Inhibition of benzoyl peroxide/Cu2+-dependent lipid peroxidation by manganese

1. Formation of peroxides by benxoyl peroxide (BPO) and CuCl2 was examined in the human red blood cell ghost. 2. Amounts of peroxides formed increased with the amount of the ghost solution added. 3. Of all the cations tested only manganese ion inhibited the formation of peroxides in BPO-CuCl2 reaction system. 4. The formation of peroxides was inhibited approx. 50% with 0.4 microM manganese. 5. The inhibitory manner of manganese was non-competitive against copper.     

Nonsurgical collection of embryos in Shiba goats

A method of nonsurgical embryo collection in the Shiba goat, a native Japanese miniature goat breeding nonseasonally, was developed. The apparatus used for flushing the uterus was made on the model of the two-way catheter for cows. Embryo collection was performed on days 5 to 7 in 37 females superovulated with PMSG and hCG and resulted in successful recovery of 69 embryos in 19 females (51.4%). The average number of embryos collected from each successful female was 3.6. The recovery rate of embryos calculated on the basis of the number of embryos recovered and corpora lutea observed by culdoscopy in 15 successful females was 89.5%. This nonsurgical method seem to be efficient enough for collecting morulae and blastocysts in Shiba goats.     

Are fluorescent bodies of Y-spermatozoa detectable in common with mammalan species?

An attempt was made to detect the fluorescent bodies (F-body), using Quinacrine mustard (Q-M) staining in the spermatozoa from eight mammalian species (human, bull, boar, dog, rabbit, rat, mouse, and mastomys) as well as in the cock (used as negative control). Sperm suspension, prepared after rinsing by repeated centrifugation with phosphate buffered saline (PBS), was either stained with Q-M for 24 h or treated with protease and then stained with Q-M for 60 min. The final concentration of Q-M in the mixed staining sperm suspension was 0.025 mg/ml. The examination using a reflecting fluorescent microscope revealed that the F-body found in human sperm was also present in the sperm of all the mammals but not in the cock after 24 h of staining. The enzyme-treated specimens showed higher incidences of F-bodies than specimens stained for 24 h without enzymatic digestion. These findings strongly suggest that the F-body is commonly present in the spermatozoa of many mammalian species.     

Production of normal piglets from microsurgically split morulae and blastocysts

The embryo splitting technique was applied to pig embryos, and the developmental ability of the split embryos was examined by means of in vitro culture and transfer. Morulae, early blastocysts and blastocysts were collected from Landrace x Large White F(1) gilts which had been mated to Duroc boars. The embryos were bisected with a fine glass or alloy (PtIr) needle after the softening of zonae pellucidae. The halved-embryos, which had either been placed in zonae pellucidae or not, were transferred to recipient gilts immediately after the micromanipulation (Experiment 1) or after cultivation for 15 to 20 h (Experiment 2). In Experiment 1, two fetuses were obtained from one of three recipients which had received 12 half-embryos. In Experiment 2, three of five recipients became pregnant, and in one recipient, seven piglets of a litter were obtained from 12 zona-free half-embryos produced from the original seven blastocysts. The results obtained indicate that a simple method not requiring the encasing of split embryos into zonae pellucidae is satisfactory to produce viable half-embryos.     

Detection of tetrodotoxin by thin-layer chromatography/fast atom bombardment mass spectrometry

A new method for detection of tetrodotoxin (TTX) by thin-layer chromatography/fast atom bombardment (FAB) mass spectrometry was developed. TTX and/or related substances were separated by TLC on LHP-K high-performance precoated plates, with a solvent system of pyridine:ethyl acetate:acetic acid:water (15:5:3:4). The plates were subjected to positive FAB mass spectrometry, under scanning within a mass range from m/z 100 to 500. TTX was identified by selected ion-monitored chromatograms at m/z 320 (M + H)+ and 302 (M + H - H2O)+, along with full scan positive ion FAB mass spectrometry. The limit of detection for TTX was about 0.1 micrograms. TTX was also detected by cellulose acetate membrane electrophoresis/FAB mass spectrometry.     

Sexing of half-embryos produced by microsurgical bisection of mouse morulae and production of chimeric mouse of defined sex composition by aggregating two sexed half-embryos

Using the halved morulae of mice obtained with microsurgical technique, the following two experiments were performed. 1) Sexing of half-embryos by chromosomal analysis and transfer of the half-embryos after determining the sex of the other monozygotic half. One half of the bisected embryo was cultured in Colcemid solution (0.04 micrograms/ml) to be ensured for chromosomal preparation. More than 50% (152/270) of the blastulated embryos from the halves could be sexed by direct sex chromosome analysis. Thirty-nine of the half-embryos of which the co-twin halves were sexed, were transplanted in to the uterine horns of 18 pseudopregnant mice, and twelve became pregnant. The autopsies of them on Day 18 to 20 of pregnancy, revealed the presence of 16 fetuses. The morphological sex of these fetuses thus obtained coincided completely with the previous judgement based on the chromosomal sexing. 2) Production of chimeras of defined sex composition by aggregating two half-morulae of defined sex. Out of 147 pairs of half-morulae of two different strains (ICR and C3H/He), which were replaced in pairs into empty zona pellucidae, 107 (72.8%) were aggregated successfully and developed in vitro into full expanding blastocysts of typical form. Among the 107 aggregate blastocysts, 31 were sexed for both component embryos by chromosomal analysis on the co-twin half-embryos. When these 31 blastocysts were transferred, 11 living offspring including 4 chimeras were obtained. Transfer of 12 male-male and 5 female-female aggregate blastocysts resulted in 8 males and 1 female, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of cytosolic aspartate aminotransferase accompanying modification of Trp 48 by N-bromosuccinimide

Reaction of N-bromosuccinimide with pig heart cytosolic aspartate aminotransferase led to loss of the enzymatic activity. Chemical analysis indicated the modification of two tryptophan residues. At a low ratio of N-bromosuccinimide to enzyme, oxidation of Trp 122 occurred without affecting the enzymatic activity. Increase in the ratio resulted in the oxidation of Trp 48 with a concomitant decrease in enzyme activity. The modified enzyme did not react with substrates and their analogs. Trp 48 is not within the active site but in the hinge region linking the large domain of the enzyme to the small domain that shows dynamic movement upon binding substrates. The present result suggests that oxidation of Trp 48 may impair the structural integrity of the interdomain interface.     

Structural study on the active site of porcine pepsin and Rhizopus chinensis acid protease. Spin labeling with diazoketone reagents

To investigate the active site structures of porcine pepsin and Rhizopus chinensis acid protease (RAP), spin label techniques were applied for these enzymes. Comparison of spin labeled porcine pepsin and RAP suggested that the active site cleft of porcine pepsin was narrower at the top, but wider at the bottom than that of RAP. Addition of pepstatin restricted the motion of the labeled nitroxide radicals. Under alkaline conditions, the enzymes changed their conformation discontinuously and irreversibly to open the active site clefts and to lose the binding ability for pepstatin. The denaturation points of both the enzymes were determined to be pH 6.2.     

The survival of whole and bisected rabbit morulae after cryopreservation by the vitrification method

The survival of whole and bisected rabbit morulae cryopreserved by the vitrification method was investigated. The embryos were loaded in a column of vitrification solution (VS, a mixture of 25% glycerol and 25% 1, 2-propanediol in PBS+16% calf serum), which was located between two columns of 1 M sucrose solution in a plastic straw. The embryos were frozen by being plunged into liquid nitrogen and thawed in a water bath at 20 degrees C. Two methods of loading embryos into straws were used: the single and double column vitrification solution methods. The embryonic survival rates between these two methods were compared. Seventy-one (86.6%) out of 82 morulae vitrified in double column straws developed into the blastocyst stage in vitro. Eleven (18.3%) live fetuses were obtained after the transfer of 60 frozen-thawed morulae to four recipients. By contrast, the survival rate (36.5%, 27 74 ) of embryos vitrified in the single column straws was significantly lower (P<0.05). The vitrification solution of the single column straws became opaque, indicating ice-crystal formation, upon thawing in 5 of 11 straws, which was assumed to have damaged the embryos. More than 80% (29 36 ) of the bisected morulae frozen and thawed in the double column straws developed to the blastocyst stage in vitro when cryoprotectant was diluted stepwise with 1 M and 0.25 M sucrose solution. When the cryoprotectant was removed by one-step dilution with 1 M sucrose solution, swelling in blastomeres was observed and the development rate of the recovered embryos decreased (45.8%, 11 24 ). These results indicate that the vitrification method employed in our experiment is not only efficient for the cryopreservation of rabbit morulae, but it can also be used for the preservation of bisected rabbit morulae, which had not been successful using previous methods.