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.     

Mesenchymal stem cell adhesion to cardiac microvascular endothelium: activators and mechanisms

Circulating stem cells home within the myocardium, probably as the first step of a tissue regeneration process. This step requires adhesion to cardiac microvascular endothelium (CMVE). In this study, we studied mechanisms of adhesion between CMVE and mesenchymal stem cells (MSCs). Adhesion was studied in vitro and in vivo. Isolated 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled rat MSCs were allowed to adhere to cultured CMVE in static and dynamic conditions. Either CMVE or MSCs were pretreated with cytokines [IL-1beta, IL-3, IL-6, stem cell factor, stromal cell-derived factor-1, or TNF-alpha, 10 ng/ml]. Control or TNF-alpha-treated MSCs were injected intracavitarily in rat hearts in vivo. In baseline in vitro conditions, the number of MSCs that adhered to CMVE was highly dependent on the flow rate of the superfusing medium but remained significant at venous and capillary shear stress amplitudes. Activation of both CMVE and MSCs with TNF-alpha or IL-1beta before adhesion concentration dependently increased adhesion of MSCs at each studied level of shear stress. Consistently, in vivo, activation of MSCs with TNF-alpha before injection significantly enhanced cardiac homing of MSCs. TNF-alpha-induced adhesion could be completely blocked by pretreating either CMVE or MSCs with anti-VCAM-1 monoclonal antibodies but not by anti-ICAM-1 antibodies. Adhesion of circulating MSCs in the heart appears to be an endothelium-dependent process and is sensitive to modulation by activators of both MSCs and endothelium. Inflammation and the expression of VCAM-1 but not ICAM-1 on both cell types have a regulatory effect on MSC homing in the heart.     

Polo-like kinase-1 controls proteasome-dependent degradation of Claspin during checkpoint recovery

DNA-damage checkpoints maintain genomic integrity by mediating a cell-cycle delay in response to genotoxic stress or stalled replication forks. In response to damage, the checkpoint kinase ATR phosphorylates and activates its effector kinase Chk1 in a process that critically depends on Claspin . However, it is not known how exactly this kinase cascade is silenced. Here we demonstrate that the abundance of Claspin is regulated through proteasomal degradation. In response to DNA damage, Claspin is transiently stabilized, and its expression depends on Chk1 kinase activity. In addition, we show that Claspin is degraded upon mitotic entry, a process that depends on the beta-TrCP-SCF ubiquitin ligase and Polo-like kinase-1 (Plk1). We demonstrate that Claspin interacts with both beta-TrCP and Plk1 and that inactivation of these components or the beta-TrCP recognition motif in Claspin prevents its mitotic degradation. Interestingly, expression of a nondegradable Claspin mutant inhibits recovery from a DNA-damage-induced checkpoint arrest. Thus, we conclude that Claspin levels are tightly regulated, both during unperturbed cell cycles and after DNA damage. Moreover, our data demonstrate that the degradation of Claspin at the onset of mitosis is an essential step for the recovery of a cell from a DNA-damage-induced cell-cycle arrest.     

Dietary Restraint and Control Over “Wanting” Following Consumption of “Forbidden” Food

Eating behavior can be influenced by the rewarding value of food, i.e., “liking” and “wanting.” The objective of this study was to assess in normal‐weight dietary restrained (NR) vs. unrestrained (NU) eaters how rewarding value of food is affected by satiety, and by eating a nonhealthy perceived, dessert‐specific food vs. a healthy perceived, neutral food (chocolate mousse vs. cottage cheese). Subjects (24NR age = 25.0 ± 8.2 years, BMI = 22.3 ± 2.1 kg/m²; 26NU age = 24.8 ± 8.0 years, BMI = 22.1 ± 1.7 kg/m²) came to the university twice, fasted (randomized crossover design). Per test‐session “liking” and “wanting” for 72 items divided in six categories (bread, filling, drinks, dessert, sweets, stationery (placebo)) was measured, before and after consumption of chocolate mousse/cottage cheese, matched for energy content (5.6 kJ/g) and individual daily energy requirements (10%). Chocolate mousse was liked more than cottage cheese (P < 0.05). After consumption of chocolate mousse or cottage cheese, appetite and “liking” vs. placebo were decreased in NR and NU (P < 0.03), whereas “wanting” was only decreased in NR vs. NU (P ≤ 0.01). In NR vs. NU “wanting” was specifically decreased after chocolate mousse vs. cottage cheese; this decrease concerned especially “wanting” for bread and filling (P < 0.05). To conclude, despite similar decreases in appetite and “liking” after a meal in NR and NU, NR decrease “wanting” in contrast to NU. NR decrease “wanting” specifically for a nonhealthy perceived, “delicious,” dessert‐specific food vs. a nutritional identical, yet healthy perceived, slightly less “delicious,” “neutral” food. A healthy perceived food may thus impose greater risk for control of energy intake in NR.     

Reverse MAPPIT detects disruptors of protein-protein interactions in human cells

Reverse mammalian protein-protein interaction trap (MAPPIT) is a mammalian reverse two-hybrid technology. The method is adapted from the forward MAPPIT technique, a two-hybrid complementation system in which the interaction between a bait-fusion protein and a prey-fusion protein restores ligand-dependent cytokine receptor signaling. In the reverse mode described in detail here, a positive readout is generated on disruption of the designated protein-protein interactions. Reverse MAPPIT functions in intact human cells, facilitating simultaneous analysis of disruption, toxicity and permeability of the tested compounds, making it particularly suitable for screening for molecules that target therapeutically interesting protein-protein interactions or for mapping the interaction interface between proteins. The total handling time of a typical reverse MAPPIT experiment is approximately 9 h and is spread over 4-5 d.     

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.     

A sensitive and inexpensive yeast bioassay for the mycotoxin zearalenone and other compounds with estrogenic activity

Zearalenone (ZON) is a nonsteroidal estrogenic mycotoxin produced by plant-pathogenic species of Fusarium. As a consequence of infection with Fusarium culmorum and Fusarium graminearum, ZON can be found in cereals and derived food products. Since ZON is suspected to be a cause of human disease, including premature puberty syndrome, as well as hyperestrogenism in farm animals, several countries have established monitoring programs and guidelines for ZON levels in grain intended for human consumption and animal feed. We developed a low-cost method for monitoring ZON contamination in grain based on a sensitive yeast bioassay. The indicator Saccharomyces cerevisiae strain YZRM7 is unable to grow unless an engineered pyrimidine biosynthetic gene is activated by the expressed human estrogen receptor in the presence of exogenous estrogenic substances. Deletion of the genes encoding ATP-binding cassette (ABC) transporters Pdr5p and Snq2p increases net ZON uptake synergistically. Less than 1 microg of ZON per liter of medium is sufficient to allow growth of the indicator strain. To prevent interference with pyrimidines potentially present in biological samples, we also disrupted the genes FUR1 and URK1, blocking the pyrimidine salvage pathway. The bioassay strain YZRM7 allows qualitative detection and quantification of total estrogenic activity in cereal extracts without requiring further cleanup steps. Its high sensitivity makes this assay suitable for low-cost monitoring of contamination of maize and small grain cereals with estrogenic Fusarium mycotxins.     

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.