Ethoxyformylation of histidine residues in equine growth hormone

Reactivity of histidine residues in equine growth hormone to ethoxyformic anhydride was studied. The existence of two kinetically different sets was demonstrated: one of them including only the slow reacting histidine 169 (k = 0.164 min-1) and the other containing fast reacting histidines 19 and 21 (k = 0.892 min-1). A correlation between the decrease in the capacity to compete with 125I-labeled hormone for rat liver binding sites and the degree of ethoxyformylation of the fast group was found. Circular dichroism studies indicated no significant conformational changes in the protein with all three residues modified. These results fully agree with those obtained for bovine growth hormone which is further evidence supporting the vinculation of histidines 19 and/or 21 with the binding site of these hormones to their specific receptors.     

The lepidopteran mitochondrial control region: structure and evolution

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies-- although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.      

Glutamine repeats and inherited neurodegenerative diseases: molecular aspects

Several dominantly inherited, late onset, neurodegenerative diseases are due to expansion of CAG repeats, leading to expansion of glutamine repeats in the affected proteins. These proteins are of very different sizes and, with one exception, show no sequence homology to known proteins or to each other; their functions are unknown. In some, the glutamine repeat starts near the N-terminus, in another near the middle and in another near the C-terminus, but regardless of these differences, no disease has been observed in individuals with fewer than 37 repeats, and absence of disease has never been found in those with more than 41 repeats. Protein constructs with more than 41 repeats are toxic to E. coli and to CHO cells in culture, and they elicit ataxia in transgenic mice. These observations argue in favour of a distinct change of structure associated with elongation beyond 37–41 glutamine repeats. The review describes experiments designed to find out what these structures might be and how they could influence the properties of the proteins of which they form part. Poly- -glutamines form pleated sheets of β-strands held together by hydrogen bonds between their amides. Incorporation of glutamine repeats into a small protein of known structure made it associate irreversibly into oligomers. That association took place during the folding of the protein molecules and led to their becoming firmly interlocked by either strand- or domain-swapping. Thermodynamic considerations suggest that elongation of glutamine repeats beyond a certain length may lead to a phase change from random coils to hydrogen-bonded hairpins. Possible mechanisms of expansion of CAG repeats are discussed in the light of looped DNA model structures.     

Development of cell surface saccharides on embryonic pancreatic cells

Using a battery of seven lectin-ferritin conjugates as probes for cell surface glycoconjugates, we have studied the pattern of plasmalemmal differentiation of cells in the embryonic rat pancreas from day 15 in utero to the early postpartum stage. Our results indicate that differentiation of plasmalemmal glycoconjugates on acinar, endocrine, and centroacinar cells is temporally correlated with development and is unique for each cell type, as indicated by lectin-ferritin binding. Specifically, (a) expression of adult cell surface saccharide phenotype can be detected on presumptive acinar cells as early as 15 d in utero, as indicated by soybean agglutinin binding, and precedes development of intracellular organelles characteristic of mature acinar cells; (b) maturation of the plasmalemma of acinar cells is reached after intracellular cytodifferentiation is completed, as indicated by appearance of Con A and fucoselectin binding sites only at day 19 of development; conversely, maturation of the endocrine cell plasmalemma is accompanied by "loss" (masking) of ricinus communis II agglutinin receptors; and (c) binding sites for fucose lectins and for soybean agglutinin are absent on endocrine and centroacinar cells at all stages examined. We conclude that acinar, centroacinar, and endocrine cells develop from a common progenitor cell(s) whose plasmalemmal carbohydrate composition resembles most closely that of the adult centroacinar cell. Finally, appearance of acinar lumina beginning at approximately 17 d in utero is accompanied by differenetiation of apical and basolateral plasmalemmal domains of epithelial cells, as indicated by enhanced binding of several lectin-ferritin conjugates to the apical plasmalemmal, a pattern that persists from this stage through adult life.     

Beyond Motor Scheme: A Supramodal Distributed Representation in the Action-Observation Network

The representation of actions within the action-observation network is thought to rely on a distributed functional organization. Furthermore, recent findings indicate that the action-observation network encodes not merely the observed motor act, but rather a representation that is independent from a specific sensory modality or sensory experience. In the present study, we wished to determine to what extent this distributed and ‘more abstract’ representation of action is truly supramodal, i.e. shares a common coding across sensory modalities. To this aim, a pattern recognition approach was employed to analyze neural responses in sighted and congenitally blind subjects during visual and/or auditory presentation of hand-made actions. Multivoxel pattern analyses-based classifiers discriminated action from non-action stimuli across sensory conditions (visual and auditory) and experimental groups (blind and sighted). Moreover, these classifiers labeled as ‘action’ the pattern of neural responses evoked during actual motor execution. Interestingly, discriminative information for the action/non action classification was located in a bilateral, but left-prevalent, network that strongly overlaps with brain regions known to form the action-observation network and the human mirror system. The ability to identify action features with a multivoxel pattern analyses-based classifier in both sighted and blind individuals and independently from the sensory modality conveying the stimuli clearly supports the hypothesis of a supramodal, distributed functional representation of actions, mainly within the action-observation network.     

Translating niche features: Modelling differential exposure of Argentine reptiles to global climate change

Global climate change affects the distributions of ectotherms and may be the cause of several conservation problems, such as great displacement of climatic suitable spaces for species and, consequently, important reductions of the extent of liveable places, threatening the existence of many of them. Species exposure (and hence vulnerability) to global climate change is linked to features of their climatic niches (such as the relative position of the inhabited localities of each species in the climatic space), and therefore to characteristics of their geographic ranges (such as the extent of the distributions or altitudinal range inhabited by the species). In order to analyze the pattern of response of Argentine reptiles to global climate change, we ran phylogenetic generalized least squares models using species exposure to global climate change as a response variable, and (i) niche properties (breadth and position of the species in the climate space) and (ii) general features of the distribution of species (maximum latitude, altitudinal range, maximum elevation, distributional range and proximity to the most important dispersal barrier) as predictors. Our results suggest that the best way to explain climate change exposure is by combining breadth and position of climatic niche of the species or combining geographic features that are indicators of both niche characteristics. Our best model shows that in our study area, species with the narrowest distributional ranges that also inhabit the highest elevations are the most exposed to the effects of global climate change. In this sense, reptile species from Yungas, Puna and Andes ecoregions could be especially vulnerable to the effects of climate change. We believe that these types of models may represent an interesting tool for determining species and places particularly threatened by the effects of global climate change, which should be strongly considered in conservation planning.     

Detection of hepatitis C virus RNA by reverse-transcriptase and polymerase chain reaction: clinical applications of quantitative analysis

Polymerase chain reaction (PCR) applications to diagnostics allowed the detection of viral nucleic acids in expected and unexpected clinical circumstances. This has raised some scepticism on the practical usefulness of PCR in the routine laboratory and emphasized the need for quantitative analysis. We addressed this question detecting HCV-RNA by a single step RT-PCR in serum samples from 50 patients with chronic non-A, non-B hepatitis included in clinical trials for recombinant alpha-interferon therapy. We obtained at least 5 serum specimens from each patient (baseline, during and after therapy samples) during an 18-month mean follow-up (range 12-45 months). RT-PCR was performed on total RNA extracted from 100 microl serum aliquots using primers for the highly conserved 5'NCR of HCV-RNA and 35 amplification cycles. PCR products were analyzed by agarose gel electrophoresis and Southern blot hybridization against a P(32)-oligonucleotide probe. Sensitivity was evaluated in separate experiments on tenfold dilutions of a reference Chimp serum containing 10(6) CID(50)/ml. The overall sensitivity of our assay ranged between 10(2) and 10(3) genome Eq./ml. We establish a semiquantitative score system to evaluate viremia levels: 2 = HCV-RNA levels >10(4) genome Eq./ml; 1 = levels between 10(3) and 10(4) g.Eq./ml; 0 = levels less than 10(2) g.Eq/ml. The reproducibility of this scoring system was confirmed testing repeatedly in duplicate end-point dilutions of positive serum samples. A statistically significant relation was observed between elevated HCV-RNA and ALT values (83.8%, chi-square 159.963 P < 0.001). Response to IFN therapy was significantly better in patients with lower baseline HCV-RNA levels. A time relation was found between flare-ups of serum HCV-RNA levels and ALT elevations higher than 3 x normal values viremia elevations coincident or occurring about 1 month earlier than ALT elevations. This finding suggests that immuno pathogenesis might be responsible of HCV-induced liver damage as in chronic hepatitis B where identical relations were observed between viremia and ALT serum levels. In conclusion, single-step HCV-RNA RT-PCR can be a specific and reproducible semiquantitative assay and provides useful diagnostic informations for therapeutic decision making and monitoring of HCV-infected patients.     

Structural and immunological aspects of Polybia scutellaris Antigen 5

Vespid venoms contain Antigen 5, an important allergen whose primary structure and immunological behavior have been extensively studied from venoms of vespids of the Northern Hemisphere. We report herein structural and immunological aspects of Antigen 5 from Polybia scutellaris subspecies rioplatensis (vulgar name: camoati) found in South America. Mast cell degranulation, histamine release, and IgE induction experiments performed in mice allow us to suggest that P. scutellaris Antigen 5 is a variant with reduced IgE response and anaphylactic activity. Sequence data indicate that the protein has a 72.5-90.3% similarity to that of members of the vespid Antigen 5 family with an already known primary structure. Moreover, results suggest that the protein-a new member of an extracellular protein superfamily-could be a good candidate for immunotherapy related to vespid allergy.