To investigate the channel properties of the mammalian type 3 ryanodine receptor (RyR3), we have cloned the RyR3 cDNA from rabbit uterus by reverse transcriptase-polymerase chain reaction and expressed the cDNA in HEK293 cells. Immunoblotting studies showed that the cloned RyR3 was indistinguishable from the native mammalian RyR3 in molecular size and immunoreactivity. Ca 2؉ release measurements using the fluorescence Ca 2؉ indicator fluo 3 revealed that the cloned RyR3 functioned as a caffeine-and ryanodine-sensitive Ca 2؉ Ryanodine receptors are a family of intracellular Ca 2ϩ release channels that were originally identified in the sarcoplasmic reticulum (SR) 1 of striated muscles. To date, three members of this family have been identified in mammalian tissues, namely the skeletal muscle (RyR1), the cardiac muscle (RyR2), and the brain (RyR3) ryanodine receptor. These proteins are the products of different genes and share 66 -70% amino acid sequence identity (1-5). Earlier studies using RNA blot analysis revealed that the expression patterns of these isoforms were very different (6). RyR1 was predominantly expressed in skeletal muscle, whereas RyR2 was mainly expressed in heart and brain. The expression of RyR3 was detected in smooth muscle tissues and certain regions of the brain. However, results of recent ribonuclease protection assay demonstrate that all three RyR isoforms are widely and differentially expressed (7). These studies also indicate that most tissues express more than one RyR isoform. For example, skeletal muscles express both RyR1 and RyR3, although RyR3 is expressed at a much lower level than that of RyR1.

No abstract

How seizures start is a major question in epilepsy research. Preictal EEG changes occur in both human patients and animal models, but their underlying mechanisms and relationship with seizure initiation remain unknown. Here we demonstrate the existence, in the hippocampal CA1 region, of a preictal state characterized by the progressive and global increase in neuronal activity associated with a widespread buildup of low-amplitude high-frequency activity (HFA) (Ͼ100 Hz) and reduction in system complexity. HFA is generated by the firing of neurons, mainly pyramidal cells, at much lower frequencies. Individual cycles of HFA are generated by the near-synchronous (within ϳ5 ms) firing of small numbers of pyramidal cells. The presence of HFA in the low-calcium model implicates nonsynaptic synchronization; the presence of very similar HFA in the high-potassium model shows that it does not depend on an absence of synaptic transmission. Immediately before seizure onset, CA1 is in a state of high sensitivity in which weak depolarizing or synchronizing perturbations can trigger seizures. Transition to seizure is characterized by a rapid expansion and fusion of the neuronal populations responsible for HFA, associated with a progressive slowing of HFA, leading to a single, massive, hypersynchronous cluster generating the high-amplitude low-frequency activity of the seizure.

The results show that PE can estimate the sevoflurane drug effect more effectively than AE. This method could be applied to design a new electroencephalographic monitoring system to estimate sevoflurane anesthetic drug effect.

FcγRIIb (CD32B, Online Mendelian Inheritance in Man 604590), an IgG FcR with a tyrosine-based inhibitory motif, plays a critical role in the balance of tolerance and autoimmunity in murine models. However, the high degree of homology between FcγRIIb and FcγRIIa in humans and the lack of specific Abs to differentiate them have hampered study of the normal expression profile of FcγRIIb and its potential dysregulation in autoimmune diseases such as systemic lupus erythematosus (SLE). Using our newly developed anti-FcγRIIb mAb 4F5 which does not react with FcγRIIa, we found that FcγRIIb is expressed on the cell surface of circulating B lymphocytes, monocytes, neutrophils, myeloid dendritic cells (DCs), and at very low levels on plasmacytoid DCs from some donors. Normal donors with the less frequent 2B.4 promoter haplotype have higher FcγRIIb expression on monocytes, neutrophils, and myeloid DCs similar to that reported for B lymphocytes, indicating that FcγRIIb expression on both myeloid and lymphoid cells is regulated by the naturally occurring regulatory single nucleotide polymorphisms in the FCGR2B promoter. FcγRIIb expression in normal controls is up-regulated on memory B lymphocytes compared with naive B lymphocytes. In contrast, in active SLE, FcγRIIb is significantly down-regulated on both memory and plasma B lymphocytes compared with naive and memory/plasma B lymphocytes from normals. Similar down-regulation of FcγRIIb on myeloid-lineage cells in SLE was not seen. Our studies demonstrate the constitutive regulation of FcγRIIb by natural gene polymorphisms and the acquired dysregulation in SLE autoimmunity, which may identify opportunities for using this receptor as a therapeutic target.

HighlightbHLH115 functions downstream of the E3 ligase BRUTUS and positively controls the expression of the iron-deficiency responsive bHLH transcription factors POPEYE and bHLH38/39/100/101 for iron homeostasis in Arabidopsis.

FcγRIIb, the immunoreceptor tyrosine-based inhibitory motif-containing receptor for IgG (Mendelian Inheritance in Man no. 604590), plays an important role in maintaining the homeostasis of immune responses. We have identified 10 novel single-nucleotide polymorphisms in the promoter region of human FCGR2B gene and characterized two functionally distinct haplotypes in its proximal promoter. In luciferase reporter assays, the less frequent promoter haplotype leads to increased expression of the reporter gene in both B lymphoid and myeloid cell lines under constitutive and stimulated conditions. Four independent genome-wide scans support linkage of the human FcγR region to the systemic lupus erythematosus (SLE; Online Mendelian Inheritance in Man no. 152700) phenotype. Our case-control study in 600 Caucasians indicates a significant association of the less frequent FCGR2B promoter haplotype with the SLE phenotype (odds ratio = 1.65; p = 0.0054). The FCGR2B haplotype has no linkage disequilibrium with previously identified FCGR2A and FCGR3A polymorphisms, and after adjustment for FCGR2A and FCGR3A, FCGR2B showed a persistent association with SLE (odds ratio = 1.72; p = 0.0083). These results suggest that an expression variant of FCGR2B is a risk factor for human lupus and implicate FCGR2B in disease pathogenesis.

Results. The nonsynonymous C-to-T transition in the first cytoplasmic exon, originally reported in the Raji cell line, was not found in either the AfricanAmerican or the Caucasian population, but a nonsynonymous T-to-C transition at nucleotide 775 in exon 4 of FCGR2B, which changes isoleucine to threonine at residue 187 in the transmembrane domain, was significantly more common in African Americans. Using the Fc␥RIIb-negative mouse B cell line IIA1.6, we expressed both allelic forms as both full-length and truncated cytoplasmic domain constructs. The FCGR2B-187T allele mediated a higher level of CD19 dephosphorylation (P ‫؍‬ 0.029) and a greater degree of inhibition of the calcium response (P ‫؍‬ 0.003) when co-engaged with BCR than did FCGR2B-187I, independent of the presence of the ITIM. In contrast, Fc␥RIIb modulation of BCR-induced and anti-Fas antibody-induced cell death rates were similar in IIA1.6 cells expressing either the 187I or the 187T allelic form.Conclusion. The differential activity of FCGR2B alleles suggests a novel mechanism of Fc␥RIIb regulation that may influence the risk of autoimmune disease.The low-affinity, inhibitory IgG receptor Fc␥ receptor IIb (Fc␥RIIb) is expressed on B cells and most myeloid lineage cells. The observation that FCGR2B-deficient mice display elevated Ig levels in response to both thymus-dependent and thymus-independent antigens (1), and that, in the context of the B6 genetic background, FCGR2B-deficient mice develop a lupuslike autoimmune disease (2-4), has focused attention on FCGR2B as a candidate gene for autoimmunity.The structural basis for Fc␥RIIb function includes multiple elements involved in inhibitory or apoptotic signaling. Co-ligation of Fc␥RIIb with B cell antigen receptor (BCR) leads to inhibition of B cell activation (5,6) and causes apoptosis (7). This co-ligation results in the selective dephosphorylation of CD19 tyrosines, the tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM) in the Fc␥RIIb cytoplasmic domain, and the recruitment of SH2-containing inositol 5-phosphatase (SHIP), causing a block in calcium influx (6,(8)(9)(10)(11). Although the C-terminal 16 cytoplasmic domain residues are required for Fc␥RIIb association with and enhanced tyrosine phosphorylation of SHIP (12), Fc␥RIIb-mediated de-