Inhibitory Effect of Calmodulin Antagonists and Calcium Antagonists on Fertilization-Induced Cyanide-Insensitive Respiration in Sea Urchin Eggs

During initial several minutes after fertilization, sea urchin eggs exhibited high rate of respiration which was only slightly inhibited by cyanide. This cyanide-insensitive respiration was inhibited by calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalensulfonamide hydrochloride (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and chlorpromazine, which were added within 1 min after insemination. The inhibitory effect of W-7 on cyanide-insensitive respiration was higher than that of W-5. Cyanide-sensitive respiration of fertilized eggs observed after this initial period was not inhibited by these compounds. Ca²⁺ influx in eggs just after fertilization was inhibited by calcium antagonists but was rather enhanced by calmodulin antagonists. Fertilization-induced stimulation of cyanide-insensitive respiration probably results from calmodulin-dependent reactions which are activated by Ca²⁺ influx.     

Alicyclic acid metabolism in plants 3. Fate of 14C-shikimate and 14C-quinate in mung bean plants

Young mung bean plants (Phaseolus mungo) were exposed to ¹⁴C-shikimateor ¹⁴C-quinate in the light. After 8 or 23.5 hr of incubationat 25°C, radioactivities in free and bound amino acids,organic acids, soluble and insoluble carbohydrates, ether-solublefraction and lignin were determined. Shikimic and quinic acidswere separated by the combined use of paper-chromatography andcolumn chromatography. Specific activity of formed quinate orshikimate was only slightly lower than that of fed shikimateor quinate. Specific activities of phenylalanine, tyrosine andbound tryptophan were high as compared with those of non-aromaticamino acids. Discussion is focused upon the interconversionbetween shikimate and quinate, and their roles in the biosynthesisof aromatic amino acids. (Received November 15, 1968; )     

Stem Nodules in Aeschynomene indica and Their Capacity of Nitrogen Fixation

There has been no report on stem nodules with nitrogen fixing activity. Aeschynomene indica produce nodules on the aerial parts, and the stem nodules proved to be capable of considerable nitrogen fixation [C₂H₄ produced c. 4 μmol (g fresh weight)^-1 h^-1]. The stem nodules embed reddish tissues, and a rod-shaped bacterium was isolated from the tissues. The bacterium was ascertained to form root and stem nodules on seedlings of A. indica.     

Partial Synchronization of Carrot Cell Culture by Auxin Deprivation

A strain of carrot cells (Daucus carota cv. Kintoki) grew exponentially in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D, 1 mg/1) with a doubling time of about 2 days. When those cells were transferred to a medium lacking 2,4-D, they continued to grow at almost the same rate for about a week. When the cells were again transferred to the auxin-free medium, the rate of cell division gradually decreased. After the cell division had ceased, cells were returned to the ordinary 2,4-D medium. A burst of cell divisions occurred after about 2 days. Timing of DNA synthesis and of mitosis suggested that the cells had been arrested at G₁ phase. In a medium containing indoleacetic acid instead of 2,4-D, the auxin was rapidly degraded and the culture was similarly synchronized as in the auxin-omitted medium.     

Application of Fluorescent Antibody for Detecting Capsular Substances in Staphylococcus aureus

A fluorescent antibody technique was developed for the determination of the capsular-type of strains of Staphylococcus aureus. It compared favourably with the method using serum-soft agar (Yoshida 1972). With the new technique, many populations of encapsulated and unencapsulated strains were investigated. Of 1421 fresh isolates of Staph. aureus , 54 were encapsulated and among these 54·8% and 48·1% were mono- and polyvalent, respectively. Capsular-type antigens A and B were found in 92·5% and 44·4% of strains respectively; capsular-types C and D were found relatively infrequently. In the other group, of unencapsulated strains, capsular-type antigen production was demonstrated in 125 out of 163 strains examined. Mono- and polyvalent capsular-types (A and B antigen producing strains) comprised 77·6% and 22·4%. respectively. In these capsular-types A and B were found in 54·4% and 62·4%, respectively: capsular-type antigen C and D producing strains were again infrequent. These results indicate that a majority of ordinary Staph . aureus strains produce capsular-type antigens although isolation of the encapsulated strains is infrequent.     

2. Alicyclic acid metabolism in plants Enzymes related to the shikimate pathway in developing mung bean seedlings

A study was made of chages in the activities of enzymes relatedto the biosynthesis of aromatic compounds in etiolated mungbean seedlings during their growth. Shikimate: NADP oxidoreductaseactivity in the root-shoot axes increased rapidly to attainits highest activity the 4th day after sowing, and remainedat that level over the experimental period of 7 days. 5-Dehydroquinatehydro-lyase activity continuously increased for at least 7 days.In the cotyledons, a gradual decrease in the activities of theseenzymes occurred. Phenylalanine ammonia-lyase activity in root-shootaxes gradually increased showing a maximum on the 6th day. Thehighest specific activity, on a protein basis, of this enzymewas seen in the initial stage of growth. In the cotyledons,a rise in total activity appeared on the 2nd day. Tyrosine ammonia-lyaseactivity was very low as compared with phenylalanine ammonia-lyase.The enzyme activities of light-germinated seedlings were comparedto those of dark-germinated seedlings on the 7th day. Lighthad practically no significant effect on the appearance of shikimate:NADP oxidoreductase and 5-dehydroquinate hydro-lyase activities.On the other hand, a marked effect from the light on the riseof phenylalanine ammonia-lyase and tyrosine ammonia-lyase activitieswas found, especially in the epicotyl-plumules. The results are discussed with respect to the metabolism ofalicyclic acids such as shikimic acid in the developing mungbean seedlings. ¹This work was partly supported by a grant-in-aid from the Ministryof Education.     

Age-Related Alterations in the Biochemical and Functional Properties of the Bladder in Type 2 Diabetic GK Rats

Previous studies have demonstrated that experimental type 1 diabetes induced by streptozotocin causes alterations in the biochemical and functional properties of several receptor systems in the rat bladder. However, the exact mechanism involved in the pathophysiology of voiding dysfunction in type 2 diabetic patients is unknown. Because the GK rat is a widely accepted genetically determined rodent model for human type 2 diabetes, we investigated diabetes-induced changes in the bladder smooth muscle of the GK rats at several time points. Male GK rats and age-matched Wistar rats, as controls, were maintained for 4, 8, 16, and 32 weeks. Contractile responses to KCl, carbachol, ATP, and electrical field stimulation (EFS) were measured by using the isolated muscle bath techniques. Acetylcholine (ACh) release induced by EFS from bladder muscle strips was measured by using high-performance liquid chromatography coupled with a microdialysis procedure. Maximum contractile responses to carbachol and ATP, the release of ACh, and tissue sorbitol levels were similar in bladders from GK and control rats until 8 weeks of age. At 16 weeks of age, however, the contractile responses to carbachol and ATP, and tissue sorbitol levels were increased, and the EFS-induced ACh release was decreased in GK rats compared with controls. Although the maximum contractile responses to EFS were unchanged until 16 weeks of age, they were decreased in 32-week-old GK rats, compared with controls. Our data indicate the presence of age-related alterations in the biochemical and functional properties of the bladder in type 2 diabetic GK rats.     

A METABOLIC PATHWAY OF ISOCITRIC ACID IN COTYLEDONS OF A BEAN, VIGNA SESQUIPEDALIS

1. It has been established that the absence of isocitric dehydrogenaseactivity in cotyledons of Vigna sesquipedalisin the germinationstage is due to the lack of endogenous TPN. It is unlikely thatthe TCA cycle in the cotyledon is operative.
2. The activitiesof enzymes of the glyoxylate cycle, a modifiedTCA cycle, weremeasured through the germination stage. Theiractivities wereconsiderably higher in the cotyledon tissuethan in the hypocotyl.A dominant metabolic pathway of isocitricacid in the cotyledonmay be the glyoxylate cycle.
3. DPN kinase which produces TPNfrom DPN, ATP and Mg⁺⁺ was localizedin the supernatant portionof cell components in the cotyledon.Its activity was low. DPNkinase serves as a control factorfor the cotyledon metabolism.

(Received April 17, 1961; )