Degradation of phytate in the gut of pigs ‐ pathway of gastrointestinal inositol phosphate hydrolysis and enzymes involved

The present study gives an overview on the whole mechanism of phytate degradation in the gut and the enzymes involved. Based on the similarity of the human and pigs gut, the study was carried out in pigs as model for humans. To differentiate between intrinsic feed phytases and endogenous phytases hydrolysing phytate in the gut, two diets, one high (control diet) and the other one very low in intrinsic feed phytases (phytase inactivated diet) were applied. In the chyme of stomach, small intestine and colon inositol phosphate isomers and activities of phytases and alkaline phosphatases were determined. In parallel total tract phytate degradation and apparent phosphorus digestibility were assessed. In the stomach chyme of pigs fed the control diet, comparable high phytase activity and strong phytate degradation were observed. The predominant phytate hydrolysis products were inositol phosphates, typically formed by plant phytases. For the phytase inactivated diet, comparable very low phytase activity and almost no phytate degradation in the stomach were determined. In the small intestine and colon, high activity of alkaline phosphatases and low activity of phytases were observed, irrespective of the diet fed. In the colon, stronger phytate degradation for the phytase inactivated diet than for the control diet was detected. Phytate degradation throughout the whole gut was nearly complete and very similar for both diets while the apparent availability of total phosphorus was significantly higher for the pigs fed the control diet than the phytase inactivated diet. The pathway of inositol phosphate hydrolysis in the gut has been elucidated.     

Arginine mediated purification of trehalose-6-phosphate synthase (TPS) from Candida utilis: Its characterization and regulation

Background

Trehalose is the most important multifunctional, non-reducing disaccharide found in nature. It is synthesized in yeast by an enzyme complex: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP).

Methods

In the present study TPS is purified using a new methodology from Candida utilis cells by inclusion of 100 mM l-arginine during cell lysis and in the mobile phase of high performance gel filtration liquid chromatography (HPGFLC).

Results

An electrophoretically homogenous TPS that was purified was a 60 kDa protein with 22.1 fold purification having a specific activity of 2.03 U/mg. Alignment of the N-terminal sequence with TPS from Saccharomyces cerevisiae confirmed the 60 kDa protein to be TPS. Optimum activity of TPS was observed at a protein concentration of 1 μg, at a temperature of 37 °C and pH 8.5. Aggregation mediated enzyme regulation was indicated. Metal cofactors, especially MnCl₂, MgCl₂ and ZnSO₄, acted as stimulators. Metal chelators like CDTA and EGTA stimulated enzyme activity. Among the four glucosyl donors, the highest V_max and lowest K_m values were calculated as 2.96 U/mg and 1.36 mM when adenosine di phosphate synthase (ADPG) was used as substrate. Among the glucosyl acceptors, glucose-6-phosphate (G-6-P) showed maximum activity followed by fructose-6-phosphate (F-6-P). Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity with different glucosyl donors.

General significance

Substrate specificity, V_max and K_m values provided an insight into an altered trehalose metabolic pathway in the C. utilis strain where ADPG is the preferred substrate rather than the usual substrate uridine diphosphaphate glucose (UDPG). The present work employs a new purification strategy as well as highlights an altered pathway in C. utilis.     

Serine/threonine phosphatases regulate platelet αIIbβ3 integrin receptor outside-in signaling mechanisms and clot retraction

Aims

We studied the role of serine/threonine phosphatases (PSTPs) on α_IIbβ₃ signaling and the potential selectivity of the level of PSTP inhibition with okadaic acid (OA) on α_IIbβ₃ signaling for regulation of platelet aggregation and clot retraction.

Main methods

We used washed platelets from normal donors and OA as inhibitor of PSTPs. Clot retraction was induced by 1 U/mL of thrombin. Reorganized cytoskeleton was isolated from Triton X-100 lysed platelets. The presence of proteins incorporated to the cytoskeleton was assayed by immunoblotting with specific antibodies.

Key findings

We found that both 100 and 500 nM OA blocked platelet mediated clot retraction. In contrast, only 500 nM OA inhibited thrombin-induced inside-out α_IIbβ₃ activation, platelet aggregation, and cytoskeletal reorganization. Among markers of α_IIbβ₃ outside-in signaling, 500 nM OA inhibited the incorporation to the cytoskeleton of syk, src, and FAK (Focal Adhesion Kinase) tyrosine kinases and the incorporation and phosphorylation at Tyr⁷⁵⁹ of the β₃ chain of α_IIbβ₃, while 100 nM OA only inhibited the FAK translocation and its tyrosine phosphorylation.

Significance

The level of inhibition of PSTPs by low or high OA concentration (33% and 73% inhibition, respectively) in intact whole cells differentially regulates platelet aggregation and integrin signaling, but have a common effect in blocking clot retraction. The latter may be associated with the presence of phosphorylated FAK in the cytoskeleton. This study reveals a novel target for anti-platelet treatment to block clot retraction without affecting the platelet hemostatic function by a partial inhibition of PSTPs.     

Shrinkage-induced Activation of the Na+/H+ Exchanger in Ehrlich Ascites Tumor Cells: Mechanisms Involved in the Activation and a Role for the Exchanger in Cell Volume Regulation

Amiloride-sensitive, Na⁺-dependent, DIDS-insensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells. This was assessed using the fluorescent pH-sensitive probe 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel increase in the amiloride-sensitive unidirectional Na⁺ influx is also observed. This indicates that hypertonic challenge activates a Na⁺/H⁺ exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge, and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes in cellular pH _i , but the overall picture confirms the results from cell suspensions. Shrinkage-induced alkalinization and recovery of cellular pH after an acid load, is strongly reduced in ATP-depleted cells. Furthermore, it is inhibited by chelerythrine and H-7, inhibitors of protein kinase C (PKC). In contrast, Calyculin A, an inhibitor of protein phosphatases PP1 and PP2A, stimulates shrinkage-induced alkalinization. Osmotic activation of the exchanger is unaffected by removal of calcium from the experimental medium, and by buffering of intracellular free calcium with BAPTA. At 25 mm HCO⁻ ₃, but not in nominally HCO⁻ ₃-free medium, Na⁺/H⁺ exchange contributes significantly to regulatory volume increase in Ehrlich cells. Under isotonic conditions, the Na⁺/H⁺ exchanger is activated by ionomycin, an effect which may be secondary to ionomycin-induced cell shrinkage. Received: 2 March 1995/Revised: 29 September 1995     

Transit of hormonal and EGF receptor-dependent signals through cholesterol-rich membranes

The functional consequences of changes in membrane lipid composition that coincide with malignant growth are poorly understood. Sufficient data have been acquired from studies of lipid binding proteins, post-translational modifications of signaling proteins, and biochemical inhibition of lipidogenic pathways to indicate that growth and survival pathways might be substantially re-directed by alterations in the lipid content of membranes. Cholesterol and glycosphingolipids segregate into membrane patches that exhibit a liquid-ordered state in comparison to membrane domains containing relatively lower amounts of these classes of lipids. These "lipid raft" structures, which may vary in size and stability in different cell types, both accumulate and exclude signaling proteins and have been implicated in signal transduction through a number of cancer-relevant pathways. In prostate cancer cells, signaling from epidermal growth factor receptor (EGFR) to the serine-threonine kinase Akt1, as well as from IL-6 to STAT3, have been demonstrated to be influenced by experimental interventions that target cholesterol homeostasis. The recent finding that classical steroid hormone receptors also reside in these microdomains, and thus may function within these structures in a signaling capacity independent of their role as nuclear factors, suggests a novel means of cross-talk between receptor tyrosine kinase-derived and steroidogenic signals. Potential points of intersection between components of the EGFR family of receptor tyrosine kinases and androgen receptor signaling pathways, which may be sensitive to disruptions in cholesterol metabolism, are discussed. Understanding the manner in which these pathways converge within cholesterol-rich membranes may present new avenues for therapeutic intervention in hormone-dependent cancers.     

The involvement of protein kinases and protein phosphatases in signal transduction during neurotransmitter-induced stimulation of root water-pumping activity

In detached roots of etiolated maize (Zea mays L.) seedlings, neurotransmitters, adrenalin and noradrenalin, stimulated exudation by increasing the root pressure due to activation of its metabolic component. In these treatments, the osmotic pressure of the exudate was somewhat reduced. In contrast, a temperature coefficient Q₁₀ was increased, which as in accordance with the increase of the absolute value of the metabolic component and its proportion in the total root pressure. To obtain some information about transmitting the signals induced by adrenalin and noradrenalin action on water transport, we used two inhibitors of the most important and universal elements of signaling pathways, staurosporine (the inhibitor of protein kinases) and okadaic acid (the inhibitor of protein phosphatases). In control roots, staurosporine markedly slowed and okadaic acid accelerated exudation. In the presence of staurosporine in the incubation medium, a stimulatory effect of both neurotransmitters was completely abolished and the rate of exudation became even below the control value. Okadaic acid exerted an opposite action: it augmented markedly stimulatory effects of both neurotrasmitters. The data obtained indicated the involvement of protein kinases and protein phosphatases in transduction of signals induced by adrenalin and noradrenalin, which stimulated root water-pumping activity.     

Discovery of potent PTP1B inhibitors via structure-based drug design,synthesis and in vitro bioassay of Norathyriol derivatives

Protein tyrosine phosphatase 1B (PTP1B) has recently been identified as a potential target of Norathyriol. Unfortunately, Norathyriol is not a potent PTP1B inhibitor, which somewhat hinders its further application. Based on the fact that no study on the relationship of chemical structure and PTP1B inhibitory activity of Norathyriol has been reported so far, we attempted to perform structural optimization so as to improve the potency for PTP1B. Via structure-based drug design (SBDD), a rational strategy based on the binding mode of Norathyriol to PTP1B, we designed 26 derivatives with substitutions at the four phenolic hydroxyl groups of Norathyriol. By chemical synthesis and in vitro bioassay, we identified seven PTP1B inhibitors that were more potent than Norathyriol, of which XWJ24 showed the highest potency (IC₅₀: 0.6 μM). We also found out that XWJ24 was a competitive inhibitor and showed the 4.5-fold selectivity over its close homolog, TC-PTP. Through molecular docking of XWJ24 against PTP1B, we highlighted the essential role of its hydrogen bond with Asp181 for PTP1B inhibition and identified a potential halogen bond with Asp48 that was not observed for Norathyriol. The current data indicate that our SBDD strategy is effective to discover potent PTP1B-targeted Norathyriol derivatives, and XWJ24 is a promising lead compound for further development.     

Structural insight into effector proteins of Gram-negative bacterial pathogens that modulate the phosphoproteome of their host

Invading pathogens manipulate cellular process of the host cell to establish a safe replicative niche. To this end they secrete a spectrum of proteins called effectors that modify cellular environment through a variety of mechanisms. One of the most important mechanisms is the manipulation of cellular signaling through modifications of the cellular phosphoproteome. Phosphorylation/dephosphorylation plays a pivotal role in eukaryotic cell signaling, with ∼500 different kinases and ∼130 phosphatases in the human genome. Pathogens affect the phosphoproteome either directly through the action of bacterial effectors, and/or indirectly through downstream effects of host proteins modified by the effectors. Here we review the current knowledge of the structure, catalytic mechanism and function of bacterial effectors that modify directly the phosphorylation state of host proteins. These effectors belong to four enzyme classes: kinases, phosphatases, phospholyases and serine/threonine acetylases.     

谷氨酸受体可逆磷酸化及其功能

谷氨酸受体(GluRs)C端区存在被多种蛋白激酶磷酸化的位点,同时又能被多种蛋白磷酸酶去磷酸化,磷酸化的结果可使Ca²⁺内流增加,增强GluRs功能;去磷酸化作用则相反.正常情况下GluRs可逆磷酸化处于一种动态平衡状态,在突触可塑性机制如长时程增强(LTP)中起重要作用,而在病理状态如缺血性脑损伤中,这种平衡失衡加重兴奋性神经元损伤.