Concentrated polymer brushes do not induce the expression of inflammatory and angiogeneic genes in human umbilical vein endothelial cells

Objective

When polymer brushes are applied as the inner coating for artificial blood vessels, they may induce unwanted responses in vascular endothelial cells continuously exposed to the polymer surface. Accordingly, we have examined the in vitro effect of non-biofouling concentrated polymer brushes (CPBs) on pro-inflammatory and angiogenic responses of human umbilical vein endothelial cells (HUVECs).

Results

Micro-patterned CPBs were prepared on silicon wafers using biocompatible polymers, poly(poly(ethylene glycol)methyl ether methacrylate) (PPEGMA) and poly(2-hydroxyethyl methacrylate) (PHEMA). HUVECs were cultured on PPEGMA-CPBs and PHEMA-CPBs with different channel widths (20, 50, and 80 µm) and analyzed for mRNA expression of the pro-inflammatory cytokines IL-6 and IL-8 and angiogeneic vascular endothelial growth factor (VEGF). Irrespective of channel width, PHEMA-CPBs reduced the expression of all target genes, whereas PPEGMA-CPBs reduced VEGF and did not affect IL-6 and IL-8 levels.

Conclusion

Micro-patterned CPBs, irrespective of chemical structure or adhesion area, do not induce the expression of important pro-inflammatory and angiogenic mediators in endothelial cells.

     

4-Aryl-5-carbamoyl-3-isoxazolols as competitive antagonists of insect GABA receptors: Synthesis,biological activity,and molecular docking studies

Competitive antagonists (CAs) of ionotropic GABA receptors (GABARs) reportedly exhibit insecticidal activity and have potential for development as novel insecticides for overcoming emerging resistance to traditional GABAR-targeting insecticides. Our previous studies demonstrated that 4,5-disubstituted 3-isoxazolols or 3-isothiazolols are an important class of insect GABAR CAs. In the present study, we synthesized a series of 4-aryl-5-carbamoyl-3-isoxazolols and examined their antagonism of insect GABARs expressed in Xenopus oocytes. Several of these 3-isoxazolols exhibited potent antagonistic activities against housefly and common cutworm GABARs, with IC₅₀ values in the low-micromolar range in both receptors. 4-(3-Amino-4-methylphenyl)-5-carbamoyl-3-isoxazolol (3u) displayed the highest antagonism, with IC₅₀ values of 2.0 and 0.9?μM in housefly and common cutworm GABARs, respectively. Most of the synthesized 3-isoxazolols showed moderate larvicidal activities against common cutworms, with more than 50% mortality at 100?μg/g. These results indicate that 4-monocyclic aryl-5-carbamoyl-3-isoxazolol is a promising scaffold for insect GABAR CA discovery and provide important information for the design and development of GABAR-targeting insecticides with a novel mode of action.     

'Working' cardiomyocytes exhibiting plateau action potentials from human placenta-derived extraembryonic mesodermal cells

The clinical application of cell transplantation for severe heart failure is a promising strategy to improve impaired cardiac function. Recently, an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and embryonic stem cells, have become important candidates for cell sources for cardiac repair. In the present study, we focused on the placenta as a cell source. Cells from the chorionic plate in the fetal portion of the human placenta were obtained after delivery by the primary culture method, and the cells generated in this study had the Y sex chromosome, indicating that the cells were derived from the fetus. The cells potentially expressed 'working' cardiomyocyte-specific genes such as cardiac myosin heavy chain 7beta, atrial myosin light chain, cardiac alpha-actin by gene chip analysis, and Csx/Nkx2.5, GATA4 by RT-PCR, cardiac troponin-I and connexin 43 by immunohistochemistry. These cells were able to differentiate into cardiomyocytes. Cardiac troponin-I and connexin 43 displayed a discontinuous pattern of localization at intercellular contact sites after cardiomyogenic differentiation, suggesting that the chorionic mesoderm contained a large number of cells with cardiomyogenic potential. The cells began spontaneously beating 3 days after co-cultivation with murine fetal cardiomyocytes and the frequency of beating cells reached a maximum on day 10. The contraction of the cardiomyocytes was rhythmical and synchronous, suggesting the presence of electrical communication between the cells. Placenta-derived human fetal cells may be useful for patients who cannot supply bone marrow cells but want to receive stem cell-based cardiac therapy.     

Phosphorylation of adult type Sept5 (CDCrel-1) by cyclin-dependent kinase 5 inhibits interaction with syntaxin-1

Increasing evidence implicates cyclin-dependent kinase 5 (Cdk5) in neuronal synaptic function. We searched for Cdk5 substrates in synaptosomal fractions prepared from mouse brains. Mass spectrometric analysis after two-dimensional SDS-PAGE identified several synaptic proteins phosphorylated by Cdk5-p35; one protein identified was Sept5 (CDCrel-1). Although septins were isolated originally as cell division-related proteins in yeast, Sept5 is expressed predominantly in neurons and is implicated in exocytosis. We confirmed that Sept5 is phosphorylated by Cdk5-p35 in vitro and identified Ser17 of adult type Sept5 (Sept5_v1) as a major phosphorylation site. We found that Ser17 of Sept5_v1 is phosphorylated in mouse brains. Coimmunoprecipitation from synaptosomal fractions and glutathione S-transferase-syntaxin-1A pulldown assays of Sept5_v1 expressed in COS-7 cells showed that phosphorylation of Sept5_v1 by Cdk5-p35 decreases the binding to syntaxin-1. These results indicate that the interaction of Sept5 with syntaxin-1 is regulated by the phosphorylation of Sept5_v1 at Ser17 by Cdk5-p35.     

Applicability of a segmental bioelectrical impedance analysis for predicting the whole body skeletal muscle volume.

This study aimed to test the hypothesis that a segmental bioelectrical impedance (BI) analysis can predict whole body skeletal muscle (SM) volume more accurately than a whole body BI analysis. Thirty males (19-34 yr) participated in this study. They were divided into validation (n = 20) and cross-validation groups (n = 10). The BI values were obtained using two methods: whole body BI analysis, which determines impedance between the wrist and ankle; and segmental BI analysis, which determines the impedance of every body segment in both sides of the upper arm, lower arm, upper leg and lower leg, and five parts of the trunk. Using a magnetic resonance imaging method, whole body SM volume was determined as a reference (SMV(MRI)). Simple and multiple regression analyses were applied to (length)(2)/Z (BI index) for the whole body and for every body segment, respectively, to develop the prediction equations of SMV(MRI). In the validation group, there were no significant differences between the measured and estimated SMV and no systematic errors in either BI analysis. In the cross-validation group, the whole body BI analysis produced systematic errors and resulted in the overestimation of SMV(MRI), but the segmental BI analysis was cross-validated. In the pooled data, the segmental BI analysis produced a prediction equation, which involves the BI indexes of the trunk and upper thigh as independent variables, with a SE of estimation of 1,693.8 cm(3) (6.1%). Thus the findings obtained here indicated that the segmental BI analysis is superior to the whole body BI analysis for estimating SMV(MRI).     

Mutations in tetrapyrrole biosynthesis pathway uncouple nuclear WUSCHEL expression from de novo shoot development in Arabidopsis

Plant Cell, Tissue and Organ Culture (PCTOC) - The effect of several parameters on trans-resveratrol extracellular production in Vitis vinifera cv Monastrell suspension cultured cells elicited with...     

Glutamate synthesis via photoreduction of NADP+ by photostable chlorophyllide coupled with polyethylene-glycol

Chlorophyllide a was coupled with alpha-(3-aminopropyl)-omega-methoxypoly(oxyethylene) (PEG-NH2) to form a PEG-chlorophyllide conjugate through an acid-amide bond. The conjugate catalyzed the reduction of methylviologen in the presence of 2-mercaptoethanol. It also catalyzed the photoreduction of NADP+ or NAD+ in the presence of ascorbate as an electron donor and ferredoxin-NADP+ reductase as the coupling enzyme. Utilizing the reducing power of NADPH generated by PEG-chlorophyllide conjugate under illumination, glutamate was synthesized from 2-oxoglutarate and NH4+ in the presence of glutamate dehydrogenase. PEG-chlorophyllide conjugate was quite stable toward light illumination compared with chlorophyll a. The increase in the molecular weight of PEG in the PEG-chlorophyllide conjugates was accompanied by the enhancement of photostability of the conjugate and also by the increased solubility in the aqueous solution.     

Induction of 2-amino-D2-thiazoline-4-carboxylic acid hydrolase and N-carbamoyl-l-cysteine amidohydrolase by S-compounds in Pseudomonas putida AJ3865

The induction of 2-amino-Delta(2)-thiazoline-4-carboxylic acid hydrolase (ATCase) and N-carbamoylcysteine amidohydrolase (NCCase), both of which are involved in the conversion step of 2-amino-Delta(2)-thiazoline carboxylic acid (ATC) to cysteine, was studied with Pseudomonas putida AJ3865. We found that L-ATC induced L-ATCase and L-NCCase, but that D-ATC induced only L-NCCase, whereas L- or D-NCC and thiazoline derivatives did not induce both enzymes. The bacterium showed neither D-ATCase nor D-NCCase activities, indicating that the role of L-ATC and D-ATC was different in the enzyme induction. We also found new inducers, d- and l-methionine, S-methyl-L-cysteine, cysteic acid, and 2-aminoethane sulfonic acid. However, the induction level of both enzymes by new inducers was much lower than those by L-ATC and D-ATC. Furthermore, the induction rate of both enzymes was synergistically increased only under a combination of D,L-ATC and new inducers. S-Compounds, however, such as new inducers except S-methyl-L-cysteine, inhibited both enzyme activities. This is the first report on the new inducers, synergistic induction, and the new inhibitors of L-ATCase and L-NCCase.     

Brap2 functions as a cytoplasmic retention protein for p21 during monocyte differentiation

The cell cycle inhibitor p21 plays an important role in monocytic cell differentiation, during which it translocates from the nucleus to cytoplasm. This process involves the negative regulation of the p21 nuclear localization signal (NLS). Here, we sought to determine the relationship between the cytoplasmic translocation of p21 and another molecule, Brap2, a cytoplasmic protein which binds the NLS of BRCA1 and was recently reported to inactivate KSR in the Ras-activating signal pathway under the name of IMP. We report that p21 and Brap2 directly interact, both in vitro and in vivo, in a manner requiring the NLS of p21 and the C-terminal portion of Brap2. When it is cotransfected with Brap2, p21 is expressed in the cytoplasm. Monocytic differentiation of the promyelomonocytic cell lines U937 and HL60 is associated with the upregulation of Brap2 expression concomitantly with the upregulation and cytoplasmic relocalization of p21. Our results underscore the role played by Brap2 in the process of cytoplasmic translocation of p21 during monocyte differentiation.