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M C Neville

Publications and source records attributed to M C Neville.

At least 73 records · Page 4Linked to original sources
PubMed1983

Measurement of steady state protein degradation in cultured human muscle cells.

Double label techniques for measurement of protein turnover in cultured cells are described. In the isotope withdrawal method protein in cultured muscle is labeled with two isotopes of the same amino acid for 24 to 100 h, followed by exposure to fresh medium containing one isotope only at the same specific activity for an additional 24 to 48 h. In the isotope addition method the order of addition of single and double-labeled media is reversed. After incubation the ratio of the two isotopes in the cell protein is a function of the incubation time and the degradation rate constant KD; KD can readily be calculated using a graphical or iterative method. In mixed cultures of human muscle with initial incubation ranging 24 to 159 h, the KD's obtained from various incubation times were similar. Both the isotope withdrawal and the isotope addition methods gave a KD value of 0.018 h-1 similar to values obtained by two different single isotope methods which monitor the appearance of free isotope in the medium of previously labeled cells. There were no differences of KD values obtained in cultures of muscle from normal patients and those with denervation, inflammatory myopathies, or nonspecific myopathic biopsy changes. When proteins were separated by gel electrophoresis, those of molecular weight greater than 60,000 had higher average KD values as compared to lower molecular weight proteins. The double isotope labeling method has the advantage of being easily applied to cultures with small numbers of cells and is potentially useful in obtaining the degradation rates of individual cellular proteins. The major disadvantages are (1) in their present form the methods can be used only in steady state cultures and (2) they require rather long (24 h) labeling times.

Cells, Cultured↗
PubMed1983

Secretion of calcium into milk: review.

Milk calcium exists in bound and ionized forms. Bound calcium is associated both with casein micelles and complexed to citrate and phosphate. Ionized calcium in milk is 1 to 4 millimolar, at least 1000 times its postulated concentration in the mammary alveolar cell. For this reason active transport mechanisms are necessary for transfer of this nutrient to the lumen of the mammary alveolus. Evidence that the major active transport system is a calcium adenosine triphosphatase residing in the membrane of the Golgi secretory vesicle is summarized. This adenosine triphosphatase appears to be activated by calcium concentrations in the micromolar range, to require magnesium ions, and to operate by phosphorylation of a 100,000 dalton enzyme intermediate. Metabolic processes are required to maintain a low concentration of calcium within the cytosol of the mammary alveolar cell. Because no evidence for sodium/calcium exchange could be found in the mammary gland of the lactating mouse, we suggest that these processes operate through a calcium adenosine triphosphatase in the basolateral membrane of the cell. Decreased calcium in the alveolar lumina decreased the integrity of the barrier between blood and milk. It is postulated from observations in other secretory systems that an increase in cystolic activity calcium may play a role in lactogenesis.

Animals↗
PubMed1983

Ionized calcium in human milk determined with a calcium-selective electrode.

We measured the concentration of ionized calcium in human milk with a Radiometer ionized calcium analyzer at 37 degrees C. This instrument is designed to use simultaneous outputs from a pH electrode and a calcium ion-selective electrode to obtain the true ionized calcium in blood plasma. Because both the ionic strength and the pH of human milk differ significantly from that of plasma, we used a calibration curve that corrected for these variables. No other substances in milk interfered with the electrode response to Ca2+. The Ca2+ concentration of milk decreased and the pH increased as CO2 was lost to the atmosphere. Samples collected in glass capillaries, which minimized CO2 loss, contained 2.84 (SD 0.56) mmol of Ca2+ per liter at pH 6.98. We emphasize the importance of maintaining physiological CO2 concentrations when Ca2+ is being measured in milk.

Calcium↗
PubMed1982

Calcium fluxes in mouse mammary tissue in vitro: intracellular and extracellular calcium pools.

1. The total Ca content of the mammary gland increased from about 2 to 12 mumole/g tissue during the transition from pregnancy to lactation in the mouse. In tissue from lactating mice at least two thirds of the total Ca exchanged with external Ca in 6 hr. There was little non-exchangeable Ca in tissues from pregnant mice.2. At 37 degrees C the time courses of influx and efflux of (45)Ca in lactating tissues could be analysed by assuming three exponential components with rate constants of about 0.3, 0.06 and 0.005 min(-1) and containing, respectively, 1.7, 1.5 and 4.7 mumole (45)Ca/g tissue at the steady state.3. The rapidly effluxing component showed the time- and temperature-dependence characteristic of bulk-phase-limited diffusion through the extracellular space. The diffusion coefficient was about one quarter of the self-diffusion coefficient of Ca in aqueous solution, consistent with a tortuosity factor of about 2. A portion of the Ca in this component was displaced by La(3+). The amount remaining in the presence of 3 mm-La(3+) was close to that expected for free extracellular Ca. The rapid component was therefore interpreted as originating from an extracellular compartment containing both free and bound Ca.4. The rate of efflux of the intermediate component was slowed by a factor of ten when the temperature was decreased from 37 to 0 degrees C giving a Q(10) of 2.7, expected for membrane transport. The slow component present at 37 degrees C was not displaced by EGTA or La(3+), suggesting that it is not localized extracellularly. It was not apparent in the 0 degrees C efflux curves.5. The biphasic time course of uptake of ionophore (A23187)-releasable (45)Ca in particulate fractions obtained by homogenization and centrifugation of tissues which had been incubated with the isotope was consistent with the hypothesis that the two slower components of (45)Ca flux originate from intracellular compartments. Mitochondrial uptake probably did not contribute significantly to Ca exchange in these tissues.6. (45)Calcium fluxes in mammary tissues from pregnant mice also showed three components with rate constants similar to those found in tissues from lactating mice. The amount of Ca in each component was much smaller than in lactating tissue when compared on the basis of tissue weight.7. We conclude from these studies that: (i) intra- and extracellular Ca pools in mammary tissue can be distinguished on the basis of the temperature dependence of their fluxes and (ii) the transition from pregnancy to lactation is accompanied by large increases in both intra- and extracellular Ca pools in mammary alveolar cells.

Animals↗
PubMed1981

ATP-dependent calcium transport by a Golgi-enriched membrane fraction from mouse mammary gland.

Crude particulate preparations from the mammary glands of lactating mice were shown to transport calcium against a concentration gradient in the presence of ATP and mitochondrial inhibitors. Density gradient centrifugation with both sucrose and Percoll gradients indicated the presence of ATP-dependent transport in more than one membrane fraction. A Golgi-enriched membrane fraction possessed the highest specific activity of calcium transport. Digitonin, which increases the permeability of plasma membranes to calcium, did not affect this process. The Golgi fraction contained a 100,000 Dalton protein whose phosphorylation by gamma-[32P]-ATP was enhanced by a micromolar concentrations of free calcium. The phosphorylation was acid-stable and hydroxylamine-sensitive. These properties suggest that Golgi membranes in an activity secreting mammary epithelium possess a calcium transport system which resembles the calcium ATPase present in the sarcoplasmic reticulum of skeletal muscle.

Adenosine Triphosphate↗
PubMed1981

Ionized calcium in milk and the integrity of the mammary epithelium in the goat.

1. Injection of citrate or EGTA solutions into the lumen of the mammary gland of goats in quantities sufficient to reduce ionized calcium to less than one-tenth of normal, led to increases in milk concentrations of Na and Cl and decreases in K and lactose. 2. Subsequent milk yields were decreased in glands treated with citrate but not in those treated with EGTA. 3. Blood-milk potential difference decreased (i.e. towards zero) in glands in which citrate was present. 4. In goats milked hourly with the aid of oxytocin, milk Na and Cl concentrations increased while K and lactose decreased; there was no apparent decrease in Ca2+ concentration. 5. It is suggested that ionized calcium in milk is essential to preserve the integrity of the mammary epithelium during lactation.

Animals↗
PubMed1980

K+ influx components in ascites cells: the effects of agents interacting with the (Na+ + K+)-pump.

Several agents known to interact with the (Na+ + K+)-pump were tested for their effects on the components of steady-state K+ flux in ascites cells. 86Rb+ was used as a tracer for K+, and influx was differentiated into a ouabain-inhibitable "pump" component, a Cl--dependent and furosemide-sensitive "exchange" component, and a residual "leak" flux. All agents tested (ouabain, quercetin, oligomycin, phosphate) affected both the "pump" flux and the Cl--linked flux. These findings suggest a linkage between the activity of the Na/K ATPase and the Cl--dependent K+ exchange flux. In the discussion we point out that the mechanism of this linkage could be direct; e.g., Cl--dependent exchange may represent a mode of operation of the Na/K ATPase. However, data from this and other systems tend to suggest an indirect linkage between the Na+ pump and a KCl symporter, perhaps via a change in the level of intracellular ATP.

Animals↗
PubMed1979

Antianaphylactic agents. 1. 2-(Acylamino)oxazoles.

The synthesis and biological properties of 35 2-(acylamino)oxazoles are described. The majority of the compounds inhibit the release of slow-reacting substance of anaphylaxis (SRS-A) in vitro from sensitized guinea pig chopped lung. In addition, several of the compounds inhibited the release of SRS-A from passively sensitized human chopped lung and protected guinea pigs from the effects of anaphylaxis in a modified Herxheimer test.

Anaphylaxis↗
PubMed1979

The secretion of calcium and phosphorus into milk.

1. The time course of appearance of radioactivity in milk was studied following close-arterial infusion of labelled phosphate, Ca or leucine into the mammary artery of lactating goats. Maximum activities were reached at 1.5 hr in all milk fractions including inorganic soluble phosphate, inorganic colloidal phosphate, casein P, soluble Ca, protein-associated Ca and casein. 2. At 0.5 hr, labelling of the soluble and colloidal phosphate fractions was significantly higher than that of the casein P. 3. Recovery of 32P or 47Ca 3 or more hours after infusion into the cistern of the mammary glands was 98% or greater, indicating that the mammary epithelium is virtually impermeable to [32P]phosphate and 47Ca in the milk to blood direction. 4. Ca and P failed to enter milk in excess of the normal secretion rate when the milk was diluted with isosmotic sucrose given by intraductal injection. 5. These data suggest that milk Ca and phosphate in their various forms are secreted, like protein and lactose, by exocytosis of Golgi vesicles. Unless a paracellular pathway is present, as in oxytocin-treated animals, the milk concentrations are maintained by virtue of the impermeability of the mammary epithelium to these substances.

Animals↗
PubMed1979

The extracellular compartments of frog skeletal muscle.

1. Detailed studies of solute efflux from frog sartorius muscle and single muscle fibres were carried out in order to characterize a 'special region' (Harris, 1963) in the extracellular space of muscle and determine whether this 'special region' is the sarcoplasmic reticulum. 2. The efflux of radioactive Na, Cl, glusose, 3-O-methylglucose, xylose, glycine, leucine, cycloleucine, Rb, K, inulin (mol. wt. 5000) and dextran (mol. wt. 17,000) from previously loaded muscles was studied. In all cases except dextran the curve had three components, a rapid (A) component which could be equated with efflux from the extracellular space proper, a slow (C) component representing cellular solute and an intermediate (B) component. The distribution space for the B component was 8% of muscle volume in summer frogs and 12% in winter frogs and appeared to be equal for all compounds studied. We tested the hypothesis that the B component originated from the sarcoplasmic reticulum. 3. The C component was missing from the dextran curves. Both dextran and inulin entered the compartment of origin of the B component (compartment B) to the same extent as small molecules. 4. For all compounds studies, the efflux rate constant for the A component could be predicted from the diffusion coefficient. For the B component the efflux rate constant was 6--10 times slower than that for the A component but was still proportional to the diffusion coefficient for the solute in question. 5. When Na and sucrose efflux from single fibres was followed, a B component was usually observed. The average distribution space for this component was small, averaging 1.5% of fibre volume. There was no difference between the average efflux rate constants for Na and sucrose. 6. In an appendix, the constraints placed on the properties of a hypothetical channel between the sarcoplasmic reticulum and the T-system by the linear electrical parameters of frog skeletal muscle are derived. It is shown that the conductance of such a channel must be less than 0.06 x 10(-3) mohs/cm2 of fibre membrane. 7. The conductance between compartment B and the extracellular space can be calculated from the efflux rate constants for Na, K and Cl. The value obtained was 5 x 10(-3) mhos/cm2 of fibre membrane or 100 times the limiting value for the conductance of the T-SR junction. 8. The finding that there is a B component in the efflux curves for large molecular weight substances like inulin and dextran and the small size of the B component in efflux curves from single muscle fibres indicate that the 'speical region' of the extra-cellular space of frog muscle is not the sarcoplasmic reticulum. This conclusion is confirmed by a calculation of the conductance between the B compartment and the extracellular space. The value obtained is incompatible with predicted electrical properteis of the SR-T-tubule junction...

Animals↗
PubMed1979

Extracellular space of frog skeletal muscle in vivo and in vitro: relation to proton magnetic resonance relaxation times.

1. The Na and Cl distribution spaces of freshly isolated frog muscles are 16.7 and 12.6%, respectively. These values increase to 25.6 and 23.3%, respectively, on incubation. 2. The extracellular components of both Na and Cl efflux curves are significantly smaller in freshly isolated muscles (approximately 12%) than in incubated muscles (approximately 18%). The fast exchanging A component of the extracellular space is increased more by incubation than the more slowly exchanging B component. 3. The proton magnetic resonance (p.m.r.) transverse relaxation curve for the water of freshly isolated frog muscles did not show the long, slowly relaxing tail present in curves from muscles incubated in Ringer solution. 4. When muscles were incubated in hypertonic solutions the p.m.r. transverse relaxation curves could be resolved into three components whose sizes were consistent with the components present in the sodium and chloride efflux curves. The non-exponentiality of the p.m.r. transverse relaxation curve therfore appears to arise from water in both the A and B extracellular compartments of muscle. 5. Efflux analysis indicated that the cellular Na content of both freshly isolated and incubated frog muscle is similar to that predicted by others (Lev, 1964; Armstrong & Lee, 1971; Lee & Armstrong, 1974) from measurements of intracellular Na ion activity using Na-sensitive micro-electrodes. The remainder of the tissue Na was found in the more rapidly exchanging extracellular compartments. The results of these experiments are inconsistent with the presence of a substantial fraction of bound Na in frog muscle. 6. These experiments show that muscle extracellular space is smaller in vivo than in vitro. Efflux analysis is suggested as the most accurate method of assessing extra-cellular components.

Animals↗
PubMed1977

Control of amino acid transport in the mammary gland of the pregnant mouse.

The regulation of the uptake of the amino acid analog alpha-aminoisobutyric acid was studied in diced mammary glands from pregnant mice. Stimulation of uptake by insulin was not prevented by inhibitors of protein synthesis; protein synthesis inhibitors decreased uptake by 20%; this response occurred more promptly in insulin-treated tissues. Elimination of extracellular amino acids led to a substantial increase in transport which was not abolished by inhibitors of protein synthesis. These results indicate that insulin does not increase amino acid transport in this system by altering synthesis and degradation of transport protein. They are consistent with a model in which the activity of the existing amino acid transport protein in subject to negative feedback regulation from the intracellular amino acid pool.

Aminoisobutyric Acids↗