Damaged articular cartilage, caused by traumatic injury or degenerative diseases, has a limited regenerative capacity and frequently leads to the onset of osteoarthritis. As a promising strategy for the successful regeneration of long-lasting hyaline cartilage, tissue engineering has received increasing recognition. In this study, we attempted to design a novel type of porous chitosan scaffold, containing transforming growth factor-β1 (TGF-β1), to enhance chondrogenesis. First, to achieve a sustained release of TGF-β1, chitosan microspheres loaded with TGF-β1 (MS-TGFs) were prepared by the emulsion method, in the presence of tripolyphosphate; with an identical manner, microspheres loaded with BSA, a model protein, were also prepared. Both microspheres containing TGF-β1and BSA had spherical shapes with a size ranging from 0.2 to 1.5 μm. From the release experiments, it was found that both proteins were slowly released from the microspheres over 5 days in a PBS solution (pH 7.4), in which the release rate of TGF-β1 was much lower than that of BSA. Second, MS-TGFs were seeded onto the porous chitosan scaffold, prepared by the freeze-drying method, to observe the effect on the proliferation and differentiation of chondrocytes. It was obviously demonstrated from in vitro tests that, compared to the scaffold without MS-TGF, the scaffold containing MS-TGF significantly augments the cell proliferation and production of extracellular matrix, indicating the role of TGF-β1 released from the microspheres. These results suggest that the chitosan scaffold containing MS-TGF possesses a promising potential as an implant to treat cartilage defects.

betaig-h3 is an extracellular matrix protein and its expression is highly induced by TGF-beta and it has also been suggested to play important roles in skin wound healing. In this paper, we demonstrate that betaig-h3 is present in the papillary layer of dermis and synthesized in the basal keratinocytes in vivo and its expression is induced by TGF-beta in normal human keratinocytes (NHEK) and HaCaT cells. betaig-h3 mediates not only adhesion and spreading of keratinocytes but also supports migration and proliferation. These activities are mediated through interacting with alpha3beta1 integrin. Previously identified two alpha3beta1 integrin-interacting motifs of betaig-h3, EPDIM, and NKDIL, are responsible for these activities. The results suggest that betaig-h3 may regulate keratinocyte functions in normal skin and potentially during wound-healing process.

A three-dimensional, porous collagen/chitosan complex sponge was prepared to closely simulate basic extracellular matrix (ECM) constitutes, collagen and glycosaminoglycan. The complex sponge was prepared by a lyophilization method and had the regular network with highly porous structure, suitable for cell adhesion and growth. The pores were well interconnected, and their distribution was fairly homogeneous. The complex sponge was crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to increase its biological stability and enhance its mechanical properties. The crosslinking medium had a great effect on the inner structure of the sponge. The homogeneous, porous structure of the sponge was remarkably collapsed in an aqueous crosslinking medium. However, the morphology of the sponge remained almost intact in a water/ethanol mixture crosslinking milieu. Mechanical properties of the collagen/chitosan sponge were significantly enhanced by EDC-mediated crosslinking. The potential of the sponge as a scaffold for tissue engineering was investigated using a Chinese hamster ovary cell (CHO-K1) line.

Lysophosphatidic acid (LPA) is a lipid metabolite that induces the activation of mitogen-activated protein kinase (MAPK) through binding to the G protein-coupled receptor in a number of cell lines and cultures. Recent studies have revealed that LPA is able to rapidly induce the phosphorylation of MAPK through an epidermal growth factor (EGF) receptor-dependent pathway. We investigated the role of the EGF receptor in the signaling pathway initiated by LPA stimulation in nerve growth factor (NGF)-responsive PC12 cells well known to transiently retract their own neurites upon LPA stimulation. LPA-stimulated MAPK signaling was suppressed by the selective EGF receptor inhibitor and in the dominant negative mutant EGF receptor cell line. As in the EGF signaling pathway, the complex of EGF receptor with adapter proteins Shc and Sos was formed in response to LPA stimulation, suggesting there is an intracellular mechanism for transactivation. A neurite retraction assay was also performed to examine the role of the EGF receptor in PC12 cell differentiation, which related to the involvement of LPA-induced neurite retraction. These results suggest that the receptor tyrosine kinase can be activated in a ligand-independent manner through intracellular crosstalk between the signaling pathways.

Purpose. To develop a non-viral gene delivery system in the form ofan oil-in-water (o/w) lipid emulsionMethod. Cationic lipid emulsions were formulated with soybean oil,1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP) as acationic emulsifier and other co-emulsifiers. The physicalcharacteristics of the lipid emulsion and the emulsion/DNA complex weredetermined. The in vitro transfection efficiency of the emulsion/DNAcomplex was determined in the presence of up to 90% serum.

Although renal oncocytoma is currently considered to be a rare, benign tumor occurring in the renal cortex, there have been increasing reports that tumors initially diagnosed as renal oncocytoma tend to have malignant potential and metastatic potential. The mechanisms by which some tumors initially diagnosed as renal oncocytoma become malignant tumors remain to be determined. However, it is extremely important to differentially diagnose renal oncocytoma from malignant renal cell carcinoma because the treatment modality of this benign tumor is completely different from that of the malignant tumor. In order to identify any specific molecular marker(s) of renal oncocytoma, we have performed ''Differential Display of mRNA'' technique using mRNA extracted from normal kidney tissue and renal oncocytoma obtained from 37 year-old patient. The clinical and pathological studies of this patient showed the typical characteristics of oncocytoma, including abundant mitochondria in cytoplasm. DNA now cytometry showed normal diploidy in this oncocytoma. Twenty different polymerase chain reaction (PCR) primer sets were used to compare gene expression patterns of normal and tumor tissues. Under our experimental conditions,. the differential display of mRNA technique produced approximately 55 up-regulated and 35 down-regulated bands each representing partial cDNA fragments. Among these differentially expressed cDNA fragments, we examined the cDNA sequences of 20 prominent polymerase chain reaction bands showing downregulated expression patterns in RNA from the oncocytoma tissues. The DNA sequence comparison analysis using Genbank data base revealed that most clones had unknown genes. However, one clone showed 98% identity to the yi43f06.s1 clone, which is expressed in the female placenta at birth. Two other clones showed 70% identity to yh8810.ri clone and human IFNAR gene for interferone alpha receptor. Northern blot analysis using these PCR products as a probe confirmed that the differentially detected PCR bands were not artifacts, but indeed reflected the differential expression of each mRNA during tumorigenesis. We are in the process of further characterizing these genes.

We previously isolated an avian erythroblastosis virus, AEV-GEE35, in which the complete extracellular and transmembrane domains of the v-erbB oncoprotein were replaced with sequences from the gag and env proteins. The GEE35 virus was capable of transforming both fibroblasts and erythroblasts as efficiently as wild-type v-erbB. Analysis of the v-erbB proteins encoded by GEE35 revealed two proteins of similar molecular weights of approximately 130,000 Da. One of these proteins was an N-linked glycosylated membrane protein, whereas the other was a cytoplasmic protein. Biochemical characterization of these two proteins revealed that the transmembrane protein has the v-erbB domain outside the cell, such that it no longer had access to its tyrosine kinase substrates. This implies that it is the cytoplasmically located v-erbB-encoded protein that is responsible for the efficient transforming ability of this virus.