Differential localization and androgen sensitivity of prostatic nuclear protein kinases in euchromatin and heterochromatin fractions

We have examined the distribution and androgenic regulation of protein kinases and phosphoproteins in euchromatin and heterochromatin fractions of rat ventral prostate chromatin. Available procedures to prepare euchromatin and heterochromatin fractions were found to result in the loss of various chromatin-associated protein kinases even though there was no gross change in the gel electrophoretic profile of proteins in these fractions. This loss was prevented by the addition of 0.5 mM phenylmethylsulfonyl fluoride throughout the preparative procedures, which indicates that the protein kinases associated with the chromatin may be particularly susceptible to proteolytic degradation during further subfractionation. By utilizing an improved method for fractionation of chromatin, we have demonstrated a marked enrichment of protein kinase activity (towards phosvitin and endogenous chromosomal proteins) in the euchromatin fraction as compared with heterochromatin. Both of these fractions were also examined for the incorporation of 32P into two main classes of nonhistone proteins (namely, H2SO4-soluble and -insoluble nonhistones). The amount of 32P incorporated into heterochromatin-associated proteins of both classes was markedly less than that in the euchromatin-associated proteins. Protein kinase activities (especially those active towards phosvitin and nonhistone proteins) in the euchromatin fraction as compared with the heterochromatin were significantly reduced within 24 h after androgenic deprivation in the animal. The decreased phosphorylation of nonhistone proteins could be attributed to the loss of endogenous protein kinase activity. The results indicate that not only are chromatin-associated protein phosphokinases preferentially localized in euchromatin fractions but also that these euchromatin-associated protein kinases display the greatest sensitivity to androgenic status of the animal.     

p63蛋白在软骨母细胞瘤中的表达及其价值探讨

目的探讨p63蛋白在软骨母细胞瘤中的表达水平及其诊断价值。方法 采用免疫组织化学法(Maxvision)技术对25例软骨母细胞瘤、52例骨巨细胞瘤、10例动脉瘤样骨囊肿和20例腱鞘巨细胞瘤中p63和S-100p蛋白的表达水平进行分析。结果 p63蛋白在软骨母细胞瘤中呈单核细胞的胞核阳性,阳性率为88.0%(22/25)。而骨巨细胞瘤中除呈单核细胞的胞核阳性,少数多核巨细胞的胞核亦呈阳性,阳性表达率为92.3%(48/52)。p63蛋白在软骨母细胞瘤和骨巨细胞瘤中的表达两者比较差异无统计学意义(P>0.05),而S-100p在软骨母细胞瘤和骨巨细胞瘤中阳性表达率分别为88.0%(22/25)和1.9%(1/52),两者差异有统计学意义(P<0.05)。p63蛋白和S-100p在动脉瘤样骨囊肿和腱鞘巨细胞瘤中均不表达。结论 p63蛋白在软骨母细胞瘤和骨巨细胞瘤中均呈高表达,并有相似的免疫表型,提示两者在发生分化上可能均来自相同的起源细胞或肿瘤干细胞。p63蛋白结合S-100p联合应用对鉴别软骨母细胞瘤及其相关性病变具有一定辅助诊断意义。     

Is DARPP-32 a potential therapeutic target?

Signaling pathways play important roles in the coordination and integration of a myriad cellular functions. Because of widespread interest in the dopaminergic pathways, the protein dopamine and cyclic adenosine 3′,5′-monophosphate-regulated phosphoprotein with molecular weight of 32 kDa, known by the acronym DARPP-32, occupies a central role in the biology of dopaminoceptive neurons in the central and peripheral nervous system (PNS). Its involvement has been demonstrated in many neural phenomena, including physiologic and pathologic neuroplasticity to drug effects and cognition. However, DARPP-32 has also been identified in non-neuronal tissues and its level of expression has been associated with the malignant level of some types of cancer, via modulation of cell survival and differentiation. This review considers some of these apparently compartmentalized functions of DARPP-32 and its potential as a therapeutic target.     

一种磷酸化蛋白质组定性定量分析方法的考察

目的 对用于磷酸化蛋白质的相对定量分析的一种基于双向差示凝胶电泳(2-D DIGE)技术和Pro-Q Diamond 荧光染色的方法进行考察.方法 采用DIGE技术,从Pro-Q Diamond染色后对Cy2、Cy5荧光强度的影响、Pro-QDiamond染色过程中采用的试剂对CyDye标记的蛋白质的影响、去离子水对DIGE扫描结果的影响、CyDye染料扫描的稳定性影响等方面,考察了该方法的有效性和特异性.结果 在Pro-Q Diamond染色前对DIGE凝胶进行处理的过程中,有多种因素影响荧光强度变化,以致在进行pro-Q Diamond染色后,凝胶点的荧光强度变化无法区分是染色造成的灰度值变化还是CyDye染料自身的变化,从而使通过荧光强度变化来定量检测磷酸化蛋白质变得困难.结论 不宜采用对同一DIGE凝胶进行Pro-Q Diamond再染色检测磷酸化蛋白质的方法进行比较蛋白质组学的定量分析,为能同时展示出胶上的磷酸化蛋白质和总蛋白质,可通过DIGE实验与Pro-Q Diamond分开运行,再通过DIGE扫描图谱与Pro-Q Diamond染色图谱进行匹配的方法进行改进.     

Inhibition of hydroxyapatite growth by casein,a potential salivary phosphoprotein homologue

Salivary phosphoproteins are essential in tooth mineral regulation but are often overlooked in vitro. This study aimed to evaluate the effect of casein, as a salivary phosphoprotein homologue, on the deposition and growth of hydroxyapatite (HA) on tooth surfaces. Hydroxyapatite growth was quantified using seeded crystal systems. Artificial saliva (AS) containing HA powder and 0, 10, 20, 50, or 100 μg ml^?1 of casein, or 100 μg ml^?1 of dephosphorylated casein (Dcasein), was incubated for 0–8 h at 37°C, pH 7.2. Calcium concentrations were measured using atomic absorption spectroscopy (AAS). Surface precipitation of HA on bovine enamel and dentine blocks, incubated in similar conditions for 7 d, was examined using field emission scanning electron microscopy (FE‐SEM) and transmission electron microscopy (TEM) with selected area electron diffraction (SAED). Casein adsorption was assessed using modified Lowry assays and zeta‐potential measurements. The AAS results revealed a concentration‐dependent inhibition of calcium consumption. Hydroxyapatite precipitation occurred when no casein was present, whereas precipitation of HA was apparently completely inhibited in casein‐containing groups. Adsorption data demonstrated increasingly negative zeta‐potential with increased casein concentration and an affinity constant similar to proline‐rich proteins with Langmuir modelling. Casein inhibited the deposition and growth of HA primarily through the binding of esterized phosphate to HA active sites, indicating its potential as a mineral‐regulating salivary phosphoprotein homologue in vitro.     

Phosphoryl transfer reaction regulated by amino acid side chains: A model for phosphoproteins

Through a kinetic study of the reaction of phosphoamino acids as incubated in alcohol, it was found that the inter- and intramolecular phosphoryl transfer reactions were regiospecific and stereoselective. First, the phosphoryl transfer reaction required the regio-specifically neighboring α-carboxy group activation of amino acid but not 8-carboxy group. The intramolecular side chain catalytic effects relative to hydrogen for N-phosphoamino acids compared to N-phosphoglycine were a 119- to 4-times enhancement of the phosphoryl transfer reaction, respectively. Secondly, the intramolecular N→O phosphoryl transfer migration was highly stereoselective, since the reaction rate constant of phosphoallothreonine relative to its diastereomeric threonine was reduced to half. The pentacoordinate transition states modulated by the amino acid side chains were demonstrated by the formation rates of intramolecular pentacoordinate spiro mixed anhydride compounds. © Munksgaard 1996.     

Measuring the biological effect of presurgical metformin treatment in endometrial cancer

Background:

Preclinical studies in endometrial cancer (EC) show that metformin reduces cellular proliferation by PI3K-AKT-mTOR inhibition. We tested the hypothesis that short-term presurgical metformin reduces cellular proliferation in atypical endometrial hyperplasia (AEH) and endometrioid EC, and assessed the feasibility of using phosphorylated PI3K-AKT-mTOR proteins as tissue end points.

Methods:

Women with AEH or EC received metformin 850 mg twice a day or no drug in the presurgical window between diagnosis and hysterectomy. Before and after the window, tissue samples were obtained; serum markers of insulin resistance (e.g. homeostasis model of assessment of insulin resistance index) were determined; and anthropometrics measured (e.g. BMI). Cell proliferation (Ki-67) and PI3K-AKT-mTOR phosphostatus were assessed by immunohistochemistry and scored blinded to treatment.

Results:

Twenty-eight metformin-treated and 12 untreated patients, well matched for age and BMI, completed the study. Metformin treatment (median 20 days, range 7–34) was associated with a 17.2% reduction in tumour Ki-67 (95% CI −27.4, −7.0, P=0.002), in a dose-dependent manner. Tumour PI3K-AKT-mTOR protein phosphostatus varied but the effects were not significant after adjusting for changes in controls.

Conclusions:

Short-term metformin was associated with reduced Ki-67 expression in EC. Changes in tumour PI3K-AKT-mTOR protein phosphostatus were seen in both groups. Future studies should address the variability attributed to different sampling techniques including devascularisation of the uterus at hysterectomy.     

Phospho-accepting proteins in bovine sera

Summary Phospho-accepting proteins in bovine sera have been detected by the use of immobilized protein kinase from rat muscle and (³²P)-ATP in an in vitro system. A partial biochemical characterization points to the generation of typical phosphoproteins. Differences in the phosphorylation pattern between fetal serum and calf serum as demonstrated by electrophoresis in the presence of dodecylsulfate are described.

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Zusammenfassung Mit Hilfe einer immobilisierten Proteinkinase aus Rattenmuskel und (³²P)-ATP konnten in vitro typische Phosphoproteine des Rinderserums erzeugt werden, wie eine partielle biochemische Charakterisierung des Produktes ergab. Die Auftrennung phosphorylierter Seren im Polyacrylamidgel in der Gegenwart von Natriumdodecylsulfat ergibt eindeutige Unterschiede zwischen fötalem und Kälberserum, die beschrieben werden.

     

A lasting change in protein phosphorylation associated with septal kindling

Male Holtzman rats stimulated through a right medial septal electrode displayed prominent hippocampal afterdischarges and the progressive development of kindled seizures. Hippocampal synaptic plasma membranes of kindled rats showed a reduction of³²P incorporation into a protein of approximately 50,000 daltons. This change was not present in controls that received twice as many stimulations as kindled rats with a timing that did not lead to kindling. In kindled rats, reduction of³²P incorporation persisted at least 2 months without further stimulation.