Block of sodium channels by internal mono- and divalent guanidinium analogues. Modulation by sodium ion concentration.

We have investigated the block of squid axon sodium channels by mono- and divalent guanidinium analogues. The action of these compounds on steady state sodium currents was independent of the presence or absence of the normal inactivation process. Block by both mono- and divalent analogues was voltage-dependent, but was a steeper function of potential for divalent molecules. The voltage-dependence could not, in general, be reproduced by a simple model based on Boltzmann's equation. Inhibition of steady state currents by guanidinium ions with 50 mM internal sodium was reasonably well described by a 1:1 drug/channel binding function. Increasing the internal sodium ion concentration increased both the degree and voltage-dependence of current inhibition. This is in sharp contrast to the decrease in inactivation caused by internal sodium. Changes in the external sodium concentration had very little effect on drug block. These results are consistent with a model of the sodium channel as a multi-ion pore. Only a small increase in block can be produced by increased internal sodium in a three-barrier two-site model, but a four-barrier three-site model can reproduce these experimental findings. The implications of these results for physical models of inactivation are discussed.     

Permeability of the squid giant axon to organic cations and small nonelectrolytes

Summary The permeability of the Na channel of squid giant axon to organic cations and small nonelectrolytes was studied. The compounds tested were guanidinium, formamidinium, and¹⁴C-labeled urea, formamide, thiourea, and acetone. Permeability was calculated from measurements of reversal potential and influx on internally perfused, voltage clamped squid axons. The project had two objectives: (1) to determine whether different methods of measuring the permeability of organic cations yield similar values and (2) to see whether neutral analogs of the organic cations can permeate the Na channel. Our results show that the permeability ratio of sodium to a test ion depends upon the ionic composition of the solution used. This finding is consistent with the view put forward previously that the Na channel can contain more than one ion at a time. In addition, we found that the uncharged analogs of permeant cations are not measurably permeant through the Na channel, but instead probably pass through the lipid bilayer.     

Protein phosphorylation in plant mitochondria

Protein kinase activity was detected in osmotically lysed mitochondria isolated from etiolated seedlings of corn, pea, soybean, and wheat, as well as from potato tubers. Ther kinase(s) phosphorylated both endogenous polypeptides and exogenous, nonmitochondrial proteins when supplied with ATP and Mg²⁺. Eight to fifteen endogenous mitochondrial polypeptides were phosphorylated. The major mitochondrial polypeptide labeled in all species migrated during denaturing electrophoresis with an apparent monomeric molecular weight of 47,000. Incorporation of phosphate into endogenous proteins appeared to be biphasic, being most rapid during the first 1 to 2 minutes but slower thereafter. The kinase activity was greatest at neutral and alkaline pH values and utilized ATP with a K_m of approximately 200 micromolar. The kinase was markedly inhibited by CaCl₂ but was essentially unaffected by NaF, calmodulin, oligomycin, or cAMP. These data suggest that plant mitochondrial protein phosphorylation may be similar to protein phosphorylation in animal mitochondria.     

Fatty-acid composition of lipids and physicochemical characteristics of membrane fractions and the membrane of endoplasmic reticulum of thymus and Pliss' lymphosarcoma

The paper is concerned with composition of neutral lipids and phospholipids, the regularity of lipid bilayer and structural reorganization of plasma membranes, and membranes of smooth and rough cell reticulum of thymus and Pliss lymphosarcoma are studied at linear and stationary growth phase. No qualitative differences are found in the fatty-acid composition of lipid membranes in normal and tumour cells. In plasma membranes of phospholipids and in membranes of smooth reticulum of tumour cells the unsaturated lipid component increases in the process of growth, the cholesterin/phospholipids ratio decreases, fluidity of the lipid bilayer diminishes and structural heterogeneity of these membranes rises while in membranes of rough reticulum the saturation of lipids increases, but the cholesterin/phospholipids ratio does not change. The temperatures of structural reorganization also does not change, which evidences for a less structural lability of membranes of rough reticulum as compared with other membranes.     

Roles of Long-range Electrostatic Domain Interactions and K+ in Phosphoenzyme Transition of Ca2+-ATPase

Sarcoplasmic reticulum Ca²⁺-ATPase couples the motions and rearrangements of three cytoplasmic domains (A, P, and N) with Ca²⁺ transport. We explored the role of electrostatic force in the domain dynamics in a rate-limiting phosphoenzyme (EP) transition by a systematic approach combining electrostatic screening with salts, computer analysis of electric fields in crystal structures, and mutations. Low KCl concentration activated and increasing salt above 0.1 m inhibited the EP transition. A plot of the logarithm of the transition rate versus the square of the mean activity coefficient of the protein gave a linear relationship allowing division of the activation energy into an electrostatic component and a non-electrostatic component in which the screenable electrostatic forces are shielded by salt. Results show that the structural change in the transition is sterically restricted, but that strong electrostatic forces, when K⁺ is specifically bound at the P domain, come into play to accelerate the reaction. Electric field analysis revealed long-range electrostatic interactions between the N and P domains around their hinge. Mutations of the residues directly involved and other charged residues at the hinge disrupted in parallel the electric field and the structural transition. Favorable electrostatics evidently provides a low energy path for the critical N domain motion toward the P domain, overcoming steric restriction. The systematic approach employed here is, in general, a powerful tool for understanding the structural mechanisms of enzymes.     

Molinate biodegradation in soils: natural attenuation versus bioaugmentation

The aims of the present study were to assess the potential of natural attenuation or bioaugmentation to reduce soil molinate contamination in paddy field soils and the impact of these bioremediation strategies on the composition of soil indigenous microbiota. A molinate mineralizing culture (mixed culture DC) was used as inoculum in the bioaugmentation assays. Significantly higher removal of molinate was observed in bioaugmentation than in natural attenuation microcosms (63 and 39 %, respectively) after 42 days of incubation at 22 °C. In the bioaugmentation assays, the impact of Gulosibacter molinativorax ON4^T on molinate depletion was observed since the gene encoding the enzyme responsible for the initial molinate breakdown (harboured by that actinobacterium) was only detected in inoculated microcosms. Nevertheless, the exogenous mixed culture DC did not overgrow as the heterotrophic counts of the bioaugmentation microcosms were not significantly different from those of natural attenuation and controls. Moreover, the actinobacterial clone libraries generated from the bioaugmentation microcosms did not include any 16S rRNA gene sequences with significant similarity to that of G. molinativorax ON4^T. The multivariate analysis of the 16S rRNA DGGE patterns of the soil microcosm suggested that the activity of mixed culture DC did not affect the soil bacterial community structure since the DGGE patterns of the bioaugmentation microcosms clustered with those of natural attenuation and controls. Although both bioremediation approaches removed molinate without indigenous microbiota perturbation, the results suggested that bioaugmentation with mixed culture DC was more effective to treat soils contaminated with molinate.     

Can optimal resource allocation models explain why ectotherms grow larger in cold?

Basically all organisms can be classified as determinate growers if their growth stops or almost stops at maturation, or indeterminate growers if growth is still intense after maturation. Adult size for determinate growers is relatively well defined, whereas in indeterminate growers usually two measures are used: size at maturation and asymptotic size. The latter term is in fact not a direct measure but a parameter of a specific growth equation, most often Bertalanffy's growth curve. At a given food level, the growth rate in determinate growers depends under given food level on physiological constraints as well as on investments in repair and other mechanisms that improve future survival. The growth rate in indeterminate growers consists of two phases: juvenile and adult. The mechanisms determining the juvenile growth rate are similar to those in determinate growers, whereas allocation to reproduction (dependent on external mortality rate) seems to be the main factor limiting adult growth. Optimal resource allocation models can explain the temperature-size rule (stating that usually ectotherms grow slower in cold but attain larger size) if the exponents of functions describing the size-dependence of the resource acquisition and metabolic rates change with temperature or mortality increases with temperature. Emerging data support both assumptions. The results obtained with the aid of optimization models represent just a rule and not a law: it is possible to find the ranges of production parameters and mortality rates for which the temperature-size rule does not hold.     

Electroencephalographic Correlates of Mental Performance of Emotional Personal and Scenic Situations: I. Characteristics of Local Synchronization

An electroencephalogram (EEG) was recorded in 19 standard derivations in two groups of subjects (student actors and nonactors) during emotionally positive and negative mental recalls of personal experiences (test 1) or mental reproduction of scenic situations (test 2), as well as during mental count of time. Statistical comparison of EEG spectral power estimations in the frequency bands , , ₁, ₂, ₁, and ₂ showed that the induction of emotionally positive and emotionally negative states led to statistically significant changes in the EEG absolute power (local synchronization) simultaneously in many derivations and frequency bands. Analysis of all possible comparisons and changes in frequency bands showed that bilateral prefrontal and temporal cortical areas are most active during the internal induction of emotional states. Emotionally positive states were characterized, predominantly, by higher local EEG power than emotionally negative states. The EEG power changes accompanying internal induction of emotionally negative and positive states were of the same order in the case of reproduction of a presented situation (scenic material), whereas, in the case of recall of personal experience, emotionally negative states were accompanied by substantially weaker EEG spectral changes than emotionally positive states. The internal induction of emotional states on the basis of scenic material was accompanied by a pronounced increase in the power of the EEG range over the whole cortical surface. EEG reactions to induction of emotional states were generally stronger in actors than in nonactors. In case of emotional induction on the basis of scenic material, qualitative differences in the EEG reactions were also revealed between the groups. The findings are discussed in comparison with known data of investigations into regional cerebral blood flow during internal emotional induction and EEG studies of internal and external emotional induction.     

Deletions of any single residues in Glu40-Ser48 loop connecting a domain and the first transmembrane helix of sarcoplasmic reticulum Ca(2+)-ATPase result in almost complete inhibition of conformational transition and hydrolysis of phosphoenzyme intermediate

Possible roles of the Glu40-Ser48 loop connecting A domain and the first transmembrane helix (M1) in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) were explored by mutagenesis. Deletions of any single residues in this loop caused almost complete loss of Ca(2+)-ATPase activity, while their substitutions had no or only slight effects. Single deletions or substitutions in the adjacent N- and C-terminal regions of the loop (His32-Asn39 and Leu49-Ile54) had no or only slight effects except two specific substitutions of Asn39 found in SERCA2b in Darier's disease pedigrees. All the single deletion mutants for the Glu40-Ser48 loop and the specific Asn39 mutants formed phosphoenzyme intermediate (EP) from ATP, but their isomeric transition from ADP-sensitive EP (E1P) to ADP-insensitive EP (E2P) was almost completely or strongly inhibited. Hydrolysis of E2P formed from Pi was also dramatically slowed in these deletion mutants. On the other hand, the rates of the Ca(2+)-induced enzyme activation and subsequent E1P formation from ATP were not altered by the deletions and substitutions. The results indicate that the Glu40-Ser48 loop, with its appropriate length (but not with specific residues) and with its appropriate junction to A domain, is a critical element for the E1P to E2P transition and formation of the proper structure of E2P, therefore, most likely for the large rotational movement of A domain and resulting in its association with P and N domains. Results further suggest that the loop functions to coordinate this movement of A domain and the unique motion of M1 during the E1P to E2P transition.