Transcellular transport of fluorescein dextran through an arterial endothelial cell monolayer

Transcellular transport of fluorescein dextran (FD) of various molecular weights (4K, 10K, 20K, 70K and 150K daltons) through porcine arterial endothelial cells cultured on a type I collagen gel supported by a dacron sheet was studied and compared with the transport of low density lipoprotein labeled with rhodamine B (RB-LDL) described previously (Hashida et al., Cell Struct. Funct. 11, 31-42, 1986). The rate of FD transport through the monolayer depended on the size of the FD. FD transport was not temperature-dependent and was not a saturable process. Our findings show that FD transport differs from RB-LDL transport which is temperature- and dose-dependent. The mechanism of the transport of FD is compared with that of RB-LDL.     

Two ANGUSTIFOLIA genes regulate gametophore and sporophyte development in Physcomitrella patens

In Arabidopsis thaliana the ANGUSTIFOLIA (AN) gene regulates the width of leaves by controlling the diffuse growth of leaf cells in the medio‐lateral direction. In the genome of the moss Physcomitrella patens, we found two normal ANs (PpAN1‐1 and 1‐2). Both PpAN1 genes complemented the A. thaliana an‐1 mutant phenotypes. An analysis of spatiotemporal promoter activity of each PpAN1 gene, using transgenic lines that contained each PpAN1‐promoter– uidA (GUS) gene, showed that both promoters are mainly active in the stems of haploid gametophores and in the middle to basal region of the young sporophyte that develops into the seta and foot. Analyses of the knockout lines for PpAN1‐1 and PpAN1‐2 genes suggested that these genes have partially redundant functions and regulate gametophore height by controlling diffuse cell growth in gametophore stems. In addition, the seta and foot were shorter and thicker in diploid sporophytes, suggesting that cell elongation was reduced in the longitudinal direction, whereas no defects were detected in tip‐growing protonemata. These results indicate that both PpAN1 genes in P. patens function in diffuse growth of the haploid and diploid generations but not in tip growth. To visualize microtubule distribution in gametophore cells of P. patens, transformed lines expressing P. patens α‐tubulin fused to sGFP were generated. Contrary to expectations, the orientation of microtubules in the tips of gametophores in the PpAN1‐1/1‐2 double‐knockout lines was unchanged. The relationships among diffuse cell growth, cortical microtubules and AN proteins are discussed.     

Hepatocyte-specific distribution of catalase and its inhibitory effect on hepatic ischemia/reperfusion injury in mice

To explore the possibility of using catalase for the treatment of reactive oxygen species (ROS)-mediated injuries, the pharmacokinetics of bovine liver catalase (CAT) labeled with ¹¹¹In was investigated in mice. At a dose of 0.1 mg/kg, more than 70% of ¹¹¹In-CAT was recovered in the liver within 10 min after intravenous injection. In addition, ¹¹¹In-CAT was predominantly recovered from the parenchymal cells (PC) in the liver. Increasing the dose retarded the hepatic uptake of ¹¹¹In-CAT, suggesting saturation of the uptake process. This cell-specific uptake could not be inhibited by coadministration of various compounds which are known to be taken up by liver PC, indicating that the uptake mechanism of CAT by PC is very specific to this compound. The preventive effect of CAT on a hepatic ischemia/reperfusion injury was examined in mice by measuring the GOT and GPT levels in plasma. A bolus injection of CAT at 5 min prior to the reperfusion attenuated the increase in the levels of these indicators in a dose-dependent manner. These results suggest that catalase can be used for various hepatic injuries caused by ROS.     

Genotypic characteristics of a Mycobacterium sp. isolated from yellowtail Seriola quinqueradiata and striped jack Pseudocaranx dentex in Japan

In Japan, a Mycobacterium marinum‐like mycobacterium was isolated from the yellowtail, Seriola quinqueradiata. The species was identified as M. marinum by a commercial mycobacterial DNA‐DNA hybridization kit. Nevertheless, PCR restriction analysis of the DNA of its RNA polymerase β‐subunit gene definitively showed that this Mycobacterium sp. was M. ulcerans. PCR analysis revealed the genotypic characteristics of M. ulcerans in the Mycobacterium sp., only the mup053 gene sequence being absent, as has been found previously in other piscine mycobacteria such as M. marinum strains DL240490 and DL045 and M. pseudoshottsii. With one exception, this Mycobacterium sp. and M. pseudoshottsii had identical 16S rRNA gene sequences, which is also probably true of M. marinum strains DL240490 and DL045. Similarly, according to comparisons of the 16S rRNA gene, ITS region, and hsp65 gene sequences, this Mycobacterium sp. is more closely related to M. pseudoshottsii than to M. ulcerans or M. marinum. A PCR product of approximately 2000 bp was amplified from region of difference 9 in the Mycobacterium sp. The nucleotide sequence revealed insertion of IS2404, the sequence of which is 1366 bp long. The novel single nucleotide polymorphisms identified in this region distinguished this Mycobacterium sp. from M. marinum strain DL240490 and M. pseudoshottsii. The present findings raise the possibility that these species have a common ancestor. Further studies are required to improve our understanding of the relationship between their geographical origin and genetic diversity.     

Involvement of serum mannan binding proteins and mannose receptors in uptake of mannosylated liposomes by macrophages

The roles of serum mannan binding protein (MBP) and the mannose receptor in the cellular uptake of mannosylated liposomes (Man-liposomes) by macrophages were studied. Man-liposomes were prepared by incorporating cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiomannosylethyl)amino)butyl)formamide (Man-C4-Chol) into small unilamellar long circulating liposomes consisting of cholesterol (Chol) and distearoyl phosphatidylcholine (DSPC). In the in vitro cellular uptake study with cultured mouse peritoneal macrophages, [(3)H]Man-liposomes were taken up to a great extent, whereas no significant uptake was observed for [(3)H]cholesterol and DSPC liposomes without Man-C4-Chol (Bare-liposomes). The uptake of [(3)H]Man-liposomes was dose- and temperature-dependent and inhibited by an excess of mannosylated bovine serum albumin, suggesting their specific uptake via membrane mannose receptor-mediated endocytosis. Furthermore, it was demonstrated that (111)In-MBP binds strongly to Man-liposomes based on the recognition of Man-C4-Chol and markedly enhanced their uptake by macrophages. These results are supported by confocal laser microscopic images. In addition, in vivo hepatic uptake of (111)In-MBP was enhanced by Man-liposomes. On the other hand, the uptake of Man-liposomes was significantly reduced by preincubation with serum and further with MBP-depleted serum suggesting inhibitory effects of serum proteins such as albumin on mannose receptor-mediated endocytosis. The involvement of serum-type MBP and membrane mannose receptors in the uptake of Man-liposomes is thus suggested.     

Liver uptake and hepato-biliary transfer of galactosylated proteins in rats are determined by the extent of galactosylation

The effect of molecular mass and surface density of galactose residues on hepatic uptake and subsequent biliary excretion of galactosylated proteins was investigated in rats. Several proteins with different molecular weights (15-70 kDa) and different numbers of galactose units were synthesized and radiolabeled with 111In. Galactosylated proteins were administered i.v. to anaesthetized rats and samples of plasma and bile were collected for 3 h. Liver was harvested at the end of the experiments and the radioactivity of all samples was measured. Galactosylated proteins accumulated primarily in the liver and 2-10% of the administered dose appeared in the bile, mainly in undegraded form. The hepatic uptake clearance (Cl liver) and biliary excretion rate constant (kbile) of galactosylated proteins were calculated. No direct effect of molecular weight was observed, however, on increasing the galactose density, Cl liver increased from about 4 to 400 ml/h whereas kbile gradually decreased from about 0.057 to 0.007 (h-1). In conclusion, both hepatic uptake and biliary excretion of galactosylated proteins were found to be affected by the extent of galactosylation.     

Tissue-specific replicating capacity of a chimeric poliovirus that carries the internal ribosome entry site of hepatitis C virus in a new mouse model transgenic for the human poliovirus receptor

Nucleotides (nt) 108 to 742 of an infectious cDNA clone of poliovirus (PV) Mahoney strain, including the corresponding region of the internal ribosome entry site (IRES), was replaced by nt 28 to 710 of hepatitis C virus (HCV) cDNA corresponding to the whole HCV IRES. A chimeric PV (2A-369) was generated by transfecting mammalian cells with an RNA transcribed in vitro from the cDNA. To examine replicating capacity of virus 2A-369 in the brain and liver of a mouse model for poliomyelitis, a new mouse model (MPVRTg25-61) that is transgenic for human PV receptor (hPVR; CD155) was generated in order to obtain a higher expression level of hPVR in the liver than those of hPVRTg mouse lines generated by us so far. The transgene used was constructed by combining a putative regulatory region of the mouse PVR homolog and the whole structural region of the hPVR gene. Virus 2A-369 replicated well in the liver of MPVRTg25-61 but not in the brain, whereas control Mahoney virus replicated well both in the liver and in the brain. The data suggest that the HCV IRES works more efficiently in the liver than in the brain and that PV IRES works well both in the liver and in the brain. The results support the notion that tissue-specific activity of IRES may be reflected in tissue tropism of a virus whose specific translation initiation is driven by IRES, that is, an IRES-dependent virus tropism.     

Detection of one milliattomole of ferritin by novel and ultrasensitive enzyme immunoassay

A novel and ultrasensitive enzyme immunoassay (immune complex transfer two-site enzyme immunoassay) for ferritin is described. Ferritin was reacted simultaneously with affinity-purified dinitrophenyl biotinyl anti-ferritin IgG and affinity-purified anti-ferritin Fab'-beta-D-galactosidase conjugate. The complex formed of the three components was trapped onto affinity-purified (anti-dinitrophenyl group) IgG-coated polystyrene balls. After eliminating excess conjugate by washing, the complex was eluted from the polystyrene balls with an excess of epsilon N-dinitrophenyl-L-lysine and transferred to streptavidin-coated polystyrene balls. The beta-D-galactosidase activity bound to streptavidin-coated polystyrene balls was assayed by fluorometry. Nonspecifically bound beta-D-galactosidase activity was remarkably lowered but there was much less decrease in specifically bound beta-D-galactosidase activity. As a result, the detection limit of ferritin was lowered to 1 milliattomole (1 x 10(-21) mol, 600 molecules as calculated from Avogadro's number). This technique will be useful for measuring, for example, antigens in single cells.     

Binding of FAD to cytochrome b558 is facilitated during activation of the phagocyte NADPH oxidase, leading to superoxide production

The superoxide-producing phagocyte NADPH oxidase can be reconstituted in a cell-free system. The activity of NADPH oxidase is dependent on FAD, but the physiological status of FAD in the oxidase is not fully elucidated. To clarify the role of FAD in NADPH oxidase, FAD-free full-length recombinant p47(phox), p67(phox), p40(phox), and Rac were prepared, and the activity was reconstituted with these proteins and purified cytochrome b(558) (cyt b(558)) with different amounts of FAD. A remarkably high activity, over 100 micromol/s/micromol heme, was obtained in the oxidase with purified cyt b(558), ternary complex (p47-p67-p40(phox)), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b, the dissociation constant K(d) of FAD in cyt b(558) of reconstituted oxidase was estimated as nearly 1 nm. We also examined addition of FAD on the assembly process in reconstituted oxidase. The activity was remarkably enhanced when FAD was present during assembly process, and the efficacy of incorporating FAD into the vacant FAD site in purified cyt b(558) increased, compared when FAD was added after assembly processes. The absorption spectra of reconstituted oxidase under anaerobiosis showed that incorporation of FAD into cyt b(558) recovered electron flow from NADPH to heme. From both K(d) values of FAD and the amount of incorporated FAD in cyt b(558) of reconstituted oxidase, in combination with spectra, we propose the model in which the K(d) values of FAD in cyt b(558) is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase.