CT01 Sigma-AldrichMTT Cell Growth Assay Kit

Ovalicin, a secondary metabolite produced by an entomopathogenic fungus Metarhizium anisopliae var. anisopliae, is currently used as an efficient biological control agent against various agricultural insect pests, but not so many biological activities have been investigated. To assess whether ovalicin has potential in ameliorating atopic dermatitis-related symptoms in mice, we first sensitized skin in the dorsal neck of Balb/c mice using compound 48/80, and scrutinized whether the compound affected the atopic dermatitis-related symptoms. The results revealed that ovalicin significantly reduces scratching behavior in a concentration-dependent fashion. Moreover, the treatment inhibits the levels of the degranulation of mast cells by 65%, and levels of histamine release by 51% at a concentration of 10 µg/ml. Together, the present data strongly suggest that ovalicin elicits potential anti-atopic activities in mice.

Human aldehyde dehydrogenases (ALDHs) comprise a family of 17 homologous enzymes that metabolize different biogenic and exogenic aldehydes. To date, there are relatively few general ALDH inhibitors that can be used to probe the contribution of this class of enzymes to particular metabolic pathways. Here, we report the discovery of a general class of ALDH inhibitors with a common mechanism of action. The combined data from kinetic studies, mass spectrometric measurements, and crystallographic analyses demonstrate that these inhibitors undergo an enzyme-mediated ?-elimination reaction generating a vinyl ketone intermediate that covalently modifies the active site cysteine residue present in these enzymes. The studies described here can provide the basis for rational approach to design ALDH isoenzyme-specific inhibitors as research tools and perhaps as drugs, to address diseases such as cancer where increased ALDH activity is associated with a cellular phenotype.

It is known that cells suffer a chronic hypoxic condition during the development of proximal tubulointerstitial disease. However, it is accepted that extracellular-superoxide dismutase (EC-SOD) protects the cells from oxidative stress. The purpose of this study was to elucidate the regulation of EC-SOD expression in cells under hypoxia. The results show that the expressions of EC-SOD mRNA and protein in cobalt chloride (CoCl(2))-treated COS7 cells decreased in a dose- and time-dependent manner, whereas the expressions of other SOD isoforms (Cu/Zn-SOD and Mn-SOD) were not changed. The down-regulation of EC-SOD mRNA was suppressed by pre-treatment with the antioxidant trolox and the p38 mitogen-activated protein kinase (p38-MAPK) inhibitor SB203580. It is concluded that the expression of EC-SOD is decreased through ROS and p38-MAPK signalling cascades and that the down-regulation of EC-SOD leads to a decrease in the resistance to oxidative stress of COS7 cells under hypoxia induced by CoCl(2).

Photosynthetic organisms have the unique ability to transform light energy into reducing power. We study the requirements for photosynthesis in the alpha-proteobacterium Rhodobacter sphaeroides. Global gene expression analysis found that approximately 50 uncharacterized genes were regulated by changes in light intensity and O2 tension, similar to the expression of genes known to be required for photosynthetic growth of this bacterium. These uncharacterized genes included RSP4157 to -4159, which appeared to be cotranscribed and map to plasmid P004. A mutant containing a polar insertion in RSP4157, CT01, was able to grow via photosynthesis under autotrophic conditions using H2 as an electron donor and CO2 as a carbon source. However, CT01 was unable to grow photoheterotrophically in a succinate-based medium unless compounds that could be used to recycle reducing power (the external electron acceptor dimethyl sulfoxide (DMSO) or CO2 were provided. This suggests that the insertion in RSP4157 caused a defect in recycling reducing power during photosynthetic growth when a fixed carbon source was present. CT01 had decreased levels of RNA for genes encoding putative glycolate degradation functions. We found that exogenous glycolate also rescued photoheterotrophic growth of CT01, leading us to propose that CO2 produced from glycolate metabolism can be used by the Calvin cycle to recycle reducing power generated in the photosynthetic apparatus. The ability of glycolate, CO2, or DMSO to support photoheterotrophic growth of CT01 suggests that one or more products of RSP4157 to -4159 serve a previously unknown role in recycling reducing power under photosynthetic conditions.

Preclinical studies were conducted in mice and rabbits to evaluate biodistribution/persistence and potential integration of plasmid DNA (pDNA) after intramuscular administration of a poloxamer-formulated pDNAbased vaccine, VCL-CT01, encoding gB, pp65, and IE1 human cytomegalovirus (hCMV) immunogens. Tissue distribution in mice vaccinated with VCL-CT01 was compared with that in mice vaccinated with a phosphate- buffered saline (PBS)-formulated control pDNA vaccine. Residual pDNA copy number (PCN), in selected tissues collected on days 3, 30, and 60 after vaccination, was measured by quantitative polymerase chain reaction. In VCL-CT01-vaccinated mice and in control pDNA-vaccinated mice, pDNA was below the limit of detection by day 60 in all tissues except the injection site. Clearance of pDNA from the injection site was slower in VCL-CT01-vaccinated mice compared with PBS-pDNA-vaccinated mice. An integration study was conducted in rabbits to determine whether pDNA integration into the genome of the vaccinated animal contributed to pDNA persistence. Residual pDNA in VCL-CT01-injected rabbit muscle collected 60 days after vaccination (geometric mean of 1085 PCN/microg total DNA) was comparable to that observed in VCL-CT01- injected mouse muscle (geometric mean of 1471 PCN/microg total DNA) collected at the same time point. pDNA integration was not detectable by column agarose gel electrophoresis despite the persistence of pDNA at the injection site 60 days after vaccination. Therefore the risk of genomic integration of hCMV pDNA formulated with poloxamer was considered negligible.

We studied the serum levels of 1,25-dihydroxyvitamin D [1,25(OH)2D (Vit D)] in patients with renal cell carcinoma (RCC) and the influence of 1,25(OH)2D3 (Vit D3) on gap junctional intercellular communication (GJIC) during carcinogenesis. The serum Vit D levels were measured by a competitive protein-binding assay using the chromatographic method. Using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay, noncytotoxic concentrations of Vit D3 and the tumor promoters N-nitrosodimethylamine (NDMA) and N-ethyl-N-hydroxyethylnitrosamine (EHEN) were tested against cultured human renal proximal tubular cells (HRPTCs). GJIC function was assayed by the scrape-loading dye transfer technique. Cx43 mRNA expression was also examined by the reverse transcriptase-polymerase chain reaction (RT-PCR). Serum Vit D levels in patients with RCC were lower than those in controls (p< 0.001). Patients with T3 to T4 (rapid-growth) tumors had lower levels of Vit D than did patients with T1 to T2 (slow-growth) tumors (p < 0.001). Vit D3 enhanced the GJIC function of HRPTCs (p < 0.05), whereas NDMA and EHEN suppressed it (p < 0.05). When the cells were treated with tumor promoters and Vit D3 simultaneously, the GJIC functions remained at pretreatment levels. We also demonstrated Cx43 mRNA expression in RPTECs treated with EHEN and VitD3 simultaneously. These data suggest that a decrease in the serum Vit D level is one of the risk factors for development and progression of RCC, and Vit D3 may prevent RCC by preserving GJIC during carcinogenesis.

A rapid colorimetric microtiter assay has been developed to detect cytotoxic lymphokines produced by human lymphocytes activated with lectins or tumor cells. The viability of lymphotoxin-treated target cells was detected using a tetrazolium dye that is reduced to a blue formazan by living but not dead cells. The amount of dye formed was quantitated using a microplate spectrophotometer (ELISA plate reader) and visual observations confirmed the amount of formazan dye produced was directly proportional to the number of viable target cells. The advantages of using this colorimetric method are that it requires no washing steps or radioisotopes and its precision and rapidity. Optimal conditions were established using the murine L929 and human ESH -5L cell lines as target cells for detecting lymphotoxins produced by human lymphocytes. The data indicate that the L929 cell line was 10-50-fold more sensitive than the ESH -5L line to the lytic activity of cytotoxins produced by human phytohemagglutinin-P-activated T lymphocytes, or the cytotoxins produced by peripheral blood lymphocytes stimulated with various tumor cell lines. This assay system was also useful in detecting antibodies capable of neutralizing lymphotoxin activity and thus should be a suitable method to aid in the molecular characterization of these lymphokines.