Green vegetable drink consumption protects peripheral lymphocytes DNA damage in Korean smokers

Smoking increases indices of free radical-mediated damage of DNA which are potential underlying processes in the pathogenesis of many diseases. In this study, we evaluated whether 8 weeks of green vegetable drink (Angelica keiskei based juice) supplementation to smokers can be protective against lymphocytic DNA damage. Twenty smokers were given 240 ml of commercially available green vegetable drink every day for 8 weeks. The DNA damage was determined using single cell gel electrophoresis (COMET assay) and the damage was quantified by measuring tail length (TL), tail moment (TM), and percent DNA in tail. Eight weeks of green vegetable drink consumption resulted in a significant in lymphocytes DNA damage in all three measurements; TL, TM and % DNA in tail. These results support the hypothesis that green vegetable drink exerts a cancer-protective effect via a decrease in oxidative damage to DNA in humans.     

Safety and protective effect of a disinfectant (STEL water) for white spot syndrome viral infection in shrimp

The efficacy of STEL water for protection against white spot syndrome virus (WSSV) infection was evaluated using shrimp. The LC50 of residual chlorine (Cl-) in STEL water for brood-stock and 2-mo-old shrimp were 2.3 and 3.2 ppm, respectively. All 2-month-old shrimp raised in seawater containing more than 40 microl 2l(-1) of a WSSV-infected tissue homogenate died within 3 d post-exposure (dpe). Thus, a 10-fold dose of 400 microl 2 l(-1) was used in the disinfection tests. Low concentrations of STEL water effectively prevented mortality of shrimp at this challenge dose. All 2-month-old shrimp exposed to seawater with 400 microl of viral homogenate disinfected with STEL water at Cl- concentrations over 0.125 ppm for 1 and 10 min, lived until 5 dpe. With 5-mo-old shrimp, all positive control shrimps died within 3 dpe, whereas most shrimp reared in seawater disinfected with STEL water for 1 h before addition of homogenate lived until 5 dpe. Results suggested that continuous disinfection of seawater with STEL water may be effective for preventing WSSV infection in shrimp.     

Evaluation of Antigen Retrieval Buffer Systems

The introduction of antigen retrieval (AR) techniques has dramatically improved the sensitivity of immunohistochemical detection of various antigens in formalin-fixed, paraffin-embedded tissues. The microwave-heating and pressure-cooking procedures are the most effective AR methods reported to date. Although extensive efforts have been made to optimize AR procedures using these two methods, previous studies have not led to a standard protocol applicable to all antibodies derived from different clones. In this study we have investigated the optimal AR buffer conditions for 29 antibodies that are in common use for diagnostic purposes in hospitals worldwide. Borate (pH 8.0) and Tris buffer (pH 9.5) yielded the highest retrieved antigen immunoreactivity against most antibodies as compared to other buffers tested. In addition, the microwave pressure-cooking gave better results than microwave-heating alone. Therefore, borate (pH 8.0) or Tris (pH 9.5) buffer used in conjunction with the pressure-cooking procedure is strongly recommended for standard routine use.     

Analysis of substance P in rat brain by means of immunoaffinity capture and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry

Interest in the analysis of low abundance neuropeptides particularly using matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF-MS) is increasing because these neuropeptides are essential to the mechanism of transportation and the metabolism. This article describes an immunoprecipitation procedure that is suitable for MALDI-MS analysis of substance P (SP), a neuropeptide, in rat brain tissues. Substance P was precipitated from brain tissue extracts by immunoprecipitation with antibodies directed against SP, and are analyzed by MALDI-TOF-MS. Mass spectrometric analysis showed a singly charged [M+H]+ ion peak that corresponded to the SP molecular mass and was observed with a detection error of 1.6%. The average mass errors between the observed and theoretical molecular mass were within the 0.11 Da range. Capillary zone electrophoresis analysis was subsequently performed, and the effects of the different separation parameters were examined. Beginning with milligram quantities of brain tissue, picomole quantities of SP could be detected using this method.     

Lipid raft proteome reveals ATP synthase complex in the cell surface

Since detergent-resistant lipid rafts are involved in pathogen invasion, cholesterol homeostasis, angiogenesis, neurodegenerative diseases and signal transduction, protein identification in the rafts could provide important information to study their function. Here, we analyzed detergent-resistant raft proteins isolated from rat liver by capillary liquid chromatography-tandem mass spectrometry. Out of 196 proteins identified, 32% belonged to the raft or plasma membrane, 24% to mitochondrial, 20% to microsomal, 7% to miscellaneous, and 17% are unknown proteins. For example, membrane-bound receptors, trimeric GTP-binding proteins, ATP-binding cassette transporters, and glycosylphosphatidylinositol-anchored proteins were identified in this analysis. Unexpectedly, there were many mitochondrial proteins, raising a new issue for the presence of mitochondrial rafts or the localization of mitochondrial proteins into plasma membrane rafts. We confirmed that ATP synthase alpha and beta were expressed on the surface of the plasma membrane in HepG2 hepatocytes by immunofluorescence, cell surface biotinylation, and cellular fractionation. They had two distinct biochemical properties, detergent insolubility and low density, suggesting that the ATP synthase complex might be located in plasma membrane rafts as well as in the mitochondria.     

Proteome analysis of rice blast fungus (Magnaporthe grisea) proteome during appressorium formation

We used two-dimensional gel electrophoresis (2-DE) to identify the proteins that are induced in the rice blast fungus Magnaporthe grisea during appressorium formation. Proteins were extracted from conidia that had germinated on hydrophilic glass plates or from germinated and appressoria-forming conidia on leaf wax-coated hydrophobic glass plates after 4, 8, and 12 h of incubation. Differentially expressed protein spots during appressorium formation were confirmed from gels after 2-DE analysis where proteins had been labeled with (35)S methionine and stained with silver. Internal amino acid sequencing identified five proteins among several proteins induced during appressorium formation. Two denoted as M. grisea proteasome homolgues (MgP1 and MgP5) were 20S proteasome alpha subunits. The remaining three were scytalone dehydratase (SCD), and serine carboxypeptidase Y (CPY). None of the five have been reported previously in the rice blast fungus apart from SCD. We further investigated the role the alpha subunit of 20S proteasome plays in appressorium formation. We confirmed by Western blot analysis that MgP5 is highly expressed during appressorium formation and found that it is also markedly induced by nitrogen- and carbon-starvation, in particular by the former. These observations suggest that the 20S proteasome may be involved in remobilizing storage proteins, which then help to build the appressorium. Thus, fungal proteome analysis may provide important clues about developmental changes such as the generation of the appressorium.     

Purification and cDNA cloning of a cecropin-like peptide from the great wax moth, Galleria mellonella

A cecropin-like antimicrobial peptide, Gm cecropin, was purified from hemolymph of larvae of the wax moth, Galleria mellonella, immunized against E. coli, and its antibacterial activity was examined in a radial diffusion assay. The molecular mass of Gm cecropin was 4,160.69 Da by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis. The full-length cDNA of the Gm cecropin precursor was cloned by a combination of RT-PCR, based on the N-terminal sequence obtained by Edman degradation, and 5'-RACE-PCR. Analysis of the cDNA showed that cecropin is synthesized as a prepropeptide, with a putative 22-residue signal peptide, a 4-residue propeptide and a 39-residue mature peptide with a calculated mass of 4,344.18 Da the difference between the calculated and measured masses suggests that Gm cecropin is a 37-residue peptide generated by removal of the C-terminal residue and amidation.     

Developmental changes in inhibin-alpha gene expression in the mouse testis

Inhibin is a gonadal hormone which is composed of an alpha-subunit and one of two related beta-subunits (betaA, betaB). Inhibin is important for pituitary FSH regulation, normal follicle development and maintenance of the estrous cycle in the female, whereas the role of inhibin in the male is less clear. Thus, we examined the expression of the inhibin-alpha gene in testis during sexual maturation in male mice, to try to gain insight into its functions in the male. Male mice of the ICR strain attained fertility at 6 weeks of age, and histological analysis revealed that a functional testis was formed, with seminiferous tubules which contain mature sperm and with an abundant population of Leydig cells. Parallel with this sexual maturation, inhibin-alpha subunit protein synthesis increased, whereas synthesis of the activin betaA and activin betaB followed with a delayed time course. Inhibin-alpha mRNA also increased during this critical period, and this corresponded to a change in the methylation status of the inhibin-alpha gene. Taken together, our data reveal that activation of inhibin-alpha gene during testis development correlated with the histological maturation of the testis and the acquisition of fertility in male mice.