Altered Phosphorylation of Rat Dentine Phosphoproteins by Fluoride In Vivo

Dentine phosphoproteins have been proposed to have an important role in mineralization. This study focused on the influence of fluoride on the biochemical composition of dentine phosphoproteins and attempts to relate changes to the altered mineralization witnessed during fluorosis. Wistar rats were rendered fluorotic by the administration of 20 ppm sodium fluoride in their drinking water ad libitum, a nonfluorotic group received double-distilled, deionized water only. After 17 weeks, the teeth showed signs of fluorosis. The incisors were removed, split longitudinally, and the pulps were removed. Teeth were powdered and demineralized in 10% EDTA with protease inhibitors, after which the organic matrix was extracted with 4 M guanidinium chloride. Phosphoproteins were selectively precipitated from the soluble extract by the addition of 1.0 M calcium chloride and further purified by anion exchange chromatography. SDS-PAGE revealed two protein bands with molecular weights of 130 kDa and 66 kDa in the nonfluorotic fraction and 116 kDa and 66 kDa in the fluorotic fraction. Western blotting analysis identified the 66 kDa band as α₂-HS glycoprotein which co-precipitated with phosphoproteins. Electroelution of the protein bands was performed with subsequent biochemical analyses. Phosphate content was determined for each protein band and was detectable in the 116 kDa and 130 kDa bands from the fluorotic and nonfluorotic samples, respectively, with a decreased level noted in the 116 kDa band. The presence of phosphate and the amino acid analysis of these bands suggested their identity to be dentine phosphoproteins. No changes in the ratio of amino acids was detected in fluorotic samples. The fluoride-induced alterations to the biochemical structure of dentine phosphoproteins would appear to influence the phosphorylation of these macromolecules only, possibly affecting posttranslational events. Such alterations may play a role in disrupting the patterns of mineralization seen during fluorosis. Received: 14 November 1997 / Accepted: 9 July 1998     

Abnormalities of cAMP signaling in affective disorders: implication for pathophysiology and treatment

Objective: During the last decade, much attention has been given to the role of signal transduction pathways in affective disorders. This review describes the possible role of the cAMP signaling in such disorders.

Methods: Among the components of cAMP signaling, this review focuses on the cAMP-dependent phosphorylation system. We analyzed the basic components of the cAMP-dependent phosphorylation system and the preclinical evidence supporting their involvement in the biochemical action of antidepressants and mood stabilizers. The clinical data available until now, concerning the possible link between the cAMP-dependent phosphorylation system and the pathophysiology of affective disorders, are also reviewed.

Results: The studies herein presented demonstrated that the levels and the activity of cAMP-dependent protein kinase are altered by antidepressants and mood stabilizers. Furthermore, these medications are able to modify the phosphorylation state, as well as the levels of some of the cAMP-dependent protein kinase substrates. More recently, clinical studies have reported abnormalities in the cAMP-dependent phosphorylation system in both peripheral cells and the postmortem brain of patients with affective disorders.

Conclusions: Overall, these studies support an involvement of cAMP signaling in affective disorders. The precise knowledge of the findings has the potential to improve the understanding of pharmacotherapy and to provide directions for the development of novel biochemical and genetic research strategies on the pathogenesis of affective disorders.     

酸性核糖体磷酸化蛋白P(0-2)的研究进展

酸性核糖体磷酸化蛋白P0、P1、P2是位于真核生物核糖体60S大亚基上的三种核糖体磷酸化蛋白,它们在核糖体上共同组成一个独特的向外侧凸出的五聚体复合物核糖体茎区,此茎区与核糖体上的28 SrRNA的一个保守结构域共同形成一个GTPase相关位点,并在蛋白质翻译延伸过程起着重要的作用。此外,酸性核糖体磷酸化蛋白P0、P1、P2还与细胞凋亡、肿瘤和免疫性疾病的发生、发展密切相关。本文就这三个蛋白结构、功能及其相关作用机制作一综述。     

Regulation and subcellular localization of the microtubule-destabilizing stathmin family phosphoproteins in cortical neurons

Stathmin is a ubiquitous cytosolic phosphoprotein, preferentially expressed in the nervous system, and the generic element of a protein family that includes the neural-specific proteins SCG10, SCLIP, and RB3 and its splice variants, RB3' and RB3". All phosphoproteins of the family share with stathmin its tubulin binding and microtubule (MT)-destabilizing activities. To understand better the specific roles of these proteins in neuronal cells, we performed a comparative study of their expression, regulation, and intracellular distribution in embryonic cortical neurons in culture. We found that stathmin is highly expressed ( approximately 0.25% of total proteins) and uniformly present in the various neuronal compartments (cell body, dendrites, axon, growth cones). It appeared mainly unphosphorylated or weakly phosphorylated on one site, and antisera to specific phosphorylated sites (serines 16, 25, or 38) did not reveal a differential regulation of its phosphorylation among neuronal cell compartments. However, they revealed a subpopulation of cells in which stathmin was highly phosphorylated on serine 16, possibly by CaM kinase II also active in a similar subpopulation. The other proteins of the stathmin family are expressed about 100-fold less than stathmin in partially distinct neuronal populations, RB3 being detected in only about 20% of neurons in culture. In contrast to stathmin, they are each mostly concentrated at the Golgi apparatus and are also present along dendrites and axons, including growth cones. Altogether, our results suggest that the different members of the stathmin family have complementary, at least partially distinct functions in neuronal cell regulation, in particular in relation to MT dynamics.     

骨桥蛋白与整合素β3在脑胶质瘤中的表达及其意义

目的探讨骨桥蛋白（OPN）、整合素β3与胶质瘤恶性程度的关系,及OPN与整合素β3之间的相关性.方法采用免疫组化染色、逆转录-聚合酶链反应（RT-PCR）及免疫印迹法（Western B1ot）等方法检测正常人脑组织和胶质瘤组织中OPN、整合素β3的mRNA及蛋白表达.结果正常脑组织中无OPN、整合素β3表达,而胶质瘤细胞膜、细胞质及血管内皮细胞中有表达.随着脑胶质瘤病理分级的增高,OPN与整合素β3的表达显著性增高（P＜0.05）.OPN与整合素β3的mRNA（r=0.990,P＜0.05）及蛋白（=0.943,P＜0.05）表达均呈正相关.结论OPN与整合素β3在人脑胶质瘤组织中的表达强度与其恶性程度相关,且OPN和整合素β3的表达之间有内在联系.     

Effect of various forms of training and stimulation on the incorporation of 32P into nuclear phosphoproteins of the rat brain

Incorporation of ³²P into acid-extractable nuclear proteins was measured in the hippocampus, caudate nucleus, rest of the brain, and liver of rats submitted to various different behavioral treatments in a shuttle-box. After 5 min of classical conditioning, of avoidance without CS-US pairing, and of avoidance with CS-US pairing (standard shuttle avoidance), there was an increased ³²P uptake by acid-extractable nuclear proteins in the hippocampus and caudate nucleus. The effect disappeared between 5 and 25 min of training. After 25 min of buzzers alone, or of footshocks alone, a similar ³²P uptake change was noted in the same brain structures, which raises doubts as to the specificity of the phenomenon in terms of learning mechanisms.     

Membrane phosphoproteins of rat hippocampus: Sensitivity to tetanic stimulation and enkephalin

Hippocampal slices are electrically stimulated in the perforant path with a pulse-train, which can lead to long-term potentiation (LTP). Of the thus stimulated slices, subcellular fractions are prepared and used in an endogenous protein phosphorylation assay. A phosphoprotein band which was reported earlier to be sensitive to electric stimulation as well as to methionine-enkephalin is now further analyzed: it consists of two phosphoproteins only slightly differing in molecular weight: 50,000 Mr (50 K) and 52,000 Mr (52 K), but having distinct biochemical properties and subcellular localization. Their IEP is dissimilar (3.5-4.3 and 5.3, respectively), they display different sensitivity towards calcium when tested in the phosphorylation assay, but are both cAMP-independently phosphorylated. Only one of them responds to tetanic stimulation with an increased phosphorylation post hoc. This protein, the 52 K component, is localized in synaptic membranes. Moreover, this protein also responds to incubation of slices with methionine-enkephalin. The phosphorylation of the 50 K component is not influenced by electric stimulation, nor by incubations with neuropeptides; its phosphorylation takes place in material sedimenting with the mitochondrial cell fractions and is strongly calcium- and calmodulin-dependent.     

The role of cell type-specific responses in IFN-β therapy of multiple sclerosis

The mechanism of IFN-β therapy in relapsing-remitting multiple sclerosis (RRMS) is not well understood, but induction of apoptosis in specific leukocyte subsets is likely to be important. Enhanced expression of TNFSF10 or TNF-related apoptosis-inducing ligand (TRAIL) mRNA in unseparated leukocytes has been put forward as a therapeutic response marker, but it is unclear which leukocyte subsets express TRAIL. We investigated the basis of TRAIL expression in response to IFN-β by studying activation of STATs 1, 3, and 5, p38 MAPK, and NF-κB in different leukocyte subsets of patients with RRMS. Monocytes, B cells, and T cells showed substantial differences in the activation of p38 and the STATs in response to i.m. injection of IFN-β1a or stimulation in vitro. Induction of cell-surface TRAIL, analyzed in nine leukocyte subsets, was observed only on monocytes and granulocytes and correlated with the activation of p38 and/or NF-κB in these subsets only, in agreement with previous work in fibroblasts showing that the induction of TRAIL in response to IFN-β depends on the activation of p38 and NF-κB as well as STATs 1 and 2. We propose that, in myeloid cells, the differential activation of p38 and NF-κB and induction of TRAIL, which sensitizes cells to apoptosis, can help to explain differences in responsiveness to IFN-β therapy among patients with RRMS and, furthermore, that such differential patterns of activation and expression may also be important in understanding the therapeutic responses to IFN-α/β in hepatitis and cancer.     

VASP在结肠癌组织中的表达及其对预后的影响

目的 探讨血管扩张刺激磷蛋白(vasodilator stimulated phosphoprotein,VASP)在结肠癌中的表达及对预后的影响.方法 收集手术切除的60例结肠癌组织及对应癌旁组织,采用免疫组织化学法检测VASP蛋白的表达情况.随访3年,采用x2检验分析VASP蛋白与临床病理资料间的相关性,Kaplan-Meier生存曲线分析VASP蛋白表达对患者预后的影响.结果 VASP在结肠癌组织中表达水平高于对应癌旁组织(P＜0.05).结肠癌组织中VASP蛋白阳性表达与肿瘤直径增大(≥5 cm)、淋巴结转移及高TNM分期(Ⅲ～Ⅳ期)呈正相关(均P＜0.05).VASP蛋白阳性表达的患者3年总生存率及无瘤生存率均低于VASP蛋白表达阴性患者(均P＜0.05).Cox多因素回归分析证实,VASP阳性表达是结肠癌患者不良预后的独立危险因素之一(P ＜0.05).结论 VASP在结肠癌组织中表达上调,高水平VASP蛋白表达与肿瘤恶性临床病理特征及不良预后有关,VASP可能成为结肠癌患者诊断和预后评价的有效分子标志物.     

兔牙本质磷酸蛋白稳定性分析

目的：探讨兔牙本质磷酸蛋白（Dentin phosphoproteins,DPP）的稳定性机制。方法：通过钙离子沉淀法从兔切牙牙本质非胶原蛋白（Dentin non-collagenous proteins,NCP）中提取DPP，并应用凝胶过滤及离子交换层析技术纯化磷酸蛋白，同时观察多种蛋白酶抑制剂对DPP降解的抑制作用。结果：钙离子能够启动DPP的降解系统，而该作用可被巯基蛋白酶抑制剂N－顺丁烯二酰亚胺（NEM）抑制，从而阻止DPP分解。结论：提示NCPs中可能存在钙离子依赖性蛋白水解酶体系。