Efficient,one step and cultivar independent shoot organogenesis of potato

An efficient, one step and genotype independent protocol of shoot organogenesis was developed from leaf and internodal explants taken from microshoots of different cultivars of potato (Solanum tuberosum L.). Initially, microshoots were cultured on basal Murashige and Skoog medium additionally supplemented with 10 µM AgNO₃ (MS1 medium) to achieve healthy shoot growth required to get the quality explants. Shoot organogenesis was induced from both types of explants (leaf and internodal) on MS1 medium variously supplemented with 6-benzyladenine (BA) and gibberellic acid (GA₃). Maximum explants were induced shoot organogenesis on MS1 medium supplemented with 10 µM BA and 15.0 µM GA₃ from both the cultivars namely ‘Kufri Chipsona 1’ and ‘Kufri Pukhraj’. Among the types of explants used, better response was observed from internodal segments as compared to leafs. This optimized medium combination was found to be equally effective for all the eight cultivars tested namely ‘Kufri Pukhraj’, ‘Kufri Chipsona 1’, ‘Kufri Chipsona 2’, ‘Kufri Jyoti’, ‘Kufri Surya’, ‘Kufri Chandramukhi’, ‘Kufri Khyati’ and ‘Desiree’. The clonal uniformity of the regenerated shoots was confirmed using random amplified polymorphic DNA and inter-simple sequence repeats markers.     

Reactive Oxygen Species Activity and Antioxidant Properties of Fusarium Infected Bananas

Fusarium infection of bananas is a global problem that threatens the production of bananas. This study looks at the effects of the infection upon the reactive oxygen species (ROS) system, as well as the induced antioxidant properties in the roots, stems, leaves and fruits. Results show that there is a greater amount of damage in infected tissue samples as opposed to non‐infected. The damage was observed to be higher in the root samples. ROS assays were divided into two classes: ROS assays and ROS‐scavenging assays. Of the ROS assays, lipoxygenase was observed to be higher in the infected samples, while peroxidase (POD) and polyphenol oxidase (PPO) were significantly higher in infected stem, leaf and fruit samples. Among root samples, there was no significant difference in POD activity and PPO was lower in infected samples. Induction of ROS is important for the hypersensitive response (HR) to function properly. The ROS‐scavenging enzymes, namely ascorbate peroxidase, guaiacol peroxidase and superoxide dismutase, exhibited higher levels in the infected tissue. This is most likely to counter the build‐up of the ROS enzymes and to prevent further cell death. The increase in ROS‐scavenging assays also correlates with higher antioxidant properties as antioxidants play a critical role in regulating the HR free radicals.     

Impact of single nucleotide polymorphisms in the OGG1 and XRCC1 genes on modulation of DNA damage in pesticide-exposed agricultural workers in Punjab,North-West India

Abstract

Pesticide-induced DNA damage is primarily repaired by base excision repair (BER) pathway. However, polymorphism in DNA repair genes may modulate individual’s DNA repair capacity (DRC) leading to increased genotoxicity and adverse health effects. Our first study in North-West Indian population aimed to evaluate the impact of OGG1 rs1052133 (Ser326Cys; C1245G), XRCC1 rs1799782 (Arg194Trp; C26304T) and XRCC1 rs25487 (Arg399Gln; G28152A) polymorphisms on the modulation of pesticide-induced DNA damage in a total of 450 subjects (225 pesticide-exposed agricultural workers and 225 age- and sex-matched controls). DNA damage was estimated by alkaline comet assay using silver-staining method. Genotyping was carried out by PCR-RFLP using site-specific restriction enzymes. Mann-Whitney U-test revealed elevation in DNA damage parameters (p?<?0.01) in pesticide-exposed agricultural workers than controls. Chi-square test showed significant (p?<?0.05) differences in the XRCC1 Arg194Trp (C26304T) and Arg399Gln (G28152A) genotypes among two groups. Multivariate logistic-regression analysis revealed that heterozygous genotypes of OGG1 rs1052133 (326Ser/Cys; 1245CA), XRCC1 rs1799782 (194Arg/Trp; 26304CT) and XRCC1 rs25487 (399Arg/Gln; 2815GA) were positively associated (p?<?0.05) with elevated DNA damage parameters in pesticide-exposed agricultural workers. Our results strongly indicate significant positive association of variant OGG1 and XRCC1 genotypes with reduced DRC and higher pesticide-induced DNA damage in North-West Indian agricultural workers.     

Agonist-activated glucagon receptors are deubiquitinated at early endosomes by two distinct deubiquitinases to facilitate Rab4a-dependent recycling

The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein–coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule–binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.     

Characterization of extracellular proteins produced by Aeromonas hydrophila AH-1

Aeromonas hydrophila is a ubiquitous Gram-negative bacterium which can cause motile aeromonad septicemia in both fish and humans. A. hydrophila secretes many extracellular proteins associated with pathogenicity and environmental adaptability. In this study, an extracellular proteome map of A. hydrophila AH-1 was constructed. The major extracellular virulence factors were characterized by comparing the proteomes of various deletion mutants with that of the wild type. The results suggested that serine protease was involved in the processing of a toxin and secreted enzymes such as hemolysin, glycerophospholipid-cholesterol acyltransferase and metalloprotease. We also showed that expressions of polar and lateral flagellins were under the control of temperature, FlhA, LafK, and RpoN. In addition, three novel proteins (potential effector proteins including one ExoT-like protein) were revealed to be secreted via the type III secretion system (TTSS) of A. hydrophila AH-1. Another novel finding was the demonstration of a crosstalk between the lateral flagellar system and the TTSS in A. hydrophila. These results showed that proteomics is a powerful tool for characterizing virulence factors. The construction of proteome maps will provide a valuable means of finding potential candidates for developing suitable diagnostics and therapeutics for this emerging pathogen.     

Inhibitory effect of curcumin and its natural analogues on genotoxicity of heterocyclic amines from cooked food

Curcumin (C) and its natural analogues demethoxycurcumin (dmC) and bisdemethoxycurcumin (bdmC), known for their potent anti-inflammatory, antioxidant, antimutagenic and anticarcinogenic effects, were tested for their possible inhibitory effects against seven cooked food mutagens (heterocyclic amines): 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), in both TA98 and TA100 strains of Salmonella typhimurium using Ames Salmonella/reversion assay in the presence of Aroclor induced rat liver S9 homogenate. In the present investigations, curcumin as well as its two natural analogues i.e., dmC and bdmC were found to be highly effective in suppressing genotoxicity of all the tested cooked food mutagens in a dose-dependent manner, in both the frame shift (TA98) as well as base pair mutation sensitive (TA100) strains of S. typhimurium. However, bdmC appeared to be a relatively less active antimutagen compared to C and dmC. More than 80% inhibition of mutagenicity was observed at 200 microg/plate in case of C and dmC in both TA98 and TA100 against all tested cooked food mutagens. Where as, bdmC showed 39-79% inhibition in TA100 and 60-80% inhibition in TA98, at a dose of 200 microg/plate. These findings warrant further biochemical, enzymatic and in vivo investigations in animal models as well as in humans to establish the chemoprotective effect of these agents against mutagenic heterocyclic amines found in cooked food.     

Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides

Natural killer (NK) cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. We previously reported that the binding of a common MHC class I molecule in the rhesus macaque, Mamu-A1*002, to the inhibitory receptor Mamu-KIR3DL05 is stabilized by certain simian immunodeficiency virus (SIV) peptides, but not by others. Here we investigated the functional implications of these interactions by testing SIV peptides bound by Mamu-A1*002 for the ability to modulate Mamu-KIR3DL05⁺ NK cell responses. Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 suppressed the cytolytic activity of primary Mamu-KIR3DL05⁺ NK cells, including three immunodominant CD8⁺ T cell epitopes previously shown to stabilize Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. Substitutions at C-terminal positions changed inhibitory peptides into disinhibitory peptides, and vice versa, without altering binding to Mamu-A1*002. The functional effects of these peptide variants on NK cell responses also corresponded to their effects on Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. In assays with mixtures of inhibitory and disinhibitory peptides, low concentrations of inhibitory peptides dominated to suppress NK cell responses. Consistent with the inhibition of Mamu-KIR3DL05⁺ NK cells by viral epitopes presented by Mamu-A1*002, SIV replication was significantly higher in Mamu-A1*002⁺ CD4⁺ lymphocytes co-cultured with Mamu-KIR3DL05⁺ NK cells than with Mamu-KIR3DL05^- NK cells. These results demonstrate that viral peptides can differentially affect NK cell responses by modulating MHC class I interactions with inhibitory KIRs, and provide a mechanism by which immunodeficiency viruses may evade NK cell responses.     

The structural basis of chain length control in Rv1086

In Mycobacterium tuberculosis, two related Z-prenyl diphosphate synthases, E,Z-farnesyl diphosphate synthase (Rv1086) and decaprenyl diphosphate synthase (Rv2361c), work in series to synthesize decaprenyl phosphate (C₅₀) from isopentenyl diphosphate and E-geranyl diphosphate. Decaprenyl phosphate plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan; thus, its synthesis has attracted considerable interest as a potential therapeutic target. Rv1086 is a unique prenyl diphosphate synthase in that it adds only one isoprene unit to geranyl diphosphate, generating the 15-carbon product (E,Z-farnesyl diphosphate). Rv2361c then adds a further seven isoprene units to E,Z-farnesyl diphosphate in a processive manner to generate the 50-carbon prenyl diphosphate, which is then dephosphorylated to generate a carrier for activated sugars. The molecular basis for chain-length discrimination by Rv1086 during synthesis is unknown. We also report the structure of apo Rv1086 with citronellyl diphosphate bound and with the product mimic E,E-farnesyl diphosphate bound. We report the structures of Rv2361c in the apo form, with isopentenyl diphosphate bound and with a substrate analogue, citronellyl diphosphate. The structures confirm the enzymes are very closely related. Detailed comparison reveals structural differences that account for chain-length control in Rv1086. We have tested this hypothesis and have identified a double mutant of Rv1086 that makes a range of longer lipid chains.     

Differential proline metabolism in vegetative and reproductive tissues determine drought tolerance in chickpea

Proline is emerging as a critical component of drought tolerance and fine tuning of its metabolism under stress affects the plants sensitivity and response to stress. Thus the study was carried out to analyse the effect of water deficit on the proline content and principal enzymes involved in its synthesis (Δ¹-pyrolline-carboxylate synthetase) and catabolism (proline dehydrogenase) at different developmental stages and in different organs (roots, nodules, leaves, pod wall, and seeds) of two chickpea (Cicer arietinum L.) cultivars differing in drought tolerance (drought tolerant ICC4958 and drought sensitive ILC3279). It was observed that increased Δ¹-pyrolline-carboxylate synthetase activity under moderate stress in roots and nodules of ICC4958 caused an increase in proline content during initiation of reproductive development whereas increased proline dehydrogenase activity in nodules and leaves at this period helped to maintain reducing power and energy supply in tissues and proper seed development as seed biomass increased consistently up to maturity. On the other hand, roots and nodules of ILC3279 responded to stress by increasing proline content after the developmental phase of reproductive organs was over (near maturity) which negatively affected the response of pod wall to stress. Concurrent increase in activities of Δ¹-pyrolline-carboxylate synthetase and proline dehydrogenase in pod wall of ILC3279 aggravated the oxidative stress and affected seed development as seed biomass initially increased rapidly under stress but was unaffected near maturity.     

Pre-culturing islets with mesenchymal stromal cells using a direct contact configuration is beneficial for transplantation outcome in diabetic mice

Background aimsWe recently showed that co-transplantation of mesenchymal stromal cells (MSCs) improves islet function and revascularization in vivo. Pre-transplant islet culture is associated with the loss of islet cells. MSCs may enhance islet cell survival or function by direct cell contact mechanisms and soluble mediators. We investigated the capacity of MSCs to improve islet cell survival or β-cell function in vitro using direct and indirect contact islet-MSC configurations. We also investigated whether pre-culturing islets with MSCs improves islet transplantation outcome.MethodsThe effect of pre-culturing islets with MSCs on islet function in vitro was investigated by measuring glucose-stimulated insulin secretion. The endothelial cell density of fresh islets and islets cultured with or without MSCs was determined by immunohistochemistry. The efficacy of transplanted islets was tested in vivo using a syngeneic streptozotocin-diabetic minimal islet mass model. Graft function was investigated by monitoring blood glucose concentrations.ResultsIndirect islet-MSC co-culture configurations did not improve islet function in vitro. Pre-culturing islets using a direct contact MSC monolayer configuration improved glucose-stimulated insulin secretion in vitro, which correlated with superior islet graft function in vivo. MSC pre-culture had no effect on islet endothelial cell number in vitro or in vivo.ConclusionsPre-culturing islets with MSCs using a direct contact configuration maintains functional β-cell mass in vitro and the capacity of cultured islets to reverse hyperglycemia in diabetic mice.