Vascular endothelial growth factor-A (VEGF-A) is a critical angiogenic factor which is mainly secreted from podocytes and epithelial cells in kidney and plays an important role in renal pathophysiology. In recent years, functions of different isoforms of VEGF-A and the new secretion approach via extracellular vesicles (EVs) have been identified. Thus, further understanding are needed for the role of VEGF-A and its isoforms in renal injury and repair. In this review, we summarized the expression, secretion and regulation of VEGF-A, its biological function, and the role of different isoforms of VEGF-A in the development of different renal diseases. Meanwhile, the research progress of VEGF-A as diagnostic marker and therapeutic target for renal diseases were discussed.
Humans ; Kidney/metabolism* ; Kidney Diseases ; Protein Isoforms/metabolism* ; Vascular Endothelial Growth Factor A/physiology*

The methylcytosine dioxygenases TET proteins (TET1, TET2, and TET3) play important regulatory roles in neural function. In this study, we investigated the role of TET proteins in neuronal differentiation using Neuro2a cells as a model. We observed that knockdown of TET1, TET2 or TET3 promoted neuronal differentiation of Neuro2a cells, and their overexpression inhibited VPA (valproic acid)-induced neuronal differentiation, suggesting all three TET proteins negatively regulate neuronal differentiation of Neuro2a cells. Interestingly, the inducing activity of TET protein is independent of its enzymatic activity. Our previous studies have demonstrated that srGAP3 can negatively regulate neuronal differentiation of Neuro2a cells. Furthermore, we revealed that TET1 could positively regulate srGAP3 expression independent of its catalytic activity, and srGAP3 is required for TET-mediated neuronal differentiation of Neuro2a cells. The results presented here may facilitate better understanding of the role of TET proteins in neuronal differentiation, and provide a possible therapy target for neuroblastoma.
Animals ; Catalytic Domain ; Cell Differentiation ; drug effects ; physiology ; Cell Line, Tumor ; DNA-Binding Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Enzyme Inhibitors ; pharmacology ; GTPase-Activating Proteins ; genetics ; metabolism ; Immunohistochemistry ; Mice ; Microscopy, Fluorescence ; Neuroblastoma ; metabolism ; pathology ; Protein Isoforms ; antagonists & inhibitors ; genetics ; metabolism ; Proto-Oncogene Proteins ; antagonists & inhibitors ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; metabolism ; Valproic Acid ; pharmacology

BACKGROUNDHyperinsulinemia, insulin-like growth factor (IGF)-I and -II (IGF-II) are associated with increased risk of endometrial carcinoma. Insulin receptor isoform A (IR-A) is more frequently expressed in endometrial carcinoma than in normal endometrial tissues. To better understand their roles in endometrial carcinoma, we investigated the effects of insulin, IGF-I, and IGF-II in endometrial carcinomas cells with different IR-A expression levels.

METHODSTo explore the role of IR-A in mediating the activity of IGF-I, IGF-II, and insulin, we investigate the cellular proliferation of endometrial carcinoma cell lines RL95-2 and RL95-2-IR-A by MTS assays. Then we examined the protein kinase Akt phosphorylation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in both cell lines by Western blotting. The effect of IGF-II and AG1024 on cell cycle progression and apoptosis was assessed by flowcytometry. To examine whether the effects of IGFs were mediated by IR-A, we blocked IGF-I receptor (IGF-IR) in both cell lines using AG1024, an IGF-IR-specific inhibitor.

RESULTSIGF-I and IGF-II significantly enhanced proliferation of both cell lines (P < 0.05). By contrast, insulin significantly increased proliferation of RL95-2-IR-A cells only (P < 0.05). IGF-I and IGF-II significantly increased pAkt levels in RL95-2 cells and pERK1/2 levels in RL95-2-IR-A cells (all, P < 0.05). Insulin increased pERK1/2 levels in RL95-2-IR-A cells only (P < 0.05). LY294002 and PD98059 inhibited the specific signaling activities and cellular proliferation. After AG1024 pretreatment, neither IGF-I nor IGF-II affected pAkt levels in RL95-2 cells. IGF-II, but not IGF-I, increased pERK1/2 levels in RL95-2-IR-A cells. After AG1024 pretreatment, the proliferation rate and DNA content corresponding to the S phase increased and apoptosis decreased significantly in IGF-II-treated RL95-2-IR-A cells only (P < 0.05).

CONCLUSIONSThe proliferation effect of insulin is mediated by IR-A. When IR-A dominates in a cell line, IGF-II activated cell proliferation mainly through the ERK1/2 pathway. On the other hand, IGF-II activated cell proliferation mainly through the Akt pathway. IR-A can at least partly mediate the proliferative and anti-apoptotic effects of IGF-II through the ERK1/2 pathway.

Antigens, CD ; physiology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Endometrial Neoplasms ; metabolism ; pathology ; Female ; Humans ; Insulin ; pharmacology ; Insulin-Like Growth Factor I ; pharmacology ; Insulin-Like Growth Factor II ; pharmacology ; Intracellular Space ; metabolism ; Protein Isoforms ; metabolism ; Receptor, Insulin ; physiology ; Signal Transduction ; drug effects

OBJECTIVETo investigate the roles of the hepatitis B virus (HBV)-encoded X protein (HBx), including the full-length and truncated isoforms, and in conjunction with the host-encoded damaged DNA binding protein 1 (DDB1) in HBV replication.

METHODSRecominant expression plasmids carrying the wild-type HBV genome (pGEM-HBV1.2) or with deletion of the full-length HBx protein (pHBV-deltaX), or carrying the full-length HBx protein (pSI-X) or the HBx1to101 (pSI-X1to101) or HBx43to154 (pSI-X43to154) isoforms were constructed for transfection into HepG2 cells. The pcDNA6.2-GW/EmGFP-miR (DDB1-miRNA) vector was constructed for silencing of the DDB1 gene in co-transfected HepG2 cells. At 72 h after transfections, DDB1 silencing was confirmed by western blot analysis and real-time quantitive reverse transcription PCR, HBV DNA copies number was assessed by real time PCR, and levels of hepatitis B surface antigen (HbsAg) and hepatitis B e antigen (HbeAg) were determined by ELISA. Differences between groups was statistically analyzed by single-factor analysis of variance and the t-test.

RESULTSTransfection with pHBV-deltaX led to reductions in DDB1 mRNA (to 52.74% of that in the wild-type pGEM-HBV1.2 transfected cells), HBV replication (to 55.49%), HBsAg level (48.05%), and HBeAg level (46.22%). Co-transfection with pSI-X or pSI-X43to154, but not with pSI-X1to101, restored the pHBV-deltaX-induced reductions in DDB1 mRNA, HBV replication, HBsAg and HBeAg to wild-type levels. The quantity of DDB1 mRNA was approximately parallel with the quantity of HBV DNA copies in all the HepG2 transfection groups.

CONCLUSIONThe COOH-terminal amino acids of HBx are required for HBV replication in hepatocytes, possibly involving the host-encoded DDB1 protein.

DNA-Binding Proteins ; metabolism ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; metabolism ; physiology ; Host-Pathogen Interactions ; Humans ; Protein Isoforms ; metabolism ; Trans-Activators ; metabolism ; Transfection ; Virus Replication

Autophagy-related protein 8 (Atg8) is an essential component of autophagy formation and encystment of cyst-forming parasites, and some protozoa, such as, Acanthamoeba, Entamoeba, and Dictyostelium, have been reported to possess a type of Atg8. In this study, an isoform of Atg8 was identified and characterized in Acanthamoeba castellanii (AcAtg8b). AcAtg8b protein was found to encode 132 amino acids and to be longer than AcAtg8 protein, which encoded 117 amino acids. Real-time PCR analysis showed high expression levels of AcAtg8b and AcAtg8 during encystation. Fluorescence microscopy demonstrated that AcAtg8b is involved in the formation of the autophagosomal membrane. Chemically synthesized siRNA against AcAtg8b reduced the encystation efficiency of Acanthamoeba, confirming that AcAtg8b, like AcAtg8, is an essential component of cyst formation in Acanthamoeba. Our findings suggest that Acanthamoeba has doubled the number of Atg8 gene copies to ensure the successful encystation for survival when 1 copy is lost. These 2 types of Atg8 identified in Acanthamoeba provide important information regarding autophagy formation, encystation mechanism, and survival of primitive, cyst-forming protozoan parasites.
Acanthamoeba castellanii/cytology/*genetics/physiology ; Amebiasis/*parasitology ; Amino Acid Sequence ; Autophagy ; Cell Membrane/metabolism ; DNA, Protozoan/chemistry/genetics ; Gene Dosage ; Gene Silencing ; Genes, Reporter ; Humans ; Molecular Sequence Data ; Phagosomes/metabolism ; Protein Isoforms ; Protozoan Proteins/*genetics/metabolism ; RNA, Messenger/genetics ; RNA, Protozoan/genetics ; RNA, Small Interfering/chemical synthesis/genetics ; Recombinant Fusion Proteins ; Sequence Alignment

Plant betaine aldehyde dehydrogenase (BADH) is a physiologically important enzyme in response to salt or drought stress. In this study, two BADH genes (PeBADH1 and PeBADH2) were cloned from Populus euphratica. Both PeBADH1 and PeBADH2 genes encode the proteins of 503 amino acid residues, with a calculated molecular mass of 54.93 kDa and 54.90 kDa, respectively. Reverse transcription PCR showed the divergence of expression pattern between the PeBADH1 and PeBADH2 genes in P. euphratica. The recombinant PeBADH1 and PeBADH2 proteins were overexpressed in E. coli, and purified by Ni-affinity chromatography. The PeBADH2 protein had 1.5-fold higher enzymatic activity towards the substrate aldehyde than PeBADH1 protein. The PeBADH1 protein revealed higher thermal stability than PeBADH2 protein. These results indicated obvious functional divergence between the PeBADH1 and PeBADH2 genes.
Amino Acid Sequence ; Betaine-Aldehyde Dehydrogenase ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Plant ; physiology ; Molecular Sequence Data ; Plant Proteins ; biosynthesis ; chemistry ; genetics ; Populus ; genetics ; Protein Isoforms ; chemistry ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Substrate Specificity

Cellular senescence is an irreversible cell cycle arrest triggered by the activation of oncogenes or mitogenic signaling as well as the enforced expression of tumor suppressors such as p53, p16(INK4A) and promyelocytic leukemia protein (PML) in normal cells. E2F-binding protein 1 (E2FBP1), a transcription regulator for E2F, induces PML reduction and suppresses the formation of PML-nuclear bodies, whereas the down-regulation of E2FBP1 provokes the PML-dependent premature senescence in human normal fibroblasts. Here we report that the depletion of E2FBP1 induces the accumulation of PML through the Ras-dependent activation of MAP kinase signaling. The cellular levels of p16(INK4A) and p53 are elevated during premature senescence induced by depletion of E2FBP1, and the depletion of p16(INK4A), but not p53 rescued senescent cells from growth arrest. Therefore, the premature senescence induced by E2FBP1 depletion is achieved through the p16(INK4A)-Rb pathway. Similar to human normal fibroblasts, the growth inhibition induced by E2FBP1 depletion is also observed in human tumor cells with intact p16(INK4A) and Rb. These results suggest that E2FBP1 functions as a critical antagonist to the p16(INK4A)-Rb tumor suppressor machinery by regulating PML stability.
Cell Line, Tumor ; Cells, Cultured ; Cellular Senescence ; genetics ; physiology ; Cyclin-Dependent Kinase Inhibitor p16 ; antagonists & inhibitors ; genetics ; physiology ; DNA-Binding Proteins ; deficiency ; genetics ; physiology ; Down-Regulation ; Fibroblasts ; Gene Expression Regulation ; Humans ; Intranuclear Inclusion Bodies ; metabolism ; MAP Kinase Signaling System ; Nuclear Proteins ; genetics ; metabolism ; physiology ; Promyelocytic Leukemia Protein ; Protein Isoforms ; Protein Stability ; RNA Interference ; Retinoblastoma Protein ; antagonists & inhibitors ; genetics ; physiology ; Transcription Factors ; deficiency ; genetics ; metabolism ; physiology ; Transfection ; Tumor Suppressor Protein p53 ; physiology ; Tumor Suppressor Proteins ; genetics ; metabolism ; physiology ; Ubiquitination ; ras Proteins ; metabolism

The purpose of this study is to determine 1) whether morphine postconditiong (MPostC) can attenuate the intercellular adhesion molecules-1 (ICAM-1) expression after reoxygenation injury and 2) the subtype(s) of the opioid receptors (ORs) that are involved with MPostC. Human umbilical vein endothelial cells (HUVECs) were subjected to 6 hr anoxia followed by 12 hr reoxygenation. Three morphine concentrations (0.3, 3, 30 microM) were used to evaluate the protective effect of MPostC. We also investigated blockading the OR subtypes' effects on MPostC by using three antagonists (a micro-OR antagonist naloxone, a kappa-OR antagonist nor-binaltorphimine, and a delta-OR antagonist naltrindole) and the inhibitor of protein kinase C (PKC) chelerythrine. As results, the ICAM-1 expression was significantly reduced in the MPostC (3, 30 microM) groups compared to the control group at 1, 6, 9, and 12 hours reoxygenation time. As a consequence, neutrophil adhesion was also decreased after MPostC. These effects were abolished by coadministering chelerythrine, nor-binaltorphimine or naltrindole, but not with naloxone. In conclusion, it is assumed that MPostC could attenuate the expression of ICAM-1 on endothelial cells during reoxygenation via the kappa and delta-OR (opioid receptor)-specific pathway, and this also involves a PKC-dependent pathway.
Animals ; Benzophenanthridines/pharmacology ; Endothelial Cells/cytology/*drug effects/*metabolism ; Endothelium, Vascular/cytology ; Humans ; Intercellular Adhesion Molecule-1/genetics/*metabolism ; Morphine/*pharmacology ; Naloxone/pharmacology ; Naltrexone/analogs & derivatives/pharmacology ; Narcotic Antagonists/pharmacology ; Narcotics/*pharmacology ; Protein Isoforms/metabolism ; Protein Kinase C/antagonists & inhibitors/metabolism ; Receptors, Opioid/metabolism ; Reperfusion Injury/*metabolism ; Signal Transduction/physiology ; Umbilical Veins/cytology

OBJECTIVETo study the changes in endogenous neuregulin-1 (Nrg1) expression in the anterior pituitary of female Wistar-Furth rats in different phases of the estrous cycle.

METHODSFemale Wistar-Furth rats during estrous cycles were used. RT-PCR was employed to study the changes in the expression of Nrg1 isoforms and their cognate receptors ErbB-2 and ErbB-4 in the anterior pituitary in different phases of the estrous cycle. Western blotting was used to detect Nrg1 expression at the protein level. Immunofluorescence staining was used to identify hypophyseal cells expressing Nrg1 and observe the localization and distribution of Nrg1 and functional phosphorylation of ErbB-4. The co-expression of Nrg1 and ErbB-4 in the anterior pituitary of Rhesus monkey was also investigated.

RESULTSSome of the Nrg1 isoforms, especially type III Nrg1s, were expressed at a higher level during the estrous cycle I (E1) and estrous cycle II (E2), a result consistent with that of Western blotting for samples of the anterior pituitaries collected at these phases. Immunofluorescence staining identified the gonadotrophs as the main source of Nrg1, and showed an extensive distribution of Nrg1 in the anterior pituitary in E1 and E2 phases accompanied by apparent phosphorylated activation of ErbB-4. Adjacent distribution of Nrg1- and ErbB-4-positive cells was also observed in the anterior pituitary of male Rhesus monkeys.

CONCLUSIONOur results provide evidence for the expression of multiple Nrg1 isoforms and the presence of Nrg1/ErbB-4 signaling in the anterior pituitary of female Wistar-Furth rats. This signaling demonstrates an estrous cycle phase-related pattern. Additionally, Nrg1/ErbB-4-based juxtacrine signaling may exist in the anterior pituitary of male non-human primate.

Animals ; Estrous Cycle ; physiology ; Female ; Macaca mulatta ; Male ; Neuregulin-1 ; metabolism ; Phosphorylation ; Pituitary Gland ; metabolism ; Protein Isoforms ; metabolism ; Rats ; Rats, Inbred WF ; Receptor, Epidermal Growth Factor ; metabolism ; Receptor, ErbB-4

OBJECTIVETo investigate the expression and functional role of the small conductance Ca(2+)-activated K(+) channels in human atrial myocytes.

METHODSWe collected the right atrial appendage tissues from 8 patients with congenital heart defect with sinus rhythm undergoing open-heart surgery. Immunohistochemistry was performed to identify the expression of 3 isoforms of SK channel (SK1, SK2 and SK3). Using the classical two-step enzymatic isolation method, perforated patch clamp and conventional voltage-clamp techniques were performed to record the action potentials (APs) and the whole-cell Ca(2+)-activated K(+) current (I(K, Ca)) in the single atrial myocyte. We compared the changes in action potential duration (APD) before and after the application of a specific SK channels blocker apamin (100 nmol/L).

RESULTSHuman atrial myocytes showed positivity for all the SK1, SK2 and SK3 isoform channels. Patch-clamp recording confirmed the presence of I(K,Ca), and apamin significantly prolonged APD at 90% repolarization (APD(90)), but produced no obvious effect on APD(50).

CONCLUSIONThe three isoforms of SK channels are all expressed in human atrial myocytes. SK channels play a prominent role in the late phase of repolarization in human atrial myocytes, which is distinct from their functional roles in neurons where they mediate the process of afterhyperpolarization following APs.

Action Potentials ; physiology ; Adolescent ; Atrial Appendage ; cytology ; Cells, Cultured ; Female ; Humans ; Male ; Myocytes, Cardiac ; metabolism ; Patch-Clamp Techniques ; Protein Isoforms ; metabolism ; physiology ; Small-Conductance Calcium-Activated Potassium Channels ; metabolism ; physiology