Simultaneous LC-MS/MS quantification of P-glycoprotein and cytochrome P450 probe substrates and their metabolites in DBS and plasma.

BACKGROUND: An LC-MS/MS method has been developed for the simultaneous quantification of P-glycoprotein (P-gp) and cytochrome P450 (CYP) probe substrates and their Phase I metabolites in DBS and plasma. P-gp (fexofenadine) and CYP-specific substrates (caffeine for CYP1A2, bupropion for CYP2B6, flurbiprofen for CYP2C9, omeprazole for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4) and their metabolites were extracted from DBS (10 µl) using methanol. Analytes were separated on a reversed-phase LC column followed by SRM detection within a 6 min run time. RESULTS: The method was fully validated over the expected clinical concentration range for all substances tested, in both DBS and plasma. The method has been successfully applied to a PK study where healthy male volunteers received a low dose cocktail of the here described P-gp and CYP probes. Good correlation was observed between capillary DBS and venous plasma drug concentrations. CONCLUSION: Due to its low-invasiveness, simple sample collection and minimal sample preparation, DBS represents a suitable method to simultaneously monitor in vivo activities of P-gp and CYP.

Impact of genetic polymorphisms in cytomegalovirus glycoprotein B on outcomes in solid-organ transplant recipients with cytomegalovirus disease.

BACKGROUND:It is unknown whether specific viral polymorphisms affect in vivo therapeutic response in patients with cytomegalovirus (CMV) disease. Polymorphisms in the CMV glycoprotein B (gB) gene allow discrimination of 4 distinct genotypes (gB1-gB4). We assessed the influence of gB genotypes on the clinical and virologic outcome of CMV disease. METHODS:Solid-organ transplant recipients enrolled in a multicenter trial of CMV disease treatment (VICTOR study) were included in this study. CMV gB genotyping was performed using quantitative real-time polymerase chain reaction at day 0 (start of antiviral therapy). RESULTS:Among 239 patients with CMV disease, the prevalence of gB strain types was 26% for gB1, 10% for gB2, 10% for gB3, and 5% for gB4, whereas mixed infections were present in 49%. Donor-seropositive/recipient-seropositive patients were more likely to have mixed gB infection than donor-seropositive/recipient-seronegative patients (40% vs. 12%; P = .001). Median baseline viral loads were higher and time to viral eradication was longer ( P = .006 and P = .026 , respectively) for mixed infection versus infection with a single genotype. In a multivariate model, mixed gB infection was a significant predictor of failure to eradicate virus by day 21 (mixed vs single genotype; odds ratio, 2.66; 95% confidence interval, 1.31-5.38; P = .007 ) after controlling for baseline viral load, CMV serostatus at baseline, ganciclovir resistance, and antiviral treatment. No effect of gB genotype was seen on virologic or clinical CMV recurrence. CONCLUSIONS:No specific gB genotype appears to confer a specific CMV virulence advantage. However, mixed gB genotype infections are associated with higher viral loads and delayed viral clearance.

Viral envelope glycoprotein processing by proprotein convertases.

The proprotein convertases (PCs) are a family of nine mammalian enzymes that play key roles in the maintenance of cell homeostasis by activating or inactivating proteins via limited proteolysis under temporal and spatial control. A wide range of pathogens, including major human pathogenic viruses can hijack cellular PCs for their own purposes. In particular, productive infection with many enveloped viruses critically depends on the processing of their fusion-active viral envelope glycoproteins by cellular PCs. Based on their crucial role in virus-host interaction, PCs can be important determinants for viral pathogenesis and represent promising targets of therapeutic antiviral intervention. In the present review we will cover basic aspects and recent developments of PC-mediated maturation of viral envelope glycoproteins of selected medically important viruses. The molecular mechanisms underlying the recognition of PCs by viral glycoproteins will be described, including recent findings demonstrating differential PC-recognition of viral and cellular substrates. We will further discuss a possible scenario how viruses during co-evolution with their hosts adapted their glycoproteins to modulate the activity of cellular PCs for their own benefit and discuss the consequences for virus-host interaction and pathogenesis. Particular attention will be given to past and current efforts to evaluate cellular PCs as targets for antiviral therapeutic intervention, with emphasis on emerging highly pathogenic viruses for which no efficacious drugs or vaccines are currently available.

The fourth transmembrane segment of the Na,K-ATPase alpha subunit: a systematic mutagenesis study.

The Na,K-ATPase is a major ion-motive ATPase of the P-type family responsible for many aspects of cellular homeostasis. To determine the structure of the pathway for cations across the transmembrane portion of the Na,K-ATPase, we mutated 24 residues of the fourth transmembrane segment into cysteine and studied their function and accessibility by exposure to the sulfhydryl reagent 2-aminoethyl-methanethiosulfonate. Accessibility was also examined after treatment with palytoxin, which transforms the Na,K-pump into a cation channel. Of the 24 tested cysteine mutants, seven had no or a much reduced transport function. In particular cysteine mutants of the highly conserved "PEG" motif had a strongly reduced activity. However, most of the non-functional mutants could still be transformed by palytoxin as well as all of the functional mutants. Accessibility, determined as a 2-aminoethyl-methanethiosulfonate-induced reduction of the transport activity or as inhibition of the membrane conductance after palytoxin treatment, was observed for the following positions: Phe(323), Ile(322), Gly(326), Ala(330), Pro(333), Glu(334), and Gly(335). In accordance with a structural model of the Na,K-ATPase obtained by homology modeling with the two published structures of sarcoplasmic and endoplasmic reticulum calcium ATPase (Protein Data Bank codes 1EUL and 1IWO), the results suggest the presence of a cation pathway along the side of the fourth transmembrane segment that faces the space between transmembrane segments 5 and 6. The phenylalanine residue in position 323 has a critical position at the outer mouth of the cation pathway. The residues thought to form the cation binding site II ((333)PEGL) are also part of the accessible wall of the cation pathway opened by palytoxin through the Na,K-pump.

Inhibition of glial cell proinflammatory activities by peroxisome proliferator-activated receptor gamma agonist confers partial protection during antimyelin oligodendrocyte glycoprotein demyelination in vitro.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a member of the nuclear hormone superfamily originally characterized as a regulator of adipocyte differentiation and lipid metabolism. In addition, PPAR-gamma has important immunomodulatory functions. If the effect of PPAR-gamma's activation in T-cell-mediated demyelination has been recently demonstrated, nothing is known about the role of PPAR-gamma in antibody-induced demyelination in the absence of T-cell interactions and monocyte/macrophage activation. Therefore, we investigated PPAR-gamma's involvement by using an in vitro model of inflammatory demyelination in three-dimensional aggregating rat brain cell cultures. We found that PPAR-gamma was not constitutively expressed in these cultures but was strongly up-regulated following demyelination mediated by antibodies directed against myelin oligodendrocyte glycoprotein (MOG) in the presence of complement. Pioglitazone, a selective PPAR-gamma agonist, partially protected aggregates from anti-MOG demyelination. Heat shock responses and the expression of the proinflammatory cytokine tumor necrosis factor-alpha were diminished by pioglitazone treatment. Therefore, pioglitazone protection seems to be linked to an inhibition of glial cell proinflammatory activities following anti-MOG induced demyelination. We show that PPAR-gamma agonists act not only on T cells but also on antibody-mediated demyelination. This may represent a significant benefit in treating multiple sclerosis patients.

Quantifying degradation rates of transmembrane receptor kinases.

Transmembrane receptor-kinases are widespread throughout eukaryotes and their activities are known to regulate all kinds of cellular responses in diverse organs and cell types. In order to guarantee the correct amplitude and duration of signals, receptor levels at the cellular surface need to be tightly controlled. The regulation of receptor degradation is the most direct way to achieve this and elaborate mechanisms are in place to control this process. Therefore, the rate of receptor degradation is a parameter of central importance for understanding the dynamics of a signal transduction cascade. Unfortunately, degradation of transmembrane receptors is a complicated multistep process that involves internalization from the plasma membrane, invagination into the lumen of endosomal compartments, and finally fusion with the vacuole for degradation by vacuolar proteases. Therefore, degradation should be measured in an as noninvasive way as possible, such as not to interfere with the complicated transport processes. Here, a method for minimally invasive, in vivo turn-over measurements in intact organs is provided. This technique was used for quantifying the turn-over rates of the Brassinosteroid receptor kinase BRI1 (BRASSINOSTEROID INSENSITIVE 1) in Arabidopsis thaliana root meristems. Pulse-chase expression of a fluorescently labeled BRI1 variant was used and its turn-over rate was determined by quantitative confocal microscopy. This method is well suited to measure turn-over of transmembrane kinases, but can evidently be extended to measure turn-over of any types of transmembrane proteins.

NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly.

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.

Effect of the Contents and Form of Rabies Glycoprotein on the Potency of Rabies Vaccination in Cattle

One of the methods used for controlling cattle rabies in Brazil consists of vaccination. Sometimes, however, rabies occurs in cattle supposedly protected. Since rabies vaccine batches are officially controlled by tests performed on laboratory animals, it is questionable whether the minimal mandatory requirements really correspond to immunogenicity in the target species. We have analyzed the association among potencies of rabies vaccines tested by the NIH test, the contents and form (free-soluble or virus-attached) of rabies glycoprotein (G) in the vaccine batches, and the virus-neutralizing antibodies (VNA) titers elicited in cattle. No correlation was found between G contents in the vaccine batches and the NIH values, whatever the presentation of G. There was no correlation either between NIH values and VNA titers elicited in cattle. There was, however, a positive correlation (r = 0.8681; p = 0.0001) between the amounts of virion-attached G present in the vaccine batches and VNA elicited in cattle. This was not observed when the same analysis was performed with total-glycoprotein or free-soluble glycoprotein. The study demonstrated that NIH values can not predict the effect of the immunogen in cattle. On the other hand, the quantification of virus-attached rabies glycoprotein has a strong correlation with VNA elicited in cattle.

Isoelectric focusing of alpha-1 acid glycoprotein (orosomucoid) in immobilized pH-gradients with 8M urea: detection of its desialylated variants using an alkaline phosphatase-linked secondary antibody system.

A method allowing a clear separation of the different variants of desialylated alpha 1-acid glycoprotein (orosomucoid) has been developed using isoelectric focusing in immobilized pH gradients, supplemented with 8 M urea and 2% v/v 2-mercaptoethanol. Immunoblotting with two antibody-steps afforded high sensitivity and permitted the detection of about 700 pg of alpha 1-acid glycoprotein in a 20 microL plasma sample diluted 1:28 672. A one year old bloodstrain, kept at room temperature, could easily be phenotyped.

Carcinoembryonic antigen (CEA): demonstration of a partial identity between CEA and a normal glycoprotein.

The carcinoembryonic antigen of the human digestive tract (CEA), described by Gold as a glycoprotein specific for digestive carcinomatous and foetal tissues, was found to have common antigenic determinants with a glycoprotein of smaller size extracted from normal adult tissues. This observation suggests that only a part of the CEA molecule carries the onco-foetal specificity. It also has practical implications regarding the radioimmunoassay for CEA used for the diagnosis of certain carcinomas.

Ezrin-Radixin-Moesin-binding Sequence of PSGL-1 Glycoprotein Regulates Leukocyte Rolling on Selectins and Activation of Extracellular Signal-regulated Kinases.

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the capture (tethering) of free-flowing leukocytes and subsequent rolling on selectins. PSGL-1 interactions with endothelial selectins activate Src kinases and spleen tyrosine kinase (Syk), leading to α(L)β(2) integrin-dependent leukocyte slow rolling, which promotes leukocyte recruitment into tissues. In addition, but through a distinct pathway, PSGL-1 engagement activates ERK. Because ezrin, radixin and moesin proteins (ERMs) link PSGL-1 to actin cytoskeleton and because they serve as adaptor molecules between PSGL-1 and Syk, we examined the role of PSGL-1 ERM-binding sequence (EBS) on cell capture, rolling, and signaling through Syk and MAPK pathways. We carried out mutational analysis and observed that deletion of EBS severely reduced 32D leukocyte tethering and rolling on L-, P-, and E-selectin and slightly increased rolling velocity. Alanine substitution of Arg-337 and Lys-338 showed that these residues play a key role in supporting leukocyte tethering and rolling on selectins. Importantly, EBS deletion or Arg-337 and Lys-338 mutations abrogated PSGL-1-induced ERK activation, whereas they did not prevent Syk phosphorylation or E-selectin-induced leukocyte slow rolling. These studies demonstrate that PSGL-1 EBS plays a critical role in recruiting leukocytes on selectins and in activating the MAPK pathway, whereas it is dispensable to phosphorylate Syk and to lead to α(L)β(2)-dependent leukocyte slow rolling.