Social memory,amnesia, and autism: Brain oxytocin secretion is regulated by NAD+ metabolites and single nucleotide polymorphisms of CD38

Previously, we demonstrated that CD38, a transmembrane protein with ADP-ribosyl cyclase activity, plays a critical role in mouse social behavior by regulating the release of oxytocin (OXT), which is essential for mutual recognition. When CD38 was disrupted, social amnesia was observed in Cd38 knockout mice. The autism spectrum disorders (ASDs), characterized by defects in reciprocal social interaction and communication, occur either sporadically or in a familial pattern. However, the etiology of ASDs remains largely unknown. Therefore, the theoretical basis for pharmacological treatments has not been established. Hence, there is a rationale for investigating single nucleotide polymorphisms (SNPs) in the human CD38 gene in ASD subjects. We found several SNPs in this gene. The SNP rs3796863 (C > A) was associated with high-functioning autism (HFA) in American samples from the Autism Gene Resource Exchange. Although this finding was partially confirmed in low-functioning autism subjects in Israel, it has not been replicated in Japanese HFA subjects. The second SNP of interest, rs1800561 (4693C > T), leads to the substitution of an arginine (R) at codon 140 by tryptophan (W; R140W) in CD38. This mutation was found in four probands of ASD and in family members of three pedigrees with variable levels of ASD or ASD traits. The plasma levels of OXT in ASD subjects with the R140W allele were lower than those in ASD subjects lacking this allele. The OXT levels were unchanged in healthy subjects with or without this mutation. One proband with the R140W allele receiving intranasal OXT for approximately 3 years showed improvement in areas of social approach, eye contact and communication behaviors, emotion, irritability, and aggression. Five other ASD subjects with mental deficits received nasal OXT for various periods; three subjects showed improved symptoms, while two showed little or no effect. These results suggest that SNPs in CD38 may be possible risk factors for ASD by abrogating OXT function and that some ASD subjects can be treated with OXT in preliminary clinical trials.     

Anti-inflammatory effect of proteoglycan and progesterone on human uterine cervical fibroblasts

AimsThe aim of this study was to compare the anti-inflammatory effect of proteoglycan (PG) with that of progesterone (P) in the cultured fibroblasts from human uterine cervix.Main methodsAfter obtaining informed consent, the cervix was collected from normal women undergoing total hysterectomy. The cervix was cultured until fibroblasts proliferated and had grown to confluence, then, the fibroblasts were stimulated by lipopolysaccharide (LPS) with or without PG, P and a combination of both; they were cultured for 24–48 h. The anti-inflammatory effects of PG and P were evaluated by the suppression of IL-6 or IL-8 secretion. The expression of the IL-6 or IL-8 gene and the expression of their protein were determined by real-time PCR, and ELISA, respectively. Activation of Toll-like receptor (TLR) 4 was evaluated by Western blotting.Key findingsLPS markedly enhanced gene and protein expression of IL-6 and IL-8 in human uterine cervical fibroblasts. The up-regulation of the IL-6 or IL-8 gene and protein expression by LPS was significantly suppressed with PG, P and a combination of both. Western blotting revealed that combination of PG and P showed more potent inhibition on LPS-stimulated TLR4 induction than that seen by each.SignificanceThis study showed that both PG and P have an inhibitory effect on LPS-induced inflammation. This anti-inflammatory effect of PG and P was augmented by co-administration of both, suggesting for the first time that PG has an anti-inflammatory effect on human uterine cervical fibroblasts.     

Serial assembly of Thermus megaplasmid DNA in the genome of Bacillus subtilis 168: a BAC-based domino method applied to DNA with a high GC content

Bacillus subtilis is the only bacterium-based host able to clone giant DNA above 1000 kbp. DNA previously handled by this host was limited to that with GC content similar to or lower than that of the B. subtilis genome. To expand the target DNA range to higher GC content, we tried to clone a pTT27 megaplasmid (257 kbp, 69% of G+C) from Thermus thermophilus. To facilitate the reconstruction process, we subcloned pTT27 in a bacterial artificial chromosome (BAC) vector of Escherichia coli. Owing to the ability of BAC to carry around 100 kbp DNA, only 4 clones were needed to cover the pTT27 and conduct step-by-step assembly in the B. subtilis genome. The full length of 257 kbp was reconstructed through 3 intermediary lengths (108, 153, and 226 kbp), despite an unexpected difficulty in the maintenance of DNA >200 kbp. Retrieval of these four pTT27 segments from the B. subtilis genome by genetic transfer to a plasmid pLS20 was attempted. A stable plasmid clone was obtained only for the 108 and 153 kbp intermediates. The B. subtilis genome was demonstrated to accommodate large DNA with a high GC content, but may be restricted to less than 200 kbp by unidentified mechanisms.     

Molecular Properties and Extracellular Processing of the Lipase of Staphylococcus warneri M

Staphylococcus warneri M exhibited extracellular lipase activity. By zymogram analysis of extracellular proteins, multiple bands were detected and the profiles changed depending on the bacterial growth phase. N-terminal amino acid sequences of three bands (N1-N3) were determined. From the genome library of S. warneri M whole DNA, the gene-directing lipase activity (named gehC(WM)) was cloned and characterized. The gehC(WM )gene encoded a protein (GehC(WM)), whose calculated molecular mass was 83.4 kDa, and the sequence was similar to the other staphylococcal lipases. Though two lipases have been known from S. warneri 863, GehC(WM) differs from both of them, indicating that this enzyme is the third extracellular lipase of the S. warneri strain. The N-terminal sequences of the N1-N3 polypeptides completely coincided with the deduced amino acid sequences in GehC(WM). GehC(WM) was predicted to be a prepro-protein. In vitro processing and protein sequencing suggested that pro-GehC(WM) is possibly processed by extracellular glutamyl endopeptidase, PROM. Inductively coupled plasma-atomic emission spectrometer analysis showed that purified his-tagged mature GehC(WM) possessed zinc ion. A gehC(WM) knockout mutant was constructed by insertion of an erythromycin resistance gene into the gehC(WM). Zymogram and immunoblot analyses of the gehC(WM )mutant indicated that GehC(WM) was a major extracellular lipase of S. warneri M.     

Type IV pilin is glycosylated in Pseudomonas syringae pv. tabaci 6605 and is required for surface motility and virulence

Type IV pilin (PilA) is a major constituent of pilus and is required for bacterial biofilm formation, surface motility and virulence. It is known that mature PilA is produced by cleavage of the short leader sequence of the pilin precursor, followed by methylation of N-terminal phenylalanine. The molecular mass of the PilA mature protein from the tobacco bacterial pathogen Pseudomonas syringae pv. tabaci 6605 (Pta 6605) has been predicted to be 12 329 Da from its deduced amino acid sequence. Previously, we have detected PilA as an approximately 13-kDa protein by immunoblot analysis with anti-PilA-specific antibody. In addition, we found the putative oligosaccharide-transferase gene tfpO downstream of pilA. These findings suggest that PilA in Pta 6605 is glycosylated. The defective mutant of tfpO (ΔtfpO) shows reductions in pilin molecular mass, surface motility and virulence towards host tobacco plants. Thus, pilin glycan plays important roles in bacterial motility and virulence. The genetic region around pilA was compared among P. syringae pathovars. The tfpO gene exists in some strains of pathovars tabaci, syringae, lachrymans, mori, actinidiae, maculicola and P. savastanoi pv. savastanoi. However, some strains of pathovars tabaci, syringae, glycinea, tomato, aesculi and oryzae do not possess tfpO, and the existence of tfpO is independent of the classification of pathovars/strains in P. syringae. Interestingly, the PilA amino acid sequences in tfpO-possessing strains show higher homology with each other than with tfpO-nonpossessing strains. These results suggest that tfpO and pilA might co-evolve in certain specific bacterial strains.     

Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K

Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤ 50 °C) was 10 °C higher than that for CITase-T3040 (≤ 40 °C); the k_cat/K_M value of CITase-598K was approximately two times higher (32.2 s^− 1 mM^− 1) than that of CITase-T3040 (17.8 s^− 1 mM^− 1). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.     

Saturated fatty acid and TLR signaling link β cell dysfunction and islet inflammation

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Highlights? Palmitate induces β cell dysfunction by activating inflammatory processes in islets ? β cells sense palmitate via the TLR4 pathway and recruit M1 macrophages to islets ? M1 macrophages play a pivotal role in palmitate-induced β cell dysfunction ? M1 macrophages and inflammation also play a role in β cell dysfunction in T2D models     

T-type Ca2+ channel blockers prevent cardiac cell hypertrophy through an inhibition of calcineurin-NFAT3 activation as well as L-type Ca2+ channel blockers

T-type Ca2+ channels (TCCs) are involved in cardiac cell growth and proliferation in cultured cardiomyocytes. Underlying molecular mechanisms are not well understood. In this study, we investigated the role of TCCs in signal transduction in cardiac hypertrophy compared with L-type Ca2+ channels (LCCs). Cardiomyocytes dissociated from neonatal mouse ventricles were cultured until stabilization. Cell hypertrophy was induced by reapplication of 1% fatal bovine serum (FBS) following a period (24 h) of FBS depletion. Cell surface area increased from 862+/-73 microm2 to 2153+/-131 microm2 by FBS stimulation in control (250+/-1.8%). T-type Ca2+ current (I(CaT)) was inhibited dose-dependently by kurtoxin (KT) and efonidipine (ED) with IC50 0.07 microM and 3.2 microM, respectively in whole-cell voltage clamp. On the other hand, 1 microM KT which inhibits I(CaT) over 90% did not effect on L-type Ca2+ current (I(CaL)). 10 microM ED had the ability of I(CaL) blockade as well as that of I(CaT) blockade. 3 microM nisoldipine (ND) suppressed I(CaL) by over 80%. The increase in cell surface area following reapplication of FBS as observed in control (250+/-1.8%) was significantly reduced in the presence of 1 microM KT (216+/-1.2%) and virtually abolished in the presence of 10 microM ED (97+/-0.8%) and 3 microM ND (80+/-1.1%). Hypertrophy was associated with an increase in BNP mRNA of 316+/-3.6% in control and this increase was reduced as well in the presence of 1 microM KT (254+/-1.8%) and almost abolished in the presence of 10 microM ED (116+/-1.1%) and 3 muM ND (93+/-0.8%). Immunolabeling showed that translocation of nuclear factor of activated T cells (NFAT3) into the nucleus in response to FBS stimulation was markedly inhibited by either KT or ED as well as ND. Calcineurin phosphatase activity was upregulated 2.2-fold by FBS, but KT, ED and ND decreased this upregulation (1.7-fold, 0.8-fold, and 0.7-fold with KT, ED and ND respectively). These results suggest that blockade of Ca2+ entry into cardiomyocytes via TCCs may block pathophysiological signaling pathways leading to hypertrophy as well as via LCCs. The mechanism may be the inhibition of calcineurin-mediated NFAT3 activation resulting in prevention of its translocation into the nucleus.     

The novel mutant scl of the medaka fish, Oryzias latipes, shows no secondary sex characters

A new mutant that has neither male nor female secondary sex characters was found in the medaka, Oryzias latipes. Both XX and XY mature mutants had gonads with many spermatozoa, but spawning did not occur when the mutants were paired with normal males or normal females. F1 progeny were successfully obtained by artificial insemination using unfertilized eggs from wild-type females and spermatozoa of the XY mutant. The mutant phenotype did not occur in the F1 progeny from this cross. Incrossing among the F1 progeny produced 17 mutant offspring out of 68 progeny (25%), demonstrating that the mutant phenotype is caused by a single recessive mutation. This mutant was named scl (sex character-less). Because papillary processes, a male secondary sex character, were induced in the XY mutants by androgen administration, it seems that the androgen receptor is functioning normally. We found a loss-of-function type mutation in the P450c17 gene of the mutant; this gene encodes a steroidogenic enzyme required for the production of estrogen and androgen. The scl phenotype was completely linked to the mutant genotype of P450c17, strongly suggesting that mutation at the P450c17 locus is responsible for the scl mutant phenotype.