Mechanism of T5-induced DNA polymerase. II. Characterization of the dead-end complex.

We have shown that bacteriophage T5-induced DNA polymerase replicates short primer-templates (400 to 600 nucleotides long) at a rapid rate initially, followed by a slower rate sustained for much longer periods (Das, S. K., and Fujimura, R. K. (1977) J. Biol. Chem. 252, 8700-8707). In order to explain the slower steady rate and the results of polymer-challenge experiments, we conjectured the presence of a "dead-end complex" formed by the enzyme with the primer-template at the end of the primer elongation process. In this communication we present evidence which indicates that the presumed complex shows a first order kinetics of decay with a half-life of 3.5 min at 37 degrees. Energies of activation for the steady phase of synthesis and the decay of the dead-end complex were both found to be about 23 kcal/mol. This indicates that the dissociation of the aforesaid complex might be the rate-limiting step during the steady phase of synthesis. Correlation between the salt-induced reduction in the half-life of the complex and the increase in the steady rate of synthesis is in agreement with the above mentioned possibility.     

Mechanism of T5-induced DNA polymerase. I. Replication of short primer templates.

Bacteriophage T5-induced DNA polymerase shows an initial phase of rapid synthesis, followed by a slower steady rate for much longer periods, with short DNA primer-templates (400 to 600 nucleotides long), in vitro. On extrapolating the line of steady rate back to 0 min, an intercept is obtained on the ordinate. With large DNA primer-templates, such as denatured T5 DNA (average chain length approximately 50,000 bases), the rate of synthesis remains constant and is equal to the initial rate obtained with short primer-templates. The zero time intercept was proportional to the amount of enzyme used and independent of temperature. Polymer challenge experiments indicate that the initial phase of rapid synthesis can be attributed to the processive mode of synthesis by T5 DNA polymerase. After synthesizing a stretch of DNA processively for about 200 nucleotide residues, the enzyme apparently forms a "dead-end complex" with the primer-templates used and must dissociate from the primer-template in order to resume synthesis. The average size of the product made processively, during various phase of synthesis, remains invariant and is in good agreement with the size of the zero time intercept per enzyme molecule.     

Changes in Electrophoretic Patterns of Oocyte Proteins during Oocyte Maturation in Oryzias latipes

Changes in the two-dimensional SDS-electrophoretic patterns of extracts of maturing denuded oocytes of the medaka ( Oryzias latipes ) were surveyed. In oocytes without follicular constituents several proteins became detectable in the area between the acidic and slightly basic proteins on the two-dimensional electrophoretograms, while a few of the protein spots disappeared during the process of oocyte maturation. The former proteins were detected also in oocytes that were induced to mature in vivo without breakdown of the germinal vesicle. Several proteins newly observed in extracts of post-vitellogenic oocytes during maturation after breakdown of the germinal vesicle were also identified by two-dimensional electrophoresis. Of several proteins that exhibited noticeable changes in maturing oocytes, only one spot incorporated ¹⁴C-labeled amino acid during maturation, suggesting that post-translational modification of many proteins occurred during oocyte maturation.     

Information, discrimination and divergence in cytology. III. Optimization of classification of Papanicolaou smears

The performance of a cytology laboratory can be objectively quantitated as the total discrimination, a defined quantity of information. The total discrimination is dependent on the number of categories used in gynecologic cytology and on the corresponding histologic states; over-classification results in a higher rate of misinformation and reduced total discrimination. Total divergence is another measure of the association between cytologic categories and histologic states; in contrast to the total discrimination, the total divergence does not require a one-to-one correspondence between the cytologic categories and the histologic states. Using data from the Gynecologic Cytology Laboratory of the University of Minnesota, the total discrimination was maximized when gynecologic cytology used three categories of diagnosis, consisting of (1) normal, atypical benign or reactive atypia, (2) cervical intraepithelial neoplasia (CIN) and (3) all malignancies. The use of four categories, (1) normal, atypical benign or reactive atypia, (2) mild or moderate dysplasia, (3) severe dysplasia or squamous carcinoma in situ and (4) all malignancies, was almost equally informative. Observations on the total divergences resulted in similar conclusions. These findings generally support the recommendation of the consensus workshop sponsored by the National Cancer Institute (the Bethesda System nomenclature) to group all degrees of CIN into two large categories.     

Effect of glucose on protein phosphorylation in rat pancreatic islets

Isolated rat pancreatic islets, incubated in the presence of extracellular ³²Pi to steady state ³²P incorporation into cellular phosphopeptides, were exposed to glucose for 10 min. Glucose (16.7 mM) significantly stimulated the phosphorylation of six phosphoproteins with molecular weights of 15,000, 35,000, 49,000, 64,000, 93,000 and 138,000. Mannoheptulose (16.7 mM) markedly inhibited glucose-stimulated phosphorylation of these six phosphoproteins. This protein phosphorylation might be important in mediating glucose-stimulated insulin release.     

Anti-oxidation activity of ethanol extracts from natural thalli of lichens

Screening test on anti-oxidation activity using 1,1-diphenyl-2-picrylhydrazyl(DPPH) was performed for 99 ethanol extracts of 85 species of natural thalli of lichens in order to find novel anti-oxidation compounds.The 17 extracts of natural thalli showed high anti-oxidation activity.Among them,the activities of extracts from Hypogymnia vittata,Peltigera aphthosa,Nephromopsis ornata,Pseudevernia furfuracea,Cladonia vulcani and Peltigera elizabethae were higher.Extracts of Peltigera spp.showed higher activity than those of other genera.The ethanol extract of P.aphthosa had been separated into ethyl acetate-soluble and water-soluble fractions.Two anti-oxidative spots were found only in the water-soluble fractions by thin-layer chromatography.The compound in the lower spot had the same Rf value,UV spectrum,and color as authentic solorinine that was previously found as a unique quaternary ammonium compound from Peltigera spp.We now report that the hydrophilic lichen substance,solorinine showed a nearly same anti-oxidation activity(EC50=120?mol/Lol/L) as standard antioxidant Trolox(EC50=150?mol/L).     

(4-Piperidinyl)-piperazine: A new platform for acetyl-CoA carboxylase inhibitors

Acetyl-CoA carboxylases (ACCs), the rate limiting enzymes in de novo lipid synthesis, play important roles in modulating energy metabolism. The inhibition of ACC has demonstrated promising therapeutic potential for treating obesity and type 2 diabetes mellitus in transgenic mice and preclinical animal models. We describe herein the synthesis and structure–activity relationships of a series of disubstituted (4-piperidinyl)-piperazine derivatives as a new platform for ACC1/2 non-selective inhibitors.