Hyperbaric oxygen induces VEGF expression through ERK, JNK and c-Jun/AP-1 activation in human umbilical vein endothelial cells

Summary Hyperbaric oxygen (HBO) is increasingly used in a number of areas of medical practice, such as selected problem infections and wounds. The beneficial effects of HBO in treating ischemia-related wounds may be mediated by stimulating angiogenesis. We sought to investigate VEGF, the main angiogenic regulator, regulated by HBO in human umbilical vein endothelial cells (HUVECs). In this study, we found that VEGF was up regulated both at mRNA and protein levels in HUVECs treated with HBO dose- and time-dependently. Since there are several AP-1 sites in the VEGF promoter, and the c-Jun/AP-1 is activated through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and extracellular signal regulated kinase (ERK), we further examined the c-Jun, JNK and ERK that might be involved in the VEGF induced by HBO. The VEGF mRNA induced by HBO was blocked by both PD98059 and SP600125, the ERK and JNK inhibitors respectively. HBO induced phospho-ERK and phospho-JNK expressions within 15 min. We further demonstrated that c-Jun phosphorylation was induced within 60 min of HBO treatment. HBO also induced the nuclear AP-1 binding ability within 30–60 min, but the AP-1 induction was blocked by treatment with either the ERK or JNK inhibitor. To verify that the VEGF expression induced by HBO is through the AP-1 trans-activation and VEGF promoter, both the VEGF promoter and AP-1 driving luciferase activity were found increased by the cells treated with HBO. The c-Jun mRNA, which is also driven by AP-1, was also induced by HBO, and the induction of c-Jun was blocked by ERK and JNK inhibitors. We suggest that VEGF induced by HBO is through c-Jun/AP-1 activation, and through simultaneous activation of ERK and JNK pathways.     

Mechanical Stretch Induces Apoptosis Regulator TRB3 in Cultured Cardiomyocytes and Volume-Overloaded Heart

The expression of TRB3 (tribbles 3), an apoptosis regulated gene, increases during endoplasmic reticulum (ER) stress. How mechanical stress affects the regulation of TRB3 in cardiomyocytes during apoptosis is not fully understood. An in vivo model of aorta-caval shunt in adult rats demonstrated the increased TRB3 protein expression in the myocardium. The tumor necrosis factor-alpha (TNF-α) antagonist etanercept reversed the TRB3 protein expression and cardiomyocyte apoptosis induced by AV shunt. An in vitro model of cyclic stretch in neonatal rats was also used to investigate TRB3 expression. We hypothesized that cardiomyocyte apoptosis induced by cyclic stretch is TRB3 dependent. Neonatal rat cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. Cyclic stretch significantly increased TRB3 protein and mRNA expression. Addition of c-jun N-terminal kinase (JNK) inhibitor SP600125, TNF-α antibody and etanercept 30 min before stretch reversed the induction of TRB3 protein induced by stretch. Cyclic stretch induced the DNA-binding activity of growth arrest and DNA damaged inducible gene-153 (GADD153) by electrophoretic mobility shift assay. SP600125, JNK siRNA, TNF-α antibody and etanercept abolished the binding activity induced by stretch. TRB3 promoter activity was enhanced by stretch and TRB3-mut plasmid, SP600125, TNF-α antibody and etanercept attenuated TRB3 promoter activity induced by stretch. Exogenous administration of TNF-α recombinant protein to the non-stretched cardiomyocytes increased TRB3 protein expression similar to that seen after stretch. Cyclic stretch induced cardiomyocyte apoptosis is inhibited by TRB3 siRNA and etanercept. The stretch-induced TRB3 is mediated by TNF-α、JNK and GADD153 pathway. These results indicate that TRB3 plays an important role in stretch-induced cardiomyocyte apoptosis.     

3D-Assisted Quantitative Assessment of Orbital Volume Using an Open-Source Software Platform in a Taiwanese Population

Orbital volume evaluation is an important part of pre-operative assessments in orbital trauma and congenital deformity patients. The availability of the affordable, open-source software, OsiriX, as a tool for preoperative planning increased the popularity of radiological assessments by the surgeon. A volume calculation method based on 3D volume rendering-assisted region-of-interest computation was used to determine the normal orbital volume in Taiwanese patients after reorientation to the Frankfurt plane. Method one utilized 3D points for intuitive orbital rim outlining. The mean normal orbital volume for left and right orbits was 24.3±1.51 ml and 24.7±1.17 ml in male and 21.0±1.21 ml and 21.1±1.30 ml in female subjects. Another method (method two) based on the bilateral orbital lateral rim was also used to calculate orbital volume and compared with method one. The mean normal orbital volume for left and right orbits was 19.0±1.68 ml and 19.1±1.45 ml in male and 16.0±1.01 ml and 16.1±0.92 ml in female subjects. The inter-rater reliability and intra-rater measurement accuracy between users for both methods was found to be acceptable for orbital volume calculations. 3D-assisted quantification of orbital volume is a feasible technique for orbital volume assessment. The normal orbital volume can be used as controls in cases of unilateral orbital reconstruction with a mean size discrepancy of less than 3.1±2.03% in females and 2.7±1.32% in males. The OsiriX software can be used reliably by the individual surgeon as a comprehensive preoperative planning and imaging tool for orbital volume measurement and computed tomography reorientation.     

A single gene encoding vitellogenin in the sea urchin Strongylocentrotus purpuratus: sequence at the 5'' end.

The synthesis of vitellogenin (yolk protein precursor) in the sea urchin, Strongylocentrotus purpuratus, is unique in that both males and females produce a high level of the protein. In this paper we show that this organism also is unique in possessing only a single vitellogenin gene. Like the genes that encode analogous proteins in vertebrates, the sea urchin gene is large, about 19 kb in length. The sequence surrounding the 5' end of the gene revealed several other similarities to vertebrate vitellogenin genes: the signal sequence is exceptionally short and has a sequence similar to those from frog and chick; there is a canonical TATA box at -32; and there is a sequence closely resembling the estrogen-responsive element at -207.     

Anxiety- and depressive-like responses and c-<Emphasis Type="Italic">fos</Emphasis> activity in preproenkephalin knockout mice: Oversensitivity hypothesis of enkephalin deficit-induced posttraumatic stress disorder

The present study used the preproenkephalin knockout (ppENK) mice to test whether the endogenous enkephalins deficit could facilitate the anxiety- and depressive-like symptoms of posttraumatic stress disorder (PTSD). On Day 1, sixteen wildtype (WT) and sixteen ppENK male mice were given a 3 mA or no footshock treatment for 10 seconds in the footshock apparatus, respectively. On Days 2, 7, and 13, all mice were given situational reminders for 1 min per trial, and the freezing response was assessed. On Day 14, all mice were tested in the open field test, elevated plus maze, light/dark avoidance test, and forced swim test. Two hours after the last test, brain tissues were stained to examine c-fos expression in specific brain areas. The present results showed that the conditioned freezing response was significant for different genotypes (ppENK vs WT). The conditioned freezing effect of the ppENK mice was stronger than those of the WT mice. On Day 14, the ppENK mice showed more anxiety- and depressive-like responses than WT mice. The magnitude of Fos immunolabeling was also significantly greater in the primary motor cortex, bed nucleus of the stria terminalis-lateral division, bed nucleus of the stria terminalis-supracapsular division, paraventricular hypothalamic nucleus-lateral magnocellular part, central nucleus of the amygdala, and basolateral nucleus of the amygdala in ppENK mice compared with WT mice. In summary, animals with an endogenous deficit in enkephalins might be more sensitive to PTSD-like aversive stimuli and elicit stronger anxiety and depressive PTSD symptoms, suggesting an oversensitivity hypothesis of enkephalin deficit-induced PTSD.     

Mesenchymal stem cells are superior to angiogenic growth factor genes for improving myocardial performance in the mouse model of acute myocardial infarction

Summary Both cell therapy and angiogenic growth factor gene therapy have been applied to animal studies and clinical trials. Little is known about the direct comparison between cell therapy and angiogenic growth factor gene therapy. The goal of this study was to compare the effects of human bone marrow-derived mesenchymal stem cells (hMSCs) transplantation and injection of angiogenic growth factor genes in a model of acute myocardial infarction in mice. The hMSCs were obtained from adult human bone marrow and expanded in vitro. The purity and characteristics of hMSCs were identified by flow cytometry and immunophenotyping. Immediately after ligation of the left anterior descending coronary artery in male severe combined immunodeficient (SCID) mice, culture-expanded hMSCs or angiogenic growth factor genes were injected intramuscularly at the left anterior free wall. The engrafted hMSCs were positive for cardiac marker, desmin. Infarct size was significantly smaller in the hMSCs-treated group than in the angiopoietin-1 (Ang-1) or vascular endothelial growth factor (VEGF)-treated group at day 28 after infarction. hMSCs transplantation was better in decreasing left ventricular end-diastolic dimension and increasing fractional shortening than Ang1 or VEGF gene therapy. Capillary density was markedly increased after hMSCs transplantation than Ang1 and VEGF gene therapy. In conclusion, intramyocardial transplantation of hMSCs improves cardiac function after acute myocardial infarction through enhancement of angiogenesis and myogenesis in the ischemic myocardium. hMSCs are superior to angiogenic growth factor genes for improving myocardial performance in the mouse model of acute myocardial infarction. Transplantation of MSCs may become the future therapy for acute myocardial infarction for myocardial regeneration.     

Caenorhabditis elegans FOS-1 and JUN-1 Regulate plc-1 Expression in the Spermatheca to Control Ovulation

Fos and Jun are components of activator protein-1 (AP-1) and play crucial roles in the regulation of many cellular, developmental, and physiological processes. Caenorhabditis elegans fos-1 has been shown to act in uterine and vulval development. Here, we provide evidence that C. elegans fos-1 and jun-1 control ovulation, a tightly regulated rhythmic program in animals. Knockdown of fos-1 or jun-1 blocks dilation of the distal spermathecal valve, a critical step for the entry of mature oocytes into the spermatheca for fertilization. Furthermore, fos-1 and jun-1 regulate the spermathecal-specific expression of plc-1, a gene that encodes a phospholipase C (PLC) isozyme that is rate-limiting for inositol triphosphate production and ovulation, and overexpression of PLC-1 rescues the ovulation defect in fos-1(RNAi) worms. Unlike fos-1, regulation of ovulation by jun-1 requires genetic interactions with eri-1 and lin-15B, which are involved in the RNA interference pathway and chromatin remodeling, respectively. At least two isoforms of jun-1 are coexpressed with fos-1b in the spermatheca, and different AP-1 dimers formed between these isoforms have distinct effects on the activation of a reporter gene. These findings uncover a novel role for FOS-1 and JUN-1 in the reproductive system and establish C. elegans as a model for studying AP-1 dimerization.     

In vitro activity of mastoparan-AF alone and in combination with clinically used antibiotics against multiple-antibiotic-resistant Escherichia coli isolates from animals

The in vitro activity of mastoparan-AF, an amphipathic antimicrobial peptide isolated from the hornet venom of Vespa affinis, alone and in combination with various clinically used antibiotics, was investigated against 21 Escherichia coli isolates/strains. Most E. coli isolates tested were detected containing multiple-antimicrobial resistance genes. Antimicrobial activity of mastoparan-AF was measured by MIC, MBC, time-kill kinetic assay and chequerboard titration method. Mastoparan-AF exhibited potent antimicrobial activity against most multiple-antibiotic-resistant E. coli isolates at the concentrations ranging from 4 to 16 μg/ml. Combination studies showed that mastoparan-AF acts synergistically with certain antibiotics, i.e., cephalothin or gentamicin, against some multiple-antibiotic-resistant E. coli isolates. In conclusion, mastoparan-AF alone or in combination with other antibiotics could be promising as alternatives for combating multiple-antibiotic-resistant E. coli in future clinical applications.     

Comparative peptide binding studies of the PABC domains from the ubiquitin-protein isopeptide ligase HYD and poly(A)-binding protein. Implications for HYD function

The PABC domain is a peptide-binding domain that is specifically found in poly(A)-binding protein (PABP) and a HECT ubiquitin-protein isopeptide ligase (E3) known as HYD (hyperplastic discs), EDD (E3 isolated by differential display), or Rat100. The PABC domain of PABP recruits various regulatory proteins and translation factors to poly(A) mRNAs through binding of a conserved 12-amino acid peptide motif, PAM2 (PABP-interacting motif 2). In contrast, little is known about the specificity or function of the domain from HYD. Here, we used isothermal calorimetry and surface plasmon resonance titrations to show that the PABC domain of HYD binds PAM2 peptides with micromolar affinity. NMR chemical shift perturbations were used to map the peptide-binding site in the PABC domain of HYD. The structural features of binding are very similar to those of the interactions with the domain of PABP, which explains the overlapping peptide specificity and binding affinity. We identified the anti-proliferative Tob proteins as potential binding partners of HYD. This was confirmed by glutathione S-transferase pulldown and immunoprecipitation experiments demonstrating the interaction with full-length Tob2. Altogether, our results point to a role of the PABC domain as a protein-protein interaction domain that brings together the processes of translation, ubiquitin-mediated protein degradation, and cell cycle control.