Null mutations in Drosophila N-acetylglucosaminyltransferase I produce defects in locomotion and a reduced life span

UDP-GlcNAc:alpha3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I (encoded by Mgat1) controls the synthesis of hybrid, complex, and paucimannose N-glycans. Mice make hybrid and complex N-glycans but little or no paucimannose N-glycans. In contrast, Drosophila melanogaster and Caenorhabditis elegans make paucimannose N-glycans but little or no hybrid or complex N-glycans. To determine the functional requirement for beta1,2-N-acetylglucosaminyltransferase I in Drosophila, we generated null mutations by imprecise excision of a nearby transposable element. Extracts from Mgat1(1)/Mgat1(1) null mutants showed no beta1,2-N-acetylglucosaminyltransferase I enzyme activity. Moreover, mass spectrometric analysis of these extracts showed dramatic changes in N-glycans compatible with lack of beta1,2-N-acetylglucosaminyltransferase I activity. Interestingly, Mgat1(1)/Mgat1(1) null mutants are viable but exhibit pronounced defects in adult locomotory activity when compared with Mgat1(1)/CyO-GFP heterozygotes or wild type flies. In addition, in null mutants males are sterile and have a severely reduced mean and maximum life span. Microscopic examination of mutant adult fly brains showed the presence of fused beta lobes. The removal of both maternal and zygotic Mgat1 also gave rise to embryos that no longer express the horseradish peroxidase antigen within the central nervous system. Taken together, the data indicate that beta1,2-N-acetylglucosaminyltransferase I-dependent N-glycans are required for locomotory activity, life span, and brain development in Drosophila.     

Potent and cellularly active 4-aminoimidazole inhibitors of cyclin-dependent kinase 5/p25 for the treatment of Alzheimer’s disease

Utilizing structure-based drug design, a 4-aminoimidazole heterocyclic core was synthesized as a replacement for a 2-aminothiazole due to potential metabolically mediated toxicity. The synthetic route utilized allowed for ready synthesis of 1-substituted-4-aminoimidazoles. SAR exploration resulted in the identification of a novel cis-substituted cyclobutyl group that gave improved enzyme and cellular potency against cdk5/p25 with up to 30-fold selectivity over cdk2/cyclin E.     

Dendritic Spikes Amplify the Synaptic Signal to Enhance Detection of Motion in a Simulation of the Direction-Selective Ganglion Cell

The On-Off direction-selective ganglion cell (DSGC) in mammalian retinas responds most strongly to a stimulus moving in a specific direction. The DSGC initiates spikes in its dendritic tree, which are thought to propagate to the soma with high probability. Both dendritic and somatic spikes in the DSGC display strong directional tuning, whereas somatic PSPs (postsynaptic potentials) are only weakly directional, indicating that spike generation includes marked enhancement of the directional signal. We used a realistic computational model based on anatomical and physiological measurements to determine the source of the enhancement. Our results indicate that the DSGC dendritic tree is partitioned into separate electrotonic regions, each summing its local excitatory and inhibitory synaptic inputs to initiate spikes. Within each local region the local spike threshold nonlinearly amplifies the preferred response over the null response on the basis of PSP amplitude. Using inhibitory conductances previously measured in DSGCs, the simulation results showed that inhibition is only sufficient to prevent spike initiation and cannot affect spike propagation. Therefore, inhibition will only act locally within the dendritic arbor. We identified the role of three mechanisms that generate directional selectivity (DS) in the local dendritic regions. First, a mechanism for DS intrinsic to the dendritic structure of the DSGC enhances DS on the null side of the cell''s dendritic tree and weakens it on the preferred side. Second, spatially offset postsynaptic inhibition generates robust DS in the isolated dendritic tips but weak DS near the soma. Third, presynaptic DS is apparently necessary because it is more robust across the dendritic tree. The pre- and postsynaptic mechanisms together can overcome the local intrinsic DS. These local dendritic mechanisms can perform independent nonlinear computations to make a decision, and there could be analogous mechanisms within cortical circuitry.     

SELDI-TOF MS of quadruplicate urine and serum samples to evaluate changes related to storage conditions

Proteomic profiling with SELDI-TOF MS has facilitated the discovery of disease-specific protein profiles. However, multicenter studies are often hindered by the logistics required for prompt deep-freezing of samples in liquid nitrogen or dry ice within the clinic setting prior to shipping. We report high concordance between MS profiles within sets of quadruplicate split urine and serum samples deep-frozen at 0, 2, 6, and 24 h after sample collection. Gage R&R results confirm that deep-freezing times are not a statistically significant source of SELDI-TOF MS variability for either blood or urine.     

Functional post-translational proteomics approach to study the role of N-glycans in the development of Caenorhabditis elegans

Glycosylation is one of the most common post-translational protein modifications. Carbohydrate-mediated interactions between cells and their environment are important in differentiation, embryogenesis, inflammation, cancer and metastasis and other processes. Humans and mice with mutations that prevent normal N-glycosylation show multi-systemic defects in embryogenesis, thereby proving that these molecules are essential for normal development; however, a large number of proteins undergo defective glycosylation in these human and mouse mutants, and it is therefore difficult to determine the precise molecular roles of specific N-glycans on individual proteins. We describe here a 'functional post-translational proteomics' approach that is designed to determine the role of N-glycans on individual glycoproteins in the development of Caenorhabditis elegans.