Dephosphorylation of glycogen synthase in rat heart extracts by E. coli alkaline phosphatase

Summary Regulation of the dephosphorylation of glycogen synthase in extracts from rat heart has been studied by adding exogenous phosphatase to the extract. These experiments were possible only because the endogenous protein phosphatase activity of the extract could be inhibited by KF under conditions where alkaline phosphatase activity was not. The concentration of substrate (glycogen synthase from the heart extract) and catalyst (purified E. coli alkaline phosphatase) could be varied independently, by adding known amounts of alkaline phosphatase to the KF-containing heart extracts. Alkaline phosphatase could completely dephosphorylate glycogen synthase while phosphorylase was unchanged. The rate of dephosphorylation was proportional to both the concentration of alkaline phosphatase added to the tissue extract and the amount of glycogen synthase in the extract. The K_m for glycogen synthase was close to the concentration found in heart tissue. The K_m and the maximum rate of dephosphorylation were both dependent on the phosphorylation state of the glycogen synthase. Less phosphorylated enzyme forms were dephosphorylated faster. These results indicate the necessity for precise control of many variables in studying the rate of glycogen synthase dephosphorylation.Alkaline phosphatase-catalyzed dephosphorylation could be inhibited by physiological concentrations of glycogen. Glycogen synthase dephosphorylation in extracts from fasted-refed rats was less sensitive to glycogen inhibition than in extracts from normal animals. The phosphorylation state of the glycogen synthase in these animals was assessed by kinetic studies to show that differences in phosphorylation state probably could not account for the observations. Fasting led to a decreased rate of dephosphorylation of glycogen synthase due to both an apparent change in kinetic properties of glycogen synthase as a substrate for alkaline phosphatase, and an increased inhibitory effect of glycogen. Stable modifications of glycogen synthase caused by altered nutritional states in the animals are thought to produce these effects.%GSI represents the percentage of glycogen synthase activity that is active without glucose 6-P.     

Structural dependence of oligonucleotide photooxidation

Oxidative photosensitization was used to characterize the conformational-dependent reactivity of various structures formed by oligonucleotides 14-15 nucleotides in length. The rate and product composition from a single hit process was analyzed using quantitative ion exchange chromatography under native and denaturing conditions. The primary damage incurred under aerobic acetone sensitization was base oxidation that, in turn, would induce strand scission upon a secondary treatment with piperidine. The reactive intermediates of this process were not consistent with diffusible radical species or singlet oxygen, as indicated by isotope and quenching studies. Derivatization was most likely initiated through a type I photoprocess with a direct interaction between DNA bases and excited state acetone preceding an irreversible oxidation step. This dominant reaction demonstrated no obvious sequence or site specificity for initial modification; the relative reactivity among the oligonucleotides did not correspond to any simple trend of base composition or near neighbor analysis. Likewise, the steric requirements of base modification allowed for similar rates of oxidation for single-strand, helical, and aberrant forms of DNA. Hybridization of the most reactive oligonucleotides, however, did suppress their relative single-strand vs double-strand reactivity by as much as fourfold.     

Cell surface receptors for CCN proteins

The CCN family (CYR61; CTGF; NOV; CCN1–6; WISP1–3) of matricellular proteins in mammals is comprised of six homologous members that play important roles in development, inflammation, tissue repair, and a broad range of pathological processes including fibrosis and cancer. Despite considerable effort to search for a high affinity CCN-specific receptor akin to growth factor receptors, no such receptor has been found. Rather, CCNs bind several groups of multi-ligand receptors as characteristic of other matricellular proteins. The most extensively documented among CCN-binding receptors are integrins, including α_vβ₃, α_vβ₅, α₅β₁, α₆β₁, α_IIbβ₃, α_Mβ₂, and α_Dβ₂, which mediate diverse CCN functions in various cell types. CCNs also bind cell surface heparan sulfate proteoglycans (HSPGs), low density liproprotein receptor-related proteins (LRPs), and the cation-independent mannose-6-phosphate (M6P) receptor, which are endocytic receptors that may also serve as co-receptors in cooperation with other cell surface receptors. CCNs have also been reported to bind FGFR-2, Notch, RANK, and TrkA, potentially altering the affinities of these receptors for their ligands. The ability of CCNs to bind a multitude of receptors in various cell types may account for the remarkable versatility of their functions, and underscore the diverse signaling pathways that mediate their activities.     

Determination of seven unconjugated steroids in the blood and seminal plasma of the fertile male rabbit.

Seven unconjugated steroids were measured in the blood and seminal plasmas of fertile male rabbits by radioimmunoassay. The blood plasma testosterone concentration was 4--5 times that of the seminal plasma. Dehydroepiandrosterone, estrone and 17beta-estradiol were found in measurable amounts in the blood plasma; however, these steroid levels were slightly lower in seminal plasma. Androstenedione and 5alpha-dihydrotestosterone were present in equal quantities in both the seminal and blood plasmas. By contrast, seminal plasma pregnenolone level was about twice that of the blood plasma. The determination of seminal plasma steroids may lend itself as a complementary assessment to blood steroid determinations for the evaluation of the normal function of various reproductive organs.     

EFFECTS OF SOIL NITROGEN ON POLLEN PRODUCTION,POLLEN GRAIN SIZE,AND POLLEN PERFORMANCE IN CUCURBITA PEPO (CUCURBITACEAE)

To determine the effects of soil nitrogen on pollen production, pollen size, and pollen performance, two cultivars of zucchini (Cucurbita pepo) were grown under two nitrogen regimes in an experimental garden. The two cultivars were true breeding for alternative alleles for a one gene trait, ovary color. The soil nitrogen treatment had a significant effect on most measures of reproductive output through the female function. The nitrogen treatment did not affect the number of staminate flowers per plant but did have an effect on the number of pollen grains per staminate flower and the mean pollen grain size. A pollen mixture experiment revealed that pollen produced by plants in the high nitrogen treatment sired significantly more seeds than pollen from low nitrogen plants. Moreover, we found that the high nitrogen pollen sired even a greater percentage of seeds in the region of the fruit (ovary) previously shown to be fertilized by the fastest growing pollen tubes. Thus, the difference in the number of seeds sired by pollen from the two nitrogen treatments is due to differences in pollen performance. We conclude that spatial heterogeneity in soil nitrogen can influence the paternity of seeds in a plant population.     

Trophic control of lung development by sympathetic neurons: effects of neonatal sympathectomy with 6-hydroxydopamine

The onset of peripheral sympathetic neuronal function is thought to provide trophic regulatory signals for development of adrenergic target tissues. In the current study, we examined the effects on lung development of neonatal sympathectomy with 6-hydroxydopamine. The completeness of the lesion and effectiveness in reducing sympathetic input to the tissue were confirmed by direct measurement of norepinephrine levels and turnover. Despite the denervation, no evidence of beta-receptor up-regulation was found; in fact, receptor binding sites tended to be reduced throughout development. The cyclic AMP response to isoproterenol challenge was initially suppressed in the lesioned animals, but became supersensitive even in the face of reduced receptor binding capabilities. Evidence was also obtained for ontogenetic abnormalities in the ornithine decarboxylase/polyamine system, which is partially controlled by beta-adrenergic input and which regulates macromolecule synthesis in replicating and differentiating cells. Eventually, the alterations were reflected in aberrant developmental patterns of DNA, RNA and protein in the lung. These results indicate that sympathetic neurons influence the biochemical development of the lung and may serve to program permanently the relationships among receptor sites, receptor coupling to cellular function, and control of cell maturation.     

Purification and N-terminal sequence of two tartrate-resistant acid phosphatases type-5 from the hairy cell leukemia spleen

Tartrate-resistant acid phosphatases types 5a and 5b were purified from human hairy cell leukemia spleen by sequential chromatography on Phenyl-Sepharose, CM-Sepharose, concanavalin A-Sepharose, FPLC Superose-12 and FPLC Mono-S. The purification over the original tissue extract was 1150- and 3300-fold, with a yield of 2.1% and 2.5%, respectively. Gel filtration indicated an Mr of about 30000 for both forms. There was a N-terminal sequence identity between the two enzymes. However, they appeared to be different as assessed by cation exchange chromatography and amino acid composition.     

Soluble ACE2-mediated cell entry of SARS-CoV-2 via interaction with proteins related to the renin-angiotensin system

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