Downregulation of Wnt/β-catenin signaling causes degeneration of hippocampal neurons in vivo

The possibility that the degeneration of hippocampal neurons can be caused by mis-regulation of Wnt/β-catenin signaling was tested. Downregulation of Wnt signaling by the inducible expression of Axin, ICAT, and dnTcf4E causes degeneration of hippocampal neurons, while upregulation of Wnt signaling by the inducible expression of Dvl and β-catenin has a negligible effect. Treatment with ICG-001, a small molecule known to inhibit Wnt signaling, causes degeneration of hippocampal neurons, while the treatment with a JNK specific inhibitor does not show any effect. The results from LDH and TUNEL assays suggest that degeneration occurs via apoptotic processes. Inhibition of Wnt signaling reduced IGF-1 expression and the addition of IGF-1 blocked degeneration, which suggests that downregulation of IGF-1/Akt signaling is partially responsible for the degeneration. Inducible expression of Axin in the hippocampal neurons isolated from Axin2P-rtTA/pBI-EGFP-Axin double transgenic mice also causes degeneration. Consistent with the findings, these mice had more neuronal cell death in hippocampus and had differences in contextual conditioning upon the inducible expression of Axin. In summary, our data strongly support the idea that downregulation of Wnt/β-catenin signaling causes degeneration of hippocampal neurons in vivo and may be a cause of neurodegenerative diseases related to an anxiety related response.     

Ischemia-induced increase of stiffness of αB-crystallin/HSPB2-deficient myocardium

The two small heat shock proteins (sHSPs), B-crystallin and HSPB2, have been shown to translocate within a few minutes of cardiac ischemia from the cytosol to myofibrils; and it has been suggested that their chaperone-like properties might protect myofibrillar proteins from unfolding or aggregation during stress conditions. Further evidence of an important role for HSPs in muscle function is provided by the fact that mutations of the B-crystallin gene cause myopathy and cardiomyopathy. In the present study, we subjected isolated papillary muscles of B-crystallin/HSPB2-deficient mice to simulated ischemia and reperfusion. During ischemia in B-crystallin/HSPB2-deficient muscles, the development of contracture started earlier and reached a higher value compared to the wildtype mice. The recovery of contracture of B-crystallin/HSPB2-deficient muscles was also attenuated during the simulated reperfusion period. However, twitch force was not significantly altered at any time of the experiment. This suggests that during ischemic insults, B-crystallin/HSPB2 may not be important for the contraction process itself, but rather serve to maintain muscular elasticity.     

C/EBPα inhibits proliferation of breast cancer cells via a novel pathway of miR-134/CREB

C/EBPα plays an important role in the modulation of cell proliferation, differentiation or apoptosis in various tissues. Most recently, reduced expression of C/EBPα and growth inhibitory effect was found in primary mammary carcinomas. However, the underlying mechanism is still not fully aware. Here, we firstly identified miR-134 as a target of C/EBPα in MCF7 breast cancer cell lines. C/EBPα overexpression promoted miR-134 expression, causing suppression of apoptosis- protective genes CREB and Bcl-2, and resulted in the proliferation inhibition of MCF7 cells. Moreover, anti-miR-134 rescued the proliferation inhibition of MCF7 cells and the suppression of anti-apoptotic genes CREB and Bcl-2 caused by C/EBPα overexpression. Collectively, C/EBPα inhibited cell growth in breast cancer cells via a novel pathway miR-134/CREB.     

Role of α2/δ subunit in the development of morphine-induced rewarding effect and behavioral sensitization

Previous data demonstrate that L-type voltage-gated calcium channel (VGCC) blockers, which bind to α₁ subunits of VGCC to suppress Ca²⁺ entry into cells, inhibit the development of psychological dependence on drugs of abuse, suggesting the upregulation of L-type VGCC in the development of psychological dependence. However, there are few available data on changes of the auxiliary subunit α₂/δ modifying L-type VGCC under such conditions. We therefore investigated here the role of α₂/δ subunits of VGCCs in the brain of mouse after repeated treatment with morphine. The treatment with morphine increased α₂/δ subunit expression in the frontal cortex and the limbic forebrain of mice showing rewarding effect and sensitization to hyperlocomotion by morphine. The morphine-induced behavioral sensitization and place preference were also suppressed by gabapentin, which binds to an exofacial epitope of the α₂/δ auxiliary subunits of VGCCs. These findings indicate that the upregulation of α₂/δ subunit as well as α₁ subunits of VGCC in the frontal cortex and the limbic forebrain plays a critical role in development of morphine-induced rewarding effect and behavioral sensitization following neuronal plasticity.     

Effect of chitosan properties on the membrane strength of chitosan/alginate microcapsules

INTRODUCTION　　 With the success of transplanting porcine islet cells encapsulated in microcap-sules,substituting the function of pancreas in diabetic rats,microcapsules with cal-cium alginate gel beads as core,polylysine and chitosan as coating membrane…     

Inhibition of Lupus by Genetic Alteration of the Interferon-α/β Receptor

Type I interferons (IFN-αβ) are immunoregulatory cytokines that promote both innate and adaptive immune responses. Although they have been implicated in human SLE, recent studies in mice have helped solidify this connection. By using lupus-prone mice with knockout of the IFN-αβ receptor, we and others have documented that lack of IFN-αβ leads to a marked reduction in disease manifestations, including autoantibody production, target organ damage and mortality. Furthermore, IFN-αβ was found to potentially contribute to several levels of disease pathogenesis. These included the differentiation and activation of dendritic cells, the activation and proliferation of T cells, T cell survival and the activation and survival of autoantibody-producing B cells. These findings strongly support the targeting of IFN-αβ in SLE and suggest that definition of the specific pathways critical for disease induction will be important for optimal intervention.     

Tissue Engineering of Ligament/tendon-Part II-Importance of Biochemical and Mechanical Factors on the Neogenesis of Ligament

1 IntroductionLigaments and tendons are bands of parallel fibers, made of dense connective tissue, that play an important role in mediating normal movement and stability of joints. Injury to these structures can cause significant joint instability, which may lead to injury of others tissues and the development of degenerative joint diseases. Tissue engineering offers the potential to improve the reconstruction of tendons and ligaments. The concept is based on the manipulation of cellular and m…     

Molting-associated suppression of symbiont population and up-regulation of antimicrobial activity in the midgut symbiotic organ of the Riptortus–Burkholderia symbiosis

The majority of insects possess symbiotic bacteria. Since symbiont titers can affect host phenotypes of biological importance, host insects are expected to evolve some mechanisms for regulating symbiont population. Here we report that, in the Riptortus–Burkholderia gut symbiosis, titers of the beneficial symbiont transiently decrease at the pre-molt stages in host development. This molting-associated suppression of the symbiont population is coincident with the increase of antimicrobial activity in the symbiotic midgut, which is observed in both symbiotic and aposymbiotic insects. Two genes, pyrrhocoricin-like antimicrobial peptide and c-type lysozyme, exhibit significantly increased expression in the symbiotic midgut at the pre-molt stages. These results suggest that the molting-associated up-regulation of antimicrobial activity in the symbiotic midgut represents a physiological mechanism of the host insect to regulate symbiosis, which is presumably for defending molting insects against injury and infection and/or for allocating symbiont-derived energy and resources to host molting.     

Characterization of IncA/C conjugative plasmid harboring blaTEM-52 and blaCTX-M-15 extended-spectrum β-lactamases in clinical isolates of Escherichia coli in Tunisia

To characterize the extended-spectrum β-lactamases (ESBLs) as well as their genetic environment in different isolates of Escherichia coli from patients with repeated urinary tract infections, large multidrug resistance (MDR) plasmids have been found. Definitive evidence for the presence of an A/C incompatibility complex (IncA/C) plasmid in the MDR isolates was provided by the probing of plasmids extracted from the clinical isolates. Conjugation experiments showed that bla genes were transferred by conjugation from the ten E. coli clinical isolates to E. coli XL1-Blue recipient. A comparative restriction fragment length polymorphism (RFLP) analysis of these plasmids showed that they are genetically similar, while the overall similarity of these plasmids supports the likelihood of recent movements among these E. coli isolates. Polymerase chain reaction (PCR) amplification and sequencing of the amplicons showed that the IncA/C plasmids harbor two ESBLs, identified as TEM-52 and CTX-M-15. Analysis of the plasmid DNA surrounding the bla _CTX-M-15 gene in the clinical isolates under study revealed a partially truncated fragment of ISEcp1 tnpA transposase. This result indicates the variety of genetic events that have enabled associations between ISEcp1 sequences and bla _CTX-M-15 genes in these clinical isolates.     

Regulation of hemagglutinin/protease expression by the VarS/VarA–CsrA/B/C/D system in Vibrio cholerae

In this study, through the analysis of Vibrio cholerae 2740-80 mutant strains produced by the cholera toxin subunit B gene containing Mariner-based transposon, we found that disruption of the varS gene, a member of the recently reported sensory system VarS/VarA–CsrA/B/C/D, resulted in altered expression of hemagglutinin/protease A. To further investigate the connection between VarS and HapA, we generated an additional varS mutant, V. cholerae 2740-80-VS, and examined the effect of this mutation on expression of HapA and of genes in the VarS/VarA–CsrA/B/C/D system. 2740-80-VS showed decreased expression of varS, csrB/C, hapR, and hapA along with increased biofilm production. Interestingly, expression of the alternative sigma factor σ^s, which is important for adaptation to environmental stress, was also decreased in this mutant. These results indicate that the VarS/VarA–CsrA/B/C/D system is involved in the control of HapA expression and biofilm production in V. cholerae 2740-80 through HapR regulation, and also that VarS/VarA controls expression of σ^s for HapA regulation.     

Association of -308G/A and -238G/A polymorphisms of TNF-α and osteosarcoma risk

Objective: As a proinflammatory cytokine, TNF-α is associated with increased risk of osteosarcoma (OS). Our study aimed to explore the association of TNF-α polymorphisms and OS susceptibility in the Han Chinese population. Methods: 80 OS patients and 99 healthy people, matched on the age and sex, participated in the study. Genotyping was conducted by the method of polymerase chain reaction-restricted fragment length polymorphisms (PCR-RFLP). Then logistic regression was used to evaluate the effects of TNF-α polymorphisms (-308G/A and -238G/A) on the pathology of OS. Results: The frequency of AA genotype in -308G/A locus in the cases was significantly higher than that of the healthy group (20.0% vs. 6.1%). Patients with OS were more likely to possess AA genotype of -308G/A locus (OR=4.00, 95% CI=1.41-11.38). For the patients with A allele, the risk for OS increased 0.62 fold (OR=1.62, 95% CI=1.04-2.50). There was no remarkable relationship of -238G/A polymorphisms and OS susceptibility. In addition, we found that patients with G-A and A-A haplotypes was much higher in the cases than that of control group (68.0% and 25.0%, 53.0% and 38.9%, respectively). A-G haplotype appeared to increase the risk for OS (OR=1.93, 95% CI=1.13-2.94). Conclusion: The AA genotype of -308G/A locus of TNF-α gene was a risk factor for OS, however there was no correlation between -238G/A of TNF-α and OS.     

Silencing of CEMIP suppresses Wnt/β-catenin/Snail signaling transduction and inhibits EMT program of colorectal cancer cells

Cell migration inducing hyaluronan binding protein (CEMIP) is a hyaluronic acid binding protein, the abnormal elevation of which is suggested as a contributor in the carcinogenesis of colorectal cancer (CRC). Cancer cells lose their adhesive properties and acquire an enhanced mobility by undergoing epithelial-mesenchymal transition (EMT). This study is performed to investigate whether and how CEMIP orchestrates the EMT process of CRC cells. To avoid the unexpected off-target effects possibly caused by one single shRNA, two shRNAs targeting different mRNA regions of CEMIP gene were used to knock down the mRNA and protein expression of CEMIP. Our data showed that the proliferation, migration and invasion of two CRC cell lines, HCT116 and SW480 cells, were inhibited by CEMIP shRNA. We here defined EMT as the complete or partial loss of E-cadherin and zona occludens protein 1 (ZO-1) (epithelial markers) and the gain of Vimentin and N-cadherin (mesenchymal markers), and found that the EMT process was attenuated in CEMIP-silenced SW480 cells. Snail, a direct target of β-catenin/T cell factor complex, is known to activate the EMT program during cancer metastasis. CEMIP shRNA was further found to suppress the Wnt/β-catenin/Snail signaling transduction in CRC cells as manifested by the decreased nuclear β-catenin and Snail. Collectively, our work demonstrates that CEMIP contributes to metastatic phenotype of CRC cells in vitro.     

Enzymatic properties of t-PA/α2-macroglobulin complexes

Complexes between single chain tissue plasminogen activator (sc-t-PA) or two chain tissue plasminogen activator (tc-t-PA) with α₂-macroglobulin have been made from the purified constituents with α₂-macroglobulin in excess. After separation of the free tissue plasminogen activator (t-PA), some of the properties of the complexes have been evaluated. The specific activities, measured as quotient between t-PA activity and t-PA antigen, are similar for the complexes as compared to the free t-PA derivatives. Indeed, the specific activity for sc-t-PA/α₂-macroglobulin complex is even slightly higher than for free sc-t-PA, most likely because of complexation to α₂-macroglobulin interferes with the sc-t-PA antigen analysis. The enzymatic properties of the complexes in activating plasminogen were investigated using different plasminogen concentrations, either in the presence of soluble fibrin or in its absence. All reactions obeyed Michaelis-Mentens kinetics. The k_cat values were similar in all reactions (0.025–0.044 s⁻¹). However, K_m for sc-t-PA/α₂-macroglobulin was 16 μmol/l in the absence of fibrin and 16 nmol/l in its presence. Thus, a 1000-fold decrease in K_m was observed by addition of fibrin. With tc-t-PA/α₂-macroglobulin complex the K_m in the absence of fibrin was 2.1 μmol/l as compared to 18 nmol/l in its presence. This corresponds to a decrease in K_m of approximately 120-fold. Inactivation of sc-t-PA/α₂-macroglobulin complex by α₂-antiplasmin was found to be somewhat slower than the reaction with free t-PA. However, the reaction rate of sc-t-PA/α₂-macroglobulin by PAI-1 was about 1000-fold slower as compared to the reaction with free t-PA. Furthermore, the time course of the reaction suggests a clearly reversible reaction. The physiologic implications or existence of t-PA/α₂-macroglobulin complex in vivo are still unknown.     

Effect of Liquid Crystal Properties on the Hemocompatibility of Polymer/Liquid Crystal Compositc Membranes

Aswehaveknown,theirmersurfaceofvesselsseemsverysmoothymacroscopicallyandiscoveredwithaliquidmatrixWhichembedallkindsofsacchproteinandsacchlipids.ThesacchProteinandsacchlipidsmoleculesarealwaysmovementintheliquidmatrixandtheinnermembraneofvesselsisamacroscopicallysmoothandmicroscopicallyphase-separatingstructUre.Inouropion,thesemaybemainreasonwhythevesselhasanexcellentamicoagulability.Inaddition,itiswidelyrecognizedthatthecellmembraneisthebilayermembranecomposedbylipidsandprotein.Lipidsmolecu…     

Is persistent activity of calcium/calmodulin-dependent kinase required for the maintenance of LTP?

Calcium/calmodulin-dependent protein kinase II (CaMKII) is concentrated in the postsynaptic density (PSD) and plays an important role in the induction of long-term potentiation (LTP). Because this kinase is persistently activated after the induction, its activity could also be important for LTP maintenance. Experimental tests of this hypothesis, however, have given conflicting results. In this paper we further explore the role of postsynaptic CaMKII in induction and maintenance of LTP. Postsynaptic application of a CaMKII inhibitor [autocamtide-3 derived peptide inhibitor (AC3-I), 2 mM] blocked LTP induction but had no detectable affect on N-methyl-D-aspartate (NMDA)-mediated synaptic transmission, indicating that the primary function of CaMKII in LTP is downstream from NMDA channel function. We next explored various methodological factors that could account for conflicting results on the effect of CaMKII inhibitors on LTP maintenance. In contrast to our previous work, we now carried out experiments at higher temperature (33 degrees C), used slices from adult animals, and induced LTP using a tetanic stimulation. However, we still found that LTP maintenance was not affected by postsynaptic application of AC3-I. Furthermore the inhibitor did not block LTP maintenance under conditions designed to enhance the Ca(2+)-dependent activity of protein phosphatases 1 and 2B (elevated Ca(2+), calmodulin, and an inhibitor of protein kinase A). We also tested the possibility that CaMKII inhibitor might not be able to affect CaMKII once it was inserted into the PSD. In whole-brain extracts, AC3-I blocked autophosphorylation of both soluble and particulate/PSD CaMKII with similar potencies although the potency of the inhibitor toward other CaMKII substrates varied. Thus we were unable to demonstrate a functional role of persistent Ca(2+)-independent CaMKII activity in LTP maintenance. Possible explanations of the data are discussed.     

Cryptorchidism-induced CFTR down-regulation results in disruption of testicular tight junctions through up-regulation of NF-κB/COX-2/PGE2

STUDY QUESTION Does elevated temperature-induced cystic fibrosis transmembrane conductance regulator (CFTR) down-regulation in Sertoli cells in cryptorchid testis disrupt testicular tight junctions (TJs) through the nuclear factor kappa B (NF-κB)/cyclooxygenase-2 (COX-2)/prostaglandin E(2) (PGE(2)) pathway? SUMMARY ANSWER Our results suggest that CFTR may be involved in regulating testicular TJs and the blood-testis barrier (BTB) through its negative regulation of the NF-κB/COX-2/PGE(2) pathway in Sertoli cells, a defect of which may result in the spermatogenesis defect in cryptorchidism. WHAT IS KNOWN ALREADY Cryptorchidism, or undescended testes, is known to result in defective spermatogenesis. Although an elevated testicular temperature is regarded as an important factor affecting spermatogenesis in cryptorchidism, the exact mechanism remains elusive. It is known that the expression of functional CFTR is temperature sensitive. Our previous study has demonstrated that CFTR negatively regulates NF-κB/COX-2/PGE(2) in bronchial epithelial cells. Disruption of TJs by COX-2/PGE(2) has been found in tumour cells. STUDY DESIGN AND METHODS Expression of CFTR, NF-κB, COX-2 and TJ proteins was examined in the testes of a surgical-induced cryptorchidism mouse model and a testicular hyperthermia mouse model, as well as in control or CFTR-inhibited/knocked down primary rat Sertoli cells. PGE(2) production was measured by ELISA. Sertoli cell barrier function was determined by transepethelial resistance (TER) measurements in rat Sertoli cell primary cultures. BTB integrity in the cryptorchidism model was monitored by examining tracker dye injected into seminiferous tubules. MAIN RESULTS Down-regulation of CFTR accompanied by activation of NF-κB, up-regulation of COX-2 and down-regulation of TJ proteins, including ZO-1 and occludin, was observed in a cryptorchidism mouse model. BTB leakage revealed impaired BTB integrity in cryptorchid testes, confirming the destruction of TJs. The inverse correlation of CFTR and COX-2 was further confirmed in a mouse testis hyperthermia model and CFTR knockout mouse model. Culturing primary Sertoli cells at 37°C, which mimics the pathological condition of cryptorchidism, led to a significant decrease in CFTR and increase in COX-2 expression and PGE(2) production compared with the culture at the physiological 32°C. Inhibition or knockdown of CFTR led to increased COX-2 but decreased ZO-1 and occludin expression in Sertoli cells, which could be mimicked by PGE(2), but reversed by NF-κB or COX-2 inhibitor, suggesting that the regulation of TJs by CFTR is mediated by a NF-κB/COX-2/PGE(2) pathway. Inhibition of CFTR or administration of PGE(2) significantly decreased Sertoli cell TER. LIMITATIONS This study has tested only the CFTR/NF-κB/COX-2/PGE(2) pathway in mouse testes in vivo and in rat Sertoli cells in vitro, and thus, it has some limitations. Further investigations in other species, especially humans, are needed. WIDER IMPLICATIONS OF THE FINDINGS Our study may shed more light on one of the aspects of the complicated underlying mechanisms of defective spermatogenesis induced by cryptorchidism. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Basic Research Program of China (2012 CB944900), Natural Sciences Foundation of China (30870933), Li Ka Shing Institute of Health Sciences, the Focused Investment Scheme of the Chinese University of Hong Kong and Morningside Foundation. The authors declare no conflict of interest.