Hypoxia and stem cell‐based engineering of mesenchymal tissues

Stem cells have the ability for prolonged self‐renewal and differentiation into mature cells of various lineages, which makes them important cell sources for tissue engineering applications. Their remarkable ability to replenish and differentiate in vivo is regulated by both intrinsic and extrinsic cellular mechanisms. The anatomical location where the stem cells reside, known as the “stem cell niche or microenvironment,” provides signals conducive to the maintenance of definitive stem cell properties. Physiological condition including oxygen tension is an important component of the stem cell microenvironment and has been shown to play a role in regulating both embryonic and adult stem cells. This review focuses on oxygen as a signaling molecule and the way it regulates the stem cells' development into mesenchymal tissues in vitro. The physiological relevance of low oxygen tension as an environmental parameter that uniquely benefits stem cells' expansion and maintenance is described along with recent findings on the regulatory effects of oxygen on embryonic stem cells and adult mesenchymal stem cells. The relevance to tissue engineering is discussed in the context of the need to specifically regulate the oxygen content in the cellular microenvironment in order to optimize in vitro tissue development. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry

Disordered or unstructured regions of proteins, while often very important biologically, can pose significant challenges for resonance assignment and three‐dimensional structure determination of the ordered regions of proteins by NMR methods. In this article, we demonstrate the application of ¹H/²H exchange mass spectrometry (DXMS) for the rapid identification of disordered segments of proteins and design of protein constructs that are more suitable for structural analysis by NMR. In this benchmark study, DXMS is applied to five NMR protein targets chosen from the Northeast Structural Genomics project. These data were then used to design optimized constructs for three partially disordered proteins. Truncated proteins obtained by deletion of disordered N‐ and C‐terminal tails were evaluated using ¹H‐¹⁵N HSQC and ¹H‐¹⁵N heteronuclear NOE NMR experiments to assess their structural integrity. These constructs provide significantly improved NMR spectra, with minimal structural perturbations to the ordered regions of the protein structure. As a representative example, we compare the solution structures of the full length and DXMS‐based truncated construct for a 77‐residue partially disordered DUF896 family protein YnzC from Bacillus subtilis, where deletion of the disordered residues (ca. 40% of the protein) does not affect the native structure. In addition, we demonstrate that throughput of the DXMS process can be increased by analyzing mixtures of up to four proteins without reducing the sequence coverage for each protein. Our results demonstrate that DXMS can serve as a central component of a process for optimizing protein constructs for NMR structure determination. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Alterations in the expression of the Atp7a gene in the early postnatal development of the mosaic mutant mice (Atp7amo-ms) – An animal model for Menkes disease

Copper is a trace element that is essential for the normal growth and development of all living organisms. In mammals, the ATP7A Cu-transporting ATPase is a key protein that is required for the maintenance of copper homeostasis. In both humans and mice, the ATP7A protein is coded by the X-linked ATP7A/Atp7a gene. Disturbances in copper metabolism caused by mutations in the ATP7A/Atp7a gene lead to severe metabolic syndromes Menkes disease in humans and the lethal mottled phenotype in mice. Mosaic is one of numerous mottled mutations and may serve as a model for a severe Menkes disease variant. In Menkes patients, mutations in the ATP7A gene often result in a decreased level of the normal ATP7A protein. The aim of this study was to analyse the expression of the Atp7a gene in mosaic mutants in early postnatal development, a critical period for starting copper supplementation therapy in both Menkes patients and mutant mice. Using real-time quantitative RT-PCR, we analysed the expression of the Atp7a gene in the brain, kidney and liver of newborn (P0.5) and suckling (P14) mice. Our results indicate that in mosaic P0.5 mutants, the Atp7a mRNA level is decreased in all analysed organs in comparison with wild-type animals. In two week-old mutants, a significant decrease was observed only in the kidney. In contrast, their hepatic level of Atp7a tended to be higher than in wild-type mice. We speculate that disturbance in the expression of the Atp7a gene and, consequently, change in the copper concentration of the organs, may contribute to the early fatal outcome of mosaic males.     

Genetic inactivation of NPC1L1 protects against sitosterolemia in mice lacking ABCG5/ABCG8

Mice lacking Niemann-Pick C1-Like 1 (NPC1L1) (NPC1L1(-/-)mice) exhibit a defect in intestinal absorption of cholesterol and phytosterols. However, wild-type (WT) mice do not efficiently absorb and accumulate phytosterols either. Cell-based studies show that NPC1L1 is a much weaker transporter for phytosterols than cholesterol. In this study, we examined the role of NPC1L1 in phytosterol and cholesterol trafficking in mice lacking ATP-binding cassette (ABC) transporters G5 and G8 (G5/G8(-/-) mice). G5/G8(-/-) mice develop sitosterolemia, a genetic disorder characterized by the accumulation of phytosterols in blood and tissues. We found that mice lacking ABCG5/G8 and NPC1L1 [triple knockout (TKO) mice] did not accumulate phytosterols in plasma and the liver. TKO mice, like G5/G8(-/-) mice, still had a defect in hepatobiliary cholesterol secretion, which was consistent with TKO versus NPC1L1(-/-) mice exhibiting a 52% reduction in fecal cholesterol excretion. Because fractional cholesterol absorption was reduced similarly in NPC1L1(-/-) and TKO mice, by subtracting fecal cholesterol excretion in TKO mice from NPC1L1(-/-) mice, we estimated that a 25g NPC1L1(-/-) mouse may secrete about 4 mumol of cholesterol daily via the G5/G8 pathway. In conclusion, NPC1L1 is essential for phytosterols to enter the body in mice.     

Singlet oxygen generation from the decomposition of alpha-linolenic acid hydroperoxide by cytochrome c and lactoperoxidase

Generation of singlet oxygen is first investigated in the decomposition of polyunsaturated lipid peroxide, alpha-linolenic acid hydroperoxide (LAOOH), by heme-proteins such as cytochrome c and lactoperoxidase. Chemiluminescence and electron spin resonance methods are used to confirm the singlet oxygen generation and quantify its yield. Decomposition products of LAOOH are characterized by HPLC-ESI-MS, which suggests that singlet oxygen is produced via the decomposition of a linear tetraoxide intermediate (Russell's mechanism). Free radicals formed in the decomposition are also identified by the electron spin resonance technique, and the results show that peroxyl, alkyl, and epoxyalkyl radicals are involved. The changes of cytochrome c and lactoperoxidase in the reaction are monitored by UV-visible spectroscopy, revealing the action of a monoelectronic and two-electronic oxidation for cytochrome c and lactoperoxidase, respectively. These results suggest that cytochrome c causes a homolytic reaction of LAOOH, generating alkoxyl radical and then peroxyl radical, which in turn releases singlet oxygen following the Russell mechanism, whereas lactoperoxidase leads to a heterolytic reaction of LAOOH, and the resulting ferryl porphyryl radical of lactoperoxidase abstracts the hydrogen atom from LAOOH to give peroxyl radical and then singlet oxygen. This observation would be important for a better understanding of the damage mechanism of cell membrane or lipoprotein by singlet oxygen and various radicals generated in the peroxidation and decomposition of lipids induced by heme-proteins.     

Effects of granulosa cell mitochondria transfer on the early development of bovine embryos in vitro

The objective of this study was to determine the effect of exogenous mitochondria obtained from granulosa cells on the development of bovine embryos in vitro. We classified cumulus oocyte complexes (COCs) as good (G)- and poor (P)-quality oocytes based on cytoplasmic appearance and cumulus characteristics, and assessed mtDNA copy numbers in the G and P oocytes with real-time polymerase chain reaction (PCR). The mitochondria were isolated by fractionation and suspended in mitochondria injection buffer (MIB). Part one of the experiment consisted of the following treatments: (1) G-oocytes + sperm, (2) P-oocytes + mitochondria + MIB + sperm, (3) P-oocytes + MIB + sperm, and (4) P-oocytes + sperm. In part 2, oocytes were parthenogenetically activated. The treatments were: (1) G-oocytes, (2) P-oocytes + mitochondria + MIB, (3) P-oocytes + MIB, and (4) P-oocytes alone. The results indicated a significant difference in mtDNA copy number between G (361 113 +/- 147 114) and P (198 293 +/- 174 178) oocytes (p < 0.01). The rates of morula, blastocyst, and hatched blastocysts derived from P-oocytes + mitochondria were similar to those of G-oocytes, but significantly higher than P-oocytes without exogenous mitochondria in both the ICSI and parthenogenetic activation experiments. We found no difference in blastomere numbers between G-oocytes and P-oocytes + mitochondria in either experiment, but blastomere numbers in these two groups were significantly higher than in P-oocyte groups without exogenous mitochondria. These data suggest that mtDNA content is very important for early embryo development. Furthermore, the transfer of mitochondria from the same breed may improve embryo quality during preimplantation development.     

The connectivity structure,giant strong component and centrality of metabolic networks

MOTIVATION: Structural and functional analysis of genome-based large-scale metabolic networks is important for understanding the design principles and regulation of the metabolism at a system level. The metabolic network is conventionally considered to be highly integrated and very complex. A rational reduction of the metabolic network to its core structure and a deeper understanding of its functional modules are important. RESULTS: In this work, we show that the metabolites in a metabolic network are far from fully connected. A connectivity structure consisting of four major subsets of metabolites and reactions, i.e. a fully connected sub-network, a substrate subset, a product subset and an isolated subset is found to exist in metabolic networks of 65 fully sequenced organisms. The largest fully connected part of a metabolic network, called 'the giant strong component (GSC)', represents the most complicated part and the core of the network and has the feature of scale-free networks. The average path length of the whole network is primarily determined by that of the GSC. For most of the organisms, GSC normally contains less than one-third of the nodes of the network. This connectivity structure is very similar to the 'bow-tie' structure of World Wide Web. Our results indicate that the bow-tie structure may be common for large-scale directed networks. More importantly, the uncovered structure feature makes a structural and functional analysis of large-scale metabolic network more amenable. As shown in this work, comparing the closeness centrality of the nodes in the GSC can identify the most central metabolites of a metabolic network. To quantitatively characterize the overall connection structure of the GSC we introduced the term 'overall closeness centralization index (OCCI)'. OCCI correlates well with the average path length of the GSC and is a useful parameter for a system-level comparison of metabolic networks of different organisms. SUPPLEMENTARY INFORMATION: http://genome.gbf.de/bioinformatics/     

DGGE analysis of 16S rDNA of ammonia-oxidizing bacteria in chemical–biological flocculation and chemical coagulation systems

Microbial community DNA was extracted from activated sludge samples taken from a chemical bioflocculation process and a chemical coagulation process in Shanghai, China. 16S rDNA of ammonia-oxidizing bacteria (AOB)was amplified by nested polymerase chain reaction and fingerprinted by denaturing gradient gel electrophoresis for microbial structure analysis. The Shannon diversity index of each sample was determined. The results indicated that the microbial structure of AOB in chemical bioflocculation process was comparable at two operational conditions. The ammonia-oxidizing bacterial communities were similar in three channels of the chemical bioflocculation process and in three serial tanks in the chemical coagulation process at the same condition. The diversity of microbial structures in the chemical bioflocculation process was higher than in the chemical coagulation process, in which the microbial structure was similar to that in the influent. Although the microbial study provides insights to the nitrification removal, higher microbial diversity of AOB does not necessarily mean higher ammonia oxidization. Molecular analysis should be combined with chemical assays to optimize operational conditions.     

Ecotoxicity of soils contaminated with industrial and domestic wastewater in western shenyang,China

Soil samples were collected from 7 sites in the up-, mid-and down-reach along and nearby the wastewater irrigation channel, western Shenyang of China. The concentrations of selected pollutants (mineral oil, PAHs - polycycle aromatic hydrocarbons and Cd) were determined by UV spectrometer, HPLC and AAS (atomic adsorption spectrometer) spectrometer, respectively. Toxicity effects of soils were evaluated by seedling emergence test with root length of wheat as the end-point and by earthworms test with the mortality rate and inhibition rates of body weight as endpoints. Results showed accumulation of pollutants for most soils with concentration of 200.2 mg.kg⁻¹∼1600 mg.kg⁻¹ for mineral oil, 0.33 mg.kg⁻¹∼1.81 mg.kg⁻¹ for Cd and 900.16 mg.kg⁻¹ ∼ 2737.91 mg.kg⁻¹ for PAHs. The inhibition rates of root elongation were from −20% up to 40 %, and mortality rates of earthworms ranged from 0%∼40% from the exposure period of two weeks to eight weeks by sampling interval of two weeks, the inhibition rates of earthworm growth were from −19.36% to 34.53%, showing effects of stimulation at 2 weeks to an increasing effects of inhibition at 4, 6 and 8 weeks, respectively. Mortality rates correlated with the loss of body weight of earthworms.

This study indicated the potential risk of pollutants of environmental low content in soil by the determination of selected chemicals combined with toxicity indexes.

     

Cytokine-induced stable neuronal differentiation of human bone marrow mesenchymal stem cells in a serum/feeder cell-free condition

The characteristics and multilineage differentiation potential of bone marrow mesenchymal stem cells (BM MSC) remain controversial. This study aimed to characterize human BM MSC isolated by plastic adherent or antibody selection and their neuronal differentiation potential using growth factors or chemical inducing agents. MSC were found to express low levels of neuronal markers: neurofilament-M, beta tubulin III, and neuron specific enolase. Under a serum- and feeder cell-free condition, basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor induced neuronal morphology in MSC. In addition to the above markers, these cells expressed neurotransmitters or associated proteins: gamma-aminobutyric acid, tyrosine hydroxylase and serotonin. These changes were maintained for up to 3 months in all bone marrow specimens (N = 6). In contrast, butylated hydroxyanisole and dimethylsulfoxide were unable to induce sustained neuronal differentiation. Our results show that MSC isolated by two different procedures produced identical lineage differentiation with defined growth factors in a serum- and feeder cell-free condition.