The serine/threonine kinase SAD regulates neural functions such as axon/dendrite polarization and neurotransmitter release. In the vertebrate central nervous system, SAD-B, a homolog of Caenorhabditis elegans SAD-1, is associated with synaptic vesicles and the active zone cytomatrix in nerve terminals. However, the distribution of SAD-B in the peripheral nervous system remains elusive. Here, we show that SAD-B is specifically localized to neuromuscular junctions. Although the active zone protein bassoon showed a punctated signal indicating its localization to motor end plates, SAD-B shows relatively diffuse localization indicating its association with both the active zone and synaptic vesicles. Therefore, SAD kinase may regulate neurotransmitter release from motor end plates in a similar manner to its regulation of neurotransmitter release in the central nervous system.

BackgroundTranscription factor nuclear factor-κB (NF-κB) plays a key role in the regulation of immune responses to inflammation. However, convenient assay systems to quantitate the NF-κB activity level in a timely manner are not available in the setting of clinical laboratories. Therefore, we developed a novel and high-throughput quantitative assay based on fluorescence correlation spectroscopy (FCS) to detect the NF-κB activity level in cellular nuclear extracts and evaluated the performance of this method. The basic principle of this assay is to calculate the binding fraction of NF-κB to fluorescent-labeled DNA probes, which contain NF-κB binding sites.MethodsNon-fluorescent competitive probes are employed to normalize the influence of the viscosity of the nuclear extracts between samples and to eliminate the influence of nonspecific binding of the fluorescent probes. To confirm accurate quantitation, human recombinant NF-κB p50 was mixed into U937 cell nuclear extracts, and the binding fraction of the fluorescent probes to NF-κB in the mixture was calculated for quantitation. To evaluate whether this method can be applied to measure the NF-κB activity in human lymphocytes, the NF-κB activity levels of systemic inflammatory response syndrome patients during perioperative periods were measured.ResultsThe percentage recovery was 88.9%. The coefficients of variation of the intra-assay were approximately 10%. NF-κB activity levels during the perioperative period can were successfully measured. The assay time for the FCS measurement was within 20 minutes.ConclusionsThis assay system can be used to quantitate NF-κB activity levels in a timely manner in the setting of hospital laboratories.

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