Simultaneous detection of C-reactive protein and other cardiac markers in human plasma using micromosaic immunoassays and self-regulating microfluidic networks

We show a proof-of-concept in which we combine our previously published concepts of micromosaic immunoassays (microMIAs) with self-regulating microfluidic networks (microFNs) to detect C-reactive protein (CRP) and other cardiac markers such as myoglobin (Mb) and cardiac Troponin I (cTnI). The microFNs are microfabricated in Si, have a well-defined surface chemistry, and are affixed to a bibulous material so as to self-regulate the displacement of an aliquot of liquid through the microFNs using capillary forces. An open section of the channels of the microFNs is covered with a hydrophobic poly(dimethylsiloxane) (PDMS) slab that acts as the substrate for a solid-phase immunoassay. Here, individual assays are conducted using independent channels. These assays are "sequential": series of samples, reagents, and buffers are displaced one after the other over the PDMS surface, and, as these assays are conducted under "microfluidic" conditions, they are fast to perform, very economical in their use of reagents, extremely integrated, and yield high-quality signals. The combinatorial character of microMIAs is exploited to optimize the assay parameters for detecting CRP. In particular, we found it optimal to deposit the capture antibody for CRP on PDMS at a concentration between 20 and 500 microg ml(-1) in PBS in 1 min and to detect captured CRP in 2 min using a detection antibody having a concentration in PBS of 120 microg ml(-1). With this method, CRP is quantitatively detected within 10 min in one microliter of human plasma down to concentrations of 30 ng ml(-1), which suggests the possibility to detect CRP at clinically relevant concentrations for the management of coronary heart disease (CHD) and systemic inflammation.     

A family-based approach reveals the function of residues in the nuclear receptor ligand-binding domain

Literature studies, 3D structure data, and a series of sequence analysis techniques were combined to reveal important residues in the structure and function of the ligand-binding domain of nuclear hormone receptors. A structure-based multiple sequence alignment allowed for the seamless combination of data from many different studies on different receptors into one single functional model. It was recently shown that a combined analysis of sequence entropy and variability can divide residues in five classes; (1) the main function or active site, (2) support for the main function, (3) signal transduction, (4) modulator or ligand binding and (5) the rest. Mutation data extracted from the literature and intermolecular contacts observed in nuclear receptor structures were analyzed in view of this classification and showed that the main function or active site residues of the nuclear receptor ligand-binding domain are involved in cofactor recruitment. Furthermore, the sequence entropy-variability analysis identified the presence of signal transduction residues that are located between the ligand, cofactor and dimer sites, suggesting communication between these regulatory binding sites. Experimental and computational results agreed well for most residues for which mutation data and intermolecular contact data were available. This allows us to predict the role of the residues for which no functional data is available yet. This study illustrates the power of family-based approaches towards the analysis of protein function, and it points out the problems and possibilities presented by the massive amounts of data that are becoming available in the "omics era". The results shed light on the nuclear receptor family that is involved in processes ranging from cancer to infertility, and that is one of the more important targets in the pharmaceutical industry.     

Can extinction rates be estimated without fossils?

There is considerable interest in the possibility of using molecular phylogenies to estimate extinction rates. The present study aims at assessing the statistical performance of the birth-death model fitting approach to estimate speciation and extinction rates by comparison to the approach considering fossil data. A simulation-based approach was used. The diversification of a large number of lineages was simulated under a wide range of speciation and extinction rate values. The estimators obtained with fossils performed better than those without fossils. In the absence of fossils (e.g. with a molecular phylogeny), the speciation rate was correctly estimated in a wide range of situations; the bias of the corresponding estimator was close to zero for the largest trees. However, this estimator was substantially biased when the simulated extinction rate was high. On the other hand the estimator of extinction rate was biased in a wide range of situations. Surprisingly, this bias was lesser with medium-sized trees. Some recommendations for interpreting results from a diversification analysis are given.     

High variety of different simian T-cell leukemia virus type 1 strains in chimpanzees (Pan troglodytes verus) of the Taï National Park, Côte d'Ivoire

We found human T-cell leukemia virus type 1- and simian T-cell leukemia virus type 1 (STLV-1)-related infections in 5 of 10 chimpanzees originating from three groups of wild chimpanzees. The new virus isolates showed a surprising heterogeneity not only in comparison to STLV-1 described previously in other primate species but also between the different chimpanzee groups, within a group, or even between strains isolated from an individual animal. The interdisciplinary combination of virology, molecular epidemiology, and long-term behavioral studies suggests that the primary route of infection might be interspecies transmission from other primates, such as red colobus monkeys, that are hunted and consumed by chimpanzees.     

Ecological correlates and evolutionary divergence in the skull of turtles: a geometric morphometric assessment

Resource use and phylogeny are often correlated with morphological variation. Moreover, because biological shapes are often complex and evolve depending on several internal constraints, they must be assessed using integrative methods. We analyzed the morphological variation of the turtle skull in the context of an adaptive radiation. Our focus are turtles of the superfamily Testudinoidea, which are remarkably diverse, both in number of species and in ecology. In this study, we depict morphological variation in the turtle skull in three dimensions with respect to diet, phylogeny, and habitat using modern geometric morphometrics. Our study revealed that morphological specialization was related to both diet and habitat. Morphological variation is decomposed in regard of both resource use (habitat and diet) and phylogeny. Feeding mode depending on environment was suggested as a key factor determining morphological evolution and diversification of turtle skulls. Diet (especially durophagy) leads to parallel morphologies in different clades. Phylogeny seemed to constrain only localized features of the skull and remained of minor influence, because overall morphotypes, closely correlated with ecological factors, occurred in both clades. In conclusion, the adaptive radiation of the Testudinoidea is revealed to demonstrate a clear relationship between the skull shape and life style.     

Oxidized SOD1 alters proteasome activities in vitro and in the cortex of SOD1 overexpressing mice

Premature ageing, one of the characteristics of Down syndrome (DS), may involve oxidative stress and impairment of proteasome activity. Transgenic mice overexpressing the human copper/zinc superoxide dismutase (SOD1) gene are one of the first murine models for DS and it has been shown that SOD1 overexpression might be either deleterious or beneficial. Here, we show a reduction in proteasome activities in the cortex of SOD1 transgenic mice and an associated increase in the content of oxidized SOD1 protein. As we demonstrate that in vitro oxidized SOD can inhibit purified proteasome peptidase activities, modified SOD1 might be partially responsible for proteasome inhibition shown in SOD1 transgenic mice.     

Major O-glycans from the nest of Vespula germanica contain phospho-ethanolamine

We describe here the structural deciphering of four wasp O-glycans. Following purification of a mixture of glycoproteins from nests of the common wasp Vespula germanica L. (Hymenoptera), their substituting O-glycans were liberated by reducing β-elimination and characterised using a combination of high resolution NMR and mass spectrometry analyses. Besides ubiquitously found in the insect cells GalNAc-ol and Gal(β1-3)GalNAc-ol compounds, two novel O-glycans carrying a 2-aminoethyl phosphate group were described for the first time here. We suggest that they present the following structures: Etn-P-(O→6)-GalNAc-ol and Etn-P-(O→6)-[Gal(β1-3)]GalNAc-ol. In conjunction with previous studies, these results suggest that a 2-aminoethyl phosphate group may act as an alternative to sialic acid for conferring charges to glycoproteins.     

Pertussis toxin-sensitive modulation of glutamate transport by endothelin-1 type A receptors in glioma cells

Endothelin-1 (ET-1) is a 21 amino acids peptide that exerts several biological activities through interaction with specific G-protein coupled receptors. Increased ET-1 expression is frequently associated with pathological situations involving alterations in glutamate levels. In the present study, a brief exposure to ET-1 was found to increase aspartate uptake in C6 glioma cells, which endogenously express the neuronal glutamate transporter EAAC1 (pEC50 of 9.89). The stimulatory effect of ET-1 mediated by ETA receptors corresponds to a 62% increase in the Vmax with no modification of the affinity for the substrate. While protein kinase C activity is known to participate in the regulation of EAAC1, the effect of ET-1 on the glutamate uptake was found to be independent of this kinase activation. In contrast, the inactivation of Go/i type G-protein dependent signaling with pertussis toxin was found to impair ET-1-mediated regulation of EAAC1. An examination of the cell surface expression of EAAC1 by protein biotinylation studies or by confocal analysis of immuno-fluorescence staining demonstrated that ET-1 stimulates EAAC1 translocation to the cell surface. Hence, the disruption of the cytoskeleton with cytochalasin D prevented ET-1-stimulated aspartate uptake. Together, the data presented in the current study suggest that ET-1 participates in the acute regulation of glutamate transport in glioma cells. Considering the documented role of glutamate excitotoxicity in the development of brain tumors, endothelinergic system constitutes a putative target for the pharmacological control of glutamate transmission at the vicinity of glioma cells.     

Varicella-zoster virus IE63 protein phosphorylation by roscovitine-sensitive cyclin-dependent kinases modulates its cellular localization and activity

During the first stage of Varicella-Zoster virus (VZV) infection, IE63 (immediate early 63 protein) is mostly expressed in the nucleus and also slightly in the cytoplasm, and during latency, IE63 localizes in the cytoplasm quite exclusively. Because phosphorylation is known to regulate various cellular mechanisms, we investigated the impact of phosphorylation by roscovitine-sensitive cyclin-dependent kinase (RSC) on the localization and functional properties of IE63. We demonstrated first that IE63 was phosphorylated on Ser-224 in vitro by CDK1 and CDK5 but not by CDK2, CDK7, or CDK9. Furthermore, by using roscovitine and CDK1 inhibitor III (CiIII), we showed that CDK1 phosphorylated IE63 on Ser-224 in vivo. By mutagenesis and the use of inhibitors, we demonstrated that phosphorylation on Ser-224 was important for the correct localization of the protein. Indeed, the substitution of these residues by alanine led to an exclusive nuclear localization of the protein, whereas mutations into glutamic acid did not modify its subcellular distribution. When transfected or VZV-infected cells were treated with roscovitine or CiIII, an exclusive nuclear localization of IE63 was also observed. By using a transfection assay, we also showed that phosphorylation on Ser-224 and Thr-222 was essential for the down-regulation of the basal activity of the VZV DNA polymerase gene promoter. Similarly, roscovitine and CiIII impaired these properties of the wild-type form of IE63. These observations clearly demonstrated the importance of CDK1-mediated IE63 phosphorylation for a correct distribution of IE63 between both cellular compartments and for its repressive activity toward the promoter tested.     

Tumor-derived multiple chaperone enrichment by free-solution isoelectric focusing yields potent antitumor vaccines

We have utilized a free-solution/isoelectric focusing technique (FS-IEF) to obtain fractions rich in multiple chaperone proteins from clarified A20 tumor lysates. Vaccines prepared from chaperone-rich fractions are capable of providing protective immunity in mice subsequently challenged intravenously with the same A20 B cell leukemia cells. This protection is at least equal to that provided by purified, tumor-derived heat-shock protein 70, which was the best chaperone immunogen in our hands against this aggressive murine leukemia model. Dosage escalation studies, however, revealed that increasing vaccine dosages actually abrogated the protective effects. The physical nature of the enriched chaperones indicates that they are associated in complexes, which may have implications for their function. FS-IEF is relatively simple, rapid, and efficient, thus making combined multi-chaperone therapy feasible. Received: 11 May 2000 / Accepted: 13 June 2000