Osteogenic extracellular matrix sheet for bone tissue regeneration.

The application of extracellular matrix (ECM) sheets without a scaffold is not extensively reported in bone regenerative medicine. The aim of the present study was to demonstrate that an osteogenic ECM sheet (OECMS) can retain ECM integrity and growth factors to enhance bone formation in a rat non-union model. OECMS was produced from osteogenic cell sheets (OCS). Collagen and growth factor [bone morphogenetic protein 2 (BMP-2), vascular endothelial growth factors (VFGFs), basic fibroblast growth factor (bFGF) and transforming growth factor ß1 (TGF-ß1)] concentrations in the OECMS were quantified by enzyme-linked immunosorbent assay (ELISA). Next, hydroxyapatite (HA) constructs combined with OECMSs were implanted subcutaneously into the rats' backs to evaluate their osteoinductive capacity by histological evaluation. In addition, OECMSs were implanted in a rat femoral non-union model. 18 male Fischer 344 inbred rats were divided into OECMS and control groups. Fracture healing was evaluated by radiological and histological analyses at 2, 5 and 8 weeks and biological analysis at 8 weeks. Collagen I and growth factors were retained in the OECMSs. Osteoid formation was identified in the HA combined with OECMS at 4 weeks. Enhanced bone regeneration at the non-union of the OECMS group was confirmed at 5 and 8 weeks. Biomechanical testing revealed a significantly higher maximum bending load in the OECMS group as compared to the control group at 8 weeks. The results demonstrated that OECMS retained BMP-2 and TGF-ß1 and high osteoinductive and osteoconductive capacity. As such, OECMS represents a potential new scaffold-free material for bone tissue engineering.

Emx2 is a dose-dependent negative regulator of Sox2 telencephalic enhancers.

The transcription factor Sox2 is essential for neural stem cells (NSC) maintenance in the hippocampus and in vitro. The transcription factor Emx2 is also critical for hippocampal development and NSC self-renewal. Searching for 'modifier' genes affecting the Sox2 deficiency phenotype in mouse, we observed that loss of one Emx2 allele substantially increased the telencephalic ß-geo (LacZ) expression of a transgene driven by the 5' or 3' Sox2 enhancer. Reciprocally, Emx2 overexpression in NSC cultures inhibited the activity of the same transgene. In vivo, loss of one Emx2 allele increased Sox2 levels in the medial telencephalic wall, including the hippocampal primordium. In hypomorphic Sox2 mutants, retaining a single 'weak' Sox2 allele, Emx2 deficiency substantially rescued hippocampal radial glia stem cells and neurogenesis, indicating that Emx2 functionally interacts with Sox2 at the stem cell level. Electrophoresis mobility shift assays and transfection indicated that Emx2 represses the activities of both Sox2 enhancers. Emx2 bound to overlapping Emx2/POU-binding sites, preventing binding of the POU transcriptional activator Brn2. Additionally, Emx2 directly interacted with Brn2 without binding to DNA. These data imply that Emx2 may perform part of its functions by negatively modulating Sox2 in specific brain areas, thus controlling important aspects of NSC function in development.

The gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4 and Sox-2.

UTF1 is a transcriptional coactivator which has recently been isolated and found to be expressed mainly in pluripotent embryonic stem (ES) cells (A. Okuda, A. Fukushima, M. Nishimoto, et al., EMBO J. 17:2019-2032, 1998). To gain insight into the regulatory network of gene expression in ES cells, we have characterized the regulatory elements governing UTF1 gene expression. The results indicate that the UTF1 gene is one of the target genes of an embryonic octamer binding transcription factor, Oct-3/4. UTF1 expression is, like the FGF-4 gene, regulated by the synergistic action of Oct-3/4 and another embryonic factor, Sox-2, implying that the requirement for Sox-2 by Oct-3/4 is not limited to the FGF-4 enhancer but is rather a general mechanism of activation for Oct-3/4. Our biochemical analyses, however, also reveal one distinct difference between these two regulatory elements: unlike the FGF-4 enhancer, the UTF1 regulatory element can, by its one-base difference from the canonical octamer-binding sequence, selectively recruit the complex comprising Oct-3/4 and Sox-2 and preclude the binding of the transcriptionally inactive complex containing Oct-1 or Oct-6. Furthermore, our analyses reveal that these properties are dictated by the unique ability of the Oct-3/4 POU-homeodomain that recognizes a variant of the Octamer motif in the UTF1 regulatory element.

Structure and expression of the rat c-jun messenger RNA: tissue distribution and increase during chemical hepatocarcinogenesis.

c-jun is the cellular homologue of the recently isolated nuclear oncogene v-jun. This protooncogene encodes the cellular transcription factor AP-1. We have isolated the complementary DNA clone of rat c-jun mRNA. The rat c-jun complementary DNA clone encodes 334 amino acid residues, the sequence of which shows about 98, 96, and 81% homologies with mouse, human, and chicken c-jun products, respectively. The c-jun mRNA is highly expressed in the lung and slightly expressed in the brain. The amount of mRNA is even smaller in the liver and is not detected in the spleen. We have also determined c-jun expression during chemical hepatocarcinogenesis and demonstrated increased expression of mRNA in the precancerous lesion, hyperplastic nodules, as well as in the primary hepatocellular carcinomas.

Mechanism of selective killing by dilinoleoylglycerol of cells transformed by the E1A gene of adenovirus type 12.

Rat 3Y1 fibroblasts transformed by the E1A gene of adenovirus type 12 (E1A-3Y1 cells) are highly sensitive to the cell-killing effect of 1,3-dilinoleoylglycerol (DLG) administered in a culture medium, whereas the parental 3Y1 cells are less sensitive (H. Shimura et al., Cancer Res., 48: 578-583, 1988). The selective cytotoxicity of DLG to E1A-3Y1 cells was markedly reduced by the simultaneous administration of nonspecific antioxidants such as vitamin E, butylated hydroxytoluene, and ascorbic acid. Specific scavengers for oxygen radicals had no effect. Lipoxygenase inhibitors (nordihydroguaiaretic acid, esculetin, and baicalein) reduced the DLG-mediated selective cytotoxicity, whereas cyclooxygenase inhibitors (acetylsalicylic acid and indomethacin) showed no effect. The intracellular and extracellular contents of the products from lipid peroxidation as measured by the thiobarbituric acid test were significantly greater in E1A-3Y1 cells than in the parental 3Y1 cells. In comparison with DLG, linoleic acid and monolinoleoylglycerol were equally toxic to E1A-3Y1 and parental 3Y1, and trilinoleoylglycerol was weakly toxic to both types of cells. Scanning electron microscopy revealed that numerous holes about 0.2 micron in diameter were scattered all over the surface of the E1A-3Y1 cells after treating the cultures with DLG. These results suggest that; (a) the DLG-mediated cytotoxicity to the E1A-transformed cells is attributable to lipid peroxidation; (b) the structural property of DLG is essential to the E1A specificity of cytotoxicity; and finally (c) the destruction of the cell membrane is the basis of cytotoxicity of DLG.

The effects of sodium fluoride on the resorptive activity of isolated osteoclasts.

It is not clear to what extent the increased bone mass observed in vertebral trabecular bone of fluoride-treated mammals is a consequence of effects of fluoride on the number and activity of osteoclasts or of osteoblasts. In the present communication, we have analyzed the effects of NaF on the activity of isolated rabbit osteoclasts cultured on thin slices of devitalized compact bovine bone. Osteoclastic resorption was quantitated by counting the number of resorption lacunae and measuring their surface area and their depth using scanning electron microscopy. Our results show that NaF in concentrations of 0.5-1.0 mM decreased the number of resorption lacunae made by individual osteoclasts and decreased the resorbed area per osteoclast. We argue that the concentration of fluoride in these experiments may be within the range "seen" by osteoclasts in mammals treated for prolonged periods with approximately 1 mg of NaF/kg body weight (bw) per day.

Expression of calcitonin receptors during osteoclast differentiation in mouse metatarsals.

Metatarsal bones of 15-day-old mouse embryos contain proliferative tartrate-resistant acid phosphatase (TRAP) negative (-) osteoclast progenitors that progressively differentiate into multinucleated TRAP positive (+) osteoclasts. Using histochemical and autoradiographic techniques, we have examined the expression of calcitonin receptors during osteoclast differentiation in mouse metatarsals. Fresh mouse metatarsals from embryos aged 14-17 days and metatarsals from 15-day-old embryos cultured for 1, 2, 3, and 6 days were stained for TRAP. Calcitonin binding to osteoclasts and their precursors was studied by incubating metatarsals with [125I]salmon calcitonin (sCT) and quantitating grain counts from autoradiographs of tissue sections. Calcitonin receptors first appear on nonproliferating osteoclast precursors, most often just after or simultaneously with the development of TRAP activity. The effect of sCT on the development of TRAP+ mononuclear preosteoclasts was examined by culturing 15-day-old metatarsals in the continuous presence of 5 mU sCT for periods of up to 3 days and quantitating the number of TRAP+ mononuclear preosteoclasts that develop. Calcitonin did not affect the differentiation of osteoclasts up to the stage of the TRAP+ mononuclear preosteoclast.

NBP1 (Nap1 binding protein 1), an essential gene for G2/M transition of Saccharomyces cerevisiae, encodes a protein of distinct sub-nuclear localization.

Nap1p is identified in mammalian cell extract by its intrinsic activity to facilitate nucleosome assembly in vitro in the physiological ionic condition. The homologous proteins are present in most eukaryotes, and their functional analyses in vitro have suggested that they are necessary to keep proper nucleosome structures in transcription and replication. This protein is also identified for its interaction with Clb2p in vitro. To address the function of Nap1p in vivo, we have surveyed for proteins to interact with Nap1p by two-hybrid system and obtained two genes, NBP1 and NBP2 (Nap1 Binding Protein 1 and 2). NBP1 is an essential gene and encodes a novel protein consisting of 319 amino acids, with a coiled-coil structure in the center of the predicted amino acid sequence. Several A-kinase dependent phosphorylation sites and Cdc28p kinase-dependent sites are also observed. By isolating the temperature-sensitive mutant, we demonstrate that the nuclear division at a non-permissive temperature is delayed and that the population of cells with a large bud carrying a single nucleus with a short spindle are increased. This mutant also confers resistance against benomyl, a microtubule-destabilizing agent. Judging from the green fluorescent protein (GFP) signal fused with Nbp1p, this protein localizes in the nucleus as one or two tiny dots.

Association of high molecular weight DNA fragmentation with apoptotic or non-apoptotic cell death induced by calcium ionophore.

Calcium ionophore (A23187)-induced high molecular weight (HMW) and internucleosomal DNA fragmentation were investigated in human leukemia cell lines. An apoptosis-sensitive cell line, HL-60, showed HMW, internucleosomal DNA fragmentation and morphological changes of apoptosis by A23187. MOLT-4, which is resistant to apoptosis, exhibited only HMW DNA fragmentation and died of necrosis under the same conditions. Autodigestion experiments suggested the endonucleolytic activity to cause HMW fragmentation in the cytoplasm of both cell lines. The activity was more dependent on Mg2+ than Ca2+ in HL-60, whereas it was Ca(2+)-dependent in MOLT-4. These results suggest that HMW DNA fragmentation is not specific to apoptosis.

Constitutive endonuclease to induce high molecular weight or internucleosomal DNA fragmentation in freshly isolated leukemia cells.

Using an autodigestion method, we investigated endogenous endonuclease(s) in leukemia cells freshly obtained from pediatric patients with various types of leukemia. Endonucleolytic activity was found to cause both high molecular weight and internucleosomal DNA fragmentation at a neutral pH in whole cell lysates of all common acute lymphoblastic leukemia (cALL) blasts, which was Mg2+-dependent and Ca2+-independent. Whole lysates from most acute myeloblastic leukemia (AML) cells possessed similar endonuclease activity, but both Mg2+ and Ca2+ were required for the activity. Our results suggest that leukemia cells of different lineages have distinct constitutive endonucleases, which may play a role in the occurrence of apoptosis in these cells.

Pyrrolidine dithiocarbamate, a potent inhibitor of nuclear factor kappa B (NF-kappa B) activation, prevents apoptosis in human promyelocytic leukemia HL-60 cells and thymocytes.

We examined the effect of pyrrolidine dithiocarbamate (PDTC), which potently blocks the activation of nuclear factor kappa B (NF-kappa B), on the induction of apoptosis by a variety of agents. Treatment of a human promyelocytic leukemia cell line, HL-60, with 10 micrograms/mL etoposide or 2 microM 1-beta-D-arabinofuranosylcytosine induced NF-kappa B activation within 1 hr and subsequently caused apoptosis within 3-4 hr. The simultaneous addition of 50-500 microM PDTC with these agents blocked NF-kappa B activation and completely abrogated both morphologically apoptotic changes and internucleosomal DNA fragmentation for up to 6 hr. However, PDTC failed to inhibit the endonuclease activity contained in the whole cell lysates. The inhibitory effect of PDTC was also observed in etoposide- and dexamethasone-induced apoptosis in human thymocytes at a concentration of 1-10 microM. Since PDTC has both antioxidant and metal-ion chelating activities, we tested the effects of N-acetyl-L-cysteine (NAC) (antioxidant) or o-phenanthroline (OP) (metal-ion chelator) on the induction of apoptosis. Pretreatment of HL-60 cells or thymocytes with 100-500 microM OP for 2 hr, but not 10-60 mM NAC, suppressed subsequent occurrence of apoptosis induced by etoposide. These results suggest that the activation of NF-kappa B plays an important role in the apoptotic process of human hematopoietic cells.

Augmentation by aphidicolin of 1-beta-D-arabinofuranosylcytosine-induced c-jun and NF-kappa B activation in a human myeloid leukemia cell line: correlation with apoptosis.

1-beta-D-arabinofuranosylcytosine (ara-C) (2 microM) can induce apoptosis in a human myeloid leukemia cell line, U937, after 4 h of incubation. Pretreatment of cells with aphidicolin (2 microM) augments ara-C-induced apoptosis, since it was first observed at 0.4 microM ara-C and became more intense at 2 and 10 microM. Although aphidicolin itself had a marginal effect on c-jun expression, it significantly augmented ara-C induced c-jun upregulation by shortening the lag time and lowering ara-C concentrations necessary for the induction of detectable c-jun transcripts. Aphidicolin and ara-C acted synergistically to increase NF-kappa B DNA binding activity as determined by an electrophoretic mobility shift assay. Expression of c-myc was slightly increased through the DNA degradative phase, and was then downregulated. Thus, the activation of NF-kappa B and c-jun expression seems to be well correlated with the potentiation by aphidicolin of ara-C-induced apoptosis.

Dependency of the efficiency of transformation by simian-virus-40 on the proliferative state of cultured fibroblasts at the time of virus inoculation.

We previously demonstrated that the efficiency of cell transformation by simian virus 40 (SV40) is reduced when proliferating rat 3Y1 cells are inoculated with SV40 as compared with when density-arrested cells are inoculated. In this study, we characterized in more detail the cellular state at the time of virus inoculation that affects the susceptibility to SV40 transformation. When density-arrested 3Y1 cells were stimulated to progress one round of cell cycle by refeeding with medium containing high serum, they began to reduce the susceptibility to SV40 transformation before entering S phase. After re-entry into the non-proliferative state, prolonged maintenance of cells in the non-proliferative state was necessary for restoration of the susceptibility to SV40 transformation. Such dependency of the transformation efficiency on the cellular state at the time of SV40 inoculation paralleled with that of the expression of T antigen in nuclei, while virus adsorption and degradation of the DNA of the adsorbed virus was not dependent on the cellular state. We conclude that after inoculation of proliferating cells with SV40, an unidentified event between the virus adsorption and the accumulation of T antigen protein in nuclei is blocked and that this blockage causes the inhibition of the initiation of transformation.

Cytotoxic and cytostatic effects of polyphenols against rat 3y1 fibroblasts transformed by e1a gene of human adenovirus-type-12.

Toxicity of polyphenols against rat 3Y1 fibroblasts and the cells transformed by human adenovirus (Ad12-3Y1), its EIA gene (EIA-3Y1), or simian virus 40 (SV-3Y1) was examined. Among the diphenol compounds examined, pyrocatechol (o-diphenol) and hydroquinone (p-diphenol) showed selective toxicity against Ad12-3Y1 and EIA-3Y1 cells, while resorcinol (m-diphenol) showed a much weaker non-specific toxicity against these cells. Another o-diphenol (dopamine) and triphenols (gallic acid and pyrogallol) were less toxic but showed selective toxicity. At lower concentrations where they were not toxic, all polyphenols attenuated toxicity of phosphatidylcholine against EIA-3Y1 cells. Among antioxidants examined, ascorbic acid reduced the toxicity of pyrocatechol, but alpha-tocopherol and butyrated hydroxytoluene did not. Oxidation of pyrocatechol was not enhanced in the presence of 3Y1 or EIA-3Y1 cells and their homogenates. These results suggest that the selective toxicity of polyphenols against Ad12-3Y1 and E1A-3Y1 cells is not related to their oxidation velocity but other factors such as the activity of active oxygen-scavenging enzymes.

E1a-3y1 cell-specific toxicity of tea polyphenols and their killing mechanism.

To screen carcinostatic components in foodstuffs, the toxicity of tea polyphenols was compared between rat 3Y1 diploid fibroblasts and a variety of their virally transformed cells. Among tea polyphenols tested, epigallocatechin gallate killed 3Y1 cells transformed by E1A gene of human adenovirus type 12 (E1A-3Y1 cells) at a 100 times lower concentration than the parental 3Y1 cells. Epigallocatechin gallate also exerted a strong E1A-3Y1 cell-specific toxicity, while epicatechin and epicatechin gallate did not. When the activity of three antioxidant enzymes was compared between 3Y1 and its transformants, catalase activity was markedly low in the latter, especially in E1A-3Y1 cells, and the substrate of the enzyme, hydrogen peroxide, exerted a toxicity specific to this cell line. Then the inhibitory activities of various chemicals on E1A-3Y1 cell-specific toxicity of phospholipids or catechol were examined. Among lipoxygenase inhibitors, all of the polyphenolic compounds inhibited the toxicity of phospholipids, but not a nonpolyphenolic inhibitor (clofibrate). Two phospholipase A,inhibitors (dexamethasone and quinacrine) did not inhibit the toxicity. These results indicate that the triphenol structure of the B ring is essential for the E1A-3Y1 cell-specific toxicity of tea polyphenols, and that the decrease in catalase activity is partially responsible for the higher sensitivity of E1A-3Y1 cells against the polyphenols. The inhibitory effect of polyphenolic lipoxygenase inhibitors is ascribed at least in part to their antioxidant activities.

Reversible proliferation arrest of rat 3y1 fibroblasts and selective killing of simian-virus transformed derivation of 3y1 by short-chain Fatty-acids.

We examined the effect of saturated fatty acids with carbon number from 2 to 10 on proliferation and survival of rat untransformed fibroblast 3Y1 cells and its transformed derivation induced by Simian virus 40 (SV-3Y1). Acetic acid (C2) had no effect on proliferation of these cells. Among medium-chain fatty acids (C6-C10), caproic acid (C6) showed the lowest proliferation inhibitory effect. Caprylic (C8), pelargonic (C9), and capric acid (C10) exerted killing activity to both 3Y1 cells and SV-3Y1 cells, and the toxicity increased with the elongation of their alkyl chains. The toxicity was a little greater to 3Y1 cells than to SV-3Y1 cells. In contrast, short-chain fatty acids caused reversible proliferation arrest in 3Y1 cells at 25 mM in propionic (C3), 5 mM in butyric (C4) and 10 mM in valeric (C5) acids. These short-chain fatty acids arrested 3Y1 cells not only in the GI phase but also in the G2 phase of the cell cycle. To SV-3Y1 cells, however, these short-chain fatty acids were cytotoxic. The cytotoxicity to SV-3Y1 cells was the greatest in butyric acid among short-chain fatty acid tested. These results suggest that short-chain fatty acids exert ploliferation-arresting activity against normal cells while exerting killing activity to the transformed cells.

Optimal perfusion flow rate for the brain during deep hypothermic cardiopulmonary bypass at 20 degrees C. An experimental study.

The relationship between the perfusion flow rate and cerebral oxygen consumption during deep hypothermic cardiopulmonary bypass at 20 degrees C was investigated in dogs. In 10 dogs the perfusion flow rate was decreased in steps from 100 to 60, 30, and 15 ml/kg/min every 30 minutes. Although cerebral blood flow decreased as perfusion flow rate decreased, the ratio of cerebral blood flow to the perfusion flow rate increased significantly (p less than 0.05) at a perfusion flow rate of 15 ml/kg/min compared to that at a perfusion flow rate of 100 or 60 ml/kg/min. The arterial-sagittal sinus blood oxygen content difference increased as perfusion flow rate decreased. Consequently, cerebral oxygen consumption did not vary significantly at perfusion flow rates of 100 (0.48 +/- 0.10), 60 (0.43 +/- 0.14), and 30 ml/kg/min (0.44 +/- 0.12 ml/100 gm/min), and it decreased significantly to 0.31 +/- 0.22 ml/100 gm/min at a perfusion flow rate of 15 ml/kg/min. In five dogs the perfusion flow rate was decreased in one step from 100 to 15 ml/kg/min, and after 60 minutes' perfusion at a perfusion flow rate of 15 ml/kg/min, the perfusion flow rate was returned to 100 ml/kg/min. Cerebral oxygen consumption decreased significantly during 60 minutes' perfusion at a perfusion flow rate of 15 ml/kg/min and did not return to its initial value after the perfusion flow rate was returned to 100 ml/kg/min. These data indicate that the optimal perfusion flow rate for the brain during deep hypothermic cardiopulmonary bypass at 20 degrees C appears to be 30 ml/kg/min, with a possible oxygen debt in the brain resulting in anaerobic metabolism if the perfusion flow rate is kept at 15 ml/kg/min or less.

Identification of the specific labile sites in the 180 kDa catalytic polypeptide of the Drosophila melanogaster DNA polymerase alpha-primase complex.

The immunoaffinity-purified DNA polymerase alpha-primase complex from Drosophila melanogaster Kc cells contains three high molecular weight polypeptides besides the 180 kDa catalytic polypeptide. These polypeptides are immunologically cross-reactive with the 180 kDa polypeptide. When the immunoaffinity-purified complex was kept at 4 degrees C for about four weeks, the amounts of the three polypeptides increased, while the 180 kDa polypeptide completely disappeared. Sodium bisulfite inhibited the decrease in the 180 kDa polypeptide. The N-terminal amino acid sequences of all the polypeptides were all assigned to ones present in a portion close to the N-terminus of the 180 kDa polypeptide. The N-terminal residue of all the three polypeptides was Ser. The cleavage sites were Phe130-Ser131, Thr180-Ser181, and Phe237-Ser238. These results show that the three polypeptides are cleavage products of the 180 kDa catalytic polypeptide, the cleavage occurring at specific labile sites including a Ser residue. The amino acid residues at the sites are quite different from those (Lys-Lys) in the human 180 kDa catalytic polypeptide.

Pancreatoduodenectomy for periampullary cancer associated with celiac occlusion: a case report.

A 62-year-old man with atherosclerotic occlusion of the celiac axis underwent pancreatoduodenectomy because of cancer of the common bile duct. Preoperative angiography showed that the arterial blood supply to the celiac viscera was sustained through the gastroduodenal artery via the pancreaticoduodenal arcades from the superior mesenteric artery. Therefore reconstruction of the celiac circulation was attempted before division of the gastroduodenal artery. An autograft of the saphenous vein was placed between the infrarenal aorta and the common hepatic artery. The postoperative course was uneventful. Patency of the graft was demonstrated angiographically on the 50th postoperative day.