蚊期和红内期持续表达绿色荧光蛋白的重组约氏疟原虫

目的 构建蚊期、红内期持续表达绿色荧光蛋白（GFP）的约氏疟原虫BY265株。 方法 用SacⅡ酶酶切含有伯氏疟原虫ssu-rrna基因和GFP基因的重组质粒pl0017使之线性化,用电转化的方法将该重组质粒转化入红内期约氏疟原虫BY265株,获BY265-EGFP重组疟原虫,尾静脉注射感染昆明小鼠,24～30 h后用乙胺嘧啶饲喂小鼠5～6 d,鼠尾静脉采血涂片观察原虫感染率。以疟原虫基因组DNA为模板,PCR鉴定转染重组质粒pl0017的约氏疟原虫。斯氏按蚊叮咬感染BY265-EGFP重组疟原虫的小鼠,按蚊血餐后第7天和第16天解剖蚊胃和唾液腺,观察疟原虫能否在蚊体内正常发育。 结果 荧光显微镜观察到呈绿色荧光的红内期各期形态正常的约氏疟原虫。PCR检测结果表明,GFP和ssu-rrna基因已成功整合到约氏疟原虫基因组中。按蚊感染实验证实重组BY265株能在蚊体内正常发育。 结论 构建了蚊期、红内期持续表达绿色荧光蛋白的约氏疟原虫。     

A review of neurohormone GPCRs present in the fruitfly Drosophila melanogaster and the honey bee Apis mellifera

G protein-coupled receptor (GPCR) genes are large gene families in every animal, sometimes making up to 1-2% of the animal's genome. Of all insect GPCRs, the neurohormone (neuropeptide, protein hormone, biogenic amine) GPCRs are especially important, because they, together with their ligands, occupy a high hierarchic position in the physiology of insects and steer crucial processes such as development, reproduction, and behavior. In this paper, we give a review of our current knowledge on Drosophila melanogaster GPCRs and use this information to annotate the neurohormone GPCR genes present in the recently sequenced genome from the honey bee Apis mellifera. We found 35 neuropeptide receptor genes in the honey bee (44 in Drosophila) and two genes, coding for leucine-rich repeats-containing protein hormone GPCRs (4 in Drosophila). In addition, the honey bee has 19 biogenic amine receptor genes (21 in Drosophila). The larger numbers of neurohormone receptors in Drosophila are probably due to gene duplications that occurred during recent evolution of the fly. Our analyses also yielded the likely ligands for 40 of the 56 honey bee neurohormone GPCRs identified in this study. In addition, we made some interesting observations on neurohormone GPCR evolution and the evolution and co-evolution of their ligands. For neuropeptide and protein hormone GPCRs, there appears to be a general co-evolution between receptors and their ligands. This is in contrast to biogenic amine GPCRs, where evolutionarily unrelated GPCRs often bind to the same biogenic amine, suggesting frequent ligand exchanges ("ligand hops") during GPCR evolution.     

CD16+ monocytes exposed to HIV promote highly efficient viral replication upon differentiation into macrophages and interaction with T cells

The CD16+ subset of monocytes is dramatically expanded in peripheral blood during progression to AIDS, but its contribution to HIV pathogenesis is unknown. Here, we demonstrate that CD16+ but not CD16- monocytes promote high levels of HIV replication upon differentiation into macrophages and interaction with T cells. Conjugates formed between CD16+ monocyte-derived macrophages and T cells are major sites of viral replication. Furthermore, similar monocyte-T cell conjugates detected in peripheral blood of HIV-infected patients harbor HIV DNA. Thus, expansion of CD16+ monocytes during HIV infection and their subsequent recruitment into tissues such as lymph nodes, brain, and intestine may contribute to HIV dissemination and establishment of productive infection in T cells.     

Silk-elastinlike protein polymers for matrix-mediated cancer gene therapy

Silk-elastinlike protein polymers (SELPs) are recombinant polymers designed from silk fibroin and mammalian elastin amino acid repeats. These are versatile materials that have been examined as controlled release systems for intratumoral gene delivery. SELP hydrogels comprise monodisperse and tunable polymers that have the capability to control and localize the release and expression of plasmid DNA and viruses. This article reviews recent developments in the synthesis and characterization of SELP hydrogels and their use for matrix-mediated gene delivery.     

Mouse cortical inhibitory neuron type that coexpresses somatostatin and calretinin

Mammalian cortex contains a diversity of inhibitory neuron types, each with distinct morphological, immunochemical, and/or physiological properties. In rat cortex, chemical markers distinguish at least four distinct and nonoverlapping neuron classes based on expression of parvalbumin (PV), somatostatin (SST), calretinin (CR), and cholecystokinin (CCK). It has generally been assumed that these classifications should also apply to other rodent species. In mouse cortex, however, we found significant colocalization of SST and CR in inhibitory neurons; about 30% of SST-positive cells contained CR, and about 33% of CR-positive cells contained SST across frontal, somatosensory (S1), and visual cortex (V1). The SST and CR colocalized cells were concentrated in layer 2/3. We further characterized morphological and physiological properties of the mouse cortical inhibitory neuron types that express SST by using "GIN" transgenic mice, in which GFP is expressed in a subset of SST inhibitory neurons (see Oliva et al. [2000] J Neurosci 20:3354-3368). Generally, both SST/CR+ cells and SST/CR- cells exhibited morphological features of Martinotti cells as described in rat cortex, and they also had similar accommodating spike-firing patterns. However, they differed significantly in quantitative comparisons of morphology and spike shapes. SST/CR+ cells had more horizontally extended dendritic fields and more primary process than did SST/CR- cells; and SST/CR- cells had narrower action potential widths and faster afterhyperpolarization than did SST/CR+ cells. Thus, our data show an important species difference in the chemical distinction of inhibitory neuron subtypes, and indicate that colocalization of CR in SST cells correlates with different morphological and physiological features.     

基于用GFP标记的大肠杆菌构建大鼠可视化细菌性前列腺感染模型

目的采用绿色荧光蛋白(green fluorescent protein,GFP)标记的大肠杆菌构建大鼠细菌性前列腺炎模型,观察细菌在组织中的侵袭路径和机体免疫反应,并建立感染菌在体直接定量的分析方法。方法于分组大鼠左侧前列腺背叶注入大肠杆菌K88-GFP液(1.49×109cfu.ml-1)0.05 ml,采用腺体表面荧光成像及组织切片荧光显微镜观察,研究荧光菌的感染侵袭过程以及机体的免疫反应,建立可视化细菌性前列腺炎模型。结果①通过表面拍照和病理切片得出细菌在腺体内主要侵袭途径,以及组织的病理变化过程。②测定从感染腺体内提取分离的GFP荧光强度,初步建立直接测定感染菌数量的方法。结论利用GFP标记的细菌可以构建可视化细菌性前列腺炎模型,利用荧光成像技术可以标定感染的程度并进行直接细菌计量,为进一步开展抗菌药PK/PD研究提供新思路。     

Method for single-cell microarray analysis and application to gene-expression profiling of GABAergic neuron progenitors

The mammalian central nervous system is populated with various types of neurons and glia. To investigate the functions and development of individual cells requires gene-expression analysis at the single-cell level. Here, we developed a microarray-based method for the gene-expression profiling of single cells and tested it for GABAergic neuron progenitors. Single GABAergic neuron progenitors were collected from the neocortex of GAD67-GFP knock-in mice by dissociation followed by the aspiration of GFP-positive cells. Complementary DNA from the single cells was amplified by a method in which Super SMART PCR and T7 RNA polymerase amplification were combined at a optimized condition. The cRNA was subjected to microarray hybridization and analysis, which yielded reliable and reproducible results.     

Cell-cell interaction modulates neuroectodermal specification of embryonic stem cells

The controlled differentiation of embryonic stem (ES) cells is of utmost interest to their clinical, biotechnological, and basic science use. Many investigators have combinatorially assessed the role of specific soluble factors and extracellular matrices in guiding ES cell fate, yet the interaction between neighboring cells in these heterogeneous cultures has been poorly defined due to a lack of conventional tools to specifically uncouple these variables. Herein, we explored the role of cell-cell interactions during neuroectodermal specification of ES cells using a microfabricated cell pair array. We tracked differentiation events in situ, using an ES cell line expressing green fluorescent protein (GFP) under the regulation of the Sox1 gene promoter, an early marker of neuroectodermal germ cell commitment in the adult forebrain. We observed that a previously specified Sox1-GFP+ cell could induce the specification of an undifferentiated ES cell. This induction was modulated by the two cells being in contact and was dependent on the age of previously specified cell prior to coculture. A screen of candidate cell adhesion molecules revealed that the expression of connexin (Cx)-43 correlated with the age-dependent effect of cell contact in cell pair experiments. ES cells deficient in Cx-43 showed aberrant neuroectodermal specification and lineage commitment, highlighting the importance of gap junctional signaling in the development of this germ layer. Moreover, this study demonstrates the integration of microscale culture techniques to explore the biology of ES cells and gain insight into relevant developmental processes otherwise undefined due to bulk culture methods.     

Three genes of Citrus tristeza virus are dispensable for infection and movement throughout some varieties of citrus trees

Citrus tristeza virus (CTV), a member of the Closteroviridae, possesses a 19.3-kb positive-stranded RNA genome that is organized into twelve open reading frames (ORFs). The CTV genome contains two sets of conserved genes, which are characteristic of this virus group, the replication gene block (ORF 1a and 1b) and the quintuple gene block (p6, HSP70 h, p61, CPm, and CP). With the exception of the p6 gene, they are required for replication and virion assembly. CTV contains five additional genes, p33, p18, p13, p20 and p23, in the 3' half of the genome, some of which (p33, p18 and p13) are not conserved among other members of this virus group, and have been proposed to have evolved for specific interactions with the citrus host. In the present study, the requirements for systemic infection of citrus trees of p33, p6, p18, p13 and p20 were examined. Viral mutants with a deletion in the p6 or the p20 ORF failed to infect citrus plants systemically, suggesting their possible roles in virus translocation/systemic infection. However, we found that deletions within the p33, p18 or p13 ORF individually resulted in no significant loss of ability of the virus to infect, multiply, and spread throughout citrus trees. Furthermore, deletions in the p33, p18 and p13 genes in all possible combinations including deletions in all three genes allowed the virus to systemically invade citrus trees. Green fluorescent protein-tagged CTV variants with deletions in the p33 ORF or the p33, p18 and p13 ORFs demonstrated that the movement and distribution of these deletion mutants were similar to that of the wild-type virus.     

The ORF3-encoded proteins of vitiviruses GVA and GVB induce tubule-like and punctate structures during virus infection and localize to the plasmodesmata

The genomic RNA of vitiviruses contains 5 open reading frames (ORF). ORF3 encodes a protein to which the function of a movement protein (MP) was assigned, based on sequence homology with other viral proteins. The aim of the research described in this paper was to gain further insight in distribution profile of the ORF3 product encoded by the vitiviruses Grapevine virus A (GVA) and Grapevine virus B (GVB). Expression of the GVA MP-GFP fusion protein via the virus genome in Nicotiana benthamiana leaves resulted in the formation of irregular spots and fibrous network structures on the outermost periphery of epidermal cells. Expression of GVA MP-GFP and GVB MP-GFP was involved in the formation of the tubule-like and punctate structures on the periphery of N. benthamiana and Vitis vinifera protoplasts. Co-expression of the GVA MP-GFP and GVA MP-RFP in protoplasts resulted in co-localization of these proteins into the same punctate structures, indicating that the MP is not accumulated randomly onto the cell surface, but targeted to particular sites at the cell periphery, where punctate and tubule-like structures are likely formed. With the use of cytoskeleton and secretory pathway inhibitors, we showed that the cytoskeletal elements are not likely to be involved in targeting of the MP-GFP to the punctate cellular structures. In addition to MP, a functional coat protein was found to be essential for virus spread within inoculated leaves.