Oscillations of membrane potential in L cells

Summary Effects of divalent cations on oscillations of membrane potentials (i.e., spontaneous repetitive hyperpolarizing responses) and on hyperpolarizing responses induced by electrical stimuli as well as on resting potentials were studied in large nondividing L cells. Deprivation of Ca²⁺ from the external medium inhibited these hyperpolarizing responses accompanying slight depolarization of the resting potential. Sr²⁺ or Mn²⁺ applied to the external medium in place of Ca²⁺ was able to substitute for Ca²⁺ in the generation of hyperpolarizing responses, while Mg²⁺, Ba²⁺ or La³⁺ suppressed hyperpolarizing responses. The addition of A23187 to the bathing medium or intracellular injection of Ca²⁺, Sr²⁺, Mn²⁺ or La³⁺ induced membrane hyperpolarization. When the external Ca²⁺, Sr²⁺ or Mn²⁺ concentration was increased, the resting potential also hyperpolarized, in a saturating manner. The amplitude of maximum hyperpolarization produced by high external Ca²⁺ was of the same order of magnitude as those of hyperpolarizing responses and was dependent on the external K⁺ concentration. In the light of these experimental observations, it was deduced that the K⁺ conductance increase associated with the hyperpolarizing excitation is the result of an increase in the intracellular concentration of free Ca²⁺ mainly derived from the external solution.     

Relationships among the three light-induced absorbance changes related to the reaction-center of photosystem II

The relationships among X₅₉₁, Cyt-b₅₅₉ and C-550 in the primaryphotoact of PS-II were analysed by examining the effects ofvarious inhibitory substances and treatments on the light-inducedabsorbance changes of these components. The results were fully explainable by the scheme previouslypresented by Huzisige, in which two photoreactions are involvedin PS-II. Our conclusion is that X₅₉₁ acts as the electron acceptorfor one of the photoreactions in PS-II. (Received October 23, 1978; )     

Orientation of the protonmotive force in membrane vesicles of escherichia coli

Membrane vesicles of Escherichia coli can be produced by 2 different methods: lysis of intact cells by passage through a French pressure cell or by osmotic rupturing of spheroplasts. The membrane of vesicles produced by the former method is everted relative to the orientation of the inner membrane in vivo. Using NADH, D-lactate, reduced phenazine methosulfate, or ATP these vesicles produce protonmotive forces, acid and positive inside, as determined using flow dialysis to measured the distribution of the weak base methylamine and the lipophilic anion thiocyanate. The vesicles accumulate calcium using the same energy sources, most likely by a calcium/proton antiport. Calcium accumulation, therefore, is presumably indicative of a proton gradient, acid inside. The latter type of vesicle, on the other hand, exhibits D-lactate-dependent proline transport but does not accumulate calcium with D-lactate as an energy source. NADH oxidation or ATP hydrolysis, however, will drive the transport of calcium but not proline in these vesicles. Oxidation of NADH or hydrolysis of ATP simultaneous with oxidation of D-lactate does not result in either calcium or proline transport. These results suggest that the vesicles are a patchwork or mosiac, in which certain enzyme complexes have an orientation opposite to that found in vivo, resulting in the formation of electrochemical proton gradients with an orientation opposite to that found in the intact cell. Other complexes retain their original orientation, making it possible to set up simultaneous proton fluxes in both directions, causing an apparent uncoupling of energy-linked processes. That the vesicles are capable of generating protonmotive forces of the opposite polarity was demonstrated by measurements of the distribution of acetate and methylamine (to measure the ΔpH) and thiocyanate (to measure the Δψ).     

Kinetics of substrate utilization in adenine-limited chemostat cultures of Bacillus subtilis KYA741.

The specific rates of limiting substrate utilization were investigated in adenine- or glucose-limited chemostat cultures of Bacillus subtilis KYA741, an adenine-requiring strain, at 37 degrees C. With the glucose-limited cultures, the specific rate of glucose consumption versus dilution rate gave a linear relationship from which the true growth yield and maintenance coefficient were determined to be 0.09 mg of bacteria per mg of glucose and 0.2 mg of glucose per mg of bacteria per h, respectively. With the adenine-limited cultures, adenine as the limiting substrate was not completely consumed at lower dilution rates (e.g., D less than 0.1), unlike in the glucose-limited cultures. When a linear relationship of specific rate of adenine consumption versus dilution rate was extrapolated to zero dilution rate, a negative value for the specific rate of adenine consumption, -0.01 mg of adenine per mg of bacteria per h, was obtained, giving a true growth yield for adenine of 5.2 mg of bacteria per mg of adenine. On the other hand, the maintenance coefficient of oxygen uptake gave a positive value of 8.1 x 10(-3) mmol/mg of bacteria per h. Based on previous results showing that adenine is resupplied by lysing cells, we developed kinetic models of adenine utilization and cell growth that gave a good estimation of the peculiar behavior of cell growth and adenine utilization in adenine-limited chemostat cultures.     

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig

Summary Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K⁺ (E _K). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at nearE _K, indicating that both time-dependent and voltagegated currents were due to K⁺ conductances. Both outward and inward K⁺ currents were suppressed by extracellular application of Ba²⁺, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs⁺ blocked the inward current but not the outward current. The conductance of inward K⁺ currents, but not outward K⁺ currents, became larger with increasing extracellular K⁺ concentration. A Ca²⁺-mobilizing acid secretagogue, carbachol, and a Ca²⁺ ionophore, ionomycin, brought about activation of another type of outward K⁺ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca²⁺ chelation. Quinine preferentially inhibited this K⁺ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K⁺ channels, inward rectifier and outward rectifier, and that the cells have Ca²⁺-activated K⁺ channels which might be involved in acid secretion under stimulation by Ca²⁺-mobilizing secretagogues.     

Three short fragments of Rts1 DNA are responsible for the temperature-sensitive growth phenotype (Tsg) of host bacteria.

Rts1 is a multiphenotype drug resistance factor, and one of its phenotypes is temperature-sensitive growth (Tsg) of host bacteria. A 3.65-kb fragment from Rts1 DNA was shown to cause the Tsg phenotype in host cells. This tsg fragment was split by a restriction enzyme, HincII, into four fragments. Two of these fragments were called HincII-S (short) and HincII-L (long), respectively. Each of these two fragments conferred the Tsg phenotype, indicating that, in fact, these two independent regions were responsible for the Tsg phenotype. The HincII-S 783-bp and HincII-L 1,479-bp fragments were sequenced. The region in the HincII-S fragment to which the Tsg phenotype was attributed was narrowed to a 146-bp (nucleotides 1 to 146) fragment by various restriction enzyme digestions. Further digestion of the 146-bp fragment with Bal 31 suggested that the 116-bp (nucleotides 9 to 124) fragment is the minimum sequence required for Tsg. On the other hand, in the HincII-L fragment, a fragment of 249 bp (nucleotides 1210 to 1458) and a fragment of 321 bp (nucleotides 1942 to 2262) contained separate temperature-sensitive growth activity. None of three tsg fragments contained open reading frames. The 249-bp fragment had very weak Tsg activity, while the 321-bp fragment had no Tsg activity. On the other hand, when these two fragments were together in the pUC19 vector, they exhibited very strong Tsg activity equivalent to that of the original 1,479-bp fragment. In addition, two of the 249-bp fragments gave similar, strong Tsg activity. The HincII-L 1,479-bp fragment contained an open reading frame for kanamycin resistance which was found between nucleotides 1423 and 2238. This kanamycin resistance gene sequence was different from that of the reported kanamycin resistance gene of Tn903 at 12 positions which were deduced to change seven amino acids.     

Generation of Probucol Radicals and Their Reduction by Ascorbate and Dihydrolipoic Acid in Human Low Density Lipoproteins

Probucol, 4.4'-[(1-methylethylidene)bis(thio)]bis-[2,6-bis(1.1-dimethyl)phenol], is a lipid regulating drug whose therapeutic potential depends on its antioxidant properties. Probucol and x-tocopherol were quantitatively compared in their ability to scavenge peroxyl radicals generatcd by the thermal decomposition of the lipid-soluble azo-initiator 2,2'-azo-bis(2,4-dimethyl-valeronitrile), AMVN, in dioleoylphos-phatidylcholine (DOPC) liposomes. Probucol showed 15-times lower peroxyl radical scavenging efficiency than x-tocopherol as measured by the effects on AMVN-induced luminol-dependent chemiluminescence. We suggest that probucol cannot protect x-tocopherol against its loss in the course of oxidation, although probucol is known to prevent lipid peroxidation in membranes and lipoproteins. In human low density lipoproteins (LDL) ESR signals of the probucol phenoxyl radical were detected upon incubation with lipoxygenase + linolenic acid or AMVN. Ascorbate was shown to reduce probucol radicals. Dihydro-lipoic acid alone was not able to reduce the probucol radical but in the presence of both ascorbate and dihydrolipoic acid a synergistic effect of a stepwise reduction was observed. This resulted from ascorbate-dependent reduction of probucol radicals and dihydrolipoic acid-dependent reduction of ascorbyl radicals. The oxidized form of dihydrolipoic acid, thioctic acid, did not affect probucol radicals either in the presence or in the absence of ascorbate.     

Population dynamics of coniferous and broad-leaved trees in a Japanese temperate mixed forest

The growth and survival of coniferous and broad-leaved trees were followed over a 5-yr period in a temperate old-growth mixed forest in Japan, and dynamic features of the forest were studied in relation to the life history of the dominants, the coniferous Abies homolepis and the broad-leaved Fagus crenata. During this period, the gap formation rate was 31m² ha^?1yr^?1, the mortality of trees > 2m high was 1.7%/yr, and the rate of loss in basal area 1.4%/yr. These values were much higher than the recruitment, 0.3%/yr, and the total growth of surviving and new trees, 0.6%/yr, owing to the inhibition of regeneration by understorey dwarf bamboo (Sasa borealis). A transition matrix model based on DBH size classes predicts that the basal area of the forest will decrease by 14% in 50 yr, but that the DBH distribution of trees > 10 cm diameter will change little. Equilibrium DBH distributions assuming recruitment being equal to mortality, were quite different between broad-leaved and coniferous trees, reflecting different survivorship curves of the two dominants. The composition and structure of the forest may change depending on the pattern and frequency of disturbances, or episodic events, notably the synchronous death of Sasa borealis.