Quantitative characterization of protein tertiary motifs

Dimerization of 2-naphthalenecarbonitrile (2-NpCN) mediated by cucurbit[8]uril (CB[8]) has been investigated employing the density functional theory. Different structures of 2-NpCN dimers were generated by combining monomers in anti-head-to-head (A), anti-head-to-tail (B) and syn-head-to-tail (C) fashion. All these dimeric structures possess rigid cube-like architecture. On confinement within the CB[8] dimer A turns out to be the lowest energy structure. Calculated ¹H NMR spectra revealed that the 2-NpCN dimer exhibits large shielding for aromatic protons consistent with the experiment. The protons attached to cubane moiety on the other hand, led to down-field signals. Dimerization mediated with CB[8] cavitand is further accompanied by the frequency up-shift (blue shift) of methylene stretching vibration in its infrared spectra.

Peptidomimetic 2-cyanopyrrolidines as potent selective cathepsin L inhibitors

Cathepsins have been found to have important physiological roles. The implication of cathepsin L in various types of cancers is well established. In a search for selective cathepsin L inhibitors as anticancer agents, a series of 2-cyanoprrolidine peptidomimetics, carrying a nitrile group as warhead, were designed. Two series of compounds, one with a benzyl moiety and a second with an isobutyl moiety at P₂ position of the enzyme were synthesized. The synthesized compounds were evaluated for inhibitory activity against human cathepsin L and cathepsin B. Although, none of the compounds showed promising inhibitory activity, (E)N-{(S)1-[(S)2-cyano-1-pyrrolidinecarbonyl]-3-methylbutyl}-2,3-diphenylacrylamide (24) with an isobutyl moiety at P₂ was found to show selectivity as a cathepsin L inhibitor (Ki 5.3 μM for cathepsin L and Ki > 100 μM for cathepsin B). This compound could act as a new lead for the further development of improved inhibitors within this inhibitor type.     

Purification and partial characterization of a sperm motility-inhibitory protein of ram cauda epididymal plasma

Mammalian sperm remain quiescent but fertile for several weeks in cauda epididymis. Although several sperm quiescent factors of epididymal plasma have been identified in goat, pig and cattle; however, little is known in sheep. In the present study, purification and characterization of a novel sperm quiescent protein of ovine cauda epididymal plasma (CEP) was carried out. The sperm quiescent protein was partially purified by hydroxyapatite gel adsorption chromatography followed by DEAE-sepharose® anion exchange chromatography. In the latter, the sperm quiescent activity was eluted both in 0.05 and 0.2 M potassium phosphate buffer (pH 7.5) fractions having a predominant protein of about 80 and 70 kDa with 87% and 63% homogeneity, respectively. The proteins were designated as motility-inhibitory factor of sheep I and II (MIFS-I and II), respectively. Significant (about 60%) inhibition of sperm motility was observed following treatment of cauda epididymal sperm with 6 and 12 µg/mL of partially purified MIFS-I and II, respectively. Specific activities of the partially purified MIFS-I and II were 563 and 261 U/mg of protein, while the fold-purification of the activity were 5119 and 2373, respectively. Both the proteins were heat-labile and the activity was completely lost following incubation at 100°C for 5 min. Further, the partially purified MIFS-I (5 µg/mL) caused significant reduction in in vitro sperm capacitation and slight decline in tyrosine phosphorylated p72 and p52 proteins; however the protein was nontoxic to sperm. Mass spectrometric analysis of MIFS-I revealed significant identity with human semaphorin 3D. Both dot blot and western blot analysis demonstrated cross-reactivity of MIFS-I with polyclonal anti-human SEMA3D antibody. It was concluded that the MIFS-I of ovine CEP was putative ovine semaphorin 3D protein having potent sperm quiescent and decapacitating activities and it possibly acts through inhibition of protein tyrosine phosphorylation.     

MADD/DENN splice variant of the IG20 gene is a negative regulator of caspase-8 activation. Knockdown enhances TRAIL-induced apoptosis of cancer cells

The MADD variant of the IG20 gene is necessary and sufficient for cancer cell survival. Abrogation of MADD, but not the other IG20 splice variants, can render cancer cells more susceptible to spontaneous as well as TRAIL (tumor necrosis factor alpha-related apoptosis-inducing ligand)-induced apoptosis. Both types of apoptosis in cells devoid of MADD can be inhibited by expression of CrmA or dominant-negative FADD, thereby suggesting that endogenous MADD may be targeting caspase-8 activation. Immunoprecipitation studies showed that MADD down-modulation could lead to caspase-8 activation at the death receptors without an apparent increase in the recruitment of death-inducing signaling complex components such as FADD. Further, we found that MADD can directly interact with death receptors, but not with either caspase-8 or FADD, and can inhibit caspase-8 activation. These results clearly demonstrate the importance of MADD in the control of cancer cell survival/death and in conferring significant resistance to TRAIL-induced apoptosis. In addition, our results indicate the therapeutic potential of MADD abrogation in enhancing TRAIL-induced selective apoptosis of cancer cells.     

Statistics of catalytic domain sequences in protein kinases vs non-kinases

The catalytic regions of Protein Kinases are known to have similarity in primary chains. However, it is not known whether there is a signature profile specific to a particular catalytic region? Whether the signature profile, if any, is unique to a protein kinases family in a particular species or in a group of species? We have attempted analyzing some of these aspects by statistical data mining using an authentic and exhaustive database of Protein Kinases. The results reveal interesting features and provide some new directions to look at their applications.     

Polymerase chain reaction analysis of transgenic plants contaminated byAgrobacterium

Agrobacterium-mediated genetic transformation is a method of choice for the development of transgenic plants. The presence of latentAgrobacterium that multiplies in the plant tissue in spite of antibiotic application confounds the results obtained by polymerase chain reaction (PCR) analysis of putative transgenic plants. The presence ofAgrobacterium can be confirmed by amplification of eitherAgrobacterium chromosomal genes or genes present out of transfer DNA (T-DNA) in the binary vector. However, the transgenic nature ofAgrobacterium-contaminated transgenic plants cannot be confirmed by PCR. Here we report a simple protocol for PCR analysis ofAgrobacterium-contaminated transgenic plants. This protocol is based on denaturation and renaturation of DNA. The contaminating plasmid vector becomes double-stranded after renaturation and is cut by a restriction enzyme having site(s) within the PCR amplicon. As a result, amplification by PCR is not possible. The genomic DNA with a few copies of the transgene remains single-stranded and unaffected by the restriction enzyme, leading to amplification by PCR. This protocol has been successfully tested with 4 different binary vectors and 3Agrobacterium tumefaciens strains: EHA105, LBA4404, and GV3101.