Quantitative proteomic analysis of cell wall and plasma membrane fractions from multidrug-resistant Acinetobacter baumannii

Acinetobacter baumannii is a Gram-negative, nonmotile aerobic bacterium that has emerged as an important nosocomial pathogen. Multidrug-resistant (MDR) A. baumannii is difficult to treat with antibiotics, and treatment failure in infected patients is of great concern in clinical settings. To investigate proteome regulation in A. baumannii under antibiotic stress conditions, quantitative membrane proteomic analyses of a clinical MDR A. baumannii strain cultured in subminimal inhibitory concentrations of tetracycline and imipenem were performed using a combination of label-free (one-dimensional electrophoresis-liquid chromatography-tandem mass spectrometry) and label (isobaric tag for relative and absolute quantitation) approaches. In total, 484 proteins were identified, and 302 were classified as outer membrane, periplasmic, or plasma membrane proteins. The clinical A. baumannii strain DU202 responded specifically and induced different cell wall and membrane protein sets that provided resistance to the antibiotics. The induction of resistance-nodulation-cell division transporters and protein kinases, and the repression of outer membrane proteins were common responses in the presence of tetracycline and imipenem. Induction of a tetracycline resistant pump, ribosomal proteins, and iron-uptake transporters appeared to be dependent on tetracycline conditions, whereas β-lactamase and penicillin-binding proteins appeared to be dependent on imipenem conditions. These results suggest that combined liquid chromatography-based proteomic approaches can be used to identify cell wall and membrane proteins involved in the antibiotic resistance of A. baumannii.     

The Effect of Ultrasonificated Extracts of <Emphasis Type="Italic">Spirulina maxima</Emphasis> on the Anticancer Activity

The effect of ultrasonic extraction on extraction yields, cytotoxicity, and anticancer activity of Spirulina maxima was investigated in this study. Optimal extraction conditions were determined as 60 kHz frequency at 60°C for 30 min with 120 W intensity, which resulted in 19.3% of extraction yields and 19.1% of cytotoxicity on normal human cells. Yields from conventional water and ethanol extraction were 15.8% at 100°C and 8.3% at 80°C, respectively. It was found that the extracts obtained by ultrasonic extraction process selectively inhibited the digestive-related cancer cell lines, such as human stomach cancer cells, having 89% of the highest inhibition ratio and 4.5 of the highest selectivity. In adding 0.5 mg/mL of the extract, human promyelocytic leukemia cells' cell differentiation was increased 1.72 times over that of the control. Expression level of B cell lymphoma-2 from Hep3B cell was also effectively suppressed by the extract obtained at 60 kHz and 60°C, leading to the inhibition of the early step of carcinogenesis. This work suggests that anticancer activity of the extracts is due to water-soluble polysaccharides rather than proteins and is further supported by the result that the ultrasonification extraction process can efficiently extract relatively intact polysaccharides rather than digesting the proteins in S. maxima by matrix assisted laser desorption ionization-time of flight and high performance size exclusion chromatography chromatogram analyses. Therefore, ultrasonic extraction increases both extraction yield and the biological activity of S. maxima extracts, which might be useful as an alternative natural anticancer agent in the medical and food industries.     

Proteome analysis of Thermococcus onnurineus NA1 reveals the expression of hydrogen gene cluster under carboxydotrophic growth

Thermococcus onnurineus NA1 is a typical sulfur-reducing hyperthermophilic archaeon. Genome sequence analysis has shown that T. onnurineus NA1 retains the metabolic pathways necessary not only for organotrophic, but also for carboxydotrophic, growth. T. onnurineus NA1 carboxydotrophic growth may result in hydrogen production, as this archaeon produces hydrogen during oxidizing formate. In this study, we profiled the proteome of T. onnurineus NA1 cultured under carboxydotrophic conditions using CO as an electron donor by the SDS-PAGE/LC-MS/MS method. A total of 1395 proteins were identified by two independent proteomic analyses, which corresponds to ~71% of the total predicted open reading frames. To our knowledge, this level of identification coverage exceeds those of other global proteome profiling studies in Archaea. Furthermore, the biological functions of the identified proteins were predicted and cognate enzymes were mapped to the appropriate metabolic pathways. More than 90% of the genes belonging to hydrogenase gene clusters such as Mbx, Sulf-I, Mbh, Hyg4-I, Hyg4-II, and Hyf4-III were expressed during CO culture. This means that hydrogenases induced under carboxydotrophic conditions surpass those induced under an organotrophic condition (yeast extract-peptone-sulfur). Our data suggest that hydrogen production is caused by the expression and functional assembly of T. onnurineus NA1 hydrogenase genes during culture in the presence of CO.     

Proteomic characterization of the Pseudomonas putida KT2440 global response to a monocyclic aromatic compound by iTRAQ analysis and 1DE-MudPIT

Pseudomonas putida KT2440 is a metabolically versatile soil bacterium. To examine the effects of an aromatic compound on the proteome of this bacterium, cytosolic proteins induced by the presence of benzoate and succinate were analyzed using two liquid chromatography (LC)-based proteomic approaches: an isobaric tag for relative and absolute quantitation (iTRAQ) for quantitative analysis and one-dimensional gel electrophoresis/multidimensional protein identification technology (1-DE MudPIT) for protein identification. In total, 1286 proteins were identified by 1-DE MudPIT; this represents around 23.3% of the total proteome. In contrast, 570 proteins were identified and quantified by iTRAQ analysis. Of these, 55 and 52 proteins were up- and down-regulated, respectively, in the presence of benzoate. The proteins up-regulated included benzoate degradation enzymes, chemotaxis-related proteins, and ABC transporters. Enzymes related to nitrogen metabolism and pyruvate metabolism were down-regulated. These data suggest that a combination of 1-DE MudPIT and iTRAQ is an appropriate method for comprehensive proteomic analysis of biodegradative bacteria.     

Probing rhodopsin-transducin interactions by surface modification and mass spectrometry

The interactions of rhodopsin and the alpha-subunit of transducin (G(t)) have been mapped using a surface modification "footprinting" approach in conjunction with mass spectrometric analysis employing a synthetic peptide corresponding to C-terminal residues 340-350 of the alpha-subunit of G(t), G(t)alpha(340-350). Membrane preparations of unactivated (Rh) and light-activated rhodopsin (Rh*), each in the presence or absence of G(t)alpha(340-350), were acetylated with the water-soluble reagent sulfosuccinimidyl acetate, and the extent of the acetylation was determined by mass spectrometry. By comparing the differences in acetylation among Rh, Rh*, and the Rh-G(t)alpha(340-350) and Rh*-G(t)alpha(340-350) complexes, we demonstrate that the surface exposure of the acetylation sites was reduced by the conformational change associated with light activation, and that binding of G(t)alpha(340-350) blocks acetylation sites on cytoplasmic loops 1, 2, and 4 of Rh*. In addition, we show evidence of interaction between the end of the C-terminal tail of rhodopsin and G(t)alpha in the unactivated state of rhodopsin.     

Toxicity bioassay in Sprague-Dawley rats exposed to 20 kHz triangular magnetic field for 90 days

Sprague-Dawley rats (10 each of male and female per group for sham and magnetic field exposed) were exposed in a carrousel irradiator to 20 kHz intermediate frequency (IF) magnetic field at 6.25 microT rms for 8 h/day, 5 days/week for 90 days. Urine analysis (pH, serum glucose, protein, ketone bodies, RBC, WBC, bilirubin, urobilinogen, and specific gravity), blood analysis [WBC, RBC, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), thrombocyte count, and leucocyte count], blood biochemistry (total protein, blood urea nitrogen, creatinine, glucose, total bilirubin, total cholesterol, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and lactate dehydrogenase), and histopathological analysis for organs such as liver, kidney, testis, ovary, spleen, brain, heart, and lung were performed on day 90. Results showed no significant differences in the above analyses between IF magnetic field exposed and sham control rats. Therefore, we conclude that there were no significant toxicities in rats exposed to 20 kHz IF triangular magnetic field-exposure for 90 days.     

Proteome Analysis of Cellular Response of Pseudomonas putida KT2440 to Tetracycline Stress

Tetracycline-induced proteome of Pseudomonas putida KT2440 was analyzed by 2-D gel electrophoresis and matrix-assisted laser desorption ionization–time of flight/mass spectrum (NALDI-TOF/MS) in order to understand cellular response to tetracycline. Of the proteins upregulated in a culture medium containing subinhibitory concentration of tetracycline (50 μg/mL), we identified 38 proteins from cytosol and precipitated fractions by peptide mass fingerprinting and mass spectrum/mass spectrum analysis. Various amino acids ABC transporters, a ribose ABC transporter, and a sulfate ABC transporter were found to be upregulated. Protein synthesis-related proteins, stress proteins, energy metabolic enzymes, and unknown proteins were also strongly induced. Of the identified upregulated proteins, several proteins (isocitrate lyase, branched-chain amino acid ABC transporter, superoxide dismutase, etc.) were also upregulated under phenol-induced stress condition. These results demonstrate that tetracycline at a high concentration induced comprehensive stress in P. putida KT2440 and the global induction of proteins related to bacteria survival. Proteome analysis was found to be a useful tool for the elucidation of antibiotic-induced proteins in the present study.     

Cys155 of 27 kDa maize gamma-zein is a key amino acid to improve its in vitro digestibility

Twenty-seven kilodalton gamma-zein is a subclass of the maize zein storage proteins and, due to its localization at the protein body periphery, is critical to digestibility characteristics of all zeins. This protein had low in vitro digestibility, presumably due to its high Cys content (7.35 mol%) that is similar to the hard-to-digest analogous sorghum protein, gamma-kafirin. Therefore, each of the conserved disulfide-bonded Cys' was mutated to create C144A, C148A, C155A, and C156A maize gamma-zein mutants. The C155A showed a remarkable increase in digestibility to proteases - pepsin, chymotrypsin, and trypsin. A high conservation of this Cys among cereal gamma-prolamins indicates the utility of this finding.     

Spermatotoxic effects of α-chlorohydrin in rats

This study was conducted to investigate the potential effects of α-chlorohydrin (ACH) on epididymal function and antioxidant system in male rats. The test chemical was administered to male rats by gavage at doses of 0, 3, 10, and 30 mg/kg/day for 7 days. Twenty-four male rats were randomly assigned to four experimental groups, with six rats in each group. Spermatotoxicity was assessed by measurement of reproductive organ weight, testicular sperm head count, epididymal sperm motility and morphology, histopathologic examination, and oxidative damage analysis in rats. At 30 mg/kg/day, an increase in the incidence of clinical signs, epididymis weight, and gross necropsy findings of the epididymis, a decrease in the sperm motility, and an increased incidence of histopathological changes of the epididymis were observed in a dose-dependent manner. At 10 mg/kg/day, an increased incidence of clinical signs and histopathological changes and decreased sperm motility were observed. In the oxidative damage analysis, an increase in the malondialdehyde concentration and a decrease in the glutathione content and glutathione peroxidase and catalase activities in the epididymal tissue were detected at ≥3 mg/kg/day. The results show that graded doses of ACH elicit depletion of the antioxidant defense system and that the spermatotoxicity of ACH may be due to the induction of oxidative stress.     

Analysis of expressed sequence tags from the antarctic psychrophilic green algae, Pyramimonas gelidicola

Expressed sequence tags (ESTs) from the Antarctic green algae Pyramimonas gelidicola were analyzed to obtain molecular information on cold acclimation of psychrophilic microorganisms. A total of 2,112 EST clones were sequenced, generating 222 contigs and 219 singletons, and 200 contigs and 391 singletons from control (4 degrees C) and cold-shock conditions (-2 degrees C), respectively. The complete EST sequences were deposited to the DDBJ EST database (http:// www.ddbj.nig.ac.jp/index-e.html) and the nucleotide sequences reported in this study are available in the DDBJ/EMBL/ GenBank. These EST databases of Antarctic green algae can be used in a wide range of studies on psychrophilic genes expressed by polar microorganisms.