Changes in intracellular location of DNA polymerase-α during oocyte maturation of the toad,bufo bufo japonicus

Intracellular location of DNA polymerase-α during oocyte maturation of the toad was studied. Quantitative and qualitative changes in the activity of DNA polymerase-α were not observed during the maturational process. Nearly all activity was found in isolated germinal vesicles from full grown oocytes and in enucleated mature oocytes. The cytoplasmic DNA polymerase-α of mature oocytes was recovered at buoyant densities equivalent to microsome by isopycnic centrifugation. These findings indicate that DNA polymerase-α in the germinal vesicle is released into the cytoplasm and binds to the endoplasmic reticulum when the germinal vesicle breaks down.     

Comparison of cytochrome P-450 species which catalyze the hydroxylations of the aromatic ring of estradiol and estradiol 17-sulfate

For identification of microsomal cytochrome P-450 (P-450) enzymes which catalyze 2- or 4-hydroxylations of estrogens in the rat liver, estradiol (E₂) and estradiol 17-sulfate (E₂-17-S) were selected as the substrates and incubated with various kinds of purified P-450 enzymes: PB-1, PB-2, PB-4 and PB-5 obtained from phenobarbital-treated male rats (Sprague-Dawley); MC-1 and MC-5 from 3-methylcholanthrene-treated male rats; and UT-1, UT-2, UT-4 and UT-5 from untreated animals. The reactions were carried out under the P-450-reconstructed system, and the resulting products were determined by HPLC using electrochemical detection. All the enzymes tested were shown to have varying degrees of catalytic activities for 2-hydroxylation of the two substrates; UT-1 and UT-2 had the highest activity. Of the induced P-450 enzymes, PB-2 and MC-1 showed fairly high catalytic activity for 4-hydroxylation of E₂. The P-450 enzymes obtained from the untreated male rats, especially UT-4, showed the highest catalytic activity for 4-hydroxylation of the two substrates. From these results and also from kinetic experiments, the P-450 enzymes which catalyze 2- and 4-hydroxylations of estrogen were considered to be different species. A part of E₂ was converted to such metabolites as estrone and those having a hydroxyl group at positions 6β, 15 or 16, each production of which was estimated to be catalyzed by single or multiple P-450s.     

Mechanism of induction of cellular DNA synthesis by the adenovirus E1A 12S cDNA product

The mechanism of induction of DNA synthesis in quiescent rat 3Y1 cells by the adenovirus E1A gene was investigated using the 3Y1 derivative cell lines g12-21, gn12RB1, and gn12RB2. The g12-21 cells express the E1A 12S cDNA and the latter two cells express both the E1A 12S cDNA and the human retinoblastoma susceptibility (Rb) gene at different levels in response to dexamethasone (dex). The cDNA sequences of E1A-inducible cell cycle-dependent genes, clone 3 and clone 16, were isolated by differential screening of a cDNA library constructed from dex-treated g12-21 cells. The quiescent 3Y1 cells induced c-fos and c-myc expression within 2 h after serum stimulation and expressed clone 16 and clone 3 transiently at around 8 h before the onset of DNA synthesis (10 h). In contrast, the quiescent g12-21 cells treated with dex expressed a high level of E1A at 6 to 8 h after treatment and expressed clone 16 and clone 3 at around 8 h without stimulation of c-fos and c-myc expression, suggesting that E1A bypasses the cell cycle early in G1. The half-maximal rate of DNA synthesis was reached in a much shorter time in dex-treated g12-21 cells (12 h) than in serum-treated 3Y1 cells (18 h), suggesting that E1A also bypasses the cell cycle at the G1/S boundary. The gn12RB1 and gn12RB2 cells were unable to induce DNA synthesis in response to dex presumably due to lower levels of E1A expression, although gn12RB2 but not gn12RB1 cells could express clone 16 and clone 3. These results suggest that the level of E1A required for bypass at the G1/S boundary is higher than that required early in G1.     

Molecular basis for the interaction of histamine with the histamine H2 receptor.

We undertook these studies to characterize the molecular basis of the interaction of histamine with the H2 receptor. Key areas of homology in the structures of the histamine H2 and beta 2 adrenergic receptor suggested specific transmembrane amino acids that might be important for binding of histamine. A third transmembrane aspartic acid of the histamine receptor (Asp98), thought to serve as a counter anion that interacts with the cationic amine moiety of histamine, was mutated to Asn98, and the mutated receptor was expressed in Hepa cells. Removal of the negatively charged amino acid abolished both binding of the H2 receptor antagonist [methyl-3H]tiotidine and histamine stimulated increases in cellular cAMP content. Mutation of a fifth transmembrane aspartic acid (Asp186) to Ala186 or Asn186 by itself or in conjunction with mutation of another fifth transmembrane amino acid (Thr190 to Ala190) resulted in a loss of [methyl-3H] tiotidine binding, although the generation of cAMP in response to histamine was maintained. The histamine receptor with only a Thr190 to Ala190 or Cys190 mutation retained the ability to bind [methyl-3H]tiotidine, but both the affinity and efficacy of binding were reduced. These data lead us to propose a model for histamine binding in which Asp98 is essential for histamine binding and action, Asp186 defines H2 selectivity, and Thr190 is important in establishing the kinetics of histamine binding, but is not essential for H2 selectivity.     

Changes in Electrophoretic Patterns of Oocyte Proteins during Oocyte Maturation in Oryzias latipes

Changes in the two-dimensional SDS-electrophoretic patterns of extracts of maturing denuded oocytes of the medaka ( Oryzias latipes ) were surveyed. In oocytes without follicular constituents several proteins became detectable in the area between the acidic and slightly basic proteins on the two-dimensional electrophoretograms, while a few of the protein spots disappeared during the process of oocyte maturation. The former proteins were detected also in oocytes that were induced to mature in vivo without breakdown of the germinal vesicle. Several proteins newly observed in extracts of post-vitellogenic oocytes during maturation after breakdown of the germinal vesicle were also identified by two-dimensional electrophoresis. Of several proteins that exhibited noticeable changes in maturing oocytes, only one spot incorporated ¹⁴C-labeled amino acid during maturation, suggesting that post-translational modification of many proteins occurred during oocyte maturation.     

Studies on the constituents of the seeds of Cassia obtusifolia: The structures of two new lactones,isotoralactone and cassialactone

Torosachrysone and two new naphthalenic lactones, isotoralactone and cassialactone, were isolated from the seeds of Cassia obtusifolia. Their structures were established as 9,10-dihydroxy-7-methoxy-3-methylene-¹H-naphtho(2,3-c)dihydropyrone-1-one and 8-methoxy-4-methyl-1-oxo-4,10,11-trihydroxy-naphtho(2,3-c)oxepin, respectively.     

Detection of the Nicotiana rustica chloroplast genome coding for the large subunit of Fraction I protein in a somatic hybrid in which only the N. tabacum chloroplast genome appeared to have been expressed

Nine plants were produced from anthers of a somatic hybrid which had been obtained by fusion of Nicotiana tabacum L. and N. rustica L. protoplants. As determined by electrofocusing, the Fraction I protein of the original somatic hybrid had largesubunit polypeptides exclusively of the N. tabacum type. Two of the plants regenerated from anthers contained Fraction-I-protein large subunits exclusively of the N. rustica type. Since each plant was regenerated from a single cell, the somatic hybrid must have had cells containing both the N. tabacum and N. rustica chloroplast genome although the latter was not expressed. Possibilities to account for this non-expression of a chloroplast genome in the somatic hybrid are discussed.     

Collection of rice phloem sap from stylets of homopterous insects severed by YAG laser

Pure phloem sap from rice plant was collected by a method similarto the aphid technique using leafhoppers and planthoppers instead.A Yttrium-Aluminium Garnet laser was used to sever the insectstylets. Sap exuded from the plant through the stylet for upto 3 hr at a rate of 0.2 µl/hr. Analysis of the sap forsugars revealed that the only carbohydrate present was sucrose;its content was estimated to be 17% and remained at this levelfor 3 hr. (Received April 16, 1980; )     

The maximum leaf surface temperatures of the higher plants observed in the Inland Sea area

The maximum leaf surface temperatures (MLSTs) of 126 species of higher plants were measured by means of an infrared thermometer, in the Inland Sea area, southwest of Honshu-Island, Japan, where plants suffered from severe environmental conditions due to an abnormally small amount of precipitation during the summer of 1978. The MLSTs of plants in the summer of 1978 were greater than or equal to those of 1979, when the environmental conditions were not so severe. The MLST measured in this study was 50.4 C for a non-succulent plant (Liriodendron), and 53.1 C for a succulent plant (Agave). Plants with different life forms appeared to have different MLSTs. The average of the MLSTs of conifers deciduous trees, and evergreens were 36.4, 37.7, and 40.3 C, respectively. This order corresponds to the distribution of forests from high to low, latitudes. Also the MLSTs were higher for woody plants than for herbaceous plants. Relatively high leaf temperatures were observed for climbing plants, both herbaceous and woody. Plants with narrow leaves had lower leaf surface temperatures than those with borad leaves. Herbaceous dicotyledonous plants actively growing at the end of the summer of 1978, in full sun at Hiroshima Castle were exclusively those with relatively high leaf temperatures.