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S B Andrews

Publications and source records attributed to S B Andrews.

At least 37 records · Page 2Linked to original sources
PubMed1993

Characterization of spontaneous and action potential-induced calcium transients in developing myotubes in vitro.

We have investigated the onset and maturation of action potential- and calcium-induced calcium release from the sarcoplasmic reticulum during the differentiation of excitation-contraction coupling in skeletal muscle. Microfluorometry and video imaging of cultured myotubes loaded with the fluorescent calcium indicator fluo-3 revealed the dynamics, time course, and physiological properties of calcium transients as well as their changes during development. Spontaneous and stimulated contractions in well-differentiated myotubes are accompanied by brief (200-500 ms) increases in the concentration of free cytoplasmic calcium. These transients are modulated by sub-threshold concentrations of caffeine, resulting in a plateau of elevated calcium. Two novel types of calcium transients were observed in non-contracting myotubes. 1) Fast localized transients (FLTs) are radially restricted focal release events that occur spontaneously within the myoplasm at various densities and frequencies. 2) Upon addition of caffeine, propagating calcium waves are generated (35-70 microns/s velocity), which are accompanied by contractures. Aside from caffeine sensitivity, calcium waves and contraction-related sustained release events are similar in amplitude and duration, as well as in their inactivation and refractory properties. Thus, these transients may represent calcium-induced calcium release in quiescent and active myotubes, respectively. Following one calcium-induced calcium release event, myotubes become refractory to new calcium-induced transients; however, action potential-induced transients and FLTs are not blocked. This suggests that these transients occur by distinct release mechanisms and that dual modes of calcium release exist prior to the coupling of calcium release to excitation.

Action Potentials↗
PubMed1993

Measurement of low calcium concentrations in cryosectioned cells by parallel-EELS mapping.

Electron energy loss spectroscopy (EELS) in the scanning transmission electron microscope provides a high sensitivity for microanalysis of certain important biological elements such as calcium whose physiological concentrations in cells are rather low. Application of parallel-EELS mapping to the analysis of freeze-dried cryosections of rapidly frozen tissue provides a means of detecting small amounts of calcium in structures with diameter approximately 50 nm. Detector pattern noise due to channel gain variations can be reduced by acquiring difference spectra at each pixel. By segmenting nitrogen maps that reflect the structure through the protein distribution it is possible to sum spectra from specific compartments. These are then processed by fitting reference spectra for the Ca L23-edge and the carbon background. It has been found that useful data can be collected at 100 keV beam energy from freeze-dried cryosections of cerebellar cortex cut to nominal thickness of 100 nm. The analysis results in a sensitivity of +/- 0.4 mmol Ca/kg dry weight with a total acquisition time of 400 s, a significant improvement over that achievable with energy-dispersive X-ray spectroscopy.

Animals↗
PubMed1993

Triad formation: organization and function of the sarcoplasmic reticulum calcium release channel and triadin in normal and dysgenic muscle in vitro.

Excitation-contraction (E-C) coupling is thought to involve close interactions between the calcium release channel (ryanodine receptor; RyR) of the sarcoplasmic reticulum (SR) and the dihydropyridine receptor (DHPR) alpha 1 subunit in the T-tubule membrane. Triadin, a 95-kD protein isolated from heavy SR, binds both the RyR and DHPR and may thus participate in E-C coupling or in interactions responsible for the formation of SR/T-tubule junctions. Immunofluorescence labeling of normal mouse myotubes shows that the RyR and triadin co-aggregate with the DHPR in punctate clusters upon formation of functional junctions. Dysgenic myotubes with a deficiency in the alpha 1 subunit of the DHPR show reduced expression and clustering of RyR and triadin; however, both proteins are still capable of forming clusters and attaining mature cross-striated distributions. Thus, the molecular organization of the RyR and triadin in the terminal cisternae of SR as well as its association with the T-tubules are independent of interactions with the DHPR alpha 1 subunit. Analysis of calcium transients in dysgenic myotubes with fluorescent calcium indicators reveals spontaneous and caffeine-induced calcium release from intracellular stores similar to those of normal muscle; however, depolarization-induced calcium release is absent. Thus, characteristic calcium release properties of the RyR do not require interactions with the DHPR; neither do they require the normal organization of the RyR in the terminal SR cisternae. In hybrids of dysgenic myotubes fused with normal cells, both action potential-induced calcium transients and the normal clustered organization of the RyR are restored in regions expressing the DHPR alpha 1 subunit.

Amino Acid Sequence↗
PubMed1992

Coordinated development of myofibrils, sarcoplasmic reticulum and transverse tubules in normal and dysgenic mouse skeletal muscle, in vivo and in vitro.

We studied the development of transverse (T)-tubules and sarcoplasmic reticulum (SR) in relationship to myofibrillogenesis in normal and dysgenic (mdg/mdg) mouse skeletal muscle by immunofluorescent labeling of specific membrane and myofibrillar proteins. At E16 the development of the myofibrils and membranes in dysgenic and normal diaphragm was indistinguishable, including well developed myofibrils, a delicate network of T-tubules, and a prominent SR which was not yet cross-striated. In diaphragms of E18 dysgenic mice, both the number and size of muscle fibers and myofibrillar organization were deficient in comparison to normal diaphragms, as previously reported. T-tubule labeling was abnormal, showing only scattered tubules and fragments. However, many muscle fibers displayed cross striation of sarcomeric proteins and SR comparable to normal muscle. In cultured myotubes, cross-striated organization of sarcomeric proteins proceeded essentially in two stages: first around the Z-line and later in the A-band. Sarcomeric organization of the SR coincided with the first stage, while the appearance of T-tubules in the mature transverse orientation occurred infrequently, only after A-band maturation. In culture, myofibrillar and membrane organization was equivalent in normal and dysgenic muscle at the earlier stage of development, but half as many dysgenic myotubes reached the later stage as compared to normal. We conclude that the mdg mutation has little effect on the initial stage of membrane and myofibril development and that the deficiencies often seen at later stages result indirectly from the previously described absence of dihydropyridine receptor function in the mutant.

Animals↗
PubMed1992

Characterization of biological macromolecules by combined mass mapping and electron energy-loss spectroscopy.

The combination of scanning transmission electron microscopy (STEM) and parallel-detection energy-loss spectroscopy (EELS) was used to detect specific bound elements within macromolecules and macromolecular assemblies prepared by direct freezing. After cryotransferring and freeze-drying in situ, samples were re-cooled to liquid nitrogen temperature and low-dose (about 10(3) e/nm2) digital dark-field images were obtained with single-electron sensitivity using a beam energy of approximately 100 keV and a probe current of approximately 5 pA. These maps provided a means of characterizing the molecular weights of the structures at low dose. The probe current was subsequently increased to about 5 nA in order to perform elemental analysis. The 320 copper atoms in a keyhole limpet haemocyanin molecule (mol.wt = 8 MDa) were detected with a sensitivity of +/- 30 atoms in an acquisition time of 200 s. Phosphorus was detected in an approximately 10-nm length of single-stranded RNA contained in a tobacco mosaic virus particle (mol.wt = 130 kDa/nm) with a sensitivity of +/- 25 atoms. Near single-atom sensitivity was achieved for the detection of iron in one haemoglobin molecule (mol.wt = 65 kDa, containing four Fe atoms). Such detection limits are only feasible if special processing methods are employed, as is demonstrated by the use of the second-difference acquisition technique and multiple least-squares fitting of reference spectra. Moreover, an extremely high electron dose (about 10(10) e/nm2) is required resulting in mass loss that may be attributable to 'knock-on' radiation damage.

Animals↗
PubMed1991

Cryofixation of vascular endothelium.

Cryofixation refers to the immobilization of tissue components by the rapid removal of heat from the specimen, so that the structure is interred and stabilized in a natural embedding medium, namely, frozen (amorphous or microcrystalline) tissue water. Cryofixation is now often used as a complement to the more traditional fixation methods, especially when the cell structure is delicate or dynamic and may be inaccurately preserved by the slow selective action of chemical fixatives. Vascular endothelial cells are specialized for transcellular transport and for the regulation of blood flow and composition. The dynamic and labile subcellular organization of these cells, presumably reflecting these functional specializations, makes them ideal candidates for cryofixation. Several different types of endothelial cells were directly frozen at temperatures below 20 degrees Kelvin by pressing them against a liquid-helium-cooled block. These samples were subsequently processed for structural analysis by freeze-substitution. Detailed rationales, designs, and protocols are described for both freezing and freeze-substitution. Electron micrographs of cryofixed arterial and venous capillaries (rete mirabile of the American eel), iliac vein (rabbit), and cultured endothelium from the iliac vein (human) reveal that the organization of the characteristic intracellular membrane system of endothelial vesicles is qualitatively similar to that seen in chemically fixed endothelium, especially with regard to the interconnection of clusters of individual vesicles to form elaborate networks. The luminal and abluminal networks are not in communication, at least not in static images. Quantitatively, however, most directly frozen endothelial cells have far fewer vesicular profiles than comparable glutaraldehyde-fixed cells. The differences can be explained by presuming that the rapid action of cryofixation (approximately 1 msec) gives a more accurate picture of the vesicular network because it captures the transient structure of labile or dynamic membranes.

Animals↗
PubMed1991

Analysis of directly frozen macromolecules and tissues in the field-emission STEM.

A VG Microscopes HB501 field-emission high-resolution scanning transmission electron microscope (STEM) was used to image and analyse rapidly frozen, isolated macromolecules and small organelles in tissue cryosections. Dark-field images were obtained from frozen-hydrated microtubules demonstrating that sufficient contrast is available to reveal structural information. The samples were subsequently freeze-dried in the STEM and low-dose (approximately 10(3) e/nm2) dark-field mass maps were recorded with single electron sensitivity. Elemental analysis of individual macromolecules was achievable at high dose using parallel-detection electron energy-loss spectroscopy, albeit with some structural degradation. Detection of copper (320 atoms) in di-decameric haemocyanin molecules was easily within the limits of sensitivity. Elemental analysis of hydrated cryosections is limited by radiation damage to a resolution of approximately 1 micron2. For freeze-dried sections, however, the high probe current and stable cold stage of the HB501 STEM allow energy-dispersive X-ray (EDX) microanalysis of low elemental concentrations in highly localized subcellular volumes. EDX spectra from cryosections of cerebellar cortex show that a 100-s analysis time is sufficient to quantify the calcium content of 400-nm2 regions within Purkinje cell dendrites with an uncertainty of +/- 2 mmol/kg dry weight, equivalent to +/- 12 atoms.

Animals↗
PubMed1989

Co-localization of the myelin-associated glycoprotein and the microfilament components, F-actin and spectrin, in Schwann cells of myelinated nerve fibres.

The myelin-associated glycoprotein (MAG) is an intrinsic membrane protein that is specific for myelinating cells. MAG has been proposed to function in the PNS as an adhesion molecule involved in Schwann cell-axon contact and maintenance of cytoplasmic channels within the myelin sheath. In this report we show that the microfilament components, F-actin and spectrin, co-localize with MAG in periaxonal membranes, Schmidt-Lanterman incisures, paranodal myelin loops, and inner and outer mesaxons of myelinating Schwann cells. F-actin was localized light microscopically by rhodamine-labelled phallicidin binding. Spectrin and MAG were localized by light microscopic and ultrastructural immunocytochemistry. The findings indicate that plasma membrane linkage of F-actin in Schwann cells is likely to occur via spectrin, and raise the possibility that microfilaments interact with the cytoplasmic domain of MAG. An interaction between MAG and microfilaments would be consistent with the proposed function of MAG as an adhesion molecule.

Actins↗
PubMed1989

The myelin-associated glycoprotein is enriched in multivesicular bodies and periaxonal membranes of actively myelinating oligodendrocytes.

The myelin-associated glycoprotein (MAG) is a member of the immunoglobulin gene superfamily that is selectively expressed by myelin-forming cells. A developmentally regulated, alternative splicing of a single MAG transcript produces two MAG polypeptides (72 and 67 kD) in the central nervous system (CNS). MAG occurs predominantly as the 67-kD polypeptide in the peripheral nervous system (PNS). This study determined the subcellular localization of CNS MAG at different postnatal times when the 72-kD form (7-d) and 67-kD form (adult) are quantitatively abundant. These distributions were also compared to those of MAG in the PNS. In adult rat, MAG is selectively enriched in periaxonal membranes of CNS myelin internodes. This restricted distribution differs from that in PNS myelin internodes where MAG is also enriched in paranodal loops, Schmidt-Lanterman incisures, and mesaxon membranes. In 7-d-old rat CNS, MAG was associated with periaxonal membranes during axonal ensheathment and enriched in Golgi membranes and cytoplasmic organelles having the appearance of multivesicular bodies (MVBs). MAG-enriched MVBs were found in oligodendrocyte perinuclear regions, in processes extending to myelin internodes, and along the myelin internode in outer tongue processes and paranodal loops. MAG-enriched MVBs were not found in oligodendrocytes from adult animals or in myelinating Schwann cells. These findings raise the possibility that the 72-kD MAG polypeptide is associated with receptor-mediated endocytosis of components from the periaxonal space or axolemma during active stages of myelination.

Aging↗
PubMed1988

Three-dimensional organization of the plasmalemmal vesicular system in directly frozen capillaries of the rete mirabile in the swim bladder of the eel.

Several recent studies comparing chemically fixed and cryofixed endothelium have indicated that glutaraldehyde fixation may result in increases in the population of "vesicles" in the cytoplasm. Other reports based on ultrathin serial-section reconstruction of chemically fixed endothelium have revealed that the vesicular system is comprised of interconnected membranous compartments, which are ultimately continuous with either cell surface but do not extend across the endothelial cell. In this study, we have investigated the three-dimensional organization of the vesicular system in directly frozen, freeze-substituted capillaries of the rete mirabile from the swim bladder of the eel, specifically using the same block of embedded capillaries in which frozen capillaries had previously been found to contain less "vesicles" than chemically fixed capillaries. The results show that essentially all vesicles remain interconnected with each other and are part of two separate sets of invaginations from the luminal and abluminal cell surface like in chemically fixed tissue. Any increase in vesicle number resulting from glutaraldehyde fixation does not affect the overall three-dimensional organization of the vesicular system in these endothelial cells.

Air Sacs↗
PubMed1988

Quantitative X-ray mapping of biological cryosections.

The potential for applying X-ray mapping to the elemental microanalysis of biological cryosections is discussed. Methods are described for acquiring and processing data, including use of the top-hat digital filter to remove the average effects of the background contribution. Practical considerations for X-ray mapping are discussed in terms of typical counts per pixel and minimum detectability which depends on the number of pixels chosen to integrate the signal. These aspects are illustrated with elemental maps (Na, P, K, Ca and Fe) from freeze-dried cryosections of mouse cerebellar cortex. A calcium sensitivity in the range 0.5 to 2.5 mmol/kg wet weight of tissue is demonstrated. The correction for overlap of potassium K beta and calcium K alpha is demonstrated with X-ray maps from cryosectioned synaptosomes of squid optic lobe. Quantitative results obtained using internal standards to determine wet weight concentrations are in reasonable agreement with expected values. Alternate schemes applicable to X-ray maps for determining the dry mass concentration, such as the peak/continuum (Hall method), are also discussed.

Electron Probe Microanalysis↗
PubMed1988

Activity-dependent accumulation of calcium in Purkinje cell dendritic spines.

The calcium content of synapses of parallel fibers on Purkinje cell dendritic spines was determined by electron probe x-ray microanalysis of freeze-dried cryosections from directly frozen slices of mouse cerebellar cortex. In fresh slices frozen within 20-30 sec of excision, calcium concentrations ranging from 0.8 to 18.6 mmol/kg of dry weight were measured in cisterns of smooth endoplasmic reticulum within Purkinje cell dendritic spines. The average calcium content of spine cisterns in rapidly excised slices (6.7 +/- 0.6 mmol/kg of dry weight +/- SEM) was higher than the average calcium content of spine cisterns in brain slices incubated without stimulation for 1-2 hr before direct freezing (2.5 +/- 0.4 mmol/kg of dry weight). Depolarization of incubated cerebellar slices by isotonic 55 mM KCl resulted in the accumulation within spine cisterns of very high amounts of calcium or isotonically substituted strontium, both derived from the extracellular fluid. These results suggest that one function of spine cisterns is to sequester free calcium that enters the spine through ligand-gated or voltage-gated channels during synaptic transmission.

Animals↗
PubMed1988

Cellular and subcellular distribution of 2',3'-cyclic nucleotide 3'-phosphodiesterase and its mRNA in the rat central nervous system.

The 2',3'-cyclic nucleotide 3'-phosphodiesterases (CNPs) are closely related oligodendrocyte proteins whose in vivo function is unknown. To identify subcellular sites of CNP function, the distribution of CNP and CNP mRNA was determined in tissue sections from rats of various developmental ages. Our results indicate that CNP gene products were expressed exclusively by oligodendrocytes in the CNS. CNP mRNA was concentrated around oligodendrocyte perinuclear regions during all stages of myelination. Developmentally, initial detection of CNP mRNA closely paralleled initial detection of its translation products. In electron micrographs of immunostained ultrathin cryosections, CNP was associated with oligodendrocyte membranes during the earliest phase of axonal ensheathment. In more mature fibers, immunocytochemistry established that the CNPs are not major components of compact myelin but are concentrated within specific regions of the oligodendrocyte and myelin internode. These include (a) the plasma membrane of oligodendrocytes and their processes, (b) the periaxonal membrane and inner mesaxon, (c) the outer tongue process, (d) the paranodal myelin loops, and (e) the "incisure-like" membranes found in many larger CNS myelin sheaths. A cytoplasmic pool of CNP was also detected in oligodendrocyte perikarya and larger oligodendrocyte processes. CNP was also enriched in similar locations in myelinated fibers of the PNS.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed1987

Ultrastructure of capillaries in the red body (rete mirabile) of the eel swim bladder.

The retea mirabilia are paired capillary organs located on the dorsal surface of the swimbladder of the common eel. They consist of bundles of closely apposed capillary segments which function in countercurrent exchange of gases and other solutes and concentrate oxygen in the swim bladder. Ultrastructural features of the afferent arterial capillaries and efferent venous capillaries were studied by scanning EM of corrosion casts and critical-point dried retes and transmission EM of thin sections and freeze-fractured retes. A loose association between endothelial cells in the venous capillaries is indicated by penetration of casting material into interendothelial clefts and the appearance of clefts bounded by cytoplasmic flaps in exposed critical-point dried specimens. In thin sections, open gaps between venous endothelial cells are bounded by cytoplasmic processes. Sections through arterial capillaries exhibit tight occluding junctions joining endothelial cells together and these can be seen in freeze-fracture replicas to extend without interruption along the length of the arterial capillaries. These studies indicate the absence of open or hydraulically conductive pathways across the arterial capillary walls and that they probably constitute a rate-limiting barrier in countercurrent exchange of solutes.

Air Sacs↗
PubMed1987

Distribution of calcium and potassium in presynaptic nerve terminals from cerebellar cortex.

The elemental composition of the presynaptic nerve terminals in rapidly frozen synapses of the cerebellar molecular layer was determined by electron probe x-ray microanalysis and elemental imaging of characteristic x-rays. Elemental imaging of thin freeze-dried cryosections from fresh cerebellar slices frozen within 20 sec of removal from the brain showed normal concentrations of potassium (95 +/- 6 mmol/liter wet tissue +/- SEM) and calcium (0.8 +/- 0.4 mmol/liter) in whole presynaptic terminals, even though mitochondrial and nonmitochondrial sites containing up to 30 mmol of calcium per liter were present elsewhere in the neuropil. Quantitative electron probe analysis of synaptic vesicle clusters and intraterminal mitochondria indicated that their calcium concentrations were 0.4 +/- 0.1 and 1.2 +/- 0.2 mmol/liter, respectively. The low calcium content of presynaptic organelles was confirmed by the absence of detectable deposits in preparations freeze-substituted so as to stabilize calcium content. Similar experiments were carried out on cerebellar slices rapidly frozen after incubation in vitro. The distribution of potassium and calcium in presynaptic terminals of resting and depolarized (55 mM potassium) slices was qualitatively and quantitatively similar to that in freshly excised cortex, although resting slices lacked the few calcium-rich sites that appeared in other areas of the neuropil after stimulation. The calcium concentrations in whole terminals, synaptic vesicles, and mitochondria of resting slices were 1.4 +/- 0.7, 0.7 +/- 0.2, and 0.9 +/- 0.2 mmol/liter, respectively. Thus, amounts of calcium typical of storage organelles in other tissues are not present within cerebellar synaptic vesicles, suggesting that they have a limited role in calcium storage and release.

Animals↗
PubMed1984

Rubidium uptake in single cells.

Rubidium uptake was measured in single erythroid and myeloid cells of rabbit by means of X-ray microanalysis. It was found in the nucleated bone marrow cells that after incubation in rubidium the sums of potassium and rubidium concentrations were similar to the original potassium concentrations, indicating that there was one-to-one replacement of potassium by rubidium. Although the nuclear potassium and rubidium concentrations were higher than those in the cytoplasm, the nuclear and cytoplasmic ratios of K/Rb were similar. This implies that the potassium in both compartments exchanged freely with rubidium. In the erythroid line of cells there was a continuous reduction of potassium transport activity during the maturation process as indicated by the decrease in rubidium uptake rates. The uptake was measured in seven groups of cell types that could be distinguished on the basis of morphology and chemical composition. The order of the groups from high to low rubidium uptake were: esosinophilic myelocyte greater than early erythroblast and thin-rimmed erythroblast greater than late erythroblast greater than early bone marrow red cell greater than late bone marrow red cell greater than peripheral blood red cell. Thus, there is a continuous decrease in rubidium transport as the erythroid cells mature.

Animals↗