Interspecific hybridization between Nicotiana repanda Willd. and N. tabacum L. through the pollen irradiation technique and the egg cell irradiation technique

Summary Two techniques were useful in overcoming hybrid inviability between N. repanda and N. tabacum. These techniques combine gamma-ray irradiation to pollen or to egg cells (in ovules) with in vitro culture of fertilized ovules. When in vitro culture of fertilized ovules from in situ hybridization of N. repanda x N. tabacum was combined without gamma-ray irradiation to pollen or to egg cells (in ovules), all of the resulting seedlings developed chlorosis and died. Furthermore, in the case of in situ hybridization of N. repanda x N. tabacum with gamma-ray irradiated N. tabacum pollen, no viable seeds were obtained. By using both techniques, combining gamma-ray irradiation to N. tabacum pollen or to egg cells in (N. repanda ovules) with in vitro culture of fertilized ovules, we were successful in obtaining flowering hybrid plants. Thus, it appears that it may be possible to overcome hybrid inviability to a certain extent using both the pollen irradiation technique and the egg cell irradiation technique, i.e., gamma-ray irradiation to pollen or to egg cells (in ovules) before pollination and in vitro culture of fertilized ovules.The research reported in this paper is in partial fulfillment of PhD requirements for the senior author     

Localization of receptors for atrial natriuretic polypeptide (ANP) in the glomerulus: in vitro electron microscopic autoradiographical investigation using 125I-labeled ANP

The localization of receptors for atrial natriuretic polypeptide (ANP) in the glomerulus of the rat was studied by electron microscopic autoradiography using 125I-labeled ANP. A total of 1,134 silver grains counted in 30 glomeruli were distributed as follows: processes of the podocytes (36.4%), cell bodies of the podocytes (14.8%), basement membrane (12.3%), endothelial cells (6.3%), mesangial cells (7.7%) and others, for example the vascular spaces (22.5%). This finding indicates that ANP binding sites are mainly localized on the foot processes of the podocytes in the glomerulus.     

Effects of denaturation with HCl on the immunological staining of bromodeoxyuridine incorporated into DNA

Immunochemical procedures for detection of BrdUrd incorporated into DNA require a denaturation step of DNA. Denaturation with HCl is widely used for flow cytometric analysis of the cell cycle and for histological preparations. This brief communication describes an attempt to standardize a denaturation procedure with HCl. Various denaturation conditions at 20 degrees C were examined for human promyelocytic leukemia cells (HL-60 cells) fixed in ethanol. After denaturation of DNA, the cells were stained by an indirect immunofluorescence method using a commercially available monoclonal anti-BrdUrd antibody or by propidium iodide. The relative fluorescence intensities of stained BrdUrd and double-stranded DNA were altered reciprocally by changing HCl concentration and/or denaturation time. Treatment with 4N HCl for 10-20 min at 20 degrees C allowed denaturation of more than 80% of DNA and the maximum BrdUrd-linked immunofluorescence. Under this condition, the coefficient of variation of the DNA histograms remained relatively small.     

An improved and rapid HPLC-EC method for the isocratic separation of amino acid neurotransmitters from brain tissue and microdialysis perfusates

An improved, HPLC with electrochemical detection method for the isocratic separation and determination of amino acids from post-mortem brain tissue and from microdialysates of awake-behaving animals is described. Optimal conditions that maximize stability, resolution, and sensitivity were determined for the pre-column derivatization of amino acids using o-phthalaldehyde and B-mercaptoethanol. Ten different amino acids including aspartate, glutamate, taurine, tyrosine and GABA were effectively resolved within 18 min. Tissue measurements from caudate, globus pallidus and substantia nigra showed regional variations in amino acid content. Microdialysis of the striatum yielded significant amounts of all amino acids examined, including GABA, from only 25 microliter of perfusate.     

Changes in Crassulacean Acid Metabolism of Kalanchoe blossfeldiana by Different Nitrogen Sources

Kalanchoe blossfeldiana Poelln. cv. Hikan (a Crassulacean acidmetabolism (CAM) plant) was grown in pots containing soil for6 months and then cultured in nutrient solution containing 10mM nitrate or ammonium as a sole nitrogen source for 2 or 3months, under a long-day (16 h) condition. Plant growth was better in the nitrate medium. Leaves of thenitrate-grown plants showed greater diurnal fluctuations intitratable acidity and malate content than those of the ammonium-grownplants. The diurnal patterns in CO₂ exchange of nitrate-grownplants were basically similar for both groups, but the amountof net CO₂ uptake at night was twice as large in the nitrate-grownplants. The leaves of the nitrate-grown plants had 1.3 to 2.5times higher activities of phosphoenolpyruvate carboxylase (PEPC),phosphofructokinase (PFK) and NAD glycelaldehyde-3-phosphatedehydrogenase (G3PDH). These results indicate that K. blossfeldianagrown in nitrate medium showed more CAM activity than thosein ammonium medium. (Received August 13, 1987; Accepted February 22, 1988)     

Arabinogalactan-Proteins from Primary and Mature Roots of Radish (Raphanus sativus L.)

Organ-specific variations in blood group H-like activity were observed in developing radish plants. A temporary increase in serological activity was found to occur in the roots at the earlier stages of development. Arabinogalactan-proteins (AGPs) were isolated from primary and mature roots, and investigated for changes in their physicochemical properties, structure, and serological activities. These root AGPs were composed mainly of l-arabinose and d-galactose but were distinguishable from each other in their contents of l-fucose as well as of protein and hydroxyproline. The structures of the carbohydrate moieties of the root AGPs were essentially similar to those of AGPs isolated from seeds and mature leaves in that they consisted of consecutive (1→3)-linked β-d-galactosyl backbone chains having side chains of (1→6)-linked β-d-galactosyl residues, to which α-l-arabinofuranosyl residues were attached in the outer regions. One prominent feature of the primary root AGPs was that they contained appreciable amounts of l-fucose, which was presumably responsible for expression of the serological activity. In their immunological reactions with rabbit anti-radish leaf AGP antibody, the root AGPs were shown to share common antigenic determinant(s) with those of seed and leaf AGPs.     

Purification and characterization of c-Ki-ras p21 from bovine brain crude membranes

In the present studies, we attempted to purify the native molecular forms of the c-ras proteins (c-ras p21s) from bovine brain crude membranes and separated at least three GTP-binding proteins (G proteins) cross-reactive with the antibody recognizing all of Ha-, Ki-, and N-ras p21s. Among them, one G protein with a Mr of about 21,000 was highly purified and characterized. The Mr 21,000 G protein bound maximally about 0.6 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 30 nM. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by pretreatment with N-ethylmaleimide. Mr 21,000 G protein hydrolyzed GTP to liberate Pi with a turnover number of about 0.01 min-1. Mr 21,000 G protein was not copurified with the beta gamma subunits of the G proteins regulatory for adenylate cyclase. Mr 21,000 G protein was not recognized by the antibody against the ADP-ribosylation factor for Gs. The peptide map of Mr 21,000 G protein was different from those of the G proteins with Mr values of 25,000 and 20,000, designated as smg p25A and rho p20, respectively, which we have recently purified from bovine brain crude membranes. The partial amino acid sequence of Mr 21,000 G protein was identical with that of human c-Ki-ras 2B p21. These results indicate that Mr 21,000 G protein is bovine brain c-Ki-ras 2B p21 and that c-Ki-ras 2B p21 is present in bovine brain membranes.     

Seedling recruitment ofChamaecyparis obtusa andSciadopitys verticillata in different microenvironments in an old-growthSciadopitys verticillata forest

Seedling recruitment ofChamecyparis obtusa andSciadopitys verticillata in different microenvironments in an old-growthS. verticillata forest was studied for five years. Exposed mineral soil beneath a gap, which was newly created by a typhoon, supported the best seedling emergence forC. obtusa. S. verticillata seemed to require no specific microenvironment for its seedling emergence. Mortality of seedlings was higher for both species beneath a closed canopy, where all new seedlings ofC. obtusa died within a year after their emergence. Beneath the gap, mortality of seedlings was generally lower for both species, regardless of the presence of litter on the ground. The occurrence of saplings was more frequent forC. obtusa beneath the gaps and forS. verticillata beneath a closed canopy. Difference of seedling recruitment behaviour could maintain the coexistence ofC. obtusa andS. verticillata in the canopy layer of this forest in a non-equilibrium state caused by occasional disturbance.