Degradation of HMG-CoA reductase in vitro. Cleavage in the membrane domain by a membrane-bound cysteine protease

We have recently shown that the endoplasmic reticulum (ER) membrane protein, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is cleaved in isolated membrane fractions enriched for endoplasmic reticulum. Importantly, the cleavage rate is accelerated when the membranes are prepared from cells that have been pretreated with mevalonate or sterols, physiological regulators of the degradation process in vivo (McGee, T. P., Cheng, H. H., Kumagai, H., Omura, S., and Simoni, R. D. (1996) J. Biol. Chem. 271, 25630-25638). In the current study, we further characterize this in vitro cleavage of HMG-CoA reductase. E64, a specific inhibitor of cysteine-proteases, inhibits HMG-CoA reductase cleavage in vitro. In contrast, lactacystin, an inhibitor of the proteasome, inhibits HMG-CoA reductase degradation in vivo but does not inhibit the in vitro cleavage. Purified ER fractions contain lactacystin-sensitive and E64-insensitive proteasome activity as measured by succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin hydrolysis. We removed the proteasome from purified ER fractions by solubilization with heptylthioglucoside and observed that the detergent extracted, proteasome-depleted membrane fractions retain regulated cleavage of HMG-CoA reductase. This indicates that ER-associated proteasome is not involved in degradation of HMG-CoA reductase in vitro. In order to determine the site(s) of proteolysis of HMG-CoA reductase in vitro, four antisera were prepared against peptide sequences representing various domains of HMG-CoA reductase and used for detection of proteolytic intermediates. The sizes and antibody reactivity of the intermediates suggest that HMG-CoA reductase is cleaved in the in vitro degradation system near the span 8 membrane region, which links the N-terminal membrane domain to the C-terminal catalytic domain of the protein. We conclude that HMG-CoA reductase can be cleaved in the membrane-span 8 region by a cysteine protease(s) tightly associated with ER membranes.     

Molecular characterization of a globally distributed lineage of serotype 12F Streptococcus pneumoniae causing invasive disease

These studies have identified a major genetic lineage of capsule serotype 12F Streptococcus pneumoniae, which has maintained two different types of the pneumococcal surface protein A (PspA) virulence factor and caused invasive disease in geographically disjoint locations. Twenty outbreak strains from a Texas jail and Maryland day care center and 16 reference strains from Texas, Maryland, Washington, Michigan, Oklahoma, Missouri, Alaska, and Australia were examined. Although the Texas and Maryland outbreak strains were indistinguishable by IS1167 and boxA genotyping procedures, all strains examined were members of a genetically similar lineage. The microevolutionary history of pspA differed from that of the overall genetic background of the strains. Taken together, these findings suggested that the Texas and Maryland outbreaks were caused by different clones of a major genetic lineage of serotype 12F pneumococci, within which at least one PspA has been acquired via localized genetic recombination.     

In vitro induction of differentiation by ginsenoides in F9 teratocarcinoma cells

The aim of this study was to determine the ability of the ginsenosides, extracts of Panax ginseng C.A. Meyer, to cause differentiation of F9 teratocarcinoma stem cells as a model system. F9 stem cells cultured in the presence of the ginsenosides together with dibutyryl cyclic AMP (dbcAMP) became parietal endoderm-like cells. Moreover, the expression of differentiation marker genes, such as laminin B1 and type IV collagen, was increased after treatment with the ginsenosides. Among the various purified ginsenosides, Rh1 and Rh2 were the most effective at causing differentiation of F9 cells. Since ginsenosides and glucocorticoid hormone have similar chemical structures, we examined the possibility of the involvement of a glucocorticoid receptor (GR) in the differentiation process induced by the ginsenosides. According to Southwestern blot analysis, a 94 kDa protein regarded as a GR was detected in F9 cells cultured in the medium containing the ginsenosides Rh1 or Rh2. In addition, F9 stem cells treated with the ginsenosides Rh1 or Rh2 and with RU486, a glucocorticoid antagonist with a high affinity for the GR, did not differentiate into endoderm cells morphologically, and the expression of laminin B1 gene was not induced in these cells. In a gel mobility shift assay, protein factors capable of binding to the glucocorticoid responsive element (GRE) specifically were detected in nuclear extracts of the ginsenoside-treated F9 cells. Moreover, overexpression of GR by cotransfection of GR expression vector and GRE-luciferase vector enhanced the transactivation activity of GRE promoter in the presence of ginsenosides Rh1 or Rh2 and was further augmented by dbcAMP. In addition, ginsenosides Rh1 and Rh2 bound to a GR assessed by whole-cell binding assay, even though the specific binding affinity was weaker compared to dexamethasone. Based on these data, we suggest that the ginsenosides Rh1 and Rh2 cause the differentiation of F9 cells and the effects of ginsenosides might be exerted via binding with a GR or its analogous nuclear receptor.     

Optimisation of osmotic dehydration process of carrot cubes in mixtures of sucrose and sodium chloride solutions

In the optimisation of the osmotic dehydration process of the carrot cubes in mixtures of sucrose and sodium chloride by response surface methodology, using face-centred central composite design (CCF), it was shown that the independent process variables for osmotic dehydration process were osmotic solution concentrations (5–15% w/v sodium chloride in 50 °Brix sucrose syrup), temperature (35–55 °C) and process duration (120–240 min). Statistical analysis of results showed that the linear terms of all the process variables have a significant effect on all the responses. The optimum osmotic dehydration process conditions for maximum water loss, minimum solute gain, maximum retention of colour, and sensory score were: 50 °Brix + 15% w/v sodium chloride solution, 54.8 °C solution temperature and 120 min process duration.     

Therapeutic Intervention with Anti-Complement Component 5 Antibody Does Not Reduce NASH but Does Attenuate Atherosclerosis and MIF Concentrations in Ldlr-/-.Leiden Mice

Background: Chronic inflammation is an important driver in the progression of non-alcoholic steatohepatitis (NASH) and atherosclerosis. The complement system, one of the first lines of defense in innate immunity, has been implicated in both diseases. However, the potential therapeutic value of complement inhibition in the ongoing disease remains unclear. Methods: After 20 weeks of high-fat diet (HFD) feeding, obese Ldlr-/-.Leiden mice were treated twice a week with an established anti-C5 antibody (BB5.1) or vehicle control. A separate group of mice was kept on a chow diet as a healthy reference. After 12 weeks of treatment, NASH was analyzed histopathologically, and genome-wide hepatic gene expression was analyzed by next-generation sequencing and pathway analysis. Atherosclerotic lesion area and severity were quantified histopathologically in the aortic roots. Results: Anti-C5 treatment considerably reduced complement system activity in plasma and MAC deposition in the liver but did not affect NASH. Anti-C5 did, however, reduce the development of atherosclerosis, limiting the total lesion size and severity independently of an effect on plasma cholesterol but with reductions in oxidized LDL (oxLDL) and macrophage migration inhibitory factor (MIF). Conclusion: We show, for the first time, that treatment with an anti-C5 antibody in advanced stages of NASH is not sufficient to reduce the disease, while therapeutic intervention against established atherosclerosis is beneficial to limit further progression.     

Thermodynamic,spectroscopic, and molecular characterization of rice straw biomass for use as biofuel feedstock

India is one of the leading producers of rice in the world. Gasification and pyrolysis are two thermochemical processes. In the gasification process, biomass is transformed into syngas, which serves as an energy source. This conversion occurs under high temperatures with a carefully regulated and restricted air supply. On the other hand, pyrolysis, which transpires at lower temperatures without the presence of air, generates pyrolysis oil as a by-product. This oil can be further refined into liquid fuels. For the purpose of investigating the feasibility of biofuel production, the current study involved the characterization of rice straw biomass using various techniques such as thermogravimetry differential thermal analysis (TG/DTA), Fourier transform infrared (FTIR), carbon, hydrogen, nitrogen, sulphur and oxygen (CHNS/O), inductively coupled plasma optical emission spectroscopy (ICP-OES), among others. These analytical methods were employed to assess the potential of rice straw biomass for the production of biofuels. The existence of a significant amount of cellulose (32.1%), volatiles (approximately 67.06%), and high heating value (HHV) (13.18 MJKg⁻¹) in rice straw inferred their capability to be used as feedstocks in the production of biofuel. The activation energy of approximately 173.20 KJ/Mol (Flynn Wall Ozawa [FWO]) indicated the viability of the burning process. From master plot (Z(α)) analysis, the experimental curve was seen passing through different theoretical curves, indicating the complex nature of the pyrolysis process for rice straw.     

Effect of contamination on rise velocity of bubble swarms at moderate Reynolds numbers

In this work, hydrodynamics of contaminated bubble swarms is numerically investigated using the free surface cell model combined with the spherical stagnant cap model. The governing field equations are solved numerically to elucidate the effect of Reynolds number, gas holdup and degree of contamination on the hydrodynamic behavior of bubble swarms. New extensive results are reported over the range of conditions as follows: Reynolds number, Re: 1–200, bubble holdup, Φ: 0.1–0.5, and stagnant cap angle, α: 0–180°. Finally, the effects of these parameters on streamlines and vorticity contours, surface pressure and vorticity distributions and on drag coefficients are discussed in detail. Briefly, the drag coefficients decrease with the decreasing stagnant cap angle and/or the decreasing bubble hold up and/or the increasing Reynolds number; whereas the ratio of the pressure and friction drag coefficients exhibits mixed trends with respect to these parameters.     

A mechanistic study of beta-adrenoceptor antagonists on ethanol-induced gastric damage

BACKGROUND: In the first weeks of life there are important maturational changes in the central nervous system in many species in energy metabolism, synapse number, and concentration of neuronal excitatory receptors. METHODS: Four groups of 10 piglets (aged 1, 2, 4, and 10 weeks) underwent 1 hour of deep hypothermic circulatory arrest at 15 degrees C, with cooling and rewarming on cardiopulmonary bypass. Cerebral blood flow and metabolic rate measurements and electroencephalographic recordings were obtained from 5 animals per group. The remaining animals underwent cerebral magnetic resonance spectroscopy. RESULTS: Preoperative cerebral blood flow and glucose consumption were higher at 4 and 10 weeks than at 1 and 2 weeks. Cerebral adenosine triphosphate content decreased more rapidly during deep hypothermic circulatory arrest at 4 and 10 weeks. Phosphocreatine recovery was greater at 30 minutes of reperfusion at 10 weeks compared with 1 week. Recovery of cerebral phosphocreatine/ adenosine triphosphate ratio and intracellular pH was remarkably uniform at all ages. Latency to recovery of electroencephalographic activity decreased with increasing age (p = 0.04). CONCLUSIONS: Differences in acute recovery of brain energy metabolism and electroencephalogram after cardiopulmonary bypass and 1 hour of deep hypothermic circulatory arrest in piglets between 1 and 10 weeks of age are small. Further studies are required to correlate these acute findings with subsequent neurologic outcome.     

Intrusions in Alzheimer's disease in immediate and delayed memory as a function of presentation modality

Young and elderly participants, and participants with Alzheimer's disease (AD) were compared via the suffix paradigm, where a not-to-be-recalled item is appended onto sequences to be immediately recalled. This task was followed by delayed tasks. In immediate recall, AD subjects showed both extralist and suffix intrusions. Recall of auditorily as compared with visually presented stimuli was superior, with the difference increasing in older subjects. The auditory but not the visual suffix produced an end-of-sequence decrement, which was greater in AD than in other groups. After delay, the elderly and young showed virtually perfect performance. The AD participants showed relatively high performance; however, extralist intrusions were frequent, resulting in a relatively low hit rate. As in immediate recall, intrusions showed specificity for AD, and in this paradigm appeared to be a marker differentiating AD and normal subjects. However, the sample size limits the power and generalizability of these findings.