One novel dichromato-bridged Nickel(II) polymeric complex: formation of metal-oxygen chain structure

A novel one-dimensional Ni(II) coordination polymer [Ni(C₆H₈N₂)₂Cr₂O₇]_n (1) has been synthesised and characterised by elemental analyses, spectroscopic and single crystal X-ray diffraction study. Single crystal X-ray analysis revealed that the [Ni(C₆H₈N₂)₂]²⁺ cations are connected by μ_1,5 dichromato bridges forming infinite zig-zag chain along b-axis.     

Dimeric galectin-1 induces surface exposure of phosphatidylserine and phagocytic recognition of leukocytes without inducing apoptosis

We report that human galectin-1 (dGal-1), a small dimeric beta-galactoside-binding protein, induces phosphatidylserine (PS) exposure, measured by Annexin V staining, on human promyelocytic HL-60 cells, T leukemic MOLT-4 cells, and fMet-Leu-Phe-activated, but not resting, human neutrophils. This effect of dGal-1 on HL-60 and MOLT-4 cells is enhanced by pretreatment of the cells with neuraminidase, but treatment of resting neutrophils with neuraminidase does not enhance their sensitivity to dGal-1. Although the induction of staining with Annexin V is often associated with apoptosis, the dGal-1-treated HL-60 cells, MOLT-4 cells, and activated neutrophils do not undergo apoptosis, and there is no detectable DNA fragmentation. HL-60 and MOLT-4 cells treated with dGal-1 continue to grow normally. By contrast, camptothecin-treated HL-60 cells, etoposide-treated MOLT-4 cells, and anti-Fas-treated neutrophils exhibit extensive DNA fragmentation and/or cell death. Lactose inhibits the dGal-1-induced effects, indicating that dGal-1-induced signaling requires binding to cell surface beta-galactosides. The dimeric form of Gal-1 is required for signaling, because a monomeric mutant form of Gal-1, termed mGal-1, binds to cells but does not cause these effects. Importantly, dGal-1, but not mGal-1, treatment of HL-60 cells and activated human neutrophils significantly promotes their phagocytosis by activated mouse macrophages. These dGal-1-induced effects are distinguishable from apoptosis, but like apoptotic agents, prepare cells for phagocytic removal. Such effects of dGal-1 may contribute to leukocyte homeostasis.     

Thyroid Hormone Influences Antioxidant Defense System in Adult Rat Brain

The objective of the current study was to find out whether thyroid hormone influences antioxidant defense parameters of rat brain. Several oxidative stress and antioxidant defense parameters of mitochondrial (MF) and post-mitochondrial (PMF) fractions of cerebral cortex (CC) of adult rats were compared among euthyroid (control), hypothyroid [6-n-propylthiouracil (PTU)-challenged], and hyperthyroid (T3-treatment to PTU-challenged rats) states. Oxidative stress parameters, such as thiobarbituric acid-reactive substances (TBA-RS) and protein carbonyl content (PC), in MF declined following PTU challenge in comparison to euthyroid rats. On the other hand, when PTU-challenged rats were treated with T3, a significant increase in the level of oxidative stress parameters in MF was recorded. Hydrogen peroxide content of MF as well as PMF of CC was elevated by PTU-challenge and brought to normal level by subsequent treatment of T3. Although mitochondrial glutathione (reduced or oxidized) status did not change following PTU challenge, a significant reduction in oxidized glutathione (GSSG) level was noticed in PMF following the treatment. T3 administration to PTU-challenged rats had no effect on mitochondrial glutathione status. Total and CN-resistant superoxide dismutase (SOD) activities in MF of CC augmented following PTU challenge. CN-resistant SOD activity did not change when PTU-challenged rats were treated with T3. Although CN-sensitive SOD activity of PMF remained unaltered in response to PTU challenge, its activity increased when PTU-challenged rats were treated with T3. Catalase activity in PMF of CC of PTU-challenged rats increased, whereas the activity was decreased when hypothyroid rats were treated with T3. Similarly, total and Se-dependent glutathione peroxidase (GPx) activities of MF increased following PTU challenge and reduced following administration of T3. Se-independent GPx activity of MF and PMF and glutathione reductase activity of PMF decreased following PTU challenge and did not change further when rats were treated with T3. On the other hand, glutathione S-transferase activity of MF and PMF of CC did not change following PTU challenge but decreased below detectable level following T3 treatment. Results of the current investigation suggest that antioxidant defense parameters of adult rat brain are considerably influenced by thyroid states of the body.     

Ku heterodimer binds to both ends of the Werner protein and functional interaction occurs at the Werner N-terminus

The human Werner syndrome protein, WRN, is a member of the RecQ helicase family and contains 3′→5′ helicase and 3′→5′ exonuclease activities. Recently, we showed that the exonuclease activity of WRN is greatly stimulated by the human Ku heterodimer protein. We have now mapped this interaction physically and functionally. The Ku70 subunit specifically interacts with the N-terminus (amino acids 1–368) of WRN, while the Ku80 subunit interacts with its C-terminus (amino acids 940– 1432). Binding between Ku70 and the N-terminus of WRN (amino acids 1–368) is sufficient for stimulation of WRN exonuclease activity. A mutant Ku heterodimer of full-length Ku80 and truncated Ku70 (amino acids 430–542) interacts with C-WRN but not with N-WRN and cannot stimulate WRN exonuclease activity. This emphasizes the functional significance of the interaction between the N-terminus of WRN and Ku70. The interaction between Ku80 and the C-terminus of WRN may modulate some other, as yet unknown, function. The strong interaction between Ku and WRN suggests that these two proteins function together in one or more pathways of DNA metabolism.     

Analysis of the human replication protein A:Rad52 complex: evidence for crosstalk between RPA32, RPA70, Rad52 and DNA

The eukaryotic single-stranded DNA-binding protein, replication protein A (RPA), is essential for DNA replication, and plays important roles in DNA repair and DNA recombination. Rad52 and RPA, along with other members of the Rad52 epistasis group of genes, repair double-stranded DNA breaks (DSBs). Two repair pathways involve RPA and Rad52, homologous recombination and single-strand annealing. Two binding sites for Rad52 have been identified on RPA. They include the previously identified C-terminal domain (CTD) of RPA32 (residues 224-271) and the newly identified domain containing residues 169-326 of RPA70. A region on Rad52, which includes residues 218-303, binds RPA70 as well as RPA32. The N-terminal region of RPA32 does not appear to play a role in the formation of the RPA:Rad52 complex. It appears that the RPA32CTD can substitute for RPA70 in binding Rad52. Sequence homology between RPA32 and RPA70 was used to identify a putative Rad52-binding site on RPA70 that is located near DNA-binding domains A and B. Rad52 binding to RPA increases ssDNA affinity significantly. Mutations in DBD-D on RPA32 show that this domain is primarily responsible for the ssDNA binding enhancement. RPA binding to Rad52 inhibits the higher-order self-association of Rad52 rings. Implications for these results for the "hand-off" mechanism between protein-protein partners, including Rad51, in homologous recombination and single-strand annealing are discussed.     

Glutathione S-Transferase Isoenzymes from Streptomyces griseus

An inducible, cytosolic glutathione S-transferase (GST) was purified from Streptomyces griseus. GST isoenzymes with pI values of 6.8 and 7.9 used standard GST substrates including 1-chloro-2,4-dinitrobenzene. GST had subunit and native M_rs of 24 and 48, respectively, and the N-terminal sequence SMILXYWDIIRGLPAH.     

X-ray structure of Na-ASP-2, a pathogenesis-related-1 protein from the nematode parasite, Necator americanus, and a vaccine antigen for human hookworm infection

Human hookworm infection is a major cause of anemia and malnutrition of adults and children in the developing world. As part of on-going efforts to control hookworm infection, The Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective L3 larval stages of the parasite, including a family of pathogenesis-related (PR) proteins known as the Ancylostoma-secreted proteins (ASPs). A novel crystal structure of Na-ASP-2, a PR-1 protein secreted by infective larvae of the human hookworm Necator americanus, has been solved to resolution limits of 1.68 A and to an R-factor of 17% using the recombinant protein expressed in and secreted by Pichia pastoris. The overall fold of Na-ASP-2 is a three-layer alphabetaalpha sandwich flanked by an N-terminal loop and a short, cysteine-rich C terminus. Our structure reveals a large central cavity that is flanked by His129 and Glu106, two residues that are well conserved in all parasitic nematode L3 ASPs. Na-ASP-2 has structural and charge similarities to chemokines, which suggests that Na-ASP-2 may be an extra-cellular ligand of an unknown receptor. Na-ASP-2 is a useful homology model for NIF, a natural antagonistic ligand of CR3 receptor. From these modeling studies, possible binding modes were predicted. In addition, this first structure of a PR-1 protein from parasitic helminths may shed light on the molecular basis of host-parasite interactions.     

Contributions of Ca2+ to galectin-1-induced exposure of phosphatidylserine on activated neutrophils

Apoptotic cells redistribute phosphatidylserine (PS) to the cell surface by both Ca(2+)-dependent and -independent mechanisms. Binding of dimeric galectin-1 (dGal-1) to glycoconjugates on N-formyl-Met-Leu-Phe (fMLP)-activated neutrophils exposes PS and facilitates neutrophil phagocytosis by macrophages, yet it does not initiate apoptosis. We asked whether dGal-1 initiated Ca(2+) fluxes that are required to redistribute PS to the surface of activated neutrophils. Prolonged occupancy by dGal-1 was required to maximally mobilize PS to the surfaces of fMLP-activated neutrophils. Like fMLP, dGal-1 rapidly elevated cytosolic Ca(2+) levels in Fluo-4-loaded neutrophils. An initial Ca(2+) mobilization from intracellular stores was followed by movement of extracellular Ca(2+) to the cytosolic compartment, with return to basal Ca(2+) levels within 10 min. Chelation of extracellular Ca(2+) did not prevent PS mobilization. Chelation of intracellular Ca(2+) revealed that fMLP and dGal-1 independently release Ca(2+) from intracellular stores that cooperate to induce optimal redistribution of PS. Ca(2+) mobilization by ionomycin did not permit dGal-1 to mobilize PS, indicating that fMLP initiated both Ca(2+)-dependent and -independent signals that facilitated dGal-1-induced exposure of PS. dGal-1 elevated cytosolic Ca(2+) and mobilized PS through a pathway that required action of Src kinases and phospholipase Cgamma. These results demonstrate that transient Ca(2+) fluxes contribute to a sustained redistribution of PS on neutrophils activated with fMLP and dGal-1.     

Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity

Werner syndrome (WS) is a human premature aging disorder characterized by chromosomal instability. The cellular defects of WS presumably reflect compromised or aberrant function of a DNA metabolic pathway that under normal circumstances confers stability to the genome. We report a novel interaction of the WRN gene product with the human 5' flap endonuclease/5'-3' exonuclease (FEN-1), a DNA structure-specific nuclease implicated in DNA replication, recombination and repair. WS protein (WRN) dramatically stimulates the rate of FEN-1 cleavage of a 5' flap DNA substrate. The WRN-FEN-1 functional interaction is independent of WRN catalytic function and mediated by a 144 amino acid domain of WRN that shares homology with RecQ DNA helicases. A physical interaction between WRN and FEN-1 is demonstrated by their co-immunoprecipitation from HeLa cell lysate and affinity pull-down experiments using a recombinant C-terminal fragment of WRN. The underlying defect of WS is discussed in light of the evidence for the interaction between WRN and FEN-1.     

Evaluation of tomato genotypes for resistance to whitefly (Bemisia tabaci Gennadius) and tomato leaf curl virus in eastern India

Tomato production in the tropics is threatened by whitefly infestation and tomato leaf curl virus (ToLCV) causing severe economic losses. No stable resistance to these biotic challenges has been identified in eastern India. Therefore, initial screening of 19 advance breeding lines of tomato was carried out during the year 2016–17. Based on the whitefly population per leaf and tomato leaf curl disease severity, eight tomato genotypes were selected for final screening during the year 2017–18. Morphological leaf traits and biochemical parameters in tomato leaf were assessed in selected genotypes and considered as potential mediators of resistance. Significant variation was observed for whitefly infestation and ToLCV disease severity among the tomato genotypes tested. Higher leaf trichome density with narrow and thinner leaves and higher total phenol content in leaf emerged as reliable morphological and biochemical markers for early selection of tomato genotype resistant to whitefly and ToLCV disease. The resistance of tomato genotypes 2016/Res-1, 2015/Res-5, 2014/Res-1 and 2014/Res-4 to both whitefly and ToLCV disease is based on antixenotic properties and they could be utilized in future breeding to enhance stable resistance against these biotic challenges.