Novel voltage clamp to record small, fast currents from ion channels expressed in Xenopus oocytes.

The present report describes a novel technique for voltage-clamping amphibian oocytes in which part of the membrane is isolated by a vaseline gap and the cytoplasmic fluid is exchanged by cutting or permeabilizing the remaining membrane. The main features of this open-oocyte, vaseline-gap voltage clamp are: (a) low current noise (1 nA at 3 kHz), (b) control of the ionic composition of both the internal and external media, (c) fast time resolution (20-100 microseconds time constant of decay of the capacity transient) and (d) stable recordings for several hours. These features allow reliable measurements of tail or gating currents and the new method is especially suitable when either of these currents must be measured to test the effects of mutations introduced into the cDNAs of cloned ion channels.     

Acylphosphatase from human skeletal muscle: purification, some properties and levels in normal and myopathic muscles

Human skeletal muscle acylphosphatase was purified by immunoaffinity chromatography using anti-horse muscle acylphosphatase antibodies. The three forms of the enzyme present in human muscle are very similar to those found in muscles of other animal species. The two main forms, Hu 1 and Hu 3, were also characterized with respect to molecular weight and some kinetic properties. Levels of acylphosphatase activity were measured in specimens of muscle from normals and from patients with various forms of muscular dystrophies and other myopathies. Acylphosphatase activity appears to be lower in all myopathic forms considered than in controls, and seems to be correlated with percentage of Ca2+ activation of (Ca2+ + Mg2+)-ATPase.     

Effect of Fructose Supplementation on Sorbitol Accumulation and myolnositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Concentrations

Aldose reductase activity is increased in neuroblastoma cells grown in media containing 30 mM fructose and/or 30 mM glucose. Neuroblastoma cells cultured in media supplemented with increased concentrations of glucose and fructose amass greater amounts of sorbitol than do cells exposed to media containing only high glucose concentrations. The increase in sorbitol content is dependent on the fructose and glucose concentration in the media. The increase in sorbitol content caused by exposing neuroblastoma cells to media containing 30 mM glucose/30 mM fructose is due to a protein synthesis sensitive mechanism and not to an alteration in the redox state. The addition of sorbinil to media containing 30 mM glucose blocks the increase in sorbitol content. In contrast, sorbinil treatment of media containing 30 mM glucose/30 mM fructose does not totally block the increase in sorbitol levels. myo-Inositol accumulation and incorporation into inositol phospholipids and intracellular myo-inositol content are decreased in cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose compared to cells cultured in unsupplemented media or media containing 30 mM fructose. However, maximal depletion of myo-inositol accumulation and intracellular content occurs earlier in cells exposed to media containing 30 mM glucose/30 mM fructose than in cells exposed to media supplemented with 30 mM glucose. Sorbinil treatment of media containing 30 mM glucose/30 mM fructose maintains cellular myo-inositol accumulation and incorporation into phospholipids at near normal levels. myo-Inositol content in neuroblastoma cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose recovers within 72 h when the cells are transferred to unsupplemented media or media containing 30 mM fructose. In contrast, the sorbitol content of cells previously exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose then transferred into media containing 30 mM fructose remains elevated compared to the sorbitol content of cells transferred into unsupplemented media. These data suggest that fructose may be activating or increasing sorbinil-resistant aldose reductase activity as well as partially blocking sorbitol dehydrogenase activity. The presence of increased concentrations of fructose in combination with increased glucose levels may enhance alterations in cell metabolism and properties due to increased sorbitol levels.     

Abnormal human sarcoplasmic reticulum Ca2+ release channels in malignant hyperthermic skeletal muscle.

Single sarcoplasmic reticulum (SR) Ca2+ release channels were reconstituted from normal and malignant hyperthermic (MH) human skeletal muscle biopsies (2-5 g samples). Conduction, gating properties, and myoplasmic Ca2+ dependence of human SR Ca2+ release channels were similar to those in other species (rabbit, pig). The MH diagnostic procedure distinguishes three phenotypes (normal, MH-equivocal, and MH-susceptible) on the basis of muscle contracture sensitivity to caffeine and/or halothane. Single channel studies reveal that human MH muscles (both MH phenotypes) contain SR Ca2+ release channels with abnormally greater caffeine sensitivity. Muscles from MH-equivocal and MH-susceptible patients appear to contain channels with the same abnormality. Further, our data (n = 115, 21 channels, 11 patients) reveals that human MH muscles (both phenotypes) may contain two populations of SR Ca2+ release channels, possibly corresponding to normal and abnormal isoforms. Thus, whole cell phenotypic variation (MH-equivocal vs. MH-susceptible) arises in muscles containing channels with similar caffeine sensitivity suggesting that human MH does not arise from a single defect. These results have important ramifications concerning (a) correlation of functional and genetic MH studies, (b) identification of other, yet to be determined, factors which may influence MH expression, and (c) characterization of normal SR Ca2+ release channel function by exploring genetic channel defects.     

Negative supercoiling of DNA by gyrase is inhibited in Salmonella enterica serovar Typhimurium during adaptation to acid stress

DNA in intracellular Salmonella enterica serovar Typhimurium relaxes during growth in the acidified (pH 4–5) macrophage vacuole and DNA relaxation correlates with the upregulation of Salmonella genes involved in adaptation to the macrophage environment. Bacterial ATP levels did not increase during adaptation to acid pH unless the bacterium was deficient in MgtC, a cytoplasmic‐membrane‐located inhibitor of proton‐driven F₁F₀ ATP synthase activity. Inhibiting ATP binding by DNA gyrase and topo IV with novobiocin enhanced the effect of low pH on DNA relaxation. Bacteria expressing novobiocin‐resistant (Nov^R) derivatives of gyrase or topo IV also exhibited DNA relaxation at acid pH, although further relaxation with novobiocin was not seen in the strain with Nov^R gyrase. Thus, inhibition of the negative supercoiling activity of gyrase was the primary cause of enhanced DNA relaxation in drug‐treated bacteria. The Salmonella cytosol reaches pH 5–6 in response to an external pH of 4–5: the ATP‐dependent DNA supercoiling activity of purified gyrase was progressively inhibited by lowering the pH in this range, as was the ATP‐dependent DNA relaxation activity of topo IV. We propose that DNA relaxation in Salmonella within macrophage is due to acid‐mediated impairment of the negative supercoiling activity of gyrase.     

Occluding junctions in a cultured transporting epithelium: Structural and functional heterogeneity

Summary MDCK cells (epithelioid of renal origin) form monolayers which are structurally and functionally similar to transporting epithelia. One of these similarities is the ability to form occluding junctions and act as permeability barriers. This article studies the junctions of MDCK monolayers formed on a permeable and transparent support (a disk of nylon cloth coated with collagen) by combining two different approaches: (i)Scanning of the electric field: the disk is mounted as a flat sheet between two Lucite chambers and pulses of 20–50 A cm^–2 are passed across. The apical surface of the monolayer is then scanned with a microelectrode to detect those points where the current is flowing. This shows that the occluding junctions of this preparation are not homogeneous, but contain long segments of high resistance, intercalated with sites of high conductance. (ii)Freeze fracture electron microscopy: the junctions are composed of regions of eight to ten strands intercalated with others where the strands are reduced to one or two ridges. The sites of high conductance may correspond to those segments where the number of junctional strands is reduced to 1 or 2. It is concluded that the occluding junctions of MDCK monolayers are functionally and morphologically heterogeneous, with tight regions intermixed with leaky ones.     

Expression of chromosome 21 specific sequences in normal and Down''s syndrome tissues.

Using RNA isolated from age and sex matched normal and Down's Syndrome foetal liver and brain tissues, Northern blots were prepared and probed with 4 chromosome 21 specific sequences. The results show that no consistent pattern of expression emerges when Down's Syndrome tissue is compared with normal tissue but the results are very different from the 3/2 ratio of expression which may be expected. Two sequences 21.3 and 26C show only minor differences in expression in trisomy 21 liver samples but significant changes in their expression pattern when normal and Down's Syndrome brain samples are compared. The other sequences, JG77 and JG90 show a 5 fold higher degree of expression in Down's brain but when liver samples are compared one of these sequences shows equal levels of expression in normal and Down's Syndrome samples and the other shows a decrease in expression level in Down's Syndrome samples.     

Functional studies of Ca2+ channels from plasmalemma and sarcoplasmic reticulum membranes in muscle cells

Physiological and biochemical studies (channel characteristics, intracellular Ca2+ determinations and, channel purification, cloning and expression) of the different components involved in the regulation of intercellular Ca2+ have provided new information about their specific role. Recent information favors a major role for plasmalemma Ca2+ channels in E-C coupling of cardiac muscle, while a major role for sarcoplasmic reticulum Ca2+ release channels (ryanodine receptors) is proposed for E-C coupling of skeletal muscle. In smooth muscle, both plasmalemma and sarcoplasmic reticulum (IP3 receptors) Ca2+ channels are involved in E-C coupling. These studies will be comparatively discussed for skeletal, cardiac and smooth muscle cells.     

Studies on synthesis and degradation rates and some molecular properties of guinea-pig muscle acylphosphatase.

Acylphosphatase (acylphosphate phosphohydrolase, EC 3.6.1.7) was purified from guinea-pig muscle by a procedure involving immuno-affinity chromatography and a subsequent ion-exchange chromatography. This purification technique gave an overall yield of about 60% and permitted the isolation of three molecular forms with acylphosphatase activity, with a distribution greatly resembling those found in horse and turkey muscle. The main form appears to be very similar to the corresponding form in horse and turkey muscle, as indicated by amino acid composition, end-group analysis, the presence of glutathione as a mixed disulphide in almost the same stoichiometric ratio and kinetic analysis. From turnover data, the main form of acylphosphatase in guinea-pig muscle exhibits a degradation constant of 0.10 day-1, corresponding to a half-life of 6.8 days. These values are very close to those found for muscle total soluble proteins.