Application of glucosylceramide-based liposomes increased the ceramide content in a three-dimensional cultured skin epidermis.

Ceramide is an intercellular lipid of the stratum corneum and is one of the most important components of the epidermal permeability barrier. Glucosylceramide (GlcCer), a ceramide precursor, was applied to three-dimensional skin culture to regulate ceramide. GlcCer/dimyristoylphosphatidylcholine (DMPC) = 4/4 (molar ratio and GlcCer/DMPC/dimyristoylphosphatidylglycerol (DMPG) = 4/4/1(molar ratio) liposomes were prepared by the thin-layer method. The particle diameters of GlcCer/DMPC and GlcCer/DMPC/DMPG liposomes were 124.0 ± 0.6 and 119.3 ± 18.9 nm, and the zeta potentials were 1.3 ± 0.3 and -19.9 ± 0.3 mV, respectively. Stability of these GlcCer liposomes was measured by transmission light scattering. Transmission light scattering of neutrally charged GlcCer (GlcCer/DMPC) liposomes increased in a time dependent manner. In contrast, negatively charged GlcCer (GlcCer/DMPC/DMPG) liposomes were not changed. ß-Glucocerebrosidase activity was measured in a cultured human skin model. Results confirmed that the cultured human skin model has ß-glucocerebrosidase activity. GlcCer/DMPC/DMPG liposomes were applied to the three-dimensional cultured human skin model, and ceramide NS, NP, AS, and AP were extracted from it. The various extracted ceramides were separated by high-performance thin-layer chromatography and quantified by a densitometer. The amount of ceramide AS only in the cultured skin model was significantly higher with the application of GlcCer-based liposomes than that of the nonapplication group, and was also dose dependent. Thus, GlcCer-based liposomes are useful for enriching the ceramide AS levels in a three-dimensional cultured skin model.

Effect of hyaluronan tetrasaccharides on epidermal differentiation in normal human epidermal keratinocytes.

OBJECTIVE: Hyaluronan (HA) plays a role in keratinocyte proliferation and differentiation. In addition, HA has been shown to have different biological activities depending on its molecular weight. It has been reported that HA-mediated CD44 activation regulates keratinocyte differentiation. Therefore, the aim of this study was to investigate the influence of HA tetrasaccharides (HA4) on the regulation of keratinocyte differentiation, CD44 gene expression and CD44-phosphorylated protein in human keratinocytes, and compare HA4 with high molecular weight HA. METHODS: Normal human epidermal keratinocytes (NHEKs) were treated at doses of 1 µg mL(-1) HA or HA oligosaccharides (HA4). After treatment, cell viability was checked using an MTT (3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Each differentiation marker and CD44 mRNA expression was detected by real-time PCR. Each differentiation marker and CD44-phosphorylated protein was assessed by Western blotting. RESULTS: Hyaluronan and HA4 showed no cytotoxicity up to a dose of 1 µg mL(-1) . On day 3 after HA4 treatment, each differentiation marker mRNA and K10 protein level was higher than that of the control. On day 9, late differentiation marker mRNA and protein levels were increased with HA and HA4 treatment. In addition, HA4 treatment increased the expression of CD44 mRNA, CD44-phosphorylated protein and intracellular calcium concentrations. HA4 enhanced keratinocyte differentiation and increased CD44-phosphorylated protein levels. CONCLUSION: HA4 may induce epidermal differentiation through phosphorylation of CD44.

Increase in ceramide level after application of various sizes of sphingomyelin liposomes to a cultured human skin model.

Sphingomyelin-based liposomes (SPM-L) that were sized (or not) by extrusion through a filter with pores of 100, 200, or 400 nm were applied to a three-dimensional cultured human skin model in order to evaluate which size of SPM-L was most effective at increasing its ceramide level. The diameters of the SPM-L in PBS were 102.7, 181.0, 224.0, and 380.1 nm. The diameters of the liposomes in the culture medium were 117.5, 199.2, 242.1, and 749.8 nm. The diameter of the small liposomes (<200 nm in diameter) did not change much, at least for 7 days. SPM-L in saline or culture medium were applied to the basal layer side or stratum corneum side of the cultured skin model, and ceramide II, III, V, and VI were then extracted from it. The extracted ceramide molecules were separated by HPTLC, and the concentration of each type of ceramide was quantified using a densitometer. When the small SPM-L (110 or 190 nm in diameter) were applied to the basal layer side, the levels of ceramide III and V were increased. When they were applied to the stratum corneum side, the levels of ceramide II, III, V, and VI were significantly increased compared to those of the PBS group, especially after the application of the small SPM-L (110 nm in diameter). Thus, the application of small SPM-L was useful for increasing the ceramide II, III, V, and VI levels of a cultured human skin model.

Focal segmental glomerular sclerosis, a type of intractable chronic glomerulonephritis, is a stem cell disorder.

The etiopathogenesis of focal and segmental glomerular sclerosis (FGS) remains unknown. Using a new animal model for FGS (FGS mouse), we demonstrate here that bone marrow transplantation from normal mice to FGS mice with a high grade of proteinuria (+ + +) ameliorates FGS, and that the transplantation of bone marrow cells or purified hemopoietic stem cells (HSCs) from FGS mice induces FGS in normal mice. These findings strongly suggest that FGS is a stem cell disorder; the abnormalities may be genetically programmed at the level of HSCs.

Peroxisomal cholesterol synthesis in vivo: accumulation of 4-methyl intermediate sterols after aminotriazole inhibition of cholesterol synthesis.

To clarify the importance and pathway of peroxisomal cholesterol synthesis in vivo, we have examined whether or not 4,4-dimethyl-5 alpha-cholest-8-en-3 beta-ol and 4 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol are accumulated in hepatic peroxisomes of aminotriazole-treated rats (we have shown that these intermediate steroids accumulate in rat liver when cholesterol synthesis is inhibited by aminotriazole: Hashimoto, F. and Hayashi, H. (1991) Biochim. Biophys. Acta 1086, 115). Differential centrifugation and Nycodenz gradient centrifugation showed that these intermediate steroids were localized in peroxisomes and microsomes. Cholestyramine (3-hydroxy-3-methylglutaryl-CoA reductase activator) pretreatment of aminotriazole-treated rats increased the contents of the intermediate steroids in both peroxisomes and microsomes. In peroxisomes, both 4 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol and 4,4-dimethyl-5 alpha-cholest-8-en-3 beta-ol were increased to about 3 times the control (aminotriazole-treated rat), and they were predominantly (about 70%) recovered in the membrane fraction after treatment with 0.05% deoxycholate or 100 mM Na2CO3. Gemfibrozil (peroxisomal proliferator) pretreatment enhanced the contents of 4 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol and 4,4-dimethyl-5 alpha-cholest-8-en-3 beta-ol of peroxisomes to 4.5 times and 37 times the control, respectively. The effects of aminotriazole, cholestyramine and gemfibrozil on the intermediate contents were different between peroxisomes and microsomes. We suggest that peroxisomes in addition to microsomes participate in cholesterol synthesis in vivo, and the biosynthetic pathway includes 4 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol and 4,4-dimethyl-5 alpha-cholest-8-en-3 beta-ol.

Identification of intermediates after inhibition of cholesterol synthesis by aminotriazole treatment in vivo.

Cholesterol synthesis from mevalonate is inhibited by aminotriazole treatment in vivo. We tried to identify intermediates accumulated in liver of aminotriazole-treated rats. At 6 h after the aminotriazole treatment, the liver was excised. Sterols were extracted from it, and subjected to capillary gas-liquid chromatography, high-performance liquid chromatography, gas-liquid chromatography linked to mass spectrometry and gas-liquid chromatography linked to Fourier-transform infrared spectrometry. It was found that 4 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol and 4,4-dimethyl-5 alpha-cholest- 8-en-3 beta-ol were accumulated in the liver, mainly as the free forms. The contents of the former and the latter were increased to 25- and 64-times the control values, respectively. In another experiment, [2-13C]mevalonate was injected at 2 h after aminotriazole treatment, and 4 h later the liver was excised. The sterols extracted from the liver were subjected to gas-liquid chromatography linked to mass spectrometry. Specific fragment ions reflecting the incorporation of [13C] mevalonate were detected in the mass spectra of the intermediate sterols. Accumulation of 4 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol and 4,4-dimethyl-5 alpha-cholest-8-en-3 beta-ol after aminotriazole treatment suggests that elimination of the 4 alpha-methyl group from 4-methyl intermediate sterols is inhibited by aminotriazole.

Significance of catalase in peroxisomal fatty acyl-CoA beta-oxidation.

Catalase activity was inhibited by aminotriazole administration to rats in order to evaluate the influence of catalase on the peroxisomal fatty acyl-CoA beta-oxidation system. 2 h after the administration of aminotriazole, peroxisomes were prepared from rat liver, and the activities of catalase, the beta-oxidation system and individual enzymes of beta-oxidation (fatty acyl-CoA oxidase, crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase) were determined. Catalase activity was decreased to about 2% of the control. Among the individual enzymes of the beta-oxidation system, thiolase activity was decreased to 67%, but the activities of fatty acyl-CoA oxidase, crotonase and beta-hydroxybutyryl-CoA dehydrogenase were almost unchanged. The activity of the peroxisomal beta-oxidation system was assayed by measuring palmitoyl-CoA-dependent NADH formation, and the activity of the purified peroxisome preparation was found to be almost unaffected by the administration of aminotriazole. The activity of the system in the aminotriazole-treated preparation was, however, significantly decreased to 55% by addition of 0.1 mM H2O2 to the incubation mixture. Hydrogen peroxide (0.1 mM) reduced the thiolase activity of the aminotriazole-treated peroxisomes to approx. 40%, but did not affect the other activities of the system. Thiolase activity of the control preparation was decreased to 70% by addition of hydrogen peroxide (0.1 mM). The half-life of 0.1 mM H2O2 added to the thiolase assay mixture was 2.8 min in the case of aminotriazole-treated peroxisomes, and 4 s in control peroxisomes. The ultraviolet spectrum of acetoacetyl-CoA (substrate of thiolase) was clearly changed by addition of 0.1 mM H2O2 to the thiolase assay mixture without the enzyme preparation; the absorption bands at around 233 nm (possibly due to the thioester bond of acetoacetyl-CoA) and at around 303 nm (due to formation of the enolate ion) were both significantly decreased. These results suggest that H2O2 accumulated in peroxisomes after aminotriazole treatment may modify both thiolase and its substrate, and consequently suppress the fatty acyl-CoA beta-oxidation. Therefore, catalase may protect thiolase and its substrate, 3-ketoacyl-CoA, by removing H2O2, which is abundantly produced during peroxisomal enzyme reactions.

Changes in polyamine-oxidizing capacity of peroxisomes under various physiological conditions in rats.

Rat liver peroxisomal polyamine oxidase activity was determined under various physiological conditions by using the peroxidase method with phenol and 4-aminoantipyrine. N1-Acetylpolyamines such as N1-acetylspermine and N1-acetylspermidine were better substrates than the free polyamines. The polyamine oxidase activity in rat peroxisomes increased significantly when cell proliferation was high. The activity began to appear in fetal liver at the 16th approximately 18th day of pregnancy and peaked in neonatal liver on the first day (approx. 1.7-times higher than in adult liver). In regenerating rat liver, only polyamine oxidase activity among the peroxisomal enzymes tested was increased considerably 12 h after partial hepatectomy (approx. 2.8-fold over the control liver). Finally, the enzyme activity was significantly increased by administration of clofibrate, a peroxisome proliferator, which also causes hepatomegaly. In all cases, the increase in polyamine oxidase activity was not more than 3-fold. Since the level of polyamine oxidase activity in the normal liver is more than adequate in relation to the level of the substrates, the slight but significant increase under conditions of cell proliferation may have a role in modulating levels of polyamines in the proliferating liver tissue.

Transient eosinophilia associated with pancreatitis and pseudocyst formation.

Eosinophilia is frequently associated with allergic rhinitis, asthma, drug reactions, parasitic infections, malignant neoplasms, collagen vascular diseases, skin diseases, and pulmonary infiltrates. It has been infrequently described in conjunction with pancreatic diseases and not before, to my knowledge, with pseudocyst formation. A patient with alcohol-related pancreatitis manifested a transient eosinophilia during development of a massive pancreatic pseudocyst. Although he was atopic, with a greatly elevated serum IgE level, there was no recent contact with the specific allergen to which he was sensitized. This constellation of alcohol-related pancreatitis with pseudocyst formation, atopy with elevated serum IgE level, and transient eosinophilia is an interesting coincidence.

Use of a semiquantitative microscopic method for detecting bacteriuria.

We evaluated a microscopic examination for bacteriuria, performed by experienced observers and by 25 house officers who were less practiced in the technique. Simulated urine samples containing known concentrations of Escherichia coli with or without polymorphonuclear leukocytes were examined uncentrifuged in a wet mount with a methylene blue stain. The experienced observers were able to determine whether a sample contained greater or less than 10(5) organisms per milliliter with a high degree of sensitivity and specificity. The house officers demonstrated a lessened discrimination, and leukocytes in the samples altered their decisions as to whether samples contained significant numbers of bacteria. Experienced observers can successfully use the microscopic examination as a rapid and inexpensive diagnostic tool to diagnose significant bacteriuria.

Neuroleptic malignant syndrome and dopaminergic blockade.

After receiving 90 mg of haloperidol and 100 mg of chlorpromazine hydrochloride within 25 hours, a 29-year-old man was found to have neuroleptic malignant syndrome (NMS), characterized by the acute onset of hyperpyrexia, extreme muscular rigidity, autonomic instability, and coma. Subsequently, rhabdomyolysis developed, with myoglobinuric renal failure and bilateral anterior tibial compartment syndromes. The patient's initial neuroleptic levels were in the therapeutic and nontoxic ranges. He was treated with supportive measures and his clinical improvement was paralleled by decreased neuroleptic levels, a return toward normal of an elevated prolactin level, and an increased responsiveness to a dopamine hydrochloride infusion. This supports an association between NMS and dopamine receptor blockade.

Enhancing effects of cyclosporin A on hematopoietic progenitors: possible role of CD8+ T cells as negative regulators.

To elucidate the mechanism by which the administration of cyclosporin A (Cy A) improves a certain type of anemia, Cy A was orally administered to normal mice; hematopoietic activity, natural killer (NK) cell activity, and natural suppressor (NS) cell activity in the bone marrow (BM) were then examined. Hematopoietic colony formation was significantly enhanced by both 7-day and 21-day treatment with Cy A. Cy A did not, however, affect the total cell count or the numbers of macrophages, granulocytes, or B cells in the BM. Neither did Cy A affect the in vitro colony formation of the purified hematopoietic progenitors. These results suggest that Cy A acts on the negative regulators such as NK cells, NS cells, and CD8+ T cells. Since both NK and NS activity were also enhanced by Cy A treatment, these cells were not candidates. CD4+ or CD8+ T cells in the thymus, peripheral blood (PBL), and BM decreased as a result of the treatment. Therefore, to further examine the involvement of CD4+ or CD8+ T cells, hematopoietic colony formation was evaluated using bone marrow cells (BMCs) from mice purged of CD4+ and/or CD8+ cells. A significant enhancement of erythroid and multipotent colonies was observed in the BM of the CD8+ cell-purged mice. On the other hand, a significant enhancement of myeloid colonies was found in the BM of the CD4+ cell-purged mice. These findings suggest that single-positive T cells (particularly CD8+ T cells), which are diminished in number after Cy A treatment, are involved in the negative regulation of hematopoiesis.

Force-induced osteoclast apoptosis in vivo is accompanied by elevation in transforming growth factor beta and osteoprotegerin expression.

The mechanism controlling the disappearance of osteoclasts from bone surfaces after bone resorption in vivo is largely unknown. This is because there is no suitable experimental system to trace the final fate of osteoclasts. Here, we used an experimental model of tooth movement in rats to show that preexisting osteoclasts disappeared from the bone surface through apoptosis during a force-induced rapid shift from bone resorption to formation. On the distal alveolar bone surface of the maxillary molar in growing rats, many mature osteoclasts were present. When light tensional force was applied to the bone surface through an orthodontic appliance, these preexisting osteoclasts gradually disappeared. One day after the application of force, about 24% of the osteoclasts exhibited apoptotic morphology and the proportion of apoptotic cells was increased to 41% by day 2, then decreased afterward. These changes were undetectable on the control distal alveolar bone surface, which is free from tensional force. As shown by in situ hybridization, a marked increase in transforming growth factor beta1 (TGF-beta1) and osteoprotegerin (OPG) messenger RNA (mRNA) was observed in the stretched cells on the tensioned distal bone surface, simultaneously with the loss of osteoclasts. Both of these factors are known to have a negative effect on osteoclast recruitment and survival. As early as 2 days after force application, some of these stretched cells were identified as cuboidal osteoblasts showing intense signals for both factors. Our data suggest there may be a sequential link in tensional force applied on the bone lining cells, up-regulation of TGF-beta1/OPG, and disappearance of osteoclasts.

A common downstream signaling activity of osteoclast survival factors that prevent nitric oxide-promoted osteoclast apoptosis.

Treatment with NO-releaser NOC18 significantly promoted apoptosis in murine osteoclast-like cells, with a transient increase in caspase-3-like protease activity. In contrast, the apoptosis was protected against by caspase inhibitors, most efficiently with the broadly acting caspase specific inhibitor z-Asp-CH2-DCB, indicating involvement of multiple caspases in progression of the apoptosis. Among osteoclast survival factors examined, calcitonin completely protected against morphologically defined-apoptosis and the increase of caspase-3-like protease activity. The effect of calcitonin was mimicked by treatment of cells with (Bu)2cAMP and forskolin, and abolished by protein kinase-A inhibitor H-89. Independently from the PKA activation, colony stimulating factor-1, interleukin-1beta and the receptor activator of NF-kappaB ligand also protected against the apoptosis but were less effective than calcitonin. All survival factors investigated inhibited conversion of procaspases-3 and -9 to their mature forms in the cells. Thus, downstream antiapoptotic signaling activity from each factor overlapped in inhibition of caspases. However, how this was attained seemed to be different from each other. Typically, only colony stimulating factor-1 up-regulated expression of endogenous caspase inhibitor protein, X-linked inhibitor of apoptosis (XIAP), in the osteoclast-like cells.

Changes in isoprenoid lipid synthesis by gemfibrozil and clofibric acid in rat hepatocytes.

We studied whether gemfibrozil and clofibric acid alter isoprenoid lipid synthesis in rat hepatocytes. After incubation of the cells with the agent for 74 hr, [(14)C]acetate or [(3)H]mevalonate was added, and the cells were further incubated for 4 hr. Gemfibrozil and clofibric acid increased ubiquinone synthesis from [(14)C]acetate and [(3)H]mevalonate. The effect of gemfibrozil was greater than that of clofibric acid. Also, gemfibrozil decreased dolichol synthesis from [(14)C]acetate and [(3)H]mevalonate. However, clofibric acid increased dolichol synthesis from [(3)H]mevalonate. Gemfibrozil decreased cholesterol synthesis from [(14)C]acetate and [(3)H]mevalonate. Clofibric acid decreased cholesterol synthesis from [(14)C]acetate, but did not affect synthesis from [(3)H]mevalonate. These results suggest that both agents, at different rates, activate the synthetic pathway of ubiquinone, at least from mevalonate. Gemfibrozil may inhibit the synthetic pathway of dolichol, at least from mevalonate. Contrary to gemfibrozil, clofibric acid may activate the synthetic pathway of dolichol from mevalonate. Gemfibrozil may inhibit the synthetic pathway of cholesterol from mevalonate in addition to the pathway from acetate to mevalonate inhibited by both agents.

Effect of gemfibrozil on lipid biosynthesis from acetyl-CoA derived from peroxisomal beta-oxidation.

The effect of gemfibrozil, a peroxisome proliferator, on lipid biosynthesis from acetyl-CoA derived from peroxisomal beta-oxidation was studied. The specific activity of the peroxisomal fatty acyl-CoA beta-oxidation system of rats fed a chow containing 0.2% gemfibrozil for 2 weeks was approximately five times higher than that of control rats. When [1-14C]lignoceric acid, a very-long-chain fatty acid which is degraded exclusively by the peroxisomal beta-oxidation system at first, was injected into rats treated with gemfibrozil, radioactivity and content of bile acid in the bile were enhanced to approximately 2.2 and 3.5 times the control, respectively. Gemfibrozil increased the radioactivity and content of chenodeoxycholic acid more than that of cholic acid. The incorporation of radioactivity into cholesterol in the bile was as much as 4.5 times greater than the control, and content was 2.6 times greater. In the liver, incorporation of [14C]lignoceric acid into the simple lipids phosphatidylethanolamine and phosphatidylcholine was unaffected by gemfibrozil. The radioactivity and content of cholesterol separated from the simple lipids were also virtually unaffected. However, the specific activities of 3-hydroxy-3-methylglutararyl-CoA reductase (rate-limiting enzyme of cholesterol synthesis) of peroxisomes and microsomes were remarkably stimulated by gemfibrozil treatment. These results suggest that biosyntheses of cholesterol and bile acid from acetyl-CoA derived from peroxisomal beta-oxidation are stimulated by gemfibrozil, due at least in part to activation of the peroxisomal beta-oxidation system and 3-hydroxy-3-methylglutaryl-CoA reductase of peroxisomes and/or microsomes. Most peroxisomal proliferators (e.g. clofibrate) have been known to inhibit 3-hydroxy-3-methylglutaryl-CoA reductase activity. Therefore, gemfibrozil is expected to be a very useful tool for elucidating the relationship between peroxisomes and the biosyntheses of cholesterol and bile acid.

Induction of donor-specific T cell anergy by portal venous injection of allogeneic cells.

The mechanisms behind tolerance induction by portal venous (pv) injection of allogeneic cells are investigated. When a hematopoietic stem cell (HSC)-enriched population of BALB/c bone marrow was pv injected into C57BL/6 mice, the response of the T cells in the B6 mice to BALB/c alloantigens in mixed lymphocyte reaction (MLR) decreased until day 4 after the injection. Neither clonal deletion of V beta 11+ T cell nor donor-specific suppressor activity was observed. When recipient T cells were separated into CD4+ and CD8+ cells, only the CD8+ cell population showed donor-specific tolerance. The donor cells were trapped and retained in the host liver. MHC class I antigens were highly expressed on the trapped cells whereas class II antigens or B7 costimulatory molecules were not. The tolerance to BALB/c alloantigens in MLR was obtained also by the pv injection of Meth A, a BALB/c-derived sarcoma cell line. However, tolerance was not induced by the pv injection of B7-transfected Meth A cells. In addition to MLR, tolerance was also observed in DTH responses, and this was also due to the unresponsiveness of CD8+ cells to the donor alloantigens. However, the BALB/c-specific DTH responses were not suppressed after the pv injection of B7-transfected Meth A cells. These results strongly suggest that the tolerance induced by pv injection of allogeneic cells is due to clonal anergy generated by the absence of costimulatory signals in the interaction between donor-specific CD8+ T cells and donor hematopoietic cells trapped in the host liver.

Significance of catalase in peroxisomal fatty acyl-CoA beta-oxidation: NADH oxidation by acetoacetyl-CoA and H2O2.

To clarify the significance of catalase in peroxisomes, we have examined the effect of aminotriazole treatment of rats on the activity of beta-hydroxybutyryl-CoA dehydrogenase in liver peroxisomes. When the effect of H2O2 on the dehydrogenase activity was examined using an extract of liver peroxisomes from aminotriazole-treated rats, the acetoacetyl-CoA-dependent oxidation of NADH was found to increase considerably on the addition of dilute H2O2. Such an effect of H2O2 was not seen on the beta-hydroxybutyryl-CoA-dependent reduction of NAD nor with extracts from untreated animals. We then noticed that similar NADH oxidation was caused non-enzymatically by a mixture of acetoacetyl-CoA and H2O2. The oxidation was dependent on both acetoacetyl-CoA and H2O2, and was blocked by scavengers of oxyradicals such as ascorbate and ethanol. Degradation products formed during the reaction of acetoacetyl-CoA with H2O2 had no NADH oxidizing activity, indicating that effective oxidant(s) were generated during the reaction of H2O2 with acetoacetyl-CoA. No other fatty acyl-CoA so far examined nor acetoacetate could replace acetoacetyl-CoA in this reaction. Therefore, if H2O2 were to be accumulated in peroxisomes, it would decrease both NADH and acetoacetyl-CoA, thus affecting the fatty acyl-CoA beta-oxidation system. These results, together with our previous finding that peroxisomal thiolase was significantly inactivated by H2O2 [Hashimoto, F. & Hayashi, H. (1987) Biochim. Biophys. Acta 921, 142-150] suggest that the role of catalase in peroxisomes is at least in part to protect the fatty acyl-CoA beta-oxidation system from the deleterious action of H2O2.

Time-dependent changes of collagen crosslinks in the socket after tooth extraction in rabbits.

Time-dependent changes of the reducible collagen crosslinks in the healing tissue of rabbit tooth extraction wounds were analyzed chromatographically. The ratio of dihydroxylysinonorleucine to hydroxylysinonorleucine in the collagen from normal alveolar bone was 4.4. This value increased about four times, on the 10th day after tooth extraction, coinciding with the phase of active woven bone formation, and then decreased rapidly toward a normal value on the 14th day after tooth extraction. The data suggest that active biosynthesis and fibrillogenesis of bone collagen precede the morphological completion of lamellar bone formation.

Force-induced rapid changes in cell fate at midpalatal suture cartilage of growing rats.

The application of expansional force induces replacement of the cartilaginous tissue with bone at the midpalatal suture of growing rats. We examined the early cellular events evoked by force by analyzing the expression of proliferating cell nuclear antigen (PCNA), an operational marker of cell proliferation, and of several bone matrix proteins. A rectangular orthodontic appliance was set between the right and left upper molars of four-week-old rats, with 50 g of initial expansional force. Two days after application of the force, the pre-existing cartilage was separated laterally. Mesenchymal cells with stretched shapes were arranged parallel to the expansional force and filled the center of the suture. Only a few of these stretched cells exhibited nuclear accumulation of PCNA. In contrast, many polygonal mesenchymal cells distributed along the inner lateral side of the cartilaginous tissue exhibited strong immunoreactivity for PCNA. Localization of alkaline phosphatase activity overlapped into this proliferating cell zone. Nascent extracellular matrix under the proliferating cells was positive for osteocalcin, indicating commencement of active bone formation. These findings indicated that, among mesenchymal cells subjected to expansional forces, only cells located on the inner side of the cartilaginous tissue proliferate and differentiate into osteoblasts. In agreement with rapid bone growth progression, apoptosis was also observed in the zone of proliferating cells, as measured by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assays.