Mice lacking the IFN-γ receptor or fyn develop severe experimental autoimmune uveoretinitis characterized by different immune responses

Endogenous interferon (IFN)- negatively regulates experimental autoimmune uveoretinitis (EAU), a Th1-mediated disease. Although it is well known that IFN- exerts its effects by binding to the IFN- receptor (IFN-R), the role that IFN-R plays in the development of EAU has not been investigated. Fyn has been reported to inhibit Th2 differentiation. We aimed to investigate how endogenous IFN-R and fyn, which influence Th1/Th2 differentiation, participate in the development of EAU. Sex-matched 6- to 10-week-old C57BL/6 wild-type (WT), IFN-R knockout (GRKO) and fyn knockout (fyn KO) mice were compared. Mice were immunized subcutaneously with human interphotoreceptor retinoid-binding protein peptide 1–20 emulsified in Freunds complete adjuvant together with an intraperitoneal injection of Bordetella pertussis toxin. Three weeks later, mice were sacrificed, and their eyes and spleens were harvested for histopathologic analyses and examination of cellular immune responses, respectively. Cellular immune responses were evaluated by measuring the proliferative responses and cytokine production [interleukin (IL)-4, IL-5, IL-6, IL-13, IFN- and tumor necrosis factor (TNF)-] of splenocytes. The incidence of EAU was 40.0% in WT mice, 59.3% in GRKO mice and 78.6% in fyn KO mice. The average EAU score was 0.294 in WT mice, 0.917 in GRKO mice and 1.063 in fyn KO mice. Upon EAU induction, significant infiltration of eosinophils into the eyes was observed in GRKO and fyn KO mice compared to WT mice. Splenocytes from GRKO mice proliferated against the antigen and a mitogen more vigorously than those from WT and fyn KO mice. Stimulation of splenocytes with the antigen induced a higher production of IL-4, IL-6, IL-13 and IFN- in GRKO mice compared to WT and fyn KO mice. In contrast, IL-5 and TNF- were most abundantly produced by splenocytes from fyn KO mice compared to WT and GRKO mice. The incidence and mean severity of EAU were significantly higher in GRKO and fyn KO mice than in WT mice, suggesting that endogenous IFN-R and fyn negatively regulate the development of EAU. The different cytokine production patterns by the GRKO and fyn KO mice indicate that the negative regulatory mechanism mediated by IFN-R and fyn may differ.     

Fission yeast homolog of neuronal calcium sensor-1 (Ncs1p) regulates sporulation and confers calcium tolerance

The neuronal calcium sensor (NCS) proteins (e.g. recoverin, neurocalcins, and frequenin) are expressed at highest levels in excitable cells, and some of them regulate desensitization of G protein-coupled receptors. Here we present NMR analysis and genetic functional studies of an NCS homolog in fission yeast (Ncs1p). Ncs1p binds three Ca2+ ions at saturation with an apparent affinity of 2 microm and Hill coefficient of 1.9. Analysis of NMR and fluorescence spectra of Ncs1p revealed significant Ca2+-induced protein conformational changes indicative of a Ca2+-myristoyl switch. The amino-terminal myristoyl group is sequestered inside a hydrophobic cavity of the Ca2+-free protein and becomes solvent-exposed in the Ca2+-bound protein. Subcellular fractionation experiments showed that myristoylation and Ca2+ binding by Ncs1p are essential for its translocation from cytoplasm to membranes. The ncs1 deletion mutant (ncs1Delta) showed two distinct phenotypes: nutrition-insensitive sexual development and a growth defect at high levels of extracellular Ca2+ (0.1 m CaCl(2)). Analysis of Ncs1p mutants lacking myristoylation (Ncs1p(G2A)) or deficient in Ca2+ binding (Ncs1p(E84Q/E120Q/E168Q)) revealed that Ca2+ binding was essential for both phenotypes, while myristoylation was less critical. Exogenous cAMP, a key regulator for sexual development, suppressed conjugation and sporulation of ncs1Delta, suggesting involvement of Ncs1p in the adenylate cyclase pathway turned on by the glucose-sensing G protein-coupled receptor Git3p. Starvation-independent sexual development of ncs1Delta was also complemented by retinal recoverin, which controls Ca2+-regulated desensitization of rhodopsin. In contrast, the Ca2+-intolerance of ncs1Delta was not affected by cAMP or recoverin, suggesting that the two ncs1Delta phenotypes are mechanistically independent. We propose that Schizosaccharomyces pombe Ncs1p negatively regulates sporulation perhaps by controlling Ca2+-dependent desensitization of Git3p.     

Solution structures of the N-terminal domain of yeast calmodulin: Ca2+-dependent conformational change and its functional implication

We have determined solution structures of the N-terminal half domain (N-domain) of yeast calmodulin (YCM0-N, residues 1-77) in the apo and Ca(2+)-saturated forms by NMR spectroscopy. The Ca(2+)-binding sites of YCM0-N consist of a pair of helix-loop-helix motifs (EF-hands), in which the loops are linked by a short beta-sheet. The binding of two Ca(2+) causes large rearrangement of the four alpha-helices and exposes the hydrophobic surface as observed for vertebrate calmodulin (CaM). Within the observed overall conformational similarity in the peptide backbone, several significant conformational differences were observed between the two proteins, which originated from the 38% disagreement in amino acid sequences. The beta-sheet in apo YCM0-N is strongly twisted compared with that in the N-domain of CaM, while it turns to the normal more stable conformation on Ca(2+) binding. YCM0-N shows higher cooperativity in Ca(2+) binding than the N-domain of CaM, and the observed conformational change of the beta-sheet is a possible cause of the highly cooperative Ca(2+) binding. The hydrophobic surface on Ca(2+)-saturated YCM0-N appears less flexible due to the replacements of Met51, Met71, and Val55 in the hydrophobic surface of CaM with Leu51, Leu71, and Ile55, which is thought to be one of reasons for the poor activation of target enzymes by yeast CaM.     

Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region.

A contiguous 111,402-nucleotide sequence corresponding to the 0 to 2.4 min region of the E. coli chromosome was determined as a first step to complete structural analysis of the genome. The resulting sequence was used to predict open reading frames and to search for sequence similarity against the PIR protein database. A number of novel genes were found whose predicted protein sequences showed significant homology with known proteins from various organisms, including several clusters of genes similar to those involved in fatty acid metabolism in bacteria (e.g., betT, baiF) and higher organisms, iron transport (sfuA, B, C) in Serratia marcescens, and symbiotic nitrogen fixation or electron transport (fixA, B, C, X) in Azorhizobium caulinodans. In addition, several genes and IS elements that had been mapped but not sequenced (e.g., leuA, B, C, D) were identified. We estimate that about 90 genes are represented in this region of the chromosome with little spacer.     

Effectiveness of Trivalent Inactivated Influenza Vaccine in Children Estimated by a Test-Negative Case-Control Design Study Based on Influenza Rapid Diagnostic Test Results

We assessed vaccine effectiveness (VE) against medically attended, laboratory-confirmed influenza in children 6 months to 15 years of age in 22 hospitals in Japan during the 2013–14 season. Our study was conducted according to a test-negative case-control design based on influenza rapid diagnostic test (IRDT) results. Outpatients who came to our clinics with a fever of 38°C or over and had undergone an IRDT were enrolled in this study. Patients with positive IRDT results were recorded as cases, and patients with negative results were recorded as controls. Between November 2013 and March 2014, a total of 4727 pediatric patients (6 months to 15 years of age) were enrolled: 876 were positive for influenza A, 66 for A(H1N1)pdm09 and in the other 810 the subtype was unknown; 1405 were positive for influenza B; and 2445 were negative for influenza. Overall VE was 46% (95% confidence interval [CI], 39–52). Adjusted VE against influenza A, influenza A(H1N1)pdm09, and influenza B was 63% (95% CI, 56–69), 77% (95% CI, 59–87), and 26% (95% CI, 14–36), respectively. Influenza vaccine was not effective against either influenza A or influenza B in infants 6 to 11 months of age. Two doses of influenza vaccine provided better protection against influenza A infection than a single dose did. VE against hospitalization influenza A infection was 76%. Influenza vaccine was effective against influenza A, especially against influenza A(H1N1)pdm09, but was much less effective against influenza B.     

The inhibitors of cyclin-dependent kinases and GSK-3β enhance osteoclastogenesis

Osteoclasts are multinucleated cells with bone resorption activity that is crucial for bone remodeling. RANK‐RANKL (receptor activator of nuclear factor κB ligand) signaling has been shown as a main signal pathway for osteoclast differentiation. However, the molecular mechanism and the factors regulating osteoclastogenesis remain to be fully understood. In this study, we performed a chemical genetic screen, and identified a Cdks/GSK-3β (cyclin-dependent kinases/glycogen synthase kinase 3β) inhibitor, kenpaullone, and two Cdks inhibitors, olomoucine and roscovitine, all of which significantly enhance osteoclastogenesis of RAW264.7 cells by upregulating NFATc1 (nuclear factor of activated T cells, cytoplasmic 1) levels. We also determined that the all three compounds increase the number of osteoclast differentiated from murine bone marrow cells. Furthermore, the three inhibitors, especially kenpaullone, promoted maturation of cathepsin K, suggesting that the resorption activity of the resultant osteoclasts is also activated. Our findings indicate that inhibition of GSK-3β and/or Cdks enhance osteoclastogenesis by modulating the RANK–RANKL signaling pathway.     

Atkinsiella parasitica sp. nov. isolated from a rotifer,Brachionus plicatilis

A new species ofAtkinsiella (Lagenidiales, Oomycetes),Atkinsiella parasitica is described and illustrated. It was isolated from the eggs and bodies of a rotifer,Brachionus plicatilis, with fungal infection in 1992. The rotifer was reared in a hatchery as the first food supply for seed production of crustaceans and fishes. The fungus is characterized by producing monoplanetic, lateral biflagellate zoospores, and infrequently branched discharge tubes. Optimum temperature for the fungus was 25°C. The fungus was considered an obligate marine fungus, because its growth was observed only on PYGS medium including seawater.     

Studies on the reaction intermediate of protocatechuate 3,4-dioxygenase. Formation of enzyme-product complex.

The nature of the oxygenated intermediate observed (Fujisawa, H., Hiromi, K., Uyeda, M., Okuno, S., Nozaki, M. and Hayaishi, O. (1972) J. Biol. Chem. 247, 4422--4428) during the reaction of protocatechuate 3,4-dioxygenase (protocatechuate:oxygen 3,4-oxidoreductase (decyclizing), EC 1.13.11.3) was investigated. 3,4-Dihydroxyphenylpropionic acid and 3,4-dihydroxyphenylacetic acid were used as substrates of the enzyme to slow down the rate of the reaction. The enzyme reactions were performed under conditions where the concentration of the organic substrate was lower than those of the enzyme and oxygen in the reaction mixture. The reactions were stopped before completion by the addition of hydrochloric acid or guanidine hydrochloride and then the organic compounds were extracted from the reaction mixture to be analyzed. The qualitative analyses by thin-layer chromatography revealed that there was no species other than the organic substrate and the enzymatic reaction end-product during reaction. The quantitative spectrophotometric analyses revealed that the organic substrate which had participated in the formation of the oxygenated intermediate existed as a species indistinguishable from the reaction end-product, indicating that the oxygenated intermediate was not a simple complex of oxygen, substrate and the enzyme, i.e., a ternary complex, but a species rather close to a binary complex of product and the enzyme.     

Molecular cloning of a unique CMP-sialic acid synthetase that effectively utilizes both deaminoneuraminic acid (KDN) and N-acetylneuraminic acid (Neu5Ac) as substrates

2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a sialic acid (Sia) that is ubiquitously expressed in vertebrates during normal development and tumorigenesis. Its expression is thought to be regulated by multiple biosynthetic steps catalyzed by several enzymes, including CMP-Sia synthetase. Using crude enzyme preparations, it was shown that mammalian CMP-Sia synthetases had very low activity to synthesize CMP-KDN from KDN and CTP, and the corresponding enzyme from rainbow trout testis had high activity to synthesize both CMP-KDN and CMP-N-acetylneuraminic acid (Neu5Ac) (Terada et al. [1993] J. Biol. Chem., 268, 2640-2648). To demonstrate if the unique substrate specificity found in the crude trout enzyme is conveyed by a single enzyme, cDNA cloning of trout CMP-Sia synthetase was carried out by PCR-based strategy. The trout enzyme was shown to consist of 432 amino acids with two potential nuclear localization signals, and the cDNA sequence displayed 53.8% identity to that of the murine enzyme. Based on the Vmax/Km values, the recombinant trout enzyme had high activity toward both KDN and Neu5Ac (1.1 versus 0.68 min(-1)). In contrast, the recombinant murine enzyme had 15 times lower activity toward KDN than Neu5Ac (0.23 versus 3.5 min(-1)). Northern blot analysis suggested that several sizes of the mRNA are expressed in testis, ovary, and liver in a tissue-specific manner. These results indicate that at least one cloned enzyme has the ability to utilize both KDN and Neu5Ac as substrates efficiently and is useful for the production of CMP-KDN.     

Interleukin-1β Induces Substance P Release from Primary Afferent Neurons Through the Cyclooxygenase-2 System

Substance P (SP) is synthesized in the dorsal root ganglion (DRG) and released from primary afferent neurons to convey information regarding noxious stimuli. The effects of the proinflammatory cytokine interleukin-1 (IL-1) beta on the release of SP were investigated using primary cultured rat DRG cells. Recombinant mouse IL-1beta added to the cells at 0.1 ng/ml increased the SP-like immunoreactivity (SPLI) in the culture medium after incubation for 6 h by approximately 50% as compared with that of nontreated DRG cells. The effect of IL-1beta was Ca(2+)-dependent and significantly inhibited by 100 ng/ml IL-1 receptor-specific antagonist (IL-1r antagonist), cyclooxygenase (COX) inhibitors such as 0.1 mM aspirin, 1 microg/ml indomethacin, and 1 microM NS-398 (specific for COX-2), and 1 microM dexamethasone. Furthermore, a 1-h incubation with IL-1beta markedly increased the inducible COX-2 mRNA level, which was inhibited by an IL-1r antagonist and dexamethasone, whereas IL-1beta showed no effect on the level of constitutive COX-1 mRNA. These observations indicated that IL-1beta induced the release of SP from the DRG cells via specific IL-1 receptors, the mechanism of which might involve prostanoid systems produced by COX-2. This could be responsible for the hyperalgesic action with reference to inflammatory pain in the primary afferent neuron to spinal cord pathway.