Benzyladenine-Enhanced Growth of Attached Young Bean Leaves. Studies with Inhibitors of Nucleic Acid and Protein Synthesis

Benzyladenine (BA) stimulated division but not expansion ofmesophyll cells and repressed chlorophyll accumulation in attachedyoung bean leaves. Even in the presence of fluorodeoxyuridine(FUdR) or mitomycin C which causes complete suppression of BA-inducedincrease in DNA content, BA increased RNA and protein contentsand fresh weight, but decreased chlorophyll accumulation. Moreover,BA n the presence of FUdR induced marked cell expansion. Inthe presence of a-amanitin (AM), BA did not produce any changein DNA content, fresh weight or cell size. All of the BA effectswere observed even in the presence of fluorouracil (FU) plusthymidine (TdR). AM and cycloheximide added 0–12 h effectively inhibitedBA-stimulated cell division but showed no effect if added at18 h. FU plus TdR added 0–18 h had almostno effect onthe cell number at 24 h. These results indicate that BA stimulates the mRNA synthesisnecessary for induction of cell division, and that the synthesisof cytoplasmic rRNA is not always necessary for BA-stimulatedcell division, and moreover, that BA stimulates expansion growthof cells in which DNA synthesis is suppressed. (Received August 16, 1982; Accepted March 31, 1983)     

METHOD FOR COMBINED ULTRASTRUCTURAL AND BIOCHEMICAL ANALYSIS OF NEURAL TISSUE

No significant change was found in the electrolytes and lipids of the brain analyzed after glutaraldehyde fixation by perfusion of laboratory animals; such fixation also satisfactorily preserves neural tissues for electron microscopy. The brains of normal and tumor-bearing C3H mice, Wistar rats, and New Zealand rabbits were studied. Little difference was found in the dry weight and the content of sodium, potassium, total lipid and lipid fractions, and in the sulfate space (S³⁵O₄) between specimens from unperfused and perfused animals, whether normal or tumor-bearing. The results suggest the possibility of using selected regions of the nervous system, dissected after fixation, for chemical study and at the same time characterizing similar regions morphologically with the electron microscope.     

Isolation of Glutamyltaurine from Bovine Brains and Proof of Its γ-Linkage by the B/E Linked Scan SIMS Technique

We isolated a glutamyltaurine from bovine brains and determined its structure as gamma-glutamyltaurine (gamma-Glu-Tau; glutaurine) by use of a new mass spectrometric technique [B/E linked scan sputtered ion mass spectrometry (SIMS)], which we have recently shown to be useful for distinguishing the gamma- from the alpha-isomer of glutamyl-dipeptides. Neither the alpha-isomer of glutamyltaurine nor any aspartyltaurines could be detected in bovine brain.     

Metabolism of exogenous galactosylceramide in the twitcher mouse brain

The in vivo metabolism of galactosylceramide (gal-cer) in normal mice and in twitcher mice, a model of human GLD, was examined following intracerebral administration of gal-cer containing [1-¹⁴C]stearic acid. In normal mice, gal-cer was hydrolyzed to ceramide within 6 hours and ceramide was hydrolyzed to sphingosine and fatty acid. Most of the released fatty acid was immediately incorporated into other lipids. About 75% of injected gal-cer was hydrolyzed 80 hours after the injection, while in the twitcher mouse, only 17% of gal-cer was hydrolyzed. These results show that degradation of gal-cer is impaired in the twitcher mouse brain, but contradict to the fact that there was no evidence of any accumulation of gal-cer in the brain. This discrepancy may be due to the different sorting routes of biosynthesized and exogenously-administered gal-cer in the mouse brain. Most of the biosynthesized gal-cer is incorporated into myelin, while the injected gal-cer is incorporated into lysosomes.     

Utilization of Anhydrous Aluminum Phosphate as a Sole Source of Phosphorous by a Selected Carrot Cell Line

Two variants of carrot (Daucus carota L. cv. MS Yonsun) celllines, which had been selected with Al-phosphate as a sole sourceof phosphorous, were characterized on their mechanisms of phosphate-utilizationfrom Al-phosphate. Both cell lines excreted citrate into themedium. The amount of citrate excretion was highly correlatedwith cell growth in the presence of Al-phosphate. There wasabout a 1 to 1 correlation between solublized-Al and excreted-citratein the medium during cell growth. These results suggest that1) the citrate could chelate with Al, at a 1 to 1 ratio, inAl-phosphate, 2) the citrate-chelated Al remains outside thecells, and 3) solublized phosphate from Al-phosphate is utilizedfor the growth of carrot cells. The characteristics of the selectedcells were very stable, since the rate of citrate-excretionshowed no change after subculturing 25 passages without Al-phosphate. (Received August 7, 1989; Accepted October 19, 1989)     

Unusual Branching in the Seedlings of Lotus japonicus--Gibberellins Reveal the Nitrogen-sensitive Cell Divisions within the Pericycle on Roots

We studied the effects of several plant-growth regulators onthe induction of nodule-like structures on roots of Lotus japonicus,which has been proposed as a candidate for a leguminous plantfor molecular genetic analysis. Contrary to our expectations,the addition of gibberellin A₃ (GA₃) at concentrations of 10^-4M to 10^-4 M resulted in the formation of nodule-like structureson roots when seedlings were plated on nitrogen-free Fahraeusagar medium. GA₄ also induced such outgrowths but was less activethan GA₃. Application of an inhibitor of auxin transport, N-(1-naphthyl)-phthalamicacid (NPA) and of kinetin, which have been reported to inducepseudonodules in other legumes, had no effect on L. japonicus.Microscopic observations showed that GA₃-induced nodule-likestructures were caused by cell divisions within the pericycleon the roots. In addition, the outgrowths elicited by GA₃ couldbe completely suppressed by the addition of 15 mM potassiumnitrate or ammonium nitrate. These results show that the pericyclecells of the roots of L. japonicus are specifically sensitiveto gibberellins and that potential for cell division might bemodulated by nitrogen compounds. We also examined the effectsof ancymidol and uniconazole [S-3307; (E)-1-(4-chIorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol],two synthetic plant-growth retardants. Both compounds at 3 x10^-5 M significantly increased the number of stunted lateralroots. The unusual branching could not be counteracted by theexogenous addition of GA₃ but by 10^-6 M brassinolide. We discussthe physiological role of brassinolide in the initiation oflateral roots. (Received August 4, 1995; Accepted March 11, 1996)     

The Excessive Production of Indole-3-Acetic Acid and Its Significance in Studies of the Biosynthesis of This Regulator of Plant Growth and Development

Because of the importance of indole-3-acetic acid (IAA) in thegrowth and development of plants, extensive studies of the biosynthesisof IAA have been performed during the four decades since thediscovery of IAA as a plant hormone. The pathway for the biosynthesisof IAA in plants remains, however, to be unelucidated, eventhough studies within the past decade have revealed unexpectedaspects of such biosynthesis. By contrast, two pathways to IAAhave been characterized in bacteria at the molecular level:the indole-3-acetamide (IAM) pathway (L-tryptophan     

Efficient Conversion of L-Tryptophan to Indole-3-Acetic Acid and/or Tryptophol by Some Species of Rhizoctonia

In a study of various phytopathogenic fungi, we found that fungithat belong to the genus Rhizoctonia produce IAA efficientlyfrom tryptophan. R. solani Kühn MAFF-305219, in particular,produced large amounts of tryptophol (Tol), which was assumedto be a specific by-product of the indole-3-pyruvate (IPy) pathway,in addition to IAA. Therefore, this fungus seemed suitable foranalysis of the function and the regulation of the biosynthesisof auxin by a fungal pathogen. Under normal aerobic conditions,the ratio of IAA to Tol synthesized by this strain was higherthan that under less aerobic conditions. In metabolic studieswith various indole derivatives, R. solani converted L-tryptophanand indole-3-acetaldehyde to IAA and Tol, but other indole derivativeswere scarcely metabolized. These results suggest that both IAAand Tol are synthesized from tryptophan through the IPy pathwayin Rhizoctonia. (Received May 27, 1996; Accepted July 8, 1996)     

Induction of Manganese Superoxide Dismutase by Cytokines and Lipopolysaccharide in Cultured Mouse Astrocytes

Abstract: To determine whether cytokines or lipopolysaccharide (LPS) are involved in the induction of superoxide dismutase (SOD) in the nervous system, we examined the effects of these substances on the levels of SOD in cultured mouse astrocytes. Treatment of astrocytes with 10² to 10⁴ U/ml tumor necrosis factor-α for 3 days increased the levels of Mn SOD in a dose- and time-dependent manner to as much as six times the level under nontreated conditions. Treatment with 1.0 µg/ml LPS for 3 days elicited a fourfold increase in levels of Mn SOD, and the effect of LPS was also dose dependent. Furthermore, Mn SOD in astrocytes was induced by a 3-day exposure to interleukin-1α at concentrations of 10² or 10³ U/ml. However, these stimuli had no effect on levels of copper-zinc SOD (Cu/Zn SOD) in astrocytes. By contrast, interferon-γ did not change the levels of either Mn or Cu/Zn SOD in the cells. The results indicate that the selective induction of Mn SOD by cytokines and LPS, which has been observed in nonnervous tissues, may also occur in nervous tissues. The induction of Mn SOD may represent a mechanism for protection of cells from oxidative stress.     

Mutation analysis of a Sandhoff disease patient in the Maronite community in Cyprus

Sandhoff disease occurs in the Christian Maronite community in Cyprus, a community that established over a thousand years ago. Nowadays, this community comprises less than 1% of the whole population, and has been culturally and socially isolated. Cultured fibroblasts from a patient from this inbred group showed a -hexosaminidase subunit mRNA of apparently the normal size but of reduced quantity. A mutational analysis of cDNA obtained by polymerase chain reaction amplification of mRNA showed a deletion of A at nt 76 (counted from A of the initiation codon, ATG). The deletion results in a frame shift and a premature termination within 20 amino acids from the N-terminus of the normal mature enzyme protein. The patient was homozygous for the deletion. The 5-end of the gene showed many discrepancies from the previously published sequence. We consider that these differences are probably polymorphisms of little functional significance, because the patient's fibroblasts generate decreased but stable mRNA and because some of these base changes were also found in the genes from control fibroblasts. An extensive evaluation of the prevalence of this mutant allele in this community is being initiated.