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.     

Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.      

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.     

Growth of Phytomonas serpens in a Defined Medium; Nutritional Requirements

ABSTRACT. Three strains of Phytomonas serpens two from tomatoes, Lycopersicon esculentum one from the insect Phtia picta (Hemiptera, Coreidae), were cultivated in a chemically defined medium developed from a defined medium for cultivating insect flagellates. Besides organic growth factors required by other insect trypanosomatids this flagellate requires, serine and inositol. Glutamine stimulates growth, and, surprisingly, does not require heme.     

Membrane Skeletal Proteins and Their Integral Membrane Protein Anchors are Targets for Tyrosine and Threonine Kinases in Euglena

ABSTRACT. Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [³²P]-orthophosphate or γ-[³²P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.     

Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi

Borrelia burgdorferi is a spirochete pathogen transmitted among warm- blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.