A simple, precise method for determining the carbon stable isotope ratio of total dissolved inorganic C (ΣCO2) in freshwater samples is described. Water samples are packed in airtight glass bottles of known inner volume (∼70 ml) with no air bubbles. Subsequently, a headspace of 5.0 ml is created inside each bottle with pure helium gas, and each sample is acidified by adding 0.5 ml of a CO2‐free, 6.0 N HCl solution. After the original dissolved CO2 has equilibrated with the headspace gas, a portion of this headspace gas is subsampled and injected into the GC/C/IRMS (gas chromatograph/combustion furnace/isotope‐ratio mass spectrometer) system to determine the carbon isotope ratio of the CO2. The isotope ratio of CO2 remaining in the liquid phase is calculated by temperature‐dependent isotope discrimination between gas and aqueous phases. The isotope ratio of ΣCO2 of the original sample is then derived assuming isotope mass balance. The analytical precision of this method is ±0.1‰. The method enables a single operator to determine the isotopic ratio in at least 60 lake‐water samples within 3 d of sampling.

We used the patch-clamp technique to clarify the nature of ion channels in renal mesangial cells in culture. In the cell-attached mode most patches were silent in the absence of agonists. In some patches a 25-pS nonselective channel was observed. This 25-pS cation channel was consistently observed in inside-out patches, and it was activated by intracellular Ca2+. Excised patch experiments also revealed the existence of a 40-pS K+ channel, which was activated by intracellular Ca2+. This 40-pS K+ channel was observed infrequently in the cell-attached mode. The activities of both channels were increased by arginine vasopressin or angiotensin II, resulting from an increase in intracellular Ca2+ concentration.

Apart from a catalytic domain, the alkaline protease of Pseudomonas aeruginosa has a novel parallel beta-helix domain stabilized through Ca2+ binding. In order to clarify the importance of the beta-helix structure in maturation of the enzyme, aspartic residue D-356 or D-365 in the Ca2+ binding sequence motif was replaced with L-alanine, and the catalytic activity of each mutant was assayed. These mutants did not show any proteolytic activity, although the composition of their polypeptide chains was the same as that of the wild type except for the mutated alanine residue. These results suggest that D-356 and D-365 are important in control of the beta-helix folding induced by Ca2+ binding and that incomplete beta-helix folding due to the lack of their side-chains affects the maturation of the enzyme in the long-range order.

Several isoforms of Na+/H+ exchanger (NHE-1-5) have been identified. LLC-PK1 clone 4 (CL4) expresses the amiloride-sensitive type of NHE predominantly in the basolateral membrane, which is believed to be NHE-1. It is not clear whether CL4 expresses NHE in the apical membrane and which side of NHE is encoded by the NHE-1 mRNA. Using acidified CL4 cells on the filter membrane, we examined Na(+)-dependent pH recovery of the apical and basolateral membranes separately. Na+ applied to the apical membrane recovered cell pH. Na(+)-dependent pH recovery in the apical membrane was not inhibited by SITS, DIDS, or contralateral amiloride. Li+ but not K+, chol+, or NMG+ could replace Na+. These data are consistent with the presence of NHE in the apical membrane. Transfection with an antisense oligonucleotide corresponding to the 5' terminal site of NHE-1 cDNA of CL4 decreased NHE activity in the basolateral membrane but not in the apical membrane. We conclude that CL4 expresses NHE activities in both apical and basolateral membranes and that NHE-1 mRNA encodes NHE only in the basolateral membrane.

however, the problem still remains open. The recent development of techniques for removal of the cell wall by lysozyme and for isolation of several structures of Bacillus megaterium provided an opportunity for a new approach to this aspect of the gram reaction.Protoplasts of strain KM of B. megaterium were isolated by the method of Weibull (J. Bacteriol., 66, 688, 1953). Cytoplasmic membranes (Vennes and Gerhardt, Science, 124, 535, 1956) and intracellular particles in turn were prepared from protoplasts by treating them with "Versene" and deoxyribonuclease and separating the fractions by differential centrifugation. Cell walls were obtained essentially by the procedure of