Cryopreservation of wild mouse spermatozoa

Spermatozoa of wild mice from China, Czechoslovakia, Denmark, India, Japan and Switzerland were frozen and stored at -196 degrees C. After thawing, intact oocytes were inseminated in vitro with relatively high motility frozen-thawed mouse spermatozoa from Czechoslovakia, Denmark and India, while oocytes with a partially dissected zona were inseminated with low motility frozen-thawed spermatozoa from China, Japan and Switzerland. Embryos developing to the 2-cell stage from oocytes fertilized with frozen-thawed spermatozoa were transferred to the oviducts of female recipients on the first day of pseudopregnancy (day when a vaginal plug was confirmed). Successful embryo development to the 2-cell stage was 46 to 67%. Offspring resulted from 17 to 51% of these transferred 2-cell embryos.     

Lipase modulator protein (LimL) of Pseudomonas sp. strain 109.

Plasmids containing a Pseudomonas sp. strain 109 extracellular lipase gene (lipL) lacking NH2-terminal sequence and a lipase modulator gene (limL) lacking the NH2-terminal hydrophobic region were constructed and expressed independently in Escherichia coli by using the T7 promoter expression vector system. Recombinant LipL (rLipL) was produced as inclusion bodies, whereas recombinant LimL (rLimL) was present as a soluble protein. During in vitro renaturation of the purified rLipL inclusion bodies after they had been dissolved in 8 M urea, addition of rLimL was essential to solubilize and modulate rLipL. The solubility and activity of rLipL were influenced by the rLimL/rLipL molar ratio; the highest level of solubility was obtained at an rLimL/rLipL ratio of 4:5, whereas the highest activity level was obtained at an rLimL/rLipL ratio of 4:1. After renaturation, rLipL and rLimL were coprecipitated with anti-rLipL antibody, indicating the formation of an rLipL-rLimL complex. Activity of the native lipase purified from Pseudomonas sp. strain 109 was also inhibited by rLimL. By Western blotting (immunoblotting) with anti-rLimL antibody, native LimL was detected in Pseudomonas cells solubilized by sarcosyl treatment. LimL was purified from Pseudomonas sp. strain 109, and the NH2-terminal amino acid sequence was determined to be NH2-Leu-Glu-Pro-Ser-Pro-Ala-Pro-. We propose that to prevent membrane degradation, LimL weakens lipase activity inside the cell, especially in the periplasm, in addition to modulating lipase folding.     

Effects of light-dark cycles on the circadian conidiation rhythm inNeurospora crassa

The effects of 24 hr light-dark cycles on the circadian conidiation rhythm inNeurospora crassa were compared among will-typefrq ⁺ and clock mutantsfrq ⁺,frq ³,frq ⁷,frq ⁹ andfrq ¹¹. The minimum length of the light period necessary for complete entrainment to the light-dark cycles was almost 2 hr infrq ⁺,frq ³ andfrq ⁷ strains. The minimum duration of the dark period necessary for the appearance of circadian conidiation was almost 4 hr in all of the strains except thefrq ¹¹ strain. The phase of the conidiation rhythm was dependent on the light to dark transition in thefrq ¹ strain in all light-dark cycles examined and in thefrq ⁺ andfrq ³ strains when the light period was shorter than 16 hr. In contrast, the phase of thefrq ⁷ strain was dependent on the light to dark transition when the light period was shorter than 10 hr.     

Aldosterone biosynthesis by a reconstituted cytochrome P-45011 beta system

[3H]Corticosterone was incubated with cytochrome P-45011 beta purified to electrophoretic homogeneity from bovine adrenocortical mitochondria, and the reaction products were analyzed by high performance liquid chromatography. The production of aldosterone (21.2 pmol/nmol P-450/min) and 18-hydroxycorticosterone (1.17 nmol/nmol P-450/min) was observed. When lipidic extracts from mitochondria of bovine adrenocortical zona glomerulosa were added to the reaction mixture, the rate of production of aldosterone was increased 28-fold. When [3H]18-hydroxycorticosterone was incubated with cytochrome P-45011 beta, the amount of aldosterone produced was 55.7 pmol/nmol P-450/min in the absence of the lipidic extracts and the enhancing effect of the lipidic extracts was 4-fold.     

Inhibition by Mg2+ of the interaction of Ca2+ with spin-labeled g2 bound to myosin.

According to the measurement of ESR spectrum, Ca2+ induced conformational change of spin-labeled g2 bound to myosin in the presence of 1 mM Mg2+. The half-maximal changes were observed at pCa 6.8 and at pCa 3.7. Spin-labeled phosphorylated g2 bound to myosin showed one transition at pCa 4.5, which shifted to pCa 6.5 after the dephosphorylation with E. coli alkaliphosphatase.     

Purified proton conductor in proton translocating adenosine triphosphatase of a thermophilic bacterium.

1. The membrane-integrated portion (TF0) of the proton translocating ATPase complex (TF0-F1) of the thermophilic bacterium PS3 was highly purified. Its proton-conducting activity was investigated in vesicles reconstituted from TF0 and phospholipids (TF0 vesicles). 2. The rate of proton conduction through TF0 was proportional to the membrane potential imposed (6H+ uptake/s/TF0 molecule with 103 mV at pH 8.0). The pH profile of the rate revealed that a proton, not a hydroxy ion, was the true substrate conducted and that there was a monoprotic proton binding site in TF0 (pKa = 6.8). The temperature coefficient of proton conductance of TF0 showed a considerable variation depending on the phospholipids of the vesicles with respective transition temperatures. 3. Passive proton conduction through TF0 was inhibited stoichiometrically by addition of either the soluble ATPase portion (TF1) of TF0-F1, or an energy transfer inhibitor dicyclohexylcarbodiimide or an antibody against TF0. 4. The proton conductance of TF0 was concluded to represent its intrinsic activity in the original TF0-F1 complex.     

Enzymic mechanism of starch breakdown in germinating rice seeds : 11. Ultrastructural changes in scutellar epithelium

The ultrastructural changes occurring in the scutellar epithelium cells of rice seeds have been studied during germination and early seedling growth. During this time, several prominent structural changes occur, including (a) formation, development, and proliferation of organelles such as mitochondria, rough endoplasmic reticulum, free ribosomes, and Golgi apparatus; (b) folded structural modification of plasmamembranes in later stages; and (c) conspicuous decrease in lipid-storing spherosomes. Glyoxysome-like electron dense particles are detectable but their formation is much less prominent. It is conceivable that all these structural changes are related to the enhancement of the metabolic activities of the epithelial cells including the synthesis of hydrolytic enzymes such as α-amylase and their secretion into the endosperm tissues. Some enzyme activities characteristic of mitochondria and glyoxysomes have been determined using the crude scutellar extracts, and the results dealing with the low activities of the glyoxylate cycle enzymes and palmitoyl-coenzyme A oxidase appear to indicate that fatty acid breakdown is possibly via mitochondrial β-oxidation, although we reserve a definitive conclusion on the glyoxysomes being nonfunctional in fatty acid oxidation in rice seedlings.     

Protection by picolinamide,a novel inhibitor of poly (ADP-ribose) synthetase,against both streptozotocin-induced depression of proinsulin synthesis and reduction of NAD content in pancreatic islets

Picolinamide, 2-pyridinecarboxylic acid amide, was found to be a strong inhibitor of poly (ADP-ribose) synthetase of nuclei from rat pancreatic islet cells. Another experiment using isolated pancreatic islets of rats showed that picolinamide protects against streptozotocin-induced depression of proinsulin synthesis as well as against streptozotocin-induced reduction of NAD content. The protection by picolinamide against the NAD depression was considered to be due to the blockage of an increased degradation of NAD mediated by a streptozotocin-induced increase in poly (ADP-ribose) synthetase activity. A possible mechanism of streptozotocin diabetes and its prevention is discussed.     

Enzymic mechanisms of starch breakdown in germinating rice seeds: 7. Amylase formation in the epithelium

The time sequence analysis of the starch digestion pattern of the thin sectioned germinating rice (Oryza sativa L.) seed specimens using the starch film method showed that at the initial stage amylase activity was almost exclusively localized in the epithelium septum between the scutellum and endosperm. Starch breakdown in the endosperm tissues began afterward; amylase activity in the aleurone layers was detectable only after 2 days. Polyacrylamide gel electrofocusing (pH 4 to 6) revealed nearly the same zymogram patterns between endosperm and scutellum extracts, although additional amylase bands appeared in the endosperm extracts at later germination stages (4 to 6 days). These are presumably attributable to the newly synthesized enzyme molecules in the aleurone cells.     

A sulfotransferase model: Covalent participation of a macrocyclic oxime in the hydrolysis of 2,4-dinitrophenyl sulfate

The liberation of 2,4-dinitrophenolate ion from 2,4-dinitrophenyl sulfate (DNPS) in aqueous organic solvent with 0.1 N sodium hydroxide was accelerated upon addition of an equimolar amount of Oxime-I (10-hydroxy-11-hydroxyimino[20]-paracyclophane) to the sulfate ester. Oxime-I was found to undergo covalent participation at the oxime group to afford oxime O-sulfonate. The rate acceleration with Oxime-I was larger than that with β-CD (cycloheptaamylose). The catalytic efficiency of Oxime-I has been ascribed primarily to the tighter inclusion of the substrate ester into the more hydrophobic Oxime-I cavity provided by the effective apolar paracyclophane skeleton, as well as to the greater nucleophilicity of the oxime group than of the hydroxyl group in β-CD. Consequently, Oxime-I may be considered as a conventional model for arylsulfatases and sulfotransferases, providing the effective binding process for the substrate.