Proteins associated with reproductive disorders in testes of human erythropoietin gene-harboring transgenic boars

To investigate reproductive disorder in human erythropoietin (EPO)-expressing pig, we performed comparative proteomic analyses of testicular tissues from human erythropoietin (hEPO) gene-harboring transgenic pigs and wild type pigs born from natural conception. In hEPO TG pigs, we found relatively low sperm motility and higher death rate indicating impaired sperm development. Consistently, plasma concentration of testosterone was significantly lower in the transgenic post-pubertal boars compared with wild type boars. Normalized protein spots showing higher than 2-fold differential expression intensity in two-dimensional polyacrylamide gel electrophoresis were selected for matrix associated laser desorption/ionization time-to-flight mass spectrometry analysis. Specific proteins were identified by searching the NCBI protein sequence databases. Among 55 proteins selected, 12 proteins were identified as those differentially expressed between transgenic and wild type pigs. Three downregulated proteins (β-globin, carbonyl reductase 1, and peroxiredoxin 6) and nine upregulated proteins (cytoskeletal β-actin, α 2,3-sialyltransferase, apolipoprotein A-I, tubulin α-1A chain, tropomodulin 3, thioredoxin, heat shock Protein 70.2, ch4/domains of swine IgM, and albumin), all of which are closely related to apoptosis and cytoskeletal development, were found in the transgenic boar testes. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay confirmed the increased occurrence of apoptosis in the transgenic boar testes compared with the wild type boar testes. Reproductive defects of the hEPO-expressing transgenic pigs may be caused by the abnormal expression of the genes identified in this study.     

Molecular characterization of a 2-Cys peroxiredoxin induced by abiotic stress in mungbean

A mungbean low temperature-inducible VrPrx1 encoding 2-Cys peroxiredoxin (2-Cys Prx) was cloned by subtractive suppression hybridization. The deduced VrPrx1 amino acid sequence showed highest sequence homology to 2-Cys Prxs of Phaseolus vulgaris (95%), Pisum sativum (89%), and Arabidopsis thaliana (87%). VrPrx1 RNA and protein levels were increased by low temperature, hydrogen peroxide (H₂O₂), and wounding but decreased by high salinity, drought, and exogenous abscisic acid. Recombinant His-tagged VrPrx1 recombinant protein protected DNA and glutamine synthetase activity from degradation via the thiol/Fe(III) oxygen mixed-function oxidation system, and exhibited peroxidase activity to H₂O₂ in the presence of the reducing agent dithiothreitol (DTT) in vitro. The oxidized dimers and oligomers of the VrPrx1 recombinant protein were reduced to monomers by DTT or thioredoxin. Subcellular localization studies confirmed that VrPrx1-GFP was targeted to the plastid. To evaluate the function of VrPrx1 in planta, the antioxidant activities and photosynthetic efficiency were investigated in VrPrx1-overexpressing Arabidopsis plants. VrPrx1 ectopic expression conferred improved photosynthetic efficiency under oxidative stress conditions. Hence, mungbean VrPrx1 may play an important role in protecting the photosynthetic apparatus against oxidative and abiotic stress conditions.     

Fermentation strategies for 1,3-propanediol production from glycerol using a genetically engineered Klebsiella pneumoniae strain to eliminate by-product formation

We generated a genetically engineered Klebsiella pneumoniae strain (AK-VOT) to eliminate by-product formation during production of 1,3-propanediol (1,3-PD) from glycerol. In the present study, the glycerol-metabolizing properties of the recombinant strain were examined during fermentation in a 5 L bioreactor. As expected, by-product formation was completely absent (except for acetate) when the AK-VOT strain fermented glycerol. However, 1,3-PD productivity was severely reduced owing to a delay in cell growth attributable to a low rate of glycerol consumption. This problem was solved by establishing a two-stage process separating cell growth from 1,3-PD production. In addition, nutrient co-supplementation, especially with starch, significantly increased 1,3-PD production from glycerol during fed-batch fermentation by AK-VOT in the absence of by-product formation.     

RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity

RraA is a protein inhibitor of RNase E (Rne), which catalyzes the endoribonucleolytic cleavage of a large proportion of RNAs in Escherichia coli. The antibiotic-producing bacterium Streptomyces coelicolor also contains homologs of RNase E and RraA, designated as RNase ES (Rns), RraAS1, and RraAS2, respectively. Here, we report that RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity. Analyses of the steady-state level of RNase E substrates indicated that coexpression of RraAS2 in E. coli cells overproducing Rns effectively inhibits the ribonucleolytic activity of full-length RNase ES, but its inhibitory effects were moderate or undetectable on other truncated forms of Rns, in which the N- or/and C-terminal scaffold domain was deleted. In addition, RraAS2 more efficiently inhibited the in vitro ribonucleolytic activity of RNase ES than that of a truncated form containing the catalytic domain only. Coimmunoprecipitation and in vivo cross-linking experiments further showed necessity of both scaffold domains of RNase ES for high-affinity binding of RraAS2 to the enzyme, resulting in decreased RNA-binding capacity of RNase ES. Our results indicate that RraAS2 is a protein inhibitor of RNase ES and provide clues to how this inhibitor affects the ribonucleolytic activity of RNase ES.     

Antibacterial effects of <Emphasis Type="Italic">N</Emphasis>-acetylcysteine against endodontic pathogens

The success of endodontic treatment depends on the eradication of microorganisms from the root canal system and the prevention of reinfection. The purpose of this investigation was to evaluate the antibacterial and antibiofilm efficacy of N-acetylcysteine (NAC), an antioxidant mucolytic agent, as an intracanal medicament against selected endodontic pathogens. Minimum inhibitory concentrations (MICs) of NAC for Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus mutans, and Enterococcus faecalis were determined using the broth microdilution method. NAC showed antibacterial activity, with MIC values of 0.78–1.56 mg/ml. The effect of NAC on biofilm formation of each bacterium and a multispecies culture consisting of the four bacterial species was assessed by crystal violet staining. NAC significantly inhibited biofilm formation by all the monospecies and multispecies bacteria at minimum concentrations of 0.78–3.13 mg/ml. The efficacy of NAC for biofilm disruption was evaluated by scanning electron microscopy and ATP-bioluminescence quantification using mature multispecies biofilms. Preformed mature multispecies biofilms on saliva-coated hydroxyapatite disks were disrupted within 10 min by treatment with NAC at concentrations of 25 mg/ml or higher. After 24 h of treatment, the viability of mature biofilms was reduced by > 99% compared with the control. Moreover, the biofilm disrupting activity of NAC was significantly higher than that of saturated calcium hydroxide or 2% chlorhexidine solution. Within the limitations of this in vitro study, we conclude that NAC has excellent antibacterial and antibiofilm efficacy against endodontic pathogens and may be used as an alternative intracanal medicament in root canal therapies.     

siRNA screen of the human signaling proteome identifies the PtdIns(3,4,5)P<Subscript>3</Subscript>-mTOR signaling pathway as a primary regulator of transferrin uptake

Background  

Iron uptake via endocytosis of iron-transferrin-transferrin receptor complexes is a rate-limiting step for cell growth, viability and proliferation in tumor cells as well as non-transformed cells such as activated lymphocytes. Signaling pathways that regulate transferrin uptake have not yet been identified.     

Molecular characterization of the soybean L-asparaginase gene induced by low temperature stress

L-asparaginase (EC 3.5.1.1) catalyzes the hydrolysis of the amide group of L-asparagine, releasing aspartate and NH4+. We isolated a low temperature-inducible cDNA sequence encoding L-asparaginase from soybean leaves. The full-length L-asparaginase cDNA, designated GmASP1, contains an open reading frame of 1,258 bp coding for a protein of 326 amino acids. Genomic DNA blotting and fluorescence in situ hybridization showed that the soybean genome has two copies of GmASP1. GmASP1 mRNA was induced by low temperature, ABA and NaCl, but not by heat shock or drought stress. E. coli cells expressing recombinant GmASP1 had 3-fold increased L-asparaginase activity. A possible function of L-asparaginase in the early response to low temperature stress is discussed.     

Development of predictive mathematical model for the growth kinetics of Staphylococcus aureus by response surface model

A response surface model was developed for predicting the growth rates of Staphylococcus aureus in tryptic soy broth (TSB) medium as a function of combined effects of temperature, pH, and NaCl. The TSB containing six different concentrations of NaCl (0, 2, 4, 6, 8, and 10%) was adjusted to an initial of six different pH levels (pH 4, 5, 6, 7, 8, 9, and 10) and incubated at 10, 20, 30, and 40 degrees C. In all experimental variables, the primary growth curves were well (r2=0.9000 to 0.9975) fitted to a Gompertz equation to obtain growth rates. The secondary response surface model for natural logarithm transformations of growth rates as a function of combined effects of temperature, pH, and NaCl was obtained by SAS's general linear analysis. The predicted growth rates of the S. aureus were generally decreased by basic (pH 9-10) or acidic (pH 5-6) conditions and higher NaCl concentrations. The response surface model was identified as an appropriate secondary model for growth rates on the basis of correlation coefficient (r=0.9703), determination coefficient (r2=0.9415), mean square error (MSE=0.0185), bias factor (B(f)=1.0216), and accuracy factor (A(f)=1.2583). Therefore, the developed secondary model proved reliable for predictions of the combined effect of temperature, NaCl, and pH on growth rates for S. aureus in TSB medium.     

Ferulic acid supplementation prevents trimethyltin-induced cognitive deficits in mice

This study's objective was to clarify the ameliorative effects ferulic acid (4-hydroxy-3-methoxycinnamic acid) has against cognitive deficits and ChAT activation in trimethyltin (TMT) induced, memory injured mice following a 28-d ferulic acid treatment. After administering TMT for 3 d, each mouse performed Y-maze and passive avoidance tests to check immediate working memory performance and cognitive function. The results showed that ferulic acid administration attenuated TMT-induced memory injury and a decline in ChAT activity in the mice. This suggests that ferulic acid might be useful for preventing cognitive dysfunction as well as for boosting the activation of ChAT in dementia.