Glutathione-mediated response to acid stress in the probiotic bacterium, Lactobacillus salivarius

Lactobacillus salivarius, a probiotic bacterium, encounters acidic conditions in its passage through the gastrointestinal tract of human and animal hosts. We studied the effect of a rapid downshift in extracellular pH from 6.5 to 4 on cell growth. The maximum growth rate was higher in low pH medium with glutathione supplementation than without. Cells developed a GSH-mediated acid-tolerance response and, when grown with 0.5 mM GSH, reached a higher final density than with other conditions. These findings suggest that the increased growth rate is caused by uptake of GSH which acts as a nutrient source as well as having protective functions, allowing for continued growth.     

Buffer optimization for high resolution of human lung cancer tissue proteins by two-dimensional gel electrophoresis

A problem in proteomic analysis of lung cancer tissue is the presence of complex components of different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma, and adenocarcinoma). The efficient solubilization of protein components before two-dimensional electrophoresis (2-DE) is a very critical. Poor solubilization has been associated with a failure to detect proteins and diffuse, streaked and/or trailing protein spots. Here, we have optimized the solubilization of human lung cancer tissue to increase protein resolution. Isoelectric focusing (IEF) rehydration buffer containing a thiourea–urea mixture provided superior resolution, whereas a buffer without thiourea yielded consistently poor results. In addition, IEF rehydration buffers containing CHAPS and DTT gave superior resolution, whereas buffers containing Nonidet P-40 (NP-40) and/or Triton X-100 did not. A tributylphosphine-containing buffer gave consistently poor results. Using optimized conditions, we used 2-D gel analysis of human lung cancer tissue to identify 11 differentially-expressed protein spots by MALDI-mass spectrometry. This study provides a methodological tool to study the complex mammalian proteomes.     

Synthesis of veratraldehyde from veratryl alcohol by lignin peroxidase with in situ electrogeneration of hydrogen peroxide in an electrochemical reactor

We report the synthesis of veratraldehyde from veratryl alcohol by Phanerochaete chrysosporium lignin peroxidase with in situ electrogeneration of hydrogen peroxide in an electroenzymatic reactor. The effects of operating parameters such as enzyme level, pH, and electrical potential on the efficiency of veratryl alcohol oxidation were investigated. Furthermore, we compared direct addition of hydrogen peroxide with electrogeneration of the material during enzymatic oxidation of veratryl alcohol. The electroenzymatic method using in situ-generated hydrogen peroxide was found to be effective for oxidation of veratryl alcohol by lignin peroxidase. The new method may be easily applied to biodegradation systems.     

Influence of gel dimensions on resolution and sample throughput on two-dimensional gels

To achieve high throughput and economical format of 2-D PAGE, comparison between gel size and resolution was conducted on human breast carcinoma cell line (MCF-7/AZ) proteins. SDS gel length showed a weaker influence of separation length on resolution in the second dimension, and there was little benefit of separation distances greater than 15 to 19 cm. IPG strip separation distances were very important with dramatic increase in resolution of longer gels compared with smaller gels, and maximal resolution was obtained using 18- and 24-cm IPG strips. Loading optimal amount of proteins on 2-D gels can also increase the number of detected spots. Therefore, taken together, compromise 2-D gels are crucial for higher capacity and higher throughput.     

Prunus mume extract exerts antioxidant activities and suppressive effect of melanogenesis under the stimulation by alpha-melanocyte stimulating hormone in B16-F10 melanoma cells

In the current study, we examined the antioxidant and skin-whitening properties of Prunus mume extract (PME). The ability of PME to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was investigated in vitro. At a concentration of 1000 μg/mL, PME neutralized >45% free radical activity. Cell viability assessment with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that at concentrations <1500 μg/mL, PME does not exert cytotoxic effects on murine B16 melanoma (B16) cells. Morphological analysis disclosed that melanin production is inhibited in B16 cells treated with 250 nM α-melanocyte-stimulating hormone (α-MSH) and PME. We conclude that fruit extracts of P. mume exert a skin-whitening effect by inhibiting melanin production via regulation of melanogenesis-associated protein expression in melanocytes.