Basal insulin, glucagon, and growth hormone replacement

Conclusions drawn from the pancreatic (or islet) clamp technique (suppression of endogenous insulin, glucagon, and growth hormone secretion with somatostatin and replacement of basal hormone levels by intravenous infusion) are critically dependent on the biological appropriateness of the selected doses of the replaced hormones. To assess the appropriateness of representative doses we infused saline alone, insulin (initially 0.20 mU.kg(-1).min(-1)) alone, glucagon (1.0 ng.kg(-1).min(-1)) alone, and growth hormone (3.0 ng.kg(-1).min(-1)) alone intravenously for 4 h in 13 healthy individuals. That dose of insulin raised plasma insulin concentrations approximately threefold, suppressed glucose production, and drove plasma glucose concentrations down to subphysiological levels (65 +/- 3 mg/dl, P < 0.0001 vs. saline), resulting in nearly complete suppression of insulin secretion (P < 0.0001) and stimulation of glucagon (P = 0.0059) and epinephrine (P = 0.0009) secretion. An insulin dose of 0.15 mU.kg(-1).min(-1) caused similar effects, but a dose of 0.10 mU.kg(-1).min(-1) did not. The glucagon and growth hormone infusions did not alter plasma glucose levels or those of glucoregulatory factors. Thus, insulin "replacement" doses of 0.20 and even 0.15 mU.kg(-1).min(-1) are excessive, and conclusions drawn from the pancreatic clamp technique using such doses may need to be reassessed.     

Long-chain Acylcarnitines Reduce Lung Function by Inhibiting Pulmonary Surfactant

The role of mitochondrial energy metabolism in maintaining lung function is not understood. We previously observed reduced lung function in mice lacking the fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase (LCAD). Here, we demonstrate that long-chain acylcarnitines, a class of lipids secreted by mitochondria when metabolism is inhibited, accumulate at the air-fluid interface in LCAD^−/− lungs. Acylcarnitine accumulation is exacerbated by stress such as influenza infection or by dietary supplementation with l-carnitine. Long-chain acylcarnitines co-localize with pulmonary surfactant, a unique film of phospholipids and proteins that reduces surface tension and prevents alveolar collapse during breathing. In vitro, the long-chain species palmitoylcarnitine directly inhibits the surface adsorption of pulmonary surfactant as well as its ability to reduce surface tension. Treatment of LCAD^−/− mice with mildronate, a drug that inhibits carnitine synthesis, eliminates acylcarnitines and improves lung function. Finally, acylcarnitines are detectable in normal human lavage fluid. Thus, long-chain acylcarnitines may represent a risk factor for lung injury in humans with dysfunctional fatty acid oxidation.     

Synthesis and biological evaluation of estradiol linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD) conjugates as potential anticancer agents

A series of new estradiol linked pyrrolo[2,1-c][1,4]benzodiazepine (E(2)-PBD) conjugates (3a-f, 4a-f and 5a-f) with different linker architectures including a triazole moiety have been designed and synthesized. All the 18 compounds have been evaluated for their anticancer activity and it is observed that some of the compounds particularly 4c-e and 5c,d exhibited significant anticancer activity. The detailed biological aspects relating to the cell cycle effects and tubulin depolymerization activity have been examined with a view to understand the mechanism of action of these conjugates. Among all these conjugates, one of the compound 5c could be considered as the most effective compound particularly against MCF-7 breast cancer cell line.     

Solid Phase and Solution Phase Synthesis of Gamma Amino Acid Homo-Oligomers and Mixed Oligomers

Abstract  

An efficient stepwise synthesis of homo-oligomers and mixed oligomers of gabapentin and pregabalin on solid support using Fmoc-protected derivatives and HBTU/HOBt/DIEA as coupling agent is described. The synthesis was also carried out using solution phase methodology. The Gpn/Pgn homo oligomers and mixed oligomers forms C₉ helix in solution as determined by NMR study. Chiral as well as achiral gamma amino acids were used for the synthesis of oligomers in order to investigate the secondary structural preferences.     

Up-regulation of MUC2 and IL-1β expression in human colonic epithelial cells by Shigella and its interaction with mucins

Background

The entire gastrointestinal tract is protected by a mucous layer, which contains complex glycoproteins called mucins. MUC2 is one such mucin that protects the colonic mucosa from invading microbes. The initial interaction between microbes and mucins is an important step for microbial pathogenesis. Hence, it was of interest to investigate the relationship between host (mucin) and pathogen interaction, including Shigella induced expression of MUC2 and IL-1β during shigellosis.

Methods

The mucin-Shigella interaction was revealed by an in vitro mucin-binding assay. Invasion of Shigella dysenteriae into HT-29 cells was analyzed by Transmission electron microscopy. Shigella induced mucin and IL-1β expression were analyzed by RT-PCR and Immunofluorescence.

Results

The clinical isolates of Shigella were found to be virulent by a congo-red binding assay. The in vitro mucin-binding assay revealed both Shigella dysenteriae and Shigella flexneri have binding affinity in the increasing order of: guinea pig small intestinal mucinShigella dysenteriae into HT-29 cells occurs within 2 hours. Interestingly, in Shigella dysenteriae infected conditions, significant increases in mRNA expression of MUC2 and IL-1β were observed in a time dependent manner. Further, immunofluorescence analysis of MUC2 shows more positive cells in Shigella dysenteriae treated cells than untreated cells.

Conclusions

Our study concludes that the Shigella species specifically binds to guinea pig colonic mucin, but not to guinea pig small intestinal mucin. The guinea pig colonic mucin showed a greater binding parameter (R), and more saturable binding, suggesting the presence of a finite number of receptor binding sites in the colonic mucin of the host. In addition, modification of mucins with TFMS and sodium metaperiodate significantly reduced mucin-bacterial binding; suggesting that the mucin-Shigella interaction occurs through carbohydrate epitopes on the mucin backbones. Overproduction of MUC2 may alter adherence and invasion of Shigella dysenteriae into human colonic epithelial cells.     

Attempts to delineate the relative contributions of changes in hydrophobicity and packing to changes in stability of ribonuclease S mutants

While the hydrophobic driving force is thought to be a major contributor to protein stability, it is difficult to experimentally dissect out its contribution to the overall free energy of folding. We have made large to small substitutions of buried hydrophobic residues at positions 8 and 13 in the peptide/protein complex, RNase-S, and have characterized the structures by X-ray crystallography. The thermodynamics of association of these mutant S peptides with S protein was measured in the presence of different concentrations of methanol and ethanol. The reduction in the strength of the hydrophobic driving force in the presence of these organic solvents was estimated from surface-tension data as well as from the dependence of the DeltaC(p) of protein/peptide binding on the alcohol concentration. The data indicated a decrease in the strength of the hydrophobic driving force of about 30-40% over a 0-30% range of the alcohol concentration. We observe that large to small substitutions destabilize the protein. However, the amount of destabilization, relative to the wild type, is independent of the alcohol concentration over the range of alcohol concentrations studied. The data clearly indicate that decreased stability of the mutants is primarily due to the loss of packing interactions rather than a reduced hydrophobic driving force and suggest a value of the hydrophobic driving force of less than 18 cal mol(-)(1) A(2).     

Quinazolinone linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD) conjugates: Design,synthesis and biological evaluation as potential anticancer agents

A series of novel quinazolinone linked pyrrolobenzodiazepine (PBD) conjugates were synthesized. These compounds 4a–f and 5a–f were prepared in good yields by linking C-8 of DC-81 with quinazolinone moiety through different alkane spacers. These conjugates were tested for anticancer activity against 11 human cancer cell lines and found to be very potent anticancer agents with GI₅₀ values in the range of <0.1–26.2 μM. Among all the PBD conjugates, one of the conjugate 5c was tested against a panel of 60 human cancer cells. This compound showed activity for individual cancer cell lines with GI₅₀ values of <0.1 μM. The thermal denaturation studies exhibited effective DNA binding ability compared to DC-81 and these results are further supported by molecular modeling studies. The detailed biological aspects of these conjugates on A375 cell line were studied. It was observed that compounds 4b and 5c induced the release of cytochrome c, activation of caspase-3, cleavage of PARP and subsequent cell death. Further, these compounds when treated with A375 cells showed the characteristic features of apoptosis like enhancement in the levels of p53, p21 and p27 inhibition of cyclin dependent kinase-2 (CDK2) and suppression of NF-κB. Moreover, these two compounds 4b and 5c control the cell proliferation by regulating anti-apoptotic genes like (B-cell lymphoma 2) Bcl-2. Therefore, the data generated suggests that these PBD conjugates activate p53 and inhibit NF-κB and thereby these compounds could be promising anticancer agents with better therapeutic potential for the suppression of tumours.