Biomedical subjects
Publications and source records attributed to F Sato.
Tandem addition of the two carbon-titanium bonds of a titanacyclopentadiene to the ester group of the same molecule affords carbocycles (shown schematically): The metal portion of the metallacycle has been replaced with a carbon fragment. Depending on the starting diyne, mono-, bi-, or tricyclic compounds can be obtained.
Two sublines of B16 melanoma cells, F10 and BL6, are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. We found a new member of the GTPase superfamily, namely TIB929, which displayed an induction of expression in BL6 cells. It conserved three consensus sequences for GTP-binding site motifs and showed a significant homology to the yeast Gtr2 gene throughout the coding sequence. TIB929 was expressed ubiquitously in human tumor cells, with a marked expression in highly metastatic cells. TIB929 was mapped on mouse chromosome 4D, syntenic to human chromosome 1p. The results suggested an involvement of TIB929 in malignant progression.
A collaborative study was conducted to evaluate whether a replicative DNA synthesis (RDS) test using the rat liver can detect nongenotoxic (Ames-negative) hepatocarcinogens with three or seven daily administrations at dose-levels effective in long-term bioassays. The assay methods were well-validated by the 14 participants. Of six compounds tested, carbon tetrachloride (50 and 100 mg/kg), clofibrate (125 and 250 mg/kg), diethylstilbestrol (0.125 and 0.25 mg/kg) and urethane (100 mg/kg) gave positive results, methyl carbamate (200 and 400 mg/kg) exerted equivocal effects, and D,L-ethionine (125 mg/kg) failed to elevate RDS. These findings suggest that the RDS test can detect many nongenotoxic rat hepatocarcinogens with short-term administration at dose-levels used in long-term bioassays.
After a brief exposure to supra-saturating light, leaves of a tobacco transformant, in which chloroplastic NAD(P)H dehydrogenase (NDH) was defective, showed more severe photoinhibition than the wild-type, when judged by the parameter of chlorophyll fluorescence Fv/Fm. Repeated application of supra-saturating light eventually resulted in chlorosis in the NDH-defective mutant, while the wild-type sustained less photodamage and was able to recover from it. The mechanism of the phenomena is discussed with respect to the potential role of NDH in photosynthesis.
The crystal structure of tobacco PR-5d, an antifungal thaumatin-like protein isolated from cultured tobacco cells, was determined at the resolution of 1.8 A. The structure consists of 208 amino acid residues and 89 water molecules with a crystallographic R-factor of 0.169. The model has good stereochemistry, with respective root-mean-square deviations from the ideal values for bond and angle distances of 0.007 A and 1.542 degrees. Of the homologous PR-5 proteins, only those with antifungal activity had a common motif, a negatively charged surface cleft. This cleft is at the boundary between domains I and II, with a bottom part consisting of a three-stranded antiparallel beta-sheet in domain I. The acidic residues located in the hollow of the cleft form the beta-sheet region. Sequence and secondary structure analyses showed that the amino acid residues comprising the acidic cleft of PR-5d are conserved among other antifungal PR-5 proteins. This is the first report on the high-resolution crystal structure of an antifungal PR-5 protein. This structure provides insight into the function of pathogenesis-related proteins.
We report here that the 12-lipoxygenase metabolite of arachidonic acid, 12-hydroxy-5Z, 8Z, 10E, 14Z, eicosatetraenoic acid (12-HETE), stimulates cAMP production in human fibroblasts among various cultured cell lines tested. Although 12-HETE seemed to stimulate the phospholipase C (PLC)-protein kinase C (PKC) system, inhibitors against PLC and PKC did not reduce the cAMP production induced by 12-HETE, indicating that the activation of PLC-PKC system is not positively coupled with the stimulation of cAMP production. On the other hand, the cAMP production induced by 12-HETE was dependent on the Ca2+/calmodulin system in the cells. The results suggest that 12-HETE specifically stimulates Ca2+/calmodulin-dependent adenylyl cyclase to increase cAMP level in the fibroblasts.
Hepatic cells isolated from an adult rat liver, consisting of small hepatocytes (SHs), mature hepatocytes (MHs), liver epithelial cells (LECs), Kupffer cells, sinusoidal endothelial cells, and stellate cells, were cultured in a medium supplemented with 10% fetal bovine serum, 10 mmol/L nicotinamide, 1 mmol/L ascorbic acid 2-phosphate, 10 ng/mL epidermal growth factor, and 1% dimethyl sulfoxide. The SHs rapidly proliferated and formed a colony. About 10% of cytokeratin 8 (CK8)-positive cells formed SH colonies. All SHs at day 10 immunocytochemically showed positivity for albumin, transferrin, CK8, and CK18, which are markers for hepatocytes. In contrast, alpha-fetoprotein (AFP)-, CK14-, OC2-, and glutathione S-transferase placental type (GST-P)-positive cells, which are thought to be markers for hepatic immature cells, were rarely observed. At day 20 some cells in the colonies were positive for AFP, CK7, CK19, and GST-P. LECs and stellate cells proliferated and surrounded the colonies. About 2 weeks after plating, piled up cells were often observed on the SH colonies. In those colonies LECs and stellate cells invaded under the colonies. The invasion of the cells and gradual deposits of extracellular matrix (ECM) such as type I collagen, type IV collagen, and laminin induced alteration of the shape of the SHs from relatively flat to cuboidal or rectangular. With the cellular structural changes, the expression of albumin, connexin 32 (Cx32), and tryptophan 2,3-dioxygenase (TO) messenger RNAs increased. In addition, overlapping nonparenchymal cells (NPCs) on the piled up cells induced the formation of duct- or cyst-like structures consisting of MHs. In the present experiment we showed that SHs could differentiate to MHs by interacting with NPCs and ECM. Thus, SHs may be "committed progenitor cells" that can further differentiate into MHs.
Aortic dissection is a rare but devastating complication of cardiac surgery. Adequate and early diagnosis of intraoperative aortic dissection and quick therapeutic decision making are the keys for saving patients in such cases. We describe the case of a 68-year-old man referred for CABG and mitral valve replacement with severe calcification of the ascending aorta. Intra-operative transesophageal echocardiography was useful for diagnosis of intra-operative aortic dissection and malperfusion of the true lumen. Immediate switching of the arterial perfusion site established flow in the true lumen with prompt subsidence of the expanded false lumen. CABG, mitral valve replacement and graft replacement of the ascending aorta could be simultaneously performed in this patient.
Topical vitamin D3 has relatively recently been introduced for the treatment of psoriasis. Synthetic vitamin D3 analogues with a high potential for inducing differentiation of cells, but with a low hypercalcemic effect have recently been developed. One such synthetic analogue of 1,25-dihydroxyvitamin D3 (calcitriol), 22-oxacalcitriol (OCT), is a novel agent for the topical treatment of psoriasis. The activity of OCT in vitro was investigated and compared with that of a series of vitamin D3 analogues as to their ability to inhibit murine T lymphocyte proliferation stimulated by con-A, to suppress IL-6 and IL-8 production by keratinocytes stimulated with IL-1alpha and TNFalpha, and to inhibit AP-1- and NFkappaB-dependent reporter gene expression. OCT inhibited the proliferation of lymphocytes and suppressed IL-8 and IL-6 production by keratinocytes to the same extent as the other vitamin D3 analogues. It also inhibited AP-1- and NFkappaB-controlled luciferase activity to the same extent as the other vitamin D3 analogues, which demonstrates its mechanism of action in the suppression of inflammatory processes.
The hypolipidemic effect of NK-104 and its mechanisms of action (effects on hepatic sterol synthesis, low density lipoprotein (LDL)-receptor expression and very low density lipoprotein (VLDL) secretion) were studied in guinea pigs using simvastatin as a reference substance. There was a dose-dependent and significant reduction of both plasma total cholesterol (17.4, 24.5 and 45.3% at 0.3, 1 and 3 mg/kg, respectively) and triglycerides (21.1 and 32.2% at 1 and 3 mg/kg, respectively) after 14-day administration of NK-104. Simvastatin at 30 mg/kg lowered plasma total cholesterol (25.0%) but not triglyceride levels. NK-104 (3 mg/kg) and simvastatin (30 mg/kg) inhibited hepatic sterol synthesis by approximately 80%, 3 h after dosing, and enhanced LDL receptor binding-capacity of liver membranes 1.5-fold after 14-day dosing. The former group accelerated LDL clearance somewhat more markedly than the latter, and increased fractional catabolic rate 1.8-fold (vs. 1.4-fold). Furthermore, only the NK-104 (3 mg/kg) suppressed VLDL secretion into the liver perfusate (triglyceride. 19.9%; apoB, 24.2%) with extensive reduction of hepatic sterol synthesis caused by prolonged action. These results indicate that NK-104 and simvastatin at 10 times the dosage of the former, similarly enhances hepatic LDL receptor; however, only NK-104 with prolonged action suppresses VLDL secretion to show higher cholesterol-lowering potency and triglyceride-reducing effect.
Chloroplast replication in cultured cells of tobacco (Nicotiana tabacum cv. Samsun NN) was investigated by electron microscopy in comparison with that of green leaves. The structure of chloroplasts in cultured cells changed conspicuously during cell growth especially in photoautotrophic cells. The frequency of dumbbell-shaped chloroplasts (intermediate of chloroplast division) was the maximum (about 23% of total chloroplast) in photoautotrophic cells at 3 days after inoculation, before the cells had started to grow. By contrast, in photomixotrophically cultured cells, the highest frequency of dividing chloroplasts was observed at the early exponential phase (about 7 days after inoculation). The dividing chloroplast was hardly detected in green leaves even at a young stage. The advantages of cultured cells for the study of chloroplast replication and ultrastructural development are discussed.
Lymph node metastasis is a strong independent prognostic factor for oesophageal cancer. The expression of matrix metalloproteinases (MMPs) and reduction of E-cadherin correlate with lymph node metastasis of oesophageal cancer. We previously reported that the expression of vascular endothelial growth factor (VEGF) is associated with lymph node metastasis. This study was designed to determine whether VEGF, MMP-9 and E-cadherin expression is stable or changes in the process of lymph node metastasis of oesophageal cancer. Using immunohistochemistry, we detected VEGF, MMP-9 and E-cadherin expression in paraffin-embedded specimens of oesophageal squamous cell carcinoma. We classified 134 primary tumours and 174 nodal metastases using two different criteria: the absence [Group N(-)] or presence [Group N(+)] of nodal metastasis, and the stage of metastasis--Early Stage (cancer cells < 50% of lymph node) or Late Stage (> or = 50%)--and compared the expression among two groups and among two stages. The expression rates of Group N(-), Group N(+), Early Stage and Late Stage are as follows: VEGF (49%, 74%, 60%, 33%), MMP-9 (76%, 65%, 95%, 69%) and E-cadherin (49%, 24%, 55%, 38%). VEGF expression was down-regulated in Late Stage lymph node metastasis, while MMP-9 expression was elevated in Early Stage metastasis. E-cadherin expression is restored somewhat in Early Stage metastasis, but suppressed again in Late Stage metastasis. These data suggest that the expression of VEGF, MMP-9 and E-cadherin each change in the process of lymph node metastasis in oesophageal cancer, and that the patterns of change are different.
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Higher plants accumulate several kinds of "pathogenesis-related (PR)" proteins in response to infection by pathogens such as fungi or viruses. Gene expression of one group of PR proteins is known to be mediated by phytohormone ethylene. Here we describe the signal transduction system from the ethylene receptor ETR to transcription factors, ERFs. Ethylene-inducible PR genes are expressed constitutively in roots and cultured cells even when are not infected. We discuss the mechanisms of this pathogen-independent expression of PR genes and describe recent findings in the study of molecular mechanisms of antifungal activities of the PR proteins. Genes of PR-1 and -5 proteins have now been identified in the genomes of various species of organisms, including humans and nematodes. PR proteins may contribute to the innate immunity of plants as well as to that of other organisms.
AIMS/BACKGROUND: We report in this study that, 10 mM nicotinamide can stimulate the proliferation of primary rat hepatocytes in serum-free Dulbecco's modified Eagle's medium supplemented with 10 ng/ml epidermal growth factor and that small hepatocyte colonies appear from 4 to 5 days after plating. We examined the effects of nicotinamide-related agents on the growth and differentiation of primary rat hepatocytes and on the appearance of small hepatocyte colonies. METHODS: As nicotinamide is an aqueous vitamin named niacin and known to act as an inhibitor of poly (ADP-ribose) polymerase (PARP), we therefore chose to examine the effects on hepatocytes of three nicotinamide-related agents, nicotinic acid (NA) which is also a niacin, 3-aminobenzamide (3-AB) which is a strong inhibitor of PARP but is not a niacin, and 3-acetylpyridine (3-AP) which is a weak inhibitor of PARP and also not a niacin. To examine their effects on the growth of the cells and on the formation of the colony, immunocytochemistry for BrdU was carried out. Expression of albumin, tryptophan 2,3-dioxygenase (TO), and connexin 32 (Cx32) mRNAs were used as marks of hepatic differentiation. Intracellular NAD+ content was also measured. RESULTS: At concentration of 10 mM, NA could not enhance the proliferation of mature hepatocytes but induced the appearance of small hepatocyte colonies. At concentration of 5 mM, 3-AB enhanced the proliferation of the hepatocytes but did not induce small hepatocyte colonies. On the other hand, although 10 mM 3-AP remarkably inhibited the DNA synthesis of the cells, the expression not only of albumin but also of TO and Cx32 mRNAs in the cells was well maintained for more than one week. The intracellular NAD+ concentration was correlated with the proliferation of the hepatocytes. CONCLUSION: These results suggest that the intracellular NAD+ content may be correlated with the proliferation of primary hepatocytes and that the supplementation of niacin in the medium may be important for the appearance of small hepatocyte colonies.
It has been suggested that a variety of stresses on animals may accelerate their production of superoxide. Racehorses are considered to be exposed to substantial oxide stress due to transport and exercise for training and racing. To determine the effect of exercise and transport on racehorses in terms of superoxide and antioxidative ability, changes in the superoxide-scavenging ability of equine serum were observed using electron spin resonance (ESR). Changes in the concentration of lipid peroxide, which is produced in equine serum by superoxide, were also examined. The analysis revealed that lipid peroxide concentrations increased as a result of stress from exercise and transport. On the other hand, the superoxide-scavenging ability of equine serum showed a decline during transport, which is in sharp contrast to the increase seen immediately after a race due to the severe load exerted in exercise.
Adenosine, a purine nucleoside, plays a variety of roles in cardiovascular and ventilatory control, and may be a marker of tissue hypoxia. There is, however, no direct evidence of an increase in plasma or in tissue levels of adenosine during moderate hypoxia in humans. We measured the plasma concentrations of adenosine in an artery and the median cubital vein simultaneously in 12 normal volunteers, and also in the internal jugular vein in seven of them during normoxia and moderate hypoxia (SaO2 = 80%, 20 min) with or without dipyridamole (0.6 mg/kg) pretreatment. Dipyridamole was expected to block reuptake of adenosine by red blood cells and vascular endothelial cells so that the plasma level of adenosine would more likely reflect the tissue level. Blood was sampled with an appropriate stopping solution, and adenosine was measured with a high-pressure liquid chromatographic (HPLC)-fluorometric technique. The plasma concentration of adenosine did not rise either in the artery or in the vein at any phase of hypoxia without the dipyridamole pretreatment. However, when subjects were pretreated with dipyridamole, the plasma concentration of adenosine increased significantly and markedly in a time-dependent manner during hypoxia in the vein, but not in the artery. The adenosine level rose from 20. 7 +/- 2.5 nM (mean +/- SE) during normoxia to 50.7 +/- 10.7 nM at 20 min of hypoxia, and returned to the baseline level in the recovery phase. The plasma concentration of adenosine in the jugular vein did not change during hypoxia either with or without dipyridamole pretreatment. These data provide evidence that in humans, the local production of adenosine increases during moderate hypoxia in forearm tissue, although this is not reflected in plasma unless the subject is pretreated with dipyridamole.
The entire cDNA sequences were determined by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques for equine copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD) through the use of total RNA extracted from the testis of an adult Thoroughbred. The results revealed a protein coding region for equine Cu/Zn-SOD with bases totaling 465 bp, accompanied by an estimated 154 residues of amino acids. As for equine Mn-SOD, its coding region contained a total of 669 bp and an estimated 222 residues of amino acids. Further, the expression of Cu/Zn-SOD and Mn-SOD genes were confirmed in the equine tissues by RT-PCR and in situ hybridization.