Ultrastructural distribution of endogenous IgGs in the glomerular wall of control and diabetic rats

Summary Endogenous IgG molecules were revealed with high resolutionem over the glomerular wall in renal tissues sampled from short and longterm control and streptozotocin induced diabetic rats by applying the protein A-gold immunocytochemical approach. In tissues from control animals, IgG antigenic sites were revealed on the subendothelial side of the basement membrane, the epithelial side being only weakly labelled. In contrast, in longterm diabetic animals IgG antigenic sites were present throughout the entire thickness of the basement membrane, and in patches closely associated with the plasma membrane of the epithelial cells. Deposits of basement membrane-like material present in the mesangial area were also highly labelled for IgG. Numerous intensely labelled lysosome-like structures were present in the epithelial cells. Morphometrical evaluation of the distribution of the labelling over the basement membrane confirmed these observations. In control animals a peak of labelling was found at 30 nm from the endothelial cell region corresponding to the subendothelial side of the lamina densa. In longterm diabetic animals the labelling was more uniformly distributed throughout the entire thickness of the basement membrane. These data were correlated to biochemical determinations of proteinuria and IgG excretion in urine samples from the control and the diabetic animals. These results suggest that in normal conditions the lamina densa may represent the main barrier for the restriction of the passage of IgGs through the glomerular wall. Modifications at that level occur during diabetes leading to or participating in the loss of the selective permeability of the basement membrane.     

Immunocytochemical localization of parathormone in the mammalian parathyroid gland using the protein A-gold technique

Summary Electron-microscopic immunocytochemistry for the demonstration of parathormone in parathyroid chief cells was performed in adult male rats, gerbils, mice, and dogs, using the protein A-gold technique. Protein A-gold particles were detected over both large and small secretory granules in all the animals examined. In the former, they were concentrated not only over type-I granules with a large core, but also over type-II granules with a small core. They were also located over atypical granules, including heterogeneously dense granules, granules having vesicles in a finely particulate core, and distorted granules. All labelled secretory granules were characterized by the presence of a clear halo of varying width around the core. Occasionally, Golgi cisternae as well as Golgi vacuoles with a finely particular content were also labelled. The labelling of the secretory granules was strong in dogs, moderate in rats and gerbils, and weak in mice. In addition, it was more intense in the non-osmicated preparations than in the osmicated preparations. The frequency of both types of large granules showed species differences. The possible factors involved in these differences are discussed.     

FINE STRUCTURE OF THE MYOEPITHELIUM OF THE ECCRINE SWEAT GLANDS OF MAN

The secretory coils of glutaraldehyde-osmium tetroxide-fixed and Epon-Araldite-embedded eccrine sweat glands from the palms of young men were studied with the electron microscope. The myoepithelial cells lie on the epithelial side of the basement membrane and abut other epithelial elements directly. The irregularly serrated base of the cell has dense thickenings along the plasma membrane which alternate with zones bearing pits; the smooth apical surface lacks dense thickenings, is studded with pits, and conjoined to secretory cells by occasional desmosomes. Masses of myofilaments, 50 A in diameter, fill most of the cell and are associated with irregular dense zones. In cross-section the arrangement of the myofilaments seems identical with that of the I band of striated muscle, and the dense zone has typical Z band structure. A few microtubules and cytoplasmic cores bearing profiles of the endoplasmic reticulum, filamentous mitochondria, and glycogen granules penetrate the fibrillar masses and run parallel to the oriented myofilaments. In the perinuclear zone, Golgi membranes, rough- and smooth-surfaced elements of the endoplasmic reticulum, mitochondria, glycogen, microtubules, lipid, pigment, and dense granules are variable components in the cytoplasm. The interrelationships of the myoepithelial cells with the secretory cells suggest that the former may act as regulators, controlling the flow of metabolites to the secretory epithelium.     

Non-expression of insulin-like growth factor-I receptor is associated with apoptosis: an ultrastructural study on rat ameloblasts

Insulin-like growth factor-I (IGF-I) is a pleiotrophic polypeptide which appears to have roles both as a circulating endocrine hormone and as a locally synthesized paracrine or autocrine tissue factor. IGF-I plays a major role in regulating the growth of cells in vivo and in vitro and initiates metabolic and mitogenic processes in a wide variety of cell types by binding to specific type I receptors in the plasma membrane. In this study, we report the distribution of IGF-I receptors in odontogenic cells at the ultrastructural level using the high resolution protein A-gold technique. In the pre-secretory stage, very little gold label was visible over the ameloblasts and odontoblasts. During the secretory stage the label was mostly seen in association with the cell membranes and endoplasmic reticulum of the ameloblasts. Lysosome-like elements in the post-secretory stage were labelled as well as multivesicular dense bodies. Very little labelling was encountered in the ameloblasts in the transitional stage, where apoptotic bodies were clearly visible. The maturation stage also exhibited labelling of the secretory-like granules in the distal surface. The presence of gold particles over the plasma membrane is an indication that IGF-I receptor is a membrane-bound receptor. Furthermore, the intracellular distribution of the label over the endoplasmic reticulum supports the local synthesis of the IGF-I receptor. The absence of labelling over the transitional ameloblasts suggests that the transitional stage may require the non-expression of IGF-I as a prerequiste or even a trigger for apoptosis. This revised version was published online in November 2006 with corrections to the Cover Date.     

Concentration of amylase along its secretory pathway in the pancreatic acinar cell as revealed by high resolution immunocytochemistry

Summary The modified protein A-gold immunocytochemical technique was applied to the localization of amylase in rat pancreatic acinar cells. Due to the good ultrastructural preservation of the cellular organelles obtained on glutaraldehyde-fixed, osmium tetroxide-postfixed tissue, the labelling was detected with high resolution over the cisternae of the rough endoplasmic reticulum (RER), the Golgi apparatus, the condensing vacuoles, the immature pre-zymogen granules, and the mature zymogen granules. Over the Golgi area, the labelling was present over the transitional elements of the endoplasmic reticulum, some of the smooth vesicular structures at thecis- andtrans-faces and all the different Golgi cisternae. The acid phosphatase-positive rigidtrans-cisternae as well as the coated vesicles were either negative or weakly labelled. Quantitative evaluations of the degree of labelling demonstrated an increasing intensity which progresses from the RER, through the Golgi, to the zymogen granules and have identified the sites where protein concentration occurs. The results obtained have thus demonstrated that amylase is processed through the conventional RER-Golgi-granule secretory pathway in the pancreatic acinar cells. In addition a concomitance has been found between some sites where protein concentration occurs: thetrans-most Golgi cisternae, the condensing vacuoles, the pre- and the mature zymogen granules, and the presence of actin at the level of the limiting membranes of these same organelles as reported previously (Bendayan, 1983). This suggests that beside their possible role in transport and release of secretory products, contractile proteins may also be involved in the process of protein concentration.     

前包埋阳离子胶体金标记技术定量检测硬脑膜毛细血管阴离子场方法的探讨

介绍一种定量观察血管内皮细胞表面阴离子场的技术方法。以研究毛细血管腔面阴离子场与血管通透性的关系。用前包埋程序以阳郭胶体金（CCG）做为探针,标记硬脑膜毛细血管内皮腔面的阴离子场,在电子显微镜下摄影计数内皮细胞表面标记的金颗粒,在硬脑膜毛细血管腔面,微绒毛和质膜小泡的表面均见CCG标记,而不带电荷的BSA－胶体金呈阴性结果,前包埋阳离子胶体金标记技术可特异性地显示硬脑膜血管内皮细胞表面的阴离子场,并可做定量研究,标记微环境,血管开口大小和复染可能影响标记结果。     

Immunoelectron microscopic localization of serine: pyruvate aminotransferase in rat eosinophile leukocytes

Localization of serine: pyruvate aminotransferase [EC 2.6.1.51]; SPT in rat eosinophile leukocytes was investigated by protein A-gold technique. Thin sections of rat intestine were incubated with anti-SPT, followed by protein A-gold complex. Labelling with gold particles was seen on the specific granules of eosinophile leukocytes, in which 78% of the gold particles were localized on their paracrystalline cores and 22% on matrix, indicating that the main intragranular sites of SPT are the core. Other cell organelles such as nucleus and mitochondria were not labelled specifically. Quantitative analysis of labelling density in the subcellular compartments also confirmed that SPT is present exclusively in the specific granules.     

Association of secreted insulin with particular domains of the pancreatic B-cell plasma membrane: the actin-rich microvilli.

Association of insulin with the plasma membrane of the pancreatic B-cell was revealed using the high-resolution protein A-gold immunocytochemical approach. The labeling was found to be heterogeneously distributed, the plasma membrane of the actin-rich microvilli being preferentially labeled. Morphometrical evaluation of labeling intensities confirmed the microvilli location of the insulin binding sites. This membrane domain, which also displays the glucose transporter, therefore appears to play important functional roles in the secretory activity of the B-cell. That the autocrine influence insulin exerts on its own secretion is receptor mediated through these insulin binding sites remains, however, to be determined.     

Topology of morphologically detectable protein and cholesterol in membranes of polypeptide-secreting cells

The freeze-fracture morphology of intracellular and plasma membranes in endocrine and exocrine polypeptide-secreting cells has been studied to detect changes while these membranes interact during secretion. A qualitative and quantitative evaluation of intramembrane particles and filipin binding as indicators of protein and cholesterol content of the membranes, respectively, reveals the following changes. From the forming of the maturing pole of the Golgi complex, membranes lose morphologically detectable protein and gain morphologically detectable cholesterol. The protein-poor, cholesterol-rich secretory granule membrane then interacts with a richly particulate plasma membrane in endocrine cells and with a moderately particulate luminal membrane in exocrine cells. The site of interaction between secretory granule and plasma membrane is characterized by a local clearing of intramembrane particles; by contrast, filipin-binding sites revealing cholesterol are present in this area. In exocrine cells, the fused secretory granule, which is initially rich in filipin-cholesterol complexes and poor in particles, appears to lose progressively its filipin labelling to resemble the poorly labelled luminal membrane. These findings, although they cannot be interpreted definitely at present, clearly show impressive changes of membrane structure along the secretory pathway and suggest that a corresponding degree of functional specialization is needed for proper interaction to occur.     

Immunocytochemical localization of heparan sulfate proteoglycan in human decidual cell secretory bodies and placental fibrinoid

A distinct ultrastructural feature of human decidual cells is the presence of membrane-bound secretory bodies, 0.3-0.5 micron in diameter, located within club-shaped processes at the cell periphery. These secretory bodies contain 30-60 nm electron-dense granules. Using specific antibody and the protein A-gold technique, we examined the localization of heparan sulfate proteoglycan in human decidual cells. Morphometric analysis of gold particles in cellular compartments was performed with a Zeiss Videoplan computer system. Immuno-gold staining was present in the decidual cell cytoplasm and the extracellular space, especially in the zone of the external lamina. Gold particles, indicating the locale of heparan sulfate proteoglycan, were concentrated over the electron-dense granular material within decidual secretory bodies contained in club-shaped processes at the cell periphery. Immunolabeling of placental fibrinoid was also observed. This report provides the first identification of a specific molecular constituent of decidual secretory bodies and indicates a role for these structures in secretion of the peri-decidual cell extracellular matrix.     

Immunocytochemical electron microscopic study and Western blot analysis of paramyosin in different invertebrate muscle cell types of the fruit flyDrosophila melanogaster, the earthwormEisenia foetida, and the snailHelix aspersa

Summary The presence and distribution pattern of paramyosin have been examined in different invertebrate muscle cell types by means of Western blot analysis and electron microscopy immunogold labelling. the muscles studied were: transversely striated muscle with continuous Z lines (flight muscle fromDrosophila melanogaster), transversely striated muscle with discontinuous Z lines (heart muscle from the snailHelix aspersa), obliquely striated body wall muscle from the earthwormEisenia foetida, and smooth muscles (retractor muscle from the snail and pseudoheart outer muscular layer from the earthworm). Paramyosin-like immunoreactivity was localized in thick filaments of all muscles studied. Immunogold particle density was similar along the whole thick filament length in insect flight muscle but it predominated in filament tips of fusiform thick filaments in both snail heart and earthworm body wall musculature when these filaments were observed in longitudinal sections. In obliquely sectioned thick filaments, immunolabelling was more abundant at the sites where filaments disappeared from the section. These results agree with the notion that paramyosin extended along the whole filament length, but that it can only be immunolabelled when it is not covered by myosin. In all muscles examined, immunolabelling density was lower in cross-sectioned myofilaments than in longitudinally sectioned myofilaments. This suggests that paramyosin does not form a continuous filament. The results of a semiquantitative analysis of paramyosin-like immunoreactivity indicated that it was more abundant in striated than in smooth muscles, and that, within striated muscles, transversely striated muscles contain more paramyosin than obliquely striated muscles.     

Ultrastructural localization of Tamm-Horsfall glycoprotein (THP) in rat kidney as revealed by protein A-gold immunocytochemistry

Summary The present study describes the intracellular distribution of Tamm-Horsfall protein (THP) in rat kidney. The localization was determined by immunoelectron microscopy using the protein A-gold technique. Various fixation and embedding protocols were evaluated for this purpose. Brief perfusion fixation (3 min) with 1% glutaraldehyde and embedding in a highly hydrophilic glycol methacrylate-polyester mixture were most appropriate for antigen-antibody recognition and structural preservation. The overall tissue distribution of THP was evaluated by indirect immunofluorescence microscopy; reaction was strong along the entire thick ascending limb of the loop of Henle (TAL) with enhanced fluorescence in the apical cytoplasm. On the electron microscopic level immunogold labelling was concentrated over numerous membrane-bound vesicles which form a compartment in the apical cytoplasm. The Golgi region was consistently labelled, whereas the plasma membranes revealed only sporadic labelling at the luminal side, and basolateral membranes were mostly unlabelled. Quantitative evaluation of the gold labelling, which was separately done for the inner stripe, outer stripe and cortical TAL, consistently showed the highest particle density in the apical cytoplasm. Middle and basal levels in the TAL cells were only moderately labelled. The results are discussed with respect to the current opinion which describes THP as a membrane glycoprotein. We speculate that the accumulation of THP in the apical vesicular compartment of TAL cells indicates a storage site of the protein, possibly prior to extrusion via exocytosis of the vesicle contents.     

黑缘烟蟋螽丝腺结构

【目的】蟋螽是直翅目中唯一具有吐丝筑巢行为的类群。本研究旨在探讨蟋螽丝腺的结构特点。【方法】应用解剖学观察、免疫荧光、苏木精-伊红染色、PAS苏木精染色、扫描电镜和透射电镜等方法从细胞水平对黑缘烟蟋螽Capnogryllacris nigromarginata丝腺的显微与超微结构进行了观察。【结果】黑缘烟蟋螽丝腺由导管和腺泡构成。腺泡由鞘细胞延伸形成的结缔组织鞘包围。腺泡的主体有4种细胞,分别为Ⅰ型分泌细胞、Ⅱ型分泌细胞、围细胞和腔细胞。Ⅰ型和Ⅱ型分泌细胞为大的腺细胞,形状不规则。分泌细胞细胞核很大,胞质内有大量的内质网和分泌颗粒。Ⅰ型分泌细胞靠近腺泡中心,PAS-苏木精染色表明Ⅰ型分泌细胞内含糖蛋白,Ⅱ型分泌细胞在腺泡外周,位于Ⅰ型分泌细胞与围细胞或结缔组织鞘之间。腔细胞分散在分泌细胞之间,包围形成胞外运输分泌物的通道。围细胞与鞘细胞接触,具有由细胞膜内陷形成的微绒毛腔,胞质内有大量的线粒体。围细胞微绒毛腔与腔细胞包围的细胞外运输通道相连,分泌细胞分泌的颗粒聚集在分泌细胞和胞外运输通道之间的连接处,并将分泌物排出至胞外运输通道。多个腺泡的胞外运输通道汇集到由单层细胞组成的丝腺导管。单层导管细胞靠近管腔外围具有规则排列的质膜内陷和大量伸长的线粒体;靠近管腔的一侧具连续的细胞膜突起,在导管壁的表皮下紧密排列。【结论】黑缘烟蟋螽丝腺分泌细胞分为Ⅰ型分泌细胞和Ⅱ型分泌细胞。分泌物质产生及分泌过程依次经过分泌细胞、腔细胞包围的胞外通道、分支导管、总导管和唾窦。其中在腺泡细胞之间,分泌物向外运输过程中,围细胞微绒毛腔的微丝束可能对分泌物的外排提供推动力。     

Ultrastructural localization of Tamm-Horsfall glycoprotein (THP) in rat kidney as revealed by protein A-gold immunocytochemistry

The present study describes the intracellular distribution of Tamm-Horsfall protein (THP) in rat kidney. The localization was determined by immunoelectron microscopy using the protein A-gold technique. Various fixation and embedding protocols were evaluated for this purpose. Brief perfusion fixation (3 min) with 1% glutaraldehyde and embedding in a highly hydrophilic glycol methacrylate-polyester mixture were most appropriate for antigen-antibody recognition and structural preservation. The overall tissue distribution of THP was evaluated by indirect immunofluorescence microscopy; reaction was strong along the entire thick ascending limb of the loop of Henle (TAL) with enhanced fluorescence in the apical cytoplasm. On the electron microscopic level immunogold labelling was concentrated over numerous membrane-bound vesicles which form a compartment in the apical cytoplasm. The Golgi region was consistently labelled, whereas the plasma membranes revealed only sporadic labelling at the luminal side, and basolateral membranes were mostly unlabelled. Quantitative evaluation of the gold labelling, which was separately done for the inner stripe, outer stripe and cortical TAL, consistently showed the highest particle density in the apical cytoplasm. Middle and basal levels in the TAL cells were only moderately labelled. The results are discussed with respect to the current opinion which describes THP as a membrane glycoprotein. We speculate that the accumulation of THP in the apical vesicular compartment of TAL cells indicates a storage site of the protein, possibly prior to extrusion via exocytosis of the vesicle contents.     

Ultrastructure of muscle cells in the adductor of the boring clam Tridacna crocea

The ultrastructure of the adductor muscle of the boring clam (Tridacna crocea) was investigated. The adductor was composed of opaque and translucent portions. The opaque portion contained smooth muscle cells; the translucent portion contained obliquely striated cells. Smooth muscle cells were classified, according to the statistically analyzed diameters of their thick myofilaments, into two types, S-1 and S-2. S-1 cells had thick myofilaments, 50–60 nm in diameter. S-2 cells had thick myofilaments of two sizes, about 55–65 nm and 85–100 nm in diameter, respectively. Obliquely striated muscle cells in the translucent portion were also classified into two types: O-1 cells, with thick myofilaments 30–35 nm in diameter, and O-2 cells, with myofilaments of 50–60 nm.     

Immunocytochemical localization of parathyroid hormone in hamster parathyroid gland

The immunocytochemical localization of parathyroid hormone was examined in the hamster parathyroid gland by using the protein A-gold technique. Protein A-gold particles were concentrated over secretory granules, large secretory granules thought to be storage granules and Golgi vacuoles. No protein A-gold particles were detected over large vacuolar bodies and cisternae of the granular endoplasmic reticulum.     

Binding sites of Ulex europaeus-lectin I in human parotid gland

Summary Twenty non-neoplastic parotid glands (removed during neck dissection for regional tumours) were examined for cellular and subcellular binding sites of Ulex europaeus-lectin I (UEA-I), a lectin reported to be specific for -L-fucose. For light microscopy, an extended peroxidase-antiperoxidase method was applied; for the evaluation of the subcellular localization of bound lectin, three of these glands were examined following immunocryoultramicrotomy and staining by the protein A-gold technique.In addition to the known cytoplasmic affinity of UEA-I for capillary endothelium, acinar cells bound the lectin within the cytoplasmic compartment; the number and distribution of stained acinar cells varied among individuals. Furthermore, cytomembrane-bound labelling that occurred most markedly at the luminar surface was observed in striated-duct epithelium.Using the electron microscope, protein A-gold particles were seen in zymogen granules and in Golgi cisternae of serous acinar cells; primary saliva secreted in the lumina exhibited strong labelling; serous acinar cells had binding sites on their cell membranes, striated-duct epithelium had binding sites on its surface membrane and in the vicinity of apical vesicles. Our results show that UEA-I is a useful tool for the study of the structure and functional states of the parotid gland epithelium and its associated pathological alterations.Dedicated to Prof. Dr. med. G. Seifert on the occasion of his 65th birthday     

Fine structure of the Malpighian tubules of chironomus larva in relation to glycogen storage and fate of hemoglobin

The larval Malpighian tubules of Chironomus tentans were studied using light and electron microscopy. The tubules are composed of two cell types: primary and stellate cells. Both cell types lack muscles, tracheoles, and laminate crystals in the cytoplasm and mitochondria in the microvilli. The primary cells exhibit long, wide basal membrane infoldings associated with mitochondria. They have a number of canaliculi and long, closely packed microvilli. The stellate cells possess shorter interconnecting basal infoldings and shorter, well-spaced microvilli. Both cell types are linked by septate and gap junctions. They have cytoplasmic processes and pedicels which enclose narrow slits between them and that are apposed to a basal lamella. In the 'fed' larva, the cells are stuffed with glycogen which is depleted in the 'starved' larva. Both cell types are involved in the vesicular transport of biliverdin. The presence of coated vesicles, tubular elements and various forms of lysosomes in the primary cells suggests they transport and break down functional hemoglobin. Structural modification of basal infoldings, canaliculi and microvilli is strongly correlated with increased secretory activity of the Malpighian tubules in 'fed' versus 'starved' larva.     

Cell types differing in thick myofilament diameter in obliquely striated muscle of a polychaete annelid, Neanthes

The fine structure of the obliquely striated muscle cells of the longitudinal muscle of an annelid was investigated. The characteristics of myofilaments and cell components, such as sarcoplasmic reticular system (S.R.), T-systems and J-rods corresponded to those previously reported, but it was noted for the first time that the cells could be classified into two types with respect to the diameters of their thick myofilaments. In one type, the thick myofilaments were about 29 nm in diameter (A-type) and in the other they were about 41 nm in diameter (B-type). Most of the obliquely striated muscles described to date have been composed of a single type of cell, but we found two types of cell mixed together in the longitudinal muscle. The A-type cells with slender thick myofilaments were distributed mainly in the inner part of the muscle and the B-type cells with broader thick myofilaments were distributed in the outer part.