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Mutations of the genes encoding T‐cell receptor (TCR)‐proximal signaling molecules, such as ZAP‐70, can be causative of immunological diseases ranging from T‐cell immunodeficiency to T‐cell–mediated autoimmune disease. For example, SKG mice, which carry a hypomorphic point mutation of the Zap‐70 gene, spontaneously develop T‐cell–mediated autoimmune arthritis immunopathologically similar to human rheumatoid arthritis (RA). The Zap‐70 mutation alters the sensitivity of developing T cells to thymic positive/negative selection by self‐peptides/MHC complexes, shifting self‐reactive TCR repertoire to include a dominant arthritogenic specificity and also affecting thymic development and function of autoimmune suppressive regulatory T (Treg) cells. Polyclonal self‐reactive T cells, including potentially arthritogenic T cells, thus produced by the thymus recognize self‐peptide/MHC complexes on antigen‐presenting cells (APCs) in the periphery and stimulate them to produce cytokines including IL‐6 to drive the arthritogenic T cells to differentiate into arthritogenic T‐helper 17 (Th17) cells. Insufficient Treg suppression or activation of APCs via microbial and other environmental stimuli evokes arthritis by activating granulocyte‐macrophage colony‐stimulating factor‐secreting effector Th17 cells, mediating chronic bone‐destructive joint inflammation by activating myeloid cells, innate lymphoid cells, and synoviocytes in the joint. These findings obtained from the study of SKG mouse arthritis are instrumental in understanding how arthritogenic T cells are produced, become activated, and differentiate into effector T cells mediating arthritis, and may help devising therapeutic measures targeting autoimmune pathogenic Th17 cells or autoimmune‐suppressing Treg cells to treat and prevent RA. [ABSTRACT FROM AUTHOR]

Aim: The syndrome of periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) is a common inflammatory disease that presents with periodic fever. We aimed to establish more specific diagnostic criteria for PFAPA based on the clinical characteristics of PFAPA patients in our directory. Method: The clinical, laboratory, genetic, and family history details of 257 Japanese PFAPA patients treated at our and other affiliated hospitals between April 2000 and April 2018 were analyzed along with quantitative measurements of the number of CD64 molecules on neutrophils, and the levels of serum inflammatory cytokines. The sensitivity and specificity of the criteria were calculated for several diseases. Results: Because recurrent fevers were crucial findings, they were defined as the required criterion. Tonsillitis/pharyngitis with white moss were important accompanying signs. Other symptoms associated with febrile episodes were cervical lymphadenitis with tenderness, aphthous stomatitis, sore throat, vomiting, and headache but not cough. A total of 159 (62%) patients had a family history of recurrent fevers, indicating autosomal dominant inheritance. C‐reactive protein levels were extremely elevated during febrile attacks but normal in attack‐free periods. Serum immunoglobulin D levels were high in 72 of the 199 tested patients. Oral glucocorticoid and cimetidine were extremely effective in all and 51.6% of the patients, respectively. We defined the above as supportive criteria. These criteria were sensitive and specific enough to distinguish PFAPA from other recurrent fever diseases. Raised serum interferon‐γ levels and remarkable CD64 expression on neutrophils during flare‐ups were recognized, indicating they contributed to diagnosis. Conclusion: Our new criteria are useful for diagnosing PFAPA. [ABSTRACT FROM AUTHOR]

Ca2+/calmodulin-dependent protein kinase (CaMK)II is highly expressed in the CNS and mediates activity-dependent neuronal plasticity. Four CaMKII isoforms, α, β, γ and δ, have a large number of splicing variants. Here we identified isoforms of CaMKII in the rat substantia nigra (SN). Northern blot and RT–PCR analyses revealed that the γ and δ isoform mRNAs with several splicing variants were predominantly expressed in SN. Immunoblot analysis indicated that the major isoforms were γA, γC, δ1 and δ3. An immunohistochemical study also confirmed the preferential localization of γ and δ isoforms in SN dopaminergic neurons. In dopaminergic neurons, immunoreactivity against anti-CaMKIIδ1–4 antibody was detected in both nucleus and cytoplasm, in contrast to the predominant expression of γ isoforms in the cytoplasm. Furthermore, we showed expression of brain-derived neurotrophic factor (BDNF) mRNAs with exons II and IV in SN. Taken together with our previous observations, the results suggest that the CaMKIIδ3 isoform is involved in the expression of BDNF in the SN. [ABSTRACT FROM AUTHOR]

Dopamine D2 receptor (D2R) has known to activate Ca2+/calmodulin-dependent protein phosphatase, calcineurin by increasing in the intracellular Ca2+. We previously showed that D2LR (long isoform) and D2SR (short isoform) enhanced SRE and NF-κB, and conversely suppressed CRE transcriptional activities in NG108-15 cells stably expressed with these receptors (NGD2LR and NGD2SR). In this study, to investigate whether activation of calcineurin is involved in the transcriptional regulations through D2R, we evaluated effect of cyclosporin A, a selective calcineurin inhibitor, on them in NGD2LR and NGD2SR cells using luciferase reporter gene assay. We first confirmed that D2LR activates calcineurin in NG108-15 cells by measurement of dephosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein Mr 32 000 (DARPP-32) at threonin 34 by immunoblot analysis with its phospho-specific antibody. Cyclosporin A treatment showed no change in suppression of forskolin-induced CRE activation or activation of SRE but significantly attenuated NF-κB activation by D2LR stimulation in NGD2LR cells. Interestingly, D2SR-induced NF-κB activation, which was weaker than that by D2LR stimulation, was not affected by cyclosporin A treatment in NGD2SR cells. Furthermore, D2SR stimulation did not cause dephosphorylation of DARPP-32 at threonin 34. Taken together, D2SR and D2LR may employ different signaling pathway on intracellular Ca2+ mobilization, thereby showing different NF-κB activation in the calcineurin-dependent manner. [ABSTRACT FROM AUTHOR]

To investigate whether the third cytoplasmic loop and the C-terminal cytoplasmic tail of dopamine D2 receptor (D2R) are involved in extracellular signal-regulated kinase (ERK) acti- vation and subsequent regulation of transcription factors, we established NG108-15 cells stably expressing D2LR and D2SR deleted 40 amino acid residues in the third cytoplasmic loop (NGD2LR-3rd-dele and NGD2SR-3rd-dele) or the C-terminal cytoplasmic tail (NGD2LR-C-dele and NGD2SR-C-dele) and evaluated these receptors' functions using luci-ferase reporter gene assay. Immunocytochemical studies showed similar intracellular distributions of D2LR-3rd-dele and D2SR-3rd-dele to D2LR and D2SR, respectively. Quinpirole- induced inhibition of forskolin-induced cyclic AMP responsive element (CRE) activation was not affected by the deletion of 40 amino acid residues. However, nuclear factor-kappa B (NF-κB) activation by D2R-3rd-dele was largely attenuated compared to that by D2R. Similarly, ERK or serum-responsive element (SRE) activation by quinpirole treatment was totally abolished in NGD2R-3rd-dele cells. Moreover, D2R-C-dele was diffusely distributed or clustered in the cell bodies and lost the receptor functions. Taken together, the 40 amino acid residues in the third cytoplasmic loop are essential for the ERK activation but not for inhibition of adenylyl cyclase through Gi/o proteins. In addition, the C-terminal cytoplasmic tail is essential for membrane association of D2Rs to elicit the receptor functions. [ABSTRACT FROM AUTHOR]

We previously showed that dopamine receptors D1R and D2R expressed in NG108-15 cells activated protein kinase A and extracellular signal-regulated kinase (ERK) respectively, resulting in differential activation of nuclear factor (NF)-κB activity. To investigate whether other dopamine receptor subtypes regulate NF-κB, we established NG108-15 cells stably expressing D3R, D4R and D5R (NGD3R, NGD4R and NGD5R). D5R stimulation with SKF 38393 decreased NF-κB luciferase reporter activity in NGD5R cells, similar to D1R stimulation in NGD1R cells. However, D3R or D4R stimulation with quinpirole showed no change in NF-κB-Luci activity, although forskolin-induced cyclic AMP responsive element-Luci activation was attenuated by quinpirole treatment in NGD2LR, NGD3R and NGD4R cells. As expected, activation of ERK or serum responsive element- luciferase reporter not observed following stimulation with quinpirole in D3R- or D4R-expressing cells. We further examined the effects of haloperidol and risperidone, which are typical and atypical antipsychotic drugs respectively, on NF-κB activity by gel shift assay in mouse frontal cortex. Haloperidol treatment slightly attenuated basal NF-κB activity. By contrast, risperidone treatment enhanced NF-κB activity. Taken together, D2R and D1R/D5R had opposite effects on NF-κB activity in NG108-15 cells. Risperidone up-regulated and haloperidol down-regulated NF-κB activity in mouse brain. This effect may be related to the atypical antipsychotic properties of risperidone. [ABSTRACT FROM AUTHOR]

Abstract The dopamine D[sub 2] receptor (D2R) is target for antipsychotic drugs and associated with several neuropsychiatric disorders. D2R has a long third cytoplasmic loop and a short carboxyl-terminal cytoplasmic tail. It exists as two alternatively spliced isoforms, termed D2LR and D2SR, which differ in the presence and absence, respectively, of a 29 amino acid insert in the third cytoplasmic loop. To evaluate the differential roles of the two D2R isoforms, we transfected both isoforms into NG108-15 cells and observed their subcellular localization by a confocal laser scanning light microscope. D2SR was predominantly localized at the plasma membrane, whereas D2LR was mostly retained in the perinuclear region around the Golgi apparatus. Using a yeast two hybrid system with a mouse brain cDNA library and coimmunoprecipitation assay, we found that heart-type fatty acid binding protein (H-FABP) interacts with D2LR but not with D2SR. H-FABP is a cytosolic protein involved in binding and transport of fatty acids. Overexpressed H-FABP and endogenous H-FABP were colocalized with the intracellular D2LR in NG108-15 cells. Furthermore, in the rat striatum, H-FABP was detected in the D2R-expressing neurons. From these results, H-FABP is associated with D2LR, and may thereby modulate the subcellular localization and function of D2LR. [ABSTRACT FROM AUTHOR]

Abstract To investigate transcriptional regulation by dopamine receptors, we established NG108-15 cells stably expressing D1R, D2LR and D2SR (NGD1R, NGD2LR and NGD2SR) and evaluated the effects of these receptors on NF-κB, SRE and CRE activity using luciferase reporter constructs. Stimulation with quinpirole, a selective D2R agonist, increased NF-κB and SRE activity but decreased CRE activity in both NGD2R cell lines. By contrast, stimulation with SKF 38393, a selective D1R agonist, decreased NF-κB and SRE activity but increased CRE activity in NGD1R cells. Stimulation with forskolin and overexpression of constitutively active PKA suppressed NF-κB activity, likely due to D1R stimulation. D2R stimulation activated ERK, and treatment with U1026, a selective MEK inhibitor, eliminated D2R-induced NF-κB activation. D2R stimulation also activated the neural cell adhesion molecule (NCAM) promoter, which includes a potential NF-κB site. Furthermore, by transfecting constitutively active CaM KII and MEKK, and dominant negative p38 MAPK, we show that the NCAM promoter is positively regulated by CaM KII but negatively regulated by p38 MAPK. These results indicate that D2R-induced NF-κB activation through ERK may be involved in activation of the NCAM promoter, and additionally that other protein kinases such as CaM KII and p38 MAPK also regulate NCAM expression. [ABSTRACT FROM AUTHOR]

We identified the isoforms of Ca[sup 2+]/calmodulin-dependent protein kinase II (CaM kinase II) subunits in rat striatum. All four subunits of CaM kinase II α, β, γ and δ were detected including the isoforms of αB, γA, γA', γA.B, δ3 and δ7 with nuclear localization signal. We established NG108-15 cells with the stably expressed dopamine D2L receptor (D2LR, long form), which is an alternative splicing variant. The cells were termed NGD2L. Immunostaining demonstrated that D2LR was localized in plasma membranes. Calcium imaging with fluo-3 AM revealed that quinpirole, a D2R agonist, increased the intracellular Ca[sup 2+], which was blocked by treatment with sulpiride and pertussis toxin in NGD2L cells, but not in mock cells. Furthermore, stimulation of D2LR with quinpirole in NGD2L cells activated the nuclear isoform of CaM kinase II. Stimulation of D2LR increased the expression of exon III- and IV-BDNF mRNA. Overexpression of CaM kinase II δ3 increased exon IV- but not exon III-BDNF mRNA. These results suggest that D2R is involved in the activation of the nuclear isoform of CaM kinase II and thereby in stimulation of gene expression through Ca[sup 2+] signaling. [ABSTRACT FROM AUTHOR]

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) occurs in astrocytes as well as in neurons in brain. We have reported that CaM kinase II is involved in the regulation of cytoskeletal proteins and gene expression in astrocytes. In this study, we identified all isoforms of CaM kinase II in astrocytes and examined their subcellular localization. When we amplified the isoforms of four subunits by RT-PCR followed by the “nested” PCR, totally 10 isoforms were obtained. Immunoblot analyses with five types of antibodies against CaM kinase II indicated that the most abundant isoform was δ2. Immunostaining suggested that the δ2 isoform was localized predominantly at the Golgi apparatus. The localization of the δ2 isoform at the Golgi apparatus was also observed in NG108-15 cells. We overexpressed all isoforms that contained the nuclear localization signal to examine their nuclear targeting in NG108-15 cells. In contrast to the αB and δ3 isoforms that entered the nucleus, as reported, the γA isoform was excluded from the nucleus in the transfected NG108-15 cells. These results suggest that the 15-amino acid insertion following the nuclear localization signal inhibits the nuclear targeting of the γA isoform. [ABSTRACT FROM AUTHOR]

We have reported that the delta3 isoform of Ca[sup2plus]/calmodulin-dependent protein kinase II (CaM kinase II) is abundant in the nucleus in cerebellar granule cells. To examine the possibility that the nuclear isoforms of CaM kinase II are involved in the expression of brain-derived neurotrophic factor (BDNF), we transiently overexpressed the delta3 isoform in NG108-15 cells. The quantitative RT-PCR analysis revealed that rat cerebellum and NG108-15 cells expressed the exon IV-containing mRNA of BDNF (exon IV-BDNF mRNA) more than the exon III-BDNF mRNA. Treatment of NG108-15 cells with Bay K 8644 increased both exon III- and exon IV-BDNF mRNAs, and overexpression of the delta3 isoform potentiated the expression of the exon IV-BDNF mRNA. The potentiation was not observed in the cells that were overexpressed with either the d1 isoform, a nonnuclear isoform, or the inactive mutant of the delta3 isoform. We constructed the luciferase reporter gene following the promoter upstream of exon IV and confirmed that overexpression of the delta3 isoform increased luciferase gene expression. Double-immuno-staining of NG108-15 cells with the antibodies to CaM kinase II and BDNF clearly showed that BDNF was highly expressed in the cells that were overexpressed with the delta3 isoform or the alphaB isoform, another nuclear isoform of CaM kinase II. These results suggest that the nuclear isoforms of CaM kinase II are involved in the expression of BDNF. [ABSTRACT FROM AUTHOR]

Abstract : To examine the physiological roles of the δ subunit of Ca2+ /calmodulin-dependent protein kinase ∥ (CaM kinase ∥δ) in brain, we examined the localization of CaM kinase ∥δ in the rat brain. A specific antibody to CaM kinase ∥δ1-δ4 isoforms was prepared by immunizing rabbits with a synthesized peptide corresponding to the unique carboxyl-terminal end of these isoforms. The prepared antibody did not recognize the α, β, and γ subunits, which were each overexpressed in NG108-15 cells. Immunoblot analysis on various regions and the nuclear fractions from rat brains suggested that some isoforms of CaM kinase ∥δ1-δ4 were abundant in the nucleus in the cerebellum. Total RNA from the cerebellum was analyzed by RT-PCR with a primer pair from variable domain 1 to variable domain 2. We detected the three PCR products δ3.1, δ3.4, and δ3 that contained the nuclear localization signal. These CaM kinase ∥δ3 isoforms were localized in the nuclei in transfected NG108-15 cells. Immunohistochemical study suggested the existence of these isoforms in the nuclei in cerebellar granule cells. These results suggest that CaM kinase ∥δ3 isoforms are involved in nuclear Ca2+ signaling in cerebellar granule cells. [ABSTRACT FROM AUTHOR]

Summary: Nyctinasty is the circadian rhythmic nastic movement of leguminous plants in response to the onset of darkness, a unique and intriguing phenomenon that has attracted attention for centuries. The movement itself is caused by the asymmetric volume change of motor cells between the adaxial and abaxial sides of the leaflet. Recently, we identified the ion channels responsible for the volume change of motor cells during the leaf‐opening process of Samanea saman; the asymmetric expression of SsSLAH1, which is under the control of SsCCA1, was found to play a key role in this process. Here, we summarize the history of the study of nyctinasty, our current results and several insights for further study. [ABSTRACT FROM AUTHOR]

Abstract: Background: Although the incidence of neonatal sepsis is decreasing, neonatal sepsis remains a severe life‐threatening disease. No current biochemical marker can provide perfect diagnostic accuracy for neonatal sepsis. The aim of this study was therefore to evaluate the accuracy of presepsin (P‐SEP) as a novel biomarker of bacterial infection for neonatal sepsis diagnosis. Methods: We prospectively studied newborns with sepsis (sepsis group; n = 13) during the first 30 days after birth and compared them with control preterm newborns (control group; n = 18). In addition, we evaluated term newborns with some clinical signs of early onset sepsis (non‐sepsis term group; n = 35). Results: P‐SEP in the sepsis group was significantly higher than in the control group (P < 0.001) The area under the curve for P‐SEP was 0.868 (95%CI: 0.71–1.00). A P‐SEP cut‐off of 795 pg/mL was established, with 85% sensitivity and 89% specificity. The positive and negative predictive values were 85% and 89%, respectively. In the non‐sepsis term group, P‐SEP had better stability than white blood cells and C‐reactive protein for 3 days after birth. Conclusions: P‐SEP can better discriminate between infections and non‐infectious inflammatory conditions than the currently used biomarkers. [ABSTRACT FROM AUTHOR]

Multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) including CaMKI, II and IV, are thought to regulate a variety of neuronal functions. Unlike CaMKII, which is regulated by autophosphorylation, CaMKI as well as CaMKIV are activated by CaMKK. In this study, we examined the cellular and subcellular localization of CaMKIδ, a recently identified fourth isoform of CaMKI, in the mature brain. In situ hybridization analysis demonstrated wide expression of CaMKIδ mRNA in the adult mouse brain with prominent expression in the hippocampal pyramidal cells. FLAG-tagged CaMKIδ was localized at the cytoplasm and neurites without nuclear immunoreactivity in approximately 80% of the transfected primary hippocampal neurons. The stimulation with either KCl depolarization or glutamate triggered the nuclear localization of FLAG-tagged CaMKIδ by two-fold with a peak at 1 min. In contrast, the catalytically inactive mutants of CaMKIδ remained cytoplasmic without nuclear translocation during KCl depolarization, indicating the requirement of its activation for the nuclear translocation. Furthermore, we showed that immunoprecipitated CaMKIδ could phosphorylate cAMP response element binding protein (CREB)α in vitro and that the over-expression of CaMKIδ enhanced GAL4-CREB-luciferase activity in PC12 cells stimulated by KCl depolarization. Our present study provides the first evidence for the possible involvement of CaMKIδ in nuclear functions through its nuclear translocation in response to stimuli that trigger intracellular Ca2+ influx. [ABSTRACT FROM AUTHOR]