Control of cocklebur seed germination by nitrogenous compounds : Nitrite, nitrate, hydroxylamine, thiourea, azide and cyanide

Germination of non-dormant upper cocklebur (Xanthium pinsylvanicumWallr.) seeds was stimulated by not only CS(NH₂)₂ but also NH₂OH,KCN and NaN₃. This stimulation was not via the enhancement ofaerobic C₂H₄ production. NH₂OH, KCN and NaN₃ in certain concentrationspromoted the initial growth of axial and/or cotyledonary parts,but the degree of growth promotion by NH₂OH, NaN₃ and KCN wasslight compared with that by CS(NH₂)₂. As in the case of CS(NH₂)₂,however, the germinationstimulating effect of NH₂OH disappearedrapidly as the preceding imbibition period was prolonged. Incontrast, KCN and NaN₃ were still effective in stimulating thegermination of aged seeds maintained on a water substratum,as previously seen with anaerobiosis. Anaerobic induction wasenhanced not only by NaN₃ and KCN but also by NH₂OH, KNO₃, KNO₂CO(NH₂)₂ and CS(NH₂)₂ applied during the anaerobic treatment,but without causing an increase in anaerobic production of C₂H₄.Furthermore, KCN and NaN₃, given prior to the anaerobic treatmentacted additively with anaerobic induction. The germination-stimulatingactions of nitrogenous compounds are discussed in comparisonwith those of C₂H₄ and anaerobiosis. (Received May 6, 1978; )     

Poly(adenosine diphosphate ribose) polymerase activity in neuronal and glial nuclei from bovine cerebrum

Two different preparations isolated from beef cerebrum have been used to compare the polyadenosine diphosphate ribose (polyADPR) polymerase activities in neuronal and glial nuclei: (1) nuclear suspensions (with or without DNase I treatment), and (2) 1 M NaCl nuclear extracts (soluble enzyme). The DNAse I treatment of nuclei and the solubilization of polyADPR polymerase by 1 M NaCl enhances the polyADPR polymerase activity. The polyADPR polymerase activity is similar in neuronal and glial nuclear suspensions, while the neuronal soluble enzyme activity is significantly higher than that of the glial soluble enzyme. Evidence is presented that the difference in soluble enzyme activities is not due to the effects of DNA or degrading enzymes. Some activating factor(s) seem to be present in neuronal soluble extracts, while both inhibiting and activating factor(s) seem to be present in glial soluble extracts.     

Studies on the control of cell division in a green alga,Ankistrodemus gracilids (Chlorococcales)

The proliferation patterns of the green alga,Ankistrodesmus gracilis were investigated by electron microscopy. The multichotomically dividing phase, namely the type I phase was characterized by multichotomical nuclear division, whereas the dichotomically dividing phase, i.e., the type II phase was related to dichotomical nuclear division. Type I phase was controlled by a lower cell density and a diurnal light-dark rhythm of LD 14:10. Type II phase was induced by a cell density of more than 1.0×10⁷ cells/ml. Independent of the 2 types of proliferation patterns, the diurnal light-dark rhythms related to the autospore release from a mother cell. The diurnal light-dark rhythm of LD 14∶10 induced the scattered release of autospores. On the other hand, a rhythm of LD 18∶6 induced the release of a couple of tightly adhered autospores from a mother cell which had been observed apparently as a type II cell by light microscopy.     

Studies on the control of cell division in a green alga,Ankistrodesmus gracilis (Chlorococcales)

The effects of various kinds of nitrogen compounds on the proliferation patterns ofAnkistrodesmus gracilis were examined. Among the tested compounds, the addition of peptone, arginine or glutamine to the N-free medium was the most effective on cell growth. The effectiveness was also shown in media containing one of several kinds of amino acid. In both peptone and arginine media the alga proliferated with multiple fission type of cell division, whereas the cultures grown in glutamine or serine medium contained 2-celled colonies at high frequencies. The latter was an efficient culture condition for cell reproduction with binary fission. The evidence for the behavior of nuclear and cytoplasmic division in these growth patterns were obtained from observations of thin sectioned materials.     

Heparin-binding (fibroblast) growth factors are potential autocrine regulators of esophageal epithelial cell proliferation

Summary A serum-free culture system supplemented with neural tissue extract for normal and tumor human esophagi was applied to the culture of mouse esophageal epithelium. Similar to mouse mesenchyme and skin epithelium, esophageal epithelial lines (MEE) emerged after serial culture. The cells had an apparent unlimited life span but retained morphology and other characteristics of normal epithelial cells. The cells formed a small cyst consisting of keratined squamous epithelium in syngenic hosts. A screen for growth factors that stimulated growth of the nonmalignant MEE cells in the absence of neural extract revealed that epidermal growth factor (EGF) and heparin-binding (fibroblast) growth factors (HBGF) were most effective. An HBGF-like activity was apparent in extracts of rapidly proliferating but not quiescent MEE cells at low or confluent densities. A cloned cell line (MEE/C8) was selected from MEE cell cultures in the absence of neural extract. MEE/C8 cells proliferated independent of either EGF or HBGF at rates equal to MEE cells, cell extracts exhibited HBGF-like activity at all stages of proliferation, and the cells formed large invasive tumors in syngenic hosts. The HBGF-like activity present in extracts of tumorigenic MEE/C8 and proliferating nonmalignant MEE cells had properties similar to HBGF-1 (acidic fibroblast growth factor). These results constitute a cultured mouse esophageal epithelial cell model for study of conversion of immortalized premalignant cells to malignant cells, and suggest that conversion from a state of cell cycle-dependent autocrine expression of one or more members of the HBGF family to a state of constitutive expression correlates with and may contribute to malignancy. The work was supported in part by grants CA37589 and DK35310 to Dr. McKeehan, from the National Cancer Institute, Bethesda, MD.     

Genetic differentiation between two types of dark chub,Zacco temmincki,in Japan

Two types of the dark chub,Zacco temmincki, collected from 10 river systems in Japan were genetically characterized at 27 protein coding loci using starch-gel electrophoresis. They were fixed for different alleles at 13 loci. No hybrid individuals were observed, even in specimens collected in stations where both types appear sympatrically, indicating that each type of the dark chub represents a distinct species.     

Effect of 24,25-dihydroxyvitamin D3 in osteoclasts.

Previous results demonstrated that the administration of pharmacological doses of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) to animals reduces bone resorption and increases bone volume with a decrease in osteoclast number. In order to clarify whether 24,25(OH)2D3 has an effect to inhibit osteoclastic bone resorption, the effect of 24,25(OH)2D3 on the formation and function of osteoclastic cells was examined in vitro. Treatment of hemopoietic blast cells, which are progenitors of osteoclasts, with parathyroid hormone (PTH) or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) stimulated the formation of osteoclast-like multinucleated cells in a dose-dependent manner. Although 24,25(OH)2D3 in itself had little effect on osteoclast-like multinucleated cells formation, it inhibited the stimulatory effect of PTH on the formation of osteoclastic cells. In addition, 24,25(OH)2D3 also inhibited the stimulation of resorption pit formation by osteoclasts under stimulation with PTH. In contrast, 1,25(OH)2D3 stimulated the formation and function of osteoclastic cells even at low concentrations, and the effect was additive to PTH. These results could not be explained by either an agonistic or antagonistic effect of 24,25(OH)2D3 on 1,25(OH)2D3, and are consistent with the assumption that 24,25(OH)2D3 has a unique inhibitory effect on the formation and function of osteoclasts. Because 24,25(OH)2D3 is shown to stimulate the degradation of 1,25(OH)2D3 and because the formation of 24,25(OH)2D3 is stimulated by 1,25(OH)2D3 not only in the kidney but also in many of its target tissues, including bone, the inhibitory effect of 24,25(OH)2D3 on osteoclastic bone resorption may play a role in the local modulation of the actions of osteotropic hormones in bone.     

Secretory expression of a glutamic-acid-specific endopeptidase (SPase) from Staphylococcus aureus ATCC12600 in Bacillus subtilis

Summary In order to obtain a large quantity of glutamic-acid-specific endopeptidase of Staphylococcus aureus ATCC12600 (SPase) without cultivating its pathogenic host bacterium, expression plasmids enabling secretion of SPase from Bacillus subtilis were constructed by inserting the SPase gene into B. subtilis-Escherichia coli shuttle vectors. B. subtilis harbouring a simple recombinant plasmid containing the coding and the 5-flanking regions of SPase in the shuttle vector pHY300PLK secreted 22 mg/l of SPase into the medium. As this level was lower than that of the natural strain (45 mg/l), we tried to increase the expression level by constructing a series of hybrid plasmids with the following features: (1) the terminator sequence of the alkaline protease gene from B. subtilis, (2) the promoter and the leader sequences of the -amylase gene or of alkaline protease gene from B. amyloliquefaciens, (3) the vector pHY300PLK and the fused vector of pHY300PLK and pUB110. By using a variety of hybrid plasmids, the resulting transformants secreted SPase at levels of 33–120 mg/l. The recombinant SPase isolated from the medium was indistinguishable from the natural one with respect to its behaviour on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting as well as its enzyme activity.Correspondence to: S. Kakudo     

Production of recombinant human glucagon in the form of a fusion protein in Escherichia coli; recovery of glucagon by sequence-specific digestion

Summary Recombinant human glucagon was succesfully produced with a high level of expression in Escherichia coli as a fusion protein with human interferon . The synthetic gene was designed to release glucagon, which does not contain glutamic acid residues, from fusion protein with the Staphylococcus aureus strain V8 protease that specifically cleaves the peptide bond on the carboxyl side of the glutamic acid residue. The resulting glucagon was purified to homogeneity by a combination of C₁₈ reverse-phase HPLC and ion-exchange HPLC. The yield of intact glucagon obtained from 11 of culture was approximately 12 mg. The structure of recombinant human glucagon was confirmed by HPLC and amino acid composition/sequence analyses. Offprint requests to: J. Ishizaki