Epilepsy,Behavioral Abnormalities,and Physiological Comorbidities in Syntaxin-Binding Protein 1 (STXBP1) Mutant Zebrafish

Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1), are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b) have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing “dark-flash” visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations.     

In vitro interaction between mecillinam and piperacillin-tazobactam in the presence of azithromycin against members of the Enterobacteriaceae family and Pseudomonas aeruginosa

Mecillinam was tested in vitro alone or in combination with piperacillin-tazobactam and azithromycin against representative species of the Enterobacteriaceae family and Pseudomonas aeruginosa to extend its antibacterial spectrum, and to protect mecillinam from inactivating enzymes taking advantage of the presence of tazobactam. Drug interactions were studied by microdilution method, by selection of spontaneous resistant mutants on agar plates containing the drugs in combination and by time kill experiments. Against Enterobacteriaceae mecillinam and piperacillin-tazobactam showed synergistic interaction in 24/60 tests carried out by microdilution technology, in 4/16 by selecting resistant mutants and in 5/9 by time-kill experiments. P. aeruginosa reacted indifferently to the drug combinations, with few exceptions, when azithromycin was present a reduction of the MICs were recorded. Mecillinam reacted favourably in vitro in combination with piperacillin-tazobactam against not only strains included in its antibacterial spectrum but also against resistant Morganella morganii, Proteus spp and P. aeruginosa. The addition of azithromycin (8 mg/L) was beneficial for the drug combination increasing the bactericidal effect in the great majority of the cases. Only systematic in vivo studies may establish the clinical significance and benefits of the present observations.     

The endocytic pathway followed by the keratinocyte growth factor receptor

Keratinocyte growth factor (KGF/FGF7) acts specifically on epithelial cells and regulates their proliferation and differentiation. It binds to and activates a receptor tyrosine kinase, the KGF receptor (KGFR), which is a splicing variant of the fibroblast growth factor receptor 2. The endocytic pathway followed by KGF and its receptor was analyzed here using immunofluorescence and confocal microscopy. After 10 min of internalization at 37 degrees C, both KGF and its receptor were localized in early endosomes, and after 30-60 min of endocytosis ligand and receptor were seen to reach perinuclear late endosomes and not the recycling endosomal compartment. Parallel western blot analysis revealed that KGFRs were tyrosine phosphorylated both at early and late steps of internalization, suggesting that KGF and KGFR remain associated in active complexes through the endocytic pathway. Pulse-chase experiments showed that the internalized KGFRs underwent degradation detectable at 1 h of endocytosis at 37 degrees C, indicating that KGFRs are functionally downregulated.     

Cloning and characterization of additional members of the G protein-coupled receptor family

A search of the expressed sequence tag (EST) database retrieved a human cDNA sequence which partially encoded a novel G protein-coupled receptor (GPCR) GPR26. A human genomic DNA fragment encoding a partial open reading frame (ORF) and a rat cDNA encoding the full length ORF of GPR26 were obtained by library screening. The rat GPR26 cDNA encoded a protein of 317 amino acids, most similar (albeit distantly related) to the serotonin 5-HT(5A) and gastrin releasing hormone BB2 receptors. GPR26 mRNA expression analysis revealed signals in the striatum, pons, cerebellum and cortex. HEK293 and Rh7777 cells transfected with GPR26 cDNA displayed high basal cAMP levels, slow growth rate of clonal populations and derangements of normal cell shape. We also used a sequence reported only in the patent literature encoding GPR57 (a.k.a. HNHCI32) to PCR amplify a DNA fragment which was used to screen a human genomic library. This resulted in the cloning of a genomic fragment containing a pseudogene, psiGPR57, with a 99.6% nucleotide identity to GPR57. Based on shared sequence identities, the receptor encoded by GPR57 was predicted to belong to a novel subfamily of GPCRs together with GPR58 (a.k.a. phBL5, reported only in the patent literature), putative neurotransmitter receptor (PNR) and a 5-HT(4) pseudogene. Analysis of this subfamily revealed greatest identities (approximately 56%) between the receptors encoded by GPR57 and GPR58, each with shared identities of approximately 40% with PNR. Furthermore, psiGPR57, GPR58, PNR and the 5-HT(4) pseudogene were mapped in a cluster localized to chromosome 6q22-24. PNR and GPR58 were expressed in COS cells, however no specific binding was observed for various serotonin receptor-specific ligands.     

Expression of functional mGlu5 metabotropic glutamate receptors in human melanocytes

Cultured human melanocytes express mGlu5 metabotropic glutamate (mGlu) receptors, as shown by RT-PCR, immunocytochemistry, Western blot analysis, and measurement of agonist-stimulated polyphosphoinositide hydrolysis. The mGlu5 receptor agonists (S)-3, 5-dihydroxyphenylglycine and quisqualate increased [(3)H-methyl]thymidine incorporation and melanocyte proliferation in subconfluent cultures, but impaired cell viability in confluent cultures. Both effects were prevented by 2-methyl-6-(2-phenyl-1-ethynyl)-pyridine, a potent and highly selective mGlu5 receptor antagonist. Agonists of other mGlu receptor subtypes (such as the mGlu2/3 receptor agonist, 2S,2'R,3'R-2-2', 3'-dicarboxycyclopropylglycine, or the mGlu4/6/7/8 receptor agonist, L-2-amino-4-phosphonobutanoate) or selective agonists of ionotropic glutamate receptors (N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate, and kainate) did not affect melanocyte proliferation or viability. The presence of a receptor for glutamate, the major excitatory neurotransmitter, in human melanocytes is intriguing. mGlu5 receptors may be involved in the control of melanocyte proliferation (and perhaps in other functions), but harbor a potential toxicity and may therefore contribute to cell damage under pathological conditions.     

Ubiquitination differentially regulates clathrin-dependent internalization of protease-activated receptor-1

Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is irreversibly activated by proteolysis. Consequently, PAR1 trafficking is critical for the fidelity of thrombin signaling. PAR1 displays constitutive and agonist-induced internalization, which are clathrin and dynamin dependent but are independent of arrestins. The clathrin adaptor AP2 (adaptor protein complex-2) is critical for constitutive but not for activated PAR1 internalization. In this study, we show that ubiquitination negatively regulates PAR1 constitutive internalization and specifies a distinct clathrin adaptor requirement for activated receptor internalization. PAR1 is basally ubiquitinated and deubiquitinated after activation. A PAR1 lysineless mutant signaled normally but was not ubiquitinated. Constitutive internalization of ubiquitin (Ub)-deficient PAR1 was markedly increased and inhibited by the fusion of Ub to the cytoplasmic tail. Ub-deficient PAR1 constitutive internalization was AP2 dependent like the wild-type receptor. However, unlike wild-type PAR1, AP2 was required for the internalization of activated Ub-deficient receptor, suggesting that the internalization of ubiquitinated PAR1 requires different endocytic machinery. These studies reveal a novel function for ubiquitination in the regulation of GPCR internalization.