Resolution of bovine brain calcineurin subunits: stimulatory effect of subunit B on subunit A phosphatase activity

Calcineurin was dissociated into subunits A and B by SDS and the dissociated subunits were separated by Sephadex G-100 column chromatography in SDS. The phosphatase activity was associated with the A subunit and was detected only in the presence of MnCl2 of the various divalent cations tested. The Mn2+-dependent phosphatase of A subunit was stimulated (4-5-fold) by calmodulin. The subunit B increased only modestly Mn2+ stimulated phosphatase activity of subunit A but markedly increased it when assay also contained calmodulin. These results support the view that subunit B plays an important role in Mn2+/calmodulin regulation of subunit A phosphatase activity. They also lend further support to our earlier postulate ([1984] FEBS Lett. 169, 251-255) that Mn2+ is a powerful regulator of calcineurin phosphatase.     

Asplenetin,a flavone and its glycoside from Launaea asplenifolia

A new flavone, asplenetin, has been isolated from Launea asplenifolia and characterized as 5,7,3′,4′,5′-pentahydroxy-3-(3-methylbutyl)flavone. Its glycoside, asplenetin 5-O-neohesperidoside, is also reported.     

Kinetic properties of IAA oxidase from mung bean cotyledons

A crude enzyme preparation from mung bean cotyledons was separated into peroxidative and non-peroxidative IAA oxidase on a DEAE-cellulose column. Both fractions differed in their pH optima, K_m and V_max. The K_m and V_max of non-peroxidative IAA oxidase were higher than those of peroxidative IAA oxidase. Peroxidative IAA oxidase showed a linear increase in absorption at 247 and 254 nm after a short lag of 2–3 min. The addition of catalytic amounts of hydrogen peroxide eliminated the lag period and also enhanced the rate of IAA degradation. The non-peroxidative IAA oxidase fraction, however, did not exhibit any significant increase in absorption at 247 and 254 nm and showed a lag period of 5 min which was not affected by hydrogen peroxide. Instead, addition of the same catalytic amount of hydrogen peroxide inhibited the rate of IAA degradation. The peroxidative IAA oxidase fraction exhibited the reaction kinetics characteristic of peroxidase-catalysed IAA degradation. The rate of IAA oxidation by purified non-peroxidative IAA oxidase was very low. The slow rate of catalysis shown by non-peroxidative IAA oxidase appears to be due to the presence of inhibitor(s).     

NMR studies of intracellular sodium ions in mammalian cardiac myocytes

The unambiguous measurement of intracellular sodium ion [Na+]i by the noninvasive NMR technique offers a new opportunity to monitor precisely the maintenance and fluctuations of [Na+]i levels in intact cells and tissues. The anionic frequency shift reagent, dysprosium (III) tripolyphosphate, which does not permeate intact cells, when added to suspensions of intact adult rat cardiac myocytes, alters the NMR frequency of extracellular sodium ions, [Na+]o, leaving that of intracellular ions, [Na+]i, unaffected. Using 23Na NMR in conjunction with this shift reagent, we have determined NMR-visible intracellular Na+ ion concentration in a suspension of isolated cardiac myocytes under standard conditions with insulin and Ca2+ in the extracellular medium to be 8.8 +/- 1.2 mmol/liter of cells (n = 4). This value is comparable to that measured by intracellular ion-selective microelectrodes in heart tissue. Cardiac myocytes incubated for several hours in insulin-deficient, Ca2+-containing medium prior to NMR measurement exhibited a somewhat lower [Na+]i value of 6.9 +/- 0.5 mmol/liter of cells (n = 3). Reversible Na+ loading of the cells by manipulation of extracellular calcium levels is readily measured by the NMR technique. Incubation of myocytes in a Ca2+-free, insulin-containing medium causes a 3-fold increase in [Na+]i to a level of 22.8 +/- 2.6 mmol/liter of cells (n = 10). In contrast to cells with insulin, insulin-deficient myocytes exhibit a markedly lower level of [Na+]i of only 14.6 +/- 2.0 mmol/liter of cells (n = 4) in Ca2+-free medium. These observations suggest that insulin may stimulate a pathway for Na+ influx in heart cells.     

Dexamethasone-induced phospholipase A2-inhibitory proteins (PLIP) influenced by the H-2 histocompatibility region

Recent work has indicated that the H-2 histocompatibility complex on chromosome 17 influences the degree of glucocorticoid-induced teratogenicity and anti-inflammatory response. Since both of these hormonal actions appear to be mediated by the induction of phospholipase A2-inhibitory proteins (PLIP), the influence of the H-2 complex on the induction of PLIP by glucocorticoids in thymocytes and embryonic palates has been investigated. Analysis of dexamethasone-induced PLIP by Sephadex G-100 revealed four peaks of mol wt 55,000, 40,000, 28,000 and 15,000 in mouse thymocytes and from one to three of these PLIPs in mouse embryonic palates. The 55,000 mol wt PLIP comprised 50-60% of the total activity. The total amount of dexamethasone-induced PLIP is significantly higher in B10.A (H-2a) thymocytes than that in thymocytes of their congenic resistant partners, B10 (H-2b). The induced level of PLIP in the embryonic palates treated with dexamethasone is also significantly higher in the H-2a congenic strains with either the A or B background (AWy or B10.A) than that in their resistant partners (A.BY or B10). Thus, both susceptibility to glucocorticoid-induced cleft palate and the production of PLIP by this hormone are influenced by the H-2 complex.     

Selection of diverse genotypes for heterosis in yield and response in toria (Brassica campestris L.)

Summary Thirty-one genotypes of toria (Brassica campestris L.) were grown in twelve environments and subsequently analysed in order to select potential parents which expressed diversity for both 12 different characters (estimated by Mahalanobis' D² technique) and response to the environments (estimated on the basis of negative correlation between deviations in seed yield of a pair of genotypes from their respective environmental means). Coefficients of determination (r²) were also used to measure the reliability of correlation, which is the basis of diversity of response. Stability parameters (b and S²d) and mean seed yield were also considered in selecting potential parents. On the basis of these criteria, three pairs of genotypes (ITSA and TCSU-1, TCSU-7 and TH-8 and Ludhiana Composite-1 and TH-4) are recommended to be used as parents for hybridization programmes so that heterosis both in seed yield and response may be exploited.     

Detection of antimycin-binding subunits of Complex III by photoaffinity-labeling with an azido derivative of antimycin

Deformamidoazidoantimycin A (DAA), a photoactive derivative of antimycin A containing an azido group substituting for the formamido group attached to the phenyl ring, was synthesized. The ultraviolet spectrum of DAA was almost identical to that of antimycin A, indicating little alteration of the electronic structure of the substituted phenyl ring by the azido substitution. However, the inhibitory effectiveness of DAA toward ubiquinol-cytochromec reductase (Complex III) purified from bovine heart (K _i =ca. 0.5 µM) was considerably less than that of antimycin (K _i 3 pM), indicating a direct rather than a supporting role of the formamido group in the inhibitory activity of antimycin. Exposure of purified Complex III to [³H]DAA plus ultraviolet light caused a major labeling by tritium of SDS-PAGE band 7 (m=13 kDa by SDS-PAGE) and lesser but significant labeling of bands 3, 6, 8, and 9. Pretreatment of Complex III with antimycin greatly suppressed the labeling of bands 5, 6, and 7 but caused an apparent increased labeling of bands 8 and 9 by [³H]DAA, respectively. The labeling of band 7 by [³H]DAA also was strongly suppressed by reduction of Complex III by either sodium borohybride or ascorbate. Based on magnitude of labeling by [³H]DAA and the degree of suppression of labeling by antimycin, the protein of band 7 qualified as the principal component for specific binding of antimycin with the protein of band 6 (m=16 kDa) showing a lesser but significant amount of specific binding.     

Callus initiation and plant regeneration in ragi (Eleusine coracana Gaertn.)

Callus was successfully initiated on root, mesocotyl and leaf base segments of 3- to 4-day-old seedlings of ragi (Eleusine coracana Gaertn.). 2,4-D along with casein hydrolysate for Murashige and Skoog's basal medium was found to be most effective for callus initiation and maintenance. Mesocotyl and leaf base tissue derived calli gave shoot buds in medium in which the 2,4-D concentration was lowered.     

Identification of a phosphoenolpyruvate:fructose 1-phosphotransferase system in Azospirillum brasilense

An inducible phosphoenolpyruvate:fructose phosphotransferase system has been detected in Azospirillum brasilense, which requires a minimum of two components of the crude extracts for activity: (i) a soluble fraction (enzyme I) and (ii) a membrane fraction (enzyme II). The uninduced cells neither show any uptake of fructose nor express activity of either of these two enzyme fractions. C-1 of fructose is the site of phosphorylation. This phosphotransferase system does not accept glucose as a substrate for phosphorylation.