Partial cloning and sequencing of a cDNA encoding bonnet monkey (<i>Macaca radiata</i>) oviduct specific protein

900-903Based on the complete nucleic acid sequence of human estrogen dependent oviductal protein and deduced amino acid sequence, potential antigenic site of the protein was identified. Two oligonucleotide primers were designed to specifically amplify the region which includes this antigenic site. With the expectation that the human, and monkey oviductins would have high nucleotide sequence homology, Bonnet monkey oviduct along with endometrium was obtained on day 5, 9, 12 and 22 of the cycle. Using RT PCR correct sized PCR product was detected in oviduct taken from day 9 and 12 of the cycle. PCR product was cloned into pBluescript KS [+] and nucleic acid sequence determined. A 96% homology to human, Baboon and rhesus monkey estrogen induced glycoprotein, and a 84-88% homology to other mammalian oviductal protein was noted, thus confirming the authencity of cDNA clone for monkey fallopian tube specific protein

Structural and immunological characterisation of partially reduced chicken riboflavin carrier protein

166-170To improve the accessibility of highly folded chicken riboflavin carrier protein (cRCP) towards proteolytic enzymes, partial disulphide reduction of the protein was carried out. The structural and immunological alteration following reduction and alkylation of two out of the total nine disulphide bonds (2S-CMRCP) of cRCP was studied. The ability of the protein to interact with riboflavin was completely abolished with no change in its far UV, CD spectra. Monoclonal antibodies directed towards the assembled epitopes of cRCP also interacted with 2S-CMRCP to a similar extent. However, sandwich assays with the mAbs revealed that the topography of the epitopes of 2S-CMRCP is subtly altered. 2S-CMRCP was more accessible to chymotrypsin and two peptides were obtained by chymotryptic digestion of 2S-CMRCP

Fertilization, embryonic development and oviductal environment: Role of estrogen induced oviductal glycoprotein

1043-1055Mammalian oviduct is the physiological site for sperm capacitation, gamete fertilization and early embryonic development. The secretory cells lining the lumen of the mammalian oviduct synthesize and secrete high molecular weight glycoprotein (OGP) in response to estrogen. The protein has been shown to interact with gametes and early embryo. Several key functions have been postulated particularly its role in pre-implantation events which would have far reaching implications in assisted reproductive technology and in the development of non-hormonal contraceptive vaccine. The intention of this article is to discuss the current status of the protein and analyze how far the postulated function of OGP has been borne out by the available data

Development of sesbania mosaic virus nanoparticles for imaging

The capsids of viruses have a high degree of symmetry. Therefore, virus nanoparticles (VNPs) can be programmed to display many imaging agents precisely. Plant VNPs are biocompatible, biodegradable and non-infectious to mammals. We have carried out bioconjugation of sesbania mosaic virus (SeMV), a well characterized plant virus, with fluorophores using reactive lysine-N-hydroxysuccinimide ester and cysteine-maleimide chemistries. Monitoring of cellular internalization of labelled SeMV nanoparticles (NPs) by confocal microscopy and flow cytometry showed that the particles have a natural preference for entry into MDA-MB-231 (breast cancer) cells, although they could also enter various other cell lines. The fluorescence of SeMV NPs labelled via the cysteines with Cy5.5 dye was found to be more stable and was detectable with greater sensitivity than that of particles labelled via the lysines with Alexa Fluor. Live-cell imaging using SeMV internally labelled with Cy5.5 showed that it could bind to MDA-MB-231 cells in less than 5 minutes and enter the cells within 15 minutes. The particles undergo endolysosomal degradation by 6 h as evidenced by their co-localization with LAMP-1. Far-western blot analysis with a HeLa cell membrane protein fraction showed that SeMV interacts with 54-, 35- and 33-kDa proteins, which were identified by mass spectrometry as vimentin, voltage-dependent anion-selective channel protein (VDAC1), and annexin A2 isoform 2 (ANXA2), respectively, suggesting that the particles may bind and enter the cell through these proteins. The results presented here demonstrate that the SeMV NPs provide a new platform technology that could be used to develop in vivo imaging and targeted drug delivery agents for cancer diagnosis and therapy

Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles

The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the beta H-beta I loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies-D6F10 (targeting abrin), anti-a-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies