Biocontrol Potential of Soybean Bacterial Endophytes Against Charcoal Rot Fungus, <Emphasis Type="Italic">Rhizoctonia bataticola</Emphasis>

A total of 137 bacterial isolates from surface sterilized root, stem, and nodule tissues of soybean were screened for their antifungal activity against major phytopathogens like Rhizoctonia bataticola, Macrophomina phaseolina, Fusarium udam, and Sclerotium rolfsii. Nine bacterial endophytes suppressed the pathogens under in vitro plate assay. These were characterized biochemically and identified at the genus level based on their partial sequence analysis of 16S rDNA. Eight of the isolates belonged to Bacillus and one to Paenibacillus. The phylogenetic relationship among the selected isolates was studied and phylogenetic trees were generated. The selected isolates were screened for biocontrol traits like production of hydrogen cyanide (HCN), siderophore, hydrolytic enzymes, antibiotics, and plant growth promoting traits like indole 3-acetic acid production, phosphate solubilization, and nitrogen fixation. A modified assessment scheme was used to select the most efficient biocontrol isolates Paenibacillus sp. HKA-15 (HKA-15) and Bacillus sp. HKA-121 (HKA-121) as potential candidates for charcoal rot biocontrol as well as soybean plant growth promotion.     

Hyperthermia-induced Hsp90·eNOS Preserves Mitochondrial Respiration in Hyperglycemic Endothelial Cells by Down-regulating Glut-1 and Up-regulating G6PD Activity

Uncoupling of NO production from NADPH oxidation by endothelial nitric-oxide synthase (eNOS) is enhanced in hyperglycemic endothelium, potentially due to dissociation of heat shock proteins 90 (Hsp90), and cellular glucose homeostasis is enhanced by a ROS-induced positive feed back mechanism. In this study we investigated how such an uncoupling impacts oxygen metabolism and how the oxidative phosphorylation can be preserved by heat shock (42 °C for 2 h, hyperthermia) in bovine aortic endothelial cells. Normal and heat-shocked bovine aortic endothelial cells were exposed to normoglycemia (NG, 5.0 mm) or hyperglycemia (30 mm). With hyperglycemia treatment, O₂ consumption rate was reduced (from V_O2max = 7.51 ± 0.54 to 2.35 ± 0.27 mm Hg/min/10⁶ cells), whereas in heat-shocked cells, O₂ consumption rate remained unaltered (8.19 ± 1.01 mm Hg/min/10 × 10⁶ cells). Heat shock was found to enhance Hsp90/endothelial NOS interactions and produce higher NO. Moreover, ROS generation in the hyperglycemic condition was also reduced in heat-shocked cells. Interestingly, glucose uptake was reduced in heat-shocked cells as a result of decrease in Glut-1 protein level. Glucose phosphate dehydrogenase activity that gives rise to NADPH generation was increased by hyperthermia, and mitochondrial oxidative metabolism was preserved. In conclusion, the present study provides a novel mechanism wherein the reduced oxidative stress in heat-shocked hyperglycemic cells down-regulates Glut-1 and glucose uptake, and fine-tuning of this pathway may be a potential approach to use for therapeutic benefit of diabetes mellitus.     

Structures of host range-controlling regions of the capsids of canine and feline parvoviruses and mutants

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) differ in their ability to infect dogs and dog cells. Canine cell infection is a specific property of CPV and depends on the ability of the virus to bind the canine transferrin receptor (TfR), as well as other unidentified factors. Three regions in the capsid structure, located around VP2 residues 93, 300, and 323, can all influence canine TfR binding and canine cell infection. These regions were compared in the CPV and FPV capsid structures that have been determined, as well as in two new structures of CPV capsids that contain substitutions of the VP2 Asn-93 to Asp and Arg, respectively. The new structures, determined by X-ray crystallography to 3.2 and 3.3 A resolutions, respectively, clearly showed differences in the interactions of residue 93 with an adjacent loop on the capsid surface. Each of the three regions show small differences in structure, but each appears to be structurally independent of the others, and the changes likely act together to affect the ability of the capsid to bind the canine TfR and to infect canine cells. This emphasizes the complex nature of capsid alterations that change the virus-cell interaction to allow infection of cells from different hosts.     

Novel particulate spin probe for targeted determination of oxygen in cells and tissues

The synthesis and characterization of a new lithium octa-n-butoxy-substituted naphthalocyanine radical probe (LiNc-BuO) and its use in the determination of concentration of oxygen (oximetry) by electron paramagnetic resonance (EPR) spectroscopy are reported. The probe is synthesized as a needle-shaped microcrystalline particulate. The particulate shows a single-line EPR spectrum that is highly exchange-narrowed with a line-width of 210 mG. The EPR line-width is sensitive to molecular oxygen showing a linear relationship between the line-width and concentration of oxygen (pO(2)) with a sensitivity of 8.5 mG/mmHg. We studied a variety of physicochemical and biological properties of LiNc-BuO particulates to evaluate the suitability of the probe for in vivo oximetry. The probe is unaffected by biological oxidoreductants, stable in tissues for several months, and can be successfully internalized in cells. We used this probe to monitor changes in concentration of oxygen in the normal muscle and RIF-1 tumor tissue of mice as a function of tumor growth. The data showed a rapid decrease in the tumor pO(2) with increase of tumor volume. Human arterial smooth muscle cells, upon internalization of the LiNc-BuO probe, showed a marked oxygen gradient across the cell membrane. In summary, the newly synthesized octa-n-butoxy derivative of lithium naphthalocyanine has unique properties that are useful for determining oxygen concentration in chemical and biological systems by EPR spectroscopy and also for magnetic tagging of cells.     

Heat shock regulates the respiration of cardiac H9c2 cells through upregulation of nitric oxide synthase

Mild and nonlethal heat shock (i.e., hyperthermia) is known to protect the myocardium and cardiomyocytes against ischemic injury. In the present study, we have shown that heat shock regulates the respiration of cultured neonatal cardiomyocytes (cardiac H9c2 cells) through activation of nitric oxide synthase (NOS). The respiration of cultured cardiac H9c2 cells subjected to mild heat shock at 42 degrees C for 1 h was decreased compared with that of control. The O2 concentration at which the rate of O2 consumption is reduced to 50% was increased in heat-shocked cells, indicating a lowering of O2 affinity in the mitochondria. Western blot analyses showed a fourfold increase in the expression of heat shock protein (HSP) 90 and a twofold increase in endothelial NOS (eNOS) expression in the heat-shocked cells. Immunoblots of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) in the immunoprecipitate of HSP90 of heat-shocked cells showed that there was a sevenfold increase in eNOS and no changes in iNOS and nNOS. Confocal microscopic analysis of cells stained with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate showed higher levels of NO production in the heat-shocked cells than in control cells. The results indicate that heat shock-induced HSP90 forms a complex with eNOS and activates it to increase NO concentration in the cardiac H9c2 cells. The generated NO competitively binds to the complexes of the respiratory chain of the mitochondria to downregulate O2 consumption in heat-shocked cells. On the basis of these results, we conclude that myocardial protection by hyperthermia occurs at least partly by the pathway of HSP90-mediated NO production, leading to subsequent attenuation of cellular respiration.     

On the structural significance of the linkage region constituents of N-glycoproteins: an X-ray crystallographic investigation using models and analogs

The linkage region constituents, namely, 2-acetamido-2-deoxy-beta-D-glucopyranose and asparagine are conserved in the N-glycoproteins of all the eukaryotes. The present work is aimed at understanding the reasons for the occurrence of GlcNAc and Asn as the linkage region constituents. A total of six sugar amides have been designed as models and analogs of the linkage region and their crystal structures have been solved. This is the first report on the X-ray crystallographic investigation of the effect of systematic changes in the linkage sugar as well as its aglycon moiety on the N-glycosidic torsion, psi(N) (O5-C1-N1-C1(')). This also forms the first report on the crystal structure of a model of L-RhabetaAsn, a variant linkage found in the surface layer glycoprotein of Bacillus stearothermophillus. Among the models and analogs examined, the acetamido derivatives of Man and Xyl, the linkage sugars of O-glycoproteins, show a psi(N) value of -114.5 degrees and -121.2 degrees, respectively, deviating maximum from the value of -89.8 degrees reported for the model compound GlcNAcbetaNHAc. The L-Rha and Gal derivatives also show noticeable deviations. The psi(N) values, -89.5 degrees and -91.0 degrees, of the propionamide derivatives of Glc and GlcNAc (analogs of GlcbetaGln and GlcNAcbetaGln, respectively) agree well with those (-93.8 degrees and -89.8 degrees ) reported for their corresponding acetamide derivatives suggesting Gln could serve as well as Asn as the linkage region amino acid. However, the rotational freedom about the additional C-C bond would lead to altered rigidity of the linkage region. An analysis of packing reveals that the molecular assembly of these compounds is driven by different infinite and finite chains of hydrogen bonds. The double pillaring of hydrogen bonds involving the amide groups at C1 and C2 is seen as a unique packing feature characteristic of beta-1-N-acyl derivatives of GlcNAc. Based on the findings of the present study, it is speculated that the linkage region constituents of the eukaryotic N-glycoproteins appear to fulfill three essential structural requirements: rigidity, planarity, and linearity and these are met by the trisaccharide core and Asn at the linkage region.     

RPE65 gene: multiplex PCR and mutation screening in patients from India with retinal degenerative diseases

We used multiplex PCR followed by sequencing to screen for mutations in the 14 exons of theRPE65 gene in early-hildhood-onset autosomal recessive retinitis pigmentosa (arRP) and Leber’s congenital amaurosis (LCA) patients. The RPE65 protein is believed to play an important role in the metabolism of vitamin A in the visual cycle and mutations identified in the gene could have implications for vitamin A-based therapeutic intervention. We were able to identify a homozygous mutation (AAT → AAG) in exon 9 in an arRP patient and a heterozygous missense transversion (AAT → AAG) also in exon 9 of an LCA patient. We also identified a polymorphism in exon 10 (GAG → GAA) in an arRP as well as an LCA patient. Mutation screening would be greatly facilitated by multiplex PCR which could cut down costs, labour and time involved. The nucleotide changes observed in this study could bede novo. Though a larger study has been undertaken, from the preliminary results it appears that in India theRPE65 gene seems to be less involved in causation of LCA.     

Effect of metformin on nitric oxide synthase in genetically obese (ob/ob) mice.

Genetically obese (ob/ob) mice were employed for the study of the effect of metformin on activity and expression of nitric oxide synthase (NOS ) in vitro and in vivo. For in vitro analysis, mouse liver extracts were used. For the in vivo study, (ob/ob) and their control litter mates (ob/c) mice were injected with specified amounts of metformin and the expression of NOS in the adipose tissue and hypothalamus was measured by Western blotting. Results show that metformin exhibited a biphasic effect on NOS activity in vitro. Expression of metformin was differentially altered in the hypothalamus and adipose tissues of the normal and ob/ob animals that were treated with metformin. Further, a significant decrease in food intake occurred in the (ob/ob) mice that received metformin. This decrease in food intake was not accompanied by changes in serum glucose. At inhibitory concentrations, hypothalamic NOS expression changes differentially in normal and ob/ob mice. In normal mice, metformin stimulated NOS expression, while in ob/ob mice there was an inhibition. NOS expression increased in brown adipose tissue of metformin treated control mice, while no such increase was observed in ob/ob mice. No effect of metformin was observed in white adipose tissue of control or obese mice. Thus, metformin may produce anorectic effects through modulation of NOS.     

In vitro establishment and multiplication of Nymphaea hybrid ‘James Brydon’

Rhizome tips were the most suitable explants for in vitro plant regeneration and multiplication of Nymphaea hybrid James Brydon on Murashige and Skoog medium containing different concentrations and combinations of indole-3-acetic acid, 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA), kinetin, 2-isopentenyladenine (2iP) and gibberellic acid (GA₃). A combination of 2iP, BA, and NAA strongly favored induction of shoot buds and shoot proliferation. Pretreatment of shoot cultures at 8°C for 30 days or with 14.4 or 28.9 M GA₃ for 15 days did not improve shoot multiplication. A 16-h photoperiod with photosynthetic photon flux of 30 mol m^-2 s^-1 was found to be the optimum light condition for shoot growth and multiplication. Multiple shoots produced well developed root systems within 4 weeks after transfer to a plant growth regulator-free medium containing activated charcoal.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - MS Murashige and Skoog medium - NAA 1-naphthaleneacetic acid