Theory of allosteric effects in serine proteases.

The classical Botts-Morales theory for the action of a modifier on the catalytic properties of an enzyme has been extended to deal with allosteric effects in serine proteases. The exact analytical solution derived for the linkage scheme at steady state provides a rigorous framework for the study of many biologically relevant systems, including enzymes activated by monovalent cations and cofactor-controlled protease-zymogen interactions in blood coagulation. When the enzyme obeys Michaelis-Menten kinetics, the exact solution of the kinetic linkage scheme simplifies considerably. Of particular importance for practical applications is a simple equation expressing the dependence of the specificity constant of the enzyme, kcat/Km, on the concentration of the modifier, from which the equilibrium binding constant for the formation of the enzyme-modifier complex can be estimated. Analysis of the allosteric changes in thrombin activity induced by thrombomodulin and Na+ in terms of this equation yields accurate determinations of the equilibrium binding constants for both effectors.     

Anti-idiotypic antiserum to monoclonal anti-Sm inhibits the autoantigen-induced proliferative response

Anti-idiotypic sera were produced in BALB/c mice against three established monoclonal anti-Sm antibodies. Inhibition assays showed that the anti-idiotypic antibodies recognized determinants that were present on all three monoclonal antibodies but not on normal mouse IgG from unimmunized BALB/c mice or myeloma proteins. Normal (+/+) and autoimmune (lpr/lpr) MRL/MpJ or C3H/HeJ mice were immunized with Sm in complete Freund's adjuvant. Immune T cells from the draining lymph nodes proliferated in response to the addition of Sm in vitro. Anti-idiotypic serum added to these cultures inhibited the proliferative response by 50 to 70%, whereas normal BALB/c serum had no effect. This inhibition of proliferation was antigen specific, because the anti-idiotypic serum did not inhibit the T cell proliferative response to an irrelevant antigen, TNP-KLH, or ovalbumin. Kinetic studies showed that the anti-idiotypic serum inhibited an early event in antigen-induced proliferative response, because the addition of serum late in culture did not cause any significant reduction in proliferation. The reduced proliferative response was due to direct action of the anti-idiotypic serum on the Lyt-1+, 2- T cell population.     

α5-nAChR modulates melanoma growth through the Notch1 signaling pathway

The roles of α5-nicotinic acetylcholine receptors (α5-nAChRs) in various types of solid cancer have been reported; however, its role in melanoma remains unknown. We knocked down α5-nAChR expression in melanoma cells to investigate the role of α5-nAChR in the proliferation, migration, and invasion of melanoma cells, and its effect on downstream signaling pathways. Using immunohistochemical analysis, we determined that α5-nAChR expression is significantly increased in human melanoma tissues and cell lines compared with normal human skin tissues. Knocking down α5-nAChR expression in melanoma cells in culture significantly inhibited the proliferation, migration, and invasiveness of melanoma cell lines. Specifically, knockdown of α5-nAChR inhibited PI3K-AKT and ERK1/2 signaling activity. Moreover, we confirmed that the Notch1 signaling pathway is the downstream target of α5-nAChR in melanoma. Our findings suggest that α5-nAChR plays a critical role in melanoma development and progression, and that targeting α5-nAChR may be a strategy for melanoma treatment.     

Rat liver histidyl-tRNA synthetase. Purification and inhibition by the myositis-specific anti-Jo-1 autoantibody

Myositis is an autoimmune inflammatory muscle disease of unknown etiology. We demonstrate directly that the antigen to the myositis-specific anti-Jo-1 antibody is histidyl-tRNA synthetase. The anti-Jo-1 antibody inhibits human HeLa and rat liver histidyl-tRNA synthetase. Using conventional and immunoaffinity chromatography with immobilized anti-Jo-1 antibody, we have purified rat liver histidyl-tRNA synthetase which has a subunit M_r 64,000 and an estimated native M_r suggesting an α₂ structure. The evidence indicates that the Jo-1 antigen is histidyl-tRNA synthetase, and that some of the histidyl-tRNA synthetase structure are conserved across species.     

Agnogene Deletion in a Novel Pathogenic JC Virus Isolate Impairs VP1 Expression and Virion Production

Infection of glial cells by the human polyomavirus JC (JCV) causes progressive multifocal leukoencephalopathy (PML). JCV Encephalopathy (JCVE) is a newly identified disease characterized by JCV infection of cortical pyramidal neurons. The virus JCV_CPN associated with JCVE contains a unique 143 base pair deletion in the agnogene. Contrary to most JCV brain isolates, JCV_CPN has an archetype-like regulatory region (RR) usually found in kidney strains. This provided us with the unique opportunity to determine for the first time how each of these regions contributed to the phenotype of JCV_CPN. We characterized the replication of JCV_CPN compared to the prototype virus JCV_Mad-1 in kidney, glial and neuronal cell lines. We found that JCV_CPN is capable of replicating viral DNA in all cell lines tested, but is unable to establish persistent infection seen with JCV_Mad-1. JCV_CPN does not have an increased ability to replicate in the neuronal cell line tested. To determine whether this phenotype results from the archetype-like RR or the agnogene deletion, we generated chimeric viruses between JCV_CPN of JCV_Mad-1. We found that the deletion in the agnogene is the predominant cause of the inability of the virus to maintain a persistent infection, with the introduction of a full length agnogene, either with or without agnoprotein expression, rescues the replication of JCV_CPN. Studying this naturally occurring pathogenic variant of JCV provides a valuable tool for understanding the functions of the agnogene and RR form in JCV replication.