Biomedical subjects
R C Thomas
Publications and source records attributed to R C Thomas.
Simple, rapid and sensitive high-performance liquid chromatographic procedure for the quantitation of itazigrel in human plasma or serum.
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Behaviour of cows in cubicles and its possible relationship with laminitis in replacement dairy heifers.
The behaviour of cows in cubicles was studied in two dairy herds which were under the same ownership and had similar buildings and management systems. One of the herds had an annual problem of lameness due to laminitis leading to solar ulceration in the replacement first lactation heifers. There were considerable behavioural differences between the cows in the two herds. In the problem herd the heifers and cows stood for significantly longer, more heifers were seen not to use the cubicles, and there were more examples of aberrant behaviour than in the other herd. Less straw was used for cubicle bedding in the problem herd and when the amount used was increased to one bale per 10 cows per day no new cases of laminitis and solar ulceration occurred in the heifers.
Cell growth factors. Bicarbonate and pHi response.
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Experience with a metered-dose inhaler with a spacer in the pediatric emergency department.
Studies of adults suggest that metered-dose inhalers with spacers are as effective as hand-held nebulizers for bronchodilator delivery. We studied 13 children with acute asthma. They received two puffs every 2 minutes from metered-dose inhalers with spacers (range, 4 to 14 puffs) titrated until improvement stopped. Peak expiratory flow increased 34% for metered-dose inhalers with spacers after the first 2 puffs and increased 87% for metered-dose inhalers with spacers after dose titration. After titration, respiratory rate decreased by 12%, heart rate increased by 2%, and breath sounds improved in 92% of the patients. We concluded that the metered-dose inhalers with spacers are an effective device for the treatment of asthma in the pediatric emergency department and that the use of metered-dose inhalers with spacers with titration can achieve significant bronchodilation in the treatment of patients with acute asthma.
A simple method for the characterization of photoproducts from DNA applied to the cis-syn thymine dimer.
A method for the unambiguous characterization of DNA photoproducts has been developed. It does not require radio-labelled DNA, or specialized techniques. In the preliminary step, UV-irradiated DNA is hydrolysed to its constituent bases and photoproducts. The photoproducts are then separated from nucleic acid bases using low pressure ion-exchange chromatography on a Dowex 1 x 8 200-400, and an Amberlite CG-50-H+ column. Further separation and purification of photoproducts is carried out on the HPLC Whatman Partisil ODS2 column, using 15% MeOH. The procedure is simple, reproducible and versatile.
Proton channels in snail neurones. Does calcium entry mimic the effects of proton influx?
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The intrinsic intracellular H+ buffering power of snail neurones.
1. We measured intracellular pH (pHi) in snail neurones using pH-sensitive glass microelectrodes. We then calculated the intracellular buffering power (beta i) from the pHi changes associated with the influx or efflux of a variety of weak acids or bases. 2. The weak acid anions butyrate and propionate (20 mM) gave similar values for beta i but those measured using 20 mM-acetate were on average twice as great. 3. Although solutions were nominally CO2-free, blockage of pHi regulation with SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) increased the sizes of the pHi changes upon weak acid addition and removal. The corresponding measured values of beta i were on average 26% lower with SITS than without. 4. With pHi regulation blocked, the use of 2.7% CO2 to measure beta i gave beta i values similar to those measured with butyrate or propionate. These values were about 50% less than those previously measured in snail neurones using CO2. 5. beta i values calculated from the pHi changes due to the removal of 5 mM of the weak bases trimethylamine, procaine and NH4Cl were all similar and comparable to those measured using butyrate or propionate. Removing the influence of pHi regulation on the undershoots after NH4Cl removal was found to decrease the apparent measured values of beta i by 10%. 6. Combining all the data (except the values obtained using CO2 and acetate), and adjusting for the errors due to pHi regulation reducing the sizes of the pHi changes, we found that the mean value for beta i was 10.4 +/- 0.6 mM (+/- S.E.M.) at a mean pHi of 7.36 +/- 0.05. 7. We also investigated the relationship between beta i and pHi using ionophoretic acid injection. By means of step-wise injections, with pHi regulation blocked, we found that at normal pHi levels beta i remained relatively constant. However, at a pHi of less than about 6.8 beta i increased with decreasing pHi.
Apparent proton influx via channels: perhaps pHi decrease in depolarised snail neurones is caused by calcium entry.
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Proton channels in snail neurons studied with surface pH glass microelectrodes.
Surface pH was recorded on voltage-clamped snail neurons with Hinke-type glass microelectrodes. During stepwise depolarization from -50 mV to +40 mV the surface pH usually increased at first and then fell rapidly during each 7.5 sec voltage step. The pH changes appeared to be due to proton passage through voltage-sensitive channels. The pH changes were inhibited by cadmium and zinc, and were not as sensitive to external pH as to internal pH. Changes in internal pH lagged behind surface pH. Changes in external calcium had little effect on the surface pH increases seen with small depolarizations. These results confirm earlier conclusions that depolarization opens proton-permeable channels.
Changes in the surface pH of voltage-clamped snail neurones apparently caused by H+ fluxes through a channel.
1. The surface and intracellular pH of snail neurones was recorded with microelectrodes while the membrane potential was reduced in 10 mV steps for a few seconds each or to positive values for periods of several minutes. 2. Depolarizations to positive membrane potentials caused rapid falls in surface pH (pHs) which varied from cell to cell and from one point to another on the surface of the same cell. 3. When pHi was normal or alkaline, the first few 10 mV steps of depolarization often caused a small pHs increase which changed to a decrease as the depolarization increased. The threshold potential at which the pHs increase changed to a decrease varied with pHi in a linear manner, so that at acid pHi values the threshold potential approached the normal resting potential. There was good agreement between the threshold and H+ equilibrium potentials calculated from pHi and pHs. 4. The size of the pHs decrease observed at a given pHi and depolarization depended on extracellular buffering power in a non-linear manner. Solutions buffered with 20 mM-NaHCO3 had similar surface buffering power to CO2-free solutions buffered with only 1-2 mM-HEPES, pH 7.5. 5. In 1 mM-HEPES pHs changes were larger, and pHi increases slower, than those seen in cells depolarized to the same potential in 20 mM-HEPES. The slowing of the rate of pHi increase suggests that the pHs changes occur all over the cell surface, and not only at the recording site. 6. With long-lasting depolarizations the size of the pHs decrease was proportional to the rate of pHi increase and thus, assuming a constant intracellular buffering power, to the rate of efflux of H+. 7. The results provide further evidence that snail neurones possess a channel permeable to H+ which is opened on depolarization. H+ efflux through this channel could cause rapid acidification of a confined extracellular space.
A microelectrode study of the mechanisms of L-lactate entry into and release from frog sartorius muscle.
1. Changes in intracellular pH and intracellular anion levels were monitored in frog sartorius muscle fibres during exposure to extracellular L-lactate, using ion-sensitive microelectrodes. 2. Resting intracellular pH (pHi) in 20 mmol l-1 HEPES buffer was 7.18 +/- 0.015 (S.E. of mean, n = 62). Exposure to an extracellular solution at pH 6.5 buffered with 20 mmol l-1 3-(N-morpholino)propanesulphonic acid (MOPS) resulted in a slow intracellular acidification. 3. A reversible decrease in pHi and an increase in intracellular anion levels was observed when L-lactate replaced chloride in equimolar amounts. The increase in intracellular anion level is consistent with intracellular accumulation of L-lactate ion. 4. The rate and steady-state change in pHi and anion level was a function of both extracellular pH and L-lactate concentration, providing evidence for the coupled movement of lactate and proton equivalents. 5. The initial rate of uptake of L-lactate, as measured by the change of pHi, was a non-linear function of the extracellular L-lactate concentration at extracellular pH 6.8 and 7.35. 6. No saturation was observed with concentrations of L-lactate between 5 and 60 mmol l-1 at pH 7.35 and 2.5 and 40 mmol l-1 at pH 6.8. 7. The non-linear relationship between the initial rate of change in pHi and extracellular L-lactate was well fitted by a curve defining uptake as the sum of a carrier process displaying Michaelis-Menten kinetics and a passive diffusion component. The apparent Km of the carrier was 10 mmol l-1 at pHo 7.35 and 4 mmol l-1 at pHo 6.8. 8. The initial rate of change of pHi in the presence of L-lactate was significantly inhibited 39.1 +/- 6.2% by 2-5 mmol l-1 alpha-cyano-4-hydroxycinnamate (n = 9; P less than 0.05, paired t test). 9. alpha-Cyano-4-hydroxycinnamate had no detectable effect on the initial rate of change of pHi induced by propionate exposure. 10. The initial rate of change of pHi induced by L-lactate was not affected by 20-100 mumol l-1 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS). 11. We conclude that L-lactate crosses the membrane of the frog sartorius muscle with proton equivalents via (1) a carrier-mediated process, and (2) passive diffusion of lactic acid. In the physiological range of L-lactate concentrations and pH the transport process dominates.
Spectinomycin modification. IV. The synthesis of 3'-aminomethyldihydrospectinomycins via spectinomycin 3'-cyanohydrins.
The C-3'-carbonyl group of N-protected spectinomycin is converted into the corresponding aminomethylalcohols via the intermediacy of cyanohydrins. Methodology for the selective synthesis of either epimer with retention of protection in the aminocyclitol ring provides valuable synthetic intermediates for the preparation of analogs of this important antibiotic. The new methodology provides an efficient synthesis of the highly active 3'-aminomethyldihydrospectinomycins.
Spectinomycin modification. V. The synthesis and biological activity of spectinomycin analogs with ring-expanded sugars.
Tiffeneau-Demjanov rearrangement of 3'-(R)-N,N'-dibenzyloxycarbonyl-3'-aminomethyl-dihydrospectinom ycin results in ring expansion affording the homologous analog with a seven-membered sugar ring. In stark contrast, attempted rearrangement of the 3'-S-isomer leads only to epoxide formation. Deprotection of the ring-expanded homolog gives homospectinomycin. The synthesis and biological activity of this interesting new member of the spectinomycin series and the derived dihydrohomospectinomycin is detailed in this paper.
Voltage-dependent intracellular pH in Helix aspersa neurones.
1. The intracellular pH (pHi) of large nerve cells from the mollusc, Helix aspersa, was measured with pH-sensitive micro-electrodes. Cells were held under voltage clamp and the effect on pHi of different holding potentials was determined. 2. Depolarization of the cell from the resting potential (about -50 mV) to -10 mV produced a fall in pHi that could be reduced by bathing the cell in nominally Ca2+-free saline. 3. At positive holding potentials pHi increased to a steady level that depended upon the electrochemical gradient for H+ across the cell membrane; it shifted by about 1 unit when the external pH was increased from 7 to 8 (or when the membrane potential increased by 58 mV, Thomas & Meech, 1982). 4. The depolarization-induced increase in H+ permeability was insensitive to SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid, 20 microM), which blocks pHi regulation at the resting potential in these cells (Thomas, 1976). When pHi was displaced from a steady level by ionophoretic injection of HCl, there was a rapid recovery at depolarized potentials even in the presence of SITS. The H+ pathway appeared to be little affected by prolonged periods at positive membrane potentials. 5. The depolarization-induced H+ efflux was insensitive to the metabolic inhibitor CCmP (carbonyl cyanide-m-chlorophenylhydrazone, 20 microM) and persisted in cells bathed in pH-buffered n-methyl glucamine-gluconate. It was also insensitive to DCCD (N, N'-dicyclohexylcarbodiimide, 10-100 microM) and oligomycin (2-10 micrograms/ml). 6. The H+ pathway could be fully blocked by 1 mM-ZnCl2, 1 mM-LaCl3, 1 mM-CuCl2, 2 mM-CdCl2 or 10 mM-CoCl2. Other divalent ions such as BaCl2 (10 mM) produced a block at membrane potentials near 0 mV but the block was released at more positive potentials. Low levels of LaCl3 (0.1 mM), the organic Ca2+ channel antagonist D600 (100 mg/ml) and high levels of the K+ channel blocker TEA (50 mM) all had similar effects to Ba2+. 7. The K+ channel blocker 4-aminopyridine (10 mM), which blocks H+ currents in perfused Lymnaea neurones (Byerly, Meech & Moody, 1984), has a complex action.(ABSTRACT TRUNCATED AT 400 WORDS)
Extracellular acidification at the surface of depolarized voltage-clamped snail neurones detected with eccentric combination pH microelectrodes.
A new design of double micropipette was used to measure intracellular pH, membrane potential, and surface pH of superfused snail neurones. A third double micropipette was used to control the membrane potential via a CsCl-filled barrel and inject HCl iontophoretically. In one series of experiments the surface pH fell by up to one-third of a pH unit when the membrane potential was clamped to 20 mV, pHi was initially 6.7, and extracellular pH was about 7.4 in a medium buffered either with 2 mM HEPES or 2.7% CO2 and 20 mM bicarbonate. In a second series in which surface pH was observed during brief depolarizations to different potentials with different pHi, the potential at which the surface began to acidify varied with pHi with a slope of 32 mV per pH unit. The results confirm that H+ ions leave depolarized snail neurones if the electrochemical gradient is favourable and show that CO2-bicarbonate buffered solutions have a low effective extracellular buffering power for rapid additions of acid.
New method for calculating pHi from accurately measured changes in pHi induced by a weak acid and base.
From the combined Henderson-Hasselbalch relations for a weak acid and a weak base, we derive an expression for steady-state intracellular pH(pHi). The derivation requires only accurate measurements of the pHi changes when the two electrolytes are applied, rather than absolute values. The method is based on the assumption of equilibrium of the neutral forms of the two compounds across the plasma membrane, that is, absence of permeation of the charged forms. It is also assumed that no significant pHi regulation takes place, that there is no significant permeability to intracellular buffers and that intracellular buffering power, over the measured span of pHi, is the same. This precludes the use of CO2/bicarbonate buffered systems. We find that under our experimental conditions steady-state pHi values calculated in this way agree closely with those measured directly with pH-sensitive microelectrodes both in snail neurones and crab muscle. The method would allow the intracellular calibration of other pHi measuring techniques.
A dual mechanism for intracellular pH regulation by leech neurones.
Neutral-carrier pH-sensitive micro-electrodes were used to investigate intracellular pH (pHi) in leech neurones. When used in snail neurones such electrodes gave very similar pHi values to those recorded simultaneously by recessed-tip glass micro-electrodes. Leech Retzius neurones superfused with a pH 7.4 HCO3--free physiological saline were found to have a pHi of 7.3, too high to be explained by a passive distribution of H+ or OH-. To investigate pHi regulation the pHi was decreased by one of three methods: by exposure to propionate, by adding and then removing NH4Cl or by exposure to CO2. Acidification by any method was followed by a recovery to normal pHi values within 15-20 min. In HCO3--free solutions, pHi recovery from acidification was blocked by removing external Na or by amiloride (2 mM). In solutions buffered with 2% CO2 and 11 mM-HCO3-, amiloride slowed but did not block pHi recovery. The anion exchange inhibitor SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid) also slowed pHi recovery in the presence of HCO3-. In CO2/HCO3- solution the removal of external Na either slowed or blocked pHi recovery, and blocked it completely in the presence of amiloride. We conclude that in HCO3--free solutions pHi regulation is by a Na-H exchange system; but in the presence of HCO3- there is an additional mechanism which is probably a Na-dependent Cl-HCO3 exchanger.