Plasma membrane vesicles of human umbilical cord mesenchymal stem cells ameliorate acetaminophen-induced damage in HepG2 cells: a novel stem cell therapy.

BACKGROUND: Acetaminophen (APAP) overdose is the common cause of acute liver failure (ALF) due to the oxidative damage of multiple cellular components. This study aimed to investigate whether plasma membrane vesicles (PMVs) from human umbilical cord mesenchymal stem cells (hUCMSCs) could be exploited as a novel stem cell therapy for APAP-induced liver injury. METHODS: PMVs from hUCMSCs were prepared with an improved procedure including a chemical enucleation step followed by a mechanical extrusion. PMVs of hUCMSCs were characterized and supplemented to hepatocyte cultures. Rescue of APAP-induced hepatocyte damage was evaluated. RESULTS: The hUCMSCs displayed typical fibroblastic morphology and multipotency when cultivated under adipogenic, osteogenic, or chondrogenic conditions. PMVs of hUCMSCs maintained the stem cell phenotype, including the presence of CD13, CD29, CD44, CD73, and HLA-ABC, but the absence of CD45, CD117, CD31, CD34, and HLA-DR on the plasma membrane surface. RT-PCR and transcriptomic analyses showed that PMVs were similar to hUCMSCs in terms of mRNA profile, including the expression of stemness genes GATA4/5/6, Nanog, and Oct1/2/4. GO term analysis showed that the most prominent reduced transcripts in PMVs belong to integral membrane components, extracellular vesicular exosome, and extracellular matrix. Immunofluorescence labeling/staining and confocal microscopy assays showed that PMVs enclosed cellular organelles, including mitochondria, lysosomes, proteasomes, and endoplasmic reticula. Incorporation of the fusogenic VSV-G viral membrane glycoprotein stimulated the endosomal release of PMV contents into the cytoplasm. Further, the addition of PMVs and a mitochondrial-targeted antioxidant Mito-Tempo into cultures of APAP-treated HepG2 cells resulted in reduced cell death, enhanced viability, and increased mitochondrial membrane potential. Lastly, this study demonstrated that the redox state and activities of aminotransferases were restored in APAP-treated HepG2 cells. CONCLUSIONS: The results suggest that PMVs from hUCMSCs could be used as a novel stem cell therapy for the treatment of APAP-induced liver injury.

Application of the yeast-surface-display system for orally administered salmon calcitonin and safety assessment.

High manufacturing costs and oral delivery are the constraints in clinical application of calcitonin. We selected surface-displayed Saccharomyces cerevisiae as a low-cost and safe carrier for oral delivery of salmon calcitonin (sCT). The sCT DNA fragment, optimized according to the codon preference of S. cerevisiae, was synthesized and cloned into the plasmid M-pYD1 to yield recombinant yAGA2-sCT, which was induced to express sCT by galactose for 0, 12, and 24 h. sCT expression was detected on the cell surface by indirect immunofluorescence and peaked at 12 h. About 65% recombinants expressed sCT on flow cytometry. The in vivo and in vitro activity of recombinant sCT was determined by detecting bioactivity of antiosteoclastic absorption on bone wafers and orally administering yAGA2-sCT to Wistar rats, respectively. For safety assessment of yAGA2-sCT, we observed abnormalities, morbidity, and mortality and determined body weight, serum chemistry parameters, hematological parameters, and organ weight. In vitro bioactivity of the recombinant sCT was similar to that of commercial sCT, Miacalcic; oral administration of 5 g/kg yAGA2-sCT induced a long-term hypocalcemic effect in Wistar rats and no adverse effects. This study demonstrates that yAGA2-sCT anchoring sCT protein on a S. cerevisiae surface has potential for low-cost and safe oral delivery of sCT.

[Application of bioinformatics analysis of signal peptide in the identification of Neurospora crassa phyA gene].

OBJECTIVE: To identify the function of the gene encoding Neurospora crassa EAA33149.1 protein which has 46.85% similarity with Aspergillus niger phA gene. METHODS: The bioinformatics analysis was conducted using the prediction algorithms SignalP v3.0, arginine and lysine propeptide cleavage sites in eukaryotic protein sequence prediction algorithms ProP 1.0 server, transmembrane domain prediction algorithms Tmpred and TMHMM v2.0, potential GP I-anchor sites prediction algorithm big-P I Predictor and the subcellular protein location prediction algorithms TargetP v1.01. According to the analysis results, the gene was cloned into Saccharomyces cerevisiae. RESULTS: The signal peptide, the cleavage site and the secretion pathway were determined, and the expressed recombinant protein with 54,000 displayed phytase activity. CONCLUSION: The gene has been identified to encode N. crassa phyA.

FT-IR methodology for quality control of arabinogalactan protein (AGP) extracted from green tea (Camellia sinensis ).

A rapid methodology of quality control was developed for arabinogalactan proteins (AGP) extracted and purified from green tea. Using the vectorial angle method and IR spectrum analysis, the 1200-800 cm(-1) region in second-derivative IR spectra was determined as the key fingerprinting region of green tea AGP, with the 1090-900 cm(-1) region reflecting their conservative and common characteristics. In fact, the key monosaccharides, galactose (Gal) and arabinose (Ara), were shown to have intense peaks at about 1075 and 1045 cm(-1), respectively, and uronic acids at about 1018 cm(-1) in second-derivative IR spectra. The variable region was identified to be at about 1134-1094 and 900-819 cm(-1) and was probably due to compositional and structural differences between AGPs. The constructed methodology was tested on green tea AGP extracted by three treatments and purified to apparent homogeneity as water-extracted Camellia sinensis AGP (CSW-AGP), pectinase-extracted C. sinensis AGP (CSP-AGP), and trypsin-extracted C. sinensis AGP (CST-AGP) with an Ara/Gal ratio of 1.37, 1.57, and 1.82, respectively. Regarding in vitro antioxidant activity, the AGPs (CSW-AGP and CST-AGP) with higher similarity (closer cos theta values calculated for second-derivative IR spectra) exhibited a similar ability of chelating ferrous ions and had a similar capability for scavenging hydroxyl radicals. In conclusion, the combination of second-derivative IR spectrum analysis and the vectorial angle method has allowed a successful characterization of green tea AGPs and was shown to be suitable for their compositional and activity discrimination and rapid quality evaluation.

Effects of hepatitis B virus S protein on human sperm function.

BACKGROUND: Hepatitis B virus (HBV) has been determined to exist in semen and male germ cells from patients with chronic HBV infection, but no data are yet available on the impact of HBV S protein (HBs), the main component of HBV envelop protein, on the human reproductive system. The purpose of this article was to investigate the effect of HBs on human sperm function. METHODS: Sperm motility analyses, sperm penetration assays, mitochondrial membrane potential assays, immunolocalizations with confocal microscopy and flow cytometry analyses were performed. RESULTS: HBs reduced sperm motility in a dose- and time-dependent manner and caused the loss of sperm mitochondrial membrane potential. HBs-HBs monoclonal antibody (MAb) complex apparently aggravated such impairments. After 4 h incubation with HBs at concentrations of 25, 50, 100 microg/ml, the percentages of sperm motility a+b significantly decreased compared with the control (P < 0.01). The fertilization rate and the fertilizing index in HBs-treated group were 40% and 0.57, respectively, which were significantly lower than 90% and 1.6, respectively, in the control (P < 0.01). The asialoglycoprotein receptor (ASGP-R) and HBs were found to localize mainly on the postacrosomal region. Both ASGP-R MAb and asialofoetuin, a high-affinity ligand of ASGP-R, inhibited the HBs-caused loss of sperm motility and mitochondrial membrane potential. CONCLUSIONS: HBs had adverse effects on human sperm function, and ASGP-R may play a role in the uptake of HBs into sperm cells, as demonstrated by the competitive inhibition of ASGP-R MAb or asialofoetuin, resulting in diminished impairment caused by HBs.