Kinetic study on starch hydrolysis using a glucose/maltose sensing system

Summary A dual-enzyme electrode flow injection system that can simultaneously determine glucose and maltose is used for an on-line study of starch hydrolyses catalysed by amylases. With the working system, determinations can be made every 2 minutes. A 10 L sample size with recycled back-flow minimises any loss of the reaction medium. The production, growth and decay of glucose and maltose concentrations during starch hydrolysis under various enzymatic conditions can thus be closely monitored, making it useful for the study of the catalytic kinetics of amylases and in screening and analysing enzyme systems.     

N2O Evolution by Green Algae

Evidence is presented here that axenic cultures of Chlorella, Scenedesmus, Coelastrum, and Chlorococcum spp. evolve N₂O when grown on NO₂⁻, showing that the Chlorophyceae are a source of N₂O in aquatic systems.     

Effect of pyridine compounds on ammonia oxidation by autotrophic nitrifying bacteria andMethylosinus trichosporium OB3b

Ammonia oxidation, as measured by nitrite production, was inhibited by 2-chloro-6-trichloromethyl-pyridine (nitrapyrin, N-serve) in the methane-oxidizing bacterium,Methylosinus trichosporium OB3b, and the autotrophic nitrifying organisms,Nitrosococcus oceanus andNitrosomonas marina. 6-Chloropicolinic acid, a hydrolysis product of nitrapyrin, was ineffective as an inhibitor of ammonia oxidation by either the methanotroph or the autotrophs. Picolinic acid (2-carboxy-pyridine), in contrast, inhibited nitrification by the methane-oxidizing bacterium but not by the autotrophic cultures. Picolinic acid may provide a means for differentiating ammonia oxidation attributable to methanotrophs from that resulting from autotrophs in environmental studies.     

Variations in short term products of inorganic carbon fixation in exponential and stationary phase cultures of Aphanocapsa 6308

Aphanocapsa 6308 metabolizes both NaHCO₃ and Na₂CO₃. The short term incorporation (5-s) metabolic pattern and the patterns of incorporation of bicarbonate for exponential versus stationary phase cultures differ, however. Cells were equilibrated for 10 min in air and distilled water prior to injection of either NaH¹⁴CO₃ at pH 8.0, or Na₂ ¹⁴CO₃ at pH 11.0. Hot ethanol extracts were analyzed via paper chromatography and autoradiography for products of CO₂ fixation. At 5 s, malate (51.5%) predominates slightly as a primary bicarbonate fixation product over 3-phosphoglycerate (40.3%); 3-phosphoglycerate is the primary product of carbonate fixation. At 60 s, the carbonate and bicarbonate labelling patterns are similar. Cells in stationary phase fix in 5 s a greater proportion of bicarbonate into malate (36% vs. 14% for 3-phosphoglycerate) than do cells in exponential growth. Likewise, 60 s incorporations show a large amount of bicarbonate fixed into aspartate (30.9%) in stationary phase cells over that of exponential phase (11.6%). These data suggest an operative C₄ pathway for purposes not related to carbohydrate synthesis but rather as compensation for the incomplete tricarboxylic acid cycle in cyanobacteria. The enhancement of both aspartate fixation and CO₂ fixation into citrulline in stationary phase correlates with an increase in cyanophycin granule production which requires both aspartate and arginine.Nonstandard Abbreviations 3-PGA 3-phosphoglyceric acid - TCA tricarboxylic acid     

Lipid-lipid and lipid-protein interactions in chromaffin granule membranes: A spin label ESR study

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This ‘stiffening’ effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35°C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This corresponds to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10°C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.     

Leishmania mexicana: Immunology and histopathology in C3H mice

C3H mice were infected subcutaneously with 10⁵ promastigotes of Leishmania mexicana and subsequent lesions were examined at 3, 5, and 8 months. All animals developed persistent nonulcerating nodules of variable size which did not metastasize. The nodules contained amastigotes with a mononuclear infiltrate of histiocytes, lymphocytes, and plasma cells, but without formation of tuberculoid-type granulomas. Neutrophils and eosinophils were also encountered in some cases. Specific antileishmanial antibodies and delayed-type hypersensitivity to leishmanial antigen were present at 3, 5, and 8 months postinfection. L. mexicana infection in C3H mice differs from classic self-healing cutaneous leishmaniasis by the pesistence of nonhealing, nonulcerating, nonmetastasizing lesions, despite evidence of cellular and humoral immunity.     

Mutation in continuous cultures of Schizosaccharomyces Pombe. I. Dependence of the kinetics of mutation accumulation upon the growth-limiting nutrilite

The rate of spontaneous mutation to resistance to the 12,13-epoxytrichothecene trichodermin has been determined under different growth limiting conditions in continuous cultures of the microbial eukaryote Schizosaccharomyces pombe. In agreement with data obtained in bacterial systems by previous workers the kinetics observed for the accumulation of mutations is found to be dependent upon the nutrient used to limit the growth of the population. Under conditions of glucose-limitation mutation accumulation is directly proportional to the rate of cell division, while under histidine-limitation it is proportional to chronological time. Various possible explanations for these observations are discussed.     

Transforming growth factor-β inhibition of interleukin-1 activity involves down-regulation of interleukin-1 receptors on chondrocytes

Interleukin-1 (IL-1) plays an important role in cartilage destruction associated with inflammatory and degenerative arthritis because of its ability to induce matrix degrading enzymes. Previously, we have shown that the IL-1-induced chondrocyte protease activity was inhibited by transforming growth factor-β (TGF-β). In this paper, we show that TGF-β inhibits the IL-1-induced synthesis of collagenase and stromelysin by reducing the steady-state mRNA levels in rabbit articular chondrocytes. We further demonstrate that TGF-β-treated chondrocytes show reduced ¹²⁵I-IL-1 binding that returns to a normal level when TGF-β is removed from the culture medium. The inhibitory effect of TGF-β is observed for both naturally occurring as well as fibroblast growth factor (FGF)-inducible binding sites (receptors). Scatchard analysis of receptor—ligand interactions demonstrate that the reduced binding is due to a reduction in the number of receptors for IL-1 and is not due to changes in affinity. Affinity cross-linking studies suggest that control chondrocytes contain two major cross-linked bands of M_r =116 and 80 kDa and a minor band of M_r =100 kDa. FGF-treated cells show enhanced levels of all the bands, plus an additional 200-kDa band. TGF-β treatment of chondrocytes results in the reduction of all of these bands in both control as well as FGF-induced cells. These observations suggest that the ability of TGF-β to down-regulate the IL-1 receptor may be a mechanism by which it exerts its effects in antagonizing the IL-1 activity on chondrocytes.     

Identification of an epithelial protein related to the desmosome and intermediate filament network

Using a mAb, referred to as 08L, we have identified a protein, of M(r) approximately 140,000, associated with desmosomes of epithelial cells. The 08L antibody stained the intracellular side of lateral cell margins of monolayer epithelial cells but did not stain cell margins free of cell contact. Immunoelectron microscopy revealed that the 08L antigen was localized to the cytosolic surface of the desmosomal plaque near points of intermediate filament convergence with apparently little staining of the desmosomal plaque proper. Western blots revealed the 08L antigen to be a protein, of M(r) approximately 140,000, found in the Triton-X 100 insoluble pellet. High salt-containing buffers extracted the 08L antigen from the insoluble material. Examination of the assembly of 08L to the desmosome complex, in cells grown in low confluent culture or in calcium-switch assays, by double immunofluorescence with 08L and anti-desmoplakin antibody, revealed that 08L was recruited to morphologically identifiable desmosomes. 08L antigen may exist in a cytosolic pool prior to assembly to the cell surface. The solubility of 08L in low calcium and normal calcium conditions, however, was similar. 08L association to the desmosome was correlated with increased organization of the intermediate filament network. We suggest that the 08L antigen may be involved in the organization and stabilization of the desmosome-IF complexes of epithelia.