Home About us Contact
|
Home About us Contact | ||
|
|
||
Whole Cells (whole + cell)
Terms modified by Whole Cells Selected AbstractsHIGH-RESOLUTION MAGIC ANGLE SPINNING NMR ANALYSIS OF WHOLE CELLS OF CHAETOCEROS MUELLERI (BACILLARIOPHYCEAE) AND COMPARISON WITH 13C-NMR AND DISTORTIONLESS ENHANCEMENT BY POLARIZATION TRANSFER 13C-NMR ANALYSIS OF LIPOPHILIC EXTRACTS,JOURNAL OF PHYCOLOGY, Issue 3 2004Matilde S. Chauton Lipid composition in extracted samples of Chaetoceros muelleri Lemmermann was studied with 13C-NMR and distortionless enhancement by polarization transfer (DEPT) 13C-NMR, resulting in well-resolved 13C-NMR spectra with characteristic resonance signals from carboxylic, olefinic, glyceryl, methylene, and methyl groups. The application of a DEPT pulse sequence aided in the assignment of methylene and methine groups. Resonance signals were compared with literature references, and signal assignment included important unsaturated fatty acids such as eicosapentaenoic and docosahexaenoic and also phospholipids and glycerols. Results from the extracted samples were used to assign resonance signals in a high-resolution magic angle spinning (HR MAS) DEPT 13C spectrum from whole cells of C. muelleri. The NMR analysis on whole cells yielded equally good information on fatty acids and also revealed signals from carbohydrates and amino acids. Broad resonance signals and peak overlapping can be a problem in whole cell analysis, but we found that application of HR MAS gave a well-resolved spectrum. The chemical shift of metabolites in an NMR spectrum depends on the actual environment of nuclei during analysis, and some differences could therefore be expected between extracted and whole cell samples. The shift differences were small, and assignment from analysis of lipophilic extract could be used to identify peaks in the whole cell spectrum. HR MAS 13C-NMR therefore offers a possibility for broad-range metabolic profiling directly on whole cells, simultaneously detecting metabolites that are otherwise not detected in the same analytical set up and avoiding tedious extraction procedures. [source] Enantioselective Synthesis of l -Homophenylalanine by Whole Cells of Recombinant Escherichia coli Expressing l -Aminoacylase and N -Acylamino Acid Racemase Genes from Deinococcus radiodurans BCRC12827BIOTECHNOLOGY PROGRESS, Issue 6 2006Shih-Kuang Hsu l -Homophenylalanine (l -HPA) is a chiral unnatural amino acid used in the synthesis of angiotensin converting enzyme inhibitors and many pharmaceuticals. To develop a bioconversion process with dynamic resolution of N -acylamino acids for the l -HPA production, N -acylamino acid racemase (NAAAR) and l -aminoacylase (LAA) genes were cloned from Deinococcus radiodurans BCRC12827 and expressed in Escherichia coli XLIBlue. The recombinant enzymes were purified by nickel-chelate chromatography, and their biochemical properties were determined. The NAAAR had high racemization activity toward chiral N -acetyl-homophenylalanine (NAc-HPA). The LAA exhibited strict l -enantioselection to hydrolyze the NAc- l -HPA. A stirred glass vessel containing transformed E. coli cells expressing D. radiodurans NAAAR and LAA was used for the conversion of NAc- d -HPA to l -HPA. Unbalance activities of LAA and NAAAR were found in E. coli cell coexpressing laa and naaar genes, which resulted in the accumulation of an intermediate, NAc- l -HPA, in the early stage of conversion and a low productivity of 0.83 mmol l -HPA/L h. The results indicated that low activity of LAA present in the biomass is the rate-limiting factor in l -HPA production. In the case of two whole cells with separately expressed enzyme, the enzymatic activities of LAA and NAAAR could be balanced by changing the loading of individual cells. When the activities of two enzymes were fixed at 3600 U/L, 99.9% yield of l -HPA could be reached in 1 h, with a productivity of 10 mmol l -HPA/L h. The cells can be reused at least six cycles at a conversion yield of more than 96%. This is the first NAAAR/LAA process using NAc-HPA as substrate and recombinant whole cells containing Deinococcus enzymes as catalysts for the production of l -HPA to be reported. [source] Anticancerogenic effect of a novel chiroinositol-containing polysaccharide from Bifidobacterium bifidum BGN4FEMS MICROBIOLOGY LETTERS, Issue 2 2004Hyun Ju You Abstract Strains of bifidobacteria have many health-promotion effects. Whole cells or cytoplasm extracts of Bifidobacterium bifidum BGN4, isolated from human feces, inhibited the growth of several cancer cell lines. The polysaccharide fraction (BB-pol) extracted from B. bifidum BGN4 had a novel composition, comprising chiroinositol, rhamnose, glucose, galactose, and ribose. Three human colon cancer cell lines were treated with BB-pol: HT-29, HCT-116, and Caco-2. Trypan blue exclusion assay and BrdU incorporation assay showed that BB-pol inhibited the growth of HT-29 and HCT-116 cells but did not inhibit the growth of Caco-2 cells. [source] Identification of repertoires of surface antigens on leukemias using an antibody microarrayPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2003Larissa Belov Abstract We have previously described a microarray of cluster of differentiation (CD) antibodies that enables concurrent determination of more than 60 CD antigens on leukocytes. This procedure does not require protein purification or labeling, or a secondary detection system. Whole cells are captured by a microarray of 10 nL antibody dots immobilized on a nitrocellulose film on a microscope slide. Distinct patterns of cell binding are observed for different leukemias or lymphomas. These haematological malignancies arise from precursor cells of T- or B-lymphocytic, or myeloid lineages of hematopoiesis. The dot patterns obtained from patients are distinct from those of peripheral blood leukocytes from normal subjects. This microarray technology has recently undergone a number of refinements. The microarray now contains more CD antibodies, and a scanner for imaging dot patterns and software for data analysis provide an extensive immunophenotype sufficient for diagnosis of common leukemias. The technology is being evaluated for diagnosis of leukemias with parallel use of conventional diagnostic criteria. [source] Scaling-up of complex whole-cell bioconversions in conventional and non-conventional mediaBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Marco P.C. Marques Abstract The use of whole cells is becoming a more common approach in pharmaceutical and agrochemical industries in order to obtain pure compounds with fewer production steps, higher yields, and cleaner processes, as compared to those achieved with traditional strategies. Whole cells are often used as enzymes pools, in particular when multi-step reactions and/or co-factor regeneration are envisaged. Nonetheless, published information on the scale-up of such systems both in aqueous and in two-phase aqueous,organic systems is relatively scarce. The present work aims to evaluate suitable scale-up criteria in conventional and non-conventional medium for a whole-cell bioconversion that uses resting cells of Mycobacterium sp. NRRL B-3805 to cleave the side chain of ,-sitosterol, a poorly water-soluble substrate. The experiments were performed in 24-well microtiter plates and in 250,mL shaken flasks as orbital stirred systems, and in 300,mL stirred tanks as mechanically stirred systems. Results show that productivity yields were similar in all scales tested, when maintaining oxygen mass transfer coefficients constant in aqueous systems, or when maintaining constant volumetric power consumption in aqueous,organic two-phase systems. Biotechnol. Bioeng. 2010;106: 619,626. © 2010 Wiley Periodicals, Inc. [source] Strong immunostimulation in murine immune cells by Lactobacillus rhamnosus GG DNA containing novel oligodeoxynucleotide patternCELLULAR MICROBIOLOGY, Issue 3 2005Iliyan D. Iliev Summary Whole cells, cell wall components and some soluble factors from Lactobacillus rhamnosus GG (LGG) are known to invoke immune responses as they interact with animal and human immune cells. In the present study, we found that chromosomal DNA from LGG is a potent inducer of splenic B cell proliferation, CD86/CD69 expression and cytokine production in mice. In the genomic DNA of LGG we discovered TTTCGTTT oligodeoxynucleotide (ODN) ID35, which has a potent activity in a number of immunostimulatory assays. Phosphorothioate backbone is not required for the activity of ID35. The ODN ID35 showed levels of activity comparable with those induced by the murine prototype ODN 1826 in B cell proliferation, CD86/CD69 expression, interleukin (IL)-6, IL-12, IL-18, interferon gamma (IFN-,) and tumour necrosis factor alpha (TNF-,) mRNA expression and IFN-,/IL-12p70 protein production assays. Additionally, ID35 appeared to be equally active in both murine and human immune cells. These stimulatory effects are due to TTTCGTTT motif located in the 5, end of ID35. In this study we demonstrate for a first time that, DNA from LGG is a factor of immunobiotic activity. Furthermore, ODN ID35 is the first ODN, with such a strong immunostimulatory activity to be found in immunobiotic bacterial DNA. [source] Spontaneous recurrent network activity in organotypic rat hippocampal slicesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2005Majid H. Mohajerani Abstract Organotypic hippocampal slices were prepared from postnatal day 4 rats and maintained in culture for >6 weeks. Cultured slices exhibited from 12 days in vitro spontaneous events which closely resembled giant depolarizing potentials (GDPs) recorded in neonatal hippocampal slices. GDP-like events occurred over the entire hippocampus with a delay of 30,60 ms between two adjacent regions as demonstrated by pair recordings from CA3,CA3, CA3,CA1 and interneurone,CA3 pyramidal cells. As in acute slices, spontaneous recurrent events were generated by the interplay of GABA and glutamate acting on AMPA receptors as they were reversibly blocked by bicuculline and 6,7-dinitroquinoxaline-2,3-dione but not by dl -2-amino-5-phosphonopentaoic acid. The equilibrium potentials for GABA measured in whole cell and gramicidin-perforated patch from interconnected interneurones,CA3 pyramidal cells were ,70 and ,56 mV, respectively. The resting membrane potential estimated from the reversal of N -methyl- d -aspartate-induced single-channel currents in cell-attach experiments was ,75 mV. In spite of its depolarizing action, in the majority of cases GABA was still inhibitory as it blocked the firing of principal cells. The increased level of glutamatergic connectivity certainly contributed to network synchronization and to the development of interictal discharges after prolonged exposure to bicuculline. In spite of its inhibitory action, in a minority of cells GABA was still depolarizing and excitatory as it was able to bring principal cells to fire, suggesting that a certain degree of immaturity is still present in cultured slices. This was in line with the transient bicuculline-induced block of GDPs and with the isoguvacine-induced increase of GDP frequency. [source] Mechanisms Associated with the Negative Inotropic Effect of Deuterium Oxide in Single Rat Ventricular MyocytesEXPERIMENTAL PHYSIOLOGY, Issue 2 2000K. Hongo Deuterium oxide (D2O) is known to cause a negative inotropic effect in muscle although the mechanisms associated with this response in cardiac muscle are not well understood. We studied the effects of D2O in single rat ventricular myocytes in order to characterise the mechanisms associated with its negative inotropic effect and to assess its possible use as an acute modulator of microtubules. D2O rapidly reduced the magnitude of contraction in rat ventricular myocytes, and there was some recovery of contraction in the presence of D2O. Colchicine, an agent known to depolymerise microtubules, did not modify the effect of D2O. D2O decreased the L-type Ca2+ current (ICa), measured under whole cell and perforated patch clamp conditions. Slowing of the time to peak and a delay in inactivation of ICa were observed. Intracellular calcium ([Ca2+]i) and sodium ([Na+]i) were measured using the fluorescent indicators fura-2 and SBFI, respectively. The fall in contraction upon exposure to D2O was not associated with a fall in the [Ca2+]i transient; this response is indicative of a reduction in myofilament Ca2+ sensitivity. Both the [Ca2+]i transient and [Na+]i increased during the partial recovery of contraction in the presence of D2O. We conclude that a decrease in the myofilament sensitivity for Ca2+ and a reduction in Ca2+ influx via ICa are principally responsible for the negative inotropic effect of D2O in cardiac muscle. We found no evidence to explain the negative inotropic effect of D2O in terms of microtubule proliferation. In addition we suggest that acute application of D2O is not a useful procedure for the investigation of the role of microtubules in excitation-contraction coupling in cardiac muscle. [source] Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actinFEBS JOURNAL, Issue 22 2007Anastasia V. Pivovarova Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F-actin, but effectively prevents aggregation of thermally denatured F-actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548,1553], and supposed that Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27,3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. All the methods show that the size of actin,Hsp27-3D complexes decreases with increasing Hsp27-3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27-3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of ,,16 nm, a sedimentation coefficient of 17,20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27-3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions. [source] Membrane orientation of laminin binding proteinFEBS JOURNAL, Issue 18 2003An extracellular matrix bridging molecule of Leishmania donovani Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m -iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end. [source] Differential effects of arachidonoyl trifluoromethyl ketone on arachidonic acid release and lipid mediator biosynthesis by human neutrophilsFEBS JOURNAL, Issue 15 2002Evidence for different arachidonate pools The goal of this study was to determine the effects of a putative specific cytosolic phospholipase A2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), on arachidonic acid (AA) release and lipid mediator biosynthesis by ionophore-stimulated human neutrophils. Initial studies indicated that AACOCF3 at concentrations 0,10 µm did not affect AA release from neutrophils. In contrast, AACOCF3 potently inhibited leukotriene B4 formation by ionophore-stimulated neutrophils (IC50 , 2.5 µm). Likewise, AACOCF3 significantly inhibited the biosynthesis of platelet activating factor. In cell-free assay systems, 10 µm AACOCF3 inhibited 5-lipoxygenase and CoA-independent transacylase activities. [3H]AA labeling studies indicated thatthe specific activities of cell-associated AA mimicked that of leukotriene B4 and PtdCho/PtdIns, while the specific activities of AA released into the supernatant fluid closely mimicked that of PtdEtn. Taken together, these data argue for the existence of segregated pools of arachidonate in human neutrophils. One pool of AA is linked to lipid mediator biosynthesis while another pool provides free AA that is released from cells. Additionally, the data suggest that AACOCF3 is also an inhibitor of CoA-independent transacylase and 5-lipoxygenase. Thus, caution should be exercised in using AACOCF3 as an inhibitor of cytosolic phospholipase A2 in whole cell assays because of the complexity of AA metabolism. [source] Alpha-fetoprotein-specific transfer factors downregulate alpha-fetoprotein expression and specifically induce apoptosis in Bel7402 alpha-fetoprotein-positive hepatocarcinoma cellsHEPATOLOGY RESEARCH, Issue 7 2007Hui Zhang Aim:, To investigate the mechanisms of AFP-specific transfer factors (AFP-TF) in induced Bel7402 cells apoptosis. Further, we investigate the interaction between AFP-TF and AFP in the apoptosis. Methods:, Bel7402 and HepG2 AFP-positive hepatocarcinoma cell lines, SK-Hep-1 AFP-negative hepatocarcinoma cell line and Changliver normal liver cell line are used. Cell viability is evaluated by MTT assay and apoptosis is measured by Hoechst33342 staining and TUNEL assay. FACS is used to analyze the cell cycle. AFP expression is examined by RT-PCR, Western blotting and immunocytochemistry. The interaction between AFP-TF and AFP in the apoptosis is investigated by addition of AFP in cultures or AFP transfection in Bel7402 cells prior to AFP-TF treatment. Mitochondrial membrane potential (,,m) and intracellular Ca2+ concentration are respectively measured by Rhodamine123 and Fluo-3 AM Ester. Western blotting detects the involvement of several apoptosis-related proteins. Finally, caspase-3 and Caspase-9 activity are respectively examined. Results:, AFP-TF can induce apoptosis in Bel7402 and HepG2 AFP-positive hepatocarcinoma cells, but not SK-Hep-1 and Changliver cells. AFP-mRNA level changes little in apoptotic Bel7402 cells; while AFP expression is downregulated and uniformly dispersed throughout the whole cell. Addition of exogenous AFP or overexpression of intracellular AFP can reduce such apoptotic effect. Besides, apoptotic Bel7402 cells show a disruption of ,,m, an immediate elevation of Ca2+ concentration, a prominently decreased ratio of bcl-2 to bax, a release of cytochrome c from mitochondria to cytosol, and ultimately an activation of caspase-9 and caspase-3. Conclusion:, AFP-TF induced Bel7402 cells apoptosis is mitochondrial-dependent and is mediated by the interaction of AFP-TF with intracellular AFP. [source] Acinetobacter bioreporter assessing heavy metals toxicityJOURNAL OF BASIC MICROBIOLOGY, Issue 5 2006Desouky Abd-El-Haleem Dr. This work was conducted to employ a whole cell-based biosensor to monitor toxicity of heavy metals in water and wastewater. An isolate of industrial wastewater bacterium, Acinetobacter sp. DF4, was genetically modified with lux reporter gene to create a novel bioluminescent bacterial strain, designated as DF4/PUTK2. This bioreporter can investigate the toxicity through light inhibition due to cell death or metabolic burden and the specific stress effects of the tested soluble materials simultaneously. The use of Acinetobacter DF4/PUTK2 as a bioluminescent reporter for heavy metal toxicity testing and for the application of wastewater treatment influent toxicity screening is presented in this study. Among eight heavy metals tested, the bioluminescence of DF4/PUTK2 was most sensitive to Zn, Cd, Fe, Co, Cr followed by Cu in order of decreasing sensitivity. The same pattern of sensitivity was observed when several contaminated water and wastewater effluents were assayed. This work suggested that luxCDABE -marked Acinetobacter bacterium DF4/PUTK2 can be used to bioassay the ecotoxicity of wastewater and effluent samples contaminated with heavy metals. Using this assay, it is possible to pre-select the more toxic samples for further chemical analysis and to discard wastewater samples with low or no inhibition because they are not toxic to the environment. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Ionic Basis for Action Potential Prolongation by Phenylephrine in Canine Epicardial MyocytesJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 1 2000RICHARD B. ROBINSON Ph.D. Phenylephrine Action on Repolarization. Introduction: In canine ventricle, ,-adrenergic agonists prolong action potential duration (APD) without any effect on the action potential notch, suggesting that, in this species, the effect on repolarization might he independent of inhibition of Ito. The present study investigated the action of the ,-adrenergic agonist phenylephrine on the action potential and the repolarizing currents Ito and IK in isolated canine epicardial myocytes. Methods and Results: Isolated cells from canine epicardial tissue, and Purkinje fibers, were studied with the whole cell, voltage clamp method. Phenylephrine 0.1 ,M increased APD by 13%± 4% at 90% repolarization without affecting the notch or amplitude. Under voltage clamp, concentrations of phenylephrine as high as 10 ,M had no effect on Itp in canine epicardial myocytes. However, Ito of isolated canine Purkinje myocytes was reduced to 69%± 7% of control by 1 ,M phenylephrine. Further studies in canine epicardial myocytes revealed an action of phenylephrine to inhibit Ik, and in particular IKs Using a voltage protocol that included a two-step repolarization to separate IKs and IKr tail components, the largely 1Ke, component was not significantly affected by 1 ,M phenylephrine, whereas the largely IKs component was reduced to 81%± 5% of control value. Conclusion: ,-Adrenergic prolongation of repolarization in canine epicardium does not result from inhibition of Ito. Rather, it appears that reduction of IKs contributes to the action of phenylephrine. The unresponsiveness of epicardial Ito is not a general characteristic of the canine heart, because Purkinje myocyte Ito was inhibited, suggesting regional differences in the molecular basis of lto, and/or a-adrenergic signaling in the canine heart. [source] Analysis of nuclear proteome in C57 mouse liver tissue by a nano-flow 2-D-LC,ESI-MS/MS approachJOURNAL OF SEPARATION SCIENCE, JSS, Issue 17 2006Jie Zhang Abstract The analysis of whole cell or tissue extracts is too complex for current protein identification technology and not suitable for the study of proteins with low copy levels. To concentrate and enrich low abundance proteins, organelle proteomics is a promising strategy. This approach can not only reduce the protein sample complexity but also provide information about protein location in cells, organs, or tissues under analysis. Nano-flow two-dimensional strong-cation exchange chromatography (SCX),RPLC,ESI-MS/MS is an ideal platform for analyzing organelle extracts because of its advantages of sample non-bias, low amounts of sample required, powerful separation capability, and high detection sensitivity. In this study, we apply nano-scale multidimensional protein identification technology to the analysis of C57 mouse liver nuclear proteins. Organelle isolation has been optimized to obtain highly pure nuclei. Evaluation of nucleus integrity and purity has been performed to demonstrate the effectiveness of the optimized isolation procedure. The extracted nuclear proteins were identified by five independent nano-flow on-line SCX,RPLC,ESI-MS/MS analyses to improve the proteome coverage. Finally, a total of 462 proteins were identified. Corresponding analyses of protein molecular mass and pI distribution and biological function categorization have been undertaken to further validate our identification strategy. [source] Quantitative evaluation of shunts in solar cells by lock-in thermographyPROGRESS IN PHOTOVOLTAICS: RESEARCH & APPLICATIONS, Issue 8 2003O. Breitenstein Abstract Infrared lock-in thermography allows to image shunts very sensitively in all kinds of solar cells and also to measure dark currents flowing in certain regions of the cell quantitatively. After a summary of the physical basis of lock-in thermography and its practical realization, four types of quantitative measurements are described: local I,V characteristics measured thermally up to a constant factor (LIVT); the quantitative measurement of the current through a local shunt; the evaluation of the influence of shunts on the efficiency of a cell as a function of the illumination intensity; and the mapping of the ideality factor n and the saturation current density J0 over the whole cell. The investigation of a typical multicrystalline solar cell shows that the shunts are predominantly responsible for deterioration of the low-light-level performance of the cell, and that variations of the injection current density related to crystal defects are predominantly determined by variation of J0 rather than of n. Copyright © 2003 John Wiley & Sons, Ltd. [source] Flow cytometry-assisted purification and proteomic analysis of the corticotropes dense-core secretory granulesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 18 2008Daniel J. Gauthier Abstract The field of organellar proteomics has emerged as an attempt to minimize the complexity of the proteomics data obtained from whole cell and tissue extracts while maximizing the resolution on the protein composition of a single subcellular compartment. Standard methods involve lengthy density-based gradient and/or immunoaffinity purification steps followed by extraction, 1-DE or 2-DE, gel staining, in-gel tryptic digestion, and protein identification by MS. In this paper, we present an alternate approach to purify subcellular organelles containing a fluorescent reporter molecule. The gel-free procedure involves fluorescence-assisted sorting of the secretory granules followed by gentle extraction in a buffer compatible with tryptic digestion and MS. Once the subcellular organelle labeled, this procedure can be done in a single day, requires no major modification to any instrumentation and can be readily adapted to the study of other organelles. When applied to corticotrope secretory granules, it led to a much enriched granular fraction from which numerous proteins could be identified through MS. [source] Proteomics of ischemia/reperfusion injury in rabbit myocardium reveals alterations to proteins of essential functional systemsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2005Melanie Y. White Abstract Brief periods of myocardial ischemia prior to timely reperfusion result in prolonged, yet reversible, contractile dysfunction of the myocardium, or "myocardial stunning". It has been hypothesized that the delayed recovery of contractile function in stunned myocardium reflects damage to one or a few key sarcomeric proteins. However, damage to such proteins does not explain observed physiological alterations to myocardial oxygen consumption and ATP requirements observed following myocardial stunning, and therefore the impact of alterations to additional functional groups is unresolved. We utilized two-dimensional gel electrophoresis and mass spectrometry to identify changes to the protein profiles in whole cell, cytosolic- and myofilament-enriched subcellular fractions from isolated, perfused rabbit hearts following 15 min or 60 min low-flow (1 mL/min) ischemia. Comparative gel analysis revealed 53 protein spot differences (> 1.5-fold difference in visible abundance) in reperfused myocardium. The majority of changes were observed to proteins from four functional groups: (i) the sarcomere and cytoskeleton, notably myosin light chain-2 and troponin C; (ii) redox regulation, in particular several components of the NADH ubiquinone oxidoreductase complex; (iii) energy metabolism, encompassing creatine kinase; and (iv) the stress response. Protein differences appeared to be the result of isoelectric point shifts most probably resulting from chemical modifications, and molecular mass shifts resulting from proteolytic or physical fragmentation. This is consistent with our hypothesis that the time course for the onset of injury associated with myocardial stunning is too brief to be mediated by large changes to gene/protein expression, but rather that more subtle, rapid and potentially transient changes are occurring to the proteome. The physical manifestation of stunned myocardium is therefore the likely result of the summed functional impairment resulting from these multiple changes, rather than a result of damage to a single key protein. [source] Enhancement of the innate immune system and disease-resistant activity in Cyprinus carpio by oral administration of ,-glucan and whole cell yeastAQUACULTURE RESEARCH, Issue 6 2010Ayyaru Gopalakannan Abstract The effects of dietary ,- (1,3) glucan and whole cell yeast (Sacharomyces uvarum) on the immune response and disease resistance to Aeromonas hydrophila were investigated in Cyprinus carpio. ,-(1,3) glucan was extracted from the yeast. Both ,-(1,3) glucan and whole yeast were incorporated into the diet at 1% level and fed to common carp C. carpio for a period of 60 days. Control and treated fish were exposed to A. hydrophila on the 30th and the 60th day of the experimental period. Dietary supplementation of glucan significantly increased the white blood cell count in fish on the 60th day (2.91±0.04 × 104), and the highest nuetrophil nitro blue tetrazolium (NBT) activity was also observed in glucan-fed fish (30th day). A consistent increase in neutrophil (NBT) activity was also observed in whole cell fed fish until the end of the experiment. Similarly, ,-(1,3) glucan and whole cell yeast enhanced the serum lysozyme activity from the 15th day onwards but higher activity was reported on the 30th day in glucan and the 60th day in whole cell yeast-fed fish. Suplementation of ,-(1,3) glucan protected the fish from A. hydrophila infection. Nearly 75,80% of the fish survived pathogen exposure (relative percentage survival). However, only 54,60% survival was observed in the whole cell-fed fish. ,-(1,3) glucan and whole cell yeast protect the fish from pathogens by enhancing the cellular and humoral immune response in C. carpio. [source] KMUP-1 activates BKCa channels in basilar artery myocytes via cyclic nucleotide-dependent protein kinasesBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2005Bin-Nan Wu This study investigated whether KMUP-1, a synthetic xanthine-based derivative, augments the delayed-rectifier potassium (KDR)- or large-conductance Ca2+ -activated potassium (BKCa) channel activity in rat basilar arteries through protein kinase-dependent and -independent mechanisms. Cerebral smooth muscle cells were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K+ - and Ca2+ channel activities. KMUP-1 (1 ,M) had no effect on the KDR current but dramatically enhanced BKCa channel activity. This increased BKCa current activity was abolished by charybdotoxin (100 nM) and iberiotoxin (100 nM). Like KMUP-1, the membrane-permeable analogs of cGMP (8-Br-cGMP) and cAMP (8-Br-cAMP) enhanced the BKCa current. BKCa current activation by KMUP-1 was markedly inhibited by a soluble guanylate cyclase inhibitor (ODQ 10 ,M), an adenylate cyclase inhibitor (SQ 22536 10 ,M), competitive antagonists of cGMP and cAMP (Rp-cGMP, 100 ,M and Rp-cAMP, 100 ,M), and cGMP- and cAMP-dependent protein kinase inhibitors (KT5823, 300 nM and KT5720, 300 nM). Voltage-dependent L-type Ca2+ current was significantly suppressed by KMUP-1 (1 ,M), and nearly abolished by a calcium channel blocker (nifedipine, 1 ,M). In conclusion, KMUP-1 stimulates BKCa currents by enhancing the activity of cGMP-dependent protein kinase, and in part this is due to increasing cAMP-dependent protein kinase. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and the relaxation of cerebral arteries. British Journal of Pharmacology (2005) 146, 862,871. doi:10.1038/sj.bjp.0706387 [source] Characterisation of the binding of [3H]-SB-674042, a novel nonpeptide antagonist, to the human orexin-1 receptorBRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2004Christopher J Langmead This study characterises the binding of a novel nonpeptide antagonist radioligand, [3H]SB-674042 (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone), to the human orexin-1 (OX1) receptor stably expressed in Chinese hamster ovary (CHO) cells in both a whole cell assay and in a cell membrane-based scintillation proximity assay (SPA) format. Specific binding of [3H]SB-674042 was saturable in both whole cell and membrane formats. Analyses suggested a single high-affinity site, with Kd values of 3.76±0.45 and 5.03±0.31 nM, and corresponding Bmax values of 30.8±1.8 and 34.4±2.0 pmol mg protein,1, in whole cell and membrane formats, respectively. Kinetic studies yielded similar Kd values. Competition studies in whole cells revealed that the native orexin peptides display a low affinity for the OX1 receptor, with orexin-A displaying a ,five-fold higher affinity than orexin-B (Ki values of 318±158 and 1516±597 nM, respectively). SB-334867, SB-408124 (1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) and SB-410220 (1-(5,8-difluoro-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) all displayed high affinity for the OX1 receptor in both whole cell (Ki values 99±18, 57±8.3 and 19±4.5 nM, respectively) and membrane (Ki values 38±3.6, 27±4.1 and 4.5±0.2 nM, respectively) formats. Calcium mobilisation studies showed that SB-334867, SB-408124 and SB-410220 are all functional antagonists of the OX1 receptor, with potencies in line with their affinities, as measured in the radioligand binding assays, and with approximately 50-fold selectivity over the orexin-2 receptor. These studies indicate that [3H]SB-674042 is a specific, high-affinity radioligand for the OX1 receptor. The availability of this radioligand will be a valuable tool with which to investigate the physiological functions of OX1 receptors. British Journal of Pharmacology (2004) 141, 340,346. doi:10.1038/sj.bjp.0705610 [source] Enhancement of the NAD(P)(H) Pool in Saccharomyces cerevisiaeENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2008A. Knepper Abstract Asymmetric biosyntheses allow for an efficient production of chiral building blocks. The application of whole cells as biocatalysts for asymmetric syntheses is advantageous because they already contain the essential coenzymes NAD(H) or NADP(H), which additionally can be regenerated in the cells. Unfortunately, reduced catalytic activity compared to the oxidoreductase activity is observed in many cases during whole-cell biotransformation. This may be caused by low intracellular coenzyme pool sizes and/or a decline in intracellular coenzyme concentrations. To enhance the intracellular coenzyme pool sizes, the effects of the precursor metabolites adenine and nicotinic acid on the intracellular accumulation of NAD(H) and NADP(H) were studied in Saccharomyces cerevisiae. Based on the results of simple batch experiments with different precursor additions, fed-batch processes for the production of yeast cells with enhanced NAD(H) or enhanced NADP(H) pool sizes were developed. Supplementation of the feed medium with 95,mM adenine and 9.5,mM nicotinic acid resulted in an increase of the intracellular NAD(H) concentration by a factor of 10 at the end of the fed-batch process compared to the reference process. The final NAD(H) concentration remains unchanged if the feed medium was solely supplemented with 95,mM adenine, but intracellular NADP(H) was increased by a factor of 4. The effects of NADP(H) pool sizes on the asymmetric reduction of ethyl-4-chloro acetoacetate (CAAE) to the corresponding (S)-4-chloro-3-hydroxybutanoate (S-CHBE) was evaluated with S.,cerevisiae,FasB,His6 as an example. An intracellular threshold concentration above 0.07,mM NADP(H) was sufficient to increase the biocatalytic S-CHBE productivity by 25,% compared to lower intracellular NADP(H) concentrations. [source] Bacterial and archaeal populations associated with freshwater ferromanganous micronodules and sedimentsENVIRONMENTAL MICROBIOLOGY, Issue 1 2001Lisa Y. Stein Biology is believed to play a large role in the cycling of iron and manganese in many freshwater environments, but specific microbial groups indigenous to these systems have not been well characterized. To investigate the populations of Bacteria and Archaea associated with metal-rich sediments from Green Bay, WI, we extracted nucleic acids and analysed the phylogenetic relationships of cloned 16S rRNA genes. Because nucleic acids have not been routinely extracted from metal-rich samples, we investigated the bias inherent in DNA extraction and gene amplification from pure MnO2 using defined populations of whole cells or naked DNA. From the sediments, we screened for manganese-oxidizing bacteria using indicator media and found three isolates that were capable of manganese oxidation. In the phylogenetic analysis of bacterial 16S rRNA gene clones, we found two groups related to known metal-oxidizing genera, Leptothrix of the ,-Proteobacteria and Hyphomicrobium of the ,-Proteobacteria, and a Fe(III)-reducing group related to the Magnetospirillum genus of the ,-Proteobacteria. Groups related to the metal-reducing ,-Proteobacteria constituted 22% of the gene clones. In addition, gene sequences from one group of methanogens and a group of Crenarchaeota, identified in the archaeal gene clone library, were related to those found previously in Lake Michigan sediments. [source] Differential inhibition in vivo of ammonia monooxygenase, soluble methane monooxygenase and membrane-associated methane monooxygenase by phenylacetyleneENVIRONMENTAL MICROBIOLOGY, Issue 5 2000Sonny Lontoh Phenylacetylene was investigated as a differential inhibitor of ammonia monooxygenase (AMO), soluble methane monooxygenase (sMMO) and membrane-associated or particulate methane monooxygenase (pMMO) in vivo. At phenylacetylene concentrations >,1 µM, whole-cell AMO activity in Nitrosomonas europaea was completely inhibited. Phenylacetylene concentrations above 100 µM inhibited more than 90% of sMMO activity in Methylococcus capsulatus Bath and Methylosinus trichosporium OB3b. In contrast, activity of pMMO in M. trichosporium OB3b, M. capsulatus Bath, Methylomicrobium album BG8, Methylobacter marinus A45 and Methylomonas strain MN was still measurable at phenylacetylene concentrations up to 1000 µM. AMO of Nitrosococcus oceanus has more sequence similarity to pMMO than to AMO of N. europaea. Correspondingly, AMO in N. oceanus was also measurable in the presence of 1000 µM phenylacetylene. Measurement of oxygen uptake indicated that phenylacetylene acted as a specific and mechanistic-based inhibitor of whole-cell sMMO activity; inactivation of sMMO was irreversible, time dependent, first order and required catalytic turnover. Corresponding measurement of oxygen uptake in whole cells of methanotrophs expressing pMMO showed that pMMO activity was inhibited by phenylacetylene, but only if methane was already being oxidized, and then only at much higher concentrations of phenylacetylene and at lower rates compared with sMMO. As phenylacetylene has a high solubility and low volatility, it may prove to be useful for monitoring methanotrophic and nitrifying activity as well as identifying the form of MMO predominantly expressed in situ. [source] Soluble LDL-R are formed by cell surface cleavage in response to phorbol estersFEBS JOURNAL, Issue 3 2004Michael J. Begg A 140-kDa soluble form of the low density lipoprotein (LDL) receptor has been isolated from the culture medium of HepG2 cells and a number of other cell types. It is produced from the 160-kDa mature LDL receptor by a proteolytic cleavage, which is stimulated in the presence of 4,-phorbol 12-myristate 13-acetate (PMA), leading to the release of a soluble fragment that constitutes the bulk of the extracellular domain of the LDL receptor. By labeling HepG2 cells with [35S]methionine and chasing in the presence of PMA, we demonstrated that up to 20% of LDL-receptors were released into the medium in a 2-h period. Simultaneously, the level of labeled cellular receptors was reduced by 30% in those cells treated with PMA compared to untreated cells, as was the total number of cell surface LDL-receptors assayed by the binding of 125I-labeled antibody to whole cells. To determine if endocytosis was required for cleavage, internalization-defective LDL-receptors were created by mutagenesis or deletion of the NPXY internalization signal, transfected into Chinese hamster ovary cells, and assayed for cleavage in the presence and absence of PMA. Cleavage was significantly greater in the case of the mutant receptors than for wild-type receptors, both in the absence and presence of PMA. Similar results were seen in human skin fibroblasts homozygous for each of the internalization-defective LDL receptor phenotypes. LDL receptor cleavage was inhibited by the hydoxamate-based inhibitor TAPI, indicating the resemblance of the LDL receptor cleavage mechanism to that of other surface released membrane proteins. [source] Pigments and proteins in green bacterial chlorosomes studied by matrix-assisted laser desorption ionization mass spectrometryFEBS JOURNAL, Issue 2 2000Søren Persson We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for mass determination of pigments and proteins in chlorosomes, the light-harvesting organelles from the photosynthetic green sulfur bacterium Chlorobium tepidum. By applying a small volume (1 µL) of a concentrated suspension of isolated chlorosomes directly to the target of the mass spectrometer we have been able to detect bacteriochlorophyll a and all the major homologs of bacteriochlorophyll c. The peak heights of the different bacteriochlorophyll c homologs in the MALDI spectra were proportional to peak areas obtained from HPLC analysis of the same sample. The same result was also obtained when whole cells of Chl. tepidum were applied to the target, indicating that MALDI-MS can provide a rapid method for obtaining a semiquantitative determination or finger-print of the bacteriochlorophyll homologs in a small amount of green bacterial cells. In addition to information on pigments, the MALDI spectra also contained peaks from chlorosome proteins. Thus we have been able with high precision to confirm the molecular masses of the chlorosome proteins CsmA and CsmE which have been previously determined by conventional biochemical and genetic methods, and demonstrate the presence of truncated versions of CsmA and CsmB. [source] Microbial desulfurization of gasoline by free whole-cells of Rhodococcus erythropolis XPFEMS MICROBIOLOGY LETTERS, Issue 2 2006Bo Yu Abstract Rhodococcus erythropolis XP could grow well with condensed thiophenes, mono-thiophenic compounds and mercaptans present in gasoline. Rhodococcus erythropolis XP was also capable of efficiently degrading the condensed thiophenes in resting cell as well as biphasic reactions in which n -octane served as a model oil phase. Free whole cells of R. erythropolis XP were adopted to desulfurize fluid catalytic cracking (FCC) and straight-run (SR) gasoline oils. About 30% of the sulfur content of FCC gasoline and 85% of sulfur in SR gasoline were reduced, respectively. Gas chromatography analysis with atomic emission detection also showed depletion of sulfur compounds in SR gasoline. Rhodococcus erythropolis XP could partly resist the toxicity of gasoline and had an application potential to biodesulfurization of gasoline. [source] Biocatalytic aldehyde reduction using tailor-made whole-cell catalysts: a novel synthesis of the aroma chemical cinnamyl alcohol,FLAVOUR AND FRAGRANCE JOURNAL, Issue 3 2007Francoise Chamouleau Abstract A biocatalytic method for the synthesis of the aroma chemical cinnamyl alcohol by means of a wholecell-catalysed reduction of cinnamyl aldehyde has been developed. As a biocatalyst, recombinant whole cells overexpressing an alcohol dehydrogenase from Lactobacillus kefir and a glucose dehydrogenase from Thermoplasma acidophilum have been used in combination with d- glucose as co-substrate. The reduction process proceeds with a conversion of 98%, even at a high substrate input of 166 g/l cinnamyl aldehyde, and gives a yield of 77% of the desired product, cinnamyl alcohol. In addition to high product concentrations, further advantages of this approach are the use of a low-cost whole-cell catalyst, the high purity of the product, as well as the fact that there is no need for the addition of external co-factor in the biotransformation step. Copyright © 2007 John Wiley & Sons, Ltd. [source] Facile Synthesis of Enantiopure 4-Substituted 2-Hydroxy-4- butyrolactones using a Robust Fusarium LactonaseADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 17 2009Bing Chen Abstract A facile chemo-enzymatic process has been developed for producing stereoisomers of 4-substituted 2-hydroxy-4-butyrolactones with good to excellent enantioselectivity. This process involves an easy separation of the diastereoisomers by column chromatography and efficient enzymatic resolution by whole cells of Escherichia coli JM109 expressing Fusarium proliferatum lactonase gene. This biocatalyst shows strong tolerance towards different substrate structures and at least three out four possible isomers could be obtained in excellent enantiomeric purity. Different substrate concentrations (10,mM,200,mM) were examined, giving a substrate to catalyst ratio of up to 26:1. This general and efficient enzymatic process provides access to stereoisomers of 4-substituted 2-hydroxy-4-butyrolactones readily and cost-effectively. The stereochemical assignments were conducted systematically based on NMR, X-ray diffraction and circular dichroism, leading to further understanding of the enzyme's stereoselectivity. [source] Biocatalytic Asymmetric Dihydroxylation of Conjugated Mono- and Poly-alkenes to Yield Enantiopure Cyclic cis -DiolsADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 7-8 2005Derek Abstract Dioxygenase-catalysed asymmetric dihydroxylation, of a series of conjugated monoalkenes and polyenes, was found to yield the corresponding monols and 1,2-dihydrodiols. The diol metabolites were obtained from monosubstituted, gem -disubstituted, cis -disubstituted, and trisubstituted alkene substrates, using whole cells of Pseudomonas putida strains containing toluene and naphthalene dioxygenases. Dioxygenase selection and alkene type were established as important factors, in the preference for dioxygenase-catalysed 1,2-dihydroxylation of conjugated alkene or arene groups, and monohydroxylation at benzylic or allylic centres. Competition from allylic hydroxylation of methyl groups was observed only when naphthalene dioxygenase was used as biocatalyst. The structures, enantiomeric excess values and absolute configurations of the bioproducts, were determined by a combination of stereochemical correlation, spectroscopy (NMR and CD) and X-ray diffraction methods. cis -1,2-Diol metabolites from arenes, cyclic alkenes and dienes were generally observed to be enantiopure (>98% ee), while 1,2-diols from acyclic alkenes had lower enantiomeric excess values (<88% ee). The enantiopure cis -diol metabolite of a gem -disubstituted fulvene was used as precursor in a new chemoenzymatic route to a novel C2 -symmetrical ketone. [source] | |||||||||||||||||||||||||||||||||||||