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PubMed1998

In vivo autopolysialylation and localization of the polysialyltransferases PST and STX.

A select group of mammalian proteins have been shown to possess alpha2,8-polysialylated oligosaccharide chains. The best studied of these proteins is the neural cell adhesion molecule (NCAM). Polysialylation of NCAM has been shown to decrease NCAM-dependent and independent cell adhesion. PST (ST8Sia IV) and STX (ST8Sia II) are the two polysialyltransferases responsible for NCAM polysialylation. Recent studies revealed that PST itself is autopolysialylated in vitro (Muhlenhoff, M., Eckhardt, M., Bethe, A., Frosch, M., and Gerardy-Schahn, R. (1996) EMBO J. 15, 6943-6950). Here we report studies on the biosynthesis and localization of the PST and STX polysialyltransferases. Both PST and STX are expressed as high molecular mass, polydisperse forms that are associated with the cell and found soluble in the medium. Analysis of these high molecular mass forms by glycosidase digestion and serial immunoprecipitation/immunoblot experiments demonstrated that PST and STX are autopolysialylated in vivo. Indirect immunofluorescence microscopy and immunoprecipitation analyses demonstrated that autopolysialylated PST and STX are localized in the Golgi, on the cell surface, and in the extracellular space. The cell surface and extracellular localization of these polysialylated polysialyltransferases suggest that their polysialic acid chains, like those of NCAM, may modulate cell interactions.

Animals↗
PubMed2004

Fatty acid synthase as a tumor marker: its extracellular expression in human breast cancer.

Overexpression of fatty acid synthase (FAS EC 2.3.1.85) is associated with certain cancers and therefore is a putative tumor marker. The presence of FAS in patients with breast, prostate, colon, ovarian, and other cancers has been reported. The mechanism of FAS overexpression in malignancies remains unknown. Here, we show that FAS is released into the extracellular space in cancer cells. The extracellular FAS are present in various immunoreactive forms, and show different expression patterns in various cancer cells. In serum of breast cancer patients, the FAS is a small molecule similar to the form in breast cancer cell lysate but not conditioned medium of cultured cells. The extracellular expression of FAS in breast cancer cells is time dependent and may be hormone independent. These results indicate that the FAS are an ordered cellular response of a living cell and actively exclude excess intracellular FAS molecules from the cell. This phenomenon is up-regulated in breast and may be in other cancer cells as well. Significant elevation of FAS was detected in serum of breast cancer patients compared to healthy subjects. In comparison with CA27.29, no correlation between these two tumor markers was found. Thus, the extracellular FAS may serve as a potential diagnostic and prognostic marker.

Biomarkers, Tumor↗
PubMed1997

Evaluation of extracellular lipid peroxidation in brain cortex of anaesthetized rats by microdialysis perfusion and high-performance liquid chromatography with fluorimetric detection.

A method for in vivo evaluation of lipid peroxidation in the extracellular space of anaesthetized rat brain cortex was developed. This method involved the use of microdialysis perfusion and high-performance liquid chromatography. The microdialysates, eluted from implanted probes, were reacted with thiobarbituric acid (TBA) prior to analysis by an HPLC system equipped with a fluorescence detector (excitation and emission wavelengths were 515 and 550 nm, respectively). Lipid peroxidation in the extracellular space was evaluated as the concentration of malondialdehyde, a lipid peroxidation end product which reacts with TBA to form a fluorescent conjugate. Significantly increased production of malondialdehyde following hydrogen peroxide perfusion (0.03%, 0.3% at a flow-rate of 1 microl/min) was observed in the brain cortex of anaesthetized rats.

Anesthesia↗
PubMed1979

The evidence of production or activation of a natriuretic factor in the liver.

The natriuretic activity was measured with the aid of bioassay in blood flowing out from the brain, kidney and liver before and after the expansion of extracellular space in dogs. In another experiments in rats the volume natriuresis and the natriuretic activity in blood were measured in controls and in animals with carbon tetrachloride liver dystrophy and pharmacological stimulation (dehydrocholic acid, Ca pantothenate, cyanocobalamin) of liver functions. In dogs, the level of natriuretic activity in blood flowing out of the liver before and after the expansion of extracellular fluid volume was significantly higher than that in arterial blood. In contrast, the natriuretic activity was not found in the blood flowing out of the brain and the kidney. In rats the experimental dystrophy of the liver decreased the content of a natriuretic factor in blood plasma and the expansion of extracellular space did not produce any natriuretic effect as compared to control rats. After pharmacological stimulation of the liver by dehydrocholic acid, Ca pantothenate and cyanocobalamine the volume expansion led to a significant increase of the excretion of sodium by the kidney and to the increase of the content of a natriuretic factor in blood plasma as compared to control rats. These facts are considered to support the view that a natriuretic factor is either synthesized or activated by the liver.

Animals↗
PubMed2004

Water transport in the brain: role of cotransporters.

It is generally accepted that cotransporters transport water in addition to their normal substrates, although the precise mechanism is debated; both active and passive modes of transport have been suggested. The magnitude of the water flux mediated by cotransporters may well be significant: both the number of cotransporters per cell and the unit water permeability are high. For example, the Na(+)-glutamate cotransporter (EAAT1) has a unit water permeability one tenth of that of aquaporin (AQP) 1. Cotransporters are widely distributed in the brain and participate in several vital functions: inorganic ions are transported by K(+)-Cl(-) and Na(+)-K(+)-Cl(-) cotransporters, neurotransmitters are reabsorbed from the synaptic cleft by Na(+)-dependent cotransporters located on glial cells and neurons, and metabolites such as lactate are removed from the extracellular space by means of H(+)-lactate cotransporters. We have previously determined water transport capacities for these cotransporters in model systems (Xenopus oocytes, cell cultures, and in vitro preparations), and will discuss their role in water homeostasis of the astroglial cell under both normo- and pathophysiologal situations. Astroglia is a polarized cell with EAAT localized at the end facing the neuropil while the end abutting the circulation is rich in AQP4. The water transport properties of EAAT suggest a new model for volume homeostasis of the extracellular space during neural activity.

Amino Acid Transport System X-AG↗
PubMed1990

The intracellular movement and cycling of ricin.

The binding, internalization and recycling of the plant toxin ricin, was studied using electron microscopy and biochemical techniques. For the electron microscope study, ricin was visualized using a gold-labeled second antibody, in the cells of the EJ human bladder carcinoma line growing in monolayer culture. The labeled antibody/toxin complex was found to enter the cell in coated pits and to accumulate in endosomes and to a lesser extent in vesicles associated with the Golgi system. The complex recycled to the cell surface partly in uncoated vesicles, but largely in multivesicular bodies which appeared to exocytose their contents to the extracellular space. Twenty hours after the initial contact with ricin as much as 50% of the cellular label was found on the cell surface mainly associated with shed vesicles. When cells were treated with unlabeled ricin holotoxin and then after 20 h stained post-fixation, ricin molecules, partly associated with vesicles, were present on the cell surface. Biochemical studies showed that ricin was internalized by cells and then released in an intact form to the extracellular space. It was found that less than 10% of the released material had been degraded during its passage through the cells, which is in accord with the low level of label found in the lysosomal system during the morphological study.

Antitoxins↗
PubMed1990

Molecular and cellular characterization of the glial roof plate of the spinal cord and optic tectum: a possible role for a proteoglycan in the development of an axon barrier.

Certain types of glial structures, located at strategic positions along axon pathways, may provide the mechanical and/or chemical elements for the construction of barriers which can grossly direct the elongation of axons during development. The roof plate, a putative axon barrier, is located along the dorsal midline of the developing spinal cord and may be important for the guidance of the commissural and dorsal column axons. We examined the roof plate to determine the developmental morphology of the region and to determine which molecules were correlated with the barrier function when axons were growing nearby. Light and electron microscopic observations of the roof plate revealed that this glial domain undergoes a dramatic change in shape from a "wedge" with large extracellular spaces between the cell apices at E12.5 to a thin, dense septum with reduced extracellular space at E15.5. Immunocytochemical techniques demonstrated that highly sialylated neural cell adhesion molecule (N-CAM), the carbohydrate recognized by L2 monoclonal antibody, cholinesterase, stage-specific embryonic antigen 1, and a ligand that binds tetragonolobus purpureas agglutinin are expressed by the roof plate. These molecules, however, were also found in other regions of the spinal cord which are permissive or attractive to axon growth. A molecule which is unique to the roof plate when axons grow close to, but do not cross, the dorsal midline is a glycosaminoglycan (GAG), keratan sulfate. Keratan sulfate is also present in the tectal midline and in other noninnervated regions such as the outer epidermis and developing cartilage. Our data suggest that keratan sulfate, alone or in combination with other molecules expressed by the roof plate, may be responsible, in part, for the inhibition of axon elongation through the roof plate in the embryonic spinal cord.

Animals↗
PubMed2004

Subcellular topography of neuronal Abeta peptide in APPxPS1 transgenic mice.

In transgenic mice expressing human mutant beta-amyloid precursor protein (APP) and mutant presenilin-1 (PS1), Abeta antibodies labeled granules, about 1 microm in diameter, in the perikaryon of neurons clustered in the isocortex, hippocampus, amygdala, thalamus, and brainstem. The granules were present before the onset of Abeta deposits; their number increased up to 9 months and decreased in 15-month-old animals. They were immunostained by antibodies against Abeta 40, Abeta 42, and APP C-terminal region. In double immunofluorescence experiments, the intracellular Abeta co-localized with lysosome markers and less frequently with MG160, a Golgi marker. Abeta accumulation correlated with an increased volume of lysosomes and Golgi apparatus, while the volume of endoplasmic reticulum and early endosomes did not change. Some granules were immunolabeled with an antibody against flotillin-1, a raft marker. At electron microscopy, Abeta, APP-C terminal, cathepsin D, and flotillin-1 epitopes were found in the lumen of multivesicular bodies. This study shows that Abeta peptide and APP C-terminal region accumulate in multivesicular bodies containing lysosomal enzymes, while APP N-terminus is excluded from them. Multivesicular bodies could secondarily liberate their content in the extracellular space as suggested by the association of cathepsin D with Abeta peptide in the extracellular space.

Amyloid beta-Peptides↗
PubMed2006

G protein betagamma-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties.

Neurotransmitters are thought to be released as quanta, where synaptic vesicles deliver packets of neurotransmitter to the synaptic cleft by fusion with the plasma membrane. However, synaptic vesicles may undergo incomplete fusion. We provide evidence that G protein-coupled receptors inhibit release by causing such incomplete fusion. 5-hydroxytryptamine (5-HT) receptor signaling potently inhibits excitatory postsynaptic currents (EPSCs) between lamprey reticulospinal axons and their postsynaptic targets by a direct action on the vesicle fusion machinery. We show that 5-HT receptor-mediated presynaptic inhibition, at this synapse, involves a reduction in EPSC quantal size. Quantal size was measured directly by comparing unitary quantal amplitudes of paired EPSCs before and during 5-HT application and indirectly by determining the effect of 5-HT on the relationship between mean-evoked EPSC amplitude and variance. Results from FM dye-labeling experiments indicate that 5-HT prevents full fusion of vesicles. 5-HT reduces FM1-43 staining of vesicles with a similar efficacy to its effect on the EPSC. However, destaining of FM1-43-labeled vesicles is abolished by lower concentrations of 5-HT that leave a substantial EPSC. The use of a water-soluble membrane impermeant quenching agent in the extracellular space reduced FM1-43 fluorescence during stimulation in 5-HT. Thus vesicles contact the extracellular space during inhibition of synaptic transmission by 5-HT. We conclude that 5-HT, via free Gbetagamma, prevents the collapse of synaptic vesicles into the presynaptic membrane.

Animals↗
PubMed2005

Extracellular trafficking of myocilin in human trabecular meshwork cells.

Myocilin (MYOC) is a protein with a broad expression pattern, but unknown function. MYOC associates with intracellular structures that are consistent with secretory vesicles, however, in most cell types studied, MYOC is limited to the intracellular compartment. In the trabecular meshwork, MYOC associates with intracellular vesicles, but is also found in the extracellular space. The purpose of the present study was to better understand the mechanism of extracellular transport of MYOC in trabecular meshwork cells. Using a biochemical approach, we found that MYOC localizes intracellularly to both the cytosolic and particulate fractions. When intracellular membranes were separated over a linear sucrose gradient, MYOC equilibrated in a fraction less dense than traditional secretory vesicles and lysosomes. In pulse-labeling experiments that followed nascent MYOC over time, the characteristic doublet observed for MYOC by SDS-PAGE did not change, even in the presence of brefeldin A; indicating that MYOC is not glycosylated and is not released via a traditional secretory mechanism. When conditioned media from human trabecular meshwork cells were examined, both native and recombinant MYOC associated with an extracellular membrane population having biochemical characteristics of exosomes, and containing the major histocompatibility complex class II antigen, HLA-DR. The association of MYOC with exosome-like membranes appeared to be specific, on the extracellular face, and reversible. Taken together, data suggest that MYOC appears in the extracellular space of trabecular meshwork cells by an unconventional mechanism, likely associated with exosome-like vesicles.

Adenoviridae↗
PubMed1983

Morphology and permeability of junctional complexes in maturing ameloblasts of rat incisors.

Thin sections of newborn rat incisors were examined by tracer experiments and freeze-fracture replication in order to clarify the morphology and permeability of ameloblast junctional complexes in the maturation stage. Ameloblast junctional complexes consisted of gaps and tight junctions at the proximal and distal ends. Whereas the proximal junctional complexes sealed extracellular spaces incompletely, the distal ones formed complete, belt-like barriers around the cell. Tight junctions of these junctional complexes, however, were composed of both continuous and discontinuous rows of particles with various spaces among them. Intravenously injected horseradish peroxidase (HRP) reached the enamel surface through the extracellular spaces among ameloblasts and was absorbed by ameloblasts of the ruffled borders. Pinocytosis and transcellular migration of HRP could not be demonstrated in maturing ameloblasts except in the ruffled border zone.

Ameloblasts↗
PubMed2005

Measuring the size of the extracellular fluid space using bromide, iohexol, and sodium dilution.

There is a need to find methods to assess the size of the extracellular fluid (ECF) volume without involving radioactive tracers. For this purpose, we applied 3 methods for measuring the ECF volume in 10 male volunteers (mean age, 34 yr). Steady-state plasma bromide concentration (control) was compared to the results of kinetic analysis of plasma iohexol and to kinetic analysis of the dilution of serum sodium after IV infusion of 1 L of isotonic mannitol. The volume of distribution of these tracers was used to indicate the ECF volume. The results disclosed statistically significant correlations between the results of all 3 methods, although the average sodium dilution showed 0.7 L lower values than iohexol and 1.4 L lower than bromide. All three methods correlated significantly with body weight. The percentage of the body weight indicated by the methods was 18.3% (3.1%) for sodium, 19.6% (1.0%) for iohexol, and 20.5% (1.1%) for bromide. We conclude that sodium dilution may be performed at bedside but iohexol and bromide showed less intersubject variability. Iohexol simultaneously measures the glomerular filtration rate and should be a viable clinical option if the hospital performs routine assessments of kidney function using this tracer.

Adult↗
PubMed1983

An SEM analysis of neural crest migration in the mouse.

The cellular morphology and migratory pathways of the trunk neural crest are described in normal mouse embryos, and in embryos homozygous for Patch in which neural crest derivatives develop abnormally. Trunk neural crest cells initially appear in 8 1/2-day embryos as a unique cell population on the dorsal neural tube surface and are relatively rounded. Once they begin to migrate the cells flatten and orient somewhat tangentially to the neural tube, and advance ventrad between the somites and neural tube. At the onset of migration neural crest cells extend lamellipodia onto the surface of the tube while detaching their trailing processes from the lumenal surface. The basal lamina on the dorsal neural tube is discontinuous when cell migration begins in this region. As development proceeds, the basal lamina gradually becomes continuous from a lateral to dorsal direction and neural crest emigration is progressively confined to the narrowing region of discontinuous basal lamina. Cell separation from the neural tube ceases concomitant with completion of a continuous basement membrane. Preliminary observations of the mutant embryos reveal that abnormal extracellular spaces appear and patterns of crest migration are subsequently altered. We conclude that the extracellular matrix, extracellular spaces and basement membranes may delimit crest migration in the mouse.

Animals↗
PubMed1975

Simultaneous efflux of K+ and Na+ from frog sartorius muscle freed of extracellular fluids: evidence for rapidly exchanging Na+ from the cells.

After removal of radioactivity trapped in the extracellular space and correcting for the contribution of connective tissue elements, the K+ -efflux curve of frog sartorius muscles becomes a perfect straight line in a semilogarithmic plot. The simultaneously recorded Na+-efflux curve from the same muscles remains strongly curved, and can be resolved into a slow fraction (which conventionally has been regarded as representing the entire cell Na+) and at least one fast fraction. The fast fraction of Na+ could not have originated from a sarcoplasmic reticulum or any other extracellular space extensions; otherwise a similar fast fraction should exist for K+. The data agree with the interpretation that it is the fast fraction that is rate limiting by cell permeability and the slow fraction by desorption from intracellular adsorption sites.

Adsorption↗
PubMed1997

Effects of different perfusion medium on the extracellular basal concentration of dopamine in striatum and medial prefrontal cortex: a zero-net flux microdialysis study.

The zero-net flux microdialysis method was used to determine (1) the basal concentration of dopamine (DA) in the extracellular space, and (2) the in vivo recovery of Da in the striatum and the medial prefrontal cortex by using three different kinds of perfusion medium. They were, (a) commercial Ringer's solution, (b) low Ca2+ Ringer's solution, and (c) artificial cerebrospinal fluid (aCSF). Our results not only support previous findings that the high Ca2+ concentration in the perfusion medium could increase the baseline concentration of DA in the dialysate, which was collected from extracellular space through dialysis probe; but also provides evidence that this baseline increase was primarily due to an increase of basal DA concentration, and not from the increase of the in vivo recovery. Additionally, there was no significant difference in the basal DA concentration by using either commercial Ringer's solution or aCSF. This indicates that both commercial Ringer's solution and aCSF are suitable as good perfusion medium to determine the basal DA in the rat's brain.

Animals↗
PubMed2001

S100: a multigenic family of calcium-modulated proteins of the EF-hand type with intracellular and extracellular functional roles.

S100 is a multigenic family of non-ubiquitous Ca(2+)-modulated proteins of the EF-hand type expressed in vertebrates exclusively and implicated in intracellular and extracellular regulatory activities. Within cells, most of S100 members exist in the form of antiparallelly packed homodimers (in some cases heterodimers), capable of functionally crossbridging two homologous or heterologous target proteins in a Ca(2+)-dependent (and, in some instances, Ca(2+)-independent) manner. S100 oligomers can also form, under the non-reducing conditions found in the extracellular space and/or within cells upon changes in the cell redox status. Within cells, S100 proteins have been implicated in the regulation of protein phosphorylation, some enzyme activities, the dynamics of cytoskeleton components, transcription factors, Ca(2+) homeostasis, and cell proliferation and differentiation. Certain S100 members are released into the extracellular space by an unknown mechanism. Extracellular S100 proteins stimulate neuronal survival and/or differentiation and astrocyte proliferation, cause neuronal death via apoptosis, and stimulate (in some cases) or inhibit (in other cases) the activity of inflammatory cells. A cell surface receptor, RAGE, has been identified on inflammatory cells and neurons for S100A12 and S100B, which transduces S100A12 and S100B effects. It is not known whether RAGE is a universal S100 receptor, S100 members interact with other cell surface receptors, or S100 protein interaction with other extracellular factors specifies the biological effects of a given S100 protein on a target cell. The variety of intracellular target proteins of S100 proteins and, in some cases, of a single S100 protein, and the cell specificity of expression of certain S100 members suggest that these proteins might have a role in the fine regulation of effector proteins and/or specific steps of signaling pathways/cellular functions. Future analyses should discriminate between functionally relevant S100 interactions with target proteins and in vitro observations devoid of physiological importance.

Animals↗
PubMed1995

Morphometric analysis of gap junctions in nonpregnant and term pregnant human myometrium.

BACKGROUND AND METHODS: Some structures in the human myometrium, namely; caveolae, extracellular space and gap junctions were quantified in tissues from nonpregnant women, women who were not in labor, and women in spontaneous labor or oxytocin-induced labor, in order to reveal morphologic differences that would contribute to explaining the events leading to labor. Transmission electron micrographs taken from tissues from twenty-four pregnant and ten nonpregnant women were studied. RESULTS: Differences were observed in cell growth which was accompanied by enlargement of the extracellular space, and in gap junctions. Gap junctions were demonstrated in nonpregnant women, and even in one postmenopausal woman. Their frequency and size increased gradually between groups of pregnant women in the order of non-labor, oxytocin-induced labor and spontaneous labor. The increase in size of the junctions was less than the increase in number. CONCLUSIONS: Gap junctions are most frequently found in human myometrial cells in spontaneous labor, which strongly suggests a role for these structures in the termination of pregnancy. The effect of oxytocin on the appearance of gap junctions was negligible; hence, the contribution of oxytocin to establish myometrial contractile synchronicity is thought to be minor. Caveolae are probably not relevant for the initiation of labor as their distribution did not differ between the groups.

Adolescent↗
PubMed2006

Shaping of monocyte and macrophage function by adenosine receptors.

Adenosine is an endogenous purine nucleoside that, following its release into the extracellular space, binds to specific adenosine receptors expressed on the cell surface. Adenosine appears in the extracellular space under metabolically stressful conditions, which are associated with ischemia, inflammation, and cell damage. There are 4 types of adenosine receptors (A(1), A(2A), A(2B) and A(3)) and all adenosine receptors are members of the G protein-coupled family of receptors. Adenosine receptors are expressed on monocytes and macrophages and through these receptors adenosine modulates monocyte and macrophage function. Since monocytes and macrophages are activated by the same danger signals that cause accumulation of extracellular adenosine, adenosine receptors expressed on macrophages represent a sensor system that provide monocytes and macrophages with information about the stressful environment. Adenosine receptors, thus, allow monocytes and macrophages to fine-tune their responses to stressful stimuli. Here, we review the consequences of adenosine receptor activation on monocyte/macrophage function. We will detail the effect of stimulating the various adenosine receptor subtypes on macrophage differentiation/proliferation, phagocytosis, and tissue factor (TF) expression. We will also summarize our knowledge of how adenosine impacts the production of extracellular mediators secreted by monocytes and macrophages in response to toll-like receptor (TLR) ligands and other inflammatory stimuli. Specifically, we will delineate how adenosine affects the production of superoxide, nitric oxide (NO), tumor necrosis factor-alpha, interleukin (IL)-12, IL-10, and vascular endothelial growth factor (VEGF). A deeper insight into the regulation of monocyte and macrophage function by adenosine receptors should assist in developing new therapies for inflammatory diseases.

Adenosine↗