Fabrication of Biomimetic Bone Tissue Using Mesenchymal Stem Cell-Derived Three-Dimensional Constructs Incorporating Endothelial Cells

The development of technologies to promote vascularization of engineered tissue would drive major developments in tissue engineering and regenerative medicine. Recently, we succeeded in fabricating three-dimensional (3D) cell constructs composed of mesenchymal stem cells (MSCs). However, the majority of cells within the constructs underwent necrosis due to a lack of nutrients and oxygen. We hypothesized that incorporation of vascular endothelial cells would improve the cell survival rate and aid in the fabrication of biomimetic bone tissues in vitro. The purpose of this study was to assess the impact of endothelial cells combined with the MSC constructs (MSC/HUVEC constructs) during short- and long-term culture. When human umbilical vein endothelial cells (HUVECs) were incorporated into the cell constructs, cell viability and growth factor production were increased after 7 days. Furthermore, HUVECs were observed to proliferate and self-organize into reticulate porous structures by interacting with the MSCs. After long-term culture, MSC/HUVEC constructs formed abundant mineralized matrices compared with those composed of MSCs alone. Transmission electron microscopy and qualitative analysis revealed that the mineralized matrices comprised porous cancellous bone-like tissues. These results demonstrate that highly biomimetic bone tissue can be fabricated in vitro by 3D MSC constructs incorporated with HUVECs.     

Effects of nonselective cation channels and PI3K on endothelin-1-induced PYK2 tyrosine phosphorylation in C6 glioma cells

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca²⁺-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) in C6 glioma cells. In the present study, we investigated the effects of NSCCs on the ET-1-induced proline-rich tyrosine kinase 2 (PYK2) phosphorylation in C6 glioma cells. In addition, we examined the effects of phosphoinositide 3-kinase (PI3K) on the ET-1-induced NSCCs activation and PYK2 phosphorylation. The PI3K inhibitors wortmannin and LY-294002 inhibited ET-1-induced Ca²⁺ influx through NSCC-2 but not NSCC-1. On the other hand, addition of these inhibitors after stimulation with ET-1 failed to suppress Ca²⁺ influx through NSCC-2. PYK2 phosphorylation was abolished by blocking Ca²⁺ influx through NSCCs. The PI3K inhibitors blocked the NSCC-2-dependent part of ET-1-induced PYK2 phosphorylation. These results indicate that 1) NSCC-2 is stimulated by ET-1 via a PI3K-dependent cascade, whereas NSCC-1 is stimulated via a PI3K-independent cascade; 2) PI3K seems to be required for the activation of the Ca²⁺ entry, but not for its maintenance; 3) Ca²⁺ influx through NSCC-1 and NSCC-2 plays an essential role in ET-1-induced PYK2 phosphorylation; and 4) PI3K is involved in the ET-1-induced PYK2 phosphorylation that depends on the Ca²⁺ influx through NSCC-2. endothelin; phosphoinositide 3-kinase; nonselective cation channel; proline-rich tyrosine kinase 2; glioma cell     

Cell-free synthesis of mitochondrial ATPase inhibitor precursor and its transport into yeast mitochondria

ATPase inhibitor protein, which blocks mitochondrial ATPase activity by forming an enzyme-inhibitor complex, was found to be synthesized as a larger precursor in a cell-free translation system directed by yeast mRNA. Other protein factors, which stabilize latent ATPase by binding to the enzyme-inhibitor complex, were also found to be formed as larger precursors. The precursor of ATPase inhibitor protein was transported into isolated yeast mitochondria and was cleaved to the mature peptide in the mitochondria. Impaired mitochondria lacking phosphorylation activity could not convert the precursor to the mature form. Neither antimycin A nor oligomycin alone exhibited a marked effect on the transport-processing of the precursor by intact mitochondria. However, when antimycin A was added with oligomycin, the transport-processing was markedly inhibited. The processing was also strongly inhibited by an uncoupler, carbonylcyanide p-trifluoro-methoxyphenyl hydrazone. The inhibition by the uncoupler was not relieved by ATP added externally. It is concluded that the transport-processing of precursor proteins requires intact mitochondria with a potential difference across the inner membrane.     

Northern hybridization analysis of VH gene expression in murine monoclonal antibodies directed to cancer-associated ganglioside antigens having various sialic acid linkages

The expression of the VH genes in 46 murine hybridoma cells that secrete mAb directed to the cancer-associated carbohydrate Ag, especially acidic glycolipids such as gangliosides and sulfated glycoplipids, was analyzed by Northern hybridization of poly(A)+ RNA of hybridoma with cDNA probes for nine VH gene families. Different hybridomas tended to express VH genes of the same family when the cognate Ag had the same or similar carbohydrate structures; i.e., the VH genes of the J558 family (group 1) were preferentially expressed in the mAb directed to various gangliosides that have NeuAc alpha (or NeuGc alpha) 2-3 and/or 2-8 linkage (71%), the most common linkage of sialic acid residues in the gangliosides of higher animals, and the hybridomas directed to sulfated glycolipids also expressed mainly the VH genes of the J558 family (80%). In contrast, the five mAb directed to various gangliosides with NeuAc alpha 2-6 linkage were exclusively encoded by the VH genes of Q52 family (group 2, 100%), and three antibodies directed to gangliosides with a NeuAc alpha 2-9 linkage all expressed genes of J606 family (group 6, 100%). The VH family usage was largely correlated with the linkage of sialic acid residues in the cognate carbohydrate Ag, but was not correlated at all with the difference in the fine specificities toward the core neutral carbohydrate chain, to which the sialic acid residues were attached. These findings suggest that the VH gene family in these anticarbohydrate antibodies is selected, depending primarily on the linkage of the sialic acid residues in carbohydrate Ag; these residues form the immunodominant sugar residue in the respective antigenic determinant.     

Primary structure of rat insulin-like growth factor-I and its biological activities

Rat insulin-like growth factor-I (IGF-I), a serum polypeptide with growth promoting activity, was isolated from rat serum by a combination of acid/ethanol extraction, affinity chromatography, and a series of reversed phase high performance liquid chromatography, cation exchange, and reversed phase. All peptide fragments produced by chymotrypsin digestion of reduced and carboxymethylated rat IGF-I were amino acid sequenced and compared with the sequence of human IGF-I. Three out of 70 of the rat amino acid residues differed from those of human IGF-I as follows: Asp20----Pro, Ser35----Ile and Ala67----Thr. Purified rat IGF-I cross-reacted with polyclonal anti-human IGF-I antibody 75% as compared to human IGF-I, but it cross-reacted only 3% with monoclonal anti-human IGF-I antibody. Thus, it is possible to monitor the metabolic fate of human IGF-I, when injected into rats, without interference by endogenous rat IGF-I. Rat IGF-I showed 65% activity in the radioreceptor, 28.6% activity in the lipogenesis and 22.5% activity in the free fatty acid release inhibition assays as compared to human IGF-I on a protein quantity basis.     

Construction of hybrid photosynthetic units using peripheral and core antennae from two different species of photosynthetic bacteria: detection of the energy transfer from bacteriochlorophyll a in LH2 to bacteriochlorophyll b in LH1

Typical purple bacterial photosynthetic units consist of supra-molecular arrays of peripheral (LH2) and core (LH1-RC) antenna complexes. Recent atomic force microscopy pictures of photosynthetic units in intact membranes have revealed that the architecture of these units is variable (Scheuring et al. (2005) Biochim Bhiophys Acta 1712:109–127). In this study, we describe methods for the construction of heterologous photosynthetic units in lipid-bilayers from mixtures of purified LH2 (from Rhodopseudomonas acidophila) and LH1-RC (from Rhodopseudomonas viridis) core complexes. The architecture of these reconstituted photosynthetic units can be varied by controlling ratio of added LH2 to core complexes. The arrangement of the complexes was visualized by electron-microscopy in combination with Fourier analysis. The regular trigonal array of the core complexes seen in the native photosynthetic membrane could be regenerated in the reconstituted membranes by temperature cycling. In the presence of added LH2 complexes, this trigonal symmetry was replaced with orthorhombic symmetry. The small lattice lengths for the latter suggest that the constituent unit of the orthorhombic lattice is the LH2. Fluorescence and fluorescence-excitation spectroscopy was applied to the set of the reconstituted membranes prepared with various proportions of LH2 to core complexes. Remarkably, even though the LH2 complexes contain bacteriochlorophyll a, and the core complexes contain bacteriochlorophyll b, it was possible to demonstrate energy transfer from LH2 to the core complexes. These experiments provide a first step along the path toward investigating how changing the architecture of purple bacterial photosynthetic units affects the overall efficiency of light-harvesting.