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.     

Entry of bovine viral diarrhea virus into ovine cells occurs through clathrin-dependent endocytosis and low pH-dependent fusion

Although mechanisms of bovine viral diarrhea virus (BVDV) entry into bovine cells have been elucidated, little is known concerning pestivirus entry and receptor usage in ovine cells. In this study, we determined the entry mechanisms of BVDV-1 and BVDV-2 in sheep fetal thymus cells. Both BVDV-1 and BVDV-2 infections were inhibited completely by chlorpromazine, β-methyl cyclodextrin, sucrose, bafilomycin A1, chloroquine, and ammonium chloride. Simultaneous presence of reducing agent and low pH resulted in marked loss of BVDV infectivity. Moreover, BVDV was unable to fuse with ovine cell membrane by the presence of reducing agent or low pH alone, while combination of both led to fusion at low efficiency. Furthermore, sheep fetal thymus cells acutely infected with BVDV-1 or BVDV-2 were found protected from heterologous BVDV infection. Taken together, our results showed for the first time that entry of both BVDV-1 and BVDV-2 into ovine cells occurred through clathrin-dependent endocytosis, endosomal acidification, and low pH-dependent fusion following an activation step, besides suggesting the involvement of a common ovine cellular receptor during attachment and entry.     

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.     

Mercuric chloride inhibits the in vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells

Glutamate is removed mainly by astrocytes from the extracellular fluid via high-affinity astroglial Na⁺-dependent excitatory amino acid transporters, glutamate/aspartate transporter (GLAST), and glutamate transporter-1 (GLT-1). Mercuric chloride (HgCl₂) is a highly toxic compound that inhibits glutamate uptake in astrocytes, resulting in excessive extracellular glutamate accumulation, leading to excitotoxicity and neuronal cell death. The mechanisms associated with the inhibitory effects of HgCl₂ on glutamate uptake are unknown. This study examines the effects of HgCl₂ on the transport of ³H-d-aspartate, a nonmetabolizable glutamate analog, using Chinese hamster ovary cells (CHO) transfected with two glutamate transporter subtypes, GLAST (EAAT1) and GLT-1 (EAAT2), as a model system. Additionally, studies were undertaken to determine the effects of HgCl₂ on mRNA and protein levels of these transporters. The results indicate that (1) HgCl₂ leads to significant (p<0.001) inhibition of glutamate uptake via both transporters, but is a more potent inhibitor of glutamate transport via GLAST and (2) the effect of HgCl₂ on inhibition of glutamate uptake in transfected CHO cells is not associated with changes in transporter protein levels despite a significant decrease in mRNA expression; thus, (3) HgCl₂ inhibition is most likely related to its direct binding to the functional thiol groups of the transporters and interference with their uptake function.     

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.     

Utility of thiol-cross-linked fluorescent dye labeled terminators for DNA sequencing

Dipivaloyl-5-carboxyfluorescein N-hydroxysuccinimidyl ester 1 and 5-propargylamino-2',3'-dideoxyuridine triphosphate 5 were modified with maleimide, haloacetamide, and sulfhydryl reactive functional groups to participate in cross-conjugation reactions via sulfide bonds to generate fluorescently labeled, thioether cross-conjugated terminators 10 and 11. Their DNA sequencing potential was compared with an amide cross-conjugated terminator 13, synthesized by directly coupling 5-carboxyfluorescein NHS ester with 18-ddUTP 9. These terminators (10, 11, and 13) in combination with the Thermo Sequenase II DNA polymerase, in thermal cycle sequencing experiments, revealed that the thioether cross-conjugated terminator 10 and amide cross-conjugated terminator 13 served as good terminating substrates, generating satisfactory single-color gel images and electropherograms, while the other thioether cross-conjugated and maleimide derived one 11 underwent unexpected pH and temperature induced decomposition without showing fluorescent signatures for incorporation.     

Development and validation of multiplex-PCR assay for simultaneous detection of rare alleles of <Emphasis Type="Italic">crtRB1</Emphasis> and <Emphasis Type="Italic">lcyE</Emphasis> governing higher accumulation of provitamin A in maize

Vitamin A deficiency is a widely prevalent health disorder among millions of people worldwide. Introgression of crtRB1 and lcyE favourable alleles that enhance concentration of provitamin A in maize endosperm have been employed in maize biofortification programmes. To make marker-assisted selection (MAS) more effective, we have developed rapid and convenient multiplex-polymerase chain reaction (PCR) assay to simultaneously discover the allelic combinations among the segregants. Validation of the multiplex assay was done in two backcross-derived populations developed using elite inbreds viz., HKI193-1 and HKI193-2 carrying unfavourable alleles of crtRB1 (296 bp) and lcyE (300 bp) and HarvestPlus inbreds viz., HP704-22 and HP704-23 possessing favourable alleles of crtRB1 (543 bp) and lcyE (650 bp). We also standardized the uniplex-PCR assays for both the genes that gave robust and reproducible results in sub-tropical populations. Gel profiles of BC₁F₁, BC₂F₁ and BC₂F₂ revealed that these assays identified the backcross progenies homo-or hetero-zygous for the favourable- or unfavourable-alleles. Multiplex-PCR assay also precisely confirmed the results of individual uniplex assays in different backcross generations. Cost and time analyses showed that multiplex-PCR assay has potential to save 41% of cost, and 50% of time compared to two uniplex assays in a MAS programme. It has also saved 50% of the manpower. The multiplex assay possesses significant advantage over uniplex assays and enhances the efficiency of selection. This is the first report of development and validation of multiplex-PCR assay of crtRB1 and lcyE for utilization in maize biofortification programme.