Tests of the carrier model for ion transport by nonactin and trinactin

The fluxes of K⁺ and NH₄⁺ carried by nonactin and trinactin across thin lipid membranes have been measured as functions of ion activity, electric potential and time. In agreement with the predictions of a version of the carrier model in common use, the shape of the initial current-voltage relation is independent of the activity of the electrolyte, $\text{a}{}_{\mathrm{<mtext>i</mtext>}}$ while the ratio of the initial conductance, $\text{G}{}_0$, to the steady-state conductance, $\text{G}{}_{\mathrm{\infty }}$, increases according to $\text{G}{}_0\text{/G}{}_{\mathrm{\infty }}\text{=}\text{const}{}_1\text{+ const}{}_2\text{× a}{}_{\mathrm{<mtext>i</mtext>}}$. For trinactin the data presented allow the estimation of the rate constants of the carrier process (in the limit of zero potential) in a manner which does not assume any particular variation with potential for the constants. Using empirically determined functions of potential, a complete set of values is also available for nonactin. The curve fitting which is necessary is described in the following paper. The data presently available for valinomycin are sufficient neither to test the model nor to determine a complete set of constants.     

Catalytic function of thymidylate synthase is confined to S phase due to its association with replitase

Catalytic activity of thymidylate synthase, as measured $\text{in}\text{,}\text{vivo}$, is tightly linked to S phase of the cell cycle in Chinese hamster embryo fibroblast cells. This activity, as measured $\text{in}\text{,}\text{vitro}$, is found in all parts of the cell cycle. Thymidylate synthase activity in nuclear (karyoplast) extracts increased as the cells progressed from $\text{G}{}_0\text{G}{}_1$ to S phase. This enzymatic activity in the nuclei of S phase cells is associated with the multienzyme complex (replitase) that also contained DNA polymerase and other enzymes of DNA replication and precursor synthesis. The degree of association of thymidylate synthase with replitase, which increased co-ordinately as the cells progressed from $\text{G}{}_0\text{G}{}_1$ phase to S phase, coincided strongly with the level of $\text{in}\text{,}\text{vivo}$ activity of the enzyme.     

Influence of duration of incubation on zooplankton respiration and excretion results

Respiration (O), ammonium (NH₄), phosphate (PO₄), total nitrogen (N_T) and phosphorus (P_T) excretions were measured on mixed zooplankton during 3-, 6-, 9-, 12-, 21-, and 24-h incubation periods at 20–23 C. The excretion rates of PO₄, N_T. and P_T decrease during a 21-h period, while rates of respiration and excretion of NH{IN4} are constant. The percentage of inorganic nitrogen excreted increases regularly from 3 h (30–40% of total nitrogen) to 21 h (70–80%) and it could be either due to a bacterial activity which was measured or to a decrease with time of organic nitrogen excreted because of starvation. $\text{O}\text{N}{}_{\mathrm{T}}$, $\text{O}\text{PO}{}_4$, $\text{O}\text{P}{}_{\mathrm{T}}$, and $\text{NH}{}_4\text{PO}{}_4$ ratios increase during the first 9 h of incubation; the percentage of inorganic phosphorus excreted is higher at the very beginning and then remains constant from 6 to 24 h. $\text{O}\text{NH}{}_4$ and $\text{N}{}_{\mathrm{T}}\text{P}{}_{\mathrm{T}}$ ratios are constant during a 24-h term, which makes them useful metabolic indexes.     

Galactose oxidase action on GM1 ganglioside in micellar and vesicular dispersions

G_M1 ganglioside was dispersed in different membrane-mimicking systems and the effect of dispersion on G_M1 oxidation by galactose oxidase was studied. The following membrane-mimicking systems were used: homogeneous micelles of G_M1; mixed micelles (at different proportions of constituents) of G_M1 with either G_D1a ganglioside (which is resistant to the enzyme), or the non-ionic detergent Triton X-100, or bovine serum albumin; small unilamellar vesicles of egg phosphatidylcholine (PC), containing various proportions of G_M1. As a reference substrate not involved in membranous systems and freely interacting with the enzyme, the oligosaccharide portion of G_M1 (DesG_M1) was employed.The apparent $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the enzyme was dramatically dependent on the type of G_M1 dispersion. The lowest value was recorded on homogeneous micelles of G_M1 and on mixed G_M1-G_D1a micelles. From this value, the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ increased 2-fold with G_M1-bovine serum albumin lipoprotein micelles, up to 1400-fold with mixed G_M1-Triton X-100 (optimal molar ratio, 1:13.8) micelles, and up to 14 000-fold on PC vesicles containing 8 mol% G_M1 (this proportion was optimal for enzyme activity on vesicles). The activity developed on these latter vesicles turned out to be still greater (2-fold) than that displayed on DesG_M1. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ had very similar values in all different membrane systems; in contrast, it was markedly greater on DesG_M1. Both Triton X-100 micelles and PC vesicles did not appreciably alter the kinetics of galactose oxidase action on pure galactose, indicating that the above effects are dependent on the intrinsic characteristics of the membrane-like systems containing gangliosides.     

The cell cycle time of the haemocytes in the insect Calliphora vicina

The cell cycle time of Calliphora vicina prohaemocytes was examined using the labelled mitoses method after the administration of a pulse of H³-thymidine. The total cycle time occupied 9.1 hr, while $\text{G}{}_1\text{+}\text{1}\text{2}\text{M}$, S and $\text{G}{}_2\text{+}\text{1}\text{2}\text{M}$ occupied 1.6 hr, 2.7 hr and 4.8 hr respectively.     

Interaction of lymphocytes and macrophage cell line cells (M1 cells). I. Functional maturation and appearance of Fc receptors im M1 cells

M₁ cells, which are cell line cells established from myeloid leukemia cells of the SL strain mouse, can differentiate from blast cells ($\text{M}{}_1{}^{\mathrm{?}}$) to mature macrophages ($\text{M}{}_1{}^{\mathrm{+}}$) within 48 hr, when they are cultured with conditioned medium (CM) obtained from murine embryonic fibroblasts. While $\text{M}{}_1{}^{\mathrm{?}}$ cells have no phagocytic activity nor Fc receptor (FcR), $\text{M}{}_1{}^{\mathrm{+}}$ cells possess both characteristics. The appearance of FcR is temperature-dependent and inhibited by a metabolic inhibitor, cycloheximide. FcR on $\text{M}{}_1{}^{\mathrm{+}}$ cells is resistant to trypsin and pronase. $\text{M}{}_1{}^{\mathrm{+}}$ cells improve the viability of macrophage-depleted SL splenic lymphocytes and restore the in vitro secondary plaque forming cell response of macrophage-depleted spleen cells to particulate and soluble antigens. $\text{M}{}_1{}^{\mathrm{?}}$ cells lack this macrophage-substituting capacity. Mm₁ cells, mutant cells from M₁ cells, having FcR and higher phagocytic activity than $\text{M}{}_1{}^{\mathrm{+}}$ cells, are also devoid of this capacity.     

Hydration of Escherichia coli lipids: Deuterium T1 relaxation time studies of phosphatidylglycerol,phosphatidylethanolamine and phosphatidylcholine

The hydration properties of Escherichia coli lipids (phosphatidylglycerol, phosphatidylethanolamine) and synthetic $\text{1,2-}\text{dioleoyl}\text{-sn-}\text{glycero-3-phosphocholine}$ in H₂O/²H₂O mixtures (9:1, v/v) were investigated with ²H-NMR. Comparison of the ²H₂O spin lattice relaxation time ($\text{T}{}_1$) as a function of the water content revealed a remarkable quantitative similarity of all three lipid-H₂O systems. Two distinct hydration regions could be discerned in the $\text{T}{}_1$ relaxation time profile. (1) A minimum of 11–16 water molecules was needed to form a primary hydration shell, characterized by an average relaxation time of $\text{T}{}_1\text{≈ 90}\text{ms}$. (2) Additional water was found to be in exchange with the primary hydration shell. The exchange process could be described in terms of a two-site exchange model, assuming rapid exchange between bulk water with $\text{T}{}_1\text{= 500}\text{ms}$ and hydration water with $\text{T}{}_1\text{= 80–120}\text{ms}$. Analysis of the linewidth and the residual quadrupole splitting (at low water content) confirmed the size of the primary hydration layer. However, each lipid-water system exhibited a somewhat different linewidth behavior, and a detailed molecular interpretation appeared to be preposterous.     

Deoxyribose 5-phosphate aldolase: an enzyme that peaks in the G2 phase of rat hepatoma cells.

The activity of deoxyribose 5-P aldolase (2-deoxy-d-ribose 5-phosphate acetaldehyde lyase, EC 4.1.2.4) increases three- to fourfold in cultures of rat hepatoma cells that have reached stationary growth and have stopped in the G₂ phase of the cell cycle. We have shown that in cell cultures synchronized by three independent methods the enzyme activity peaks in late G₂, followed by a rapid decline during mitosis. Two compounds, dibutyryl cAMP and isoproterenol, that appear to block these cells in G₂, also cause a two- to fourfold increase in deoxyribose 5-P aldolase activity. It is suggested that this enzyme may play a role in lowering the pools of deoxyribonucleotides in the late $\text{G}{}_2\text{M}$ phase of the cell cycle.     

Time lag in a model of a biochemical reaction sequence with end product inhibition

The model studied is that of Goodwin, in which all but one of the reactions obey linear kinetics, while the end-product inhibits the first reaction in a term of Michaelis-Menten form, with Hill coefficient ?:

$\text{z=}\text{∫}\text{?∞}\text{t}\text{x}{}_{\mathrm{n}}\text{(T)G(t?T)dt}$

The results obtained relate to time lag in the off diagonal terms in these equations. The time lag is taken in distributed form, for example replacing x_n in the first equation by

$\text{dx}{}_{\mathrm{t}}\text{dt}\text{=k}{}_1\text{x}{}_{\mathrm{t??}}\text{1?b}{}_1\text{x}{}_{\mathrm{t}}\text{, i=2, …n.}$

For any non-negative G, time lag in these terms can not destabilize the equilibrium point in the case ? = 1. For a particular class of functions G one can obtain some insight into the consequences of time lag by relating the model to that with a longer loop of reactions. Then known results can be used for general ? and n.     

The effect of prostaglandins E1 and E2 on the human erythrocyte as monitored by spin labels

The effects of the prostaglandins PGE₁ and PGE₂ on the deformability of the human erythrocyte were studied using spin-labeled erythrocytes. Two magnetic resonance parameters were measured: (1) The orientation relaxation time, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, for the erythrocyte, and (2) the order parameter, S, for a fatty acid spin label bound to the membrane. Prostaglandins PGE₁ and PGE₂ exhibited opposite effects on both $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S. PGE₂ made the cell less deformable (increases of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S) and PGE₁ made the erythrocyte more deformable (decrease of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S).     

Separation of solubilized alpha and beta adrenergic receptors by affinity chromatography.

Isoelectric focusing in the presence of Nonidet P-40 splits human chromatographically pure γ globin chains into two bands of isoelectric points 6.95 and 6.85, respectively. The comparison of the relative proportions of the two bands with the ratios between the G_γ and A_γ non allelic chains of human fetal hemoglobin suggests that the band at pI 6.95 corresponds to G_γ and the band at pI 6.85 corresponds to the A_γ chain; the latter is the only band present in a patient with Greek type hereditary persistence of fetal hemoglobin, producing only A_γ chains. Fluorography of electrofocusing-separated radioactive γ globin chains synthesized by thalassemic reticulocytes indicates that the relative $\text{G}{}_{\mathrm{\gamma }}\text{A}{}_{\mathrm{\gamma }}$ synthetic ratios are similar to the relative amounts of G_γ and A_γ chains accumulated in the erythrocytes, suggesting that the activities for the G_γ and A_γ mRNAs decay at roughly similar rates.     

Significance of O2 availability and cycling on the respiratory burst response of human PMN's exposed to cytochrome c and superoxide dismutase

In many instances the effect of superoxide ($\text{O}{}_2{}^{\mathrm{?}}$) trapping agents in suppressing the net rate of O₂ consumption of activated PMN's is not in accordance with theoretical expectations. We offer here an alternate explanation to those previously presented by Segal and Meshulam (FEBS Letters 100, 27–32) and Babior (Biochem. Biophys. Res. Comm. 91, 222–226). The paradoxical results previously presented can be explained by recognizing that shortly after activation of resting cells an O₂ diffusion layer is established at or near the outer surface of these cells. The presence of this diffusion layer can markedly alter the anticipated stochiometric relationship between $\text{O}{}_2{}^{\mathrm{?}}$ trapped and apparent O₂ consumed by these cells when they are exposed to $\text{O}{}_2{}^{\mathrm{?}}$ trapping agents.     

Comformation and absolute configuration of -methyllanthionine

If the bicyclic peptide ring proposed by Gross $\text{et}$$\text{al}$. (1,2) does in fact exist in nisin and related antibiotics, then the unusual β-methyllanthionine component must be significantly distorted from its conformation in the free state, as determined by x-ray structure analysis. The torsion angles about the SC_β bonds are 50–100° from the torsion angles in models of the sulfur-bridged peptide ring proposed for nisin. The chirality of the methylated β-carbon atom is (S). The conformation of the amino acid differs from that of $\text{meso}$-lanthionine only by a 180° rotation of a carboxyl group about the $\text{C}{}_{\mathrm{\alpha }}{}^{\mathrm{D}}\text{C}{}_{\mathrm{\beta }}\text{(CH}{}_3\text{)}$ bond.     

Soluble and enzymatically stable (Na++K+)-ATPase from mammalian kidney consisting predominantly of protomer αβ-units: Preparation,assay and reconstitution of active Na+, K+transport

Soluble $\text{(Na}{}^{\mathrm{+}}\text{+K}{}^{\mathrm{+}}\text{)-ATPase}$ consisting predominantly of αβ-units with $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ below 170 000 was prepared by incubating pure membrane-bound $\text{(Na}{}^{\mathrm{+}}\text{+K}{}^{\mathrm{+}}\text{)-ATPase}$ (35–48 μmol P_i/min per mg protein) from the outer renal medulla with the non-ionic detergent dodecyloctaethyleneglycol monoether (C₁₂E₈). $\text{(Na}{}^{\mathrm{+}}\text{+K}{}^{\mathrm{+}}\text{)-ATPase}$ and potassium phosphatase remained fully active in the detergent solution at C₁₂E₈/protein ratios of 2.5–3, at which 50–70% of the membrane protein was solubilized. The soluble protomeric $\text{(Na}{}^{\mathrm{+}}\text{+K}{}^{\mathrm{+}}\text{)-ATPase}$ was reconstituted to Na⁺, K⁺ pumps in phospholipid vesicles by the freeze-thaw sonication procedure. Protein solubilization was complete at C₁₂E₈/protein ratios of 5–6, at the expense of partial inactivation, but $\text{(Na}{}^{\mathrm{+}}\text{+K}{}^{\mathrm{+}}\text{)-ATPase}$ and potassium phosphatase could be reactivated after binding of C₁₂E₈ to Bio-Beads SM2. At C₁₂E₈/protein ratios higher than 6 the activities were irreversibly lost. Inactivation could be explained by delipidation. It was not due to subunit dissociation since only small changes in sedimentation velocities were seen when the C₁₂E₈/protein ratio was increased from 2.9 to 46. As determined immediately after solubilization, $\text{S}{}_{\mathrm{<mtext>20,</mtext><mtext>w</mtext>}}$ was 7.4 S for the fully active $\text{(Na}{}^{\mathrm{+}}\text{+K}{}^{\mathrm{+}}\text{)-ATPase}$, 7.3 S for the partially active particle, and 6.5 S for the inactive particle at high C₁₂E₈/protein ratios. The maximum molecular masses determined by analytical ultracentrifugation were 141 000–170 000 dalton for these protein particles. Secondary aggregation occurred during column chromatography, with formation of enzymatically active $\text{(αβ)}{}_2\text{-}\text{dimers}$ or $\text{(αβ)}{}_3\text{-}\text{trimers}$ with $\text{S}{}_{\mathrm{<mtext>20,</mtext><mtext>w</mtext>}}\text{=10–12}$ S and apparent molecular masses in the range 273 000–386 000 daltons. This may reflect non-specific time-dependent aggregation of the detergent micelles.     

The polymorphic phase behaviour and miscibility properties of synthetic phosphatidylethanolamines

(1) The polymorphic phase behaviour of aqueous dispersions of various synthetic phosphatidylethanolamines, both singly and in mixtures, has been investigated by ³¹P-NMR. (2) $\text{14:0}\text{14:0}$ PE remains in the lamellar phase up to 90°C. $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibits a lamellar to hexagonal (H_II) transition between 60°C and 63°C. For $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE, the lamellar to hexagonal (H_II) transition occurs between 7 and 12°C, whereas for $\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}$ PE, the hexagonal (H_II) phase is the preferred structure above ?15°C. (3) Mixtures of $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibit near-ideal miscibility behaviour. For mixtures of $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{14:0}\text{14:0}$ PE there is evidence of fluid-solid immiscibility at temperatures below the gel-liquid crystalline transition temperature of the $\text{14:0}\text{14:0}$ PE component. Mixtures of $\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibit complex phase behaviour involving limited fluid-solid immiscibility at low temperatures and formation of a phase allowing isotropic motional averaging at higher temperatures. (4) ³¹P-NMR provides a graphic method for investigating the miscibility properties of mixed PE systems.     

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans

(1) $\text{H}$⁺/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O₂, NO^?₂ or N₂O. Under optimal H⁺-translocation conditions, the ratios $\text{H}{}^{\mathrm{+}}\text{O}$, $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO^?₂ or N₂O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were ?0.84, ?2.33 and ?1.90, respectively. (3) The $\text{H}{}^{\mathrm{+}}\text{oxidant}$ ratios, mentioned in item 2, were not altered in the presence of $\text{valinomycin}\text{K}{}^{\mathrm{+}}$ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O₂, NO^?₂ and N₂O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO^?₂, N₂O or O₂ pass two H⁺-translocating sites. The $\text{H}{}^{\mathrm{+}}\text{electron}$ acceptor ratios predicted by this scheme are in good agreement with the experimental values.     

Histone phosphorylation in late interphase and mitosis

Histone phosphorylation in late interphase has been investigated employing cells synchronized by the isoleucine-deprivation method, followed by resynchronization at the $\text{G}{}_1\text{S}$ boundary using hydroxyurea. Phosphorylation occurred in both f1 and f2a2 as cells synchronously entered S phase following removal of hydroxyurea. The relative rates of phosphorylation of both species of histone increased in G₂-rich and metaphase-rich cultures. A small amount of histone f3 phosphorylation was also observed in M-rich cultures which was not seen in G₁, S, or G₂-rich cultures. It is concluded that f1 phosphorylation is not dependent on continous DNA replication. These experiments suggest consideration of the concept that f1 phosphorylation is initiated as a preparation for impending cell division.     

Cockroaches on a treadmill: Aerobic running

Five species of cockroach were tested on a miniature treadmill at three velocities as O₂ consumption () was measured: Gromphadorhina chopardi, Blaberus discoidalis, Eublaberus posticus, Byrsotria fumagata and Periplaneta americana. All cockroaches showed a classical aerobic response to running: increased rapidly from a resting rate to a steady-state on-response varied from under 30 s to 3 min. Recovery after exercise was rapid as well; $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ off-response varied from under 30 s to 6 min. These times are faster or similar to mammalian values. varied directly with velocity as in running mammals, birds and reptiles. during steady-state running was only 4–12 times higher than at rest. Running is energetically much less costly per unit time than flying, but the cost of transport per unit distance is much more expensive for pedestrians. The minimal cost of transport (M_run), the lowest necessary to transport a given mass a specific distance, is high in cockroaches due to their small size. The new data suggest that insects may be less economical than comparable sized vertebrates.