Biomedical subjects
Publications and source records attributed to S S Miller.
Despite many attractive hypotheses, neonatal necrotising enterocolitis (NNEC) remains a disease of unknown aetiology. By means of the immunofluorescent direct sandwich technique we have demonstrated immune complex deposition in the small vessels of the diseased bowel wall. We believe this is the first report of evidence of an immunological reaction occurring in this disease.
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The value of a system for reducing the number of women with breast lumps who consent unnecessarily to mastectomy was assessed. Sixty-one patients with breast lumps were divided preoperatively into three groups with benign, doubtful, and malignant lumps according to clinical, mammographic and ultrasound criteria. On the basis of these criteria written consent was requested from 29 patients for mastectomy and from 32 for only excision of the lump. Fourteen of the 29 patients who gave consent for mastectomy had carcinomas, and none of the 32 patients consenting to only lump excision. In an attempt to improve further on these results the same 61 patients were analysed retrospectively. Criteria based on age and the results of clinical examination and mammography were devised. By using these criteria only 19 women would have had to give consent for mastectomy. This new policy, which was devised to spare many women the stress of consenting unnecessarily to mastectomy, requires to be tested further in a much larger series of patients.
Rats offered a nutritionally balanced and complete liquid diet containing 35% of energy as ethanol, 12% as fat, 21% as protein, and the balance as carbohydrate consumed greater than 9 g/kg ethanol after 10 days. Rats displayed signs of physical dependence and tolerance while showing a net gain in weight. Physical dependence was indicated by severe intensity of the following signs during withdrawal from ethanol: Muscle rigidity; tail tremors; caudal tremors; and general tremors. Severity of these signs reached a maximum intensity by 19 h after withdrawal of ethanol. Tolerance was exhibited by chronically treated rats as measured by significantly reduced time off the belt after 7 days. Concentrations of ethanol in blood were documented on selected mornings and were observed to increase. These data suggest that physical dependence and tolerance can be induced through voluntary consumption of ethanol by rats and without nutritional compromises or weight loss.
Nutritionally complete diets formulated according to American Institute of Nutrition guidelines were used to make rats dependent upon ethanol. When intubated with a diet-ethanol solution for four days maintained initial body weight. When forced to consume the solution as the sole source of nutrients and water for nineteen days, rats gained weight. All animals developed severe withdrawal signs as measured by the intensity of tremors and spastic rigidity. The diet ingredients did not alter the absorption of the ethanol. The results demonstrate that physical dependence on ethanol can be induced in the rat without nutritional impairment.
The role of the retinal pigment epithelium in regulating transport between the choriocapillariss and sensory retina was studied by examining the diffusion fluorescent across the intact rabbit retinal pigment epithelium and the retinal pigment epithelium damaged by intravenous injection of sodium iodate. Destruction of the tight junctions of the retinal pigment epithelium allowed free diffusion of fluorescein from the choroid into the retina. The electrical characteristics of the cat retinal pigment epithelium which play an important role in governing transport of substances across the retinal pigment epithelium were studied. The apical membrane of the cat retinal pigment epithelium resembled that of the frog in having high sensitivity to apical K+, greater sensitivity to apical Na+, and less sensitivity to apical HCO3-. The apical membrane was sensitive to oubain which produced a rapid fall in the transepithelial potential. Previous studies of the electrical properties of the retinal epithelium in cold blooded preparations are summarized with emphasis on the important interactions between the retinal pigment epithelium and sensory retina and the implications that these studies have with regard to the regulation of photoreceptor function.
Net taurine transport across the frog retinal pigment epithelium-choroid was measured as a function of extracellular potassium concentration, [K+]o. The net rate of retina-to-choroid transport increased monotonically as [K+]o increased from 0.2 mM to 2 mM on the apical (neural retinal) side of the tissue. No further increase was observed when [k+]o was elevated to 5 mM. The [K+]o changes that modulate taurine transport approximate the light-induced [K+]o changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium. The taurine-potassium interaction was studied by using rubidium as a substitute for potassium and measuring active rubidium transport as a function of extracellular taurine concentration. An increase in apical taurine concentration, from 0.2 mM to 2 mM, produced a threefold increase in active rubidium transport, retina to choroid. Net taurine transport can also be altered by relatively large, 55 mM, changes in [Na+]o. Apical ouabain, 10(-4) M, inhibited active taurine, rubidium, and potassium transport; in the case of taurine, this inhibition is most likely due to a decrease in the sodium electrochemical gradient. In sum, these results suggest that the apical membrane contains a taurine, sodium co-transport mechanism whose rate is modulated, indirectly, through the sodium pump. This pump has previously been shown to be electrogenic and located on the apical membrane, and its rate is modulated, indirectly, by the taurine co-transport mechanism.
We compared the initiation specificities in vitro of deoxyribonucleic acid-dependent ribonucleic acid polymerases purified from two cyanobacteria, Fremyella diplosiphon and Anacystis nidulans, and from Escherichia coli. A restriction fragment made from lambda deoxyribonucleic acid was used as a template. The cyanobacterial and E. coli ribonucleic acid polymerases recognized the same lambda promoters but exhibited different sensitivities to the inhibitor heparin, suggsesting differences in the structure of the initiation complexes.
It was previously shown that ouabain decreases the potential difference across an in vitro preparation of bullfrog retinal pigment epithelium (RPE) when applied to the apical, but not the basal, membrane and that the net basal-to-apical Na+ transport is also inhibited by apical ouabain. The suggested the presence of a Na+ - K+ pump on the apical membrane of the RPE. In the present experiments, intracellular recordings from RPE cells show that this pump is electrogenic and contributes approximately - 10 mV to the apical membrane potential (VAP). Apical ouabain depolarized VAP in two phases. The initial, fast phase was due to the removal of the direct, electrogenic component. In the first one minute of th response to ouabain, VAP depolarized at an average rate of 4.4 +/- 0.42 mV/min (n = 10, mean +/- SEM) and VAP depolarized an average of 9.6 +/- 0.5 mV during the entire fast phase. A slow phase of membrane depolarization, due to ionic gradients running down across both membranes, continued for hours at a much slower rate, 0.4 mV/min. Using a simple diffusion model and K+-specific microelectrodes, it was possible to infer that the onset of the ouabain-induced depolarization coincided with the arrival of ouabain molecules at the apical membrane. This result must occur if ouabain affects an electrogenic pump. Other metabolic inhibitors, such as DNP and cold, also produced a fast depolarization of the apical membrane. For a decrease in temperature of congruent to 10 degrees C, the average depolarization of the apical membrane was 7.1 +/- 3.4 mV (n = 5) and the average decrease in transepithelial potential was 3.9 +/- 0.3 mV (n = 10). These changes in potential were much larger than could be explained by the effect of temperature on an RT/F electrodiffusion factor. Cooling the tissue inhibited the same mechanism as ouabain, since prior exposure to ouabain greatly reduced the magnitude of the cold effect. Bathing the tissue in 0 mM [K+] solution for 2 hr inhibited the electrogenic pump, and subsequent re-introduction of 2 mM [K+] solution produced a rapid membrane hyperpolarization. We conclude that the electrogenic nature of this pump is important to retinal function, since its contribution to the apical membrane potential is likely to affect the transport of ions, metabolites, and fluid across the RPE.
We have studied the hyperpolarizing, electrogenic pump located on the apical membrane of the retinal pigment epithelium (RPE) in an vitro preparation of bullfrog RPE-choroid. Changes in RPE [K+]i alter the current produced by this pump. Increasing [K+]o in the solution perfusing the basal membrane increases RPE [K+]i (measured with a K+-specific microelectrode), and also depolarizes the apical membrane. The depolarization is due to a decrease in electrogenic pump current flowing across the apical membrane resistance, since it is abolished when the pump is inhibited by apical ouabain, by cooling the tissue, or by 0 mM [K+]o outside the apical membrane. Removal of Cl- from the solution perfusing the basal membrane abolishes the K+-evoked apical depolarization by preventing the entry of K+ (as KCl) into the cell. We conclude that the increase in [K+]i causes the decrease in pump current. This result is consistent with the finding that [K+]i is a competitive inhibitor of the Na+ - K+ pump in red blood cells. It is possible that the light-evoked changes in [K+]o in the distal retina could alter RPE [K+]i, and thus could affect the pump from both sides of the apical membrane. Any change in pump current is likely to influence retinal function, since this pump helps to determine the composition of the photoreceptor extracellular space.
We have purified and characterized a DNA-dependent RNA polymerase from the blue-green alga Fremyella diplosiphon. This enzyme, purified by gel filtration, DEAE-cellulose chromatography, and glycerol gradient centrifugation, is comprised of five polypeptide subunits. Their masses are 161,000, 134,000, 91,000, 72,000, and 41,000 daltons. Preparative electrophoresis of the purified enzyme on nondenaturing gels separates the 41,000-dalton polypeptide from the rest of the enzyme. The enzyme is extremely labile in the presence of a variety of salts of strong acids and bases; the purification procedure was devised to avoid exposure to such compounds.
An in vitro preparation of a mammalian retinal pigment epithelium (RPE)-choroid was developed, that of the cat, in order to study the RPE in isolation from the neural retina. The purpose of these initial experiments was to evaluate the electrical characteristics of the tissue and the ionic properties of the RPE apical membrane. They were designed so that results would be directly comparable to those previously obtained on a more extensively studied cold-blooded preparation, the bullfrog. The electrical characteristics of the best cat tissues were similar to those routinely obtained in bullfrog, whereas the average cat apical membrane potential was 20 mV more depolarized than that of frog. The apical membrane of cat resembled the frog's in having a large sensitivity to potassium, but it had a relatively smaller sensitivity to bicarbonate and a relatively larger sensitivity to sodium. The cat, like the frog, also had a ouabain-sensitive mechanism on its apical membrane that directly contributed to membrane potential. Two factors contributing to the lower apical membrane potential in cat were the higher potassium concentration of the mammalian salt solution and the sodium conductance of the apical membrane.
The isolated pigment epithelium and choroid of frog was mounted in a chamber so that the apical surfaces of the epithelial cells and the choroid were exposed to separate solutions. The apical membrane of these cells was penetrated with microelectrodes and the mean apical membrane potential was --88 mV. The basal membrane potential was depolarized by the amount of the transepithelial potential (8--20 mV). Changes in apical and basal cell membrane voltage were produced by changing ion concentrations on one or both sides of the tissue. Although these voltage changes were altered by shunting and changes in membrane resistance, it was possible to estimate apical and basal cell membrane and shunt resistance, and the relative ionic conductance Ti of each membrane. For the apical membrane: TK approximately equal to 0.52, THCO3 approximately equal to 0.39 and TNa approximately equal to 0.05, and its specific resistance was estimated to be 6000--7000 omega cm2. For the basal membrane: TK approximately equal to 0.90 and its specific resistance was estimated to be 400--1200 omega cm2. From the basal potassium voltage responses the intracellular potassium concentration was estimated at 110 mM. The shunt resistance consisted of two pathways: a paracellular one, due to the junctional complexes and another, around the edge of the tissue, due to the imperfect nature of the mechanical seal. In well-sealed tissues, the specific resistance of the shunt was about ten times the apical plus basal membrane specific resistances. This epithelium, therefore, should be considered "tight". The shunt pathway did not distinguish between anions (HCO--3, Cl--, methylsulfate, isethionate) but did distinguish between Na+ and K+.
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Pigment epithelial cell membrane potentials were measured in an in vitro frog retina-pigment epithelium-choroid preparation. Light stimuli hyperpolarized the apical membrane of the pigment epithelium. This hyperpolarization was the source of the c-wave of the electroretinogram. Light stimuli also decreased retinal extracellular potassium ion concentration, [K+]O, with the same time course as the apical hyperpolarization. It is suggested that the pigment epithelial hyperpolarization, which causes the c-wave, results directly from the light-evoked decrease in retinal [K+]O.
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