Competition between protein folding and aggregation with molecular chaperones in crowded solutions: insight from mesoscopic simulations

The living cell is inherently crowded with proteins and macromolecules. To avoid aggregation of denatured proteins in the living cell, molecular chaperones play important roles. Here we introduce a simple model to describe crowded protein solutions with chaperone-like species based on a dynamic density functional theory. As predicted by others, our simulations show that macromolecular crowding enhances the association of proteins and chaperones. However, when the intrinsic folding rate of the protein is slow, it is possible that crowding also enhances aggregation of proteins. The results of simulation suggest that, when the concentration of the crowding agent is as high as that in the cell, the association of the protein and unbound chaperone becomes correlated with the aggregation process, and that the protein-bound chaperones efficiently destroy the potential nuclei of aggregates and thus prevent the aggregation.     

Interferon-gamma production and host protective response against Mycobacterium tuberculosis in mice lacking both IL-12p40 and IL-18

Interferon (IFN)-gamma plays an essential role in host defense against infection with Mycobacterium tuberculosis, and its synthesis is critically regulated by interleukin (IL)-12, IL-18 and the recently identified IL-23. The present study was designed to determine the roles of these cytokines in IFN-gamma-mediated host defenses against M. tuberculosis. For this purpose, we compared host protective responses in IL-12p40 and IL-18 double-knockout (DKO) mice (which lacked both IL-12/IL-18 and also IL-23) and IFN-gamma gene-disrupted (GKO) mice. DKO mice were more resistant to the infection than GKO mice, as indicated by their extended survival and reduced live colony numbers in spleen, liver and lung. IFN-gamma was detected by ELISA in liver and lung homogenates, but not in spleen and serum, and in all organs by RT-PCR in DKO mice at comparable or reduced levels to those in wild-type mice. IFN-gamma production was reduced by depletion of CD4+ T cells, but not of natural killer (NK), NKT, gammadeltaT and dendritic cells. Neutralization of IFN-gamma or TNF-alpha by specific monoclonal antibodies (mAbs) significantly shortened the survival time of the infected DKO mice. Furthermore, anti-TNF-alpha mAb partially attenuated IFN-gamma synthesis in the liver of these mice. Finally, the expression level of inducible nitric oxide synthase (iNOS) mRNA in the spleen, liver and lung was considerable in DKO mice but only marginal or undetected in GKO mice. Our results indicate the presence of IL-12-, IL-18- and IL-23-independent host protective responses against mycobacterial infection mediated by IFN-gamma, which was secreted from helper T cells.     

Validation of a simplified method for determination of cimetidine in human plasma and urine by liquid chromatography with ultraviolet detection

A HPLC method was developed for determination of cimetidine in human plasma and urine. Plasma samples were alkalinized followed by liquid extraction with water-saturated ethyl acetate then evaporated under nitrogen. The extracts were reconstituted in mobile phase and injected onto a C(18) reversed-phase column; UV detection was set at 228 nm. Urine samples were diluted with an internal standard/mobile phase mixture (1:9) prior to injection. The lower limit of quantification in plasma and urine were 100 ng/ml and 10 microg/ml, respectively; intra- and inter-day coefficients of variation were 相似文献   

Effect of electrostatic interactions on the binding of charged substrate to GroEL studied by highly sensitive fluorescence correlation spectroscopy

The binding processes of GroEL with apo cytochrome c (apo-cyt c) and disulfide-reduced apo alpha-lactalbumin (rLA) in homogeneous solution at low concentration were analyzed by fluorescence correlation spectroscopy (FCS) with extremely high sensitivity. Although apo-cyt c, a positively charged substrate, was tightly bound to GroEL in both the absence and the presence of 200 mM KCl, the strength of the binding was changed with varying salt concentration. Results from experiments when two different salts (KCl or MgCl(2)) were titrated into a sample solution containing GroEL and apo-cyt c clearly showed that the binding strength decreased with increasing salt concentration. On the other hand, the binding affinity of GroEL for rLA, a negatively charged substrate, increased by adding of 200 mM KCl. These results indicate that electrostatic interactions substantially contribute to the binding interactions by manipulating the binding affinity of charged substrates.     

Dectin-1 is not required for the host defense to Cryptococcus neoformans

Dectin-1 is known as a sole receptor for beta-glucan, a major cell wall component of fungal microorganisms. In the current study, we examined the role of this molecule in the host defense to Cryptococcus neoformans, an opportunistic fungal pathogen in AIDS patients. There was no significant difference in the clinical course and cytokine production between dectin-1 gene-deficient and control mice. These results indicate that dectin-1 is not likely essential for the development of host protective responses to C. neoformans.     

Delayed Expression of Both GABABR1 and GABABR2 Subunits in Murine Hippocampal Dentate Gyrus After a Single Systemic Injection of Trimethyltin

Neurochemical Research - Trimethyltin (TMT) has been used as a cytotoxin to neurons rather than glial cells in the mammalian hippocampus. The systemic administration of TMT led to declined...     

Suppressive effects of electrochemically reduced water on matrix metalloproteinase-2 activities and in vitro invasion of human fibrosarcoma HT1080 cells

It has been demonstrated that hydrogen peroxide (H(2)O(2)) is directly associated with elevated matrix metalloproteinase-2 (MMP-2) expression in several cell lines. Electrochemically reduced water (ERW), produced near the cathode during electrolysis, and scavenges intracellular H(2)O(2) in human fibrosarcoma HT1080 cells. RT-PCR and zymography analyses revealed that when HT1080 cells were treated with ERW, the gene expression of MMP-2 and membrane type 1 MMP and activation of MMP-2 was repressed, resulting in decreased invasion of the cells into matrigel. ERW also inhibited H(2)O(2)-induced MMP-2 upregulation. To investigate signal transduction involved in MMP-2 downregulation, mitogen-activated protein kinase (MAPK)-specific inhibitors, SB203580 (p38 MAPK inhibitor), PD98059 (MAPK/extracellular regulated kinase kinase 1 inhibitor) and c-Jun NH(2)-terminal kinase inhibitor II, were used to block the MAPK signal cascade. MMP-2 gene expression was only inhibited by SB203580 treatment, suggesting a pivotal role of p38 MAPK in regulation of MMP-2 gene expression. Western blot analysis showed that ERW downregulated the phosphorylation of p38 both in H(2)O(2)-treated and untreated HT1080 cells. These results indicate that the inhibitory effect of ERW on tumor invasion is due to, at least in part, its antioxidative effect.     

Production of double repeated B subunit of Shiga toxin?2e at high levels in transgenic lettuce plants as vaccine material for porcine edema disease

Pig edema disease is a bacterial disease caused by enterohemorrhagic Escherichia coli. E.?coli produces Shiga toxin?2e (Stx2e), which is composed of one A subunit (Stx2eA) and five B subunits (Stx2eB). We previously reported production of Stx2eB in lettuce plants as a potential edible vaccine (Matsui et?al. in Biosci Biotechnol Biochem 73:1628-1634, 2009). However, the accumulation level was very low, and it was necessary to improve expression of Stx2eB for potential use of this plant-based vaccine. Therefore, in this study, we optimized the Stx2eB expression cassette and found that a double repeated Stx2eB (2× Stx2eB) accumulates to higher levels than a single Stx2eB in cultured tobacco cells. Furthermore, a linker peptide between the two Stx2eB moieties played an important role in maximizing the effects of the double repeat. Finally, we generated transgenic lettuce plants expressing 2× Stx2eB with a suitable linker peptide that accumulate as much as 80?mg per 100?g fresh weight, a level that will allow us to use these transgenic lettuce plants practically to generate vaccine material.