Identification and optimization of small molecule antagonists of vasoactive intestinal peptide receptor-1 (VIPR1)

Identification, synthesis and structure-activity relationship of small-molecule VIPR1 antagonists encompassing two chemical series are described.     

Coherent ontogenic dynamics of geraniol acetyltransferase activity and geranyl acetate concentration in flowers and leaves of aroma grass Cymbopogon martinii var. Motia

We have investigated the correlation between the concentration of geranyl acetate (GA) and acetyl CoA: geraniol acetyltransferase (GAAT) activity in palamarosa (Cymbopogon martini var. Motia) inflorescence and leaves at their different physiological stages. The results on GA concentration and the GAAT activity have been expressed on per gram fresh weight, per spikelet pair or leaf and per unit area of the phylloplane also incase of leaf. The percentage of geranyl acetate and geraniol in the volatile oil has also been considered. GA concentration was found to be highest in unopened floral spikelets and on the decline in fully open spikelets matching the trend of GAAT activity. Similarly, highest concentration of GA and maximum GAAT activity were found in leaves at mid-stage of development (stage II). The regression analysis curve between GA concentration (mg gFw⁻¹) and GAAT activity (IU 10⁻³ gFw⁻¹) gave an estimate of correlation coefficient (at 95% confidence) value of 0.79 for flowers and 0.92 for leaf. The results suggest that volatile ester (like geranyl acetate) synthesis in foliage and flowers of the aroma oil plant is controlled by the existent catalytic levels GAAT rather than the availability of geraniol. The study also indicates that the GAAT to be a good target to over-express for improvement of oil quality in terms of GA linked to fruit-fresh olfactory note of the oil.     

Development of a mutant of Trichoderma citrinoviride for enhanced production of cellulases

Considering importance of a microbial strain capable of increased cellulases production and insensitive to catabolite repression for industrial use, we have developed a mutant strain of Trichoderma citrinoviride by multiple exposures to EMS and ethidium bromide. The mutant produced 0.63, 3.12, 8.22 and 1.94 IU ml(-1) FPase, endoglucanase, beta-glucosidase and cellobiase, respectively. These levels were, respectively, 2.14, 2.10, 4.09 and 1.73 fold higher than those in parent strain. Glucose (upto 20 mM) did not repress enzyme production by the mutant under submerged fermentation conditions. In vitro activity assay with partially purified cellulase showed lack of inhibition by glucose. Interestingly, the partially purified endoglucanase and beta-glucosidase were activated by 2.0 fold and 2.6 fold, respectively, by 20 mM and 30 mM ethanol in the assay mixture. Genetic distinction of the mutant was revealed by the presence of two unique amplicans in comparative DNA fingerprinting performed using 20 random primers.     

Down Regulation of Genes Involved in T Cell Polarity and Motility during the Induction of Heart Allograft Tolerance by Allochimeric MHC I

Background

The allochimeric MHC class I molecule [α1h1/u]-RT1.Aa that contains donor-type (Wistar Furth, WF; RT1u) epitopes displayed on recipient-type (ACI, RT1a) administered in conjunction with sub-therapeutic dose of cyclosporine (CsA) induces indefinite survival of heterotopic cardiac allografts in rat model. In vascularized transplantation models, the spleen contributes to graft rejection by generating alloantigen reactive T cells. The immune response in allograft rejection involves a cascade of molecular events leading to the formation of immunological synapses between T cells and the antigen-presenting cells.

Methodology/Principal Findings

To elucidate the molecular pathways involved in the immunosuppressive function of allochimeric molecule we performed microarray and quantitative RTPCR analyses of gene expression profile of splenic T cells from untreated, CsA treated, and allochimeric molecule + subtherapeutic dose of CsA treated animals at day 1, 3 and 7 of post transplantation. Allochimeric molecule treatment caused down regulation of genes involved in actin filament polymerization (RhoA and Rac1), cell adhesion (Catna1, Vcam and CD9), vacuolar transport (RhoB, Cln8 and ATP6v1b2), and MAPK pathway (Spred1 and Dusp6) involved in tubulin cytoskeleton reorganization and interaction between actin and microtubule cytoskeleton. All these genes are involved in T cell polarity and motility, i.e., their ability to move, scan and to form functional immunological synapse with antigen presenting cells (APCs).

Conclusions

These results indicate that the immunosuppressive function of allochimeric molecule may depend on the impairment of T cells'' movement and scanning ability, and possibly also the formation of immunological synapse. We believe that these novel findings may have important clinical implications for organ transplantation.     

Mutational Analysis of the Candida albicans Ammonium Permease Mep2p Reveals Residues Required for Ammonium Transport and Signaling

The ammonium permease Mep2p mediates ammonium uptake and also induces filamentous growth in the human-pathogenic yeast Candida albicans in response to nitrogen limitation. The C-terminal cytoplasmic tail of Mep2p contains a signaling domain that is not required for ammonium transport but is essential for Mep2p-dependent morphogenesis. Progressive C-terminal truncations showed Y433 to be the last amino acid that is essential for the induction of filamentous growth, thereby delimiting the Mep2p signaling domain. To understand in more detail how the signaling activity of Mep2p is regulated by ammonium availability and transport, we mutated conserved amino acid residues that have been implicated in ammonium binding or uptake. Mutation of D180, which has been proposed to mediate initial contact with extracellular ammonium, or the pore-lining residues H188 and H342 abolished Mep2p expression, indicating that these residues are important for protein stability. Mutation of F239, which together with F126 is thought to form an extracytosolic gate to the conductance channel, abolished both ammonium uptake and Mep2p-dependent filament formation, despite proper localization of the protein. On the other hand, mutation of W167, which is assumed to participate with Y122, F126, and S243 in the recruitment and coordination of the ammonium ion at the extracytosolic side of the cell membrane, also abolished filament formation without having a strong impact on ammonium transport, demonstrating that extracellular alterations in Mep2p can affect intracellular signaling. Mutation of Y122 reduced ammonium uptake much more strongly than mutation of W167 but still allowed efficient filament formation, indicating that the signaling activity of Mep2p is not directly correlated with its transport activity. These results provide important insights into ammonium transport and control of morphogenesis by Mep2p in C. albicans.     

Substitutions of 2S and 7U chromosomes of Aegilops kotschyi in wheat enhance grain iron and zinc concentration

Biofortification through genetic manipulation is the best approach for improving micronutrient content of the staple food crops to alleviate hidden hunger, namely, the deficiency of Fe and Zn affecting more than two billion people worldwide. An interspecific hybridization was made between T. aestivum line Chinese Spring (CS) and Aegilops kotschyi accession 3790 selected for high grain iron and zinc concentration. The CS × Ae. kotschyi F₁ hybrid with low chromosome pairing was highly male and female sterile. This was backcrossed with wheat cultivars to get seed set. The selfed BC₁F₁ and BC₂F₁ plants with high grain iron and zinc concentration were selected in subsequent generations. The selected derivatives showed 60–136% enhanced grain iron and zinc concentration and 50–120% increased iron and zinc content per seed as compared to the recipient wheat cultivars. Thirteen cytologically stable, fertile and agronomically superior plants with high grain iron and zinc concentrations were selected for molecular characterization. The application of anchored wheat SSR markers, transferable to Ae. kotschyi, to the high grain iron and zinc containing derivatives indicated introgression of group 2 and group 7 chromosomes of Ae. kotschyi. GISH and FISH analysis of some derivatives confirmed the substitution of chromosomes 2S and 7U for their homoeologues of the A genome, suggesting that some of the genes controlling high grain micronutrient content in the Ae. kotschyi accession are on these chromosomes.     

Rubella vaccine-induced cellular immunity: evidence of associations with polymorphisms in the Toll-like, vitamin A and D receptors, and innate immune response genes

Toll-like, vitamin A and D receptors and other innate proteins participate in various immune functions. We determined whether innate gene-sequence variations are associated with rubella vaccine-induced cytokine immune responses. We genotyped 714 healthy children (11–19 years of age) after two doses of rubella-containing vaccine for 148 candidate SNP markers. Rubella virus-induced cytokines were measured by ELISA. Twenty-two significant associations (range of P values 0.002–0.048) were found between SNPs in the vitamin A receptor family (RARA, RARB, TOP2B and RARG), vitamin D receptor and downstream mediator of vitamin D signaling (RXRA) genes and rubella virus-specific (IFN-γ, IL-2, IL-10, TNF-α, and GM-CSF) cytokine immune responses. A TLR3 gene promoter region SNP (rs5743305, −8441A > T) was associated with rubella-specific GM-CSF secretion. Importantly, SNPs in the TRIM5 gene coding regions, rs3740996 (His43Tyr) and rs10838525 (Gln136Arg), were associated with an allele dose-related secretion of rubella virus-specific TNF-α and IL-2/GM-CSF, respectively, and have been previously shown to have functional consequences regarding the antiviral activity and susceptibility to HIV-1 infection. We identified associations between individual SNPs and haplotypes in, or involving, the RIG-I (DDX58) gene and rubella-specific TNF-α secretion. This is the first paper to present evidence that polymorphisms in the TLR, vitamin A, vitamin D receptor, and innate immunity genes can influence adaptive cytokine responses to rubella vaccination.     

A comparative study on the graft copolymerization of acrylic acid onto rayon fibre by a ceric ion redox system and a γ-radiation method

Functionalization of rayon fibre has been carried out by grafting acrylic acid (AAC) both by a chemical method using a Ce⁴⁺-HNO₃ redox initiator and by a mutual irradiation (γ-rays) method. The reaction conditions affecting the grafting percentage have been optimized for both methods, and the results are compared. The maximum percentage of grafting (50%) by the chemical method was obtained utilizing 18.24 × 10⁻³ moles/L of ceric ammonium nitrate (CAN), 39.68 × 10⁻² moles/L of HNO₃, and 104.08 × 10⁻² moles/L of AAc in 20 mL of water at 45 °C for120 min. For the radiation method, the maximum grafting percentage (60%) was higher, and the product was obtained under milder reaction conditions using a lower concentration of AAc (69.38 × 10⁻² moles/L) in 10 mL of water at an optimum total dose of 0.932 kGy. Swelling studies showed higher swelling for the grafted rayon fibre in water (854.54%) as compared to the pristine fibre (407%), while dye uptake studies revealed poor uptake of the dye (crystal violet) by the grafted fibre in comparison with the pristine fibre. The graft copolymers were characterized by IR, TGA, and scanning electron micrographic methods. Grafted fibre, prepared by the radiation-induced method, showed better thermal behaviour. Comparison of the two methods revealed that the radiation method of grafting of acrylic acid onto rayon fibre is a better method of grafting in comparison with the chemical method.     

Regulation of the apolipoprotein B in heterozygous hypobeta-lipoproteinemic knock-out mice expressing truncated apoB,B81. Low production and enhanced clearance of apoB cause low levels of apoB

Meat quality of pigs is dependent on biochemical and biophysical processes in the time course post mortem (p.m.) and is associated with the intracellular Ca2+ homeostasis. However, there is little known about changes in the Ca2+ transporting proteins controlling the Ca2+ uptake of sarcoplasmic reticulum (SR) in the time course p.m. In this study changes in the Ca2+ transporting proteins were investigated in homogenates of longissimus muscles of 4 malignant hyperthermia susceptible (MHS) and 6 malignant hyperthermia resistant (MHR) Pietrain pigs. Muscle samples were obtained at different time intervals: biopsy 2 h prior slaughtering and from the carcass immediately after exsanguination (0 h), 45 min, 4 h, and 22 h p.m. The SR Ca2+ uptake rate was measured immediately after homogenization with closed calcium release channel (CRC), with opened CRC and without manipulation of CRC. Additionally the SR Ca2+ ATPase activity was determined.The results show: (i) The ability of SR to sequester Ca2+ declined to about 60% in the first 45 min p.m. in MHS samples irrespective of CRC state, whereas in MHR samples this decline was about 5%; (ii) Ca2+ uptake and Ca2+ ATPase activity were not different between the biopsy and 0 h samples, i.e. the stress of slaughter was of no immediate influence; (iii) The Ca2+ ATPase activity of the SR declined at about the same rate as the Ca2+ uptake in both MHS and MHR pig samples in the course of time p.m.; (iv) In samples, taken immediately after exsanguination, the Ca2+ ATPase activity of MHS pigs was higher than that of MHR pigs. However, in samples taken 4 h p.m. Ca2+ ATPase activity of MHS pigs has declined to about 30% of the value at 0 h; (v) The CRC can be closed and opened in all samples up to 22 h p.m. and seems to be fully functional at all sampling times; (vi) The CRC of MHS pigs is almost fully open, whereas the CRC of MHR pigs is only partially open at all sampling times; (vii) The permeability of the SR membrane to Ca2+ (determined as the ratio of SR Ca2+ ATPase with and without ionophore A23187) is the same in both MHS and MHR and did not change with ongoing time; (viii) No uncoupling of uptake from ATP hydrolysis occurred up to 4 h p.m., but the coupling differed between MHS and MHR for all time intervals with lower values for MHS pigs. The results suggest that the decreasing Ca2+ uptake rate of homogenates, sampled at different times p.m., is essentially caused by changes in the Ca2+ pump and not by changes in the CRC or an increased phospholipid membrane permeability to Ca2+.