eggs. From 15 study sites as many as 10475 larvae of different stages and pupae were collected and reared in laboratory.Exorista rossica [Tachinidae: Diptera] was recorded to parasitize upto 8.42 per cent of larvae compared to 0.89 percent byCompsilura sp.Tetrastichus sp. [Eulophidae: Hymenoptera] was observed to be most dominant of the 6 pupal parasites, accounting for 33.41 percent of the measured parasitism, followed byPimpla sp. [Ichneumonidae: Hymenoptera] andTheronia atalantae atalantae [Ichneumonidae: Hymenoptera] which parasitized 6.84 and 4.03 per cent of pupae respectively.Brachymeria intermedia Nees [Chalcididae: Hymenoptera] was recorded to parasitize upto 2.98 per cent whereasBrachymeria lasus Walker recorded for the first time in Kashmir was found to parasitize up to 2.01 per cent, but was not widely distributed in the State.     

Application of SSR markers for molecular characterization of hybrid parents and purity assessment of ICPH 2438 hybrid of pigeonpea [<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millspaugh]

With an objective of achieving a breakthrough in the productivity of pigeonpea, a hybrid breeding technology based on elements of the cytoplasmic-nuclear male-sterility (CMS) system and partial natural out-crossing has recently been developed. However, there is no molecular diversity information available on parental lines of hybrids being generated at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT). This study deals with the use of 148 simple sequence repeat (SSR) markers, including 32 novel markers reported here for the first time, on 159 A (cytoplasmic male sterile), B (maintainer) and R (fertility restorer) lines. In total, 41 (27.7%) markers showed polymorphism with 2 to 6 (average 2.6) alleles and 0.01 to 0.81 (average 0.34) polymorphism information content (PIC) value. Of these polymorphic markers, 22 SSR markers showed polymorphism between A (ICPA 2039) and R (ICPR 2438) lines of the commercial hybrid (ICPH 2438); however, only 21 of these SSR markers showed the same profile between A (ICPA 2039) and B (ICPB 2039) lines. Finally, two SSR markers, CCB4 and CCttc006, were found most suitable for purity assessment of hybrid seeds of the ICPH 2438 hybrid. The utility of these two diagnostic SSR markers has been demonstrated by using seed lots of this hybrid from two sources, ICRISAT and Mahabeej. It is anticipated that molecular diversity information generated on parental lines of hybrids under development, and identification of the two most suitable markers for testing the purity of hybrid seeds of ICPH 2438, will facilitate the pigeonpea hybrid breeding programme.     

Atbarapur (Hoshiarpur district,Punjab), the Acheulian of the Siwalik Range within the South Asian context

The largest collection of Acheulian artefacts in the Siwalik region is from the site of Atbarapur in north-western India. The artefacts occur in reworked sediments of the Pinjore Formation, starting with the onset of the Pleistocene and continuing at places in this region till 0.6 Ma. The technical study shows two similar “chaînes opératoires”: one based on cobbles for making small flakes and the second based on boulders for large flakes. Both are short and simple: cores are not prepared and each of them produced about seven flakes. Handaxes and cleavers, typical Acheulian tools, are made on the large flakes, often struck from the ventral face of larger flakes (Kombewa method) or from split boulders. The technology compares well with the Lower Pleistocene Acheulian of peninsular India, but with slightly more refined bifaces. It also compares with assemblages from Africa and East Asia: Atbarapur stands as a milestone on the diffusion route(s) of the Acheulian.