The ATP depletion process of human erythrocytes studied by freeze fracturing

By the freeze-fracture method it is shown that metabolic depletion of erythrocytes affects three levels of cell organization: the microstructural (erythrocyte form), microstructural (micro-relief of erythrocyte surface) and ultrastructural (ultrastructural state of erythrocyte plasma membranes) ones. As it is established, the size of spikes on the echinocyte surface and that of membrane vesicles budding from a cell coincide with each other. The structural modification of the membrane precedes the stage of erythrocyte crenation. The following model of vesicle budding process is suggested: reduction of ATP level and dephosphorylation of actin-spectrin network--structural modification of the protein and lipid membrane phases with the formation of regions disconnected from the spectrin framework--protrusion of these anomalous regions in the form of spikes--budding of spikes as spherical vesicles.     

A new statistical method for identifying interconnections between neuronal network elements

A new method is proposed to analyse dependencies in point processes, which takes into account specific character of neuronal activity. Simulation modelling of neuronal network revealed that the estimated weight of connection depends monotonically on the value of the model synaptic strength. In contrast to the crosscorrelation, the method allows for nonlinear interconnections and does not require point processes to be stationary and samples to be large. Examples are presented of the method's application to neurophysiological data analysis.     

A modified method of isolation of "tight" 70S ribosomes from Escherichia coli highly active at different stages of the elongation cycle

The functional activity of the wide-spread "tight" 70S ribosomes is usually equal to 55-80%. We show here that the inactive fraction of this type of ribosomes is virtually blocked by residual endogenous RNA's. These RNA's are shown to be removable by introducing an additional stage in the isolation procedure including: 1. short heating (15 min, 37 degrees C) of "tight" 70S under dissociation conditions, i. e. in a buffer containing 3 mM MgCl2 and 200 mM NH4Cl; 2. washing off endogenous RNA's on a sucrose density gradient in the same buffer; 3. final selection of purified "tight" 70S on the sucrose gradient containing 5 mM MgCl2 and 50 mM NH4Cl. "Tight" 70S ribosomes isolated by such a procedure are 90-100% active with respect to tRNA binding (including the factor-dependent one), peptide bond synthesis and translocation.     

Interaction of tRNA with ribosomes

Definition of the site of tRNA-binding to ribosomes is suggested on the basis of a free energy of tRNA-ribosome interaction. From this point of view disagreements that have arisen in recent years concerning the numbers of tRNA binding sites on the ribosome, their distribution between subunits, the properties of the third site E in ribosomes and the compatibility of new experimental data with different models of elongation cycle are discussed. The observation of the third site in the ribosome (messenger independent and with a presumably exit function) is not a refutation but an extension of Watson's model of translating ribosome.     

Binding of the yeast phenylalanine tRNA with Escherichia coli ribosomes. Effect of the removal of a modified base from the 3'-end of the anticodon on codon-anticodon interaction

Phe-tRNAPhe+Y and N-acetyl-Phe-tRNAPhe+Y from yeast interact with prokaryotic 30S subunits and 70S ribosomes with slightly lower affinity than respective tRNA's of E. coli (decrease of standard free energy change of interaction less than 10%). The removal of Y-base from Phe-tRNAPhe+Y results in two orders of magnitude decrease of association constant of Phe-tRNAPh-Ye with P site of the 30S X poly(U) complex and one ordef of magnitude or more of that with A site. The same modification decreases the association constants of Phe-tRNAPhe-Y and N-acetyl-Phe-tRNAPhe-Y 60 and 15 times respectively with P site of the 70S X poly(U) complex. In the absence of poly(U) the affinity of N-acetyl-Phe-tRNAPhe-Y to P-site of 70S ribosome was 20-fold lower than that of native N-acetyl-Phe-tRNAPhe+Y. The sign of interaction enthalpy of N-acetyl-Phe-tRNAPhe+/-Y and Phe-tRNAPhe-Y changes below 6-7 degrees C exposing the hydrophobic part of P-site interactions. Similar removal of Y-base does not change both the enthalpy of interaction with P-site and magnesium concentration dependence.     

Extension of Watson's model for the elongation cycle of protein biosynthesis

The scheme for the elongation cycle of protein biosynthesis is proposed based on modern quantitative data on the interactions of mRNA and different functional forms of tRNA with 70S ribosomes and their 30S and 50S subunits. This scheme takes into account recently discovered third ribosomal (E) site with presumable exit function. The E site is introduced into 70S ribosome by its 50S subunit, the codon-anticodon interaction does not take place at the E site, and the affinity of tRNA for the E site is considerably lower than that for the P site. On the other hand, the P and A sites are located mainly on a 30S subunit, the codon-anticodon interactions being realized on both these sites. An mRNA molecule is placed exclusively on a 30S subunit where it makes U-turn. The proposed scheme does not contradict to any data but includes all main postulates of the initial Watson's model (J. D. Watson, Bull. Soc. Chim. Biol. 46, 1399 (1964), and is considered as a natural extension of the later according to modern experimental data.     

Big flies, small repeats: the "Thr-Gly" region of the period gene in Diptera

The region of the clock gene period (per) that encodes a repetitive tract of threonine-glycine (Thr-Gly) pairs has been compared between Dipteran species both within and outside the Drosophilidae. All the non- Drosophilidae sequences in this region are short and present a remarkably stable picture compared to the Drosophilidae, in which the region is much larger and extremely variable, both in size and composition. The accelerated evolution in the repetitive region of the Drosophilidae appears to be mainly due to an expansion of two ancestral repeats, one encoding a Thr-Gly dipeptide and the other a pentapeptide rich in serine, glycine, and asparagine or threonine. In some drosophilids the expansion involves a duplication of the pentapeptide sequence, but in Drosophila pseudoobscura both the dipeptide and the pentapeptide repeats are present in larger numbers. In the nondrosophilids, however, the pentapeptide sequence is represented by one copy and the dipeptide by two copies. These observations fulfill some of the predictions of recent theoretical models that have simulated the evolution of repetitive sequences.      

Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene

We have analyzed the conserved regions of the gene coding for the circumsporozoite protein (CSP) in 12 species of Plasmodium, the malaria parasite. The closest evolutionary relative of P. falciparum, the agent of malignant human malaria, is P. reichenowi, a chimpanzee parasite. This is consistent with the hypothesis that P. falciparum is an ancient human parasite, associated with humans since the divergence of the hominids from their closest hominoid relatives. Three other human Plasmodium species are each genetically indistinguishable from species parasitic to nonhuman primates; that is, for the DNA sequences included in our analysis, the differences between species are not greater than the differences between strains of the human species. The human P. malariae is indistinguishable from P. brasilianum, and P. vivax is indistinguishable from P. simium; P. brasilianum and P. simium are parasitic to New World monkeys. The human P. vivax-like is indistinguishable from P. simiovale, a parasite of Old World macaques. We conjecture that P. malariae, P. vivax, and P. vivax-like are evolutionarily recent human parasites, the first two at least acquired only within the last several thousand years, and perhaps within the last few hundred years, after the expansion of human populations in South America following the European colonizations. We estimate the rate of evolution of the conserved regions of the CSP gene as 2.46 x 10(-9) per site per year. The divergence between the P. falciparum and P. reichenowi lineages is accordingly dated 8.9 Myr ago. The divergence between the three lineages leading to the human parasites is very ancient, about 100 Myr old between P. malariae and P. vivax (and P. vivax-like) and about 165 Myr old between P. falciparum and the other two.      

Excitation of System I and System II in Photosynthesis as a Possible Control Mechanism of Glycolate Metabolism in the Blue-Green Alga Anacystis nidulans

Photosynthetic enhancement studies performed at 619 nm (excitation of Systems I and II) and at 446 nm (mainly excitation of System I) revealed an 18% photosynthetic enhancement simultaneously with a 31% reduction in glycolate excretion. This observation supports the hypothesis that some glycolate may be consumed in an oxidation process associated with System I when System II is poorly excited and the supply of electrons from the water splitting process of photosynthesis is low.