Naokatu Arakaki

Native and recombinant human hepatocyte growth factors are highly potent promoters of DNA synthesis in both human and rat hepatocytes.

Human hepatocyte growth factor (hHGF) has recently been expressed as a recombinant polypeptide from Chinese hampster ovary cell transfectants. Using a primary rat hepatocyte bioassay, we have tested the biological activity of recombinant hHGF and compared it with native hHGF. Dose-response curves were almost identical, with half-maximal stimulation of DNA synthesis at 1-2 ng/ml (equivalent to approximately 10 pM). S-phase labeling index was similarly enhanced and numerous mitotic cells were observed. Recombinant and native hHGF also stimulated DNA synthesis and S-phase labeling index in primary adult human hepatocytes. Human cells were more responsive than rat hepatocytes, with recombinant hHGF slightly more potent than native hHGF (half-maximal stimulation 0.3 and 0.6 ng/ml, respectively). Since HGF levels rise in patients with fulminant hepatic failure and in animals after partial hepatectomy or administration of hepatotoxins, situations where liver regeneration occurs, HGF is suggested to play a key role in regulation of hepatic growth. The high potency of the factor on human hepatocytes reinforces its candidacy as a critical mitogen in human liver growth. The availability of a recombinant hHGF opens the way for in vivo experimental studies and to the possibility of using hHGF as a clinical therapeutic agent, either alone or in combination with other factors.

Alkyl gallates, intensifiers of beta-lactam susceptibility in methicillin-resistant Staphylococcus aureus.

ABSTRACT We found that ethyl gallate purified from a dried pod of tara (Caesalpinia spinosa) intensified β-lactam susceptibility in methicillin-resistant and methicillin-sensitive strains of Staphylococcus aureus (MRSA and MSSA strains, respectively). This compound and several known alkyl gallates were tested with MRSA and MSSA strains to gain new insights into their structural functions in relation to antimicrobial and β-lactam susceptibility-intensifying activities. The maximum activity of alkyl gallates against MRSA and MSSA strains occurred at 1-nonyl and 1-decyl gallate, with an MIC at which 90% of the isolates tested were inhibited of 15.6 μg/ml. At concentrations lower than the MIC, alkyl gallates synergistically elevated the susceptibility of MRSA and MSSA strains to β-lactam antibiotics. Such a synergistic activity of the alkyl gallates appears to be specific for β-lactam antibiotics, because no significant changes were observed in the MICs of other classes of antibiotics examined in this study. The length of the alkyl chain was also associated with the modifying activity of the alkyl gallates, and the optimum length was C5 to C6. The present work clearly demonstrates that the length of the alkyl chain has a key role in the elevation of susceptibility to β-lactam antibiotics.

Involvement of Oxidative Stress in Tumor Cytotoxic Activity of Hepatocyte Growth Factor/Scatter Factor

In this study, we show thatN-acetylcysteine (NAC), a precursor of glutathione and an intracellular free radical scavenger, almost completely prevented hepatocyte growth factor (HGF)-suppressed growth of Sarcoma 180 and Meth A cells, and HGF-induced apoptosis, assessed by DNA fragmentation, and increase in caspase-3 activity, in Sarcoma 180 cells. The reduced form of glutathione also prevented HGF-suppressed growth of the cells as effective as NAC. Ascorbic acid partially prevented the effect of HGF, but other antioxidants such as superoxide dismutase, catalase, and vitamin E, and the free radical spin trapsN-t-butyl-α-phenylnitrone and 3,3,5,5-tetramethyl-1-pyrroline-1-oxide did not have protective effects. HGF caused morphological changes of the cells, many cells showing condensation and rounding, and enhanced the generation of intracellular reactive oxygen species (ROS) as judged by flow cytometric analysis using 2′,7′-dichlorofluorescein diacetate. NAC completely prevented both HGF-induced morphological changes and the enhancement of ROS generation in the cells. However, NAC did not prevent the HGF-induced scattering of Madin-Darby canine kidney cells. To our knowledge, this is the first report that HGF stimulates the production of ROS, and our results suggest the involvement of oxidative stress in the mechanism by which HGF induces growth suppression of tumor cells.

Identification of the N-terminal residue of the heavy chain of both native and recombinant human hepatocyte growth factor.

The N-terminal amino acid of the heavy chain of native (purified from human plasma) and recombinant human hepatocyte growth factor (hHGF) was determined by analyses of amino acid composition and sequence of peptide fragments derived by enzymatic cleavage, peptide mapping, and fast atom bombardment mass spectrometry. Our results indicate that the N-terminal amino acid of the heavy chain of hHGF, both native and recombinant, is pyroglutamate, derived from glutamine at the 32nd residue from the initiation methionine.

Cell-surface H+-ATP synthase as a potential molecular target for anti-obesity drugs

Here we show that the cell‐surface expression of the alpha subunit of H+‐ATP synthase is markedly increased during adipocyte differentiation. Treatment of differentiated adipocytes with small molecule inhibitors of H+‐ATP synthase or antibodies against alpha and beta subunits of H+‐ATP synthase leads to a decrease in cytosolic lipid droplet accumulation. Apolipoprotein A‐I, which has been shown to bind to the ectopic β‐chain of H+‐ATP synthase and inhibit the activity of cell‐surface H+‐ATP synthase, also was found to inhibit cytosolic lipid accumulation. These results suggest that the cell‐surface H+‐ATP synthase has a previously unsuspected role in lipid metabolism in adipocytes.

Expression of HGF/SF in mesothelioma cell lines and its effects on cell motility, proliferation and morphology

The expression of hepatocyte growth factor/scatter factor (HGF/SF) was studied in 12 mesothelioma cell lines characterized by either an epithelioid or a fibroblast-like phenotype. Conditioned media from these lines were analysed by bioassay and ELISA, and HGF/SF was detected in three cell lines, all with a fibroblast-like or mixed morphology. None of eight epithelioid cell lines expressed the factor. Thus, for these cell lines, the ability to secrete HGF/SF correlated with the cell phenotype. Following on from these observations, two cell lines, BR and BT, with a fibroblast-like and an epithelioid phenotype, respectively, were further investigated. Both cell lines expressed the Met receptor but only BR secreted HGF/SF. Both cell lines responded to exogenous HGF/SF treatment by a change of morphology but in different ways: BR became more elongated and bipolar, while BT formed more spread-out cell colonies. HGF/SF acted as a paracrine effector on the epithelioid BT cells and stimulated both cell-spreading and proliferation. Interestingly, BT cells spread but did not scatter in response to exogenous HGF/SF. In contrast BR cells showed only some stimulation of cell motility with HGF/SF and no increase in cell proliferation was observed. Because HGF/SF was previously found in the pleural effusion fluids of patients with malignant mesothelioma and in paraffin-embedded tumour tissues, it is concluded that HGF/SF may well stimulate the growth and spread of malignant mesothelioma in vivo by paracrine and/or autocrine mechanisms.

Hepatocyte growth factor/scatter factor is present in most pleural effusion fluids from cancer patients.

Pleural effusion samples were obtained from 55 patients with malignant disease, including patients with primary lung cancers and those with a variety of other tumours metastatic to the pleura. The effusions were assayed for the presence of hepatocyte growth factor/scatter factor (HGF/SF), by both ELISA and bioassay. The presence of malignant cells in the effusions was also assessed. Detectable amounts of the factor, as judged by both criteria, were found in over 90% of all the effusions, including those from patients with a wide variety of carcinomas and also lymphomas. A wide range of HGF/SF levels were found for all tumour classes, some effusions containing high levels above 4 ng ml-1. It is concluded that tumours within the pleura and adjacent lung tissue are usually exposed to biologically significant levels of HGF/SF.

Possible Role of Mitochondrial Remodelling on Cellular Triacylglycerol Accumulation

Mitochondrial fusion and fission processes play a role in a variety of cell functions, including energy metabolism, cell differentiation and programmed cell death. Still, it is not clear how these processes contribute to the cell functions. Here, we investigated the role of mitochondrial remodelling on lipid metabolism in adipocytes. In 3T3-L1 pre-adipocytes, the morphology of mitochondria is organized as a continuous reticulum. Upon differentiation of adipocytes manifested by cellular triacylglycerol (TG) accumulation, mitochondrial morphology altered from filamentous to fragmented and/or punctate structures. When the mitochondrial fusion was induced in adipocytes by silencing of mitochondrial fission proteins including Fis1 and Drp1, the cellular TG content was decreased. In contrast, the silencing of mitochondrial fusion proteins including mitofusin 2 and Opa1 increased the cellular TG content followed by fragmentation of mitochondria. It also appears that polyphenolic phytochemicals, negative regulators of lipid accumulation, have mitochondrial fusion activity and that there is a good correlation between mitochondrial fusion activity and the cellular TG accumulation-reducing activity of the phytochemicals. These results suggest that cellular TG accumulation is regulated, at least in part, via mitochondrial fusion and fission processes.

Prostaglandin E2 Predominantly Induces Production of Hepatocyte Growth Factor/Scatter Factor in Human Dental Pulp in Acute Inflammation

Hepatocyte growth factor (HGF), which is also known as the scatter factor, is a broad-spectrum and multifunctional cytokine, mediates epithelial-mesenchyme interaction, and is shown to be involved in the development and regeneration of various tissues, including tooth. Here, we report that HGF was present in adult human dental pulps, and its levels increased during acute inflammation of the tissue. Levels of HGF mRNA in dental pulps also increased with inflammation, as determined by reverse-transcription/polymerase chain-reaction. The production of HGF in fibroblasts from dental pulps in culture was dose-dependently stimulated by inflammatory cytokines such as interleukin (IL)-la and tumor necrosis factor (TNF)-a, and by prostaglandin (PG) E2, as determined by an enzyme-linked immunosorbent assay. We also showed that indomethacin did not affect the increase in HGF production by the cells with IL-la, TNF-a, and PGE2 . The levels of HGF mRNA in the cells were simultaneously increased by these stimulants, as determined by Northern blotting. Since the production of PGs is known to increase at the beginning of inflammation, PGE2 may be involved in the regeneration of dental pulps by the induction of HGF expression after inflammation.

Pigment epithelium-derived factor binds to cell-surface F1-ATP synthase

Pigment epithelium‐derived factor (PEDF), a potent blocker of angiogenesis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to an as yet unknown protein on the surfaces of endothelial cells. Given that protein fingerprinting suggested a match of a ∼ 60 kDa PEDF‐binding protein in bovine retina with Bos taurus F1‐ATP synthase β‐subunit, and that F1Fo‐ATP synthase components have been identified recently as cell‐surface receptors, we examined the direct binding of PEDF to F1. Size‐exclusion ultrafiltration assays showed that recombinant human PEDF formed a complex with recombinant yeast F1. Real‐time binding as determined by surface plasmon resonance demonstrated that yeast F1 interacted specifically and reversibly with human PEDF. Kinetic evaluations revealed high binding affinity for PEDF, in agreement with PEDF affinities for endothelial cell surfaces. PEDF blocked interactions between F1 and angiostatin, another antiangiogenic factor, suggesting overlapping PEDF‐binding and angiostatin‐binding sites on F1. Surfaces of endothelial cells exhibited affinity for PEDF‐binding proteins of ∼ 60 kDa. Antibodies to F1β‐subunit specifically captured PEDF‐binding components in endothelial plasma membranes. The extracellular ATP synthesis activity of endothelial cells was examined in the presence of PEDF. PEDF significantly reduced the amount of extracellular ATP produced by endothelial cells, in agreement with direct interactions between cell‐surface ATP synthase and PEDF. In addition to demonstrating that PEDF binds to cell‐surface F1, these results show that PEDF is a ligand for endothelial cell‐surface F1Fo‐ATP synthase. They suggest that PEDF‐mediated inhibition of ATP synthase may form part of the biochemical mechanisms by which PEDF exerts its antiangiogenic activity.