Regulation of human autoimmune regulator (AIRE) gene translation by miR-220b

Although mutations of autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), presenting a wide spectrum of many characteristic and non-characteristic clinical features, some patients lack AIRE gene mutations. Therefore, something other than a mutation, such as dysregulation of AIRE gene, may be a causal factor for APECED or its related diseases. However, regulatory mechanisms for AIRE gene expression and/or translation have still remained elusive. We found that IL-2-stimulated CD4⁺ T (IL-2T) cells showed a high expression of AIRE gene, but very low AIRE protein production, while Epstein–Barr virus-transformed B (EBV-B) cells express both AIRE gene and AIRE protein. By using microarray analysis, we could identify miR-220b as a possible regulatory mechanism for AIRE gene translation in IL-2T cells. Here we report that miR-220b significantly reduced the expression of AIRE protein in AIRE gene with 3′UTR region transfected 293T cells, whereas no alteration of AIRE protein production was observed in the open reading frame of AIRE gene alone transfected cells. In addition, anti-miR-220b reversed the inhibitory function of miR-220b for the expression of AIRE protein in AIRE gene with 3′UTR region transfected cells. Moreover, when AIRE gene transfected cells with mutated 3′UTR were transfected with miR-220b, no reduction of AIRE protein production was observed. Taken together, it was concluded that miR-220b inhibited the AIRE gene translation through the 3′UTR region of AIRE gene, indicating that miR-220b could serve as a regulator for human AIRE gene translation.     

Quantitative analysis of cortical actin filaments during polar body formation in starfish oocytes

Polar body formation is an extremely unequal cell division. In order to understand the mechanism of polar body formation, morphological changes at the animal pole were investigated in living oocytes of the starfish, Asterina pectinifera, and the amounts of cortical actin filaments were quantitatively estimated after staining the maturing oocytes with fluorescently-labeled phallotoxins using a computer and image-processing software. Formation of a bulge, which is presumed to become a polar body, and the anaphase separation of chromosomes occurred simultaneously. When the bulge became large, one group of chromatids moved into the bulge. The dividing furrow then formed and finally a polar body formed. Just at the time of bulge formation, the intensity of the fluorescence produced by the actin filaments at the top of the animal pole began to decrease, and subsequently the intensity at the top fell to half of the original value. On the other hand, the fluorescence intensity at the base of the bulge increased gradually. This actin accumulation at the base created a dividing furrow around the top of the animal pole as the bulge grew. Even when the polar body formation was inhibited mechanically, a similar pattern of actin deficiency and accumulation in the cortex near the animal pole was observed. This indicates that such regulation of filamentous actin can take place without bulging. Therefore, polar body formation is initiated by the bulging of the cortex weakened by actin deficiency and followed by contraction of the base of the bulge reinforced by actin accumulation.     

Involvement of sphingosine-1-phosphate in glutamate secretion in hippocampal neurons

Neuronal activity greatly influences the formation and stabilization of synapses. Although receptors for sphingosine-1-phosphate (S1P), a lipid mediator regulating diverse cellular processes, are abundant in the central nervous system, neuron-specific functions of S1P remain largely undefined. Here, we report two novel actions of S1P using primary hippocampal neurons as a model system: (i) as a secretagogue where S1P triggers glutamate secretion and (ii) as an enhancer where S1P potentiates depolarization-evoked glutamate secretion. Sphingosine kinase 1 (SK1), a key enzyme for S1P production, was enriched in functional puncta of hippocampal neurons. Silencing SK1 expression by small interfering RNA as well as SK1 inhibition by dimethylsphingosine resulted in a strong inhibition of depolarization-evoked glutamate secretion. Fluorescence recovery after photobleaching analysis showed translocation of SK1 from cytosol to membranes at the puncta during depolarization, which resulted in subsequent accumulation of S1P within cells. Fluorescent resonance energy transfer analysis demonstrated that the S1P(1) receptor at the puncta was activated during depolarization and that depolarization-induced S1P(1) receptor activation was inhibited in SK1-knock-down cells. Importantly, exogenously added S1P at a nanomolar concentration by itself elicited glutamate secretion from hippocampal cells even when the Na(+)-channel was blocked by tetrodotoxin, suggesting that S1P acts on presynaptic membranes. Furthermore, exogenous S1P at a picomolar level potentiated depolarization-evoked secretion in the neurons. These findings indicate that S1P, through its autocrine action, facilitates glutamate secretion in hippocampal neurons both by secretagogue and enhancer actions and may be involved in mechanisms underlying regulation of synaptic transmission.     

Modulation of Fas-FasL related apoptosis by PBN in the early phases of choline deficient diet-mediated hepatocarcinogenesis in rats

This study focused on the detection of apoptosis related events in very early phases of choline-deficient (CD)-induced hepatocarcinogenesis (at 2-5 weeks). Flow cytometry of isolated intact primary hepatocytes from CD diet fed rats indicated increased expression of the apoptosis-associated protein Fas. Increased apoptosis in CD-treated livers was confirmed by Western blot analyses of caspases and cytochrome c. This study was also able to detect differences in apoptotic events following phenyl butyl nitrone (PBN) treatment. Fas expression was inhibited by PBN, indicating that PBN is anti-apoptotic. It is speculated that in the early stages of CD-induced hepatotoxicity, PBN is involved in inhibiting pro-inflammatory factor-driven apoptosis of normal hepatocytes, which protects against the initiation of carcinogenesis. The CD diet model is also considered as a model for non-alcoholic steatohepatitis (NASH) in humans and early expression of Fas could also be a good index of the progression of NASH.     

Establishment of Tools for Neurogenetic Analysis of Sexual Behavior in the Silkmoth,Bombyx mori

Background

Silkmoth, Bombyx mori, is an ideal model insect for investigating the neural mechanisms underlying sex pheromone-induced innate behavior. Although transgenic techniques and the GAL4/UAS system are well established in the silkmoth, genetic tools useful for investigating brain function at the neural circuit level have been lacking.

Results

In the present study, we established silkmoth strains in which we could visualize neural projections (UAS-mCD8GFP) and cell nucleus positions (UAS-GFP.nls), and manipulate neural excitability by thermal stimulation (UAS-dTrpA1). In these strains, neural projections and nucleus position were reliably labeled with green fluorescent protein in a GAL4-dependent manner. Further, the behavior of silkworm larvae and adults could be controlled by GAL4-dependent misexpression of dTrpA1. Ubiquitous dTrpA1 misexpression led both silkmoth larvae and adults to exhibit seizure-like phenotypes in a heat stimulation-dependent manner. Furthermore, dTrpA1 misexpression in the sex pheromone receptor neurons of male silkmoths allowed us to control male sexual behavior by changing the temperature. Thermally stimulated male silkmoths exhibited full sexual behavior, including wing-flapping, orientation, and attempted copulation, and precisely approached a thermal source in a manner similar to male silkmoths stimulated with the sex pheromone.

Conclusion

These findings indicate that a thermogenetic approach using dTrpA1 is feasible in Lepidopteran insects and thermogenetic analysis of innate behavior is applicable in the silkmoth. These tools are essential for elucidating the relationships between neural circuits and function using neurogenetic methods.     

Functional role of residue 193 (chymotrypsin numbering) in serine proteases: influence of side chain length and beta-branching on the catalytic activity of blood coagulation factor XIa

In serine proteases, Gly193 (chymotrypsin numbering) is conserved with rare exception. Mutants of blood coagulation proteases have been reported with Glu, Ala, Arg or Val substitutions for Gly193. To further understand the role of Gly193 in protease activity, we replaced it with Ala or Val in coagulation factor XIa (FXIa). For comparison to the reported FXIa Glu193 mutant, we prepared FXIa with Asp (short side chain) or Lys (opposite charge) substitutions. Binding of p-aminobenzamidine (pAB) and diisopropylfluorphosphate (DFP) were impaired 1.6-36-fold and 35-478-fold, respectively, indicating distortion of, or altered accessibility to, the S1 and oxyanion-binding sites. Val or Asp substitutions caused the most impairment. Salt bridge formation between the amino terminus of the mature protease moiety at Ile16 and Asp194, essential for catalysis, was impaired 1.4-4-fold. Mutations reduced catalytic efficiency of tripeptide substrate hydrolysis 6-280-fold, with Val or Asp causing the most impairment. Further studies were directed toward macromolecular interactions with the FXIa mutants. kcat for factor IX activation was reduced 8-fold for Ala and 400-1100-fold for other mutants, while binding of the inhibitors antithrombin and amyloid beta-precursor protein Kunitz domain (APPI) was impaired 13-2300-fold and 22-27000-fold, respectively. The data indicate that beta-branching of the side chain of residue 193 is deleterious for interactions with pAB, DFP and amidolytic substrates, situations where no S2'-P2' interactions are involved. When an S2'-P2' interaction is involved (factor IX, antithrombin, APPI), beta-branching and increased side chain length are detrimental. Molecular models indicate that the mutants have impaired S2' binding sites and that beta-branching causes steric conflicts with the FXIa 140-loop, which could perturb the local tertiary structure of the protease domain. In conclusion, enzyme activity is impaired in FXIa when Gly193 is replaced by a non-Gly residue, and residues with side chains that branch at the beta-carbon have the greatest effect on catalysis and binding of substrates.     

Allele-selective effect of PA28 in MHC class I antigen processing

PA28 is an IFN-gamma-inducible proteasome activator and its genetic ablation causes complete loss of processing of certain Ags, but not all of them. The reason why this occurs and how PA28 influences the formation of peptide repertoires for MHC class I molecules remains unknown. In this study, we show the allele-specific role of PA28 in Ag processing. Retrovirus-transduced overexpression of PA28alpha decreased expression of K(d) (D(d)) while it increased K(b) and L(d) on the cell surface. By contrast, overexpression of PA28alphaDeltaC5, a mutant carrying a deletion of its five C-terminal residues and capable of attenuating the activity of endogenous PA28, produced the opposite effect on expression of those MHC class I molecules. Moreover, knockdown of both PA28alpha and beta by small-interfering RNA profoundly augmented expression of K(d) and D(d), but not of L(d), on the cell surface. Finally, we found that PA28-associated proteasome preferentially digested within epitopic sequences of K(d), although correct C-terminal flankings were removed, which in turn hampered production of K(d) ligands. Our results indicate that whereas PA28 negatively influences processing of K(d) (D(d)) ligands, thereby, down-regulating Ag presentation by those MHC class I molecules, it also efficiently produces K(b) (L(d)) epitopes, leading to up-regulation of the MHC molecules.     

The interaction of the Rieske iron-sulfur protein with occupants of the Qo-site of the bc1 complex, probed by electron spin echo envelope modulation.

The bifurcated reaction at the Q(o)-site of the bc(1) complex provides the mechanistic basis of the proton pumping activity through which the complex conserves redox energy in the proton gradient. Structural information about the binding of quinone at the site is lacking, because the site is vacant in crystals of the native complexes. We now report the first structural characterization of the interaction of the native quinone occupant with the Rieske iron-sulfur protein in the bc(1) complex of Rhodobacter sphaeroides, using high resolution EPR. We have compared the binding configuration in the presence of quinone with the known structures for the complex with stigmatellin and myxothiazol. We have shown by using EPR and orientation-selective electron spin echo envelope modulation (ESEEM) measurements of the iron-sulfur protein that when quinone is present in the site, the isotropic hyperfine constant of one of the N(delta) atoms of a liganding histidine of the [2Fe-2S] cluster is similar to that observed when stigmatellin is present and different from the configuration in the presence of myxothiazol. The spectra also show complementary differences in nitrogen quadrupole splittings in some orientations. We suggest that the EPR characteristics, the ESEEM spectra, and the hyperfine couplings reflect a similar interaction between the iron-sulfur protein and the quinone or stigmatellin and that the N(delta) involved is that of a histidine (equivalent to His-161 in the chicken mitochondrial complex) that forms both a ligand to the cluster and a hydrogen bond with a carbonyl oxygen atom of the Q(o)-site occupant.     

Molecular cloning of rat mitochondrial glutamic oxaloacetic transaminase mRNA and regulation of its expression in regenerating liver

cDNA clones for rat mitochondrial glutamic oxaloacetic transaminase (mGOT) have been isolated from a rat liver cDNA library. One of the clones, designated p501, contained a cDNA insert of 1.4 kilobase pairs in length and hybridized to a mRNA of 2.4 kilobases from rat liver.We measured mGOT mRNA content in a regenerating rat liver. In a regenerating rat liver, mGOT activity was increased and reached maximum (170% of control activity) at about 48 h following the operation. Using the cDNA of mGOT, it was revealed that the increase of mGOT in the regenerating rat liver depended on its mRNA content.     

miR-21 Gene expression triggered by AP-1 is sustained through a double-negative feedback mechanism

Methylation of cytosine residues in CpG dinucleotides plays an important role in epigenetic regulation of gene expression and chromatin structure/stability in higher eukaryotes. DNA methylation patterns are established and maintained at CpG dinucleotides by DNA methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b). In mammals and many other eukaryotes, the CpG dinucleotide is underrepresented in the genome. This loss is postulated to be the result of unrepaired deamination of cytosine and 5-methylcytosine to uracil and thymine, respectively. Two thymine glycosylases are believed to reduce the impact of 5-methylcytosine deamination. G/T mismatch-specific thymine-DNA glycosylase (Tdg) and methyl-CpG binding domain protein 4 can both excise uracil or thymine at U·G and T·G mismatches to initiate base excision repair. Here, we report the characterization of interactions between Dnmt3b and both Tdg and methyl-CpG binding domain protein 4. Our results demonstrate (1) that both Tdg and Dnmt3b are colocalized to heterochromatin and (2) reduction of T·G mismatch repair efficiency upon loss of DNA methyltransferase expression, as well as a requirement for an RNA component for correct T·G mismatch repair.