Bo-Chul Shin

Direct gene transfer into rat liver cells by in vivo electroporation

In vivo electro‐transfection efficiency and manner of transferred gene expression were investigated by fluorescence microscopic image analysis. Green fluorescent protein (GFP) gene was used as the genetic marker. Electroporation was carried out on the liver of live rats by use of disk electrodes mounted in the tips of tweezers, which were directly pressed onto the surface of a liver lobe in situ. Electroporation with eight electric pulses of 50 ms in duration at 50 V gave a good efficiency of transfection as judged by the induced GFP expression. Bright fluorescence of GFP appeared as dots, which were scattered around the area damaged by electroporation. The transfection efficiency increased as the amount of injected DNA was increased. The results indicate that the amount of induced gene expression can be controlled. Estimation of the efficiency of electro‐gene transfer using the fluorescence of GFP and digital analysis of microscopic images was useful to determine the optimum conditions for local gene therapy in tissues and organs.

p85α Gene Generates Three Isoforms of Regulatory Subunit for Phosphatidylinositol 3-Kinase (PI 3-Kinase), p50α, p55α, and p85α, with Different PI 3-Kinase Activity Elevating Responses to Insulin

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by association with a variety of tyrosine kinase receptors and intracellular tyrosine-phosphorylated substrates. We isolated a cDNA that encodes a 50-kDa regulatory subunit of PI 3-kinase with an expression cloning method using 32P-labeled insulin receptor substrate-1 (IRS-1). This 50-kDa protein contains two SH2 domains and an inter-SH2 domain of p85α, but the SH3 and bcr homology domains of p85α were replaced by a unique 6-amino acid sequence. Thus, this protein appears to be generated by alternative splicing of the p85α gene product. We suggest that this protein be called p50α. Northern blotting using a specific DNA probe corresponding to p50α revealed 6.0- and 2.8-kb bands in hepatic, brain, and renal tissues. The expression of p50α protein and its associated PI 3-kinase were detected in lysates prepared from the liver, brain, and muscle using a specific antibody against p50α. Taken together, these observations indicate that the p85α gene actually generates three protein products of 85, 55, and 50 kDa. The distributions of the three proteins (p85α, p55α, and p50α), in various rat tissues and also in various brain compartments, were found to be different. Interestingly, p50α forms a heterodimer with p110 that can as well as cannot be labeled with wortmannin, whereas p85α and p55α associate only with p110 that can be wortmannin-labeled. Furthermore, p50α exhibits a markedly higher capacity for activation of associated PI 3-kinase via insulin stimulation and has a higher affinity for tyrosine-phosphorylated IRS-1 than the other isoforms. Considering the high level of p50α expression in the liver and its marked responsiveness to insulin, p50α appears to play an important role in the activation of hepatic PI 3-kinase. Each of the three α isoforms has a different function and may have specific roles in various tissues.

Colocalization of tight junction proteins, occludin and ZO-1, and glucose transporter GLUT1 in cells of the blood-ocular barrier in the mouse eye

Abstract The facilitative glucose transporter GLUT1 is abundant in cells of the blood-ocular barrier and serves as a glucose transport mechanism in the barrier. To see the relationship between the glucose transfer function and junctional proteins in the barrier, we examined the localization of GLUT1 and the tight junction proteins, occludin and ZO-1, in the mouse eye. Their localization in the retina, ciliary body, and iris was visualized by double-immunofluorescence microscopy and immunogold electron microscopy. Occludin and ZO-1 were colocalized at tight junctions of the cells of the barrier: retinal pigment epithelial cells, non-pigmented epithelial cells of the ciliary body, and endothelial cells of GLUT1-positive blood vessels. Occludin was restricted to these cells of the barrier. ZO-1 was found, in addition, in sites not functioning as a barrier: the outer limiting membrane in the retina, in the cell border between pigmented and non-pigmented epithelial cells in the ciliary body, and GLUT1-negative blood vessels. These observations show that localization of occludin is restricted to tight junctions of cells of the barrier, whereas ZO-1 is more widely distributed.

Immunolocalization of GLUT1 and connexin 26 in the rat placenta

Abstract.Interhemal membrane in the rat placenta is composed of three trophoblastic layers and endothelial cells. GLUT1, an isoform of the facilitated-diffusion glucose transporter, is abundant in the cells of the placental barrier, i.e., syncytiotrophoblastic layers I and II. GLUT1 is localized at the plasma membranes of the maternal-blood side of syncytiotrophoblastic layer I, and of the fetal-blood side of syncytiotrophoblastic layer II. Double-immunofluorescence microscopy has shown that connexin 26 is present between these GLUT1-positive sites, i.e., between syncytiotrophoblastic layers I and II. Immunogold electron microscopy has revealed that connexin 26 is localized in the gap junctions connecting the two layers. Connexin 26 in these layers therefore makes them functionally a single syncytial layer for the transfer of small molecules such as glucose in the rat placental barrier. These results suggest that glucose transfer in the rat placental barrier is carried out as follows: GLUT1 is used for the entry of glucose into the cytoplasm of syncytiotrophoblastic layer I, connexin 26 for the transfer of glucose from syncytiotrophoblastic layer I to syncytiotrophoblastic layer II, and GLUT1 for the exit of glucose to the fetal circulation.

Connexin 43 and the glucose transporter, GLUT1, in the ciliary body of the rat

To investigate the relationship between the gap junction protein connexin 43 and the glucose transporter GLUT1, their localization was visualized by double-immunofluorescence microscopy using frozen sections as well as immunogold staining of ultrathin frozen sections. In pigmented epithelial cells, most of the GLUT1 was localized along the plasma membrane facing the blood vessels, whereas in non-pigmented epithelial cells. it was present along the plasma membrane facing the aqueous humor. Connexin 43 was abundant in the ciliary body and localized mainly in the gap junctions connecting the pigmented and non-pigmented epithelial cells. Localization of GLUT1 and connexin 43 in the blood-aqueous barrier suggests that GLUT1, connexin 43, and GLUT1 disposed in this order could be a machinery responsible for the transport of glucose across the blood-aqueous barrier.