Calcium-Binding Properties of the Mitochondrial Channel-Forming Hydrophobic Component

A hydrophobic, low-molecular weight component extracted from mitochondria forms aCa²⁺-activated ion channel in black-lipid membranes (Mironova et al., 1997). At pH 8.3–8.5, thecomponent has a high-affinity binding site for Ca²⁺ with a K_d of 8 × 10^–6 M, while at pH7.5 this K_d was decreased to 9 × 10^–5 M. B_max for the Ca²⁺-binding site did not changesignificantly with pH. In the range studied, 0.2 ± 0.06 mmol Ca²⁺/g component were boundor one calcium ion to eight molecules of the component. The Ca²⁺ binding was stronglydecreased by 50–100 mM Na⁺, but not by K⁺. Treatment of mitochondria withCaCl₂ priorto ethanolic extraction resulted in a high level of Ca²⁺-binding capacity of the partially purifiedcomponent. Cyclosporin A, a specific inhibitor of the mitochondrial permeability transition,when added to the mitochondrial suspension, decreased the Ca²⁺-binding activity of thepurified extract severalfold. The calcium-binding capability of the partially purified componentcorrelates with its calcium-channel activity. This indicates that the channel-forming componentmight be involved in the permeability transition that stimulates its formation.     

杨树顶芽细胞内质网与其他膜系统的结构联系及其在休眠过程中的变化

杨树（Ｐｏｐｕｌｕｓ ｄｅｌｔｏｉｄｅｓ Ｂａｒｔｒ．ｅｘ Ｍａｒｓｈ）顶芽分生组织细胞经一种改良的高锰酸钾固定法固定后,显示出一种十分清晰的内膜结构,尤其展现了内质网与其他膜系统存在一种结构上的密切联系。一些与核膜相连接的内质网伸展到细胞质中与线粒体、质体及高尔基体发生联系,可延伸到质膜。还有些内质网的一端与一个细胞的核膜相连结,其另一端穿过胞间连丝与邻近的另一个细胞的核膜相连结,在两个相邻的细     

A Modification of Mayer's Tannic Acid-Ferric Chloride Staining Method for Demonstrating Cellular Membranous Systems for Light Microscopy

To observe cellular membranous systems under a light microscope, we modified Mayer's tannic acid-ferric chloride stain method by adding a treatment with hematoxylin after the original procedure. We used the modified tannic acid-ferric chloride (MTA-Fe) stain method to examine kidneys, liver, heart, trachea, epididymides and other organs of rats and dogs. The MTA-Fe stain clearly demonstrated the basement membrane, brush border, basolateral invaginations and cell processes in the kidneys which enabled easy differentiation of the S1 and S3 segments of proximal convoluted tubules. Our technique also demonstrated hepatic cell membranes and bile canaliculi in the liver, cross striations and longitudinal traveling of myofibrils in the heart, cilia of the epithelial cells in the trachea, and stereocilia and terminal bars in the epididymis. The MTA-Fe stain is a convenient method to visualize cellular membranous systems even for light microscopy. The stain has the advantages of using no toxic materials, simple and easy technique, little variation of staining results, and little fading for several months after staining.     

Three-dimensional ultrastructural analysis of peroxisomes in HepG2 cells

Peroxisomes in the human hepatoblastoma cell line, HepG2, exhibit distinct alterations of shape, size, and distribution, dependent on culture conditions (cell density, duration in culture, and presence of specific growth factors). Although many cells with elongated tubular peroxisomes are present in thinly seeded cultures, spherical particles forming large focal clusters are found in confluent cultures. The authors have analyzed the ultrastructure and the spatial relationship of peroxisomes of HepG2 cells at different stages of differentiation, using three-dimensional (3D)-reconstruction of ultrathin serial sections, and electronic image processing. Cells were prepared for immunofluorescence using different antibodies against peroxisomal matrix and membrane proteins, as well as for electron microscopy after the alkaline 3,3′-diaminobenzidine staining for catalase. The results indicate that the tubular peroxisomes, which can reach a length of several microns, are consistently isolated, and never form an interconnected peroxisomal reticulum. At the time of disappearance of tubular peroxisomes, rows of spherical peroxisomes, arranged like beads on a string, are observed, suggesting fission of tubular ones. In differentiated confluent cultures, clusters of several peroxisomes are seen, which, by immunofluorescence, appear as large aggregates, but after 3D reconstruction consist of single spherical and angular peroxisomes without interconnections. The majority of such mature spherical peroxisomes (but not the tubular ones) exhibit tail-like, small tubular and vesicular attachments to their surface, suggesting a close functional interaction with neighboring organelles, particularly the endoplasmic reticulum, which is often observed in close vicinity of such peroxisomes.     

Recording of calcium transient and analysis of calcium removal mechanisms in cardiac myocytes from rats and ground squirrels

With confocal microscopy, we recorded calcium transients and analyzed calcium removal rate at different temperatures in cardiac myocytes from the rat, a non-hibernator, and the ground squirrel, a hibernator. The results showed a remarkable increase of the diastolic level of calcium transients in the rat but no detectable change in the ground squirrel. Calcium transient of the ground squirrel, compared with that of the rat at the same temperature, had a shorter duration and showed a faster calcium removal. As indicated by the pharmacological effect of cyclopiazonic acid, calcium uptake by sarcoplasmic reticulum (SR) was the major mechanism of calcium removal, and was faster in the ground squirrel than in the rat. Our results confirmed the essential role of SR in hypothermia-tolerant adaptation, and negated the importance of Na-Ca exchange. We postulated the possibility to improve hypothermia-tolerance of the cardiac tissue of non-hibernating mammals.     

内质网膜复合亚基3调控内质网应激影响人畸胎瘤细胞的存活

内质网膜蛋白复合物(endoplasmic reticulum membrane complex,EMC)在跨膜蛋白质的生物发生和膜整合中发挥重要作用.内质网膜复合亚基3 (endoplasmic reticulum membrane complex 3,EMC3)是EMC的重要组成部分,但其在生殖细胞中发挥的作用未见...     

Binding of the erlin1/2 complex to the third intralumenal loop of IP3R1 triggers its ubiquitin-proteasomal degradation

Long-term activation of inositol 1,4,5-trisphosphate receptors (IP₃Rs) leads to their degradation by the ubiquitin–proteasome pathway. The first and rate-limiting step in this process is thought to be the association of conformationally active IP₃Rs with the erlin1/2 complex, an endoplasmic reticulum–located oligomer of erlin1 and erlin2 that recruits the E3 ubiquitin ligase RNF170, but the molecular determinants of this interaction remain unknown. Here, through mutation of IP₃R1, we show that the erlin1/2 complex interacts with the IP₃R1 intralumenal loop 3 (IL3), the loop between transmembrane (TM) helices 5 and 6, and in particular, with a region close to TM5, since mutation of amino acids D-2471 and R-2472 can specifically block erlin1/2 complex association. Surprisingly, we found that additional mutations in IL3 immediately adjacent to TM5 (e.g., D2465N) almost completely abolish IP₃R1 Ca²⁺ channel activity, indicating that the integrity of this region is critical to IP₃R1 function. Finally, we demonstrate that inhibition of the ubiquitin-activating enzyme UBE1 by the small-molecule inhibitor TAK-243 completely blocked IP₃R1 ubiquitination and degradation without altering erlin1/2 complex association, confirming that association of the erlin1/2 complex is the primary event that initiates IP₃R1 processing and that IP₃R1 ubiquitination mediates IP₃R1 degradation. Overall, these data localize the erlin1/2 complex–binding site on IP₃R1 to IL3 and show that the region immediately adjacent to TM5 is key to the events that facilitate channel opening.     

Battling for Ribosomes: Translational Control at the Forefront of the Antiviral Response

In the early stages of infection, gaining control of the cellular protein synthesis machinery including its ribosomes is the ultimate combat objective for a virus. To successfully replicate, viruses unequivocally need to usurp and redeploy this machinery for translation of their own mRNA. In response, the host triggers global shutdown of translation while paradoxically allowing swift synthesis of antiviral proteins as a strategy to limit collateral damage. This fundamental conflict at the level of translational control defines the outcome of infection. As part of this special issue on molecular mechanisms of early virus–host cell interactions, we review the current state of knowledge regarding translational control during viral infection with specific emphasis on protein kinase RNA-activated and mammalian target of rapamycin-mediated mechanisms. We also describe recent technological advances that will allow unprecedented insight into how viruses and host cells battle for ribosomes.     

内质网靶向纳米药物的研究进展

内质网作为真核细胞内极为重要的细胞器,在生物大分子合成与加工、物质转运、离子稳态维持、信号转导、细胞器间物质与信号交流等诸多方面发挥关键作用,其功能异常与癌症、自身免疫疾病、病原微生物感染、神经退行性疾病、糖尿病等诸多重大疾病紧密相关.随着纳米技术的不断发展,内质网靶向纳米药物的开发与应用逐渐成为生物工程、纳米医学、材...     

Ultrastructure of retrocerebral complex of the earwig, Euborellia annulipes, and rôle of aorta as a neurohaemal organ

The ultrastructure of the retrocerebral endocrine-aortal complex of the earwig, Euborellia annulipes has been studied. The space between the inner and outer stromal layers of the aorta is occupied by numerous axon terminals and pre-terminals containing large electron dense granules (NS-I) of approximately 100 to 220 nm and a few axon terminals having small granules (NS-II) of approximately 40 to 90 nm; the former appear to belong to medial neurosecretory A-cells, and the latter to the B-cells of the brain. The corpora cardiaca consist of intrinsic cells with mitochondria and multivesicular bodies. Granules of type NS-II and NS-III are observed in the axon terminals and pre-terminals in the corpora cardiaca. The NS-II are identical to those found in the aorta and are probably the secretions of the lateral B-cells. Granules of type NS-III are 40 to 120 nm and electron dense, and are intrinsic in origin. Similar granules occur in the intrinsic cells of the corpora cardiaca. E M studies have confirmed the rôle of the aorta as a neurohaemal organ for the medial neurosecretory cells, and the corpora cardiaca for the lateral neurosecretory cells of the brain. The corpora cardiaca also act as a reservoir for the intrinsic secretion. The corpus allatum is a solid body consisting of parenchymal cells with prominent nuclei, mitochondria, and endoplasmic reticulum. In between its cells are occasional glial cells and also neurosecretory as well as non-neurosecretory axons. The gland is devoid of A-cell NSM.