Experimental investigation of geologically produced antineutrinos with KamLAND.

The detection of electron antineutrinos produced by natural radioactivity in the Earth could yield important geophysical information. The Kamioka liquid scintillator antineutrino detector (KamLAND) has the sensitivity to detect electron antineutrinos produced by the decay of 238U and 232Th within the Earth. Earth composition models suggest that the radiogenic power from these isotope decays is 16 TW, approximately half of the total measured heat dissipation rate from the Earth. Here we present results from a search for geoneutrinos with KamLAND. Assuming a Th/U mass concentration ratio of 3.9, the 90 per cent confidence interval for the total number of geoneutrinos detected is 4.5 to 54.2. This result is consistent with the central value of 19 predicted by geophysical models. Although our present data have limited statistical power, they nevertheless provide by direct means an upper limit (60 TW) for the radiogenic power of U and Th in the Earth, a quantity that is currently poorly constrained.

Synthetic diamond as a pressure generator.

Synthetic diamond crystals were used as opposed anvils whose small culet surfaces were driven into contact with each other to generate high static pressures. Pressures in excess of 68 gigapascals (680 kilobars) were achieved, as determined from the fluorescence line of a ruby fragment sandwiched between the diamonds.

Onecut transcription factor OC2 is a direct target of T-bet in type-1 T-helper cells.

T-box transcription factor, T-bet, has a central role in the differentiation of T-helper (Th) progenitor cells to Th1 or Th2 effector cells, partly by regulating the expression of genes such as interferon-gamma (IFN-gamma). However, the direct target genes, especially those mediating the transcriptional network initiated by T-bet, are not yet fully understood. By combining chromatin immunoprecipitation from Th1 cells with human cytosine-phosphate-guanine-island array analysis, Onecut 2 (OC2), which encodes a member of the ONECUT class of transcriptional activators, was identified as a direct target gene of T-bet. OC2 is expressed in Th1 but not Th2 cells and reporter assays showed that T-bet transactivates OC2 transcription through putative T-bet half-sites locating -451 to -347 of OC2 promoter region. Moreover, we found that OC2 binds and transactivates human T-bet promoter. These results suggest that not only cell-extrinsic regulation via the IFN-gamma/STAT1 pathway, but also cell-intrinsic transcriptional positive feedback loop between T-bet and OC2 could be involved in Th1 development.

Activation of protein breakdown and prostaglandin E2 production in rat skeletal muscle in fever is signaled by a macrophage product distinct from interleukin 1 or other known monokines.

During sepsis or after injection of endotoxin into rats, there is a large increase in muscle protein breakdown and prostaglandin E2 (PEG2) production. Prior studies showed that partially purified interleukin 1 (IL-1) from human monocytes can stimulate these processes when added to isolated rat muscles. The availability of pure recombinant IL-1 and other monokines has allowed us to investigate the identity of the active agent in this process. Incubation of muscles with recombinant human or murine IL-1 alpha or IL-1 beta or with IL-1 plus a phorbol ester did not stimulate muscle proteolysis or PGE2 production. Homogeneous natural porcine IL-1 ("catabolin") and mouse or human IL-1 beta were also not effective in vitro. In addition, a variety of other human cytokines, including tumor necrosis factor ("cachectin"), epidermal thymocyte-activating factor, eosinophil cytotoxicity-enhancing factor, interferon-alpha, beta, and gamma, platelet-derived growth factor, and transforming growth factor (TGF) beta, which are all released by activated macrophages, TGF-alpha, or mixtures of these polypeptides, also failed to activate proteolysis or PGE2 production. By contrast, a large increase in net protein breakdown could be induced in the rat soleus by polypeptides released from porcine monocytes or by the serum from febrile cattle which had been injected with Pasteurella haemolytica or bovine rhinotracheitis virus. Therefore, a still-unidentified product of activated monocytes appears to be responsible for the negative nitrogen balance that accompanies infectious illness.

Identification of novel MUNC13-4 mutations in familial haemophagocytic lymphohistiocytosis and functional analysis of MUNC13-4-deficient cytotoxic T lymphocytes.

BACKGROUND: Familial haemophagocytic lymphohistiocytosis (FHL) has an autosomal recessive mode of inheritance and consists of at least three subtypes. FHL2 subtype with perforin (PRF1) mutation accounts for 30% of all FHL cases, while FHL with MUNC13-4 mutation was recently identified and designated as FHL3 subtype. OBJECTIVE: To examine MUNC13-4 mutations and the cytotoxic function of MUNC13-4 deficient T lymphocytes in Japanese FHL patients METHODS: Mutations of MUNC13-4 and the cytotoxicity of MUNC13-4-deficient cytotoxic T lymphocytes (CTL) were analysed in 16 Japanese families with non-FHL2 subtype. RESULTS: Five new mutations of the MUNC13-4 gene were identified in six families. The mutations were in the introns 4, 9, and 18, and exons 8 and 19. Two families had homozygous mutations, while the remaining four had compound heterozygous mutations. Cytotoxicity of MUNC13-4 deficient CTL was low compared with control CTL, but was still present. Clinically, the onset of disease tended to occur late; moreover, natural killer cell activity was not deficient in some FHL3 patients. CONCLUSIONS: MUNC13-4 mutations play a role in the development of FHL3 through a defective cytotoxic pathway.

Anti- and pro-oxidative effects of flavonoids on metal-induced lipid hydroperoxide-dependent lipid peroxidation in cultured hepatocytes loaded with alpha-linolenic acid.

Lipid hydroperoxide (LOOH)-dependent lipid peroxidation was induced in alpha-linolenic acid (LNA)-loaded hepatocytes by adding Fe, Cu, V, or Cd ions at concentrations from 20 to 500 microM. The effects of structurally related flavonoids at concentrations from 10 to 500 microM on the lipid peroxidation were examined. The results with regard to each flavonoid subclass are as follows: (i) Flavonols such as myricetin, quercetin, fisetin, and kaempferol, but not morin, showed dose-dependent antioxidative activity against metal-induced lipid peroxidation at all metal concentrations. Myricetin, quercetin, and fisetin were the most effective antioxidants, although their efficacies depended on the metal ion. Kaempferol and morin had antioxidative activity equal to the other flavonols in the presence of Cu ions, but were much less effective for the other three metal ions. (ii) Flavones, luteolin, apigenin, and chrysin were antioxidative at low Fe concentrations, but were pro-oxidative at high Fe concentrations. Luteolin exhibited antioxidative activity similar to that of catechol-containing flavonols in the presence of the other three metal ions. Apigenin and chrysin also acted as pro-oxidants with V or with all metal ions, respectively. (iii) Taxifolin, a flavanone, also showed both anti- and prooxidative activity, depending on Fe concentrations, but with other metal showed only antioxidative activity ions. (iv) Epigallocatechin, a flavanol, was antioxidative with all metal ions, and its activity was similar to that of catechol-containing flavonols. The various effects of flavonoids on metal-induced lipid peroxidation in LNA-loaded hepatocytes is discussed with regard to the change in redox potential of flavonoid-metal complexes.

Isolation and sequencing of a cDNA clone encoding 107 kDa sialoglycoprotein in rat liver lysosomal membranes.

A cDNA for 107 kDa sialoglycoprotein (LGP 107), the major protein component of rat liver lysosomal membranes, was isolated and sequenced. The 1.8 kbp cDNA contained an open reading frame encoding a polypeptide consisting of 386 amino acid residues (Mr 41,914). The deduced NH2-terminal 10-residue sequence is identical with that determined for purified LGP 107. The primary structure deduced for LGP 107 contains 20 potential N-glycosylation sites and exhibits 82.5, 43 and 60% sequence similarities to mouse LAMP-1, chicken LEP 100, and a 120-kDa human lysosomal glycoprotein, respectively. Among these lysosomal glycoproteins, the amino acid sequence of the putative transmembrane segment is highly conserved. Northern blot hybridization analysis identified a single species of LGP 107 mRNA (2.1 kbp in length) in rat liver, kidney, brain, lung, spleen, heart and pancreas, although its level in pancreas was very low.

Pituitary adenylate cyclase-activating polypeptide produces a phase shift associated with induction of mPer expression in the mouse suprachiasmatic nucleus.

The main mammalian circadian pacemaker is located in the suprachiasmatic nucleus of the hypothalamus. Clock genes such as the mouse Period gene (mPer) play a role in this core clock mechanism in the mouse. With brief light exposure during the subjective night, the photic information, which is conveyed directly to the suprachiasmatic nucleus via the retinohypothalamic tract, results in mPer1 and mPer2 expression in the suprachiasmatic nucleus. Glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP) are co-stored in the retinohypothalamic tract. Recent studies have suggested that not only glutamate but also PACAP are key players in the phase shift that occurs during subject night; however, research demonstrating a direct association between the PACAP-induced phase shift and mPer gene expression has yet to be conducted. In the present study, PACAP (200 pmol) injected into the lateral ventricle during subjective night (circadian time 16; circadian time 12, onset of locomotor activity) caused a moderate phase delay associated with moderate expression of mPer1 and only slight expression of mPer2 in the mouse suprachiasmatic nucleus. PACAP-induced mPer1 expression was also observed in the paraventricular nucleus and periventricular area of the hypothalamus. (+)MK-801 (0.5 mg/kg), an N-methyl-D-aspartate (NMDA) receptor antagonist, suppressed both the PACAP-induced phase delay and mPer1 expression. From these results we suggest that PACAP induces phase delays in the mouse circadian rhythm in association with an increase of mPer expression in the suprachiasmatic nucleus via the activation of NMDA receptors.

Receptor-mediated introduction of pepstatin-asialofetuin conjugate into lysosomes of rat hepatocytes.

Pepstatin was linked through a carboxyl group to asialofetuin (PS-ASF). An analysis by separation of hepatocytes from nonparenchymal cells showed that PS-ASF was taken up by hepatocytes, following intravenous injection into rats. After the injection of PS-ASF, pepstatin concentration in the liver reached a maximum at 2 h and then decreased. In an analysis by differential centrifugation of the liver homogenate from rats injected with PS-ASF, pepstatin showed a lysosomal type subcellular distribution pattern. Isolation studies of tritosomes clearly demonstrated the exclusive accumulation of pepstatin within the lysosomes of livers from rats given PS-ASF (at 2 h after administration). Pepstatin contained in tritosomes was in a free form, as determined by column chromatography of Sephadex G-15. The activity of cathepsin D in the livers was markedly inhibited in rats given PS-ASF. However, the treatment of rats with PS-ASF had no effect on the hepatic lysosomal degradation of endocytosed FITC-labeled asialofetuin (FITC-ASF). Introduction of PS-ASF into the hepatocytes was followed by the immediate and time-dependent excretion of free pepstatin into the bile. Quantification of pepstatin excreted into the bile revealed that the biliary excretion route can account for the disappearance of pepstatin from the liver.

Isolation and characterization of autolysosomes which appeared in rat liver after leupeptin treatment.

Autolysosomes were isolated from rat livers treated with leupeptin by a combination of differential and Percoll density gradient centrifugation techniques. The purified autolysosome fraction was verified by morphological analysis to be highly purified and to contain contaminants which were scarcely detectable. The enrichment of the lysosomal enzyme activities in the purified autolysosomes over the homogenate was 12-, 14-, 22-, and 24-fold for beta-glucuronidase, acid phosphatase, beta-N-acetylglucosaminidase, and cathepsin D, respectively. Measurement of the activity of the marker enzymes for various subcellular organelles also proved that the purified autolysosome fraction was essentially free from contamination by other organelles. When the autolysosomes isolated from rat livers treated with leupeptin for 1 h were disrupted by osmolysis, acid hydrolases were easily solubilized. Acid phosphatase, however, became membrane bound in the autolysosomes prepared at longer periods of time after the leupeptin treatment. The autolysosomes exhibited enhanced permeability of the membranes after a short duration of time after the leupeptin treatment (30 and 60 min) and became stabilized later. These changes in the properties of the autolysosomes with time after the leupeptin treatment may be interpreted as meaning that progressive rearrangement of the lysosomal constituents occurred within the autolysosomes with time after the genesis.

Appearance of autolysosomes in rat liver after leupeptin treatment.

The administration to rats of leupeptin produced prominent numbers of enlarged and irregularly shaped autolysosomes in hepatocytes. Percoll density equilibration of crude lysosomal fractions from rat livers showed that most lysosomal enzyme migrated form normal lysosomal density fractions toward higher density fractions within 30 min after leupeptin injection. The denser particles were ultrastructurally identified with the autolysosomes. In analysis by differential centrifugation of the liver homogenates, lysosomal enzymes became sedimentable with particles of greater sedimentation rate within 30 min after leupeptin injection. These changes in physical properties of lysosomes reverted to normal after 24 h, and concomitant disappearance of autolysosomes in hepatocytes was observed by electron microscopy. When the time course of distribution of leupeptin in subcellular fractions was analyzed, particle-bound leupeptin shifted with time to larger particle fractions in parallel with the shift of lysosomal enzyme distribution. Upon injection with leupeptin, cathepsin B activity was inhibited by more than 80% for about 3 h and was gradually restored to a normal level after 24 h. Leupeptin treatment caused marked delay of the degradation of endocytosed FITC-labeled asialofetuin in hepatic lysosomes, and its inhibitory effect lasted for over 9 h after the injection. It was suggested that autophagy is probably a normal process of protein degradation in hepatocytes, and because of a retarded digestion of sequestered materials, autolysosomes persisted for a long period and made up the majority of the lysosome population in the leupeptin-treated cells.

Purification, some properties, and tissue distribution of a major lysosome-associated membrane glycoprotein (r-lamp-2) of rat liver.

We previously purified and characterized a major lysosomal membrane glycoprotein (r-lamp-1) from rat liver [Akasaki et al. (1990) Chem. Pharm. Bull. 38, 2766-2770]. The present study describes the purification of another major lysosomal membrane glycoprotein (r-lamp-2) from rat liver and compares the tissue distribution of r-lamp-1 and r-lamp-2 in rats. R-lamp-2 was purified to apparent electrophoretic homogeneity from rat liver by a simple method with a protein yield of approximately 4.0 micrograms/g wet weight of liver. The purification procedure includes: preparation of tritosomal membranes, extraction of tritosomal membranes with Lubrol PX, wheat germ agglutinin (WGA)-Sepharose affinity chromatography, and monoclonal antibody-Sepharose affinity chromatography. R-lamp-2 exhibited an Mr of 96,000 on SDS-PAGE and had an acidic pI of less than 3.5. R-lamp-2 contained 52.3% carbohydrates. Its carbohydrate moieties were composed of numerous sialyl complex type N-linked oligosaccharides and small amounts of O-linked oligosaccharides. Both r-lamp-1 and r-lamp-2 were detected in all rat tissues examined by immunoblot analyses, while their apparent molecular weights differed among the tissues. Immunological quantitative analysis showed that the protein concentrations of r-lamp-2 were consistently lower than those of r-lamp-1 in all the tissues tested. There was a significant correlation with a regression coefficient of 0.86 in the tissue distribution between r-lamp-1 and r-lamp-2. A good correlation was also observed in the tissue distribution between acid phosphatase and r-lamp-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of subfractions of autolysosomes isolated from rat liver after leupeptin treatment.

Autolysosomes were isolated from leupeptin-treated rat liver (Furuno, K., Ishikawa, T., & Kato, K. (1982) J. Biochem. 91, 1943-1950). They were disrupted by hypotonic treatment and subfractionated by centrifugation in a discontinuous sucrose gradient into three distinct parts: the membranes, the soluble contents, and the insoluble remnants. The content fraction contained the bulk of the activities of lysosomal enzymes with a protein yield of about 36%. The membrane fraction, representing about 5% of protein of the autolysosomes, had a high specific activity of acid phosphatase. In SDS-polyacrylamide gel electrophoretic analysis, the autolysosomal membranes showed protein profiles similar to those of normal lysosomal membranes. The aggregates of partially digested cellular components, including organelles, in the autolysosomes were recovered as granular materials with a very high density (designated as the remnant fraction). This fraction accounted for more than half of protein of the autolysosomes but contained little of the activities of lysosomal enzymes. Lipid analyses revealed that the autolysosomes were poor in lipids because the lipid content of the insoluble remnants was very low. Measurements of the rate of protein degradation in vitro in the crude lysosomal fraction and the isolated autolysosomes from leupeptin-treated rat liver showed that proteolysis was suppressed within the autolysosomes. It was suggested that lipids of sequestered cellular components were preferentially digested within the autolysosomes due to the inhibition of proteolytic activity by leupeptin, and the resulting massive accumulation of proteins was responsible for the enhanced autolysosomal density.

Cycling of two endogenous lysosomal membrane proteins, lamp-2 and acid phosphatase, between the cell surface and lysosomes in cultured rat hepatocytes.

Our previous studies provided evidence that a 107-kDa major lysosomal membrane glycoprotein termed lamp-1 shuttles between lysosomes and the plasma membrane along the endocytic pathway in rat hepatic cells [Furuno et al. (1989) J. Biochem. 106, 708-716; Furuno et al. (1989) J. Biochem. 106, 717-722]. In the present study, we investigated the movement of a 96-kDa major lysosomal membrane glycoprotein, referred to as lamp-2, and lysosomal acid phosphatase (LAP) in the endocytic membrane transport system of cultured rat hepatocytes. Fab' fragments of anti-lamp-2 and anti-LAP antibodies conjugated with horseradish peroxidase (HRP) were used as probes to analyze quantitatively the transport of these two membrane proteins from the cell surface to lysosomes. After the addition of HRP-anti-lamp-2 and anti-LAP Fab' fragments to the culture medium, the delivery of the antibody conjugates to lysosomes was examined by cell fractionation on a Percoll density gradient. The amount of these HRP tracers in the lysosomal fraction became larger as the period of cell incubation was increased. Km values for uptake of HRP-anti-lamp-2, and LAP Fab' fragments were 0.74 and 0.62 microM, respectively, which were comparable to that of HRP-anti-lamp-1 Fab' (0.57 microM). The endocytic process of the two HRP-antibodies continued for an extended period in the cells exposed to the protein synthesis inhibitor, cycloheximide. Furthermore, we measured the transit times of HRP-anti-lamp-1, anti-lamp-2, and anti-LAP Fab' fragments from the cell surface to lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical analysis of the movement of a major lysosomal membrane glycoprotein in the endocytic membrane system.

HRP-anti LGP107Fab' and 125I-anti LGP107IgG were used as probes to study the movement of LGP107 in the endocytic membrane transport system in primary cultured hepatocytes of rats. Following the addition of HRP-anti LGP107Fab' to the culture medium, the transfer of the antibody conjugate from the cell surface of lysosomes was examined by cell fractionation on Percoll density gradients. The HRP tracer showed a bimodal subcellular distribution, in plasma membrane and lysosomal fractions. The amount of HRP found in the lysosomal fractions became larger as the period of cell incubation was increased. The rate of HRP accumulation in lysosomes was 0.13% of the administered load per hour per 10(6) cells. When cells were given 125I-anti LGP107 IgG, the antibody was not stored but was rapidly degraded in the lysosomes. The uptake of 125I-IgG by the cells, which was assessed by measuring the TCA-soluble radiolabeled degradation products released into the medium, increased proportionally to the administered concentration of the antibody and to the incubation time. The rate of uptake of the polyvalent 125I-IgG was comparable to that for the uptake of the monovalent HRP-Fab', and remained unchanged even after long exposure of the cells to a saturating concentration of the polyvalent IgG. This uptake process continued for many hours in the cells exposed to the protein synthesis inhibitor, cycloheximide. These results suggest that there is a continuous circulation of LGP107 between the cell surface and lysosomes in hepatocytes.

Isolation of chloroquine-resistant Chinese hamster V79 cell variants that are also resistant to ammonium chloride.

Chloroquine-resistant (CQr) clones (CQ-21 and CQ-22) have been isolated from mutagenized hamster lung V79 cells by exposing the cells to a high dose of chloroquine. CQ-21 and CQ-22 showed about 3-fold higher resistance to chloroquine than the parental V79 cells, and they showed specific cross-resistance to another amine, NH4Cl, which is also concentrated in lysosomes. CQr clone showed no cross-resistance to other unrelated agents. Chloroquine-induced inhibition of [125I]ricin internalization was observed in both cell lines at neutral pH, but the inhibition of uptake was less in the variant. Also, the degradation of endogenous protein was slowed in the mutant; further, treatment of cells with 30 micrograms/ml of chloroquine inhibited the degradation of endogenous proteins in the parental V79, but not in CQ-22 cells. Similar levels of acid phosphatase, beta-glucuronidase and cathepsin D were observed in V79 and CQ-22 cells, but the level of cathepsin B was lower in the mutant. Electron microscopy showed an increased number of electron-dense bodies, possibly autophagosomes/lysosomes, in the mutant cells grown for 4 days with 5 micrograms/ml of chloroquine. Similar aberrant structures were observed in the parental V79 cells treated for only 3 h with 5 micrograms/ml of chloroquine.

Morphological localization of a major lysosomal membrane glycoprotein in the endocytic membrane system.

We have raised specific polyclonal immunoglobulin G (IgG) against a major lysosomal membrane sialoglycoprotein (LGP107) taken from rat liver and have prepared a conjugate of its Fab' fragment with horseradish peroxidase (HRP-anti LGP107 Fab') as a probe for the subcellular antigen. Electron immunocytochemistry in primary cultured rat hepatocytes showed that LGP107 resided primarily within lysosomes and was associated with luminal amorphous materials as well as limiting membranes. In addition, LGP107 was shown to be substantially distributed throughout the endocytic vacuolar system. The glycoprotein was found clustered in coated pits at the cell surface and localized along the surrounding membranes in endocytic vesicles. When cultured cells were exposed to HRP-anti LGP107 Fab', the antibody which was bound to its antigen within the coated pits was internalized via a system of endocytic vesicles and transported to lysosomes. During 20 min of incubation at 37 degrees C, the HRP tracer appeared at an early stage in small vesicles and moved progressively to larger vesicles, including multivesicular bodies. After 1 h, the tracer could be clearly seen in lysosomes heterogeneous in shape and size. The existence of LGP107 in endocytic compartments and the uptake of anti LGP107 antibody by hepatocytes were not blocked by prior treatment of the cells with cycloheximide and excess amounts of anti LGP107 IgG. These data suggest that LGP107 circulates between the cell surface and lysosomes through the endocytic membrane traffic in hepatocytes.

An infant with precursor natural killer (NK) cell leukemia successfully treated with an unrelated cord blood transplantation.

Here we report a case with precursor natural killer (NK) cell leukemia successfully treated with an unrelated cord blood transplantation. A 7-month-old Japanese boy was diagnosed to have NK cell leukemia based on the existence of abnormal cells in the bone marrow with the phenotype of CD3(-) /CD4(+) /CD7(-) /CD8(-) /CD16(-) /CD33(+) /CD34(-) /CD56(+) /HLA-DR(+) /NKB1(+) / CD94(+). The leukemic cells showed few azurophilic granules in the cytoplasm and weak cytotoxic activity. Although he presented with a huge mass occupying the bilateral paranasal sinuses and hepatosplenomegaly, he achieved complete remission by the conventional chemotherapeutic regimen for acute myelogenous leukemia, followed by an unrelated cord blood transplantation. He has remained in complete remission for 14 months posttransplant. To our knowledge, this is the youngest reported case with precursor NK cell leukemia; cord blood transplantation may thus be the treatment of choice for this disease.

Possible explanations for the antagonism by nicotine against reserpine-induced depletion of monoamines in mouse brain.

The inhibitory effect of nicotine pretreatment on reserpine-induced depletion of monoamines in mouse brain was investigated. The depletion of brain monoamines by 24 h after intraperitoneal injection of reserpine (2 mg/kg) was dose-dependently inhibited by nicotine (0.3-10 mg/kg, s.c.) pretreatment 20 min before reserpine injection. This effect of nicotine was more marked on dopamine depletion than on noradrenaline or 5-hydroxytryptamine depletion. The nicotine pretreatment also inhibited the reserpine-induced hypothermia and decrease in the locomotor activity. When reserpine (2 mg/kg) was injected intraperitoneally, the inhibitory effect of nicotine (3 mg/kg, s.c.) on the reserpine-induced depletion of brain monoamines and heart noradrenaline was not antagonized by hexamethonium (8 mg/kg, s.c.) but rather potentiated by mecamylamine (2 mg/kg, s.c.). However, when reserpine (0.5 mg/kg) was injected intravenously, pretreatment with nicotine (3 mg/kg, s.c.) inhibited the reserpine-induced dopamine depletion only, and this effect of nicotine was completely blocked by mecamylamine but not by hexamethonium. These results suggest that inhibitory effect of nicotine on the intraperitoneal reserpine-induced depletion of brain monoamines is due to an inhibition of absorption of reserpine, and that central nicotinic action is also involved in the antagonism by nicotine of reserpine-induced dopamine depletion.

Cytotoxicity of food preservatives in cultured rat hepatocytes loaded with linolenic acid.

We investigated the ability of eight food preservatives to induce lipid peroxidation in normal and alpha-linolenic acid (LNA)-loaded cultured rat hepatocytes. On the addition of sodium dehydroacetate (DHA-Na), potassium sorbate (SA-K) or thiabendazole (TBZ) to the cell culture, lipid peroxidation, assessed in terms of the production of malondialdehyde (MDA), was induced in LNA-loaded cells, but not in normal cells. At the low concentrations, induction of lipid peroxidation in LNA-loaded cells was highest with TBZ, whereas at high concentrations DHA-Na greatly induced lipid peroxidation. The occurrence of lipid peroxidation in LNA-loaded cells was accompanied by a decrease in cellular GSH levels with the three preservatives and by a decrease in cellular protein-SH levels with DHA-Na and TBZ. Furthermore, cell injury, measured by the release of LDH, was produced in LNA-loaded cells exposed to DHA-Na and SA-K. The addition of TBZ caused substantial cell injury in normal cells, and even greater injury in LNA-loaded cells. The prevention of lipid peroxidation in LNA-loaded hepatocytes by addition of an antioxidant, N,N'-diphenyl-p-phenylenediamine (DPPD) almost completely prevented DHA-Na- and SA-K-induced cell injury, and reduced TBZ-induced cell injury. The addition of diphenyl (DP), o-phenylphenol (OPP) or butyl p-hydroxybenzoate (BHB) caused severe cell injury, in association with a marked decrease in cellular levels of both of GSH and protein-SH in both groups of cells. However, lipid peroxidation was not detectable in either group of cells exposed to these preservatives. Sodium propionate (PA-Na) and sodium benzoate (BA-Na) had little effect on any cytotoxic parameter in either group of cells.