Gene ontology study of methyl jasmonate-treated and non-treated hairy roots of <Emphasis Type="Italic">Panax ginseng</Emphasis> to identify genes involved in secondary metabolic pathway

The roots of Panax ginseng C.A. Meyer, known as Korean ginseng have been a valuable and important folk medicine in East Asian countries. It mainly used to maintain the homeostasis of the human body, with the presence of ginsenosides and non-saponin compounds like phenol compounds, acidic polysaccharides and polyethylene compounds. Functional genomics aid to annotate EST sequences based on gene ontology. In this study, we focused, genes which involve in secondary metabolic pathways and to visualize temporal changes of gene expression in ginseng hairy roots with methyl ester methyl jasmonate (MeJA) along with non-treated hairy roots. A 5.774 EST clones were clustered and assembled into 501 contigs and 2.955 singletons. Annotations categorized with molecular functions, biological processes, cellular compounds of gene ontological terms and biochemical functions, enzyme commission number, and metabolic pathways are assigned through Kyoto Encyclopedia of Genes and Genomes database. Comparatively, EST sequences are assigned to cellular process, metabolic process, biotic and abiotic stress stimuli, developmental and biological regulations and transports are up-regulated 2–3 fold in MeJA treated hairy roots. 46 different sub groups of enzymes found in the MeJA treated plants. These annotated ESTs represents a significant proportion of the P. ginseng and provides molecular resource for develop microarray to study genes expressions to development, metabolism and reproduction.     

Studies on the induction of -aminolevulinic acid synthetase iun mouse liver

Administration of 3,5-diethoxy carbonyl-1,4-dihydrocollidine (DDC) to mice resulted in a striking increase in the level of δ-aminolevulinic acid (ALA) synthetase in liver. Although the enzyme activity was primarily localized in mitochondria and postmicrosomal supernatant fluid, a significant level of activity was also detected in purified nuclei. The time course of induction showed a close parallelism between the bound and free enzyme activities with the former always accounting for a higher percentage of the total activity as compared to the latter. Studies with cycloheximide indicated a half-life of around 3 hr for both the bound and free ALA synthetase. Actinomycin D and hemin prevented enzyme induction when administered along with DDC, but when administered 12 hr after DDC treatment Actinomycin D did not lead to a decay of either the bound or free enzyme activity and hemin inhibited the bound enzyme activity but not the free enzyme level. The molecular sizes of the mitochondrial and cytosolic ALA synthetase(s) were found to be similar on sephadex columns.     

Vitamin E prevents deleterious effects of di (2-ethyl hexyl) phthalate, a plasticizer used in PVC blood storage bags

Vitamin E administration prevented DEHP induced deleterious effects like (i) degenerative changes in the brain and thyroid, (ii) decrease in the activity of neuronal membrane Na+ - K+ ATPase, (iii) decrease in the concentration of insulin, cortisol and TSH, and (iv) the increase in T3 and T4 in female Albino rats. The results suggest use of vitamin E to prevent harmful effects of repeated transfusion of DEHP containing blood as in thalassemia patient. The possibility of using vitamin E to prevent the harmful effects of repeated transfusion of DEHP containing blood, as in thalassemia patients, is discussed.     

Modulation by weak bases or weak acids of the pH of cell sap and phosphoenolpyruvate carboxylase activity in leaf discs of C4 plants

When leaf discs of a C₄ species, Alternanthera pungens (L.) H.B. and K. or Amaranthus hypochondriacus L., were preincubated in 7.5 m M NH₄Cl, the pH of the cell sap increased by nearly 0.3 unit, while the activity of phosphoenolpyruvate carboxylase (PEPC) about doubled compared to the cell sap from control leaf discs (preincubated in 5 m M Tricine‐KOH, pH 8.5). The sensitivity of PEPC to L ‐malate (a feedback inhibitor) decreased marginally as a result of cytosolic alkalization. The pH of the cell sap and PEPC activity decreased by nearly 0.4 unit and 50%, respectively, when leaf discs were incubated in weak organic acids such as propionic, butyric or salicylic acid. Thus, our results demonstrate a marked modulation in vivo of cell sap pH and PEPC activity in leaf discs from C₄ plants by external alkalizing or acidifying reagents. The rise in PEPC activity due to alkalization of leaf discs was not sensitive to cycloheximide, implying that cytosolic protein synthesis was not involved in the activation of PEPC. Despite the marked increase in the PEPC activity due to the base‐loading of leaf discs, the change in malate sensitivity of the enzyme was only marginal, indicating that there was no significant increase in the extent of PEPC‐phosphorylation. Besides the physiological significance, the technique of acid/ base‐loading may be an important tool for studying the regulation of PEPC in leaf discs of C₄ species, since the activity of PEPC could be enhanced apparently without phosphorylation of the enzyme.     

Probing the beta2 adrenoceptor binding site with catechol reveals differences in binding and activation by agonists and partial agonists

The beta(2) adrenergic receptor (beta(2)AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. The beta(2)AR agonist binding site is well characterized, and there is a wealth of structurally related ligands with functionally diverse properties. In the present study, we use catechol (1,2-benzenediol, a structural component of catecholamine agonists) as a molecular probe to identify mechanistic differences between beta(2)AR activation by catecholamine agonists, such as isoproterenol, and by the structurally related non-catechol partial agonist salbutamol. Using biophysical and pharmacologic approaches, we show that the aromatic ring of salbutamol binds to a different site on the beta(2)AR than the aromatic ring of catecholamines. This difference is important in receptor activation as it has been hypothesized that the aromatic ring of catecholamines plays a role in triggering receptor activation through interactions with a conserved cluster of aromatic residues in the sixth transmembrane segment by a rotamer toggle switch mechanism. Our experiments indicate that the aromatic ring of salbutamol does not activate this mechanism either directly or indirectly. Moreover, the non-catechol ring of partial agonists does not interact optimally with serine residues in the fifth transmembrane helix that have been shown to play an important role in activation by catecholamines. These results demonstrate unexpected differences in binding and activation by structurally similar agonists and partial agonists. Moreover, they provide evidence that activation of a GPCR is a multistep process that can be dissected into its component parts using agonist fragments.     

Development of a microplate-based scintillation proximity assay for MraY using a modified substrate

MraY is an established target for the discovery of antibacterial agents. The conventional assay for MraY uses radioactive substrate and analysis of products after paper chromatography or butanol extraction. Synthesis of radiolabeled substrate has been done in vitro using purified enzymes or by growing cells on radiolabeled precursors. The authors report a simple and rapid method to chemically radiolabel MraY substrate, UDP-MurNAc-pentapeptide. Specific activity obtained by this method was more than 100 times higher than the conventionally labeled substrate, and yields are high enough to support the requirements of high-throughput screening (HTS). The authors have developed a microplate-based homogeneous assay for MraY in which the product is captured on wheat germ agglutinin (WGA) scintillation proximity assay (SPA) beads. The assay was validated by showing inhibition by specific inhibitors of MraY but not by inhibitors of other enzymes of peptidoglycan synthesis. The assay uses wild-type membranes of Escherichia coli, giving it an advantage over recently described assays that need the protein to be overexpressed. In addition, it has an advantage over the high-throughput MraY-MurG coupled assay reported in the literature because it is MraY specific, and therefore hits obtained in this assay do not need further deconvolution. It has potential for use in HTS approaches to find novel inhibitors of MraY.     

The NH2 terminus of RCK1 domain regulates Ca2+-dependent BK(Ca) channel gating

Large conductance, voltage- and Ca2+-activated K+ (BK(Ca)) channels regulate blood vessel tone, synaptic transmission, and hearing owing to dual activation by membrane depolarization and intracellular Ca2+. Similar to an archeon Ca2+-activated K+ channel, MthK, each of four alpha subunits of BK(Ca) may contain two cytosolic RCK domains and eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient with one another upon Ca2+ binding to change the gating ring conformation and open the activation gate. Here we report that the conformational changes of the NH2 terminus of RCK1 (AC region) modulate BK(Ca) gating. Such modulation depends on Ca2+ occupancy and activation states, but is not directly related to the Ca2+ binding sites. These results demonstrate that AC region is important in the allosteric coupling between Ca2+ binding and channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an important step in addition to the reorientation of RCK domains leading to the opening of the BK(Ca) activation gate. Our observations are consistent with a mechanism for Ca2+-dependent activation of BK(Ca) channels such that the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca2+; Ca2+ binding and depolarization relieve this inhibition.     

A quantitative structure-activity relationship study on some HIV-1 protease inhibitors using molecular connectivity index

A quantitative structure-activity relationship (QSAR) study has been made on two different series of tetrahydropyrimidinones acting as HIV-1 protease inhibitors. A structural parameter, the first order valence molecular connectivity index ((1)chi(v)), has been used to account for the variation in the activity. The protease inhibition activity as well as the antiviral potency of the compounds are found to be significantly correlated with (1)chi(v) of P(2)/P(2') substituents attached to the two nitrogens N1 and N3, suggesting that substituents containing less electronegative and more saturated atoms, meaning thereby the less polar or more hydrophobic substituents, will be more advantageous. Further, if P(2) and P(2') are dissimilar, the former is found to be more effective than the latter. This difference is attributed to a conformational change in the enzyme that may be more favorable to P(2) binding than to P(2') binding.     

Blood profile of pigeons (Columba livia) during growth and breeding

Changes in the blood profile of domestic pigeons (Columba livia) were studied during growth and breeding cycle. Counts of erythrocytes and leucocytes, and values of mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), blood volume, plasma volume (BV), width of erythrocytes, and length, width and volume of erythrocyte nuclei of squabs almost reached adult values by the 4th week of age. During courtship and mating, while the level of plasma glucose increased, those of albumin, potassium, cholesterol, calcium and uric acid decreased. At nest-building, plasma albumin and plasma calcium increased significantly. The initial phase of incubation showed an elevation in plasma calcium and a decline in cholesterol and sodium, whereas mid-phase of incubation indicated a marked rise in cholesterol and uric acid. Terminal phase of incubation had significantly low plasma protein level. During feeding and brooding period, a significant rise in sodium, protein and glucose levels and a fall in calcium were observed. Following egg-laying, there was a significant rise in calcium and a drop in protein, haemoglobin, cholesterol, sodium and MCH values. Concomitant with the phenomenal rate of growth of squabs, their haematological indices neared adult values by the 4th week of age and during breeding activity significant changes in blood values occurred.     

Purification and Stability during Storage of Phoshoenolpyruvate Carboxylase from Leaves of Amaranthus hypochondriacus,a NAD-ME Type C4 Plant

A traditional method is reported for purification of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) from leaves of Amaranthus hypochondriacus L. with a high yield of 50 %, 135-fold purification, and specific activity of 900 mmol kg^–1(protein) s^–1. PEPC was purified from light-adapted leaves of A. hypochondriacus, involving 40–60 % ammonium sulphate fractionation, followed by chromatography on columns of DEAE-Sepharose, hydroxylapatite (HAP), and Seralose 6-B. The enzyme appeared as a single band on 10 % SDS-PAGE, with a molecular mass of about 100 kDa. Kinetic studies with purified enzyme confirmed the PEPC to be the light-form of the enzyme. Glycerol generally increased the stability of PEPC. The stability and storage of the purified enzyme was studied at temperatures of 4 °C, –20 °C, and liquid nitrogen. PEPC maintained its activity for up to 3 months upon storage with 50 % (v/v) glycerol in liquid nitrogen.