Isolation and characterization of bacteriophage T3/T7 hybrids and their use in studies on molecular basis of DNA-packaging specificity

In vitro DNA-packaging systems of bacteriophages T3 and T7 packaged homologous DNA more efficiently than heterologous DNA. Packaging of phage DNA proceeds by way of concatemeric intermediates (H. Fujisawa, J. Miyazaki, and T. Minagawa (1978), Virology 87, 394-400). The conversion of mature homologous and heterologous DNAs to concatemers was efficient in both the T3- and T7-packaging systems. In vitro complementation experiments indicate that the gene 19 product (gp19) specifies which DNA enters the capsid. To identify DNA regions recognized by the packaging systems, T3/T7 hybrids were constructed and physical maps of the hybrid DNAs were determined by restriction enzyme analysis. By comparing restriction maps and in vitro packaging of hybrid DNAs, it is concluded that the sequence responsible for specificity of DNA packaging is confined within 5% of the ends of the T3 and T7 genomes.     

Met72Thr polymorphism of pigment epithelium-derived factor gene and susceptibility to age-related macular degeneration

Age-related macular degeneration (ARMD) is the most common cause of acquired blindness among the people of occupational age. Although the pathogenesis of ARMD is not fully understood, several studies suggest a possible contribution of a genetic factor in the development and progression of ARMD. Pigment epithelium-derived factor (PEDF), a glycoprotein that belongs to the superfamily of serine protease inhibitors, was first purified from the conditioned media of human retinal pigment epithelial cells as a factor with potent neuronal differentiating activity in human retinoblastoma cells. Recently, PEDF has been shown to be a highly effective inhibitor of angiogenesis in cell culture and animal models. In addition, PEDF has been found in the vitreous, and its levels were decreased in angiogenic eye diseases, thus suggesting that a loss of PEDF in the eye is functionally important in the pathogenesis of ARMD. A functional amino acid change, a methionine to threonine polymorphism (Met72Thr polymorphism) at codon 72 in exon 3 (T/C polymorphism) of the PEDF gene, that results in the formation of BsstSI restriction site, has recently been identified. Since it is well known that a single nucleotide polymorphism and resultant amino acid change often alters the activity or expression level of the target protein, we would like to propose here a novel hypothesis that the Met72Thr polymorphism (T/C polymorphism) of PEDF gene may be a genetic marker for ARMD. Are genotype and allele frequencies of the Met72Thr polymorphism (T/C polymorphism) different between the patients with or without ARMD? Is this polymorphism associated with disease severity and progression? If the answer is yes, does this Met72Thr polymorphism regulate the vitreous levels of PEDF? These clinical studies could provide us with information whether this genetic variant of the PEDF gene could present an attractive candidate susceptibility gene for ARMD.     

Vascular endothelial growth factor acts as a pericyte mitogen under hypoxic conditions

Angiogenesis is the process by which new vascular networks are formed from preexisting capillaries. The small vessels are composed of two types of cells, namely endothelial cells (EC) and pericytes, with the former being encircled by the latter. We previously showed that hypoxia, the principal cause of angiogenesis, can induce the proliferation of pericytes as well as EC. In this report we present evidence that the hypoxic induction of pericyte growth can be ascribed at least in part to vascular endothelial growth factor (VEGF) produced by this very cell type. First, the finding that hypoxia can stimulate the proliferation of pericytes was confirmed by cultivating bovine retinal pericytes in a controlled-atmosphere culture chamber containing various concentrations of oxygen and then assaying pericyte synthesis of DNA. Second, Northern blot analysis revealed that pericyte levels of mRNA encoding VEGF increased as the atmospheric oxygen tension was decreased; this was accompanied by an increase in de novo synthesis of VEGF proteins. Third, pericytes were able to respond to exogenously added VEGF, resulting in a dose-dependent increase in viable cell numbers. Fourth, polyclonal antibodies against VEGF efficiently blocked the hypoxic induction of pericyte growth. Fifth, pericytes expressed the gene for fms-like tyrosine kinase 1 (flt1) as the predominant form of VEGF receptor, and tyrosine phosphorylation of this receptor protein was enhanced when pericytes were exposed to hypoxia, as it was when cells were exposed to VEGF. Sixth, the antisense DNA complement of flt1 mRNA abolished the hypoxia-induced stimulation of pericyte growth. Finally, exogenous VEGF stimulated the migration of pericytes in a dose-dependent manner. The results thus suggest that VEGF, which has been thought to be a specific mitogen for EC, also acts on neighboring pericytes, probably in both autocrine and paracrine manners, and that the hypoxia-induced overproduction of VEGF could promote not only EC sprouting but also the recruitment of pericytes, thereby contributing to the maturation of newly formed microvessels.     

Rho kinases regulate endothelial invasion and migration during valvuloseptal endocardial cushion tissue formation.

Rho-associated kinase (ROCK) is a downstream effector of small Rho-GTPases, and phosphorylates several substrates to regulate cell functions, including actin cytoskeletal reorganization and cellular motility. Endothelial-mesenchymal transformation (EMT) is a critical event in the formation of valves and septa during cardiogenesis. It has been reported that ROCK plays an important role in the regulation of endocardial cell differentiation and migration during mouse cardiogenesis (Zhao and Rivkees [2004] Dev. Biol. 275:183-191). Immunohistochemistry showed that, during chick cardiogenesis, ROCK1 and -2 were expressed in the transforming and migrating endothelial/mesenchymal cells in the outflow tract (OT) and atrioventricular (AV) canal regions from which valvuloseptal endocardial cushion tissue would later develop. Treatment with Y27632, a specific ROCK inhibitor, of cultured AV explants or AV endothelial monolayers of stage 14-minus heart (preactivated stage for EMT) on three-dimensional collagen gel perturbed the seeding of mesenchymal cells into the gel lattice. In these experiments, Y27632 did not suppress the expression of an early transformation marker, smooth muscle alpha-actin. Moreover, Y27632 inhibited the mesenchymal invasion in stage 14-18 AV explants, in which endothelial cells had committed to undergo EMT. ML-9, a myosin light chain kinase inhibitor, also inhibited the mesenchymal invasion in cultured AV explants. These results suggest that ROCKs have a critical role in the mesenchymal cell invasion/migration that occurs at the late onset of EMT.     

Genetic ablation of FLRT3 reveals a novel morphogenetic function for the anterior visceral endoderm in suppressing mesoderm differentiation

During early mouse development, the anterior visceral endoderm (AVE) secretes inhibitor and activator signals that are essential for establishing the anterior–posterior (AP) axis of the embryo and for restricting mesoderm formation to the posterior epiblast in the primitive streak (PS) region. Here we show that AVE cells have an additional morphogenetic function. These cells express the transmembrane protein FLRT3. Genetic ablation of FLRT3 did not affect the signaling functions of the AVE according to the normal expression pattern of Nodal and Wnt and the establishment of a proper AP patterning in the epiblast. However, FLRT3^−/− embryos showed a highly disorganized basement membrane (BM) in the AVE region. Subsequently, adjacent anterior epiblast cells displayed an epithelial-to-mesenchymal transition (EMT)-like process characterized by the loss of cell polarity, cell ingression, and the up-regulation of the EMT and the mesodermal marker genes Eomes, Brachyury/T, and FGF8. These results suggest that the AVE acts as a morphogenetic boundary to prevent EMT and mesoderm induction in the anterior epiblast by maintaining the integrity of the BM. We propose that this novel function cooperates with the signaling activities of the AVE to restrict EMT and mesoderm induction to the posterior epiblast.     

Suppression of UV-induced mutations by wild-type p53 protein in human osteosarcoma cells

We have examined whether the tumour suppressor p53 protein suppressedUV-induced mutations in the hypoxathine-guanine phosphoribosyltransferase (HPRT) gene and in the supF gene of the shuttlevector plasmid pMY189. We used human osteosarcoma Saos-LP12cells, in which wild type (wt) p53 protein was induced by treatmentwith isopopyl-(ß-D-thiogalactopyranoside. The inductionof wt p53 protein suppressed UV-induced mutations but not spontaneousmutations in the HPRT gene. The frequency of UV-induced mutationsinduced by UV-irradiation of the plasmid was also significantlylower in cells with induced wt p53 protein than in the uninducedcells. In addition, we found that frequency of G : C to A :T transition mutations which occurred at the 3' base pair ofdipyrimidine sites were significantly lower in the cells withinduced wt p53 protein than in the uninduced cells. These findingssuggestthat wt p53 protein may play roles in modulating DNArepair pathway, resulting in the suppression of UV-induced mutations. ¹To whom correspondence should be addressed     

Experimental study on osteoconductive properties of a chitosan-bonded hydroxyapatite self-hardening paste.

A developmental bone substitute, composed of a chitosan-bonded hydroxyapatite paste and having various possible applications in medical and dental treatments, was evaluated with regard to its osteoconductive properties. Radiographic examination revealed that a bone-like irregular radiopacity appeared in the region of the embedded paste. This was judged histopathologically as the formation of bone tissue with chondral tissue. These data suggest that the paste has osteoconductive properties, and may, therefore, prove clinically useful as a bioactive bone substitute.     

Acarbose is a promising therapeutic strategy for the treatment of patients with nonalcoholic steatohepatitis (NASH)

The metabolic syndrome is strongly associated with insulin resistance and has been recognized as a cluster of risk factors for cardiovascular diseases such as visceral obesity, hypertension, and diabetes. There is a growing body of evidence to show that nonalcoholic steatohepatitis (NASH) is the hepatic manifestation of insulin resistant patients with the metabolic syndrome. Indeed, insulin resistance increases adipocyte lipolysis and subsequently elevates circulating free fatty acids, thus stimulating the accumulation of fatty acids in the liver (hepatic steatosis). Fatty acids elicit reactive oxygen species generation, thereby promoting disease progression to NASH by both lipid peroxidation and inflammatory cytokine production. Postprandial hyperglycemia, one of the characteristic features of insulin resistance, also induces oxidative stress generation, being involved in dysfunction of pancreatic beta cells and vascular wall cells in the metabolic syndrome. Recently, STOP-NIDDM trial revealed that acarbose (Glucobay), an alpha-glucosidase inhibitor, improved postprandial hyperglycemia and subsequently reduced the risk of development of type 2 diabetes and newly diagnosed hypertension in patients with impaired glucose tolerance. In this study, acarbose treatment was also found to reduce body mass index and waist circumference in these patients. Furthermore, a meta-analysis of seven long-term studies has also shown that intervention with acarbose improved triglyceride levels, body weight and systolic blood pressure and subsequently prevented myocardial infarction in type 2 diabetic patients. Since acarbose improves postprandial hyperglycemia by delaying the release of glucose from complex carbohydrates in the absence of an increase in insulin secretion, the beneficial aspects of acarbose could be ascribed to improvement of insulin sensitivity in these patients. Given the pathological link between NASH and insulin resistance, we would like to hypothesize here that acarbose may become a promising therapeutic strategy for the treatment of patients with NASH. Does acarbose treatment improve steatohepatitis histologically? Is the extent of histological improvement by acarbose parallel to that of insulin sensitivity in these patients? Large clinical trials will provide us with more definite information whether acarbose treatment can improve insulin sensitivity and resultantly reduce the risk of progression of liver diseases in patients with NASH.     

Adjuvant heparinization before manipulation of artery reduces early failure in primary arteriovenous fistula for end-stage renal disease patients

Clinical and Experimental Nephrology - Arteriovenous fistula (AVF) is the most preferred vascular access for hemodialysis patients, and early failure of AVF is one of the most avoidable...