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Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved pleiotropic neuropeptide, implicated in emotional stress responses and anxiety-related disorders. Here, we examined whether our recently developed small-molecule non-peptide PACAP receptor antagonists could ameliorate anxiety-like behaviors induced by acute restraint stress in mice. The antagonists PA-9 and its derivative PA-915 improved anxiety-like behaviors in mice subjected to restraint stress. An anxiolytic effect was observed with single acute dose, suggesting their fast-acting properties. PA-915 demonstrated a statistically significant anxiolytic effect whereas fluoxetine did not. These results indicate the potential of PAC1 antagonists as a novel treatment for anxiety., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this study., (Copyright © 2022. Published by Elsevier Inc.)

Neurite outgrowth is important in neuronal circuit formation and functions, and for regeneration of neuronal networks following trauma and disease in the brain. Thus, identification and characterization of the molecules that regulate neurite outgrowth are essential for understanding how brain circuits form and function and for the development of treatment of neurological disorders. In this study, we found that structurally different lysophosphatidylethanolamine (LPE) species, palmitoyl-LPE (16:0 LPE) and stearoyl-LPE (18:0 LPE), stimulate neurite growth in cultured cortical neurons. Interestingly, YM-254890, an inhibitor of Gq/11 protein, inhibited 16:0 LPE-stimulated neurite outgrowth but not 18:0 LPE-stimulated neurite outgrowth. In contrast, pertussis toxin, an inhibitor of Gi/Go proteins, inhibited 18:0 LPE-stimulated neurite outgrowth but not 16:0 LPE-stimulated neurite outgrowth. The effects of protein kinase C inhibitors on neurite outgrowth were also different. In addition, both 16:0 LPE and 18:0 LPE activate mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2, but the effect of the MAPK inhibitor differed between the 16:0 LPE- and 18:0 LPE-treated cultures. Collectively, the results suggest that the structurally different LPE species, 16:0 LPE and 18:0 LPE stimulate neurite outgrowth through distinct signaling cascades in cultured cortical neurons and that distinct G protein-coupled receptors are involved in these processes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

CRISPR/Cas9-mediated gene knock-in in in vivo neurons using in utero electroporation is a powerful technique, but the knock-in efficiency is generally low. We previously demonstrated that co-transfection with RAD51, a key molecule of the initial step of homology-directed repair (HDR), expression vector increased EGFP knock-in efficiency in the β-actin site up to 2.5-fold in the pyramidal neurons in layer 2/3 of the somatosensory cortex of mouse brain. To further improve the efficiency, we examined the effect of inhibition of DNA ligase IV (LIG4) that is an essential molecule for non-homologous end joining (NHEJ). Co-transfection with small hairpin RNA for LIG4 (shlig4) expression vector increased the EGFP knock-in efficiency in the β-actin site up to 3.6-fold compared to the condition without shlig4. RAD51 and shlig4 expression vector co-transfection further increased the knock-in efficiency up to 4.7-fold of the control condition. These results suggest that the inhibition of LIG4 is more effective than RAD51 overexpression, and it enhances the effect of RAD51 overexpression on HDR-mediated gene knock-in in vivo neurons., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The biosynthesis of polyunsaturated fatty acids (PUFAs) in bacteria has been extensively studied. In contrast, studies of PUFA metabolism remain limited. Shewanella livingstonensis Ac10 is a psychrotrophic bacterium producing eicosapentaenoic acid (EPA), a long-chain ω-3 PUFA. This bacterium has the ability to convert exogenous docosahexaenoic acid (DHA) into EPA and incorporate both DHA and EPA into membrane phospholipids. Our previous studies revealed the importance of 2,4-dienoyl-CoA reductase in the conversion, suggesting that DHA is metabolized through a general β-oxidation pathway. Herein, to gain further insight into the conversion mechanism, we analyzed the role of acyl-CoA dehydrogenase (FadE), the first committed enzyme of the β-oxidation pathway, in DHA conversion. S. livingstonensis Ac10 has two putative FadE proteins (FadE1 and FadE2) that are highly homologous to Escherichia coli FadE. We found that FadE1 expression was induced by addition of DHA to the medium and fadE1 deletion reduced DHA conversion into EPA. Consistently, purified FadE1 exhibited dehydrogenase activity towards DHA-CoA. Moreover, its activity towards DHA- and EPA-CoAs was higher than that towards palmitoleoyl- and palmitoyl-CoAs. In contrast, fadE2 deletion did not impair DHA conversion, and purified FadE2 had higher activity towards palmitoleoyl- and palmitoyl-CoAs than towards DHA- and EPA-CoAs. These results suggest that FadE1 is the first enzyme of the β-oxidation pathway that catalyzes DHA conversion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A hyper-vesiculating Gram-negative bacterium, Shewanella vesiculosa HM13, secretes a protein of unknown function (P49) as a major cargo of the extracellular membrane vesicles (EMVs). Here, we analyzed the transport mechanism of P49 to EMVs. The P49 gene is found in a gene cluster containing the genes encoding homologs of surface glycolipid biosynthesis proteins (Wza, WecA, LptA, and Wzx), components of type II secretion system (T2SS), glycerophosphodiester phosphodiesterase (GdpD), and nitroreductase (NfnB). We disrupted the genes in this cluster and analyzed the productivity and morphology of EMVs and the localization of P49. EMV production and morphology were only moderately affected by gene disruption, demonstrating that these gene products are not essential for EMV synthesis. In contrast, the localization of P49 was significantly affected by gene disruption. The lack of homologs of the T2SS components resulted in deficiency in secretion of P49. When gdpD, wzx, lptA, and nfnB were disrupted, P49 was released to the extracellular space without being loaded to the EMVs. These results suggest that P49 is translocated across the outer membrane through the T2SS-like machinery and subsequently loaded onto EMVs through interaction with surface glycolipids of EMVs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Gene knock-in using the CRISPR/Cas9 system can be achieved in a specific population of neurons in the mouse brain, by using in utero electroporation to introduce DNA fragments into neural progenitor cells. Using this strategy, we previously knocked-in the EGFP coding sequence into the N-terminal region of the β-actin gene specifically in the pyramidal neurons in layer 2/3 of the somatosensory cortex. However, the knock-in efficiency was less than 2% of the transfected neurons. In this study, we sought to improve the knock-in efficiency using this system. First, we varied the length of the homology arms of the β-actin donor template DNA, and found that the knock-in efficiency was increased to ∼14% by extending the length of the 5' and 3' homology arms to 1.6 kb and 2.0 kb, respectively. We then tested the effect of the DNA repair protein RAD51 and the knock-in efficiency was increased up to 2.5-fold when co-transfecting with two different β-actin and a camk2a targeting EGFP knock-in modules. The RAD51 overexpression did not alter the migration of developing neurons, density or morphology of the dendritic spines compared to those in neurons not transfected with RAD51. RAD51 expression will be useful for increasing the knock-in efficiency in neurons in vivo by CRISPR/Cas9-mediated homology directed repair (HDR)., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cereblon (CRBN) was identified as a gene that causes intellectual disabilities. The encoded CRBN protein, containing 442 amino acids, is located in several organs. Cytosolic CRBN was reported to mainly act as a component of the E3 ubiquitin ligase complex. CRBN is one of the substrate receptors of the E3 ubiquitin ligase complex and promotes the degradation of targeted proteins. Studies have reported that CRBN recognizes the C-terminal region of the amyloid precursor protein (APP), a protein known for its involvement in the development of Alzheimer's disease. Although CRBN may interact with the C-terminal region of APP in mice, the CRBN-mediated degradation mechanism of human APP remains unclear. Here, we analyzed the CRBN-mediated degradation mechanism of human APP via the ubiquitin-proteasome system. Immunoprecipitation experiments showed that CRBN interacts with human full-length APP via its C-terminal region. Next, we examined CRBN-mediated degradation of APP in the ubiquitin-proteasome system. CRBN recognizes Lys 751 in human APP and ubiquitinates it in SH-SY5Y cells. Overexpression of CRBN decreased wild-type APP expression levels. In contrast, the expression level of K751R APP remained unchanged by CRBN overexpression, while knockdown of endogenous CRBN increased APP levels. As such, our results suggest that CRBN ubiquitinates Lys 751 of human APP thereby degrading it via the ubiquitin-proteasome system., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

1-Acyl-sn-glycerol-3-phosphate O-acyltransferase (PlsC) plays an essential role in the formation of phosphatidic acid, a precursor of various membrane phospholipids (PLs), in bacteria by catalyzing the introduction of an acyl group into the sn-2 position of lysophosphatidic acid. Various bacteria produce more than one PlsC. However, the physiological significance of the occurrence of multiple PlsCs is poorly understood. A psychrotrophic bacterium, Shewanella livingstonensis Ac10, which produces eicosapentaenoic acid at low temperatures, has five putative PlsCs (PlsC1-5). We previously showed that PlsC1 is responsible for the production of PLs containing an eicosapentaenoyl group. Here, we characterized another putative PlsC of this bacterium named PlsC4. We generated a plsC4-disrupted mutant and found that PLs containing 13:0 found in the parental strain were almost completely absent in the mutant. The loss of these PLs was suppressed by introduction of a plsC4-expression plasmid. PLs containing 15:0 were also drastically decreased by plsC4 disruption. Gas chromatography-mass spectrometry analysis of fatty acyl methyl esters derived from PLs of the parental strain showed that the 13:0 and 15:0 groups were an 11-methyllauroyl group and a 13-methylmyristoyl group, respectively. Phospholipase A2 treatment revealed that these fatty acyl groups were linked to the sn-2 position of PLs. Thus, PlsC4 is a new type of PlsC homolog that is responsible for the synthesis of PLs containing a branched-chain fatty acyl group at the sn-2 position and plays a clearly different role from that of PlsC1 in vivo., (Copyright © 2018 Elsevier Inc. All rights reserved.)

CD4(+) T cell activation is controlled by signaling through the T cell receptor in addition to various co-receptors, and is also affected by their interactions with effector and regulatory T cells in the microenvironment. Inflammatory bowel diseases (IBD) are caused by the persistent activation and expansion of auto-aggressive CD4(+) T cells that attack intestinal epithelial cells. However, the molecular basis for the persistent activation of CD4(+) T cells in IBD remains unclear. In this study, we investigated how the CD98 heavy chain (CD98hc, Slc3a2) affected the development of colitis in an experimental animal model. Transferring CD98hc-deficient CD4(+)CD25(-) T cells into Rag2(-/-) mice did not cause colitis accompanied by increasing Foxp3(+) inducible regulatory T cells. By comparison, CD98hc-deficient naturally occurring regulatory T cells (nTregs) had a decreased capability to suppress colitis induced by CD4(+)CD25(-) T cells, although CD98hc-deficient mice did not have a defect in the development of nTregs. Blocking CD98hc with an anti-CD98 blocking antibody prevented the development of colitis. Our results indicate that CD98hc regulates the expansion of autoimmune CD4(+) T cells in addition to controlling nTregs functions, which suggests the CD98hc as an important target molecule for establishing strategies for treating colitis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), are found in various cold-adapted microorganisms. We previously demonstrated that EPA-containing phospholipids (EPA-PLs) synthesized by the psychrotrophic bacterium Shewanella livingstonensis Ac10 support cell division, membrane biogenesis, and the production of membrane proteins at low temperatures. In this article, we demonstrate the effects of EPA-PLs on the folding and conformational transition of Omp74, a major outer membrane cold-inducible protein in this bacterium. Omp74 from an EPA-less mutant migrated differently from that of the parent strain on SDS-polyacrylamide gel, suggesting that EPA-PLs affect the conformation of Omp74 in vivo. To examine the effects of EPA-PLs on Omp74 protein folding, in vitro refolding of recombinant Omp74 was carried out with liposomes composed of 1,2-dipalmitoleoyl-sn-glycero-3-phosphoglycerol and 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (1:1 molar ratio) with or without EPA-PLs as guest lipids. SDS-PAGE analysis of liposome-reconstituted Omp74 revealed more rapid folding in the presence of EPA-PLs. CD spectroscopy of Omp74 folding kinetics at 4 °C showed that EPA-PLs accelerated β-sheet formation. These results suggest that EPA-PLs act as chemical chaperones, accelerating membrane insertion and secondary structure formation of Omp74 at low temperatures., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Phosphatidylserine (PS), a relatively abundant component of mammalian cell membranes, plays important roles in biological processes including apoptosis and cell signaling. It is believed that phosphatidyl-L-serine is the only naturally occurring PS. Here, we describe for the first time the occurrence of phosphatidyl-D-serine (D-PS) in rat cerebrum. Quantitative HPLC analysis of the derivatives of serine liberated from PS by hydrolysis revealed that the amount of D-PS was approximately 1% of the total PS in the cerebrum. Enzymatic cleavage of cerebrum PS with phospholipase D and phospholipase C resulted in the release of both isomers of serine and phosphoserine, respectively, providing additional evidence for the existence of D-PS. Free D-serine was incorporated into PS in an in vitro system using a cerebrum extract, and this activity was inhibited by EDTA, suggesting the occurrence of a divalent cation-dependent enzyme that synthesizes D-PS by a base-exchange reaction.

53BP1 plays important roles in checkpoint signaling and repair for DNA double-strand breaks. We found that a colon cancer cell line, SW48, expressed a splicing variant form of 53BP1, which lacks the residues corresponding to exons 10 and 11. Activation of ATM and phosphorylation of ATM and ATR targets occurred in SW48 cells in response to X-irradiation, and these X-ray-induced responses were not enhanced by expression of full-length 53BP1 in SW48 cells, indicating that this splicing variant fully activates the major checkpoint signaling in SW48 cells. In contrast, the expression of full-length 53BP1 in SW48 cells promoted the repair of X-ray-induced DNA damage, evidenced by faster disappearance of X-ray-induced gamma-H2AX foci, a marker for DNA damage, and less residual chromosomal aberrations after X-irradiation. We conclude that the two major roles of 53BP1, the checkpoint signaling and repair for DNA damage, can be functionally separated.

Inactivation of iscS encoding cysteine desulfurase results in a slow growth phenotype associated with the deficiency of iron-sulfur clusters, thiamine, NAD, and tRNA thionucleosides in Escherichia coli. However, the other roles of iscSin vivo are unknown. By using differential screening strategies, we identified 2 pyrimidine salvage enzymes, namely, uridine phosphorylase and cytidine deaminase, which were down-regulated in the iscS mutant. Both enzymes are positively regulated by the cAMP receptor protein (CRP). We also identified a novel protein complex, namely, YeiT-YeiA, whose expression level was decreased in the iscS mutant. The recombinant YeiT-YeiA complex exhibited NADH-dependent dihydropyrimidine dehydrogenase activity, indicating its role in pyrimidine metabolism. The presence of a CRP-binding consensus sequence on the 5'-upstream of the yeiT-YeiA gene suggests its regulation by CRP. These results provide a clue to the possible role of iscS in pyrimidine metabolism by gene regulation.

To elucidate the mechanism for the regulation of aspartate kinase (AK) via feedback inhibition, we constructed several chimeric enzymes between Bacillus subtilis AK II, a lysine-sensitive mesophilic enzyme, and Thermus flavus AK, a threonine-sensitive thermostable enzyme, each having the same alpha2beta2-type tetrameric structure. A chimeric AK, named BTT, composed of the chimeric alpha subunit that comprises of the N-terminal catalytic region from B. subtilis AK II and the C-terminal region from T. flavus, and the beta subunit from T. flavus, was inhibited only by threonine. Another chimeric enzyme, BT, which has a similar structure to that of BTT but lacks the beta subunit, having alpha2-type homo-dimeric structure, was also responsive only to threonine. However, the addition of threonine enhanced the activity of BT. These results indicate the regulatory function of C-terminal region and beta subunit in AK. BTT showed extremely high thermostability comparable to that of T. flavus, suggesting that the beta subunit also contributed to the stability of the AK.

Three related cDNAs (C184L, C184M, and C184S) were isolated from a developing mouse brain cDNA library. C184S is the 5'-end portion and C184M is the 3'-end portion, respectively, of C184L. C184S and C184M have open reading frames of 199 amino acids (ORF1) and 189 amino acids (ORF2), respectively; C184L has both ORF1 and ORF2 (dicistronic structure), but seems to translate only ORF1. Southern blot analysis suggests that all of the three related mRNAs are transcribed from the same single gene. The intervening region of C184L cDNA between ORF1 and ORF2 contained a promoter sequence for C184M mRNA, which is transcribed from the corresponding genomic sequence. Very recently, a cDNA encoding human homologue of ORF1 (human autoantigen p27) and a cDNA encoding a different mouse isoform of ORF2 (mammary tumor virus receptor) were reported. Our results indicate that the mRNAs encoding these apparently unrelated proteins are transcribed within an adjacent or overlapping area on the genome, suggesting the same origin of the two transcription units., (Copyright 1999 Academic Press.)

We show that the major membrane protein of central nervous system myelin, proteolipid protein, evolved much more rapidly than it does now more than 300 million years ago. We reason that myelin proteolipid protein evolved rapidly just after its appearance in vertebrates and that its evolutionary rate then gradually decreased. Comparison of the rates between the synonymous and nonsynonymous nucleotide substitutions for the cDNA suggests the possibility that positive selection operated on myelin proteolipid protein at least when it appeared in vertebrates.

A murine genomic clone of about 16 kilobases encompassing putatively entire bone morphogenetic protein (BMP)-4 gene has been isolated. Comparison with the cDNA clones revealed the existence of at least five exons on the 3' side of the clone. There are two alternative transcriptional start sites, one is for the first exon the other for the second, the resulting exon organization in mRNA is I-III-IV-V and II-III-IV-V, respectively. The coding region is dispersed between exons IV and V. Both promoter regions carry no canonical TATA box but carry GC rich sequences.

We measured plasma concentrations of endothelin-1 (ET-1), ET-3 and big ET-1 by sandwich-enzyme immunoassays in patients (Pt) with chronic hemodialysis (HD) (Pt-HD, n = 23) and age-matched normal subjects (NS, n = 17). In Pt-HD, plasma levels (before HD) of ET-1, ET-3 and big ET-1 were significantly higher than those in NS. Reverse-phase HPLC analysis indicated that plasma concentrations of ET-1, ET-3 and big ET-1 in both Pt-HD and NS can be precisely measured by these sandwich-enzyme immunoassays. In Pt-HD, although the plasma ET-3 or big ET-1 levels did not significantly correlate with blood pressure (BP), plasma ET-1 levels significantly (p less than 0.01) correlated with both the levels of systolic (r = 0.63) and diastolic (r = 0.54) BP. After 4-hour HD, the plasma level of ET-3, but not ET-1 or big ET-1, was significantly elevated and BP was significantly lowered. The present findings indicate that ET-1 and ET-3 play different roles in acute and chronic alterations of BP in Pt-HD.

The optimized geometry of the conformation of atoms constituting the 6-pyruvoyl tetrahydropterin molecule, the labile key intermediate of tetrahydrobiopterin biosynthesis, was obtained by molecular orbital calculations within the MINDO/3 framework. The stereostructure of the molecule showing the preferred mode for binding to sepiapterin reductase or pyruvoyl tetrahydropterin reductase was drawn in perspective. The resulting structure with the equatorial staggered configuration of the 6-1',2'-dioxopropyl (pyruvoyl) side chain indicated that O(1') and H(6) were located in the trans position around the C(6)-C(1') bond and that the two vicinal carbonyls in the side chain were fixed in the incomplete trans form. The calculation of atomic charges and LUMO coefficients of these carbonyls suggests that the C2'-carbonyl may be more reactive toward NADPH than the C1'-carbonyl in the enzymatic reaction.

A second cDNA for mouse brain 2',3'-cyclic-nucleotide 3'-phosphodiesterase (cDNAII) encoding 420 amino acids was isolated. The only difference from the cDNA obtained previously (cDNAI), which encodes 400 amino acids, was the 5'-end 53 bp. Comparison of the sequence of the mouse gene with the sequence of cDNAII indicates alternative splicing within exon 1. The 5'-end sequence of cDNAII was found in a region of the gene upstream of exon 1 and a second transcription initiation site was identified.

The mouse 2',3'-cyclic-nucleotide 3'-phosphodiesterase gene was isolated from a mouse gene library. Restriction endonuclease mapping and DNA sequencing analysis revealed that this gene is about 6 kb long and is separated into three exons by two introns. The transcription initiation site was identified. The mouse cDNA of 2374 bp was obtained and used for the screening and analysis of the gene.

cDNA clones of human myelin-associated glycoprotein were isolated and analyzed. The combination of the two overlapping cDNA clones covered the full coding region and the complete amino acid sequence was deduced. In rat and mouse, expression of the two forms of mRNA is developmentally regulated; the mRNA without exon 12 portion is expressed mainly in the actively myelinating stage of development. Although the cDNA library used here was prepared from adult human brain poly(A)+ RNA, all five clones obtained corresponded to the mRNA without exon 12 portion.

A cDNA of 1762 base pairs was obtained from a cDNA library of human brain by immunoscreening, and the nucleotide sequence of the cDNA was determined. The complete amino acid sequence of human 2',3'-cyclic-nucleotide 3'-phosphodiesterase was deduced from the nucleotide sequence of the cDNA. Human enzyme was found to contain 401 amino acids including initiation methionine and have a molecular weight of 45,098. RNA blot hybridization revealed a single mRNA band at the position of about 3000 bases. DNA blot hybridization suggested that a single-copy 2',3'-cyclic-nucleotide 3'-phosphodiesterase gene exists per haploid genome.

The structures of three forms of mouse myelin-associated glycoprotein mRNAs were determined from full-length cDNA clones. Two forms of mRNAs have been reported to be different by alternate inclusion of exon 2 and 12 in rat brain. One of the three forms of clones obtained here appeared to be a novel mRNA which lacked both the exon 2 and 12 portions, although others were identical splicing patterns to those of rat. Northern blot analysis using specific probes to mRNAs with or without the exon 2 portion in normal and quaking mouse confirmed that the splicing of exon 2 and 12 occurred independently.