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.     

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)     

Induction and Repair of Damage to DNA in Cucumber Cotyledons Irradiated with UV-B

Photoinduced lesions in DNA, namely, cyclobutane pyrimidinedimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts[(6-4)photoproducts], in cucumber cotyledons that had been irradiatedwith naturally occurring levels of UV-B (290–320 nm) werequantitated by enzyme-linked immunosorbent assays with monoclonalantibodies specific to each type of photolesion. Induction ofthese photolesions was dependent on temperature and their extentwas reduced by simultaneous irradiation with white light. Thedark repair of both types of photolesion was undetectable. Light-dependentremoval of (6-4)photoproducts was very slow, with 50% removalin 4 h. By contrast, 50% of initial CPDs were removed within15 min. Both photorepair processes were dependent on the intensityof white light and were sensitive to temperature. These resultsindicate that high photolyase activity is present in cucumbercotyledons and that repair activities in cucumber cotyledonsare different from those reported in Arabidopsis, in which (6-4)photoproductsare photorepaired more rapidly than CPDs. (Received October 13, 1995; Accepted December 28, 1995)     

Transient expression of bile-duct-specific cytokeratin in fetal mouse hepatocytes

The differentiation of hepatocytes and biliary epithelial cells has been histochemically analyzed with anti-calf cytokeratin antiserum in the fetal mouse liver. Almost all young fetal hepatocytes transiently express bile-duct-specific cytokeratin; subsequently, the strong staining of the cytokeratin is confined to progenitor cells of intrahepatic biliary epithelial cells around portal veins. These results suggest that all fetal hepatocytes are bi-potent in terms of the differentiation of mature hepatocytes and intrahepatic bile-duct cells, and that the microenvironment around portal veins plays an important role in bile-duct differentiation. Large periportal hepatocytes continue to stain weakly for cytokeratin until 2 weeks after birth, although the number of positive hepatocytes decreases with development. The differentiation of bile ducts from periportal hepatocytes may continue for 2 weeks after birth.     

Isolation of cDNA for a Plasma Membrane H+-ATPase from Guard Cells of Vicia faba L.

cDNA encoding the plasma membrane H⁺-ATPase of guard cells ofVicia faba L. was isolated. The clone encoded a 105-kDa polypeptide(956 amino acids) that was 79–85% identical in terms ofamino acid sequence to other plant H⁺-ATPases. High levels ofmRNA explain the high H⁺-ATPase activity of these plasma membranes. (Received December 24, 1994; Accepted April 12, 1995)     

Relationship between functional castration and alpha-fetoprotein produced by hepatoma-bearing female rats.

In rats bearing Morris hepatoma No. 7777, serum levels of alpha fetoprotein (AFP) increased as tumour size increased. Hepatoma-bearing females remained in dioestrus once the tumour exceeded a cross-sectional area of 12 cm2. The following changes were seen in tumour-bearing rats compared to control animals: uterine wet weight was significantly decreased, the uterine epithelial mucosa was lower, castration cells developed in the anterior pituitary, and there was a 3- to 23-fold elevation in serum oestrogen levels. We conclude that circulating oestrogen is bound to AFP in hepatoma-bearing female rats and that in this bound state it is unable to exert its normal physiological actions.     

Structure and function of psychrophilic alanine racemase

We describe the structure and function of psychrophilic alanine racemases from Bacillus psychrosaccharolyticus and Pseudomonas fluorescens. These enzymes showed high catalytic activities even at 0°C and were extremely labile at temperatures over 35°C. The enzymes were also found to be less resistant to organic solvents than alanine racemases from thermophilic and mesophilic bacteria, both in vivo and in vitro. Both enzymes have a dimeric structure and contain 2 mol of pyridoxal 5′-phosphate (PLP) per mol as a coenzyme. The enzyme from B. psychrosaccharolyticus was found to have a markedly large K_m value (5.0 μM) for PLP in comparison with other reported alanine racemases, and was stable at temperatures up to 50°C in the presence of excess amounts of PLP. The dissociation of PLP from the P. fluorescens enzyme may trigger the unfolding of the secondary structure. The enzyme from B. psychrosaccharolyticus has a distinguishing hydrophilic region around residue no. 150 in its deduced amino acid sequence, whereas the corresponding regions of other Bacillus alanine racemases are hydrophobic. The position of this region in the three dimensional structure of this enzyme was predicted to be in a surface loop surrounding the active site. This hydrophilic region may interact with solvent, reduce the compactness of the active site, and destabilize the enzyme.