Function of the SmpB tail in transfer-messenger RNA translation revealed by a nucleus-encoded form

Stalled bacterial ribosomes are freed when they switch to the translation of transfer-messenger RNA (tmRNA). This process requires the tmRNA-binding and ribosome-binding cofactor SmpB, a beta-barrel protein with a protruding C-terminal tail of unresolved structure. Some plastid genomes encode tmRNA, but smpB genes have only been reported from bacteria. Here we identify smpB in the nuclear genomes of both a diatom and a red alga encoding a signal for import into the plastid, where mature SmpB could activate tmRNA. Diatom SmpB was active for tmRNA translation with bacterial components in vivo and in vitro, although less so than Escherichia coli SmpB. The tail-truncated diatom SmpB, the hypothetical product of a misspliced mRNA, was inactive in vivo. Tail-truncated E. coli SmpB was likewise inactive for tmRNA translation but was still able to bind ribosomes, and its affinity for tmRNA was only slightly diminished. This work suggests that SmpB is a universal cofactor of tmRNA. It also reveals a tail-dependent role for SmpB in tmRNA translation that supersedes a simple role of linking tmRNA to the ribosome, which the SmpB body alone could provide.     

Cytotoxic and apoptotic activities of Amorphophallus campanulatus (Roxb.) Bl. tuber extracts against human colon carcinoma cell line HCT-15

Colorectal cancer is one of the leading causes of cancer death worldwide and is the third most common form of malignancy in both men and women. Several possible colon cancer chemopreventive agents are found in edible plants. Amorphophallus campanulatus (Roxb.) Blume (family: Araceae) is a tuber crop, largely cultivated throughout the plains of India for using its corm as food. This tuber has also been traditionally used for the treatment of abdominal tumors, liver diseases, piles etc. The aim of this study was to evaluate the dose-dependent cytotoxic and apoptosis inducing effects of the sub fractions of A. campanulatus tuber methanolic extract (ACME) viz. petroleum ether fraction (PEF), chloroform fraction (CHF), ethyl acetate fraction (EAF) and methanolic fraction (MEF) on the colon cancer cell line, HCT-15. Antiproliferative effects of the sub fractions of ACME were studied by MTT assay. Apoptotic activity was assessed by DAPI, Annexin V-FITC and JC-1 fluorescent staining. The chemotherapeutic drug, 5-flurouracil (5-FU) was used as positive drug control. The sub fractions of ACME significantly inhibited the proliferation of HCT-15 cells in a dose-dependent manner. In addition, the extracts were found to induce apoptosis and were confirmed by DAPI, Annexin V-FITC and JC-1 fluorescent staining. A pronounced results of cytotoxic and apoptotic activities were observed in the cells treated with 5-FU and CHF, whereas, EAF and MEF treated cells exhibited a moderate result and the least effect was observed in PEF treated cells. Our results suggested that, among the sub fractions of ACME, CHF had potent cytotoxic and apoptotic activity and thus it could be explored as a novel target for anticancer drug development. Furthermore, these findings confirm that the sub fractions of ACME dose-dependently suppress the proliferation of HCT-15 cells by inducing apoptosis.     

Synthesis and optimization of a novel series of HCV NS3 protease inhibitors: 4-Arylproline analogs

In this report we describe the synthesis and evaluation of diverse 4-arylproline analogs as HCV NS3 protease inhibitors. Introduction of this novel P2 moiety opened up new SAR and, in combination with a synthetic approach providing a versatile handle, allowed for efficient exploitation of this novel series of NS3 protease inhibitors. Multiple structural modifications of the aryl group at the 4-proline, guided by structural analysis, led to the identification of analogs which were very potent in both enzymatic and cell based assays. The impact of this systematic SAR on different drug properties is reported.     

The effect of duration and time of food availability on the photoperiodic response in the male house sparrow, Passer domesticus

We investigated the effects of food availability on the seasonal testicular growth in the photoperiodic house sparrow (Passer domesticus). Two experiments were performed, each lasting 4 weeks. In experiment 1, sparrows were exposed to natural (NDL; group 1), short (8L:16D; group 2) and long (16L:8D; groups 3-5) day lengths with access to food ad libitum (groups 1-3) or for 10 h (zeitgeber time (zt) 0-10, group 4; zt 0 is the time of light onset) or for 8 h (zt 8-16, group 5). Testes recrudesced under long, but not short and natural, day lengths, and the recrudescence under long days was influenced by the duration of food availability. In experiment 2, the sparrows were exposed to short (8L:16D, group 1) and long (16L:8D, groups 2-5) day lengths with access to food ad libitum (for groups 1 and 2) or for 6 h (for groups 3-5) at different times during the 16 h light period (group 3- zt 0-6, group 4- zt 5-11, group 5- zt 10-16). As the expected, the testes recrudesced only under long lengths, but the photoinduction was variable among the 4 groups. The testes grew to full size in groups 2 and 3 that received food either ad libitum or for 6 h at zt 0-6, but to sub-maximal size in the groups that received 6 h food either at zt 5-11 (group 4) or at zt 10-16 (group 5). Altogether, these results support the idea that the photoperiodic regulation of reproduction in a seasonally breeding species is influenced both by the duration and the time of food availability.     

Isolation and Characterization of Brassinosteroids from Leaves of Camellia sinensis (L.) O. Kuntze

Brassinosteroids are of ubiquitous occurrence in plants and elicit a wide spectrum of physiological responses. In our study, brassinosteroids were isolated and identified in topmost dormant leaves of tea plants. Six brassinosteriods, i.e. 6-deoxocastasterone, 24-epibrassinolide,3-dehydroteasterone, typhasterol, 3-deoxotyphasterol and 28-homodolicholide, were isolated and identified by GC–MS. All the brassinosteroids identified belong to important components of early and late C6 oxidation pathways proposed for brassinosteroids biosynthesis in plants. It suggests that both pathways are operating in tea to produce brassinolide, the most active brassinosteroid biologically.     

The severe acute respiratory syndrome coronavirus Nsp15 protein is an endoribonuclease that prefers manganese as a cofactor

Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has endoribonuclease activity that preferentially cleaved 5' of uridylates of RNAs. Blocking either the 5' or 3' terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn(2+) at 2 to 10 mM was needed for optimal endoribonuclease activity, but Mg(2+) and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn(2+) needed for endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus endoribonuclease.     

Atomic Structure of GRK5 Reveals Distinct Structural Features Novel for G Protein-coupled Receptor Kinases

G protein-coupled receptor kinases (GRKs) are members of the protein kinase A, G, and C families (AGC) and play a central role in mediating G protein-coupled receptor phosphorylation and desensitization. One member of the family, GRK5, has been implicated in several human pathologies, including heart failure, hypertension, cancer, diabetes, and Alzheimer disease. To gain mechanistic insight into GRK5 function, we determined a crystal structure of full-length human GRK5 at 1.8 Å resolution. GRK5 in complex with the ATP analog 5′-adenylyl β,γ-imidodiphosphate or the nucleoside sangivamycin crystallized as a monomer. The C-terminal tail (C-tail) of AGC kinase domains is a highly conserved feature that is divided into three segments as follows: the C-lobe tether, the active-site tether (AST), and the N-lobe tether (NLT). This domain is fully resolved in GRK5 and reveals novel interactions with the nucleotide and N-lobe. Similar to other AGC kinases, the GRK5 AST is an integral part of the nucleotide-binding pocket, a feature not observed in other GRKs. The AST also mediates contact between the kinase N- and C-lobes facilitating closure of the kinase domain. The GRK5 NLT is largely displaced from its previously observed position in other GRKs. Moreover, although the autophosphorylation sites in the NLT are >20 Å away from the catalytic cleft, they are capable of rapid cis-autophosphorylation suggesting high mobility of this region. In summary, we provide a snapshot of GRK5 in a partially closed state, where structural elements of the kinase domain C-tail are aligned to form novel interactions to the nucleotide and N-lobe not previously observed in other GRKs.