Short-Term PCB (Aroclor 1254) Toxicity on Few Phosphatases in Mice Brain

The present communication reports the dose and duration dependent toxicity of a PCB, Aroclor 1254, to a few ion dependent ATPases, Acid phosphatase, Alkaline phosphatase and Glucose-6-phosphatase in the whole brain tissue of mice. Two groups of mice were subjected to two sublethal doses (0.1 and 1 mg kgbw⁻¹ day⁻¹) of PCB orally and exposed for 4, 8 or12 days. A separate control group received the corn oil vehicle for the same exposure times. The observed results indicated exposure duration dependent changes in the enzymatic levels in the brain. The results suggest that the alteration in the enzymatic activity was possibly due to imposed oxidative stress generated by Aroclor 1254 on membrane-bound ion-dependent ATPases and other phosphatases in the brain tissue.     

Integrative Physiology: Defined Novel Metabolic Roles of Osteocalcin

The prevailing model of osteology is that bones constantly undergo a remodeling process, and that the differentiation and functions of osteoblasts are partially regulated by leptin through different central hypothalamic pathways. The finding that bone remodeling is regulated by leptin suggested possible endocrinal effects of bones on energy metabolism. Recently, a reciprocal relationship between bones and energy metabolism was determined whereby leptin influences osteoblast functions and, in turn, the osteoblast-derived protein osteocalcin influences energy metabolism. The metabolic effects of bones are caused by the release of osteocalcin into the circulation in an uncarboxylated form due to incomplete γ-carboxylation. In this regard, the Esp gene encoding osteotesticular protein tyrosine phosphatase is particularly interesting because it may regulate γ-carboxylation of osteocalcin. Novel metabolic roles of osteocalcin have been identified, including increased insulin secretion and sensitivity, increased energy expenditure, fat mass reduction, and mitochondrial proliferation and functional enhancement. To date, only a positive correlation between osteocalcin and energy metabolism in humans has been detected, leaving causal effects unresolved. Further research topics include: identification of the osteocalcin receptor; the nature of osteocalcin regulation in other pathways regulating metabolism; crosstalk between nutrition, osteocalcin, and energy metabolism; and potential applications in the treatment of metabolic diseases.     

听觉毛细胞死亡的分子调控

听觉毛细胞损伤是引起听力损失最常见的原因,本综述主要介绍几个信号通路,包括炎症反应、丝裂原活化蛋白激酶信号传导途径、氧化性应激等,涉及毛细胞发生毒性反应或应激反应后的程序性和坏死性死亡,揭示了引起听觉毛细胞损伤的信号通路的相互作用,以及避免听觉毛细胞因凋亡或非凋亡途径死亡的治疗干预策略。     

近红外双荧光系统在磷酸酯酶筛选中的应用

目的应用奥德赛（Odyssey）近红外双荧光系统筛选去甲基化酶KDM3A去磷酸化的磷酸酯酶。方法使用磷酸酯酶干扰质粒转染HEK293T细胞,使用磷酸化的KDM3A抗体对细胞进行杂交,近红外荧光染料DRAQ5标记细胞核,利用奥德赛（Odyssey）近红外双荧光系统对细胞进行荧光扫描,荧光强度的比值可代表细胞内相对KDM3A磷酸化水平,进而筛选出相关的磷酸酯酶。结果磷酸酯酶广谱抑制剂冈崎酸OA处理转染后的细胞,近红外双荧光扫描系统可以检测到KDM3A的磷酸化,初步筛选出4个可能参与KDM3A去磷酸化的磷酸酯酶亚基。结论近红外双荧光系统可以应用于磷酸酯酶的初步筛选。     

Why does troponin I have so many phosphorylation sites? Fact and fancy

We discuss a current controversy regarding the relative role of phosphorylation sites on cardiac troponin I (cTnI) (Fig. 1) in physiological and patho-physiological cardiac function. Studies with mouse models and in vitro studies indicate that multi-site phosphorylations are involved in both control of maximum tension and sarcomeric responsiveness to Ca²⁺. Thus one hypothesis is that cardiac function reflects a balance of cTnI phosphorylations and a tilt in this balance may be maladaptive in acquired and genetic disorders of the heart. Studies on human heart samples taken mainly at end-stage heart failure, and in depth proteomic analysis of human and rat heart samples demonstrate that Ser23/Ser24 are the major and perhaps the only sites likely to be relevant to control cardiac function. Thus functional significance of Ser23/Ser24 phosphorylation is taken as fact, whereas the function of some other sites is treated as fancy. Maybe the extremes will meet: in any case we both agree that further work needs to be carried out with relatively large mammals and with determination of the time course of changes in phosphorylation to identify transient modifications that may be relevant at a beat-to-beat basis. Moreover, we agree that the changes and effects of cTnI phosphorylation need to be fully integrated into the effects of other phosphorylations in the cardiac myocyte.     

Functional defects in troponin and the systems biology of heart failure

R. J. Solaro and E. M. Burkart. Functional Defects in Troponin and the Systems Biology of Heart Failure.Journal of Molecular and Cellular Cardiology (2002) 34, 689-693.     

Differential modulation of pharmacologically distinct components of Ca2+ currents by protein kinase C activators and phosphatase inhibitors in nerve-growth-factor-differentiated rat pheochromocytoma (PC12) cells

We have studied the effects of protein kinase C (PKC) activators 4-phorbol 12-myristate 13-acetate (4-PMA) and 1-oleoyl-2-acetylglycerol (OAG) and of phosphatase inhibitors (okadaic acid and calyculin A) on voltage-activated Ca²⁺ and K⁺ channels in nerve growth factor-(NGF)-differentiated pheochromocytoma (PC12) cells. Whole-cell Ba²⁺ and K⁺ currents were recorded at room temperature with the patch-clamp technique. By using -conotoxin (CgTX) and isradipine, two specific Ca²⁺ channel blockers, we found three types of Ba²⁺ currents (I _Ba): (1) a -CgTX-sensitive I _Ba; (2) an isradipine-sensitive I _Ba; and (3) a -CgTX plus isradipine-resistant I _Ba. The external application of 4 -PMA or OAG down-modulated the isradipine-sensitive I _Ba whereas the two other I _Ba were not affected. 4-PMA-induced inhibition of I _Ba was prevented by staurosporine (a protein kinase inhibitor) and PKC (19–31) (a specific PKC inhibitor). The delayed rectifier K⁺ current (I _K) was unaffected by PKC activators. Both okadaic acid and calyculin A affected the components of the I _Ba in different manners. The presence of okadaic acid decreased the isradipine-sensitive I _Ba more than the -CgTX-sensitive I _Ba. The -CgTX plus isradipine-resistant I _Ba was not affected. Calyculin A down-modulated all three components of I _Ba to a similar degree. Our results suggest a differential modulation of voltage-activated Ca²⁺ and K⁺ channels by the PKC signalling pathway in NGF-differentiated PC12 cells.     

Enzymes of protein and phosphate catabolism in rat bone

A study was made of several enzymatic activities in rat femur and tibia. Quantitative assays were developed for the determination of acid and alkaline phosphatase, lactate dehydrogenase, proteolytic activity, leucine aminopeptidase, glycylglycine dipeptidase, glutamate dehydrogenase, aspartate transaminase, and alanine transaminase. Some properties of these enzymes were studied using metaphyseal homogenates. The distribution of activity in the metaphysis, diaphysis, and marrow was determined. The activity per mg of DNA was highest in the metaphysis and lowest in marrow. The activities in general were 40–70% lower in the diaphysis than in the metaphysis. Metaphyseal enzymatic activity was the same in 29- and 49-day-old rats but lower in 82-day-old rats. Proteolytic activity at pH 8.0 differed from the other activities studied being higher in marrow than in bone and increasing markedly with age.

---

Zusammenfassung Verschiedene Enzymaktivitäten in Femur und Tibia der Ratte wurden untersucht. Es wurden quantitative Prüfmethoden für die Bestimmung von saurer und alkalischer Phosphatase, Lactatdehydrogenase, proteolytischer Aktivität, Leucinaminopeptidase, Glycylglycin-Dipeptidase, Glutamatdehydrogenase, Aspartat-Transaminase und Alanin-Transaminase entwickelt. Einige Eigenschaften dieser Enzyme wurden in Metaphysenhomogenaten untersucht. Die Verteilung der Enzymaktiväten in Metaphyse, Diaphyse und Knochenmark wurde bestimmt. Die Aktivität pro mg DNA war am höchsten in der Metaphyse, am niedrigsten im Knochenmark. Allgemein waren die Aktivitäten 40–70% tiefer in der Diaphyse als in der Metaphyse. Die metaphysäre enzymatische Aktivität war dieselbe bei 29 und 49 Tage alten Ratten, hingegen tiefer bei 82 Tage alten Ratten. Die proteolytische Aktivität bei pH 8 war von anderen untersuchten Aktivitäten verschieden; sic war höher im Knochenmark als im Knochen und zeigte deutliche Zunahme mit dem Alter.

---

Résumé Les activités de différentes encymes du fémur et de la tibia du rat étaient étudiées. Pour mesurer acide et alkaline phosphatase, lactatdehydrogenase, activité protéolytique, leucinaminopéptidase, glycylglycindipéptidase, glutamatdehydrogenase, aspartate transaminase et alanintransaminase, une méthode quantitative était developpé. Quelques réactions characteristiques étaient examinées das n des homogénates de la métaphyse. La distribution des encymes dans la métaphyse, diaphyse et dans la moelee osseuse était étudié. L'activité la plus grande per mg DNA était observé dans la métaphyse, celle la plus basse dans la moelle osseuse. En générale les activités étaient 40–70% abaissées dans la diaphyse comparé avec la métaphyse. L'activité encymatique de la métaphyse n'était pas différente l'une de l'autre chez les rats agés de 29 et 49 jours mais elle diminuait chez celles de 82 jours. L'activité protéolytique a pH 8 ne correspondait pas avec les autres encymes: elle était plus haut dans la moelle osseuse que dans l'os et augmentait visiblement avec l'age.

     

Okadaic acid modulates exocytotic and transporter-dependent release of dopamine in bovine retina in vitro

Bovine retinas were isolated for the study of the modulation of exocytotic and transporter-dependent release of dopamine (DA) in vitro. Endogenous DA was measured in the medium using HPLC with electrochemical detection under successive incubations with transfers in fresh medium every 30 min.As expected, potassium caused a calcium-dependent exocytotic liberation of DA. Amphetamine or tyramine induced a calcium-independent release by reversing DA transport across the plasma membrane. Okadaic acid, a specific inhibitor of phosphatases 1 and 2A, induced a slight but significant DA release in the absence of calcium. Furthermore, the toxin increased potassium-, amphetamine-or tyramine-induced DA release independently of extracellular calcium. In addition, okadaic acid completely annulled the ability of a calcium-free extracellular environment to inhibit the potassium-induced DA release. Finally, the toxin prevented the time-dependent decline in the efficacies of amphetamine or tyramine to release DA.In agreement with proposed schemes described for rat striatum, the results of the present study confirmed the existence of distinct release modes of DA in bovine retina. The results obtained with okadaic acid suggest that phosphatase 1 and/or phosphatase 2A constitute part of a direct or indirect mechanism to inhibit both exocytotic and transporter-dependent DA release.