Effects of Glyphosate on the Shikimate Pathway and Regulation of Phenylalanine Ammonia-lyase in Cryptomeria and Perilla Cell Suspension Cultures

Treatment of Cryptomeria and Perilla cell suspension cultureswith glyphosate resulted in a marked suppression of the formationof flavans and caffeic acid derivatives, respectively, whileit caused only a slight decline in the cell growth. In contrastwith 3-deoxy-D-arabino-heptulosonate (DAHP) synthase-Mn isozyme,DAHP synthase-Co isozyme from Cryptomeria and Perilla cellswas much more sensitive to inhibition by glyphosate. The additionof 1 to 2 mM glyphosate caused an accumulation of shikimateand quinate and a reduction of L-phenylalanine in both cellcultures. The inhibition of phenylalanine ammonia-lyase (PAL)activity by glyphosate was reversed by exogenously suppliedL-phenylalanine to near the control level. Cycloheximide andactinomycin D nullified the recovery by exogenous L-phenylalanineon PAL activity. L-Phenylalanine itself promoted PAL activityto some extent. No recovery of PAL activity in L--aminooxy-ß-phenylpropionate(L-AOPP)-treated cell cultures could be observed by the additionof L-phenylalanine. Therefore, L-AOPP seems to inhibit the formationof PAL, though it has been considered a competitive inhibitor. ³Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai 980, Japan. (Received October 28, 1985; Accepted March 13, 1986)     

Identification of jasmonic acid in Mimosa pudica and its inhibitory effect on auxin- and light-induced opening of the pulvinules

Jasmonic acid was identified from Mimosa pudica L. plants by mass spectrometry, high performance liquid chromatography and thin layer chromatography. Effects of authentic jasmonic acid on pulvinule movement and transpiration of the pinnae were compared with those of abscisic acid. Jasmonic acid and abscisic acid each at 10⁻⁵ M inhibited both auxin- and light-induced opening of the pulvinules. A closure-inducing activity of jasmonic acid at 10⁻⁴ M was greater than that of abscisic acid at 10⁻⁴ M. Pinnae transpiration was reduced by 10⁻⁵ M abscisic acid but not by 10⁻⁴ M jasmonic acid.     

Determination of 9α,11β-prostaglandin F2 by stereospecific antibody in various rat tissues

In view of the recent finding that prostaglandin D₂ is stereospecifically converted to 9α,11β-prostaglandin F₂, an isomer of prostaglandin F₂α, a highly specific and sensitive radioimmunoassay for 9α,11β-prostaglandin F₂ was developed and applied to determine the content of this prostaglandin in various rat tissues. Antisera against 9α-11β-prostaglandin F₂ were raised in rabbits immunized with the bovine serum albumin conjugate, and [³H]9α,11β-prostaglandin F₂ was enzymatically prepared from [³H]prostaglandin D₂. The assay detected 9α,11β-prostaglandin F₂ over the range of 20 pg to 1 ng, and the antiserum showed less than 0.04% cross-section with prostaglandin F₂α, prostaglandin F₂β and 9β,11β-prostaglandin F₂. To avoid postmortem changes, tissues were frozen in liquid nitrogen immediately after removal. The basal level of 9α,11β-prostaglandin F₂ was hardly detectable in various tissues of the rat examined, including spleen, lung, liver and brain; although it was found to be 0.31 ± 0.06 ng/g wet weight in the small intestine. During convulsion induced by pentylenetetrazole, enormous amounts of prostaglandin D₂ (ca. 180 ng/g wet weight) and prostaglandin F₂α (ca. 70 ng/g) were produced in the brain; however, 9α,11β-prostaglandin F₂ was detected neither there nor in the blood. This result demonstrates that the conversion to 9α,11β-prostaglandin F₂ is a minor pathway, if one at all, of prostaglandin D₂ metabolism in the rat brain.     

Conversion of 4-aminobutyraldehyde to γ-aminobutyric acid in striatum treated with semicarbazide and kainic acid

4-Aminobutyraldehyde (ABAL) has been shown to cross the blood-brain barrier and to be converted rapidly to -aminobutyric acid (GABA) in various regions of the brain. In this paper, the formation of GABA from ABAL was studied with striatum that had suffered a lesion to GABA synthesis via glutamic acid decarboxylase (GAD). The GABA formation from ABAL was invariably observed in striatum in which GAD was severely inhibited by semicarbazide or kainic acid. Thus, this is another pathway for GABA formation.     

Plasmodium falciparum: Attenuation by irradiation

The effect of irradiation on the in vitro growth of Plasmodium falciparum was investigated. The cultured malarial parasites at selected stages of development were exposed to gamma rays and the sensitivity of each stage was determined. The stages most sensitive to irradiation were the ring forms and the early trophozoites; late trophozoites were relatively insensitive. The greatest resistance was shown when parasites were irradiated at a time of transition from the late trophozoite and schizont stages to young ring forms. The characteristics of radiosensitive variation in the parasite cycle resembled that of mammalian cells. Growth curves of parasites exposed to doses of irradiation upto 150 gray had the same slope as nonirradiated controls but parasites which were exposed to 200 gray exhibited a growth curve which was less steep than that for parasites in other groups. Less than 10 organisms survived from the 10⁶ parasites exposed to this high dose of irradiation; the possibility exists of obtaining radiation-attenuated P. falciparum.     

Replication of Deoxyribonucleic Acid in Escherichia coli C Mutants Temperature Sensitive in the Initiation of Chromosome Replication

An Escherichia coli HF4704S mutant temperature sensitive in deoxyribonucleic acid (DNA) synthesis and different from any previously characterized mutant was isolated. The mutated gene in this strain was designated dnaH. The mutant could grow normally at 27 C but not at 43 C, and DNA synthesis continued for an hour at a decreasing rate and then ceased. After temperature shift-up, the increased amount of DNA was 40 to 50%. When the culture was incubated at 43 C for 70 min and then transferred to 27 C, DNA synthesis resumed after about 50 min, initiating synchronously at a fixed region on the bacterial chromosome. The initiation step in DNA replication sensitive to 30 mug of chloramphenicol per ml occurs synchronously before the resumption of DNA replication after the temperature shift-down, being completed about 30 min before the start of DNA replication. When the cells incubated at 27 C in the presence of 30 mug of chloramphenicol per ml after the temperature shift-down to 27 C were transferred to 43 C with simultaneous removal of the antibiotic, no resumption of DNA replication was observed. When the culture was returned to 43 C after being released from high-temperature inhibition at 30 min before the start of DNA replication, no recovery replication was observed; whereas at 20 min, the recovery of replication was observed. These results indicated that HF4704S was temperature sensitive in the initiation of DNA replication. Analysis of HF4704S, by an interrupted conjugation experiment, indicated that gene dnaH was located at about 64 min on the E. coli C linkage map. In E. coli S1814 (a K-12 derivative), which was a dnaH(ts) transductant from HF4704S (C strain) with phage P1, the mutated gene (dnaH) was demonstrated to be closely linked to the thyA marker by conjugation and P1 transduction experiments and to be distinct from genes dnaA through dnaG.     

Electron microscopic histochemistry of acetylcholinesterase of rat brain by Karnovsky's method

Summary Karnovsky's electron microscopic acetylcholinesterase method was successfully applied to rat brain fixed by vascular perfusion with either 2% glutataldehyde or 4% formaldehyde. 2% glutaraldehyde showed better fine structure but worse preservation of the enzyme than 4% formaldehyde.In the neuropil of the caudate nucleus, locus coeruleus and dorsal nucleus of the vagus, AChE activity was most intensely demonstrated on the plasma membranes of preterminal axons and somewhat less strongly on those of axon terminals and contacting dendritic branches. The axoplasm and synaptic vesicles were usually negative, while the cytoplasm and neurotubules of the dendritic branches showed some activity. In the nodule and uvula of the cerebellum moderate activity was exhibited on the synaptic contacts between the mossy fiber endings and granule cell dendrites. In the hypothalamus and other autonomic regions the characteristic coexistence of AChE and granulated vesicles of axon terminals could be demonstrated.In the perikaryon of positive nerve cells, AChE was observed strongly in the cytoplasm, disseminated irregularly or attached to the endoplasmic reticulum, while it was absent in the mitochondria and lysosomal dense bodies.     

Conformation of the fowl protamine, galline, and its binding properties to DNA

1. CD spectra showed that the fowl protamine, galline, has an unordered structure rich in reverse turns in neutral solution. Eight reverse turns were predicted to be present in the galline molecule on the basis of its amino acid sequence. Spectrophotometric analyses revealed that galline efficiently bound to DNA in 0.25 mM EDTA/10 mM Tricine-HCl, pH 7.4, but hardly so in 30 mM NaCl/3 mM sodium citrate, pH 7.0. Citrate ions bound specifically to the galline molecule, causing a conformational change in it. As a result, galline could not interact with DNA. 2. The concentration of unbound galline in a mixture of DNA and galline in 100 mM NaCl/50 mM Tricine-HCl, pH 7.4, at 37 C was determined by measurement of the intrinsic fluorescence of tyrosine residues of galline in the supernatant after ultracentrifugation of the mixture. The Scatchard plot showed positive co-operativity in the binding of galline to DNA and the binding parameters were determined: the co-operative binding constant (Kc) = 3.3 X 10(7)M-1, the co-operativity factor (q) = 800, and the number of nucleotides of DNA occupied by one galline molecule (n) = 28. The Kc and q values were intermediate between those for clupeine Z from herring sperm and S-methyl protamine from boar sperm. That is, the binding constants of protamine as to DNA decrease in the order of herring, fowl, and boar, while the co-operativities in binding increase in that order.     

Reversion of the apical hook of pea in the dark and its promotion by a red light pulse.

At the developmental stage at which the apical hook passed the 3rd and 4th nodes, dark-grown seedlings of pea ( Pisum sativum L. cv. Progress No.9) opened the hook upright and then formed a new hook above the node nearly in the opposite direction to the previous one. In cv. Alaska, in contrast, many (about 84%) seedlings closed the hook in the original direction after they partially (up to about 110°) opened it at the 3rd node, thus doing a wagging movement, while a small percentage (about 16%) of the seedlings reversed the hook direction. Exposure to red light of cv. Alaska seedlings for 10 min increased the percentage of the hook reversion up to 71% or more. The hook reversion was never observed except when the hook part passed the nodes, suggesting the involvement of the nodes in the phenomenon.     

ATP-Dependent Depolymerization of Cortical Microtubules by an Extract in Tobacco BY-2 Cells

An extract of tobacco BY-2 cells, prepared by rupturing protoplastsby exposure to centrifugal force, depolymerized cortical microtubuleson protoplast ghosts of BY-2 cells in an ATP-dependent manner.Depolymerization of cortical microtubules by the cell extractdid not occur in the presence of taxol. The extract failed todepolymerize microtubules of phragmoplasts and spindles isolatedfrom BY-2 cells. The activity was detected in a fraction precipitatedbetween 30–50% saturation with ammonium sulfate. Theseresults suggest the presence in the cell extract of a proteinfactor(s) that depolymerizes cortical microtubules in an ATP-dependentmanner. The importance of this factor(s) in the premitotic disappearanceof cortical microtubules is discussed. (Received April 25, 1990; Accepted September 6, 1990)