Cloning and characterization of a SnRK1-encoding gene from <Emphasis Type="Italic">Malus hupehensis</Emphasis> Rehd. and heterologous expression in tomato

Sucrose non-fermenting-1-related protein kinase-1 (SnRK1) plays an important role in metabolic regulation in plant. To understand the molecular mechanism of amino acids and carbohydrate metabolism in Malus hupehensis Rehd. var. pinyiensis Jiang (Pingyi Tiancha, PYTC), a full-length cDNA clone encoding homologue of SnRK1 was isolated from PYTC by Rapid Amplification of cDNA Ends (RACE). The clone, designated as MhSnRK1, contains 2063 nucleotides with an open reading frame of 1548 nucleotides. The deduced 515 amino acids showed high identities with other plant SnRK1 genes. Quantitative real-time PCR analysis revealed this gene was expressed in roots, stems and leaves. Exposing seedlings to nitrate caused and initial decrease in expression of the MhSnRK1 gene in roots, leaves and stems in short term. Ectopic expression of MhSnRK1 in tomato mainly resulted in higher starch content in leaf and red-ripening fruit than wild-type plants. This result supports the hypothesis that overexpression of SnRK1 causes the accumulation of starch in plant cells. All the results suggest that MhSnRK1 may play important roles in carbohydrate and amino acid metabolisms.     

Autoantibody to vasoactive intestinal peptide in human circulation

In a radioassay for Vasoactive Intestinal Peptide (VIP)-binding, eight out of 33 plasma samples from healthy human subjects exhibited specific binding ranging from 2.6% to 46.7% of total [125 I]VIP. This binding was competitively displaced by unlabeled VIP. The structurally homologous peptides, Peptide Histidine Isoleucine (PHI) and secretin, were, respectively, 72-fold and 413-fold less potent than VIP in displacing bound [125 I]VIP, whereas the unrelated peptides, neurotensin, eledoisin, bombesin and metenkephalin, were without effect on the binding. The antibody nature of the VIP-binding factor was suggested by its precipitation with ammonium sulfate, attenuation after absorption with Staphylococcus aureus preparations, precipitation with antisera against human IgG and IgM, and coelution with standard IgG and IgM on anion-exchange and high-performance gel-filtration columns. Pepsin treatment of purified IgG fraction yielded a VIP-binding species with apparent molecular weight of 108 +/- 13 kDa that was precipitated by antiserum against the F(ab)2 fragment of the IgG molecule. These results demonstrate the existence in some human plasmas of an autoantibody that binds VIP.     

Purification and properties of cathepsin D from the mammary glands of lactating rabbits

Cathepsin D was purified from the lactating rabbit mammary gland by a rapid procedure, which included fractionation with (NH4)2SO4, acid precipitation, double affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100, resulting in approximately 360-fold purification of the enzyme over the homogenate and approximately 16% recovery. After isoelectric focusing, the enzyme dissociated into four (pI 5.8, 6.3, 6.5 and 7.2) multiple forms, but appeared homogeneous on polyacrylamide gel electrophoresis. Cathepsin D has a Mr of 45 kDa as determined by Sephadex G-100 column chromatography. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme gave a single protein band, corresponding to Mr of 45 kDa. The amino acid composition of the enzyme is similar to that of cathepsins D from other tissues. A single N-terminal amino acid was glycine. Cathepsin D contains 6.4% carbohydrates consisting of mannose, galactose, fucose and glucosamine at a ratio of 3:9:2:2. Cathepsin D is inhibited by pepstatin with Ki of 2.5 X 10(-9) M and irreversibly by N-diazoacetyl-N'-2.4-dinitrophenyl-ethylene diamine. The enzyme hydrolyzes bovine hemoglobin with the maximal activity at pH 3.0 with Km = 10(-5) M and HLeu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe with Km = 4 X 10(-5) M and Rcat = 0.95 s-1. The major cleavage sites were Leu15-Tyr16, Phe24-Phe25 and Phe25-Tyr26 during hydrolysis of the oxidized insulin B-chain by cathepsin D.     

Effect of nucleoside triphosphates on cyclic nucleotide phosphodiesterase: role of protein modulators

The effects of nucleoside triphosphates (ATP and GTP) on phosphodiesterase (PDE) of brain and outer segments of the retina enriched or devoid of protein modulators were studied. In the case of retinal outer segment PDE the enzyme activity was considerably inhibited by both nucleosides only when the enzyme was separated from the inhibitor. In case of brain PDE, on the contrary, the effect of the nucleosides was much more pronounced in the enzyme preparation coupled with the protein activator, calmodulin. The latter when added to brain PDE devoid of the activator in the presence of ATP and GTP considerably reduced the enzyme activity. An addition of the inhibitor simultaneously with GTP to the purified PDE of outer segments increased the PDE activity. The constants for the inhibition of brain PDE coupled with calmodulin and retinal outer segment PDE separated from the inhibitor by ATP and GTP were determined.     

Growth and water relations in a central North Carolina population of <Emphasis Type="Italic">Microstegium vimineum</Emphasis> (Trin.) A. Camus

The ability of an invasive plant to occupy new areas is often attributed to both morphological and physiological plasticities that allow them to remain viable over a wide range of environmental conditions. Studies addressing the ecological requirements of Microstegium vimineum often consider soil moisture or soil moisture along with other factors as important explanatory components for the establishment and persistence of this invasive monocot. However, controlled studies specifically targeting water relations in M. vimineum are needed. Therefore, the purpose of this study was to determine how different water availabilities influence the growth and physiological performance of M. vimineum. This study utilized experimental microcosms to achieve different water availabilities including low soil moisture (<15% water), moderate soil moisture (ca. 20–30%), and flooded conditions. While both flooded and low soil moisture resulted in diminished growth, M. vimineum still survived under these conditions. Physiological processes including C₄ metabolism, minimum stress under low water conditions, and the ability to increase tissue rigidity may confer some advantages to M. vimineum during periods of limiting water conditions. Similarly, the proportionally low root biomass, shallow root structure, and its ability to maintain stable water relations during flooding and/or soil waterlogging may facilitate M. vimineum’s ability to invade mesic habitats. It is likely, therefore, that the capacity to tolerate both low soil moistures and flooded conditions has enhanced the ability of M. vimineum to populate disturbed systems in central North Carolina.     

Activity of cAMP-dependent protein kinases and cAMP-binding proteins from rat renal cytosol upon dehydration

The activity of cAMP-dependent protein kinases, cAMP binding and the spectrum of cAMP-binding proteins in renal papillary cytosol of intact rats and of rats kept on a water-deprived diet for 24 hours were investigated. It was found that the stimulation of protein kinases by 10(-6) M cAMP in the experimental group was significantly higher than in the control one. On DEAE-cellulose chromatography, the position of peaks of the specific cAMP binding corresponded to those of the regulatory cAMP-dependent protein kinases type I and II. Under these conditions, more than 80% of the binding activity in intact animals was localized in peak II, whereas in rats kept on a water-deprived diet over 60% of the binding activity was localized in peak I. The total binding activity of cytosol in experimental animals remained unchanged is compared to intact rats. It is suggested that in renal papilla dehydration is accompanied by the induction of synthesis of regulatory subunits of cAMP-dependent protein kinase type I.     

Soluble, prolonged-acting insulin derivatives. II. Degree of protraction and crystallizability of insulins substituted in positions A17, B8, B13, B27 and B30

It has previously been found that insulins, to which positive charge has been added by substitutions in position B30, thus raising the isoelectric point towards pH 7, had a prolonged action when injected as slightly acidic solutions because such derivatives crystallize very readily upon neutralization. Positive charge has now been added by substituting the B13 and A17 glutamic acid residues with glutamines and B27 threonine with lysine or arginine. These substitutions were introduced by site-specific mutagenesis in a gene coding for a single-chain insulin precursor. By tryptic transpeptidation the single-chain precursors were transformed to the double-chain insulin structure, concomitantly with incorporation of residue B30. Thus insulins combining B13 glutamine, A17 glutamine and B27 lysine or arginine with B30 threonine, threonine amide or lysine amide were synthesized. The time course of blood glucose lowering effect and the absorption were studied after subcutaneous injection in rabbits and pigs. The prolonged action of B30-substituted insulins was markedly enhanced by B27 lysine or arginine substitutions and by B13 glutamine. The B27 residue is located on the surface of the hexamer, so a basic residue in this position presumably promotes the packing of hexamers at neutral pH. The B13 residues cluster in the centre of the hexamer. When the electrostatic repulsive forces from six glutamic acid residues are abolished by substitution with glutamine, a stabilization of the hexamer can be envisaged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diazepam tolerance: effect of age,regular sedation,and alcohol.

The dose of intravenous diazepam required for sedation was estimated in a series of 78 patients aged 17-85 years given the drug for dental and endoscopic procedures. Multiple regression analysis showed a significant correlation (r = 0.71; p less than 0.001) between dose and age, body weight, the taking of regular sedation, and the taking of more than 40 g alcohol daily, but there were no differences in the doses required between men and women, smokers and non-smokers, inpatients and outpatients, or dental and endoscopy patients. Patients aged 80 required an average dose of 10 mg and patients aged 20 an average dose of 30 mg, and the dose required was much higher in those receiving regular sedation or having a high alcohol intake. Plasma total and free diazepam concentrations were measured in the second half of the series of patients (n = 37). Plasma concentrations required for sedation fell twofold to threefold between the ages of 20 and 80 and were significantly higher in those taking regular sedation or alcohol. Differences in the acute response to diazepam appeared to be due to differences in the sensitivity of the central nervous system (pharmacodynamic tolerance) rather than to differences in pharmacokinetic factors.     

Effect of ascorbic acid on the binding of 3H-GABA and 3H-glutamic acid to synaptosomes of the rat cerebral cortex

The effects of different concentrations of L-ascorbic acid (Asc) on Na+-dependent binding of 3H-GABA and 3H-DL-glutamic acid to rat brain cortical synaptosomes were studied. Asc, at a concentration nearly equal to brain extracellular one (3 X 10(-4) M), had no effect on specific and nonspecific 5H-GABA binding. At higher concentrations (10(-3) M) Asc strongly inhibited, and at lower concentrations (10(-6) M) considerably stimulated 3H-GABA binding. At a concentration of 10(-5)-10(-3) M Asc tended to decrease 3H-DL-glutamic acid binding.     

Range of the antigenic specificity of peptidoglycan in Staphylococcus aureus compared to other representatives of the Staphylococcus genus

In investigations based on the use of a highly sensitive test system permitting the detection of normal human antibodies to S. aureus peptidoglycan, the antigenic relationships between the peptidoglycans of S. aureus and other representatives of the genus Staphylococcus have been studied. Among other staphylococcal species, S. simulans, S. xylosus, S. hyicus, S. cohnii, S. hyicus s. s. chromogenes have been found to possess peptidoglycans most closely related to S. aureus peptidoglycans, while S. warneri and S. epidermidis peptidoglycans have proved to be least closely related to it.