Slow Exponential Phase Research Articles

An Anti-microbial Terpenoid Fraction from Gymnema sylvestre Induces Flip-flop of Fluorescent-Phospholipid Analogs in Model Membrane.

Therapeutic potential of Gymnema sylvestre on diverse cell types is predominantly due to a variety of terpenoids and their derivatives. However, their bioavailability becomes limited due to poor solubility and lower lipophilic properties, provoking the search for novel membranotropic terpenoids and their mechanism of action. A terpenoid fraction purified from Gymnema sylvestre exhibited broad spectrum antimicrobial activity against both Gram positive and Gram negative bacteria with IC50 ˂ 0.1mg/ml. Evaluation of its membranotropic effect in vitro on reconstituted model membrane revealed that the fraction induced flip-flop of fluorescent phospholipid analogs across the lipid bilayer. The terpenoid-induced lipid flipping was biphasic with a fast linear phase (rate constant (k1) = 3 to 5S-1) and a second slow exponential phase (rate constant (k2) = (4 to 9) × 10-3S-1). The lipid-flippase activity of the terpenoid fraction showed concentration and incubation-dependent cooperativity, indicating their lipophilic nature and membrane-destabilizing activity that facilitated lipid translocation. For the first time, our study reveals the flippase activity of a terpenoid fraction of Gymnema sylvestre that could be further explored for their membrane-mediated pharmacological properties. Graphical Abstract.

Reduced Heme Levels Underlie the Exponential Growth Defect of the Shewanella oneidensis hfq Mutant

The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA) function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA), the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.

Studying the folding process of the acylphosphatase from Sulfolobus solfataricus. A comparative analysis with other proteins from the same superfamily.

The folding process of the acylphosphatase from Sulfolobus solfataricus (Sso AcP) has been followed, starting from the fully unfolded state, using a variety of spectroscopic probes, including intrinsic fluorescence, circular dichroism, and ANS binding. The results indicate that an ensemble of partially folded or misfolded species form rapidly on the submillisecond time scale after initiation of folding. This conformational ensemble produces a pronounced downward curvature in the Chevron plot, appears to possess a content of secondary structure similar to that of the native state, as revealed by far-UV circular dichroism, and appears to have surface-exposed hydrophobic clusters, as indicated by the ability of this ensemble to bind to 8-anilino-1-naphthalenesulfonic acid (ANS). Sso AcP folds from this conformational state with a rate constant of ca. 5 s(-1) at pH 5.5 and 37 degrees C. A minor slow exponential phase detected during folding (rate constant of 0.2 s(-1) under these conditions) is accelerated by cyclophilin A and is absent in a mutant of Sso AcP in which alanine replaces the proline residue at position 50. This indicates that for a lower fraction of Sso AcP molecules the folding process is rate-limited by the cis-trans isomerism of the peptide bond preceding Pro50. A comparative analysis with four other homologous proteins from the acylphosphatase superfamily shows that sequence hydrophobicity is an important determinant of the conformational stability of partially folded states that may accumulate during folding of a protein. A low net charge and a high propensity to form alpha-helical structure also emerge as possibly important determinants of the stability of partially folded states. A significant correlation is also observed between folding rate and hydrophobic content of the sequence within this superfamily, lending support to the idea that sequence hydrophobicity, in addition to relative contact order and conformational stability of the native state, is a key determinant of folding rate.

Plasma-kallikrein clearance during liver regeneration after partial hepatectomy in the rat.

The liver clears circulating plasma-kallikrein through a receptor-mediated endocytosis process: an initial fast phase is followed by a slow exponential phase. To determine whether the clearance rate of plasma-kallikrein is affected during liver regeneration, we perfused isolated rat livers with rat plasma-kallikrein (rPK) at 0, 1, 2, 3 and 7 days after partial hepatectomy or sham operation. Liver regeneration was followed by the expression of the proliferating-cell nuclear antigen (PCNA) labeling index. The serum concentration of alpha2-macroglobulin, an acute phase protein in rats, was measured. At day 1, the fast phase of rPK clearance rate increased in hepatectomized rats when compared with day 0 (4.9+/-0.4 and 3.7+/-0.4 mU/g liver min, p<0.05). However, at day 2, the rPK fast phase clearance rate dropped significantly (2.6+/-0.2, p<0.05), when compared with day 1. No difference was found among the sham groups at different days of hepatectomy. These changes seem to be independent of the acute phase reaction. The regenerative liver weight increased continuously during the observation period. PCNA expression increased significantly after hepatectomy, with maximal PCNA-labeling indices at days 1 and 2, declining thereafter. The rPK fast phase clearance rate changes during liver regeneration, with a zenith occurring when PCNA labeling index is maximal (day 1) and a nadir occurring at the mitotic phase (day 2).

Kinetics of the phosphorylation of Na,K-ATPase by inorganic phosphate detected by a fluorescence method.

Phosphorylation by Pi of the Na,K-ATPase from rabbit kidney in the absence of Na+ ions but in the presence of Mg2+ ions has been studied. In the absence of K+ ions, unphosphorylated and phosphorylated states induce different fluorescence levels in the membrane-bound styryl dye RH421, and hence transitions between the two states were monitored. Transient kinetic studies of phosphorylation were initiated by manual addition of Pi or by photochemical release of Pi from 1-(2-nitrophenyl)ethyl phosphate (caged Pi) using laser flash photolysis at 308 nm. Equilibrium studies of phosphorylation showed that the apparent Km for Pi was 23.0 +/- 0.3 microM (mean +/- sem) at pH 7.1 and 21 degrees C. The dye fluorescence increased in a biphasic manner on addition of 500 microM Pi to the enzyme: a rapid phase (t 1/2 < 1 s) and a slower exponential phase at 0.059 +/- 0.003 s-1. The rate of the rapid phase was studied by fast concentration-jump experiments and exhibited first-order kinetics in Pi up to 60 microM. Fluorescence records vs time were exponential, and a plot of the rate constant versus [Pi] had a slope of 1.47 x 10(5) M-1 s-1 and ordinate [Pi] = 0) intercept of 3.1 s-1. Addition of 50 mM NaCl to the phosphorylated enzyme induced an exponential decay in the dye fluorescence from which a rate constant of 0.10 +/- 0.005 s-1 was determined. These data were interpreted in terms of transformations between conformational states E1 and E2, and the phosphorylated state P-E2 defined in the Post-Albers mechanism of the Na,K-ATPase [Läuger, P., (1991) Electrogenic Ion Pumps, Sinauer Associates Inc., Sunderland, MA] as follows: [formula: see text] The RH421 fluorescence of state P-E2 was studied over the pH range 6-8.5. Fluorescence was greatest at pH 8.5 and lowest at pH 6.0 in a simple binding isotherm with pK 7.5. The apparent Km for Pi rose cooperatively with increasing pH (pKa 8.6 and a Hill coefficient of 2). Therefore in the absence of monovalent metal ions, occupation of the cation (K+) binding sites by protons promotes phosphorylation by Pi.

Effects of O2 and CO2 on Nonsteady-State Photosynthesis (Further Evidence for Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Limitation).

The effects of CO2 and O2 on nonsteady-state photosynthesis following an increase in photosynthetic photon flux density (PPFD) were examined in Spinacia oleracea to investigate the hypotheses that (a) a slow exponential phase (the ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] phase) of nonsteady-state photosynthesis is primarily limited by Rubisco activity and (b) Rubisco activation involves two sequential, light-dependent processes as described in a previous study (I.E. Woodrow, K.A. Mott [1992] Plant Physiol 99: 298-303). Photosynthesis was found to be sensitive to O2 during the Rubisco phase in the approach of photosynthesis to steady state. Analyses of this sensitivity to O2 showed that the control coefficient for Rubisco was approximately equal to 1 during this phase, suggesting that Rubisco was the primary limitation to photosynthesis. O2 had almost no effect on the kinetics (described using a relaxation time, [tau] of the Rubisco phase for leaves starting in darkness or for leaves starting in low PPFD, but [tau] was substantially higher in the former case. CO2 was found to affect both the rate of photosynthesis and the magnitude of [tau] for the Rubisco phase. The [tau] value for the Rubisco phase was found to be negatively correlated with intercellular CO2 concentration (ci), and leaves starting in darkness had higher values of [tau] at any ci than leaves starting in low PPFD. The effects of CO2 and O2 on the Rubisco phase are consistent with the existence of two sequential, light-dependent processes in the activation of Rubisco if neither process is sensitive to O2 and only the second process is sensitive to CO2. The implications of the data for the mechanism of Rubisco activation and for the effects of stomatal conductance on nonsteady-state photosynthesis are discussed.

Actions of perchlorate ions on rat soleus muscle fibres.

1. The effects of perchlorate (ClO4-) on contraction have been studied in rat soleus muscle fibres using (i) potassium (K+) contracture and (ii) two-microelectrode-point voltage clamp techniques. 2. Membrane potentials (Vm) at all external [K+] were 3-5 mV more negative in ClO4-. The hyperpolarization could not be attributed to a change in Na+, K+, or Cl- permeability, or to an effect on the Na(+)-K+ pump. 3. ClO4- shifts the voltage dependence of tension activation, and contraction threshold, to more negative membrane potentials without altering maximum tension. Consequently, twitches and submaximal K+ contractures were potentiated, whereas tetanic contractions and 200 mM-K+ contractures were unaltered. 4. The decay of K+ contractures during steady depolarization with ClO4- developed a slow exponential phase with an average time constant of 6.05 +/- 0.76 min at -38 mV, and 1.68 +/- 0.15 min at -19 mV. This slow component was (a) under the rapid control of the surface Vm and (b) did not depend on external Ca2+. 5. Inactivation of E-C coupling was measured with a test 200 mM-K+ depolarization following 3-10 min depolarizations in conditioning solutions containing 20-120 mM-K+. ClO4- induced a negative shift in the curve-relating test K+ contracture amplitude to conditioning Vm but did not alter the rate of repriming of tension upon repolarization. 6. The results suggest that ClO4- increases the amount of activator produced during depolarization and thus allows the slow inactivation step in excitation-contraction (E-C) coupling to be reflected in the decay of K+ contracture tension. 7. A 'perchlorate contracture', which did not depend on the activation of E-C coupling, was observed. The contracture depended on external Ca2+, but not on voltage-dependent Ca2+ channels or Na(+)-Ca2+ exchange.

Evidence for the existence of three types of potassium channels in the frog Ranvier node membrane.

1. In voltage clamped myelinated fibres, the K+ current was recorded in high-K+ media to allow analysis without complications due to K+ accumulation. 2. After a depolarization, the tail of K+ current following repolarization decreases in two phases: a fast phase lasting about 20 msec and a slow exponential phase lasting several hundred milliseconds. When the duration of the depolarization is increased, the amplitude at time zero of the fast phase increases (activation of the conductance) and then decreases slowly (inactivation of the conductance). Simultaneously, the amplitude of the slow phase, extrapolated to time zero of repolarization, increases slowly and reaches a steady-state level (about 20% of the maximum instantaneous current) after about 600 msec of depolarization. 3. The fast phase of the tail current is blocked by external application of 4-aminopyridine (4-AP) (KD = 10(-5)M). The slow phase is unaltered by 4-AP (10(-7)-10(-2)M). 4. In the presence of 4-AP (10-3M), the remaining slow K+ current, activated by depolarizations, does not inactivate. 5. During depolarizations and repolarizations, the conductance of the slow current (GKs) varies exponentially. The steady-state value of the slow conductance and its time constant of activation vary with voltage. The variation of the slow conductance with time and voltage can be described by a closed-open mode, assuming that each channel is gated by one particle. The activation kinetics of the slow current is unaltered by long lasting (500 msec) prepolarizations. 6. The fast K+ conductance, calculated from the fast tail current, is fully inactivated at the end of a 3 min depolarization to 0 mV. 7. The fast K+ conductance can be decomposed into two components: one component (GKf1) activating between -80 and -30 mV and inactivating very slowly (tau = 45 sec at E = 0 mV); one component (GKf2) activating between -40 mV and +30 mV and inactivating slowly (tau = 2 sec at E = 0 mV). t = 12 degrees C. 8. The maximum slow and fast conductances increase with [K]0. While the maximum fast conductance tends to saturate at high external K+ concentrations, the maximum slow conductance shows no sign of saturation. 9. A comparison between motor and sensory fibres shows that, while the amplitude of maximum slow and fast conductances are identical for both types of fibres, the amplitude of fast-1 conductance is larger and consequently the amplitude of fast-2 is smaller in motor than in sensory fibres. The different spike frequency adaptations observed on both types of fibres are discussed in relation to these different relative fast conductances amplitudes. 10. It is concluded that the K+ conductance of the nodal membrane is composed of three components (GKS, GKf1 and GKf2) corresponding to three different and distinct types of K+ channels.

Stomach Emptying in the Newborn

An error was made in the formula we used for measuring gastric emptying in a recent issue.1 The formula should be: See Equation in the PDF File See Image in the PDF File See Table in the PDF File The new formula does not change our conclusion that the pattern of gastric emptying in neonates is characterized by an initial rapid phase followed by a slower exponential phase. Similarly there were no significant differences between any of the four positions. The time taken for half and three quarters of the test meal to leave the stomach is longer than in the previous study but the differences were not significant in any of the four positions.

Heavy meromyosin Mg-ATPase: presteady-state and steady-state Hplus release.

The presteady state and steady state release of protons during hydrolysis of ATP by myosin has been studied by using a proteolytic subfragment of myosin, heavy meromyosin, isolated from rabbit skeletal muscle. Under the conditions of 0.5 M KCl, 9 mM MgCl(2), pH 8, and 25 degrees , the rate of the H(+) initial burst is proportional to the concentration of ATP added, and shows no plateau even when the concentration of ATP added is increased up to 750 muM. The apparent second order rate constant is approximately 7 x 10(5) M(-1) sec(-1). When the concentration of ATP added is equal to or less than the concentration of heavy meromyosin catalytic sites, the H(+) initial burst is followed by a slow exponential release of H(+) with a rate constant of 0.017 sec(-1). This rate constant is equal to the steady state rate constant calculated for each heavy meromyosin catalytic site, suggesting that both sites split ATP independently. The magnitude of the H(+) released per ATP bound is always 0.4 in the initial burst, and 0.6 in the slow exponential phase, which equals to a total of 1 H(+) released per ATP hydrolyzed. On the basis of these observations, it is concluded that the H(+) initial burst may well represent a fast ionization of H(+) due to a conformational change as a result of nucleotide binding.

Slow exponential growth of Escherichia coli in presence of p-fluorophenylalanine. Effect of the analog on aromatic biosynthesis

1. 1. A new growth phase of Escherichia coli B in the presence of p-fluorophenylalanine is described. This is a slow exponential growth phase which follows the linear growth induced by the analog, and which continues for several generations until supplanted by a resistant mutant. A graphic analysis of the complete growth cycle is given. 2. 2. The p-fluorophenylalanine is incorporated exponentially during the slow exponential growth, without alteration, as a partial substitute for phenylalanine. 3. 3. Extensive filamentation of the organisms occurs during linear growth. Cell division is resumed in the slow exponential phase. 4. 4. The phenomena of inhibition of mass increase and inhibition of cell division are shown to be separable. Cell-division inhibition appears dependent on tyrosine deprivation or interference with tyrosine metabolism. It is postulated that dl -p- fluorophenylalanine interferes with aromatic biosynthesis by inhibiting enzymes early in the pathway and by partially repressing the synthesis of these enzymes. 5. 5. A new interpretation is made of the nature of the resistant mutant.