Prophylactic administration of HPA-1a-specific antibodies prevents fetal/neonatal alloimmune thrombocytopenia in mice.

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal alloantibodies directed against paternally inherited human platelet alloantigens (HPAs) present on the surface of fetal and neonatal platelets. There are currently no approved therapies for the prevention of FNAIT. We report herein the ability of 2 human HPA-1a-specific therapeutic candidates, one a polyclonal, and the other a monoclonal antibody, to prevent alloimmunization in a novel preclinical mouse model of FNAIT.

Design and preclinical characterization of ALXN1210: A novel anti-C5 antibody with extended duration of action.

Eculizumab, a monoclonal antibody (mAb) directed against complement protein C5, is considered to be the current standard of care for patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome. This study describes the generation and preclinical attributes of ALXN1210, a new long-acting anti-C5 mAb, obtained through select modifications to eculizumab to both largely abolish target-mediated drug disposition (TMDD) and increase recycling efficiency via the neonatal Fc receptor (FcRn). To attenuate the effect of TMDD on plasma terminal half-life (t1/2), histidine substitutions were engineered into the complementarity-determining regions of eculizumab to enhance the dissociation rate of the mAb:C5 complex in the acidic early endosome relative to the slightly basic pH of blood.

Structural Basis for Eculizumab-Mediated Inhibition of the Complement Terminal Pathway.

Eculizumab is a humanized mAb approved for treatment of patients with paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Eculizumab binds complement component C5 and prevents its cleavage by C5 convertases, inhibiting release of both the proinflammatory metabolite C5a and formation of the membrane attack complex via C5b. In this study, we present the crystal structure of the complex between C5 and a Fab fragment with the same sequence as eculizumab at a resolution of 4.

Deciphering protein kinase specificity through large-scale analysis of yeast phosphorylation site motifs.

Phosphorylation is a universal mechanism for regulating cell behavior in eukaryotes. Although protein kinases target short linear sequence motifs on their substrates, the rules for kinase substrate recognition are not completely understood. We used a rapid peptide screening approach to determine consensus phosphorylation site motifs targeted by 61 of the 122 kinases in Saccharomyces cerevisiae.

Substrate discrimination among mitogen-activated protein kinases through distinct docking sequence motifs.

Mitogen-activated protein kinases (MAPKs) mediate cellular responses to a wide variety of extracellular stimuli. MAPK signal transduction cascades are tightly regulated, and individual MAPKs display exquisite specificity in recognition of their target substrates. All MAPK family members share a common phosphorylation site motif, raising questions as to how substrate specificity is achieved.

The role of membrane and vesicular monoamine transporters in the neurotoxic and hypothermic effects of 1-methyl-4-(2'-aminophenyl)-1,2,3,6-tetrahydropyridine (2'-NH(2)-MPTP).

The neurotoxin 1-methyl-4-(2'-aminophenyl)-1,2,3,6-tetrahydropyridine (2'-NH(2)-MPTP) damages forebrain serotonin (5-HT) and norepinephrine (NE) nerve terminals while sparing striatal dopaminergic innervation. Previous studies suggest that 2'-NH(2)-MPTP acts by a mechanism that involves uptake by the plasma membrane 5-HT and NE transporters. The present investigation further explores the molecular mechanism of 2'-NH(2)-MPTP with regard to cellular transport and effects on body temperature.

A faster way to make GFP-based biosensors: two new transposons for creating multicolored libraries of fluorescent fusion proteins.

Background: There are now several ways to generate fluorescent fusion proteins by randomly inserting DNA encoding the Green Fluorescent Protein (GFP) into another protein's coding sequence. These approaches can be used to map regions in a protein that are permissive for GFP insertion or to create novel biosensors. While remarkably useful, the current insertional strategies have two major limitations: (1) they only produce one kind, or color, of fluorescent fusion protein and (2) one half of all GFP insertions within the target coding sequence are in the wrong orientation.

A new way to rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction.

Background: The jellyfish green fluorescent protein (GFP) can be inserted into the middle of another protein to produce a functional, fluorescent fusion protein. Finding permissive sites for insertion, however, can be difficult. Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins.