Expression analysis on 14-3-3 proteins in regenerative liver following partial hepatectomy

Abstract 14-3-3 proteins play a vital part in the regulation of cell cycle and apoptosis as signaling integration points. During liver regeneration, the quiescent hepatocytes go through hypertrophy and proliferation to restore liver weight. Therefore, we speculated that 14-3-3 proteins regulate the progression of liver regeneration. In this study, we analyzed the expression patterns of 14-3-3 proteins during liver regeneration of rat to provide an insight into the regenerative mechanism using western blotting. Only four isoforms (γ, ε, σ and τ/θ) of the 14-3-3 proteins were expressed in regenerative liver after partial hepatectomy (PH). The dual effects, the significant down-regulation of 14-3-3ε and the significant up-regulation of 14-3-3τ/θ at 2 h after PH, might play particularly important roles in S-phase entry. The significant peaks of 14-3-3σ at 30 h and of ε and τ/θ at 24 h might be closely related not only to the G2/M transition but also to the size of hepatocytes. Possibly, the peak of 14-3-3ε expression seen at 168 h plays critical roles in the termination of liver regeneration by inhibiting cellular proliferation.

The sensitivity of auxiliary examinations in different stages of sporadic Creutzfeldt-Jakob disease / 解放军医学杂志

Objective To analyze the sensitivity of auxiliary examinations in different periods of sporadic Creutzfeldt-Jakob disease (sCJD).Methods The clinical data of 53 sCJD patients were retrospectively analyzed including the different stages of skull diffusion-weighted magnetic resonance imaging (DWI),24-hour ambulatory electroencephalogram (EEG),18F-FDG PET/CT (PET-CT)and cerebrospinal fluid 14-3-3 protein.When calculating the sensitivity of an auxiliary examination,the diagnostic criteria were defined by combining the specific clinical manifestations with two or more positive results of other auxiliary examinations.Results There were 24,53 and 22 sCJD patients,respectively,met the criterion of early (E),middle (M) and later (L) stage of disease (some patients fit 2 or 3 stages).The sensitivity ofDWl (E:58.3％ M:85.4％,L:94.7％),EEG (E:45.8％,M:62.7％,L:77.8％),14-3-3 protein in cerebrospinal fluid (E:11.1％,M:52.9％) and PET-CT (E:80％,M:100％) increased gradually with disease progression,The sensitivity of PET-CT was higher than the other auxiliary examinations for E and M stages;no PET-CT was conducted in L stage.High signal regions mainly distributed in the cortex in E and M stages,but in L stage,no significant difference was found on the distribution of high signal regions between cortex and basal ganglia.Conclusions The sensitivities of the auxiliary examinations were different for sCJD patients in different stages.Reexaminations in different periods may improve the sensitivity for sCJD diagnosis.The sensitivity of PET-CT was high,and the combination of PET-CT and other auxiliary examinations may play a key role in the diagnosis of sCJD.

The Effects of 14-3-3 Phosphorylation Induced by JNK on Ischemic Brain Injury in Rats / 天津医药

Objective To investigate the effects of 14-3-3 phosphorylation (p-14-3-3) induced by C-Jun N-termi-nal kinase (JNK) on ischemic brain injury in rats. Methods Twenty rats were divided into 4 groups:sham operation group, ischemia-reperfusion group, SP600125 group and solvent control group. The rat model of cerebral ischemia was established. The p-14-3-3, the binding of 14-3-3 and Bax and the protein expression of Bax in cytoplasm and mitochondria in hippo-campal CA1 region were detected by immunoprecipitation (IP) and immunoblotting 12-hour after ischemia-reperfusion in four groups. Results Compared with the sham operation group, protein expression levels of p-14-3-3 in cytoplasm and Bax in mitochondria were significantly increased, the binding of 14-3-3 and Bax was significantly decreased in ischemia-re-perfusion group, solvent control group and SP600125 group. The protein expressions of p-14-3-3 and Bax were significantly lower in SP600125 group than those of ischemia-reperfusion group and solvent control group. The binding of 14-3-3 and Bax was significantly higher in SP600125 group than that of ischemia-reperfusion group and solvent control group (P <0.05). Conclusion 14-3-3 phosphorylation induced by JNK plays important effects on ischemic brain injury in rats.

Human 14-3-3γ gene transfer may protect dopaminergic neurons in rat of Parkinson's disease / 中华老年医学杂志

Objective To explore the effects of 14-3-3 γ gene on dopaminergic neurons of PD rat model.Methods 6 hydroxydopamine (6-OHDA) was injected into the corpus striatum of 60 SD rats to establish PD model.A week later,Ad/14-3-3 γ was injected into the corpus striatum of 16 rats,while PBS and Ad-LacZ were injected into the corpus striatum of 16 and 28 rats,respectively,as control.X-gal dyeing was utilized to examine the LacZ reporter gene expression in the corpus striatum at 3 day,2 week and 6 week.Real-time PCR was utilized to test the expression level of 14-3-3 γmRNA at 2 weeks after Ad/14-3-3 γ injection.Immunohistochemistry technique was used to detect TH positive neurons and fibers in the corpus striatum and substantial nigra.Western blotting was performed to check the expression of 14-3-3 γ in the corpus striatum at 2 weeks and caspase-3 at 6 veeks after Ad/14-3-3 γ injection.High performance liquid chromatography (HPLC) method was used to examine the contents of DA and DOPAC in the corpus striatum.The rats underwent rotational ethological examination at 1,2 and 6 weeks after the second injection.Results The expression of β-gal,which showed the successful LacZ gene transfection,was found in the corpus striatum of LacZ groups.The 14-3-3 γ mRNA and protein expression in the corpus striatum were significantly higher in the Ad/14-3-3 γ group than in the other two groups.The TH-positive cell ratio of substania nigra to contralateral area in the lesion side was 0.44±0.17 and the optical density (OD) of TH-positive fibers was 0.62±0.14 in the Ad/14-3-3 γ group,both higher than those in PBS group (0.16±0.13 and 0.36±0.15) and LacZ group (0.15±0.09 and 0.30±0.11) (all P＜0.01).The contents of DA and DOPAC in the lesion sides of corpus striatum were increased in the Ad/14-3-3 γ group [(248± 116)ng/g and (752±177) ng/g] than in PBS group [(106±35) ng/g and (724±159) ng/g] and LacZ group [(136±49) ng/g and (765±163) ng/g] (P＜0.01).The DA and DOPAC ratios of the lesion side of corpus striatum to the contralateral side were 0.37±0.14 and 0.38±0.17 in the Ad/14-3-3 γgroup,higher than those in PBS group (0.15±0.08 and 0.13±0.10,respectively) and LacZ group (0.19±0.11 and 0.16±0.09 (all P＜0.01).The expression level of caspase-3 was decreased in Ad/14-3-3 γ group than in PBS and LacZ groups at 6 weeks after the second injection.The turns/min induced by apomorphine in Ad/14-3-3 γ group were 9.4 ± 2.5 at 1 week after the Ad/14-3-3 γinjection,and reduced to 4.6±2.2 at 6 weeks later.While in PBS and LacZ group,the turns were 14.5±4.9 and 13.8±3.5 at 1 week after the second injection,6 weeks later rised to 18.7±5.2 and 20.6± 6.7 respectively,significantly higher than those in Ad/14-3-3 γ group (all P＜0.01).Conclusions 14-3-3γ gene transfer has a protective effect on the dopaminergic neurons and it may be a promising candidate gene for curing PD.

Progression of 14-3-3σ in nasopharyngeal carcinoma / 国际肿瘤学杂志

14-3-3σ,an vital tumor suppressor which is regulated by p53,plays a key role in cell cycle regulation, apoptosis, migration and proliferation, affecting tumor formation, invasion and metastasis. The methylation inactivation of 14-3-3σ is widely recognized as one of the mechanisms of tumorigenesis,and be associated with the metastasis of NPC.

14-3-3epsilon protein increases matrix metalloproteinase-2 gene expression via p38 MAPK signaling in NIH3T3 fibroblast cells

One of the 14-3-3 protein isoforms, 14-3-3epsilon, was previously shown to be increased during skin aging. We suggest here a possible role for the 14-3-3epsilon protein in skin aging by providing evidence that 14-3-3epsilon increases the expression of the matrix-metalloproteinase (MMP)-2 gene in NIH3T3 fibroblast cells. Expression of the 14-3-3epsilon gene in NIH3T3 cells primarily up-regulated the expression of the MMP-2 gene at the transcriptional level by inducing specific DNA binding proteins bound to an upstream region of the MMP-2 promoter from -1,629 to -1,612. Inhibition of endogenous 14-3-3epsilon gene expression by RNA interference also decreased endogenous MMP-2 gene expression. Furthermore, up-regulation of the MMP-2 gene by 14-3-3epsilon was suppressed by expression of a dominant-negative mutant of p38 MAP kinase. These findings strongly suggest that increased expression of 14-3-3epsilon contributes to remodeling of extracellular matrix in skin through increasing MMP-2 gene expression via p38 MAP kinase signaling.

Steroid-Responsive Recurrent Encephalopathy Associated with Subacute Thyroiditis

BACKGROUND: Steroid-responsive encephalopathy associated with subacute thyroiditis has, to our knowledge, not been reported previously. CASE REPORT: A 49-year-old woman was found collapsed and brought to our institution with decreased mentality, dysarthria, and gait disturbance. Brain magnetic resonance imaging and angiography were normal but blood tests revealed thyroid-autoantibody-negative thyrotoxicosis. Results of a (99m)technetium-pertechnetate scan were compatible with the thyrotoxic phase of subacute thyroiditis. 14-3-3 proteins were detected in cerebrospinal fluid. Her mental status began to improve from the day following steroid administration. Recurrent encephalopathy was found 2 months after the initial admission, which was also effectively treated with steroid. CONCLUSIONS:We speculate that steroid-responsive recurrent encephalopathy associated with subacute thyroiditis is a subtype of Hashimoto's encephalopathy, and consider that steroid treatment should not be delayed in suspected patients.

Relationship between 14-3-3β expression of classic type of Kaposi's sarcoma in Xinjiang and its proliferation and apoptosis / 肿瘤

Objective: To explore the relationship between 14-3 -3β expression and proliferation and apoptosis of classic type of Kaposi's sarcoma (KS) in Xinjiang. Methods: Immunohistochemical Envision 2-step method was used to detect 14-3-3β expression in 10 cases of classic type of KS and 12 cases of normal skin tissues from healthy subjects. Cell proliferating index (PI) was measured. Cell apoptotic index (AI) was determined by the TdT-mediated dUTP nick end labeling (TUNEL) method. Results: The positive expression rate of 14-3-3β protein in KS tissues was significantly higher than that in normal skin tissues (P <0.001). The degree of positive reaction was significantly different between KS and normal skin tissues (t = 14.661, P <0.01). The PI and AI of KS cells were significantly higher than that of normal skin tissues(t = 5.427 and 2.712,P <0.05). The expression of 14-3-3β positively correlated with PI and AI (r = 0.770 and 0.879, P < 0.01). Conclusion: Over-expression of 14-3-3β is related with the loss of control of proliferation of classic type of Kaposi's sarcoma in Xinjiang. The apoptosis of KS is related with multiple pathways and regulates by several negative regulating factors.

Expression of 14-3-3 proteins and Bax in the placenta of preeclampsia / 대한산부인과학회지

OBJECTIVE: Purpose of our study was to examine the expression level of 14-3-3 proteins and Bcl-2 family and to estimate the interaction between 14-3-3 proteins and Bcl-2 family in normal and preeclamptic placenta. METHODS: Placental tissues were sampled from preeclampsia with caesarean delivery (n=5) and normal pregnant women with caesarean delivery (n=5). Western blot and immunoprecipitation related Western blotting were performed on all placental samples. Unpaired Student t-test was used to determine the statistical significance. RESULTS: Western blot analysis revealed that the expression of Bax and 14-3-3 zeta was significantly greater in the preeclamptic placenta than in the normal placenta. Immunoprecipitation related Western blotting revealed that the interaction between 14-3-3 zeta and Bax was significantly less in the preeclamptic placenta than in the normal placenta. CONCLUSION: Increased expression of Bax and reduced interaction (between) 14-3-3 zeta and Bax in preeclamptic placenta might influence pathogenesis or sequelae of preeclampsia. Further study is needed to identify the trigger that induces dissociation of Bax from 14-3-3 proteins.

14-3-3 proteins are associated with GLUT4 in rat adipocytes / 基础医学与临床

Objective To investigate the interaction between glucose transporter 4(GLUT4) and the(14-3-3) proteins in rat adipocytes.Methods Isolated primary adipocytes were obtained from the epididymal fat pads of male Sprague-Dawley rats by collagenase Ⅰ digestion.Total cell extract as well as three fractions,referred to as Fraction T,H and L,were obtained from rat adipocytes using hypotonic lysis and glycerol gradient velocity sedimentation.Immunoadsorption of GLUT4 with Trisacryl beads cross-linked with 1F8,a specific monoclonal antibody to GLUT4,was conducted in total cell extract and the three fractions and followed by elution and Western blot analysis.Co-immunoprecipitation of(14-3-3) proteins and GLUT4 was demonstrated by detecting(14-3-3) in the eluates.Results(14-3-3) proteins were co-immunoprecipitated with GLUT4 either in the total adipocyte extract, or in each of the three fractions mentioned above.The co-precipitated(14-3-3)s were identified as two bands in Western blot analysis,the molecular weights of which were approximately 29kDa and 60kDa respectively,indicating that they might be in the state of homo-or hetero-dimers while interacting with GLUT4.Conclusion There is a physiological interaction between(143-3) proteins and GLUT4 in rat adipocyte.

Expression of the 14-3-3 sigma Protein and Methylation Status of the 14-3-3 sigma gene in Biliary Neoplasms

BACKGROUND: The 14-3-3 sigma (sigma) protein has a negative regulatory role in the cell cycle progression of the. Down-regulation or overexpression of the 14-3-3 sigma protein has been reported in various human cancers. METHODS: Immunohistochemistry for the 14-3-3 sigma protein was performed in non-neoplastic bile duct cells, intraductal papillary neoplasms of the liver (IPNL), mass-forming intrahepatic cholangiocarcinomas (ICC) and non-papillary extrahepatic cholangiocarcinomas (ECC). We investigated the methylation status of the 14-3-3 sigma gene in 45 cases of these 3 tumor groups. RESULTS: The non-neoplastic bile duct cells demonstrated negative or weakly positive cytoplasmic immunoreactivity for the 14-3-3 sigma protein and no methylation of the 14-3-3 sigma gene. Overexpression as well as negative immunoreactivity associated with hypermethylation of the 14-3-3 sigma protein was observed in 16 (69.6%) of 23 cases of IPNL, in 21 (63.6%) of 33 cases of mass-forming ICC and in 27 (71.1%) of 38 cases of non-papillary ECC. Negative immunoreactivity was increased in the invasive IPNL (4/6, 66.7%), as well as in the poorly differentiated cases of mass-forming ICC (8/12, 66.7%) and the non-papillary ECC (5/8, 62.5%). CONCLUSIONS: The similar rates for the abnormal expression of the 14-3-3 sigma protein among the three groups of biliary neoplasms indicate its general association with biliary carcinogenesis. Furthermore, the loss of the 14-3-3 sigma protein may be involved in the tumor progression and differentiation in the biliary carcinogenesis.

zVAD-fmk, unlike BocD-fmk, does not inhibit caspase-6 acting on 14-3-3/Bad pathway in apoptosis of p815 mastocytoma cells

In a preliminary study, we found that benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD- fmk), unlike Boc-aspartyl(OMe)-fluoromethylketone (BocD-fmk), at usual dosage could not prevent genistein-induced apoptosis of p815 mastocytoma cells. This study was undertaken to reveal the mechanism underlying the incapability of zVAD-fmk in preventing this type of apoptosis. We observed that 14-3-3 protein level was reduced in genistein-treated cells and that BocD-fmk but not zVAD-fmk prevented the reduction of 14-3-3 protein level and the release of Bad from 14-3-3. We also demonstrated that truncated Bad to Bcl-xL interaction in genistein- treated cells was prevented by BocD-fmk but not by zVAD-fmk treatment. Our data indicate that BocD- fmk, compared to zVAD-fmk, has a certain preference for inhibiting 14-3-3/Bad signalling pathway. We also elucidated that this differential efficacy of BocD-fmk and zVAD-fmk resulted from the different effect in inhibiting caspase-6 and that co-treatment of zVAD-fmk and caspase-6 specific inhibitor substantially prevented genistein-induced apoptosis. Our data shows that caspase-6 plays a role on Bad/14-3-3 pathway in genistein-induced apoptosis of p815 cells, and that the usual dose of zVAD-fmk, in contrast to BocD-fmk, did not prevent caspase-6 acting on 14-3-3/Bad-mediated event.

Expression and diagnostic value of 14-3-3 protein in brains of patients with Creutzfeldt-Jakob disease / 中华神经科杂志

Objectives To investigate the expression and diagnostic value of 14-3-3 protein in brains of patients with sporadic Creutzfeldt-Jakob disease(sCJD).Methods 14-3-3 protein was immunohistochemically analyzed in tissue from the frontal lobe of 5 patients with sCJD and 4 non-CJD eases Using 14-3-3 ?and ?antibodies with reference to the results of KB,GFAP and PrP detection.Results The expressions of 14-3-3 protein in five brains of sCJD were more obviously,mostly in gray matters and astrocytes in three cases.The concentration was related to PrP deposition type,but not related to prion protein genotype.Except few expression of 14-3-3 protein in neurous of two cases of acute contusion,there were no expression in the other two cases in control group.Conclusions The expression of 14-3-3 protein in brain is useful to pathological diagnosis of CJD.