Actin-independent behavior and membrane deformation exhibited by the four-transmembrane protein M6a

M6a is a four-transmembrane protein that is abundantly expressed in the nervous system. Previous studies have shown that over-expression of this protein induces various cellular protrusions, such as neurites, filopodia, and dendritic spines. In this detailed characterization of M6a-induced structures, we found their varied and peculiar characteristics. Notably, the M6a-induced protrusions were mostly devoid of actin filaments or microtubules and exhibited free random vibrating motion. Moreover, when an antibody bound to M6a, the membrane-wrapped protrusions were suddenly disrupted, leading to perturbation of the surrounding membrane dynamics involving phosphoinositide signaling. During single-molecule analysis, M6a exhibited cytoskeleton-independent movement and became selectively entrapped along the cell perimeter in an actin-independent manner. These observations highlight the unusual characteristics of M6a, which may have a significant yet unappreciated role in biological systems.     

HVJ-envelope vector for gene transfer into central nervous system

To overcome some problems of virus vectors, we developed a novel non-viral vector system, the HVJ-envelope vector (HVJ-E). In this study, we investigated the feasibility of gene transfer into the CNS using the HVJ-E both in vitro and in vivo. Using the Venus reporter gene, fluorescence could be detected in cultured rat cerebral cortex neurons and glial cells. In vivo, the reporter gene (Venus) was successfully transfected into the rat brain by direct injection into the thalamus, intraventricular injection, or intrathecal injection, without inducing immunological change. When the vector was injected after transient occlusion of the middle cerebral artery, fluorescence due to EGFP gene or luciferase activity could be detected only in the injured hemisphere. Finally, luciferase activity was markedly enhanced by the addition of 50 U/ml heparin (P<0.01). Development of efficient HVJ-E for gene transfer into the CNS will be useful for research and clinical gene therapy.     

Vasohibin prevents arterial neointimal formation through angiogenesis inhibition

Vasohibin is a VEGF-inducible angiogenesis inhibitor in vascular endothelium. Here we examined the presence of vasohibin in human arterial wall, and found it in endothelium of adventitial microvessels in atherosclerotic lesion. Adventitial angiogenesis is involved in the progression of neointimal formation. Even in the presence of endogenous angiogenesis inhibitors, pathological angiogenesis persists. However, the supplementation of exogenous angiogenesis inhibitors can prevent pathological angiogenesis. We evaluated the potential role of vasohibin in neointimal formation. Adenovirus-mediated human vasohibin gene transfer in mouse liver resulted in the release of vasohibin in plasma and exhibited anti-angiogenic effects at remote sites. This gene transfer inhibited adventitial angiogenesis, macrophage infiltration, and neointimal formation after cuff placement on mouse femoral artery. Vasohibin exhibited no direct effect on migration and proliferation of smooth muscle cells. Thus, vasohibin has an activity to prevent neointimal formation by inhibiting adventitial angiogenesis.     

cDNA microarray analyses reveal candidate marker genes for the detection of ascidian disease in Korea

A serious disease of the ascidian Halocynthia roretzi has been spread extensively among Korean aquaculture sites. To reveal the cause of the disease and establish a monitoring system for it, we constructed a cDNA microarray spotted with 2,688 cDNAs derived from H. roretzi hemocyte cDNA libraries to detect genes differentially expressed in hemocytes between diseased and non-diseased ascidians. We detected 21 genes showing increased expression and 16 genes showing decreased expression in hemocytes from diseased ascidians compared with those from non-diseased ascidians. RT-PCR analyses confirmed that the expression levels of genes encoding astacin, lysozyme, ribosomal protein PO, and ubiquitin-ribosomal protein L40e fusion protein were increased in hemocytes from diseased ascidians, while those of genes encoding HSP40, HSP70, fibronectin, carboxypeptidase and lactate dehydrogenase were decreased. These genes were expressed not only in hemocytes but also in various other tissues in ascidians. Furthermore, the expression of glutathione-S transferase omega, which is known to be up-regulated in H. roretzi hemocytes during inflammatory responses, was strongly increased in hemocytes from diseased ascidians. These gene expression profiles suggest that immune and inflammatory reactions occur in the hemocytes of diseased ascidians. These genes will be good markers for detecting and monitoring this disease of ascidians in Korean aquaculture sites.     

Dbp9p, a member of the DEAD box protein family, exhibits DNA helicase activity

The yeast Dbp9p is a member of the DEAD box family of RNA helicases, which are thought to be involved in RNA metabolism. Dbp9p seems to function in ribosomal RNA biogenesis, but it has not been biochemically characterized. To analyze the enzymatic characteristics of the protein, we expressed a recombinant Dbp9p in Escherichia coli and purified it to homogeneity. The purified protein exhibited RNA unwinding and binding activity in the absence of NTP, and this activity was abolished by a mutation in the RNA-binding domain. We then characterized the ATPase activity of Dbp9p with respect to cofactor specificity; the activity was found to be severely inhibited by yeast total RNA and moderately inhibited by poly(U), poly(A), and poly(C) but to be stimulated by yeast genomic DNA and salmon sperm DNA. In addition, Dbp9p exhibited DNA-DNA and DNA-RNA helicase activity in the presence of ATP. These results indicate that Dbp9p has biochemical characteristics unique among DEAD box proteins.     

Involvement of signaling of VEGF and TGF-β in differentiation of sinusoidal endothelial cells during culture of fetal rat liver cells

Embryonic development of the liver is closely associated with vascular organization. However, little is known about the mechanisms of vascular differentiation during liver development. Our previous study showed that the maturation of sinusoidal endothelial cells (SECs) occurred during embryonic day 13.0 (E13.0) to E15.0. To improve our understanding of SEC differentiation, we examined here the expression of maturation markers, SE-1 and stabilin-2, in fetal livers and also attempted to establish an in vitro SEC differentiation system by culturing E13.5 fetal liver cells. Immunohistochemical examination of SE-1 and stabilin-2 expression during fetal rat liver development revealed that these differentiation markers were co-expressed in SECs in the late stage of liver development, although stabilin-2 was expressed in almost all vascular endothelial cells in the early stage. Liver cells from the E13.5 rat fetus were cultured in EBM-2 medium containing vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGF-β1) and VEGF plus SB-431542 (an inhibitor of the TGF-β1 receptor, activin receptor-like kinase 5 [ALK-5]). In vitro SEC differentiation, as indicated by the appearance of cells co-expressing SE-1 and stabilin-2 and of cells with cytoplasmic fenestrae in endothelial sheets, was induced by the addition of both VEGF and SB-431542, an inhibitor of the phosphorylation of Smad2/3 but not that of Smad1/5/8 in the cultured cells. These results indicate for the first time that both VEGF signaling and the blocking of the ALK-5-Smad2/3 signal pathway are important for the fetal differentiation of SECs.     

Transgenic rats overexpressing the human MrgX3 gene show cataracts and an abnormal skin phenotype

The human MrgX3 gene, belonging to the mrgs/SNSRs (mas related genes/sensory neuron specific receptors) family, was overexpressed in transgenic rats using the actin promoter. Two animal lines showed cataracts with liquification/degeneration and swelling of the lens fiber cells. The transient epidermal desquamation was observed in line with higher gene expression. Histopathology of the transgenic rats showed acanthosis and focal parakeratosis. In the epidermis, there was an increase in cellular keratin 14, keratin 10, and loricrin, as well as PGP 9.5 in innervating nerve fibers. These phenotypes accompanied an increase in the number of proliferating cells. These results suggest that overexpression of the human MrgX3 gene causes a disturbance of the normal cell-differentiation process.     

Changes of aldolase A and B messenger RNA levels in rat liver during azo-dye-induced hepatocarcinogenesis

The expression of aldolase A and B mRNAs during azo-dye-induced carcinogenesis in rat liver was examined. After feeding the dye for 18 weeks, the level of aldolase A mRNA increased to about 11 times that in a normal liver, with the concomitant decrease of aldolase B mRNA level to about 25% of that in a normal liver. These changes did not occur progressively during the carcinogenesis, but occurred as an additional phase after 4 week-feeding of the azo-dye. At this stage, the levels of aldolase A and B mRNAs were about 7 times and 45% of that in a normal liver, respectively. This biphasic pattern in the aldolase isozyme expression in the azo-dye-fed rat liver is discussed together with the kinetic data of the enzyme activity.