Are there gap junctions between chief (glomus,type I) cells in the carotid body chemoreceptor? A review

Since the dye- and electronic couplings between the carotid body chief cells have been demonstrated, the detection and localization of the gap junctions in the carotid body is crucial to understanding the functional mechanism of chemoreception. However, conventional electron microscopy has been unsuccessful in unquestionably detecting ultrastructural features equivalent to the gap junctions, such as close (2 nm in width) membrane appositions in ultrathin sections and aggregations of intramembranous particles in freeze-fracture replicas of the carotid body. We previously reported using a modified electron microscopic study by chemically fixed and subsequent rapid freezing and freeze-substitution method a number of close membrane appositions comparable to the gap junctions. However, we later found that the freeze-substitution also induces numerous close apposition of the membrane in sites where the gap junctions are not known to occur, indicating that the modified electron microscopy by freeze-substitution is not always confirmative in the detection of the gap junction. With regard to the molecular evidence for the gap junction in the carotid body, there have so far been few data on the immunohistochemical demonstration on connexin 32 and 43 in cultured chief cells, but not in the in situ cells.     

Dynamics of subcellular organelles, growth hormone, Rab3B, SNAP-25, and syntaxin in rat pituitary cells caused by growth hormone releasing hormone and somatostatin

Rab3B is involved in the exocytosis of synaptic vesicles and secretory granules in the central nervous system and the anterior pituitary cells. The aim of this study was to elucidate both the role of rab3B in GH secretion and the mutual relationship of rab3B and SNARE proteins. Adult male rats were injected intravenously with 10 microg of growth hormone releasing hormone (GHRH) or 10 microg of somatostatin (SRIF). Untreated rats were used as controls, and their pituitary glands were sectioned for histochemical examination. Rab3B is localized on the limiting membrane of the secretory granules and the cytosol. Confocal laser scanning microscopic observation of immunohistochemical double staining of rab3B and GH revealed that immunoreactivity of rab3B increased in GHRH-treated rats and decreased in SRIF-treated rats. Confocal laser scanning microscopic observation of immunohistochemical double staining of SNAP-25, syntaxin, and rab3B revealed the co-localization of rab3B and these SNARE proteins in GHRH-treated rats, and their dissociation in SRIF-treated rats. These results suggest that rab3B plays a principal role in GH secretion in the anterior pituitary cells and that SNAP-25 and syntaxin act as co-workers with rab3B in the functional regulation of GH secretion.     

Distribution of neurotrophin and TrK receptor-like immunoreactivity in the adrenal gland of birds

The occurrence and localization of neurotrophins and their specific TrK receptor-like proteins in the adrenal gland of chicken, duck and ostrich were examined by immunohistochemical methods. In all species studied NGF-, TrK A- and TrK C-like immunoreactivity was observed in neurons and fibers of adrenal ganglia. Thin TrK A- and TrK C-like immunoreactive fibers were also observed among chromaffin cells. NT-3-like immunoreactivity was detected in chromaffin cells as revealed by the double immunolabelings NT-3/chromogranin A and NT-3/DbetaH. The interrenal tissue never showed IR to any neurotrophins and TrK tested, and none of the adrenal structures displayed immunoreactivity to BDNF and TrK B. Double immunolabelings NGF/TrK A, NGF/TrK C and TrK A/TrK C showed colocalization in some neurons and fibers in adrenal ganglia. In adrenal glands of the species studied, the distribution of neurotrophins and TrK receptors could suggest an involvement of NT-3 on neuronal populations innervating adrenal ganglia by means of its high affinity receptor TrK C and low affinity receptor TrK A. In addition, NGF could be utilized by neuronal populations of adrenal ganglia through its preferential receptor TrK A by an autocrine or paracrine modality of action.     

Cryoultramicrotomy versus plastic embedding: comparative immunocytochemistry of rat anterior pituitary cells

The anterior pituitary of the rat is used as a model for the study of the effects of freezing or plastic embedding on the maintenance of antigenicity. Rat anterior pituitaries are fixed in 2.5% glutaraldehyde in 0.1 m phosphate buffer pH 7.4. Some of the blocks are post-fixed before being divided into two lots. One batch is frozen, while the other is dehydrated and embedded. The indirect antibody enzyme method is applied to ultrathin sections obtained by cryoultramicrotomy after freezing or by sectioning after embedding. All six pituitary hormones are detected by both methods. Comparison shows that the morphological characteristics are identical for both techniques, though ultrastructural preservation is better after embedding. Immunoreactivity is found in secretory granules and sometimes in the endoplasmic reticulum. Osmium postfixation may reduce or even abolish antigenicity in plastic-embedded tissue. After cryoultramicrotomy, however, even after osmium fixation, antibody may be used 1000 times more diluted than after plastic embedding. Embedding preserves ultrastructure and limited antigenicity while the use of cryoultramicrotomy is a far more sensitive technique.     

定位胶结构定位在机床装配技术中的应用分析

文中介绍了定位胶结构定位这种新型的定位形式,并与机床行业中传统的定位方式进行比较。通过具体的用胶案例分析,比较了不同定位方式对机床某些性能参数的影响,充分论证了高精度定位胶结构定位在机床装配中的优势所在。     

Junctional complex revisited by high-resolution scanning electron microscopy

The present study correlates the ultrastructural morphology of junctional complexes as revealed by transmission electron microscopy (TEM) with that observed by high-resolution scanning electron microscopy (HRSEM), thanks to a new modification of the osmium tetroxide maceration technique. The removal of all cytoplasmic organelles by this technique allows the inspection of the inner side of the plasmalemma. With this treatment, a continuous band of tightly packed particles is observed at the most apical portion of lateral membranes. Just below this band, irregular clusters of apparently identical particles are placed all around the cellular contour. The topographical correspondence among these clusters and spot desmosomes seen by TEM identifies them as desmosomes. The continuous band seems to represent the combination of both zonulae, occludens and adherens. Regarding the nature of the particles, we suppose that they probably consist of peripheral membrane proteins clustered at the cytoplasmic surface of intercellular junctions and involved in the linkage between cytoskeleton and plasmalemma.     

电子汽车衡常见故障与预防

本文主要以举例的方式介绍电子汽车衡常见故障及排除方法,再从实际工作出发如何去预防故障的发生。     

Experiments and quantitative analysis of frequency division multiplexing confocal fluorescence microscopy with UV excitation

In this paper, we experimentally demonstrated a two-channel frequency division multiplexing confocal fluorescence microscopy system using a UV laser as the excitation source. In our two-channel frequency division multiplexing confocal fluorescence system, the incoming laser beam was divided into two beams and each beam was modulated with an individual carrier frequency. These two laser beams were then spatially combined with a small angle and focused into two diffraction-limited spots on the targeted cell (rat neural cell) surface to generate fluorescent signal. As a result, the fluorescent signals from two spots of the rat neural cell surface can be demodulated and distinguished during data processing. Furthermore, a quantitative analysis on the cross-talk among different frequencies was provided as well. The experimental results confirm that the two-channel frequency division multiplexing confocal fluorescence technology can not only maintain the high spatial resolution, but also realize the multiple points detection simultaneously with high temporal resolution (within millisecond level range), which benefits the dynamic studies of living biological cells.     

Complementary freeze fracture replication: An example of its use in the study of horizontal cell gap junctions of the carp retina

Dark-adapted carp retinas were fixed with glutaraldehyde, frozen in liquid nitrogen slush, and their complementary replicas were harvested using a bipolar-vacuum evaporater. On montage pictures, plasmic (PFF) and ectoplasmic fracture faces (EFF) of the external horizontal cells (EHC) were marked, and PFF of gap junction (GJ) areas were scrutinized by virtue of particulate aggregation visible at relatively low magnification. Summation of digitized values obtained from a complementary pair of replicas was taken to represent all existent GJ areas in an examined EHC surface. A similar method was applied to large horizontal processes (LHP) that are located in the inner-most part of the internal nuclear layer and currently accepted to be extensions of EHC. The occurrence of fractured faces of plasma membrane was smaller in EHC than LHP. The percentage of GJ areas was about ten times larger in EHC than LHP. Examining complementary replicas at relatively low magnification appears to be useful for facilitating quantitative analysis of intercellular coupling by GJ structures.     

A review of rapid microwave fixation technology: Its expanding niche in morphologic studies

Microwave (MW) fixation methods are important because excellent preservation of both cell structure and antigenicity can be attained several orders of magnitude faster than by routine chemical fixation methods. However, because of the limitations of commercial MW ovens, fixation results are often irreproducible. We present a standardization protocol for MW fixation in household MW ovens that emphasizes magnetron warm-up; the use of a water load during sample irradiation, of an agar/saline/Giemsa model to evaluate uniformity of irradiation within the MW cavity, and of specimen containers with one dimension less than 1.5 cm; and fast specimen handling to prevent conductive heating artifacts after irradiation. We describe a prototypic MW device that improves the precision of sample irradiation and fixes blocks of tissue and cells in suspension in milliseconds. The solutions used to immerse the specimen during irradiation influence the specimen morphology. Aldehyde- or osmium-containing solutions used simultaneously with MW irradiation resulted in the best morphologic preservation of specimens up to 1 cm³. Using MW fixation methods and a postembedding, ultrastructural immunogold-labeling approach, we have localized granule chymase and histamine in rat mast cells and amylase in rat parotid acinar cells.     

Intercellular junctions and the application of microscopical techniques: the cardiac gap junction as a case model

Intercellular junctions are fundamental to the interactions between cells. By means of these junctions, the activities of the individual cells that make up tissues are co-ordinated, enabling each tissue system to function as an integrated whole. In this review, the work of the authors on one specific type of junction—the cardiac gap junction—is presented as a case model to illustrate how the application of a range of microscopical methods, as part of a multidisciplinary approach, can help extend our understanding of cell junctions and their functions. In the heart, gap junctions form the low-resistance pathways for rapid impulse conduction and propagation, enabling synchronous stimulation of myocyte contraction. Gap junctions also form pathways for direct intercellular communication, a function of particular importance for morphogenetic signalling during development. The work discussed demonstrates some of the applications of techniques in electron microscopy, immunocytochemistry and confocal scanning laser microscopy to the understanding of the structural basis of the function of gap junctions in the normal adult heart, the developing heart and the diseased heart. Freeze-fracture electron microscopy of heart tissue prepared by rapid freezing techniques, in which excision-related structural damage to the cells is minimized or avoided, makes it possible to deduce the structure of the functioning gap junction in vivo. Gap junctions in hearts that are beating normally in the living animal until the very instant of freezing consist of connexons (transmembrane channels) organized in a quasicrystalline arrangement, not a ‘random’ arrangement as proposed in the original hypothesis on the structural correlates of gap junction function. Alterations in connexon arrangement occur in response to ischaemia and hypoxia, though the relationship of these to gap-junctional permeability is indirect. To obtain probes for mapping the distribution of gap junctions in cardiac tissue, polyclonal antisera to synthetic peptides matching portions of the sequence of connexin43, the major gap-junctional protein reported in the heart, were raised. The specificity of the antisera was confirmed by dot blotting, Western blotting and by immunogold labelling of isolated gap junctions. One antiserum (that raised to residues 131–142) was found to be particularly effective as a cytochemical probe. An immunofluorescence labelling procedure for use with confocal scanning laser microscopy was developed to enable the three-dimensional precision mapping of gap junctions through thick slices of cardiac tissue. By exploiting the serial optical sectioning ability of the confocal microscope, we have succeeded in (1) elucidating the organization of gap junctions at the intercalated disc, (2) establishing temporal and spatial patterns of gap-junctional protein expression in embryogenesis that correlate with functional differentiation in subsets of cardiac cells, and (3) demonstrating abnormalities of gap-junction distribution and quantity that may contribute to the genesis of arrhythmias in ischaemic heart disease.