Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.     

簇生沿丝伞液体发酵培养及其发酵物体内抗肿瘤活性研究

采用中心组合旋转设计法确定了簇生沿丝伞液体深层培养菌丝体的最佳方案。采用H22荷瘤小鼠进行体内抗肿瘤试验,对簇生沿丝伞发酵物的抗肿瘤活性进行了探究。结果表明,当培养基配方为麦芽糖59.27g/L,蛋白胨:酵母粉=1:1为8.04g/L,磷酸二氢钾:硫酸镁:硫酸铵=1:1:1为2.27g/L时,生物量达到最高,为15.06g/L。簇生沿丝伞发酵物的抑瘤率与剂量存在明显的量效关系,200mg/kg时,抑瘤率达最高,为54.71%,且白细胞数和IL-2的含量与对照组相比均显著增高。     

骨形态发生蛋白9基因敲除小鼠的构建

目的运用CRISR/Cas9技术敲除小鼠基因组中Bmp9基因片段,构建Bmp9基因敲除小鼠。方法根据Bmp9基因的外显子序列,设计一段sgRNA并合成。sgRNA体外转录后和Cas9mRNA混合后显微注射受精卵细胞,注射后的受精卵细胞移植至受体动物获得子代小鼠。提取子代小鼠基因组DNA测序鉴定其基因型。基因型鉴定正确的小鼠与野生型交配后筛选纯合子小鼠。同时取纯合子小鼠心脏、肝、脾、肺、肾,匀浆后提取总RNA和总蛋白,通过qPCR、WB和免疫组化检测BMP9在各组织中的表达。结果设计并合成20bp的sgRNA并进行体外转录,显微注射并回植后得到基因突变小鼠,连续交配后得F2代纯合子。测序结果显示,突变小鼠存在两种基因型,一种为5bp缺失突变,另一种为13bp缺失并伴有1bp插入突变。与野生型C57BL/6相比,qPCR、WB和免疫组化结果均表明基因敲除小鼠肝中BMP9表达显著降低。结论利用CRISPR/Cas9技术成功构建出了BMP9基因敲除小鼠。     

一种皮肤型狼疮小鼠模型的鉴定

目的通过对老龄系统性红斑狼疮小鼠(TC)皮肤症状的观察和鉴定,探讨其是否可以作为一种研究皮肤型狼疮的小鼠模型。方法观察TC小鼠出现皮肤症状的年龄,对比同龄小鼠与C57BL/6小鼠的皮肤症状,ELISA法检测小鼠血清抗dsDNA抗体及抗ANA抗体,HE染色检测小鼠皮肤病理学症状。结果 2/3的TC小鼠出现毛发脱失、皮肤溃烂等症状,其病变最早发生于40周龄,皮肤病变的狼疮小鼠血清抗dsDNA抗体和抗ANA抗体滴度明显高于C57BL/6小鼠(P<0.05);HE染色结果显示狼疮小鼠角质层缺失,上皮层连续性中断,真皮层大量淋巴细胞浸润。结论狼疮小鼠在40周后会出现皮肤病变,显示该小鼠可作为一种研究慢性皮肤型狼疮的疾病模型。     

The Protein-disulfide Isomerase ERp57 Regulates the Steady-state Levels of the Prion Protein

Although the accumulation of a misfolded and protease-resistant form of the prion protein (PrP) is a key event in prion pathogenesis, the cellular factors involved in its folding and quality control are poorly understood. PrP is a glycosylated and disulfide-bonded protein synthesized at the endoplasmic reticulum (ER). The ER foldase ERp57 (also known as Grp58) is highly expressed in the brain of sporadic and infectious forms of prion-related disorders. ERp57 is a disulfide isomerase involved in the folding of a subset of glycoproteins in the ER as part of the calnexin/calreticulin cycle. Here, we show that levels of ERp57 increase mainly in neurons of Creutzfeldt-Jacob patients. Using gain- and loss-of-function approaches in cell culture, we demonstrate that ERp57 expression controls the maturation and total levels of wild-type PrP and mutant forms associated with human disease. In addition, we found that PrP physically interacts with ERp57, and also with the closest family member PDIA1, but not ERp72. Furthermore, we generated a conditional knock-out mouse for ERp57 in the nervous system and detected a reduction in the steady-state levels of the mono- and nonglycosylated forms of PrP in the brain. In contrast, ERp57 transgenic mice showed increased levels of endogenous PrP. Unexpectedly, ERp57 expression did not affect the susceptibility of cells to ER stress in vitro and in vivo. This study identifies ERp57 as a new modulator of PrP levels and may help with understanding the consequences of ERp57 up-regulation observed in human disease.     

Lithospermic acid attenuates 1-methyl-4-phenylpyridine-induced neurotoxicity by blocking neuronal apoptotic and neuroinflammatory pathways

Background

Parkinson’s disease is the second most common neurodegenerative disorders after Alzheimer’s disease. The main cause of the disease is the massive degeneration of dopaminergic neurons in the substantia nigra. Neuronal apoptosis and neuroinflammation are thought to be the key contributors to the neuronal degeneration.

Results

Both CATH.a cells and ICR mice were treated with 1-methyl-4-phenylpyridin (MPP⁺) to induce neurotoxicity in vitro and in vivo. Western blotting and immunohistochemistry were also used to analyse neurotoxicity, neuroinflammation and aberrant neurogenesis in vivo. The experiment in CATH.a cells showed that the treatment of MPP⁺ impaired intake of cell membrane and activated caspase system, suggesting that the neurotoxic mechanisms of MPP⁺ might include both necrosis and apoptosis. Pretreatment of lithospermic acid might prevent these toxicities. Lithospermic acid possesses specific inhibitory effect on caspase 3. In mitochondria, MPP⁺ caused mitochondrial depolarization and induced endoplasmic reticulum stress via increasing expression of chaperone protein, GRP-78. All the effects mentioned above were reduced by lithospermic acid. In animal model, the immunohistochemistry of mice brain sections revealed that MPP⁺ decreased the amount of dopaminergic neurons, enhanced microglia activation, promoted astrogliosis in both substantia nigra and hippocampus, and MPP⁺ provoked the aberrant neurogenesis in hippocampus. Lithospermic acid significantly attenuates all of these effects induced by MPP⁺.

Conclusions

Lithospermic acid is a potential candidate drug for the novel therapeutic intervention on Parkinson’s disease.     

The in vivo distribution of Cr-labeled Trypanosoma cruzi in mice

The optimal conditions for labeling Trypanosoma cruzi culture forms with ⁵¹CrO₄²⁻ were determined. Labeled trypanosomes or labeled human red blood cells (RBCs) were injected intravenously into normal C3H(He) female mice and the rate of clearance and organ distribution of the isotope were observed over a 30 h period. It was found that trypanosomes and xenogeneic RBCs were cleared rapidly from the peripheral blood and accumulated primarily in the liver, spleen, lungs and kidneys. A difference was noted in accumulation of trypanosomes and RBCs in these mice.     

Reproductive and neurobehavioral effects of maternal exposure to piperonyl butoxide (PBO) in F1‐generation mice

Female mice were exposed maternally to piperonyl butoxide (PBO) through diet to provide levels of 0 (control), 0.015, 0.03, and 0.06% during gestation and lactation periods, and selected reproductive and neurobehavioral parameters were measured in F₁ generation. There was no adverse effect of PBO on litter size, litter weight, or sex ratio at birth. The average body weights of offspring showed no significant effects of PBO treatment through the lactation period in both sexes except for the low‐dose group of females on PND 21. With respect to behavioral developmental parameters, swimming direction of female offspring on PND 7 was significantly accelerated in the low‐dose group (p = 0.022). Exploratory behavior examination in male offspring indicated that total distance and movement time shortened significantly in dose‐related manners (p = 0.0138 and 0.00231, respectively), average time of rearing lengthened significantly in a dose‐related manner (p = 0.00814), and the frequencies of mice with urination was increased significantly in a dose‐related manner (p < 0.05). For spontaneous behavior examination, the average time of movement in males and average time of rearing in females showed slightly dose‐related effects in the F₁ generation. The dose levels of PBO in the present study produced some adverse effects in neurobehavioral parameters in mice.     

Natural selection drives altitudinal divergence at the albumin locus in deer mice, Peromyscus maniculatus

In populations that are distributed across steep environmental gradients, the potential for local adaptation is largely determined by the spatial scale of fitness variation relative to dispersal distance. Since altitudinal gradients are generally characterized by dramatic ecological transitions over relatively short linear distances, adaptive divergence across such gradients will typically require especially strong selection to counterbalance the homogenizing effect of gene flow. Here we report the results of a study that was designed to test for evidence of adaptive divergence across an altitudinal gradient in a natural population of deer mice, Peromyscus maniculatus. We conducted a multilocus survey of allozyme variation across a steep altitudinal gradient in the southern Rocky Mountains that spanned several distinct biomes, from prairie grassland to alpine tundra. As a control for the effects of altitude, we also surveyed the same loci in mice sampled along a latitudinal transect through the prairie grassland that ran perpendicular to the east-west altitudinal transect. We used a coalescent-based simulation model to identify loci that deviated from neutral expectations, and we then assessed whether locus-specific patterns of variation were nonrandom with respect to altitude. Results indicated that the albumin locus (Alb) reflects a history of diversifying selection across the altitudinal gradient. This conclusion is supported by two main lines of evidence: (1) Alb was characterized by levels of divergence across the altitudinal transect that exceeded neutral expectations in two consecutive years of sampling (in contrast to the spatial pattern of variation across the latitudinal transect), and (2) levels of divergence at the Alb locus exhibited a positive association with altitudinal distance in both years (in contrast to the pattern observed at unlinked loci). We conclude that clinal variation at the Alb locus reflects a balance between gene flow and diversifying selection that results from elevational changes in fitness rankings among alternative genotypes.     

Touchscreen learning deficits in Ube3a,Ts65Dn and Mecp2 mouse models of neurodevelopmental disorders with intellectual disabilities

Mutant mouse models of neurodevelopmental disorders with intellectual disabilities provide useful translational research tools, especially in cases where robust cognitive deficits are reproducibly detected. However, motor, sensory and/or health issues consequent to the mutation may introduce artifacts that preclude testing in some standard cognitive assays. Touchscreen learning and memory tasks in small operant chambers have the potential to circumvent these confounds. Here we use touchscreen visual discrimination learning to evaluate performance in the maternally derived Ube3a mouse model of Angelman syndrome, the Ts65Dn trisomy mouse model of Down syndrome, and the Mecp2^Bird mouse model of Rett syndrome. Significant deficits in acquisition of a 2‐choice visual discrimination task were detected in both Ube3a and Ts65Dn mice. Procedural control measures showed no genotype differences during pretraining phases or during acquisition. Mecp2 males did not survive long enough for touchscreen training, consistent with previous reports. Most Mecp2 females failed on pretraining criteria. Significant impairments on Morris water maze spatial learning were detected in both Ube3a and Ts65Dn, replicating previous findings. Abnormalities on rotarod in Ube3a, and on open field in Ts65Dn, replicating previous findings, may have contributed to the observed acquisition deficits and swim speed abnormalities during water maze performance. In contrast, these motor phenotypes do not appear to have affected touchscreen procedural abilities during pretraining or visual discrimination training. Our findings of slower touchscreen learning in 2 mouse models of neurodevelopmental disorders with intellectual disabilities indicate that operant tasks offer promising outcome measures for the preclinical discovery of effective pharmacological therapeutics.