No abstract

A single chain Fv fragment (scFv) of the murine monoclonal antibody 11-1G10 was constructed by directly joining the C-terminal residue of the VH domain to the Nterminal residue of V L . 11-1G10 is an anti-idiotype and competes with the antigen, influenza virus neuraminidase (NA), for binding to the NC41 antibody. The scFv formed stable trimers with three active antigen combining sites for NC41 Fab fragments. We propose that trimeric scFvs may be the preferred conformation for directly linked VR-VL molecules, which contrasts the formation of scFv dimers (diabodies) when the V H and V L domains are joined by short flexible linkers of between 5-10 residues. BIAcore biosensor binding experiments showed that the trimeric scFv showed an expected increase in binding affinity, due to avidity, compared to the monomeric 15-residue linked scFv. The increase in avidity of scFv trimers offers advantages for imaging and immunotherapy.

Activation of the heterotrimeric G protein Gq causes cardiomyocyte hypertrophy in vivo and in cell models. Our previous studies have shown that responses to activated Gq in cardiomyocytes are mediated exclusively by phospholipase Cβ1b (PLCβ1b), because only this PLCβ subtype localizes at the cardiac sarcolemma. In the current study, we investigated the proteins involved in targeting PLCβ1b to the sarcolemma in neonatal rat cardiomyocytes. PLCβ1b, but not PLCβ1a, coimmunoprecipitated with the high-MW scaffolding protein SH3 and ankyrin repeat protein 3 (Shank3), as well as the known Shank3-interacting protein α-fodrin. The 32-aa splice-variant-specific C-terminal tail of PLCβ1b also associated with Shank3 and α-fodrin, indicating that PLCβ1b binds via the C-terminal sequence. Shank3 colocalized with PLCβ1b at the sarcolemma, and both proteins were enriched in the light membrane fractions. Knockdown of Shank3 using siRNA reduced PLC activation and downstream hypertrophic responses, demonstrating the importance of sarcolemmal localization for PLC signaling. These data indicate that PLCβ1b associates with a Shank3 complex at the cardiac sarcolemma via its splice-variant-specific C-terminal tail. Sarcolemmmal localization is central to PLC activation and subsequent downstream signaling following Gq-coupled receptor activation.

Folate biosynthesis remains a key target for antimicrobial therapy. Folate is an essential vitamin (vitamin B9) that is required for many one-carbon transfer reactions and is a critical precursor for the biosynthesis of purines, pyrimidines, and amino acids. Unlike higher eukaryotes that scavenge preformed folates, prokaryotic and lower eukaryotic microorganisms are dependent on several enzymes for the de novo biosynthesis of folate. One of these enzymes, dihydropteroate synthase (DHPS), is the target of the first chemically-synthesized antimicrobial agents, the sulfadrugs, which date back to the 1940s. Others are essential enzymes that remain to be explored as drug targets. Resistance to the sulfadrugs rapidly emerges due to the ability of the microbe to alter its susceptibility to the drug by various means. Recently a number of new structures of the enzymes in the pathway has become available. We review the recent literature relating to these targets (the enzymes: GTP cyclohydrolase (GTP-CH); 7,8-dihydroneopterin aldolase (DHNA), 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), dihydropteroate synthase (DHPS), dihydrofolate synthase (DHFS)), their mode of action and how current drugs may modulate this on a structural level. Furthermore, these data advance our understanding of the emergence of drug resistance and may aid efforts and play a major role in the design of new, more effective compounds as antimicrobial agents. To this end we also review the recent literature in the development of inhibitors of these enzymes. Future progress in this key area has the potential to benefit the war against devastating organisms such as drug-resistant Staphylococcus aureus and Plasmodium falciparum.