Various Tissues Of Mice Research Articles

Studies on Silver Nanoparticle–Glycyrrhizic Acid Complex as a Radioprotector and an Adjuvant in Radiotherapy UnderIn VivoConditions

Silver nanoparticles (SN) of particle size of less than 50 nm were dispersed in an aqueous solution of Pluronic F127 and complexed with the phytoceutical, glyzyrrhizic acid (GLY). Radioprotecting ability of the obtained nanoparticle-glyzyrrhizic acid complex (SN-GLY) was evaluated in an in vivo model using Swiss albino mice. The potential of the complex as an adjuvant during radiotherapy was also analyzed in tumor-bearing mice. The administration of SN-GLY, SN, and GLY protected the hemopoetic and gastrointestinal system against radiation-induced damages as revealed by the total white blood cell count, bone marrow cellularity, endogenous spleen colony formation, levels of cellular antioxidants, and histopathologcal examination of gastrointestinal tract. Oral administration of SN-GLY, SN, and GLY 1 hour before a sublethal dose of radiation exposure reduced the radiation-induced depletion of cellular antioxidants and lipid peroxidation in various tissues of mice. Survival of animals following exposure to a lethal dose of gamma radiation was also improved. It was also found that the oral administration of the complex to tumor-bearing mice before 4 Gy gamma irradiation resulted in a faster tumor regression.

Identification of human and murine sulfotransferases able to activate hydroxylated metabolites of methyleugenol to mutagens in Salmonella typhimurium and detection of associated DNA adducts using UPLC-MS/MS methods

Methyleugenol, a secondary metabolite present in many herbal spices, is carcinogenic in various tissues of mice and rats but negative in standard in vitro mutagenicity tests. Several observations indicate that hydroxylation followed by sulfation is an important activation pathway in the carcinogenicity and DNA adduct formation by methyleugenol and other alkenylbenzenes in animal models. However, sulfation is not taken into account in standard in vitro tests. Therefore, we have studied whether expression of murine or human sulfotransferases (SULTs) in the target strain, Salmonella typhimurium TA100, leads to the activation of hydroxylated metabolites of methyleugenol [(+)-1'-hydroxymethyleugenol, (-)-1'-hydroxymethyleugenol and (E)-3'-hydroxymethylisoeugenol]. Human SULT1A1 (a form expressed at high levels in many tissues) and SULT1C2 (expressed primarily in foetal tissues) activated all three compounds even at very low substrate concentrations. At higher concentrations, activation was also observed with human SULT1A2 and SULT1E1. Murine Sult1a1 required higher substrate concentrations than its human orthologue. Other SULT forms (human 1A3, 1C1, 1C3, 2A1 and 2B1b as well as murine 1d1) did not activate any methyleugenol metabolites studied. Furthermore, we developed isotope-dilution mass-spectrometric methods for the sensitive and specific detection of DNA adducts formed by methyleugenol metabolites. All three hydroxylated metabolites formed the same DNA adducts in S. typhimurium TA100-hSULT1A1: high levels of N (2)-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine and modest levels of N (6)-(trans-methylisoeugenol-3'-yl)-2'-deoxyadenosine. Adduct levels correlated with the mutagenic effects induced. No adducts were formed by the test compounds in the SULT-deficient standard strain TA100. In conclusion, several methyleugenol metabolites are activated to DNA-reactive mutagens in S. typhimurium upon incorporation of appropriate sulfation capacity. We have identified human and murine SULT forms able to catalyse this activation. Methods were developed that may be utilised to analyse DNA samples from human tissues specifically for the possible presence of methyleugenol adducts.

Amelioration of γ-radiation induced genomic insult and oxidative stress in whole body irradiated Swiss albino mice by sesamol

Sesamol (3,4-methylene dioxyphenol) protected mice from adverse effects of whole body gamma-irradiation. Studies indicated that oral administration of sesamol (SM), 1 hour prior to whole body gamma-irradiation (2–6 Gy), significantly protected cellular DNA in various tissues of mice, as evident from alkaline comet assay. Whole body exposure of mice to a sublethal dose of 6 Gy gamma radiation resulted in oxidative damage in various tissues of mice, and radiation-induced peroxidative damage and depletion of antioxidants such as GSH, GPx in tissues such as liver, kidney and brain. Administration of SM prior to radiation exposure resulted in prevention of radiation-induced peroxidation of membrane lipids and depletion of cellular antioxidants in these tissues. The administration of SM significantly reduced the mortality of the animals following exposure to a lethal dose of 10 Gy whole body gamma-radiation and improved the recovery of the body weight. Thus the studies suggest that oral administration of SM could offer protection to mice from the deleterious effects of ionising radiation.

Estimation of the postmortem duration of mouse tissue by electron spin resonance spectroscopy.

Electron spin resonance (ESR) method is a simple method for detecting various free radicals simultaneously and directly. However, ESR spin trap method is unsuited to analyze weak ESR signals in organs because of water-induced dielectric loss (WIDL). To minimize WIDL occurring in biotissues and to improve detection sensitivity to free radicals in tissues, ESR cuvette was modified and used with 5,5-dimethtyl-1-pyrroline N-oxide (DMPO). The tissue samples were mouse brain, hart, lung, liver, kidney, pancreas, muscle, skin, and whole blood, where various ESR spin adduct signals including DMPO-ascorbyl radical (AsA∗), DMPO-superoxide anion radical (OOH), and DMPO-hydrogen radical (H) signal were detected. Postmortem changes in DMPO-AsA∗ and DMPO-OOH were observed in various tissues of mouse. The signal peak of spin adduct was monitored until the 205th day postmortem. DMPO-AsA∗ in liver (y = 113.8–40.7 log (day), R1 = −0.779, R2 = 0.6, P < .001) was found to linearly decrease with the logarithm of postmortem duration days. Therefore, DMPO-AsA∗ signal may be suitable for detecting an oxidation stress tracer from tissue in comparison with other spin adduct signal on ESR spin trap method.

Changes in 2′-deoxycytidine levels in various tissues of tumor-bearing mice

The nucleoside 2'-deoxycytidine (dCyd) increases in the plasma of cancer patients with poor prognoses. 5-fluorouracil (5FU) is one of the anti-cancer agents used in chemotherapy for patients whose plasma dCyd is elevated. We examined the free dCyd level in various tissues of mice, with and without tumors, and in mice with and without the administration of 5FU or of dCyd, and investigated the effects of dCyd in tumor-bearing animals. SP2/0-Ag14 mouse myeloma cells were transplanted subcutaneously into mice and 5FU or dCyd was administered intraperitoneally. Free dCyd was measured in blood and tissues by HPLC at two time points, once when mouse body weight was maximally decreased (1 day after the last administration of 5FU, day 16) and again when it returned to control level at 1 week after the last 5FU treatment (day 22). Results showed that in tumor-bearing mice, the level of dCyd (per g wet weight) increased in the spleen. The change in liver weight caused by the administration of 5FU correlated with the level of dCyd in the liver. Notably, the relative tumor volume and tumor weight was decreased in dCyd-treated animals in comparison with controls. In conclusion, the levels of dCyd were markedly altered in the tissues of the reticuloendothelial and lymphatic systems, such as liver and spleen, and dCyd apparently had the ability to inhibit tumor growth in the body.

Dietary isoflavone increases insulin-like growth factor-I production, thereby promoting hair growth in mice

Sensory neurons release calcitonin gene-related peptide (CGRP) upon activation. We previously demonstrated that CGRP increases insulin-like growth factor-I (IGF-I) production in various tissues of mice including the skin. We demonstrated that isoflavone increases the CGRP synthesis in the dorsal root ganglion (DRG) neurons in rats. Since IGF-I plays a critical role in hair growth, we hypothesized that isoflavones may promote hair growth by increasing the IGF-I production in hair follicles. We examined this hypothesis using wild-type (WT) and CGRP-knockout (CGRP −/−) mice. Isoflavone significantly increased the CGRP mRNA levels in DRG neurons isolated from WT mice ( P<.01). Administration of isoflavone for 3 weeks increased the dermal levels of CGRP, IGF-I and IGF-I mRNA in WT mice, but not in CGRP −/− mice. Isoflavone administration increased the immunohistochemical expression of IGF-I in hair follicle dermal papilla cells in WT mice. Significant enhancements of hair follicle morphogenesis, hair regrowth, and hair pigmentation were also observed in WT mice administered isoflavone. However, none of these effects in WT mice were observed in CGRP −/− mice. These observations strongly suggest that isoflavone might increase IGF-I production in the hair follicle dermal papilla cells in mice through increasing CGRP production in the sensory neurons, thereby promoting hair growth associated with melanogenesis in mice.

Cell Lineage metastability in Gfi1-deficient mouse intestinal epithelium

Elucidating the mechanisms determining multipotent progenitor cell fate remains a fundamental project of contemporary biology. Various tissues of mice and men with defects in the zinc-finger transcriptional repressor Gfi1 have dramatic perturbations in the proportions of their differentiated cell types. In Gfi1-deficient intestinal epithelium there is a shift from mucous and Paneth towards enteroendocrine cells, leading to the proposal that Gfi1 functions in the allocation of the progeny derived from a hypothetical common granulocytic progenitor. However, studies of clones have yielded no evidence of such a common progenitor prompting us to investigate alternate mechanisms explaining the Gfi1-deficient phenotype. We report that mucous and Paneth but not enteroendocrine lineage cells normally express Gfi1. Sporadic mucous and Paneth lineage cells in the crypts of Gfi1-deficient mice aberrantly express the pro-enteroendocrine transcription factor Neurog3, indicating that stable repression of Neurog3 in these lineages requires Gfi1. Importantly, we also find mucous and Paneth lineage cells in various stages of cellular reprogramming into the enteroendocrine lineage in Gfi1-deficient mice. We propose that mucous and Paneth cell lineage metastability, rather than reallocation at the level of a hypothetical common granulocytic progenitor, is responsible for the shifts in cell type proportions observed in Gfi1-deficient intestinal epithelium.

Redox atlas of the mouse: Immunohistochemical detection of glutaredoxin-, peroxiredoxin-, and thioredoxin-family proteins in various tissues of the laboratory mouse

Background Oxidoreductases of the thioredoxin family of proteins have been thoroughly studied in numerous cellular and animal models mimicking human diseases. Despite of their well documented role in various disease conditions, no systematic information on the presence of these proteins is available. Methods Here, we have systematically analyzed the presence of some of the major constituents of the glutaredoxin (Grx)-, peroxiredoxin (Prx)-, and thioredoxin (Trx)-systems, i.e. Grx1, Grx2, Grx3 (TXNL-2/PICOT), Grx5, nucleoredoxin (Nrx), Prx1, Prx2, Prx3, Prx4, Prx5, Prx6, Trx1, thioredoxin reductase 1 (TrxR1), Trx2, TrxR2, and γ-glutamyl cysteine synthetase (γ-GCS) in various tissues of the mouse using immunohistochemistry. Results The identification of the Trx family proteins in the central nervous system, sensory organs, digestive system, lymphatic system, reproductive system, urinary system, respiratory system, endocrine system, skin, heart, and muscle revealed a number of significant differences between these proteins with respect to their distribution in these tissues. Conclusion Our results imply more specific functions and interactions between the proteins of this family than previously assumed. General significance Crucial functions of Trx family proteins have been demonstrated in various disease conditions. A detailed overview on their distribution in various tissues will be helpful to fully comprehend their potential role and the interactions of these proteins in the most thoroughly studied model for human diseases—the laboratory mouse. This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.

Major Urinary Protein-1 Increases Energy Expenditure and Improves Glucose Intolerance through Enhancing Mitochondrial Function in Skeletal Muscle of Diabetic Mice

Major urinary protein-1 (MUP-1) is a low molecular weight secreted protein produced predominantly from the liver. Structurally it belongs to the lipocalin family, which carries small hydrophobic ligands such as pheromones. However, the physiological functions of MUP-1 remain poorly understood. Here we provide evidence demonstrating that MUP-1 is an important player in regulating energy expenditure and metabolism in mice. Both microarray and real-time PCR analysis demonstrated that the MUP-1 mRNA abundance in the liver of db/db obese mice was reduced by approximately 30-fold compared with their lean littermates, whereas this change was partially reversed by treatment with the insulin-sensitizing drug rosiglitazone. In both dietary and genetic obese mice, the circulating concentrations of MUP-1 were markedly decreased compared with the lean controls. Chronic elevation of circulating MUP-1 in db/db mice, using an osmotic pump-based protein delivery system, increased energy expenditure and locomotor activity, raised core body temperature, and decreased glucose intolerance as well as insulin resistance. At the molecular level, MUP-1-mediated improvement in metabolic profiles was accompanied by increased expression of genes involved in mitochondrial biogenesis, elevated mitochondrial oxidative capacity, decreased triglyceride accumulation, and enhanced insulin-evoked Akt signaling in skeletal muscle but not in liver. Altogether, these findings raise the possibility that MUP-1 deficiency might contribute to the metabolic dysregulation in obese/diabetic mice, and suggest that the beneficial metabolic effects of MUP-1 are attributed in part to its ability in increasing mitochondrial function in skeletal muscle.

Caloric Restriction Stimulates Revascularization in Response to Ischemia via Adiponectin-mediated Activation of Endothelial Nitric-oxide Synthase

Caloric restriction (CR) can extend longevity and modulate the features of obesity-related metabolic and vascular diseases. However, the functional roles of CR in regulation of revascularization in response to ischemia have not been examined. Here we investigated whether CR modulates vascular response by employing a murine hindlimb ischemia model. Wild-type (WT) mice were randomly divided into two groups that were fed either ad libitum (AL) or CR (65% of the diet consumption of AL). Four weeks later, mice were subjected to unilateral hindlimb ischemic surgery. Body weight of WT mice fed CR (CR-WT) was decreased by 26% compared with WT mice fed AL (AL-WT). Revascularization of ischemic hindlimb relative to the contralateral limb was accelerated in CR-WT compared with AL-WT as evaluated by laser Doppler blood flow and capillary density analyses. CR-WT mice had significantly higher plasma levels of the fat-derived hormone adiponectin compared with AL-WT mice. In contrast to WT mice, CR did not affect the revascularization of ischemic limbs of adiponectin-deficient (APN-KO) mice. CR stimulated the phosphorylation of endothelial nitric-oxide synthase (eNOS) in the ischemic limbs of WT mice. CR increased plasma adiponectin levels in eNOS-KO mice but did not stimulate limb perfusion in this strain. CR-WT mice showed enhanced phosphorylation of AMP-activated protein kinase (AMPK) in ischemic muscle, and administration of AMPK inhibitor compound C abolished CR-induced increase in limb perfusion and eNOS phosphorylation in WT mice. Our observations indicate that CR can promote revascularization in response to tissue ischemia via an AMPK-eNOS-dependent mechanism that is mediated by adiponectin.

Vascular Inflammation, Insulin Resistance, and Reduced Nitric Oxide Production Precede the Onset of Peripheral Insulin Resistance

Obesity causes inflammation and insulin resistance in the vasculature as well as in tissues involved in glucose metabolism such as liver, muscle, and adipose tissue. To investigate the relative susceptibility of vascular tissue to these effects, we determined the time course over which inflammation and insulin resistance develops in various tissues of mice with diet-induced obesity (DIO) and compared these tissue-based responses to changes in circulating inflammatory markers. Adult male C57BL/6 mice were fed either a control low-fat diet (LF; 10% saturated fat) or a high-fat diet (HF, 60% saturated fat) for durations ranging between 1 to 14 weeks. Cellular inflammation and insulin resistance were assessed by measuring phospho-IkappaBalpha and insulin-induced phosphorylation of Akt, respectively, in extracts of thoracic aorta, liver, skeletal muscle, and visceral fat. As expected, HF feeding induced rapid increases of body weight, fat mass, and fasting insulin levels compared to controls, each of which achieved statistical significance within 4 weeks. Whereas plasma markers of inflammation became elevated relatively late in the course of DIO (eg, serum amyloid A [SAA], by Week 14), levels of phospho-IkappaBalpha in aortic lysates were elevated by 2-fold within the first week. The early onset of vascular inflammation was accompanied by biochemical evidence of both endothelial dysfunction (reduced nitric oxide production; induction of intracellular adhesion molecule-1 and vascular cell adhesion molecule-1) and insulin resistance (impaired insulin-induced phosphorylation of Akt and eNOS). Although inflammation and insulin resistance were also detected in skeletal muscle and liver of HF-fed animals, these responses were observed much later (between 4 and 8 weeks of HF feeding), and they were not detected in visceral adipose tissue until 14 weeks. During obesity induced by HF feeding, inflammation and insulin resistance develop in the vasculature well before these responses are detected in muscle, liver, or adipose tissue. This observation suggests that the vasculature is more susceptible than other tissues to the deleterious effects of nutrient overload.

Effects of naturally occurring coumarins on hepatic drug-metabolizing enzymes inmice

Cytochromes P450 (P450s) and glutathione S-transferases (GSTs) constitute two important enzyme families involved in carcinogen metabolism. Generally, P450s play activation or detoxifying roles while GSTs act primarily as detoxifying enzymes. We previously demonstrated that oral administration of the linear furanocoumarins, isopimpinellin and imperatorin, modulated P450 and GST activities in various tissues of mice. The purpose of the present study was to compare a broader range of naturally occurring coumarins (simple coumarins, and furanocoumarins of the linear and angular type) for their abilities to modulate hepatic drug-metabolizing enzymes when administered orally to mice. We now report that all of the different coumarins tested (coumarin, limettin, auraptene, angelicin, bergamottin, imperatorin and isopimpinellin) induced hepatic GST activities, whereas the linear furanocoumarins possessed the greatest abilities to induce hepatic P450 activities, in particular P450 2B and 3A. In both cases, this corresponded to an increase in protein expression of the enzymes. Induction of P4502B10, 3A11, and 2C9 by xenobiotics often is a result of activation of the pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR). Using a pregnane X receptor reporter system, our results demonstrated that isopimpinellin activated both PXR and its human ortholog SXR by recruiting coactivator SRC-1 in transfected cells. In CAR transfection assays, isopimpinellin counteracted the inhibitory effect of androstanol on full-length mCAR, a Gal4-mCAR ligand-binding domain fusion, and restored coactivator binding. Orally administered isopimpinellin induced hepatic mRNA expression of Cyp2b10, Cyp3a11, and GSTain CAR(+/+) wild-type mice. In contrast, the induction of Cyp2b10 mRNA by isopimpinellin was attenuated in the CAR(−/−) mice, suggesting that isopimpinellin induces Cyp2b10 via the CAR receptor. Overall, the current data indicate that naturally occurring coumarins have diverse activities in terms of inducing various xenobiotic metabolizing enzymes based on their chemical structure.

Effects of gpx4 Haploid Insufficiency on GPx4 Activity, Selenium Concentration, and Paraquat-Induced Protein Oxidation in Murine Tissues

Selenium-dependent glutathione peroxidase-4 (GPx4) catalyzes the reduction of phospholipid hydroperoxides. Because a full gpx4 knockout is embryonic lethal, we examined the effect of deletion of one copy of gpx4 on the activities of three selenoperoxidases (GPx1, GPx3, and GPx4), selenium concentrations, and pro-oxidant-induced protein oxidation in various tissues of mice. A total of 32 gpx4 hemizygous (GPx4+/-) and wild-type (WT) mice (8- to 10-weeks old; 16 males and 16 females) were fed a selenium-adequate diet and given an intraperitoneal injection of paraquat (PQ; 24 mg/kg body wt) or phosphate-buffered saline (PBS). All mice were euthanized 4 hrs after injection to collect tissues for analyses. In PBS-treated mice, GPx4 activities in lung, liver, kidney, and testes of GPx4+/- mice were 24-39% lower (P < 0.05) than in WT mice. Among PQ-treated mice, only testis GPx4 activity in GPx4+/- mice was significantly lower (54% P < 0.05) than WT mice. Selenium concentration in testes, but not in other tissues, was reduced (34% P < 0.05) in GPx4+/- mice compared with WT mice, irrespective of treatment. Tissue GPx1 activities and plasma GPx3 and alanine aminotransferase (ALT) activities were unaffected by PQ treatment or gpx4 hemizygosity. Total protein carbonyl was elevated (73% P < 0.05) by PQ only in lung, and this effect of PQ was independent of genotypes. In conclusion, gpx4 haploid insufficiency reduced GPx4 activities and/or selenium concentrations, but had no effect on pro-oxidant-induced protein oxidation in various tissues of mice.

Specific point mutations may not accumulate with aging in the mouse mitochondrial DNA control region

Increasing evidence suggests that mitochondrial function declines during aging in various tissues and in a wide range of organisms. This correlates with an age-dependent large accumulation of specific point mutations in the mtDNA control region that was reported recently in human fibroblast and skeletal muscle. However, evaluations of aging-related mtDNA mutations in other model animal systems. In this study, we analyzed mtDNA control regions of brain, skeletal muscle, heart, and other tissues from aged mice, in search of specific point mutations. A 948-bp fragment covering the entire mtDNA control region from various tissues of mice at the age of 25–26 months was sequenced. The sequence analysis was accomplished with a newly developed program Mutation Quantifier, which was able to accurately detect mutations with frequencies as low as 3%. Probably due to the relative shorter life-span, unlike what has been reported in human mtDNA, our results indicated there might be no significant accumulation of specific mutations in mouse mtDNA control region during aging.

Induction of NAD(P)H:quinone Reductase by Probucol: A Possible Mechanism for Protection against Chemical Carcinogenesis and Toxicity

Dietary antioxidants protect laboratory animals against induction of tumours by a variety of chemical carcinogens. Among possible mechanism, protection against chemical carcinogenesis could be mediated via antioxidant-dependent induction of detoxifying enzymes, including quinone reductase and glutathione S-transferase (GSH transferase). Probucol is used cholesterol-lowering drug used in the clinic, with pronounced antioxidant effect that protect against chemical carcinogenesis and toxicity. In the present study we therefore examined the ability of probucol to induce activities of quinone reductase in the cytosolic fractions of various tissues of mice. Quinone reductase activity was increased significantly in 6 of 8 tissues examined from probucol-fed mice. The greatest proportionate increase, to 1.8 times control levels, was observed in liver. Probucol also increased quinone reductase activities of forestomach, heart, kidney, lungs and spleen. Quinone reductase is a major enzyme of xenobiotic metabolism that carries out obligatory two-electron reductions and thereby protects cells against toxicity of quinones. It is induced in many tissues coordinately with other enzymes that protect against electrophilic toxicity. The protective effects of probucol appear to be due, at least in part, to the ability of this antioxidant to increase the activities in rodent tissues of several enzymes involved in the non-oxidative metabolism of a wide variety of xenobiotics. The induction of such enzyme, quinone reductase by probucol suggests the potential value of this compound as a protective agent against chemical carcinogenesis and other forms of electrophilic toxicity. The significance of these results can be implicated in relation to cancer chemopreventive effects of probucol in various target organs.

Nephrin is an important component of the barrier system in the testis.

Nephrin, a gene product of the congenital nephrotic syndrome of the Finnish type (NPHS1), is a 1242-residue putative transmembrane protein of the immunoglobulin family of cell adhesion molecules. The expression of this gene is localized in rat and human glomerular epithelial cells. Here we report the expression of nephrin in various tissues other than the kidneys in mice. The expression of nephrin mRNA in various tissues of mice, including the kidneys, testes, spleen, thymus and brain, were first investigated by the RT-PCR method, and it was shown that a high level of nephrin mRNA could be detected in the testes of mice 1-6 weeks old. In situ hybridization revealed the expression of the nephrin gene in the Sertoli cells. Additionally, immunofluorescent staining studies indicated that nephrin was colocalized with anchoring protein ZO-1 in the mouse testis. From these results, it is inferred that nephrin is an important component of the barrier system in testes.

Site-specific Phosphorylation of Tau Accompanied by Activation of Mitogen-activated Protein Kinase (MAPK) in Brains of Niemann-Pick Type C Mice

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol in most tissues and progressive neurodegeneration with the formation of neurofibrillary tangles. Neurofibrillary tangles are composed of paired helical filaments (PHF), a major component of which is the hyperphosphorylated tau. In this study we used NPC heterozygous and NPC homozygous mouse brains to investigate the molecular mechanism responsible for tauopathy in NPC. Immunoblot analysis using anti-tau antibodies (Tau-1, PHF-1, AT-180, and AT-100) revealed site-specific phosphorylation of tau at Ser-396 and Ser-404 in the brains of NPC homozygous mice. Mitogen-activated protein kinase, a potential serine kinase known to phosphorylate tau, was activated, whereas other serine kinases such as glycogen synthase kinase-3beta and cyclin-dependent kinase 5 were inactive. Morphological examination demonstrated that a number of neurons, the perikarya of which strongly immunostained with PHF-1, exhibited polymorphorous cytoplasmic inclusion bodies and multi-concentric lamellar-like bodies. Importantly, the accumulation of intracellular cholesterol in NPC mouse brains was determined to be a function of age. From these results we conclude that abnormal cholesterol metabolism due to the genetic mutation in NPC1 may be responsible for activation of the mitogen-activated protein kinase-signaling pathway and site-specific phosphorylation of tau in vivo, leading to tauopathy in NPC.

Tissue-specific and PPAR-regulated expression of fatty acid transport-related proteins in various tissues of mouse

Tissue-specific and PPAR-regulated expression of fatty acid transport-related proteins in various tissues of mouse

Correlation between DNA or protein adducts and benzo[a]pyrene diol epoxide I-triglyceride adduct detected in vitro and in vivo.

In this study, we demonstrated the in vitro and in vivo formation of carcinogen-lipid adduct and its correlation with DNA or protein adducts. The lipids from serum or hepatocyte membranes of Sprague-Dawley rats, human serum and standard major lipids were in vitro reacted with benzo[a]pyrene (B[a]P) and B[a]P metabolites. 7, 8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE-I), an ultimate carcinogenic form of B[a]P, was covalently bound to triglyceride (TG). BPDE-I-TG adducts isolated by thin-layer chromatography (TLC) were further detected by high-performance liquid chromatography. TGs, including triolein, tripalmitin and tristearin, showed positive reactions with BPDE-I. However, cholesterol, phospholipids (phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidyl-inositol and sphingomyelin) and non-esterified fatty acids (palmitic acid, oleic acid, linoleic acid and stearic acid) did not react with BPDE-I. In addition, other B[a]P metabolites (B[a]P-phenols and -diols) did not react with TG. TG appeared to be the most reactive lipid yet studied with respect to its ability to form an adduct with BPDE-I. There was a clear-cut dose-related formation of [1,3-(3)H]BPDE-I-lipid adducts in vitro between TG and [1,3-(3)H]BPDE-I. In an animal study, BPDE-I-TG was also formed in the serum of rats orally treated with B[a]P (25 mg/rat). Also, obvious correlations between [(3)H]B[a]P related-biomolecule adducts (DNA or protein) or lipid damage and the BPDE-I-TG adducts were obtained in various tissues of mice i.p. treated with [(3)H]B[a]P. These data suggest that TG can form an adduct with BPDE-I, as do other macromolecules (DNA, RNA and protein). Therefore, a carcinogen-lipid adduct would be a useful biomarker for chemical carcinogenesis research and cancer risk assessment.

Chat, a Cas/HEF1-associated Adaptor Protein That Integrates Multiple Signaling Pathways

Cas (Crk-associated substrate) and HEF1 (human enhancer of filamentation) are related adaptor proteins that function in integrin-mediated cell adhesion and antigen receptor signaling pathways. We report here a molecular cloning of Chat (Cas/HEF1-associated signal transducer) that associates with Cas and HEF1. Chat is a 78-kDa signaling molecule with an N-terminal SH2 domain and is expressed in a wide range of tissues. In hematopoietic cells, a 115-kDa isoform of Chat (Chat-H) was specifically expressed. Chat is associated with Cas in brain, and Chat-H is associated with HEF1 in splenocytes. Deletion analyses revealed that Chat and Cas are associated with each other by their C-terminal domains. Treatment of PC12 cells with epidermal growth factor or nerve growth factor increased the phosphorylation level of Chat. This increase was suppressed by an inhibitor of mitogen-activated protein (MAP) kinase kinase, PD98059, suggesting the phosphorylation of Chat by MAP kinase. In Chat-overexpressed COS7 cells, the activity of c-Jun N-terminal kinase was up-regulated. After the epidermal growth factor stimulation, Chat and Cas were colocalized with actin filaments at ruffling membranes. These findings suggest that Chat transduces signals of tyrosine kinases and MAP kinase to Cas signaling pathway.