Molecular determinants of voltage-dependent slow inactivation of the Ca2+ channel.

Ba(2+) current through the L-type Ca(2+) channel inactivates essentially by voltage-dependent mechanisms with fast and slow kinetics. Here we found that slow inactivation is mediated by an annular determinant composed of hydrophobic amino acids located near the cytoplasmic ends of transmembrane segments S6 of each repeat of the alpha(1C) subunit. We have determined the molecular requirements that completely obstruct slow inactivation. Critical interventions include simultaneous substitution of A752T in IIS6, V1165T in IIIS6, and I1475T in IVS6, each preventing in additive manner a considerable fraction of Ba(2+) current from inactivation. In addition, it requires the S405I mutation in segment IS6. The fractional inhibition of slow inactivation in tested mutants caused an acceleration of fast inactivation, suggesting that fast and slow inactivation mechanisms are linked. The channel lacking slow inactivation showed approximately 45% of the sustained Ba(2+) or Ca(2+) current with no indication of decay. The remaining fraction of the current was inactivated with a single-exponential decay (pi(f) approximately 10 ms), completely recovered from inactivation within 100 ms and did not exhibit Ca(2+)-dependent inactivation properties. No voltage-dependent characteristics were significantly changed, consistent with the C-type inactivation model suggesting constriction of the pore as the main mechanism possibly targeted by Ca(2+) sensors of inactivation.     

Plasmid content in Yersinia pestis strains of different origin

Plasmid content in 242 Yersinia pestis strains from various natural plague foci of the U.S.S.R. and other countries was studied. Of these strains, 172 (71%) were shown to carry three plasmids described previously of about 6, 45-50 and 60 MDa, respectively. Twenty strains (8%) from different foci harboured additional cryptic plasmids, most often of about 20 mDa in size. Plasmid pPst displayed considerable constancy of its molecular mass. On the contrary, size variations of pCad (45-49 MDa) and, especially, pFra (60-190 MDa) were found. Molecular mass of these plasmids correlated with the host strain origin.     

Application of three-dimensional molecular hydrophobicity potential to the analysis of spatial organization of membrane domains in proteins: I. Hydrophobic properties of transmembrane segments of Na+, K+-ATPase

A new computer-aided molecular modeling approach based on the concept of three-dimensional (3D) molecular hydrophobicity potential has been developed to calculate the spatial organization of intramembrane domains in proteins. The method has been tested by calculating the arrangement of membrane-spanning segments in the photoreaction center ofRhodopseudomonas viridis and comparing the results obtained with those derived from the X-ray data. We have applied this computational procedure to the analysis of interhelical packing in membrane moiety of Na⁺, K⁺-ATPase. The work consists of three parts. In Part I, 3D distributions of electrostatic and molecular hydrophobicity potentials on the surfaces of transmembrane helical peptides were computed and visualized. The hydrophobic and electrostatic properties of helices are discussed from the point of view of their possible arrangement within the protein molecule. Interlocation of helical segments connected with short extramembrane loops found by means of optimization of their hydrophobic/hydrophilic contacts is considered in Part II. The most probable 3D model of packing of helical peptides in the membrane domain of Na⁺, K⁺-ATPase is discussed in the final part of the work.     

Increased number of nucleoli in the salivary gland cells of Drosophila melanogaster under conditions of rDNA dose compensation

A considerable increase in the number of nucleoli non-associted with the nucleolar organizer (NO) was shown in the salivary gland cells of Drosophila melanogaster mutants, heterozygous for a deficiency of NO. The frequency of formation of additional nucleoli increased with the raising of the chromosome polyteny level. By means of in situ hybridization we showed that in the mutant and the wildtype polytene cells the ribosomal DNA (rDNA) of these unlawful nucleoli included ribosomal gene repeats (18S+28S) with two types of insertions: ivs-I and ivs-II Such additional nucleoli can be attached to varying sites of the polytene chromosomes containing type I insertion sequences.     

Evaluation of quantitative parameters of the interaction of antibody-bearing liposomes with target antigens

The model system for the analysis of targeted liposomes is proposed--the layer of protein antigen adsorbed on polystyrene wells. Antibodies were treated with palmitoyl chloride and liposomes were produced by the cholate dialysis method in the presence of the modified protein (7 X 10(-4) mol protein/mol lipid). Affinity of antibody-bearing liposomes to the antigen on the surface of Multiwell plates was studied, and apparent dissociation constant value was estimated: KD was in the range 1.5 to 5 X 10(-9) M liposomes. Sequential transfers of liposomes in antigen-coated plates revealed that the high-affinity fraction of liposomes is adsorbed first. The bound fraction has 1.7-times-higher protein content. For effective in vivo targeting it would be necessary to have high-affinity liposomes and a high concentration of the target antigen.     

Serotonin-like immunoreactivity in the optic lobes of three insect species

The cellular localization of 5-HT in the optic lobes of three insect species was assayed with the use of antibodies raised against 5-HT. In Schistocerca, Periplaneta, and Calliphora all neuropil regions of the optic lobe, the lamina, medulla and lobula, contain 5-HT-immunoreactive varicose fibres in different patterns, like columns and layers. Such fibres also connect the lobula to neuropil in the lateral protocerebrum. In Calliphora also 5-HT-positive fibres of the medulla and lobula plate have projections to the lateral protocerebrum, whereas the origin of the lamina fibres is not certain. In all species the processes displaying 5-HT-like immunoreactivity appear to be derived from a relatively small number of cell bodies, each neuron thus having processes over a large volume of the neuropil of the optic lobe in different layers.     

Volume-dependent regulation of ion transport and membrane phosphorylation in human and rat erythrocytes

Summary Osmotic swelling of human and rat erythrocytes does not induce regulatory volume decrease. Regulatory volume increase was observed in shrunken erythrocytes of rats only. This reaction was blocked by the inhibitors of Na⁺/H⁺ exchange. Cytoplasmic acidification in erythrocytes of both species increases the amiloride-inhibited component of²²Na influx by five- to eight-fold. Both the osmotic and isosmotic shrinkage of rat erythrocytes results in the 10- to 30-fold increase of amiloride-inhibited²²Na influx and a two-fold increase of furosemide-inhibited⁸⁶Rb influx. We failed to indicate any significant changes of these ion transport systems in shrunken human erythrocytes. The shrinking of quin 2-loaded human and rat erythrocytes results in the two- to threefold increase of the rate of⁴⁵Ca influx, which is completely blocked by amiloride. The dependence of volume-induced²²Na influx in rat erythrocytes and⁴⁵Ca influx in human erythrocytes on amiloride concentration does not differ. The rate of⁴⁵Ca influx in resealed ghosts was reduced by one order of magnitude when intravesicular potassium and sodium were replaced by choline. It is assumed that the erythrocyte shrinkage increases the rate of a nonselective Ca _o ²⁺ (Na _i ⁺ , K _i ⁺ ) exchange. Erythrocyte shrinking does not induce significant phosphorylation of membrane protein but increases the³²P incorporation in diphosphoinositides. The effect of shrinkage on the³²P labeling of phosphoinositides is diminished after addition of amiloride. It is assumed that volume-induced phosphoinositide response plays an essential role in the mechanism of the activation of transmembrane ion movements.     

Control of Photosystem II in spinach leaves by continuous light and by light pulses given in the dark

The light-induced induction of components of non-photochemical quenching of chlorophyll fluorescence which are distinguished by different rates of dark relaxation (qNf, rapidly relaxing and qNs, slowly relaxing or not relaxing at all in the presence brief saturating light pulses which interrupt darkness at low frequencies) was studied in leaves of spinach.After dark adaptation of the leaves, a fast relaxing component developed in low light only after a lag phase. Quenching increased towards a maximum with increasing photon flux density. This fast component of quenching was identified as energy-dependent quenching qE. It required formation of an appreciable transthylakoid pH and was insignificant when darkened spinach leaves received 1 s pulses of light every 30 s even though zeaxanthin was formed from violaxanthin under these conditions.Another quenching component termed qNs developed in low light without a lag phase. It was not dependent on a transthylakoid pH gradient, decayed exponentially with a long half time of relaxation and was about 20% of total quenching irrespective of light intensity. When darkened leaves were flashed at frequencies higher than 0.004 Hz with 1 s light pulses, this quenching also appeared. Its extent was very considerable, and it did not require formation of zeaxanthin. Relaxation was accelerated by far-red light, and this acceleration was abolished by NaF.We suggest that qNs is the result of a so-called state transition, in which LHC II moves after its phosphorylation from fluorescent PS II to nonfluorescent PS I. This state transition was capable of decreasing in darkened leaves the potential maximum quantum efficiency of electron flow through Photosystem II by about 20%.Abbreviations PFD photon flux density - PS photosystem