Transcellular transport of lipoprotein through arterial endothelial cells in monolayer culture

To study the mechanism of lipoprotein transport through arterial endothelial cells, porcine endothelial cells were cultured on gelated type I collagen supported by a dacron sheet, and the transport of low density lipoprotein (LDL) labeled with rhodamine B isothiocyanate (RB-LDL) through the cells was measured. Light and scanning electron microscopy showed that the cells on the gel were confluent. There was little RB-LDL transport through the endothelial monolayer at 0 degrees C. RB-LDL transport through the monolayer at 37 degrees C was dose-dependent saturable at 0.4 mg protein/ml. The transport was energy-dependent, since its rate was affected by temperature and was inhibited by a combination of 2-deoxyglucose (50 mM) and NaN3 (10 mM). RB-LDL was shown not to be degraded during transport.     

PEGylated catalase prevents metastatic tumor growth aggravated by tumor removal

Although surgical removal is a primary option for treating tumors, it can lead to the increased growth of metastatic tumors. Because surgical procedures may generate reactive oxygen species (ROS), known promoters of tumor metastasis and growth, we investigated whether PEGylated catalase (PEG-catalase, plasma half-life of 13.6 h) was able to prevent this after surgical removal of a footpad tumor in mice. Murine melanoma cells labeled with the firefly luciferase gene were used to monitor the distribution of tumor cells. After inoculation into the footpad, tumor cells were found in the lung, and the number increased with time. The surgical removal of the footpad tumor significantly (p < 0.05) increased the number of metastatic tumor cells and the level of plasma lipoperoxides. An intravenous injection of PEG-catalase significantly (p < 0.05) suppressed the metastatic tumor growth as well as the peroxidation. Quantitative RT-PCR and Western blot analyses indicated that PEG-catalase markedly reduced the increase in the expression of epidermal growth factor receptor. These findings indicate that the removal of tumor produces ROS, which then aggravate metastatic tumor growth by activating several growth factors. PEG-catalase can effectively prevent this metastatic tumor growth by detoxifying the ROS.     

Presentation of functional foreign peptides on the surface of SV40 virus-like particles

Viral capsids of simian virus 40 (SV40) are highly efficient gene delivery vehicles that infect a broad range of cells and tissues. To develop a controlled, cell type-specific delivery system, we sought to display foreign peptides on the capsid surface by genetically manipulating the major capsid protein Vp1. Here we report the identification of two sites within the surface loops of Vp1 that can accommodate foreign peptides in such a way that the foreign peptides are displayed on the surface of the virus-like particles (VLPs) without interfering with VLP assembly or the packaging of viral DNA. Insertion of Flag-tags but not RGD integrin-binding motifs at these sites strongly inhibited cell attachment of VLPs, which normally associate with host cells through cell surface molecules such as major histocompatibility complex (MHC) class I and ganglioside GM1. Instead, VLPs carrying the RGD motifs bound to integrin in vitro and to the cell surface in an RGD-dependent manner. Thus, insertion of foreign sequences into the surface loops of Vp1 can reduce natural virus-cell interactions and even confer an ability to bind to a new target receptor. This study demonstrates the potential usefulness of this strategy for the development of novel delivery vehicles with different cell tropisms.     

Enhanced gene expression in lung by a stabilized lipoplex using sodium chloride for complex formation

BACKGROUND: In this study, we investigated the in vivo gene transfection efficacy of a 'surface charge regulated' (SCR) lipoplex, dispersed in the presence of an essential amount of NaCl during lipoplex formation. METHODS: SCR lipoplexes were prepared and their physicochemical properties were analyzed. After intravenous (i.v.) administration, transfection efficacy, distribution characteristics, and liver toxicity were evaluated in mice. RESULTS: At NaCl concentrations of 10 mM, the particle sizes of the SCR lipoplexes were about 120 nm and were compatible with a conventional lipoplex. However, fluorescent resonance energy transfer analysis revealed that cationic liposomes in the SCR lipoplexes increased fusion. After i.v. administration, the transfection activity in the lung of the SCR lipoplex (10 mM NaCl solution in the lipoplex) was approximately 10-fold higher than that of the conventional lipoplex. Pharmacokinetic studies demonstrated a higher distribution in lung by the SCR lipoplex. When the gene expression levels of the SCR lipoplex and conventional lipoplex were compared, the SCR lipoplex at a dose of 30 microg was compatible with that of the conventional lipoplex at a dose of 50 microg. A significantly higher serum alanine aminotransferase (ALT) activity and TNFalpha concentration was observed by the conventional lipoplex (pDNA dose; 50 microg), but this was not the case for the SCR lipoplex (pDNA dose; 30 microg). CONCLUSIONS: We demonstrated that the SCR lipoplex could enhance the transfection efficacy in the lung without increasing the liver toxicity. Hence, the information will be valuable for the future use, design, and development of lipoplexes for in vivo applications.     

Enhanced SUMOylation in polyglutamine diseases

Small ubiquitin-like modifiers (SUMOs) are proteins homologous to ubiquitin that possibly regulate intranuclear protein localization, nuclear transport, and ubiquitination. We examined patients of DRPLA, SCA1, MJD, and Huntington's disease and found that neurons in affected regions of the brain react strongly to SUMO-1, a family member of SUMOs. Western blot with a transgenic mouse expressing mutant ataxin-1 showed the increase of SUMOylated proteins in the cerebellar cortex, which we named ESCA1 and ESCA2. These results indicated activation of SUMO-1 system in polyglutamine diseases and predicted its involvement in the pathology.     

Adrenomedullin release in the rat mesenteric resistance artery

Adrenomedullin (AM) is a potent vasodilator peptide whose major source is the vascular wall. In the present study, the mechanism of release of AM was investigated in the rat mesenteric resistance artery. The isolated mesenteric vascular bed was perfused with Krebs solution at a constant flow rate (5 ml/min) and AM in the perfusate was measured by a highly sensitive enzyme immunoassay (Immunoenzymometric assay; IEMA) method. In preparations without endothelium, spontaneous release of AM was detected in the perfusate (68.7+/-5.8 fmol/ml, n=45). Periarterial nerve stimulation (PNS, 4 and 8 Hz) caused 11.4+/-3.9% (4 Hz) and 9.1+/-3.5% (8 Hz) decreases in the spontaneous release of AM. Removal of Ca2+ from the medium did not affect the spontaneous AM release, but abolished the PNS-induced inhibition of spontaneous AM release. Perfusion of 10nM calcitonin gene-related peptide (CGRP) or 0.1 microM capsaicin (inducer of CGRP release) inhibited significantly the spontaneous AM release. PNS (8 Hz)-induced inhibition of spontaneous AM release was antagonized by CGRP(8-37) (CGRP receptor antagonist). These results suggest that AM is mainly released from vascular smooth muscle cells of the rat mesenteric artery and endogenous or exogenous CGRP inhibits AM release.     

Pharmacokinetics and disposition characteristics of recombinant decorin after intravenous injection into mice

The pharmacokinetics and disposition characteristics of recombinant decorin after intravenous administration were investigated in mice. Following bolus injection of 111In-labeled decorin at doses of 0.02 and 0.1 mg/kg, radioactivity rapidly disappeared from the circulation and approximately 70% of the dose accumulated in liver within 10 min. 111In-labeled decorin was preferentially localized in hepatic nonparenchymal cells. At a higher dose of 1 mg/kg, clearance from the circulation and hepatic uptake of [111In]decorin were slower than at lower doses. Both the accumulation in other tissues and urinary excretion of [111In]decorin were 5% or less. Pharmacokinetic analysis demonstrated that hepatic uptake clearance was large and accounted almost completely for total body clearance; in addition the clearance values decreased as the dose increased, suggesting that the hepatic uptake of decorin is mediated by a specific mechanism which becomes saturated at higher doses. In competitive inhibition experiments, hepatic uptake of 111In-labeled decorin was partially inhibited (about 20-30%) by several sulfated glycans such as glycosaminoglycans and dextran sulfate and by mannosylated bovine serum albumin (BSA), mannan and mannose to a lesser extent (about 10%). On the other hand, polyinosinic acid, polycytidylic acid and succinylated BSA were ineffective, suggesting that the scavenger receptor for polyanions in the liver is not involved in the hepatic uptake of decorin. A basic protein, protamine, and a ligand of the apoE receptor, lactoferrin, also had no effect. Taken together, the present results have demonstrated that recombinant decorin is rapidly eliminated from the blood circulation through extensive uptake by the liver, primarily by the nonparenchymal cells, following systemic administration. The sugar structure and mannose residue in decorin have also been suggested to play an important role in the hepatic uptake of decorin. These findings provide useful information for the development of decorin as a therapeutic agent.     

The hydrophobic repeated domain of the Clostridium cellulovorans cellulose-binding protein (CbpA) has specific interactions with endoglucanases.

We overexpressed one of the hydrophobic repeated domains (HBDs) (110 amino acid residues) of the cellulose-binding protein (CbpA) from Clostridium cellulovorans by making a hybrid protein with the Escherichia coli maltose-binding protein (MalE). The HBD was purified to homogeneity, and interactions between the HBD and endoglucanases were analyzed by a novel interaction Western blotting (immunoblotting) method. The HBD had specific interactions with endoglucanases (EngB and EngD) from C. cellulovorans. These results indicated that the HBD was an endoglucanase binding site of CbpA.     

Bovine thymus poly(adenosine diphosphate ribose) polymerase.

About 1,300-fold purification of poly(adenosine diphosphate ribose) polymerase has been achieved from the extract of bovine thymus with a recovery of 10 to 20%. The final preparation has a purity of 99%, and the enzyme is composed of a single peptide with a molecular weight of 130,000. The purified enzyme required NAD+, Mg2+, a thiol compound, DNA, and histones for full activity. Whereas DNA is essential for activation of the enzyme, histones are not. The observed stimulation of the reaction by histones is shown to be due to masking of the inhibitory effect of contaminating denartured DNA in native DNA preparation. The concentration of DNA required for half-maximal enzyme activity (apparent Km for DNA) is proportional to the concentration of enzyme in the reaction mixture. The minimum estimation of the number of nucleotide pairs of DNA required for half-maximal activation of one enzyme molecule is 220 to 240 for bulk of calf thymus DNA, while the value is 10 for a calf thymus DNA fraction, "active DNA," which was separated from the enzyme fraction in a stage of the purification. These results suggest that the enzyme is activated by binding to a specific site on calf thymus DNA. The apparent Km for NAD+ and the maximum velocity of the enzyme are estimated to be 60 micrometer and 0.91 mumolper min per mg, respectively.     

Molecular mechanism of the inhibitory effect of cobalt ion on thermolysin activity and the suppressive effect of calcium ion on the cobalt ion-dependent inactivation of thermolysin

Thermolysin activity in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide (FAGLA) and FA-l-leucyl-l-alanine amide (FALAA) was examined at various Co(2+) and Ca(2+) concentrations. It decreased to 28% with increasing [Co(2+)] up to 18 mM. The Co(2+)-dependent inactivation was in part suppressed by adding Ca(2+) ion up to 0.5 mM, but 33% of the activity remained to be inactivated even with a sufficient concentration of Ca(2+) (>0.5 mM). The Co(2+)-dependent inactivation was shown to be composed of Ca(2+)-sensitive and Ca(2+)-insensitive parts. In the latter part which is observed at [Ca(2+)] >0.5 mM, Co(2+) plays as a competitive inhibitor. On the other hand, the Co(2+)-dependent inactivation in the Ca(2+)-sensitive part observed at [Ca(2+)] <0.5 mM proceeds time-dependently following second-order kinetics, and the time-course is in good agreement with that of decrease in the thermolysin band due to autolysis in SDS-PAGE. This indicates that Co(2+) accelerates the autolysis. Here, we describe the co-regulation of thermolysin activity by Co(2+) and Ca(2+) ions and propose a molecular mechanism for the inhibition of thermolysin by Co(2+) and suppressive effect of Ca(2+) on the Co(2+)-dependent inhibition. Co(2+) ion inhibits thermolysin activity not only as a competitive inhibitor but also promoting the autolysis.