Expression changes of microtubule associated protein 1B in the brain of Fmr1 knockout mice

Objective To explore the regulatory effect of fragile X mental retardation protein （FMRP） on the translation of microtubule associated protein 1B （MAP1B）. Methods The expressions of MAP1B protein and MAP1B mRNA in the brains of 1-week and 6-week old fragile X mental retardation-1 （FmrI） knockout （KO） mice were investigated by immunohistochemistry, Western blot, and in situ hybridization, with the age-matched wild type mice （WT） as controls. Results The mean optical density （MOD） of MAP1B was significantly decreased in each brain region in KO6W compared with WT6W, whereas in KO1W, this decrease was only found in the hippocampus and cerebellum. MAP1B in 6-week mice was much less than that in 1-week mice of the same genotype. The results of Western blot and in situ hybridization showed that MAP1B protein and MAP1B mRNA were significantly decreased in the hippocampus of both KO1W and KO6W. Conclusion The decreased MAP1B protein and MAP1B mRNA in the Fmrl knockout mice indicate that FMRP may positively regulate the expression of MAP1B.

Determination of trans-resveratrol in mouse liver by high performance liquid chromatography

To develop a sensitive high performance liquid chromatography （HPLC） assay for the determination of trans-resveratrol in mouse liver. The whole liver of a mouse was removed from the body, homogenated, and extracted by ethyl acetate. The organic layer was isolated and evaporated to dryness, the residue was reconstituted in 0.2 mL mobile phase for centrifugation, and 50 uL of the supernatant was injected into the/-IPLC instrument. The sample was separated on a Shimadzu ODS column （150 mm × 4.6 mm, 5 um） at 35 ℃ and detected by ultraviolet （UV） detector at the wavelength of 305 nm. The mobile phase consisted of methanol and 0.1 mol/L acetic acid （4：6, v/v） with the flow-rote at 1 mL/min. The limit of detection was 3.0 ng/g in liver homogenate with a signal/noise ratio of 3：1. The linear range of the calibration curve was 5.0-120.0 ng/g. The mean recoveries at the concentrations of 6, 10 and 80 ng/g were 102%, 96.0% and 91.5%, respectively. The RSDs for inter- and intra-day assays were less than 5%. Compared with other reported methods, this method was faster and more sensitive. It was also proved to be of good linearity, selectivity, accuracy and precision, and can be efficiently applied to the pharmacoldnetic study of trans-resveratrol in mouse liver.

Effect of Leukemia Vaccine on the Macrophage of Mice

Objective: To evaluate the e?ect of the prepared leukemia vaccine on the cytotoxicity of macrophage of C57BL/6 mice. Methods: The model of mice bearing leukemia was established and three types of leukemia vaccine were prepared before they were administered on the mice respectively. The cy- totxicity of M? derived from the mice was measured after the active immunotherapy or prevention for 2–4 weeks later by using MTT colorimetric method and compared with the control group. Results: (1) With the growth of leukemia cells in the mice, the cytotoxicity of M? was seriously depressed; (2) The administration of leukemia vaccine prepared from inactivated leukemic cells, incomplete Freund’s adjuvant (IFA) and cytokines, such as rGM-CSF, rIL-2 and rIL-6, promoted the cellular immunity of mice bearing leukemia, more e?cient than leukemia vaccine from either inactivated leukemic cells and IFA or inactive leukemic cells per se. Conclusion: The leukemia vaccine prepared with inactive leukemic cells, IFA and cytokines could activate the non-speci?c cellular immunity such as M?, as well as, antigen present, immune surveillance and so on, and this activation constructed a base for further activate T lymphocyte. Naturally, this will certainly have a promising future in the therapy against hematopietic tumor.

Study on the Gene Transcription Level of Hippocampus NGF in C_(57) Mouse Development

β-Nerve growth factor (β NGF) mRNA levels in hippocampa from C57BL/6J mice of different ages were compared by semi-quantitative RT-PCR method, taking β-actin gene as an internal control. The levels of 6-and 12-month-old C57 mice were much lower compared with that of the l-month-old mouse. However, there was no significant difference between these two groups. This result suggests that gene transcription level of hippocampus β NGF in a C57, mouse descends evidently as it grows up. Base T at Site 294 of β NGF cDNA was substituted by C in the C57, mouse, as revealed by DNA sequencing for the first time. Nevertheless, this polymorphism of nucleotide did not make any difference in amino acid composition.

Radioprotective Effect of Lyophyllum Decastes and the Effect on Immunological Functions in Irradiated Mice

In this study, to explore the radiation protection effects of Lyophyllum Decastes Sing （LDS）, a hot distilled-water extract of LDS was orally administered at a dosage of 250mg/kg every other day for a period of 2 weeks in irradiated mice. An automatic blood cell counter was used to measure white blood cells （lymphocytes, monocyte, and granulocytes） one day before X-ray irradiation, and 3 hours, 12 hours, 24 hours, 3 days, 7 days, 15 days and 30 days after irradiation. The Dunnett test was used to examine statistical significance of differences. The peripheral blood cell counts in the Lyophyllum-administered non-irradiation group revealed an increase in the numbers of ieukocytes, lymphocytes and monocytes. For 2 Gy whole body radiation, a significant statistical difference was found between the X-ray group and the Lyophyllum plus X-ray group in the numbers of leukocytes, lymphocytes and monocytes. The results suggest that Lyophyllum restrains blood cell-count falling after irradiation, which is probably mediated at least in part by hemopoietic function, and NK and LAK activities seems to play a role in preventing secondary irffections associated with irradiation.

TypeⅠinositol 1, 4, 5-triphosphate receptors increase in kidney of mice with fulminant hepatic failure

AIM： To delineate the mechanisms of renal vasoconstriction in hepatorenal syndrome （HRS）, we investigated the expression of type I inositol 1, 4, 5-triphosphate receptors （IP3R I） of kidney in mice with fulminant hepatic failure （FHF）. METHODS： FHF was induced by lipopolysaccharide （LPS） in D-galactosamine （GAIN） sensitized BALB/c mice. There were 20 mice in normal saline （NS）-treated group, 20 mice in LPS-treated group, 20 mice in GaIN- treated group, and 60 mice in GalN/LPS-treated group （FHF group）. Liver and kidney tissues were obtained at 2, 6, and 9 h after administration. The liver and kidney specimens were stained with hematoxylin-eosin for studying morphological changes under light microscope. The expression of IP3R I in kidney tissue was tested by immunohistochemistry, Western blot and reverse transcription （RT）-PCR. RESULTS： Kidney tissues were morphologically normal at all time points in all groups. IP3R I proteins were found localized in the plasma region of glomerular mesangial cells （GMC） and vascular smooth muscle cells （VSMC） in kidney by immunohistochemical staining. In kidney of mice with FHF at 6 h and 9 h IP3R I staining was upregulated. Results from Western blot demonstrated consistent and significant increment of IP3R I expression in mice with FHF at 6 h and 9 h （t = 3.16, P 〈 0.05; t = 5.43, P 〈 0.01）. Furthermore, we evaluated IP3R I mRNA expression by RT-PCR and observed marked upregulation of IP3R I mRNA in FHF samples at 2 h, 6 h and 9 h compared to controls （t = 2.97, P 〈 0.05; t = 4.42, P 〈 0.01; t = 3.81, P 〈 0.01）. CONCLUSION： The expression of IP3R I protein increased in GMC and renal VSMC of mice with FHF, possibly caused by up-regulation of IP3R I mRNA.

Embryonic stem cells develop into hepatocytes after intrasplenic transplantation in CCl_4-treated mice

AIM： To transplant undifferentiated embryonic stem （ES） cells into the spleens of carbon tetrachloride （CCl4）-treated mice to determine their ability to differentiate into hepatocytes in the liver. METHODS： CCh, 0.5 mL/kg body weight, was injected into the peritoneum of C57BL/6 mice twice a week for 5 wk. In group 1 （n = 12）, 1 × 10^5 undifferentiated ES cells （0.1 mL of 1 × 10^6/mL solution）, genetically labeled with GFP, were transplanted into the spleens 1 d after the second injection. Group 2 mice （n = 12） were injected with 0.2 mL of saline twice a week, instead of CCh, and the same amount of ES cells was transplanted into the spleens. Group 3 mice （n = 6） were treated with CCh and injected with 0.1 mL of saline into the spleen, instead of ES cells. Histochemical analyses of the livers were performed on post-transplantation d （PD） 10, 20, and 30. RESULTS： Considerable numbers of GFP-immunopositive cells were found in the periportal regions in group 1 mice （CCh-treated） on PD 10, however, not in those untreated with CCh （group 2）. The GFP-positive cells were also immunopositive for albumin （ALB）, alpha-1 antitrypsin, cytokeratin 18, and hepatocyte nuclear factor 4 alpha on PD 20. Interestingly, most of the GFP-positive cells were immunopositive for DLK, a hepatoblast marker, on PD 10. Although very few ES-derived cells were demonstrated immunohistologically in the livers of group 1 mice on PD 30, improvements in liver fibrosis were observed. Unexpectedly, liver tumor formation was not observed in any of the mice that received ES cell transplantation during the experimental period CONCLUSION： Undifferentiated ES cells developed into hepatocyte-like cells with appropriate integration into tissue, without uncontrolled cell growth.

Involvement of aquaporins in a mouse model of rotavirus diarrhea

Rotavirus diarrhea is a major worldwide cause of infantile gastroenteritis; however, the mechanism responsible for intestinal fluid loss remains unclear. Water transfer across the intestinal epithelial membrane seems to occur because of aquaporins(AQPs). Accumulating evidence indicates that alterations in AQPs may play an important role in pathogenesis. Here, we focus on changes in AQPs in a mouse model of rotavirus diarrhea. In the present study, 32 of 35 mice developed diarrhea and mild dehydration within 24 hours after infection with rotavirus strain SA11. Intestinal epithelial cells demonstrated cytoplasmic vacuolation, malaligned villi, and atrophy. AQP1 expression was significantly attenuated in the ileum and colon in comparison with controls; likewise, AQP4 and-8 protein expression were significantly decreased in the colon of rotavirus diarrhea-infected mice. In contrast, AQP3 protein expression was significantly increased in the colon of rotavirus-infected mice in comparison with controls. These results indicate that rotavirus diarrhea is associated with the downregulation of AQP1,-4, and-8 expression. Therefore, AQPs play an important role in rotavirus diarrhea.

Chromatin-binding in vivo of the erythroid kruppel-like factor,EKLF,in the murine globin loci

EKLF is an erythroid-specific, zinc finger-containing transcription factor essential for the activation of the mammalian beta globin gene in erythroid cells of definitive lineage. We have prepared a polyclonal anti-mouse EKLF antibody suitable for Western blotting and immunoprecipitation （IP） qualities, and used it to define the expression patterns of the EKLF protein during mouse erythroid development. We have also used this antibody for the chromatin-immunoprecipitation （CHIP） assay. EKLF was found to bind in vivo at both the mouse beta-major-globin promoter and the HS2 site of beta-LCR in the mouse erythroleukemia cells （MEL） in a DMSO-inducible manner. The DMSO-induced bindings of EKLF as well as three other proteins, namely, RNA polymerase Ⅱ, acetylated histone H3, and methylated histone H3, were not abolished but significantly lowered in CB3, a MEL-derived cell line with null-expression of p45/NF-E2, an erythroid-enriched factor needed for activation of the mammalian globin loci. Interestingly, binding of EKLF in vivo was also detected in the mouse alpha-like globin locus, at the adult alpha globin promoter and its far upstream regulatory element alpha-MRE （HS26）. This study provides direct evidence for EKLF-binding in vivo at the major regulatory elements of the mouse beta-like globin gene clusters the data also have interesting implications with respect to the role of EKLF-chromatin interaction in mammalian globin gene regulation.

Gene therapy for allergic rhinitis with recombinant adenovirus vector containing CTLA4Ig in mice

Objective: To examine the role of recombinant adenovirus vector containing CTLA4Ig gene(Ad-CTLA4Ig) in the treatment of induced allergic rhinitis in mice.Methods: Allergic rhinitis was induced by sensitizing and challenging with ovalbumin(OVA).Ad-CTLA4Ig was intraperitoneally injected 30 min before OVA challenge.Adenovirus vector without inserted CTLA4Ig cDNA served as the control.The symptoms and morphological changes of nasal mucosa of each group were observed, and the serum levels of IgE against OVA were detected with ELISA.Results: There were no obvious symptoms and pathological changes in Ad-CTLA4Ig treated group, in which the serum OVA-specific IgE levels were significantly lower than that in control groups(P< 0.05).Conclusion: Ad-CTLA4Ig prevents and treats allergic rhinitis of mice,implying the possibility of the usage of Ad-CTLA4Ig against allergic rhinitis in clinic in future.

Generation of the regulatory protein rtTA transgenic mice

AIM: To translate Tet-on system into a conditional mouse model, in which hepatitis B or C virus (HBV or HCV) gene could be spatiotemporally expressed to overcome 'immune tolerance' formed during the embryonic development and 'immune escape' against hepatitis virus antigen(s), an effector mouse, carrying the reverse tetracycline-responsive transcriptional activator (rtTA) gene under the tight control of liver-specific human apoE promoter, is required to be generated. METHODS: To address this end, rtTA fragment amplified by PCR was effectively inserted into the vector of pLiv.7 containing apoE promoter to create the rtTA expressing vector, I.e., pApoE-rtTA. ApoE-rtTA transgenic fragment (-6.9 kb) released from pApoE-rtTA was transferred into mice by pronucleus injection, followed by obtaining one transgene (+) founder animal from microinjection through PCR and Southern blot analysis.RESULTS: rtTA transgene which could be transmitted to subsequent generation (F1) derived from founder was expressed in a liver-specific fashion. CONCLUSION: Taken together, these findings demonstrate that rtTA transgenic mice, in which rtTA expression is appropriately targeted to the murine liver, are successfully produced, which lays a solid foundation to 'off-on-off' regulate expression of target gene (s) (e.g., HBV and/or HCV) in transgenic mice mediated by Tet-on system.

Characterization of Natural Killer Cells in the Liver of Young Mice

OBJECTIVE To determine the quantity and quality of liver NK cells from young and adult mice and compare their characteristics. METHODS C57BL/6 mice were used at 2 weeks （young） and 8 weeks （adult） of age. The percentage and absolute number of NK cells in the liver and spleen were analyzed. The cytotoxicity of NK cells in the liver and spleen against various targets were detected by a 4 h ^51Cr-release method. FACScan was used to analyze the expression of CD69, Mac-l1 Ly49C/I and CD94 on the NK cells. Perforin mRNA levels were analyzed by the reverse transcription polymerase chain reaction （RT-PCR）. RESUTLS The percentages of NK cells in the liver of young and adult mice were similar （11.9%＋1.7% vs. 9.9%＋1.6%, P〉0.05）, but the absolute number per liver weight was higher in the young animals （11.6＋2.5×10^5/g vs. 3.4＋0.8×10^5/g, P〈0.05）. The level of NK cytotoxicity was extremely high in the liver of young compared to adult mice （71.0%＋5.5% vs. 23.8%＋4.4%, P〈0.05）, but this difference was not observed in the spleen. Phenotypes of the liver NK cells from young and adult mice were completely different from each other. The liver NK cells from young mice were CD69^high Mac-1^low Ly49C/ I^low, whereas NK cells from older mice displayed inverse antigen levels （CD- 69^low Mac-1^high Ly49C/I^high）. The expression levels of other NK cell-related markers were similar in both groups.The perforin mRNA level in the liver lymphocytes from young mice was consistently greater compared to adult mice. CONCLUSION From 2 to 8 weeks C57BU6 mice liver NK cells undergo age-associated changes. At 2 weeks of age the liver NK cells showed a high level of NK cytotoxicity and a unique phenotype which was not apparent at 8 weeks of age.

Uptake of albumin nanoparticle surface modified with glycyrrhizin by primary cultured rat hepatocytes

AIM: To investigate the uptake difference between bovine serum albumin nanoparticle (BSA-NP) and bovine serum albumin nanoparticles with their surface modified byglycyrrhizin (BSA-NP-GL) and to develop a novel hepatocyte targeting BSA-NP-GL based on active targeting technology mediated by specific binding site of GL on rat cellular membrane. METHODS: Calcein loaded bovine serum albumin nanoparticles (Cal-BSA-NP) were prepared by desolvation process. Glycyrrhizin was conjugated to the surface reactive amino groups (SRAG) of Cal-BSA-NP by sodium periodate oxidization, which resulted in calcein-loaded bovine serum albumin nanoparticles with their surface modified by glycyrrhizin (Cal-BSA-NP-GL). The morphology of the two types of prepared nanoparticles (NP) was observed by transmission electron microscopy. The diameter of NP was measured with a laser particle size analyzer. The interaction between Cal-BSA-NP-GL and primary cultured hepatocytes was studied through cellular uptake experiments. The uptake amount of Cal-BSA-NPGL and Cal-BSA-NP by rat hepatocytes was determinedby fluorospectrophotometry. Uptake characteristics were investigated through experiments of competitive inhibition of specific binding site of GL. RESULTS: Both Cal-BSA-NP-GL and Cal-BSA-NP had regular spherical surfaces. The average diameter of CalBSA-NP-GL and Cal-BSA-NP was 77 and 79 nm respectively. The uptake amount of the two NP by hepatocytes reached its maximum at 2 h after incubation. The uptake amount of Cal-BSA-NP-GL by rat hepatocytes was 4.43-fold higher than that of Cal-BSA-NP. There was a significant difference in the uptake of Cal-BSA-NP-GL and Cal-BSA-NP by hepatocytes (P＜0.01). The uptake of Cal-BSA-NP-GL was inhibited when GL was added previously to isolated rat hepatocytes, and the uptake of Cal-BSA-NP was not affected by GL.CONCLUSION: A binding site of GL is present on the surface of rat hepatocytes, BSA-NP-GL may be internalized via this site by hepatocytes and can be used as a drug carrier for active targeting of delivery drugs to hepatocytes.

Fatty Acid Synthase and Hormone-sensitive Lipase Expression in Liver Are Involved in Zinc-α2-glycoprotein-induced Body Fat Loss in Obese Mice

Objective To explore the effects of zinc-0t2-glycoprotein （ZAG） on body weight and body fat in high-fat-diet （HF1））-induced obesity in mice and the possible mechanism. Methods Thirty-six male mice were fed with standard food （SF） （n=9） and HFD （n=27）, respectively. Five weeks later, 9 mice fed with HFD were subjected to ZAG expression plasmid DNA transfection by liposome transfection method, and another 9 mice to negative control plasmid transfection. Two weeks later, serum ZAG level in the mice was assayed by Western blot, and the effects of ZAG over-expression on body weight, body fat, serum biochemical indexes, and adipose tissue of obese mice were evaluated. The mRNA expressions of fatty acid synthase （FAS） and hormone sensitive lipase （HSL） in liver tissue were deterlnined by reverse transcription-polymerase chain reaction. Results Serum ZAG level significantly lowered in simple HFD-fed mice in comparison to SF-fed mice （0.51±0.10 AU vs. 0.75±0.07 AU, P〈0.01）. Further statistical analysis demonstrated that ZAG level was negatively correlated with body weight （r =-0.56, P〈0.001）, epididymal fat mass （r=-0.67, P〈O. 001）, percentage of epididymal fat （r= 0.65, P〈0.001）, and increased weight （r= 0.57, P〈0.001） in simple SF- and HFD fed mice. ZAG over-expression in obese mice reduced body weight and the percentage of epididyreal fat. Furthermore, FAS mRNA expression decreased （P〈0.01） and HSL mRNA expression increased （P〈0.001） in the liver in ZAG over-expressing mice. Conclusions ZAG is closely related to obesity. Serum ZAG level is inversely correlated with body weight and percentage of body fat. The action of ZAG is associated with reduced FAS expression and increased HSL expression in the liver of obese mice.

Astragalus polysaccharides can regulate cytokine and P-glycoprotein expression in H22 tumor-bearing mice

AIM:To investigate the adjunct anticancer effect of Astragalus polysaccharides in H22 tumor-bearing mice.METHODS:To establish a solid tumor model,5.0 × 10 6 /mL H22 hepatoma cells were inoculated subcutaneously into the right armpit region of Kunming mice(6-12 wk old,18-22 g).When the tumors reached a size of 100 mm 3,the animals were treated as indicated,and the mice were randomly assigned to seven groups(n = 10 each).After ten days of treatment,blood samples were collected from mouse eyes,and serum was harvested by centrifugation.Mice were sacrificed,and the whole body,tumor,spleen and thymus were weighed immediately.The rate of tumor inhibition and organ indexes were calculated.The expression levels of serum cytokines,P-glycoprotein(P-GP) and multidrug resistance(MDR) 1 mRNA in tumor tissues were detected using enzyme-linked immunosorbent assay,Western blotting,and quantitative myeloid-derived suppressor cells reverse transcription-polymerase chain reaction,respectively.RESULTS:The tumor inhibition rates in the treatment groups of Adriamycin(ADM) + Astragalus polysaccharides(APS)(50 mg/kg),ADM + APS(100 mg/kg),and ADM + APS(200 mg/kg) were significantly higher than in the ADM group(72.88% vs 60.36%,P = 0.013;73.40% vs 60.36%,P = 0.010;77.57% vs 60.36%,P = 0.001).The spleen indexes of the above groups were also significantly higher than in the ADM group(0.65 ± 0.22 vs 0.39 ± 0.17,P = 0.023;0.62 ± 0.34 vs 0.39 ± 0.17,P = 0.022;0.67 ± 0.20 vs 0.39 ± 0.17,P = 0.012),and the thymus indexes of the ADM + APS(100 mg/kg) and ADM + APS(200 mg/kg) groups were significantly higher than in the ADM group(0.20 ± 0.06 vs 0.13 ± 0.04,P = 0.029;0.47 ± 0.12 vs 0.13 ± 0.04,P = 0.000).APS was found to exert a synergistic antitumor effect with ADM and to alleviate the decrease in the sizes of the spleen and thymus induced by AMD.The expression of interleukin-1α(IL-1α),IL-2,IL-6,and tumor necrosis factor-α(TNF-α) was significantly higher in the ADM + APS(50 mg/kg),ADM + APS(100 mg/kg) and ADM + APS(200 mg/kg) groups than in the ADM group;and IL-10 was significantly lower in the above groups than in the ADM group.APS could increase IL-1α,IL-2,IL-6,and TNF-α expression and decrease IL-10 levels.Compared with the ADM group,APS treatment at a dose of 50-200 mg/kg could downregulate MDR1 mRNA expression in a dose-dependent manner(0.48 ± 0.13 vs 4.26 ± 1.51,P = 0.000;0.36 ± 0.03 vs 4.26 ± 1.51,P = 0.000;0.21 ± 0.04 vs 4.26 ± 1.51,P = 0.000).The expression level of P-GP was significantly lower in the ADM + APS(200 mg/kg) group than in the ADM group(137.35 ± 9.20 mg/kg vs 282.19 ± 20.54 mg/kg,P = 0.023).CONCLUSION:APS exerts a synergistic anti-tumor effect with ADM in H22 tumor-bearing mice.This may be related to its ability to enhance the expression of IL1α,IL-2,IL-6,and TNF-α,decrease IL-10,and downregulate MDR1 mRNA and P-GP expression levels.

Defective maintenance of intracellular Ca^(2+) homeostasis is linked to increased muscle fatigability in the MG29 null mice

Mitsugumin 29 (MG29) is a transmembrane protein that is normally found in the triad junction of skeletal muscle. Our previous studies have shown that targeted deletion of mg29 from the skeletal muscle resulted in abnormality of the triad junction structure, and also increased susceptibility to muscle fatigue. To elucidate the basis of these effects, we investigated the properties of Ca2+-uptake and -release in toxin-skinned Extensor Digitorium Longus (EDL) muscle fibers from control and mg29 knockout mice. Compared with the control muscle, submaximal Ca2+-uptake into the sarcoplasmic reticulum (SR) was slower and the storage of Ca2+ inside the SR was less in the mutant muscle, due to increased leakage process of Ca2+ movement across the SR. The leakage pathway is associated with the increased sensitivity of Ca2+/caffeine -induced Ca2+ release to myoplasmic Ca2+. Therefore, the increased fatigability of mutant EDL muscles can result from a combination of a slowing of Ca2+ uptake, modification of Ca2+-induced Ca2+ release (CICR), and a reduction in total SR Ca2+ content.

Hyperhomocysteinemia independently causes and promotes atherosclerosis in LDL receptor-deficient mice

Background Hyperhomocysteine is an independent risk factor of coronary heart disease （CHD）. However, whether hyperhomocys teine affects the progression of atherosclerosis is unclear. In the present study, we examined the effect of hyperhomocysteine on the forma tion of atherosclerosis in low-density lipoprotein receptor-deficient （LDLr ） mice. Methods Forty-eight 7-week-old LDLr/ mice were assigned to the following groups： mice fed a standard rodent diet （control group）, mice fed a high-methionine diet （high-methionine group）, mice fed a high-fat diet （high-fat group）, and mice fed a diet high in both methionine and fat （high-methionine and high-fat group）. At the age of 19, 23, and 27 weeks, four mice at each interval in every group were sacrificed. Results At the end of the study, mice did not show atherosclerotic lesions in the aortic sinus and aortic surface until 27 weeks old in the control group. However, atherosclerotic lesions developed in the other three groups at 19 weeks. The amount of atherosclerotic lesions on the aortic surface was lower in the high-methionine group than in the high-fat group （P 〈 0.001）. Atherosclerotic lesions on the aortic surface in the high-methionine and high-fat group were the most severe. The mean area of atherosclerotic lesions in the aortic sinus compared with atherosclerotic lesions on the aortic surface was lower in the high-methionine group than in the high-fat group （P 〈 0.001）. Atherosclerotic lesions in the aortic sinus in the high-methionine and high-fat group were the most severe. Conclusions Homocysteinemia accelerates atherosclerotic lesions and induces early atherosclerosis independently in LDLrmice. Reducing the level of homocysteinemia may be beneficial for prevention and treatment of CHD.

Expression of Mouse MT-1 as a Fusion Protein in Anabaena sp. PCC 7120

To produce mouse metallothionein-1 (mMT-I ) in cyanobacterium Anabaena sp. PCC 7120, a novel Escherichia co/i-cyanobacterium shuttle fusion expression vector, pKG-MT, was constructed. Via this vector, mMT-I cDNA which was fused with a carboxyl terminal extension of the 26 kD glutathione-S-trans-ferase (GST) containing a thrombin specific site was expressed in Anabaena under the control of tac promoter. SDS-polyacrylamid gel electrophoresis (SDS-PAGE) showed that the fusion protein GST-MT was expressed in the transgenic Anabaena sp. PCC 7120 after induction with isopropylthio-β-D-galactoside (IPTG). Glutatione-S-transferase metallothionein (GST-MT) was purified from the crude extracts by affinity chromatography on immobilized glutathione and mMT- I was obtained by digesting the fusion protein with thrombin on column and gel filtration on Sephadex G-50. SDS-PAGE demonstrated that the purified mMT- I was the desired protein. The result of ELISA for the purified mMT-I showed that the recovery of mMT- I from the transgenic cyanobacterium was about 0.6 mg/g fresh weight. According to the data of atomic absorption assay, metal-binding activity of the purified mMT-I was almost the same as that of wild type MT.

Lipocalin-2 Test in Distinguishing Acute Lung Injury Cases from Septic Mice Without Acute Lung Injury

Objective To explore whether the amount of lipocalin-2 in the biofluid could reflect the onset of sepsis-induced acute lung injury(ALI) in mice. Methods Lipopolysaccharide(LPS, 10 mg/kg) injection or cecal ligation and puncture(CLP) was performed to induce severe sepsis and ALI in C57 BL/6 male mice randomly divided into 5 groups(n=10 in each group): group A(intraperitoneal LPS injection), group B(intravenous LPS injection via tail vein), group C(CLP with 25% of the cecum ligated), group D(CLP with 75% of the cecum ligated), and the control group(6 sham-operation controls plus 4 saline controls). All the mice received volume resuscitation. Measurements of pulmonary morphological and functional alterations were used to identify the presence of experimental ALI. The expressions of lipocalin-2 and interleukin(IL)-6 in serum, bronchoalveolar lavage fluid(BALF), and lung tissue were quantified at both protein and mRNA levels. The overall abilities of lipocalin-2 and IL-6 tests to diagnose sepsis-induced ALI were evaluated by generating receiver operator characteristic curves(ROC) and computing area under curve(AUC). Results In both group B and group D, most of the "main features" of experimental ALI were reproduced in mice, while group A and group C showed septic syndrome without definite evidence for the presence of ALI. Compared with septic mice without ALI(group A+group C), lipocalin-2 protein expression in septic mice with ALI(group B+group D) was significantly up-regulated in BALF(P<0.01) and in serum(P<0.01), and mRNA expression boosted in lung tissues(all P<0.05). Lipocalin-2 tests performed better than IL-6 tests in recognizing sepsis-induced ALI cases, evidenced by the larger AUC of the former(BALF tests, 0.8800 versus 0.6625; serum tests, 0.8500 versus 0.7000). Using a dual cutoff system to diagnose sepsis-induced ALI, BALF lipocalin-2 test exhibited the highest positive likelihood ratio(13.000) and the lowest negative likelihood ratio(0.077) among the tests of lipocalin-2 and IL-6 in blood and BALF. A statistically significant correlation was found between lipocalin-2 concentration in BALF and that in serum(Spearman r=0.8803,P<0.0001). Conclusions Lipocalin-2 expression is significantly up-regulated in septic ALI mice compared with those without ALI. Lipocalin-2 tests with a dual cutoff system could be an effective tool in distinguishing experimental ALI cases.

Osteopontin expression is associated with hepatopathologic changes in Schistosoma japonicum infected mice

AIM:To investigate osteopontin expression and its association with hepatopathologic changes in BALB/C mice infected with Schistosoma japonicum.METHODS:The schistosomal hepatopathologic mouse model was established by abdominal infection with schistosomal cercaria.Liver samples were obtained from mice sacrif iced at 6,8,10,14,and 18 wk after in-fection.Liver histopathological changes were observed with hematoxylin-eosin and Masson trichrome staining.The expression of osteopontin was determined with im-munohistochemistry,reverse transcription-polymerase chain reaction,and Western blotting.The expressionof α-smooth muscle actin(α-SMA)and transforming growth factor-β1(TGF-β1)were determined by im-munohistochemistry.Correlations of osteopontin ex-pression with other variables(α-SMA,TGF-β1,hepato-pathologic features including granuloma formation and degree of liver f ibrosis)were analyzed.RESULTS:Typical schistosomal hepatopathologic changes were induced in the animals.Dynamic changes in the expression of osteopontin were observed at week 6.The expression increased,peaked at week 10(P<0.01),and then gradually decreased.Positive correla-tions between osteopontin expression and α-SMA(r=0.720,P<0.01),TGF-β1(r=0.905,P <0.01),granu-loma formation(r=0.875,P<0.01),and degree of liver f ibrosis(r=0.858,P<0.01)were also observed.CONCLUSION:Osteopontin may play an important role in schistosomal hepatopathology and may promote granuloma formation and liver fi brosis through an un-explored mechanism.