Imaging of immobilized enzyme spots by scanning chemiluminescence microscopy with electrophoretic injection

Scanning chemiluminescence microscopy (SCLM) with electrophoretic injection was developed and applied to visualize enzyme reactions localized in an enzyme microspot. The SCLM uses a tapered glass capillary as a probe for injecting a small amount of luminol onto the substrate to generate localized chemiluminescence. The electrophoretic injection by application of a constant current between the inside and outside of the capillary enabled the continuous and controllable injection of a minute quantity of luminol in the range of 0.1 pmol/s. The image of enzyme activity in a monolayer spot of horseradish peroxidase was obtained by using the electrophoretic injection-based SCLM system.     

Differential level in co-down-modulation of CD4 and CXCR4 primed by HIV-1 gp120 in response to phorbol ester, PMA, among HIV-1 isolates

HIV-1 enters cells through interacting with cell surface molecules such as CD4 and chemokine receptors. We generated recombinant soluble gp120s derived from T-cell line-tropic (T-tropic) and macrophage-tropic (M-tropic) HIV-1 strains using a baculovirus expression system and investigated the association of CD4-gp120 complex with the chemokine receptor and/or other surface molecule(s). For monitoring the co-down-modulations of the CD4-gp120 complex, a cytoplasmic domain deletion mutant (tailless CD4), which is not capable of undergoing down-modulation by itself in response to phorbol ester PMA, was used. Our studies revealed both cell-type and HIV-1 strain-specific differences. We found that T-tropic gp120s were capable of priming co-down-modulation with tailless CD4 by interacting with CXCR4, whereas M-tropic SF162 gp120 could not after PMA treatment even in the presence of CCR5. Among the T-tropic HIV-1 envelopes, IIIB gp120 was the most potent. Furthermore, the ability of gp120 to prime the PMA induced co-down-modulation of tailless CD4 appeared to be dependent on the concentration of the principal coreceptor CXCR4. Nevertheless, the observation that IIIB gp120 strongly primed tailless CD4 co-down-modulation on human osteosarcoma HOS cells that express undetectable levels of surface CXCR4 raised the possibility that membrane component(s) other than those recently identified can be involved in down-modulation of the CD4/gp120 complexes.     

Isolation of 48 genetic markers appropriate for high throughput genotyping of inbred rat strains by B1 repetitive sequence-representational difference analysis

Mapping of genetic suppressors, modifiers, and quantitative trait loci (QTLs) requires genetic markers that can be efficiently and inexpensively genotyped for a large number of individuals. To isolate rat genetic markers suitable for this purpose, representational difference analysis (RDA) was performed with amplicons prepared by PCR with the B1 repetitive sequence used as the primer (B1-amplicons). In total, 48 polymorphic DNA fragments were isolated by five series of RDA, subtracting the B1-amplicons prepared from an ACI/N (ACI) rat from those prepared from BUF/Nac (BUF), and vice versa. All the polymorphic fragments detected ``presence-or-absence' polymorphisms with B1-amplicons prepared from ACI, BUF, and their F₂ progeny, and each fragment was linkage mapped. Dot-blotting amplicons onto filters at a high density and hybridization of the filters with these B1-RDA markers made it possible to genotype a large number of rats simultaneously for multiple loci. These B1-RDA markers were polymorphic between two given inbred strains of rat at frequencies between 30% and 70%. This is the first report on the isolation of B1-RDA markers among inbred strains of rats. Received: 15 July 1998 / Accepted: 18 August 1998     

Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

We evaluated whether dexamethasone augments the osteogenic capability of bone marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic bone formation induced by bone morphogenetic protein-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without 10-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic bone formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic bone formation model, bone formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2.Highlights: 1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2.     

Epigenetic regulation of autophagy by the methyltransferase EZH2 through an MTOR-dependent pathway

Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the autophagy regulation machinery has been widely studied, the key epigenetic control of autophagy process still remains unknown. Here we report that the methyltransferase EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) epigenetically represses several negative regulators of the MTOR (mechanistic target of rapamycin [serine/threonine kinase]) pathway, such as TSC2, RHOA, DEPTOR, FKBP11, RGS16 and GPI. EZH2 was recruited to these genes promoters via MTA2 (metastasis associated 1 family, member 2), a component of the nucleosome remodeling and histone deacetylase (NuRD) complex. MTA2 was identified as a new chromatin binding protein whose association with chromatin facilitated the subsequent recruitment of EZH2 to silenced targeted genes, especially TSC2. Downregulation of TSC2 (tuberous sclerosis 2) by EZH2 elicited MTOR activation, which in turn modulated subsequent MTOR pathway-related events, including inhibition of autophagy. In human colorectal carcinoma (CRC) tissues, the expression of MTA2 and EZH2 correlated negatively with expression of TSC2, which reveals a novel link among epigenetic regulation, the MTOR pathway, autophagy induction, and tumorigenesis.     

A Food-Derived Flavonoid Luteolin Protects against Angiotensin II-Induced Cardiac Remodeling

Oxidative stress has been implicated in cardiac remodeling (cardiac fibrosis and hypertrophy), which impairs cardiac function and metabolism; therefore, it is anticipated antioxidative compounds will have protective properties against cardiac remodeling. Luteolin (3’,4’,5,7-tetrahydroxyflavone), a widely distributed flavonoid found in many herbal extracts including celery, green pepper, perilla leaves and seeds, and chamomile, is a known to be a potent antioxidant and was previously demonstrated to exert an antifibrotic effect in the lungs and the liver. In this study, we clearly demonstrate that oral pretreatment with the higher-luteolin diet (0.035% (wt/wt)) protected against cardiac fibrosis and hypertrophy as well as a hyperoxidative state in Ang II-infused rats. In cardiac tissue, increased gene expression levels of TGFβ1, CTGF, Nox2, Nox4, ANP, and BNP induced by Ang II were restored by oral pretreatment of this high-luteolin diet. In cultured rat cardiac fibroblasts, H₂O₂-induced TGFβ1 expression and the phosphorylation of JNK were suppressed by luteolin pretreatment. In conclusion, food-derived luteolin has protective actions against Ang II-induced cardiac remodeling, which could be mediated through attenuation of oxidative stress.     

Bisleuconothine A,an eburnane–aspidosperma bisindole alkaloid from Leuconotis griffithii

A new bisindole alkaloid, bisleuconothine A (1) consisting of an eburnane–aspidosperma type skeleton, was isolated from the bark of Leuconotis griffithii. The structure including absolute stereochemistry was elucidated on the basis of 2D NMR data and X-ray analysis. Bisleuconothine A (1) showed cell growth inhibitory activity against various human cancer cell lines.     

Genetic characterization of mutants resistant to the antiauxin p-chlorophenoxyisobutyric acid reveals that AAR3, a gene encoding a DCN1-like protein, regulates responses to the synthetic auxin 2,4-dichlorophenoxyacetic acid in Arabidopsis roots

To isolate novel auxin-responsive mutants in Arabidopsis (Arabidopsis thaliana), we screened mutants for root growth resistance to a putative antiauxin, p-chlorophenoxyisobutyric acid (PCIB), which inhibits auxin action by interfering the upstream auxin-signaling events. Eleven PCIB-resistant mutants were obtained. Genetic mapping indicates that the mutations are located in at least five independent loci, including two known auxin-related loci, TRANSPORT INHIBITOR RESPONSE1 and Arabidopsis CULLIN1. antiauxin-resistant mutants (aars) aar3-1, aar4, and aar5 were also resistant to 2,4-dichlorophenoxyacetic acid as shown by a root growth assay. Positional cloning of aar3-1 revealed that the AAR3 gene encodes a protein with a domain of unknown function (DUF298), which has not previously been implicated in auxin signaling. The protein has a putative nuclear localization signal and shares homology with the DEFECTIVE IN CULLIN NEDDYLATION-1 protein through the DUF298 domain. The results also indicate that PCIB can facilitate the identification of factors involved in auxin or auxin-related signaling.     

The Functional Interrelationship between Gap Junctions and Fenestrae in Endothelial Cells of the Liver Organoid

Functional intact liver organoid can be reconstructed in a radial-flow bioreactor when human hepatocellular carcinoma (FLC-5), mouse immortalized sinusoidal endothelial M1 (SEC) and A7 (HSC) hepatic stellate cell lines are cocultured. The structural and functional characteristics of the reconstructed organoid closely resemble the in vivo liver situation. Previous liver organoid studies indicated that cell-to-cell communications might be an important factor for the functional and structural integrity of the reconstructed organoid, including the expression of fenestrae. Therefore, we examined the possible relationship between functional intact gap junctional intercellular communication (GJIC) and fenestrae dynamics in M1-SEC cells. The fine morphology of liver organoid was studied in the presence of (1) irsogladine maleate (IM), (2) oleamide and (3) oleamide followed by IM treatment. Fine ultrastructural changes were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and compared with control liver organoid data. TEM revealed that oleamide affected the integrity of cell-to-cell contacts predominantly in FLC-5 hepatocytes. SEM observation showed the presence of fenestrae on M1-SEC cells; however, oleamide inhibited fenestrae expression on the surface of endothelial cells. Interestingly, fenestrae reappeared when IM was added after initial oleamide exposure. GJIC mediates the number of fenestrae in endothelial cells of the liver organoid.     

Low-intensity pulsed ultrasound (LIPUS) induces RANKL, MCP-1, and MIP-1beta expression in osteoblasts through the angiotensin II type 1 receptor

Constant mechanical stress is essential for the maintenance of bone mass and strength, which is achieved through the cooperative functions of osteoblasts and osteoclasts. However, it has not been fully elucidated how these cell types mediate mechanical signals. Low-intensity pulsed ultrasound (LIPUS) therapy is a recently developed method for application of mechanical stress, and is used clinically to promote bone fracture healing. In the present study, we applied LIPUS to osteoblasts at different stages of maturation and analyzed their chemokine and cytokine expression. In comparison with their immature counterparts, mature osteoblasts expressed significantly higher levels of mRNAs for the receptor activator of nuclear factor kappa B ligand (RANKL), monocyte chemoattractant protein (MCP)-1, and macrophage-inflammatory protein (MIP)-1beta after a few hours of LIPUS treatment. Intriguingly, protein and mRNA expression of angiotensin II type 1 receptor (AT1), a known mechanoreceptor in cardiomyocytes, was detected in osteoblasts, and the level of expression increased significantly during cell maturation. Furthermore, LIPUS-induced extracellular signal-regulated kinase (ERK) phosphorylation and RANKL/chemokine expression was abrogated by a specific AT1 inhibitor. Thus, AT1 may play one of the essential roles in bone metabolism as a mechanoreceptor of osteoblasts.