Dynamic observation and quantification of type I/II collagen in chondrogenesis of mesenchymal stem cells by second‐order susceptibility microscopy

Second‐order susceptibility (SOS) microscopy is used to image and characterize chondrogenesis in cultured human mesenchymal stem cells. SOS analysis shows that the SOS tensor ratios can be used to characterize type I and II collagens in living tissues and that both collagen types are produced at the onset of chondrogenesis. Time‐lapse analysis shows a modulation of extracellular matrix results in a higher rate in increase of type II collagen, as compared to type I collagen. With time, type II collagen content stabilizes at the composition of 70% of total collagen content. SOS microscopy can be used to continuously and noninvasively monitor the production of collagens I and II. With additional development, this technique can be developed into an effective quality control tool for monitoring extracellular matrix production in engineered tissues.      

Epidermal growth factor and transforming growth factor-beta1 enhance HK-2 cell migration through a synergistic increase of matrix metalloproteinase and sustained activation of ERK signaling pathway

Epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1), upregulated in renal diseases, have a combinational effect on epithelial-mesenchymal transformation (EMT) of renal proximal tubular cells. The aim of this study was to examine the mechanism regarding the combinational effect of EGF and TGF-beta1 on cell migration following EMT. The results demonstrated that EGF (10 ng/ml) and TGF-beta1 (3 ng/ml) synergistically increased cell migration, accompanied by an increase in matrix metalloproteinase-9 (MMP-9) gene expression, production and activity. Inhibition of MMP-9 production and activity by an MMP-2/MMP-9-specific inhibitor blocked the synergistic effect of EGF and TGF-beta1 on cell migration. The kinetic profile of extracellular signal-regulated kinase (ERK) signals demonstrated that ERK1/2 activation was rapidly and strongly induced by EGF but delayed and less marked by TGF-beta1 stimulation. In contrast, co-administration of EGF and TGF-beta1 caused an early pronounced and persistent ERK1/2 activation. Inhibition of the ERK1/2 activity by PD98059 abrogated the synergistic effect of EGF and TGF-beta1 on cell migration, MMP-9 production and activity, indicating that EGF and TGF-beta1 converged at the ERK signaling pathway to mediate cell migration. This study demonstrates that EGF and TGF-beta1 synergistically stimulate proximal tubular cell migration through the increased MMP-9 function and enhanced ERK1/2 activation.     

HIV-1 Nef assembles a Src family kinase-ZAP-70/Syk-PI3K cascade to downregulate cell-surface MHC-I

HIV-1 Nef, which is required for the efficient onset of AIDS, enhances viral replication and infectivity by exerting multiple effects on infected cells. Nef downregulates cell-surface MHC-I molecules by an uncharacterized PI3K pathway requiring the actions of two Nef motifs-EEEE(65) and PXXP(75). We report that the Nef EEEE(65) targeting motif enables Nef PXXP(75) to bind and activate a trans-Golgi network-localized Src family tyrosine kinase (SFK). The Nef/SFK complex then recruits and phosphorylates the tyrosine kinase ZAP-70, which binds class I PI3K to trigger MHC-I downregulation in primary CD4+ T cells. In promonocytic cells, Nef/SFK recruits the ZAP-70 homolog Syk to downregulate MHC-I, implicating this PI3K pathway in multiple HIV-1 reservoirs. Isoform-specific PI3K inhibitors repress MHC-I downregulation, identifying them as potential therapeutic agents to combat HIV-1. The discovery of this Nef-SFK-ZAP-70/Syk-PI3K signaling pathway explains the hierarchal role of the Nef motifs in effecting immunoevasion.     

An in vivo study of common carp (Cyprinus carpio L.) liver during prolonged hypoxia

Hypoxia induced apoptosis has been studied extensively in many mammalian cell lines but there are only a few studies using whole animal models. We investigated the response of the intact liver to hypoxia in a hypoxia tolerant fish, the carp (Cyprinus carpio, L). We exposed carp to hypoxia for up to 42 days, using oxygen level (0.5 mgO₂/L) that were slightly higher than the critical oxygen level of carp. There was extensive DNA damage in liver cells, especially during the first week of exposure, indicated by a massive TUNEL signal. However there was no change in cell proliferation, cell number or size, no increase in caspase-3 activity, no increase in single stranded DNA and this, combined with a number of other observations, led us to conclude there was no increase in apoptosis in the liver during hypoxia. There was up-regulation of some anti-apoptotic genes and proteins (Bcl-2, HSP70, p27) and down-regulation of some pro-apoptotic genes (Tetraspanin 5 and Cell death activator). The cells appeared to enter cell cycle arrest, presumably to allow repair of damaged DNA. As there was no change in cell proliferation and cell number, the damaged cells were not entering apoptosis and must have recovered during prolonged hypoxia.     

Expression of human BK ion channels in Sf9 cells, their purification using metal affinity chromatography, and functional reconstitution into planar lipid bilayers

This report describes a procedure for purification of large conductance calcium-activated potassium (BK, maxi-K) channels using immobilised metal affinity chromatography (IMAC) under non-denaturing conditions. An amino-terminal histidine fusion tag was added to hSlo, the human BK channel, and expressed in Sf9 insect cells. Following IMAC purification and production of proteoliposomes, protein function was assessed electrophysiologically in planar bilayer lipid membranes. Single channel openings had conductances of 250-300 pS and were inhibited by paxilline, demonstrating that the BK channels remained functional following IMAC purification. This method to obtain functional human ion channels will be useful in assays to screen potential pharmaceuticals.     

Analysis of a Major Phenolic Glucoside and Biochemistry Compounds from Curculigo Orchioides Gaertn

Background:Orcinol-β-D-glucoside, which is also known as orcinol glucoside, is a major phenolic glucoside compound in the rhizome of the Curculigo orchioides Gaertn. This compound has many medicinal properties such as being antioxidant, immunomodulatory, antiosteoporosis, stress relief, antidepressant, etc.Methods:Determination of reducing sugar content by Bertrand’s method, determination of lipid content by Soxhlet method, determination of vitamin C content by iodine titration, determination of enzyme activity catalase by titration with KMnO4. Quantification of Orcinol-β-D-glucoside was conducted by HPLC analysis.Results:The Orcinol-β-D-glucoside of C. orchioides in Thuy Bang mountain was highest. Besides, the content of reducing sugars, vitamin C, enzyme catalase, and lipids of C. orchioides differed significantly among sites. In which, the reducing sugar and vitamin C of C. orchioides in Ngu Binh mountain was highest. Whereas, enzyme catalase was also highest in Thuy Bang mountain. However, the lipid content of C. orchioides was also highest in Huong Tho mountain.Conclusion:The result will contribute to providing the scientific basis for the selection of breeding, planting, development of C. orchioides in Thua Thien Hue province, as well as other provinces in Vietnam and opening new research directions for applications in the future.Key Words: Antioxidants, Hypoxidaceae, Medicinal Plants, Vietnam