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B S Spooner

Publications and source records attributed to B S Spooner.

At least 55 records · Page 3Linked to original sources
PubMed1989

Effects of microgravity on liposome-reconstituted cardiac gap junction channeling activity.

Effects of microgravity on cardiac gap junction channeling activity were investigated aboard NASA zero-gravity aircraft. Liposome-reconstituted gap junctions were assayed for channel function during free-fall, and the data were compared with channeling at 1 g. Control experiments tested for 0 g effects on the structural stability of liposomes, and on the enzyme-substrate signalling system of the assay. The results demonstrate that short periods of microgravity do not perturb reconstituted cardiac gap junction channeling activity.

Animals↗
PubMed1989

beta-Xyloside effects on basal lamina structure and anionic site distribution the embryonic mouse submandibular salivary gland.

beta-D-Xyloside is a proteoglycan biosynthesis inhibitor. Previous studies on embryonic salivary glands have demonstrated that 0.5 mM beta-xyloside (1) inhibits proteoglycan synthesis by 50%; (2) severely depresses sulphated glycosaminoglycan deposition at the basal epithelial surface, and (3) dramatically inhibits epithelial branching morphogenesis. Electron microscopy revealed a conventional three-layered basal lamina that is altered in the presence of beta-xyloside by a 35% reduction in the number of tannic acid-resolved particles in the lamina densa. Basal lamina anionic sites, resolved with ruthenium red (RR) and polyethyleneimine (PEI) cationic probes, were also reduced in the presence of beta-xyloside. PEI particles were reduced by 28%, and RR particles by 24%, per two-dimensional unit of basal lamina. These beta-xyloside effects on anionic sites are consistent with an hypothesis that sulphated glycosaminoglycans account for 50% of the basal lamina anionic sites and a predicted 25% decrease in anionic sites in the presence of beta-xyloside.

Animals↗
PubMed1989

Embryonic salivary gland epithelial branching activity is experimentally independent of epithelial expansion activity.

Embryonic mouse submandibular salivary gland rudiments undergo morphogenesis in organ culture, characterized by extensive epithelial growth and expansion and repetitive branching activity. Tunicamycin, at a concentration of 25 ng/ml culture medium, decreases the degree of net protein accumulation by 83% and the degree of epithelial expansion by 70% compared to controls, over a 48-hr culture tenure. These decreases correlate with reduced incorporation of [3H]thymidine into DNA. Nevertheless, epithelial branching activity is uncompromised, undergoing an approximately 10-fold increase in lobe numbers, in both controls and tunicamycin-treated rudiments, during the same 48-hr period. The effect is most striking during the 24- to 48-hr culture interval, when controls and tunicamycin-treated rudiments each triple their lobe numbers and controls approximately double epithelial area, while tunicamycin virtually stops all epithelial expansion.

Animals↗
PubMed1988

Reconstitution of cardiac gap junction channeling activity into liposomes: a functional assay for gap junctions.

Cardiac gap junctions were reconstituted into liposomes. To determine if reconstitution resulted in membrane channel formation, we developed an assay for channel function that used a liposome-entrapped peroxidase to detect entry of a substrate into the liposome. The data demonstrate, for the first time, that reconstituted gap junctions from heart are capable of channel-forming activity in artificial membranes.

Animals↗
PubMed1987

An autoradiographic analysis of N-linked glycoconjugates in embryonic salivary gland morphogenesis.

The synthesis, deposition, and loss of mannose-bearing glycoconjugates during branching morphogenesis of embryonic mouse salivary glands has been evaluated. Day 13 embryonic mouse salivary glands were cultured for 44 hr, pulse labeled 4 hr with [3H]mannose, then fixed after 0, 2, 4, 8, or 24 hr of chase in nonradioactive medium, and processed for autoradiography. Light microscopic autoradiograms of sectioned rudiments reveal extensive label within the epithelium, little label over the mesenchyme, and a concentration of radioactivity at the basal surface of the epithelium. Autoradiograms of "chased" rudiments reveal a) no detectable loss of label from the epithelium or the basal epithelial surface over the first 8 hr, and b) significant label loss by 24 hr of chase at the basal epithelial surface, while moderate amounts of radioactivity remain throughout the rest of the epithelium. The [3H]bound material is insensitive to chondroitinase ABC, a glycosaminoglycan degradative enzyme, but is sensitive to tunicamycin presence in the culture medium. Earlier studies showed that embryonic mouse salivary glands cultured in medium containing tunicamycin (25 ng/ml) continued normal epithelial branching while epithelial growth was inhibited. The present autoradiographic studies of [3H]mannose-labeled rudiments demonstrate that tunicamycin causes a significant decrease in radioactivity, relative to controls. Thus, our results suggest that epithelial branching activity is independent of control levels of mannose-containing/tunicamycin-sensitive, glycoconjugate deposition.

Animals↗
PubMed1986

Basal lamina anionic sites in the embryonic submandibular salivary gland: resolution and distribution using ruthenium red and polyethyleneimine as cationic probes.

The basal lamina of the embryonic submandibular epithelium is a dynamic compartment of the extracellular matrix required for branching morphogenesis. A transmission electron microscopy (TEM) structural analysis of the basal lamina, at a time of intense branching activity, was conducted, comparing standard glutaraldehyde-fixed preparations with ones that included tannic acid in the primary fixative, and comparing anionic site resolution and distribution with two cationic probes, ruthenium red (RR) and polyethyleneimine (PEI). Standard TEM revealed a conventional basal lamina structure, with a lamina densa, a lamina lucida interna and a lamina lucida externa. Fine filaments emanated from the lamina densa, traversing both lamina lucidae. Tannic acid revealed approximately 35 nm diameter electron-dense particles in the lamina densa with a spacing repeat of approximately 45 nm. Basal lamina anionic sites were resolved as approximately 26 nm diameter RR-particles and approximately 50 nm diameter PEI-particles, present in the lamina lucida interna and associated with the lamina lucida externa. RR-particle linear spacing was 70 nm in the externa and 50 nm in the interna, while the PEI-particle spacing repeat was 90 nm in both compartments. Binding of both probes was blocked by testicular hyaluronidase or chondroitinase treatment, a result suggesting that the anionic sites were chondroitin sulfate proteoglycan, hyaluronic acid, or both. The greater particle spacing observed with PEI was not simply a physical limitation resulting from the average PEI particle diameter being almost twice that of RR particles, since PEI-resolved anionic sites on interstitial collagen were much more closely spaced (approximately 60 nm) than RR-resolved sites (approximately 105 nm).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Sulfated glycosaminoglycan deposition and processing at the basal epithelial surface in branching and beta-D-xyloside-inhibited embryonic salivary glands.

We investigated whether the inhibition of proteoglycan synthesis and salivary branching morphogenesis by beta-D-xyloside was related to the deposition and processing of newly synthesized glycosaminoglycans at the basal epithelial surface that correlates with normal branching activity. Forty eight-hour cultures of control and 0.5 mM beta-xyloside-treated submandibular rudiments were labeled for 2 hr with [35S]sulfate and fixed and processed for autoradiography, immediately or after 2, 4, 6, or 8 hr of postlabeling chase in nonradioactive medium. The data demonstrated that deposition of chondroitin sulfate-rich material at the basal epithelial surface was strikingly reduced in beta-xyloside-treated rudiments, while patterns of label loss during postlabeling chase were not altered.

Animals↗
PubMed1984

Precardiac mesoderm differentiation in vitro. Actin-isotype synthetic transitions, myofibrillogenesis, initiation of heartbeat, and the possible involvement of collagen.

The differentiation of precardiac mesoderm into beating heart tissue was examined during explant culture. Explanted tissue forms tubular heart-like vesicles and initiates rhythmic contractility within 18-24 h in vitro, a developmental time-course approximating that observed during in vivo development. Electron-microscopic observations reveal that beating heart cells are rich in cytoplasmic myofibrils in varying degrees of order, with some regions containing highly organized myofibrillar arrays. The analysis of actin-isotype biosynthesis, using metabolic labeling with [35S]-methionine and isoelectric-focusing resolution of the synthesized radioactive polypeptides, demonstrates that the initiation of cardiac alpha-actin synthesis and the pattern of transition in the synthesis of alpha-, beta-, and gamma-actin isotypes is equivalent to the initiation time and pattern observed in vivo. A possible collagen involvement in the differentiation process was investigated by assessing the effects of collagen-synthesis inhibitors on the development of the explant cultures. Two different agents, alpha, alpha'-dipyridyl and L-azetidine-2-carboxylic acid, exhibited a dose-dependent ability to inhibit the formation of beating heart tissue. When examined by electron microscopy, the nonbeating tissue exhibited a drastic depression of myofibrillogenesis, but otherwise appeared healthy. Further examination of the effect of L-azetidine-2-carboxylic acid demonstrated that the inhibition of myofibril formation and heartbeat was correlated with a 60% inhibition of native collagen synthesis; however, the time-course and pattern of actin-isotype biosynthesis was completely unaffected. The data suggest a possible involvement in heart differentiation that is necessary for either the synthesis of non-actin cardiac contractile proteins or the assembly of cardiac contractile proteins into myofibrils.

Actins↗
PubMed1983

Immunological detection of microtubule poison-induced conformational changes in tubulin.

The interaction of tubulin-microtubule poison complexes with anti-tubulin antisera has been investigated using radioimmunoassay. The binding of the major antiserum used in this study to tubulin does not interfere with the binding of colchicine to the tubulin or affect the decay of the colchicine-binding activity of the tubulin. Conversely, if colchicine is incubated with the tubulin, forming tubulin-colchicine complexes, the tubulin-colchicine complexes are less efficient competitors for antibody-binding sites than tubulin alone. This is the result of the formation of specific colchicine-tubulin complexes, since tubulin, incubated with lumicolchicine or isocolchicine, behaves as if the tubulin were incubated alone in the radioimmunoassay. When tubulin is incubated with other microtubule poisons, podophyllotoxin or vinblastine, the tubulin-drug complexes have diminished ability to compete with tubulin as did the tubulin-colchicine complexes. These changes observed in the binding of tubulin-microtubule poison complexes to anti-tubulin antisera in a tubulin radioimmunoassay suggest that the binding of colchicine, podophyllotoxin, or vinblastine to tubulin induces subtle conformational changes on the surface of the tubulin dimer involving antigenic determinant sites.

Animals↗
PubMed1983

Preparation and reactions of an iodinated imidoester reagent with actin and alpha-actinin.

The chemical iodination of an imidoester (methyl-p-hydroxybenzimidate, Wood et al. (1975) Anal. Biochem. 68, 339) and subsequent coupling of iodinated imidoester (IIE) to protein is an indirect method of iodinating proteins that is specific for the epsilon amino group of lysine residues and maintains the positive charge on the amino group at physiological pH. Purification of the IIE from chloramine-T and free iodine by benzene extraction eliminates the need for isoelectric precipitation and produces a more time- and cost-efficient IIE preparation and purification protocol. The separation of free from protein-bound label by chromatography, using centrifugal elution, provides a separation method that is rapid and efficient, without the generation of large volumes of radioactive wastes characteristic of conventional chromatographic and dialysis methods. To optimize the parameters of labeling protein with IIE, a systematic assessment of the effects of pH, reactant concentrations, and reaction time was made using purified cardiac actin and gizzard alpha-actinin. The parameters were defined to achieve an average labeling ratio of one IIE per protein polypeptide. The data demonstrate that both proteins appear to be labeled at the same rate and define several determining factors that limit the rate and extent of IIE incorporation into protein.

Actinin↗
PubMed1983

Proteoglycan and glycosaminoglycan synthesis in embryonic mouse salivary glands: effects of beta-D-xyloside, an inhibitor of branching morphogenesis.

The proteoglycans and glycosaminoglycans synthesized by embryonic mouse salivary glands during normal morphogenesis and in the presence of beta-xyloside, an inhibitor of branching morphogenesis, have been partially characterized. Control and rho-nitrophenyl-beta-D-xyloside-treated salivary rudiments synthesize proteoglycans that are qualitatively similar, based on mobility on Sepharose CL-4B under dissociative conditions and glycosaminoglycan composition. However, beta-xyloside inhibits total proteoglycan-associated glycosaminoglycan synthesis by 50%, and also stimulates synthesis of large amounts of free chondroitin (dermatan) sulfate. This free glycosaminoglycan accounts for the threefold stimulation of total glycosaminoglycan synthesis in beta-xyloside-treated cultures. Several observations suggest that the disruption of proteoglycan synthesis rather than the presence of large amounts of free glycosaminoglycan is responsible for the inhibition of branching morphogenesis. (a) We have been unable to inhibit branching activity by adding large amounts of chondroitin (dermatan) sulfate, extracted from beta-xyloside-treated cultures, to the medium of salivary rudiments undergoing morphogenesis. (b) In the range of 0.1-0.4 mM beta-xyloside, the dose-dependent inhibition of branching morphogenesis is directly correlated with the inhibition of proteoglycan synthesis. The stimulation of free glycosaminoglycan synthesis is independent of dose in this range, since stimulation is maximal even at the lowest concentration used, 0.1 mM. The data strongly suggest that the inhibition of branching morphogenesis is caused by the disruption of proteoglycan synthesis in beta-xyloside-treated salivary glands.

Animals↗
PubMed1982

Immunofluorescence comparisons of anti-actin specificity.

The abilities of antibody populations against brain actin and two immunogenic forms of cardiac actin to react with sarcomeric muscle actin and cytoplasmic non-muscle actin were tested by indirect immunofluorescence, by using isolated skeletal muscle myofibrils and cultured non-neuronal dorsal root ganglion cells as the test systems. All three antibody preparations stained the I-bands of myofibrils, a result that demonstrated the presence of antigenic determinants shared among skeletal, cardiac, and brain actins. However, although antibodies against cytoplasmic brain actin stained the stress fibers of cultured cells, those against glutaraldehyde cross-linked cardiac actin did not, a result that implies that cardiac actin possesses determinants common to sarcomeric actins but not present on cytoplasmic actin. Finally, antibodies against SDS-treated cardiac actin readily stained the stress fibers of cultured cells, in contrast to those against glutaraldehyde cross-linked cardiac actin, a result that suggests that the state of the original immunogen can affect the actin type specificity of the resulting antibody population.

Actins↗
PubMed1981

Distribution of tubulin and actin in neurites and growth cones of differentiating nerve cells.

Embryonic chick nerve cells, from dissociated dorsal root ganglia, were cultured on polylysine substrata and examined for tubulin and actin distribution by indirect immunofluorescence. Antibodies generated against chick brain tubulin produced specific fluorescence in growth cones, neurites, and cell bodies without revealing distribution differences or substructure in the nerve cells. However, at reduced antitubulin concentrations, differences were resolved. Tubulin fluorescence remained uniform and intense in neurites and cell bodies, but exhibited reduced intensity and patterning in growth cones. Nonneuronal cells in the cultures served as controls for typical cytoplasmic tubulin fluorescence distribution. Straining controls demonstrated that fluorescence resulted from tubulin-antitubulin binding. Analogous studies, using antibodies generated against chick brain actin, demonstrated distribution differences at reduced antiactin concentrations, including "hot spots" of intense fluorescence in growth cones and a paucity of fluorescence in neurites.

Actins↗