Arp3 is required during preimplantation development of the mouse embryo

The role of Arp3 in mouse development was investigated utilizing a gene trap mutation in the Arp3 gene. Heterozygous Arp3(WT/GT) mice are normal, however, homozygous Arp3(GT/GT) embryos die at blastocyst stage. Earlier embryonic stages appear unaffected by the mutation, probably due to maternal Arp3 protein. Mutant blastocysts isolated at E3.5 fail to continue development in vitro, lack outgrowth of trophoblast-like cells in culture and express reduced levels of the trophoblast marker Cdx2, while markers for inner cell mass continue to be present. The recessive embryonic lethal phenotype indicates that Arp3 plays a vital role for early mouse development, possibly when trophoblast cells become critical for implantation.     

广泛表达人载脂蛋白E3转基因小鼠的建立

目的建立人载脂蛋白E3（apolipoprotein E3,ApoE3）转基因小鼠,研究该基因在多种组织中表达水平的变化对动物的影响,探索该基因的功能。方法RT-PCR法克隆人ApoE3基因,把该基因插入CMV启动子下游,构建转基因表达载体,通过显微注射法建立转ApoE3基因C57BL／6J小鼠。并利用特异引物PCR法鉴定转基因小鼠的基因型,Western blot检测基因表达水平。通过生化指标分析初步鉴定ApoE3基因的功能。结果建立了2个系的高表达人ApoE3转基因小鼠。结论成功建立了CMV启动子启动的高表达人ApoE3基因转基因小鼠,为进一步探索该基因的功能奠定了基础。     

Dependence of interfacial properties of normal and transformed 3T3 cell membranes on treatment with factors modifying proliferation

Interfacial properties of the outer cell membrane of normal and transformed in vitro cultures of mouse 3T3 cells have been investigated. The contact angles of sessile drops on dried cell preparations were measured and the interfacial tensions derived using the thermodynamic approach introduced by Neumann. Interfacial tensions were found to be within an order of magnitude of those determined for other cell and model membranes. Treatment of cells with calf serum, a stimulant to proliferation, resulted in a decrease in the interfacial tension of normal and transformed cells, whereas use of concanavalin A and its succinylated derivative lead to an increase of interfacial tensions of both cell types. These and further results show a detailed correlation between the growth-regulating effects and the effects on interfacial properties of these proliferation-modifying factors. An inter-pretation of the results of serum depression of the interfacial tension in terms of a binding equilibrium dependent on the concentration of humoral growth factors in the medium is attempted.     

Stimulatory Effects of Exogenous Thyroid Hormone and Growth Hormone on sn-Glycerol-3-Phosphate Dehydrogenase Activity in the Genetic-Hypothyroid Mutant Mouse Cerebellum: Restricted to the Second 20 Days of Postnatal Life

We recently reported that by postnatal day 40 the activity of sn-glycerol-3-phosphate dehydrogenase (GPDH) was significantly depressed in the cerebellum of genetic-hypothyroid mutant mice. This mutant mouse-GPDH combination was used in the present study to define the critical time period during which thyroid hormone (T4) and growth hormone (GH) are essential for maturation of Bergmann glial cells. Our findings are that (a) induction of GPDH activity in the Bergmann glial cell is dependent on T4, (b) T4 is most effective when administered during the second 20 days of postnatal life, (c) the effect of GH on GPDH activity is complementary to or synergistic with that of T4, and (d) Bergmann glial cells and radial glial fibers of the mutant mice contain immunoreactive GPDH following various hormonal treatments. These results suggest that T4 is indispensable for the maturation of Bergmann glial cells.     

稳定表达小鼠CD40L的NIH3T3细胞系的建立及其在B细胞培养和激活中的应用

该研究构建小鼠CD40L真核表达重组质粒pcDNA3.1-mCD40L,通过电转法将重组质粒转至NIH3T3细胞中。利用G418对转染后细胞进行压力筛选,获得稳定转染细胞株。提取稳定转染细胞株RNA,通过RT-PCR法检测Neo基因的mRNA表达情况。分离稳定转染细胞上清,利用ELISA法检测小鼠CD40L蛋白水平的表达情况。RT-PCR结果显示,Neo基因能够在稳定转染细胞中表达,ELISA结果显示,获得的稳定转染细胞株NIH3T3-mCD40L细胞上清中CD40L的表达量高达1.286 ng/mL。进一步活性研究表明,该细胞系能够在体外与IL-2和IL-21共同作用培养B细胞至14天,并刺激B细胞产生特异性抗体。该细胞系的成功构建,为利用体外B细胞分离培养和活化法分离特异性单克隆抗体奠定了良好的基础。     

A knockout-first model of H3f3a gene targeting leads to developmental lethality

Histone variant H3.3 is encoded by two genes, H3f3a and H3f3b, which can be expressed differentially depending on tissue type. Previous work in our lab has shown that knockout of H3f3b causes some neonatal lethality and infertility in mice, and chromosomal defects in mouse embryonic fibroblasts (MEFs). Studies of H3f3a and H3f3b null mice by others have produced generally similar phenotypes to what we found in our H3f3b nulls, but the relative impacts of the loss of either H3f3a or H3f3b have varied depending on the approach and genetic background. Here we used a knockout-first approach to target the H3f3a gene for inactivation in C57BL6 mice. Homozygous H3f3a targeting produced a lethal phenotype at or before birth. E13.5 null embryos had some potential morphological differences from WT littermates including smaller size and reduced head size. An E18.5 null embryo was smaller than its control littermates with several potential defects including small head and brain size as well as small lungs, which would be consistent with a late gestation lethal phenotype. Despite a reduction in H3.3 and total H3 protein levels, the only histone H3 post-translational modification in the small panel assessed that was significantly altered was the unique H3.3 mark phospho-Serine31, which was consistently increased in null neurospheres. H3f3a null neurospheres also exhibited consistent gene expression changes including in protocadherins. Overall, our findings are consistent with the model that there are differential, cell-type-specific contributions of H3f3a and H3f3b to H3.3 functions in epigenetic and developmental processes.     

Coxsackievirus B3-Induced Chronic Myocarditis in Mouse: Use of Whole Blood Culture to Study the Activation of TNFα-Producing Cells

The pathogenesis of CVB3-induced chronic myocarditis remains unknown. Activated monocytes and macrophages may maintain ongoing inflammation during a persistent CVB3 infection and possibly represent the major mechanism leading to chronic myocarditis. We decided to study the activation status of cells by studying TNFα secretion in vitro using whole blood culture in CVB3-induced murine chronic myocarditis. Seven DBA/2 +/+ mice and 18 NMRI nu/nu mice were inoculated intraperitoneally with 5 × 10⁵ pfu of CVB3, and mice were mock-infected. Thirty-one days post-infection, all mice were sacrificed, blood samples were obtained from the heart, and the heart was removed. Enteroviral genomic detection by RT-PCR, virus isolation and histological analysis of heart samples were performed. Heparinized whole blood (25 μl) was cultured for 4 hr and 24 hr in sterile 96 well-plate containing 225 μl RPMI in the presence or the absence of activators (LPS + PHA). The TNFα levels in the whole blood from mock-infected DBA/2 (n = 4) and NMRI nu/nu mice (n = 5) were not different. A moderate increase of TNFα was observed in three out of five DBA/2 mice with negative CVB3 that had no histological abnormalities in myocardium. An increased level of TNFα was found in the sole DBA/2 mouse with positive CVB3 detection and chronic myocarditis. An increased level of TNFα was found in one out of nine NMRI nu/nu mice with positive CVB3 detection and chronic myocarditis and in one out of seven mice with positive CVB3 detection exempt of lesions in myocardium. In other infected mice, the level of TNFα was normal. Enteroviral genome was not detected in the blood from infected mice at 31 days post-infection. The increased TNFα level in some mice may be designed for a beneficial inflammatory and immune response, however, an exaggerated release may be associated with an adverse effect. The normal TNFα level in whole blood cultures from mice with chronic myocarditis does not exclude enhanced cytokine production at infected loci such as myocardial tissue. This is the first report to use whole blood cultures to study the production of cytokines in virus-induced disease in a small animal model.     

Immunohistochemical study of caspase-3-expressing cells within the pancreas of non-obese diabetic mice during cyclophosphamide-accelerated diabetes

During insulin-dependent diabetes mellitus, immune cells infiltrate pancreatic islets progressively and mediate beta cell destruction over a prolonged asymptomatic prediabetic period. Apoptosis may be a major mechanism of beta cell loss during the disease. This process involves a proteolytic cascade in which upstream procaspases are activated which themselves activate downstream caspases, including caspase-3, a key enzyme involved in the terminal apoptotic cascade. Here dual-label immunohistochemistry was employed to examine the intra-islet expression, distribution and cellular sources of active caspase-3 in the non-obese diabetic (NOD) mouse given cyclophosphamide to accelerate diabetes. NOD mice were treated at day 95 and caspase-3 expression was studied at days 0, 4, 7, 11 and 14. Its expression was also correlated with advancing disease and compared with age-matched NOD mice treated with diluent alone. At day 0 (=day 95), caspase-3 immunolabelling was observed in several peri-islet and intra-islet macrophages, but not in CD4 and CD8 cells and only extremely rarely in beta cells. At day 4, only a few beta cells weakly expressed the enzyme, in the absence of significant insulitis. At day 7, caspase-3 expression was observed in a small proportion of intra-islet macrophages. At day 11, there was a marked increase in the number of intra-islet macrophages positive for caspase-3 while only a few CD4 cells expressed the enzyme. At day 14, caspase-3 labelling became prominent in a significant proportion of macrophages. Only a few CD4 and CD8 cells expressed the enzyme. Capase-3 labelling was also present in a proportion of macrophages in perivascular and exocrine regions. Surprisingly, beta cell labelling of caspase-3 at days 11 and 14 was rare. At this stage of heightened beta cell loss, a proportion of intra-islet interleukin-1-positive cells coexpressed the enzyme. Caspase-3 was also observed in numerous Fas-positive cells in heavily infiltrated islets. During this late stage, only a proportion of caspase-3-positive cells contained apoptotic nuclei, as judged by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). We conclude that during cyclophosphamide-accelerated diabetes in the NOD mouse, the predominant immunolabelling of caspase-3 in intra-islet macrophages suggests that apoptosis of macrophages may be an important mechanism for its elimination. The virtual absence of caspase-3 immunolabelling in most beta cells even during heightened beta cell loss supports their rapid clearance following their death during insulin-dependent diabetes mellitus.     

Molecular cloning of a murine glycerol-3-phosphate acyltransferase-like protein 1 (xGPAT1)

A novel murine glycerol-3-phosphate acyltransferase-like protein 1 (named xGPAT1) has been cloned. The mouse xGPAT1 gene is located on mouse Chromosome 2, spans >19 kb, and consists of at least 23 exons. The protein is 32% identical and 72% similar to mouse mitochondrial GPAT (mtGPAT) on the amino acid level. Sequencing analysis confirmed that xGPAT1 has a 2403-bp open reading frame (ORF) that encodes an 801-amino acid protein with an estimated molecular mass of 89.1 kDa. A hydropathy plot of the deduced xGPAT1 protein showed a high degree of similarity with that of the mtGPAT protein. Using 5′-rapid amplification of cDNA ends, two alternate, untranslated exon 1 (1a and b) isoforms were obtained, generating variants xGPAT1-v1 and xGPAT1–v2. xGPAT1-v1 is expressed in mouse heart, liver, spleen, kidney and murine inner medullary collecting duct 3 (mIMCD3) cells, while xGPAT1-v2 is expressed in mouse liver, spleen, kidney, white and brown adipose tissues and 3T3-L1 pre- and post-adipocytes. xGPAT1 was distributed in the membrane fraction and showed GPAT activity when epitope-tagged xGPAT1 was expressed in Chinese hamster ovary (CHO)-K1 cells.     

小鼠Smad3基因的克隆及其在小鼠组织中的表达

采用PCR获得的Smad3cDNA片段作为探针筛选小鼠脑cDNA文库 .克隆了小鼠全长的Smad3基因 .对小鼠Smad3基因的全编码区进行了序列测定 .结果表明 ,小鼠SMAD3与人SMAD3氨基酸同源性高达 99% .与小鼠Smad2基因相比 ,碱基同源性高达 91 8% .Northern杂交显示 ,Smad3基因在小鼠胚胎发育和各成体器官中普遍表达 .原位杂交显示 ,Smad3基因表达在小鼠胚胎期E16 5d的软骨、骨髓和皮肤角质细胞中     

Phosphoprotein phosphate activity at the outer surface of intact normal and transformed 3T3 fibroblasts

Using ³²P-labeled phosphocasein or phosphohistones as exogenous substrates it was possible to detect a phosphoprotein phosphate activity on the outer surface of intact normal and transformed 3T3 fibroblasts. Incubation of monolayers of intact cells in buffered salt solution with the radioactively labeled substrate resulted in the release of alkali-labile ³²P counts into the surrounding medium. The reaction was: (a) linear with time (at least up to 20 min); (b) proportional to the cell density; (c) dependent on the temperature and pH of the incubation medium; (d) stimulated by K⁺; and (e) inhibited by sodium fluoride, inorganic pyrophosphate, zinc chloride and relatively impermeant sulfhydryl reagents. Less than 2% of the externally located phosphoprotein phosphatase activity was detectable in pooled cell-free washings of the intact cell monolayer. Phosphocasein did not cause any detectable leakage of intracellular lactate dehydrogenase or soluble phosphoprotein phosphatase activity into the external medium; incubation of the cells with phosphohistones, on the other hand, resulted in appreaciable leakage of both these cytoplasmic activities. Neoplastic transformation was associated with a nearly two-fold decrease in the activity of the surface phosphoprotein phosphatase. Addition of serum to either non-transformed 3T3 or spontaneously transformed 3T6 cells resulted in a rapid and remarkable drop in the cell surface dephosphorylating activity. Acrylamide gel electrophoresis of the dephosphorylated casein or histone substrate revealed no proteolytic degradation or change in electrophoretic mobility. The intact cells showed no damage upon microscopic examination as a result of exposure to phosphocasein or phosphohistones.     

Metallothionein-3 and neuronal nitric oxide synthase levels in brains from the Tg2576 mouse model of Alzheimer's disease

Using antiserum against the recombinant isoform 3 of mouse brain metallothionein (MT3), the amount of MT3 protein was determined in whole brain homogenates from the Tg2576 transgenic mouse model of Alzheimer's Disease. Twenty-two month old transgenic positive mice showed a 27% decrease of MT3 normalized to the total protein in the extracts compared to same age, control transgenic negative mice. Metallothioneins bind seven molar equivalents of divalent metal ions per mole of protein so metal levels also were measured in these whole brain extracts using inductively coupled plasma atomic absorption (ICP-AA) spectrometry. No significant difference was observed for any metal assayed. Because neuronal nitric oxide synthase (nNOS) is involved in neurodegenerative disease and nitric oxide specifically interacts with MT3, the concentration and total nNOS activity also were evaluated. The transgenic positive mice showed a decrease of 28% in nNOS protein compared to the same age transgenic negative mice. Normalized to the amount of nNOS protein, total NOS activity was higher in the transgenic positive mice. These data showed that protein levels of both MT3 and nNOS were reduced in transgenic positive mice that show many characteristics of Alzheimer's Disease. In vitro studies suggested that MT3 was not a likely candidate for directly affecting nNOS activity in the brain.     

DNA variants in the human RAB3A gene are not associated with autism

Mutation screening of the RAB3A gene in 47 individuals with autism provided no evidence that DNA variants in this gene are associated with autism. Since Rab3a constitutive knockout mice react to novel stimuli with hyperactivity, a further search for association of RAB3A DNA variants with other neurobehavioral disorders such as attention deficit/hyperactivity disorder appears justified.     

Peripheral neuropathy in the Twitcher mouse involves the activation of axonal caspase 3

Infantile Krabbe disease results in the accumulation of lipid-raft-associated galactosylsphingosine (psychosine), demyelination, neurodegeneration and premature death. Recently, axonopathy has been depicted as a contributing factor in the progression of neurodegeneration in the Twitcher mouse, a bona fide mouse model of Krabbe disease. Analysis of the temporal-expression profile of MBP (myelin basic protein) isoforms showed unexpected increases of the 14, 17 and 18.5 kDa isoforms in the sciatic nerve of 1-week-old Twitcher mice, suggesting an abnormal regulation of the myelination process during early postnatal life in this mutant. Our studies showed an elevated activation of the pro-apoptotic protease caspase 3 in sciatic nerves of 15- and 30-day-old Twitcher mice, in parallel with increasing demyelination. Interestingly, while active caspase 3 was clearly contained in peripheral axons at all ages, we found no evidence of caspase accumulation in the soma of corresponding mutant spinal cord motor neurons. Furthermore, active caspase 3 was found not only in unmyelinated axons, but also in myelinated axons of the mutant sciatic nerve. These results suggest that axonal caspase activation occurs before demyelination and following a dying-back pattern. Finally, we showed that psychosine was sufficient to activate caspase 3 in motor neuronal cells in vitro in the absence of myelinating glia. Taken together, these findings indicate that degenerating mechanisms actively and specifically mediate axonal dysfunction in Krabbe disease and support the idea that psychosine is a pathogenic sphingolipid sufficient to cause axonal defects independently of demyelination.     

胰腺p-STAT3在急性胰腺炎危重演变中的表达变化

目的：检测信号转导与转录激活因子3（STAT3）在不同程度胰腺炎模型小鼠胰腺组织中表达的变化,探讨其在急性胰腺炎危重演变中的作用。方法：48只健康雄性balb/c小鼠随机分为3组（n=16）：对照组（Con）、轻症急性胰腺炎（MAP）组、重症急性胰腺炎（SAP）组。Con组腹腔注射0.9% NaCl;MAP组腹腔注射雨蛙素;SAP组腹腔注射雨蛙素联合脂多糖;分别于造模后2 h、6 h检测血清淀粉酶的活性;分离胰腺、称重,计算胰腺湿重比;检测肺组织髓过氧化物酶（MPO）活性,评估炎细胞浸润肺组织的程度;HE染色切片,光镜下观察胰腺、肺组织病理学改变; Western blot法检测磷酸化STAT3（p-STAT3）的变化。结果：与Con组比较, MAP组和SAP组在各时间点血清淀粉酶活性和胰腺组织湿重比均升高（P<0.05）;肺组织MPO活性显著升高（P<0.05）,且SAP组肺MPO含量明显高于MAP组（P<0.01）。MAP组和SAP组,在造模后2 h,胰腺和肺均可见不同程度的病理学改变; SAP组在造模后2 h胰腺p-STAT3的表达最高,6 h表达有所减弱;MAP组各时间点仅有微量表达;Con组在各时间点为阴性表达。结论：p-STAT3在轻症急性胰腺炎和重症急性胰腺炎模型小鼠胰腺中的表达差异明显,说明重症急性胰腺炎的重症化与STAT3的活化关系密切;抑制STAT3活化将成为阻止急性胰腺炎重症化的靶点之一。     

Mint3 is dispensable for pancreatic and kidney functions in mice

Munc-18 interacting protein 3 (Mint3) is an activator of hypoxia-inducible factor-1 in cancer cells, macrophages, and cancer-associated fibroblasts under pathological conditions. However, exactly which cells highly express Mint3 in vivo and whether Mint3 depletion affects their physiological functions remain unclear. Here, we surveyed mouse tissues for specific expression of Mint3 by comparing Mint3 expression in wild-type and Mint3-knockout mice. Interestingly, immunohistochemical analyses revealed that Mint3 was highly expressed in islet cells of the pancreas, distal tubular epithelia of the kidney, choroid plexus ependymal cells of the cerebrum, medullary cells of the adrenal gland, and epithelial cells of the seminal gland. We also studied whether Mint3 depletion affects the physiological functions of the islets and kidneys. Mint3-knockout mice did not show any abnormalities in glucose-tolerance and urine-biochemical tests, indicating that Mint3 depletion was compensated for in these organs. Thus, loss of Mint3 might be compensated in the islets and kidneys under physiological conditions in mice.     

Identification of mouse brain proteins associated with isoform 3 of metallothionein

Using immunological approaches and mass spectrometry, five proteins associated with metallothionein-3 in mouse brains have been identified. Metallothionein-3 and associated proteins were isolated using immunoaffinity chromatography over immobilized anti-mouse brain MT3 antibody. Proteins in the recovered pool were separated by SDS-polyacrylamide gel electrophoresis, and distinct bands were excised and the proteins digested using trypsin. Peptides were extracted and analyzed using electrospray ionization mass spectrometry. Initial identification was done comparing the identified peptide mass:charge ratios to the MASCOT database. Confirmation of proteins was accomplished by sequencing of selected peptides using tandem mass spectrometry and comparison to the MASCOT database. The proteins were heat-shock protein 84 (mouse variant of heat-shock protein 90), heat-shock protein 70, dihydropyrimidinase-like protein 2, creatine kinase, and beta actin. Independently using antibodies against metallothionein-3, creatine kinase, and heat-shock protein 84 showed that all three proteins were coimmunoprecipitated from whole mouse brain homogenates with each of the three antibodies. Mixing purified samples of metallothionein and human brain creatine kinase also generated a complex that could be immunoprecipitated either by anti-metallothionein-3 or anticreatine kinase antibody. These data are consistent with metallothionein-3 being present in the mouse brain as part of a multiprotein complex providing new functional information for understanding the role of metallothionein-3 in neuronal physiology.