α2-Macroglobulin secretion enhanced in rat hepatocytes by partially characterized factor from Kupffer cells

Isolated rat Kupffer cells produced a factor which stimulated the synthesis of ₂-macroglobulin (2M) in primary cultured rat hepatocytes. Although Kupffer cells placed in culture produced the factor without stimulation by lipopolysaccharide (LPS), the LPS-stimulated cells produced larger amounts of the factor. On the other hand, the production of the factor was inhibited by addition of actinomycin D. The induction of2M synthesis by cultured hepatocytes was enhanced in the presence of dexamethasone (Dex), in that hepatic synthesis of2M increased by addition of the factor alone and with Dex 1.5 and three- to four-fold, respectively. The factor was nondialyzable and stable at 60°C for 30 min. When the factor was fractionated using the molecular sieve method, the activity recovered in the fraction had a molecular weight of over 30,000.     

Cartilage regeneration using slow release of bone morphogenetic protein-2 from a gelatin sponge to treat experimental canine tracheomalacia: a preliminary report

We investigated whether saber sheath-type tracheomalacia could be treated by the slow release of bone morphogenetic protein (BMP)-2 from a gelatin sponge. A 1 cm gap was made in the middle portion of each of 10 consecutive tracheal cartilage rings in the canine cervix (control group, n = 3), then a gelatin sponge containing 12 microg of BMP-2 solution was implanted in the gap (12 microg group, n = 3). In another group (120 microg + P group, n = 3), the implanted gelatin sponge contained 120 microg of BMP-2 solution, and the gap was covered with periosteum. All of the control dogs developed saber sheath-type tracheomalacia, whereas tracheomalacia was not observed in the 12 microg and 120 microg + P groups. In the 12 microg group, fibrous cartilage was observed at the ends of the cartilage stumps. In the 120 microg + P group, newly formed bone and cartilage were observed to form a bridge between the cartilage stumps. The regeneration of cartilage or bone induced by the slow release of BMP-2 from a gelatin sponge might be useful for treatment of tracheomalacia.     

Glutathione redox regulates lipopolysaccharide-induced IL-12 production through p38 mitogen-activated protein kinase activation in human monocytes: role of glutathione redox in IFN-gamma priming of IL-12 production

We examined whether changes in intracellular reduced (GSH) or oxidized (GSSG) glutathione of human monocytes regulate lipopolysaccharide (LPS)-induced IL-12 production and defined the molecular mechanism that underlies glutathione redox regulation. Monocytes exposed to glutathione reduced form ethyl ester (GSH-OEt) or maleic acid diethyl ester (DEM) increased or decreased the intracellular GSH/GSSG ratio, respectively. LPS-induced IL-12 production and p38 mitogen-activated protein (MAP) kinase activation were enhanced by GSH-OEt but suppressed by DEM. Selective p38 inhibitors showed that p38 promoted GSH-OEt-enhanced IL-12 production. Furthermore, IFN-gamma priming increased the GSH/GSSG ratio and enhanced IL-12 production through p38, and DEM negated the priming effect of IFN-gamma on p38 activation and IL-12 production as well as on the GSH/GSSG ratio. These findings reveal that glutathione redox regulates LPS-induced IL-12 production from monocytes through p38 MAP kinase activation and that the priming effect of IFN-gamma on IL-12 production is partly a result of the glutathione redox balance.     

Association of Pasteurella multocida toxin with vimentin

To help understand the molecular mechanisms of Pasteurella multocida toxin (PMT) action, we searched for a cellular protein interacting with PMT. The ligand overlay assay revealed a 60-kDa cellular protein that binds to a region from the 840th to 985th amino acids of the toxin. This protein was identified as vimentin by peptide mass fingerprinting. The N-terminal head domain of vimentin was further found to be responsible for the binding to the toxin.     

N-Glycosylation contributes to the limited cross-reactivity between hemagglutinin neuraminidase proteins of human parainfluenza virus type 4A and 4B

cDNAs encoding human parainfluenza virus type 4B (hPIV-4B) hemagglutinin neuraminidase (HN) protein were cloned and the nucleotide sequences were determined. A high degree of identity (81.4%) was observed between the nucleotide sequences of hPIV-4A and -4B HN proteins, and an 87.3% identity was found between the deduced amino acid sequences. This degree of identity is considered to be greater than immunological similarity between hPIV-4A and -4B HN proteins determined using monoclonal antibodies. To elucidate the causes of the antigenic difference between HN proteins of hPIV-4A and -4B, we constructed three cDNAs of hPIV-4B HN whose potential N-glycosylation sites were partially or completely the same as in hPIV-4A HN cDNA. We compared the antigenicity of the expressed wild-type and mutant proteins, and found that the antigenicities of the mutant hPIV-4B HN proteins were more similar to the hPIV-4A HN protein than to the non-mutant hPIV-4B HN protein. This study indicated that the antigenic diversity between hPIV-4A and -4B was partly caused by deletion or creation of glycosylation sites, showing that the point mutations resulting in deletion or creation of glycosylation sites is one of the initial steps leading to the division of virus into subtypes. Received: 21 January 2000     

Confocal imaging of biofilm formation process using fluoroprobed Escherichia coli and fluoro-stained exopolysaccharide

We developed a novel method of evaluating biofilm architecture on a synthetic material using green fluorescent protein-expressing Escherichia coli and red fluorescence staining of exopolysaccharides. Confocal laser scanning microscopy observation revealed the time course of the change in the in situ three-dimensional structural features of biofilm on a polyurethane film without structural destruction: initially adhered cells are grown to form cellular aggregates and secrete exopolysaccharides. These cells were spottily distributed on the surface at an early incubation time but fused to form a vertically grown biofilm with incubation time. Fluorescence intensity, which is a measure of the number of cells, determined using a fluorometer and biofilm thickness determined from confocal laser scanning microscopy vertical images were found to be effective for quantification of time-dependent growth of biofilms. The curli (surface-located fibers specifically binding to fibronectin and laminin)-producing Escherichia coli strain, YMel, significantly proliferated on fibronectin-coated polyurethane, whereas the curli-deficient isogenic mutant, YMel-1, did not. The understanding of biofilm architecture in molecular and morphological events and new fluorescence microscopic techniques may help in the logical surface design of biomaterials with a high antibacterial potential.     

Controlled release by biodegradable hydrogels enhances the ectopic bone formation of bone morphogenetic protein

The objective of this study is to develop a carrier for the controlled release of bone morphogenetic protein-2 (BMP-2) suitable for enhancement of the bone regeneration activity. Hydrogels with different water contents were prepared through glutaraldehyde crosslinking of gelatin with an isoelectric point of 9.0 under varied reaction conditions. Following subcutaneous implantation of the gelatin hydrogels incorporating 125I-labeled BMP-2 into the back of mice, the in vivo retention period of BMP-2 prolonged with a decrease in the water content of hydrogels used, although every time period was much longer than that of BMP-2 solution injection. Ectopic bone formation studies demonstrated that the alkaline phosphatase (ALP) activity and osteocalcin content around the implanted site of BMP-2-incorporated gelatin hydrogels were significantly high compared with those around the injected site of BMP-2 solution. The values became maximum for the gelatin hydrogel incorporating BMP-2 with a middle period of BMP-2 retention, while bone formation was histologically observed around the hydrogel incorporating BMP-2. The ALP activity was significantly higher than that of the collagen sponge incorporating BMP-2. We concluded that the controlled release technology of BMP-2 for a certain time period was essential to induce the potential activity for bone formation.     

Role of apoptosis controlled by cytochrome c released from mitochondria for luteal function in human granulosa cells

PROBLEM: The aim of this study was to investigate the relationship between apoptosis by the mitochondrial pathway and luteal function in human granulosa cells. METHOD OF STUDY: Granulosa cells were obtained by ultrasound-guided follicular aspiration from patients undergoing in vitro fertilization and embryo transfer. After the addition of RU486, cells were stained with a mitochondria-specific fluorescent dye, MitoTracker Red CM x Ros. Using flow cytometry and National Institute of Health image, the mitochondrial fluorescent area was measured. After staining with Hoechst 33258 dye, the number of apoptotic bodies per 1000 cells were counted at random on photomicrographs. Homogenates were used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis using antibodies against cytochrome c or caspase-3. RESULTS: The incidence of apoptotic bodies increased and the mitochondrial membrane potential decreased time dependently. The opposite effect was observed dose dependently with RU486 treatment. Western blot analysis showed increased cytochrome c expression, after treatment with 1-2 microg/mL of RU486 which then decreased with 5-10 microg/mL of RU486. Caspase-3 expression increased dose dependently with RU486. CONCLUSIONS: These results suggest that the activation of caspase-3 caused by cytochrome c released from mitochondria plays an important role in apoptosis-related luteal function in human granulosa cells.     

Preparation of blood-compatible hollow fibers from a polymer alloy composed of polysulfone and 2-methacryloyloxyethyl phosphorylcholine polymer

Blood-compatible hollow fibers were successfully prepared from a polymer alloy composed of polysulfone (PSf) and the 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer. To improve the hydrophilicity, fouling-resistance characteristics, and blood compatibility of the PSf hollow fiber in a hemodialyzer, an MPC polymer that can be blended with PSf was synthesized in order to prepare the polymer alloy (PSf/MPC polymer). The contents of the MPC polymer blended in the PSf were 7 and 15 wt%. The PSf/MPC polymer hollow fiber could be prepared by both wet and dry-wet processing methods. The hollow fiber took an asymmetric structure, that is, the hollow-fiber membrane had a dense skin layer on the porous sponge-like structure. The mechanical strength was higher than that of conventional PSf hollow fibers for hemodialysis. The surface characterization of the PSf/MPC polymer hollow fiber by x-ray photoelectron spectroscopy revealed that the MPC units were concentrated at the surface. The permeability for solutes through the PSf/MPC polymer hollow fibers was measured for 4 h. The permeabilities of both a low-molecular-weight compound and protein were greater than those of the PSf hollow fibers. The amount of adsorbed protein was lower on the PSf/MPC polymer hollow fiber when compared to that of the PSf hollow fiber. Moreover, platelet adhesion was also effectively inhibited on the PSf/MPC polymer hollow fiber. Based on these results, the addition of the MPC polymer to the PSf is a very useful method to improve the functions and blood compatibility of the hollow fiber.