A specific uncoupler-binding protein in Tetrahymena pyriformis and Paracoccus denitrificans

The uncoupler of mitochondrial oxidative phosphorylation, 2-nitro-4-azido-carbonylcyanide phenylhydrazone (N₃CCP) which is capable of photoaffinity labeling has been used to examine the effect of uncouplers on the energy conserving membranes of Paracoccus denitrificans and Tetrahymena pyriformis. The N₃CCP uncouples respiration in P. denitrificans and T. pyriformis cells with $\text{U}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values of 1.05 μM and 0.24 μM, respectively. Binding studies show the presence of 0.65 ± 0.05 high affinity sites per cytochrome a with a K_d of 0.5 ± 0.1 μM in P. denitrificans membranes and 1.4 ± 0.2 sites per cytochrome a₂ with a K_d of 0.4 ± 0.1 μM in T. pyriformis membranes. Irradiation of [³H]-N₃CCP bound to the membranes leads to a covalent linking of the radioactive uncoupler to a peptide of 10–15 kdaltons as analyzed by SDS-polyacrylamide gel electrophoresis. It is concluded that these two microbial systems contain a specific high affinity uncoupler binding site very similar to that of mammalian mitochondria (Katre, N.V. and Wilson, D.F. (1978) Arch. Biochem. Biophys. 191, 647–656).     

Subunit organization of Euglena chromatin

Digestion of $\text{Euglena}$ nuclei or extracted chromatin with micrococcal nuclease results in the identification of a repeating structure. The DNA repeat size, analyzed on agarose and polyacrylamide gels, is found to be 225±13 base pairs. DNase I digestion produces a serie of fragments multiples of roughly 10 bases. Eventhough pressure shearing is necessary to disrupt the though pellicule of the phytoflagellate, we confirm that, in $\text{Euglena}$, chromatin organization is similar to that of other eukaryotes.     

The study of multiple polymerization equilibria by glass bead exclusion chromatography with allowance for thermodynamic non-ideality effects

Two related aspects are explored of the frontal exclusion chromatography of proteins employing controlled-pore glass beads as the stationary phase. First, it is shown theoretically that, despite the absence of osmotic shrinkage effects previously encountered with Sephadex matrices, the experimentally measurable partition coefficient of a single non-associating solute will be dependent on its concentration due to the differing ratios of activity coefficients in mobile and stationary phases at different total concentrations. The effect is demonstrated with results obtained using ovalbumin in phosphate buffer of pH 7.4, and is Shown to be consistent (up to a solute concentration of 5 $\text{g}\text{litre}$) with theoretical prediction formulated in terms of a single virial coefficient. Secondly, it is shown for self-associating systems that it is possible to determine the monomer concentration as a function of total concentration, provided the stationary phase is selected to ensure exclusion of all oligomeric species except monomer: the relation derived for this purpose accounts for the concentrationdependence of the partition coefficient of monomer, again as a first approximation involving one virial coefficient. Such information on the monomer concentration permits elucidation of the polymerization characteristics of the system in terms of the types of species present and the relevant equilibrium constants. The feasibility of the method, its likely sources of error and the relative contribution of the non-ideality effect are investigated using bovine glutamate dehydrogenase (up to a total concentration of 5.4 $\text{g}\text{litre}$) in phosphate buffer of pH 6.9. This system was selected since comparison was possible with results obtained by other methods, which have established the enzyme polymerization pattern as an isodesmic indefinite self-association. The isodesmic equilibrium constant of 1.5 ± 0.3 $\text{litre}\text{g}$ found in this work is in reasonable agreement with previous findings.     

The dimer-monomer equilibrium constant for [125I]β nerve growth factor

The equilibrium constant for $\text{[}{}^{125}\text{I]β}$ nerve growth factor was determined using polyacrylamide gel electrophoresis to separate the monomer and dimer. Various concentrations of the radiolabelled nerve growth factor were incubated for 24 and 48 hours. The equilibrium constants obtained for both incubation periods were the same, 3.2 ± 1.4 × 10^?11$\text{M}$ and 2.6 ± 1.6 × 10^?11$\text{M}$, respectively. Thus, at physiological concentrations the β nerve growth factor is in the dimeric form almost exculsively.     

Kinetics of testosterone metabolism in normal postmenopausal women and women with breast cancer

The constant infusion and single injection techniques were utilized to study the kinetics of ³H-testosterone (T) metabolism in postmenopausal women with and without breast cancer. The metabolic clearance rates (mean ± SEM) for normal postmenopausal women were $\text{578 ± 82}\text{and}\text{644 ±128}\text{1}\text{24}\text{h}$ as obtained by the constant infusion and single injection techniques, respectively. The corresponding results for the women with breast cancer (patients) are $\text{644 ± 25}\text{and}\text{617 ± 106}\text{1}\text{24}\text{h}$. The single injection technique yielded values for rate constants (units) and volumes of distribution (1); k₁ = 37.5 ± 1.6 for the normals and 34.5 ±1.9 for the patients. K₂ = 76.6 ± 5.1 for the normals and 71.1 ± 1.6 for the patients, V₁ = 7.9 ± 2.2 for the normals and 8.7 ± 1.4 for the patients and V₂ = 7.0 ± 1.5 for the normals and 6.4 ± 1.2 for the patients. The constant infusion technique yielded values for the conversion ratios for the transformation of T to several products; 4-androstene-3,17-dione/T of 0.02 ± 0.003 for normals and 0.03± 0.002 for patients, 5α-dihydrotestosterone/T of 0.02 ± 0.002 for normals and 0.03 ± 0.002 for patients, estrone/T of 0.04 ± 0.01 for normals and 0.04 ± 0.01 for patients, estradiol-17β/T of 0.02 ± 0.005 for normals and 0.03 ± 0.005 for patients and estrone sulfate/T of O.16 ± 0.02 for normals and 0.24 ± 0.06 for patients. The T plasma concentrations and production rates were similar for the two groups of subjects. Hence there were no significant differences between the normals and the patients for all the kinetic parameters. It was determined that all the estradiol being produced in postmenopausal women could be coming from circulating T.     

Adenylate cyclase and cyclic AMP through the cell cycle of Tetrahymena.

Adenylate cyclase activity and 3′, 5′ cyclic adenosinemonophosphate (cAMP) have been followed through the heat-synchronized cell cycle of Tetrahymena pyriformis. While the specific activity of adenylate cyclase remained essentially constant throughout the cycle, cAMP oscillated (between 10 and 50 pmoles/mg protein) through two cycles. Minima were observed at each division ($\text{D}\text{S}$ border) and maxima at each $\text{S}\text{G}\text{2}$ border. Each heat shock caused slight temporary reduction in cyclase activity. Further observations suggest to us that adenylate cyclase shows conformational changes in response to temperature-induced alterations and to changes in lipid composition of membranes.     

Generation of small plasmids from colicin E1 factor carrying genes for galactose utilization and ampicillin resistance,.

Ampicillin-resistant colonies that did not utilize galactose appeared sporadically in cultures of galactose genedeleted $\text{Escherichia coli}$ K-12 cells containing colicin E1 factor carrying genes for galactose utilization and ampicillin resistance. Most of these colonies contained small plasmid DNAs. These plasmids existed as monomer DNAs within $\text{E. coli}$ K-12 cells and formed a series of covalently closed circular DNA molecules ranging in size from 6.3 × 10⁶ to 15.1 × 10⁶ daltons. The use of these plasmid DNAs was discussed.     

Dopamine-stimulated cyclic AMP and binding of [3H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10^?8 M and was half-maximal at $\text{7.9±3.4·10}{}^{\mathrm{?7}}\text{M}$. The increase at 1·10^?5 M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10^?9 M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10^?5 M dopamine was $\text{2.3±0.9·10}{}^{\mathrm{?6}}\text{M}$. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ value for high affinity binding sites was $\text{0.43±0.1·10}{}^{\mathrm{?7}}\text{M}$ and $\text{4.7±1.6·10}{}^{\mathrm{?7}}\text{M}$ for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was $\text{1.2±0.4·10}{}^{\mathrm{/t-7}}\text{M}$ noradrenaline, 2·10^/t-7 M epinine, $\text{4.1±1.8·10}{}^{\mathrm{/t-6}}\text{M}$ fluphenazine, $\text{8.0±1.6·10}{}^{\mathrm{/t-6}}\text{M}$ haloperidol, $\text{4.2±1.2·10}{}^{\mathrm{?6}}\text{M}$cis-flupenthixol, $\text{2.7±0.4·10}{}^{\mathrm{?5}}\text{M}$trans-flupenthixol, $\text{>1·10}{}^{\mathrm{?5}}\text{M}$ apomorphine, sulpiride, naloxone and isoproterenol.     

An ultrastructural examination of everted rat jejunum

Male, albino, Sprague-Dawley rats were sacrificed by cervical separation. Segments of jejunum were excised, everted and examined with the electron microscope. Examination of tissue fixed immediately after eversion revealed the following changes as compared to non-everted segments fixed $\text{in}$$\text{situ}$ and $\text{in}$$\text{vitro}$: 1) an increase in the length of microvilli from (mean ± S. E.) 0.991 ± 0.011μ for normal tissue to 1.389 ± 0.023μ for everted tissue, 2) an increase in width of microvilli from (mean ± S. E.) 0.089 ± 0.001μ for normal tissue to 0.097 ± 0.001μ for everted tissue, 3) an increase in length and number of lateral membrane interdigitations, and 4) the appearance of intercellular “lakes” in the lateral spaces. The above changes are in those structures hypothesized to be involved with salt and water transport across epithelia and may reflect altered transport rates $\text{in}$$\text{vitro}$ as compared to $\text{in}$$\text{vivo}$.     

Preparation of multivesicular liposomes

A novel type of liposome, named here multivesicular liposomes, was prepared by evaporation of organic solvents from chloroform-ether spherules suspended in water. Within each spherule were numerous water droplets that contained solutes to be trapped in liposomes upon solvent evaporation. Liposome preparations of different average diameters were made, varying from $\text{29 ± 10 μ}\text{m}$ to $\text{5.6 ± 1.7 μ}\text{m}$. The liposomes were morphologically characterized by light microscopy and transmission electron microscopy. Materials successfully trapped within the liposomes ranged in molecular size from glucose to nucleic acids. Extremely high percentages of encapsulation (up to 89%) were achieved.     

Minimal functional unit for transport and enzyme activities of (Na+ + K+)-ATPase as determined by radiation inactivation

Frozen aqueous suspensions of partially purified membrane-bound renal (Na⁺ + K⁺)-ATPase have been irradiated at –135°C with high-energy electrons. (Na⁺ + K⁺)-ATPase and K⁺-phosphatase activities are inactivated exponentially with apparent target sizes of $\text{184 ± 4 kDa and 125 ± 3 kDa}$, respectively. These values are significantly lower then found previously from irradiation of lyophilized membranes. After reconstitution of irradiated (Na⁺ + K⁺)-ATPase into phospholipid vesicles the following transport functions have been measured and target sizes calculated from the exponential inactivation curves: ATP-dependent Na⁺?K⁺ exchange, $\text{201 ± 4}\text{kDa}$; (ATP + P_i)-activated Rb⁺?Rb⁺ exchange, $\text{206 ± 7}\text{kDa}$ and ATP-independent Rb⁺?Rb⁺ exchange, $\text{117 ± 4}\text{kDa}$. The apparent size of the α-chain, judged by disappearance of Coomassie stain on SDS-gels, lies between 115 and 141 kDa. That for the β-glycoprotein, though clearly smaller, could not be estimated. We draw the following conclusions: (1) The simplest interpretation of the results is that the minimal functional unit for (Na⁺ + K⁺)-ATPase is αβ. (2) The inactivation target size for (Na⁺ + K⁺)-dependent ATP hydrolysis is the same as for ATP-dependent pumping of Na⁺ and K⁺. (3) The target sizes, for K⁺-phosphatase (125 kDa) and ATP-independent Rb⁺?Rb⁺ exchange (117 kDa) are indistinguishable from that of the α-chain itself, suggesting that cation binding sites and transport pathways, and the $\text{p-}\text{nitrophenyl}$ phosphate binding site are located exclusively on the α-chain. (4) ATP-dependent activities appear to depend on the integrity of an αβ complex.     

Enteric release of vasoactive intestinal peptide after a peptone meal in the dog

Vasoactive intestinal peptide (VIP) concentrations were measured by radioimmunoassay in plasma from portal and peripheral venous blood obtained from six alert, non-anesthetized dogs before and after gastric infusion of a 10% peptone meal. Mean basal portal and cephalic vein plasma VIP concentrations were $\text{42 ± 11.7}$ and $\text{42 ± 8.0}\text{(S.E.M.) pg/ml}$, respectively. No significant changes in peripheral venous plasma VIP concentrations were noted after the peptone meal throughout the duration of the collection period. In contrast, however, the mean VIP concentration in portal plasma increased promptly after the peptone meal with a peak of $\text{79 ± 8.2}\text{pg/ml}$ ($\text{P < 0.02}$) occurring 8 min after infusion of the meal. This was followed by a gradual decline in portal plasma VIP levels, with a return to prefeeding concentrations at 60 min ($\text{44 ± 6.3}\text{pg/ml}$). Results of these studies demonstrate that following gastric infusion of a peptone meal in the dog, portal, but not peripheral, plasma VIP concentrations increase significantly. Failure to detect augmentation of peripheral vein VIP levels after the meal is probably due to hepatic clearance of VIP.     

Cellular pH and water permeability control in frog urinary bladder a possible action on the water pathway

Mucosal acidification (from pH 8.1 to 6.0) reversibly inhibited the hydroosmotic responses to oxytocin, cyclic AMP and 8-bromo-cyclic AMP in frog urinary bladder. These inhibitory effects were only observed in the presence of a permeant buffer in the apical medium and could also be elicited by CO₂ bubbling, even when the mucosal pH was clamped at 8.1. Acid pH reduced the oxytocin-induced net water flux faster than norepinephrine or oxytocin removal and the difference was especially important at low temperature. The time course of recovery from acid pH inhibition was, at 20°C, similar to that of the hormonal action, but when the medium temperature was reduced to 6–7°C, the recovery from acid pH inhibition paradoxically became faster while the oxytocin action was markedly slowed down ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of changes in net water fluxes (expressed in min): oxytocin addition at 20°C, $\text{6.2 ± 0.9}$; at 6°C, $\text{24 ± 3}$; oxytocin removal at 20°C, $\text{4.7 ± 0.8}$; at 6°C, $\text{22 ± 3}$; pH inhibition at 20°C, $\text{2.6 ± 0.2}$; at 6°C $\text{2.5 ± 0.2}$; recovery from pH 6 at 20°C, $\text{6.5 ± 0.9}$; at 6°C, $\text{2.7 ± 0.3}$). These results can be explained by accepting two main loci sensitive to medium acidification: (1) the cyclase system and (2) an intracellular, temperature-independent, post-cyclic AMP site. The fact that the intramembranous particle aggregates associated with the oxytocin-induced water permeability increase did not disappear after the flow inhibition by acid pH at low temperature suggests that the second effect could be located at the water channel itself.     

Biosynthesis of 2-methylalkanes in the crickets Nemobius fasciatus and Gryllus pennsylvanicus.

The biosynthesis of 2-methylalkanes was studied in the crickets $\text{Nemobius}\text{fasciatus}$ and $\text{Gryllus}\text{pennsylvanicus}$. Labelled acetate, valine, and isobutyric acid were incorporated into the cuticular hydrocarbon of $\text{N.}\text{fasciatus}$ at levels of 6.0 ± 1, 6.5 ± 2, and 1.5 ± 0.7 percent respectively. The hydrocarbons of this insect are 20 percent 2-methylalkanes, primarily of even numbered carbon chain lengths, and 80% n-alkanes. Of the label incorporated into the hydrocarbon fraction, 28 ± 2 percent of sodium [1-¹⁴C] acetate, 98 ± 1 percent of L-[G-³H] valine, and 75 ± 10 percent of [1-¹⁴C] isobutyric acid were incorporated into the 2-methylalkanes. This suggests that valine is converted to isobutyric acid and is incorporated into the even numbered carbon chain length 2-methylalkanes during the initial stages of chain elongation. Similar data obtained in $\text{G.}\text{pennsylvanicus}$ suggests that leucine is converted to isovaleric acid which is then incorporated into the odd numbered carbon chain length 2-methylalkanes.     

Amphotericin B-induced sodium transport and water flow across rabbit corneal epithelium

We have determined fluid translocation across the cellular layers lining the cornea by measuring changes in corneal transparency. The loss of 1.3 μ1/cm² fluid from the stroma causes an increase of +1% in transparency. Amphotericin B ($\text{2 · 10}{}^{-6}\text{M}$) when added to the tear side (=mucosal side) of the epithelium causes a rapid increase in potential difference of $\text{12.3 ± 0.7}\text{mV}\text{(}\text{mean}\text{±}\text{S.E.}\text{, n=6}$) followed by a slower increase of $\text{18.6 ± 1.5}\text{mV}$. The electrical resistance is reduced from $\text{3.2 ± 0.3}\text{k}\text{Ω ·}\text{cm}{}^2\text{to}\text{0.6 ± 0.1}\text{k}\text{Ω ·}\text{cm}{}^2$. The resulting increase in calculated short circuit current is accompanied by a decrease in transparency at a rate of $\text{3.6 ± 1.0\%}\text{per h}$, corresponding to an uptake of fluid by the cornea of $\text{4.7 μ}\text{l}\text{·}\text{cm}{}^{-2}\text{·}\text{h}{}^{-1}$. Replacement of the fluid bathing the endothelial side of the cornea, in order to prevent water movement from the aqueous compartment into the stroma, did not significantly alter this uptake of fluid. Thus the epithelial fluid transport which is reported to be normally slightly secretory, becomes absorptive in the presence of amphotericin B. Serosal hypertonicity (20 mM mannitol) increases the water influx into the cornea induced by amphotericin B. These results indicate that amphotericin B induces sodium-selective channels in the epithelium leading to an accumulation of NaCl and water in the stromal layer of the cornea. Ouabain reduces the potential and calculated short circuit current in epithelia pretreated with amphotericin B. Following addition of ouabain, the NaCl and water accumulated in the stroma leak away resulting in a transient increase in transparency. Finally, a model is proposed that includes a stromal compartment involved in fluid transport and that agrees with the results presented here.     

Effect of bombesin-stimulated gastrin on gastric acid secretion in man

The effect of bombesin on gastrin release and gastric acid secretion was investigated in 10 healthy volunteers. Bombesin (0.6 μg · Kg^?1 · hr^?1) produced a significantly higher $\text{(}\text{p}\text{<}\text{0.001)}$ increase in plasma gastrin levels (86.7 11.1 pmo/1 than after a protein meal (39.6 ± 5.6 pmol1/1). The gastric acid secretory response to bombesin (12.1 ± 2.9 mEq · hr^?1) was however significantly lower $\text{(}\text{p}\text{<}\text{0.005)}$ than the maximal response produced by pentagostrin (20.9 ± 3.5 mEq · hr^?1) at the dose of 6 μg · Kg^?1. Atropine did not modify gastrin release induced by bombesin but significantly reduced gastric acid secretion $\text{(}\text{p}\text{<}\text{0.01)}$. From the data presented it may be hypothesized that less biologically active forms of gastrin and/or other peptides inhibiting the gastrin effect upon gastric acid secretion may be released by bombesin.     

Effects of lighting programs on onset of the ovulatory season in mares

Using 240 pony mares, lighting regimens were tested for their efficiency in hastening the onset of the ovulatory season. The mean number of days from January 1 to first ovulation was used as the end point. No advantage was gained by beginning a fixed lighting regimen ($\text{15.5L}\text{8.5D}$, hours light/hours dark) November 1 (66 ±8) versus December 1 (65 ±9), but beginning on January 1 was less efficient (98 ±8; controls, 132 ±5; P<0.05). In another experiment, daily three-hour interruptions of either the light phase (67 ±10) or the dark phase (71 ±11) did not significantly retard the effectiveness of a fixed regimen of $\text{15L}\text{9D}$ (54 ±5; controls, 142 ±6). A $\text{15L}\text{9D}$ regimen every other day (natural day length on alternate days) resulted in an interval (85 ±7) that was shorter (P<0.05) than for the controls and longer (not significant) than for the daily $\text{15L}\text{9D}$ regimen. When used with natural day length, a one-hour pulse of light in the evening (15 hours after sunrise) was not effective (141 ±6); a one-hour pulse in the morning 9.5 hours after sunset) was only partially effective (117 ±6). In another experiment, the interval was reduced (P<0.05) in a group with one hour of light fixed at 4:00 a.m. with natural day length (85 ±8; $\text{15L}\text{9D}$, 75 ±7; controls, 126 ±9). Results indicated that a fixed one-hour pulse of light at 4 a.m., used with natural day length, may provide an acceptable level of stimulation.     

Elimination kinetics and protein binding of theophylline in the rabbit

The detailed elimination kinetics of theophylline were studied in 27 rabbits. Each received a 10 mg/kg intravenous bolus of aminophylline. The theophylline half-life ($\text{T}\text{1}\text{2}$) was 3.8 ± 0.63 hr. The apparent volume of distribution (V_D) and total body clearance (TBC) for theophylline were 439 ± 60 ml/kg and 81.0 ± 17.3 ml/kg·hr respectively. Theophylline protein binding was determined in 10 animals. The mean bound fraction was 74.3 ± 3.9% (range, 68.3–78.0%); the fraction bound was concentration indifferent over a serum concentration range of 5–20 μgm/ml.     

Reversible redox titrations of cytochrome c and cytochrome c oxidase using detergent solubilized electrochemically generated mediator-titrants

The repetitive, $\text{reversible}$ equilibrium redox cycling of cytochrome $\text{c}$, cytochrome $\text{c}$ oxidase, or mixtures thereof has been made possible by the use of the oxidant, ferricinium ion. This ion is electrochemically generated by the use of non-ionic detergent solubilized ferrocene which is apparently incorporated as micelles and readily electron transfers with an electrode. The $\text{ferricinium-ferrocene}$ couple equilibrates rapidly with these heme proteins. Electrochemically generated benzylviologen radical cations are used as the reductant. The E^O′ values for cytochrome $\text{c}$ oxidase at pH 7.0 are 209 ± 15 mv (2e^?) and 340 ± 15 mv (2e^?).