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Herein, an efficient and scalable synthesis of 2,4-diaryl substituted chromene [43-b] pyridine derivatives, using a multicomponent reaction strategy by microwave irradiation of 4-amino 2-oxo- 2H-chromene, aromatic aldehydes, and substituted anilines is described. This synthetic strategy was found to be very useful as it follows environment benign protocol. Also it gives a good outcome in terms of yield and requires a shorter reaction time.

This thesis describes a novel approach for probing ionic conductivity of silica ionogel electrolytes on the meso- and nanoscale. These properties are important to know for the development of solid-state energy-storage devices that implement nanostructures. Silica ionogels are of specific interest because they maintain a fluidic state on the nanoscale, allowing for a high conductivity of liquid electrolytes, but on the macroscale, they maintain the stability benefits of solid electrolytes. Using single mesopores in polymer films to restrict the geometry of the ionogel to the desired size, we were able to measure the conductivity using two methods. The first method uses liquid electrolytes and makes measurements similar to how the conductivity in liquid electrolytes is found, the other method probes the solid electrolyte in a way more representative to that of energy storage devices. We concluded that the electrochemical properties of ionogel are not significantly affected by constriction to the mesoscale, meaning they can be useful in the implementation of mesoscale solid-state energy-storage devices.

Designing drug candidates exhibiting polypharmacology is one of the strategies adopted by medicinal chemists to address multifactorial diseases. Metabolic disease is one such multifactorial disorder characterized by hyperglycaemia, hypertension and dyslipidaemia among others. In this paper we report a new class of molecular framework combining the pharmacophoric features of DPP4 inhibitors with those of ACE inhibitors to afford potent dual inhibitors of DPP4 and ACE.

Aim: The mechanism of action of Annona squamosa hexane extract in mediating antihyperglycemic and antitriglyceridimic effect were investigated in this study. Materials and Methods: The effects of extract on glucose uptake, insulin receptor-β (IR-β), insulin receptor substrate-1 (IRS-1) phosphorylation and glucose transporter type 4 (GLUT4) and phosphoinositide 3-kinase (PI3 kinase) mRNA expression were studied in L6 myotubes. The in vitro mechanism of action was tested in protein-tyrosine phosphatase 1B (PTP1B), G-protein-coupled receptor 40 (GPR40), silent mating type information regulation 2 homolog 1 (SIRT1) and dipeptidyl peptidase-IV (DPP-IV) assays. The in vivo efficacy was characterized in ob/ob mice after an oral administration of the extract for 21 days. Results: The effect of extract promoted glucose uptake, IR-β and IRS-1 phosphorylation and GLUT4 and PI3 kinase mRNA upregulation in L6 myotubes. The extract inhibited PTP1B with an IC50 17.4 μg/ml and did not modulate GPR40, SIRT1 or DPP-IV activities. An oral administration of extract in ob/ob mice for 21 days improved random blood glucose, triglyceride and oral glucose tolerance. Further, the extract did not result in body weight gain before and after treatment (29.3 vs. 33.6 g) compared to rosiglitazone where significant body weight gain was observed (28.4 vs. 44.5 g; *P

SIRT1, human ortholog of yeast SIR2 protein, deacetylates histones and several other transcription factors. Recently, SIRT1 has emerged as a drug target for treating age related diseases, type II diabetes, neurodegeneration, inflammation and cancer. Here, we have optimized production of functionally active wild type full-length SIRT1 protein and its N-terminal deleted mutants. In a comparative study, we found that the region containing 192-208 amino acids towards the N-terminus is critical for right conformational folding of the protein to retain its deacetylase activity. The EC(50) and IC(50) values obtained with standard modulators showed that the SRT(748)SRT(556) can deacetylate substrate and are activated by resveratrol, whereas, deacetylase activity of all the other deletion mutants (SRT(540), SRT(532), SRT(507) and SRT(503)) was lost. We further report that the peptide substrate K(m) for SRT(748) (70+/-5.2 microM) was comparable to SRT(556) (93+/-5.4 microM). The K(m) for NAD(+) substrate was 176274 microM for SRT(748) and SRT(556), respectively. Similar substrate affinity studies demonstrate that either of the protein (SRT(748) or SRT(556)) can be utilized for screening SIRT1 modulators. We have also examined critical regions in SIRT1 required for deacetylase activity as well as kinetic analyses of SIRT1 proteins.

We report the diagnosis and follow-up of two sibs reported in 1980 with recurrent venous thromboses and protein-losing enteropathy; one sib with biopsy-proven hepatic fibrosis died at age 5. The combination of symptoms was suggestive of the recently characterized congenital disorder of glycosylation type Ib (CDG-Ib), which is caused by a deficiency of the enzyme phosphomannose isomerase (PMI). An abnormal serum transferrin isoelectric focusing (IEF) pattern and a reduced PMI activity confirmed the diagnosis of CDG-Ib. Furthermore, mutational analysis of the MPI gene revealed two missense mutations, 419 T → C (I140T) and 636 G → A (R219Q), a single base substitution in intron 5, 670 + 9G → A, as well as a polymorphism 1131A → C (V377V) in both sibs. The surviving 33-year-old sib has had no further symptoms following childhood. Short-term low-dose oral mannose supplementation improved her transferrin IEF pattern and normalized her antithrombin III activity, further substantiating the beneficial effect of mannose in CDG-Ib. When her mannose blood level was measured, she showed a lower steady-state level but a faster mannose clearance rate. These results suggest that the clinical manifestations of PMI deficiency, although serious in childhood, can improve with age, even without mannose therapy, and allow for a normal adult life. However, the long-term prognosis may vary from patient to patient.

Several herbal plants such as Chinese herb Rhizoma Coptidis have been reported to possess antidiabetic activity. Berberine is its major active constituent and functions as an insulin sensitizer and insulin secretagogue. It has been reported to modulate several signaling pathways and targets. The objective of the current study is to investigate if berberine can function as a ligand of fatty acid receptor GPR40, which stimulates glucose dependent insulin secretion. Towards this objective, a mammalian cell line with stable overexpression of GPR40 was generated and characterized. Berberine stimulated calcium mobilization with an EC(50) of 0.76 microM in this GPR40 overexpressing cell line. Further, berberine stimulated glucose dependent insulin secretion from rat pancreatic beta cell line. This suggests that berberine functions as an agonist of fatty acid receptor GPR40 and might be a novel antidiabetic mechanism of action for berberine.

In the present study, resveratrol, a polyphenolic SIRT1 activator was evaluated for its SIRT1 activation in an in vitro fluorescent based assay (EC50: 7 μM). The efficacy of resveratrol was also evaluated in ob/ob mice for its antidiabetic and associated metabolic effects. Mice aged 5–8 weeks were included in four groups; control and resveratrol at 5, 15, 50 mg/kg, b.i.d. and were dosed orally. After 4 weeks of drug treatment, body weights were noted and random blood glucose and insulin was estimated for the antidiabetic effect. Animals were also subjected to the oral glucose tolerance test to observe any improvement in the glucose excursion. Triglycerides, total cholesterol, adiponectin and free fatty acid levels were also estimated. The results showed that resveratrol exhibited significant antihyperglycemic activity with an improvement in the insulin levels compared with the control mice. There was also a significant improvement observed in the glucose excursion in the oral glucose tolerance test performed for 120 min; although an insignificant improvement in the triglycerides, total cholesterol, adiponectin and free fatty acid levels was observed at different doses of resveratrol tested. The present findings suggest that resveratrol is an antihyperglycemic agent and drugs similar to resveratrol can be considered as an effective therapeutic adjuvant for the current treatment of diabetes mellitus. Copyright © 2010 John Wiley & Sons, Ltd.

The induction of conformationally restricted N-(aryl or heteroaryl)-3-azabicyclo[3.1.0]hexane derivatives at P2 region of compounds of 2-cyanopyrrolidine class was explored to develop novel DPP-IV inhibitors. The synthesis, structure–activity relationship, and selectivity against related proteases are delineated.

We investigated the metabolic defect(s) of four children who presented with isolated cryptogenic chronic liver disease, coagulopathy, and abnormalities of several unrelated serum glycoproteins. Analysis of the patients' serum glycoproteins and fibroblasts suggest they have a novel congenital disorder of glycosylation (CDG). All had abnormal transferrin (Tf) isoelectric focusing (IEF) profiles. More detailed analysis of Tf by electrospray ionization mass spectrometry (ESI-MS) showed a plethora of abnormal glycosylations that included loss of 1-2 sialic acids and 1-2 galactose units, typical of Group II defects. Tf from two patients also lacked 1-2 entire oligosaccharide chains, typical of Group One disorders. Total serum N-glycans were analyzed by HPLC and matrix-assisted laser desorption/ionization mass spectrometry and also showed increased proportion of neutral glycan chains lacking sialic acids and galactose units. Analysis of patient fibroblasts eliminated CDG-Ia, through CDG-Ih, -IL and CDG-IId. Our results suggest that a subset of children with clinically asymptomatic, cryptogenic hypertransaminasemia and/or liver steato-fibrosis may represent a novel type of CDG-X with an unknown defect(s). Clinicians are encouraged to test such patients for abnormal Tf glycosylation by ESI-MS.

MPI encodes phosphomannose isomerase, which interconverts fructose 6-phosphate and mannose 6-phosphate (Man-6-P), used for glycoconjugate biosynthesis. MPI mutations in humans impair protein glycosylation causing congenital disorder of glycosylation Ib (CDG-Ib), but oral mannose supplements normalize glycosylation. To establish a mannose-responsive mouse model for CDG-Ib, we ablated Mpi and provided dams with mannose to rescue the anticipated defective glycosylation. Surprisingly, although glycosylation was normal, Mpi(-/-) embryos died around E11.5. Mannose supplementation even hastened their death, suggesting that man-nose was toxic. Mpi(-/-) embryos showed growth retardation and placental hyperplasia. More than 90% of Mpi(-/-) embryos failed to form yolk sac vasculature, and 35% failed chorioallantoic fusion. We generated primary embryonic fibroblasts to investigate the mechanisms leading to embryonic lethality and found that mannose caused a concentration- and time-dependent accumulation of Man 6-P in Mpi(-/-) fibroblasts. In parallel, ATP decreased by more than 70% after 24 h compared with Mpi(+/+) controls. In cell lysates, Man-6-P inhibited hexokinase (70%), phosphoglucose isomerase (65%), and glucose-6-phosphate dehydrogenase (85%), but not phosphofructokinase. Incubating intact Mpi(-/-) fibroblasts with 2-[(3)H]deoxyglucose confirmed mannose-dependent hexokinase inhibition. Our results in vitro suggest that mannose toxicity in Mpi(-/-) embryos is caused by Man-6-P accumulation, which inhibits glucose metabolism and depletes intracellular ATP. This was confirmed in E10.5 Mpi(-/-) embryos where Man-6-P increased more than 10 times, and ATP decreased by 50% compared with Mpi(+/+) littermates. Because Mpi ablation is embryonic lethal, a murine CDG-Ib model will require hypomorphic Mpi alleles.

Dietary mannose is used to treat glycosylation deficient patients with mutations in phosphomannose isomerase (PMI), but there is little information on mannose metabolism in model systems. We chose the mouse as a vertebrate model. Intravenous injection of [2-3H]mannose shows rapid equilibration with the extravascular pool and clearance t(1/2) of 28 min with 95% of the label catabolized via glycolysis in2 h. Labeled glycoproteins appear in the plasma after 30 min and increase over 3 h. Various organs incorporate [2-3H]mannose into glycoproteins with similar kinetics, indicating direct transport and utilization. Liver and intestine incorporate most of the label (75%), and the majority of the liver-derived proteins eventually appear in plasma. [2-3H]Mannose-labeled liver and intestine organ cultures secrete the majority of their labeled proteins. We also studied the long-term effects of mannose supplementation in the drinking water. It did not cause bloating, diarrhea, abnormal behavior, weight gain or loss, or increase in hemoglobin glycation. Organ weights, histology, litter size, and growth of pups were normal. Water intake of mice given 20% mannose in their water was reduced to half compared to other groups. Mannose in blood increased up to 9-fold (from 100 to 900 microM) and mannose in milk up to 7-fold (from 75 to 500 microM). [2-3H]Mannose clearance, organ distribution, and uptake kinetics and hexose content of glycoproteins in organs were similar in mannose-supplemented and non-supplemented mice. Mannose supplements had little effect on the specific activity of phosphomannomutase (Man-6-P--Man-1-P) in different organs, but specific activity of PMI in brain, intestine, muscle, heart and lung gradually increased2-fold with increasing mannose intake. Thus, long-term mannose supplementation does not appear to have adverse effects on mannose metabolism and mice safely tolerate increased mannose with no apparent ill effects.

Phosphomannose isomerase (PMI) interconverts fructose-6-P (Fru-6-P) and mannose-6-P (Man-6-P), linking energy metabolism to protein glycosylation. We have cloned the mouse Mpi cDNA, analyzed its genomic organization, and studied the expression in different tissues. The Mpi gene has eight exons covering 7.2 kb. The structure and intron-exon boundaries are essentially the same as its human ortholog with 85% amino acid identity. Mpi is alternatively spliced at the 3' end, resulting in three messages with different 3'-untranslated regions. Mpi expression is regulated at both the transcription and translation levels, with the highest expression level in testis. Rabbit antibodies prepared against mouse PMI expressed in E. coli recognize a single 47-kDa band. Immunohistochemistry of mouse tissues shows general cytosolic staining in all cells. In testis, staining is intense in round spermatids and residual bodies, moderate in pachytene spermatocytes, and weak in spermatogonia and spermatozoa. In contrast, northern blot analysis shows comparable transcripts of 1.8 and 1.6 kb in pachytene spermatocytes and round spermatids, suggesting delayed translation of PMI. The stage-specific expression of PMI in testis may be important for KDN synthesis, which requires Man-6-P, or it may be needed to ensure sufficient glycosylation precursors in cells that do not utilize glucose and instead rely on lactate and pyruvate.