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41.
Brian A. Otter Shirish A. Patil Marianne R. Spada Linda A. Jelicks Yuichi Yoshimura Akira Matsuda 《Nucleosides, nucleotides & nucleic acids》2013,32(2-4):615-635
Abstract The 5-oxo-6-methylene-pyrimidine-2,4-dione intermediate (6) that is formed when 5-acetoxy-6-acetoxymethyl-1-β-D-(5-O-acetyl-2,3-O-isopropylidene)-ribofuranosyluracil (5) is treated with sodium hydroxide undergoes cyclization at pH 14 to give 2′,3′-O-isopropylidene-5-hydroxy- O 5, 6-methanouridine (8) in good yield. Conversion of 8 into the 5-triflate ester 14 followed by reduction with [(Ph)3P]4Pd/Bu3SnH and deblocking with acetic acid then affords O 5′, 6-methanouridine (4) Conformational studies (NOE difference spectra, vicinal 1H-13C coupling constants, NOESY and CD spectra, molecular modeling) indicate that the C7-methylene group of 4 projects towards the furanose ring oxygen atom, producing a glycosyl rotation angle of about ? 160°. 相似文献
42.
A highly chitinolytic strain Penicillium ochrochloron MTCC 517 was procured from MTCC, Chandigarh, India. Culture medium supplemented with 1% chitin was found to be suitable for maximum production of chitinase. Purification of extracellular chitinase was done from the culture medium by organic solvent precipitation and DEAE-cellulose column chromatography. The chitinase was purified 6.92-fold with 29.9% yield. Molecular mass of purified chitinase was found to be 64 kDa by SDS-PAGE. The chitinase showed optimum temperature 40 °C and pH 7.0. The enzyme activity was completely inhibited by Hg2+, Zn2+, K+ and NH4+. The enzyme kinetic study of purified chitinase revealed the following characteristics, such as apparent Km 1.3 mg ml?1, Vmax 5.523 × 10?5 moles l?1 min?1 and Kcat 2.37 s?1 and catalytic efficiency 1.82 s?1 M?1. The enzyme hydrolyzed colloidal chitin, glycol chitin, chitosan, glycol chitosan, N,N′-diacetylchitobiose, p-nitrophenyl N-acetyl-β-d-glucosaminide and 4-methylumbelliferyl N-acetyl-β-d-glucosaminide. The chitinase of P. ochrochloron MTCC 517 is an exoenzyme, which gives N-acetylglucosamine as the main hydrolyzate after hydrolysis of colloidal chitin. Protoplasts with high regeneration capacity were obtained from Aspergillus niger using chitinase from P. ochrochloron MTCC 517. Since it also showed antifungal activity, P. ochrochloron MTCC 517 seems to be a promising biocontrol agent. 相似文献
43.
Shivanjali Joshi-Barr Jerome V. Karpiak Yogesh Ner Jessica H. Wen Adam J. Engler Adah Almutairi 《Journal of visualized experiments : JoVE》2013,(72)
Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10. 相似文献
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46.
Divya Vishwanath Harini Srinivasan Manjunath S. Patil Sowmya Seetarama Sachin Kumar Agrawal M. N. Dixit Kakali Dhar 《Journal of cell communication and signaling》2013,7(2):129-140
Adipocytes play a vital role in glucose metabolism. 3T3 L1 pre adipocytes after differentiation to adipocytes serve as excellent in vitro models and are useful tools in understanding the glucose metabolism. The traditional approaches adopted in pre adipocyte differentiation are lengthy exercises involving the usage of IBMX and Dexamethasone. Any effort to shorten the time of differentiation and quality expression of functional differentiation in 3T3 L1 cells in terms of enhanced Insulin sensitivity has an advantage in the drug discovery process. Thus, there is a need to develop a new effective method of differentiating the pre adipocytes to adipocytes and to use such methods for developing efficacious therapeutic molecules. We observed that a combination of Dexamethasone and Troglitazone generated differentiated adipocytes over fewer days as compared to the combination of IBMX and Dexamethasone which constitutes the standard protocol followed in our laboratory. The experiments conducted to compare the quality of differentiation yielded by various differentiating agents indicated that the lipid droplet accumulation increased by 112 % and the GLUT4 mediated glucose uptake by 137 % in cells differentiated with Troglitazone and Dexamethasone than in cells differentiated traditionally. The comparative studies conducted for evaluating efficient measurable glucose uptake by GOPOD assay, radioactive 3H-2-deoxy-D-glucose assay and by non-radioactive 6-NBDG (fluorescent analog of glucose) indicated that the non-radioactive method using 6-NBDG showed a higher signal to noise ratio than the conventional indirect glucose uptake method (GOPOD assay) and the radioactive 3H-2-deoxy-D-glucose uptake method. Differentiated 3T3 L1 cells when triggered with 2.5 ng/mL of Insulin showed 3.3 fold more glucose uptake in non-radioactive method over the radioactive 3H-2-deoxy-D-glucose uptake method. The results of this study have suggested that a combination of Dexamethasone and Troglitazone for 3T3 L1 cell differentiation helps in better quality differentiation over a short period of time with increased sensitivity to Insulin. The application of these findings for developing new methods of screening novel Insulin mimetics and for evaluating the immunological responses has been discussed. 相似文献
47.
Laren Narapareddy Derek E. Wildman Don L. Armstrong Amy Weckle Aleeca F. Bell Crystal L. Patil Suzette D. Tardif Corinna N. Ross Julienne N. Rutherford 《American journal of primatology》2020,82(3):e23101
Accumulating evidence suggests that dysregulation of placental DNA methylation (DNAm) is a mechanism linking maternal weight during pregnancy to metabolic programming outcomes. The common marmoset, Callithrix jaccus, is a platyrrhine primate species that has provided much insight into studies of the primate placenta, maternal condition, and metabolic programming, yet the relationships between maternal weight and placental DNAm are unknown. Here, we report genome-wide DNAm from term marmoset placentas using reduced representation bisulfite sequencing. We identified 74 genes whose DNAm pattern is associated with maternal weight during gestation. These genes are predominantly involved in energy metabolism and homeostasis, including the regulation of glycolytic and lipid metabolic processes pathways. 相似文献
48.
Ridim D. Mote Jyoti Yadav Surya Bansi Singh Mahak Tiwari Shinde Laxmikant V Shivprasad Patil Deepa Subramanyam 《The Journal of biological chemistry》2020,295(49):16888
Mouse embryonic stem cells (mESCs) display unique mechanical properties, including low cellular stiffness in contrast to differentiated cells, which are stiffer. We have previously shown that mESCs lacking the clathrin heavy chain (Cltc), an essential component for clathrin-mediated endocytosis (CME), display a loss of pluripotency and an enhanced expression of differentiation markers. However, it is not known whether physical properties such as cellular stiffness also change upon loss of Cltc, similar to what is seen in differentiated cells, and if so, how these altered properties specifically impact pluripotency. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking Cltc display higher Young''s modulus, indicative of greater cellular stiffness, compared with WT mESCs. The increase in stiffness was accompanied by the presence of actin stress fibers and accumulation of the inactive, phosphorylated, actin-binding protein cofilin. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Young''s modulus to values similar to those obtained with WT mESCs. However, a rescue in the expression profile of pluripotency factors was not obtained. Additionally, whereas WT mouse embryonic fibroblasts could be reprogrammed to a state of pluripotency, this was inhibited in the absence of Cltc. This indicates that the presence of active CME is essential for the pluripotency of embryonic stem cells. Additionally, whereas physical properties may serve as a simple readout of the cellular state, they may not always faithfully recapitulate the underlying molecular fate. 相似文献
49.
Shabir A. Lone A.S. Yadav A. Bajaj Ajit K. Sharma Yogesh Badkhane D.K. Raghuwanshi 《Archives Of Phytopathology And Plant Protection》2013,46(11):1327-1340
Over-utilisation and continuous depletion of medicinal plants have affected their supply and loss of genetic diversity. Hence the current study is based on conservation strategies for threatened medicinal plants with special reference to Barleria prionitis L. using in vitro and ex vitro propagation techniques. We have developed here a protocol for plant regeneration of Barleria prionitis L. We have also developed an efficient system of vegetative propagation of Barleria prionitis L. through stem cuttings using revive rooting hormones. These studies can be useful for conservation strategies of this important medicinal plant. 相似文献
50.
Vaibhav D. Bhatt Suchitra S. Dande Nitin V. Patil Chaitanya G. Joshi 《Molecular biology reports》2013,40(4):3363-3371
Rumen microorganisms play an important role in ruminant digestion and absorption of nutrients and have great potential applications in the field of rumen adjusting, food fermentation and biomass utilization etc. In order to investigate the composition of microorganisms in the rumen of camel (Camelus dromedarius), this study delves in the microbial diversity by culture-independent approach. It includes comparison of rumen samples investigated in the present study to other currently available metagenomes to reveal potential differences in rumen microbial systems. Pyrosequencing based metagenomics was applied to analyze phylogenetic and metabolic profiles by MG-RAST, a web based tool. Pyrosequencing of camel rumen sample yielded 8,979,755 nucleotides assembled to 41,905 sequence reads with an average read length of 214 nucleotides. Taxonomic analysis of metagenomic reads indicated Bacteroidetes (55.5 %), Firmicutes (22.7 %) and Proteobacteria (9.2 %) phyla as predominant camel rumen taxa. At a finer phylogenetic resolution, Bacteroides species dominated the camel rumen metagenome. Functional analysis revealed that clustering-based subsystem and carbohydrate metabolism were the most abundant SEED subsystem representing 17 and 13 % of camel metagenome, respectively. A high taxonomic and functional similarity of camel rumen was found with the cow metagenome which is not surprising given the fact that both are mammalian herbivores with similar digestive tract structures and functions. Combined pyrosequencing approach and subsystems-based annotations available in the SEED database allowed us access to understand the metabolic potential of these microbiomes. Altogether, these data suggest that agricultural and animal husbandry practices can impose significant selective pressures on the rumen microbiota regardless of rumen type. The present study provides a baseline for understanding the complexity of camel rumen microbial ecology while also highlighting striking similarities and differences when compared to other animal gastrointestinal environments. 相似文献