Effects of delayed mating on preimplantation embryos in spontaneously ovulated mice

The effects of delayed mating on mouse preimplantation embryos (78 ± 1 hours) were studied by setting up different mating periods in relation to the estimated time of spontaneous ovulation. Copulation occurred even in the late morning and early afternoon after the night of spontaneous ovulation. However, females mated in the early afternoon had no viable embryos at the time of laparotomy. Although embryonic development was not affected in the groups mated 6 or 10 hours after estimated ovulation, the percentage of degenerated embryos was increased in these groups. These results suggest that prolonged intervals between the estimated time of ovulation and mating have some deleterious effects on preimplantation embryos.     

Therapeutic efficacy of Stephania tetrandra S. Moore for treatment of neovascularization of retinal capillary (retinopathy) in diabetes--in vitro study.

The present study was designed to examine therapeutic efficacy of the root extract of Stephania Tetrandra S. Moore (STMS) (traditional Chinese medicine; Han Fang Ji) for treatment of neovascularization of the retinal capillary (retinopathy) in streptozotocin (STZ)-induced diabetic rats (STZ diabetic rats) in culture. Recently we have established the culture system in which fetal bovine serum (FBS) in Dulbecco modified Eagle medium (DMEM) induced neovascularization of the retinal capillary and choroidal capillary in normal rats in culture. STZ diabetic rats showed more neovascularization of the retinal capillary and choroidal capillary than did normal rats in culture. In this study, the retinal tissue was removed for the posterior ocular region and cultured in DMEM containing FBS. The choroidal tissue of the posterior ocular region was also removed and cultured as an internal reference. Administration of STSM (0.91, 9.1 and 91 microg/ml) significantly suppressed neovascularization of the retinal capillary in both STZ diabetic rats and normal rats in a dose-dependent manner. Similar results were obtained with the choroidal capillary; administration of STSM suppressed neovascularization of the choroidal capillary in both STZ diabetic rats and normal rats. In order to determine the component of STSM inhibiting neovascularization of the retinal capillary, tetrandrine (a major chemical constituent of STSM) was administered and neovascularization of the retinal capillary was examined in culture. The effect of tetrandrine on the choroidal capillary was also examined as an internal reference. Administration of tetrandrine (0.1, 1.0 and 10 microM) suppressed neovascularization of the retinal capillary in both STZ diabetic rats and normal rats in a dose-dependent manner. Similar results were obtained with the choroidal capillary of both STZ diabetic rats and normal rats. We infer, therefore, that STSM has a direct effect on the retinal capillary of posterior ocular region and suppresses neovascularization of retinal capillary in STZ diabetic rats through the activation of tetrandrine. These results suggest that STSM may prevent for delay the progression of retinopathy in diabetic patients.     

In vitro phosphorylation of the tumor suppressor gene RB protein by mitosis-specific histone H1 kinase

The major components of the mitosis-specific histone H1 kinase are CDC2 kinase and cyclin and the consensus amino acid sequence for phosphorylation by this enzyme has been proposed. We have noted the presence of such sequences in six sites of the tumor suppressor gene RB protein and determined whether or not RB protein is in fact phosphorylated by this kinase. Highly purified enzyme was used for this purpose. HeLa cell extracts immunoprecipitated with anti-RB antiserum as well as RB proteins expressed in E. coli cells were shown to be phosphorylated by this kinase in vitro. Synthetic peptides for the six expected sites were also phosphorylated. These results suggest the possibility that the function of RB protein is regulated by CDC2 kinase.     

Extracellular production of a heat-stable somatic antigen by Bacillus thuringiensis

Extracellular production of a heat-stable somatic antigen (HSSA) by Bacillus thuringiensis subsp. dendrolimus strain T84A1-A [flagellar (H) serotype 4a: 4b] was serologically detected. In Ouchterlony tests, the HSSA antiserum gave single precipitin lines against both untreated and heat-treated culture supernatants. These two precipitin lines fused completely. When colonies of strain T84A1-A were grown on nutrient agar plates containing the homologous HSSA antiserum, precipitin halos were formed around the colonies. Of 27 type strains of B. thuringiensis subspecies tested, only the type strains of B. thuringiensis subsp. sotto (H serotype 4a: 4b) and B. thuringiensis subsp. israelensis (H serotype 14) formed [precipitin halos on nutrient agar plates containing antiserum against the HSSA of strain T84A1-A.     

Biochemical and physiological characteristics of Xenorhabdus species,symbiotically associated with entomopathogenic nematodes including Steinernema kushidai and their pathogenicity against Spodoptera litura (Lepidoptera: Noctuidae)

The symbiotic bacterium strain, SK-1 isolated from Steinernema kushidai, a new species of entomopathogenic nematode, was compared with other strains of Xenorhabdus species. Like other Xenorhabdus nematophilus strains, this new strain is gram-negative, facultatively anaerobic, peritrichously flagellated rod and negative for catalase and nitrate reduction. It can be distinguished from the other Xenorhabdus spp. by differences in reactions to phenylalanine deaminase, no acid production from myo-inositol and utilizations of inosine, dl-malate, formate and methanol. Intra-haemocoelic injection of actual cells or liquid culture supernatant into sixth instar larvae of Spodoptera litura for either Phase I or II variants were not pathogenic. Other strains of X. nematophilus showed pathogenicity for whole cell injections. The supernatants of strain D-1 and ATCC 19061, which are symbionts of Steinernema carpocapsae were pathogenic, however pathogenicity decreased and then terminated by increases in temperature.     

Purification and Properties of 1-AminocycIopropane-l-carboxylate Synthase of Mesocarp of Cucurbita maxima Duch. Fruits

1-Aminocyclopropane-1-carboxylate (ACC) synthase, which formsAGC from S-adenosylmethionine (SAM), was purified to homogeneityfrom sliced and aged mesocarp tissue of Cucurbita maxima Duch.cv Ebisu fruits, and its enzymatic properties were determined.The specific activity of the purified enzyme was 220 mU/mg proteinat 30°C at 50 µM SAM. Native ACC synthase has a relativemolecular mass of 160 ± 10 kDa and consisted of two subunitsof about 84±3 kDa. S-adenosylhomocysteine (SAH), S-methylmethionine(SMM) and L-methionine did not serve as substrate. The enzymereaction was competitively inhibited by aminoethoxyvinylglycine(AVG) (Ki, 2.5 µM), aminooxyacetic acid (Ki, 40 µM)and SAH (Ki, 30 µM). The reaction was also strongly inhibitedby semicarbazide, and less effectively by homocysteine. Theenzyme was rapidly inactivated by its substrate, SAM in thepresence of pyridoxalphosphate (PLP), but in the absence ofPLP, SAM-induced inactivation was much slower. Inactivationdid not occur by SAH and SMM, SAM analogs without substrateactivity. Pyridoxal phosphate was an essential cofactor to beadded to a reaction mixture for maximum activity, but an enzymepreparation from which pyridoxal phosphate was removed by SephadexG-25 gel filtration exhibited one-eighth activity which wasinhibited by semicarbazide, this indicating that a small amountof pyridoxal phosphate is firmly bound to the enzyme. (Received May 6, 1986; Accepted May 20, 1986)     

Phosphorylation of five histone H1 subtypes of L5178Y cells at the exponential growth and mitotic phases

Histone H1 of cells of L5178Y, a mouse lympholeukemic cell line, consists of five molecular species designated as H1-I, II, III, IV, and V. The phosphorylation of these H1 subtypes was examined at the exponential growth phase and during mitosis, by BioRex 70 column chromatography and two-dimensional polyacrylamide gel electrophoresis. In exponentially growing cells, the degree of phosphorylation was different for each subtype. H1-II was the most highly phosphorylated, 1.8 phosphate residues per molecule, followed by H1-IV/V, 1.4, I, 0.8, and III, 0.5. In the mitotic phase, H1-II was also the most highly phosphorylated 6.0 phosphate residues per molecule, H1-IV/V, 3.5, I, 2.7, and III, 1.2. The phosphorylation started simultaneously among the subtypes after colcemid addition, and phosphorylated H1 subtypes accumulated linearly. The rate of incorporation of 32P into each H1 subtype was almost constant during colcemid treatment. During 4 h after colcemid addition, the phosphate residues incorporated into H1 did not dephosphorylated. The H1 kinase activities increased to six times higher during the colcemid treatment.     

A versatile bacterial enzyme: l-methionine γ-lyase

The properties and application of l-methionine γ-lyase [methioninase, l-methionine methanethiol-lyase (deaminating), EC 4.4.1.11], a pyridoxal 5′-phosphate enzyme, purified from Pseudomonas putida and Aeromonas sp. are presented. The enzyme has multicatalytic functions: it catalyses α,γ-elimination and γ-replacement reactions of l-methionine and its analogues (e.g. ethionine, homocysteine, O-acetylhomoserine and selenomethionine), α,β-elimination and β-replacement reactions of l-cysteine and its analogues (e.g. S-methylcysteine, O-acetylserine and Se-methylselenocysteine), deamination and γ-addition of vinylglycine, and deuterium labelling at the α and β positions of l-methionine and other straight-chain l-amino acids. These reactions are applicable to the synthesis of various optically active sulphur and selenium amino acids, preparation of deuterium or tritium labelled l-amino acids, and determination of sulphur amino acids. In addition, the enzyme shows potent anti-neoplastic activity.     

The branching of the inflorescence and vegetative shoot and taxonomy of the genusKummerowia (Leguminosae)

A study of the branching of the inflorescence and the vegetative shoot of the genusKummerowia, consisting ofK. stipulacea (Maxim.) Makino andK. striata (Thunb.) Schindler, has led to the following conclusions: (1) the inflorescences of both species are reduced compound cymes, (2) the branching system of the inflorescence ofKummerowia is not clearly different from that of the vegetative shoot and there are some transitional forms between both systems, and (3) the inflorescence ofKummerowia is different from the racemose inflorescences ofLespedeza andCampylotropis. Based on the differences found in the branching system of the inflorescence,Kummerowia is distinctly separated fromLespedeza andCampylotropis and is more correctly treated as a distinct genus from the latter two.     

Photochemical inactivation of human placental estradiol 17 beta-dehydrogenase in the presence of 2,3-butanedione

Estradiol 17 beta-dehydrogenase of human placenta was rapidly inactivated by 2,3-butanedione under u.v. light, and no protection against the inactivation was observed in the presence of sodium azide. Under ordinary laboratory illumination, the inactivation was biphasically progressed in time-dependent and concentration-dependent manners, while a partial protection from the inactivation was indicated by sodium azide. These results suggest that the inactivation mechanism of the dehydrogenase by 2,3-butanedione under laboratory illumination is different from that under u.v. light. Therefore, the inactivation under laboratory illumination proceeded by a reaction with excited singlet molecular oxygen (1 delta g or 1 sigma +g states), and that under u.v. light was caused by a reaction of substrate with triplet sensitizer. In the presence of NADP+, the inactivation of the enzyme by 2,3-butanedione was markedly reduced. The maximum protection by NADP+ was about 80% of the initial enzyme activity. Amino acid analysis of the enzyme treated with 2,3-butanedione under laboratory illumination showed that the modified enzyme contained considerably less of the following amino acids than the native enzyme: histidine, arginine, threonine, methionine, tyrosine and leucine. In addition, other dicarbonyl reagents, 1,4-dibromo-2,3-butanedione, 1-phenyl-1,2-propanedione, phenylglyoxal, 16-oxoestrone, 1,2-cyclohexanedione, 2,4-pentanedione and glyoxal were found to decrease the dehydrogenase activity in various degree.