Brain mechanisms of quantity are similar in 5-year-old children and adults

Both 5-year-old children and adults determine the quantity of a number by the use of a similar parietal lobe mechanism. Event related potentials indicate that input from Arabic digits and from dot patterns reach areas involved in determining quantity about 200 ms after input. However, voluntary key presses indicating the relation of the input to the quantity five take almost three times as long in children. The ability to trace the networks of brain areas involved in the learning of school subjects should aid in the design and testing of educational methods.

Peptide design by artificial neural networks and computer-based evolutionary search

A technique for systematic peptide variation by a combination of rational and evolutionary approaches is presented. The design scheme consists of five consecutive steps: (i) identification of a “seed peptide” with a desired activity, (ii) generation of variants selected from a physicochemical space around the seed peptide, (iii) synthesis and testing of this biased library, (iv) modeling of a quantitative sequence-activity relationship by an artificial neural network, and (v) de novo design by a computer-based evolutionary search in sequence space using the trained neural network as the fitness function. This strategy was successfully applied to the identification of novel peptides that fully prevent the positive chronotropic effect of anti-β1-adrenoreceptor autoantibodies from the serum of patients with dilated cardiomyopathy. The seed peptide, comprising 10 residues, was derived by epitope mapping from an extracellular loop of human β1-adrenoreceptor. A set of 90 peptides was synthesized and tested to provide training data for neural network development. De novo design revealed peptides with desired activities that do not match the seed peptide sequence. These results demonstrate that computer-based evolutionary searches can generate novel peptides with substantial biological activity.

Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class I presentation pathway

Mice immunized with heat shock proteins (hsps) isolated from mouse tumor cells (donor cells) produce CD8 cytotoxic T lymphocytes (CTL) that recognize donor cell peptides in association with the major histocompatibility complex (MHC) class I proteins of the responding mouse. The CTL are induced apparently because peptides noncovalently associated with the isolated hsp molecules can enter the MHC class I antigen processing pathway of professional antigen-presenting cells. Using a recombinant heat shock fusion protein with a large fragment of ovalbumin covalently linked to mycobacterial hsp70, we show here that when the soluble fusion protein was injected without adjuvant into H-2b mice, CTL were produced that recognized an ovalbumin-derived peptide, SIINFEKL, in association with Kb. The peptide is known to arise from natural processing of ovalbumin in H-2b mouse cells, and CTL from the ovalbumin-hsp70-immunized mice and a highly effective CTL clone (4G3) raised against ovalbumin-expressing EL4 tumor cells (EG7-OVA) were equally effective in terms of the concentration of SIINFEKL required for half-maximal lysis in a CTL assay. The mice were also protected against lethal challenge with ovalbumin-expressing melanoma tumor cells. Because large protein fragments or whole proteins serving as fusion partners can be cleaved into short peptides in the MHC class I processing pathway, hsp fusion proteins of the type described here are promising candidates for vaccines aimed at eliciting CD8 CTL in populations of MHC-disparate individuals.

Interaction of a transcriptional repressor with the RNA polymerase II holoenzyme plays a crucial role in repression

The yeast transcriptional repressor Tup1, tethered to DNA, represses to strikingly different degrees transcription elicited by members of two classes of activators. Repression in both cases is virtually eliminated by mutation of either member of the cyclin-kinase pair Srb10/11. In contrast, telomeric chromatin affects both classes of activators equally, and in neither case is that repression affected by mutation of Srb10/11. In vitro, Tup1 interacts with RNA polymerase II holoenzyme bearing Srb10 as well as with the separated Srb10. These and other findings indicate that at least one aspect of Tup1's action involves interaction with the RNA polymerase II holoenzyme.

Remodeling of Yeast Genome Expression in Response to Environmental Changes

We used genome-wide expression analysis to explore how gene expression in Saccharomyces cerevisiae is remodeled in response to various changes in extracellular environment, including changes in temperature, oxidation, nutrients, pH, and osmolarity. The results demonstrate that more than half of the genome is involved in various responses to environmental change and identify the global set of genes induced and repressed by each condition. These data implicate a substantial number of previously uncharacterized genes in these responses and reveal a signature common to environmental responses that involves ∼10% of yeast genes. The results of expression analysis with MSN2/MSN4 mutants support the model that the Msn2/Msn4 activators induce the common response to environmental change. These results provide a global description of the transcriptional response to environmental change and extend our understanding of the role of activators in effecting this response.

Induction of a Carbon-Starvation-Related Proteolysis in Whole Maize Plants Submitted to Light/Dark Cycles and to Extended Darkness1

Three-week-old maize (Zea mays L.) plants were submitted to light/dark cycles and to prolonged darkness to investigate the occurrence of sugar-limitation effects in different parts of the whole plant. Soluble sugars fluctuated with light/dark cycles and dropped sharply during extended darkness. Significant decreases in protein level were observed after prolonged darkness in mature roots, root tips, and young leaves. Glutamine and asparagine (Asn) changed in opposite ways, with Asn increasing in the dark. After prolonged darkness the increase in Asn accounted for most of the nitrogen released by protein breakdown. Using polyclonal antibodies against a vacuolar root protease previously described (F. James, R. Brouquisse, C. Suire, A. Pradet, P. Raymond [1996] Biochem J 320: 283–292) or the 20S proteasome, we showed that the increase in proteolytic activities was related to an enrichment of roots in the vacuolar protease, with no change in the amount of 20S proteasome in either roots or leaves. Our results show that no significant net proteolysis is induced in any part of the plant during normal light/dark cycles, although changes in metabolism and growth appear soon after the beginning of the dark period, and starvation-related proteolysis probably appears in prolonged darkness earlier in sink than in mature tissues.

Transit Peptide Mutations That Impair in Vitro and in Vivo Chloroplast Protein Import Do Not Affect Accumulation of the γ-Subunit of Chloroplast ATPase1

We have begun to take a genetic approach to study chloroplast protein import in Chlamydomonas reinhardtii by creating deletions in the transit peptide of the γ-subunit of chloroplast ATPase-coupling factor 1 (CF1-γ, encoded by AtpC) and testing their effects in vivo by transforming the altered genes into an atpC mutant, and in vitro by importing mutant precursors into isolated C. reinhardtii chloroplasts. Deletions that removed 20 or 23 amino acid residues from the center of the transit peptide reduced in vitro import to an undetectable level but did not affect CF1-γ accumulation in vivo. The CF1-γ transit peptide does have an in vivo stroma-targeting function, since chimeric genes in which the stroma-targeting domain of the plastocyanin transit peptide was replaced by the AtpC transit peptide-coding region allowed plastocyanin to accumulate in vivo. To determine whether the transit peptide deletions were impaired in in vivo stroma targeting, mutant and wild-type AtpC transit peptide-coding regions were fused to the bacterial ble gene, which confers bleomycin resistance. Although 25% of the wild-type fusion protein was associated with chloroplasts, proteins with transit peptide deletions remained almost entirely cytosolic. These results suggest that even severely impaired in vivo chloroplast protein import probably does not limit the accumulation of CF1-γ.

In vivo regulation of muscle glycogen synthase and the control of glycogen synthesis.

The activity of glycogen synthase (GSase; EC 2.4.1.11) is regulated by covalent phosphorylation. Because of this regulation, GSase has generally been considered to control the rate of glycogen synthesis. This hypothesis is examined in light of recent in vivo NMR experiments on rat and human muscle and is found to be quantitatively inconsistent with the data under conditions of glycogen synthesis. Our first experiments showed that muscle glycogen synthesis was slower in non-insulin-dependent diabetics compared to normals and that their defect was in the glucose transporter/hexokinase (GT/HK) part of the pathway. From these and other in vivo NMR results a quantitative model is proposed in which the GT/HK steps control the rate of glycogen synthesis in normal humans and rat muscle. The flux through GSase is regulated to match the proximal steps by "feed forward" to glucose 6-phosphate, which is a positive allosteric effector of all forms of GSase. Recent in vivo NMR experiments specifically designed to test the model are analyzed by metabolic control theory and it is shown quantitatively that the GT/HK step controls the rate of glycogen synthesis. Preliminary evidence favors the transporter step. Several conclusions are significant: (i) glucose transport/hexokinase controls the glycogen synthesis flux; (ii) the role of covalent phosphorylation of GSase is to adapt the activity of the enzyme to the flux and to control the metabolite levels not the flux; (iii) the quantitative data needed for inferring and testing the present model of flux control depended upon advances of in vivo NMR methods that accurately measured the concentration of glucose 6-phosphate and the rate of glycogen synthesis.

Jasmonic acid distribution and action in plants: regulation during development and response to biotic and abiotic stress.

Jasmonic acid (JA) is a naturally occurring growth regulator found in higher plants. Several physiological roles have been described for this compound (or a related compound, methyl jasmonate) during plant development and in response to biotic and abiotic stress. To accurately determine JA levels in plant tissue, we have synthesized JA containing 13C for use as an internal standard with an isotopic composition of [225]:[224] 0.98:0.02 compared with [225]:[224] 0.15:0.85 for natural material. GC analysis (flame ionization detection and MS) indicate that the internal standard is composed of 92% 2-(+/-)-[13C]JA and 8% 2-(+/-)-7-iso-[13C]JA. In soybean plants, JA levels were highest in young leaves, flowers, and fruit (highest in the pericarp). In soybean seeds and seedlings, JA levels were highest in the youngest organs including the hypocotyl hook, plumule, and 12-h axis. In soybean leaves that had been dehydrated to cause a 15% decrease in fresh weight, JA levels increased approximately 5-fold within 2 h and declined to approximately control levels by 4 h. In contrast, a lag time of 1-2 h occurred before abscisic acid accumulation reached a maximum. These results will be discussed in the context of multiple pathways for JA biosynthesis and the role of JA in plant development and responses to environmental signals.