Single cell studies of enzymatic hydrolysis of a tetramethylrhodamine labeled triglucoside in yeast.
Several hundred molecules of enzyme reaction products were detected in a single spheroplast from yeast cells incubated with a tetramethylrhodamine (TMR) labeled triglucoside, alpha-d-Glc(1-->2)alpha-d-Glc(1-->3)alpha-d-Glc-O(CH2)8CONHCH2- CH2NH- COTMR. Product detection was accomplished using capillary electrophoresis and laser induced fluorescence following the introduction of a single spheroplast into the separation capillary. The in vivo enzymatic hydrolysis of the TMR-trisaccharide involves at least two enzymes, limited by processing alpha-glucosidase I, producing TMR-disaccharide, TMR-monosaccharide, and the free TMR-linking arm. Hydrolysis was reduced by preincubation of the cells with the processing enzyme inhibitor castanospermine. Confocal laser scanning microscopy studies confirmed the uptake and internalization of fluorescent substrate. This single cell analysis methodology can be applied for the in vivo assay of any enzyme with a fluorescent substrate. (+info)
Ups and downs of protein crystallization: studies of protein crystals by high-performance capillary electrophoresis.
High-performance capillary electrophoresis is a high-technology micro-separation method. Short run time, full automation and minute amounts of sample make it a very attractive technique. In this report we describe studies of protein crystals by capillary electrophoresis. We show how high-performance capillary electrophoresis can be used effectively for rapid evaluation and examination of the protein solution used for crystallization, the protein crystals (solubilized) and surrounding mother liquor. With coated capillaries, the runs were reproducible and disturbing effects, such as electroendosmosis and interaction of the proteins with the capillary wall, were suppressed efficiently. We recommend this new technique as a powerful and routine companion to protein crystallography. (+info)
Glucocorticoid enhances interleukin-1-induced pressor response in freely moving rats through its effect on nitric oxide release.
We investigated whether changes in nitric oxide (NO) release might be responsible for the modulation by glucocorticoids of the pressor response to i.p. injection of interleukin-1beta (IL-1beta) in freely moving rats. In such rats, IL-1beta (10 microgram/kg) induced a biphasic pressor response, with a rise in the plasma concentration of NOx (NO2(-) and NO3(-): metabolites of NO) during the second phase. Systemic pretreatment with an exogenous glucocorticoid, dexamethasone (0.5 mg/kg), enhanced the second phase of the pressor response and completely suppressed the increase in plasma NOx. Treatment with Nomega-nitro-L-arginine methyl ester (L-NAME, a nonspecific NO synthase inhibitor), enhanced the pressor response while attenuating the increase in plasma NOx. After bilateral adrenalectomy, IL-1beta induced a smaller pressor response, but a larger increase in plasma NOx; dexamethasone reversed these changes. Our results suggest that endogenous NO moderates the pressor response to IL-1beta in freely moving rats, and that glucocorticoids enhance the IL-1beta-induced pressor response at least in part by reducing endogenous NO release. (+info)
Characterization of mannooligosaccharide caps in mycobacterial lipoarabinomannan by capillary electrophoresis/electrospray mass spectrometry.
A new analytical approach based on capillary electrophoresis-electrospray mass spectrometry (CE/ESI-MS) has provided new insight into the characterization of mannooligosaccharide caps from lipoarabinomannans (LAMs), which are key molecules in the immunopathogenesis of tuberculosis. This analytical approach requires oligosaccharide labeling with the fluorophore 1-aminopyrene-3,6,8-trisulfonate (APTS) by reductive amination at the reducing termini. Optimization of the separation and ionization conditions, such as the choice of capillary electrophoresis (CE) electrolyte buffers, is presented and discussed. Anionic separation of the mono and oligosaccharide APTS derivatives was finally achieved with aqueous triethylammonium formate buffer. It was found that in contrast to the triethylammonium phosphate buffer, the triethylammonium formate buffer was appropriate for CE/ESI-MS coupling analysis of APTS-carbohydrate derivatives. In this case, negative ESI-mass spectra of APTS-carbohydrate adducts showed mainly (M-2H)2-pseudomolecular ions and some sequence fragment ions allowing their non-ambiguous structural characterization at the picomolar level. This analytical approach was successfully applied to more complex mixtures of carbohydrates released by mild acid hydrolysis of the lipoarabinomannans from Mycobacterium bovis BCG. The APTS-mannooligosaccharide cap adducts were separated by CE and their structural characterization achieved by CE/ESI-MS analyses. Mannooligosaccharide caps were routinely analyzed by capillary electrophoresis-laser induced fluorescence (CE-LIF) from 50 fmol of lipoarabinomannans with mannosyl capping (ManLAMs) but sensitivity was about 50 times lower using ESI-MS detection. (+info)
Insect immunity. Isolation from the lepidopteran Heliothis virescens of a novel insect defensin with potent antifungal activity.
Lepidoptera have been reported to produce several antibacterial peptides in response to septic injury. However, in marked contrast to other insect groups, no inducible antifungal molecules had been described so far in this insect order. Surprisingly, also cysteine-rich antimicrobial peptides, which predominate in the antimicrobial defense of other insects, had not been discovered in Lepidoptera. Here we report the isolation from the hemolymph of immune induced larvae of the lepidopteran Heliothis virescens of a cysteine-rich molecule with exclusive antifungal activity. We have fully characterized this antifungal molecule, which has significant homology with the insect defensins, a large family of antibacterial peptides directed against Gram-positive strains. Interestingly, the novel peptide shows also similarities with the antifungal peptide drosomycin from Drosophila. Thus, Lepidoptera appear to have built their humoral immune response against bacteria on cecropins and attacins. In addition, we report that Lepidoptera have conferred antifungal properties to the well conserved structure of antibacterial insect defensins through amino acid replacements. (+info)
Undercarboxylation of recombinant prothrombin revealed by analysis of gamma-carboxyglutamic acid using capillary electrophoresis and laser-induced fluorescence.
The gamma-carboxyglutamic acid (Gla) content of several variants of human prothrombin has been measured by using capillary electrophoresis and laser-induced fluorescence (CE-LIF). Both plasma-derived prothrombin and recombinant prothrombin contain ten residues of Gla per molecule of protein. In contrast, a variant of human prothrombin (containing the second kringle domain of bovine prothrombin) was separated into two populations that differed in their Gla content. Direct measurement of the Gla content showed an association with the presence or absence of the calcium-dependent conformational change that is required for prothombinase function. Thus, the CE-LIF assay is useful in determining the carboxylation status of recombinant proteins. (+info)
Fast and simple purification of chemically modified hammerhead ribozymes using a lipophilic capture tag.
A new type of 5'-lipophilic capture tag is described, enabling the facile reverse phase HPLC purification of chemically modified hammerhead ribozymes (oligozymes) whilst still carrying the 2'-O-tert.-butyldimethylsilyl protection of the essential riboses. In its most convenient form, the capture tag consists of a simple diol, such as hexan-1,6-diol, which at one end is attached via a silyl residue to a highly lipophilic entity such as tocopherol (vitamin E) or cholesterol, and the other end is functionalized as a phosphoramidite. This lipophilic capture tag is added as the last residue in the solid-phase synthesis of chemically modified hammerhead ribozymes. Cleavage from the support and release of all protecting groups except for the silyl groups is achieved with ethanolamine/ethanol. The crude product is then loaded directly on to a reverse phase HPLC column. Separation of failure peaks from full length product is achieved easily using a short run time. The retarded product peak is collected, lyophilized, desilylated in the normal way and then desalted. This method removes the lipophilic capture tag yet leaves behind the hexanediol entity which helps protect the compound against degradation by 5'-exonucleases. The purity of the product as judged by analytical anion-exchange HPLC and capillary gel electrophoresis is generally better than 95% full-length, and yields of 2-4 mg from a 1 micromol scale synthesis are routine. In addition, the method can be readily scaled up, an important feature for the development of such chemically modified ribozymes as potential therapeutics. (+info)
An improved capillary electrophoresis method for measuring tissue metabolites associated with cellular energy state.
An improved method for the measurement of tissue metabolites associated with cellular energetic state by capillary electrophoresis is described. This method allows 17 compounds present in a mixture of standards to be determined simultaneously within 43 min with good reproducibility. ATP, ADP, AMP, UTP, IMP, inosine, hypoxanthine, creatine, phosphocreatine, UDP-galactose, NAD and NADH were detected in samples of either rat heart tissue or rat neonatal cardiomyocytes. This method can detect compounds at concentrations of 5 microm in samples. Recoveries for ATP and phosphocreatine added to cardiomyocyte samples were 99.4 +/- 2.1% and 103.1 +/- 3.3%, respectively (mean +/- SEM, n = 3). Our method has been comprehensively validated and is capable of measuring a wider range of tissue metabolites important in assessing cellular energy status than existing methods. (+info)