Phagosomes acquire nascent and recycling class II MHC molecules but primarily use nascent molecules in phagocytic antigen processing

Phagosomes contain class II MHC (MHC-II) and form peptide:MHC-II complexes, but the source of phagosomal MHC-II molecules is uncertain. Phagosomes may acquire nascent MHC-II or preexisting, recycling MHC-II that may be internalized from the plasma membrane. Brefeldin A (BFA) was used to deplete nascent MHC-II in murine macrophages to determine the relative contributions of nascent and recycling MHC-II molecules to phagocytic Ag processing. In addition, biotinylation of cell-surface proteins was used to assess the transport of MHC-II from the cell surface to phagosomes. BFA inhibited macrophage processing of latex bead-conjugated Ag for presentation to T cells, suggesting that nascent MHC-II molecules are important in phagocytic Ag processing. Furthermore, detection of specific peptide:MHC-II complexes in isolated phagosomes confirmed that BFA decreased formation of peptide:MHC-II complexes within phagosomes. Both flow organellometry and Western blot analysis of purified phagosomes showed that about two-thirds of phagosomal MHC-II was nascent (depleted by 3 h prior treatment with BFA) and primarily derived from intracellular sites. About one-third of phagosomal MHC-II was preexisting and primarily derived from the plasma membrane. BFA had little effect on phagosomal H2-DM or the degradation of bead-associated Ag. Thus, inhibition of phagocytic Ag processing by BFA correlated with depletion of nascent MHC-II in phagosomes and occurred despite the persistent delivery of plasma membrane-derived recycling MHC-II molecules and other Ag-processing components to phagosomes. These observations suggest that phagosomal Ag processing depends primarily on nascent MHC-II molecules delivered from intracellular sites, e.g., endocytic compartments.     

Spin-label electron spin resonance studies on the mode of anchoring and vertical location of the N-acyl chain in N-acylphosphatidylethanolamines

Electron spin resonance (ESR) studies have been performed on N-myristoyl dimyristoylphosphatidylethanolamine (N-14-DMPE) membranes using both phosphatidylcholines spin-labeled at different positions in the sn-2 acyl chain and N-acyl phosphatidylethanolamines spin-labeled in the N-acyl chain to characterize the location and mobility of the N-acyl chain in the lipid membranes. Comparison of the positional dependences of the spectral data for the two series of spin-labeled lipids suggests that the N-acyl chain is positioned at approximately the same level as the sn-2 chain of the phosphatidylcholine spin-label. Further, similar conclusions are reached when the ESR spectra of the N-acyl PE spin-labels in dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylethanolamine (DMPE) host matrixes are compared with those of phosphatidylcholine spin-labels in these two lipids. Finally, the chain ordering effect of cholesterol has also been found to be similar for the N-acyl PE spin-label and PC spin-labels, when the host matrix is either DMPC and cholesterol or N-14-DMPE and cholesterol at a 6:4 mole ratio. In both cases, the gel-to-liquid crystalline phase transition is completely abolished but cholesterol perturbs the gel-phase mobility of N-14-DMPE more readily than that of DMPC. These results demonstrate that the long N-acyl chains are anchored firmly in the hydrophobic interior of the membrane, in an orientation that is parallel to that of the O-acyl chains, and are located at nearly the same vertical position as that of the sn-2 acyl chains in the lipid bilayer. There is a high degree of dynamic compatibility between the N-acyl chains and the O-acyl chains of the lipid bilayer core, although bilayers of N-acyl phosphatidylethanolamines possess a more hydrophobic interior than phosphatidylcholine bilayers. These results provide a structural basis for rationalizing the biological properties of NAPEs.     

Measurement and control of dissolved carbon dioxide in mammalian cell culture processes using an in situ fiber optic chemical sensor

At high viable cell concentrations in large-scale mammalian cell culture processes, the accumulation of dissolved carbon dioxide (dCO(2), typically quantified as an equilibrium gas-phase concentration) becomes problematic as a result of low CO(2) removal rates at reduced surface-to-volume ratios. High dCO(2) concentrations have previously been shown to inhibit cell growth and product formation in mammalian cells and to alter the glycosylation pattern of recombinant proteins. Therefore, reliable monitoring and control of dCO(2) are important for successful large-scale operation. Off-line measurements by instruments such as blood gas analyzers (BGA) are constrained by the low frequency of data collection and cannot be used for on-line control. In a preliminary evaluation of the YSI 8500 in situ sensor, a response time (t(90%)) of 6 min, sensitivity of 0.5% CO(2) (3.6 mmHg), and linearity of measurement (R(2) = 0.9997) between the equivalent gas-phase partial pressure of 0-180 mmHg (0% and 25% CO(2)) were established. Measurements were found to be unaffected by culture pH and typical mammalian cell culture concentrations of glucose, glutamine, glutamate, lactate, and ammonium. The sensor withstood repeated sterilization and cleaning cycles. The reliability of this sensor was demonstrated in microcarrier-based Chinese hamster ovary (CHO) cell perfusion cultures at reactor scales of 30, 40, 340, and 2000 L and was successfully implemented in a dCO(2) control strategy using N(2) sparging.     

Purification and characterization of thermostable β-amylase and pullulanase from high-yielding Clostridium thermosulfurogenes SV2

Thermostable -amylase and pullulanase, secreted by the thermophilic anaerobic bacterium Clostridium thermosulfurogenes strain SV2, were purified by salting out with ammonium sulphate, DEAE-cellulose column chromatography, and gel filtration using Sephadex G-200. Maltose was identified as a major hydrolysis product of starch by -amylase, and maltotriose was identified as a major hydrolysis product of pullulan by pullulanase. The molecular masses of native -amylase and pullulanase were determined to be 180 and 100 kDa by gel filtration, and 210 and 80 kDa by SDS–PAGE, respectively. The temperature optima of purified -amylase and pullulanase were 70 and 75°C, respectively, and both enzymes were completely stable at 70°C for 2h. The presence of starch further increased the stability of both the enzymes to 80°C and both displayed a pH activity optimum of 6.0. The starch hydrolysis products formed by -amylase action had -anomeric form.     

Global Spectrum of Copy Number Variations Reveals Genome Organizational Plasticity and Proposes New Migration Routes

Global spectrum of CNVs is required to catalog variations to provide a high-resolution on the dynamics of genome-organization and human migration. In this study, we performed genome-wide genotyping using high-resolution arrays and identified 44,109 CNVs from 1,715 genomes across 12 populations. The study unraveled the force of independent evolutionary dynamics on genome-organizational plasticity across populations. We demonstrated the use of CNV tool to study human migration and identified a second major settlement establishing new migration routes in addition to existing ones.     

Effect of monocrotophos on mono amine oxidase activity in albino rats.

Monocrotophos inhibited monoamine oxidase (MAO) activity both in vitro and in vivo in liver, kidney and brain areas of albino rats. In vitro effect was more pronounced than the in vivo effect. During in vivo daily treatment with sublethal doses of Monocrotophos, the MAO activity was significantly inhibited after 1h to 7 days of treatments. Later recovery of inhibition was noticed which might be attributed to the enhanced absorption of Monocrotophos to protein and fat bodies or enhanced metabolic dispositional mechanisms. Brain areas exhibit varied responses to Monocrotophos toxicity.     

CC chemokine receptor-3 (CCR3) antagonists: improving the selectivity of DPC168 by reducing central ring lipophilicity

DPC168, a benzylpiperidine-substituted aryl urea CCR3 antagonist evaluated in clinical trials, was a relatively potent inhibitor of the 2D6 isoform of cytochrome P-450 (CYP2D6). Replacement of the cyclohexyl central ring with saturated heterocycles provided potent CCR3 antagonists with improved selectivity against CYP2D6. The favorable preclinical profile of DPC168 was maintained in an acetylpiperidine derivative, BMS-570520.     

Leptin downregulates ethanol-induced secretion of proinflammatory cytokines and growth factor

Inflammatory mediators, including cytokines and growth factors are associated with the pathology of chronic liver diseases. The aim of our present work was to study the effect of exogenous leptin and/or ethanol on the secretion of TNF-alpha, IL-6 and TGF-beta1 both in vivo and in vitro. Forty eight hours after the exposure to ethanol (500 mM) significantly elevated the secretion of TNF-alpha, IL-6 and TGF-beta1 in the cell-free culture supernatant (HepG2 and mouse HCC cell lines), which were decreased on leptin (31.2 nM) treatment. Similarly, leptin administration to ethanol (6.32 g kg(-1) body weight) fed mice for 45 days significantly lowered the concentration of these cytokines in the circulation; however, leptin alone (230 microg kg(-1) body weight i.p. administered every alternate day for the last 15 days) had no such significant effect on cytokine secretion both in vivo and in vitro conditions. We conclude that leptin is involved in the protective mechanisms that allow an organism to cope with the potentially autoaggressive effects of its immune system in alcoholic liver disease.     

Isolation, purification and properties of new restriction endonucleases from Bacillus badius and Bacillus lentus

We tentatively named two enzymes as BbaI and BleI, which were isolated and purified from Gram-positive mesophilic bacteria Bacillus badius 1458 and Bacillus lentus 1689 respectively, by ammonium sulphate precipitation, phosphocellulose and heparin-sepharose column chromatography. SDS-PAGE protein profiles for BbaI and BleI showed denatured molecular weights of 52 and 48 kDa, respectively. BbaI hydrolyzed pUC18 DNA into 1900 and 700 bp, pBR322 DNA into two fragments of 2800 and 1500 bp and Φ×174 DNA into 3800 and 1600 bp. BleI hydrolyzed pUC18 DNA into 1800 and 800 bp, pBR322 DNA into two fragments of 2700 and 1600 bp and Φ×174 DNA into 3700 and 1700 bp. The effects of temperature, ionic strength, pH and Mg²⁺ ion concentrations were studied to demonstrate some biochemical properties of BbaI and BleI. Maximum activities of these enzymes were observed at 37 °C (pH 8.0) with 100 mM NaCl and 10 mM Mg²⁺ concentrations.