Morphological and phenotypical characterization of human endothelial progenitor cells in an early stage of differentiation

The exact phenotype and lineage of endothelial progenitor cells (EPCs) are still a matter of debate and different expansion protocols are used to obtain them. In this study, EPC expansion from peripheral blood mononuclear cells was analyzed within the first week of culture. Both the adherent and suspended cells, of which the latter usually discarded, were considered. We provide, for the first time, a systematic study of EPC phenotype and functional features within the first 3 days of culture. Moreover, within the 2nd day, both cellular fractions displayed a significant increase in endothelial marker expression which correlated with EPC properties.     

An investigation of intracellular glycosylation activities in CHO cells: Effects of nucleotide sugar precursor feeding

Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub‐array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 nucleotides and 7 nucleotide sugars; were used to generate intracellular N‐glycosylation profiles. Specifically, the effects of nucleotide sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon‐γ (IFN‐γ). Galactose (±uridine), glucosamine (±uridine), and N‐acetylmannosamine (ManNAc) (±cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN‐γ sialylation as compared to the untreated control cultures. This could be directly attributed to increases in nucleotide sugar substrates, UDP‐Hex (～20‐fold), UDP‐HexNAc (6‐ to 15‐fold) and CMP‐sialic acid (30‐ to 120‐fold), respectively. Up‐regulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine + uridine and ManNAc + cytidine increased UDP‐HexNAc and CMP‐sialic acid by another two‐ to fourfold as compared to feeding sugar precursors alone. However, it did not lead to a synergistic increase in IFN‐γ sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine‐ and cytidine‐activated nucleotide sugars simultaneously, which could imply that uridine is one of the limiting substrates for nucleotide sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how nucleotide sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality. Biotechnol. Bioeng. 2010;107: 321–336. © 2010 Wiley Periodicals, Inc.     

Hypoxia associated NMDA receptor 2 subunit composition: developmental comparison between the hypoxia-tolerant subterranean mole-rat, Spalax, and the hypoxia-sensitive rat

Vertebrate brains are sensitive to oxygen depletion, which may lead to cell death. Hypoxia sensitivity originates from the high intrinsic rate of ATP consumption of brain tissue, accompanied by the release of glutamate, leading to the opening of ionotropic glutamate receptors, such as N-methyl-D-aspartate (NMDA) receptors (NMDARs). The relative expression levels of the four NMDAR-2 (NR2) subunits change during mammalian development with higher levels of units NR2B and NR2D observed during early development and correlated with hypoxic tolerance during embryonic and neonatal stages of development. Higher levels of NR2D are also abundant in brains of hypoxia tolerant species such as the crucian carp. The subterranean mole-rat, Spalax spends its life underground in sealed burrows and has developed a wide range of adaptations to this special niche including hypoxia-tolerance. In this study, we compared the in vivo mRNA expression of NR2 subunits in the brains of embryonic, neonatal and adult Spalax and rat. Our results demonstrate that under normoxic conditions, mRNA levels of NR2D are higher in Spalax than in rat at all developmental stages studied and are similar to levels in neonatal rat and in other hypoxia/anoxia tolerant species. Furthermore, under hypoxia Spalax NR2D mRNA levels increase while no response was observed in rat. Similarly, hypoxia induces an increase in mRNA levels of Spalax NR2A, claimed to promote neuronal survival. We suggest that indeed the proportional combinations of NMDAR-2 subunits contribute to the ability of the Spalax brain to cope with hypoxic environments.     

Acetylation-dependent regulation of Skp2 function

Aberrant Skp2 signaling has been implicated as a driving event in tumorigenesis. Although the underlying molecular mechanisms remain elusive, cytoplasmic Skp2 correlates with more aggressive forms of breast and prostate cancers. Here, we report that Skp2 is acetylated by p300 at K68 and K71, which is a process that can be antagonized by the SIRT3 deacetylase. Inactivation of SIRT3 leads to elevated Skp2 acetylation, which leads to increased Skp2 stability through impairment of the Cdh1-mediated proteolysis pathway. As a result, Skp2 oncogenic function is increased, whereby cells expressing an acetylation-mimetic mutant display enhanced cellular proliferation and tumorigenesis in vivo. Moreover, acetylation of Skp2 in the nuclear localization signal (NLS) promotes its cytoplasmic retention, and cytoplasmic Skp2 enhances cellular migration through ubiquitination and destruction of E-cadherin. Thus, our study identifies an acetylation-dependent regulatory mechanism governing Skp2 oncogenic function and provides insight into how cytoplasmic Skp2 controls cellular migration.     

Impact of DAG stimulation on mineral synthesis, mineral structure and osteogenic differentiation of human cord blood stem cells

It remains unexplored in what way osteogenic stimulation with dexamethasone, ascorbic acid and β-glycerol phosphate (DAG) influences the process of mineralization, the composition and structure of the assembled mineral. Therefore, we analyzed and characterized biomineralization in DAG-stimulated and unstimulated 3D human unrestricted somatic stem cell (USSC) cultures. Microspheres were analyzed by histological staining, scanning electron microscopy (SEM), semi-quantitative energy-dispersive X-ray spectroscopy (EDX), quantitative wavelength-dispersive X-ray spectroscopy (WDX), transmission electron microscopy (TEM), selected area electron diffraction (SAED) and Raman spectroscopy.Mineral material was detected by SEM and histological staining in both groups, and showed structural differences. DAG influenced the differentiation of USSCs and the formation, structure and composition of the assembled mineral. SEM showed that cells of the + DAG spheres exhibited morphological signs of osteoblast-like cells. EDX and WDX confirmed a Ca-P mineral in both groups. Overall, the mineral material found showed structural similarities to the mineral substance of bony material.     

HLA-A*02:07 is a protective allele for EBV negative and a susceptibility allele for EBV positive classical Hodgkin lymphoma in China

HLA-A2 protects from EBV+ classical Hodgkin lymphoma (cHL) in Western Europe, but it is unknown whether this protective effect also exists in the Chinese population. We investigated the association of HLA-A2 and specific common and well documented HLA-A2 subtypes with EBV stratified cHL patients (n = 161) from the northern part of China. Quantitative-PCR and sequence-based subtyping was performed to identify HLA-A2 positive samples and their subtypes. 67 (42%) of the cHL patients were EBV+. There were no significant differences in percentages of HLA-A2 positivity between cHL and controls (65% vs 66%) and between EBV+ and EBV− cHL patients (70% vs 61%). The frequency distribution of HLA-A2 subtypes was significantly different between EBV stratified cHL subgroups and controls. This difference was most striking for the HLA-A*02:07 type with a frequency of 38% in EBV+ cHL, 8% in EBV− cHL and 20% in controls. Significant differences were also observed for the HLA-A*02:07, HLA-A2 (non-02:07) and the A2-negative typings between EBV+ cHL vs controls (p = 0.028), EBV− cHL vs controls (p = 0.045) and EBV+ vs EBV− cHL cases (p = 2×10⁻⁵). In conclusion, HLA-A*02:07 is a predisposing allele for EBV+ cHL and a protective allele for EBV− cHL in the northern Chinese population.     

Bral1: "Superglue" for the extracellular matrix in the brain white matter

Bral1 is a link protein that stabilizes the binding between lecticans and hyaluronic acid and thus maintains the extracellular matrix assembly in the CNS. Bral1 is specifically located in the white matter around the nodes of Ranvier. Recent studies suggest its function in promoting saltatory neural conduction. This article reviews the current knowledge about the structure, expression and function of this link protein.