Growth,biomass production,and assimilatory characters in <Emphasis Type="Italic">Cenchrus ciliaris</Emphasis> L. Under elevated CO<Subscript>2</Subscript> condition

The effect of elevated carbon dioxide (600±50 cm³ m⁻³; C₆₀₀) on growth performance, biomass production, and photosynthesis of Cenchrus ciliaris L. cv. 3108 was studied. This crop responded significantly by plant height, leaf length and width, and biomass production under C₆₀₀. Leaf area index increased triple fold in the crops grown in the open top chamber with C₆₀₀. The biomass production in term of fresh and dry biomass accumulation increased by 134.35 (fresh) and 193.34 (dry) % over the control (C₃₆₀) condition where the crops were grown for 20 d. The rate of photosynthesis and stomatal conductance increased by 24.51 and 46.33 %, respectively, in C₆₀₀ over C₃₆₀ plants. In comparison with C₃₆₀, the rate of transpiration decreased by 6.8 % under C₆₀₀. Long-term exposure (120 d) to C₆₀₀ enhanced photosynthetic water use efficiency by 34 %. Also the contents of chlorophylls a and b significantly increased in C₆₀₀. Thus C. ciliaris grown in C₆₀₀ throughout the crop season may produce more fodder in terms of green biomass.     

Enantioselective phenylacetylene addition to aldehydes and ketones catalyzed by recyclable polymeric Zn(salen) complex

New polymeric Zn(salen) complex was employed in the enantioselective phenylacetylene addition to aldehydes and ketones to produce corresponding chiral secondary propargylic alcohols with yields (up to 96%) and enantioselectivity (up to 72%) and tertiary propargylic alcohols with yields (up to 79%) and enantioselectivity (up to 68%) at room temperature, with added advantage of four times reuse with retention of enantioselectivity.     

The Mycobacterium tuberculosis FAS-II condensing enzymes: their role in mycolic acid biosynthesis, acid-fastness, pathogenesis and in future drug development

Mycolic acids are very long-chain fatty acids representing essential components of the mycobacterial cell wall. Considering their importance, characterization of key enzymes participating in mycolic acid biosynthesis not only allows an understanding of their role in the physiology of mycobacteria, but also might lead to the identification of new drug targets. Mycolates are synthesized by at least two discrete elongation systems, the type I and type II fatty acid synthases (FAS-I and FAS-II respectively). Among the FAS-II components, the condensing enzymes that catalyse the formation of carbon-carbon bonds have received considerable interest. Four condensases participate in initiation (mtFabH), elongation (KasA and KasB) and termination (Pks13) steps, leading to full-length mycolates. We present the recent biochemical and structural data for these important enzymes. Special emphasis is given to their role in growth, intracellular survival, biofilm formation, as well as in the physiopathology of tuberculosis. Recent studies demonstrated that phosphorylation of these enzymes by mycobacterial kinases affects their activities. We propose here a model in which kinases that sense environmental changes can phosphorylate the condensing enzymes, thus representing a novel mechanism of regulating mycolic acid biosynthesis. Finally, we discuss the attractiveness of these enzymes as valid targets for future antituberculosis drug development.     

Floating granules of ranitidine hydrochloride-gelucire 43/01: Formulation optimization using factorial design

The purpose of this research was to develop and optimize a controlled-release multiunit floating system of a highly water soluble drug, ranitidine HCl, using Compritol, Gelucire 50/13, and Gelucire 43/01 as lipid carriers. Ranitidine HCl-lipid granules were prepared by the melt granulation technique and evaluated for in vitro floating and drug release. ethyl cellulose, methylcellulose, and hydroxypropyl methylcellulose were evaluated as release rate modifiers. A 3² full factorial design was used for optimization by taking the amounts of Gelucire 43/01 (X ₁) and ethyl cellulose (X ₂) as independent variables, and the percentage drug released in 1(Q₁), 5(Q₅), and 10 (Q₁₀) hours as dependent variables. The results revealed that the moderate amount of Gelucire 43/01 and ethyl cellulose provides desired release of ranitidine hydrochloride from a floating system. Batch F4 was considered optimum since it contained less Gelucire and was more similar to the theoretically predicted dissolution profile (f₂=62.43). The temperature sensitivity studies for the prepared formulations at 40°C/75% relative humidity for 3 months showed no significant change in in vitro drug release pattern. These studies indicate that the hydrophobic lipid Gelucire 43/01 can be considered an effective carrier for design of a multiunit floating drug delivery system for highly water soluble drugs such as ranitidine HCl. Published: April 13, 2007     

Exposure to serotonin adversely affects oligodendrocyte development and myelination in vitro

Serotonin (5‐hydroxytryptamine, 5‐HT) has been implicated to play critical roles in early neural development. Recent reports have suggested that perinatal exposure to selective serotonin reuptake inhibitors (SSRIs) resulted in cortical network miswiring, abnormal social behavior, callosal myelin malformation, as well as oligodendrocyte (OL) pathology in rats. To gain further insight into the cellular and molecular mechanisms underlying SSRIs‐induced OL and myelin abnormalities, we investigated the effect of 5‐HT exposure on OL development, cell death, and myelination in cell culture models. First, we showed that 5‐HT receptor 1A and 2A subtypes were expressed in OL lineages, using immunocytochemistry, Western blot, as well as intracellular Ca²⁺ measurement. We then assessed the effect of serotonin exposure on the lineage development, expression of myelin proteins, cell death, and myelination, in purified OL and neuron‐OL myelination cultures. For pure OL cultures, our results showed that 5‐HT exposure led to disturbance of OL development, as indicated by aberrant process outgrowth and reduced myelin proteins expression. At higher doses, such exposure triggered a development‐dependent cell death, as immature OLs exhibited increasing susceptibility to 5‐HT treatment compared to OL progenitor cells (OPC). We showed further that 5‐HT‐induced immature OL death was mediated at least partially via 5‐HT2A receptor, since cell death could be mimicked by 5‐HT2A receptor agonist 1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane hydrochloride, (±)‐2,5‐dimethoxy‐4‐iodoamphetamine hydrochloride, but atten‐uated by pre‐treatment with 5‐HT2A receptor antagonist ritanserin. Utilizing a neuron‐OL myelination co‐culture model, our data showed that 5‐HT exposure significantly reduced the number of myelinated internodes. In contrast to cell injury observed in pure OL cultures, 5‐HT exposure did not lead to OL death or reduced OL density in neuron‐OL co‐cultures. However, abnormal patterns of contactin‐associated protein (Caspr) clustering were observed at the sites of Node of Ranvier, suggesting that 5‐HT exposure may affect other axon‐derived factors for myelination. In summary, this is the first study to demonstrate that manipulation of serotonin levels affects OL development and myelination, which may contribute to altered neural connectivity noted in SSRIs‐treated animals.

     

Discovery of new anti-depressants from structurally novel 5-HT3 receptor antagonists: design, synthesis and pharmacological evaluation of 3-ethoxyquinoxalin-2-carboxamides

A novel series of 3-ethoxyquinoxalin-2-carboxamides were designed as per the pharmacophoric requirements of 5-HT₃ receptor antagonist using ligand-based approach. The desired carboxamides were synthesized from the key intermediate, 3-ethoxyquinoxalin-2-carboxylic acid by coupling with appropriate amines in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 1-hydroxybenzotriazole (HOBt). The 5-HT₃ receptor antagonism was evaluated in longitudinal muscle myenteric plexus preparation from guinea pig ileum against 5-HT₃ agonist, 2-methy-5-HT, which was expressed in the form of pA₂ values. Compound 6h (3-ethoxyquinoxalin-2-yl)(4-methylpiperazin-1-yl)methanone was found to be the most active compound, which expressed a pA₂ value of 7.7. In forced swim test, the compounds with higher pA₂ value exhibited good anti-depressant-like activity and compounds with lower pA₂ value failed to show activity as compared to the vehicle-treated group.     

Altered architecture of substrate binding region defines the unique specificity of UDP-GalNAc 4-epimerases

UDP-hexose 4-epimerases play a pivotal role in lipopolysaccharide (LPS) biosynthesis and Leloir pathway. These epimerases are classified into three groups based on whether they recognize nonacetylated UDP-hexoses (Group 1), both N-acetylated and nonacetylated UDP-hexoses (Group 2) or only N-acetylated UDP-hexoses (Group 3). Although the catalysis has been investigated extensively, yet a definitive model rationalizing the substrate specificity of all the three groups on a common platform is largely lacking. In this work, we present the crystal structure of WbgU, a novel UDP-hexose 4-epimerase that belongs to the Group 3. WbgU is involved in biosynthetic pathway of the unusual glycan 2-deoxy-L-altruronic acid that is found in the LPS of the pathogen Pleisomonas shigelloides. A model that defines its substrate specificity is proposed on the basis of the active site architecture. Representatives from all the three groups are then compared to rationalize their substrate specificity. This investigation reveals that the Group 3 active site architecture is markedly different from the "conserved scaffold" of the Group 1 and the Group 2 epimerases and highlights the interactions potentially responsible for the origin of specificity of the Group 3 epimerases toward N-acetylated hexoses. This study provides a platform for further engineering of the UDP-hexose 4-epimerases, leads to a deeper understanding of the LPS biosynthesis and carbohydrate recognition by proteins. It may also have implications in development of novel antibiotics and more economic synthesis of UDP-GalNAc and downstream products such as carbohydrate based vaccines.     

The C-terminal domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC is a lectin-like carbohydrate binding module

The D-arabinan-containing polymers arabinogalactan (AG) and lipoarabinomannan (LAM) are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf) transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbC(CT)) encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM). Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbC(CT) contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985) at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbC(CT), linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis.     

Complex determinants in human immunodeficiency virus type 1 envelope gp120 mediate CXCR4-dependent infection of macrophages

Host cell range, or tropism, combined with coreceptor usage defines viral phenotypes as macrophage tropic using CCR5 (M-R5), T-cell-line tropic using CXCR4 (T-X4), or dually lymphocyte and macrophage tropic using CXCR4 alone or in combination with CCR5 (D-X4 or D-R5X4). Although envelope gp120 V3 is necessary and sufficient for M-R5 and T-X4 phenotypes, the clarity of V3 as a dominant phenotypic determinant diminishes in the case of dualtropic viruses. We evaluated D-X4 phenotype, pathogenesis, and emergence of D-X4 viruses in vivo and mapped genetic determinants in gp120 that mediate use of CXCR4 on macrophages ex vivo. Viral quasispecies with D-X4 phenotypes were associated significantly with advanced CD4+-T-cell attrition and commingled with M-R5 or T-X4 viruses in postmortem thymic tissue and peripheral blood. A D-X4 phenotype required complex discontinuous genetic determinants in gp120, including charged and uncharged amino acids in V3, the V5 hypervariable domain, and novel V1/V2 regions distinct from prototypic M-R5 or T-X4 viruses. The D-X4 phenotype was associated with efficient use of CXCR4 and CD4 for fusion and entry but unrelated to levels of virion-associated gp120, indicating that gp120 conformation contributes to cell-specific tropism. The D-X4 phenotype describes a complex and heterogeneous class of envelopes that accumulate multiple amino acid changes along an evolutionary continuum. Unique gp120 determinants required for the use of CXCR4 on macrophages, in contrast to cells of lymphocytic lineage, can provide targets for development of novel strategies to block emergence of X4 quasispecies of human immunodeficiency virus type 1.