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PubMed1995

Calcium concretions in the pineal gland of aged rats: an ultrastructural and microanalytical study of their biogenesis.

The genesis of calcium concretions in aged rats was studied by means of transmission and scanning electron microscopy. The potassium pyroantimonate method, combined with X-ray microanalysis, allowed us to study the distribution of cations and calcium. Notable accumulations of calcium (associated with phosphorus) were localized in vesicles, vacuoles, lipid droplets, lipopigments, and mitochondria of dark pinealocytes. The results obtained in the present investigation suggest that these organelles are involved in the genesis of the concretions. The presence of sulfur indicates the existence of an organic matrix. We propose that genesis takes place in dark pinealocytes, which contain more calcium than light pinealocytes. Mineralization foci are sometimes associated with cellular debris and enlarge by further apposition of material. Two types of concretions, as determined by electron microscopy and confirmed by electron diffraction, could be observed: the "amorphous" type with concentric layers and the crystalline type with needle-shaped crystals. Once formed, the concretions reach the extracellular space and the cell breaks down. Possible extracellular calcification is suggested in the extracellular calcium-rich floculent material. The mineralization process is interpreted as being an age-related phenomenon and mainly a consequence of the degeneration of pinealocytes.

Aging↗
PubMed1994

Immunoelectron microscopic localization of fibronectin in cultured human keratinocytes.

We investigated the ultrastructural localization of fibronectin (FN) in cultured human keratinocytes using an improved method of peroxidase-immunoelectron microscopy. This method enabled us to visualize the precise localization of FN within the cells while preserving the morphology. FN was localized in the protein synthetic and secretory organelles, including the rough-surfaced endoplasmic reticulum, Golgi complex, multivesicular bodies and perinuclear space. It was also detected in the extracellular space, on small regions of the villous projections of cell membranes at the site of secretion and at cell-substratum contact sites. These findings confirm that human keratinocytes synthesize, secrete and deposit FN in the pericellular matrix.

Cells, Cultured↗
PubMed1988

Histologic and ultrastructural alterations of a xenografted human colon adenocarcinoma after treatment with titanocene dichloride.

The influence of the antitumor agent titanocene dichloride on the morphologic appearance of a heterotransplanted human colon adenocarcinoma was investigated. The first alterations in tumor cells manifested 12 h after administration of a single dose (40 mg/kg) and consisted of nuclear changes, such as chromatin condensation, enlargement of the nuclear envelope, structural changes of the nucleoli, and formation of segmented nuclei 12 h later; bundles of microfilaments, lipid droplets and inclusion bodies, often containing cellular debris, occurred in the cytoplasm. Intracytoplasmic virus particles of type A were detectable. They were obviously extruded into the extracellular space by a budding process and became extracellular virus particles of type C. Within 24 h after treatment, the mitotic index decreased from 2.5% to 0.3%. Whereas after administration of a single dose, recovery phenomena took place between 2 and 4 days, the severe destruction induced by 3-fold doses of titanocene dichloride (3 X 30 mg/kg), was apparently not reversible. These results confirm the tumor-inhibiting potency of titanocene dichloride against human colon adenocarcinoma and underline the discriminatory power of morphologic studies in the preclinical evaluation of cytostatic drugs against heterotransplanted human tumors.

Adenocarcinoma↗
PubMed1994

Cellular uptake of intracerebroventricularly administered biotin- or digoxigenin-labeled antisense oligodeoxynucleotides in the rat.

1. Antisense oligodeoxynucleotides (ODNs) internally labeled with biotin or digoxigenin were injected into the lateral ventricle of rats and the distribution of the labeled ODNs was examined at several timepoints following the intracerebroventricular (icv) injections. The stability of these injected antisense ODNs, which had no backbone modifications, was also studied by performing recovery experiments. 2. The most intense labeling was observed near the injection site, in periventricular areas, and in perivascular regions. Many of the labeled cells appeared to be neurons, and both the cytoplasm and the nuclei were stained. The labeled cells were detected 15 min after icv injection, demonstrating that the antisense ODNs were taken up rapidly by cells in the parenchyma. The digoxigeninated antisense ODNs were presented in both the cytoplasmic and the nuclear fractions of rat brain extracts, however, the levels appeared to be much lower in the nuclear fractions. 3. Antisense ODNs injected into the lateral ventricle seemed to follow the bulk flow of cerebrospinal fluid (CSF), i.e., from the injection site in the lateral ventricle, through the ventricular system, to the subarachnoid spaces and the perivascular spaces. From the ventricular and perivascular spaces, the antisense ODNs diffused into the extracellular space and were taken up by cells. The full-length digoxigeninated antisense ODNs were detectable within cells after only 15 min, indicating their rapid uptake. In addition, the antisense ODNs appeared to be relatively stable in the brain since the full-length digoxigeninated ODNs were still detectable after 4 hr.

Animals↗
PubMed1975

Lidoplazine and Na and K distribution in normo-and hypokalaemic rats.

The effects of lidoplazine on K-metabolism were investigated in two types of experiments. (1) K and Na concentrations were measured in plasma, heart, diaphragm and erythrocytes in normo- and hypokalaemic rats with or without chronic (1 month) lidoplazine treatment. Total body weight, heart weight, dry weight/wet weight ratios and extracellular space were also determined. (2) Radioactive K influx in the heart was measured in acute in vitro experiments with or without lidoplazine. It is demonstrated that while lidoflazine has no effect on the Na and K distribution of normokalaemic animals, it has rather a K-saving effect in conditions of severe loss of intra- and extracellular K-ions. In normal in vitro preparations the unidirectional K-influx is not affected by lidoflazine.

Animals↗
PubMed1995

Mechanism of phospholipase D activation induced by prostaglandin D2 in osteoblast-like cells: function of Ca2+/calmodulin.

Prostaglandin D2 (PGD2) stimulated the formation of choline in a dose-dependent manner in the range between 10 nM and 10 microM. The effect of PGD2 on the formation of inositol phosphates (EC50 was 20 nM) was more potent than that on the formation of choline (EC50 was 0.5 microM). The formation of choline stimulated by a combination of PGD2 and 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, was additive. Staurosporine, an inhibitor for protein kinases, enhanced the PGD2-induced formation of choline, but H-7, another inhibitor for protein kinases, had little effect. PGD2 stimulated Ca2+ influx from extracellular space dose-dependently. The depletion of extracellular Ca2+ by EGTA reduced the PGD2-induced formation of choline. W-7 and trifluoperazine dihydrochloride, antagonists of calmodulin, dose-dependently inhibited the PGD2-induced choline formation. These results strongly suggest that PGD2 activates phospholipase D in a Ca2+/calmodulin dependent manner in osteoblast-like cells, and that protein kinase C is not essential for the PGD2-induced activation of phospholipase D.

Animals↗
PubMed2025

Discrimination between vesicular and nonvesicular extracellular tRNAs and their fragments.

The extracellular space contains RNAs both inside and outside extracellular vesicles (EVs). Among RNA types, tRNAs and tRNA-derived small RNAs (tDRs) tend to be abundant and are frequently detected when performing small RNA sequencing of extracellular samples. For several applications, including answering basic biology questions and biomarker discovery, it is important to understand which specific extracellular tRNAs and tDRs are inside EVs and which are not. We have observed that EVs contain mainly full-length tRNAs, while cells also release full-length tRNAs into nonvesicular fractions. However, these nonvesicular tRNAs are fragmented by extracellular ribonucleases into nicked tRNAs, which can dissociate into tDRs both in extracellular samples and in the laboratory. It is therefore crucial to separate EVs from other nonvesicular RNA-containing extracellular carriers to prevent cross-contamination. Otherwise, extracellular tDR profiling may mix up signals coming from structurally and functionally different carrier types. Here, we provide two protocols that achieve this by: (a) density gradient separation and, (b) the use of commercial, pre-packed size-exclusion chromatography columns. The first protocol is time-consuming but achieves high resolution, while the second protocol is faster, simpler, and recommended for routine separations. Taken together, they form a solid experimental toolkit for addressing different questions related to extracellular tRNA biology or biomarker discovery.

RNA, Transfer↗
PubMed2003

Properties of the demarcation membrane system in living rat megakaryocytes.

The demarcation membrane system (DMS) is the precursor of platelet cell membranes yet little is known of its properties in living megakaryocytes. Using confocal microscopy, we now demonstrate that demarcation membranes in freshly isolated rat marrow megakaryocytes are rapidly stained by styryl membrane indicators such as di-8-ANEPPS and FM 2-10, confirming that they are invaginations of the plasma membrane and readily accessible from the extracellular space. Two-photon excitation of an extracellular indicator displayed the extensive nature of the channels formed by the DMS throughout the extranuclear volume. Under whole-cell patch clamp, the DMS is electrophysiologically contiguous with the peripheral plasma membrane such that a single capacitative component can account for the biophysical properties of all surface-connected membranes in the majority of recordings. Megakaryocyte capacitances were in the range of 64-694 pF, equivalent to 500-5500 platelets (mean value 1850). Based upon calculations for a spherical geometry, the DMS results in a 4- to 14-fold (average 8.1-fold) increase in specific membrane capacitance expressed per unit spherical surface area. This indicates a level of plasma membrane invagination comparable with mammalian skeletal muscle. Whole-cell capacitance measurements and confocal imaging of membrane-impermeant fluorescent indicators therefore represent novel approaches to monitor the DMS during megakaryocytopoiesis and thrombopoiesis.

Animals↗
PubMed1981

Pathology and pathogenesis of drusen of the optic nervehead.

This report describes the findings of a clinicopathologic correlative study of 18 cases of drusen of the optic nervehead and reports on the observations of the first ultrastructural study of this disease in the literature. From these observations, the processes of formation of drusen of the optic nervehead are proposed: abnormal axonal metabolism leads to intracellular mitochondrial calcification. Some axons rupture and mitochondria are extruded into the extracellular space. Calcium is heavily deposited in the extracellular mitochondria. Small calcified microbodies are produced and calcium continues to deposit on the surface of these nidi to form drusen. We conclude that drusen are definitely related to axonal degeneration of the optic nervehead.

Adolescent↗
PubMed1990

Transposon tagging of genes for cell-cell interactions in Myxococcus xanthus.

The prokaryote Myxococcus xanthus is a model for cell interactions important in multicellular behavior. We used the transposon TnphoA to specifically identify genes for cell-surface factors involved in cell interactions. From a library of 10,700 insertions of TnphoA, we isolated 36 that produced alkaline phosphatase activity. Three TnphoA insertions tagged cell motility genes, called cgl, which control the adventurous movement of cells. The products of the tagged cgl genes could function in trans upon other cells and were localized primarily in the cell envelope and extracellular space, consistent with TnphoA tagging genes for extracellular factors controlling motility.

Alkaline Phosphatase↗
PubMed2005

Intracellular and extracellular growth of Aspergillus fumigatus.

Aspergillus fumigatus is a filamentous fungus that can cause a life-threatening systemic mycosis in immunocompromised patients. We have studied the growth of A. fumigatus inside cultured cells, and the extracellular growth requirements (in serum). We measured the uptake of bound conidia by the cultured human type II pneumocyte cell line (A549) and a mouse macrophage cell line (J774). The extent of internalization was determined using a nystatin protection assay and by confocal microscopy. Both assays showed that A549 cells internalized 30% of bound conidia after three hours. In contrast, the value for J774 cells was 90%. In both J774 and A549 cells, conidia entered the endosomal pathway and ultimately co-localized with lysosomal markers. Lysosomes containing conidia were acidified. Internalized conidia germinated, and after 24-36 h of incubation with A549 cells, the hyphal tips of some intracellular germlings became exposed to the extracellular space. The importance of iron acquisition to extracellular growth was assessed by creating a strain of A. fumigatus in which the gene encoding the first step of hydroxamate siderophore biosynthesis, ornithine N5-oxygenase (AfusidA), was inactivated by gene replacement. Mutant strains were avirulent in a mouse model of invasive aspergillosis indicating that siderophore biosynthesis is a virulence factor in A. fumigatus.

Animals↗
PubMed2003

Tumour tissue monitoring during photodynamic and hyperthermic treatment using bioimpedance spectroscopy.

Electrical bioimpedance spectroscopy is a fast and relatively easily applicable method for tissue characterization. In the frequency range up to 10 MHz, current conduction through tissue is mainly determined by tissue structure, i.e. the extra- and intra-cellular compartments and the insulating cell membranes. Therefore, changes in the extra- and intra-cellular fluid volumes are reflected in the impedance spectra. Investigations of tumours (DS sarcoma, implanted on the hind foot dorsum of rats) during treatment with localized hyperthermia (HT), photodynamic therapy (PDT) and the combination of these two components were carried out using impedance spectroscopy in the frequency range of 37 Hz to 3.7 MHz. Data collected reveal totally different, but characteristic, behaviour patterns for the three treatments. HT caused a slow increase in conductance at high frequencies (G(HF)) and in the extracellular space index (ECSI), indicating an increase in extracellular fluid volume and in total fluid content. With PDT, G(HF) increased immediately upon commencement of irradiation and was accompanied by a distinct decrease in ECSI, indicating the development of a pronounced intracellular oedema.

Animals↗
PubMed1990

Axolemmal abnormalities in myelin mutants.

Evidence is reviewed that the paranodal axoglial junction plays important roles in the differentiation and function of myelinated axons. In myelin-deficient axons, ion flux across the axolemma is greater than that in myelinated fibers because a larger proportion of the axolemma is active during continuous, as opposed to saltatory, conduction. In addition, older myelin-deficient rats that have developed spontaneous seizures display small foci of node-like E-face particle accumulations in CNS axons as well as more diffuse regions of increased particle density and number. Assuming that the E-face particles represent sodium channels, such regions could underlie high sodium current density during activity, low threshold for excitation, and increased extracellular potassium accumulation. Depending on the degree of spontaneous channel opening, they could also represent sites of spontaneous generation of activity. The appearance of seizures and their gradual increase in frequency and severity could represent an increase in the number of such regions. In addition, diminution in the dimensions of the extracellular space during maturation would result in increased extracellular resistance, which, together with increasing axonal diameter, would tend to increase the likelihood of ephaptic interaction among neighboring axons as well as the likelihood of extracellular potassium rises to levels that could cause spontaneous activity.

Animals↗
PubMed1985

Thyroxine concentrations and secretion rates in relation to pregnancy, lactation and energy balance in goats.

Plasma thyroxine (T4) concentrations were measured during pregnancy and lactation in goats on diets with different energy concentrations to study the role of T4 secretion in metabolic adaptation to pregnancy and lactation. Thyroxine distribution space (the volume of fluid required to dissolve the total pool of circulating T4 at a concentration equal to that in the plasma) and secretion rates were determined by using 125I-labelled T4 at different stages of lactation and pregnancy. Plasma concentrations of T4 increased during lactation and reached maximum levels 4-5 months after giving birth. They then remained fairly constant for the following 2-3 months until the goats became pregnant again. Thyroxine concentrations decreased slightly during pregnancy and showed a sudden drop at the onset of lactation. Minimum concentrations were observed during the first 2-3 weeks of lactation after which time concentrations increased slowly. Average concentrations for goats on a low-energy diet were consistently lower than those in goats on a high-energy consistently lower than those in goats on a high-energy diet. The mean secretion rate was 10.4 nmol day-1 kg body wt-0.75. The distribution spaces were much lower than the total extracellular space. The average distribution space in non-pregnant and pregnant goats was 11 and 5.3% of the body weight respectively. The decrease in T4 secretion rate and T4 distribution space during pregnancy is considered to be part of a homeorhetic adaptation to the condition. The drop in T4 secretion rate at the onset of lactation appears to be a homeostatic adaptation to a decreased fuel supply, similar to the situation with fasting or energy malnutrition.

Animals↗
PubMed1992

Responses of the solitary olfactory receptor cell of the guinea pig.

Olfactory receptor cells were isolated from the nasal mucosa of the guinea pig, using enzymatic digestion and mechanical dissociation. Under a phase-contrast microscope, the cells can be readily distinguished from other tissues, because of morphological characteristics. The intracellular free calcium ion concentrations ([Ca2+]i) of these cells were determined using the Ca2+ sensitive dye fura-2 and digital imaging microscopy. In the presence of 1 microM ionomycin, calcium-ionophore, there was an irreversible increase in [Ca2+]i in all the cells. During prolonged stimulation by 10 mM n-Amyl acetate under standard conditions, 16 of the 40 isolated olfactory cells responded with [Ca2+]i. There was no evidence of any increase in [Ca2+]i in the olfactory receptor cells in nominally Ca2+ free solution and stimulated by amyl acetate. When 3 mM CaCl2 was added, the [Ca2+]i increased remarkably. When cells were stimulated with amyl acetate in a CaCl2-depleted extracellular fluid supplemented with 10 microM verapamil (an L-type voltage-dependent calcium channel antagonist), no change occurred in [Ca2+]i. The addition of CaCl2 to this fluid did not elevate [Ca2+]i, in any cell. This would suggest that the [Ca2+]i increase in the presence of amyl acetate may mainly depend on the influx of calcium from the extracellular space. Our finding that the influx of extracellular calcium into cells was suppressed by verapamil suggests that the calcium channel, which is opened by amyl acetate, is a voltage-dependent L-type Ca2+ channel.

Animals↗
PubMed1989

Immunogold localization of chondroitin 6-sulfate in the interphotoreceptor matrix of normal and RCS rats.

Localization of chondroitin 6-sulfate (6S) in the interphotoreceptor matrix (IPM) of both normal and RCS rats with inherited retinal dystrophy has been carried out using light and electron microscopic immunogold cytochemistry. In the normal rat, 6S antibody labeling was found in highest concentration at the apical surface of the retinal pigment epithelium (RPE) and between adjacent photoreceptors near their basal inner segment-outer segment junction. In the apical zone, label was localized in the IPM and toward the distal portions of the RPE apical processes. In the basal zone, label was found in the IPM and near the outer plasma membranes of inner and outer segments. Interstitial labeling, between the shafts of outer segments, occurred at much lower concentration than in the apical and basal zones. In all zones, the extent of labeling appeared to be space-dependent; it was most abundant where extracellular spaces were large, and little was present where adjacent cell membranes were contiguous. In the dystrophic rat, apical zone labeling of the RPE apical processes was minimal; instead, the relatively small amount of label that was present was localized primarily in the IPM and within encapsulated spaces of membranous whorls of debris. Label was most concentrated in the basal zone, primarily associated with the IPM within abundant interphotoreceptor spaces, and near the plasma membranes of disorganized inner and outer segments. In isolated eyecups and neural retinas, some labeling persisted after extensive buffer rinses which suggests that chondroitin 6-sulfate is a somewhat insoluble component of the IPM in the rat retina.

Animals↗
PubMed1989

Blood-tissue transport of substrates in the heart: studies by single circulation tracer dilution.

Passive capillary permeability is usually not the rate limiting step to movement of small lipid-insoluble molecules from plasma into the heart. At physiological concentrations the rate of carrier-mediated transport and or receptor binding of blood-borne substrates is often measurable in a single circulation through the coronary bed. Here I review some studies on uptake measured by the single-circulation multiple tracer dilution technique. The method consists of a sudden intra-arterial injection of a mixture of tracers followed by sequential rapid sampling of the venous outflow. Generally the tracers include a molecule confined to the intravascular space (e.g. albumin), another confined to the extracellular space and a third test tracer. The data analysis can give information about: a) capillary permeability-surface product (PS), b) endothelial cell membrane transport, c) the size of the interstitial space, d) transport across the sarcolemma, e) uptake by nerve endings, f) binding to receptors, g) rapid metabolic transformations. Studies were performed in the isolated perfused heart (dog and rabbit). Early observations on rapid tissue uptake of potassium and ouabain have recently been extended. Kinetics of unidirectional trans-sarcolemmal influx of lactate, a metabolic substrate for the myocardium and the neuronal uptake of amines, were investigated as well as the myocardium and the neuronal uptake of amines, were investigated as well as the effects of specific inhibitors for these transport mechanisms. Uptake was also measured for glucose, the amino acids L-phenylalanine, L-alanine and taurine; and for acetate and palmitate. Recent literature which proposes an important role for uptake and metabolism of adenosine by the endothelium is discussed. These studies are of interest for clinical applications such as positron emission tomography (PET) which makes use of short-life isotopes.

Amines↗
PubMed1980

Underestimation of Na permeability in muscle cells: implications for the theory of cell potential and for energy requirement of the Na pump.

A new method permits, in a complex efflux curve from living cells, identification of the fraction of labeled solute rate-limited by intracellular-extracellular exchange. Using this method, it was found that in Na+ efflux from frog voluntary muscle, the slow fraction conventionally described as rate-limited by membrane permeability is wrongly assigned. Instead, a much faster fraction, usually camouflaged by the efflux of labeled Na+ in the extracellular space, represents the exchange of intra- and extracellular Na+ rate-limited by cell surface permeation. Quantitatively, the result shows that the Na+ permeability constant has up to now been underestimated by a factor of 10. Implications of that finding for the theory of the resting potential, and the minimum energy required of resting frog muscles, are discussed.

Animals↗