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Biomedical subjects

A Ames

Publications and source records attributed to A Ames.

At least 37 records · Page 2Linked to original sources
PubMed1980

Protein turnover in retina.

Rabbit retinas were exposed in vitro to 0.5-h pulses of [3H]leucine or [14C]leucine. Some retinas were harvested promptly after labeling to measure synthesis. These were combined, in double-labeling experiments, with retinas that had been returned to unlabeled medium for a subsequent 1 h or 3.75 h to measure degradation. All of the proteins were solubilized, and separated according to size by gel electrophoresis. The gels were cut into 95 slices, and each slice was differentially counted. The amount of protein in the slice was estimated from the Coomassie blue staining, and its molecular weight from the distribution of molecular weight (MW) standards. Turnover rates of the various sizes of proteins were calculated from these data using certain well-defined assumptions. Retinal protein contained about 32 X 10(3) nmol of polypeptide per g, with a median MW of 27,000. Total synthesis was at the rate of 103 nmol/g of protein/h, with the most rapid synthesis in the 33,000--43,000 MW range, at 2 nmol/g/h for every 1000 increment in MW. Protein renewal averaged 0.52%/h, but varied directly (p < 0.0001) with MW, so that proteins of 10,000 MW were being renewed at about 0.1%/h and proteins of 140,000 MW at about 1.4%/h. Taken together, the measurements of fractional renewal and the measurements of degradation of the newly synthesized proteins demonstrated that each slice contained proteins with markedly different breakdown coefficients, and provided enough information to characterize the proteins in the slice in terms of a fast and slow subgroup. This analysis indicated that: breakdown coefficients varied much more than rates of synthesis and were therefore the prime determinant of the amount of each protein that was present; as MW increased, breakdown coefficients of the long-lived proteins increased (p < 0.0001), accounting in major part for the correlation between size and turnover; most staining bands were due to proteins with peculiarly long lifespans; the proteins with the slowest turnover of all appeared to be histones; there was an unusually rapid synthesis of a 138,000 MW polypeptide with a moderately short half-life (about 3 h).

Animals↗
PubMed1980

Synthesis and degradation of retinal proteins in darkness and during photic stimulation.

Rabbit retinas were maintained in a physiological state in vitro and exposed to 0.5-h pulses of labeled leucine. Protein synthesis was determined from incorporation of the label, and degradation from its subsequent release. The retinas were treated as test-control pairs. The control retina remained in darkness while the test retina was subjected to photic stimulation, either during labeling to determine the effect on synthesis or after labeling to determine the effect on degradation. 3H and 14C alternated as test and control labels. The two retinas were combined for solubilization. Their proteins were separated according to size by gel electrophoresis. Each gel was cut into 95 slices and each slice was differentially counted. The isolated retinas synthesized new protein rapidly and reproducibly. The average S.D. of the isotope ratios measured on gel slices from replicate experiments was 2.2% of the mean. Retinas driven by flashing light of constant intensity exhibited a marked increase in ganglion cell firing and accumulated 38% more (p = 0.005) 2-deoxyglucose than their controls kept in darkness. However, they did not differ from the controls in the incorporation of labeled leucine into total protein or into any of the protein fractions separated on the gel, the largest deviation from unity in the 95 test-control ratios being 1.7%. Continuous light and flashing light of increasing intensity also markedly affected function and energy metabolism, but had no significant effects on leucine incorporation. None of the stimuli affected degradation, though the experiments would have been less sensitive to a change in degradation that to a change in synthesis. These results indicate that the synthesis and degradation of proteins are little affected by a marked increase in functional activity.

Animals↗
PubMed1980

Nursing staff bylaws.

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Nursing Service, Hospital↗
PubMed1976

Cell volume and permeability of oxygen-and glucose-deprived retina in vitro.

Rabbit retina was deprived of O(2) and glucose in vitro for up to four hours at 37 C. Intracellular volume was measured, using inulin as an extracellular marker. After a 30-minute latency, cells swelled rapidly to more than twice normal volume while extracellular volume was unchanged. Intracellular accumulation of water was not reversed by resupply of oxygen and glucose. Permeability to small molecules was assessed with mannitol. The ratio of mannitol space to inulin space averaged 1.0 in controls. This ratio remained 1.0 up to 30 minutes of deprivation, but increased to 1.2 by 60 minutes. Permeability to large molecules was assessed from the rate of loss of isotopically labeled cell protein into the medium. There was no difference between control and deprived retinas up to three hours.

Animals↗
PubMed1976

Retina subjected to components of ischemia in vitro. Selective vulnerability and minimum lethal exposure of neurons and glia to oxygen and/or glucose deprivation and to loss of exchange with incubating medium.

Rabbit retinas were incubated at 37 C in media lacking oxygen, glucose, or both, or sealed in a small compartment without medium to convert them to a "closed system." They were then returned to control medium before being fixed for microscopy. Other retinas were incubated only in control medium and then fixed. Conversion of the retina to a closed system caused irreversible damage to all cell types within 40 minutes. Combined deprivation of oxygen and glucose also irreversibly damaged the neuronal cells within 40 minutes, but Mueller cells,the principal glial cells of the retina, were not irreversibly altered by 90 minutes of the deprivation. Deprivation of oxygen alone caused irreversible damage to receptor cells in 80 minutes, but the cells of the inner nuclear layer, ganglion cells, and Mueller cells retained normal structure for at least 180 minutes. Deprivation of glucose alone damaged receptor cells in 160 minutes and the other neuronal cells in 180 minutes, but did not irreversibly damage Mueller cells by 200 minutes.

Animals↗
PubMed1976

Protein synthesis in central nervous tissue: studies on retina in vitro.

Rabbit retinas were maintained in vitro in medium that resembled CSF but with leucine varied from 2 to 1000 microM. Both leucine and threonine were isotopically labelled. When leucine in the medium was 100-1000 microM, leucine was incorporated into protein at 2.03 +/- 0.04 (S.E.M.) mumol/g dry wt./h, a turnover per h of 0.55% of the leucine in retinal protein. Incorporation was constant for at least 7 h. It was reduced 34% when the other amino acids were omitted from the medium and 24% when they were increased 15 fold above physiological levels. When medium leucine was reduced to 2 microM with other amino acids constant, 14C-leucine incorporation fell 70% without significant change in 3H-threonine incorporation, indicating a fall in intracellular specific activity of leucine. The intracellular/extracellular concentration ratio of labelled leucine was 4:1 with medium leucine 23 microM. It fell markedly when medium leucine was reduced to 2 microM or increased to 1000 microM. The concentration ratio of labelled threonine was 15:1 with medium leucine at physiological levels but fell to 6:1 when medium leucine was increased to 1000 microM. Decarboxylation removed 1.5% of free intracellular leucine per min and, at physiological concentrations, was 7.7% the rate of protein incorporation. The ratio of protein synthesis/breakdown, estimated from changes in leucine and 7 other essential amino acids in the medium, was nearly unity. The potential of this preparation for study of CNS protein metabolism is discussed.

Amino Acids, Essential↗
PubMed1976

Transport of leucine and sodium in central nervous tissue: studies on retina in vitro.

Unidirectional leucine fluxes were measured in isolated rabbit retina maintained under steady state conditions in medium resembling CSF but with leucine varied from 2 to 20,000 microM. At physiological leucine concentration (11 microM), 1/2 time for outward transport was 88 s and intracellular fluid was cleared of isotopically labelled leucine at 2.3 ml/g dry wt./min; 1/2 time for inward transport was 16 s and interstitial fluid was cleared at 7.5 ml/g dry wt./min. The rate of leucine influx corresponded quite well with its rate of disappearance from the intracellular fluid, over a wide range of concentrations. Exchange diffusion was demonstrated for transport in both directions. There was competition by other amino acids, but no interaction between Na+ and leucine transport could be demonstrated. Kinetic analysis indicated the presence of more than one transport system for leucine. There was an unexpected fall in the efflux coefficient, with reduction in leucine concentration at the lower end of the concentration range, for which an explanation is proposed. Under control conditions, 1/2 time for efflux of intracellular 24Na+ was about 0.9 min. With intracellular Na+ increased 4 fold, 1/2 time for efflux was slightly reduced. Problems encountered in measuring fluxes in organized tissue are discussed.

Animals↗
PubMed1976

Functional homogeneity of leucine pool in retina cells.

Data on leucine metabolism in isolated rabbit retina are examined for evidence, for or against, a common intracellular pool of free leucine. Data include values for: concentrations, transport rates, degradative metabolism and protein incorporation of labelled leucine measured over a wide range of concentrations; protein incorporation of labelled threonine, measured simultaneously; and an indirect measurement of protein breakdown. The fall in labelled leucine incorporation into protein, when medium leucine was reduced below 100 microM, corresponded closely with the fall in intracellular specific activity predicted from rate of influx of labelled leucine from medium and rate of release of unlabelled leucine from protein breakdown. Protein incorporation of labelled leucine competed with decarboxylation and outward transport and reduced the free intracellular leucine in about the amounts predicted for a common pool. Implications for measurements using labelled amino acid are discussed.

Animals↗
PubMed1976

Responses to acetylcholine of ganglion cells in an isolated mammalian retina.

1. Rabbit retinas were isolated and superfused with a physiological medium. Ganglion cell activity was recorded during stimulation with focused light, and receptive fields were mapped. Receptive fields were identical to those found in vivo and did not change during a 6-h incubation. After the receptive field of a ganglion cell had been identified, acetylcholine or related agents were introduced singly or in combination into the medium, and their effect on the cell's spontaneous and light-evoked activity was observed. 2. Ganglion cells with on-center or directionally selective receptive fields were excited when ACh was added to the medium. The response to exogenous ACh was prevented by cholinergic antagonists. 3. These cells' spontaneous activity and response to light were enhanced by anticholinesterase and depressed by cholinergic antagonists. Antagonists varied in their ability to block the light-evoked response, with dihydro-beta-erythroidine the most effective. 4. Thresholds for ACh or the related agents were low, ranging from 1 to 40 muM; their effects were rapidly and completely reversed when the retina was returned to control medium. 5. In retinas incubated in medium containing 20 mM Mg2+ and 0.2 mM Ca2+, ganglion cells lost completely both their spontaneous and light-evoked activity, but retained their ability to generate action potentials in response to elevated K+. Ganglion cell activity rapidly returned to normal when the retina was returned to medium containing normal electrolytes. On-center and directionally selective cells were excited by ACh in retinas where synaptic transmission had been inhibited by 20 mM Mg2+ and 0.2 mM Ca2+. 6. The responses of on-center and directionally selective cells to ACh, to anticholinesterase, and to cholinergic antagonists in control medium indicate that the retina contains one or more synapses using ACh as a neurotransmitter. The response to ACh in retinas exposed to 20 mM Mg2+ and 0.2 mM Ca2+ suggests that at least one such synapse in on the ganglion cell itself. 7. Off-center cells were inhomogenous in their response to ACh. Although some responded just as the other classes of cell, the majority responded quite weakly and a subgroup was encountered which was entirely unaffected by even 1 mM ACh, by levels of physostigmine which inactivate virtually all retinal acetyl-cholinesterase, or by high concentrations of cholinergic antagonists. Only 2 of 20 off-cells tested in the presence of 20 mM Mg2+ and 0.2 mM Ca2+ were excited by ACh. Apparently ACh is not a primary transmitter for most off-cells.

Acetylcholine↗
PubMed1975

Dissociation of field potential from neuronal activity in the isolated retina: failure of the b-wave with normal ganglion cell response.

The b-wave of the isolated rabbit retina was compared with the ganglion cell response to light before and after modification of the retina's incubating medium. Marked diminution of the b-wave, with no reduction in ganglion cell response, was observed under three experimental conditions: (1) following a short period of anoxia; (2) following a short period in 0.2 mM Ca++; (3) in a small percentage of preparations, simply as a result of prolonged incubation in control medium. In contrast, a short period in 50 mM K+ led to a parallel fall and parallel recovery of both responses. It is apparent that under selected conditions the field potentials which constitute the b-wave are poorly correlated with the retina's neural activity.

Animals↗