A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems. Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2). However, slow maturation is still a big obstacle to the use of GFP variants for visualization. These problems are exacerbated when GFP variants are expressed at 37 degrees C and/or targeted to certain organelles. Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-). For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired. Here we describe the development of an improved version of YFP named "Venus". Venus contains a novel mutation, F46L, which at 37 degrees C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl-. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before.     

An antitumor promoter from Moringa oleifera Lam.

In the course of studies on the isolation of bioactive compounds from Philippine plants, the seeds of Moringa oleifera Lam. were examined and from the ethanol extract were isolated the new O-ethyl-4-(α- -rhamnosyloxy)benzyl carbamate (1) together with seven known compounds, 4(α- -rhamnosyloxy)-benzyl isothiocyanate (2), niazimicin (3), niazirin (4), β-sitosterol (5), glycerol-1-(9-octadecanoate) (6), 3-O-(6′-O-oleoyl-β- -glucopyranosyl)-β-sitosterol (7), and β-sitosterol-3-O-β- -glucopyranoside (8). Four of the isolates (2, 3, 7, and 8), which were obtained in relatively good yields, were tested for their potential antitumor promoting activity using an in vitro assay which tested their inhibitory effects on Epstein–Barr virus-early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). All the tested compounds showed inhibitory activity against EBV-EA activation, with compounds 2, 3 and 8 having shown very significant activities. Based on the in vitro results, niazimicin (3) was further subjected to in vivo test and found to have potent antitumor promoting activity in the two-stage carcinogenesis in mouse skin using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and TPA as tumor promoter. From these results, niazimicin (3) is proposed to be a potent chemo-preventive agent in chemical carcinogenesis.     

Differential effects of various growth factors and cytokines on the syntheses of DNA,type I collagen,laminin, fibronectin,osteonectin/secreted protein,acidic and rich in cysteine (SPARC), and alkaline phosphatase by human pulp cells in culture

The purpose of this study is to differentiate roles of several growth factors and cytokines in proliferation and differentiation of pulp cells during development and repair. In human pulp cell cultures, laminin and type I collagen levels per cell remained almost constant during the whole culture period (22 days). On the other hand, secreted protein, acidic and rich in cysteine (SPARC/osteonectin) and alkaline phosphatase (ALPase) levels markedly increased after the cultures reached confluence. Laminin and type I collagen, as well as fibronectin, stimulated the spreading of pulp cells within 1 h. Adding transforming growth factor-β (TGF-β) decreased laminin and ALPase levels, whereas it increased SPARC and fibronectin levels 3- to 10-fold. Western and Northern blots showed that TGF-β enhanced SPARC synthesis at the protein and mRNA levels. Basic fibroblast growth factor (bFGF) decreased type I collagen, laminin, SPARC, and ALPase levels without changing the fibronectin level. Platelet-derived growth factor (PDGF) selectively decreased laminin, SPARC, and ALPase levels. Epidermal growth factor (EGF) also decreased SPARC and ALPase levels. Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) decreased type I collagen and laminin levels, and abolished SPARC and ALPase syntheses. Of these peptides, bFGF and PDGF showed the greatest stimulation of [³H]thymidine incorporation into DNA. TGF-β, EGF, and TNF-α had less effect on DNA synthesis, whereas IL-1β inhibited DNA synthesis. These findings demonstrated that TGF-β, bFGF, EGF, PDGF, TNF-α, and IL-1β have characteristically different patterns of actions on DNA, laminin, type I collagen, fibronectin, ALPase, and SPARC syntheses by pulp cells. J. Cell. Physiol. 174:194–205, 1998. © 1998 Wiley-Liss, Inc.     

Prospective memory mediated by interoceptive accuracy: a psychophysiological approach

Previous studies on prospective memory (PM), defined as memory for future intentions, suggest that psychological stress enhances successful PM retrieval. However, the mechanisms underlying this notion remain poorly understood. We hypothesized that PM retrieval is achieved through interaction with autonomic nervous activity, which is mediated by the individual accuracy of interoceptive awareness, as measured by the heartbeat detection task. In this study, the relationship between cardiac reactivity and retrieval of delayed intentions was evaluated using the event-based PM task. Participants were required to detect PM target letters while engaged in an ongoing 2-back working memory task. The results demonstrated that individuals with higher PM task performance had a greater increase in heart rate on PM target presentation. Also, higher interoceptive perceivers showed better PM task performance. This pattern was not observed for working memory task performance. These findings suggest that cardiac afferent signals enhance PM retrieval, which is mediated by individual levels of interoceptive accuracy.This article is part of the themed issue ‘Interoception beyond homeostasis: affect, cognition and mental health’.     

Induction of SCEs in CHL cells by dichlorobiphenyl derivative water pollutants, 2-phenylbenzotriazole (PBTA) congeners and river water concentrates

We recently identified dichlorobiphenyl (DCB) derivatives and 2-phenylbenzotriazole (PBTA) congeners as major mutagenic constituents of the waters of the Waka River and the Yodo River system in Japan, respectively. In this study we examined sister chromatid exchange (SCE) induction by two dichlorobiphenyl derivatives, 3,3′-dichlorobenzidine (DCB, 4,4′-diamino-3,3′-dichlorobiphenyl) and 4,4′-diamino-3,3′-dichloro-5-nitrobiphenyl (5-nitro-DCB); three PBTA congeners, 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1), 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), and 2-[2-(acetylamino)amino]-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6); and water concentrates from the Waka River in Chinese hamster lung (CHL) cells. Concentration-dependent induction of SCE was found for all DCBs and PBTAs examined in the presence of S9 mix, and statistically significant increases of SCEs were detected at 2 μg per ml of medium or higher concentrations. SCE induction of MeIQx was examined to compare genotoxic activities of these water pollutants. According to the results, a ranking of the SCE-inducing potency of these compounds is the following: 5-nitro-DCB ≈ MeIQx > PBTA6 > PBTA-1 ≈ PBTA-2 > DCB.Water samples collected at a site at the Waka River showed concentration-related increases in SCEs at 6.25–18.75 ml-equivalent of river water per ml of medium with S9 mix. The concentrations of 5-nitro-DCB and DCB in the river water samples were from 2.5 to 19.4 ng/l and from 4100 to 18,900 ng/l, respectively. However, these chemicals showed only small contribution to SCE induction by the Waka River water.     

Purification and characterization of highly branched α-glucan-producing enzymes from Paenibacillus sp. PP710

Highly branched α-glucan molecules exhibit low digestibility for α-amylase and glucoamylase, and abundant in α-(1→3)-, α-(1→6)-glucosidic linkages and α-(1→6)-linked branch points where another glucosyl chain is initiated through an α-(1→3)-linkage. From a culture supernatant of Paenibacillus sp. PP710, we purified α-glucosidase (AGL) and α-amylase (AMY), which were involved in the production of highly branched α-glucan from maltodextrin. AGL catalyzed the transglucosylation reaction of a glucosyl residue to a nonreducing-end glucosyl residue by α-1,6-, α-1,4-, and α-1,3-linkages. AMY catalyzed the hydrolysis of the α-1,4-linkage and the intermolecular or intramolecular transfer of maltooligosaccharide like cyclodextrin glucanotransferase (CGTase). It also catalyzed the transfer of an α-1,4-glucosyl chain to a C3- or C4-hydroxyl group in the α-1,4- or α-1,6-linked nonreducing-end residue or the α-1,6-linked residue located in the other chains. Hence AMY was regarded as a novel enzyme. We think that the mechanism of formation of highly branched α-glucan from maltodextrin is as follows: α-1,6- and α-1,3-linked residues are generated by the transglucosylation of AGL at the nonreducing ends of glucosyl chains. Then AMY catalyzes the transfer of α-1,4-chains to C3- or C4-hydroxyl groups in the α-1,4- or α-1,6-linked residues generated by AGL. Thus the concerted reactions of both AGL and AMY are necessary to produce the highly branched α-glucan from maltodextrin.     

Design and synthesis of pyrrolo[3,2-d]pyrimidine HER2/EGFR dual inhibitors: Improvement of the physicochemical and pharmacokinetic profiles for potent in vivo anti-tumor efficacy

During the course of our studies on a novel HER2/EGFR dual inhibitor (TAK-285), we found an alternative potent pyrrolo[3,2-d]pyrimidine compound (1a). To enhance the pharmacokinetic (PK) profile of this compound, we conducted chemical modifications into its N-5 side chain and conversion of the chemically modified compounds into their salts. Among them, 2cb, the tosylate salt of compound 2c, showed potent HER2/EGFR kinase inhibitory activity (IC₅₀: 11/11 nM) and cellular growth inhibitory activity (BT-474 cell GI₅₀: 56 nM) with a good drug metabolism and PK (DMPK) profile. Furthermore, 2cb exhibited significant in vivo antitumor efficacy in both mouse and rat xenograft models with transplanted 4-1ST gastric cancer cell lines (mouse, T/C = 0%, 2cb po bid at 100 mg/kg; rat, T/C: -1%, 2cb po bid at 25 mg/kg).     

Mouse-Hamster Chimeric Prion Protein (PrP) Devoid of N-Terminal Residues 23-88 Restores Susceptibility to 22L Prions,but Not to RML Prions in PrP-Knockout Mice

Prion infection induces conformational conversion of the normal prion protein PrP^C, into the pathogenic isoform PrP^Sc, in prion diseases. It has been shown that PrP-knockout (Prnp^0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp^0/0 mice, neither developed the disease nor accumulated MHM2^ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice developed the disease with abundant accumulation of MHM2^ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2^ScΔ23-88, and that the co-expressing wild-type PrP^C can stimulate the conversion of MHM2Δ23-88 to MHM2^ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp^0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp^0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2^ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2^ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice. However, wild-type PrP^Sc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice, compared with RML- and 22L-inoculated Prnp^0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2^ScΔ23-88 without the help of the trans-acting PrP^C, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrP^C stimulates the conversion of MHM2Δ23-88 into MHM2^ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrP^C into PrP^Sc.     

Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-κB ligand (RANKL) expression in rheumatoid arthritis

Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-κB ligand (RANKL) in RA synoviocytes and CD4(+) T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4(+) T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4(+) T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-α in MH7A cells and CD4(+) T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4(+) T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.