Oral administration of bovine lactoferrin attenuates ultraviolet B-induced skin photodamage in hairless mice.

Lactoferrin (LF) is recognized as a host defensive glycoprotein, especially for newborn infants. The aim of this study was to investigate whether orally administered LF had protective activity against UV-induced skin damage in hairless mice. Transepidermal water loss and skin hydration were evaluated in nonirradiated mice, UVB-irradiated mice, and UVB-irradiated and LF-administered mice. Supplementation with LF (1,600 mg/kg per day) effectively suppressed the increase in transepidermal water loss, reduction in skin hydration, aberrant epidermal hyperplasia, and cell apoptosis induced by UV irradiation. Although no significant changes in superoxide dismutase-like activity or malondialdehyde levels were observed in the skin with both UV irradiation and LF administration, UV-stimulated IL-1ß levels in the skin were significantly suppressed by the administration of LF. Oral supplementation with LF has the potential to reduce IL-1ß levels and prevent UV-induced skin damage. Further studies are needed to elucidate the relationships between the antiinflammatory effects and skin protective function of LF.

The role of aldose reductase in sugar cataract formation: aldose reductase plays a key role in lens epithelial cell death (apoptosis).

Since aldose reductase is localized primarily in lens epithelial cells, osmotic insults induced by the accumulation of sugar alcohols occur first in these cells. To determine whether the accumulation of sugar alcohols can induce lens epithelial cell death, galactose-induced apoptosis has been investigated in dog lens epithelial cells. Dog lens epithelial cells were cultured in Dulbecco's modified Eagle's mimimum essential medium (DMEM) supplemented with 20% fetal calf serum (FCS). After reaching confluence at fifth passage, the medium was replaced with the same DMEM medium containing 50 mM D-galactose and the cells were cultured for an additional 2 weeks. Almost all of the cells cultured in galactose medium were stained positively for apoptosis with the terminal deoxynucleotidyl transferance-mediated biotin-dUTP nick end labeling (TUNEL) technique. Agarose gel electrophoresis of these cells displayed obvious DNA fragmentation, known as a ladder formation. All of these apoptotic changes were absent in similar cells cultured in galactose medium containing 1 microM of the aldose reductase inhibitor AL 1576. Addition of AL 1576 also reduced the cellular galactitol levels from 123+/-10 microgram/10(6) cells (n=5) to 3.9+/-1.9 microgram/10(6) cells (n=5). These observations confirm that galactose induced apoptosis occurs in dog lens epithelial cells. Furthermore, the prevention of apoptosis by an aldose reductase inhibitor suggests that this apoptosis is linked to the accumulation of sugar alcohols.

Effects of L-carnitine supplementation on renal anemia in poor responders to erythropoietin.

While renal anemia can be successfully treated by use of erythropoietin (EPO) in most hemodialysis (HD) patients, some patients have anemia that is refractory to treatment with a high dose of EPO. We examined whether L-carnitine treatment could raise hematocrit (Hct) levels in such patients. Fourteen HD patients who showed a poor response to EPO and no evident factors which inhibit a response to EPO were selected to receive oral L-carnitine (500 mg/day) in a 3-month trial. During the study, 36% of the patients showed Hct increases of more than 2%. Statistical analysis revealed significant increases of Hct (p = 0.003) and total iron-binding capacity (TIBC) (p = 0.050) and a significant decrease of ferritin (p = 0.005). In addition, we found that red blood cells (RBCs) in HD patients contained a comparable level of carnitine to normal controls, despite the presence of serum carnitine deficiency, and that RBC carnitine was not removed through HD, in contrast to serum carnitine. These results suggest that RBC carnitine may be essential for RBCs to perform their metabolic function in renal anemia and that oral L-carnitine treatment could improve anemia in poor responders to EPO.

A calcium-activated cation current by an alternatively spliced form of Trp3 in the heart.

To investigate a cDNA encoding cation current, we isolated an alternatively spliced form of a rat Trp3, designated Trp3sv. Trp3sv encodes 736 amino acids with a unique N terminus and six transmembrane segments. Expression of the cRNA in Xenopus oocytes was successfully performed. The cation selective current appeared after the addition of ionomycin or induced by prolonged depolarization but not by hyperpolarization. This induction was not observed by a treatment with thapsigargin, phorbol ester, or ATP. Na(+), K(+), tetraethylammonium, and divalent cations were permeable, while N-methylglucamine and chloride were nominally impermeable ions. The currents were not inhibited by flufenamate ruthenium red but nonspecifically by 2 mm Gd(3+). Northern as well as Western blot suggested lower levels of the expression observed in some organs, while reverse transcriptase-polymerase chain reaction suggested that it widely spread among various organs. Therefore, we may conclude that N-terminal spliced valiant of Trp3, Trp3sv, encodes a calcium-activated cation channel in various organs.

Oxidative DNA damage by vitamin A and its derivative via superoxide generation.

Recent intervention studies revealed that beta-carotene supplement to smokers resulted in a higher incidence of lung cancer. However, the causal mechanisms remain to be clarified. We reported here that vitamin A (retinol) and its derivative (retinal) caused cellular DNA cleavage detected by pulsed field gel electrophoresis. Retinol and retinal significantly induced 8-oxo-7,8-dihydro-2'-deoxyguanosine formation in HL-60 cells but not in H(2)O(2)-resistant HP100 cells, suggesting the involvement of H(2)O(2) in cellular DNA damage. Experiments using (32)P-labeled isolated DNA demonstrated that retinol and retinal caused Cu(II)-mediated DNA damage, which was inhibited by catalase. UV-visible spectroscopic and electron spin resonance-trapping studies revealed the generation of superoxide and carbon-centered radicals, respectively. The superoxide generation during autoxidation of retinoids was significantly correlated with the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, although the yield of carbon-centered radicals was not necessarily related to the intensity of DNA damage. These findings suggest that superoxide generated by autoxidation of retinoids was dismutated to H(2)O(2), which was responsible for DNA damage in the presence of endogenous metals. Retinol and retinal have prooxidant abilities, which might lead to carcinogenesis of the supplements of beta-carotene.

A new approach to antiplatelet therapy: inhibitor of GPIb/V/IX-vWF interaction.

Evidence has been presented that the interaction between von Willebrand factor (vWF) and its platelet membrane receptor, the GPIb/V/IX complex, plays an important role in the pathogenesis of arterial thrombosis. A monoclonal antibody against the A1 domain of vWF has been shown to inhibit thrombus formation in the animal model of arterial thrombosis. Based upon these findings, a new approach to treating arterial thrombosis has been proposed by intervening in the interaction between vWF and platelet.

[Transcatheter oily chemoembolization and intermittent hepatic artery infusion chemotherapy in the management of advanced hepatocellular carcinoma].

Between 1990 and 1997, 227 patients with hepatocellular carcinoma were treated by intrahepatic arterial injection of a Lipiodol-Epirubicin-Mitomycin C emulsion followed by intermittent hepatic artery infusion of Epirubicin, Mitomycin C and 5-FU, employing an implantable subcutaneous infusion port. A catheter was inserted percutaneously into the hepatic artery using the Seldinger technique. Objective remission was induced in 80% of the evaluable patients as evidenced by a decrease in their AFP and PIVKA II levels. These remissions were also confirmed by liver sonogram and CT scan showing decreased tumor volume. Transcatheter oily chemoembolization combined with intermittent hepatic artery infusion chemotherapy seems to be an effective treatment for unresectable hepatocellular carcinoma both for palliation of symptoms as well as prolongation of survival with good quality of life.

Characterization of cDNA encoding full-length mouse platelet glycoprotein IX.

Glycoprotein (GP) IX is a platelet membrane 20 kD protein, which is associated with GPIbalpha, GPIb beta, and GPV to form GPIb/IX/V complex. GPIb/IX/V complex is a major receptor for von Willebrand factor, which mediates platelet adhesion and aggregation under high shear stress conditions. The relevance of this receptor for hemostasis has been implicated by a congenital bleeding disorder lacking the receptor, Bernard-Soulier syndrome. All subunits for the human receptor have been cloned and characterized. However, the function of GPIX is still elusive. To obtain further information of GPIX, we have determined a cDNA sequence of mouse GPIX (811 bp). The deduced amino-acid sequence (177aa) was 71% identical to the human GPIX protein. All cysteine residues in extracytoplasmic domain and putative N-linked glycosylation site (Asn44) were conserved. Mouse GPIX contained a leucine-rich glycoprotein sequence composed of 24 amino acids, as did human GPIX.

[Chinese herbs nephropathy in the Kansai area: a warning report].

In 1993, Vanherweghem and his associates reported cases of rapidly progressive renal interstitial fibrosis in young women who were administered a slimming regimen including Chinese herbs. Subsequently, similar cases have been reported. In Japan, especially in the Kansai area, several cases of Chinese herbs nephropathy have already been reported. We experienced a patient suffering from Chinese herbs nephropathy (CHN), and further detected aristolochic acids from the Chinese herbs taken by the patient. Aristolochic acids are known to be causative agents of CHN. The danger of CHN should be noted as soon as possible and drugs containing aristolochic acids should be prohibited.

High shear stress attenuates agonist-induced, glycoprotein IIb/IIIa-mediated platelet aggregation when von Willebrand factor binding to glycoprotein Ib/IX is blocked.

High shear stress facilitates von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib/IX, causing activation of GPIIb/IIIa to induce platelet aggregation. Here we report that activated GPIIb/IIIa, even occupied by ligands, is not sufficient to mediate platelet aggregation under high shear stress conditions when vWF binding to GPIb/IX is blocked. Platelet rich plasma or washed platelet suspension supplemented with purified human fibrinogen at a concentration of 2 mg/mL were treated with an anti-vWF monoclonal antibody NMC-4 which blocks the binding of vWF to GPIb/IX. After addition of 10 mumol/L ADP, aggregation was continuously monitored under various shear stress conditions (0-108 dyne/cm2) using a cone-plate type aggregometer previously described (Ikeda Y et al J Clin Invest 1991; 87:1234). The extent of maximal aggregation of agonist-stimulated platelets in the presence of NMC-4 correlated inversely with the level of shear stress applied, with the virtual absence of aggregation at 108 dyne/cm2. Once aggregated by 10 mumol/L ADP under low shear stress (12 dyne/cm2), platelets could be disaggregated, in part, by the application of high shear stress (108 dyne/cm2), and reaggregated when shear stress was returned to 12 dyne/cm2. Flow cytometric analysis revealed that platelets stimulated with 10 mumol/L ADP at 108 dyne/cm2 bound fluorescein isothiocyanate (FITC)-labeled fibrinogen, although aggregation was absent in this experimental condition. These results demonstrate the dual effect of shear stress on platelet functions; a pro-aggregating activity that induces vWF-GPIb/IX interaction leading to platelet activation, and an anti-aggregating force to prevent the growth of platelet thrombi. It is suggested that the efficacy of vWF blockade is greater under high shear than low shear stress conditions, and that a selective inhibition of platelet functions can be possible.

Synergistic cytotoxic effect of quercetin and heat treatment in a lymphoid cell line (OZ) with low HSP70 expression.

Heat shock protein 70 (HSP70) protects cells from various injurious stimuli and is important in cell growth and differentiation. However, its expression in leukemia cells has not been analyzed systematically. We, therefore, investigated the expression of HSP70 in various types of leukemia cells and hematopoietic cell lines. Immunoblot analysis revealed that HSP72/73 were expressed at low levels in the acute lymphocytic leukemia cells and lymphoid cell lines examined. However, heat (43 C for 2 h) preferentially augmented HSP72/73 expression in lymphoid cells. This induction was partially blocked by the treatment with quercetin (10 microM for 24 h). Finally, heat shock following quercetin treatment synergistically induced apoptosis in a lymphoid cell line (OZ). These observations suggest the possibility of selective purging of lymphocytic leukemia cells by combination with quercetin and heat.

Quantitative evaluation of inflammatory cells in seasonal allergic conjunctivitis.

This study aimed to investigate the inflammatory changes occurring in the allergic conjunctiva due to different allergens. Ninety-two allergic conjunctivitis patients were divided into two groups: group I showed cedar pollen sensitivity only; group II showed sensitivities to cedar pollen and household dust or mites. Using brush cytology procedures, quantitative evaluation of inflammatory cells in the conjunctiva was made. The mean proportion of the neutrophils and lymphocytes in group II was significantly higher than the percentage of neutrophils in group I. The counts of eosinophils showed no significant difference between groups I and II. Our results suggest that the eosinophil numbers are not different between the allergic conjunctivitis groups. The neutrophilic and lymphocytic increase in allergic conjunctivitis may be due to two distinct groups of antigens.

[Traditional remedy-induced Chinese herbs nephropathy showing rapid deterioration of renal function].

A 19-year-old female was referred to our hospital for azotemia and anemia. She had been taking a health food for atopic dermatitis for about three years. Urinalysis showed proteinuria, glycosuria and microscopic hematuria. Generalized aminoaciduria was observed. Moreover, severe anemia, azotemia, hypokalemia and hypophosphatemia were also observed. Renal biopsy specimen disclosed hypocellular interstitial fibrosis and degeneration of the proximal tubular epithelial cells. No remarkable changes were observed in the glomeruli. Aristolochic acid was detected in the health food. From these findings, she was diagnosed as having Chinese herbs nephropathy (CHN). Although consumption of the food intake was stopped, her renal function deteriorated rapidly. Previously, we reported that certain kinds of Chinese herbal drugs contain aristolochic acid and that the drugs should be prohibited if aristolochic acid is identified. However, we experienced a patient of CHN arising from traditional remedy, which was not proved to be safe. It should be awared that health foods may contain aristolochic acid.

Stimulation of the activities of hepatic fatty acid oxidation enzymes by dietary fat rich in alpha-linolenic acid in rats.

The activities of hepatic fatty acid oxidation enzymes in rats fed perilla oil rich in alpha-linolenic acid (alpha-18:3) were compared with those fed saturated fats or safflower oil (the mixture of safflower oil and olive oil, 94:8, w/w) containing the same amount of polyunsaturated fatty acids with perilla oil exclusively as linoleic acid (18:2). When the rats were fed the diets containing 15% coconut, safflower, and perilla oils for 1 week, the rate of mitochondrial and peroxisomal oxidation of palmitoyl-CoA (16:0-CoA) in the liver homogenates was the highest in rats fed perilla oil. Among the rats fed the diets containing 15% palm, safflower, and perilla oils for 2 weeks, the rates of mitochondrial and peroxisomal oxidations of 16:0-, 18:2-, and alpha-18:3-CoAs were the highest in rats fed perilla oil, and the rate of oxidation of alpha-18:3-CoA by both pathways was higher than those of other acyl-CoAs in all groups. Dietary perilla oil relative to palm and safflower oils significantly increased the activities of carnitine palmitoyltransferase, acyl-CoA dehydrogenase, acyl-CoA oxidase, and 2,4-dienoyl-CoA reductase. The substrate specificity of carnitine palmitoyltransferase appeared to be responsible for differential rates of the mitochondrial oxidation of acyl-CoAs. The substrate specificity of acyl-CoA oxidase did not account for the preferential peroxisomal oxidation of alpha-18:3 relative to 18:2. The preferential mitochondrial and peroxisomal beta-oxidation of alpha-18:3-CoA relative to 16:0- and 18:2-CoAs was also confirmed in rats fed laboratory chow irrespective of the substrate/albumin ratios in the assay mixture. It was suggested that both substrate specificities and alterations in the activities of the enzymes in beta-oxidation pathway play a significant role in the regulation of the serum lipid concentrations in rats fed a diet rich in alpha-18:3.

Bone induction of hydroxyapatite combined with bone morphogenetic protein and covered with periosteum.

Using a rabbit model, we evaluated the role of the periosteum in bone induction using hydroxyapatite ceramic pellets, some of which had been coated with bone morphogenetic protein. Eighteen rabbits were divided into two groups, a control group that received pellets soaked in phosphate-buffered saline alone and another group for which the pellets had been supplemented with bone morphogenetic protein. After making a skin incision on the head of each rabbit, two caudally pedicled periosteal flaps measuring 1 cm in width and 2.5 cm in length were elevated. These flaps were wrapped around hydroxyapatite pellets manufactured from limestone and fixed with sutures. After implantation, both groups of rabbits were returned to their cages, maintained for 3, 6, or 9 weeks [every group consisted of 3 rabbits (6 pellets)], and then sacrificed. In this study, the extent of bone induction, which was measured with a dual x-ray densitometer, that resulted from covering the hydroxyapatite pellet with periosteum alone (control group) was minimal. On the contrary, since osteogenesis from the periosteum toward the pores of the pellet was observed in the bone morphogenetic protein group much more than in the control group, the usefulness of our technique was confirmed. However, active osteogenesis was observed with subperiosteal implantation of the hydroxyapatite-bone morphogenetic protein complex in the bone morphogenetic protein group, but the osteogenesis observed was not distributed over the entire pellet.

Dietary modifications of the biliary bile acid glycine:taurine ratio and activity of hepatic bile acid-CoA:amino acid N-acyltransferase (EC 2.3.1) in the rat.

Effects of dietary manipulations on the biliary bile acid glycine:taurine (G:T) ratio and the activity of hepatic bile acid-CoA:amino acid N-acyltransferase (EC 2.3.1) in the post-mitochondrial fraction of liver homogenates were examined in the rat. The G:T ratio in rats fed on the diet containing 100 g pectin/kg (2.18) was markedly higher than that in the animals fed on the diet containing 100 g cellulose/kg (0.09). The diets containing either 10 g cholesterol/kg or 5 g sodium cholate/kg, especially the latter, also increased the G:T ratio (0.77 and 2.33 respectively) compared with a control diet free of these steroids (0.34). When the saturating concentrations of taurine (20 mM) and glycine (100 mM) were the substrates, dietary pectin relative to cellulose significantly increased the activity of both taurine- and glycine-dependent bile acid-CoA:amino acid N-acyltransferase, but neither dietary bile acid nor cholesterol influenced it. In spite of the marked difference in the G:T ratio among the rats given various types of experimental diet, the bile acid-CoA:amino acid N-acyltransferase reaction produced taurine-but little glycone-conjugated bile acid when both taurine and glycine coexisted at physiological concentration ranges in the assay media. Dietary manipulations modified the hepatic taurine concentrations and the changes were inversely correlated with those in the G:T ratio. However, hepatic concentration of taurine (1.67-4.82 mumol/g) in rats given various types of experimental diet was comparable with or even higher than the reported Michaelis constant (Km) value of N-acyltransferase for this compound (0.8-2.5 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Fusion of dioleoylphosphatidylcholine vesicles induced by an amphiphilic cationic peptide and oligophosphates at neutral pH.

Peptide E5 is an analogue of the fusion peptide of influenza virus hemagglutinin and K5 is a cationic peptide which has an arrangement of electric charges complementary to that of E5. We reported that a stoichiometric mixture of E5 and K5 caused fusion of large unilamellar vesicles (LUV) of neutral phospholipids (Murata, M., Kagiwada, S., Takahashi, S. and Ohnishi, S. (1991) J. Biol. Chem. 266, 14353-14358). K5 caused fusion of LUV composed of dioleoylphosphatidylcholine (DOPC) at pH > 10, but not at neutral pH. In the presence of oligophosphates, such as 1 mM ATP, GTP, or polyphosphate, K5 caused rapid and efficient fusion of DOPC LUV at neutral pH without hydrolysis of oligophosphate groups, but another anions such as citrate, acetate, AMP, phosphate, or EDTA were ineffective. The peptide/oligophosphate-induced fusion behaviors have been investigated by a fluorescence resonance energy transfer assay for lipid mixing of LUV and negative staining electron microscopy. At higher ionic strengths ( > 0.3 M KCl) or in the presence of 5.0 mM MgCl2, the fusion was inhibited. Even at the inhibitory conditions, the association of K5 with lipid vesicles at neutral pH was directly confirmed by the Ficoll gradient assay method and by blue shifts of the tryptophan fluorescence of the peptide. A nonhydrolyzable GTP analogue, GTP gamma S, also induced fusion. These observations suggested that the electrostatic interactions between the positive and negative charges of K5 and oligophosphate, respectively, induced complex formation, triggering membrane fusion.

Dietary diacylglycerol-dependent reduction in serum triacylglycerol concentration in rats.

The effects of dietary diacylglycerol consisting of 1,3 (65.2%) and 1,2 species (32.6%) and triacylglycerol (rapeseed oil) on the serum and hepatic lipid profiles were compared in the rat. The fatty acid composition was similar between these dietary lipids. The dietary acylglycerols were added to the experimental diets so as to provide the same amounts of fatty acids (9.39%). Dietary diacylglycerol compared with triacylglycerol significantly reduced concentrations of serum triacylglycerol at 17 and 34 days of the feeding periods without influencing those of phospholipid and cholesterol. There were no significant differences in the concentrations of hepatic triacylglycerol, cholesterol and phospholipid between the two groups of rats at 34 days of the feeding period. In the second trial, triacylglycerol in the experimental diet was replaced by varying amounts of diacylglycerol while maintaining the fatty acid contents (9.39%). After 14 days of the feeding period, significant reductions in serum triacylglycerol levels were confirmed in the groups of rats fed the diets in which diacylglycerol fatty acids supplied more than 50% (50, 75 and 100%) of total dietary fatty acids. Thus, it was confirmed that dietary diacylglycerol compared with triacylglycerol exerts a potent serum triacylglycerol-lowering effect in the rat.

Isolation and characterization of macrocarpals B--G antibacterial compounds from Eucalyptus macrocarpa.

Six novel phloroglucinol dialdehyde diterpene derivatives (macrocarpals B--G), which have antibacterial activity, were isolated from leaves of Eucalyptus macrocarpa. These compounds have closely related structures, the molecular formula for B--F being C28H40O6, and that of G being C28H38O5. The structures of macrocarpals B, D, and G were analyzed by means of NMR analyses.

Soybean protein-dependent changes in triacylglycerol synthesis and concentration of diacylglycerol in the liver microsomes of fasted-refed rats.

Effects of soybean protein, casein and whole egg protein on various indices for lipid biosynthesis in the liver were compared in fasted-refed rats. Soybean protein compared to casein and whole egg protein significantly reduced activities of glucose 6-phosphate dehydrogenase and fatty acid synthetase. The protein source also slightly reduced the activity of the malic enzyme. Soybean protein compared to other proteins not only reduced the microsomal triacylglycerol but also phosphatidylcholine syntheses when the activities were measured with endogenous diacylglycerol substrate. The protein-dependent changes disappeared, if artificial dispersion of dioleoylglycerol was employed as a substrate. The concentrations of microsomal diacylglycerol and triacylglycerol in whole liver in rats fed soybean protein were lower than those fed other proteins. When the diets containing soybean protein and casein were supplemented with DL-methionine (0.5 and 0.3%, respectively) to meet the nutritional requirement of the animals, soybean protein-dependent reductions in these indices for lipid biosynthesis were still detectable but considerably attenuated. Thus, it is plausible that a soybean protein-dependent decrease in fatty acid synthesis reduced the availability of microsomal diacylglycerol substrate for triacylglycerol synthesis and in turn modified hepatic triacylglycerol concentration. The dietary availability of sulfur amino acids may, at least in part, be responsible for the consequence observed in the present study.