Search

A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells.

Tributyltin (TBT) is widely used as antifouling paints, agriculture biocides, and plastic stabilizers around the world, resulting in great pollution problem in aquatic environments. However, it has been short of the biomonitor to detect TBT in freshwater. We constructed the suppression subtractive hybridization library of Tetrahymena thermophila exposed to TBT, and screened out 101 Expressed Sequence Tags whose expressions were significantly up- or down-regulated with TBT treatment. From this, a series of genes related to the TBT toxicity were discovered, such as glutathione-S-transferase gene (down-regulated), plasma membrane Ca2+ ATPase isoforms 3 gene (up-regulated) and NgoA (up-regulated). Furthermore, their expressions under different concentrations of TBT treatment (0.5-40 ppb) were detected by real time fluorescent quantitative PCR. The differentially expressed genes of T. thermophila in response to TBT were identified, which provide the basic to make Tetrahymena as a sensitive, rapid and convenient TBT biomonitor in freshwater based on rDNA inducible expression system.

Osteosarcoma, considered as the primary cause of malignant bone tumors in children, necessitates novel therapeutic strategies to enhance overall survival rates. KAT7, a histone acetyltransferase, exerts pivotal functions in gene transcription and immune modulation. In light of this, our study identified a significant upregulation of KAT7 in the mRNA and protein levels in human osteosarcoma, boosting cell proliferation in vivo and in vitro. In addition, KAT7-mediated H3K14ac activation induced MMP14 transcription, leading to increased expression and facilitation of osteosarcoma cell metastasis. Subsequent bioinformatics analyses highlighted a correlation between KAT7 and adaptive immune responses, indicating CCL3 as a downstream target of KAT7. Mechanistically, STAT1 was found to transcriptionally upregulate CCL3 expression. Furthermore, overexpression of KAT7 suppressed CCL3 secretions, whereas knockdown of KAT7 enhanced its release. Overall, these findings underscore the oncogenic role of KAT7 in regulating immune responses for osteosarcoma treatment. • KAT7 was abundantly expressed in osteosarcoma. • KAT7 overexpression enhanced the expression of MMP14 by H3K14ac modification. • KAT7 inhibited the expression of CCL3 via JAK1-STAT1 signaling pathways. • STAT1 transcriptionally activated CCL3, thereby modulating immune responses. • KAT7 accelerated dissemination of OS cells in vivo. [ABSTRACT FROM AUTHOR]

Mutagenic screening is powerful for identifying key genes involved in developmental processes. However, such screens are successful only in lower organisms. Here, we develop a targeted genetic screening approach in mice through combining androgenetic haploid embryonic stem cells (AG-haESCs) and clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) technology. We produced a mutant semi-cloned (SC) mice pool by oocyte injection of AG-haESCs carrying constitutively expressed Cas9 and an single guide RNA (sgRNA) library targeting 72 preselected genes in one step and screened for bone-development–related genes through skeletal analysis at birth. This yielded 4 genes: Zic1 and Clec11a, which are required for bone development, and Rln1 and Irx5, which had not been previously considered. Whereas Rln1−/− mice exhibited small skeletal size only at birth, Irx5−/− mice showed skeletal abnormalities both in postnatal and adult phases due to decreased bone mass and increased bone marrow adipogenesis. Mechanistically, iroquois homeobox 5 (IRX5) promotes osteoblastogenesis and inhibits adipogenesis by suppressing peroxisome proliferator activated receptor γ (PPARγ) activation. Thus, AG-haESC-mediated functional mutagenic screening opens new avenues for genetic interrogation of developmental processes in mice. [ABSTRACT FROM AUTHOR]

Ubiquitin ligase Nrdp1 (neuregulin receptor degradation protein 1) plays important roles in multiple physiological process because it can ubiquitinate various substrates such as ErbB3, BRUCE, MyD88, C/EBPβ, and Parkin, and so forth. In addition to the physiological function, it was also found to be involved in tumor progression. It has been shown that loss of Nrdp1 enhances breast cancer cell growth. Up to now, the role of Nrdp1 in glioma has not been elucidated. Here, we reported that Nrdp1 as well as cleaved caspase 3 was lower expressed in human glioma tissues comparing with the nontumorous. And then we found that the expression of Nrdp1 and cleaved caspase 3 was increased in the treatment of Temozolomide (TMZ), a drug for glioma chemotherapy. Further investigation indicated that transient transfection of Nrdp1 significantly promoted cell apoptosis by aggravating the degradation of BRUCE and activation of caspase 3. In addition, overexpression of Nrdp1 augmented TMZ induced apoptosis by evaluating the degradation of BRUCE and the activation of caspase 3, while silencing of Nrdp1 reduced the sensitivity to the TMZ by inhibiting the degradation of BRUCE and the activation of caspase 3 in human glioma cells. These observations show that Nrdp1 is a pro-apoptotic protein in human glioma and lower expression of Nrdp1 in human glioma may promote tumor progression by reducing apoptosis, suggesting that Nrdp1 may be an important regulator in the development of human glioma. © 2014 IUBMB Life, 66(10):704-710, 2014 [ABSTRACT FROM AUTHOR]

Ras-oncogenic pathway contributes to the pathogenesis of various tumours in humans, in which mutations of three canonical genes including H-Ras, N-Ras and K-Ras are the most common events. Dysregulation of Ras signalling can be tumourigenic, especially gliomas of the central nervous system. Rap proteins are members of the small GTPase superfamily that involved in many biological processes. However, it remains largely unclear as to whether and how Rap proteins are involved in the development of multiple gliomas. We found that the levels of the protein Rap2a and the activity of Rap2a (GTP-Rap2a) were weakly expressed in glioma tissues. Overexpressed Rap2a significantly inhibited the migration and invasion of glioma cells with an increase of GTP-Rap2a. Overexpression of the dominant-active (DA-Rap2a), but not the dominant-negative (DN-Rap2a) form of Rap2a, also similarly inhibited the migration and invasion of glioma cells by reducing the phosphorylation level of AKT. In contrast, downregulation of Rap2a promoted glioma migration and invasion, and raised the phosphorylation level of AKT, whereas these effects were inhibited by PI3K-specific inhibitor, LY294002. Thus unlike the other family members of Ras, Rab2a probably serves as a tumour suppressor in the pathogenesis of glioma. [ABSTRACT FROM AUTHOR]

Purpose: We evaluated the clinical characteristic, tumor feature and immunohistochemistry factors predicting malignant pheochromocytoma. Materials and Methods: Between January 1999 and December 2008 we retrospectively reviewed the records of 136 patients with pheochromocytoma at Ruijin Hospital. We compared clinical characteristics (age, gender, symptoms and biochemical analysis), tumor features (site, weight and diameter) and the expression of 3 angiogenesis/metastasis related genes (VEGF, Cox-2 and MVD) by immunohistochemical analysis of benign vs malignant pheochromocytomas. Results: Of the 136 patients 105 (77%) had benign and 31 (23%) had malignant pheochromocytoma. Malignant tumors were larger and heavier than benign tumors, and accompanied by higher plasma metanephrine secretion (each p <0.001). Mean tumor catecholamine and preoperative 24-hour urinary metanephrine or normetanephrine were obviously higher in malignant than in benign tumors (p <0.001). Also, 25 malignant tumors (81%) were immunopositive for VEGF while only 24 benign tumors (23%) showed this characteristic (p <0.001). Microvessel density and the rate of positive staining for Cox-2 protein in malignant samples were higher than in benign samples (p <0.001). Conclusions: Several promising predictive parameters are currently available to distinguish benign from malignant pheochromocytoma. Large (5 cm or greater) or heavy (250 gm or greater) tumors, multifocal and extra-adrenal tumors, early onset postoperative hypertension and higher plasma or urine metadrenaline are high risk factors predictive of malignant pheochromocytoma. Also, expression of the 3 angiogenesis or metastasis related genes VEGF, Cox-2 and MVD helps determine the diagnosis of malignancy and suggests strict followup. [ABSTRACT FROM AUTHOR]