Insights into the structure–function relationship of brown plant hopper resistance protein,Bph14 of rice plant: a computational structural biology approach

Brown plant hopper (BPH) is one of the major destructive insect pests of rice, causing severe yield loss. Thirty-two BPH resistance genes have been identified in cultivated and wild species of rice Although, molecular mechanism of rice plant resistance against BPH studied through map-based cloning, due to non-existence of NMR/crystal structures of Bph14 protein, recognition of leucine-rich repeat (LRR) domain and its interaction with different ligands are poorly understood. Thus, in the present study, in silico approach was adopted to predict three-dimensional structure of LRR domain of Bph14 using comparative modelling approach followed by interaction study with jasmonic and salicylic acids. LRR domain along with LRR-jasmonic and salicylic acid complexes were subjected to dynamic simulation using GROMACS, individually, for energy minimisation and refinement of the structure. Final binding energy of jasmonic and salicylic acid with LRR domain was calculated using MM/PBSA. Free-energy landscape analysis revealed that overall stability of LRR domain of Bph14 is not much affected after forming complex with jasmonic and salicylic acid. MM/PBSA analysis revealed that binding affinities of LRR domain towards salicylic acid is higher as compared to jasmonic acid. Interaction study of LRR domain with salicylic acid and jasmonic acid reveals that THR987 of LRR form hydrogen bond with both complexes. Thus, THR987 plays active role in the Bph14 and phytochemical interaction for inducing resistance in rice plant against BPH. In future, Bph14 gene and phytochemicals could be used in BPH management and development of novel resistant varieties for increasing rice yield.     

Recombinant expression, purification, and kinetic characterization of chondroitinase AC and chondroitinase B from Flavobacterium heparinum

Glycosaminoglycans (GAGs) are a family of complex polysaccharides involved in a diversity of biological processes, ranging from cell signaling to blood coagulation. Chondroitin sulfate (CS) and dermatan sulfate (DS) comprise a biologically important subset of GAGs. Two of the important lyases that degrade CS/DS, chondroitinase AC (EC 4.2.2.5) and chondroitinase B (no EC number), have been isolated and cloned from Flavobacterium heparinum. In this study, we outline an improved methodology for the recombinant expression and purification of these chondroitinases, thus enabling the functional characterization of the recombinant form of the enzymes for the first time. Utilizing an N-terminal 6x histidine tag, the recombinant chondroitinases were produced by two unique expression systems, each of which can be purified to homogeneity in a single chromatographic step. The products of exhaustive digestion of chondroitin-4SO(4) and chondroitin-6SO(4) with chondroitinase AC and dermatan sulfate with chondroitinase B were analyzed by strong-anion exchange chromatography and a novel reverse-polarity capillary electrophoretic technique. In addition, the Michaelis-Menten parameters were determined for these enzymes. With chondroitin-4SO(4) as the substrate, the recombinantly expressed chondroitinase AC has a K(m) of 0.8 microM and a k(cat) of 234 s(-1). This is the first report of kinetic parameters for chondroitinase AC with this substrate. With chondroitin-6SO(4) as the substrate, the enzyme has a K(m) of 0.6 microM and a k(cat) of 480 s(-1). Recombinantly expressed chondroitinase B has a K(m) of 4.6 microM and a k(cat) of 190 s(-1) for dermatan sulfate as its substrate. Efficient recombinant expression of the chondroitinases will facilitate the structure-function characterization of these enzymes and allow for the development of the chondroitinases as enzymatic tools for the fine characterization and sequencing of CS/DS.     

Comparison of the metabolism of L-erythro- and L-threo-sphinganines and ceramides in cultured cells and in subcellular fractions

Ceramide (Cer) is a key intermediate in the synthetic and degradative pathways of sphingolipid metabolism, and is also an important second messenger. Natural Cer exists in the D-erythro configuration. Three additional, non-natural stereoisomers exist, but conflicting reports have appeared concerning their metabolism. We now compare the stereospecificity of three enzymes in the sphingolipid biosynthetic pathway, namely dihydroceramide (dihydroCer), sphingomyelin (SM) and glucosylceramide synthases, in subcellular fractions and in cultured cells. The L-erythro enantiomers of sphinganine, dihydroCer and Cer do not act as substrates for any of the three enzymes. In contrast, the diastereoisomer, L-threo-sphinganine, is acylated by dihydroCer synthase, and L-threo-dihydroCer and L-threo-Cer are both metabolized to dihydroSM and SM, respectively, but not to dihydroglucosylceramide and glucosylceramide. No significant difference was detected in the ability of SM synthase to metabolize Cer containing a short (hexanoyl) versus long acyl chain (palmitoyl), demonstrating that short-acyl chain Cers mimic their natural counterparts, at least in the sphingolipid biosynthetic pathway.     

Antioxidant role of zinc in PCB (Aroclor 1254) exposed ventral prostate of albino rats

The ability of zinc to retard oxidative processes has been recognized for many years. Polychlorinated biphenyls (PCBs) are persistent and bioaccumulative environmental toxicants. Previous study has indicated that PCBs can have deleterious effects, including oxidative stress, on various aspects of reproduction in male rats. The aim of this study was to determine the antioxidant role of zinc in PCB-exposed ventral prostate of albino rats. A group of 20 rats were treated with Aroclor 1254 (2 mg/kg body weight/day, i.p.) for 30 days. After the PCB treatment, 10 rats were treated as PCB control. The remaining 10 rats were given zinc (Zn SO(4)) (200 mg/kg body weight/day, p.o.) for 10 days. Ventral prostatic enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) were estimated in all the groups. Hydrogen peroxide (H(2)O(2)), lipid peroxidation (LPO) and ventral prostatic acid phosphatase (ACP) were also estimated. Serum hormonal profiles such as total tri-iodothyronine (T(3)), thyroxine (T(4)), thyroid stimulating hormone (TSH), testosterone, and estradiol were estimated. Ventral prostatic androgen and estrogen receptors, ventral prostatic zinc content, and serum zinc concentration were also quantified in all the groups. Antioxidant enzymes such as SOD, CAT, GPx, GST, and ACP were decreased while an increase in H(2)O(2) and LPO were observed in PCB-treated animals. Decreased serum total T(3), T(4), testosterone, estradiol and increased TSH were observed in PCB-exposed rats. Ventral prostatic androgen and estrogen receptors were also decreased significantly in PCB-exposed rats. Zinc administration restored to previous levels all parameters except ventral prostatic ACP. These results suggest that PCB induces oxidative stress in rat ventral prostate by decreasing the levels of antioxidant enzymes; the effects could be reversed by the administration of zinc. The adverse effect of PCBs (Aroclor 1254) and zinc on ventral prostate might be due to indirect action through hormonal regulation.     

Functional expression and characterization of a purine nucleobase transporter gene from Leishmania major

Leishmania major, like all the other kinetoplastid protozoa, are unable to synthesize purines and rely on purine nucleobase and nucleoside acquisition across the parasite plasma membrane by specific permeases. Although, several genes have been cloned that encode nucleoside transporters in Leishmania and Trypanosoma brucei, much less progress has been made on nucleobase transporters, especially at the molecular level. The studies reported here have cloned and expressed the first gene for a L. major nucleobase transporter, designated LmaNT3. The LmaNT3 permease shows 33% identity to L. donovani nucleoside transporter 1.1 (LdNT1.1) and is, thus, a member of the equilibrative nucleoside transporter (ENT) family. ENT family members identified to date are nucleoside transporters, some of which also transport one or several nucleobases. Functional expression studies in Xenopus laevis oocytes revealed that LmaNT3 mediates high levels of uptake of hypoxanthine, xanthine, adenine and guanine. Moreover, LmaNT3 is an high affinity transporter with K(m) values for hypoxanthine, xanthine, adenine and guanine of 16.5 +/- 1.5, 8.5 +/- 0.6, 8.5 +/- 1.1, and 8.8 +/- 4.0 microM, respectively. LmaNT3 is, thus, the first member of the ENT family identified in any organism that functions as a nucleobase rather than nucleoside or nucleoside/nucleobase transporter.     

Overexpression of manganese superoxide dismutase promotes the survival of prostate cancer cells exposed to hyperthermia

It has been hypothesized that exposure of cells to hyperthermia results in an increased flux of reactive oxygen species (ROS), primarily superoxide anion radicals, and that increasing antioxidant enzyme levels will result in protection of cells from the toxicity of these ROS. In this study, the prostate cancer cell line, PC-3, and its manganese superoxide dismutase (MnSOD)-overexpressing clones were subjected to hyperthermia (43°C, 1 h). Increased expression of MnSOD increased the mitochondrial membrane potential (MMP). Hyperthermic exposure of PC-3 cells resulted in increased ROS production, as determined by aconitase inactivation, lipid peroxidation, and H₂O₂ formation with a reduction in cell survival. In contrast, PC-3 cells overexpressing MnSOD had less ROS production, less lipid peroxidation, and greater cell survival compared to PC-3 Wt cells. Since MnSOD removes superoxide, these results suggest that superoxide free radical or its reaction products are responsible for part of the cytotoxicity associated with hyperthermia and that MnSOD can reduce cellular injury and thereby enhance heat tolerance.     

Sphingolipids are required for the stable membrane association of glycosylphosphatidylinositol-anchored proteins in yeast

Ongoing sphingolipid synthesis is specifically required in vivo for the endoplasmic reticulum (ER) to Golgi transport of glycosylphosphatidylinositol (GPI)-anchored proteins. However, the sphingolipid intermediates that are required for transport nor their role(s) have been identified. Using stereoisomers of dihydrosphingosine, together with specific inhibitors and a mutant defective for sphingolipid synthesis, we now show that ceramides and/or inositol sphingolipids are indispensable for GPI-anchored protein transport. Furthermore, in the absence of sphingolipid synthesis, a significant fraction of GPI-anchored proteins is no longer associated tightly with the ER membrane. The loose membrane association is neither because of the lack of a GPI-anchor nor because of prolonged ER retention of GPI-anchored proteins. These results indicate that ceramides and/or inositol sphingolipids are required to stabilize the association of GPI-anchored proteins with membranes. They could act either by direct involvement as membrane components or as substrates for the remodeling of GPI lipid moieties.     

Calcium-sensitive ROS-GC1 signaling outside of photoreceptors: A common theme

Until now, ROS-GC1 signal transduction system was thought to be exclusive to photoreceptors in the retina. Two recent reports, however, now show that this is not the case. In one, the ROS-GC1 signal transduction system has been identified and characterized in pinealocyte neurons. This signaling is modulated by norepinephrine. However, the response of the individual pinealocyte neuron to the norepinephrine signal depends on whether the GCAP1-linked (results in hyperpolarization) or S100-linked (results in depolarization) pathway is operational in the pinealocyte. The GCAP1-linked pathway results in hyperpolarization, while the S100-linked pathway, in depolarization. The two pathways are mutually exclusive. In the other report, the calcium-modulated ROS-GC1:GCAP1 signaling system has been discovered in mitral cells of the olfactory bulb. These findings raise the possibility that a common theme of calcium-modulated ROS-GC signaling may be utilized in a wide variety of neurosensory cells. This idea is also supported from evolutionary and functional perspectives.