ABSTRACT
This study analyzed the distribution of endophytic fungi in 3 coastal environments with different climatic, geographical, and geological characteristics: the volcanic islands of Dokdo, the East Sea, and the West Sea of Korea. The isolated fungal endophytes were characterized and analyzed with respect to the characteristics of their host environments. For this purpose, we selected common native coastal halophyte communities from three regions. Molecular identification of the fungal endophytes showed clear differences among the sampling sites and halophyte host species. Isolates were also characterized by growth at specific salinities or pH gradients, with reference to previous geographical, geological, and climate studies. Unlike the East Sea or West Sea isolates, some Dokdo Islands isolates showed endurable traits with growth in high salinity, and many showed growth under extremely alkaline conditions. A smaller proportion of West Sea coast isolates tolerate compared to the East Sea or Dokdo Islands isolates. These results suggest that these unique fungal biota developed through a close interaction between the host halophyte and their environment, even within the same halophyte species. Therefore, this study proposes the application of specific fungal resources for restoring sand dunes and salt-damaged agricultural lands and industrialization of halophytic plants.
Subject(s)
Biota , Climate , Endophytes , Fungi , Islands , Korea , Proton-Motive Force , Salinity , Salt-Tolerant PlantsABSTRACT
BACKGROUND: Karwinskia humboldtiana (Kh) is a poisonous plant of the rhamnacea family. To elucidate some of the subcellular effects of Kh toxicity, membrane fluidity and ATPase activities as hydrolytic and as proton-pumping activity were assessed in rat liver submitochondrial particles. Rats were randomly assigned into control non-treated group and groups that received 1,1.5 and 2 g/Kg body weight of dry powder of Kh fruit, respectively. Rats were euthanized at day 1 and 7 after treatment. RESULTS: Rats under Kh treatment at all dose levels tested, does not developed any neurologic symptoms. However, we detected alterations in membrane fluidity and ATPase activity. Lower dose of Kh on day 1 after treatment induced higher mitochondrial membrane fluidity than control group. This change was strongly correlated with increased ATPase activity and pH gradient driven by ATP hydrolysis. On the other hand, membrane fluidity was hardly affected on day 7 after treatment with Kh. Surprisingly, the pH gradient driven by ATPase activity was significantly higher than controls despite an diminution of the hydrolytic activity of ATPase. CONCLUSIONS: The changes in ATPase activity and pH gradient driven by ATPase activity suggest an adaptive condition whereby the fluidity of the membrane is altered.
Subject(s)
Animals , Male , Rats , Mitochondria, Liver/drug effects , Adenosine Triphosphatases/metabolism , Karwinskia/toxicity , Membrane Fluidity/drug effects , Subcellular Fractions/drug effects , Submitochondrial Particles/drug effects , Mitochondria, Liver/enzymology , Random Allocation , Rats, Sprague-Dawley , Proton-Motive Force/drug effects , Fruit/toxicityABSTRACT
OBJECTIVES: The aims of this study were to determine the erosive potential of several carbonated waters and to confirm the availability of a simple ISO protocol for screening the erosive potential of drinks. METHODS: A total of six carbonated waters were tested. Three products (Lemon-Sparkling water, Seagram, and Trevi) were domestic, and the other three (Perrier, San Pellegrino, and Rosbacher) were imported. Two kinds of carbonated drinks (Coca-Cola and Sprite) were used as controls. The erosive potential of each drink was assessed by measuring the initial pH (pH(I)), the final pH after degassing of carbon dioxide (pH(F)), and the titratable acidity to pH 5.5 (TA(5.5)) and 7.0 (TA(7.0)). The pH changes (DeltapH) caused by the addition of drinks to screening solutions were calculated according to the ISO protocol for evaluating the erosive potential of oral rinses. RESULTS: The overall erosive potential of the carbonated waters was lower than that of the control drinks. The pHI and pH(F) of the carbonated waters ranged from 3.94 to 5.84 and from 5.07 to 7.88, respectively. The Lemon-Sparkling water showed the highest erosive potential among the carbonated waters, having the lowest pH (3.94) and the highest TA(5.5) (1.67 ml). The DeltapH of all tested drinks ranged from -1.00 to 0.23. Also, the tendency of erosive potential measured by DeltapH was similar to that measured by TA(5.5). CONCLUSIONS: The carbonated waters tested in this study had a lower erosive potential than did the carbonated drinks. However, the erosive potential of domestic products was higher than that of imported products. The results of the ISO screening test could reflect the influence of the acid content as well as the pH of drinks. Therefore, this protocol could also be conveniently applied to evaluate the erosive potential of various drinks.
Subject(s)
Carbon Dioxide , Carbon , Carbonated Beverages , Carbonated Water , Hydrogen-Ion Concentration , Mass Screening , Proton-Motive Force , Tooth Erosion , WaterABSTRACT
The pancreatic islet beta-cell is uniquely specialized to couple its metabolism and rates of insulin secretion with the levels of circulating nutrient fuels, with the mitochondrial playing a central regulatory role in this process. In the beta-cell, mitochondrial activation generates an integrated signal reflecting rates of oxidativephosphorylation, Kreb's cycle flux, and anaplerosis that ultimately determines the rate of insulin exocytosis. Mitochondrial activation can be regulated by proton leak and mediated by UCP2, and by alkalinization to utilize the pH gradient to drive substrate and ion transport. Converging lines of evidence support the hypothesis that substrate cycles driven by rates of Kreb's cycle flux and by anaplerosis play an integral role in coupling responsive changes in mitochondrial metabolism with insulin secretion. The components and mechanisms that account for the integrated signal of ATP production, substrate cycling, the regulation of cellular redox state, and the production of other secondary signaling intermediates are operative in both rodent and human islet beta-cells.
Subject(s)
Humans , Adenosine Triphosphate , Cytosol , Exocytosis , Insulin , Ion Transport , Islets of Langerhans , Mitochondria , Oxidation-Reduction , Proton-Motive Force , Protons , Rodentia , Substrate CyclingABSTRACT
As part of an initial inquiry into the function of the outer membrane proteins (OMPs) of Helicobacter pylori Korean strain 51, we have conducted an extensive proteome analysis via quadrupole time of flight (Q-TOF) mass spectrometry (MS). Fifty one OMPs of H. pylori were purified using sarcosine and resolved via two-dimensional electrophoresis with immobilized pH gradient strips. The most abundant proteins were observed in the alkaline pI regions (6.0~11.0) at molecular masses between 10~100 KDa. Here, 15 spots were identified, representing 9 types of genes (KHP0852, KHP0853, KHP1353, KHP1017, KHP0172, KHP0076, KHP0617, KHP1069, KHP0614) from the sarcosin-insoluble fraction of H. pylori 51. These may be employed in the characterization of the OMPs of H. pylori 51, which will help to identify new potential target proteins for vaccine development and drug therapy.
Subject(s)
Electrophoresis , Helicobacter , Helicobacter pylori , Mass Spectrometry , Membrane Proteins , Membranes , Proteins , Proteome , Proton-Motive Force , Sarcosine , Sprains and StrainsABSTRACT
BACKGROUND/AIMS: Cerulein pancreatitis is similar to human edematous pancreatitis with dysregulation of the production and secretion of digestive enzymes, edema formation, cytoplasmic vacuolization and the death of acinar cells. We hypothesized that membrane proteins may be altered as the early event during the induction of acute pancreatitis. Present study aims to determine the differentially expressed proteins in the membranes of cerulein-treated pancreatic acinar cells. METHODS: Pancreatic acinar AR42J cells were treated with 10(-8) M cerulein for 1 hour. Membrane proteins were isolated from the cells and separated by two-dimensional electrophoresis using pH gradients of 5-8. Membrane proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. The differentially expressed proteins, whose expression levels were more or less than three-fold in cerulein-treated cells, were analyzed. RESULTS: Two differentially expressed proteins (mannan-binding lectin-associated serine protease-2, heat shock protein 60) were up-regulated while four proteins (protein disulfide isomerase, gamma-actin, isocitrate dehydrogenase 3, seven in absentia homolog 1A) were down-regulated by cerulein treatment in pancreatic acinar cells. These proteins are related to cell signaling, oxidative stress, and cytoskeleton arrangement. CONCLUSIONS: Oxidative stress may induce cerulein-induced cell injury and disturbances in defense mechanism in pancreatic acinar cells.
Subject(s)
Humans , Acinar Cells , Actins , Ceruletide , Cytoplasm , Cytoskeleton , Edema , Electrophoresis , Heat-Shock Proteins , Isocitrate Dehydrogenase , Isocitrates , Mass Spectrometry , Membrane Proteins , Membranes , Oxidative Stress , Pancreatitis , Protein Disulfide-Isomerases , Proteins , Proteome , Proton-Motive Force , SerineABSTRACT
Members of the Enterobacteriaceae family are present in the intestines of man and animals as commensals or are important disease causing agents. Bacteria bearing multidrug efflux systems (MDR) are able to survive adverse ecological niches. Multiresistant Escherichia coli and Enterobacter cloacae isolates from wholesome broiler carcasses were investigated for the presence of MDR. Lowering of Minimal Inhibitory Concentration for antimicrobials in the presence of a proton-motive force (PMF) uncoupler was tested as a potential display of the MDR phenotype. PCR amplification of the genes encoding AcrA and AcrB, components of a MDR system was performed. Diversity of each species was ascertained by Pulsed-Field Gel Electrophoresis (PFGE) of DNA digested with endonuclease XbaI. For all the isolates, except E. coli 1 and E. cloacae 9, lowering of MIC or of the growth rate in the presence of antimicrobials was observed, indicating a PMF dependent resistance mechanism. Expected products of DNA amplification with acrAB derived primers was obtained with all E. coli strains and with two of the five E. cloacae strains. Dendrogram generated shows diverse pulsetypes, confirming the genetic diversity among the strains. An important issue and related public health is the fact that different models and mechanisms of antimicrobial resistance are present in a small number of non-pathogenic strains and isolated from the same origin. These may be sources of resistance genes to others microorganisms, among them, pathogenic strains.
Os membros da família Enterobacteriaceae estão presentes no intestino do homem e dos animais como comensais ou agentes causadores de doença importantes. Bactérias multirresistentes podem possuir sistemas de efluxo multidrogas (MDR) sendo capazes de sobreviver em nichos ecológicos adversos. Escherichia coli e Enterobacter cloacae, multirresistentes, isoladas de frangos sadios foram investigadas quanto à presença de MDR. A diminuição da concentração inibitória mínima de antimicrobianos, na presença de um desacoplador da força próton motora (PMF), foi usada para detectar o fenótipo MDR. Foi realizada PCR dos genes codificadores de AcrA e AcrB, componentes de um sistema MDR. A diversidade de cada isolado foi confirmada por eletroforese em gel de campo pulsado (PFGE) usando a endonuclease XbaI. Observou-se em todos os isolados, exceto E. coli 1 e E. cloacae 9, uma diminuição das MICs ou das curvas de crescimento na presença dos antimicrobianos, indicando um mecanismo de resistência dependente da PMF. Os produtos amplificados esperados derivados de acrAB foram obtidos em todos os isolados de E. coli e em dois, dos cinco, de E. cloacae. O dendrograma gerado mostra diferentes perfis de bandas (pulsetypes), confirmando a diversidade genética entre os isolados. Uma questão importante e relacionada à saúde publica é o fato de que diferentes modelos e mecanismos de resistência aos antimicrobianos estão presentes em um número reduzidos de isolados não patogênicos e obtidos de uma mesma origem. Esses podem ser fontes de genes de resistência para outros microorganismos, entre eles, cepas patogênicas.
Subject(s)
Animals , Drug Resistance, Microbial , Endonucleases , Enterobacteriaceae Infections , Escherichia coli Infections , Enterobacteriaceae/isolation & purification , Escherichia coli/isolation & purification , In Vitro Techniques , Phenotype , Polymerase Chain Reaction , Poultry , Proton-Motive Force , Electrophoresis, Gel, Pulsed-Field , Food Samples , Methods , Methods , VirulenceABSTRACT
The protein identity of sarcosine-insoluble outer membrane proteins (OMPs) of Helicobacter pylori J99 was determined with the basic study of understanding the function of proteins. A sarcosine-insoluble OMPs was resolved by two-dimensional electrophoresis with immobilized pH gradient strips. The most abundant proteins were shown in the alkaline pI regions (6.0~11.0) with molecular masses of 10 to 100 kDa. We have performed an extensive proteome analysis by quadrupole time of flight (Q-TOF) mass spectrometry (MS). Here, of 50 spots processed, 42 spots were identified, which represented 16 genes and we newly detected 8 kinds of proteins (JHP0119, JHP0388, JHP1046, JHP1405, JHP0073, JHP0551, JHP1382, JHP0552) from the sarcosin-insoluble fraction of H. pylori J99. Those may be used to elucidate the characterization of the OMPs of H. pylori J99, which will help identify new potential target proteins for vaccine development and drug therapy.
Subject(s)
Electrophoresis , Helicobacter , Helicobacter pylori , Mass Spectrometry , Membrane Proteins , Membranes , Proteins , Proteome , Proton-Motive Force , Tandem Mass SpectrometryABSTRACT
PURPOSE: Small cell lung cancer is one of the major causes of death from cancer worldwide. To explore the expressions of global protein in small cell lung cancer cells, a proteomic approach, to identify the proteins, was used and the establishment of a protein reference map attempted. MATERIALS AND METHODS: Two-dimensional gel electrophoresis (2-DE), with subsequent analysis by mass spectrometry (MS), was applied to the study of protein identification from a small cell lung cancer cell line, NCI- H211. The cells were lysed, and the extracts subjected to isoelectric focusing, with immobilized pH gradients, followed by second dimension SDS-PAGE. The polypeptides were identified by peptide mass fingerprinting, with MALDI-TOF MS, after in-gel protein digestion. RESULTS: From silver staining of the gel, around two thousands protein spots were separated by the 2-DE. Of these protein spots visualized in the gel, one hundred and ten were identified by peptide mass fingerprinting. Different proteins, such as enzymes, cytoskeletal proteins and proteins common to eukaryotic cells, were identified. CONCLUSION: The protein expressions of the small cell lung cancer cells were analyzed to establish a protein reference map. The reference map presented here may serve as a working tool for the further study of small cell lung cancer.
Subject(s)
Humans , Cause of Death , Cell Line , Cytoskeletal Proteins , Dermatoglyphics , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Eukaryotic Cells , Isoelectric Focusing , Mass Spectrometry , Peptides , Proteolysis , Proteome , Proton-Motive Force , Silver Staining , Small Cell Lung CarcinomaABSTRACT
BACKGROUND: Psoriasis is a common, chronic, recurrent, inflammatory disease of the skin characterized by circumscribed, erythematous, thick plaques covered by silvery white scales. Although many researchers are working hard on the topic, we are still in search of its exact pathophysiology. Proteomics is a new emerging field of research for understanding cell physiology and pathophysiology of diseases, based on the protein measurement by high resolution two-dimensional gel electrophoresis followed by the protein identification and characterization by mass spectrometry. OBJECTIVE: The purpose of this study was to identify the specific epidermal proteins in patients with psoriasis. METHODS: We compared the proteome maps obtained from uninvolved and involved psoriatic epidermis by isoelectric focusing in 3-10 non-linear immobilzed pH gradients strips and two- dimensional electrophoresis. RESULTS: The significant differences in protein expressions between two groups were found as evidenced by many increased or decreased protein spots. We found that 15 spots showed changes in the involved psoriatic epidermis as compared to the uninvolved psoriatic epidermis. Protein spots with 6.8 kDa/pI 7.3, 6.9 kDa/pI 6.9, 7.3 kDa/pI 7.8, 8.8 kDa/pI 6.6, 11.5 kDa/pI 6.7, 11.6 kDa/pI 6.9, 13.1 kDa/pI 5.9, 13.2 kDa/pI 6.1, 13.8 kDa/pI 5.6, 15.2 kDa/pI 5.9, 48.3 kDa/pI 4.8, 49.7 kDa/pI 4.8, 49.9 kDa/pI 4.9, 29.7 kDa/pI 7.3 were increased in 100% of involved psoriatic epidermis and protein spots with 70.9 kDa/pI 5.3 decreased in 80% of involved psoriatic epidermis as compared to uninvolved psoriatic epidermis. CONCLUSION: The significant changes in many protein spots were observed in involved psoriatic epidermis as compared with uninvolved psoriatic epidermis. The proteomics in psoriasis may be helpful for the understanding its pathophysiology and treatment.
Subject(s)
Humans , Cell Physiological Phenomena , Electrophoresis , Electrophoresis, Gel, Two-Dimensional , Epidermis , Isoelectric Focusing , Mass Spectrometry , Proteome , Proteomics , Proton-Motive Force , Psoriasis , Skin , Weights and MeasuresABSTRACT
Effects of cadmium (Cd) intoxication on renal endosomal accumulation of organic cations (OC ) were studied in rats using 14C-tetraethylammnium (TEA) as a substrate. Cd intoxication was induced by s.c. injections of 2 mg Cd/kg/day for 2-3 weeks. Renal cortical endosomes were isolated and the endosomal acidification (acridine orange fluorescence change) and TEA uptake (Millipore filtration technique) were assessed. The TEA uptake was an uphill transport mediated by H /OC antiporter driven by the pH gradient established by H -ATPase. In endosomes of Cd-intoxicated rats, the ATP-dependent TEA uptake was markedly attenuated due to inhibition of endosomal acidification as well as H /TEA antiport. In kinetic analysis of H /TEA antiport, Vmax was reduced and Km was increased in the Cd group. Inhibition of H /TEA antiport was also observed in normal endosomes directly exposed to free Cd (but not Cd-metallothionein complex, CdMt) in vitro. These data suggest that during chronic Cd exposure, free Cd ions liberated by lysosomal degradation of CdMt in proximal tubule cells may impair the endosomal accumulation of OC by directly inhibiting the H /OC antiporter activity and indirectly by reducing the intravesicular acidification, the driving force for H /OC exchange.
Subject(s)
Animals , Rats , Biological Transport, Active , Cadmium , Cations , Citrus sinensis , Endosomes , Filtration , Fluorescence , Ion Transport , Ions , Kidney , Proton-Motive Force , Tea , TetraethylammoniumABSTRACT
Chronic exposure to cadmium (Cd) results in an inhibition of protein endocytosis in the renal proximal tubule, leading to proteinuria. In order to gain insight into the mechanism by which Cd impairs the protein endocytosis, we investigated the effect of Cd on the acidification of renal cortical endocytotic vesicles (endosomes). The endosomal acidification was assessed by measuring the pH gradient-dependent fluorescence change, using acridine orange or FITC-dextran as a probe. In renal endosomes isolated from Cd-intoxicated rats, the Vmax of ATP-driven fluorescence quenching (H -ATPase dependent intravesicular acidification) was significantly attenuated with no substantial changes in the apparent Km, indicating that the capacity of acidification was reduced. When endosomes from normal animals were directly exposed to free Cd in vitro, the Vmax was slightly reduced, whereas the Km was markedly increased, implying that the biochemical property of the H -ATPase was altered by Cd. In endosomes exposed to free Cd in vitro, the rate of dissipation of the transmembrane pH gradient after H -ATPase inhibition appeared to be significantly faster compared to that in normal endosomes, indicating that the H -conductance of the membrane was increased by Cd. These results suggest that in long-term Cd-exposed animals, free Cd ions liberated in the proximal tubular cytoplasm by lysosomal degradation of cadmium-metallothionein complex (CdMT) may impair endosomal acidification 1) by reducing the H -ATPase density in the endosomal membrane, 2) by suppressing the intrinsic H -ATPase activity, and 3) possibly by increasing the membrane conductance to H+ ion. Such effects of Cd could be responsible for the alterations of proximal tubular endocytotic activities, protein reabsorption and various transporter distributions observed in Cd-exposed cells and animals.
Subject(s)
Animals , Rats , Acridine Orange , Cadmium , Cytoplasm , Endocytosis , Endosomes , Fluorescence , Hydrogen-Ion Concentration , Ions , Kidney , Membranes , Proteinuria , Proton-Motive ForceABSTRACT
Two-dimensional gel electrophoresis (2-DE) is an essential tool of proteomics to analyse the entire set of proteins of an organism and its variation between organisms. Helicobacter pylori was tried to identify differences between strains. As the first step, whole H. pylori was lysed using high concentration urea contained lysis buffer (9.5 M Urea, 4% CHAPS, 35 mM Tris, 65 mM DTT, 0.01% SDS and 0.5% Ampholite (Bio-Rad, pH 3-10)). The extract (10 mug) was rehydrated to commercially available immobilised pH gradient (IPG) strips, then the proteins were separated according to their charges as the first dimensional separation. The IPG strips were placed on Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to separate according to molecular mass of the proteins as the second dimension. The separated protein spots were visualised by silver staining in order to compare different expression of proteins between strains. Approximately 120 spots were identified in each mini-protein electrophoresised gel, furthermore about 65 to 75 spots were regarded as identical proteins in terms of pI value and molecular weight between strains used. In addition, distinct differences were found between strains, such as 219-1, Y7 and Y14, CH150. Two representative strains were examined using strips which had pH range from 4 to 7. This strips showed a number of isoforms which were considered large spots on pH range 3-10. Furthermore, the rest of spots on pH 4-7 IPG strips appeared very distinctive compared to broad range IPG strips. 2-DE seems to be an excellent tool for analysing and identifying variations between H. pylori strains.