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Godoy A Ulloa V Rodríguez F Reinicke K Yañez AJ García Mde L Medina RA Carrasco M Barberis S Castro T Martínez F Koch X Vera JC Poblete MT Figueroa CD Peruzzo B Pérez F Nualart F 《Journal of cellular physiology》2006,207(3):614-627
It has been proposed that the enhanced metabolic activity of tumor cells is accompanied by an increased expression of facilitative hexose transporters (GLUTs). However, a previous immunohistochemical analysis of GLUT1 expression in 154 malignant human neoplasms failed to detect the GLUT1 isoform in 87 tumors. We used 146 normal human tissues and 215 tumor samples to reassess GLUT1 expression. A similar number of samples were used to compare the expression of GLUT2-6 and 9. The classical expression of GLUT1-5 in different normal human tissues was confirmed, however, we were unable to detect GLUT2 in human pancreatic islet cells. GLUT6 was principally detected in testis germinal cells and GLUT9 was localized in kidney, liver, heart, and adrenal. In tumor samples, GLUT1, 2, and 5 were the main transporters detected. GLUT1 was the most widely expressed transporter, however, 42% of the samples had very low-to-negative expression levels. GLUT2 was detected in 31% of the samples, being mainly expressed in breast, colon, and liver carcinoma. GLUT5 was detected in 27% of breast and colon adenocarcinoma, liver carcinoma, lymphomas, and testis seminoma samples. In situ RT-PCR and ultrastructural immunohistochemistry confirmed GLUT5 expression in breast cancer. GLUT6 and 9 are not clearly over-expressed in human cancer. The extensive expression of GLUT2 and 5 (glucose/fructose and fructose transporters, respectively) in malignant human tissues indicates that fructose may be a good energy substrate in tumor cells. Our functional data obtained in vitro in different tumor cells support this hypothesis. Additionally, these results suggest that fructose uptake could be used for positron emission tomography imaging and, may possibly represent a novel target for the development of therapeutic agents in different human cancers. 相似文献
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A Novel Family of Serine/Threonine Kinases Participating in Spermiogenesis 总被引:5,自引:0,他引:5
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Peter Kueng Zariana Nikolova Valentin Djonov Andrew Hemphill Valeria Rohrbach Dominik Boehlen Gisela Zuercher Anne-Catherine Andres Andrew Ziemiecki 《The Journal of cell biology》1997,139(7):1851-1859
The molecular mechanisms regulating the spectacular cytodifferentiation observed during spermiogenesis are poorly understood. We have recently identified a murine testis-specific serine kinase (tssk) 1, constituting a novel subfamily of serine/threonine kinases. Using low stringency screening we have isolated and molecularly characterized a second closely related family member, tssk 2, which is probably the orthologue of the human DGS-G gene. Expression of tssk 1 and tssk 2 was limited to the testis of sexually mature males. Immunohistochemical staining localized both kinases to the cytoplasm of late spermatids and to structures resembling residual bodies. tssk 1 and tssk 2 were absent in released sperms in the lumen of the seminiferous tubules and the epididymis, demonstrating a tight window of expression restricted to the last stages of spermatid maturation. In vitro kinase assays of immunoprecipitates containing either tssk 1 or tssk 2 revealed no autophosphorylation of the kinases, however, they led to serine phosphorylation of a coprecipitating protein of ~65 kD. A search for interacting proteins using the yeast two-hybrid system with tssk 1 and tssk 2 cDNA as baits and a prey cDNA library from mouse testis, led to the isolation of a novel cDNA, interacting specifically with both tssk 1 and tssk 2, and encoding the coprecipitated 65-kD protein phosphorylated by both kinases. Interestingly, expression of the interacting clone was also testis specific and paralleled the developmental expression observed for the kinases themselves. These results represent the first demonstration of the involvement of a distinct kinase family, the tssk serine/threonine kinases, together with a substrate in the cytodifferentiation of late spermatids to sperms. 相似文献
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Sun X Fontaine JM Rest JS Shelden EA Welsh MJ Benndorf R 《The Journal of biological chemistry》2004,279(4):2394-2402
Mammalian small heat shock proteins (sHSP) are abundant in muscles and are implicated in both muscle function and myopathies. Recently a new sHSP, HSP22 (HSPB8, H11), was identified in the human heart by its interaction with HSP27 (HSPB1). Using phylogenetic analysis we show that HSP22 is a true member of the sHSP superfamily. sHSPs interact with each other and form homo- and hetero-oligomeric complexes. The function of these complexes is poorly understood. Using gel filtration HPLC, the yeast two-hybrid method, immunoprecipitation, cross-linking, and fluorescence resonance energy transfer microscopy, we report that (i). HSP22 forms high molecular mass complexes in the heart, (ii). HSP22 interacts with itself, cvHSP (HSPB7), MKBP (HSPB2) and HSP27, and (iii). HSP22 has two binding domains (N- and C-terminal) that are specific for different binding partners. HSP22 homo-dimers are formed through N-N and N-C interactions, and HSP22-cvHSP hetero-dimers through C-C interaction. HSP22-MKBP and HSP22-HSP27 hetero-dimers involve the N and C termini of HSP22 and HSP27, respectively, but appear to require full-length protein as a binding partner. 相似文献
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Morales A Vilchis F Chávez B Chan C Robles-Díaz G Díaz-Sánchez V 《The Journal of steroid biochemistry and molecular biology》2007,107(1-2):37-41
Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) was recently identified as the first tissue-specific angiogenic molecule. EG-VEGF (the gene product of PROK-1) appears to be expressed exclusively in steroid-producing organs such as the ovary, testis, adrenals and placenta. Since the human pancreatic cells retain steroidogenic activity, in the present study we ascertained whether this angiogenic factor is expressed in normal pancreas and pancreatic adenocarcinoma. Tissue samples from normal males (n=5), normal females (n=5) and from surgically resected adenocarcinomas (n=2) were processed for RT-PCR and immunohistochemical studies. Results from semi-quantitative analysis by RT-PCR suggest a distinct expression level for EG-VEGF in the different tissue samples. The relative amount of EG-VEGF mRNA in pancreas was more abundant in female adenocarcinoma (0.89) followed by male adenocarcinoma (0.71), than normal female (0.64) and normal male (0.38). The expression of mRNA for EG-VEGF in normal tissue was significantly higher in females than in males. All samples examined showed specific immunostaining for EG-VEGF. In male preparations, the positive labeling was localized predominantly within the pancreatic islets while in female preparations the main staining was detected towards the exocrine portion. Specific immunolabeling was also observed in endothelial cells of pancreatic blood vessels. Our data provide evidence that the human pancreas expresses the EG-VEGF, a highly specific mitogen which regulates proliferation and differentiation of the vascular endothelium. The significance of this finding could be interpreted as either, EG-VEGF is not exclusive of endocrine organs, or the pancreas should be considered as a functional steroidogenic tissue. The extent of the expression of EG-VEGF appears to have a dimorphic pattern in normal and tumoral pancreatic tissue. 相似文献
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Martín-Lacave I Rojas F Bernabé R Utrilla JC Fernández-Santos JM De Miguel M Conde E 《Cell and tissue research》2002,309(3):361-368
In rats, the frequency of spontaneous C-cell tumours is very high and is both age and gender dependent. The three specific stages of neoplastic progression can be distinguished into diffuse C-cell hyperplasia, focal C-cell hyperplasia and bona fide C-cell tumours. Based on this hypothetical model of human medullary thyroid carcinoma (MTC), we carried out an immunohistochemical study using different markers (calcitonin, calcitonin gene-related peptide, somatostatin and chromogranin) to verify the existence of any relationship between their expression and the successive steps of tumour development. We found a characteristic immunohistochemical staining pattern, particularly for calcitonin and somatostatin, which distinguishes C-cell tumours from both normal and hyperplastic C cells, with no differences related to the gender of the animals under study. Specifically, a considerable heterogeneity in calcitonin expression was only displayed by C-cell carcinomas, being less pronounced in C-cell adenomas. As for somatostatin, this regulatory peptide was found only in a minority of calcitonin-positive cells in normal and hyperplastic glands. However, in some C-cell adenomas and most C-cell carcinomas nearly all calcitonin-positive cells also coexpressed somatostatin. We conclude that rat C-cell neoplasms constitute a very particular tumour entity which shares many but not all immunohistochemical features with human MTC. 相似文献
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肝细胞生成素(HPO)具有复杂的生理功能,在睾丸中的高表达提示其在生殖活动中的重要性,而不仅局限于肝再生.构建了酵母表达载体pGBKT7-HPO,采用酵母双杂交系统,以HPO为诱饵蛋白,从人睾丸cDNA文库中寻找能够与HPO相互作用的蛋白质.经过筛选、验证阳性克隆,并进行PCR、测序和序列比对,得到4种相互作用蛋白质:NADH脱氢酶1、钠/钾ATP酶β3亚基、磷脂酶C δ1以及附睾分泌蛋白.提示HPO可能参与了细胞的蛋白质合成,能量代谢等.通过对候选蛋白的研究,为探讨HPO对睾丸组织细胞功能的调节机制提供了重要的线索. 相似文献
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日本脑炎病毒E蛋白在酵母双杂交系统中的表达及其对报告基因激活作用的检测 总被引:2,自引:0,他引:2
用日本脑炎病毒(JEV)E蛋白基因片段构建酵母双杂交诱饵载体,并检测其表达产物对酵母细胞有无毒性作用及对报告基因有无激活作用。用RT—PCR从JEV感染的鼠脑中扩增出JEV E蛋白基因片段,克隆入pUCl9质粒,经测序正确后,再亚克隆入酵母双杂交诱饵载体pGBKT7中。将重组质粒导入酵母菌AHl09,检测其表达产物在酵母细胞中对报告基因有无激活作用。成功获得JEV E蛋白基因片段,表达的E蛋白对酵母菌AHl09无毒性,对报告基因亦无激活作用。为利用酵母双杂交GAL4系统3进行JEV细胞受体蛋白的研究奠定了基础。 相似文献
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Mori T Li Y Hata H Ono K Kochi H 《Biochemical and biophysical research communications》2002,296(3):530-536
Through database mining, we found a novel PEST-containing nuclear protein (PCNP). To characterize PCNP, we carried out yeast two-hybrid screening for PCNP-interacting factors. A novel Np95/ICBP90-like RING finger protein (NIRF), which possessed a ubiquitin-like domain, a PHD finger, a YDG/SRA domain and a RING finger, was identified. Interaction between PCNP and NIRF was clarified by mammalian two-hybrid system, GST pull-down assay, and nuclear co-localization. RT-PCR showed that NIRF expression is high in proliferating phase but significantly low in G0/G1 phase in normal TIG-7 and WI-38 cells, while consistently high in tumoral HT-1080 and HepG2 cells, suggesting that NIRF is involved in cell-cycle regulation. The NIRF gene resides in 9p23-24.1 that is altered in numerous types of tumors at the top of frequency. Furthermore, the NIRF gene is just within small amplicons in some tumors, suggesting that PCNP and NIRF might be involved in some aspects of tumorigenesis. 相似文献
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Cullin3 is a KLHL10-interacting protein preferentially expressed during late spermiogenesis 总被引:1,自引:0,他引:1
Kelch-like 10 (KLHL10) is a member of the BTB (Bric-a-brac, Tramtrack, and Broad-Complex)-kelch protein superfamily essential for spermiogenesis and male fertility. In a search for KLHL10-interacting proteins using a yeast two-hybrid assay, we identified Cullin3 (CUL3) as one of multiple KLHL10-interacting partners. Yeast cotransformation assays revealed that CUL3 bound the BTB/POZ domain of KLHL10. Northern blot and quantitative RT-PCR analyses demonstrated that Cul3 mRNA was preferentially expressed in the testis. In situ hybridization analysis localized Cul3 mRNA to spermatids in the adult testis. CUL3 protein was detected in elongating and elongated spermatids (steps 10-16) by immunofluorescent microscopy. The expression pattern of CUL3 resembles KLHL10. CUL3 was coimmunoprecipated with KLHL10, and KLHL10 was also detected in the CUL3 immunoprecipitants using testis lysates. These findings suggest that KLHL10, like other BTB/kelch proteins, interacts with CUL3 to form a CUL3-based ubiquitin E3 ligase that functions specifically in the testis to mediate protein ubiquitination during spermiogenesis. 相似文献
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Jing Cheng Meihua Ji Haijuan Jing Hongqi Lin 《Journal of biochemical and molecular toxicology》2023,37(5):e23310
This study aimed to explore the role of dual specificity phosphatase 12 (DUSP12) in regulating myocardial ischemia–reperfusion (I/R) injury and the underlying mechanism. The expression of DUSP12 in myocardial tissues and heat-shock protein beta-8 (HSPB8) and mitophagy-related proteins in myocardial tissues and H9c2 cells were detected by western blot analysis. The serum creatine kinase isoenzymes (CK-MB) and lactate dehydrogenase (LDH), levels of reactive oxygen species and malondialdehyde, superoxide dismutase activity in myocardial tissues and H9c2 cells, and caspase-3 activity in H9c2 cells were analyzed by corresponding assay kits. The infarct area in the rat's heart was observed by triphenyl tetrazolium chloride staining. The apoptosis of myocardial cells in myocardial tissues and H9c2 cells was detected by terminal-deoxynucleotidyl transferase dUTP-biotin nick-end labeling assay. The interaction between DUSP12 and HSPB8 was clarified by the coimmunoprecipitation assay. The transfection efficacy of si-HSPB8#1 and si-HSPB8#2 in H9c2 cells was confirmed by real-time quantitative-polymerase chain reaction and western blot analysis. As a result, DUSP12 expression was downregulated in I/R rats, which was promoted by lentivirus-expressing DUSP12. DUSP12 overexpression reduced the serum creatine kinase isoenzymes (CK-MB) and LDH, decreased the infarct area in the rat's heart, and suppressed the apoptosis and oxidative stress in myocardial tissues. DUSP12 overexpression also upregulated the expression of HSPB8 to promote mitophagy. The coimmunoprecipitation assay indicated that DUSP12 could be combined with HSPB8. In addition, DUSP12 overexpression could inhibit hypoxia/reoxygenation-elicited apoptosis as well as oxidative stress in H9c2 cells by upregulating HSPB8 expression to promote mitophagy, which was countervailed by HSPB8 deficiency. In conclusion, DUSP12 overexpression decreased the apoptosis and oxidative stress in myocardial I/R injury through HSPB8-induced mitophagy. 相似文献
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Interaction of the heart-specific LIM domain protein, FHL2, with DNA-binding nuclear protein, hNP220 总被引:3,自引:0,他引:3
Ng EK Chan KK Wong CH Tsui SK Ngai SM Lee SM Kotaka M Lee CY Waye MM Fung KP 《Journal of cellular biochemistry》2002,84(3):556-566
Using a yeast two-hybrid library screen, we have identified that the heart specific FHL2 protein, four-and-a-half LIM protein 2, interacted with human DNA-binding nuclear protein, hNP220. Domain studies by the yeast two-hybrid interaction assay revealed that the second LIM domain together with the third and the fourth LIM domains of FHL2 were responsible to the binding with hNP220. Using green fluorescent protein (GFP)-FHL2 and blue fluorescent protein (BFP)-hNP220 fusion proteins co-expressed in the same cell, we demonstrated a direct interaction between FHL2 and hNP220 in individual nucleus by two-fusion Fluorescence Resonance Energy Transfer (FRET) assay. Besides, Western blot analysis using affinity-purified anti-FHL2 antipeptide antibodies confirmed a 32-kDa protein of FHL2 in heart only. Virtually no expression of FHL2 protein was detected in brain, liver, lung, kidney, testis, skeletal muscle, and spleen. Moreover, the expression of FHL2 protein was also detectable in the human diseased heart tissues. Our results imply that FHL2 protein can shuttle between cytoplasm and nucleus and may act as a molecular adapter to form a multicomplex with hNP220 in the nucleus, thus we speculate that FHL2 may be particularly important for heart muscle differentiation and the maintenance of the heart phenotype. 相似文献
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Qian Zhang Jiang Li Michael Deavers James L Abbruzzese Linus Ho 《The journal of histochemistry and cytochemistry》2005,53(11):1371-1382
Syntaxin 17 (STX17) is a divergent member of the syntaxin family of proteins first discovered by Scheller and colleagues in a yeast two-hybrid screen designed to identify novel mammalian SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors). We recently independently identified STX17 as a novel Ras-interacting protein, but immunohistochemical studies suggested that STX17 is localized to the nucleus in normal pancreatic ductal epithelial, acinar, and islet cells in contrast to previous reports of cytoplasmic localization, albeit in other cell types. Therefore, we have conducted a more thorough survey of various human and mouse tissues to better establish the expression pattern of STX17 in different tissues and cell types. Although RT-PCR experiments demonstrate ubiquitous expression of STX17, closer examination by immunohistochemistry reveal that STX17 expression is limited to certain cell types. Furthermore, in contrast to the cytoplasmic localization previously reported in a limited number of cell types, we find that in many other cell types, syntaxin 17 can be found in the nucleus. Finally, we demonstrate that in human hepatocellular carcinoma cell lines, STX17 localization is altered relative to normal hepatocytes, although the localization of STX17 differs even between these established human cancer cell lines and fresh human hepatocellular carcinoma cells, emphasizing the caution that must be exercised in drawing conclusions from data gathered in cell lines. The sequence divergence of STX17, the unexpected nuclear localization of STX17 in many cell types, and the altered localization of STX17 in malignant cells argue for a novel function of syntaxin 17 distinct from its hypothesized role in mediating membrane fusion events. 相似文献
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Xiehong Liu Ke Liu Caiyan Li Jiaodi Cai Li Huang Huan Chen Hao Wang Jiang Zou Meidong Liu Kangkai Wang Sipin Tan Huali Zhang 《Journal of cellular physiology》2019,234(4):5117-5133
Heat-shock protein B1 (HSPB1) is a multifunctional protein that protects against oxidative stress; however, its function in antioxidant pathways remains largely unknown. Here, we sought to determine the roles of HSPB1 in H9c2 cells subjected to oxidative stress. Using nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis, we found that increased HSPB1 expression promoted the reduced states of glutathione reductase (GR), peroxiredoxin 1 (Prx1), and thioredoxin 1, whereas knockdown of HSPB1 attenuated these responses following oxidative stress. Increased HSPB1 expression promoted the activation of GR and thioredoxin reductase. Conversely, knockdown of HSPB1 attenuated these responses following oxidative stress. Importantly, overexpression of HSPB1 promoted the complex formation between HSPB1 and oxidized Prx1, leading to dephosphorylation of STE-mammalian STE20-like kinase 1 (MST1) in H9c2 cells exposed to H2O 2, whereas downregulation of HSPB1 induced the opposite results. Mechanistically, HSPB1 regulated the Hippo pathway by enhancing the dephosphorylation of MST1, resulting in reduced phosphorylation of LATS1 and Yes-associated protein (YAP). Moreover, HSPB1 regulated YAP-dependent gene expression. Thus, HSPB1 promoted the reduced state of endogenous antioxidant pathways following oxidative stress in H9c2 cells and improved the redox state of the cytoplasm via modulation of the Hippo signaling pathway. 相似文献
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Kawabe Y Seki M Seki T Wang WS Imamura O Furuichi Y Saitoh H Enomoto T 《The Journal of biological chemistry》2000,275(28):20963-20966
Werner's syndrome is a potential model of accelerated human aging. The gene responsible for Werner's syndrome encodes a protein that has a helicase domain homologous to Escherichia coli RecQ. To identify binding partners that regulate the function in concert with Wrn, we screened for proteins using the yeast two-hybrid system with mouse Wrn as bait and found three. One was a novel protein, and the other two were mouse Ubc9 and SUMO-1. Ubc9 also interacted with the mouse homologue of the Bloom's syndrome gene product, another eukaryotic RecQ-type helicase, but not mouse DNA helicase Q1/RecQL (RecQL1). Deletion experiments indicated that both proteins interacted with the N-terminal segment of Wrn (amino acid 272-514). The interaction between Wrn and SUMO-1 was weaker than that between Wrn and Ubc9. Positive interaction was observed in the heterogeneous combination of Wrn and yeast Ubc9 (yUbc9), as well as yUbc9 and SUMO-1, in the two-hybrid system. The interaction between yUbc9 and SUMO-1 was abolished by deleting the C-terminal Gly residue of SUMO-1, which is reportedly required for the formation of Ubc9-SUMO-1 thioester linkage. The interaction of Wrn and SUMO-1 was also abolished by deleting the Gly residue, indicating that the interaction of Wrn and SUMO-1 is mediated by yUbc9 in the two-hybrid system. Finally, we confirmed by immunoblotting with an anti-SUMO-1 antibody that Wrn was covalently attached with SUMO-1. 相似文献