In vitro salt tolerance of cell wall enzymes from halophytes and glycophytes

The halophyte Suaeda maritima grows optimally in high concentrations(40–60% seawater) of salt. In these conditions the concentrationof salt in the apoplast of the leaves is at least 500 mM, aconcentration which severely inhibits the activity of cytoplasmicenzymes of both glycophytes and halophytes. The in vitro salttolerance of a number of cell wall enzymes was assayed in thepresence of a range of concentrations of NaCl. There was nosignificant inhibition of the activity of galactosidase, glucosidase,peroxidase or xyloglucan endo-transglycosylase extracted fromSuaeda maritima by in vitro concentrations of NaCl up to atleast 1 M. In vitro salt tolerance of cell wall enzymes wasnot restricted to the halophyte, similar enzymes from the non-halophilicrelative Kochia tricophylla, and from the glycophytes Vignaradiata and Cicer arietinum, were inhibited little, or not atall, by the same concentrations of salt. Pectin esterase wassomewhat less tolerant, but activity at 500 mM NaCl was stillgreater than at 0 mM NaCl in both Suaeda and Vigna. It is concludedthat these enzymes of the cell wall compartment are much moresalt-tolerant than cytoplasmic enzymes of higher plants. Theresults are discussed in relation to conditions thought to pertainin the apoplast. Key words: Apoplast, cell wall enzymes, halophyte, salt tolerance, Suaeda maritima     

Does calmodulin mediate inhibition of human erythrocyte Ca(2+)-pumping ATPase by myricetin?

Myricetin, a flavonoid, potently inhibits human erythrocyte plasma membrane Ca(2+)-pumping ATPase activity: half-maximal inhibition of the basal and calmodulin-stimulated activity is obtained with 6 microM myricetin. Inhibition of the Ca(2+)-ATPase by myricetin is observed at all concentrations of free Ca2+ from 10(-8)M to 10(-5)M. The extent of inhibition of the Ca(2+)-ATPase is dependent on the time of preincubation of the plasma membranes with myricetin. Inhibition of the Ca(2+)-ATPase by myricetin is not reversed by excess calmodulin. It is concluded that calmodulin does not mediate myricetin's inhibition of human erythrocyte plasma membrane Ca(2+)-pumping ATPase.     

Premixed insulin aspart 30 (BIAsp 30) versus premixed human insulin 30 (BHI 30) in gestational diabetes mellitus: a randomized open-label controlled study

A randomized, open-label, parallel study was conducted to assess the efficacy and safety of premixed insulin aspart 30 (biphasic insulin aspart [BIAsp] 30) in managing gestational diabetes mellitus (GDM). A total of 323 women with GDM registered at a single center in India were randomly assigned to receive 6 U of either BIAsp 30 (Group A) or premixed human insulin (biphasic human insulin [BHI] 30; Group B) in a 1:1 ratio. Subjects performed home glucose monitoring and visited their care provider twice a month. The primary outcome was the degree of neonatal macrosomia (neonatal birth weight >90th percentile). Groups A and B were demographically comparable at study entry. Before labor onset, Groups A and B achieved similar degrees of fasting plasma glucose and postprandial plasma glucose control (92.97 ± 14.44 vs. 95.43 ± 18.96 and 127.59 ± 28.99 vs. 126.98 ± 29.89, respectively; both p = NS). Neonatal macrosomia frequency was 6.3% in Group A and 6.9% in Group B; however, this difference was not statistically significant. By last visit, the required insulin dose was significantly lower for Group A than Group B (19.83 ± 15.75 IU vs. 26.34 ± 23.15 IU, respectively; p = 0.006). BIAsp 30 was noninferior to BHI 30, producing comparable fetal outcomes when administered during pregnancy. Based on final doses, BIAsp 30 may offer greater treat-to-target potential for pregnant women.     

Characterisation of an ATP-dependent Ca2+ transport system in a plasma membrane enriched fraction from rat parotid

A membrane fraction enriched in plasma membrane marker enzymes K+-dependent p-nitrophenyl phosphatase, 5'-nucleotidase and alkaline phosphatase was prepared from rat parotid glands using Percoll self-forming gradient. This fraction contained an ATP-dependent CA2+ transport system which was distinct from those located on the endoplasmic reticulum and mitochondria of parotid glands. The Km for ATP was 0.57 +/- 0.07 mM (n = 3). Nucleotides other than ATP such as ADP, AMP, GTP, CTP, UTP or ITP were unable to support significant Ca2+ uptake. ATP-dependent Ca2+ uptake displayed sigmoidal kinetics with respect to free Ca2+ concentration with a Hill coefficient of 2.02. The K0.5 for Ca2+ was 44 +/- 3.1 nM (n = 3) and the average Vmax was 13.5 +/- 1.1 nmol/min per mg of protein. The pH optimum was 7.2. Trifluorperazine inhibited Ca2+ transport with half maximal inhibition observed at 30.8 microM. Complete inhibition was observed at 70 microM trifluorperazine. Exogenous calmodulin however had no effect on the rate of transport. Na+ and K+ ions activated Ca2+ transport at 20 to 30 mM ion concentrations. Higher concentrations of Na+ or K+ were inhibitory.     

Activation of rat parotid low Km cyclic AMP phosphodiesterase by isoproterenol

Approximately 94% of rat parotid cyclic AMP phosphodiesterase activity measured at a substrate concentration of 0.1 microM cyclic AMP was found in the 100,000 X g supernatant while the remaining enzyme activity was in the particulate fraction. Incubation of parotid slices with 10 microM isoproterenol resulted in approximately 40% activation of the cyclic AMP phosphodiesterase activity of the 100,000 X g supernatant. The enzyme activity in the particulate fraction was unaffected. The activation resulted from an increase in the value of the Vmax while the apparent Km (0.51 microM) was unaffected. The concentration of isoproterenol required to give half-maximal activation was 0.34 microM. The activation was rapid, became significant after 2 min and reached maximum after 30 min incubation of the parotid slices with isoproterenol. The activation of the enzyme activity by isoproterenol could be blocked by propanolol but was unaffected by cycloheximide. Dibutyryl-cyclic AMP was also effective while phenylephrine and carbamylcholine were ineffective in increasing the activity of the enzyme.     

Sodium-independent synaptosomal uptake of leucine

The uptake of leucine by rat brain synaptosomes was investigated in a Na+-free medium. It was found that leucine was transported into synaptosomes by 2 uptake processes which were both extremely sensitive to inhibition by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid. These 2 components were distinguishable from each other by kinetic analysis and by their relative sensitivity to inhibition by selected amino acids.     

Characterisation of a high affinity Ca2+-stimulated, Mg2+-dependent ATPase in the rat parotid plasma membrane

Two Ca2+-stimulated ATPase activities have been identified in the plasma membrane of rat parotid: (a) a (Ca2+ + Mg2+)-ATPase with high affinity for free Ca2+ (apparent Km = 208 nM, Vmax = 188 nmol/min per mg) and requiring micromolar concentration of Mg2+ and (b) a (Ca2+ or Mg2+)-ATPase with relatively low affinity for free Ca2+ (K0.5 = 23 microM) or free Mg2+ (K0.5 = 26 microM). The low-affinity (Ca2+ or Mg2+)-ATPase can be maximally stimulated by Ca2+ alone or Mg2+ alone. The high-affinity (Ca2+ + Mg2+)-ATPase exhibits sigmoidal kinetics with respect to ATP concentration with K0.5 = 0.4 mM and a Hill coefficient of 1.91. It displays low substrate specificity with respect to nucleotide triphosphates. Although trifluoperazine inhibits the activity of the high affinity (Ca2+ + Mg2+)-ATPase only slightly, it inhibits the activity of the low-affinity (Ca2+ or Mg2+)-ATPase quite potently with 22 microM trifluoperazine inhibiting the enzymic activity by 50%. Vanadate, inositol 1,4,5-trisphosphate, phosphatidylinositol 4,5-bisphosphate, Na+,K+ and ouabain had no effect on the activities of both ATPases. Calmodulin added to the plasma membranes does not stimulate the activities of both ATPases. The properties of the high-affinity (Ca2+ + Mg2+)-ATPase are distinctly different from those of the previously reported Ca2+-pump activity of the rat parotid plasma membrane.     

Phosphatidylinositol 4,5-biphosphate stimulates parotid endoplasmic reticulum (Ca2+ + Mg2+)-ATP'ase

The effect of various phospholipids on the (Ca2+ + Mg2+)-ATP'ase of rat parotid endoplasmic reticulum was investigated. At a concentration of 20 micrograms/ml, phosphatidylinositol 4,5-biphosphate stimulated the Ca2+-activated ATP'ase by 69%. None of the other phospholipids tested or 10 microM inositol 1,4,5-triphosphate had any significant effect on the ATP'ase. Stimulation by phosphatidylinositol 4,5-biphosphate was concentration dependent and half maximal stimulation required 8 micrograms/ml of the phospholipid. The results suggest that changes in the cellular concentration of phosphatidylinositol 4,5-biphosphate may be of importance in regulating Ca2+ transport by the endoplasmic reticulum.