European interlaboratory comparison of breath 13CO2 analysis.
The BIOMED I programme Stable Isotopes in Gastroenterology and Nutrition (SIGN) has focused upon evaluation and standardisation of stable isotope breath tests using 13C labelled substrates. The programme dealt with comparison of 13C substrates, test meals, test conditions, analysis techniques, and calculation procedures. Analytical techniques applied for 13CO2 analysis were evaluated by taking an inventory of instrumentation, calibration protocols, and analysis procedures. Two ring tests were initiated measuring 13C abundances of carbonate materials. Evaluating the data it was found that seven different models of isotope ratio mass spectrometers (IRMS) were used by the participants applying both the dual inlet system and the continuous flow configuration. Eight different brands of certified 13C reference materials were used with a 13C abundance varying from delta 13CPDB -37.2 to +2.0/1000. CO2 was liberated from certified material by three techniques and different working standards were used varying from -47.4 to +0.4/1000 in their delta 13CPDB value. The standard deviations (SDs) found for all measurements by all participants were 0.25/1000 and 0.50/1000 for two carbonates used in the ring tests. The individual variation for the single participants varied from 0.02 /1000 (dual inlet system) to 0.14/1000 (continuous flow system). The measurement of the difference between two carbonates showed a SD of 0.33/1000 calculated for all participants. Internal precision of IRMS as indicated by the specifications of the different instrument suppliers is < 0.3/1000 for continuous flow systems. In this respect it can be concluded that all participants are working well within the instrument specifications even including sample preparation. Increased overall interlaboratory variation is therefore likely to be due to non-instrumental conditions. It is possible that consistent differences in sample handling leading to isotope fractionation are the causes for interlaboratory variation. Breath analysis does not require sample preparation. As such, interlaboratory variation will be less than observed for the carbonate samples and within the range indicated as internal precision for continuous flow instruments. From this it is concluded that pure analytical interlaboratory variation is acceptable despite the many differences in instrumentation and analytical protocols. Coordinated metabolic studies appear possible, in which different European laboratories perform 13CO2 analysis. Evaluation of compatibility of the analytical systems remains advisable, however. (+info)
Direct EPR detection of the carbonate radical anion produced from peroxynitrite and carbon dioxide.
The biological effects of peroxynitrite have been recently considered to be largely dependent on its reaction with carbon dioxide, which is present in high concentrations in intra- and extracellular compartments. Peroxynitrite anion (ONOO-) reacts rapidly with carbon dioxide, forming an adduct, nitrosoperoxocarboxylate (ONOOCO2-), whose decomposition has been proposed to produce reactive intermediates such as the carbonate radical (CO-3). Here, by the use of rapid mixing continuous flow electron paramagnetic resonance (EPR), we directly detected the carbonate radical in flow mixtures of peroxynitrite with bicarbonate-carbon dioxide over the pH range of 6-9. The radical was unambiguously identified by its EPR parameters (g = 2.0113; line width = 5.5 G) and by experiments with bicarbonate labeled with 13C. In this case, the singlet EPR signal obtained with 12C bicarbonate splits into the expected doublet because of 13C (a(13C)= 11.7 G). The singlet spectrum of the unlabeled radical was invariant between pH 6 and 9, confirming that in this pH range the detected radical is the carbonate radical anion (CO-3). Importantly, in addition to contributing to the understanding of nitrosoperoxocarboxylate decomposition pathways, this is the first report unambiguously demonstrating the formation of the carbonate radical anion at physiological pHs by direct EPR spectroscopy. (+info)
Islet cell membrane antigens activate diabetogenic CD4+ T-cells in the BB/Wor rat.
Type 1 diabetes is a major histocompatibility complex (MHC) class II-associated autoimmune disease mediated by beta-cell-specific T-cells and characterized by circulating autoantibodies to beta-cell molecules. In the BB/Wor diabetes-prone (DP) rat, type 1 diabetes develops spontaneously with an incidence of >90%. BB diabetes can be adoptively transferred to naive syngeneic or MHC class II-compatible rats with islet cell-activated T-cell lines derived from diabetic BB/Wor rats. However, the target beta-cell autoantigen(s) in BB diabetes has not yet been defined. BB rat T-cell lines activated in vitro with antigen-presenting cells (APC) and BB islet cell crude membranes (CM), but not islet cell cytosol, adoptively transfer diabetes into young DP recipients. To determine if the target autoantigen is an integral or peripheral membrane protein, islet cell CM were treated with 0.5 mol/l KCl or 0.2 mol/l Na2CO3 (pH 11). Both treatments selectively extract peripheral proteins from the cell membrane without affecting the disposition of integral (transmembrane) proteins. T-cell lines activated in vitro with APC and 0.5 mol/l KCl, or pH 11 (0.2 mol/l Na2CO3)-treated islet cell CM, transferred diabetes into young DP rats. Conversely, T-cell lines activated in vitro with APC and the supernatant of 0.5 mol/l KCl-treated CM (containing extracted peripheral proteins), did not adoptively transfer diabetes. After activation in vitro with islet cell membrane antigens, the diabetes-inducing cell lines were comprised of both CD4+ CD8- T-cells and 10-30% B-cells. We conclude that a major CD4+ T-cell target autoantigen in BB diabetes is a membrane-associated beta-cell molecule with the characteristics of an integral beta-cell membrane protein. The identification of this MHC class II-restricted beta-cell target molecule will allow the design of antigen-specific intervention protocols to prevent the onset of type 1 diabetes in genetically susceptible individuals. (+info)
A 60 kDa plasma membrane protein changes its localization to autophagosome and autolysosome membranes during induction of autophagy in rat hepatoma cell line, H-4-II-E cells.
We previously reported the preparation and characterization of an antibody against membrane fraction of autolysosomes from rat liver (J. Histochem. Cytochem. 38, 1571-1581, 1990). Immunoblot analyses of total membrane fraction of a rat hepatoma cell line, H-4-II-E cells by this antibody suggested that H-4-II-E cells expressed several autolysosomal proteins, including a protein with apparent molecular weight of 60 kDa. It was suggested that this 60 kDa protein was a peripheral membrane protein, because it was eluted from the membrane by sodium carbonate treatment. We prepared an antibody against this 60 kDa protein by affinity purification method, and examined its behavior during induction of autophagy. Autophagy was induced by transferring the cells from Dulbecco's modified Eagle medium (DMEM) containing 12% fetal calf serum into Hanks' balance salt solution. In DMEM, the 60 kDa protein showed diffused immunofluorescence pattern, and immunoelectron microscopy suggested that this protein was located on the extracellular side of the plasma membrane. After inducing autophagy, the immunofluorescence configuration of the 60 kDa protein changed from the diffused pattern to a granulous one. Immunoelectron microscopy showed that the 60 kDa protein was localized on the luminal side of the limiting membrane of autolysosomes and endosomes. In the presence of bafilomycin A1 which prevents fusion between autophagosomes and lysosomes, the 60 kDa protein was localized on the limiting membrane of the autophagosomes and endosomes. These results suggest that the 60 kDa protein is transported from the plasma membrane to the autophagosome membrane through the endosomes. (+info)
Degradation of polycarbonate by a polyester-degrading strain, Amycolatopsis sp. strain HT-6.
Amycolatopsis sp. strain HT-6, a poly(tetramethylene succinate) (PTMS)-degrading actinomycete, was observed to degrade poly(tetramethylene carbonate) (PTMC). In a liquid culture with 150 mg of PTMC film, 59% degradation was achieved, but with a low yield of cell growth. On the other hand, PTMS copolymerized with a small amount of PTMC, forming a copolyester carbonate (PEC) that was completely and rapidly degraded with a high yield of cell growth. (+info)
T-1095, an inhibitor of renal Na+-glucose cotransporters, may provide a novel approach to treating diabetes.
T-1095A and T-1095 are synthetic agents derived from phlorizin, a specific inhibitor of Na+-glucose cotransporters (SGLTs). Unlike phlorizin, T-1095 is absorbed into the circulation via oral administration, is metabolized to the active form, T-1095A, and suppresses the activity of SGLTs in the kidney. Orally administered T-1095 increases urinary glucose excretion in diabetic animals, thereby decreasing blood glucose levels. Indeed, the postprandial hyperglycemia after a meal load was shown to be suppressed by this compound in streptozotocin (STZ)-induced diabetic rats. With long-term T-1095 treatment, both blood glucose and HbA1c levels were reduced in STZ-induced diabetic rats and yellow KK mice. In addition, there was amelioration of abnormal carbohydrate metabolism, i.e., hyperinsulinemia and hypertriglyceridemia, and of the development of microalbuminuria, in yellow KK mice. Thus, T-1095 may be a useful antidiabetic drug, providing a novel therapeutic approach for diabetes. (+info)
The age of the carbonates in martian meteorite ALH84001.
The age of secondary carbonate mineralization in the martian meteorite ALH84001 was determined to be 3.90 +/- 0.04 billion years by rubidium-strontium (Rb-Sr) dating and 4.04 +/- 0.10 billion years by lead-lead (Pb-Pb) dating. The Rb-Sr and Pb-Pb isochrons are defined by leachates of a mixture of high-graded carbonate (visually estimated as approximately 5 percent), whitlockite (trace), and orthopyroxene (approximately 95 percent). The carbonate formation age is contemporaneous with a period in martian history when the surface is thought to have had flowing water, but also was undergoing heavy bombardment by meteorites. Therefore, this age does not distinguish between aqueous and impact origins for the carbonates. (+info)
Antibodies against pex14p block ATP-independent binding of matrix proteins to peroxisomes in vitro.
The membrane protein Pex14p is a key component of the protein import machinery of peroxisomes. Antibodies raised against human Pex14p recognise a 66 kDa protein in sunflower glyoxysomes (HaPex14p) and immunoprecipitate in vitro-translated Arabidopsis Pex14p (AtPex14p). These antibodies inhibit the ATP-independent binding to sunflower peroxisome membranes of peroxisome targeting signal type (PTS) 1- and PTS2-targeted matrix proteins, but not an integral membrane protein. These results suggest that Pex14p functions before the ATP-dependent step of peroxisome assembly. (+info)