Interaction of NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase protein, with calmodulin and PSD-95/SAP90. A possible regulatory role in molecular clustering at synaptic sites

NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase family protein, is known to bind to C-terminal ends of N-methyl-D-aspartate receptor 2B (NR2B) through its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. We have identified that NE-dlg/SAP102 interacts with calmodulin in a Ca2+-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic alpha-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, we detected specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin with a Kd value of 44 nM. However, the binding of Ca2+/calmodulin to NE-dlg/SAP102 did not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C-terminal end of rat NR2B. We have also identified that the region near the calmodulin binding site of NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. Pull down assay revealed that NE-dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca2+ and calmodulin. These findings suggest that the Ca2+/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses.     

Cloning, genomic structure and chromosomal localization of the gene encoding mouse DNA helicase RecQ helicase protein-like 4

Five members of the RecQ helicase family, RECQL, WRN, BLM, RECQL4 and RECQL5 have been identified in humans. WRN and BLM have been demonstrated to be the responsible genes in Werner and Bloom syndromes, respectively. RECQL4 (RecQ helicase protein-like 4) was identified as a fourth member of the human RecQ helicase family bearing the helicase domain, and it was subsequently shown to be the responsible gene in Rothmund-Thomson syndrome. Here, we isolated mouse RECQL4 and determined the DNA sequence of full-length cDNA as well as the genome organization and chromosome locus. The mouse RECQL4 consists of 3651 base pairs coding 1216 amino acid residues and shares 63.4% of identical and 85.8% of homologous amino acid sequences with human RECQL4. The RECQL4 gene was localized to mouse chromosome 15D3 distal-E1 and rat chromosome 7q34 proximal. They were mapped in the region where the conserved linkage homology has been identified between the two species. Twenty-two exons dispersed over 7 kilo base pairs and all of the acceptor and donor sites for splicing of each exon conformed to the GT/AG rule. Our observations regarding mouse RECQL4 gene will contribute to functional studies on the RECQL4 products.     

Role of the Mouse Retinal Photoreceptor Ribbon Synapse in Visual Motion Processing for Optokinetic Responses

The ribbon synapse is a specialized synaptic structure in the retinal outer plexiform layer where visual signals are transmitted from photoreceptors to the bipolar and horizontal cells. This structure is considered important in high-efficiency signal transmission; however, its role in visual signal processing is unclear. In order to understand its role in visual processing, the present study utilized Pikachurin-null mutant mice that show improper formation of the photoreceptor ribbon synapse. We examined the initial and late phases of the optokinetic responses (OKRs). The initial phase was examined by measuring the open-loop eye velocity of the OKRs to sinusoidal grating patterns of various spatial frequencies moving at various temporal frequencies for 0.5 s. The mutant mice showed significant initial OKRs with a spatiotemporal frequency tuning (spatial frequency, 0.09 ± 0.01 cycles/°; temporal frequency, 1.87 ± 0.12 Hz) that was slightly different from the wild-type mice (spatial frequency, 0.11 ± 0.01 cycles/°; temporal frequency, 1.66 ± 0.12 Hz). The late phase of the OKRs was examined by measuring the slow phase eye velocity of the optokinetic nystagmus induced by the sinusoidal gratings of various spatiotemporal frequencies moving for 30 s. We found that the optimal spatial and temporal frequencies of the mutant mice (spatial frequency, 0.11 ± 0.02 cycles/°; temporal frequency, 0.81 ± 0.24 Hz) were both lower than those in the wild-type mice (spatial frequency, 0.15 ± 0.02 cycles/°; temporal frequency, 1.93 ± 0.62 Hz). These results suggest that the ribbon synapse modulates the spatiotemporal frequency tuning of visual processing along the ON pathway by which the late phase of OKRs is mediated.     

Improvement of Germ Line Transmission by Targeting β-galactosidase to Nuclei in Transgenic Mice

The Escherichia coli lacZ gene has frequently been used as a reporter in cell lineage analysis, in determining the elements regulating spatial and temporal gene expression, and in enhancer/gene trap detection of developmentally regulated genes. However, it is uncertain whether lacZ expression affects eukaryotic cell growth and development. By using a gene trap, we previously isolated the promoter, Ayu1, which is active in ES cells and in several tissues including the gonads. We used this promoter and the nuclear location signal of the SV40 large T gene to locate β-galactosidase either in the cytoplasm or the nucleus. Transgenic lines containing β-galactosidase in the cytoplasm of a wide variety of cell types did not transmit the transgene to their offspring. In contrast, transgenic mice, containing β-galactosidase in the nucleus, did transmit the transgene successfully. Interestingly, lacZ expression in the brain was more restricted when β-galactosidase activity was detected in the cytoplasm. These data suggested that cytoplasmic β-galactosidase affects certain developmental processes or gametogenesis resulting in transmission distortion of the transgene, and that this effect can be reduced by targeting β-galactosidase to the nucleus. We also found that Ayu1-driven lacZ expression in the duodenum of adult transgenic mice was sexually dimorphic, being positive in females and negative in males.     

A Sensitive Radial Diffusion Method for Detection of Esterolytic Activities of Proteases

The influence of under-nutrition (sub-maintenance feeding) and ad libitum feeding on the deposition of proteins in different subcellular sarcoplasmic fractions of red (tonic) and white (phasic) muscles of growing broilers was investigated. The relative concentration of overall sarcoplasmic proteins was lesser in red than in white muscles from ad libitum fed broilers. The content of mitochondrial proteins was slightly more and that of lysosomal and microsomal proteins and of true soluble proteins was lesser in red than in white muscles. Besides, the relative amount of some specific molecular weight proteins in each subcellular fractions differed by more than 50% between red and white muscle.

There was also conspicuous differences in the influence of under-nutrition on the proteins in red and white muscles. Some reduction in mitochondrial, lysosomal, microsomal, and soluble protein content occurred only in white muscle, whereas little change was found in subcellular fractions in red muscle from underfed broilers. The relative amount of some proteins in each subcellular fraction of both muscles remained unaffected, and that of others either decreased or increased more than 20 to 50% due to nutritional stress.