Kinetics of the renaturation of yeast tRNA3 leu.

Yeast tRNA₃^Leu is one of several tRNA molecules which can adopt a stable, biologically inactive, denatured conformation. The circular dichroism of the native and denatured conformers differs, providing the basis for the present study of the mechanism for the renaturation process. Conversion of the denatured structure to the native takes place in two steps: a rapid change occurring immediately on addition of Mg⁺⁺, followed by a slower, strongly temperature-dependent step which returns the molecule to its biologically active state. Optimal kinetic data for the second step could be obtained at 285 nm. Analysis of the time dependence of Δε₂₈₅ by the Guggenheim method demonstrated that this step follows first-order kinetics. The temperature dependence of the rate constants over the range 32–41°C yielded the following parameters for the rate-limiting step: E_a = 69 kcal/mole, ΔH^? = 69 kcal/mole, and ΔS^? = 146 cal/mole deg. Values of this magnitude are typical of order—order transitions in nucleic acids.     

Studies on the mechanism of enkephalin receptor down regulation

The association of [3H] [D-Ala2, D-Leu5] enkephalin ([3H]DADLE]) with mouse neuroblastoma cells (N4TG1) was investigated. Under identical conditions the time course, dose response curve and temperature dependence for ligand uptake were similar to those for ligand-induced receptor loss (down regulation). Uptake of [3H]DADLE was inhibited by opiate ligands as well as by the metabolic inhibitors sodium azide and 2,4 dinitrophenol. Comparison of the effects of these inhibitors on receptor binding, ligand uptake and receptor loss indicated that these cells accumulate [3H]DADLE in excess of their surface receptor number. The data suggest that receptor recycling occurs and that ligand is internalized via receptor mediated endocytosis.     

Glucose effect on respiration: possible mechanism for capacitation in guinea pig spermatozoa.

Guinea pig sperm respiration was determined in minimal capacitation medium (MCM) with different energy sources. The ZO2 observed for spermatozoa suspended in media containing pyruvate and lactate was 35.7 +/- 5.9, pyruvate alone, 27.9 +/- 3.8 and D-glucose alone 3.4 +/- 1.1. When D-glucose was added to spermatozoa rapidly respiring in media containing pyruvate as the only exogenous energy source, an immediate suppression in respiration was observed. Further reduction was caused by continued addition of D-glucose. Fructose and mannose also produced a suppression in respiratory rate. However, lactose, fucose, sucrose, L-glucose, and galactose did not alter the respiratory rate. The suppression of respiration by metabolizable sugars is paralleled by a suppression of acrosome reaction in guinea pig spermatozoa. The possibility that suppression of respiration is the mechanism for retardation of capacitation and the subsequent acrosome reaction by D-glucose and other metabolizable sugars is suggested.     

Starch and its component ratio in developing cotton leaves

During cotton leaf development, starch accumulation was characterized by an initial rise to a maximum at the second to the fourth leaf from the apex. Then, starch content progressively decreased with leaf age. Starch accumulation was inversely related to the ratio of amylopectin to amylose. Differences between leaves in this ratio resulted from variations in both amylose and amylopectin levels. Fluctuations in amylose levels were more extreme than those of amylopectin.     

Microwave treatment of intracerebral l1210 leukemia: Schedule-dependent partial reversal of the effects of methotrexate

One-GHz microwave (MW) irradiation at a power density of 5 mW/cm² was combined with methotrexate (MTX) in an attempt to treat more effectively central nervous system (CNS) L1210 leukemia in DBA/2J mice. When mice with CNS leukemia were treated with the combination of MW and MTX, there was no improvement in survival compared with a group of animals treated with MTX alone; however, the group that received MTX before the MW exposure had a significantly reduced survival time compared with the group treated with MTX alone or with the group to which MTX was administered after MW.     

Amino acid analysis at the picomole level. Application to the C-terminal sequence analysis of polypeptides.

Amino acids labelled with dimethylaminoazobenzenesulphonyl chloride can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). All 19 naturally occurring amino acids can be separated on a Zorbax ODS column by employing two different gradient systems consisting of an acetonitrile/aqueous buffer mixture. As little as 2--5 pmol of an individual dimethylaminoazobenzenesulphonyl-amino acid can be quantitatively analysed with reliability, and only 10--30 ng of the dimethylaminoazobenzenesulphonylated protein hydrolysate is needed for each complete amino acid analysis. This new technique is as sensitive as any of the current amino acid analysis methods involving ion-exchange separation plus fluorescence detection, and is technically much simpler. By the combination of this sensitive amino acid-analysing technique with carboxypeptidase, we have been able to determine the C-terminal sequence of polypeptides at the picomole level.     

Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid.

Construction and characterization of a class of multicopy plasmid cloning vehicles containing the replication system of miniplasmid P15A are described. The constructed plasmids have cleavage sites within antibiotic resistance genes for a variety of commonly employed site-specific endonucleases, permitting convenient use of the insertional inactivation procedure for the selection of clones that contain hybrid DNA molecules. Although the constructed plasmids showed DNA sequence homology with the ColE1 plasmid within the replication region, were amplifiable by chloramphenicol or spectinomycin, required DNA polymerase I for replication, and shared other replication properties with ColE1, they were nevertheless compatible with ColE1. P15A-derived plasmids were not self-transmissible and were mobilized poorly by Hfr strains; however, mobilization was complemented by the presence of a ColE1 plasmid within the same cell.