Cytotoxicity of Vibrio vulnificus cytolysin on rat peritoneal mast cells

Histamine has been thought to be a permeability enhancing factor in Vibrio vulnificus infection. The injection of living bacteria or purified V. vulnificus cytolysin (VVC) can cause lethality in mice by inducing hemoconcentration and increased vascular permeability. In the present study, we tried to identify whether histamine release causes the increased vascular permeability that is responsible for the lethal effect of VVC. Treatment of rat peritoneal mast cells with high concentrations of VVC caused the release of whole cellular histamine and lactate dehydrogenase (LDH). At concentrations less than 10 HU/ml, histamine and LDH were not released whereas preloaded 2-deoxy-D-glucose was rapidly effluxed with the concomitant decrease in cellular ATP. VVC-treated mast cells were refractory to the stimulation of histamine secretion by Compound 48/80 but remained fully responsive to Ca2+ plus GTP-gamma-S. These results indicate that histamine can be released from mast cells only when the concentration of VVC is high enough to cause the lysis of cells. At low concentrations, VVC does not induce the release of stored histamine from damaged cells. The intravenous injection of 80 HU purified VVC to rats, which can produce the calculated blood concentration of about 3 HU/ml, caused a marked increase in pulmonary vascular permeability, hemoconcentration and death. However, no increase in blood histamine level was detected. This level of VVC in rat blood was enough to cause severe hemoconcentration and lethality but might not be enough to cause cytolysis of the mast cells and resulting histamine release.     

The dopamine D4 receptor activates intracellular platelet-derived growth factor receptor β to stimulate ERK1/2

Dopamine receptors are GPCRs that play important roles in locomotion, reward, and cognitive processes. Previously, we demonstrated that this receptor transactivates PDGFRβ to modulate ERK1/2 and NMDA receptor activity. Downregulation of maturely glycosylated PDGFRβ by prolonged exposure to PDGF-BB eliminated PDGF-BB-mediated ERK1/2 activation. The DRD4-mediated ERK1/2 response was only partially blunted by PDGF-BB-mediated downregulation, but remained sensitive to the PDGFRβ kinase inhibitor tyrphostin A9. Tunicamycin prevented the N-linked glycosylation and maturation of PDGFRβ as well as its activation by PDGF-BB. However, upon tunicamycin treatment, DRD4 continued to signal to ERK1/2 in a tyrphostin A9-sensitive manner. Collectively, our observations indicate that DRD4, unlike PDGF-BB, can activate a pool of intracellularly located PDGFRβ.     

Multiple testing to establish superiority/equivalence of a new treatment compared with k standard treatments for unbalanced designs

In clinical studies, multiple superiority/equivalence testing procedures can be applied to classify a new treatment as superior, equivalent (same therapeutic effect), or inferior to each set of standard treatments. Previous stepwise approaches (Dunnett and Tamhane, 1997, Statistics in Medicine16, 2489-2506; Kwong, 2001, Journal of Statistical Planning and Inference 97, 359-366) are only appropriate for balanced designs. Unfortunately, the construction of similar tests for unbalanced designs is far more complex, with two major difficulties: (i) the ordering of test statistics for superiority may not be the same as the ordering of test statistics for equivalence; and (ii) the correlation structure of the test statistics is not equi-correlated but product-correlated. In this article, we seek to develop a two-stage testing procedure for unbalanced designs, which are very popular in clinical experiments. This procedure is a combination of step-up and single-step testing procedures, while the familywise error rate is proved to be controlled at a designated level. Furthermore, a simulation study is conducted to compare the average powers of the proposed procedure to those of the single-step procedure. In addition, a clinical example is provided to illustrate the application of the new procedure.     

Molecular simulation study of phospholipid bilayers and insights of the interactions with disaccharides

Molecular simulations of hydrated dipalmitoylphosphatidylcholine lipid bilayers have been performed for temperatures in the range of 250-450 K. The area per headgroup increases with temperature from 58 to 77 A(2). Other properties such as hydration number, alkyl tail order parameter, diffusion coefficients, and radial distribution functions exhibit a clear dependence on temperature. Simulations of bilayers have also been performed in the presence of two disaccharides, namely trehalose and sucrose, at concentrations of up to 18 wt % (lipid-free basis). The simulated area per headgroup of the bilayer is not affected by the presence of the disaccharides, suggesting that the overall structure of the bilayer remains undisturbed. The results of simulations reveal that the interaction of disaccharide molecules with the bilayer occurs at the surface of the bilayer, and it is governed by the formation of multiple hydrogen bonds to specific groups of the lipid. Disaccharide molecules are observed to adopt specific conformations to fit onto the surface topology of the bilayer, often interacting with up to three different lipids simultaneously. At high disaccharide concentrations, the results of simulations indicate that disaccharides can serve as an effective replacement for water under anhydrous conditions, which helps explain their effectiveness as lyophilization agents for liposomes and cells.     

Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a [4Fe-4S] protein

The last enzyme (LytB) of the methylerythritol phosphate pathway for isoprenoid biosynthesis catalyzes the reduction of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into isopentenyl diphosphate and dimethylallyl diphosphate. This enzyme possesses a dioxygen-sensitive [4Fe-4S] cluster. This prosthetic group was characterized in the Escherichia coli enzyme by UV/visible and electron paramagnetic resonance spectroscopy after reconstitution of the purified protein. Enzymatic activity required the presence of a reducing system such as flavodoxin/flavodoxin reductase/reduced nicotinamide adenine dinucleotide phosphate or the photoreduced deazaflavin radical.     

Synthesis and activity of novel benzoxazole derivatives of mannopeptimycin glycopeptide antibiotics

A series of benzoxazole derivatives of the mannopeptimycin glycopeptide antibiotics was synthesized via a novel benzoxazole formation reaction by treating aminophenol of mannopeptimycin-beta with an aldehyde and DDQ in DMF. Some of these derivatives (e.g., 5b, 5d, 5m, and 7b) showed good activity against Gram-(+) bacteria when compared to the parent compound mannopeptimycin-beta.     

Efficacy validation of synthesized retinol derivatives In vitro: stability,toxicity, and activity

Retinol (vitamin A) is used as an antiwrinkle agent in the cosmetics industry. However, its photo-instability makes it unsuitable for use in general cosmetic formulations. To improve the photo-stability of retinol, three derivatives (3, 4, and 5) were synthesized and their biological activities were analyzed. 1H NMR and HPLC analysis indicated that derivatives 3 and 5 were much more stable than retinol under our sunlight exposure conditions. When human adult fibroblasts were treated, the IC(50) of derivative 3 was 96 microM, which is similar to that of retinol, as determined by the MTT assay. Derivatives 4 and 5 were 2.5 and 8 times more toxic than retinol, respectively. At 1 microM treatment, like retinol, derivatives 3 and 4 were specifically active for RARalpha out of six retinoid receptors (RAR/RXRalpha, beta, gamma). Dose-dependent analysis confirmed that derivative 4 was as active as retinol and the other two derivatives were less active for RARalpha. The effect of our derivatives on the expression of collagenase, an indicator of wrinkle formation, was measured using the transient co-expression of c-Jun and RT-PCR in HaCaT cells. Collagenase promoter activity, which is increased by c-Jun expression, was reduced 42% by retinol treatment. The other derivatives inhibited collagenase promoter activity similarly. These results were further confirmed by RT-PCR analysis of the collagenase gene. Taken together, our results suggest that retinol derivative 3 is a promising antiwrinkle agent based on its higher photo-stability, lower RARalpha activity (possibly indicating reduced side effects), and similar effect on collagenase expression.     

Dynamics of living and dead bacterial cells within a mixed-species biofilm during toluene degradation in a biotrickling filter

AIMS: To study the accumulation of the bacterial living cells (LC) and dead cells (DC) in a mixed-species biofilm developed in a 3 l biotrickling filter (BTF) challenged with toluene. METHODS AND RESULTS: The bacterial LC and DC within the biofilm developed on polypropylene Pall rings in a toluene-degrading BTF were enumerated as fluoro-microscopic counts during a 62-operating day period using nucleic acid staining and the direct epifluorescence filter technique. The biofilm development could be separated into three distinct phases: (i) cell attachment, (ii) biofilm establishment and (iii) biofilm maturation. The LC were always dominant (>/=72%) in the biofilm during the establishment phase whereas the average LC fraction decreased to 51% of the total cells in the maturation phase. The concentration of LC and DC was observed to level off after 41 days at 1010 cells per ring. The biofilm thickness and the dry weight increased independently of the cell number during the maturation phase. CONCLUSIONS: After the LC reached a maximum concentration in the biofilm, the biofilm proliferation was only characterized by the accumulation of DC and organic matter. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained in the present study are of particular relevance for biofilm mathematical modelling and numerical simulations. They will also be useful to estimate the contribution of the living bacteria within the biofilm in bioprocesses.     

Physical interaction between recombinational proteins Rhp51 and Rad22 in Schizosaccharomyces pombe

In eukaryotes, Rad51 and Rad52 are two key components of homologous recombination and recombinational repair. These two proteins interact with each other. Here we investigated the role of interaction between Rhp51 and Rad22, the fission yeast homologs of Rad51 and Rad52, respectively, on the function of each protein. We identified a direct association between the two proteins and their self-interactions both in vivo and in vitro. We also determined the binding domains of each protein that mediate these interactions. To characterize the role of Rhp51-Rad22 interaction, we used random mutagenesis to identify the mutants Rhp51 and Rad22, which cannot interact each other. Interestingly, we found that mutant Rhp51 protein, which cannot interact with either Rad22 or Rti1 (G282D), lost its DNA repair ability. In contrast, mutant Rad22 proteins, which cannot specifically bind to Rhp51 (S379L and P381L), maintained their DNA repair ability. These results suggest that the interaction between Rhp51 and Rad22 is crucial for the recombinational repair function of Rhp51. However, the significance of this interaction on the function of Rad22 remains to be characterized further.