Relationship between intracellular K+ concentrations and K+ fluxes in growing and contact-inhibited cells

The K⁺ content and the K⁺ flux were measured in the cell lines ME₂ and MF₂ isolated from plasmocytoma MOPC 173. Both cell lines were shown to have the same K⁺ content and the same K⁺ steady state flux per unit of surface area.In ME₂ cells, no modification of the exchange movement was observed during contact inhibition. However, contact-inhibited cells exhibited an increased resistance to depletion, characterized by a lower K⁺ net movement.The (Na⁺ + K⁺)-ATPase measured in homogenates is poorly correlated to in vivo cation fluxes both because of the enhancement due, presumably, to the drop of K⁺ concentration on the cytoplasmic face of the membrane and because of losses during preparation which can be conspicuous, especially in contact-inhibited cells.The K⁺ net flux is considerably increased when the intracellular K⁺ level is reduced after preincubation of the cells in a K⁺-free medium. Thus, internal K⁺ seems to regulate the K⁺ influx.     

Energetics of sodium efflux from Escherichia coli

When energy-starved cells of Escherichia coli were passively loaded with 22Na+, efflux of sodium could be initiated by addition of a source of metabolic energy. Conditions were established where the source of energy was phosphate bond energy, an electrochemical proton gradient, or both. Only an electrochemical proton gradient was required for efflux from intact cells. These results are consistent with secondary exchange of Na+ for H+ catalyzed by a sodium/proton antiporter.     

The structure of rat liver mitochondria: a reevaluation

A β-N-acetylgalactosaminyltransferases (Ga1NAcT) that catalyzes the synthesis of a triglycosylceramide, GanglioTricer (Ga1NAcβ-Ga1β1-4G1c-cer), from lactosylceramide and UDP-Ga1NAc was isolated from guinea pig bone marrow. The enzyme was present in the supernatant solution obtained after homogenization of guinea pig bone marrow 12,000 × g pellet with 0.32 M sucrose containing 0.6% Triton X-100 and centrifugation at 129,000 × g. The enzyme that catalyzed the transfer of Ga1NAc to a tetraglycosylceramide (Lac-nTet-cer) was found in a membrane-bound fraction. The K_m values were 0.5 mM and 0.7 mM for the lactosylceramide and Lac-nTet-cer, respectively. 97.0% of the terminal [¹⁴C]Ga1NAc was cleaved by the action of pure β-hexosaminidase from [¹⁴C]triglycosylceramide.     

H-2 antigens incorporated into phospholipid vesicles interact specifically with allogeneic cytotoxic T lymphocytes

Subcellular fractions containing different H-2 antigens were tested for their ability to inhibit specific T cell-target cell conjugate formation. H-2-containing membrane vesicles, lentil-lectin-purified H-2 antigens solubilized with detergent (referred to in the text as high-density fraction) or incorporated into lipid vesicles, inhibited T cell-target cell conjugate formation effectively and specifically. However, two- to threefold more protein was required to inhibit T cell-target cell conjugate formation when detergent-solubilized lentil-lectin-purified H-2 antigens were tested. This suggests that a lipid matrix is advantageous for interaction with anti-H-2 T-cell receptors. Experiments were also undertaken to demonstrate specific binding of liposomes containing ¹²⁵I-labeled H-2 antigen to anti-H-2-specific cytotoxic T lymphocytes (CTLs). The binding of the ¹²⁵I-labeled H-2-containing liposomes was saturable and was specifically inhibited by unlabeled H-2 antigens. Monospecific anti-H-2 sera specifically inhibited the binding of liposomes containing H-2 antigen to the CTLs. The results suggest that a specific interaction can occur between serologically defined H-2 antigens and the receptor of anti-H-2 CTLs.     

Differential effects of detergents upon the soluble and particulate guanylate cyclases from rat lung.

The enzyme guanylate cyclase is present in both particulate and soluble form in rat lung homogenates. As previously reported, the soluble enzyme can be activated by preincubation in the presence of O₂. The inactive (nonactivated) soluble enzyme is also stimulated by nonionic detergents, in the order Tween 20 > Lubrol PX > Triton X-67 > Triton X-100. The activated enzyme, however, was inhibited by these detergents in the reverse order. Sodium deoxycholate and lysolecithin were potent inhibitors of both inactive and activated enzyme. The activity of the particulate enzyme was stimulated by Lubrol PX > Triton X-100 > Triton X-67 > Tween 20. At a low concentration of lysolecithin or deoxycholate the particulate activity was increased; however, when detergent/protein > 1, inhibition was seen. In the case of deoxycholate, the inhibition could be reversed if excess deoxycholate was removed either by chromatography or by forming mixed micelles with Lubrol PX; however, deoxycholate inhibition of the soluble enzyme was irreversible. The stimulation by detergents of the particulate enzyme was apparently the result of solubilization. The effects upon the activity of the soluble enzyme were interpreted in terms of a model which assumes two hydrophobic regions on the enzyme surface. The two regions differ in hydrophobicity with the more hydrophobic region only being exposed as a result of activation. Interaction of a nonionic detergent with the less hydrophobic region stimulates activity, while interaction with the more hydrophobic region results in inhibition.     

Activation of cyclic AMP-dependent protein kinases I and II by cyclic 3',5'-phosphates of 9-beta-d-ribofuranosylpurine and 2-beta-D-ribofuranosylbenzimidazole

Analogs of cyclic AMP lacking the 6-amino group—9-β-D-ribofuranosylpurine cyclic 3′,5′-phosphate (I)—or the 1- and 3-nitrogens as well as the 6-amino group—1-β-D-ribofuranosylbenzimidazole cyclic 3′,5′-phosphate (II)—were effective activators of both type I (cAKI) and type II (cAKII) isozymes of cAMP-dependent protein kinase. An analog with a pyrimidine ring fused to the benzimidazole ring system of II—3-β-D-ribofuranosyl-8-aminoimidazo[4,5-g]-quinazoline cyclic 3′,5′-phosphate (III)—was equipotent to I or II as an activator of cAKII but only $\text{1}\text{10}$ as potent as I or II as an activator of cAKII. The results show that neither cAKI nor cAKII requires the 6-amino group and that they may have different sensitivities to the effects of alterations in the electron distribution in the pyrimidine ring.     

Optical measurements of intracellular oxygen concentration of rat heart in vitro

The optical characteristics of hemoglobin-free perfused rat heart have been examined in detail. Ethyl hydrogen peroxide is found to convert myoglobin into “ferryl compound” in the perfused heart, as is also seen in vitro. After pretreatment with ethyl hydrogen peroxide, a typical mitochondrial absorption spectrum, similar to that of isolated rat heart mitochondria, is obtained in perfused heart. The overall absorption spectrum of the heart obtained by the aerobic to anaerobic transition is a superposition of the mitochondrial spectrum on that of myoglobin. By comparing these spectra, it is found that measurement of cytochrome a + a₃ at 605–620 nm is possible in spite of the absorbance change due to the oxygenation-deoxygenation of myoglobin, whereas the wavelength pairs for cytochrome c at 550-540 nm, cytochrome b at 562–575 nm and cytochrome a + a₃ at 445–450 nm can not be used in the heart because of interference from the absorption change of myoglobin. The partial pressure of O₂ (P₅₀) which is required for half maximal deoxygenation (or oxygenation) of myoglobin in perfused heart is found to be 2.4 mm Hg at room temperature and the Hill constant, n, is 1.1; these values are similar to those of myoglobin purified from rat heart. The steady-state O₂ titration has been performed by using absorbancy changes of myoglobin and cytochrome a + a₃ as intracellular O₂ indicators. In the perfused heart, the percentage change of oxygenation-deoxygenation of myoglobin parallels the oxidation-reduction of cytochrome a + a₃, while the mixture of purified myoglobin and isolated mitochondria shows a deviation, reflecting the difference of O₂ affinities between myoglobin and cytochrome a + a₃. The results indicate that there may be an O₂ gradient between cytosolic and mitochondrial compartments in the hemoglobin-free perfused heart. The absorption changes of myoglobin and of cytochrome a + a₃ can be measured in a single contraction-relaxation cycle. A triple beam method was introduced to eliminate the effect of light scattering changes in these measurements. The results demonstrated that myoglobin is more oxygenated during the systolic and diastolic periods and deoxygenated in the resting period, whereas cytochrome a + a₃ is more reduced in systole and diastole and oxidized in the resting state. Changing the perfusion conditions greatly alters the time course of the events which occur during the contraction-relaxation cycle of the perfused heart.     

Lymphocyte-induced production of prostaglandin E2 by tumor cells in vitro: Requirements for direct contact and lymphocyte viability

The production of prostaglandin E₂ by tumor cell lines in response to exposure to purified lymphocytes has prompted the suggestion that this phenomenon may represent a defense mechanism whereby tumors may subvert an immune response mounted against them. To further characterize this phenomenon, cell lines derived from carcinogen-induced bladder tumors and embryo fibroblasts in Fischer rats were incubated with purified lymphocytes from peripheral blood, spleen, thymus, and lymph nodes from Fischer rats under a variety of conditions, and the amount of prostaglandin E₂ (PGE₂) production was determined by radioimmunoassay. Increased numbers of blood or splenic lymphocytes were associated with the induction of increased levels of PGE₂ production by the tumor cells. However, no prostaglandin was produced by the tumor cells after exposure to thymus or lymph node lymphocytes. Irradiation of lymphocytes prior to exposure to the tumor cells led to lower levels of PGE₂ production by the tumors, as did sonication of the lymphocyte preparations prior to addition to the tumor monolayers. Separation of lymphocytes from direct contact with the tumor cells resulted in less PGE₂ production by the tumor cell lines; however, when these lymphocytes were later layered onto fresh tumor cell monolayers, PGE₂ production occurred. Results in the present study suggest that direct contact between intact, viable, functionally active lymphocytes and tumor cells is necessary for tumor cell prostaglandin production to occur. Moreover, PGE₂ production only appears to occur in response to exposure to particular populations of lymphocytes, and this may correlate with the number of specific effector or attacker lymphocytes that are present. This specificity of response to effector cell challenge may be important in probing the defense mechanisms tumor cells may have to lymphocyte challenge, as well as in gauging the efficacy of a particular cellular immune response as it may be regulated both by cells involved in effecting this response as well as by the targets in lymphocyte/tumor cell interactions.     

Initiation of copulatory behavior in castrated male rats injected with critically adjusted doses of testosterone

In castrated male rats it was possible by means of administration of adequate testosterone propionate doses to induce a state in which the males did not initiate copulatory behavior with a stimulus female displaying no (Lordotic female) or weak (Presenting female) intensity of precopulatory behavior. On the other hand, such males started to copulate and finished copulatory series when a stimulus female exhibiting the complete pattern of precopulatory behavior (Darting female) was offered. The males “prepared” in this manner displayed regularly precopulatory behavior directed toward all the stimulus females. The requisite testosterone doses differed individually. However, in the majority of cases they were in the range of 500–700 μg of testosterone injected once a week.     

Membrane lipid fatty acids and regulation of membrane-bound enzymes. Allosteric behaviour of erythrocyte Mg2+-ATPase, (Na+ + K2+)-ATPase and acetylcholineasterase from rats fed different fat-supplemented diets

Studies were carried out to determine the Hill coefficients for the inhibition by F^? of the erythrocyte membrane-bound Mg²⁺-ATPase, $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and acetylcholinesterase from rats fed with seven different diets. Five groups were fed with different natural fats or oil supplements, one with a hydrogenated fat supplement and the other with fat-free diet. The responses of the red cell fatty acids to dietary fats were recorded. The value of $\text{n}$ for the inhibition by F^? of the three enzymes revealed a particular and different behaviour in each group. Correlations between the fatty acid compositions of erythrocyte membranes and cooperativity of each enzyme were calculated. The results indicate that neither the essential fatty acid family nor the non-essential ones are particularly involved in the allosteric phenomena. The increase of the double bond index/saturation ratio of fatty acids, which is taken as indicative of membrane fluidity, was accompanied in an inverse manner by changes in allosteric transitions of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and acetylcholinesterase, whereas the Mg²⁺-ATPase was not dependent on this ratio. Diminution of membrane fluidity, carried out by in vitro increase of its cholesterol content, yields confirmatory results of this regulatory mechanism since the value of $\text{n}$ for acetylcholinesterase shifted as predicted.These facts indicate that the membrane fluidity is a physiological regulator for the allosteric behaviour of the membrane-bound enzymes and that each enzyme exhibits a particular behaviour in this phenomenon.     

The pupillary effects of oploids

Morphine's miotic action on the pupil is an easily recognizable and quantifiable effect in man. The neural pathways responsible for regulating pupil size are reasonably well defined. Yet, the mechanisms behind this and related effects of opioids on the eye in humans and laboratory animals have just begun to be explored. In this review, we have attempted to organize the available information on pupillary actions of opioids, emphasizing the dynamic nature of the responses, their species specificity, possible mechanisms of action, and the recently discovered development of tolerance to these actions. Our current knowledge regarding differences among the opioids, the effects of endogenous opioid peptides and the role of the various opiate receptor subtypes in pupillary effects is also summarized.     

Determination of source distance in the surface-feeding fish Pantodon buchholzi Pantodontidae

The African butterfly fish Pantodon buchholzi localizes its prey by means of surface waves of the water. Pantodon also responds and orientates well to artificial, short lasting prey-like signals (clicks), produced by a single air-puff or by dipping a small rod once into the water. When stimulated with clicks, which contain many frequencies, Pantodon determines the source distance (test range 5–20 cm) very precisely, regardless of stimulus amplitude, amplitude modulation and frequency band width. However, when the signal is a sine wave at a single frequency (sf) or with upward frequency modulation (ufm), the distance determination is generally impaired, i.e. the distance covered by the fish is too small to reach the wave source. However, the fish can also be tricked into moving too far by presenting it with a sine wave signal which, at a source distance of only 7 cm has a frequency modulation equivalent to a click at 15 cm. In contrast to distance determination, the ability to estimate the target angle is independent of the kind of wave signal presented. The results are discussed with respect to possible mechanisms used for prey localization.     

Inhibition of in vitro biosynthesis of n-acetylneuraminic acid by n-acyl- and n-alkyl-2-amino-2-deoxyhexoses

The biosynthesis of N-acetylneuraminic acid is markedly inhibited by 2-deoxy-2-propionamido-d-glucose (GlcNProp) and to a much lesser extent by 2-deoxy-2-propionamido-d-mannose (ManNProp), but not by 2-deoxy-2-propionamido-d-galactose and N-methylated derivatives of 2-amino-2-deoxy-d-glucose. 2-Deoxy-2-trimethylamino-d-glucose is a weak inhibitor of 2-acetamido-2-deoxy-d-mannose metabolism. When incubated in a cell-free system from rat liver, GlcNProp gives the 6-phosphate, which is converted into N-propionylneuraminic acid. Evidence is presented which shows that it is the metabolites GlcNProp-6-P and ManNProp-6-P which are the competitive inhibitors, and not GlcNProp itself.     

Structural studies on a new polysaccharide,containing d-riburonic acid,from Rhizobium meliloti IFO 13336

The structure of an extracellular, acidic polysaccharide from Rhizobium meliloti IFO 13336 was studied by a method involving successive fragmentation with specific β-d-glycanases of Flavobacterium M64. The polysaccharide is composed of repeating units of the octasaccharide shown. An acidic component was identified as d-riburonic acid.     

The effect of p-aminosalicyclic acid on iron transport and assimilation in mycobacteria

p-Aminosalicylic acid inhibits growth of Mycobacterium bovis BCG and Mycobacterium smegmatis more effectively if cells are growing with a sufficiency of iron (> 1 μg Fe/ml) in the medium than if cells are deficient in iron (<0.1 μg Fe/ml). In iron-deficient cultures formation of mycobactin, an ionophore for iron transport, is strongly inhibited by p-aminosalicylic acid. Uptake of iron into cell suspensions is also inhibited and the activity of several iron-containing enzymes declines in cells exposed to p-aminosalicylic acid during their growth. p-Aminosalicylic acid is about 50 times more effective towards a mutant of M. smegmatis which required mycobactin under iron-deficient growth conditions than towards the wild-type parent. p-Aminosalicylate is taken up into cells by an active process independent of the salicylate uptake system, possibly by the route used for assimilation of p-aminobenzoate. (This could account for why p-aminobenzoic acid, but not salicylic acid, antagonizes the action of p-aminosalicylic acid.) With iron-deficient cells, salicylate assimilation is about 50 times greater than either p-aminosalicylate or p-aminobenzoate but with iron-sufficient cells and with the mycobactin mutant salicylate uptake is negligible whereas p-aminobenzoate and p-aminosalicylate uptakes are unaffected. p-Aminosalicylic acid at 3.3 mM (500 μg/ml) partially inhibits the uptake of both p-aminobenzoate and, if it is occuring, that of salicylate as well. As p-aminosalicylic acid is always more effective when the intracellular concentration of salicylic acid is low, it probably acts as an anti-metabolite of salicylic acid, not, however, by inhibiting the conversion of salicylic acid to mycobactic, but probably somewhere along the metabolic pathway of iron uptake.     

Formation of a carboxyarabinitol bisphosphate complex with ribulose bisphosphate carboxylase/oxygenase and theoretical specific activity of the enzyme

¹⁴C-Labeled 2-carboxyarabinitol-1,5-bisphosphate was bound to both nonactivated and CO₂and Mg²⁺ activated forms of ribulose bisphosphate carboxylase/oxygenase. The complex could be precipitated with 20% polyethylene glycol and 20 mm MgCl₂ for quantitation of the moles of the affinity label bound per mole of enzyme. The [¹⁴C]carboxyarabinitol-P₂ bound to the nonactivated enzyme could be exchanged with a 100-fold excess of the unlabeled compound. With the activated enzyme the binding of [¹⁴C]carboxyarabinitol-P₂ was so tight that it did not exchange with the unlabeled compound and a binding stoichiometry of one molecule per active site was assumed. This tight binding was dependent upon pretreatment of the enzyme with both CO₂ and MgCl₂ in the same manner that enzyme activation depended on CO₂ and Mg²⁺ concentrations. Various enzyme preparations from spinach leaves tightly bound [¹⁴C]carboxyarabinitol-P₂ in proportion to their specific activities. By extrapolating to a maximum binding of 8 mol of [¹⁴C]carboxyarabinitol-P₂ per mole of this A₈B₈ enzyme a theoretical specific activity of 2.8 μmol · min^?1 · mg protein^?1 was indicated. Enzyme preparations purified from spinach leaves generally have a specific activity in the range of 1.0 to 2.3.     

1H-N.M.R. spectra of glycosaminoglycan monomers and dimers in solution in methyl sulphoxide and water

¹H-N.m.r. spectra of glycosaminoglycuronan monomers and dimers in solution in methyl sulphoxide-d₆ have been investigated; N-H and O-H resonances were observed and partially assigned. Their temperature-dependence suggests hydrogen-bonding to the solvent, with the notable exception of that of HO-4 Of sodium D-gluctironate, which was consistently downfield and relatively temperature-insensitive. The concentration-dependence ofthis signal indicates that the corresponding hydroxyl group is involved in the formation of a dimer. Signals for N-H and O-H were observed for aqueous solutions, especially at subzero temperatures.