Deletion analysis of the SUP35 gene of the yeast Saccharomyces cerevisiae reveals two non-overlapping functional regions in the encoded protein

SUP35is an omnipotent suppressor gene of Saccharomyces cerevisiae coding for a protein consisting of a C-terminal part similar to the elongation factor EF-1α and a unique N-terminal sequence of 253 amino acids. Twelve truncated versions of the SUP35 gene were generated by the deletion of fragments internal to the coding sequence. Functional studies of these deletion mutants showed that: (i) only the EF-1α-like C-terminal part of the Sup35 protein is essential for the cell viability; (ii) overexpression of either the N-terminal part of the Sup35 protein or the full-length Sup35 protein decreases translational fidelity, resulting in omnipotent suppression and reduced growth of [psi⁺] strains; (iii) expression of the C-terminal part of the Sup35 protein generates an antisuppressor phenotype; and (iv) both the N- or C-terminal segments of the Sup35 protein can bind to 80S ribosomes. Thus, the data obtained define two domains within the Sup35 protein which are responsible for different functions.     

Identification of differentially expressed genes by restriction endonuclease-based gene expression fingerprinting.

A novel method for identification of differentially expressed genes has been developed. It is based on the consecutive restriction digestions of 3' terminal cDNA fragments to produce a fingerprint of gene expression. cDNA molecules are synthesized using a biotinylated oligo(dT) primer, digested with a frequently cutting restriction endonuclease and the 3'-terminal restriction fragments are isolated using streptavidin microbeads. After amplification by PCR, cDNA fragments are immobilized again on streptavidin beads, radiolabeled and treated sequentially with a set of restriction endonucleases. The products of individual enzymatic reactions from two or more different RNA populations are resolved by polyacrylamide gel electrophoresis and compared to reveal differentially expressed genes. This strategy enabled us to identify and clone the fragments of five genes expressed differentially in murine thymus and spleen. One of the genes was found to encode terminal deoxynucleotidyl transferase; others are apparently previously unknown genes.     

Conditioned reflex transformation of unconditioned flexion of the limbs of the dog into extension

In response to electro-skin stimulation of the left forelimb eliciting its flexion,--an escape instrumental reflex was elaborated in five dogs in the form of extension of the stimulated limb (pressure on the support), i.e. a reaction antagonistic to the inborn one. The reaction was elaborated due to conditioned transformation of the unconditioned flexion: the flexion became gradually inhibited and the new form of reaction, previously not inherent in the animal increased and became stable. Such transformation of one reaction into another is connected with the reorganization of inborn coordinations.     

Direct current electric field in some teleost species: Effect of medium salinity

Summary A direct current electric field up to 3 mV/ cm was recorded in 33 sea water around the fishMyoxocephalus brandti, Hexogrammos octogrammos, Enophrys diceraus, Pleuronectes stellatus, Bathimaste r derjugini, Sebastes scorpaeniformis. The body surface potentials were positive in relation to the external and internal media; they attained 10 mV and slowly varied near the mean value at every point. The potentials at the surface points of individual skin sections adjoining the oral and branchial cavities, the anal orifice and peripheral fin sections were normally characterized by polarities opposite to those of body surface potentials (in sea water they were negative in relation to the external medium).When placed in sea water during their fresh water cycle, the salmonOncorhynchus keta and the fresh water fishSalvelinus alpinus andMisgurnus fossilis had no d.c. field.In fresh water containing less than 0.03 salt, a d.c. field up to 25 mV/cm was recorded around all the above mentioned species. The potentials had an opposite polarity to that recorded in sea water.The distribution of potentials over the fish surface depends on the species. The potentials at some points of the body surfaces were found to vary when other fish or metal objects were placed in the aquarium.The parameters of the direct current electric field generated by a whole fish and by isolated skin pieces were identical and varied by the same law with changed medium salinity. Thus it may be assumed that the d.c. electric field around the fish is produced by active electrogenic ion transport mechanisms localized in the skin.     

Purification and characterization of recombinant Pneumocystis carinii thymidylate synthase

Gatalytically active Pneumocystis carinii thymidylate synthase is expressed to the extent of about 4% of the soluble protein in Escherichia coli χ2913 harboring plasmid pUETS-1.8 (U. Edman, J. C. Edman, B. Lundgren, and D. V. Santi, Proc. Natl. Acad. Sci. USA 86, 6503–6507, 1989). Ion-exchange, affinity, hydrophobic, and reactive dye agarose chromatography steps were explored to devise a large-scale purification protocol for P. carinii thymidylate synthase. Sequential DE52, Q-Sepharose, phenyl-Sepharose, and Cibacron Blue F3GA chromatography yielded enzyme that was homogeneous by SDS-PAGE in a yield of over 50%. The sequence of the first 10 amino acid residues of the purified protein was in accord with that predicted from the DNA sequence. Isoelectric focusing gave a pI of 6.2. Kinetic analysis of the purified enzyme revealed that the the K_m values were 4.7 ± 1.3 μM for dUMP and 15.7 ± 4.3 μM for 5,10-methylenetetrahydrofolate, similar to those of many other thymidylate synthases; the κ_cat of the most active preparation was 0.8 s⁻¹. The enzyme is stable for at least 2 months when stored at −80°C in the presence of 40% glycerol, Tris-HCl, and thiol.     

Changes in the activity of the supraoptic nuclei of the hypothalamus and distal portions of the hypothalamo-hypophyseal neurosecretory system of guinea pigs under the influence of high environmental temperature]

The keeping of guinea pigs in a thermochamber at 37 degrees C for 3 days resulted in increased releasing of neurohormones of the posterior lobe of the hypophysis. By the end of the 3d day there was no substantial difference between the average logarhythm of the volume of cell nuclei of the supraoptic nucleus of experimental animals and that of intact animals. Injection of ACTH after a preliminary three-day presence of the animal in the thermochamber caused a pronounced inhibition of the hypothalamo-hypophyseal neurosecretory system (HHNS). The HHNS of animals subjected to 3-day-long continuous exposure to high temperature, in contrast to the HHNS of intact animals is not capable to increase its activity in responses to stress (immobilization).     

Magnesium supplementation and iron status among female students: The intervention study

BackgroundLiterature data indicate the benefit of magnesium (Mg) supplementation. The aim of this study was to examine the effect of short-term Mg supplementation on iron status in healthy female participants.MethodsOne hundred healthy female students of the University of Belgrade - Faculty of Pharmacy participated the study during eleven intervention days. Students ingested Mg preparations with the same dose of the active substance. The analysis included the measurement of serum iron, unsaturated iron binding capacity (UIBC), total iron binding capacity (TIBC), total Mg (tMg), ionized Mg (iMg), complete blood count, met-, carboxyand oxy-haemoglobin (metHgb, COHgb, O2Hgb). Transferrin concentrations and percentage of transferrin saturation (SAT) were calculated manually. The association among the analyzed biochemical parameters was examined using polynomial regression. A principal component analysis (PCA) was used for the evaluation of interdependence between the analyzed parameters.ResultsA statistically significant trend for change in O2Hgb (%) by tertiles of iMg concentrations was found (P = 0.029). Serum tMg reached significant positive correlation with the SAT at concentration levels greater than 0.9 mmol/L, after 11 days of intervention (R2=0.116). Ionized Mg in a concentration higher than 0.6 mmol/L is positively correlated with SAT and serum Fe (R2=0.214; 0.199, respectively). PCA revealed variability of 64.7% for two axes after 11 days.ConclusionsMg supplementation leads to an improvement in the certain iron status parameters even in individuals with optimal levels of these indices. However, caution should be exercised when supplementing Mg, and laboratory monitoring of the interaction is required.     

Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced approximately 70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca(2+)-regulated secretory events.     

Origins and evolution of eukaryotic RNA interference

Small interfering RNAs (siRNAs) and genome-encoded microRNAs (miRNAs) silence genes via complementary interactions with mRNAs. With thousands of miRNA genes identified and genome sequences of diverse eukaryotes available for comparison, the opportunity emerges for insights into the origin and evolution of RNA interference (RNAi). The miRNA repertoires of plants and animals appear to have evolved independently. However, conservation of the key proteins involved in RNAi suggests that the last common ancestor of modern eukaryotes possessed siRNA-based mechanisms. Prokaryotes have an RNAi-like defense system that is functionally analogous but not homologous to eukaryotic RNAi. The protein machinery of eukaryotic RNAi seems to have been pieced together from ancestral archaeal, bacterial and phage proteins that are involved in DNA repair and RNA processing.