Amino Acid Residue-Specific Neutralization and Nonneutralization of Hepatitis C Virus by Monoclonal Antibodies to the E2 Protein

Antibodies to epitopes in the E2 protein of hepatitis C virus (HCV) reduce the viral infectivity in vivo and in vitro. However, the virus can persist in patients in the presence of neutralizing antibodies. In this study, we generated a panel of monoclonal antibodies that bound specifically to the region between residues 427 and 446 of the E2 protein of HCV genotype 1a, and we examined their capacity to neutralize HCV in a cell culture system. Of the four monoclonal antibodies described here, two were able to neutralize the virus in a genotype 1a-specific manner. The other two failed to neutralize the virus. Moreover, one of the nonneutralizing antibodies could interfere with the neutralizing activity of a chimpanzee polyclonal antibody at E2 residues 412 to 426, as it did with an HCV-specific immune globulin preparation, which was derived from the pooled plasma of chronic hepatitis C patients. Mapping the epitope-paratope contact interfaces revealed that these functionally distinct antibodies shared binding specificity for key amino acid residues, including W⁴³⁷, L⁴³⁸, L⁴⁴¹, and F⁴⁴², within the same epitope of the E2 protein. These data suggest that the effectiveness of antibody-mediated neutralization of HCV could be deduced from the interplay between an antibody and a specific set of amino acid residues. Further understanding of the molecular mechanisms of antibody-mediated neutralization and nonneutralization should provide insights for designing a vaccine to control HCV infection in vivo.     

methBLAST and methPrimerDB: web-tools for PCR based methylation analysis

Background  

DNA methylation plays an important role in development and tumorigenesis by epigenetic modification and silencing of critical genes. The development of PCR-based methylation assays on bisulphite modified DNA heralded a breakthrough in speed and sensitivity for gene methylation analysis. Despite this technological advancement, these approaches require a cumbersome gene by gene primer design and experimental validation. Bisulphite DNA modification results in sequence alterations (all unmethylated cytosines are converted into uracils) and a general sequence complexity reduction as cytosines become underrepresented. Consequently, standard BLAST sequence homology searches cannot be applied to search for specific methylation primers.     

Urotensin II causes fatal circulatory collapse in anesthesized monkeys in vivo: a "vasoconstrictor" with a unique hemodynamic profile

Urotensin II (UII) is a vasoactive peptide that has recently emerged as a likely contributor to cardiovascular physiology and pathology. Acute infusion of UII into nonhuman primates results in circulatory collapse and death; however, the exact cause of death is not well understood. This study was undertaken to elucidate the mechanism underlying the fatal cardiovascular event on UII application in vivo in nonhuman primates. To this end, cynomolgus monkeys (n = 4) were anesthetized and tracheal intubation was performed. One internal jugular vein was cannulated for administration of drugs, and one femoral artery for recording of blood pressure and heart rate using a transonic pressure transducer. Cardiac parameters were not significantly changed after administration of 0.003 nmol/kg human UII. A bolus of human UII (0.03 nmol/kg) caused a decrease of heart rate (HR) (13%), mean blood pressure (MBP) (18%), and first-order derivative of left ventricular pressure (dP/dt) (11%). Carotid and coronary blood flow were reduced by 9% and 7%, respectively; 0.3 nmol/kg of human UII resulted in a further reduction of HR (50.3%), MBP (65%), dP/dt (45%), carotid (38%), and coronary blood flow (30%), ultimately leading to cardiovascular breakdown and death. Pulmonary pressure, however, was increased by 30%. Plasma histamine levels were found to be unaffected by administration of UII. Our results indicate that systemic administration of human UII has negative inotropic and chronotropic effects and reduces total peripheral resistance ultimately leading to severe myocardial depression, pulmonary hypertension, and fatal circulation collapse in nonhuman primates. We suggest that successful design of UII antagonists might offer a new therapeutic principle in treating cardiovascular diseases.     

Formation of nuclear splicing factor compartments is independent of lamins A/C

Nuclear lamins are major architectural elements of the mammalian cell nucleus, and they have been implicated in the functional organization of the nuclear interior, possibly by providing structural support for nuclear compartments. Colocalization studies have suggested a structural role for lamins in the formation and maintenance of pre-mRNA splicing factor compartments. Here, we have directly tested this hypothesis by analysis of embryonic fibroblasts from knock-out mice lacking A- and C-type lamins. We show that the morphology and cellular properties of splicing factor compartments are independent of A- and C-type lamins. Genetic loss of lamins A/C has no effect on the cellular distribution of several pre-mRNA splicing factors and does not affect the compartment morphology as examined by light and electron microscopy. The association of splicing factors with the nuclear matrix fraction persists in the absence of lamins A/C. Live cell microscopy demonstrates that the intranuclear positional stability of splicing factor compartments is maintained and that the exchange dynamics of SF2/ASF between the compartments and the nucleoplasm is not affected by loss of lamin A/C. Our results demonstrate that formation and maintenance of intranuclear splicing factor compartments is independent of lamins A/C, and they argue against an essential structural role of lamins A/C in splicing factor compartment morphology.     

Regulation of Rho/ROCK signaling in airway smooth muscle by membrane potential and [Ca2+]i

Recently, we have shown that Rho and Rho-activated kinase (ROCK) may become activated by high-millimolar KCl, which had previously been widely assumed to act solely through opening of voltage-dependent Ca(2+) channels. In this study, we explored in more detail the relationship between membrane depolarization, Ca(2+) currents, and activation of Rho/ROCK in bovine tracheal smooth muscle. Ca(2+) currents began to activate at membrane voltages more positive than -40 mV and were maximally activated above 0 mV; at the same time, these underwent time- and voltage-dependent inactivation. Depolarizing intact tissues by KCl challenge evoked contractions that were blocked equally, and in a nonadditive fashion, by nifedipine or by the ROCK inhibitor Y-27632. Other agents that elevate intracellular calcium concentration ([Ca(2+)](i)) by pathways independent of G protein-coupled receptors, namely the SERCA-pump inhibitor cyclopiazonic acid and the Ca(2+) ionophore A-23187, evoked contractions that were also largely reduced by Y-27632. KCl directly increased Rho and ROCK activities in a concentration-dependent fashion that paralleled closely the effect of KCl on tone and [Ca(2+)](i), as well as the voltage-dependent Ca(2+) currents that were measured over the voltage ranges that are evoked by 0-120 mM KCl. Through the use of various pharmacological inhibitors, we ruled out roles for Ca(2+)/calmodulin-dependent CaM kinase II, protein kinase C, and protein kinase A in mediating the KCl-stimulated changes in tone and Rho/ROCK activities. In conclusion, Rho is activated by elevation of [Ca(2+)](i) (although the signal transduction pathway underlying this Ca(2+) dependence is still unclear) and possibly also by membrane depolarization per se.     

Zinc induced apoptotic death of mouse dendritic cells

Zinc ions (Zn²⁺) are food components with favourable effects in infectious disease. Zn²⁺ is taken up into dendritic cells (DCs), key players in the regulation of innate and adaptive immunity. In other cell types, Zn²⁺ has been shown to stimulate the formation of ceramide, which is in turn known to trigger suicidal cell death. The present study explored whether Zn²⁺ modifies ceramide formation and survival of bone marrow derived DCs. To this end, DCs were isolated from acid sphingomyelinase knockout (asm ^−/−) and corresponding wild type (asm ^+/+) mice and treated with different concentrations of Zn²⁺. Ceramide formation was assessed with anti-ceramide antibodies in FACS and immunohistochemical analysis, sub-G1 cell population by FACS analysis, break down of phosphatidylserine asymmetry by annexin V binding, cell death by propidium iodide incorporation, metabolic cell activity by MTT assay, ROS production from dichlorofluorescein fluorescence and activation of MAPKs by Western blotting. The treatment of asm ^+/+ DCs with low Zn²⁺ concentrations (up to 100 μM) was followed by ceramide formation, increase in sub-G1 cell population and phosphatidylserine exposure, effects blunted in asm ^−/− DCs. The treatment of DCs with C2-ceramide increased the percentage of sub-G1 and apoptotic DCs from both genotypes. Zn²⁺ led to similar activation of MAPKs in asm ^+/+ and asm ^−/− DCs and did not affect ROS production. Higher concentrations of Zn²⁺ led to a marked increase of propidium iodide incorporation in DCs of both genotypes. The present study reveals that in DCs Zn²⁺ triggers ceramide formation, which in turn compromises cell survival.