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PubMed1994

Factors governing the appearance of small-mode miniature endplate currents at the snake neuromuscular junction.

At the snake neuromuscular junction, following a short period of high frequency motor nerve stimulation, a population of 'small-mode' miniature endplate currents (MEPCs) is seen. These small-mode MEPCs are believed to be due to the release of acetylcholine from incompletely refilled recycling synaptic vesicles [Searl et al., Neuroscience, 35 (1991) 145-156]. This study determines the role of the trans-vesicular membrane proton gradient in the generation of small-mode MEPCs. Preserving the trans-vesicular membrane proton gradient during synaptic vesicle exocytosis by exposure of snake nerve/muscle preparations to an extracellular pH approximately equal to the intravesicular pH, leads to an augmentation of the amplitude of the stimulation-induced small-mode MEPCs. Conversely, pretreatment of snake neuromuscular preparations with 10 mM ammonium ions, to buffer intravesicular protons and hence dissipate the trans-vesicular membrane proton gradient, leads to an inhibition of the stimulation-induced appearance of small-mode MEPCs. The data are consistent with the theory that the reduced acetylcholine content of recycled synaptic vesicles in the snake is a consequence of an incomplete restoration of the trans-vesicular membrane proton gradient following synaptic vesicle exocytosis. Thus, in recycling synaptic vesicles the limiting step in the refilling process appears to be generation of the trans-membrane proton gradient and not the transport of acetylcholine into the vesicle.

Ammonium Chloride↗

PubMed1986

Histochemical localization of acetylcholinesterase in relation to motor neurons and internuclear neurons of the cat abducens nucleus.

Three distinct patterns of AChE localization have been observed in relation to cat abducens motor neurons and internuclear neurons labelled by retrograde transport of horseradish peroxidase. First, AChE was localized predominantly within cisternae of granular endoplasmic reticulum and agranular reticulum of motor neuron somata, dendrites and axons, but was absent from internuclear neurons. AChE was also associated with saccules of the Golgi apparatus in the motor neurons, but was was absent from all other cytoplasmic organelles. Second, AChE was observed on the soma-dendritic and axonal surface membrane of the motor neurons, particularly at sites of apposition of synaptic endings of all morphological types, but was usually absent from the surface membranes of internuclear neurons. Third, AChE was associated both extracellularly and intracellularly with certain synaptic endings that contained spheroidal synaptic vesicles and that contacted both motor neurons and internuclear neurons. A similar pattern of staining of synaptic endings was observed at the neuromuscular junctions in the lateral rectus muscle. Axotomy of the VIth nerve resulted in loss of intracellular AChE associated with the Golgi apparatus and extracellular AChE on the somatic surface membrane of the motor neurons. The patterned localization of AChE contrasted with the localization of butyrylcholinesterase, which was associated predominantly with astrocytes. The findings suggest different roles of AChE as a function of the different patterns of localization.

Abducens Nerve↗

PubMed1992

Immunocytochemical localization of lysozyme and surfactant protein A in rat type II cells and extracellular surfactant forms.

Using immunogold labeling of fixed, cryosubstituted tissue sections, we compared the distribution of lysozyme, an oxidant-sensitive lamellar body protein, with that of surfactant protein A (SP-A) in rat Type II cells, extracellular surfactant forms, and alveolar macrophages. Morphometric analysis of gold particle distribution revealed that lysozyme and SP-A were present throughout the secretory and endosomal pathways of Type II cells, with prominent localization of lysozyme in the peripheral compartment of lamellar bodies. All extracellular surfactant forms were labeled for both proteins with preferential labeling of tubular myelin and unilamellar vesicles. Labeling of tubular myelin for SP-A was striking when compared with that of lamellar bodies and other extracellular surfactant forms. Lamellar body-like forms and multilamellar structures were uniformly labeled for lysozyme, suggesting that this protein is rapidly redistributed within these forms after secretion of lysozyme-laden lamellar bodies. By contrast, increased labeling for SP-A was observed over peripheral membranes of lamellar body-like forms and multilamellar structures, apparently reflecting progressive SP-A enrichment of these membranes during tubular myelin formation. The results indicate that lysozyme is an integral component of the lamellar body peripheral compartment and secreted surfactant membranes, and support the concept that lysozyme may participate in the structural organization of lung surfactant.

Animals↗

PubMed2000

Soluble neutral metallopeptidases: physiological regulators of peptide action.

Classically, the pre- and post-secretory processing of peptide signals appears to be mediated primarily by subtilisin-like peptidases in secretory vesicles and/or membrane-associated neutral endopeptidases in the extracellular environment. This article presents both biochemical and physiological evidence to support a role for soluble neutral metallopeptidases in the mediation of cell-to-cell communication by the selective generation and termination of peptide signals. These soluble peptidases have been implicated in the normal and disease-state processing of peptides involved in neurological, endocrine and cardiovascular functions. In this context, specific inhibitors of these enzymes could selectively modulate peptide levels and thus have considerable therapeutic potential. The aim of this review is to discuss the design and development of specific inhibitors of soluble neutral metallopeptidases that have been instrumental in identifying the roles of these enzymes. It will also review the evidence and present a case for the involvement of soluble neutral metallopeptidases in the regulation of peptide signalling in both central nervous system (CNS) and peripheral tissues.

Amino Acid Sequence↗

PubMed1991

Ultrastructural immunocytochemistry of the adenohypophysis in the rat: a review.

Immunocytochemistry has made great strides in the morphology of endocrine glands, especially the adenohypophysis, because the localization of hormones can be clearly demonstrated by this method in the microscopic preparations both for light and electron microscopy. In the adenohypophysis, electron microscopic immunocytochemistry is useful for identifying the producer cell of each hormone. The second contribution is its application to the cell biology of secretion mechanisms. The pituitary hormones, their precursors, derivatives, and fragments were artificially synthesized and their antibodies were produced. Using these antibodies the intracellular sites of synthesis, condensation, processing, and sorting were studied under the electron microscope. The ultrastructure of each cell organelle and its alteration due to the changing function was studied. It was proved that the intracisternal granules in the thyroidectomy cells contain thyroid-stimulating hormone (TSH). The trans-Golgi network or GERL contains a peculiar supporting structure, intracisternal skeleton. Transport of secretory granules may be performed in relation to the microtubules, actin, and some related substances. The most frequently observed mode of hormone release in the adenohypophysis is exocytosis. Sometimes multigranular exocytosis occurs. Vesiculation of membrane around the secretory granules often occur inward or outward. The inward vesiculation forms pinocytotic vesicles, through which the membrane material may be retrieved. The outward vesiculation forms vesicle-like fragments of cytoplasm being discarded to the extracellular space. By these mechanisms the surface area of the cell is maintained constantly.

Animals↗

PubMed2004

CASK is in the mammalian sperm head and is processed during epididymal maturation.

Upon adhesion to the zona pellucida or egg extracellular matrix, sperm undergo regulated exocytosis of the acrosomal vesicle. CASK is an adaptor protein that has been implicated in coupling neuronal cell adhesion to regulated exocytosis. In neurons, this scaffolding molecule is associated with several types of transmembrane receptor complexes and connects cell adhesion molecules with ion channels, the actin cytoskeleton, and the cell's exocytotic machinery. We hypothesized CASK might also be an important link between zona pellucida binding and the sperm acrosome reaction. RT-PCR experiments indicated CASK is transcribed in mouse testis. The full size (120 kDa) CASK protein was present in testis from mouse and pig. Immunoblots of mature porcine and murine sperm revealed that the 120 kDa molecule was much less abundant than in testis but the antibody also recognized a group of smaller proteins migrating at 55-65 kDa. Immunofluorescence experiments indicated both the full length and smaller CASK immunoreactive products were found only in the acrosomal region of spermatids and mature sperm and not in other testicular cell types. CASK immunofluorescence was lost following the acrosome reaction. During epididymal maturation, the abundance of the full size CASK decreased and the CASK fragments increased. These results suggest that CASK may be proteolytically processed during epididymal maturation. Because sperm acquire the ability to bind the zona pellucida, acrosome react, and fertilize eggs during epididymal maturation, CASK processing may play a role in the acquisition of these functions.

Acrosome↗

PubMed1993

Cysteine-proteinase localization in osteoclasts: an immunocytochemical study.

Cysteine-proteinases such as cathepsin B and G were localized in rat osteoclasts, by an indirect protein A-immunogold labeling technique, on post-embedded ultrathin sections. In osteoclasts, specific immunogold labeling of both anti-cathepsin B and G was localized in Golgi vesicles, lysosomes, pale vacuoles of various sizes, and the extracellular canals of ruffled borders; no immunoreactivity was seen in the cytoplasmic matrix, mitochondria, cisterns of the rough endoplasmic reticulum, or nuclei. The presence of immunolabeling of cathepsins in osteoclasts and in the subosteoclastic compartment suggests that these enzymes are involved in the extracellular degradation of collagen and other non-collagenous bone matrix proteins.

Animals↗

PubMed1986

Vitamin-D-dependent calcium-binding protein (CaBP-9K) in rat growth cartilage.

The presence of vitamin-D-dependent calcium-binding protein (CaBP-9K) in tibial growth-plate cartilage was immunohistochemically demonstrated using a specific antibody to rat duodenal CaBP-9K. The protein was found to be mainly localized in the cytoplasm of maturing chondrocytes. In hypertrophic chondrocytes, CaBP-9K concentrations decreased, and the protein was found in the cytoplasmic processes. No CaBP-specific immunoreactivity was seen in the hypertrophic chondrocytes of the lower calcified hypertrophic zone; in contrast, the protein was found in the extracellular lateral edges of longitudinal septa, i.e. where matrix vesicles are preferentially localized and where cartilage mineralization is initiated. These findings suggest that vitamin D has a direct function in this tissue. It also seems likely that CaBP-9K is an indicator of chondrocyte maturation, and that it is involved in the matrix vesicle-associated process of cartilage calcification.

Animals↗

PubMed1980

Ultrastructural localization of calcium in the mandibular condylar growth cartilage of the rat.

The potassium pyroantimonate technique was utilized for the selective subcellular localization of calcium in the mandibular condylar cartilage of 1-day-old rats. Electron dense calcium pyroantimonate precipitates were localized principally in mitochondria and at the cell membrane of the chondrocytes. In addition, small intracellular vesicles 0.1-0.2 micrometers in diameter were observed in proximity to the cell membrane of chondrocytes of the mid-hypertrophic zone. The results suggest that these vesicles were being extruded from the cell into the extracellular matrix. Energy-dispersive analysis by X-rays confirmed that calcium is the principal cation of the electron-dense precipitates.

Animals↗

PubMed2006

An ultrastructural study on the ligamentum flavum of the cervical spine in patients with ossification of the posterior longitudinal ligament.

Some histological analyses of the ossification of the posterior longitudinal ligament (OPLL) have been reported, but no ultrastructural studies of the ligamentum flavum (LF) in patients with OPLL have been published to date. To understand the pathology of the ossification of the spinal ligament, we examined, by electron microscopy, ultrastructural changes in the LF in cases of OPLL and made a comparison with the LF in cervical spondylotic myelopathy (CSM). Subjects were three men and two women with cervical OPLL who underwent longitudinal spinous process-splitting laminoplasty. During surgery, a small piece of the LF was collected from C2-C3 to C7-T1 and was then analyzed by light and electron microscopy. We observed atrophic elastic bundles with a two-layer structure and disarrangement, a partially torn area, the disappearance of microfibrils, and an enlarged interstitium with an irregular alignment of collagen fibrils. We observed some properties of a cell preceding its death: the initial phase may be the disappearance of the plasma-membrane, followed by the scattering of many organellae around its degenerated nucleus. Finally, many extracellular plasma membrane-invested particles that resemble matrix vesicles remain there without phagocytosis. These results suggest that ultrastructural abnormalities exist in the spinal ligament in cases of ossification of the spinal ligament.

Aged↗

PubMed1991

ATP-dependent leukotriene export from mastocytoma cells.

The biosynthesis of leukotrienes (LT) C4 and B4 is followed by an export of these mediators into the extracellular space. This transport was characterized using plasma membrane vesicles prepared from mastocytoma cells and identified as an ATP-dependent primary active process. The apparent Km-values were 110 nM for LTC4 and 48 microM for ATP. The transport rate was highest for LTC4, whereas LTD4, LTE4, and N-acetyl-LTE4 were transported with relative rates of 31, 12 and 8%, respectively, at a concentration of 10 nM. LTB4 transport was also dependent on ATP. LTC4 transport was inhibited by LTD4 receptor antagonists (IC50 = 1.0 microM for MK-571 and 1.3 microM for LY245769) and by the inhibitor of leukotriene biosynthesis MK-886 (IC50 = 1.8 microM). The ATP-dependent export carrier for leukotrienes in leukotriene-synthesizing cells represents a novel member of the family of ATP-dependent exit pumps.

Adenosine Triphosphate↗

PubMed1976

Light- and electron-microscopic characteristics of artrial smooth muscle cell cultures subjected to hypoxia or carbon monoxide.

Smooth muscle cells from the tunica media of piglet aortae grown under hypoxic conditions undergo the following changes: First, they become modified by partial loss of myofilaments and proliferation of organelles, which are characteristics of young primitive cells. Second, an increase in number of pinocytotic vesicles at and below the cell surface, indicating increased extracellular uptake of material, can be detected. This is followed by accumulation of Oil Red O positive intracytoplasmic granules and vacuoles as well as the subsequent formation of mount-like protrusions. The latter consist of a core of extracellular material and necrotic debris covered with a cap of viable cells. A third feature of the cells subjected to hypoxia is a conspicuous rise in the number of lysosomes. This is considered to be a manifestation of a defense mechanism of the cells to remove undesirable material from cytoplasm. Cells exposed to an atmosphere rich in carbon monoxide exhibit basically the same alterations as those grown under hypoxic conditions; however, formation of mound-like aggregates is less prominent, while the rise in the number of lysosomes is more evident than in the hypoxic cells. The above alterations are similar to changes observed in smooth muscle cells of rabbit with experimental atherosclerosis. It is suggested that whereever the arterial smooth muscle cell is subjected to adverse conditions basically the same mechanism, consisting of dedifferentiation, increased permeability and lysosomal defense reaction, takes place.

Animals↗

PubMed1984

Ultracytochemical distribution of myelin basic protein after injection into the cerebrospinal fluid. Evidence for transport through the blood-brain barrier and binding to the luminal surface of cerebral veins.

Distribution of myelin basic protein (MBP) in the central nervous system (CNS) following injection into the cerebrospinal fluid (CSF) was studied by different qualitative and quantitative immunelectron -microscopic techniques. Endogenous MBP was present in myelin sheaths in injected as well as in control animals. After injection of exogenous MBP into CSF this protein was present in the subarachnoid space, on the surface of meningeal cells, on the surface of collagen fibers, in the basement membrane of the glia limitans, in vessel walls, and in the extracellular space of spinal roots. In meningeal veins, endothelial vesicles filled with peroxidase reaction product were found on the abluminal side of endothelial cells, in the endothelial cytoplasm and sometimes opening into the vascularllumen . In addition patchy staining of the luminal surface of endothelial cells was noted, indicating binding of antigen at this location. Quantitative immunelectron microscopy (an indirect technique with rabbit anti-MBP serum as primary layer and gold-labeled anti-rabbit IgG as secondary layer) revealed highly significant MBP binding on the luminal surface of endothelial cells after injection of this antigen into the CSF. The present results indicate that MBP, when liberated in CNS is transported through the blood-brain barrier and presented on the luminal surface of endothelial cells of the cerebral and meningeal veins. This observation may be important in interpretation of pathogenesis of initial inflammatory infiltrates in experimental allergic encephalitis (EAE).

Animals↗

PubMed1996

Apoptosis: programmed cell death in fetal development.

Controlled death of cells is as much a part of embryonal development as is cell proliferation and differentiation. This cell suicide is controlled by cell genes involved in induction or prevention of programmed cell death (PCD). During embryogenesis PCD implicates cell elimination, necessary in fashioning of the body, moulding of tissues. PCD is often used synonymous with the designation apoptosis, which indicates an endogenous cell suicide program by which useless or crippled cells are eliminated. Apoptosis occurs in normal tissue turnover, in organ involution after withdrawal of trophic hormones, in distinct cells after deprivation of growth factors or specific stimuli and in cells which have undergone sublethal damage. During PCD the nucleus breaks up into DNA fragments of 180-200 kD and the endoplasmic reticulum transforms into vesicles, which are released as apoptotic bodies into the extracellular space. The apoptotic bodies, containing cell organelles and nuclear fragments, are phagocytosed by neighboring cells. Electron micrographs of embryos have revealed the presence of numerous cells with the characteristic features of apoptosis. Experiments, in which small tissue fragments were explanted to other regions, have proven that focal apoptosis is under control of genetic, hormonal and local tissue factors. Morphological analysis has shown that most of the ovarian follicles undergo during development apoptosis, resulting in follicle atresia. Only a small proportion escapes PCD. Growth factors and estrogens have been identified as follicle survival factors, androgens and gonadotropin releasing hormones are potentiating apoptosis of the follicle. An exaggerated PCD or a defective apoptosis during embryogenesis may cause developmental abnormalities. Certain viruses can inhibit apoptosis, while metabolic stress or damage of cell structures can induce apoptosis. Therefore not only viral infections, also drugs and chemical or physical injuries during embryogenesis may interfere with the balanced PCD and thus induce malformations. Drugs and therapy designs directed to modulate the apoptotic process will offer new approaches to the prevention of congenital malformations.

Animals↗

PubMed1989

Endocytosis of cationized ferritin by rat photoreceptors.

Endocytosis by rods of the rat retina was studied at the ultrastructural level with cationized ferritin. Endocytosed ferritin was found within all photoreceptor subdivisions except the outer segment. Within the cell, synaptic vesicles, lysosomes and a variety of other membrane-bound organelles including the axonal agranular reticulum contained ferritin. These findings indicate that surface membrane and materials bound to surface membrane are recycled to form synaptic vesicles, that a portion of the captured membrane and extracellular material enters a lysosomal pathway, and that endocytosed materials can be retrogradely transported to the myoid region of the photoreceptor.

Animals↗

PubMed2005

Calcification as an indicator of osteoinductive capacity of biomaterials in osteoblastic cell cultures.

Mineralized extracellular matrix formation is representative for the osteoinductive capacity of biomaterials and is often tested in vitro. Characteristics of in vitro mineralization of primary rat osteoblastic cells (bone marrow, calvaria, periosteum, fetal and adult long bone) and UMR-106 cells were compared by von Kossa staining, FTIR, X-ray diffractometry, TEM and related to parameters of early (ALP and collagen I formation) and late (osteocalcin secretion) osteoblast expression. All cultures expressed high alkaline phosphatase activity and were able to form bone apatite. However, a nodular versus diffuse mineralization pattern was observed. Bone marrow, calvaria and periosteum (early passage) derived cells mineralized restrictively on the three-dimensional area of a nodule. The extracellular matrix consisted of collagen I fibers, among matrix vesicles loaded with needle-like crystals. Long bone, late passage periosteum derived and UMR-106 cells exhibited a diffuse mineralization pattern. Needle-like crystals were observed between the cells but collagen fibers and matrix vesicles could not be detected. Secretion of osteocalcin was detected in cultures derived from bone marrow and absent in UMR-106 and long bone derived cell cultures. The present study demonstrates that dystrophic calcification can not be distinguished from cell-mediated calcification with von Kossa, FTIR and X-ray diffractometry. Primary osteoblastic cells capable of forming nodules are recommended to evaluate the osteoinductive properties of biomaterials.

Animals↗

PubMed2004

Mitochondrial dysfunction in osteoarthritis.

In osteoarthritis (OA) a time or age dependent process leads to aberrant cartilage structure which is characterized by reduced number of chondrocytes, loss of existing cartilage extracellular matrix, the production of matrix with abnormal composition and pathologic matrix calcification. Because chondrocyte matrix synthesis and mineralization are modulated by the balance between ATP generation and consumption, the mechanism by which chondrocytes generate energy have been a topic of interest. The analysis of mitochondrial respiratory chain (MRC) activity in OA chondrocytes shows a significant decrease in complexes II and III compared to normal chondrocytes. On the other hand, mitochondrial mass is increased in OA, as demonstrated by a significant rise in CS activity. Furthermore, OA cells show a reduction in the mitochondrial membrane potential (deltapsim) as demonstrated by using the fluorescent probe JC-1. OA cartilage contains high number of apoptotic chondrocytes, and mitochondria play a key role in apoptosis. Interestingly, OA cartilages show markedly elevated Bcl-2 and caspasa-3 expression. This expression is also correlated with chondrocyte apoptosis and OA lesions. The pathogenesis of OA includes elaboration of increased amounts of NO as a consequence of up-regulation of chondrocyte-inducible NO synthase induced by IL-1, TNF-alpha and other factors. NO reduces chondrocyte survival and induces cell death with morphologic changes characteristic of chondrocyte apoptosis. NO reduces the activity of complex IV and decreases the deltapsim as measured as the ratio of red/green fluorescence. Furthermore, NO induces the mRNA expression of caspase-3 and -7, and it reduces the expression of mRNA bcl-2 and the bcl-2 protein synthesis. Some studies suggest that the chondrocyte mitochondria are specialized for calcium transport and are important in the calcification of the extracellular matrix. Mineral formation has been demonstrated in matrix vesicles (MV) and within mitochondria. Direct suppression of mitochondrial respiration promoted MV-mediated mineralization in chondrocytes. Regulation of MRC may be one of the signaling pathways by which NO modulates articular cartilage matrix biosynthesis and pathologic mineralization. After age 40, the incidence of OA in humans increases progressively with increasing age. Studies show a trend to statistic significance between the age and the reduction of complex I activity of human normal chondrocytes. However, the study of relation between age and deltapsim in normal chondrocytes do not demonstrate any significant correlation. It has been reported that as the number of population doublings increased, mitochondrial DNA was degraded and the number of mitochondria per chondrocyte decline. One approach for determining the role of mitochondria in OA is to determine the effects of the MRC inhibition and to compare them with the findings in OA. Inhibition of MRC with antimycin prevents the normal ability of TGFbeta to increase excretion of Pi, thereby worsening deposition of pathologic HA crystals. In chondrocytes, the inhibition of complex IV with NaN3 modified both the deltapsim and the survival of cells inducing apoptosis. Inhibition of complex I with rotenone increases the expression and synthesis of Bcl-2 and Cox-2, both effects are similar effects to produced by IL-1 in human chondrocytes.

Journal Article↗

PubMed2000

Neuronal and glial cell biology.

Here, we review progress in our understanding of neuronal and glial cell biology during the past ten years, with an emphasis on glial cell fate specification, apoptosis, the cytoskeleton, neuronal polarity, synaptic vesicle recycling and targeting, regulation of the cytoskeleton by extracellular signals, and neuron-glia interactions.

Animals↗