Inhibition of chlorophyll biosynthesis by mercury in excised etiolated maize leaf segments during greening: effect of 2-oxoglutarate

Mercury (0.01-1.0 mM) inhibited chlorophyll formation in greening maize leaf segments. However, supplementing incubation medium with 2-oxoglutarate, maintained substantially higher level of chlorophyll in absence of metal after an initial period of 8 hr. On preincubation of leaf segments with HgCl2, per cent inhibition of chlorophyll synthesis by metal was same in the presence and absence of 2-oxoglutarate. Supply of 2-oxoglutarate (0.1-10.0 mM) exerted concentration dependent effect on chlorophyll formation in absence or presence of metal. Increase in delta-amino levulinic acid dehydratase as well as NADH-glutamate synthase activity and decrease in NADH-glutamate dehydrogenase activity by 2-oxoglutarate in the presence of Hg suggested that glutamate for delta-amino levulinic acid synthesis could be made available from NH4+ assimilation via., glutamine synthetase/glutamate synthase pathway during mercury toxicity.     

First report of leaf spot disease caused by Phoma eupyrena Sacc. on Aloe vera from Madhya Pradesh,India

A leaf spot disease caused by Phoma eupyrena Sacc. on Aloe vera was reported from Madhya Pradesh, India. Disease symptoms were observed as irregular to elongated, sunken lesion on both leaf surfaces which later turns creamish brown with maroon margin. The fungal colonies were whitish or light pale to dark grey colour. The pycnidia were glabrous and solitary, papillate, and indistinctly uniostiolate averages 100–260 μm in diameter. Conidia were ellipsoidal, 4.5–7.5 × 2.5–3.75 μm in size, with two large guttules. To the best of our knowledge, this is the first report of P. eupyrena causing leaf spot disease on A. vera in India.     

The influence of substituents (R) at N atom of thiophene-2-carbaldehyde thiosemicarbazones {(C4H3S)HCN-N(H)-C(S)NHR} on bonding, nuclearity and H-bonded networks of copper(I) complexes

The nuclearity, bonding and H-bonded networks of copper(I) halide complexes with thiophene-2-carbaldehyde thiosemicarbazones {(C₄H₃S)HC²N³-N(H)-C¹(S)N¹HR} are influenced by R substituents at N¹ atom. Thiophene-2-carbaldehyde-N¹-methyl thiosemicarbazone (HttscMe) or thiophene-2-carbaldehyde-N¹-ethyl thiosemicarbazone (HttscEt) have yielded halogen-bridged dinuclear complexes, [Cu₂(μ-X)₂(η¹-S-Htsc)₂(Ph₃P)₂] (Htsc, X: HttscMe, I, 1; Br, 2; Cl, 3; HttscEt, I, 4; Br, 5; Cl, 6), while thiophene-2-carbaldehyde-N¹-phenyl thiosemicarbazone (HttscPh) has yielded mononuclear complexes, [CuX(η¹-S-HttscPh)₂] (X, I, 7a; Br 8; Cl, 9) and a sulfur bridged dinuclear complex, [Cu₂(μ-S-HttscPh)₂(η¹-S-HttscPh)₂I₂] 7b co-existing with 7a in the same unit cell. These results are in contrast to S-bridged dimers [Cu₂(μ-S-Httsc)₂(η¹-Br)₂(Ph₃P)₂] · 2H₂O and [Cu₂(μ-S-Httsc)₂(η¹-Cl)₂(Ph₃P)₂] · 2CH₃CN obtained for R = H and X = Cl, Br (Httsc = thiophene-2-carbaldehyde thiosemicarbazone) as reported earlier. The intermolecular CH_Ph?π interaction in 1-3 (2.797 Å, 1; 3.264 Å, 2; 3.257 Å, 3) have formed linear polymers, whereas the CH_Ph?X and N³?HCH interactions in 4-6 (2.791, 2.69 Å, 5; 2.776, 2.745 Å, 6, respectively) have led to the formation of H-bonded 2D polymer. The PhN¹H?π, interactions (2.547 Å, 8, 2.599 Å, 9) have formed H-bonded dimers only. The Cu?Cu separations are 3.221-3.404 Å (1-6).     

Nature of KaiB-KaiC binding in the cyanobacterial circadian oscillator

In the cyanobacteria Synechococcus elongatus and Thermosynechococcus elongatus, the KaiA, KaiB and KaiC proteins in the presence of ATP generate a post-translational oscillator (PTO) that can be reconstituted in vitro. KaiC is the result of a gene duplication and resembles a double doughnut with N-terminal CI and C-terminal CII hexameric rings. Six ATPs are bound between subunits in both the CI and CII ring. CI harbors ATPase activity, and CII catalyzes phosphorylation and dephosphorylation at T432 and S431 with a ca. 24-h period. KaiA stimulates KaiC phosphorylation, and KaiB promotes KaiC subunit exchange and sequesters KaiA on the KaiB-KaiC interface in the final stage of the clock cycle. Studies of the PTO protein-protein interactions are convergent in terms of KaiA binding to CII but have led to two opposing models of the KaiB-KaiC interaction. Electron microscopy (EM) and small angle X-ray scattering (SAXS), together with native PAGE using full-length proteins and separate CI and CII rings, are consistent with binding of KaiB to CII. Conversely, NMR together with gel filtration chromatography and denatured PAGE using monomeric CI and CII domains support KaiB binding to CI. To resolve the existing controversy, we studied complexes between KaiB and gold-labeled, full-length KaiC with negative stain EM. The EM data clearly demonstrate that KaiB contacts the CII ring. Together with the outcomes of previous analyses, our work establishes that only CII participates in interactions with KaiA and KaiB as well as with the His kinase SasA involved in the clock output pathway.     

Eukaryotic initiation factor 2-associated glycoprotein, p67, shows differential effects on the activity of certain kinases during serum-starved conditions

Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 is the major regulatory step in the initiation of protein synthesis in mammals. P67, a cellular glycoprotein, protects phosphorylation of eIF2alpha from kinases. P67 has five conserved amino acid residues at the D251, D262, H331, E364, and E459 positions. To determine the roles of these conserved amino acid residues in eIF2alpha phosphorylation during serum-starved conditions, we constitutively expressed D251A, D262A, H331A, E364A, and E459A mutants in rat tumor hepatoma cells. We find that the point mutants D251A, H331A, and E364A lower the levels of eIF2alpha phosphorylation. These low levels of phosphorylation decrease when serum-starved cells are grown in medium containing serum. To understand the mechanism of action of the p67 mutants in eIF2alpha phosphorylation during serum-starvation, we performed detailed biochemical analyses with the D251A mutant. We find that neither the O-GlcNAc modification on the D251A mutant nor the binding of D251A mutant with eIF2gamma has significant effects on eIF2alpha phosphorylation during serum-starved conditions. However, the D251A mutant inhibits p67's activity to suppress the activity of ERK1/2. Our data suggest that both p67 and the D251A mutant bind to ERK1, thus strengthening the idea that p67 regulates the activity of ERK1. During serum-starvation conditions, both PKR and PERK are phosphorylated and the D251A mutant shows increased stability of PERK as well as a slight decrease in its activity. Altogether, our data provide evidence to suggest that p67 modulates the expression and activity of certain eIF2alpha-specific kinases.     

Cloning, expression, and purification of Brucella suis outer membrane proteins

Brucella, an aerobic, nonsporeforming, nonmotile Gram-negative coccobacillus, is a NIH/CDC category B bioterror threat agent that causes incapacitating human illness. Medical defense against the bioterror threat posed by Brucella would be strengthened by development of a human vaccine and improved diagnostic tests. Central to advancement of these goals is discovery of bacterial constituents that are immunogenic or antigenic for humans. Outer membrane proteins (OMPs) are particularly attractive for this purpose. In this study, we cloned, expressed, and purified seven predicted OMPs of Brucella suis. The recombinant proteins were fused with 6-His and V5 epitope tags at their C termini to facilitate detection and purification. The B. suis surface genes were PCR synthesized based on their ORF sequences and directly cloned into an entry vector. The recombinant entry constructs were propagated in TOP 10 cells, recombined into a destination vector, pET-DEST42, then transformed into Escherichia coli BL21 cells for IPTG-induced protein expression. The expressed recombinant proteins were confirmed with Western blot analysis using anti-6-His antibody conjugated with alkaline phosphatase. These B. suis OMPs were captured and purified using a HisGrab plate. The purified recombinant proteins were examined for their binding activity with antiserum. Serum derived from a rabbit immunized intramuscularly with dialyzed cell lysate of Brucella rough mutant WRR51. The OMPs were screened using the rabbit antiserum and purified IgG. The results suggested that recombinant B. suis OMPs were successfully cloned, expressed and purified. Some of the expressed OMPs showed high binding activity with immunized rabbit antiserum.     

Co-administration of APD668, a G protein-coupled receptor 119 agonist and linagliptin,a DPPIV inhibitor,prevents progression of steatohepatitis in mice fed on a high trans-fat diet

Non-Alcoholic SteatoHepatitis (NASH) is the more severe form of Non-Alcoholic Fatty Liver Disease (NAFLD) and is characterized by the presence of hepatic steatosis, oxidative stress, inflammation, hepatocyte injury with or without fibrosis. Recently, GPR119 receptor has emerged as a novel therapeutic target for the treatment of dyslipidemia and non-alcoholic steatohepatitis. In the present study, we investigated the effect of APD668, a GPR119 agonist alone or in combination with linagliptin, a DPPIV inhibitor on the progression of steatohepatitis in mice fed on a high trans-fat diet. In this study, monotherapy with either APD668 or linagliptin caused a reduction in the levels of ALT, AST, glucose, cholesterol and epididymal fat mass but the effect was more pronounced upon treatment with combination of both drugs.On the other hand, combined treatment of APD668 with linagliptin demonstrated a non-significant additive effect in reduction of hepatic triglyceride (?78%) and cholesterol (?56%) compared to monotherapy groups. Moreover, co-administration of APD668 and linagliptin resulted in enhanced levels of active GLP-1 with additional benefit of significant synergistic decrease in body weight gain (?19%) in mice. We speculated that the enhanced effect observed with the combination treatment could be due to either 1) direct activation of GPR119 receptors present in liver and intestine or 2) enhanced active GLP-1 levels or 3) decreased degradation of GLP-1 in-vivo through DPPIV inhibition. Therefore, these findings clearly suggest that GPR119 receptor agonists in combination with DPPIV inhibitors may represent a promising therapeutic strategy for the treatment of non-alcoholic steatohepatitis.     

Absolute Leukocyte Telomere Length in HIV-Infected and Uninfected Individuals: Evidence of Accelerated Cell Senescence in HIV-Associated Chronic Obstructive Pulmonary Disease

Combination antiretroviral therapy (cART) has extended the longevity of human immunodeficiency virus (HIV)-infected individuals. However, this has resulted in greater awareness of age-associated diseases such as chronic obstructive pulmonary disease (COPD). Accelerated cellular senescence may be responsible, but its magnitude as measured by leukocyte telomere length is unknown and its relationship to HIV-associated COPD has not yet been established. We measured absolute telomere length (aTL) in peripheral leukocytes from 231 HIV-infected adults. Comparisons were made to 691 HIV-uninfected individuals from a population-based sample. Subject quartiles of aTL were assessed for relationships with measures of HIV disease severity, airflow obstruction, and emphysema severity on computed tomographic (CT) imaging. Multivariable regression models identified factors associated with shortened aTL. Compared to HIV-uninfected subjects, the mean aTL in HIV-infected patients was markedly shorter by 27 kbp/genome (p<0.001); however, the slopes of aTL vs. age were not different (p=0.469). Patients with longer known durations of HIV infection (p=0.019) and lower nadir CD4 cell counts (p=0.023) had shorter aTL. Shorter aTL were also associated with older age (p=0.026), smoking (p=0.005), reduced forced expiratory volume in one second (p=0.030), and worse CT emphysema severity score (p=0.049). HIV-infected subjects demonstrate advanced cellular aging, yet in a cART-treated cohort, the relationship between aTL and age appears no different from that of HIV-uninfected subjects.     

Molecular docking analysis of flavonoids with aldose reductase

Diabetes mellitus is a group of metabolic disorders that has risen to become the third most common cause in humans in recent years. The development of new bioactive substances from natural sources is a relatively new area. Flavonoids are believed to have a variety of beneficial properties in nature, including anti-inflammatory, antimicrobial, anticancer, antioxidant, neuroprotective, and anti-HIV properties. 15 naturally occurring flavonoids docked with the selected target aldose reductase. We report the optimal binding of Acumitin, Agathisflavone, Agehoustin B, and alpha-Toxicarol with aldose reductase for further consideration in drug discovery for T2DM.