Biomedical subjects

S J Morris

Publications and source records attributed to S J Morris.

At least 73 records · Page 4Linked to original sources

PubMed1990

Real-time multi-wavelength fluorescence imaging of living cells.

We describe a new real-time fluorescence video microscope design for capturing intensified images of cells containing dual wavelength "ratio" dyes or multiple dyes. The microscope will perform real-time capture of two separate fluorescence emission images simultaneously, improving the time resolution of spatial distribution of fluorescence to video frame rates (30 frames/s or faster). Each emission wavelength is imaged simultaneously by one of two cameras, then digitized, background corrected and appropriately combined at standard video frame rates to be stored at high resolution on tape or digital video disk for further off-line analysis. Use of low noise, high sensitivity image intensifiers, coupled to CCD cameras produce stable, high contrast images using ultra low light levels with little persistence or bloom. The design has no moving parts in its optical train, which overcomes a number of technical difficulties encountered in the present filter wheel designs for multiple imaging. Coupled to compatible image processing software utilizing PC-AT computers, the new design can be built for a significantly lower cost than many presently available commercial machines. The system is ideal for ratio imaging applications; the software can calculate the ratio of fluorescence intensities pixel by pixel and provide the information to generate false-color images of the intensity data as well as other calculations based on the two images. Thus, it provides a powerful, inexpensive tool for studying the real-time kinetics of changes in living cells. Examples are presented for the kinetics of rapidly changing intracellular calcium detected by the calcium indicator probe indo-1 and the redistribution kinetics of multiple vital dyes placed in cells undergoing cell fusion.

Animals↗

PubMed1989

Kinetics of pH-dependent fusion between 3T3 fibroblasts expressing influenza hemagglutinin and red blood cells. Measurement by dequenching of fluorescence.

Fusion between membranes of 3T3 fibroblasts expressing hemagglutinin (HA) from the Japan strain of influenza virus and human red blood cells (RBC) was measured using an assay for lipid mixing based on the relief of self-quenching (dequenching) of fluorescence of the lipid probe octadecylrhodamine (R18). The probe was incorporated into the membrane of intact RBC at self-quenching concentrations, and the RBCs were bound to the 3T3 cells. Fusion, which allowed movement of R18 into 3T3 cell membranes, was monitored by spectrofluorometry as an increase in fluorescence. Upon lowering the pH below 5.4, the fluorescence increased after a delay of about 30 s at 37 degrees C, and leveled off within 2 min. In control experiments where R18 RBCs bound to 3T3 cells expressing the uncleaved precursor hemagglutinin (HA0) were incubated at 37 degrees C and low pH, no fluorescence increase was observed. This indicated that the R18 dequenching occurred as a result of HA-induced fusion of plasma membranes. Fusion showed a very steep pH dependence with a threshold at pH 5.4 and a maximum at pH 5.0, similar to HA-induced fusion seen previously using cell biological techniques. The fusion rate increased and the delay for the onset of fusion decreased as the temperature was raised above 20 degrees C. Low pH activation of the fusion process at 37 degrees C could be partially arrested by raising the pH after 2-10 s, but not after 15 s, indicating that the irreversible pH-activated conformational change of HA necessary for fusion was complete within about 15 s. Analysis of the data indicates that the pH-induced membrane fusion activity of HA is a highly cooperative event.

Animals↗

PubMed1989

Initial stages of influenza hemagglutinin-induced cell fusion monitored simultaneously by two fluorescent events: cytoplasmic continuity and lipid mixing.

We have monitored the mixing of both aqueous intracellular and membrane-bound fluorescent dyes during the fusion of human red blood cells to influenza hemagglutinin-expressing fibroblasts using fluorescence spectroscopy and low light, image-enhanced video microscopy. The water-soluble fluorescent dye, N-(7-nitrobenzofurazan-4-yl)taurine, was incorporated into intact human red blood cells. The fluorescence of the dye in the intact red blood cell was partially quenched by hemoglobin. The lipid fluorophore, octadecylrhodamine, was incorporated into the membrane of the same red blood cell at self-quenching concentrations (Morris, S. J., D. P. Sarkar, J. M. White, and R. Blumenthal. 1989. J. Biol. Chem. 264: 3972-3978). Fusion, which allowed movement of the water-soluble dye from the cytoplasm of the red blood cell into the hemagglutinin-expressing fibroblasts, and movement of octadecylrhodamine from membranes of red blood cell to the plasma membrane of the fibroblasts, was observed by fluorescence microscopy as a spatial relocation of dyes, and monitored by spectrofluorometry as an increase in fluorescence. Upon lowering the pH below 5.4, fluorescence increased after a delay of about 30 s at 37 degrees C, reaching a maximum within 3 min. The kinetics, pH profile, and temperature dependence were similar for both fluorescent events measured simultaneously, indicating that influenza hemagglutinin-induced fusion rapidly establishes bilayer continuity and exchange of cytoplasmic contents.

Animals↗

PubMed1987

Myelin basic protein binds heme at a specific site near the tryptophan residue.

Fluorescence of the single tryptophan residue in myelin basic protein (MBP) was excited directly at 295 nm (red-edge excitation) or at 278 nm which allows, in addition, indirect excitation by resonance energy transfer (RET) from any nearby tyrosine residues. Both red-edge excitation and the RET pathway were collisionally quenched by I- and acrylamide, but not by Cs+ or Co2+, implying that the fluorophore is in an exposed, positively charged environment. The quenching coefficients (K) for I- are 12-15 M-1 at both excitation wavelengths while coefficients for acrylamide are 15 M-1 at 278-nm and 8 M-1 at 295-nm excitation. Chloroheme, cyanoheme, and protoporphyrin IX also quench both red-edge excitation and the RET pathway with apparent quenching coefficients which are (2-5) X 10(4)-fold higher. This suggests that the mechanism of quenching now includes static in addition to collisional processes and thus that heme has a relatively high affinity for MBP. Scatchard analysis of the quenching suggests that chloroheme binds to MBP at two sites with dissociation constants (Kd) of 1.6 X 10(-8) and 2.0 X 10(-7) M and stoichiometries of 0.04:1 and 0.16:1, respectively. The hydrophobic fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate] [bis(ANS)] binds to MBP less avidly (Kd = 10(-7) M) and is rapidly displaced by chloroheme (Ki = 2 X 10(-8) M). The affinities of bis(ANS) and heme for MBP, along with the fluorescent amino acid quenching data, demonstrate that a subfraction of MBP molecules contain considerable structural specificity, implying stable long-range interactions in the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Uranyl acetate induces gel phase formation in model lipid and biological membranes.

The effect of uranyl acetate on the mesomorphic phase state of lipids in model membranes as well as in isolated biological membranes has been examined. As little as 0.8 mM (0.03% [wt/vol]) uranyl acetate induces a liquid crystal-to-gel phase transformation in egg phosphatidic acid, bovine brain phosphatidylserine, and in lysed chromaffin granule membranes. These results along with others in the literature indicate that the uranyl acetate used in samples for electron microscopy could alter membrane morphology.

Adrenal Medulla↗

PubMed1987

Patient management after endoscopic removal of the cancerous colon adenoma.

The subject of management of patients after endoscopic removal of cancerous adenomas is controversial. A retrospective review of 126 lesions in 121 patients who had had colonoscopic polypectomy of malignant lesions between 1971 and 1985 was used to determine the criteria for colon resection. Invasive cancer was identified in 80 patients, while 41 patients had carcinoma in situ. A synchronous colon cancer was found in five of the 121 patients. The patients who had carcinoma in situ had no evidence of residual tumor or metastatic disease on subsequent follow-up (colon resection in three patients and endoscopic surveillance in 38 patients). Of the 80 patients with invasive cancer, 44 had subsequent colon resection, and 34 of these had no evidence of tumor in the resected bowel or mesenteric lymph nodes. Ten patients had residual tumor, metastatic cancer to regional lymph nodes, or both. Each of the 10 had at least one of the following indications of inadequate resection or dissemination of disease to local lymph nodes (the first indication is a macroscopic evaluation, while the remaining four are all microscopic): incomplete excision, poorly differentiated tumor, invasion of the line of resection, invasion of the polyp stalk, and invasion of venous or lymphatic channels. Present recommendations for patient management after endoscopic removal of an invasive malignant adenoma should include colon resection with regional lymphadenectomy for patients with one or more of these five criteria. Patients without any of these risk factors should have early repeat endoscopic examination 3 months after initial polypectomy to evaluate the polypectomy site. Total colonoscopic examination is repeated at 1 year to ensure the surveillance program is begun with a colon without neoplasms.

Adenoma↗

PubMed1986

Effects of tricyclic antidepressant drugs on energy-linked reactions in mitochondria.

The effects of impramine and chlorimipramine on energy-linked reactions in mitochondria were characterized. Both compounds exhibited some characteristics of classical uncouplers of oxidative phosphorylation, i.e. they released respiratory control, hindered ATP synthesis, and enhanced ATPase activity of isolated rat liver mitochondria. Unlike classical uncouplers, however, these compounds only weakly stimulated proton uptake in intact mitochondria. They also exhibited unusual effects on energy-linked reactions in beef heart submitochondrial particles (SMP). Both compounds inhibited NADH oxidation in SMP in an "oligomycin-like" manner, and inhibited ATPase activity of SMP and the soluble F1-ATPase. In contrast, the drugs weakly inhibited ATPase activities of bovine adrenal gland chromaffin granules and resealed granule ghosts. The mechanisms responsible for the multiple effects on mitochondrial energy-linked processes are unclear. They may be related to the hydrophobicity of the drugs, as has been shown for other hydrophobic amines.

Adenosine Triphosphatases↗

PubMed1985

The use of cobalt ions as a collisional quencher to probe surface charge and stability of fluorescently labeled bilayer vesicles.

Co2+ quenched the fluorescence of the lipid probes NBD-phosphatidylethanolamine (NBD-PE) and lissamine-rhodamine phosphatidylethanolamine (N-Rh-PE) incorporated into lipid vesicles, according to a collisional quenching mechanism in agreement with the Stern-Vollmer law. The quenching coefficient (Q) for NBD-PE, incorporated into uncharged phosphatidylcholine (PC) vesicles was 13.8 M-1. This value was equal to the quenching coefficient of water-soluble NBD-taurine in aqueous solution, indicating that Co2+ was readily accessible to the outer surface of PC vesicles. In phosphatidylserine-phosphatidylethanolamine (PS-PE) (1:1) vesicles, quenching was also proportional to Co2+ concentration but Q was 114 mM-1, some 8000-fold smaller. Using the Gouy-Chapman-Stern model we demonstrated that the surface density of Co2+ bound to lipid was linear with Co2+ concentration in the medium up to 7%. Co2+-associated phospholipid would in turn quench NBD-PE or N-Rh-PE by collisional quenching with lateral diffusion. We investigated the ability of Co2+ to permeate PS-PE (1:1) vesicles. Co2+ quenched fluorophores on the outer surface of large unilamellar vesicles, formed by reverse-phase evaporation. In small unilamellar vesicles Co2+ quenched probes on both outer and inner surfaces, indicating rapid permeation of the ions into the vesicles. Using stopped-flow rapid mixing, we measured the rate of influx of Co2+, and correcting for surface potential using the Gouy-Chapman-Stern model, we calculated a permeability coefficient of 10(-12) cm/s for Co2+ concentrations below 300 microM. Above this concentration, there was a very steep rise in the permeability coefficient, indicating that binding of Co2+ induces defects in the bilayer of these vesicles. This may be related to the ability of the vesicles to undergo membrane fusion. A method for calculating the membrane surface potential from Co2+ quenching data is presented.

Cobalt↗

PubMed1985

Rapid kinetics of Ca2+-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles. The effect of bilayer curvature on leakage.

We have employed both small unilamellar vesicles (SUV) and large unilamellar vesicles formed by the reverse phase evaporation technique (REV) to study the initial kinetics of membrane aggregation and fusion. Stopped flow measurements of the calcium-induced changes in the turbidity of SUV and REV, formed from 1:1 (mol/mol) mixtures of bovine phosphatidylserine (PS) and Escherichia coli phosphatidylethanolamine (PE), were used to follow particle aggregation. Simultaneous measurements of the fluorescence resonance energy transfer from N-(7-nitro2,1,3-benzoxadiazol-4-yl) (NBD)-PE to rhodamine (Rho)-PE incorporated into the vesicle bilayers established that 1) both initial aggregation and fusion can be described as a bimolecular process and 2) the rate-limiting step of membrane fusion is aggregation. Thus fusion takes place in the microsecond time domain. Parallel experiments, which simultaneously measured aggregation and the dequenching of encapsulated carboxyfluorescein (CF) in the presence and absence of antifluorescein antibodies in the suspension medium, established that the small unilamellar vesicles rapidly lose their contents of CF as they fuse. On the other hand, the first few cycles of fusion of the large unilamellar vesicles are nonleaky, but leakage develops within 1-2 s as the particles grow in size. Thus the results demonstrate that the SUV are poor models for the study of nonleaky fusion, while the REV must be carefully tested before unambiguous interpretation of fusion assays involving the formation of tight complexes (such as the terbium-dipicolinic assay) can be made. NBD-PE undergoes very rapid, Ca2+-promoted changes in quantum yield which can obscure the resonance energy transfer signals. Thus data from the NBD-PE/Rho-PE energy transfer pair must be carefully scrutinized for artifacts.

Animals↗

PubMed1984

Calcium-promoted fusion of isolated chromaffin granules detected by resonance energy transfer between labeled lipids embedded in the membrane bilayer.

Chromaffin granules isolated from bovine adrenal medulla were labeled with small unilamellar vesicles (SUV) made from N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and/or N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine as donor and acceptor fluorophores. Labeling of granules could be followed by the relief of self-quenching experienced when the labels were incorporated into the granule membrane. The mechanism of incorporation seemed to be fusion of the SUV with the granule membrane although transfer of label could not be ruled out. Granule-granule fusion could be detected either by resonance energy transfer from donor to acceptor fluorophores or by donor quenching, in either of two different experimental designs: by a decrease in donor fluorescence and an increase in acceptor fluorescence when donor-labeled granules fused with acceptor-labeled granules or by an increase in donor fluorescence and a decrease in acceptor fluorescence when granules containing both probes fused with unlabeled granules. Fusion of freshly prepared granules could be initiated by millimolar concentrations of calcium; magnesium was less effective. Mg-ATP had no effect. Fusion was inhibited by potassium glutamate and a variety of organic and inorganic cations and anions, which also inhibited granule-granule aggregation to a lesser extent. The conditions for promotion and inhibition of granule-granule fusion were quite different from those reported by Knight and Baker [Knight, D. E., & Baker, P.F. (1982) J. Membr. Biol. 68, 107-140] for exocytosis of granule contents from permeabilized chromaffin cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗

PubMed1984

Effects of osmotic dehydration on the nucleotides of isolated chromaffin granules: evaluation by 31p nuclear magnetic resonance.

The 31P nuclear magnetic resonance spectrum of chromaffin granules isolated from bovine adrenal medulla, when examined at 30 degrees C in 0.3 M sucrose, shows sharply defined gamma, alpha, and beta nucleotide resonances with linewidths of 141, 58 and 90 Hz respectively. These are essentially unchanged when examined at 4 degrees C. When the granules are resuspended in 1.6 M sucrose, the linewidths of 161, 117 and 162 Hz seen at 30 degrees C reversibly broaden to greater than 1000 Hz at 4 degrees C. Addition of quinacrine produces no discernable change in the nucleotide linewidths of either preparation. The broadening at high osmolarity and low temperature appears to be due to ATP:catecholamine complex formation rather than to increased viscosity. Since purification of chromaffin granules is often performed utilizing sucrose gradients, the present results raise the question of whether dehydration may alter the structure of the granule core.

Adrenal Medulla↗

PubMed1983

Temperature-induced lysis of chromaffin granules provides evidence against the two-pool hypothesis of catecholamine storage.

The temperature-dependent release of core constituents from isolated chromaffin granules in isotonic sucrose has been a controversial and puzzling phenomenon that has been interpreted either as selective catecholamine efflux from different catecholamine pools or as temperature-dependent lysis. We have analysed the kinetics, temperature dependence and physical basis of this process. Our results demonstrate that, upon increasing the ambient temperature, chromaffin granules show a shift in their osmotic fragility to higher osmolarities, which is linearly dependent on temperature and leads to measurable lysis in 0.26 M buffered sucrose at temperatures above 12 degrees C. It is possible to demonstrate both protein and dopamine beta-hydroxylase release when lysis as a function of temperature is measured in 0.26 M buffered sucrose. Real time measurements of the lysis kinetics were recorded on cassettes and analysed by a computer program for exponential decay kinetics. It is shown that the temperature-dependent lysis proceeds in two separate phases, the fast one of which is associated with temperature-dependent shift in the osmotic fragility curve. It has no characteristics of any exponential decay kinetics. The slow phase, when followed over several hours, leads to complete lysis of the granules in a sigmoidal time course at 30 degrees C. We conclude from the absence of exponentiality that there is no basis on which to assume the existence of different catecholamine pools. The fast phase of temperature-dependent lysis can be best explained as a simple temperature-dependent increase of the granule core solution's osmotic pressure, while the slow phase is probably caused by sucrose permeation into the granules. On the basis of these results, we warn against any efflux experiments measuring the temperature-dependent transmitter release from secretory vesicles with highly concentrated core solutions.

Animals↗

PubMed1983

Characteristics and determinants of osmotic lysis in chromaffin granules.

(1) Using isolated bovine chromaffin granules, we demonstrate that osmotic lysis is not a random process and establish the osmotic pressure dependence of osmotic lysis in chromaffin granules, the so-called osmotic fragility curve. (2) We show by measuring the release of constituents of the granule core and correlating these with changes in spectroscopic parameters (turbidity and endogenous catecholamine fluorescence), that the latter can be safely used to measure lysis. (3) Within a particular granule population, noradrenaline granules lyse at higher osmolarities than adrenaline granules, suggesting a higher core osmolarity of the noradrenaline granules. (4) The size distribution of chromaffin granules as a function of lysis was determined by the use of whole mount electron microscopy. It is shown that the mean size of chromaffin granules decreases as a function of lysis. (5) On the basis of theoretical considerations three alternative models of the sequence of osmotic lysis in chromaffin granules are proposed. The experimental results best support a model which postulates that during partial osmotic lysis, granule membranes reseal into smaller vesicles after graded release of contents. The osmotic fragility would represent several cycles of lysis and resealing and would not be a reflection of the distribution of osmotic pressures in the granule population.

Animals↗

PubMed1983

Identification of a second synexin-like adrenal medullary and liver protein that enhances calcium-induced membrane aggregation.

Synexin, an approximately 47,000 Mr soluble protein isolated from adrenal medulla or liver, shows Ca2+-specific enhancement of the aggregation of chromaffin granule or other negatively charged biological or artificial membranes. We report the identification of second synexin-like protein (Mr approximately 56,000) from the same sources with similar Ca2+-specific membrane aggregation activities. However, the molecular weight, aggregation kinetics, susceptibility to protease inactivation and peptide maps of the two synexins are quite different, suggesting that they are entirely different proteins, and that the aggregation assay is only a convenient method for identifying a large number of Ca2+-specific proteins with diverse, yet to be defined activities.

Adrenal Medulla↗

PubMed1983

The chromaffin granule as a model for membrane fusion: implications for exocytosis.

Rapid freeze/freeze-fracture and thin section electron micrographic studies of the Ca2+-promoted aggregation and fusion of isolated bovine adrenal medullary chromaffin granule membranes show that the granules undergo a series of morphological changes. The contact region becomes quite extensive and the membrane curvature changes radically at the edge of the contact site. The core material retracts away from the contact site leaving an electron lucent "stripe"; however, it remains adjacent to the membrane in the non-contact areas. The pentalaminar double membrane of the contact region often shows breaks. Close examination reveals that the two granule membranes have fused and become one continuous membrane. Rearrangement of large membrane associated particles (MAPs) can be seen by freeze fracture after Ca2+-promoted granule-granule contact. The broken pentalaminar septum becomes smaller and may break down into globular structures. These observations suggest a series of reactions in which the granules first form an encounter complex, then a stable complex. The membranes within the contact region undergo lateral displacement of the proteins and phase separations of the lipids, and then fuse. Analysis of the kinetics of turbidity and fluorescence changes during granule aggregation and fusion support the main postulates of the model. The initial events of aggregation are facilitated by putative recognition proteins and K+ will promote all activities except fusion. Recent observations that several soluble proteins (synexin, and albumin) will act as fusogens are discussed in terms of the relevance to exocytosis in vivo.

Animals↗

PubMed1983

An amylase-containing subdiaphragmatic bronchopulmonary sequestration.

A single case of a subdiaphragmatic bronchopulmonary sequestrated cyst removed at laparotomy is reported. Needle aspiration of the cyst prior to removal revealed a high amylase although the serum amylase was normal. A review of the literature failed to reveal a similar case. The ultrasound, computed tomography and histopathology of this case are described.

Adult↗

PubMed1982

Calcium-promoted resonance energy transfer between fluorescently labeled proteins during aggregation of chromaffin granule membranes.

Proteins of the chromaffin granule membrane were covalently labeled in situ with sulfhydryl-specific fluorophores. Using MIANS (maleimide iodoaminonaphthyl sulfonate) as the donor and fluorescein mercury acetate or fluorescein-5-maleimide as the acceptor. Förster fluorescence resonance energy transfer (FRET) could be employed to measure the degree of inter-membrane and intra-membrane protein-protein contact upon Ca2+-induced aggregation of the membranes. The four major findings were: (1) Raising the Ca2+ concentration to approx. 500 microM causes the proteins to aggregate in the plane of the membrane. This is demonstrated by Ca2+-induced increases in the fluorescence resonance energy transfer in double labeled membranes. This effect is not protein-concentration dependent and occurs at calcium concentrations too low for granule aggregation, implying intra-membrane protein clustering or patching. To our knowledge this is the first direct demonstration of the fluid mosaic nature of subcellular organelles. (2) If two sets of granules are labeled separately, Ca2+-induced aggregation brings at least 74% of the labeled proteins into close transmembrane proximity. This effect is also observed at 10-100-fold slower rates in the absence of calcium and can be greatly reduced by depleting the granule membrane of labeled peripheral proteins. It is enhanced if the granules are aggregated by Ca2+ or K+. We conclude that (some) peripheral proteins can transfer from one membrane surface to another. (3) Aggregation of separately labeled sets of membranes by Ca2+ also produces transmembrane energy transfer since: (a) the Km for Ca2+-induced quantum transfer is in the same range as the Km for aggregation; (b) the reaction is protein-concentration dependent; (c) reversal of aggregation also (partially) reverses donor quenching. (4) A kinetic analysis of the transmembrane effect shows it to be 5-10-fold slower than aggregation itself, supporting earlier suggestions (Haynes, D.H., Kolber, M. and Morris, S.J., (1979) J. Theor. Biol. 81, 713-743) that lipid and protein rearrangements are secondary to granule membrane aggregation.

Anilino Naphthalenesulfonates↗

PubMed1982

Interactions of erythrosin B (U.S. F, D & C Red 3) with rat cortical membranes.

Erythrosin B inhibits Na,K-ATPase in rat brain tissue as demonstrated by studying glycoside binding, ATPase activity and ion fluxes. The potency of the noncompetitive inhibition of [3H]-ouabain binding by erythrosin B is influenced by glycoside concentration, monovalent cation concentration, and incubation time. [14C]-Erythrosin B binds to synaptic membranes prepared from rat cortex. Erythrosin B and some of its structural analogs inhibit both [3H]-ouabain and [14C]-erythrosin B binding, but ouabain and other glycosides do not inhibit the binding of [14C]-erythrosin B. Subcellular distributions of [3H]-ouabain and [14C]-erythrosin B binding in fractionated cortical tissue preparations are equivalent and parallel ATPase activity. The dissimilar response of [3H]-ouabain binding and [14C]-erythrosin B binding to changes in tissue preparation, incubation temperature, and partial solubilization of binding sites by deoxycholate (DOC) suggests two separate binding sites for erythrosin and ouabain to rat cortical membranes.

Animals↗