激光共聚焦扫描显微镜和透射电镜观察大鼠纹状体内谷氨酸能突触连接的对比观察

目的 探讨使用激光共聚焦扫描显微镜 (Laser scanning confocal microscope,LSCM)观察大鼠纹状体内谷氨酸能突触连接的方法的可行性.方法 12只正常大鼠分为两组,6只大鼠进行纹状体中等棘刺神经元的CM-DiI 单细胞标记,然后Ⅰ型囊泡膜谷氨酸转运体(vesicular glutamate transporter 1,VGluT1 )免疫荧光标记,LSCM层扫后三维重建,观察VGluT1阳性位点在中等棘刺神经元树突上的分布.另外6只大鼠用TEM观察不对称性突触在纹状体神经元树突上的分布.对两种方法的结果进行比较.结果 用LSCM 和TEM方法观察到的纹状体神经元上谷氨酸能突触连接分布情况一致,没有统计学差异.但LSCM更具优越性的是,可以对图像进行三维重构,从而有利于对神经元之间突触连接的空间分布观察和定量分析.结论 神经细胞荧光标记技术结合LSCM观察是考察纹状体神经元上谷氨酸能突触连接的有效方法.     

Investigation of cell adhesion to structured surfaces using total internal reflection fluorescence and confocal laser scanning microscopy

Adhesion of adherent cells on structured surfaces is influenced by the surface pattern given. Here, we designed a structured gold relief surface based on cell adhesion patterns we had previously observed. We analysed the geometric parameters and the overall distribution of focal adhesion kinase in focal adhesions on unstructured glass surfaces using optical microscopy. The basic structural elements obtained from this analysis were arranged in regular clusters that resembled the shape of a polarised migratory cell. In time-lapse studies we observed that the cells adhere preferentially to the gold pads and adopt the shape of the clusters. Staining of the actin cytoskeleton revealed that the actin filaments are aligned to the gold pads of the elementary structure.     

Developmental architecture of the nervous system in Themiste lageniformis (Sipuncula): New evidence from confocal laser scanning microscopy and gene expression

Sipuncula is a clade of unsegmented marine worms that are currently placed among the basal radiation of conspicuously segmented Annelida. Their new location provides a unique opportunity to reinvestigate the evolution and development of segmented body plans. Neural segmentation is clearly evident during ganglionic ventral nerve cord (VNC) formation across Sedentaria and Errantia, which includes the majority of annelids. However, recent studies show that some annelid taxa outside of Sedentaria and Errantia have a medullary cord, without ganglia, as adults. Importantly, neural development in these taxa is understudied and interpretation can vary widely. For example, reports in sipunculans range from no evidence of segmentation to vestigial segmentation as inferred from a few pairs of serially repeated neuronal cell bodies along the VNC. We investigated patterns of pan-neuronal, neuronal subtype, and axonal markers using immunohistochemistry and whole mount in situ hybridization (WMISH) during neural development in an indirect-developing sipunculan, Themiste lageniformis. Confocal imaging revealed two clusters of 5HT⁺ neurons, two pairs of FMRF⁺ neurons, and Tubulin⁺ peripheral neurites that appear to be serially positioned along the VNC, similar to other sipunculans, to other annelids, and to spiralian taxa outside of Annelida. WMISH of a synaptotagmin1 ortholog in T. lageniformis (Tl-syt1) showed expression throughout the centralized nervous system (CNS), including the VNC where it appears to correlate with mature 5HT⁺ and FMRF⁺ neurons. An ortholog of elav1 (Tl-elav1) showed expression in differentiated neurons of the CNS with continuous expression in the VNC, supporting evidence of a medullary cord, and refuting evidence of ontogenetic segmentation during formation of the nervous system. Thus, we conclude that sipunculans do not exhibit any signs of morphological segmentation during development.     

Simultaneous multi-parameter observation of Harringtonine-treating HL-60 cells with both two-photon and confocal laser scanning microscopy

Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb-Cephalotaxus hainanensis Li, can induce apoptosis in promyelocytic leukemia HL-60 cells. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluorescent probe Hoechst 33342, tetramethyrhodamine ethyl ester (TMRE) and Fluo 3-AM, we simultaneously observed HT-induced changes in nuclear morphology, mitochondrial membrane potential and intracellular calcium concentration ([Ca2+]i) in HL-60 cells, and developed a real-time, sensitive and invasive method for simultaneous multi-parameter observation of drug- treating living cells at the level of single cell.     

Leu-callatostatin gene expression in the blowflies Calliphora vomitoria and Lucilia cuprina studied by in situ hybridisation: comparison with Leu-callatostatin confocal laser scanning immunocytochemistry

In situ hybridisation studies using a digoxigenin-labelled DNA probe encoding the Leu-callatostatin prohormone of the blowflies Calliphora vomitoria and Lucilia cuprina have revealed a variety of neurones in the brain and thoracico-abdominal ganglion, peripheral neurosecretory neurones, and endocrine cells of the midgut. With two exceptions, the hybridising cells are the same as those previously identified in immunocytochemical studies of sections and whole-mounts using Leu-callatostatin COOH-terminal-specific antisera. Within the brain and suboesophageal ganglion, there is a variety of neurones ranging from a single pair of large cells situated in the dorsal protocerebrum, to the several pairs of neurones in the tritocerebrum, some of which, in immunocytochemical preparations, can be seen to project via axons in the cervical connective to the thoracico-abdominal ganglion. In the medulla of the optic lobes, numerous small interneurones hybridise with the probe, as do clusters of similar-sized neurones close to the roots of the ocellar nerves. These results indicate that the Leu-callatostatin neuropeptides of the brain play a variety of roles in neurotransmission and neuromodulation. There are only three pairs of Leu-callatostatin-immunoreactive neurones in the thoracico-abdominal ganglion, at least two pairs of which project axons along the median abdominal nerve to provide extensive innervation of the hindgut. The Leu-callatostatin peripheral neurosecretory cells are located in close association with both nerve and muscle fibres in the thorax. In addition to neuronal Leu-callatostatin, the presence of the peptide and its mRNA has been demonstrated in endocrine cells in the posterior part of the midgut. These observations provide an example of a named brain/gut peptide in an insect.     

Detection of radicals in single droplets of oil-in-water emulsions with the lipophilic fluorescent probe BODIPY665/676 and confocal laser scanning microscopy

Lipid oxidation is a widespread phenomenon in foods and other systems of biological origin. Detection methods for early stages of lipid oxidation are in demand to understand the progress of oxidation in space and time. The fluorescence spectrum of the nonpolar fluorescent probe BODIPY^665/676 changes upon reacting with peroxyl radicals originating from 2,2′-azobis(2,4-dimethyl)valeronitrile and tert-butoxyl radicals generated from di-tert-butylperoxide. The excitation wavelength of the main peak of BODIPY^665/676 was 675 nm in the fluorometer, and 670 nm under the microscope, and the optimum excitation wavelength for the secondary peak of BODIPY^665/676 was 580 nm. Advantages of using BODIPY^665/676 are fewer problems with autofluorescence and the possibility of combining several fluorescent probes that are excited and emitted at lower wavelengths. However, because of the spectrum of the probe, specific lasers and detectors are needed for optimal imaging under the microscope. Furthermore, BODIPY^665/676 is resistant to photobleaching at both excitation wavelengths, 670 and 580 nm. In diffusion studies, BODIPY^665/676 is highly lipophilic, remaining in the lipid phase and not diffusing into the aqueous phase or between lipid droplets.     

The musculature of Squatinella rostrum (Milne, 1886) (Rotifera: Lepadellidae) as revealed by confocal laser scanning microscopy with additional new data on its trophi and overall morphology

Wilts, E.F., Wulfken, D., Ahlrichs, W.H. and Martínez Arbizu, P. 2012. The musculature of Squatinella rostrum (Milne, 1886) (Rotifera: Lepadellidae) as revealed by confocal laser scanning microscopy with additional new data on its trophi and overall morphology.—Acta Zoologica (Stockholm) 93 : 14–27. The monogonont rotifer Squatinella rostrum was investigated with light, scanning electron and confocal laser scanning microscopy to reveal new morphological data on its inner and outer anatomy. In total, the visualized somatic musculature displays five paired longitudinal muscles (musculi longitudinales I–V) and nine circular muscles (musculi circulares I–IX). Compared to other species, S. rostrum is characterized by the absence of several longitudinal and circular muscles (e.g. musculus longitudinalis capitis, corona sphincter and pars coronalis). A reconstruction of the mastax musculature revealed a total number of seven paired and two unpaired mastax muscles. Possibly homologous somatic and mastax muscles in other, thus far investigated rotifers are discussed. Moreover, we provide a phylogenetic evaluation of the revealed morphological characters and suggest possible autapomorphic characters supporting Squatinella and Lepadellidae. Finally, we refer to some striking similarities in the morphology, ecology and way of movement of Squatinella and Bryceella that may indicate a closer relationship of both taxa.     

Structure and shear strength of microbial biofilms as determined with confocal laser scanning microscopy and fluid dynamic gauging using a novel rotating disc biofilm reactor

The cohesive strength of microbial biofilms cultivated on a rotating disc has been measured using fluid dynamic gauging (FDG). The thickness of heterotrophic mixed culture biofilms was found to depend on substrate concentration and shear force at the biofilm surface during the cultivation. For high substrate concentrations and low shear forces the biofilm thickness increased to several 100 microm within 7 days. Low substrate concentration and higher shear forces yielded thin biofilms of about 100 microm thickness. Independent from cultivation conditions and thickness of the biofilms their cohesive strength ranged between 6.0 and 7.7 N m(-2). The ratio between cohesive strength measured with FDG and shear forces applied during biofilm cultivation have ranged from 200 to 1,100. Higher concentrations of iron in the cultivation media has a positive effect on the stability of the biofilms cultivated. By using the CLSM technique a stable base biofilm with a high amount of stained EPS glycoconjugates could be visualized after gauging. The thickness of the base biofilm was about 100 microm for all biofilms cultivated and was not removable under the applied shear conditions used during FDG.     

The somatic musculature of Bryceella stylata (Milne, 1886) (Rotifera: Proalidae) as revealed by confocal laser scanning microscopy with additional new data on its trophi and overall morphology

The monogonont rotifer Bryceella stylata was investigated with light, electron and confocal laser scanning (CLSM) microscopy to provide detailed insights into its anatomy and new information for future phylogenetic analyses of the group. Results from CLSM and phalloidin staining revealed a total of six paired longitudinal muscles (musculi longitudinales I-VI) and eight circular muscles (musculi circulares I-VIII) as well a complex network of mostly fine visceral muscles. In comparison with other rotifer species that have been investigated so far, B. stylata shares the presence of the circular and longitudinal muscles: musculus longitudinalis ventralis, musculus longitudinalis lateralis inferior, musculus longitudinalis dorsalis, musculus longitudinalis capitis and musculus circumpedalis. However, the species lacks lateral and dorsolateral longitudinal muscles and some circular muscles (e.g., corona sphincter, musculus pars coronalis). With light and electron microscopy, we were able to document the precise number of pseudosegments and the arrangement of the chambers comprising the trophi elements. Furthermore, our observations revealed several new morphological characteristics, including a shield-like epidermal projection covering the dorsal antenna, an epidermal projection restricting the corona caudally and an unpaired hypopharynx with distinct shovel-like structures.     

Expression of the rice Osgrp1 promoter-Gus reporter gene is specifically associated with cell elongation/expansion and differentiation

To study the expression and regulation of a rice glycine-rich cell wall protein gene, Osgrpl, transgenic rice plants were regenerated that contain the Osgrpl promoter or its 5 deletions fused with the bacterial -glucuronidase (GUS) reporter gene. We report here a detailed histochemical analysis of the Osgrpl-Gus expression patterns in transgenic rice plants. In roots of transgenic rice plants, GUS expression was specifically located in cell elongation and differentiation regions, and no GUS expression was detectable in the apical meristem and the mature region. In shoots, GUS activity was expressed only in young leaves or in the growing basal parts of developing leaves, and little GUS activity was expressed in mature leaves or mature parts of developing leaves. In shoot apices, GUS activity was detected only in those leaf cells which were starting to expand and differentiate, and GUS expression was not detected in the apical meristem and the young meristematic leaf primordia. GUS activity was highly expressed in the young stem tissue, particularly in the developing vascular bundles and epidermis. Thus, the expression of the Osgrpl gene is closely associated with cell elongation/expansion during the post-mitotic cell differentiation process. The Osgrpl-Gus gene was also expressed in response to wounding and down-regulated by water-stress conditions in the elongation region of roots. Promoter deletion analysis indicates that both positive and negative mechanisms are involved in regulating the specific expression patterns. We propose a simple model for the developmental regulation of the Osgrpl gene expression.     

Tamoxifen-inducible gene deletion reveals a distinct cell type associated with trabecular bone, and direct regulation of PTHrP expression and chondrocyte morphology by Ihh in growth region cartilage

Indian hedgehog (Ihh) controls multiple aspects of endochondral skeletal development by signaling to both chondrocytes and perichondrial cells. Previous efforts to delineate direct effects of Ihh on chondrocytes by Col2-Cre-mediated ablation of Smoothened (Smo, encoding a transmembrane protein indispensable for Ihh signaling) has been only partially successful, due to the inability to discriminate between chondrocytes and perichondrial cells. Here we report a transgenic line (Col2-Cre) expressing under the control of the Colalpha1(II) promoter an inert form of Cre that is activatable by exogenous tamoxifen (TM); TM administration at proper times during embryogenesis induced Cre activity in chondrocytes but not in the perichondrium. By using this mouse line, we deleted Smo within subsets of chondrocytes without affecting the perichondrium and found that Smo removal led to localized disruption of the expression of parathyroid hormone-related protein (PTHrP) and the morphology of chondrocytes. Unexpectedly, TM invariably induced Cre activity in a subset of cells associated with the trabecular bone surface of long bones. These cells, when genetically marked and cultured in vitro, were capable of producing bone nodules. Expression of the Col2-Cre transgene in these cells likely reflected the endogenous Colalpha1(II) promoter activity as similar cells were found to express the IIA isoform of Colalpha1(II) mRNA endogenously. In summary, the present study has not only provided evidence that Ihh signaling directly controls PTHrP expression and chondrocyte morphology in the growth region cartilage, but has also uncovered a distinct cell type associated with the trabecular bone that appears to possess osteogenic potential.     

Transformation of peanut with a soybean vspB promoter‐uidA chimeric gene. I. Optimization of a transformation system and analysis of GUS expression in primary transgenic tissues and plants

Direct DNA delivery via microprojectile bombardment has become an established approach for gene transfer into peanut ( Arachis hypogaea L.). To optimize our transformation protocol and to simultaneously explore the function of a heterologous promoter whose activity is developmentally regulated, embryogenic cultures from three peanut cultivars were bombarded with two plasmid constructs containing a uidA gene controlled by either a soybean vegetative storage protein gene promoter or a cauliflower mosaic virus 35S promoter. We found that GUS transient expression was useful to predict stable transformation and confirmed that image analysis could provide a quick and efficient method for semi‐quantitation of transient expression. One hundred and sixty hygromycin‐resistant cell lines were recovered from and maintained on selective medium, and those tested by Southern blot analysis showed integration of the foreign gene. Over 200 transgenic plants were regenerated from 38 cell lines. More than 100 plants from 32 cell lines flowered and 79 plants from 19 cell lines produced pods. Over 1000 R₁ seeds were harvested. Analysis of expression in primary transgenic plants showed that GUS expression driven by the vspB promoter was modulated by chemical and positional information.     

Social isolation-induced increase in alpha and delta subunit gene expression is associated with a greater efficacy of ethanol on steroidogenesis and GABA receptor function

Previously we have demonstrated that social isolation of rats reduces both the cerebrocortical and plasma concentrations of 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG), and potentiates the positive effects of acute ethanol administration on the concentrations of this neurosteroid. We now show that the ethanol-induced increase in 3alpha,5alpha-TH PROG is more pronounced in the brain than in the plasma of isolated rats. The ability of ethanol to inhibit isoniazid-induced convulsions is greater in isolated rats than in group-housed animals and this effect is prevented by treatment with finasteride. Social isolation modified the effects of ethanol on the amounts of steroidogenic regulatory protein mRNA and protein in the brain. Moreover, ethanol increased the amplitude of GABA(A) receptor-mediated miniature inhibitory postsynaptic currents recorded from CA1 pyramidal neurones with greater potency in hippocampal slices prepared from socially isolated rats than in those from group-housed rats, an effect inhibited by finasteride. The amounts of the alpha(4) and delta subunits of the GABA(A) receptor in the hippocampus were increased in isolated rats as were GABA(A) receptor-mediated tonic inhibitory currents in granule cells of the dentate gyrus. These results suggest that social isolation results in changes in GABA(A) receptor expression in the brain, and in an enhancement of the stimulatory effect of ethanol on brain steroidogenesis, GABA(A) receptor function and associated behaviour.     

Cell behavior observation and gene expression analysis of melanoma associated with stromal fibroblasts in a three‐dimensional magnetic cell culture array

A three‐dimensional (3D) multicellular tumor spheroid culture array has been fabricated using a magnetic force‐based cell patterning method, analyzing the effect of stromal fibroblast on the invasive capacity of melanoma. Formation of spheroids was observed when array‐like multicellular patterns of melanoma were developed using a pin‐holder device made of magnetic soft iron and an external magnet, which enables the assembly of the magnetically labeled cells on the collagen gel‐coated surface as array‐like cell patterns. The interaction of fibroblast on the invasion of melanoma was investigated using three types of cell interaction models: (i) fibroblasts were magnetically labeled and patterned together in array with melanoma spheroids (direct‐interaction model), (ii) fibroblasts coexisting in the upper collagen gel (indirect‐interaction model) of melanoma spheroids, and (iii) fibroblast‐sheets coexisting under melanoma spheroids (fibroblast‐sheet model). The fibroblast‐sheet model has largely increased the invasive capacity of melanoma, and the promotion of adhesion, migration, and invasion were also observed. In the fibroblast‐sheet model, the expression of IL‐8 and MMP‐2 increased by 24‐fold and 2‐fold, respectively, in real time RT‐PCR compared to the absence of fibroblasts. The results presented in this study demonstrate the importance of fibroblast interaction to invasive capacity of melanoma in the 3D in vitro bioengineered tumor microenvironment. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Anthocyanins from soybean seed coat inhibit the expression of TNF-alpha-induced genes associated with ischemia/reperfusion in endothelial cell by NF-kappaB-dependent pathway and reduce rat myocardial damages incurred by ischemia and reperfusion in vivo

We examined the inhibition of the expression of some inflammatory genes associated with ischemia-reperfusion (I/R) injury by anthocyanins isolated from black soybean seed coat in tumor necrosis factor-alpha (TNF-alpha)-treated bovine aortic endothelial cells. In addition, its potential use on I/R-injury was investigated using rats subjected to 30-min occlusion of left descending coronary artery followed by 24-h reperfusion. Western blot analysis and luciferase activity assay showed that anthocyanins inhibited TNF-alpha-induced vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and cyclooxygenase-2 levels, which is through NF-kappaB-dependent pathway. Further, anthocyanins protected myocardiac injury from I/R in rats. It is suggested that anthocyanins from black soybean seed coat can be used as a useful drug to modulate cardiovascular disorder.