Photosynthesis-associated gene families: differences in response to tissue-specific and environmental factors

The endogenous small subunit of the ribulose-1,5-bisphosphate carboxylase gene rbcS and the light-harvesting chlorophyll a/b-binding protein gene (LHCP) of pea are expressed in a light-inducible manner and are active mainly in green chloroplast-containing tissue. Chimeric genes under control of the 5'-flanking sequences of the rbcS ss3.6 or LHCP AB80 genes from pea were used to study the factors relating to the issue-specific and lightinducible expression of these nuclear-encoded genes in transgenic tobacco plants. The results show that plastid development plays a crucial role in the activation of expression of these chimeric genes. Particular members of each of the above gene families respond differently to tissue-specific and environmental factors. Furthermore, the light-inducible expression directed by the 5'-flanking sequence of ss3.6 rbcSgene is not exclusively mediated by phytochrome, but probably is controlledin large part by another photoreceptor.     

Chemical glycobiology

Chemical tools have proven indispensable for studies in glycobiology. Synthetic oligosaccharides and glycoconjugates provide materials for correlating structure with function. Synthetic mimics of the complex assemblies found on cell surfaces can modulate cellular interactions and are under development as therapeutic agents. Small molecule inhibitors of carbohydrate biosynthetic and processing enzymes can block the assembly of specific oligosaccharide structures. Inhibitors of carbohydrate recognition and biosynthesis can reveal the biological functions of the carbohydrate epitope and its cognate receptors. Carbohydrate biosynthetic pathways are often amenable to interception with synthetic unnatural substrates. Such metabolic interference can block the expression of oligosaccharides or alter the structures of the sugars presented on cells. Collectively, these chemical approaches are contributing great insight into the myriad biological functions of oligosaccharides.     

法氏囊三肽囊素(Bursin)的研究Ⅰ.抗 Bursin 单克隆抗体杂交瘤细胞株的建立

以戊二醛法制备ＢｕｒｓｉｎＫＬＨ，免疫ＢＡＬＢ／ｃ小鼠，取脾细胞，以ＰＥＧ为融合剂，与ＳＰ２融合，用间接ＥＬＩＳＡ法筛选出与Ｂｕｒｓｉｎ－ＢＳＡ结合、不与ＢＳＡ结合的２Ｆ９－４克隆株，经鉴定，抗体属性为ＩｇＭ，Ｋ轻链。水溶性Ｂｕｒｓｉｎ可阻抑２Ｆ９－４对鸡法氏囊的反应，切片标本经ＰＢＳ处理，反应性消失，免疫组织化学染色于法氏囊上获得特征性阳性结果。     

A population of murine gammadelta T cells that recognize an inducible MHC class Ib molecule

Although gammadelta T cells are implicated in regulating immune responses, gammadelta T cell-ligand pairs that could mediate such regulatory functions have not been identified. Here, the expression of the major histocompatibility complex (MHC) class Ib T22 and the closely related T10 molecules is shown to be activation-induced, and they confer specificity to about 0.4% of the gammadelta T cells in normal mice. Thus, the increased expression of T22 and/or T10 might trigger immunoregulatory gammadelta T cells during immune responses. Furthermore, the fast on-rates and slow off-rates that characterize this receptor/ligand interaction would compensate for the low ligand stability and suggest a high threshold for gammadelta T cell activation.     

MHC class I deficiency: susceptibility to natural killer (NK) cells and impaired NK activity.

The role of major histocompatibility complex (MHC) class I expression in natural killer (NK) cell target recognition is controversial. Normal T cell blasts from MHC class I-deficient mutant mice were found to serve as target cells for NK cells in vitro, which suggests that MHC class I molecules are directly involved in NK cell recognition. Spleen cells from the mutant mice were deficient in their ability to lyse MHC class I-deficient target cells or NK-susceptible tumor targets, and mutant mice could not reject allogeneic bone marrow. Thus, class I molecules may participate in the positive selection or tolerance induction of NK cells.     

Gene expression in mice after high efficiency retroviral-mediated gene transfer

A retroviral expression vector (N2) containing the selectable gene, neoR, has been used to determine the optimal conditions for infecting murine hematopoietic progenitor cells at high efficiency. After infected bone marrow cells were introduced into lethally irradiated mice, the presence, stability, and expression of the vector DNA sequences were analyzed either in individual spleen foci 10 days later or in the blood, bone marrow, and spleens of mice 4 months later. When bone marrow cells were cultured in medium containing virus with titers of more than 10(6) colony-forming units per milliliter in the presence of purified murine interleukin-3, more than 85 percent of the resulting foci contained vector DNA. This proviral vector DNA was intact. Efficient expression of the neoR gene was demonstrated in most of the DNA-positive foci examined. The spleens of reconstituted animals (over a long term) contained intact "vector DNA" and the blood and bone marrow expressed the neoR gene in some animals. Thus, a retroviral vector can be used to introduce intact exogenous DNA sequences into hematopoietic stem cells with high efficiency and with substantial expression.     

犬白细胞介素-7基因对犬细小病毒DNA疫苗的免疫增强作用

【目的】白介素-7是动物体一种重要的多功能细胞因子,在促进B细胞、T细胞的形成和发育,在协同其它细胞因子提高机体免疫能力等方面发挥重要作用。本试验利用犬细小病毒VP2 DNA疫苗,在小鼠体内分析了犬白介素-7（cIL-7）基因的免疫增强作用。【方法】采用RT-PCR方法从犬脾淋巴细胞中扩增cIL-7基因,然后将cIL-7基因插入到真核表达载体pcDNA3.1A中,分别构建成与Myc/His标签融合和非融合的cIL-7真核分泌型表达载体pcDNA-cIL7/MH和pcDNA-cIL7。由磷酸钙介导将pcDNA-cIL7/MH质粒转染HEK 293T细胞使其进行瞬时表达,以Western-blot检测构建的表达载体能否介导cIL-7基因在真核细胞中进行分泌表达。用已构建的VP2表达载体pcDNA-CD5-VP2与pcDNA-cIL7载体共免疫小鼠,并设pcDNA-CD5-VP2单免疫小鼠和pcDNA-cIL7单免疫小鼠作为对照。免疫后通过ELISA方法检测抗体水平,并通过淋巴细胞增殖实验和ELISA方法分别检测免疫后35 d小鼠脾脏淋巴细胞刺激指数和γ-干扰素的表达水平。【结果】本试验扩增的cIL-7基因序列与GenBank中犬的序列完全一致。构建的表达载体能够介导cIL-7基因在HEK293T细胞中进行分泌表达。动物免疫试验结果显示,cIL-7与VP2共免疫组小鼠血清的抗体滴度和中和抗体效价分别极显著和显著高于VP2单免疫组（P＜0.01和P＜0.05）;共免疫组小鼠淋巴细胞刺激指数和γ-干扰素表达水平也显著高于VP2单免疫组（P＜0.05）。【结论】cIL-7基因可增强小鼠对VP2 DNA疫苗的免疫应答反应。     

Organogenic role of B lymphocytes in mucosal immunity

Follicle-associated epithelium (FAE) in the intestinal Peyer's patches contains M cells that deliver pathogens to organized lymphoid tissue. Development of Peyer's patches, FAE, and M cells was found to be impaired in mice that had no B cells. Transgenic expression of membrane-bound immunoglobulin M restored B cells and FAE development. The lack of M cells abrogated infection with a milk-borne retrovirus. Thus, in addition to secretion of antibodies and presentation of antigens, B cells are important for organogenesis of the mucosal immune barriers.     

Genetic ablation: targeted expression of a toxin gene causes microphthalmia in transgenic mice

Lineage-specific regulatory elements can be used to direct expression of a variety of genes to specific tissues in transgenic mice. If the hybrid constructs contain a gene encoding a cytotoxic gene product, then genetic ablation of a specific cell lineage can be achieved. We have generated six transgenic mice by introducing into fertilized eggs the mouse gamma 2-crystallin promoter fused to the coding region of the diphtheria toxin A-chain gene. Three of these mice and all the transgenic offspring analyzed were microphthalmic. The lenses of these mice displayed considerable heterogeneity: some were almost normal morphologically but reduced in size, whereas others were grossly aberrant and deficient in nuclear fiber cells. These studies indicate that programmed ablation of specific cell types can be stably transmitted through the germ line.     

Generation of mouse induced pluripotent stem cells without viral vectors

Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by introducing Oct3/4 and Sox2 with either Klf4 and c-Myc or Nanog and Lin28 using retroviruses or lentiviruses. Patient-specific iPS cells could be useful in drug discovery and regenerative medicine. However, viral integration into the host genome increases the risk of tumorigenicity. Here, we report the generation of mouse iPS cells without viral vectors. Repeated transfection of two expression plasmids, one containing the complementary DNAs (cDNAs) of Oct3/4, Sox2, and Klf4 and the other containing the c-Myc cDNA, into mouse embryonic fibroblasts resulted in iPS cells without evidence of plasmid integration, which produced teratomas when transplanted into mice and contributed to adult chimeras. The production of virus-free iPS cells, albeit from embryonic fibroblasts, addresses a critical safety concern for potential use of iPS cells in regenerative medicine.     

The role of B cells for in vivo T cell responses to a Friend virus-induced leukemia

B cells can function as antigen-presenting cells and accessory cells for T cell responses. This study evaluated the role of B cells in the induction of protective T cell immunity to a Friend murine leukemia virus (F-MuLV)-induced leukemia (FBL). B cell-deficient mice exhibited significantly reduced tumor-specific CD4+ helper and CD8+ cytotoxic T cell responses after priming with FBL or a recombinant vaccinia virus containing F-MuLV antigens. Moreover, these mice had diminished T cell responses to the vaccinia viral antigens. Tumor-primed T cells transferred into B cell-deficient mice effectively eradicated disseminated FBL. Thus, B cells appear necessary for efficient priming but not expression of tumor and viral T cell immunity.     

Matrix attachment regions included in a bicistronic vector enhances and stabilizes follistatin gene expressions in both transgenic cells and transgenic mice

In the present study, follistatin (FST) gene expression vectors with either a bicistronic gene transfer cassette alone, or a bicistron gene cassette carrying a matrix attachment region (MAR) were constructed and transfected to bovine fetal fibroblasts. Evaluations of both the integration and expression of exogenous FST indicated that the pMAR-CAG-FST-IRES-AcGFP1-polyA-MAR (pMAR-FST) vector had higher capacity to form monoclonal transgenic cells than the vector without MAR, though transient transfection and integration efficiency were similar with either construct. Remarkably, protein expression in transgenic cells with the pMAR-FST vector was significantly higher than that from the bicistronic vector. Exogenous FST was expressed in all of the pMAR-FST transgenic mice at F₀, F₁ and F₂. Total muscle growth in F₀ mice was significantly greater than in wild-type mice, with larger muscles in fore and hind limbs of transgenic mice. pMAR-FST transgenic mice were also found with more evenly distributed muscle bundles and thinner spaces between sarcolemma, which suggests a correlation between transgene expression-associated muscle development and the trend of muscle growth. In conclusion, a pMAR-FST vector, which excluded the resistant genes and frame structure, enhances and stabilizes FST gene expressions in both transfected cells and transgenic mice.     

ICR小鼠胚胎干细胞的分离、培养及鉴定

以小鼠胚胎成纤维细胞为饲养层,收集受孕3.5 d ICR小鼠的囊胚和桑椹胚进行培养,筛选纯化ES细胞集落,使其稳定传代后,对其形态学和生物学性状进行初步鉴定。结果表明,ES细胞有其典型的形态学特征:集落呈鸟巢状,边缘清楚,表面平滑,结构致密,隆起生长,细胞之间界限不清楚;单个细胞体积小、核大;对ES细胞碱性磷酸酶进行检测,在AKP底物NBT、BCIP作用下,未分化的ES细胞显微镜下为黄褐色,分化的不着色;核型鉴定表明ES细胞具有正常的二倍体核型。     

试验性趾叶炎小鼠肠组织中P物质的表达

[目的]为动物趾叶炎的预防和治疗提供免疫学基础。[方法]对昆明种小白鼠注射二磷酸组织胺制备趾叶炎小鼠病理模型,以注射生理盐水为对照。分别于给药后2、14、24、48、72 h及15、30 d取小鼠3~5 mm长的小肠组织块,检测肠组织中炎性细胞、杯状细胞、肥大细胞及其脱颗粒数、P物质表达水平的动态变化。[结果]趾叶炎小鼠的肠腺结缔组织内及其绒毛上P物质表达异常丰富,且P物质呈单根或片状走行,并与肥大细胞串联及相邻排列,随给药时间的延长,P物质表达逐渐增强,肥大细胞及其脱颗粒数也急剧增多 炎性细胞、杯状细胞、腺体数量与肥大细胞的变化趋势一致 给药72 h后,血管内壁出现裂隙,肠粘膜出现水肿。[结论]肥大细胞参与小鼠趾叶炎的发病过程,并影响小鼠肠粘膜的通透性。     

Dynamic organization of developing Purkinje cells revealed by transgene expression

The cerebellum has many properties that make it a useful model for investigating neural development. Purkinje cells, the major output neurons of the cerebellar cortex, have drawn special attention because of the availability of biochemical markers and mutants that affect their development. The spatial expression of L7, a protein specific for Purkinje cells, and L7 beta Gal, a gene expressed in transgenic mice that was constructed from the L7 promoter and the marker beta-galactosidase, delineated bands of Purkinje cells that increased in number during early postnatal development. Expression of the transgene in adult reeler mutant mice, which show inverted cortical lamination, and in primary culture showed that the initial expression of L7 is intrinsic to Purkinje cells and does not depend on extracellular signals. This may reflect an underlying developmental map in cerebellum.     

Reprogramming cellular behavior with RNA controllers responsive to endogenous proteins

Synthetic genetic devices that interface with native cellular pathways can be used to change natural networks to implement new forms of control and behavior. The engineering of gene networks has been limited by an inability to interface with native components. We describe a class of RNA control devices that overcome these limitations by coupling increased abundance of particular proteins to targeted gene expression events through the regulation of alternative RNA splicing. We engineered RNA devices that detect signaling through the nuclear factor κB and Wnt signaling pathways in human cells and rewire these pathways to produce new behaviors, thereby linking disease markers to noninvasive sensing and reprogrammed cellular fates. Our work provides a genetic platform that can build programmable sensing-actuation devices enabling autonomous control over cellular behavior.     

Altered K+ channel expression in abnormal T lymphocytes from mice with the lpr gene mutation

The observation that voltage-dependent K+ channels are required for activation of human T lymphocytes suggests that pathological conditions involving abnormal mitogen responses might be reflected in ion channel abnormalities. Gigaohm seal techniques were used to study T cells from MRL/MpJ-lpr/lpr mice; these mice develop generalized lymphoproliferation of functionally and phenotypically abnormal T cells and a disease resembling human systemic lupus erythematosus. The number and predominant type of K+ channels in T cells from these mice differ dramatically from those in T cells from control strains and a congenic strain lacking the lpr gene locus. Thus an abnormal pattern of ion channel expression has now been associated with a genetic defect in cells of the immune system.     

Transgenic mice as probes into complex systems

The transfer of genetic information into mouse embryos to stably alter the genetic constitution of mice is affording new insights into and opportunities in a wide variety of biological problems. Higher eukaryotes are composed of many interacting cells and organs. The properties of individual cell systems are often discernible only by studying natural or induced disruptions in their functions. Transgenic mice represent a new form of perturbation analysis whereby the selective expression of novel or altered genes can be used to perturb complex systems in ways that are informative about their development, their functions, and their malfunctions. The utility of this strategy is illustrated by recent research into immunological self-tolerance, oncogenes and cancer, and development.     

基础研究BXSB小鼠肾组织NF-κB的表达及意义

目的:了解NF-κB在BXSB狼疮性肾炎小鼠肾组织中的表达及意义。方法:取BXSB狼疮性肾炎小鼠(即LN组)与C57BL/6小鼠(即对照组)各10只,应用免疫组织化学SP法检测肾组织中NF-κB的表达,并与病理学指标行等级相关分析。结果:LN组小鼠肾组织中NF-κB表达较对照组显著增高(P<0.05);NF-κB在肾小球和肾小管均有表达,但以肾小管表达更为显著;肾小球NF-κB阳性细胞数、NF-κB阳性肾小管数与肾组织活动指数呈高度正相关。结论:NF-κB可能参与了LN小鼠的发病机制,检测其在肾组织中的表达可作为反映狼疮肾组织活动病变的参考指标之一。     

Adjuvant Effects of Recombinant Plasmids of Porcine IL-4 and IFN-γ to Cysticercosis cellulosae Vaccines in Mice and Pigs

To investigate the adjuvant potential of porcine IL-4 and IFN-γ in mice and pigs, the genes of porcine IL-4 and IFN-γ were cloned and the recombinant mammalian expression plasmids were constructed for in vivo expression of the cytokines. Adjuvant effects of recombinant expression plasmids of IL-4 and IFN-γ （pcDNA-IL-4, pcDNA-IFN-γ） co-administrated with Cysticercus cellulosae crude antigen or TSOL18 recombinant protein antigen have been carried out in mice and pigs, respectively. We have demonstrated that recombinant plasmids of the cytokines as an adjuvant could induce stronger immune response in mice and pigs. With the C. cellulosae parasite antigen, porcine pcDNA-IL-4 induced higher specific antibody of immunized mice than pcDNA-IFN-γ. But pcDNA-IFN-γ is significantly stronger than that of no adjuvant or empty plasmids with the antigen control group. For the TSOL18 recombinant protein antigen vaccine, pcDNA-IL-4 still had a stronger ability to enhance specific antibody in swine than pcDNA-IFN-γ （P 〈 0.01）, but the immune protective rate was lower in challenged pigs （only 68.7%）. Although pcDNA-IFN-γ showed lower specific antibody, the protection rate was very high （91%） than other group （P 〈 0.01）. This study indicated that the recombinant expression plasmids of porcine IL-4 and IFN-γ display stronger adjuvant effects to C. cellulosae vaccine, further research should be carried out for understanding of the interaction mechanism.