Identification of 4-Chloroindole-3-acetic Acid and Its Methyl Ester in Immature Seeds of Vicia amurensis (the Tribe Vicieae), and Their Absence from Three Species of Phaseoleae

The natural chlorinated auxins 4-chloroindole-3-acetic acid(4-Cl-IAA) and its methyl ester (4-Cl-IAA Me ester) were found,in addition to IAA and its Me ester, by gas chromatography-massspectrometry in immature seeds of Vicia amurensis, a Vicieaespecies. In contrast, only non-chlorinated, IAA and IAA Me esterwere present in immature seeds of three Phaseoleae species.These results are further evidence of the wide distributionof 4-Cl-IAA and its Me ester in various Vicieae. (Received October 3, 1986; Accepted December 22, 1986)     

Changes of DNA methylation level during pre-and postnatal periods in mice

DNA methylation in an adult mammalian body shows tissue-specificity. But when and how the specificity is established in the process of development has not yet been elucidated. Here we have investigated age-dependent changes in the amount of 5-methyldeoxycytidine (5mdC) that DNA of various mouse tissues contains during the late-fetal and postnatal periods, using high-performance liquid chromatography. The tissue-specificity in the 5mdC level was observed in the late-fetal stage, and the level continued to change during the subsequent periods. The most pronounced alterations were observed in brain and liver, where similar biphasic changes were seen, but at different ages. At maturation, the 5mdC levels were high in thymus, spleen and brain, intermediate in lung, and low in liver and sperm. The data demonstrate the importance of the peri- and postnatal periods in establishment of tissue-specificity in 5mdC content.     

Expression of adrenocorticotropin-releasing hormone precursor gene in placenta and other nonhypothalamic tissues in man

Adrenocorticotropin-releasing hormone (CRH) is a peptide originally isolated from the hypothalamus. Immunocytochemical and RIA studies have revealed that CRH-like peptide is also localized in human nonhypothalamic tissues and some tumors. To see if CRH is synthesized in these nonhypothalamic tissues and tumors, we examined preproCRH mRNA in these tissues by Northern blot analysis using a cloned human preproCRH gene as a probe. PreproCRH mRNA was detected in human hypothalamus, cerebral cortex, adrenal gland, placenta, pheochromocytoma, and thymic carcinoid. The content of preproCRH mRNA in placenta was apparently greater than that in the whole hypothalamus.     

Vasoinhibitory effect of NP-252, a new dihydropyridine derivative, in canine cerebral artery

The vasoinhibitory effect of NP-252, a 1,4-dihydropyridine derivative Ca++ antagonist, was examined in canine cerebral artery, and this effect was compared with that of nifedipine. NP-252 (10(-7)M) and nifedipine (10(-6) M) nearly abolished the contraction induced by addition of Ca++ to Ca(++)-free medium containing KC1. NP-252 (10(-6)M) and nifedipine (10(-6)M) attenuated the contraction produced by thromboxane A2 agonist (STA2) in normal medium, and the resultant contractions were 22% (n = 6) and 35% (n = 6) of the control contraction, respectively. The vasoinhibitory effects of NP-252 were significantly stronger than those of nifedipine in canine cerebral artery. NP-252 (10(-7) and 10(-6) M) dose-dependently attenuated nifedipine-resistant Ca(++)-contraction in the presence of STA2 in both canine cerebral and coronary arteries. The inhibitory effect of combined treatment with NP-252 (10(-6) M) and nitroglycerin (10(-6) M) on nifedipine-resistant Ca(++)-contraction in the cerebral artery was additive. These results indicate that NP-252 possesses a stronger vasoinhibitory effect than that of nifedipine in canine cerebral artery.     

Outgrowth dependency of forelimb regeneration on nerves in adult African clawed frog,Xenopus laevis

Summary The relationship between the size and shape of regenerative outgrowth and the quantity of innervation was studied in adult Xenopus laevis. The forelimbs, of which the nerve supply was artificially altered, were amputated midway through the stylopodium and were kept for 1 year. The regenerative outgrowths that formed in normal limbs with an intact nerve supply were mainly spike-shaped and occasionally rod-shaped. However, when the nerve supply to the distal part of the forelimb was augmented by surgically diverting ipsilateral sciatic nerve bundles, the quantity of innervation was increased to about two and a half times that of the normal limb. These hyperinnervated outgrowths were somewhat larger than those of the normally innervated outgrowths and the majority of them were oar-shaped, a type hardly ever encountered in normal regeneration. In contrast, when partial denervation was performed concomitantly with limb amputation, by ablation of the N. radialis at the shoulder joint, the quantity of innervation decreased to about one half that of the normal limb. The outgrowths obtained were spike-shaped in all cases, with their size being about half that of the normally innervated outgrowths. Furthermore, when both the N. radialis and N. ulnaris were ablated in the same way, the amputated limbs were mostly non-regenerative, but some of them regenerated small conical outgrowths. Based on these results, a discussion is presented concerning the relationship between a regenerative outgrowth and the innervation of the forelimb in Xenopus.     

p-nitrophenyl penta-N-acetyl-beta-chitopentaoside as a novel synthetic substrate for the colorimetric assay of lysozyme

p-Nitrophenyl beta-glycosides of N-acetylchitooligosaccharides (PNP-(GlcNAc)n n = 3-5) were examined as substrates for lysozyme [EC 3.2.1.17]. The enzyme released predominantly p-nitrophenyl N-acetyl-beta-D-glucosaminide (PNP-GlcNAc) from each substrate. Furthermore, the initial rate of PNP-GlcNAc formation in lysozyme-catalyzed hydrolysis of p-nitrophenyl penta-N-acetyl-beta-chitopentaoside (PNP-(GlcNAc)5) was about 350 and 25 times faster than those of p-nitrophenyl tri-N-acetyl-beta-chitotrioside (PNP-(GlcNAc)3) and p-nitrophenyl tetra-N-acetyl-beta-chitotetraoside (PNP-(GlcNAc)4), respectively. From these results, a new colorimetric assay method of lysozyme using PNP-(GlcNAc)5 as a substrate was developed on the basis of the determination of p-nitrophenol liberated from the substrate by lysozyme through a coupled reaction involving beta-N-acetylhexosaminidase (NAHase). The assay system gave a linear dose-response curve in the range of 2-120 micrograms of lysozyme in a 15-60 min incubation. The present assay was not significantly influenced by the ionic strength of the medium and was reproducible. This method using PNP-(GlcNAc)5 as a substrate was shown to be useful for lysozyme assay.     

Auxin-Binding Protein in Etiolated Mung Bean Seedlings: Purification and Properties of Auxin-Bindmg Protein-II

An auxin-binding protein (ABP-II) was purified from the extractof etiolated mung bean seedlings by affinity chromatographyon 2,4-D-linked Sepharose 4B and by gel filtration on Sepharose4B and Sephacryl S-200. The molecular weight was estimated tobe about 190,000 by gel filtration on Sephacryl S-200. ABP-IIgave a single band corresponding to a molecular weight of about48,000 on SDS-polyacrylamide gel electrophoresis. The dissociationconstants of ABP-II for 2,4-D determined by amrnonium sulfateprecipitation and equilibrium dialysis were 9.5?10^–6 Mand 1.1?10^–5 M, respectively. ¹⁴C-2,4-D-binding to ABP-IIwas reversible and inhibited by addition of IAA, naphthalene-1-aceticacid, 2,4,5-trichlorophenoxyacetic acid or p-chlorophenoxyisobutylicacid to the assay mixture. (Received September 5, 1984; Accepted November 5, 1984)