Nickel nitrate: a new junction permeability tracer for the study of the blood-testis barrier.

With the purpose of evaluating a new intercellular tracer, nickel-K ferrocyanide, we compared results yielded by lanthanum with information provided by nickel. This was done in the seminiferous epithelium of Holtzman rats of several postnatal ages and in a wild local seasonal breeder Galea musteloides. Tissues were studied with transmission electron microscopy and freeze-fracture replications. Nickel tracing proved to delineate cell contours more intensely and less interruptedly than lanthanum. With regard to seasonal variations in adult galea, the limits of the barrier were similar to those described in other mammals: spermatogonia, preleptotene, and leptotene spermatocytes were surrounded by the tracer in the basal compartment. The zygotenepachytenes were contained in the lumenal compartment and tracers were stopped at the inter-Sertoli cell tight junctions. During the inactive spermatogenic phase in winter, the seminiferous epithelium contained Sertoli cells and occasional germ cells, never beyond the spermatocyte stage. The tracer filled intercellular spaces, indicating that the barrier was incompetent. Some resting germ cells showed nuclear hyperchromasia, karyolysis, organelle loss, cell shrinkage, and cell fusion leading to a multinucleated cells. The inter-Sertoli tight junctions were scanty and had randomly oriented and discontinuous junctional strands. Moreover, inter-Sertoli cell gap junctions proliferated. During the active spermatogenic phase in summer, junctions were numerous. Their junctional strands were parallel to each other, and continuous.     

Effects of vitamin A deficiency on the inter-Sertoli cell tight junctions and on the germ cell population.

When 20-day-old rats are placed on a vitamin A deficient diet (VAD) for a period of 10 weeks, the seminiferous tubules are found to contain only Sertoli cells, a few residual A0, A1 spermatogonia, and preleptotene spermatocytes (PL). The type A1 spermatogonia and PL spermatocytes are arrested in their G2 phase. In VAD rats type A2-A4, intermediate (In) and B spermatogonia and all types of spermatocytes (except PL spermatocytes) and spermatids are eliminated from the seminiferous tubules. Two questions were raised in this investigation: 1) Is there, in VAD rats, any correlation between a breakdown of the blood-testis barrier (e.g., Sertoli cell tight junctions) and germ cell loss? 2) Is the disappearance of most germinal cells due to their degeneration during spermatogenesis or to a maturation depletion process resulting from an arrest of spermatogenesis at the spermatogonial stage? To investigate these questions four groups of male Sprague-Dawley rats (20-days old) were fed a VAD diet for 7 to 12 weeks. The testes were fixed by perfusion with 2.5% glutaraldehyde in 0.1 M sodium cacodylate containing 2% lanthanum nitrate, an electron opaque tracer used to test the patency of Sertoli cell tight junctions. The lanthanum permeated the intercellular space of the basal compartment but was arrested by normal inter-Sertoli cell tight junctions. The seminiferous epithelium showed numerous degenerating germ cells, some being internalized by Sertoli cells as membrane-bound phagosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extrusion of colonic epithelial cells in vitro

Rat colonic mucosae fixed in situ in Ussing chambers provided a model of the extrusion of absorptive enterocytes and less commonly of goblet and enteroendocrine cells. The cells were lost at extrusion zones midway between crypt mouths. Even in mucosae in which the number of extruding cells was large, epithelial continuity was maintained as evaluated morphologically and electrophysiologically. Beneath points of remaining contact between desquamating cells and the epithelial sheet, microfilaments of the terminal web formed band-like structures linking adjacent junctional complexes. Freeze-fracture replicas disclosed extensive macular regions of tight junction strands in the plasma membranes of desquamating cells. Tight junctions between newly neighboring cells were often irregular and often occurred beneath the terminal web region. Dithio-threitol enhanced cell loss and increased basal epithelial conductance, but histological continuity was maintained and the mucosae continued to respond typically to bradykinin. These observations suggest that during the loss of senescent enterocytes, tight junctions are maintained; old junctional elements are lost, and tight junctions are formed between remaining adjacent cells. This model offers a means to study the synthesis and turnover of tight junctions and the maintenance of the colonic epithelial barrier.     

Aberrant distribution of junctional complex components in retinoic acid receptor alpha‐deficient mice

Retinoic acid receptor alpha (RARα)‐deficient mice are sterile, with abnormalities in the progression of spermatogenesis and spermiogenesis. In this study, we investigated whether defective retinoid signaling involved at least in part, disrupted cell–cell interactions. Hypertonic fixation approaches revealed defects in the integrity of the Sertoli‐cell barrier in the tubules of RARα‐deficient testes. Dye transfer experiments further revealed that coupling between cells from the basal to adluminal compartments was aberrant. There were also differences in the expression of several known retinoic acid (RA)‐responsive genes encoding structural components of tight junctions and gap junctions. Immunostaining demonstrated a delay in the incorporation of zonula occludens (ZO‐1), a peripheral component protein of tight junctions, into the Sertoli cell tight junctions. Markedly reduced expression of connexin‐40 in mutant pachytene spermatocytes and round spermatids was found by in situ hybridization. An ectopic distribution of vimentin and disrupted cyclic expression of vimentin, which is usually tightly regulated during spermiogenesis, was found in RARα‐deficient testes at all ages examined. Thus, the specific defects in spermiogenesis in RARα‐deficient testes may correlate with a disrupted cyclic expression of RA‐responsive structural components, including vimentin, a downregulation of connexin‐40 in spermatogenic cells, and delayed assembly of ZO‐1 into Sertoli cell tight junctions. Interestingly, bioinformatic analysis revealed that many genes that are components of tight junctions and gap junctions contained potential retinoic acid response element binding sites. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Development of the choroid plexus

Mammalian choroid plexuses develop at four sites in the roof of the neural tube shortly after its closure, in the order IVth, lateral, and IIIrd ventricles. Bone morphogenetic proteins and tropomyosin are involved in early specification of these sites and in early plexus growth. Four stages of lateral ventricular plexus development have been defined, based on human and sheep fetuses; these depend mainly on the appearance of epithelial cells and presence or absence of glycogen. Other plexuses and other species are probably similar, although marsupials may lack glycogen. Choroid plexuses form one of the blood-brain barrier interfaces that control the brain's internal environment. The mechanisms involved combine a structural diffusion restraint (tight junctions between the plexus epithelial cells) and specific exchange mechanisms. In this review, it is argued that barrier mechanisms in the developing brain are different in important respects from those in the adult brain, but these differences do not necessarily reflect immaturity of the system. Absence of a barrier mechanism or presence of one not found in the adult may be a specialisation that is appropriate for that stage of brain development. Emphasis is placed on determining which mechanisms are present in the immature brain and relating them to brain development. One mechanism unique to the developing brain transfers specific proteins from blood to cerebrospinal fluid (CSF), via tubulocisternal endoplasmic reticulum in plexus epithelial cells. This results in a high concentration of proteins in early CSF. These proteins do not penetrate into brain extracellular space because of "strap" junctions between adjacent neuroependymal cells, which disappear later in development, when the protein concentration in CSF is much lower. Functions of the proteins in early CSF are discussed in terms of generation of a "colloid" osmotic pressure that expands the ventricular system as the brain grows; the proteins may also act as specific carriers and growth factors in their own right. The pathway for low molecular weight compounds, which is much more permeable in the developing choroid plexuses, appears also to be a transcellular one, rather than paracellular via tight junctions. There is thus good evidence to support a novel view of the state of development and functional significance of barrier mechanisms in the immature brain. It grows in an environment that is different from that of the rest of the fetus/neonate and that is also different in some respects from that of the adult. But these differences reflect developmental specialisation rather than immaturity.     

Cell adhesion in the process of asexual reproduction of tunicates

Cell adhesion during budding of tunicates is reviewed from the viewpoints of histology, cytology, biochemistry, and molecular biology. Two kinds of multipotent cells play important roles in bud formation and development: epithelial cells, such as the atrial epithelium of botryllids and polystyelids, and mesenchymal cells, referred to as haemoblasts. Haemoblasts are able to aggregate to form a solid mass of cells, which soon becomes a hollow vesicle. The vesicular epithelium has junctional complexes that contain adherens junctions, and, sometimes, tight junctions; both occur apicolaterally on the plasma membrane. The hollow vesicle develops into the heart, the pyloric gland and duct, the gonad, including germ cells, and even the multipotent epithelium of buds. Cell culture studies suggest that multipotent epithelial cells may be interchangeable with haemoblasts. Several kinds of calcium-dependent, galactose-binding tunicate lectins (TC-14s) have been isolated and sequenced, and have been found to facilitate both in vivo and in vitro cell aggregation and migration. Tunicate homologs of cadherin and integrin genes have recently been isolated from Botryllus and Polyandrocarpa, respectively. Their unique molecular characteristics are discussed in the context of roles that they play in cell adhesion in the process of tunicate budding.     

纳米硼酸镧对润滑油抗磨性能影响的研究

本文利用CO2超临界干燥法制备了粒径在10～70nm范围内的硼酸镧粒子,用四球机考察了其对润滑油抗磨性能的影响。结果表明,硼酸镧纳米粒子能显著提高润滑油的抗磨性,并借助扫描电子显微镜和X-射线光电子能谱初步分析了其抗磨机理。     

Tight junctions in Gerbil von Ebner's Gland: Horseradish peroxidase and freeze‐fracture studies

The permeability of tight junctions to horseradish peroxidase (HRP) and the freeze‐fracture appearance of junctional structures were investigated in the von Ebner's gland of gerbils. In the tracing study, HRP was either administered topically on the dorsal surface of tongues or injected subepithelially into the connective tissue of vallate papillae for 5–30 min. Lingual tissues containing the von Ebner's gland were sectioned and examined by light and electron microscopy. In von Ebner's glands, the reaction product for HRP was found in the intercellular and interstitial spaces, whereas HRP appeared to penetrate the tight junctions and the reaction product was localized in the lumina of serous acini. In contrast, the staining for HRP that delineated the boundary of epithelial cells was frequently observed in the superficial layers of the lingual epithelium but not the underlying tissues while applying HRP topically. Freeze‐fracture replicas of acinar cells revealed that the tight junction had a depth of 0.815 ± 0.023 μm, and 4–6 parallel strands on the protoplasmic fracture face, with a branching network of joining strands with interruptions, interconnections and high linear strand density apically, and corresponding grooves on the extracellular face. Quantitative analyses showed a greater number of strands (7.217 ± 0.326) in gerbils compared to those of acinar cells (3.86 ± 0.22) in mice. These results demonstrate that the tight junctions in the gerbil von Ebner's gland is permeable, and that specific species differences in tight junction structures may be associated with the mechanism for survival in an extremely dry environment. Microsc. Res. Tech. 78:213–219, 2015. © 2015 Wiley Periodicals, Inc.     

Effect of glutaraldehyde on the osmolarity of the buffer vehicle

The osmolarity of four different buffers (Na-cacodylate, PIPES, Na-K-phosphate and Na-Na-phosphate) at different pH-values and molarities was measured. On the addition of glutaraldehyde to these buffers, an increase in osmolarity was observed. This increase was more than could be explained by the numerical addition of the osmolar values of the glutaraldehyde and of the buffer. The increase is due to a higher buffer dissociation constant. On the addition of 5% glutaraldehyde to a 0.05 M buffer, the increase in buffer osmolarity may be as much as 107 mOsm. Graphs are given to allow the determination of K-values for these four buffers at different molarities and pH-values. The effect of different glutaraldehyde concentrations on these K-values is presented graphically. The time-dependent increase in osmolarity of glutaraldehyde solutions in water is described.     

电沉积稀土改性陶瓷涂层磨损性能研究

探讨了在电火花加工机床上沉积碳化钛金属陶瓷涂层方法,利用TiC,WC,Mo,N i粉未添加不同比例稀土元素在高压下压制并烧结了试验电极,在45#钢表面沉积了不同稀土含量的TiC陶瓷涂层,并用扫描电镜(SEM)、X射线衍射仪(XRD)、显微硬度计、环块式磨损试验机对涂层组成、组织形态进行、硬度及摩擦学性能分别进行了研究,并结合试验结果进行了理论分析。试验结果表明:用电火花放电法可沉积TiC陶瓷涂层,涂层中加入质量分数为0.5%的氧化镧后,涂层的耐磨性能较未加稀土涂层提高了3倍,摩擦因数减少10%,而加入过多的稀土镧氧化物则不利于涂层组织性能及耐磨性能的改善。稀土氧化镧对涂层的组织有改善作用,加入适量的稀土元素使得涂层致密性提高,减少涂层中的缺陷,涂层表面呈多孔结构特性。     

Ultrastructural alterations in colon absorptive cells of alloxan-induced diabetic rats submitted to long-term physical training

Absorptive cells have notable importance for proper function of the colon, absorbing water and nutrients. In type I diabetes, hyperglycemia leads to remarkable alterations in cell structure. In absorptive cells, such changes may impair the function of the organ as a whole. Also, the effects of physical training, which plays crucial role in the treatment of diabetes, are not yet known in these cells. For this reason, to analyze the changes in colon epithelial absorptive cells of diabetic rats and the effects of physical training, Wistar rats were divided into four groups: sedentary control (SC), trained control (TC), sedentary diabetic (SD), and trained diabetic (TD). The training protocol consisted of swimming for 60 min a day, 5 days per week, during 8 weeks. Colon samples were collected, processed, and evaluated by histochemical and ultrastructural techniques. Although histochemical analysis did not reveal major differences, significant morphological differences were ultrastructurally observed among groups, especially related to the structure of tight junctions, interdigitations, and microvilli, which became longer in diabetics, and whose length was reduced after physical training, as proved by statistical analysis. There were no relevant changes in organelles. Thus, the development of type I diabetes can lead to changes at ultrastructural level that, even subtle, may cause important alterations in cell function. The practice of physical training, in turn, proved to be an important ally in the treatment of such changes. However, it cannot be used singly for treating this disease, requiring the combined practice of other methods. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Lymphatic vessels in the oral cavity: different structures for the same function.

A study using a light and transmission electron microscope was performed on some structural characteristics of the lymphatic capillaries in different regions of the human oral cavity. The lymphatic capillaries of dental pulp, masticatory mucosa (gingiva and peri-implant mucosa) and lining mucosa (cheek) were examined. Our attention was focused on the morphologic characteristics of the endothelial wall in the lymphatic capillaries. In particular, the connections between endothelial cells were investigated. In the lymphatic capillaries of the dental pulp, the endothelial wall was always very complex. It frequently presented protrusions of the endothelial cells that overlapped and formed intercellular channels. These channels were thus contained by the vessel endothelial wall with their extremities opening out towards the surrounding interstitium and the vessel lumen. The endothelial wall of the lymphatic capillaries of the cheek was very smooth and thin without complex intercellular junctions. The endothelial cells were joined by end-to-end junctions and open junctions were frequently observed. Intercellular channels were also found in the endothelial wall of lymphatic capillaries of the gingiva and the peri-implant mucosa. The presence of numerous clefts represented by the open junctions in the lymphatics of the cheek and the existence of complex intercellular adhesions with the formation of intercellular channels in the endothelial wall of the lymphatic capillaries of the dental pulp and gingiva induce us to believe that these may play a role in the various mechanisms used by lymphatic capillaries to absorb interstitial fluids. These mechanisms are based on the different morpho-functional characteristics of the surrounding tissue.     

Resource-saving pneumovibrodynamic hardening of plane surfaces for repair and production processes

Experience with pneumovibrodynamic hardening of plane surfaces is systematized. In such treatment, the pressurized medium is compressed air. Data relating to pneumovibrodynamic hardening as a repair technique are presented; its characteristics are noted. This method is recommended for frictional pairs and for components in which tight sealing of junctions is required, as well as water-tightness of the contacting surfaces.     

Histochemical evidences on the chronological alterations of the hypertrophic zone of mandibular condylar cartilage

The hypertrophic chondrocytes lack the ability to proliferate, thus permitting matrix mineralization as well as vascular invasion from the bone in both the mandibular condyle and the epiphyseal cartilage. This study attempted to verify whether the histological appearance of the hypertrophic chondrocytes is in a steady state during postnatal development of the mouse mandibular condyle. Type X collagen immunohistochemistry apparently distinguished the fibrous layer described previously as the "articular zone," "articular layer," and "resting zone" from the hypertrophic zone. Interestingly, the ratio of the type X collagen-positive hypertrophic zone in the entire condyle seemed higher in the early stages but decreased in the later stages. Some apparently compacted cells in the hypertrophic zone showed proliferating cell nuclear antigen (PCNA) immunoreaction, indicating the potential for cell proliferation at the early stages. As the mice matured, in contrast, they further enlarged and assumed typical features of hypertrophic chondrocytes. Apoptotic cells were also discernible in the hypertrophic zone at the early but not later stages. Consistent with morphological configurations of hypertrophic chondrocytes, immunoreactions for alkaline phosphatase, osteopontin, and type I collagen were prominent at the later stage, but not the early stage. Cartilaginous matrices demonstrated scattered patches of mineralization at the early stage, but increased in their volume and connectivity at the later stage. Thus, the spatial and temporal occurrence of these immunoreactions as well as apoptosis likely reflect the prematurity of hypertrophying cells at the early stage, and imply a physiological relevance during the early development of the mandibular condyles.     

Rodlet cells development in the intestine of sea bass (Dicentrarchus labrax)

The enigmatic rodlet cells (RCs) are characterized by conspicuous inclusions named “rodlets”. They were discovered over 100 years ago and were considered as parasites but shortly afterward interpreted as endogenous cells. The RCs have been described in different tissues of marine and freshwater teleosts, but their origin and function remain unknown. This work was designed to an ultrastructural study on RCs development and distribution in intestinal epithelium of Dicentrarchus labrax. Three different stages of RCs development from early precursor cells to mature phase were observed, as well as a migration and finally an extrusion of their contents. In this study, the immature cells were found near the basal epithelium membrane. They were mainly identified by a rough endoplasmic reticulum with dilated cisternae, by developing rodlets and a thin fibrillar coat. The maturing RCs, localized in the middle zone of the epithelium, appeared to be undergoing a reorganization of the cell organelles. The mature RCs, placed near the free surface, showed a thick subplasmalemmar fibrillar coat. Most of the organelles were aggregated at the cell apex with a basally located nucleus. A cellular polarity was more evident. One of the most conspicuous features was the occurrence of mature rodlets club‐sac in shape orientated toward the cell apex. Adhesive junctions between surface epithelial cells and RCs, while discharging their contents, were seen. We have connected morphological figures and distribution to different stages of development in RCs, supporting the hypothesis of their secretory function. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Freeze-Fracture of physiologically identified neuromuscular junctions from single frog muscle fibers

We developed a technique for freeze-fracturing single physiologically identified neuromuscular junctions. This technique permits direct comparison of quantal content with morphological variables such as active zone length per unit terminal length for the same cell. The technique was developed to elucidate the structural basis for variability in transmitter release at the neuromuscular junction. The procedures were as follows: (1) record quantal content by conventional intracellular recordings; (2) mark cells for identification by fluorescent dye injection; (3) fix and stain endplate cholinesterase; (4) glycerinate and remove single fibers from the muscle; (5) draw endplate morphology; (6) freeze-fracture single muscle fibers; (7) examine in a transmission electron microscope; and (8) photograph and measure nerve terminal membrane ultrastructure. We found that approximately 15% of freeze-fractured single muscle fibers exhibited nerve terminal active zones. To demonstrate the usefulness of this technique, physiological and morphological information from an identified junction is presented. Freeze-fracture of identified cells has several advantages over thin sections, which cannot accurately show such things as active zone length, spacing, or intramembrane particles. This technique also has applications to the study of active zone ultrastructure in situations where neurotransmitter release is known to differ from normal levels. In addition, direct correlations between membrane structure and function can be studied in other preparations by this method.     

滚动轴承故障信号的多尺度形态学分析

数学形态分析是数字信号处理的一种非线性分析方法,滚动轴承故障信号是一种非线性非平稳信号.为了提取不同类型故障特征,利用多尺度形态学分析对滚动轴承故障振动信号建立一种不同于时频分析的信号特征描述方法.采用多尺度形态开运算得到故障信号的形态谱,定量反映了信号在不同尺度下的形态变化特征;由形态谱曲线计算形态谱熵,定量描述不同信号的形态特征.通过试验数据的分析以及与峭度和共振包络解调方法的对比,表明多尺度形态学分析方法计算效率高,特征描述准确简单,为轴承故障信号的分析、识别和分类提供了新的思路.     

Highly accurate SNR measurement using the covariance of two SEM images with the identical view

Quality of an SEM image is strongly influenced by the extent of noise. As a well-known method in the field of SEM, the covariance is applied to measure the signal-to-noise ratio (SNR). This method has potential ability for highly accurate measurement of the SNR, which is hardly known until now. If the precautions discussed in this article are adopted, that method can demonstrate its real ability. These precautions are strongly related to "proper acquisition of two images with the identical view," "alignment of an aperture diaphragm," "reduction of charging phenomena," "elimination of particular noises," and "accurate focusing," As necessary, characteristics in SEM signal and noise are investigated from a few standpoints. When using the maximum performance of this measurement, SNR of many SEM images obtained in a variety of the SEM operating conditions and specimens can be measured accurately.     

Intercellular junctions in FANFT-induced carcinomas of rat urinary bladder in tissue culture: in situ thin-section, freeze-fracture, and scanning electron microscopy studies.

This paper describes a set of simple methods for comparative light and electron microscopy studies on tissue cultured tumour cells derived from both noninvasive and invasive carcinogen-induced rat urinary bladder carcinomas. Cells are grown on Thermanox plastic coverslips and fixed in situ. Each plastic coverslip is then divided with scissors into four parts: the first is processed for light microscopy, the second for thin-section electron microscopy, the third for freeze-fracture electron microscopy, and the fourth for scanning electron microscopy. In some experiments, portions of the culture which have first been examined by light microscopy are subsequently prepared for electron microscopy. In this way, the culture conditions are kept constant and comparison of structural features (i.e. intercellular junctions) by several preparative techniques is possible. Noninvasive and invasive rat bladder tumour cells, characterized by numerous pleomorphic microvilli, have normal zonulae occludentes at the apices of lateral surfaces of tumour cells in all cultures. In some areas of invasive tumour cells, occludens junctions are focally attenuated, consisting of only one or two strands, and occasionally the strands are discontinuous. Gap junctions, type PF-1, as well as numerous demosomes are present in all cell lines. Thus, intercellular junctions in noninvasive and invasive rate bladder epithelial cell lines bear a striking resemblance to those previously described in the comparable solid primary tumours. These culture systems may be useful for studying factors which influence the formation of intercellular junctions during malignant transformation.     

Effects of fixation on electrophysiology and structure of human jejunal villi

The villi of human jejunum vary in size and shape during different functional conditions. In the base the lamina propria is isotonic with blood, in the tip hyperosmotic. Here we study electrophysiological and morphological effects of incubation in hypotonic, isotonic, or hypertonic solutions, and to test various isotonic fixatives for microscopy. Samples of jejunal mucosae, obtained during surgery in obese patients, were studied in Ussing chambers where electrical parameters were registered during incubation in Krebs solution at various osmolarities, and during fixation in formaldehyde, glutaraldehyde, or osmium tetroxide (OsO₄). The same fixatives were used for other jejunal specimens that were fixed directly for light microscopy. Morphometry was carried out to determine size and height of villi, proportion of lamina propria, and surface enlargement due to villi. Ussing chamber incubation in fluids with low osmolarity resulted in increased electrical resistance and epithelial swelling. Opposite results were obtained at high osmolality. Fixation was faster in formaldehyde than in glutaraldehyde or OsO₄. In biopsies processed directly for light microscopy the proportions of lamina propria of the mucosa, and of lamina propria of villi, were significantly larger in biopsies fixed in formaldehyde than after fixation in glutaraldehyde or OsO₄. The villus tips sometimes ended with a bleb with prominent spaces between the epithelial cells. In summary, jejunal villi swell in vitro when exposed to hypotonic solutions, and shrink in hypertonic solutions. Much of the morphological changes occurring during fixation can be related to the physiological hyperosmolar milieu in villus tips.