Mimicking a p53‐MDM2 interaction based on a stable immunoglobulin‐like domain scaffold

Antibodies recognize protein targets with great affinity and specificity. However, posttranslational modifications and the presence of intrinsic disulfide‐bonds pose difficulties for their industrial use. The immunoglobulin fold is one of the most ubiquitous folds in nature and it is found in many proteins besides antibodies. An example of a protein family with an immunoglobulin‐like fold is the Cysteine Protease Inhibitors (ICP) family I42 of the MEROPs database for protease and protease inhibitors. Members of this protein family are thermostable and do not present internal disulfide bonds. Crystal structures of several ICPs indicate that they resemble the Ig‐like domain of the human T cell co‐receptor CD8α As ICPs present 2 flexible recognition loops that vary accordingly to their targeted protease, we hypothesize that members of this protein family would be ideal to design peptide aptamers that mimic protein‐protein interactions. Herein, we use an ICP variant from Entamoeba histolytica (EhICP1) to mimic the interaction between p53 and MDM2. We found that a 13 amino‐acid peptide derived from p53 can be introduced in 2 variable loops (DE, FG) but not the third (BC). Chimeric EhICP1‐p53 form a stable complex with MDM2 at a micromolar range. Crystal structure of the EhICP1‐p53(FG)‐loop variant in complex with MDM2 reveals a swapping subdomain between 2 chimeric molecules, however, the p53 peptide interacts with MDM2 as in previous crystal structures. The structural details of the EhICP1‐p53(FG) interaction with MDM2 resemble the interaction between an antibody and MDM2.     

Body fluid homeostasis and cardiovascular adjustments during submaximal exercise: influence of chewing coca leaves

 The present study was undertaken to determine the haematological and cardiovascular status, at rest and during prolonged (1 h) submaximal exercise (approximately 70% of peak oxygen uptake) in a group (n = 12) of chronic coca users after chewing approximately 50 g of coca leaves. The results were compared to those obtained in a group (n = 12) of nonchewers. At rest, coca chewing was accompanied by a significant increase in heart rate [from 60 (SEM 4) TO 76 (SEM 3) beats · min⁻¹], in haematocrit [from 53.2 (SEM 1.2) to 55.6 (SEM 1.1)%] in haemoglobin concentration, and plasma noradrenaline concentration [from 2.8 (SEM 0.4) to 5.0 (SEM 0.5) μmol · l⁻¹]. It was calculated that coca chewing for 1 h resulted in a significant decrease in blood [−4.3 (SEM 2.2)%] and plasma [−8.7 (SEM 1.2)%] volume. During submaximal exercise, coca chewers displayed a significantly higher heart rate and mean arterial blood pressure. The exercise-induced haemoconcentration was blunted in coca chewers compared to nonchewers. It was concluded that the coca-induced fluid shift observed at rest in these coca chewers was not cumulative with that of exercise, and that the hypovolaemia induced by coca chewing at rest compromised circulatory adjustments during exercise. Accepted: 29 October 1996     

Rapid identification ofClostridium botulinum and botulinal toxin in food

Samples of green beans and mushrooms were inoculated with a toxigenic strain ofClostridium botulinum type A and incubated anaerobically at 37 °C. At various time intervals, the seeded food samples were tested for the presence of botulinal toxin andC. botulinum by an agar plating method and an enzyme-linked immunosorbent assay.C. botulinum type A that appeared as lipase-positive colonies on selective agar plates, and its elaborated toxin, were identified in all seeded food samples within 1 to 2 d. This procedure can be adapted for rapid screening of suspected food samples. This study was presented in part at the96th General Meeting of the American Society for Microbiology, New Orleans, Louisiana, May 19–23, 1996 (abstract no. P71). Part of the requirements for the MSc degree received by A. Rodriguez.     

Identification, Localization, and Expression of Two Novel Human Genes Similar to Deoxyribonuclease I

Two novel cDNAs, DNAS1L2 and DNAS1L3, are predicted to encode proteins of 299 and 305 amino acids with 56 and 46% residue identity (71 and 63% similarity), respectively, to deoxyribonuclease I (DNase I). DNAS1L2 is located on a 16p13.3 cosmid, while DNAS1L3 maps to 3p14.3–p21.1 by fluorescencein situhybridization and by PCR analysis of a radiation hybrid panel. Northern analysis revealed DNAS1L3 expression nearly exclusively in liver, while DNAS1L2 expression was detected in brain by RT-PCR. The previously defined DNL1L or DNAS1L1 is expressed highest in heart and skeletal muscle, while DNase I is expressed in the pancreas, parotid gland, and kidney. Thus, to date, four DNase I-like genes that show different tissue expression patterns are known. A comparison of DNAS1L1, DNAS1L2, and DNAS1L3 with the well-characterized DNase I suggests that the DNAS1L proteins are unlikely to be glycosylated or bind actin; however, catalytic and calcium- and DNA-binding residues are conserved, and potentially cleavable signal peptides are present among all these proteins. This analysis also identifies regions of high conservation among these proteins with no currently assigned function.     

The Expression of an Abscisic Acid-Responsive Glycine-Rich Protein Coincides with the Level of Seed Dormancy in Fagus sylvatica

By differential screening of a cDNA library constructed frompoly (A⁺) RNA of ABA-treated seeds of Fagus sylvatica L., wehave isolated an ABA-responsive clone that is present in dormantseeds and under conditions that maintain dormancy, but it tendsto disappear under conditions breaking seed dormancy. A searchof the sequence data bases showed that the clone codes for aGlycine-Rich Protein and has sequence similarity to RNA-bindingproteins. The clone, which exibits the characteristics of lea-genes,is up-regulated by ABA and down-regulated by GA3. Paclobutrazolabolishes the effect of GA³, which is restored upon additionof GA3. The possible relationship of this Glycine-Rich Proteinto seed dormancy in F. sylvatica is discussed. (Received May 23, 1997; Accepted September 22, 1997)     

Pressure effects on absorption spectra of the isolated reaction center of Photosystem II

Absorption spectra of the D1-D2-cytochrom b₅₅₉ complex at 4°C were investigated at pressures up to 300 MPa. Pressure effects were mostly reversible and independent of the detergent used (CHAPS or dodecyl--D-maltoside). Red-shifts were observed under pressure for the chlorophyll Q_y- and the -carotene S₀ S₂ bands. The relatively small Q_y-shift of approximately 0.15 cm^-1/MPa is an indication for the absence of strongly coupled chlorophyll dimers within the reaction center and supports earlier reports from low-temperature measurements (Chang HC, Jankowiak R, Reddy NRS and Small GJ (1995) Chem Phys 197: 307–321). The carotene red-shift (seen in CHAPS) is much larger (0.5 – 0.6 cm^-1/MPa) and within the range observed for excitonically coupled chlorophylls. However, since carotenes are more sensitive to changes of refractive index, we do not consider this evidence for excitonically coupled carotenes. Varying the pH and the detergent induced only small effects. Pigment exchange using high pressure instead of elevated temperature was not possible under the conditions tested.     

Localization of the insulin-like growth factor I receptor in the cerebellum and hypothalamus of adult rats: an electron microscopic study

Insulin-like growth factor I (IGF-I) is an important modulator of cell growth and plasticity in the CNS. Expression of the IGF-I receptor mRNA in brain peaks at times of active cell development perinatally and remains detectable, albeit at lower levels, in the adult. While both autoradiographic and in situ hybridization studies show a wide and specific distribution of IGF-I receptor throughout the adult rat brain, nothing is yet known about its subcellular localization, a critical issue that will help clarify the biological role of this trophic factor in the adult brain. The present study describes the subcellular localization of IGF-I receptor immunoreactivity in the cerebellar cortex and the hypothalamic arcuate nucleus by using electron microscopic immunocytochemistry. In the cerebellum, IGF-I receptor immunoreactivity is present postsynaptically in the dendrites and soma of the Purkinje cell and presynaptically in axon terminals impinging upon the Purkinje cell soma, as well as in mossy fibre rosettes in the cerebellar glomeruli. Neurons in the mediobasal hypothalamus also contain IGF-I receptors located pre- and postsynaptically. Endothelial cells, astroglial end-feet surrounding micro vessels throughout all the brain parenchyma, tanycytes of the third ventricle and oligodendrocytes in the cerebellar white matter are also rich in IGF-I receptors. These results strongly support previous observations that IGF-I is a neuromodulator in the adult brain, probably acting as both a pre- and a postsynaptic messenger. They also suggest that glial cells may be involved in the actions of IGF-I in the adult brain.