Biochemical,biophysical and molecular dynamics studies on the proteoglycan-like domain of carbonic anhydrase IX

Human carbonic anhydrase IX (hCA IX) is a tumour-associated enzyme present in a limited number of normal tissues, but overexpressed in several malignant human tumours. It is a transmembrane protein, where the extracellular region consists of a greatly investigated catalytic CA domain and a much less investigated proteoglycan-like (PG) domain. Considering its important role in tumour biology, here, we report for the first time the full characterization of the PG domain, providing insights into its structural and functional features. In particular, this domain has been produced at high yields in bacterial cells and characterized by means of biochemical, biophysical and molecular dynamics studies. Results show that it belongs to the family of intrinsically disordered proteins, being globally unfolded with only some local residual polyproline II secondary structure. The observed conformational flexibility may have several important roles in tumour progression, facilitating interactions of hCA IX with partner proteins assisting tumour spreading and progression.     

D2A sequence of the urokinase receptor induces cell growth through αvβ3 integrin and EGFR

The urokinase receptor (uPAR) stimulates cell proliferation by forming a macromolecular complex with αvβ3 integrin and the epidermal growth factor receptor (EGFR, ErbB1 or HER1) that we name the uPAR proliferasome. uPAR transactivates EGFR, which in turn mediates uPAR-initiated mitogenic signal to the cell. EGFR activation and EGFR-dependent cell growth are blocked in the absence of uPAR expression or when uPAR activity is inhibited by antibodies against either uPAR or EGFR. The mitogenic sequence of uPAR corresponds to the D2A motif present in domain 2. NMR analysis revealed that D2A synthetic peptide has a particular three-dimensional structure, which is atypical for short peptides. D2A peptide is as effective as EGF in promoting EGFR phosphorylation and cell proliferation that were inhibited by AG1478, a specific inhibitor of the tyrosine kinase activity of EGFR. Both D2A and EGF failed to induce proliferation of NR6-EGFR-K721A cells expressing a kinase-defective mutant of EGFR. Moreover, D2A peptide and EGF phosphorylate ERK demonstrating the involvement of the MAP kinase signalling pathway. Altogether, this study reveals the importance of sequence D2A of uPAR, and the interdependence of uPAR and EGFR.     

Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy

Noonan and LEOPARD syndromes are developmental disorders with overlapping features, including cardiac abnormalities, short stature and facial dysmorphia. Increased RAS signaling owing to PTPN11, SOS1 and KRAS mutations causes approximately 60% of Noonan syndrome cases, and PTPN11 mutations cause 90% of LEOPARD syndrome cases. Here, we report that 18 of 231 individuals with Noonan syndrome without known mutations (corresponding to 3% of all affected individuals) and two of six individuals with LEOPARD syndrome without PTPN11 mutations have missense mutations in RAF1, which encodes a serine-threonine kinase that activates MEK1 and MEK2. Most mutations altered a motif flanking Ser259, a residue critical for autoinhibition of RAF1 through 14-3-3 binding. Of 19 subjects with a RAF1 mutation in two hotspots, 18 (or 95%) showed hypertrophic cardiomyopathy (HCM), compared with the 18% prevalence of HCM among individuals with Noonan syndrome in general. Ectopically expressed RAF1 mutants from the two HCM hotspots had increased kinase activity and enhanced ERK activation, whereas non-HCM-associated mutants were kinase impaired. Our findings further implicate increased RAS signaling in pathological cardiomyocyte hypertrophy.     

Chromatin circles in amphibian previtellogenic oocytes

Summary Previtellogenic oocytes ofOdontophrynus americanus display hundreds of chromatin circles. Electron microscopy of spread preparations of isolated nuclei shows that the circles originate from the chromatin. The circles change their morphology and form new copies. The length of the DNA packed in the nucleosomal circles is about 2.5–3.5 m or multiples of this value. Assuming that histones need not be removed from chromatin before DNA replication³ we suggest that the circles might belong to the process of rDNA amplification.This work was supported by grants from the Brazilian CNPq, FAPESP and FEDIB.We thank Dr A. Brunner Jr for the use of the electron microscope and Dr N. Leon for his valuable help.     

The impact on microtubule network of a bracovirus IκB-like protein

Polydnavirus-encoded IκB-like proteins are similar to insect and mammalian IκB, and an immunosuppressive function in the host cells has been inferred to these proteins. Here we show that the expression of one of these IκB-like viral genes, the TnBVank1, in the Drosophila germline affects the localization of gurken, bicoid, and oskar mRNAs whose gene products are relevant for proper embryonic patterning. The altered localization of these mRNAs is suggestive of general defects in the intracellular, microtubule-based, trafficking routes. Analysis of microtubule motor proteins components such as the dynein heavy chain and the kinesin heavy chain revealed defects in the polarized microtubule network. Interestingly, the TnBVANK1 viral protein is uniformly distributed over the entire oocyte cortex, and appears to be anchored to the microtubule ends. Our data open up a very interesting issue on novel function(s) played by the ank gene family by interfering with cytoskeleton organization.     

Design of a System Dynamics Model to Analyze the Styrene Demand in the Brazilian Market

The growth of the petrochemical industry is based on end-user applications such as the automotive and construction sectors, which are the main drivers of the styrene market. However, the use of substitutes for petrochemicals is a reality and creates a competition in the applications of petroleum products. In this sense, this research aimed to design a system dynamics model to evaluate different scenarios, observing the behavior of the styrene demand over time. In the first phase of the project, a greater understanding of the issue was created and a closed loops diagram was elaborated. It was used during the second phase to design an explanatory regression validation model for the styrene demand. In phase three, a visualizing model and scenarios were designed. The scenarios themselves and the results of each scenario were evaluated. The designed and simulated scenarios aimed to evaluate the impact that the use of substitute materials and the variations in gross domestic product cause to the styrene market. The use of system dynamics together with scenario planning was efficient as different strategies for the market could be evaluated based on the simulated scenarios. A critical analysis of the model’s contribution to the decision-making within organizations concludes the study.     

Thermal ecology,activity pattern,habitat, and microhabitats used by the skink Mabuya arajara (Squamata: Scincidae) in the Araripe Plateau,northeastern Brazil

ABSTRACT

We evaluated the activity period, thermal ecology, habitats, and preference for microhabitats of the skink Mabuya arajara in a humid forest environment on the slope of the Araripe Plateau, Brazil. A total of 283 lizards were observed. The lizards were found to be diurnally active (unimodal type pattern), with peak activity during the hottest hours of the day (1100 to 1200). About habitat use, the majority of individuals (73.8% N = 209) were recorded in the narrow transitional area, in the edges of the forest; while 26.4% of individuals were observed in open areas and no observations were made in dense forest. Seven different types of microhabitats were used, with fallen palm leaves (Arecaceae) being the preferred (47.7% N = 135). Considering all lizards, microhabitat niche breadth was 3.27. Males and females presented a high overlap (0.95) in microhabitat use. The average T_c recorded for M. arajara was 32.06 ± 2.72°C. Body size (SVL) did not influence body temperature (F = 0.51, P = > 0.05). Most of the animals were observed with their bodies completely exposed (67.84%, N = 192), 18.37% (N = 52) were semi-exposed and 13.79% (N = 39) were in shelters. Among animals with some degree of sunlight exposure, 57.59% (N = 163) were in shadows, 25.10% (N = 71) were under sunlight filtered by vegetation, and 17.31% (N = 49) were under direct sunlight.A review of the ecology of Mabuya spp. shows that several features appear to be conserved among members of the genus.     

GABA transporter lysine 448: a key residue for tricyclic antidepressants interaction

The effects of three tricyclic antidepressants (TCAs) and two serotonin selective reuptake inhibitors (SSRIs) have been studied with an electrophysiological approach on Xenopus laevis oocytes expressing the rat GABA (γ-Aminobutyric-acid) transporter rGAT1. All tested TCAs and SSRIs inhibit the GABA-associated current in a dose-dependent way with low but comparable efficacy. The pre-steady-state and uncoupled currents appear substantially unaffected. The efficacy of desipramine, but not of the other drugs, is strongly increased in the lysine-glutamate or -aspartate mutants K448E and K448D. Comparison of I _max and K _0.5GABA in the absence and presence of desipramine showed that both parameters are reduced by the drug in the wild-type and in the K448E mutant. This suggests an uncompetitive inhibition, in which the drug can bind only after the substrate, an explanation in agreement with the lack of effects on the pre-steady-state and leak currents, and with the known structural data.