p53 and clock genes play an important role in memory and learning ability depression due to long-term ultraviolet A eye irradiation.

BACKGROUND: Long-term ultraviolet A (UVA) eye irradiation decreases memory and learning ability in mice. However, the underlying mechanism is still unclear. OBJECTIVES: In this study, ICR mice were used to study the effects of long-term UVA eye irradiation. METHODS: The eyes of mice were exposed to UVA from an FL20SBLB-A lamp three times a week for 1 year. Then, we analyzed memory and learning ability in the mice using water maze and step-through passive avoidance tests, and measured the levels of p53, Period2 (Per2), Clock, brain and muscle Arnt-like protein-1 (Bmal1), nicotinamide mononucleotide adenylyltransferase (NMNAT) activity, nicotinamide phosphoribosyltransferase (NAMPT) activity, nicotinamide adenine dinucleotide (NAD+), and sirtuin 1 (Sirt1) in the brains of treated and control animals. RESULTS: The results showed that the p53 level increased significantly following long-term UVA eye irradiation, whereas the levels of Period2, Bmal1, Clock, NMNAT and NAMPT activities, NAD+, and Sirt1 decreased significantly. Furthermore, we found that p53 inhibition ameliorated the UVA eye irradiation-induced depression of memory and learning ability. CONCLUSION: These results indicate that long-term UVA eye irradiation stimulates p53, inhibits the clock gene, and reduces Sirt1 production in the NAD+ constructional system, resulting in reduced memory and learning ability.

Ultraviolet B radiation to the eye induces pigmentation in the epidermis via the activation of the subunit gp91 phox of reduced nicotinamide adenine dinucleotide phosphate oxidase.

Irradiation by ultraviolet (UV)B is known to increase the number of dopamine (Dopa)-positive melanocytes in the skin. In this study, a 2.5-kJ/m(2) dose of UVB radiation was delivered by a sunlamp to the ear or the eye of wild-type C57BL/6j mice and of gp91 phox(-/-) C57BL/6j mice that had a knockout mutation of the gp91 phox subunit of reduced nicotinamide adenine dinucleotide phosphate oxidase (NADPH). The degree of change in the Dopa-positive melanocyte expression in was reduced in gp91 phox(-/-) mice given UVB irradiation to the eye, but not in those given irradiation to the ear. The plasma level of α-melanocyte-stimulating hormone (α-MSH) in the blood increased in the C57BL/6j mice after irradiation to either the eye or the ear, but it did not increase in the gp91 phox(-/-) mice given UVB irradiation to the eye. Both gp91 phox and α-MSH in the central nervous system seem to contribute to pigmentation after UVB irradiation of the eye in mice.

Oxidative stress promotes the regression of fetal liver hemopoiesis.

Although apoptosis is believed to play an important role in the ontogenetic development of animals, the molecular mechanism that triggers the regression of liver hemopoiesis during the perinatal period is not known. Apoptosis is induced by many factors such as a decrease in growth factors and increased oxygen stress. Since hepatic gamma-glutamyl transferase (GT) levels change markedly during the perinatal period in rodents, the metabolism of glutathione (GSH), a naturally occurring major antioxidant, might change significantly in and around liver cells. Hemopoietic cells but not hepatocytes exhibit significant apoptosis in thiol-free medium and the hemopoietic apoptosis can be inhibited by various thiols, such as L-cysteine, N-acetyl-L-cysteine, and GSH. The contribution of GSH levels in and around fetal liver cells in the triggering of apoptosis in hemopoietic cells is discussed.

Relaxing intraoperative natural sound blunts haemodynamic change at the emergence from propofol general anaesthesia and increases the acceptability of anaesthesia to the patient.

BACKGROUND: It is known that auditory input, such as comforting music or sound, blunts the human response to surgical stress in conscious patients under regional anaesthesia. As auditory perception has been demonstrated to remain active under general anaesthesia, playing comforting sounds to patients under general anaesthesia might also modulate the response of these patients to surgical stress. METHODS: Fifty-nine patients scheduled for laparoscopic cholecystectomy were anaesthetized with propofol general anaesthesia in combination with epidural anaesthesia. Natural sounds, chosen preoperatively by each patient as being comforting, were played to 29 patients using headphones during surgery (S group) and the remainder of the patients (n = 30) were fitted with dummy open-type headphones (N group). We compared the haemodynamic change during anaesthesia and the acceptability of anaesthetic practice between the two groups in a randomized double-blind design. RESULTS: There were no differences in haemodynamics between the S and N groups during surgery. During the emergence from anaesthesia, the mean blood pressure and heart rate gradually increased; both parameters were significantly higher in the N group than in the S group. Postoperatively, patients in the S group perceived the experience of anaesthesia as significantly more acceptable than did those in the N group. CONCLUSION: These findings indicate that allowing patients comforting background sounds during general anaesthesia may blunt haemodynamic changes upon emergence from general anaesthesia and increase the acceptability of the experience of anaesthesia.

Induction and mechanism of apoptotic cell death by propofol in HL-60 cells.

BACKGROUND: Apoptosis (programmed cell death) occurs in various physiological and pathological conditions, exhibits a characteristic mechanism of intracellular sequential reaction and may be involved in determining clinical outcome. The antioxidant activity of propofol (2,6-diisopropylphenol) together with the stimulating effect of protein kinase C suggests that propofol might have the potential to modulate apoptosis. Thus, it is of both clinical interest and biomedical importance to investigate and clarify the effect and mechanism of propofol upon the intracellular reactions underlying apoptotic cell death. METHODS: The effect of propofol on apoptosis was investigated using cultured human promyelocytic leukemia HL-60 cells. This well-characterized cell line is useful for the study of apoptosis because the various biochemical steps occurring during apoptosis have been well documented. RESULTS: Treatment of HL-60 cells with propofol resulted in growth inhibition with the formation of apoptotic bodies in a concentration-dependent manner. DNA fragmentation and ladder formation was also observed in a concentration-dependent manner. Propofol treatment resulted in activation of caspase-3, -6, -8 and -9, thereby suggesting that cell surface death receptor activation of the caspase cascade mediates propofol-induced apoptosis with consequent formation of the cleaved product of Bid (a pro-apoptotic Bcl-2 family member protein) and activation of the mitochondrial pathway with cytosolic release of cytochrome c. CONCLUSION: Propofol may induce apoptosis, which is dependent on the mechanism that activates both the cell surface death receptor pathway and the mitochondrial pathway.

Tributyltin interacts with mitochondria and induces cytochrome c release.

Although triorganotins are potent inducers of apoptosis in various cell types, the critical targets of these compounds and the mechanisms by which they lead to cell death remain to be elucidated. There are two major pathways by which apoptotic cell death occurs: one is triggered by a cytokine mediator and the other is by a mitochondrion-dependent mechanism. To elucidate the mechanism of triorganotin-induced apoptosis, we studied the effect of tributyltin on mitochondrial function. We found that moderately low doses of tributyltin decrease mitochondrial membrane potential and induce cytochrome c release by a mechanism inhibited by cyclosporine A and bongkrekic acid. Tributyltin-induced cytochrome c release is also prevented by dithiols such as dithiothreitol and 2,3-dimercaptopropanol but not by monothiols such as GSH, N-acetyl-L-cysteine, L-cysteine and 2-mercaptoethanol. Further studies with phenylarsine oxide agarose revealed that tributyltin interacts with the adenine nucleotide translocator, a functional constituent of the mitochondrial permeability transition pore, which is selectively inhibited by dithiothreitol. These results suggest that, at low doses, tributyltin interacts selectively with critical thiol residues in the adenine nucleotide translocator and opens the permeability transition pore, thereby decreasing membrane potential and releasing cytochrome c from mitochondria, a series of events consistent with established mechanistic models of apoptosis.

Nitric oxide regulates mitochondrial respiration and functions of articular chondrocytes.

OBJECTIVE: Biologic effects of nitric oxide (NO) have been shown to increase under hypoxic conditions. Because the oxygen tension in joint cavities of patients with arthritis is fairly low, biologic effects of NO would be expected to be significantly large in these compartments. This study was undertaken to investigate the effects of NO on the energy metabolism and functions of articular chondrocytes under different oxygen tension conditions. METHODS: Articular chondrocytes from rabbits were cultured under various oxygen concentrations in the presence or absence of NO and NOC18, an NO donor. Cellular respiration was measured using a Clark-type oxygen electrode. Levels of ATP in the cells were determined according to the luciferin-luciferase method. Cellular synthesis of proteoglycans was determined by measuring the incorporation of radioactivity (derived from 35S-labeled SO4) into glycosaminoglycans. Expression of stress-related proteins was evaluated by Western blotting analysis using specific antibodies. RESULTS: Respiration and ATP synthesis of cultured chondrocytes were inhibited by NO, particularly under low oxygen concentrations. The presence of either NO or specific inhibitors of mitochondrial electron transport suppressed the synthesis of proteoglycans without affecting cell viability. When exposed to NO, cellular levels of heat-shock protein 70 (hsp70) and heme oxygenase 1 (HO-1) increased markedly. The presence of inhibitors of mitochondrial electron transport also increased cellular levels of hsp70 and HO-1. CONCLUSION: These results suggest that NO generated in the joint might inhibit energy metabolism and the synthesis of proteoglycans of chondrocytes, thereby modulating pathophysiologic processes occurring in patients with arthritis.

Metabolic cooperation of ascorbic acid and glutathione in normal and vitamin C-deficient ODS rats.

Although the coordination of various antioxidants is important for the protection of organisms from oxidative stress, dynamic aspects of the interaction of endogenous antioxidants in vivo remain to be elucidated. We studied the metabolic coordination of two naturally occurring water-soluble antioxidants, ascorbic acid (AA) and reduced glutathione (GSH), in liver, kidney and plasma of control and scurvy-prone osteogenic disorder Shionogi (ODS) rats that hereditarily lack the ability to synthesize AA. When supplemented with AA, its levels in liver and kidney of ODS rats increased to similar levels of those in control rats. Hepato-renal levels of glutathione were similar with the two animal groups except for the slight increase in its hepatic levels in AA-supplemented ODS rats. Administration of L-buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, rapidly decreased the hepato-renal levels of glutathione in a biphasic manner, a rapid phase followed by a slower phase. Kinetic analysis revealed that glutathione turnover was enhanced significantly in liver mitochondria and renal cytosol of ODS rats. Administration of BSO significantly increased AA levels in the liver and kidney of control rats but decreased them in AA-supplemented ODS rats. Kinetic analysis revealed that AA is synthesized by control rat liver by some BSO-enhanced mechanism and the de novo synthesized AA is transferred to the kidney. Such a coordination of the metabolism of GSH and AA in liver and kidney is suppressed in AA-deficient ODS rats. These and other results suggest that the metabolism of AA and GSH forms a compensatory network by which oxidative stress can be decreased.

Suppressive effects of cyclosporin A and FK-506 on superoxide generation in human polymorphonuclear leukocytes primed by tumor necrosis factor alpha.

Most previous studies have found no effects of cyclosporin A and FK-506 on active oxygen generation in human polymorphonuclear leukocytes. Recently various differences in biologic properties have been reported between unprimed peripheral blood human polymorphonuclear leukocytes and tissue or primed human polymorphonuclear leukocytes. In this study, we investigated the effects of cyclosporin A and FK-506 on superoxide (O(2)(-)) generation induced by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine in human peripheral blood polymorphonuclear leukocytes primed or unprimed with tumor necrosis factor alpha. Neither cyclosporin A nor FK-506 suppressed N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced O(2)(-) generation in unprimed human polymorphonuclear leukocytes at concentrations between 0.1 nM and 10 microM, as in previous studies. Only at 1 microM of cyclosporin A and 100 nM of FK-506 were marginal suppressive effects observed. On the other hand, cyclosporin A and FK-506 both suppressed N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced O(2)(-) generation in tumor-necrosis-factor-alpha-primed human polymorphonuclear leukocytes, strongly and dose dependently, at concentrations between 1 nM and 1 microM. Neither cyclosporin A nor FK-506 influenced tyrosyl phosphorylation of 115 kDa protein, which is inducible during the priming process, suggesting that neither cyclosporin A nor FK-506 influenced the tumor-necrosis-factor-alpha-induced priming process itself, and instead modified the biologic response of primed human polymorphonuclear leukocytes.

Granulocyte-colony stimulating factor enhances anti-tumour effect of hyperthermia.

The combined effect of granulocyte-colony stimulating factor (GCSF) and hyperthermia in the treatment of experimental tumours was studied to examine the possible involvement of activated granulocytes in the antitumour effect of hyperthermia. Two weeks after transplantation of SCC VII cells (1 x 10(5)) into the instep of the left leg of C3H/HeJ male mice, the mice were given subcutaneous injections of GCSF (0.2 mg/kg) for 4 days. On day 4, hyperthermia was applied locally at 43 degrees C for 40 min. Hyperthermia inhibited the tumour growth, and this effect was enhanced by pre-treating the animals with GCSF. The numbers of circulating neutrophils in control and GCSF-treated mice were 2728 +/- 517/microl and 3124 +/- 194/microl, respectively (p = 0.53). Hyperthermia increased the number of neutrophils to 4409 +/- 700/microl (p < 0.05). Hyperthermia combined with GCSF significantly increased the number of netrophils to 5479 +/- 691/microl (p < 0.01). Chemiluminescence analysis using L-012 revealed that GCSF enhanced the generation of reactive oxygen species by about 10-fold. Glutathione contents in tumours 24 h after hyperthermia decreased by about 50% in both the hyperthermia groups with or without GCSF, as compared to those in the control. The GCSF-enhanced anti-tumour activity of hyperthermia was markedly inhibited by administration of a long-acting superoxide dismutase derivative (SM-SOD). These results suggest that GCSF activates the ability to generate active oxygen species by neutrophils and, thereby, enhances the anti-tumour effect of hyperthermia.

Cross-talk of NO, superoxide and molecular oxygen, a majesty of aerobic life.

Because nitric oxide (NO) reacts with various molecules, such as hemeproteins, superoxide and thiols including glutathione (GSH) and cysteine residues in proteins, biological effects and metabolic fate of this gaseous radical are affected by these reactants. Although the lifetime of NO is short particularly under air atmospheric conditions (where the oxygen tension is unphysiologically high), it increases significantly under physiologically low oxygen concentrations. Because oxygen tensions in human body differ from one tissue to another and change depending on their metabolism, biological activity of NO in various tissues might be affected by local oxygen tensions. To elucidate the role of NO and related radicals in the regulation of circulation and energy metabolism, their effects on arterial resistance and energy metabolism in mitochondria, mammalian cells and enteric bacteria were studied under different oxygen tensions. Kinetic analysis revealed that NO-dependent generation of cGMP in resistance arteries and their relaxation were strongly enhanced by lowering oxygen tensions in the medium. NO reversibly suppressed the respiration and ATP synthesis of isolated mitochondria and intact cells particularly under low oxygen tensions. Kinetic analysis revealed that cross-talk between NO and superoxide generated in and around endothelial cells regulates arterial resistance particularly under physiologically low oxygen tensions. NO also inhibited the respiration and ATP synthesis of E. coli particularly under low oxygen tensions. Because concentrations of NO and H+ in gastric juice are high, most ingested bacteria are effectively killed in the stomach. However, the inhibitory effects of NO on the respiration and ATP synthesis of H. pylori are extremely small. Kinetic analysis revealed that H. pylori generates the superoxide radical thereby inhibiting the bactericidal action of NO in gastric juice. Based on such observations, critical roles of the cross-talk of NO, superoxide and molecular oxygen in the regulation of energy metabolism and survival of aerobic and microaerophilic organisms are discussed.

Macrophage-derived nitric oxide induces apoptosis of rat hepatoma cells in vivo.

Although nitric oxide (NO) has been postulated to play important roles in host defense mechanisms against tumor cells, a direct evidence supporting this hypothesis is lacking. To obtain molecular insights into the antitumor action of NO, its metabolism and effect on ascites hepatoma (AH-130) cells were investigated in tumor-bearing rats. Kinetic analysis revealed that substantial amounts of nitrite and nitrate, metabolites of NO, appeared in plasma and ascites of AH-130-inoculated rats. Western blot analysis revealed that a large number of macrophages that expressed inducible type of NO synthase (iNOS) appeared in cancerous ascites, particularly during 1 to 2 weeks after inoculation of AH-130 cells. When NO generation by peritoneal macrophages increased, a significant fraction of AH-130 in ascites fluid underwent apoptosis as judged from the fragmentation of their nuclear DNA. Kinetic analysis revealed that NO strongly inhibited mitochondrial electron transport and changed calcium status in AH-130 cells, particularly under low oxygen tensions such as in cancerous ascites. Intraperitoneal injection of NO donor strongly enhanced DNA fragmentation of AH-130 cells. Antimycin A, a specific inhibitor for mitochondrial electron transport, also induced DNA fragmentation of AH-130 cells by a mechanism that was inhibited by adding ascorbate and tetramethyl-p-phenylene diamine (TMPD) as electron donors. These results indicate that NO derived from peritoneal macrophages inhibits mitochondrial electron transport and disturbs calcium homeostasis in ascites hepatoma AH-130 cells, thereby inducing their apoptosis in vivo.

A processed grain food inhibits hepatic injury in endotoxemic rats.

Although various antioxidants have been tested as therapeutics for endotoxemic subjects, the results of their efficacy are conflicting. Antioxidant biofactor (AOB) is a unique processed grain food that exhibits strong antioxidant activity (Minamiyama et al, J Nutr Sci Vitaminol, 40: 467-477, 1994). The present study was carried out to test the effect of AOB on hepatic injury in rats induced by lipopolysaccharide (LPS). Intravenous administration of LPS induced liver injury with a concomitant increase in hepatic generation of nitric oxide (NO) and 4-hydroxy-2-nonenal (HNE) modified proteins in the control group. The administration of AOB significantly inhibited the LPS-induced hepatic injury and generation of HNE-modified proteins and increased the survival rate of endotoxemic rats without affecting NO generation and plasma levels of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). AOB scavenged superoxide radicals without affecting NO production by LPS-stimulated macrophage cell line J 774.2 cells. AOB also inhibited lipid peroxidation induced by LPS in the cells. These results suggested that AOB might scavenge superoxide radicals and decrease toxic metabolites including HNE, thereby inhibiting liver injury in endotoxemic rats.

Biochemical properties of human oral polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMN) isolated from the oral cavity of healthy human volunteers, spontaneously generated superoxide, nitric oxide (NO) and other reactive oxygen species (ROS) which exhibited strong luminol chemiluminescence (LCL). To understand the physiological roles of oral PMN (OPMN), biochemical properties of the cells were analyzed. Biochemical analysis revealed that OPMN were already primed under physiological conditions. Western blot analysis revealed that they strongly expressed the inducible type of NO synthase (NOS II) and exhibited the activity to catalyze tyrosine phosphorylation of various proteins including a 115 kDa protein (cbl product). OPMN also generated H2O2 and .OH by some superoxide dismutase (SOD)-sensitive mechanism and released myeloperoxidase (MPO). Kinetic analysis using specific inhibitors revealed that OCl- generated by OPMN was predominantly responsible for the enhanced LCL. During the incubation under standard culture conditions, OPMN underwent apoptosis which proceeded more rapidly than that of the circulating PMN (CPMN). Immunochemical analysis revealed that expression of apoptosis-related gene products, such as Bcl-2, Bcl-xL and Bax, was below detectable levels with both cell types. However, caspase-3 but not caspase-1 was markedly activated in OPMN. These results indicate that the primed OPMN spontaneously generate ROS and play an important role in the defense mechanism in the oral cavity and that the generated ROS activate caspase-3 thereby inducing apoptosis of the cells.

Nitric oxide regulates energy metabolism and Bcl-2 expression in intestinal epithelial cells.

Nitric oxide (NO) inhibits the respiration of mitochondria and enteric bacteria, particularly under low O2 concentration, and induces apoptosis of various types of cells. To gain insight into the molecular role of NO in the intestine, we examined its effects on the respiration, Ca2+ status, and expression of Bcl-2 in cultured intestinal epithelial cells (IEC-6). NO reversibly inhibited the respiration of IEC-6 cells, especially under physiologically low O2 concentration. Although NO elevated cytosolic Ca2+ as determined by the fura 2 method, the cells were fairly resistant to NO. Kinetic analysis revealed that prolonged exposure to NO elevated the levels of Bcl-2 and suppressed the NO-induced changes in Ca2+ status of the cells. Because Bcl-2 possesses antiapoptotic function, toxic NO effects might appear minimally in enterocytes enriched with Bcl-2. Thus NO might effectively exhibit its antibacterial action in anaerobic intestinal lumen without inducing apoptosis of Bcl-2-enriched mucosal cells.

Thermal injury induces thymocyte apoptosis in the rat.

BACKGROUND: The thymus plays important roles in host defense, which may be impaired after burn injury. The effects of thermal injury on thymocytes were investigated in male Wistar rats. METHODS: Changes in thymus weight and content of glutathione and corticosterone were determined after burn injury. Apoptosis of thymocytes was detected by electrophoresis of DNA, and lymphocyte subsets were characterized by flow cytometry. The effects of adrenalectomy and the glucocorticoid receptor antagonist RU486 in burned animals were also studied. RESULTS: The weight of the thymus decreased progressively after burn injury, and this effect was accompanied by increases in the corticosterone concentration in plasma and the thymus and apoptosis of CD4+CD8+ thymocytes. Administration of RU486 or adrenalectomy inhibited burn-induced thymocyte apoptosis. CONCLUSION: Thermal injury increases the corticosterone concentration in plasma and the thymus and triggers thymocyte apoptosis.

Role of glutathione in nitric oxide-dependent regulation of energy metabolism in rat hepatoma cells.

Previous studies in this laboratory revealed that nitric oxide (NO) reversibly inhibits the respiration of isolated mitochondria and ascites hepatoma (AH-130) cells by an oxygen concentration-dependent mechanism. The inhibitory effect of NO on the respiration of AH-130 cells was enhanced by treating with digitonin that selectively permeabilized plasma membranes and released cytosolic low-molecular-weight compounds. Reduced glutathione (GSH) is the most abundant cytosolic thiol that easily reacts with NO. To elucidate the mechanism by which digitonin enhanced the inhibitory action of NO, the effect of GSH and related thiols was studied with AH-130 cells and their mitochondria. The inhibitory effect of NO on the respiration of digitonin-treated cells was suppressed by either GSH, L-cysteine, or N-acetylcysteine, but not by oxidized glutathione. The inhibitory effect of NO on the respiration of their mitochondria was also decreased by GSH. In contrast, the inhibitory effect of NO was markedly enhanced with AH-130 cells obtained from animals that were pretreated with L-buthionine sulfoximine (BSO), a specific inhibitor for GSH synthesis. Kinetic analysis revealed that NO dose-dependently decreased GSH levels in AH-130 cells with concomitant generation of S-nitrosothiols. Although S-nitrosoglutathione (GSNO), a slow releaser of NO, also inhibited the respiration of tumor cell mitochondria, its effect was significantly lower than that of NO. These results suggest that cellular GSH might play pivotal roles in the regulation of energy metabolism in hepatoma cells by modulating free forms of NO.

Role of glutathione and nitric oxide in the energy metabolism of rat liver mitochondria.

Previous studies have suggested that nitric oxide (NO) inhibited mitochondrial respiration. NO and/or its intermediate(s) react with various molecules, such as hemeproteins and free SH groups. The inhibitory effect of NO on mitochondrial respiration was decreased by exogenously added glutathione (GSH). However, a decrease of intramitochondrial GSH by pretreating animals with L-buthionine sulfoximine had no appreciable effect on the inhibitory effect of isolated mitochondria. Furthermore, the effect of NO was not affected by depleting free SH residues in mitochondria by N-ethylmaleimide. These results suggest that cytosolic but not intramitochondrial GSH might be an important factor that determines the NO-dependent regulation of mitochondrial energy metabolism.

Role of glutathione metabolism and apoptosis in the regression of liver hemopoiesis.

Although apoptosis has been believed to play important roles in ontogenic development of animals, the molecular mechanism that triggers the regression of liver hemopoiesis during perinatal period is not known. Apoptosis is induced by many factors, such as decrease in growth factors and increased oxygen stress. Because hepatic gamma-glutamyltransferase (GGT) changes markedly during the perinatal period of a rodent, metabolism of glutathione (GSH), a naturally occurring major antioxidant, might change significantly in and around liver cells. To know the possible involvement of apoptosis and GSH metabolism in the regression of hemopoiesis, hepatocytes and hemopoietic cells were isolated from fetal rat liver. Biochemical analysis revealed that, during the perinatal period, hepatic GGT levels transiently increased predominantly with hepatocytes, suggesting a marked change in thiol status in and around these cells. Cell culture analysis revealed that hemopoietic cells but not hepatocytes exhibited a marked apoptosis in a thiol-free medium, as judged from DNA fragmentation. The apoptosis of hemopoietic cells was inhibited by various thiols, such as L-cysteine, N-acetyl-L-cysteine (NAC), and GSH. These observations suggested that a marked change in GSH status in and around liver cells might play critical roles in triggering apoptosis of hemopoietic cells, thereby enhancing the regression of liver hemopoiesis.

Characterization of the inner enamel epithelium in the enamel-free area based on the ability to secrete enamel protein demonstrated by in situ hybridization and immunohistochemistry.

Both the expression of amelogenin mRNA and secretion of amelogenin were investigated in rat molars by in situ hybridization and immunohistochemistry. Probes were designed by multiple-labeling of oligonucleotide probes for in situ hybridization. Amelogenin mRNA first appeared in differentiating ameloblasts of the distal region and some inner enamel epithelial cells of enamel-free area (EFA cells) of the second cusp at postnatal day 0. At the same time, amelogenin protein was detected in the extracellular matrix between dentin and differentiating ameloblasts and in some EFA cells of the second cusp. At postnatal day 1-3, amelogenin was expressed in the secretory ameloblasts, and in the matrix beneath these cells. Both amelogenin mRNA and amelogenin were detected in the EFA cells and their extracellular matrix. After postnatal day 5, amelogenin mRNA and amelogenin were detected in the secretory ameoloblasts and extracellular matrix in the enamel-forming region, respectively. At this time, amelogenin mRNA was not detected in the EFA cells, but a small amount of amelogenin was found in the matrix beneath the EFA cells. These findings suggest that EFA cells differentiate into amelogenin-secreting cells, i.e. ameloblasts, but that the secretion lasts for only a short period at the early stage of tooth development.