Expression of deafness protein Tmie in postnatal developmental stages of C57BL/6J mice

Loss-of function mutations in the transmembrane inner ear expressed (Tmie/TMIE) gene have been shown to cause deafness in mice and humans (DFNB6). Previous studies report that the circling mouse can be an animal model for DFNB6. However, the expression pattern of Tmie protein in postnatal developmental stages has not been clearly revealed. In this study we tried to investigate the expression of Tmie protein in the liver, spleen, kidney, and lung, as well as in the cochlea. We examined various tissue samples from five different age groups of C57BL/6J animals. Using western blotting analysis, the expression of Tmie protein in these organs has been identified. The results show that Tmie protein expression in the cochlea has been increased in postnatal developmental stages, indicating that Tmie plays an important role in not only the development and also in the function of the cochlea. The expression pattern of Tmie in adult mouse organs such as the liver, spleen, kidney, and spleen significantly vary in adult rats. The order of Tmie expression level in mice (63 days after birth) was spleen, liver, lung, cochlea, and kidney, whereas in the adult rat it was liver, cochlea, lung, spleen, and kidney.     

Effect of Dietary Selenium Yeast Supplementation on Porcine Circovirus Type 2 (PCV2) Infections in Mice

The present study was performed to determine the protective role of dietary selenium (Se) yeast supplementation in porcine circovirus type 2 (PCV2) infected mice. Forty-eight Kun Ming female mice were randomly assigned to Se yeast group (0.3%Se +basal diet, n = 24) and control group (basal diet, n = 24). After 3 days of adaptive feeding and 15 days treatment with the experimental feed, mice were challenged by intraperitioneal injection of PCV2 at the dosage of 2000 TCID₅₀ (50% tissue culture infection dose, TCID₅₀). Serum total superoxide dismutase (SOD) activity, malondialdehyde (MDA) level, tumor necrosis factor alpha (TNF-α), C-reactive protein (CRP) and interleukin-1 beta (IL-1β) levels were measured at 5, 10, 15, 20 days post infection (dpi). The PCV2 virus load in the liver, spleen and lung, and the microscopic lesions in the liver, spleen and lung also were determined on 5, 10, 15, and 20 dpi. Dietary Se yeast supplementation decreased (Pμ0.05) the serum levels of TNF-α, but had no significant effect on the activity of SOD and the levels of MDA, CRP and IL-1β between experimental and control groups. Dietary Se yeast supplementation had little effect on the PCV2 virus load in the liver, spleen and lung. However, mice in the selenium yeast group showed a significant decrease in microscopic lesion scores in the lung and spleen compared with those in the control group (Pμ0.05). These data indicate Se yeast attenuated the PCV2 infection through altering the systemic inflammation and maintaining the normal organ morphology.     

Pharmacokinetics of the potent redox-modulating manganese porphyrin, MnTE-2-PyP(5+), in plasma and major organs of B6C3F1 mice

Mn(III) tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnTE-2-PyP(5+), a potent catalytic superoxide and peroxynitrite scavenger, has been beneficial in several oxidative stress-related diseases thus far examined. Pharmacokinetic studies are essential for the better assessment of the therapeutic potential of MnTE-2-PyP(5+) and similar compounds, as well as for the modulation of their bioavailability and toxicity. Despite high hydrophilicity, this drug entered mitochondria after a single 10 mg/kg intraperitoneal injection at levels high enough (5.1 muM; 2.95 ng/mg protein) to protect against superoxide/peroxynitrite damage. Utilizing the same analytical approach, which involves the reduction of MnTE-2-PyP(5+) followed by the exchange of Mn(2+) with Zn(2+) and HPLC/fluorescence detection of ZnTE-2-PyP(4+), we measured levels of MnTE-2-PyP(5+) in mouse plasma, liver, kidney, lung, heart, spleen, and brain over a period of 7 days after a single intraperitoneal injection of 10 mg/kg. Two B6C3F1 female mice per time point were used. The pharmacokinetic profile in plasma and organs was complex; thus a noncompartmental approach was utilized to calculate the area under the curve, c(max), t(max), and drug elimination half-time (t(1/2)). In terms of levels of MnTE-2-PyP(5+) found, the organs can be classified into three distinct groups: (1) high levels (kidney, liver, and spleen), (2) moderate levels (lung and heart), and (3) low levels (brain). The maximal levels in plasma, kidney, spleen, lung, and heart are reached within 45 min, whereas in the case of liver a prolonged absorption phase was observed, with the maximal concentration reached at 8 h. Moreover, accumulation of the drug in brain continued beyond the time of the experiment (7 days) and is likely to be driven by the presence of negatively charged phospholipids. For tissues other than brain, a slow elimination phase (single exponential decay, t(1/2)=60 to 135 h) was observed. The calculated pharmacokinetic parameters will be used to design optimal dosing regimens in future preclinical studies utilizing this and similar compounds.     

Determination of carbon monoxide (CO) in rodent tissue: effect of heme administration and environmental CO exposure

Carbon monoxide (CO), produced endogenously during heme degradation, is considered a messenger molecule in vascular and neurologic tissues. To study this role, it is important to determine CO concentration in target tissues pre- and post-perturbations. Here, we describe a sensitive and reproducible method, which is linear and accurate, and provide some examples of its application for quantitation of CO concentrations in tissues pre- and post-perturbations. Tissues from adult rats and mice were sonicated (20% w/w), and volumes representing 0.04-8 mg fresh weight (FW) were incubated at 0 degrees C for 30 min with sulfosalicylic acid. CO liberated into the headspace was quantitated by gas chromatography. Tissue CO concentrations (mean+/-SD, pmol CO/mg FW) were as follows: blood (47+/-10, 45+/-5), muscle (4+/-4, 10+/-1), kidney (5+/-2, 7+/-2), heart (6+/-3, 6+/-1), spleen (11+/-3, 6+/-1), liver (4+/-1, 5+/-1), intestine (2+/-1, 4+/-2), lung (2+/-1, 3+/-1), testes (1+/-1, 2+/-1), and brain (2+/-1, 2+/-0) in untreated rat (n=3) and mouse (n=5), respectively. Between the rat and the mouse, only CO concentrations in the muscle and spleen were significantly different (p0.05). Endogenous CO generation, after administration of heme arginate to mice (n=3), increased CO concentrations by 0-43 pmol/mg FW. Exposure of mice (n=3) to 500 ppm CO for 30 min yielded significantly elevated CO concentrations by 4-2603 pmol/mg FW in all tissues over the native state. While blood had the highest CO concentration for all conditions, muscle, kidney, heart, spleen, and liver, all rich in hemoglobin and/or other CO-binding hemoproteins, also contained substantial CO concentrations. Intestine, lung, testes, and brain contained the lowest CO concentrations.     

肝细胞生长因子mRNA在成年小鼠和人胎儿不同器官中的表达

本实验从成年小鼠和胎龄４－５月的人胎儿不同器官中分离总ＲＮＡ。经斑点印迹分析显示,肝细胞生长因子（ＨＧＦ）ｍＲＮＡ在成年ＫＭ小鼠多种器官中表达,其表达水平由高到低依次为：肺、肝、肾、卵巢、睾丸、大脑和胃;在脾、心、骨髓、小肠和骨骼肌组织中以ＨＧＦｍＲＮＡ。在胎龄４－５月的人胎儿中,ＨＧＦｍＲＮＡ表达水平由高到低依次为：大脑、肝、腮腺、胃、小肠、肾、心和骨骼肌;肺和脾组织为阴性。由此可见,ＨＧＦ在成     

The stability of methyl and ethyl phosphotriesters in DNA in vivo

C57BL male mice were injected with N-methyl-N-nitrosourea (MNUA) or N-ethyl-N-nitrosourea (ENUA) and the concentration of alkyl phosphotriesters in the DNA of lung, liver, brain, kidney, spleen and thymus determined from the extent of degradation induced in isolated DNA by alkali. The same total dose of reagent was given either as a single injection (i.p.) or by weekly injections carried out over 5-20 weeks. Methyl phosphotriesters induced in liver, lung and kidney by the single injection were lost with a half-life of about 7 days, in brain the loss was more rapid, t1/2 = 2-3 days. During the multiple injections the observed t1/2 was 16 days. Ethyl phosphotriesters formed in the DNA of lung, liver, kidney and brain were much more stable than the methyl derivatives, t1/2 = 10-15 weeks. Phosphotriesters formed in the DNA of spleen and thymus disappeared very quickly after the single injection presumably as a result of dilution due to DNA replication. No accumulation of phosphotriesters occurred in the DNA of these tissues during the multiple injections. The general pattern of the results suggests that phosphotriesters are not excised by cellular repair systems.     

小鼠组织中过氧化氢酶的活性与年龄的关系

为观察小鼠组织中过氧化氢酶的活性与年龄的关系,采用高锰酸钾滴定法测定不同年龄(1、4、18月龄)小鼠肝、肾、肺、心、脾、胃、脑组织中过氧化氢酶的活性。结果显示:小鼠过氧化氢酶在不同组织中活性不同,活性高低顺序基本表现为:肝>肾>肺>心、脾、胃>脑;小鼠肺、心、脾、胃、脑各组织中过氧化氢酶的活性在1～4月龄间随年龄增加而增加,在4～18月龄间随年龄增加而降低;小鼠肝、肾组织中过氧化氢酶的活性在1～4月龄间与年龄相关性不显著,在4～18月龄间随年龄增加而降低。结果表明,小鼠肝、肾、肺、心、脾、胃、脑等组织中过氧化氢酶的活性随年龄变化而变化,机体过氧化氢酶活性的降低与机体衰老密切相关。     

Characterization of the antioxidant status of the heterozygous manganese superoxide dismutase knockout mouse

The antioxidant status of several tissues (liver, kidney, lung, brain, heart, muscle, stomach, and spleen) from heterozygous manganese superoxide dismutase (MnSOD) mutant mice (Sod2-/+) was characterized. The activity of MnSOD was decreased (30 to 80%) in all tissues examined. The levels of mRNA coding for the major antioxidant enzymes (CuZnSOD, catalase, and glutathione peroxidase) were not significantly altered in liver, kidney, heart, lung, or brain in the Sod2-/+ mice. The activities of the enzymes were not altered in any of these tissues, with the exception of a decrease in glutathione peroxidase activity in muscle in the Sod2-/+ mice compared to the Sod2+/+ mice. Thus, there was no up-regulation of the activities of the major antioxidant enzymes to compensate for the decrease in MnSOD activity. Reduced glutathione levels were 30 to 50% lower in the lung, brain, and muscle of the Sod2-/+ mice compared to the wild-type Sod2+/+ mice. In addition, the ratio of GSH/GSSG was decreased approximately 50% in Sod2-/+ muscle, indicating that the decrease in MnSOD activity in the Sod2-/+ mice results in some degree of oxidative stress in this tissue.     

Prenatal and postnatal changes in the content and species of ferritin in rat liver

The iron and ferritin content of rat liver and the species of ferritin present were examined from 4 days before to 3 weeks after birth. 1. Total iron and ferritin iron accumulated rapidly during the last days of gestation and from the second postnatal day underwent a steady depletion. 2. The amount of iron deposited before birth in the liver of each pup varied inversely with litter size and could be increased moderately by injection of iron into the mother before mating. 3. Intraperitoneal injection of iron 1 day after birth doubled the concentration of total iron, ferritin iron and ferritin protein in the liver over the next 24h, but at 3 weeks after birth it raised the very low concentrations of iron and ferritin severalfold. 4. As shown by electrophoretic migration, ferritin and dissociated ferritin subunits prepared from the livers of rats from 4 days before to 3 weeks after birth differed from those of adult liver ferritin and were indistinguishable from those of adult kidney and spleen ferritin. Treatment with iron at 3 weeks of age induced formation of a ferritin with electrophoretic properties resembling those of adult liver. It is concluded that iron given at this stage of development may activate the genetic cistron for adult liver ferritin.     

Distribution of two urinary ribonuclease-like enzymes in human organs and body fluids

In order to determine the distribution of two human urinary RNase (RNase Us and RNase UL)-like enzymes in human tissues and body fluids, enzyme immunoassay systems were established using rabbit anti-RNase sera. The sensitivity of the assay systems was of similar order to that of radioimmunoassay systems previously reported. In the enzyme immunoassay, the cross reactivities of anti-RNase UL serum towards RNase Us, bovine kidney RNase K2, bovine RNase A, and bovine seminal RNase Vs were less than 1%. The cross reactivity of anti-RNase Us-serum towards RNase UL was less than 0.5% and cross reactivities were minimal for RNase A, RNase K2, and RNase Vs. The RNase levels in human organs and body fluids were measured by enzyme immunoassay. In milk, semen and saliva, only RNase UL-like enzyme was found. Both RNase Us- and RNase UL-like enzymes were found in kidney, stomach, and pancreas and the RNase Us/RNase UL ratios were 0.49, 1.35, and 0.34, respectively. In lung, liver, spleen, and leukocytes, most of the RNase activity was accounted for by RNase Us-like enzyme. The activity of RNase Us-like enzyme was especially high in lung, spleen, and leukocytes. The crude extracts of several tissues and body fluids were separated by phosphocellulose column chromatography and the contents of the two urinary RNase-like enzymes were determined by enzyme immunoassay. In stomach, kidney, pancreas, and serum, both enzymes were present in multiple forms. In spleen and lung, both the major RNase (RNase Us) and minor RNase (RNase UL) existed in two forms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental changes in hepatocyte growth factor mRNA and its receptor in rat liver, kidney and lung.

Hepatocyte growth factor (HGF) is a mesenchymal-derived factor which induces mitosis, cell movement and morphogenesis of tissue-like structure. We analyzed changes in HGF mRNA and its receptor, the c-met proto-oncogene product, in the liver, kidney and lung during late fetal and postnatal development in rats. In the liver, the HGF-mRNA level was very low during late gestation and in neonates, it increased remarkably and reached a maximum two weeks postnatally, to be followed by a decrease to 33% of the maximum. HGF mRNA in the kidney and lung was either undetectable or very low during late gestation and the neonatal period and increased markedly to reach a maximum, respectively, 3-4 weeks postnatally. HGF-mRNA level in the adult rat lung was fivefold higher than that in the liver and kidney. The number of HGF receptors on plasma membranes of these tissues was low in neonates but there was a rapid increase after birth and a maximum was reached within three weeks. The number of HGF receptors/ng plasma membrane protein at the maximal level was highest in the liver and lowest in the lung. c-met/HGF-receptor mRNA in the liver was also low during late-gestation or in early neonatal periods and increased postnatally. Since HGF-mRNA and HGF-receptor levels changed differently in liver, kidney and lung, the expression of HGF and its receptor may be independently regulated in each organ. However, in these organs, HGF mRNA and the HGF receptor increased within a few weeks of birth, HGF may play roles in organ growth, organ maturation and the maintenance of tissue homeostasis during the postnatal period, presumably through its potential to act as mitogen, motogen and morphogen.     

Two pathways for GM2(NeuGc) expression in mice: genetic analysis

We have reported that WHT/Ht mice express neither GM2(NeuGc) nor GM1(NeuGc) in the liver or erythrocytes due to a defect on the Ggm-2 gene, which was demonstrated to control the activity of UDP-GalNAc:GM3(NeuGc) N-acetylgalactosaminyltransferase in mouse liver, and, in addition, WHT/Ht mice do not express a detectable amount of GM2(NeuGc) but do express GM1(NeuGc) in tissues other than the liver and erythrocytes, such as the spleen, thymus, heart, lung, kidney, and testis [Nakamura et al. (1988) J. Biochem. 103, 201-208]. In order to determine whether the phenotype of WHT/Ht mice exhibiting an undetectable amount of GM2(NeuGc) in these tissues is genetically controlled or not, we analyzed the expression of gangliosides in the progeny obtained on backcross mating between (BALB/c X WHT/Ht)F1 and WHT/Ht mice, and in a GM2(NeuGc) congenic mouse, WHT.C. Concerning the expression of GM2(NeuGc) in the liver, lung, and kidney, 102 backcross mice could be segregated into two types. One type expressed a detectable amount of GM2(NeuGc) in the liver, lung, and kidney, and the other type did not. The ratio of the numbers of mice exhibiting these two types was 42: 60, indicating that the two phenotypes were genetically determined by the involvement of a single autosomal gene. Recombination as to GM2(NeuGc) expression in the liver, lung, and kidney was not detected among the 102 backcross mice. Analysis of the GM2(NeuGc) congenic mouse indicated that a detectable amount of GM2(NeuGc) was expressed in the liver, erythrocytes, lung, kidney, heart, spleen, and small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigation of thyroxine monodeiodinase activity in the liver, kidney and brown adipose tissue of foetal and neonatal rabbit

The maturation of the 5'- and 5-monodeiodinase system in liver, kidney and brown adipose tissue of rabbits, during the foetal period (from 21 days of gestation to birth) and the neonatal period (from birth to 3 weeks of life) was studied. A sudden increase of 5'- and 5-monodeiodinase activity in liver and kidney 3 days before birth was observed, falling to a nadir at day 3 after birth. Foetal and neonatal serum T4, T3 and rT3 concentration were very low and rose progressively with age, reaching adult values at about day 21. In the foetal brown adipose tissue high 5'-monodeiodinase and low 5-monodeiodinase activity was found. The 5'-monodeiodinase decreased during the first days of life whereas the 5-monodeiodinase activity remained at a low stable level until day 3 when the activities of both enzymes increased. The increase of conversion rate of T4 to T3 and rT3 in liver and kidney well correlate with the triiodothyronines concentration in serum from day 3 after birth.     

(2'-5')An-dependent endoribonuclease: enzyme levels are regulated by IFN beta, IFN gamma, and cell culture conditions

The levels of a (2'-5')An-dependent endonuclease (RNase L) were determined in extracts prepared from murine L cells and Ehrlich ascites tumor (EAT) cells by measuring specific binding of protein to a labeled derivative of (2'-5')An, (2'-5')A3[32P]pCp. RNase L levels were found to depend both on interferon (IFN) treatment and on cell growth conditions. Treatment of murine L cells and EAT cells with 100-2,000 IRU IFN beta or IFN gamma resulted in a similar 2-4-fold increase in the levels of RNase L when cells were present at low density. The levels of RNase L were also shown to increase 2-3-fold as cells approached saturation density. Serum-starved cells also displayed relatively high levels of RNase L. RNase L levels in cells maintained at high cell density did not change appreciably following treatment with IFN beta or IFN gamma. Regulation of RNase L levels by cell growth conditions as well as by IFN beta or IFN gamma treatment suggests that RNase L may play an important role in regulating the levels of cellular mRNAs as well as acting to degrade viral RNAs.     

Genomic sequence and expression profile of murine Bat1a and Nfkbil1.

In humans, susceptibility to several immunopathologic diseases maps to a conserved block encompassing the polymorphic BAT1, NFKBIL1 (IKBL) and TNF genes in the central MHC. As a pre-requisite for studies of these genes in animal models, we characterized Bat1a and Nfkbil1 in inbred mice differing in their H2 haplotype. We identified two indels and nine single nucleotide polymorphisms (SNP) upstream of Nfkbil1, one indel, nine SNP upstream of Bat1a and a synonymous SNP in exon 2 of Bat1a. H2(g7) and H2(b) mice yielded identical Bat1a and Nfkbil1 sequences. Real time PCR (RT-PCR) showed Bat1a was expressed in adult brain, heart, kidney, liver, lung, pancreas and spleen. Expression of Bat1a was higher in brain and liver of 15-day embryos compared to 1-day old mice and increased moderately in liver and lung of adult mice 2-4 h after LPS challenge. Nfkbil1 expression was low or undetetectable in all tissues and cell lines.     

Modulation of the chemokines KC and MCP-1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice

Activation of the aryl hydrocarbon receptor (AhR) by TCDD may lead to the induction of proinflammatory cytokines in various cell types and organs such as liver leading to active chronic inflammation. Here we studied the expression of the chemokines keratinocyte chemoattractant (KC) and monocyte chemoattractant protein 1 (MCP-1) in different organs of mice after exposure to TCDD. TCDD exposure led to an early and clear induction of KC in liver and spleen on day 1 which was sustained over a period of 10 days. The level of MCP-1 mRNA was induced by TCDD on day 1 in spleen, lung, kidney, and liver, which was further increased at day 7. Increase of KC and MCP-1 at day 7 in liver, thymus, kidney, adipose, and heart was associated with elevated levels of the macrophage marker F4/80, indicating the infiltration of macrophages in these organs. Induction of KC requires a functional AhR since mice with a mutation in the AhR nuclear localization domain (AhR(nls)) were found to be resistant to TCDD-induced expression of KC. These results are the first showing the induction of the chemokines KC and MCP-1 in multiple organs of mice associated with an increase of the macrophage marker F4/80 indicating the involvement in TCDD's inflammatory response like infiltration of macrophages.     

禽流感H5N1病毒感染BALB/c小鼠后多器官病变初探

为了研究禽流感H5N1病毒在各个器官的增殖和病理变化,在生物安全实验室,我们将禽流感H5N1病毒通过尾静脉接种BALB/C小鼠。结果小鼠在不经过适应的情况下,直接感染发病,甚至死亡。在观察的7天内,感染小鼠临床症状主要表现呼吸急促,体温、体重下降。尸检表现肺出血,心外膜坏死以及肝脏的坏死。组织病理检查表现心、肝、肺等多器官的病变。肺的病变伴有纤维化的弥漫性肺泡损伤;心肌外膜大量淋巴细胞浸润、坏死;肝细胞大量坏死,淋巴细胞浸润。心、肝的坏死病变在H5N1禽流感病毒相关的研究中未见报道。经过对各个组织器官的病毒载量的检测,未发现病毒在各个病变组织中的复制。免疫组化的检测,各个组织中也未检出阳性的细胞反应。因此,我们认为H5N1禽流感病毒感染小鼠引起多个器官组织的损伤,甚至死亡,不是病毒在器官的复制,而可能是病毒感染小鼠,产生炎症细胞因子的高度表达,损伤多个器官组织所致。     

Tissue distribution of avidin and streptavidin injected to mice. Effect of avidin carbohydrate, streptavidin truncation and exogenous biotin

Radioionated avidin and streptavidin were characterized for their biodistribution and tissue association in Balb/c mice, in comparison to their interaction with cells in vitro. Binding of avidin to spleen and bone-marrow cells in vitro was up to 20-fold higher than that of streptavidin, but when tested in vivo avidin clearance from blood and tissues was considerably faster than that of streptavidin. Levels of avidin at 24 h after an intravenous injection were below 1% (of the injected dose/mass tissue) in most organs. Non-glycosylated avidin was similar in its biodistribution to native avidin. Native streptavidin exhibited higher and prolonged tissue association with 5-10% levels in lung, liver, spleen, kidney and blood, whereas its truncated form showed low tissue levels (1-3%) but a remarkably high affinity to the kidney (80%). Exogenous biotin did not affect streptavidin distribution in vivo but caused a 2-7-fold increase in the retention of avidin (but not non-glycodylated avidin) in some of the organs.     

Acute sodium arsenite treatment induces Cyp2a5 but not Cyp1a1 in the C57Bl/6 mouse in a tissue (kidney) selective manner

Modulation of hepatic and extrahepatic detoxication enzymes Cyp1a1, Cyp2a5, glutathione S-transferse Ya (GSTYa) and NAD(P)H:quinone oxidoreductase (QOR) dependent catalytic activity and mRNA levels were investigated at 1, 2, or 4 days in liver, lung, or kidney of male, adult CD57 Bl/6 mice treated sc with a single dose (85 micromol/kg) of sodium arsenite (As3+). Maximum decreases of total hepatic cytochrome P450 (CYP) monooxygenase content and catalytic activities, occurring at 24 h, corresponded with maximum increases of heme oxygenase (HO-1) in all tissues, as well as maximum plasma total bilirubin. Extrahepatic increases in CYP were observed only in non-AHR dependent isozymes in the kidney, where both Cyp2a5 mRNA and catalytic activity increased maximally 24 h after treatment. In contrast, no significant changes in Cyp2b1/2-dependent PROD or mRNA activity and decreases in Cyp1a1-dependent-EROD activity were noted 1, 2, or 4 days after treatment. Increases in QOR catalytic activities were observed in all tissues examined with increased mRNA in kidney. On the other hand, GSTYa catalytic activity and mRNA increases were only detected in kidney. This study demonstrates the differential modulation of CYP, QOR, and GST-Ya, important drug metabolizing enzymes after acute As3+ administration. The induction of Cyp2a5, QOR, and GSTYa catalytic activity and gene expression occurred primarily in kidney during or shortly after conditions of oxidant stress.