A novel hirudin derivative inhibiting thrombin without bleeding for subcutaneous injection.

Currently, anticoagulants would be used to prevent thrombosis. Thrombin is an effector enzyme for haemostasis and thrombosis. We designed a direct thrombin inhibitor peptide (DTIP) using molecular simulation and homology modelling and demonstrated that the C-terminus of DTIP interacts with exosite I, and N-terminus with the activity site of thrombin, respectively. DTIP interfered with thrombin-mediated coagulation in human, rat and mouse plasma (n=10 per group) and blocked clotting in human whole blood in vitro. When administered subcutaneously, DTIP showed potent and dose-dependent extension of aPTT, PT, TT and CT in rats (n=10 per group). The antithrombotic dose of DTIP induced significantly less bleeding than bivalirudin determined by transecting distal tail assay in rats. Furthermore, DTIP reached peak blood concentration in 0.5-1 hour and did not cause increased bleeding after five days of dosing compared to dabigatran etexilate. The antithrombotic effect of DTIP was evaluated in mice using lethal pulmonary thromboembolism model and FeCl3-induced mesenteric arteriole thrombus model. DTIP (1.0 mg/kg, sc) prevented deep venous thrombosis and increased the survival rate associated with pulmonary thromboembolism from 30 % to 80 %. Intravital microscopy showed that DTIP (1.0 mg/kg, sc) decelerated mesenteric arteriole thrombosis caused by FeCl3 injury. These data establish that DTIP is a novel antithrombotic agent that could be used to prevent thrombosis without conferring an increased bleeding risk.

Retinol dehydrogenase, RDH1l, is essential for the heart development and cardiac performance in zebrafish.

BACKGROUND: Retinoic acid (RA) is a potent signaling molecule that plays pleiotropic roles in patterning, morphogenesis, and organogenesis during embryonic development. The synthesis from retinol (vitamin A) to retinoic acid requires two sequential oxidative steps. The first step involves the oxidation of retinol to retinal through the action of retinol dehydrogenases. Retinol dehydrogenases1l (RDH1l) is a novel zebrafish retinol dehydrogenase. Herein we investigated the role of zebrafish RDH1l in heart development and cardiac performance in detail. METHODS: RDH1l specific morpholino was used to reduce the function of RDH1l in zebrafish. The gene expressions were observed by using whole mount in situ hybridization. Heart rates were observed and recorded under the microscope from 24 to 72 hours post fertilization (hpf). The cardiac performance was analyzed by measuring ventricular shortening fraction (VSF). RESULTS: The knock-down of RDH1l led to abnormal neural crest cells migration and reduced numbers of neural crest cells in RDH1l morphant embryos. The reduced numbers of cardiac neural crest cells also can be seen in RDH1l morphant embryos. Furthermore, the morpholino-mediated knock-down of RDH1l resulted in the abnormal heart loop. The left-right determining genes expression pattern was altered in RDH1l morphant embryos. The impaired cardiac performance was observed in RDH1l morphant embryos. Taken together, these data demonstrate that RDH1l is essential for the heart development and cardiac performance in zebrafish. CONCLUSIONS: RDH1l plays a important role in the neural crest cells development, and then ultimately affects the heart loop and cardiac performance. These results show for the first time that an enzyme involved in the retinol to retinaldehyde conversion participate in the heart development and cardiac performance in zebrafish.

MicroRNA-200a promotes anoikis resistance and metastasis by targeting YAP1 in human breast cancer.

PURPOSE: The process of metastases involves the dissociation of cells from the primary tumor, penetration into the basement membrane, invasion, and exiting from the vasculature to seed and colonize distant tissues. miR-200a is involved in this multistep metastatic cascade. This study aimed to test the hypothesis that miR-200a promotes metastasis through increased anoikis resistance in breast cancer. EXPERIMENTAL DESIGN: Breast cancer cells transfected with mimic or inhibitor for miR-200a were assayed for anoikis in vitro. miR-200a expression was assessed by quantitative real-time PCR (qRT-PCR). Luciferase assays, colony formation assays, and animal studies were conducted to identify the targets of miR-200a and the mechanism by which it promotes anoikis resistance. RESULTS: We found that overexpression of miR-200a promotes whereas inhibition of miR-200a suppresses anoikis resistance in breast cancer cells. We identified Yes-associated protein 1 (YAP1) as a novel target of miR-200a. Our data showed that targeting of YAP1 by miR-200a resulted in decreased expression of proapoptotic proteins, which leads to anoikis resistance. Overexpression of miR-200a protected tumor cells from anoikis and promoted metastases in vivo. Furthermore, knockdown of YAP1 phenocopied the effects of miR-200a overexpression, whereas restoration of YAP1 in miR-200a overexpressed breast cancer cells reversed the effects of miR-200a on anoikis and metastasis. Remarkably, we found that YAP1 expression was inversely correlated with miR-200a expression in breast cancer clinical specimens, and miR-200a expression was associated with distant metastasis in patients with breast cancer. CONCLUSIONS: Our data suggest that miR-200a functions as anoikis suppressor and contributes to metastasis in breast cancer.

Effect of Tbx1 knock-down on cardiac performance in zebrafish.

BACKGROUND: Tbx1 is the major candidate gene for DiGeorge syndrome (DGS). Similar to defects observed in DGS patients, the structures disrupted in Tbx1(-/-) animal models are derived from the neural crest cells during development. Although the morphological phenotypes of some Tbx1 knock-down animal models have been well described, analysis of the cardiac performance is limited. Therefore, myocardial performance was explored in Tbx1 morpholino injected zebrafish embryos. METHODS: To elucidate these issues, Tbx1 specific morpholino was used to reduce the function of Tbx1 in zebrafish. The differentiation of the myocardial cells was observed using whole mount in situ hybridization. Heart rates were observed and recorded under the microscope from 24 to 72 hours post fertilization (hpf). The cardiac performance was analyzed by measuring ventricular shortening fraction and atrial shortening fraction. RESULTS: Tbx1 morpholino injected embryos were characterized by defects in the pharyngeal arches, otic vesicle, aortic arches and thymus. In addition, Tbx1 knock down reduced the amount of pharyngeal neural crest cells in zebrafish. Abnormal cardiac morphology was visible in nearly 20% of the Tbx1 morpholino injected embryos. The hearts in these embryos did not loop or loop incompletely. Importantly, cardiac performance and heart rate were reduced in Tbx1 morpholino injected embryos. CONCLUSIONS: Tbx1 might play an essential role in the development of pharyngeal neural crest cells in zebrafish. Cardiac performance is impaired by Tbx1 knock down in zebrafish.

The antithrombotic effect of a novel hirudin derivative after reconstruction of carotid artery in rabbits.

INTRODUCTION: Hirudin is a direct thrombin inhibitor that has potential mechanistic advantages over indirect inhibitors. Peptides containing the RGD motif competitively inhibit binding of fibrinogen to glycoprotein IIb/IIIa on platelets, thus inhibiting platelet aggregation. A novel hirudin derivative, recombinant RGD-hirudin (r-RGD-hirudin), was engineered by fusing the tripeptide RGD sequence to the native hirudin. We tested the antithrombotic effect of r-RGD-hirudin using a carotid artery reconstruction model in rabbits. MATERIALS AND METHODS: A fusion gene encoding r-RGD-hirudin was constructed and expressed at high levels in Pichia pastoris. Following traumatic injury and anastomosis, 42 New Zealand White rabbits were randomized to receive normal saline, abciximab, wild-type hirudin, or r-RGD-hirudin. Fibrinogen concentration, aPTT, TT, PT, and PAGm were measured prior to and following the operation. Carotid angiography and pathological examination of the anastomotic site were performed to compare patency rates among the groups. RESULTS: The r-RGD-hirudin significantly prolonged aPTT, TT, PT and inhibited PAGm following carotid anastomosis in rabbits. The median dose of r-RGD-hirudin (0.5 mg/kg) had a therapeutic effect equal to that of wild-type hirudin (1.0 mg/kg) and higher than that of abciximab (0.2 mg/kg) with regard to patency rates. CONCLUSIONS: Compared to wild-type hirudin or antiplatelet agent, the novel anticoagulant, r-RGD-hirudin was capable of inhibiting both thrombin activity and platelet aggregation, and was demonstrated to be effective in the prevention of thrombosis.

[Effects of folic acid on the development of heart of zebrafish].

OBJECTIVE: To construct the folic acid deficient model in zebrafish and observe the abnormal cardiac phenotypes, to find the optimal period for supplementing folic acid that can most effectively prevent the heart malformation induced by folic acid deficiency, and to investigate the possible mechanisms by which folic acid deficiency induces malformations of heart. METHOD: The folic acid deficient zebrafish model was constructed by using both the folic acid antagonist methotrexate (MTX) and knocking-down dhfr (dihydrofolate reductase gene). Exogenous tetrahydrofolic acid rescue experiment was performed. Folic acid was given to folic acid deficient groups in different periods. The percent of cardiac malformation, the cardiac phenotypes, the heart rate and the ventricular shortening fraction (VSF) were recorded. The out flow tract (OFT) was observed by using fluorescein micro-angiography. Whole-mount in situ hybridization and real-time PCR were performed to detect vmhc, amhc, tbx5 and nppa expressions. RESULT: About (78.00 ± 3.74)% embryos in MTX treated group and (68.00 ± 6.32)% embryos in dhfr knocking-down group had heart malformations, including the abnormal cardiac shapes, the hypogenesis of OFT and the reduced heart rate and VSF. Giving exogenous tetrahydrofolic acid rescued the above abnormalities. Given the folic acid on 8 - 12 hours post-fertilization (hpf), both the MTX treated group (20.20% ± 3.77%) and dhfr knocking-down group (43.40% ± 4.51%) showed the most significantly reduced percent of cardiac malformation and the most obviously improved cardiac development. In folic acid deficient group, the expressions of tbx5 and nppa were reduced while the expressions of vmhc and amhc appeared normal. After being given folic acid to MTX treated group and dhfr knocking-down group, the expressions of tbx5 and nppa were increased. CONCLUSIONS: The synthesis of tetrahydrofolic acid was decreased in our folic acid deficient model. Giving folic acid in the middle period, which is the early developmental stage, can best prevent the abnormal developments of hearts induced by folic acid deficiency. Folic acid deficiency did not disrupt the differentiations of myosins in ventricle and atrium. The cardiac malformations caused by folic acid deficiency were related with the reduced expressions of tbx5 and nppa.

Microplasmin degrades fibronectin and laminin at vitreoretinal interface and outer retina during enzymatic vitrectomy.

PURPOSE: To determine whether intravitreal administration of microplasmin (microPlm) will degrade fibronectin (FN) and laminin (LN) in rat retina during microPlm-induced posterior vitreous detachment (PVD). METHODS: Increasing doses of microPlm, from 0.01 U to 0.03 U, were injected into the left eyes of 60 Sprague-Dawley rats to induce PVD. The right eyes were injected with the same volume of balanced salt solution (BSS). Histochemistry, scanning electron microscopy (SEM), and phase contrast microscopy were performed after 1 day and 7 days, to assess the remnant vitreous cortex. The FN and LN level located at the vitreoretinal interface and the outer retina were detected by immunohistochemistry. RESULTS: microPlm induced complete PVD in a dose-dependent fashion, without internal limiting membrane (ILM) damage (P = 0.0001, r = -0.479). The FN and LN in the photoreceptor cell layer (PCL) were completely degraded in all microPlm-treated eyes. In eyes with complete PVD, the FN, but not the LN, was completely removed from the ILM by microPlm treatment. CONCLUSION: Intravitreal injection of microplasmin degraded FN and LN at the vitreoretinal junction as well as at the outer retina.

Progesterone promotes propagation and viability of mouse embryonic stem cells.

It has been known that estrogen-17beta stimulates proliferation of mouse embryonic stem (mES) cells. To explore the function of another steroid hormone progesterone, we used MTT method and BrdU incorporation assay to obtain growth curves, clone forming assay to detect the propagation and viability of individual mES cells, Western blot to test the expression of ES cell marker gene Oct-4, fluorescence activated cell sorter (FACS) to test cell cycle, and real-time PCR to detect the expressions of cyclins, cyclin-dependent kinases and proto-oncogenes. The results showed that progesterone promoted proliferation of mES cells. The number of clones was more in progesterone-treated group than that in the control group. The expression of pluripotency-associated transcriptional factor Oct-4 changed little after progesterone treatment as shown by Western blot, indicating that most of mES cells were in undifferentiated state. The results of FACS proved that progesterone promoted DNA synthesis in mES cells. The proportion of mES cells in S+G(2)/M phase was higher in progesterone-treated group than that in the control group. Cyclins and cyclin-dependent kinases, as well as proto-oncogenes (c-myc, c-fos) were up-regulated when cells were treated with progesterone. The results obtained indicate that progesterone promotes propagation and viability of mES cells. The up-regulation of cell cycle-related factors might contribute to the function of progesterone.

A novel hirudin derivative characterized with anti-platelet aggregations and thrombin inhibition.

BACKGROUND: Hirudin is an anti-coagulative product of the salivary glands of the medicinal leech Hirudo medicinalis. It is a powerful and specific thrombin inhibitor. Peptides containing the RGD motif competitively inhibit the binding of fibrinogen to GP IIb/IIIa on the platelets, thus inhibiting platelet aggregation. RESULTS: We have constructed a recombinant RGD-hirudin (r-RGD-hirudin) by fusing the tripeptide RGD sequence to the native hirudin (wt-hirudin). The r-RGD-hirudin was expressed at high levels in Pichia pastoris, and was purified to approximately 97% homogeneity. The specific anti-thrombin activity of purified r-RGD-hirudin is 12,000 ATU/mg, which is equivalent to wt-hirudin, but only r-RGD-hirudin can inhibit platelet aggregation. The biological effects of r-RGD-hirudin on Thrombin Time (TT), Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT), Bleeding Time (BT), maximum platelet aggregation (PAGm) induced by ADP were studied in rabbit model and compared with that of wt-hirudin. The rabbits were infused r-RGD-hirudin had prolonged TT, PT, and aPTT which were similar to that of wt-hirudin; but only r-RGD-hirudin was capable of inhibiting PAGm. Histopathological analyses showed that r-RGD-hirudin was two to three times more effective than wt-hirudin in preventing thrombosis. CONCLUSIONS: r-RGD-hirudin can potentially be used as a novel anti-coagulant for the prevention of thrombosis after carotid artery anastomosis or in other thrombotic events.

[Influence of CRX gene on development of photoreceptors in zebrafish].

OBJECTIVE: To study the development and opsin expression of zebrafish photoreceptors after CRX gene knock-down. METHODS: It was a experimental study. CRX-MOs was microinjected into the zygote of zebrafish. Seventy-two and 96 hours post fertilization, we prepared the eye histological slides, electron microscope samples and whole mount in-situ hybridizations to study the development and opsin expression. of photoreceptors. RESULTS: There were no obvious histological changes in optical microscope observation after CRX-MOs microinjection. Outer segments were developed in control and blank groups, but no obvious outer segment was found in CRX group by electron microscope observation. Opsin Rho, SWS1 and SWS2 were expressed in control and blank groups and the expression of these genes was obviously decreased in CRX group. CONCLUSION: CRX gene can influence the development and opsins expression of photoreceptors outer segments in zebrafish.

Rehmannia inhibits adipocyte differentiation and adipogenesis.

Rehmannia glutinosa, a Traditional Chinese Medicine (TCM), has been used to increase physical strength. Here, we report that Rehmannia glutinosa extract (RE) inhibits adipocyte differentiation and adipogenesis. RE impairs differentiation of 3T3-L1 preadipocytes in a dose-dependent manner. At the molecular level, treatment with RE inhibits expression of the key adipocyte differentiation regulator C/EBPbeta, as well as C/EBPalpha and the terminal marker protein 422/aP2, during differentiation of preadipocytes into adipocytes. Additionally, RE inhibits the mitotic clonal expansion (MCE) process of adipocyte differentiation, and RE prevents localization of C/EBPbeta to the centromeres. RE also prevents high fat diet (HFD) induced weight gain and adiposity in rats. Taken together, our results indicate that Rehmannia glutinosa extract inhibits preadipocyte differentiation and adipogenesis in cultured cells and in rodent models of obesity.

Disruption of tissue-type plasminogen activator gene in mice aggravated liver fibrosis.

BACKGROUND AND AIM: Tissue-type plasminogen activator (tPA) is one of the major components in the matrix proteolytic network whose role in the pathogenesis of liver fibrosis remains unknown. The aim of this study is to investigate the role of tPA in carbon tetrachloride (CCl(4))-induced liver fibrosis. METHODS: Wild-type and tPA knockout mice (8 mice per group) were injected interperitoneumly with 25% CCl(4) 2 ml/kg twice per week as CCl(4) administration groups and olive oil 2 ml/kg as controls. After 4 weeks, the livers of mice were removed under deep anesthesia and prepared for further studies such as histology, immunostaining, hydroxyproline assay, zymography and western blot analysis. RESULTS: Mice lacking tPA developed more severe morphological injury and displayed an increased deposition of collagen in the liver after CCl(4) administration compared with wild-type counterparts. Deficiency of tPA increased alpha-smooth muscle actin expression in the mice livers. On the other hand, the decrease of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) activities, metalloproteinase-13 (MMP-13) expression and a marked increase of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) expression were found in the liver of CCl(4) administrated tPA(-/-) mice compared with wild-type counterparts. CONCLUSIONS: Deficiency of tPA aggravated liver fibrosis through promoting hepatic stellate cells (HSCs) activation and inhibiting ECM degradation by decreasing MMP-2, MMP-9 activities and disrupting the balance between MMP-13 and TIMP-1.

[Effect of external retinoic acid on Tbx1 gene during zebrafish embryogenesis].

OBJECTIVE: DiGeorge/del22q11 syndrome is one of the most common genetic causes of outflow tract and aortic arch defects in human. DiGeorge/del22q11 is thought to involve an embryonic defect restricted to the pharyngeal arches and the corresponding pharyngeal pouches. Previous studies have evidenced that retinoic acid (RA) signaling is definitely indispensable for the development of the pharyngeal arches. Tbx1, one of the T-box containing genes, is proved to be the most attractive candidate gene for DiGeorge/del22q11 syndrome. However, the interaction between RA and Tbx1 has not been fully investigated. Exploring the interaction will contribute to discover the molecular pathways disrupted in DiGeorge/del22q11 syndrome, and will also be essential for understanding genetic basis for congenital heart disease. It now seems possible that genes and molecular pathways disrupted in DiGeorge syndrome will also account for some isolated cases of congenital heart disease. Accordingly, the present study aimed to extensively study the effects of external RA on the cardiac development and Tbx1 expression during zebrafish embryogenesis. METHODS: The chemical genetics approach was applied by treating zebrafish embryos with 5 x 10(-8) mol/L RA and 10(-7) mol/L RA at 12.5 hour post fertilization (hpf). The expression patterns of Tbx1 were monitored by whole-mount in situ hybridization and quantitative real-time RT-PCR, respectively. RESULTS: The zebrafish embryos treated with 5 x 10(-8) mol/L RA and 10(-7) mol/L RA for 1.5 h at 12.5 hpf exhibited selective defects of abnormal heart tube. The results of whole-mount in situ hybridization with Tbx1 RNA probe showed that Tbx1 was expressed in cardiac region, pharyngeal arches and otic vesicle during zebrafish embryogenesis. RA treatment led to a distinct spatio-temporal expression pattern for Tbx1 from that in wild type embryo. The real-time PCR analysis showed that Tbx1 expression levels were markedly reduced by RA treatment. Tbx1 expression in the pharyngeal arches and heart were obviously down regulated compared to the wild type embryos. In contrast to 5 x 10(-8) mol/L RA-treated groups, 10(-7) mol/L RA caused a more severe effect on the Tbx1 expression level. CONCLUSION: These results suggested that there was a genetic link between RA and Tbx1 during development of zebrafish embryo. RA could produce an altered Tbx1 expression pattern in zebrafish. RA may regulate the Tbx1 expression in a dose-dependant manner. RA could represent a major epigenetic factor to cause abnormal expression of Tbx1, secondarily, disrupt the pharyngeal arch and heart development.

Effect of dihydrofolate reductase gene knock-down on the expression of heart and neural crest derivatives expressed transcript 2 in zebrafish cardiac development.

BACKGROUND: Folic acid is very important for embryonic development and dihydrofolate reductase is one of the key enzymes in the process of folic acid performing its biological function. Therefore, the dysfunction of dihydrofolate reductase can inhibit the function of folic acid and finally cause the developmental malformations. In this study, we observed the abnormal cardiac phenotypes in dihydrofolate reductase (DHFR) gene knock-down zebrafish embryos, investigated the effect of DHFR on the expression of heart and neural crest derivatives expressed transcript 2 (HAND2) and explored the possible mechanism of DHFR knock-down inducing zebrafish cardiac malformations. METHODS: Morpholino oligonucleotides were microinjected into fertilized eggs to knock down the functions of DHFR or HAND2. Full length of HAND2 mRNA which was transcribed in vitro was microinjected into fertilized eggs to overexpress HAND2. The cardiac morphologies, the heart rates and the ventricular shortening fraction were observed and recorded under the microscope at 48 hours post fertilization. Whole-mount in situ hybridization and real-time PCR were performed to detect HAND2 expression. RESULTS: DHFR or HAND2 knock-down caused the cardiac malformation in zebrafish. The expression of HAND2 was obviously reduced in DHFR knock-down embryos (P < 0.05). Microinjecting HAND2 mRNA into fertilized eggs can induce HAND2 overexpression. HAND2 overexpression rescued the cardiac malformation phenotypes of DHFR knock-down embryos. CONCLUSIONS: DHFR plays a crucial role in cardiac development. The down-regulation of HAND2 caused by DHFR knock-down is the possible mechanism of DHFR knock-down inducing the cardiac malformation.

A novel anti-tissue factor monoclonal antibody with anticoagulant potency derived from synthesized multiple antigenic peptide through blocking FX combination with TF.

Tissue factor (TF) has been implicated in the pathogenesis of various thrombotic disorders. Monoclonal antibodies (mAb) that specifically target TF may have potential as antithrombotic therapy. We designed a unique TF peptide (TFP) that was specific for the binding site to factor X (FX). This peptide was used to develop TF mAb that block the coagulation cascade by interfering with the combination of FX with the TF/FVIIa complex. Chemically synthesized TFP coupled to polylysine matrix was used as multiple antigenic peptide (TF-MAP) and this was used to immunize Balb/c mice for the preparation of hybridomas. One hybridoma cell line released an antibody, named TF4A12, which had high anticoagulant potency (by dilute prothrombin time assay). Western blotting showed that TF4A12 could bind TF-MAP and the soluble TF extracellular domain (sTF(1-219)). Results of FX activation assay and amidolytic activity assay showed that the anticoagulant ability of TF4A12 is due to blocking FX, but not FVII, binding to TF. Our study identified an efficient method of developing TF mAb that could block the coagulation cascade.

[Folic acid antagonist methotrexate causes the development malformation of heart and down-regulates the BMP2b and HAS2 expressions in zebrafish].

OBJECTIVE: To study the effect of methotrexate (MTX), a folic acid antagonist which can lead to folic acid deficient, on the cardiac development and on the expressions of BMP2b and HAS2 in zebrafish. METHODS: The zebrafish embryos at 6-48 hrs post fertilization (hpf) were treated with various concentrations of MTX (0.5 x 10(-3), 1.0 x 10(-3) and 2.0 x 10(-3) M). At 48 hpf, the percentage of cardiac malformation and heart rate were recorded. The zebrafish embryos at 6-10 hpf treated with 1.5 x 10(-3) M MTX were used as the MTX treatment group. At 24 and 48 hpf the cardiac morphology was observed under a microscope. The expressions of BMP2b and HAS2 in zebrafish were detected by in situ antisense RNA hybridization and real-time PCR. RESULTS: 6-12 hpf, the early embryonic developmental stage, was a sensitive period that MTX affected cardiac formation of zebrafish. The retardant cardiac development and the evidently abnormal cardiac morphology was found in the MTX treatment group. The results of in situ antisense RNA hybridization showed that the expressions of BMP2b and HAS2 in the zebrafish heart were reduced in the MTX treatment group at 36 and 48 hpf. The real-time PCR results demonstrated that the BMP2b expression decreased at 12, 24, 36 and 48 hpf, and that the HAS2 expression decreased at 24, 36 and 48 hpf in the treatment group compared with the control group without MTX treatment. CONCLUSIONS: The inhibition of folic acid function may affect cardiac development of early embryos, resulting in a retardant development and a morphological abnormality of the heart in zebrafish, possibly by down-regulating the expressions of BMP2b and HAS2.

Bone morphogenetic protein-2 acts upstream of myocyte-specific enhancer factor 2a to control embryonic cardiac contractility.

OBJECTIVE: Cardiac contractility is regulated tightly as an extrinsic and intrinsic homeostatic mechanism to the heart. The molecular basis of the intrinsic system is largely unknown. Here, we test the hypothesis that bone morphogenetic protein-2 (BMP-2) mediates embryonic cardiac contractility upstream of myocyte-specific enhancer factor 2A (MEF2A). METHODS: The BMP-2 and MEF2A expression pattern was analyzed by RT-PCR, Western blotting, whole-mount in situ hybridization, and an in vivo transgenic approach. The cardiac phenotype of BMP-2 and MEF2A knock-down zebrafish embryos was analysed. Cardiac contractions were recorded with a video camera. Myofibrillar organization was observed with transmission electron microscopy. Gene expression profiles were performed by quantitative real-time PCR analysis. RESULTS: We demonstrate that BMP-2 and MEF2A are co-expressed in embryonic and neonatal cardiac myocytes. Furthermore, we provide evidence that BMP-2 is required for cardiac contractility in vitro and in vivo and that MEF2A expression can be activated by BMP-2 signaling in neonatal cardiomyocytes. BMP-2 is involved in the assembly of the cardiac contractile apparatus. Finally, we find that exogenous MEF2A is sufficient to rescue ventricular contractility defects in the absence of BMP-2 function. CONCLUSIONS: In all, these observations indicate that BMP-2 and MEF2A are key components of a pathway that controls the cardiac ventricular contractility and suggest that the BMP2-MEF2A pathway can offer new opportunities for the treatment of heart failure.

[FTIR characterization of the secondary structure of a staphylokinase variant (K35R) encapsulated within PLGA microspheres].

The secondary structure of a staphylokinase variant (K35R, DGR) encapsulated in poly (lactic-co-glycolic acid) (PLGA) microspheres was quantitatively examined by Fourier transform infrared (FTIR) spectroscopy. Resolution enhancement technique and Fourier deconvolution were combined with band with curve-fitting procedures to quantitate the spectral information from the amide I bands. Nine component bands were found under the broad, nearly featureless amide I bands and assigned to alpha-helix, beta-sheet, turn and irregular (random) structures. The changes of bands at 1 651 and 1 623 cm(-1) after encapsulation were discussed.

Lactacystin inhibits 3T3-L1 adipocyte differentiation through induction of CHOP-10 expression.

Hormonal induction triggers a cascade leading to the expression of CCAAT/enhancer-binding protein(C/EBP)alpha and peroxisome proliferator-activated receptor (PPAR) gamma, C/EBPalpha, and PPARgamma turns on series of adipocyte genes that give rise to the adipocyte phenotype. Previous findings indicate that C/EBPbeta, a transcriptional activator of the C/EBPalpha and PPARgamma genes, is rapidly expressed after induction, but lacks DNA-binding activity and therefore cannot activate transcription of the C/EBPalpha and PPARgamma genes early in the differentiation program. Acquisition of DNA-binding activity of C/EBPbeta occurs when CHOP-10, a dominant-negative form of C/EBP family members, is down-regulated and becomes hyperphosphorylated as preadipocytes traverse the G1-S checkpoint of mitotic clonal expansion. Evidences are presented in this report that lactacystin, a proteasome inhibitor, up-regulated the CHOP-10 expression, blocked the DNA-binding activity of C/EBPbeta, and subsequently inhibited MCE as well as adipocyte differentiation.