Soluble fibrinogen modulates neutrophil functionality through the activation of an extracellular signal-regulated kinase-dependent pathway

The integrin family not only mediates the recruitment of polymorphonuclear leukocytes (PMN) to sites of inflammation but also regulates several effector functions by binding to specific ligands. We have recently demonstrated that soluble fibrinogen (sFbg) is able to trigger an activating signal in PMN through an integrin-dependent mechanism. This activation results in degranulation, phagocytosis enhancement, and apoptosis delay. The aim of the present work was to further elucidate the molecular events that follow sFbg interaction with CD11b in human PMN, and the participation of this signaling pathway in the regulation of neutrophil functionality. We demonstrate that sFbg triggers a cascade of intracellular signals that lead to focal adhesion kinase and extracellular signal-regulated kinase 1/2 tyrosine phosphorylation. The activation of this mitogen-activated protein kinase pathway plays a central role in the sFbg modulation of secondary granule degranulation, Ab-dependent phagocytosis, and apoptosis. However, fibrinogen-induced secretory vesicle degranulation occurs independently of the signaling transduction pathways investigated herein. In the context of an inflammatory process, the intracellular signal pathway activated by sFbg may be an early event influencing the functionality of PMN.     

Molecular epidemiology of human T-lymphotropic virus type II infection in Amerindian and urban populations of the Amazon region of Brazil

Molecular characterization of human T-cell lymphotropic virus II (HTLV-II) isolates in North America and Europe has shown the existence of two principal subtypes of the virus, HTLV-IIa and HTLV-IIb. Subsequent studies on HTLV-II isolates from Brazil have suggested the existence of a unique variant phylogenetically related to HTLV-IIa but phenotypically similar to HTLV-IIb with respect to the transactivatory protein, Tax. This variant has been designated HTLV-IIc. To better clarify the variability and distribution of HTLV-II in Brazil, the viruses present in two population groups from the Amazon region were tested for the presence of HTLV-II using serological and molecular assays. The groups consisted of blood donors from three Amerindian communities and of HIV-1/HTLV-II coinfected patients residing in Belém, an urban area. Nucleotide sequences and phylogenetic analysis confirmed the presence of HTLV-IIc subtype among Amerindian populations and, for the first time, the presence of the same virus among urban groups in Belém. The isolated occurrence of the HTLV-IIc subtype among Amerindian populations in the Amazon region could be attributed to (1) the different migratory pathways and founder effect, or (2) the local origin of a proto-HTLV-II carried by Amerindian ancestors who migrated to the Amazon circa 11,000 to 13,000 years ago. These results suggest that not only is HTLV-IIc unique to this region, but that its presence in urban areas of Brazil has resulted from admixture processes during the colonization of the country.     

Specific effects of chloride on the photocycle of E194Q and E204Q mutants of bacteriorhodopsin as measured by FTIR spectroscopy

Low-temperature Fourier transform infrared spectroscopy has been used to study mutants of Glu194 and Glu204, two amino acids that are involved in proton release to the extracellular side of bacteriorhodopsin. Difference spectra of films of E194Q, E204Q, E194Q/E204Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q at 243, 277, and 293 K and several pH values were obtained by continuous illumination. A specific effect of Cl(-) ions was found for the mutants, promoting a N-like intermediate at alkaline pH and an O' intermediate at neutral or acid pH. The apparent pK(a) of Asp85 in the M intermediate was found to be decreased for E194Q in the presence of Cl(-) (pK(a) of 7.6), but it was unchanged for E204Q, as compared to wild-type. In the absence of Cl(-) (i.e., in the presence of SO(4)(2)(-)), mutation of Glu194 or of Glu204 produces M- (or M(N), M(G))-like intermediates under all of the conditions examined. The absence of N, O, and O' intermediates suggests a long-range effect of the mutation. Furthermore, it is suggested that Cl(-) acts by reaching the interior of the protein, rather than producing surface effects. The effect of low water content was also examined, in the presence of Cl(-). Similar spectra corresponding to the M(1) intermediate were found for dry samples of both mutants, indicating that the effects of the mutations or of Cl(-) ions are confined to the second part of the photocycle. The water O-H stretching data further confirms altered photocycles and the effect of Cl(-) on the accumulation of the N intermediate.     

Evaluation of filter paper collection of urine samples for detection and measurement of organic acidurias by capillary electrophoresis

There is little doubt that mental retardation has been prevented in most babies diagnosed by newborn screening programs for inborn errors and the cost-benefit ratios of these programs have been reported as highly positive. In a previous work we optimised a CE method for quick profiling of organic acidurias, which characterize a large number of inborn errors, so that it permits the separation, detection and even identification in less than 15 min of 22 organic acids in urine samples related to a wide range of metabolic disorders. In the present work we have studied the adequacy of filter paper collection of urine samples to simplify this step, always difficult in babies, when it is not performed by training personnel. The studied parameters were: media and conditions for re-extraction to give the best sensitivity and a more simple procedure when the samples are measured by CE, interferences coming from the diaper, recoveries obtained, possible correction of recoveries with creatinine and stability of the compounds. The whole method we report has the advantages of easy sample collection, easy shipping or delivery, and rapid analysis. Moreover, this method of collection and analysis allows the identification and quantitation of fumaric, methylmalonic, N-acetylaspartic, pyroglutamic and homogentisic acids, as well as glutaric acid for which screening is considered especially advisable.     

Studies on the interaction between hyaluronan and a rat colon cancer cell line

Binding studies with 125I-Tyr labelled hyaluronan (HA) on a cultured rat colon cancer cell line were performed to characterize the association of HA to tumour cells in vitro. Results show a specific and saturable binding (Kd= 1.36nM) which indicates the presence of an HA binding receptor on the tumour cells. There is a specific constant increase of cell-associated HA over time, which indicates that HA is specifically taken up by the cells through endocytosis. The binding of 125I-Tyr labelled HA was more effectively inhibited by unlabelled HA of high MW in relation to low MW species of the polysaccharide indicating that the receptor binds HA of high MW with greater affinity than low MW species. In competition experiments, the HA-binding could not be inhibited by other polysaccharides such as chondroitin sulphate and heparin. Nor could ligands for scavenger receptors and antibodies directed towards ICAM-1, CD 44 and RHAMM (Receptor for HA Mediated Motility) significantly inhibit the association of HA to tumour cells.     

Amphibian Declines in Brazil: An Overview1

Population declines have previously been reported for at least 31 amphibian species in Brazil, in the families Leptodactylidae (19), Hylidae (7), Centrolenidae (2), Dendrobatidae (2), and Bufonidae (1). In five Brazilian museum collections, we found no entries of new records dating back to at least 15 yr ago for 13 of these species. We suggest that these taxa be studied in more detail to verify their status and to generate basic ecological data. Museum data indicate that the remaining species have been recently found in areas of reported crashes, or elsewhere. Several apparent declines in Brazil can be associated with habitat loss, interspecific interactions, natural fluctuations, or lack of intensive sampling. Personal observations and field data also indicate possible declines in the states of Paraná and Ceará as well as in highlands within the Cerrado biome, in the state of Minas Gerais. Records suggest declines of montane and stream‐associated populations of Brazilian amphibians in apparently pristine habitats. Field work is necessary to confirm these cases and to examine whether factors associated with similar extinctions in other parts of the globe—such as pathogens and climate change—are also related to local disappearances. To clarify pending questions and perhaps circumvent new cases, it is important to invest in short‐ and long‐term field studies, and in the maintenance and expansion of museum collections.     

Enantiomeric LC separation of calcium antagonists on protein-based chiral stationary phases

Three chiral calcium antagonist drugs, bepridil and two dihydropyridine derivatives (nicardipine and REC 15/2375), have been successfully separated within short retention times using either the α₁-acid glycoprotein chiral stationary phase (Chiral AGP) or the ovomucoid column (Ultron ES-OVM). Aqueous buffer at defined pH is modified by the addition of an organic component (propan-2-ol, acetonitrile, ethanol) in order to modulate the retention properties of each system. The influence of pH and percentage of organic modifier on retention, selectivity, resolution, and column performance are discussed for bepridil analyzed on Chiral AGP and for the two dihydropyridines (nicardipine and REC 15/2375) analyzed on Ultron ES-OVM stationary phases. © 1993 Wiley-Liss, Inc.     

Mimotope discovery as a tool to design a vaccine against Zika and dengue viruses

Vaccine development against dengue virus is challenging because of the antibody-dependent enhancement of infection (ADE), which causes severe disease. Consecutive infections by Zika (ZIKV) and/or dengue viruses (DENV), or vaccination can predispose to ADE. Current vaccines and vaccine candidates contain the complete envelope viral protein, with epitopes that can raise antibodies causing ADE. We used the envelope dimer epitope (EDE), which induces neutralizing antibodies that do not elicit ADE, to design a vaccine against both flaviviruses. However, EDE is a discontinuous quaternary epitope that cannot be isolated from the E protein without other epitopes. Utilizing phage display, we selected three peptides that mimic the EDE. Free mimotopes were disordered and did not elicit an immune response. After their display on adeno-associated virus (AAV) capsids (VLP), they recovered their structure and were recognized by an EDE-specific antibody. Characterization by cryo-EM and enzyme-linked immunosorbent assay confirmed the correct display of a mimotope on the surface of the AAV VLP and its recognition by the specific antibody. Immunization with the AAV VLP displaying one of the mimotopes induced antibodies that recognized ZIKV and DENV. This work provides the basis for developing a Zika and dengue virus vaccine candidate that will not induce ADE.