Somatic embryogenesis in Centella asiatica L. an important medicinal and neutraceutical plant of India

Leaf segments excised from Centella asiatica, a medicinal and neutraceutical plant, produced abundant somaticembryoswhen cultured onMS mediumwith 9.29 Mkinetin in combination with 2.26 M2,4-D. Granular, white,shiny clusters of callus developed after 1 week of culture, and then formed heart and cotyledonary stage embryoson the same medium after 4 weeks. Somatic embryos matured and germinated in the presence of MS mediumcontaining 2.32 M kinetin with (2.89M) GA3. Plantlets were successfully transferred to pots containing amixture of soil and vermiculite (1:1).     

Evolving nature of the AP2 alpha-appendage hub during clathrin-coated vesicle endocytosis

Clathrin-mediated endocytosis involves the assembly of a network of proteins that select cargo, modify membrane shape and drive invagination, vesicle scission and uncoating. This network is initially assembled around adaptor protein (AP) appendage domains, which are protein interaction hubs. Using crystallography, we show that FxDxF and WVxF peptide motifs from synaptojanin bind to distinct subdomains on alpha-appendages, called 'top' and 'side' sites. Appendages use both these sites to interact with their binding partners in vitro and in vivo. Occupation of both sites simultaneously results in high-affinity reversible interactions with lone appendages (e.g. eps15 and epsin1). Proteins with multiple copies of only one type of motif bind multiple appendages and so will aid adaptor clustering. These clustered alpha(appendage)-hubs have altered properties where they can sample many different binding partners, which in turn can interact with each other and indirectly with clathrin. In the final coated vesicle, most appendage binding partners are absent and thus the functional status of the appendage domain as an interaction hub is temporal and transitory giving directionality to vesicle assembly.     

Yeast and mammalian alpha-glucosidase inhibitory constituents from Himalayan rhubarb Rheum emodi Wall.ex Meisson

The methanolic extract of rhizome of Himalayan rhubarb Rheum emodi displayed mild yeast as well as mammalian intestinal alpha-glucosidase inhibitory activity. However, further fractionation of active extract led to the isolation of several potent molecules in excellent yields, displaying varying degrees of inhibition on two test models of alpha-glucosidase. Rhapontigenin, desoxyrhapontigenin, chrysophanol-8-O-beta-d-glucopyranoside, torachrysone-8-O-beta-d-glucopyranoside displayed potent yeast alpha-glucosidase inhibition. However chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside displayed potent to moderate mammalian alpha-glucosidase inhibitory activity. Other compounds displayed mild activity on both the tests. Except desoxyrhapontigenin and rhapontigenin that increased Vmax, other compounds including crude extract decreased the Vmax significantly (p<0.02) in yeast alpha-glucosidase test. Further kinetic analysis on mammalian alpha-glucosidase inhibition showed that chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside may be classified as mixed-noncompetitive inhibitors. However, desoxyrhapontigenin and rhapontigenin may be classified as modulators of enzyme activity. Presence and position of glycoside moiety in compounds appear important for better inhibition of mammalian alpha-glucosidase. This is the first report assigning particularly, mammalian intestinal alpha-glucosidase inhibitory activity to these compounds. Chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin, desoxyrhapontigenin and rhapontigenin have been isolated in substantial yields from R. emodi for the first time. Therefore, these compounds may have value in the treatment and prevention of hyperglycemia associated diabetes mellitus.     

Analysis of substance P in rat brain by means of immunoaffinity capture and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry

Interest in the analysis of low abundance neuropeptides particularly using matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF-MS) is increasing because these neuropeptides are essential to the mechanism of transportation and the metabolism. This article describes an immunoprecipitation procedure that is suitable for MALDI-MS analysis of substance P (SP), a neuropeptide, in rat brain tissues. Substance P was precipitated from brain tissue extracts by immunoprecipitation with antibodies directed against SP, and are analyzed by MALDI-TOF-MS. Mass spectrometric analysis showed a singly charged [M+H]+ ion peak that corresponded to the SP molecular mass and was observed with a detection error of 1.6%. The average mass errors between the observed and theoretical molecular mass were within the 0.11 Da range. Capillary zone electrophoresis analysis was subsequently performed, and the effects of the different separation parameters were examined. Beginning with milligram quantities of brain tissue, picomole quantities of SP could be detected using this method.     

The synthesis of [([beta-D-ribofuranosyloxy)methyl]nucleosides

The coupling reaction of acetoxymethoxy ribofuranoside 4 with nucleic acid bases 5a-f to synthesize novel (ribofuranosyloxy)methyl uracil, thymine, cytosine, adenine, guanine derivatives 6a-g respectively in preference to the expected formation of natural nucleosides 2',3',5'-tri-O-benzoyl uridine, methyluridine, cytidine, adenosine and guanosine 7a-g is described. Detailed study of these reactions catalysed by Lewis acids TMSOTf and SnCl4 is described. TMSOTf exhibited selectivity for the formation of ribofuranosyloxy methyl derivatives 6a-g rather than 7a-g. Reason for formation of 6a-g is explained by HSAB principle.     

Differential vasoconstrictions induced by angiotensin II: role of AT1 and AT2 receptors in isolated C57BL/6J mouse blood vessels

Genetically altered mice are increasingly used as experimental models. However, ANG II responses in mouse blood vessels have not been well defined. Therefore, the aim of this study was to determine the role of ANG II in regulating major blood vessels in C57/BL6J mice with isometric force measurements. Our results showed that in mouse abdominal aorta ANG II induced a concentration-dependent contraction (EC50 4.6 nM) with a maximum contraction of 75.1 +/- 4.9% at 100 nM compared with that of 60 mM K+. Similarly, femoral artery also exhibited a contractile response of 76.0 +/- 3.4% to the maximum concentration of ANG II (100 nM). In contrast, ANG II (100 nM)-induced contraction was significantly less in carotid artery (24.5 +/- 6.6%) and only minimal (3.5 +/- 0.31%) in thoracic aorta. The nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester and the AT2 antagonist PD-123319 failed to enhance ANG II-induced contractions. However, an AT1 antagonist, losartan (10 microM), completely inhibited ANG II (100 nM) response in abdominal aorta and carotid artery. An AT1 agonist, [Sar1]-ANG II (100 nM), behaved similarly to ANG II (100 nM) in abdominal aorta and carotid artery. RT-PCR analyses showed that mouse thoracic aorta has a significantly lower AT1 mRNA level than abdominal aorta. These results demonstrate that major mouse vessels exhibit differential contractions to ANG II, possibly because of varied AT1 receptor levels.     

Ornithine Decarboxylase Activity in In Vivo and In Vitro Models of Cerebral Ischemia

Ornithine decarboxylase (ODC) is considered the rate-limiting enzyme in polyamine biosynthesis, and an increase in putrescine after central nervous system (CNS) injury appears to be involved in neuronal death. Cerebral ischemia and reperfusion trigger an active series of metabolic events, which eventually lead to neuronal death. In the present study, ODC activity was evaluated following transient focal cerebral ischemia and reperfusion in rat. The middle cerebral artery (MCA) was occluded for 2 h in male rats with an intraluminal suture technique. Animals were sacrificed between 3 and 48 h of reperfusion following MCA occlusion, and ODC activity was assayed in cortex and striatum. ODC activity was also estimated in an in vitro ischemia model using primary rat cortical neuron cultures, at 6–24 h reoxygenation following 1 h oxygen-glucose deprivation (OGD). In cortex, following ischemia, ODC activity was increased at 3 h (P < .05), reached peak levels by 6–9 h (P < .001) and returned to sham levels by 48 h reperfusion. In striatum the ODC activity followed a similar time course, but returned to basal levels by 24 h. This suggests that ODC activity is upregulated in rat CNS following transient focal ischemia and its time course of activation is region specific. In vitro, ODC activity showed a significant rise only at 24 h reoxygenation following ischemic insult. The release of lactate dehydrogenase (LDH), an indicator for cell damage, was also significantly elevated after OGD. 0.25 mM -difluoromethylornithine (DFMO) inhibited ischemia-induced ODC activity, whereas a 10-mM dose of DFMO appears to provide some neuroprotection by suppressing both ODC activity and LDH release in neuronal cultures, suggesting the involvement of polyamines in the development of neuronal cell death.     

Proinflammatory cytokines dominate the early immune response to filarial parasites

Although the early human immune response to the infective-stage larvae (L3) of Brugia malayi has not been well-characterized in vivo (because of the inability to determine the precise time of infection), the consensus has been that it must involve a predominant Th2 environment. We have set up an in vitro system to study this early immune response by culturing PBMC from unexposed individuals with live L3 of B. malayi. After 24 h of culture, T cell responses were examined by flow cytometry and by quantitative real-time RT-PCR for multiple cytokines. T cells were activated early following exposure to L3 as indicated by up-regulation of surface markers CD69 and CD71. The frequency of T cells expressing proinflammatory Th1 cytokines (IFN-gamma, TNF-alpha, GM-CSF, IL-1alpha, and IL-8) but not Th2 cytokines (IL-4, IL-5, IL-6, IL-10, and IL-13) was significantly increased in response to L3. This T cell response occurred in both the CD4 and CD8 T cell compartment and was restricted to the effector/memory pool (CD45RO(+)). This T cell response was not due to LPS activity from the parasite or from its endosymbiont, Wolbachia; moreover, it required the presence of APC as well as direct contact with live L3. Real-time RT-PCR analysis of multiple cytokines in the T cells confirmed the increased expression of proinflammatory Th1 cytokines. Up-regulation of these cytokines suggests that the primary immune response to the live infective stage of the parasite is not predominantly Th2 in nature but rather dominated by a proinflammatory response.     

Identification of a novel voltage-driven organic anion transporter present at apical membrane of renal proximal tubule

A novel transport protein with the properties of voltage-driven organic anion transport was isolated from pig kidney cortex by expression cloning in Xenopus laevis oocytes. A cDNA library was constructed from size-fractionated poly(A)+ RNA and screened for p-aminohippurate (PAH) transport in high potassium medium. A 1856-base pair cDNA encoding a 467-amino acid peptide designated as OATV1 (voltage-driven organic anion transporter 1) was isolated. The predicted amino acid sequence of OATV1 exhibited 60-65% identity to those of human, rat, rabbit, and mouse sodium-dependent phosphate cotransporter type 1 (NPT1), although OATV1 did not transport phosphate. The homology of this transporter to known members of the organic anion transporter family (OAT family) was about 25-30%. OATV1-mediated PAH transport was affected by the changes in membrane potential. The transport was Na+-independent and enhanced at high concentrations of extracellular potassium and low concentrations of extracellular chloride. Under the voltage clamp condition, extracellularly applied PAH induced outward currents in oocytes expressing OATV1. The current showed steep voltage dependence, consistent with the voltage-driven transport of PAH by OATV1. The PAH transport was inhibited by various organic anions but not by organic cations, indicating the multispecific nature of OATV1 for anionic compounds. This transport protein is localized at the apical membrane of renal proximal tubule, consistent with the proposed localization of a voltage-driven organic anion transporter. Therefore, it is proposed that OATV1 plays an important role to excrete drugs, xenobiotics, and their metabolites driven by membrane voltage through the apical membrane of the tubular epithelial cells into the urine.     

Identification of a novel system L amino acid transporter structurally distinct from heterodimeric amino acid transporters

A cDNA that encodes a novel Na+-independent neutral amino acid transporter was isolated from FLC4 human hepatocarcinoma cells by expression cloning. When expressed in Xenopus oocytes, the encoded protein designated LAT3 (L-type amino acid transporter 3) transported neutral amino acids such as l-leucine, l-isoleucine, l-valine, and l-phenylalanine. The LAT3-mediated transport was Na+-independent and inhibited by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with the properties of system L. Distinct from already known system L transporters LAT1 and LAT2, which form heterodimeric complex with 4F2 heavy chain, LAT3 was functional by itself in Xenopus oocytes. The deduced amino acid sequence of LAT3 was identical to the gene product of POV1 reported as a prostate cancer-up-regulated gene whose function was not determined, whereas it did not exhibit significant similarity to already identified transporters. The Eadie-Hofstee plots of LAT3-mediated transport were curvilinear, whereas the low affinity component is predominant at physiological plasma amino acid concentration. In addition to amino acid substrates, LAT3 recognized amino acid alcohols. The transport of l-leucine was electroneutral and mediated by a facilitated diffusion. In contrast, l-leucinol, l-valinol, and l-phenylalaninol, which have a net positive charge induced inward currents under voltage clamp, suggesting these compounds are transported by LAT3. LAT3-mediated transport was inhibited by the pretreatment with N-ethylmaleimide, consistent with the property of system L2 originally characterized in hepatocyte primary culture. Based on the substrate selectivity, affinity, and N-ethylmaleimide sensitivity, LAT3 is proposed to be a transporter subserving system L2. LAT3 should denote a new family of organic solute transporters.