Sustained delivery of exogenous melatonin influences biomarkers of oxidative stress and total antioxidant capacity in summer-stressed anestrous water buffalo (Bubalus bubalis)

High ambient temperature during summer in tropical and subtropical countries predisposes water buffaloes (Bubalus bubalis) to develop oxidative stress having antigonadotropic and antisteroidogenic actions. Melatonin is a regulator of seasonal reproduction in photoperiodic species and highly effective antioxidant and free radical scavenger. Therefore, a study was designed to evaluate the effect of sustained-release melatonin on biomarkers of oxidative stress i.e., the serum malondialdehyde (MDA) and nitric oxide (NO), and the total antioxidant capacity (TAC). For the study, postpartum buffaloes diagnosed as summer anestrus (absence of overt signs of estrus, concurrent rectal examination, and RIA for serum progesterone) were grouped as treated (single subcutaneous injection of melatonin at 18 mg/50 kg body weight dissolved in sterilized corn oil as vehicle, n = 20) and untreated (subcutaneous sterilized corn oil, n = 8). Blood sampling for estimation of serum TAC and MDA (mmol/L) and NO (μmol/L) was carried out at 4 days of interval from 8 days before treatment till 28 days after treatment or for the ensuing entire cycle length. Results showed serum TAC concentration was higher in the treatment group with a significant (P < 0.05) increasing trend, whereas MDA and NO revealed a significant (P < 0.05) decline. Serum MDA and NO were higher in control compared with those of treatment group. Moreover, buffaloes in the treatment group showed 90% estrus induction with 18.06 ± 1.57 days mean interval from treatment to the onset of estrus. These results report that melatonin has a protective effect by elevating antioxidant status and reducing oxidative stress resulting in the induction of cyclicity in summer-stressed anestrous buffaloes.     

The tRNA A76 Hydroxyl Groups Control Partitioning of the tRNA-dependent Pre- and Post-transfer Editing Pathways in Class I tRNA Synthetase

Aminoacyl-tRNA synthetases catalyze ATP-dependent covalent coupling of cognate amino acids and tRNAs for ribosomal protein synthesis. Escherichia coli isoleucyl-tRNA synthetase (IleRS) exploits both the tRNA-dependent pre- and post-transfer editing pathways to minimize errors in translation. However, the molecular mechanisms by which tRNA^Ile organizes the synthetic site to enhance pre-transfer editing, an idiosyncratic feature of IleRS, remains elusive. Here we show that tRNA^Ile affects both the synthetic and editing reactions localized within the IleRS synthetic site. In a complex with cognate tRNA, IleRS exhibits a 10-fold faster aminoacyl-AMP hydrolysis and a 10-fold drop in amino acid affinity relative to the free enzyme. Remarkably, the specificity against non-cognate valine was not improved by the presence of tRNA in either of these processes. Instead, amino acid specificity is determined by the protein component per se, whereas the tRNA promotes catalytic performance of the synthetic site, bringing about less error-prone and kinetically optimized isoleucyl-tRNA^Ile synthesis under cellular conditions. Finally, the extent to which tRNA^Ile modulates activation and pre-transfer editing is independent of the intactness of its 3′-end. This finding decouples aminoacylation and pre-transfer editing within the IleRS synthetic site and further demonstrates that the A76 hydroxyl groups participate in post-transfer editing only. The data are consistent with a model whereby the 3′-end of the tRNA remains free to sample different positions within the IleRS·tRNA complex, whereas the fine-tuning of the synthetic site is attained via conformational rearrangement of the enzyme through the interactions with the remaining parts of the tRNA body.     

Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3′ acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.     

Genetic diversity of the threatened Chinese endemic plant,Sinowilsonia henryi Hemsi. (Hamamelidaceae), revealed by inter-simple sequence repeat (ISSR) markers

Sinowilsonia henryi Hemsi., the only representative of the monotypic genus Sinowilsonia Hemsi. (Hamamelidaceae), is a threatened plant endemic to China with high phylogenetical, ecological and economical values. In the present study, inter-simple sequence repeat (ISSR) markers were employed to investigate the genetic diversity and differentiation of 214 individuals sampled from 14 populations. Fifteen selected primers yielded a total of 178 bright and discernible bands. The genetic diversity was low at the population level (h = 0.1025; I = 0.1506; PPL = 26.7%), but quite high at the species level (h = 0.2449; I = 0.3690; PPL = 72.5%). In line with the limited gene flow (N_m = 0.3537), the hierarchical analysis of molecular variance (AMOVA) revealed pronounced genetic differentiation among populations (Φ_ST = 0.6639). Furthermore, the Mantel test revealed a significant correlation between genetic and geographic distances among populations (r = 0.688, P = 0.001), indicating the role of geographic isolation in shaping its present population genetic structure. The present patterns of genetic diversity of S. henryi were assumed to result largely from its evolutionary history and geographic factors. Based on these findings, conservation strategies were proposed to preserve this threatened plant.     

Development of PEGylated serum albumin with multiple reduced thiols as a long-circulating scavenger of reactive oxygen species for the treatment of fulminant hepatic failure in mice

Reactive oxygen species (ROS) are involved in the pathophysiology of fulminant hepatic failure. Therefore, we developed polyethylene glycol-conjugated bovine serum albumin with multiple reduced thiols (PEG-BSA-SH) for the treatment of fulminant hepatic failure. As a long-circulating ROS scavenger, PEG-BSA-SH effectively scavenged highly reactive oxygen species and hydrogen peroxide in buffer solution. PEG-BSA-SH showed a long circulation time in the plasma after intravenous injection into mice. Fulminant hepatic failure was induced by intraperitoneal injection of lipopolysaccharide and d-galactosamine (LPS/d-GalN) into mice. The LPS/d-GalN-induced elevation of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels was significantly inhibited by a bolus intravenous injection of PEG-BSA-SH. Furthermore, the changes in hepatic lipid peroxide and hepatic blood flow were effectively suppressed by PEG-BSA-SH. In contrast, l-cysteine, glutathione, and dithiothreitol, three traditional reduced thiols, had no statistically significant effects on the serum levels of ALT or AST. These findings indicate that PEG-BSA-SH is a promising ROS scavenger and useful in the treatment of fulminant hepatic failure.     

Gas exchange and resource-use patterns along a Mediterranean successional gradient

Abstract. Gas exchange, leaf-nitrogen concentration and water potential were measured in early and late spring in early successional herbaceous plants occurring after cutting and after fire, and in mature woody species from the Mediterranean climax community Quercetum ilicis in central Italy. Net photosynthesis peaked in early spring in all species studied when values for temperature and light were lower but leaf-nitrogen content was higher as compared to late spring, suggesting that nitrogen more than energy input controlled photosynt-hetic rates. Herbaceous pioneer species occurring after cutting showed higher field photo synthetic capacity than evergreen climax trees and shrubs. By contrast, net photosynthesis of herbaceous species occurring in a persistent stage after fire, was in the same range as that of climax trees. This evidence suggests that carbon-gaining appears to be partly related to the dynamic stage of succession and not solely to the growth form.     

Synthesis of (1,3,4-thiadiazol-2-yl)-acrylamide derivatives as potential antitumor agents against acute leukemia cells

A lead compound with the (1,3,4-thiadiazol-2-yl)-acrylamide scaffold was discovered to have significant cytotoxicity on several tumor cell lines in an in-house cell-based screening. A total of 60 derivative compounds were then synthesized and tested in a CCK-8 cell viability assay. Some of them exhibited improved cytotoxic activities. The most potent compounds had IC₅₀ values of 1–5 μM on two acute leukemia tumor cell lines, i.e. RS4;11 and HL-60. Flow cytometry analysis of several active compounds and detection of caspase activation indicated that they induced caspase-dependent apoptosis. It was also encouraging to observe that these compounds did not have obvious cytotoxicity on normal cells, i.e. IC₅₀ > 50 μM on HEK-293T cells. Although the molecular targets of this class of compound are yet to be revealed, our current results suggest that this class of compound represents a new possibility for developing drug candidates against acute leukemia.     

Posttranslational Regulation of Human DNA Polymerase ι

Human DNA polymerases (pols) η and ι are Y-family DNA polymerase paralogs that facilitate translesion synthesis past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the proliferating cell nuclear antigen-interacting region, the Rev1-interacting region, and its ubiquitin binding motifs UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA-damaging agents such as UV light (generating UV photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand cross-links), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via Lys¹¹- and Lys⁴⁸-linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle as no polyubiquitination was observed after treatment with rotenone or antimycin A, which both inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at Lys¹¹ and Lys⁴⁸ rather than oxidative damage per se.     

Shedding of Endogenous Interleukin-6 Receptor (IL-6R) Is Governed by A Disintegrin and Metalloproteinase (ADAM) Proteases while a Full-length IL-6R Isoform Localizes to Circulating Microvesicles

Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.