Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

A Modular Mind? A Test Using Individual Data from Seven Primate Species

It has long been debated whether the mind consists of specialized and independently evolving modules, or whether and to what extent a general factor accounts for the variance in performance across different cognitive domains. In this study, we used a hierarchical Bayesian model to re-analyse individual level data collected on seven primate species (chimpanzees, bonobos, orangutans, gorillas, spider monkeys, brown capuchin monkeys and long-tailed macaques) across 17 tasks within four domains (inhibition, memory, transposition and support). Our modelling approach evidenced the existence of both a domain-specific factor and a species factor, each accounting for the same amount (17%) of the observed variance. In contrast, inter-individual differences played a minimal role. These results support the hypothesis that the mind of primates is (at least partially) modular, with domain-specific cognitive skills undergoing different evolutionary pressures in different species in response to specific ecological and social demands.     

Species of the genus Quercus in Italy: Characterization by means of leaf surface observation

Abstract

We propose a study of the main species belonging to the genus Quercus in Italy, characterized and identified by means of leaf surface observation, with special attention devoted to waxes, trichomes and stomata. Comparing our results with the classification proposed by SCHWARZ (1984), we find that species belonging to Schwarz's subgenus Quercus are recognizable because their waxes are structured in vertical scales; the two other subgenera (Sclerophyllodrys and Cerris) present smooth wax structures, their distinctive feature being the shape of the stomatal rima, which is roundish in Sclerophyllodrys and elliptical in Cerris. The study characterizes Quercus pubescens Willd. and Quercus petraea Liebl. by analyzing some morphometric traits; but the authors feel that further research is needed on these critical taxonomic entities. Lastly, the study examines forms of was degeneration correlated to the phenomenon known as oak decline.     

Structuring of H1 histone. Evidence of high-affinity binding sites for phosphate ions

Circular dichroism studies show that low concentrations of phosphate ions induce folding of the H1 histones. Sulfate and perchlorate anions have effects similar to phosphate indicating the presence on H1 histones of binding sites with high affinity for ions with tetrahedral geometry. In fact, the structuring efficiency of different ions, as determined by the midpoint value of the effect/concentration curve, is 0.05 M for NaCl, 0.005 M for NaClO4, 0.001 M for sodium phosphate and 0.0003 M for sodium sulfate on H1 histone from Chaetopterus variopedatus sperm chromatin. Phosphate shows similar folding efficiency also on calf thymus and on sea-urchin sperm H1 histones. The effect of phosphate ions on the H1 molecule is observed also by differential absorption spectroscopy in the region of absorption of amino acid side-chains. Binding studies by gel filtration chromatography on Sephadex columns show that phosphate binding occurs in the presence of structuring concentrations of sodium chloride. About 9 ATP molecules bind to H1 histones derived from non-active cell chromatins while only 3.5 ATP molecules bind to H1 derived from active somatic chromatins. The fluorescence of the tyrosine residues of Chaetopterus sperm H1 is enhanced by chloride ions and heavily quenched by phosphate ions in correlation with structuring of the molecule, demonstrating direct interactions between tyrosine residues and phosphate ions. The defined and limited number of phosphate groups bound per histone molecule, the high affinity of the interaction and the effect on the structure of the histone suggest the participation of phosphate groups in the binding of H1 histones to DNA.     

The complete amino acid sequences of cytosolic and mitochondrial aspartate aminotransferases from horse heart,and inferences on evolution of the isoenzymes

Summary We report here the complete amino acid sequences of the cytosolic and mitochondrial aspartate aminotransferases from horse heart. The two sequences can be aligned so that 48.1% of the amino acid residues are identical. The sequences have been compared with those of the cytosolic isoenzymes from pig and chicken, the mitochondrial isoenzymes from pig, chicken, rat, and human, and the enzyme fromEscherichia coli. The results suggest that the mammalian cytosolic and mitochondrial isoenzymes have evolved at equal and constant rates whereas the isoenzymes from chicken may have evolved somewhat more slowly. Based on the rate of evolution of the mammalian isoenzymes, the geneduplication event that gave rise to cytosolic and mitochondrial aspartate aminotransferases is estimated to have occurred at least 10⁹ years ago. The cytosolic and mitochondrial isoenzymes are equally related to the enzyme fromE. coli; the prokaryotic and eukaryotic enzymes diverged from one another at least 1.3×10⁹ years ago.     

Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease

The molecular basis of the high reactivity toward reducing agents of intersubunit disulfides at positions 31 and 32 of dimeric bovine seminal ribonuclease was investigated by studying in the monomeric enzyme the fast reaction kinetics with disulfides of the adjacent cysteine-31 and -32, exposed by selective reduction of the intersubunit disulfides. Negatively charged and neutral disulfide reagents were used for measuring the thiol reaction rates at neutral pH. The kinetics studied as a function of pH permitted us to define pK values for the thiols of interest and indicated the possibility of determining pK values of SH groups in proteins indirectly by measuring the kinetics of reactivity of the SH groups with a disulfide reagent. The results were compared with those obtained under identical conditions with synthetic thiol peptides and model compounds. The data indicate that the superreactivity of intersubunit disulfides of seminal ribonuclease is matched by the high reactivity at neutral pH of adjacent cysteine residues 31 and 32, as compared to all small thiol compounds tested. The synthetic hexapeptide segment of seminal ribonuclease Ac-Met-Cys-Cys-Arg-Lys-Met-OH, which includes the two cysteine residues of interest, was even more reactive. These data, and the other results reported in this paper, led to the conclusion that the superreactivity at neutral pH of cysteine residues at positions 31 and 32 of bovine seminal ribonuclease is primarily dependent on the nearby presence of positively charged groups, particularly the epsilon-NH2 of lysine-34, and is influenced by the adjacency of the two thiols and by the protein tertiary structure.     

Are seals frequently infected with avian influenza viruses?

Influenza A virus isolates of the H4N5 subtype (which has previously been detected only in birds) were recovered from harbor seals dying of viral pneumonia on the New England coast from June 1982 through March 1983. When these isolates were compared with other mammalian and avian viruses in serological assays and RNA-RNA competitive hybridization, it was found that the seal viruses were most closely related antigenically and genetically to recent avian virus strains and were readily distinguishable from mammalian viruses, including H7N7 isolates recovered from seals in 1980. Unlike any previous isolates from mammals, these recent seal viruses replicate in the intestinal tracts of ducks, a characteristic of avian viruses. The association of avian viruses with influenza outbreaks in seals suggests that transmission of avian viruses to seals is occurring in nature. Potentially, this may be an example of the adaptation of avian viruses to mammals, which would represent an intermediate step in the evolution of new mammalian strains.     

High sensitivity method for fluorofore detection in gradient polyacrylamide slab gels through excitation by laser light: application to glycoproteins stained with concanavalin A-fluorescein isothiocyanate

An easy-to-assemble apparatus for the laser-light excitation of fluorofores in polyacrylamide gels is described. The assemblage is made up of a continuous-wave ion-argon laser with adjustable power output, a beam diffuser, appropriate filters to block excitation light, and a photographic camera. With this setup a minimum 20-fold increase of sensitivity was obtained for fluorofore detection in polyacrylamide gels as compared to the more conventional uv-light excitation using a commercial preparation of Con A-FITC (concanavalin A-fluorescein isothiocyanate) as reference molecule in the gel. The same apparatus, used to analyze the Con A-positive glycoproteins contained in serum Cohn fraction IV separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed a number of fluorescent components in a wide range of relative intensities while uv-light excitation showed none. Acrylamide concentration in the gel is critical, since a working limit of between 10 and 12% has been found, above which the diffusion of Con A-FITC in the gel, necessary to label glycoprotein bands, is hampered. The system described here also permits the optimization of detection of minor components not otherwise observable by conventional light excitation, because light power, angle of incidence, and beam divergence can be adapted to analyze specific areas of the sample gel.