Enhancement of adhesion strength and cellular stiffness of osteoblasts on mirror-polished titanium surface by UV-photofunctionalization.

Ultraviolet (UV)-photofunctionalization of titanium substantially enhances the strength and quality of osseointegration by promoting osteogenic cellular attachment and proliferation. However, the mechanism underlying the initial interaction between the cells and the surface of the material remains to be elucidated, especially where the influence of surface roughness is excluded as a factor. The effect of UV-photofunctionalization on the adhesive strength and cellular stiffness of a single osteoblast and its association with the extent of cell spread, cytoskeletal development and focal adhesion assembly on a very smooth titanium surface was evaluated. Rat bone marrow-derived osteoblasts were cultured on UV-treated or untreated mirror-polished titanium disks. The mean critical shear force required to initiate detachment of a single osteoblast (n=10) was >2000nN on a UV-treated surface at 3h incubation, which was 17 times greater than that on an untreated surface. The mean total energy required to complete the detachment of osteoblasts (n=10) was consistently >60pJ on a UV-treated titanium surface after 24h culture, which was up to 42 times greater than that on an untreated surface. Cellular shear modulus, which represents cellular stiffness, was consistently greater on a UV-treated surface than on an untreated surface after 24h incubation (n=10). This strengthening of cell adhesion and cellular mechanical properties on UV-treated titanium was accompanied by enhanced cell spread and actin fiber development and increased levels of vinculin expression. These results indicate that UV-photofunctionalization substantially strengthens osteoblast retention on titanium bulk material with no topographical features, and that this is associated with enhancement of intracellular structural development during the cell adhesion process.

N-acetyl cysteine alleviates cytotoxicity of bone substitute.

Lack of cytocompatibility in bone substitutes impairs healing in surrounding bone. Adverse biological events around biomaterials may be associated with oxidative stress. We hypothesized that a clinically used inorganic bone substitute is cytotoxic to osteoblasts due to oxidative stress and that N-acetyl cysteine (NAC), an antioxidant amino acid derivative, would detoxify such material. Only 20% of rat calvaria osteoblasts were viable when cultured on commercial deproteinized bovine bone particles for 24 hr, whereas this percentage doubled on bone substitute containing NAC. Intracellular ROS levels markedly increased on and under bone substitutes, which were reduced by prior addition of NAC to materials. NAC restored suppressed alkaline phosphatase activity in the bone substitute. Proinflammatory cytokine levels from human osteoblasts on the bone substitute decreased by one-third or more with addition of NAC. NAC alleviated cytotoxicity of the bone substitute to osteoblastic viability and function, implying enhanced bone regeneration around NAC-treated inorganic biomaterials.

Purification, cDNA cloning, and characterization of a new serpin with megakaryocyte maturation activity.

A new member of the serine protease inhibitor (serpin) superfamily with megakaryocyte maturation activity was purified, and its cDNA was cloned and characterized. The predicted amino acid sequence consisting of 380 residues was unique and was 38% identical to the serpin plasminogen activator inhibitor type 2 (PAI-2). The recombinant factor expressed in Chinese hamster ovary cells showed species-specific activity on the induction of megakaryocyte maturation in vitro. When injected into mice, the factor indeed elicited an increase in the number of platelets in plasma. The sequence alignment indicated that the factor possessed a lysine residue at the P1 position, suggesting that it might function as an inhibitor of Lys-specific proteases. Although we could not show any inhibitory activities toward several known Lys-specific proteases, we detected the activity toward protease activity present in the culture supernatant of COLO 201 cells. These results suggested that the protein might influence the maturation of megakaryocytes via action as a serpin.

Phenotypic variation in human HepG2 hepatoma cells: alterations in cell growth, plasma protein synthesis and heme pathway enzymes.

1. Growth rates, morphology, plasma protein synthesis and the level of heme pathway enzymes were examined in six sublines of HepG2 cells obtained from various laboratories. 2. Five sublines represented by G2a display the known characteristics of HepG2 cell type, including morphology, plasma protein synthesis and an increase in delta-aminolevulinic acid (ALA) dehydratase activities in response to Me2SO treatment. 3. In contrast, cells of the G2f subline failed to secrete significant quantities of plasma proteins. There was also no increase in ALA dehydratase activity following Me2SO treatment. These findings suggest that G2f cells represent a variant of HepG2 cells with an altered phenotype.

delta-Aminolaevulinate synthase in human HepG2 hepatoma cells. Repression by haemin and induction by chemicals.

delta-Aminolaevulinate (ALA) synthase, the rate-limiting enzyme in haem biosynthesis in the normal liver, was examined in human HepG2 hepatoma cells. Haemin, up to 100 microM, had no effect on ALA synthase activity in vitro; it did, however, exhibit a dose-dependent inhibitory action when added to cells growing in culture (half-maximal inhibition at 1 microM). The half-life of ALA synthase activity after haemin treatment was 2 h, which was similar to that found after treatment with cycloheximide. Cells treated with actinomycin D showed a longer half-life of the enzyme activity, i.e. 4 h, compared with haemin or cycloheximide treatment. Treatment of cells with succinylacetone markedly inhibited the activity of ALA dehydratase and 59Fe incorporation into haem, but in increased ALA synthase activity. Both the haemin-induced repression and the succinylacetone-mediated de-repression of ALA synthase activity were reversible within 4 h after replacing the medium with fresh medium without the chemical. In addition to succinylacetone, dimethyl sulphoxide and 3-methylcholanthrene induced the enzyme. Induction of ALA synthase by these chemicals was also suppressed by treatment of cells with haemin. These findings indicate that the level of ALA synthase in HepG2 cells is maintained by both synthesis and degradation of the enzyme, and that the synthesis of the enzyme is regulated by the concentration of regulatory free haem in the cell.

Differential stimulation of rat lung particulate guanylate cyclase activity by atrial natriuretic peptide and sodium nitroprusside.

The effects of alpha-rat atrial natriuretic peptide (alpha-rANP) and sodium nitroprusside on the activity of rat lung particulate guanylate cyclase were examined. The particulate guanylate cyclase in partially purified rat lung membranes was stimulated by both alpha-rANP and nitroprusside. The effects of alpha-rANP and nitroprusside were, however, not additive. Diamide and N-ethylmaleimide almost completely abolished the nitroprusside-mediated stimulation, while they had only moderate effects on the alpha-rANP-mediated stimulation of the enzyme activity. ATP potentiated the enzyme stimulation by alpha-rANP, whereas it had no effect on the nitroprusside-mediated stimulation. These findings suggest that the stimulation of lung particulate guanylate cyclase activity by alpha-rANP and nitroprusside is mediated by different mechanisms.

The effects of acute-phase inducers and dimethyl sulphoxide on delta-aminolaevulinate synthase activity in human HepG2 hepatoma cells.

The effects of acute-phase inducers and dimethyl sulphoxide (Me2SO) on delta-aminolaevulinate (ALA) synthase in HepG2 cells were examined. Treatment of cells with Me2SO resulted in a significant increase in ALA synthase activity. Interleukin-6 increased ALA synthase activity only slightly, but it substantially potentiated the induction of ALA synthase by Me2SO. These data suggest that ALA synthase activity in liver is altered during acute-phase reactions.

A comparative study of the distribution of fluorescently labeled calmodulin and tubulin in the meiotic apparatus of the mouse oocyte.

The localizations of tubulin and calmodulin were investigated in the mouse oocyte during the second meiosis by fluorescently labeling and microinjecting these proteins prepared from porcine brain tissue. When injected, both tubulin and calmodulin were quickly incorporated into the preformed meiotic apparatus of the oocyte at metaphase. The localization of labeled tubulin was coincident with that of birefringence. However, the localization of labeled calmodulin was somewhat different: the fluorescence of calmodulin was intense in the polar regions of the spindle. After the chromosomes began to move, followed by parthenogenetic activation upon microinjection of a calcium buffer, these two fluorescent proteins, localized in the meiotic apparatus, moved to the interzonal region of the spindle during anaphase. At late anaphase and throughout telophase, calmodulin was excluded from the mid-bodylike structures in the interzonal region, whereas tubulin did accumulate in these structures.

The effect of dimethyl sulfoxide on heme synthesis and the acute phase reaction in human HepG2 hepatoma cells.

Effects of DMSO on heme synthesis and enzymes of the heme biosynthetic pathway were examined in human HepG2 hepatoma cells. HepG2 cells contain measurable levels of ALA synthase and ALA dehydratase, and their levels are increased after treatment of cells with DMSO. DMSO treatment also led to increases in heme content and the synthesis of haptoglobin, while it decreased the synthesis of albumin and AFP. Changes in plasma protein synthesis after DMSO treatment are characteristic of those known to occur in the acute phase reaction. These findings suggest that profound changes in heme synthesis may occur during the acute phase reaction.

Effects of dimethyl sulphoxide on the synthesis of plasma proteins in the human hepatoma HepG2. Induction of an acute-phase-like reaction.

Effects of dimethyl sulphoxide (Me2SO) on the synthesis of plasma proteins by the human hepatoma cell line HepG2 were examined. Me2SO treatment resulted in decreased synthesis of albumin and alpha-fetoprotein, and in increased synthesis of haptoglobin. Plasma-protein profiles induced by Me2SO treatment were very similar to those seen in acute-phase reactions.

Stimulation of guanylate cyclase activity by irreversible binding of atrial natriuretic peptide to its receptor.

We examined receptor binding profiles of atrial natriuretic peptide (ANP) in rat tissue using 125I-labeled alpha-rat ANP [( 125I]alpha-rANP). Specific [125I]alpha-rANP binding to its receptor was reversible following addition of unlabeled free alpha-rANP, but it became increasingly irreversible with time during incubation. Irreversible binding of alpha-rANP was observed both at 0 degrees and 25 degrees in homogenates of adrenal capsules and lungs, crude membranes of renal glomeruli, partially purified membranes of lung, solubilized membrane preparations from renal glomeruli, and intact renal glomeruli. Irreversible binding increased in a time- and temperature-dependent manner. HPLC analysis demonstrated that the irreversibly bound radioactivity, which was extracted by 1 N CH3COOH from both intact renal glomeruli and from partially purified membranes, was associated with intact [125I]alpha-rANP. Irreversibly bound alpha-rANP increased cGMP concentrations by activating guanylate cyclase activity. These findings suggest that the appearance of irreversible binding of alpha-rANP to its receptor is independent of its internalization, and may be involved in message transduction and subsequent biological responses.

Protective effect of alpha-human atrial natriuretic polypeptide (alpha-hANP) on chemical-induced pulmonary edema.

It has been established that alpha-hANP, the newly discovered peptide extracted from human cardiac atria, has potent natriuretic and hypotensive actions. Our present investigation is the first to demonstrate that alpha-hANP is capable of protecting against pulmonary edema caused by various chemicals, using isolated perfused guinea pig lung system. Lungs were perfused via pulmonary artery with Krebs-Ringer bicarbonate buffer at 5.0 ml/min, and wet weight of lungs and perfusion pressure of pulmonary artery (Pa) were monitored. Bolus injection of Triton-X or CHAPS into cannulated pulmonary artery produced edema as indicated by a massive increase in wet weight and a slight increase in Pa. Constant infusion of alpha-hANP through pulmonary artery at 200 ng/ml was effective in causing decrease in wet weight of lung. Perfusion of lung with paraquat or PGF2 alpha, and repeated bolus injection of arachidonic acid or PGE2 caused elevation in both wet weight of lung and Pa. The treatment with alpha-hANP similar to that described above also protected against edema caused by paraquat or arachidonic acid. Bolus administration of epinephrine induced a slight increase in wet weight and Pa, and alpha-hANP was effective in decreasing the elevated lung wet weight and Pa of lungs. Infusion or bolus administration of alpha-hANP into control lungs increased cGMP level in outflow perfusate as well as in lung tissue significantly. In lungs with edema which were induced by Triton-X or paraquat, there was a slight increase in cGMP level in Triton-X treated and no increase in paraquat treated lung tissues. In either cases, was there any increase in cGMP level in perfusate. The specific binding study of [125I]alpha-hANP revealed that the lack of increase in cGMP was not due to a loss of receptor in Triton-X or paraquat treated lungs. Thus our study demonstrated that alpha-hANP had a direct anti-edematic action(s) in lung which was not secondary to the systemic natriuretic and/or hypotensive action(s).

A novel 15 kDa Ca2+-binding protein present in the eggs of the sea urchin, Hemicentrotus pulcherrimus.

A novel Ca2+-binding protein, different from calmodulin, has been purified to homogeneity from the soluble cytoplasmic protein fraction of the egg of the sea urchin, Hemicentrotus pulcherrimus. This protein, designated as 15 kDa protein, shows a Ca2+-dependent mobility shift upon SDS-gel electrophoresis and has Ca2+-binding ability. This protein did not resemble the sea urchin egg calmodulin in either molecular mass or amino acid composition. The 15 kDa protein could not activate cyclic adenosine 3',5'-monophosphate-dependent phosphodiesterase from bovine brain and did not bind to fluphenazine-Sepharose 6B. Antibodies against the 15 kDa protein did not react with sea urchin egg calmodulin. These results suggest that the 15 kDa protein is a novel Ca2+-binding protein in the sea urchin egg.

Calmodulin-binding protein (55K + 17K) of sea urchin eggs has a Ca2+- and calmodulin-dependent phosphoprotein phosphatase activity.

A calmodulin-binding protein from sea urchin eggs consisting of two subunits (55 and 17K-daltons) was identified as a Ca2+-dependent phosphoprotein phosphatase similar to calcineurin in mammalian brain and to phosphatase 2B in skeletal muscle. Peptide mappings showed that the 55K subunit was different from 61K subunit of calcineurin, whereas the 17K subunit was similar to 19K subunit of calcineurin but different from calmodulin. The 55K + 17K protein of sea urchin eggs dephosphorylated 32P-inhibitor-1 in a Ca2+- and calmodulin-dependent manner. Vmax and Km for inhibitor-1 in the presence of Ca2+ and calmodulin were 2,100 pmol Pi/min/mg and 2.7 microM. Ca2+-dependent phosphatase activity for inhibitor-1 was detected in homogenates of both unfertilized and fertilized eggs, but was not detected in isolated cortices and mitotic apparatus.

Calmodulin-binding proteins in the cytosol extract of sea urchin eggs.

Calmodulin and calmodulin-binding proteins in the cytosol extract of eggs from the sea urchins Hemicentrotus pulcherrimus and Strongylocentrotus intermedius were studied in an attempt to elucidate the physiological role(s) of calmodulin in eggs. Calmodulin in the cytosol extract was found both in a free form and in complexes with other proteins, either Ca2+-dependently or Ca2+-independently. The extracts contained at least three calcium-dependent calmodulin-binding proteins. One was an NAD kinase of unknown molecular composition. The apparent molecular weights of the other two calmodulin-binding proteins were 50K and 55K + 17K daltons, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These three proteins formed complexes with calmodulin only in the presence of calcium as demonstrated by gel filtration. The 17K-dalton protein was found to be calmodulin itself; it did not dissociate from the 55K-dalton protein regardless of the presence or absence of calcium. The native molecular weights of these protein-calmodulin complexes obtained by gel filtration through a Sephacryl S-300 column were 190K for the NAD kinase, 130K for the 50K-dalton protein and 100K daltons for the 55K + 17K-dalton protein.