Functional mapping of apidaecin through secondary structure correlation

The mechanism through which apidaecin (GNNRPVYIPQPRPPHPRL) like proline rich, non-helical, antibacterial peptides penetrate into the bacterial cells is not yet clearly understood. To comprehend their transport across the bacterial cells, a detailed structure-activity correlation of apidaecin and its selected analogs is undertaken. In membrane like environment apidaecin exhibits a structural change which is mislaid in its biologically inactive P11-->Q substitution analog. This new structure, acquired by apidaecin but not by P11-->Q might be responsible for the difference in their antibacterial response. With this suggestion we explored the membrane permeation response of both by incubating them with small unilamellar vesicles (SUV). Unlike apidaecin, the P11-->Q did not induce leakage from SUV. To confirm whether this response is due to the substitution of P11-->Q of PQP motif, we chose P-ab (YVPLPNVPQPGRRPFPT), an N-terminal domain of abaecin which is homologous to apidaecin in terms of all prolines including conserved PQP, for comparison. Unlike P11-->Q but like apidaecin, P-ab also permeablized the SUV. Computational analysis also indicated that this particular mutation has a strong structural impact. These results led us to hypothesize that in bacterial environment apidaecin undergoes an ordered structural change that facilitates its entry into the bacterial membrane and also that PXP motives are important for this structural change. Apidaecin analogs not viable to organize/transform into this functionally active conformation are deleteriously affected. Adaptation to a unique conformation though insufficient (since functional binding with intracellular targets is also mandatory) seems to be an important prerequisite for the manifestation of full spectrum of antibacterial activity of apidaecin like peptides.     

In vitro Propagation and Conservation of Atropa acuminata Royle ex Lindl -An Indigenous Threatened Medicinal Plant

A micropropagation method has been developed for multiplication and conservation of Atropa acominata by induction of axillary shoot proliferation from shoot tips and nodal explants using Murashige and Skoog (MS) medium supplemented with BAP ( 1 mg I^-1) and IBA (1 mg I^-1). Revised tobacco (RT) medium with IAA (1 mg I^-1) was found most suitable for shoot elongation. Rooting was highest on full strength RT medium containing IBA (1 mg I^-1). In vitro raised plantlets were hardened and transferred to soil.     

Expression and conservation of processed copies of the RBMX gene

RBMX and RBMY are members of an ancient pair of genes located on the sex chromosomes that encode RNA-binding proteins involved in splicing. These genes have differentiated and evolved separately on the X and Y Chromosomes. RBMY has acquired a testis-specific function, whereas, as shown here, RBMX is ubiquitously expressed and is subject to X inactivation. We have also found that multiple processed copies of RBMX are present in the human genome. RBMX-like sequences (RBMXLs) located on human Chrs 1, 4, 6, 9 (9p13 and 9p24), 11, 20, and X lack introns and thus probably result from retroposition events. We found RBMXLs to be conserved in primates and great apes at corresponding chromosomal locations, indicating that they arose prior to the divergence of human. Some of the RBMXLs show insertions, deletions, and stop codons, which would probably result in nonfunctional proteins. The RBMXL on Chr 20 is deleted in some individuals. Two of the largely intact RBMXLs, located on Chrs 1 and 9p13, are expressed in different tissues and may encode novel proteins involved in splicing in a tissue-specific manner. The RBMXL located at 9p13 is specifically expressed in testis, and to a lesser extent in brain, and may therefore play a role in testis function. This autosomal, testis-specific copy of RBMX could potentially compensate for RBMX that is presumably inactivated in male germ cells, in a manner analogous to autosomal retroposed copies of other X-linked genes. Received: 19 January 2001 / Accepted: 14 March 2001     

Differential Coupling of μ-, δ-, and κ-Opioid Receptors to Gα16-Mediated Stimulation of Phospholipase C

Abstract: The μ-opioid receptor has recently been shown to stimulate phosphoinositide-specific phospholipase C via the pertussis toxin-sensitive G₁₆ protein. Given the promiscuous nature of G₁₆ and the high degree of resemblance of signaling properties of the three opioid receptors, both δ- and κ-opioid receptors are likely to activate G₁₆. Interactions of δ- and κ-opioid receptors with G₁₆ were examined by coexpressing the opioid receptors and Gα₁₆ in COS-7 cells. The δ-selective agonist [ d -Pen², d -Pen⁵]enkephalin potently stimulated the formation of inositol phosphates in cells coexpressing the δ-opioid receptor and Gα₁₆. The δ-opioid receptor-mediated stimulation of phospholipase C was absolutely dependent on the coexpression of simeter for quality control of blood units and irradiators. 13.   Transfusion 1993 ; 33 : 898 – 901 . [PubMed link] 14.   Butson MJ , Yu PK , Cheung T , et al . Dosimetry of blood irradiation with radiochromic film. Transfus Med 1999 ; 9 : 205 – 8 . [PubMed link] 15.   Nath R , Biggs PJ , Ling CC , et al . AAPM code of practice for radiotherapy accelerators: Report of AAPM Radiation Therapy Task Group No. 45. Med Phys     

Phosphorylation of Par-4 by protein kinase A is critical for apoptosis

Despite distinct dissimilarities, diverse cancers express several common protumorigenic traits. We present here evidence that the proapoptotic protein Par-4 utilizes one such common tumorigenic trait to become selectively activated and induce apoptosis in cancer cells. Elevated protein kinase A (PKA) activity noted in cancer cells activated the apoptotic function of ectopic Par-4 or its SAC (selective for apoptosis induction in cancer cells) domain, which induces apoptosis selectively in cancer cells and not in normal or immortalized cells. PKA preferentially phosphorylated Par-4 at the T155 residue within the SAC domain in cancer cells. Moreover, pharmacological-, peptide-, or small interfering RNA-mediated inhibition of PKA activity in cancer cells resulted in abrogation of both T155 phosphorylation and apoptosis by Par-4. The mechanism of activation of endogenous Par-4 was similar to that of ectopic Par-4, and in response to exogenous stimuli, endogenous Par-4 induced apoptosis by a PKA- and phosphorylated T155-dependent mechanism. Enforced elevation of PKA activity in normal cells resulted in apoptosis by the SAC domain of Par-4 in a T155-dependent manner. Together, these observations suggest that selective apoptosis of cancer cells by the SAC domain of Par-4 involves phosphorylation of T155 by PKA. These findings uncover a novel mechanism engaging PKA, a procancerous activity commonly elevated in most tumor cells, to activate the cancer selective apoptotic action of Par-4.     

Polyphosphate is involved in cell cycle progression and genomic stability in Saccharomyces cerevisiae

Polyphosphate (polyP) is a linear chain of up to hundreds of inorganic phosphate residues that is necessary for many physiological functions in all living organisms. In some bacteria, polyP supplies material to molecules such as DNA, thus playing an important role in biosynthetic processes in prokaryotes. In the present study, we set out to gain further insight into the role of polyP in eukaryotic cells. We observed that polyP amounts are cyclically regulated in Saccharomyces cerevisiae, and those mutants that cannot synthesise (vtc4Δ) or hydrolyse polyP (ppn1Δ, ppx1Δ) present impaired cell cycle progression. Further analysis revealed that polyP mutants show delayed nucleotide production and increased genomic instability. Based on these findings, we concluded that polyP not only maintains intracellular phosphate concentrations in response to fluctuations in extracellular phosphate levels, but also muffles internal cyclic phosphate fluctuations, such as those produced by the sudden demand of phosphate to synthetize deoxynucleotides just before and during DNA duplication. We propose that the presence of polyP in eukaryotic cells is required for the timely and accurate duplication of DNA.     

Identification and characterization of a novel prolyl oligopeptidase in filarial parasite Setaria cervi

A 75 kDa serine protease having prolyl oligopeptidase activity has been purified from Setaria cervi, a bovine filarial parasite. The MALDI-MS/MS analysis of the purified protein revealed 6 peptides showing nearest match S9A (prolyl oligopeptidase) family protein from Plesiocystis pacifica. The ScPOP was found to be unique compared to mammalian POP with respect to its kinetic properties. To elucidate its role, filarial parasites were exposed to specific inhibitor of POP, Z-Pro-prolinal (ZPP) for 8?h. The inhibition of POP induced calcium signaling via phospholipase c stimulation which further triggered mitochondrial mediated apoptosis in filarial parasites.