The BNIP-2 and Cdc42GAP Homology (BCH) Domain of p50RhoGAP/Cdc42GAP Sequesters RhoA from Inactivation by the Adjacent GTPase-activating Protein Domain

The BNIP-2 and Cdc42GAP homology (BCH) domain is a novel regulator for Rho GTPases, but its impact on p50-Rho GTPase-activating protein (p50RhoGAP or Cdc42GAP) in cells remains elusive. Here we show that deletion of the BCH domain from p50RhoGAP enhanced its GAP activity and caused drastic cell rounding. Introducing constitutively active RhoA or inactivating GAP domain blocked such effect, whereas replacing the BCH domain with endosome-targeting SNX3 excluded requirement of endosomal localization in regulating the GAP activity. Substitution with homologous BCH domain from Schizosaccharomyces pombe, which does not bind mammalian RhoA, also led to complete loss of suppression. Interestingly, the p50RhoGAP BCH domain only targeted RhoA, but not Cdc42 or Rac1, and it was unable to distinguish between GDP and the GTP-bound form of RhoA. Further mutagenesis revealed a RhoA-binding motif (residues 85-120), which when deleted, significantly reduced BCH inhibition on GAP-mediated cell rounding, whereas its full suppression also required an intramolecular interaction motif (residues 169-197). Therefore, BCH domain serves as a local modulator in cis to sequester RhoA from inactivation by the adjacent GAP domain, adding to a new paradigm for regulating p50RhoGAP signaling.     

Overexpression of malate dehydrogenase in transgenic tobacco leaves: enhanced malate synthesis and augmented Al-resistance

Numerous studies with transgenic plants have demonstrated that overexpression of enzymes related to organic acid metabolism under the control of CaMV 35S promoter increased organic acid exudation and Al-resistance. The synthesis of organic acids requires a large carbon skeleton supply from leaf photosynthesis. Thus, we produced transgenic tobacco overexpressing cytosolic malate dehydrogenase (MDH) cDNA from Arabidopsis thaliana (amdh) and the MDH gene from Escherichia coli (emdh), respectively, under the control of a leaf-specific light-inducible promoter (Rubisco small subunit promoter, PrbcS) in the present study. Our data indicated that an increase (120–130%) in MDH-specific activity in leaves led to an increase in malate content in the transgenic tobacco leaves and roots as well as a significant increase in root malate exudation compared with the WT plants under the acidic (pH 4.5) conditions irrespective of 300 μM Al³⁺ stress absence or presence. After being exposed to 25 μM Al³⁺ in a hydroponic solution, the transgenic plants exhibited stronger Al-tolerance than WT plants and the degree of A1 tolerance in the transgenic plants corresponded with the amount of malate secretion. When grown in an Al-stress perlite medium, the transgenic tobacco lines showed better growth than the WT plants. The results suggested that overexpression of MDH driven by the PrbcS promoter in transgenic plant leaves enhanced malate synthesis and improved Al-resistance.     

Novel miR-sc4 regulates the proliferation and migration of Schwann cells by targeting Cdk5r1

Clinical and experimental studies have shown an association between intracellular free Zn²⁺ ([Zn²⁺]_i)-dyshomeostasis and cardiac dysfunction besides [Ca²⁺]_i-dyshomeostasis. Since [Zn²⁺]_i-homeostasis is regulated through Zn²⁺-transporters depending on their subcellular distributions, one can hypothesize that any imbalance in Zn²⁺-homeostasis via alteration in Zn²⁺-transporters may be associated with the induction of ER stress and apoptosis in hypertrophic heart. We used a transverse aortic constriction (TAC) model to induce hypertrophy in young male rat heart. We confirmed the development of hypertrophy with a high ratio of heart to body weight and cardiomyocyte capacitance. The expression levels of ER stress markers GRP78, CHOP/Gadd153, and calnexin are significantly high in TAC-group in comparison to those of controls (SHAM-group). Additionally, we detected high expression levels of apoptotic status marker proteins such as the serine kinase GSK-3β, Bax-to-Bcl-2 ratio, and PUMA in TAC-group in comparison to SHAM-group. The ratios of phospho-Akt to Akt and phospho-NFκB to the NFκB are significantly higher in TAC-group than in SHAM-group. Furthermore, we observed markedly increased phospho-PKCα and PKCα levels in TAC-group. We, also for the first time, determined significantly increased ZIP7, ZIP14, and ZnT8 expressions along with decreased ZIP8 and ZnT7 levels in the heart tissue from TAC-group in comparison to SHAM-group. Furthermore, a roughly calculated total expression level of ZIPs responsible for Zn²⁺-influx into the cytosol (increased about twofold) can be also responsible for the markedly increased [Zn²⁺]_i detected in hypertrophic cardiomyocytes. Taking into consideration the role of increased [Zn²⁺]_i via decreased ER-[Zn²⁺] in the induction of ER stress in cardiomyocytes, our present data suggest that differential changes in the expression levels of Zn²⁺-transporters can underlie mechanical dysfunction, in part due to the induction of ER stress and apoptosis in hypertrophic heart via increased [Zn²⁺]_i- besides [Ca²⁺]_i-dyshomeostasis.     

Effects of SOX2 on Proliferation,Migration and Adhesion of Human Dental Pulp Stem Cells

As a key factor for cell pluripotent and self-renewing phenotypes, SOX2 has attracted scientists’ attention gradually in recent years. However, its exact effects in dental pulp stem cells (DPSCs) are still unclear. In this study, we mainly investigated whether SOX2 could affect some biological functions of DPSCs. DPSCs were isolated from the dental pulp of human impacted third molar. SOX2 overexpressing DPSCs (DPSCs-SOX2) were established through retroviral infection. The effect of SOX2 on cell proliferation, migration and adhesion ability was evaluated with CCK-8, trans-well system and fibronectin-induced cell attachment experiment respectively. Whole genome expression of DPSCs-SOX2 was analyzed with RNA microarray. Furthermore, a rescue experiment was performed with SOX2-siRNA in DPSC-SOX2 to confirm the effect of SOX2 overexpression in DPSCs. We found that SOX2 overexpression could result in the enhancement of cell proliferation, migration, and adhesion in DPSCs obviously. RNA microarray analysis indicated that some key genes in the signal pathways associated with cell cycle, migration and adhesion were upregulated in different degree, and the results were further confirmed with qPCR and western-blot. Finally, DPSC-SOX2 transfected with SOX2-siRNA showed a decrease of cell proliferation, migration and adhesion ability, which further confirmed the biological effect of SOX2 in human DPSCs. This study indicated that SOX2 could improve the cell proliferation, migration and adhesion ability of DPSCs through regulating gene expression about cell cycle, migration and adhesion, and provided a novel strategy to develop seed cells with strong proliferation, migration and adhesion ability for tissue engineering.     

Long-chain Acyl-CoA Dehydrogenase Deficiency as a Cause of Pulmonary Surfactant Dysfunction

Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD^−/− mice. LCAD^−/− mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD^−/− mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD^−/− surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD^−/− lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.