Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression

To elucidate the role of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, in tumour angiogenesis and malignant progression, an expression vector harboring human VEGF cDNA was stably transfected into three human cancer cell lines with poor VEGF productivity. Though their in vitro growth rate and intrinsic productivity of another angiogenic factor, basic fibroblast growth factor (bFGF), were not changed by transfection, those clones with higher VEGF production were endowed with tumorigenic and angiogenic potentials as follows: firstly, nontumorigenic, lung carcinoma QG90 cells having lower bFGF productivity acquired tumorigenicity as well as significant in vivo angiogenesis-inducing ability, secondly, tumorigenic colorectal carcinoma RPMI4788 cells having higher potency for bFGF production could form more vascularized solid tumour with faster growth rate and thirdly, oestrogen-dependent breast carcinoma MCF-7 cells, which did not produce detectable bFGF, acquired tumorigenicity even in the absence of oestrogen and the solid tumour growth rate was remarkably enhanced, accompanied with increased vascularization, in the presence of oestrogen. These results suggest that tumour progression closely depends on angiogenesis, and VEGF significantly contributes to malignant progression of a variety of tumour cells through its potent angiogenic activity, independent on the bFGF productivity of tumour cells.     

Cell sheet engineering: recreating tissues without biodegradable scaffolds

While tissue engineering has long been thought to possess enormous potential, conventional applications using biodegradable scaffolds have limited the field's progress, demonstrating a need for new methods. We have previously developed cell sheet engineering using temperature-responsive culture dishes in order to avoid traditional tissue engineering approaches, and their related shortcomings. Using temperature-responsive dishes, cultured cells can be harvested as intact sheets by simple temperature changes, thereby avoiding the use of proteolytic enzymes. Cell sheet engineering therefore allows for tissue regeneration by either direct transplantation of cell sheets to host tissues or the creation of three-dimensional structures via the layering of individual cell sheets. By avoiding the use of any additional materials such as carrier substrates or scaffolds, the complications associated with traditional tissue engineering approaches such as host inflammatory responses to implanted polymer materials, can be avoided. Cell sheet engineering thus presents several significant advantages and can overcome many of the problems that have previously restricted tissue engineering with biodegradable scaffolds.     

Molecular mechanism for connective tissue destruction by dipeptidyl aminopeptidase IV produced by the periodontal pathogen Porphyromonas gingivalis

Porphyromonas gingivalis is a pathogen associated with adult periodontitis. It produces dipeptidyl aminopeptidase IV (DPPIV), which may act as a virulence factor by contributing to the degradation of connective tissue. We investigated the molecular mechanism by which DPPIV contributes to the destruction of connective tissue. DPPIV itself did not show gelatinase or collagenase activity toward human type I collagen, but it promoted the activity of the host-derived matrix metalloproteinase 2 (MMP-2) (gelatinase) and MMP-1 (collagenase). DPPIV bound to fibronectin and mediated the adhesion of P. gingivalis to fibronectin. Mutant DPPIV with catalytic Ser mutagenized to Ala (DPPSA) did not accelerate the degradation of collagen and gelatin by MMPs but retained fibronectin-binding activity. The adhesion of human gingival fibroblasts and NIH 3T3 cells to fibronectin was inhibited by DPPIV. Strain 4351ADPPSA exhibited an intermediate level of virulence in mice, between that of the strain expressing wild-type DPPIV (4351ADPP) and that of the strain harboring only the plasmid vector (4351AVEC). It is suggested that both activity promoting the degradation of collagen and gelatin and binding to fibronectin are required for full virulence. These results reveal novel biological functions of DPPIV and suggest a pathological role in the progression of periodontitis.     

Thioredoxin suppresses 1-methyl-4-phenylpyridinium-induced neurotoxicity in rat PC12 cells

Thioredoxin (TRX) is a redox-active protein which plays a cytoprotective role against oxidative stress. Geranylgeranylacetone (GGA), used widely as an anti-ulcer drug, has been reported to induce TRX as well as heat shock protein 70 (HSP70) in hepatocytes and other cells. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), causes dopaminergic denervation and Parkinsonism in humans. The 1-methyl-4-phenylpyridinium ion (MPP(+)), an active metabolite of MPTP, induces cell death in a rat pheochromocytoma cell line (PC12 cells). We found that MPP(+) suppresses TRX expression in PC12 cells. Overexpression or administration of TRX attenuates MPP(+)-induced neurotoxicity on PC12 cells. Moreover, GGA induces expression of TRX and HSP70 and attenuates MPP(+)-induced toxicity in PC12 cells. These results indicate that TRX and GGA have a possible potential as new therapeutic agents for Parkinson disease.     

Cell-surface bound pertussis toxin induces polyclonal T cell responses with high levels of interferon-gamma in the absence of interleukin-12

Pertussis toxin (PTx), an exotoxin produced by Bordetella pertussis, has long been used as a mucosal adjuvant. We examined the T cell stimulatory properties of PTx in order to dissect its mechanisms of adjuvanticity. PTx or the B-oligomer of PTx (PTxB) failed to activate purified murine CD4+ or CD8+ T cells, as measured by a lack of proliferation or expression of early T cell activation markers. However, these T cells proliferated extensively in response to the toxin in the presence of syngeneic DC, and proliferation was accompanied by a high level of IFN-gamma production in the absence of IL-12. Interestingly, such responses were independent of signals mediated by MHC-TCR interaction. Both PTx and PTxB were found to bind stably to the surface of DC, and increased the adherence of DC to surrounding cells. These data suggest that polyclonal T cell responses mediated by the toxin are likely to be caused by the toxin bound on the surface of APC, either cross-linking cell surface molecules on T cells, or directly stimulating T cells together with the co-stimulatory molecules expressed on APC. B. pertussis may use this toxin as a mechanism to evade a specific immune response.     

Clinical heterogeneity of neonatal intrahepatic cholestasis caused by citrin deficiency: case reports from 16 patients

A deficiency of citrin, which is encoded by the SLC25A13 gene, causes both adult-onset type II citrullinemia (CTLN2) and neonatal intrahepatic cholestasis (NICCD). We analyzed 16 patients with NICCD to clarify the clinical features of the disease. Severe intrahepatic cholestasis with fatty liver was the most common symptom, but the accompanying clinical features were variable, namely; suspected cases of neonatal hepatitis or biliary atresia, positive results from newborn screening, tyrosinemia, failure to thrive, hemolytic anemia, bleeding tendencies and ketotic hypoglycemia. Laboratory data showed elevated serum bile acid levels, hypoproteinemia, low levels of vitamin K-dependent coagulation factors, and hypergalactosemia. Hypercitrullinemia was detected in 11 out of 15 patients examined. Most of the patients were given a lactose-free and/or medium chain triglycerides-enriched formula and lipid-soluble vitamins. The prognosis of the 16 patients is going fairy well at present, but we should observe these patients carefully to see if they manifest any symptom of CTLN2 in the future.     

Decorin expression during development of bovine skeletal muscle and its role in morphogenesis of the intramuscular connective tissue

Decorin is a small leucine-rich proteoglycan suspected of playing an important role in tissue morphogenesis. However, its role in the development of skeletal muscle is less clear. In the present study, the expression and spatial distribution of decorin in developing skeletal muscle of bovine fetuses were investigated, in order to provide a background for understanding the function of decorin in morphogenesis of the intramuscular connective tissue that supports muscle fibres. Western blot analysis showed that decorin already existed in skeletal muscle by 2.5 months of fetal development, and that decorin had a longer glycosaminoglycan chain in the early fetal stages than in later development, but its core protein was of the same size. Decorin mRNA was expressed at 1 month of fetal development, although its level was relatively low. Indirect immunofluorescence microscopy demonstrated that decorin was located in the perimysium which consisted of collagen fibres, but not in the endomysium which was composed of collagen fibril networks in fetal skeletal muscle. The relatively integrated structure of the perimysium had already formed by 2.5 months of fetal development, when muscle fibres were not tightly assembled and the surrounding endomysium was not well organized. These results suggest that decorin contributes to the formation and stabilization of collagen fibres in the perimysium that support muscle fibres assembled with myogenesis.     

Parkin localizes to the Lewy bodies of Parkinson disease and dementia with Lewy bodies

Mutations in alpha-synuclein (alpha S) and parkin cause heritable forms of Parkinson disease (PD). We hypothesized that neuronal parkin, a known E3 ubiquitin ligase, facilitates the formation of Lewy bodies (LBs), a pathological hallmark of PD. Here, we report that affinity-purified parkin antibodies labeled classical LBs in substantia nigra sections from four related human disorders: sporadic PD, inherited alphaS-linked PD, dementia with LBs (DLB), and LB-positive, parkin-linked PD. Anti-parkin antibodies also detected LBs in entorhinal and cingulate cortices from DLB brain and alphaS inclusions in sympathetic gangliocytes from sporadic PD. Double labeling with confocal microscopy of DLB midbrain sections revealed that approximately 90% of anti-alpha S-reactive LBs were also detected by a parkin antibody to amino acids 342 to 353. Accordingly, parkin proteins, including the 53-kd mature isoform, were present in affinity-isolated LBs from DLB cortex. Fluorescence resonance energy transfer and immunoelectron microscopy showed that alphaS and parkin co-localized within brainstem and cortical LBs. Biochemically, parkin appeared most enriched in cytosolic and postsynaptic fractions of adult rat brain, but also in purified, alpha S-rich presynaptic elements that additionally contained parkin's E2-binding partner, UbcH7. We conclude that parkin and UbcH7 are present with alphaS in subcellular compartments of normal brain and that parkin frequently co-localizes with alpha S aggregates in the characteristic LB inclusions of PD and DLB. These results suggest that functional parkin proteins may be required during LB formation.