Ceruloplasmin Expression Research Articles

Physical and biological effects of gellan gum on decreasing postoperative adhesion in a rat model

An antiadhesion membrane made from gellan gum was fabricated and characterized. A 12-µm-thick membrane of gellan gum was prepared and reacted with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to obtain a cross-linked membrane (G/A70) with 87% gel content and a tensile strength of 46.5 MPa. In vivo, the G/A70 membrane had 90% less tissue adhesion. Inflammation-related and extracellular matrix protein gene expression in a rat model of abdominal surgery was found by real-time quantitative reverse transcription polymerase chain reaction analysis. On day 3, after surgery, the gene expression of ceruloplasmin and type V collagen in the G/A70-treated group was 1.9 and 0.3 times that of the control group, respectively. The G/A70 membrane elicited mild inflammation but suppressed type V collagen synthesis and reduced the occurrence of tissue adhesion. These findings provide insights into the properties of gellan gum antiadhesion membranes and help to overcome problems involving tissue adhesions in surgical procedures.

Physical activity is linked to ceruloplasmin in the striatum of intact but not MPTP-treated primates

Ceruloplasmin is a protective ferroxidase. Although some studies suggest that plasma ceruloplasmin levels are raised by exercise, the impact of exercise on brain ceruloplasmin is unknown. We have examined whether striatal ceruloplasmin is raised with treadmill exercise and/or is correlated with spontaneous physical activity in rhesus monkeys. Parkinson's disease is characterized by a loss in ceruloplasmin and, similarly, Parkinson's models lead to a loss in antioxidant defenses. Exercise might protect against Parkinson's disease and is known to prevent antioxidant loss in experimental models. We have therefore examined whether treadmill exercise prevents ceruloplasmin loss in monkeys treated unilaterally with the dopaminergic neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). We found that exercise raised ceruloplasmin expression in the caudate and accumbens but not the putamen of intact monkeys. However, putamen ceruloplasmin was correlated with spontaneous activity in a home pen. MPTP alone did not cause unilateral loss of ceruloplasmin but blocked the impact of exercise on ceruloplasmin. Similarly, the correlation between putamen ceruloplasmin and activity was also lost with MPTP. MPTP elicited loss of tyrosine hydroxylase in the treated hemisphere; the remaining tyrosine hydroxylase was correlated with overall daily activity (spontaneous activity plus that induced by the treadmill). Thus, treadmill activity can raise ceruloplasmin but this impact and the link with spontaneous activity are both diminished in Parkinsonian primates. Furthermore, low overall physical activity predicts greater loss of dopaminergic phenotype in MPTP-treated primates. These data have implications for the maintenance of active lifestyles in both healthy and neurodegenerative conditions.

Potential Proteomic Biomarkers of Secondhand Smoking‐ Induced Cardiovascular Disease

Identification and characterization of proteomic changes that can serve as predictive markers of the onset and progression of secondhand smoke (SHS)‐induced disease are now in the forefront of cardiovascular (CV) research. Our objective was to identify a differential pattern of protein expression in the plasma of SHS‐exposed mice through a time course study. C57BL/6 mice were exposed for up to 48 weeks to SHS generated from 3R4F reference research cigarettes using the Teague smoking machine. Utilizing 2D‐DIGE and MS/MS, we have found a differential expression of 35 proteins in SHS‐exposed mice as compared to controls. Some of those are involved in vascular function, coagulation, or metabolism. Expression of ceruloplasmin, haptoglobin, fibrinogen, apolipoprotein E and murinoglobulin was ~2.5, 7, 3, 2 and 3.5‐fold lower than controls, respectively. The expression of alpha‐1 antitrypsin and complement C3 was ~6 and 13‐fold higher than controls, respectively. The alterations in fibrinogen, alpha‐1 antitrypsin, haptoglobin, and complement C3 are of particular interest since these have been linked to CV and lung pathology mediated by oxidative stress. Our data suggest that SHS exposure leads to changes in plasma protein expression. These changes can potentially serve as predictive markers of the onset and/or temporal progression of CV and pulmonary disease in secondhand smokers. (NIH # HL63744 to JLZ)

Protective role of macrophage-derived ceruloplasmin in inflammatory bowel disease

ObjectiveIntestinal microflora and inflammatory cell infiltrates play critical roles in the pathogenesis of acute colitis. Ceruloplasmin is an acute-phase plasma protein produced by hepatocytes and activated macrophages, and has ferroxidase...

Expression of ceruloplasmin in Down syndrome babies and their mothers

Objective To investigate the relationship between ceruloplasmin expression and Down syndrome (DS). Methods Differential protein expression in serum of six mothers with DS fetuses and six mothers with healthy fetuses was detected by two-dimensional electrophoresis and matrix assisted laser desorption ionization-mass spectrum,the results were confirmed by Western blot.The levels of serum ceruloplasmin in 11 mothers with DS fetuses,10 mothers with healthy fetuses,11 DS newborns and 10 healthy babies were detected by enzyme-linked immunosorbent assay.The difference between the two groups was compared by two-independent samples t test. Results Twenty-nine differential proteins were found in the serum of the mothers with DS fetuses; among which ceruloplasmin increased significantly compared with that in mothers with healthy fetuese with density ratio of 5.43 (t=2.7102,P＜0.05).Western blot results showed that the expression of ceruloplasmin in maternal serum with DS fetuses (0.95 ± 0.24) was higher than that of normal mothers (0.37±0.14) (t=2.9521,P＜0.05) ; while the expression of ceruloplasmin in DS babies' serum (0.74±0.03) was lower than that of normal newborns (0.89±0.06)(t=-2.9515,P＜0.05).The expression of ceruloplasmin in serum of mothers with DS fetuses [(346.5± 111.8) ng/ml] was higher than that of normal mothers [(248.6478.3) ng/ml] (t=2.301,P＜0.05) ; while the expression of ceruloplasmin in DS babies' serum [(166.1 ±55.0) ng/ml] was lower than that of normal newborns [(244.0±36.0) ng/ml] (t=-3.873,P＜0.01). Conclusions Abnormal maternal and neonatal serum ceruloplasmin level might relate to DS. Key words: Down syndrome; Ceruloplasmin; Proteomics; Infant, newborn; Pregnant women

Molecular analysis of Ceruloplasmin in a South African cohort presenting with oesophageal cancer

Oesophageal cancer (OC) is a disease characterized by the development of malignant tumors in the epithelial cells lining the oesophagus. It demonstrates marked ethnic variation, with squamous cell carcinoma (SCC) being more prevalent in the Black population and adenocarcinoma (ADC) occurring more often in Caucasians. The etiology of this complex disease has been attributed to a variety of factors, including an excess of iron (resulting in increased tumourigenesis), oesophageal injury and inflammation (due in part to Barrett's oesophagus and smoking among others). The aim of this study was to determine if genetic variations identified in the ceruloplasmin (CP) gene (implicated in iron homeostasis) contribute to OC pathogenesis or susceptibility. The study cohort consisted of 96 unrelated OC patients from the Black Xhosa-speaking South African population and 88 population-matched control individuals. The promoter and coding regions of the CP gene were analyzed for DNA sequence variation using heteroduplex single-strand conformation polymorphism (HEX-SSCP) analysis, restriction fragment length polymorphism (RFLP) analysis and semi-automated bidirectional DNA sequencing analysis. Fourteen previously described and four novel variants were identified. Statistically significant associations were revealed for two of the novel variants with OC in this study and could, therefore, potentially contribute to disease susceptibility. In silico analysis of the region of the promoter spanning the identified variants sought to identify putative transcription factor binding sites (TFBSs) that could possibly regulate the expression of CP. To our knowledge, this is the first study to examine CP with respect to OC in the Black South African population.

Lack of ceruloplasmin expression alters aspects of copper transport to the fetus and newborn, as determined in mice

Copper transport and accumulation were studied in virgin and lactating C57BL/6 mice, with and without expression of ceruloplasmin (Cp), to assess the importance of Cp to these processes. One hour after i.p. injection of tracer (64)Cu, liver and kidney accounted for 80% of the radioactivity, and mammary gland 1%, while in lactating Cp+/+ mice 2-4 days post partum, uptake by mammary gland was 9-fold higher and that of liver and other organs was decreased, with (64)Cu rapidly appearing in milk. Parallel studies in Cp-/- mice (siblings from same colony) gave virtually identical results. However, their milk contained less (64)Cu, and actual copper contents determined by furnace atomic absorption were less than half those for milk from normal dams. Liver copper concentrations of pups born to Cp-/- dams also were half those of pups from wild type dams. Copper in pup brains was unaffected; but iron concentrations were reduced. We conclude that absence of Cp, while not affecting entry of exchangeable copper from the blood into the mammary gland, does have a significant effect on the availability of this metal to the newborn through the milk and in the form of stores accumulating in gestation.

Pre-Conditioning Induces the Precocious Differentiation of Neonatal Astrocytes to Enhance Their Neuroprotective Properties

Hypoxic preconditioning reprogrammes the brain's response to subsequent H/I (hypoxia–ischaemia) injury by enhancing neuroprotective mechanisms. Given that astrocytes normally support neuronal survival and function, the purpose of the present study was to test the hypothesis that a hypoxic preconditioning stimulus would activate an adaptive astrocytic response. We analysed several functional parameters 24 h after exposing rat pups to 3 h of systemic hypoxia (8% O2). Hypoxia increased neocortical astrocyte maturation as evidenced by the loss of GFAP (glial fibrillary acidic protein)-positive cells with radial morphologies and the acquisition of multipolar GFAP-positive cells. Interestingly, many of these astrocytes had nuclear S100B. Accompanying their differentiation, there was increased expression of GFAP, GS (glutamine synthetase), EAAT-1 (excitatory amino acid transporter-1; also known as GLAST), MCT-1 (monocarboxylate transporter-1) and ceruloplasmin. A subsequent H/I insult did not result in any further astrocyte activation. Some responses were cell autonomous, as levels of GS and MCT-1 increased subsequent to hypoxia in cultured forebrain astrocytes. In contrast, the expression of GFAP, GLAST and ceruloplasmin remained unaltered. Additional experiments utilized astrocytes exposed to exogenous dbcAMP (dibutyryl-cAMP), which mimicked several aspects of the preconditioning response, to determine whether activated astrocytes could protect neurons from subsequent excitotoxic injury. dbcAMP treatment increased GS and glutamate transporter expression and function, and as hypothesized, protected neurons from glutamate excitotoxicity. Taken altogether, these results indicate that a preconditioning stimulus causes the precocious differentiation of astrocytes and increases the acquisition of multiple astrocytic functions that will contribute to the neuroprotection conferred by a sublethal preconditioning stress.

Mycobacteria-induced anaemia revisited: A molecular approach reveals the involvement of NRAMP1 and lipocalin-2, but not of hepcidin

Anaemia is a frequent complication of chronic infectious diseases but the exact mechanisms by which it develops remain to be clarified. In the present work, we used a mouse model of mycobacterial infection to study molecular alterations of iron metabolism induced by infection. We show that four weeks after infection with Mycobacterium avium BALB/c mice exhibited a moderate anaemia, which was not accompanied by an increase on hepatic hepcidin mRNA expression. Instead, infected mice presented increased mRNA expression of ferroportin ( Slc40a1), ceruloplasmin ( Cp), hemopexin ( Hpx), heme-oxygenase-1 ( Hmox1) and lipocalin-2 ( Lcn2). Both the anaemia and the mRNA expression changes of iron-related genes were largely absent in C.D2 mice which bear a functional allele of the Nramp1 gene. Data presented in this work suggest that anaemia due to a chronic mycobacterial infection may develop in the absence of elevated hepcidin expression, is influenced by Nramp1 and may involve lipocalin-2.

Ontogenetic expression and 17β-estradiol regulation of immune-related genes in early life stages of Japanese medaka ( Oryzias latipes)

Accumulating evidence suggests that environmental endocrine disrupting chemicals (EDCs) may exert adverse effects on aquatic organisms via the modulation of immune competence in addition to the endocrine system. However, to date, most studies have been undertaken only on biochemical and histopathological endpoints, and few studies have addressed the role of immune response gene transcript abundance in response to estrogen. In the present study, the ontogenetic expression of immune-related genes, including three complement components (C3-1, C3-2 and Bf/C2), two cytokines (IL-21 and type I IFN [IFN]), lysozyme (LZM), novel immune-type receptor (NITR-18), Ikaros (IK) and ceruloplasmin (CP) were characterized during different developmental periods (from 0 to 28 d post-hatch [dph]) in Japanese medaka. Furthermore, the responses of these genes to natural estrogen (i.e., 17β-estradiol [E2]) were evaluated. E2 exposure at sublethal concentrations (0.1–10 μg/L) down-regulated the gene expression of C3-1, C3-2, Bf/C2, LZM and CP, while up-regulating the expression of IL-21, IFN, NITR-18 and IK. The results demonstrate a very different trend in gene expression in fish larvae exposed to E2 when compared with the ontogenetic changes in control, suggesting that exposure to environmental chemicals with estrogenic activities may interfere with immune-related genes and thus potentially influence the susceptibility of fish to opportunistic infections. These findings confirm the ability of exogenous estrogens to elicit changes in immune-related gene expression, and broaden our understanding about the mechanisms underlying the actions of EDCs. In addition, the expression profiles of immune-related genes can be developed for use as biomarkers for future immunotoxicological studies.

Ceruloplasmin Expression in Rat Liver Cells is Attenuated by Insulin: Role of FoxO Transcription Factors

The phosphoinositide 3'-kinase (PI3 K)/Akt pathway controls the activity of a number of proteins important in the regulation of apoptosis and cell proliferation. FoxO (forkhead box, class O) transcription factors, substrates of the Ser/Thr kinase Akt, control the expression of several target genes that are crucial to the defense against oxidative stress, the regulation of cell cycle, and apoptosis in mammalian cells. Here, expression of ceruloplasmin (CP), the major copper-containing protein in blood released by the liver, was investigated. We observed a significant downregulation of CP mRNA levels after insulin treatment in H4IIE rat hepatoma cells. The PI3K inhibitor wortmannin counteracted this insulin effect on CP mRNA levels, indicating that the PI3K/Akt cascade is involved in the regulation of CP expression. Stimulation of FoxO1 was induced in H4IIE rat hepatoma cells expressing a conditionally active FoxO1 construct, resulting in significant upregulation of CP mRNA levels. This upregulation was prevented in the presence of insulin. In parallel, mRNAs of established FoxO target genes were analyzed: like CP mRNA, selenoprotein P and glucose 6-phosphatase mRNAs were upregulated by FoxO1, which was prevented by insulin. The same effects of insulin on CP mRNA levels were detected in primary rat hepatocytes. Furthermore, CP release into cell culture media was analyzed with primary hepatocytes and found to be attenuated by insulin. In line with its insulin-mimetic effects on cultured cells, Cu (2+) imitated the effect of insulin on CP expression and caused a downregulation of CP mRNA levels in rat hepatoma cells.

Role of External Loops of Human Ceruloplasmin in Copper Loading by ATP7B and Ccc2p

Ceruloplasmin is a multicopper oxidase required for correct iron homeostasis.Previously, we have identified a ceruloplasmin mutant associated with the iron overload disease aceruloplasminemia, which was unable to acquire copper from the mammalian pump ATP7B but could be produced in an enzymatically active form in yeast. Here, we report the expression of recombinant ceruloplasmin in the yeast Pichia pastoris and the study of the role of five surface-exposed loops in copper incorporation by comparing the efficiencies of mammalian ATP7B and yeast Ccc2p. The possibility to "mix and match" mammalian and yeast multicopper oxidases and copper ATPases can provide clues on the molecular features underlying the process of copper loading in multicopper oxidases.

Comparison of Macrophage Antimicrobial Responses Induced by Type II Interferons of the Goldfish (Carassius auratus L.)

Unlike mammals, bony fish have two type II interferons, IFNgamma and IFNgammarel, whose pro-inflammatory functions have not been fully characterized. To elucidate the distinct roles of these type II interferons of bony fish, we examined the effects of recombinant goldfish (rg) IFNgamma and IFNgammarel on the macrophage antimicrobial responses, immune gene expression, and their signaling pathways. Our findings indicate that rgIFNgamma and rgIFNgammarel possess unique capacities to mediate each of the above processes. Q-PCR analysis revealed similar expression of both cytokines in tissues and immune cell populations of the goldfish, although IFNgamma mRNA levels were generally higher in most tissues and cell types. Whereas rgIFNgamma had long-lasting effects on the priming of goldfish monocyte ROI production, the rgIFNgammarel had relatively short-lived ROI priming potential and eventually down-regulated the priming of ROI production induced by rgIFNgamma or rgTNFalpha2. Whereas rgIFNgamma induced relatively modest phagocytic and nitric oxide responses of goldfish macrophages, rgIFNgammarel induced significantly higher phagocytosis, iNOSA and iNOSB gene expression and nitric oxide production compared with rgIFNgamma. The rgIFNgamma and rgIFNgammarel induced different gene expression profiles in goldfish monocytes. These differences included significantly higher induction of TNFalpha2, CXCL8, ceruloplasmin, and interferon regulatory factor (IRFs) expression after activation of monocytes with rgIFNgammarel. The rgIFNgammarel was more abundant in whole cell lysates compared with rgIFNgamma. Both cytokines induced the phosphorylation of Stat1, while the nuclear localization of Stat1 was only observed following treatment of monocytes with rgIFNgamma. Our findings suggest the presence of functional segregation of the induction of macrophage antimicrobial functions by type II interferons of bony fish.

Dietary iron affects mRNA expression of porcine copper transporters

Weanling male pigs (21 days of age) were fed diets deficient (20 mg Fe/kg; L‐Fe), adequate (120 mg Fe/kg; A‐Fe) or high (520 mg Fe/kg; H‐Fe) in iron (Fe) for 32 days to evaluate the effects of dietary Fe level on copper (Cu) transporters and chaperones. Increasing dietary Fe increased Fe in proximal duodenum and liver. Liver Cu was lower in A‐Fe than in L‐Fe and H‐Fe pigs and did not differ among pigs fed L‐Fe and H‐Fe diets. Expression of liver copper transporter 1 (Ctr1) was higher in L‐Fe vs. A‐Fe pigs. Liver Atp7b was lower in both L‐Fe and H‐Fe pigs vs. A‐Fe pigs. Ceruloplasmin (Cp) expression in liver was up‐regulated in L‐Fe and H‐Fe pigs vs. A‐Fe pigs. Cp was also increased in pigs fed L‐Fe vs. H‐Fe diets. Feeding a L‐Fe diet tended to decrease Commd1 compared to feeding an A‐Fe diet. Liver cytochrome c oxidase assembly protein 17 (Cox17), antioxidant 1 (Atox1), copper chaperone for Cu/Zn superoxide dismutase (CCS), Cu/Zn superoxide dismutase (Sod1), and metallothionein 1a (Mt1a) were unaffected by dietary Fe. Duodenal Cu concentration and expression of duodenal Ctr1, Atox1, Cox17, CCS, Sod1, Atp7a, or Mt1a were not affected by dietary Fe. These data indicate that dietary Fe affects cellular Cu transporters involved in import and export of Cu to a greater extent in the liver than in duodenum. Reduced hepatic expression of Atp7b in both L‐Fe and H‐Fe pigs may explain the increased liver Cu seen in these pigs relative to A‐Fe.Grant Funding Source: Micronutrients

Characterization of plasma ceruloplasmin activity and expression following dietary copper deficiency

Ceruloplasmin (CP) is a multicopper oxidase and the most abundant copper binding protein in mammalian plasma. Loss of function mutations in humans or experimental deletion in mice result in iron overload consistent with CP's putative ferroxidase function. Prior work suggested plasma may contain multiple ferroxidases. Studies were conducted in Holtzman rats, albino mice, Cp −/− mice, and adult humans to investigate the copper‐iron interaction and further characterize CP as a biomarker. Dietary copper‐deficient (CuD) rats and mice were produced using a modified AIN‐76A diet. Results confirmed that o‐dianisidine is a better substrate than paraphenylene diamine (PPD) for assessing diamine oxidase activity of Cp. Plasma from CuD rat dams and pups, and CuD and Cp −/− mice contained no detectable Cp diamine oxidase activity. Importantly, following western blotting no detectable ferroxidase activity was observed for CuD rats, mice, or Cp −/− mice compared to robust activity for copper‐adequate (CuA) rodent controls. Immunoblot protocols detected major reductions (60–90%) in Cp protein in plasma of CuD rodents but no alteration in liver mRNA levels by qRT‐PCR. Data support that apo‐Cp is less stable than holo‐Cp. Further research is needed to explain normal plasma iron in CuD mice. Reduction in CP activity is a sensitive biomarker for copper deficiency.Supported by NIH HD‐39708 and USDA NIFA 2006‐35200‐17378.

Proteomic analysis of amniotic fluid in pregnancies with Klinefelter syndrome foetuses

Klinefelter syndrome is a sex chromosomal abnormality (47, XXY karyotype), occurring approximately in 1 in 1000 male live births. In the present study proteomic analysis was performed in twelve 2nd trimester amniotic fluid samples, eight coming from pregnancies with normal males and four with Klinefelter syndrome foetuses, as shown by routine prenatal cytogenetic analysis. Samples were analysed by 2-DE, coupled with MALDI-TOF-MS analysis. Three proteins (Ceruloplasmin, Alpha-1-antitrypsin and Zinc-alpha-2-glycoprotein) were found to be up-regulated in samples obtained from pregnancies with Klinefelter syndrome foetuses, whereas four proteins (Apolipoprotein A-I, Plasma retinol-binding protein, Gelsolin, and Vitamin D-binding protein) were down regulated when compared to proteins detected in samples from normal foetuses. The differential expression of Ceruloplasmin, Apolipoprotein A-I and Plasma retinol-binding protein was further confirmed by immunoblotting. Since these proteins are likely to cross the placenta barrier and be detected in maternal plasma they could be used as biomarkers for the non-invasive prenatal diagnosis of Klinefelter syndrome.

Ceruloplasmin Induces Polymorphonuclear Leukocyte Priming in Localized Aggressive Periodontitis

Polymorphonuclear leukocytes (PMNs) from subjects with localized aggressive periodontitis (LAgP) present multiple functional abnormalities associated with a phenotypically primed PMN phenotype. Local inflammation is characterized by hypoxia, which leads to increased production of superoxide (O(2)(-)) by PMNs. Ceruloplasmin (CP) is also induced by hypoxia and inflammation. The aim of this study was to investigate the role of CP in O(2)(-) generation in PMNs from healthy subjects and patients with LAgP. PMNs were isolated from healthy subjects and those with LAgP (N = 36). Superoxide was measured by cytochrome-C reduction at 550 nm. Intracellular CP expression was analyzed by real-time polymerase chain reaction and Western blotting. Serum levels of CP were measured by enzyme-linked immunosorbent assay. Intracellular iron ion conversion was spectrophotometrically determined by measuring the absorbance of sigma-phenanthroline at 510 nm. O(2)(-) generation was significantly higher in LAgP PMNs before and after stimulation with formyl-methionyl-leucyl-phenylalanine (100 nM). CP expression in PMNs and CP levels in serum were significantly higher in subjects with LAgP compared to the PMNs and serum samples from matched healthy donors (P <0.05). LAgP PMNs also had significantly higher levels of Fe(3+) and lower levels of Fe(2+) compared to healthy PMNs (P <0.05), suggesting increased iron conversion. Exogenous CP treatment of healthy PMNs resulted in significant increases in O(2)(-) generation and iron ion conversion similar to LAgP PMNs. LAgP PMNs are primed to express higher levels of CP, leading to hypoxia-mediated O(2)(-) generation in PMNs and increased oxidative stress and neutrophil-mediated tissue injury in LAgP.

Regulation of ceruloplasmin gene activity in mammary gland cells

Tissue-specific regulation of the expression of ceruloplasmin (CP) gene, which encodes major copper-containing extracellular glycoprotein was investigated. A decrease of the CP concentration associated with copper amounts in milk during the first 3 days of lactation was used as phenotypic index for evaluating the CP enzyme activity in the mammary gland. Computer analysis of mammalian CP gene promoter region has revealed conserved sequences of cis-elements, which potentially were capable of regulating the enzyme activity. It has been shown that changes in the nucleotide sequence of specific transcriptional factor binding sites located at 5'-end of CP gene were associated with disturbance of the regular downregulation of CP gene activity during lactation.

Modification of dietary copper levels on the early stage of tumor-promotion with propylthiouracil in a rat two-stage thyroid carcinogenesis model

To investigate the role of copper (Cu)-related cellular responses on thyroid carcinogenesis, the expression of ceruloplasmin (Cp) and metallothionein (MT)-1/2 were examined in relation to the activities of cell proliferation/apoptosis in the thyroid of rats at an early stage of tumor promotion under different dietary Cu levels. Male F344 rats were initiated with N-bis(2-hydroxypropyl)nitrosamine by single subcutaneous injection at 2800 mg/kg body weight, and 1 week later promoted with 6-propyl-2-thiouracil at 12 ppm in the drinking water for 4 weeks. Animals were fed a diet containing Cu at 0.6, 6 or 60 ppm from the time point of initiator-treatment to create marginally deficient, normal, or non-toxic supplementary levels of Cu. At both 0.6 and 60 ppm, the multiplicity of preneoplastic focal follicular cell hyperplasias (FFCHs) was decreased as compared with 6 ppm Cu, while adenomas also decreased at 0.6 ppm Cu. Both 0.6 and 60 ppm Cu levels revealed decreased Ki-67-immunoreactive proliferating cells in both FFCHs and surrounding follicles accompanied by mRNA downregulation of Cdc2a and Ccnb1, while TUNEL-positive apoptotic cells were unaltered with change of dietary Cu. Both Cp and MT-1/2 were immunolocalized in FFCHs and adenomas, with higher distribution in the latter. At both 0.6 and 60 ppm, the immunoreactivities and/or thyroidal mRNA levels of Cp and MT-1/2 were also decreased. Transcript levels of several antioxidant enzymes were up- or downregulated in the same direction at both Cu levels. Serum levels of thyroid-related hormones were unaltered at both Cu levels, except for non-significant reduction of thyroid-stimulating hormone at 0.6 ppm. These results suggest an involvement of Cp and MT-1/2 on the thyroid tumor promotion that can be suppressed by dietary Cu level through inhibition of cell proliferation associated with altered redox balance.

Japanese encephalitis virus differentially modulates the induction of multiple pro‐inflammatory mediators in human astrocytoma and astroglioma cell‐lines

Astrocytes become activated in response to many CNS pathologies. The process of astrocyte activation remains rather enigmatic and results in so-called reactive gliosis, a reaction with specific structural and functional characteristics. Astrocytes play a vital role in regulating aspects of inflammation and in the homeostatic maintenance of the CNS. However, the responses of different human astroglial cell-lines in viral encephalitis mediated inflammation are not well documented. We have shown that Japanese encephalitis virus (JEV) infection causes morphological and functional changes in astrocytic cell-lines. We have demonstrated that besides reactive oxygen species (ROS) JEV infection differentially regulated the induction pattern of IL-6, IL-1 beta and IL-8. IP-10, MCP-1, MIG and RANTES secretions in different astroglial cell-lines. The expression of different proteins such as astrocyte-specific glial fibrillary acidic protein (GFAP), the glutamate aspartate transporter/essential amino acid transporter-1 (GLAST/EAAT-1), glutamate transporter-1/essential amino acid transporter-2 (GLT-1/EAAT-2), Ceruloplasmin and Thioredoxin (TRX) expression level also differ in different human astrocyte cell-lines following infection.