Providing a microenvironment for the development of human CD34+ hematopoietic cells in SCID mice

In order to develop a convenient small-animal model that can support the differentiation of human bone-marrow-derived CD34+ cells, we transplanted SCID mice with an immortalized human stromal cell line, Lof(11–10). The Lof(11–10) cell line has been characterized to produce human cytokines capable of supporting primitive human hematopoietic cell proliferation in vitro. Intraperitoneal injection of Lof(11–10) cells into irradiated SCID mice by itself resulted in a dose-dependent survival of the mice from lethal irradiation. The radioprotective survival was reflected by an increase in the growth and number of mouse bone-marrow-derived committed hematopoietic progenitors. The Lof(11–10) cells localized to the spleen, but not to the bone marrow of these animals and resulted in detectable levels of circulating human IL-6 in their plasma. Secondary intravenous injections of either human or simian CD34+ cells into the Lof(11–10)-transplanted SCID mice resulted in engraftment of injected cells within the bone marrow of these mice. The utility of this small-animal model that allows the growth and differentiation of human CD34+ cells and its potential use in clinical gene therapy protocols are discussed.     

Using experimental evolution to probe molecular mechanisms of protein function

Directed evolution is a powerful tool for engineering protein function. The process of directed evolution involves iterative rounds of sequence diversification followed by assaying activity of variants and selection. The range of sequence variants and linked activities generated in the course of an evolution are a rich information source for investigating relationships between sequence and function. Key residue positions determining protein function, combinatorial contributors to activity and even potential functional mechanisms have been revealed in directed evolutions. The recent application of high throughput sequencing substantially increases the information that can be retrieved from directed evolution experiments. Combined with computational analysis this additional sequence information has allowed high‐resolution analysis of individual residue contributions to activity. These developments promise to significantly enhance the depth of insight that experimental evolution provides into mechanisms of protein function.     

Elevated CO<Subscript>2</Subscript> influences host plant defense response in chickpea against <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Global atmospheric concentration of CO₂ is likely to increase from 350 to 750 ppm over the next 100 years. The present studies were undertaken to understand the effects of elevated CO₂ on enzymatic activity and secondary metabolites in chickpea in relation to expression of resistance to pod borer, Helicoverpa armigera. Fifteen-day-old chickpea plants [ICCL 86111—resistant and JG 11—commercial cultivar] grown in the greenhouse were transferred to open-top chambers (OTC) and kept under 350, 550 and 750 ppm of CO₂. Twenty neonates of H. armigera were released on each plant at 7 days after shifting the pots to the OTCs. Un-infested plants were maintained as controls. After 7 days of infestation, the activities of defensive enzymes [peroxidase (POD), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL)] and amounts of total phenols and condensed tannins increased with an increase in CO₂ concentration in chickpea. The nitrogen balance index was greater in plants kept at 350 ppm CO₂ than in plants kept under ambient conditions. The H. armigera-infested plants had higher H₂O₂ content; amounts of oxalic and malic acids were greater at 750 ppm CO₂ than at 350 ppm CO₂. Plant damage was greater at 350 ppm than at 550 and 750 ppm CO₂. This information will be useful for understanding effects of increased levels of CO₂ on expression of resistance to insect pests to develop strategies to mitigate the effects of climate change.     

Proteolytic processing and inactivation of CCL2/MCP-1 by meprins

Monocyte chemotactic protein 1 (CCL2/MCP-1) is a small chemokine involved in the recruitment and trafficking of mononuclear immune cells to inflammation sites. Our studies demonstrate that the metalloendopeptidases meprin A (purified from kidney cortex), recombinant meprin α, and recombinant meprin β can all process CCL2/MCP-1. The cleavage sites were determined by amino acid sequencing and mass spectrometry analysis of the generated products, and the biological activity of the products was evaluated by chemotactic migration assay using THP-1 cells. The cleavage sites generated by the meprin isoforms revealed that meprin A and meprin α cleaved the N-terminal domain of mouse CCL2/MCP-1 at the Asn⁶ and Ala⁷ bond, resulting in significant reduction in the chemotactic activity of the cleaved CCL2/MCP-1. Meprin β was unable to cleave the N-terminus of mouse CCL2/MCP-1 but cleaved the C-terminal region between Ser⁷⁴ and Glu⁷⁵. Human CCL2/MCP-1 that lacks the murine C-terminal region was also cleaved by meprin α at the N-terminus resulting in significant loss of CCL2/MCP-1 biological activity, whereas meprin β did not affect the biological activity. These studies suggest that meprin α and meprin β may play important roles in regulating the CCL2/MCP-1 chemokine activity during inflammation.     

Purification and characterization of dolichyl-P-mannose:Man5(GlcNAc)2-PP-dolichol mannosyltransferase

The dolichyl-P-mannose:dolichyl-PP-heptasaccharide alpha-mannosyltransferase (2.4.1.130), which catalyzes the transfer of mannose from dolichyl-P-mannose to the Man5(GlcNAc)2-PP-dolichol acceptor glycolipid, was solubilized from pig aorta microsomes with 0.5% NP-40 and purified 985-fold by a variety of conventional methods. The partially purified enzyme had a pH optimum of 6.5 and required Ca2+, at an optimum concentration of 8-10 mM, for activity. Mn2+ was only 20% as effective as Ca2+, and Mg2+ was inhibitory. The mannosyltransferase activity was also inhibited by the addition of EDTA to the enzyme, but this inhibition was fully reversible by the addition of Ca2+. The enzyme was quite specific for dolichyl-P-mannose as the mannosyl donor and Man5(GlcNAc)2-PP-dolichol as the mannosyl acceptor. The Km values for dolichyl-P-mannose and the acceptor lipid Man5(GlcNAc)2-PP-dolichol were 1.8 and 1.6 microM. On Bio-Gel P-4 columns and by HPLC, the radiolabeled oligosaccharide formed during incubation of dolichyl-P-[14C]mannose and unlabeled Man5(GlcNAc)2-PP-dolichol with the purified enzyme behaved like Man6(GlcNAc)2. This octasaccharide was susceptible to digestion by endoglucosaminidase H, indicating that the newly added mannose was attached to the 6-linked mannose in an alpha 1,3-linkage. This linkage was further confirmed by acetolysis of the oligosaccharide product [i.e., Man6(GlcNAc)2], which gave a labeled disaccharide as the major product (greater than 90%).     

Modeling of Hybrid Plasmonic Ring Resonator Based on Dielectric Filled Subwavelength Metal Grating

Plasmonics - A nanophotonic dual ring Cu-SiO2-Si-Cu-SiO2 plasmonic switch with subwavelength metal grating as switching element is designed and simulated in this paper. The 2D finite element method...     

An insight into medicinal chemistry of anticancer quinoxalines

Quinoxalines are benzopyrazines containing benzene and pyrazine rings fused together. In the recent past, quinoxalines have attracted Medicinal Chemists considerably for their syntheses and chemistry due to their distinct pharmacological activities. Diverse synthetic protocols have been developed via multicomponent reactions, single pot synthesis and combinatorial approach using efficient catalysts, reagents, and nano-composites etc. Further, the versatility of the quinoxaline core and its reasonable chemical simplicity devise it extremely promising source of bioactive compounds. Therefore, a wide variety of bioactive quinoxalines has been realised as antitumour, antifungal, anti-inflammatory, antimicrobial, and antiviral agents. Already, a few of them are clinical drugs while many more are under various phases of clinical trials. Present review focuses on chemistry and pharmacology (both efficacy and safety) of quinoxalines and also provides some insight in to their structure–activity relationship.     

Adiponectin receptor 1 variants associated with lower insulin resistance in African Americans

Adiponectin has been shown to have a role in insulin resistance. However, little is known about the contribution of genetic variation in the adiponectin receptor 1 gene (ADIPOR1) in this regard. We hypothesized that variation in ADIPOR1 would be associated with significant changes in insulin resistance and tested this hypothesis in a cohort of 483 African-American adolescents. Seven single nucleotide polymorphisms (SNPs) of ADIPOR1 spanning from the promoter to the 3'-untranslated region were genotyped. We analyzed single SNPs and haplotypes for associations with insulin resistance [homeostasis model assessment of insulin resistance (HOMA-IR)] in the full cohort as well as lean (BMI < 85%) and non-lean (BMI >or= 85%) subsets. There was no evidence of ADIPOR1 variant effects on HOMA-IR in the full cohort or in the lean subset. However, in the non-lean subset, SNP +5843 (A allele), and haplotypes including SNPs -8505/-5692/+3002/+5843 (ATTA and AGTG) showed significant associations with decreased HOMA-IR after adjustment for sex, puberty, adiponectin, and waist z-score. Our findings suggest not only that ADIPOR1 variants influence insulin resistance in the presence of adiposity, but also that these variants and haplotypes are protective in African Americans.     

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside-induced AMP-activated protein kinase phosphorylation inhibits basal and insulin-stimulated glucose uptake, lipid synthesis, and fatty acid oxidation in isolated rat adipocytes

The objective of this study was to investigate the effects of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR)-induced AMP-activated protein kinase (AMPK) activation on basal and insulin-stimulated glucose and fatty acid metabolism in isolated rat adipocytes. AICAR-induced AMPK activation profoundly inhibited basal and insulin-stimulated glucose uptake, lipogenesis, glucose oxidation, and lactate production in fat cells. We also describe the novel findings that AICAR-induced AMPK phosphorylation significantly reduced palmitate (32%) and oleate uptake (41%), which was followed by a 50% reduction in palmitate oxidation despite a marked increase in AMPK and acetyl-CoA carboxylase phosphorylation. Compound C, a selective inhibitor of AMPK, not only completely prevented the inhibitory effect of AICAR on palmitate oxidation but actually caused a 2.2-fold increase in this variable. Compound C also significantly increased palmitate oxidation in the presence of inhibitory concentrations of malonyl-CoA and etomoxir indicating an increase in CPT1 activity. In contrast to skeletal muscle in which AMPK stimulates fatty acid oxidation to provide ATP as a fuel, we propose that AMPK activation inhibits lipogenesis and fatty acid oxidation in adipocytes. Inhibition of lipogenesis would conserve ATP under conditions of cellular stress, although suppression of intra-adipocyte oxidation would spare fatty acids for exportation to other tissues where their utilization is crucial for energy production. Additionally, the stimulatory effect of compound C on long chain fatty acid oxidation provides a novel pharmacological approach to promote energy dissipation in adipocytes, which may be of therapeutic importance for obesity and type II diabetes.     

In vitro regeneration of Trifolium glomeratum

In vitro regeneration of Trifolium glomeratum, a leguminous forage species, was attempted through leaf, petiole, cotyledon, hypocotyl, collar and root explants and two media combinations. Root and collar explants showed no callus induction. Medium with 0.05 mg dm⁻³ α-naphthaleneacetic acid (NAA) and 0.10 mg dm⁻³ N⁶-benzyladenine (BA) was more effective for hypocotyl explant whereas cotyledon and petiole explant were more responsive to 5.0 mg dm⁻³ NAA and 1.0 mg dm⁻³ BA. Friable, green calli obtained from petiole explant on this medium showed organogenetic potential. Modified root-inducing medium having 0.21 mg dm⁻³ indole-3-acetic acid and 2.5 % sucrose was successful for root induction and plantlets were successfully transferred to field after hardening and Rhizobium inoculation.