Transient-phase studies on the arginine kinase reaction.

1. The initial formation of arginine phosphate by arginine kinase was studied in the time range 2.8--50 ms by the quenched-flow method. 2. A transient burst phase of product formation was obtained, the amplitude of which was temperature-dependent. At 35 degrees C it was 0.64 mol arginine phosphate/mol arginine kinase and at 12 degrees C, 0.25 mol/mol. 3. These results show that for the reaction pathway of arginine kinase the rate-limiting step follows the formation of arginine phosphate on the enzyme. This is in contrast to the creatine kinase reaction where no transient phase was observed [Engelborghs, Y., Marsh, A. & Gutfreund, H. (1975) Biochem. J. 151, 47--50]. 4. The rate-limiting step on the arginine kinase reaction pathway is only slightly affected by temperature: the change in Kcat with temperature is due to a change of an equilibrium constant pertaining to at least two previous steps.     

Differential reactivity of 9-NH2-ellipticine on apurinic and apyrimidinic sites in circular DNA

Endonucleases for apurinic sites as well as chemical compounds reacting with aldehydes do not generally differentiate between apurinic and apyrimidinic sites. We have studied the effect of the apurinic site reagent, 9-NH2-ellipticine, on apyrimidinic sites enzymatically generated on PBR322 DNA and compared it to its' action on apurinic PM2 and PBR322 DNAs. In conditions where this compound induces breakage of apurinic sites, it does not display any action on apyrimidinic sites.     

Multiple coupling of neurohormone receptors with cyclic AMP and inositol phosphate production in anterior pituitary cells

Regulation of adenohypophyseal hormone secretions has been shown to involve cyclic AMP production, modulation of phosphatidyl inositol diphosphate breakdown and Ca2+ mobilization. Various neurohormone receptors are positively or negatively coupled to adenylate cyclase activity in anterior pituitary cells. The effects of these neurohormones on adenylate cyclase activity are consistent with the effect on hormone secretions, suggesting that modulation of the enzyme activity is actually involved in the regulation of adenohypophyseal secretions. Thus DA inhibits, whereas VIP stimulates adenylate cyclase activity of the same cell type, which, according to the effect of these neurohormones on prolactin secretion, appear to be lactotrophs. On the other hand, SRIF inhibits, whereas GRF stimulates the adenylate cyclase activity of another cell type, namely somatotrophs, whereas CRF appears to act on a third cell type, corticotrophs. Peripheral hormones have been shown to modulate the sensitivity of anterior pituitary cells to these neurohormones. Estradiol long-term treatment has an anti-dopaminergic effect on prolactin secretion. The steroid also suppresses the dopamine inhibition of adenylate cyclase. This effect appears selective to the DA inhibition, since AII inhibition of the enzyme is only partially reduced, whereas the somatostatin inhibition is markedly increased. Peripheral hormones seem to affect the sensitivity of adenohypophyseal cells not only by modulating the number of receptors for a given neurohormone but also by interfering with the coupling mechanisms of these receptors. AII and DA inhibit the adenylate cyclase activity of lactotroph cells. The prolactin stimulation induced by angiotensin is not consistent with the effect of the peptide on adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein band 3 phosphotyrosyl phosphatase. Purification and characterization

A phosphotyrosylprotein phosphatase has been purified from human red cell cytosol by successive DEAE cellulose, phosphocellulose and Red Procion-H3B-Sepharose chromatography. Overall purification was about 9000 with a yield of 30%. The enzyme was more than 95% pure as judged by SDS polyacrylamide gel. Its molecular weight was 17,000 and maximum activity was observed at pH 5.5. It was active towards both the phosphorylated tyrosine on the cytosolic fragment of the red cell protein band 3 and para-nitrophenyl phosphate. However the effects of ligands differ for the two substrates.     

Bafoudiosbulbins F and G, further clerodane diterpenoids from Dioscorea bulbifera L. var sativa and revised structure of Bafoudiosbulbin B

From the bulbils of Dioscorea bulbifera L. var sativa, two clerodane diterpenoids, Bafoudiosbulbins F (1) and G (2), together with five known compounds: Bafoudiosbulbins A-C, 3,5,4'-trihydroxy-3'-methoxybibenzyl, and kaempferol were isolated. Their structures were established by spectroscopic techniques, including (1)H, (13)C NMR, NOESY, ROESY, COSY, TOCSY, HSQC, and HMBC. The relative stereochemistry of compounds 1 and 2 was assigned on the basis of X-ray crystallographic diffraction analysis. Furthermore, the structure of Bafoudiosbulbin B was revised using extensive 2D NMR techniques as well as chemical transformation.     

Multiple strategies to improve sensitivity,speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection

Background  

In the past decades the rapid growth of molecular diagnostics (based on either traditional PCR or isothermal amplification technologies) meet the demand for fast and accurate testing. Although isothermal amplification technologies have the advantages of low cost requirements for instruments, the further improvement on sensitivity, speed and robustness is a prerequisite for the applications in rapid pathogen detection, especially at point-of-care diagnostics. Here, we describe and explore several strategies to improve one of the isothermal technologies, helicase-dependent amplification (HDA).     

Pharmacological rescue of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) detected by use of a novel fluorescence platform

Numerous human diseases arise because of defects in protein folding, leading to their degradation in the endoplasmic reticulum. Among them is cystic fibrosis (CF), caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR ), an epithelial anion channel. The most common mutation, F508del, disrupts CFTR folding, which blocks its trafficking to the plasma membrane. We developed a fluorescence detection platform using fluorogen-activating proteins (FAPs) to directly detect FAP-CFTR trafficking to the cell surface using a cell-impermeant probe. By using this approach, we determined the efficacy of new corrector compounds, both alone and in combination, to rescue F508del-CFTR to the plasma membrane. Combinations of correctors produced additive or synergistic effects, improving the density of mutant CFTR at the cell surface up to ninefold over a single-compound treatment. The results correlated closely with assays of stimulated anion transport performed in polarized human bronchial epithelia that endogenously express F508del-CFTR. These findings indicate that the FAP-tagged constructs faithfully report mutant CFTR correction activity and that this approach should be useful as a screening assay in diseases that impair protein trafficking to the cell surface.