Pinellic Acid Isolated from Quercetin-rich Onions has a Peroxisome Proliferator-Activated Receptor-Alpha/Gamma (PPAR-α/γ) Transactivation Activity.

Quercetin, a flavonol, is a functional compound that is abundant in onions and is known to have antioxidant and anti-inflammatory effects. Quercetin and its glucoside are known to function as peroxisome proliferator-activated receptor (PPAR) ligands and showed high PPAR-α transactivation activity but little PPAR-γ transactivation activity in some reports. In this study, we demonstrated that an aqueous extract of a quercetin-rich onion cultivar increased transactivation activities not only of PPAR-α but also of PPAR-γ. We isolated (9S,12S,13S)-(10E)-9,12,13-trihydroxyoctadec-10-enoic acid (pinellic acid) obtained from the aqueous extract using PPAR-γ transactivation as an index. Furthermore, it was revealed that pinellic acid could transactivate PPAR-α. Our findings are the first report mentioned showing that trihydroxyoctadec-10-enoic acids showed PPAR-α/γ transactivation activities.

Accelerated Idioventricular Rhythm Following Intraoral Local Anesthetic Injection During General Anesthesia.

Some anesthetic agents or adjunct medications administered during general anesthesia can cause an accelerated idioventricular rhythm (AIVR), which is associated with higher vagal tone and lower sympathetic activity. We encountered AIVR induced by vagal response to injection-related pain following local anesthetic infiltration into the oral mucosa during general anesthesia. A 48-year-old woman underwent extraction of a residual tooth root from the left maxillary sinus under general anesthesia. Routine preoperative electrocardiogram (ECG) was otherwise normal. Eight milliliters of 1% lidocaine (80 mg) with 1:100,000 epinephrine (80 µg) was infiltrated around the left maxillary molars over 20 seconds using a 23-gauge needle and firm pressure. Widened QRS complexes consistent with AIVR were observed for ∼60 seconds, followed by an atrioventricular junctional rhythm and the return of normal sinus rhythm. A cardiology consultation and 12-lead ECG in the operating room produced no additional concerns, so the operation continued with no complications. AIVR was presumably caused by activation of the trigeminocardiac reflex triggered by intense pain following rapid local anesthetic infiltration with a large gauge needle and firm pressure. Administration of local anesthetic should be performed cautiously when using a large gauge needle and avoid excessive pressure.

Stereocontrolled total synthesis of tetrodotoxin from myo-inositol and D-glucose by three routes: aspects for constructing complex multi-functionalized cyclitols with branched-chain structures.

This report describes the stereocontrolled total synthesis of the multi-functionalized cyclitol derivative, tetrodotoxin, containing eight asymmetric carbons and different types of branched-chains, from myo-inositol and D-glucose using three different methods. The tetrodotoxin derivatives possess a relatively small molecular weight but unique structural and chemical properties. Selection of the appropriate synthetic method may be useful not only for compounds related to TTX (including related derivatives), but also for other highly complex multi-functionalized cyclitols containing branched-chains.

Stereocontrolled total synthesis of tetrodotoxin from myo-inositol and D-glucose by three routes: aspects for constructing complex multi-functionalized cyclitols with branched-chain structures.

This report describes the stereocontrolled total synthesis of the multi-functionalized cyclitol derivative, tetrodotoxin, containing eight asymmetric carbons and different types of branched-chains, from myo-inositol and D-glucose using three different methods. The tetrodotoxin derivatives possess a relatively small molecular weight but unique structural and chemical properties. Selection of the appropriate synthetic method may be useful not only for compounds related to TTX (including related derivatives), but also for other highly complex multi-functionalized cyclitols containing branched-chains.

Proteolytic processing of phospholipase Czeta and [Ca2+]i oscillations during mammalian fertilization.

Phospholipase Czeta (PLCzeta) is a sperm-specific PLC capable of causing repetitive intracellular Ca2+ ([Ca2+]i) release ([Ca2+]i oscillations) in mammalian eggs. Accumulating evidence suggests that PLCzeta is the sperm factor responsible for inducing egg activation. Nevertheless, some sperm fractions devoid of 72-kDa PLCzeta showed [Ca2+]i oscillation-inducing and PLCzeta-like PLC activity (Kurokawa et al., (2005) Dev. Biol. 285, 376-392). Here, we report that PLCzeta remains functional after proteolytic cleavage at the X-Y linker region. We found that N-terminal (33 and 37 kDa) and C-terminal fragments (27 kDa), presumably the result of PLCzeta cleavage at the X-Y linker region, were present in fresh sperm as well as in sperm extracts and remained associated as functional complexes. Protease V8 cleaved 72-kDa PLCzeta into 33/37 and 27 kDa fragments, while PLC activity and [Ca2+]i oscillation-inducing activity persisted until degradation of the fragments. Immunodepletion or affinity depletion of these fragments abolished PLC activity and [Ca2+]i oscillation-inducing activity from sperm extracts. Lastly, co-expression of cRNAs encoding residues 1-361 and 362-647 of mouse PLCzeta, mimicking cleavage at the X-Y linker region, induced [Ca2+]i oscillations and embryo development in mouse eggs. Our results support the hypothesis that PLCzeta is the sole mammalian sperm factor and that its linker region may have important regulatory functions during mammalian fertilization.

De novo and salvage pathways of DNA synthesis in primary cultured neurall stem cells.

We studied the de novo and salvage pathways of DNA synthesis in sphere-forming neural stem cells obtained from mouse embryos by a neurosphere method. The former pathway needs folic acid (FA) for nucleotide biosynthesis, while the latter requires deoxyribonucleosides (dNS). We examined the proliferative activity of sphere-forming cells in E14.5 embryos by counting the number of spheres formed in media that lacked FA and/or dNS. Proliferation failure and apoptosis occurred in a deficient medium lacking of both FA and dNS. Spheres formed in the deficient medium supplemented with dNS, without FA, did not produce neuron, but rather only seem to generate astrocytes and oligodendrocytes when plated under differentiation condition in culture. On the other hand, a subpopulation of cultured cells formed spheres in the deficient medium supplemented with FA alone in an appropriate concentration, and did possess the self-renewing and multipotential characteristics of neural stem cells. Spheres formed in the media containing low dose Azathioprine and methotrexate, inhibitors of de novo DNA synthesis, were selectively prevented from producing neurons even in the presence of FA. These results suggested that activating de novo DNA synthesis was needed for neural stem cells to proliferate with multipotentiality.

Molecular identification and characterization of Xenopus egg uroplakin III, an egg raft-associated transmembrane protein that is tyrosine-phosphorylated upon fertilization.

Here we describe mass spectrometric identification, molecular cloning, and biochemical characterization of a lipid/membrane raft-associated protein that is tyrosine-phosphorylated upon Xenopus egg fertilization. This protein is homologous to mammalian uroplakin III, a member of the uroplakin family proteins (UPs) that constitute asymmetric unit membranes in the mammalian urothelial tissues, thus termed Xenopus uroplakin III (xUPIII). xUPIII contains N-linked sugars and is highly expressed in Xenopus eggs, ovary, urinary tract, and kidney. In unfertilized eggs, xUPIII is predominantly localized to the lipid/membrane rafts and exposed on the cell surface, as judged by surface biotinylation experiments and indirect immunofluorescent studies. After fertilization or hydrogen peroxide-induced egg activation, xUPIII becomes rapidly phosphorylated on tyrosine residue-249, which locates in the carboxyl-terminal cytoplasmic tail of the molecule. Raft localization and tyrosine phosphorylation of xUPIII can be reconstituted in HEK293 cells by coexpression of xUPIII, and Xenopus c-Src, a tyrosine kinase whose fertilization-induced activation in egg rafts is required for initiation of development. In mammals, UPIII is forming a complex with a tetraspanin molecule uroplakin Ib. As another tetraspanin, CD9, is known to be a critical component for sperm-egg fusion in the mouse, we have assumed that xUPIII is involved in sperm-egg interaction. An antibody against the extracellular domain of xUPIII blocks sperm-egg interaction, as judged by the occurrence of egg activation and first cell cleavage. Thus, xUPIII represents an egg raft-associated protein that is likely involved in sperm-egg interaction as well as subsequent Src-dependent intracellular events of egg activation in Xenopus.

Wild-type phenylalanine hydroxylase activity is enhanced by tetrahydrobiopterin supplementation in vivo: an implication for therapeutic basis of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency.

We previously proposed a novel disease entity, tetrahydrobiopterin (BH4)-responsive phenylalanine hydroxylase (PAH) deficiency, in which administration of BH4 reduced elevated levels of serum phenylalanine [J. Pediatr. 135 (1999) 375-378]. Subsequent reports indicate that the prevalence of BH4-responsive PAH deficiency is much higher than initially anticipated. Although growing attention surrounds treatment with BH4, little is known about the mechanism of BH4 responsiveness. An early report indicates that BH4 concentration in rat liver was 5 microM where Km for BH4 of rat PAH was estimated to be 25 microM in an oxidation experiment using a liver slice, suggesting relative insufficiency of BH4 in liver in vivo. In the present study, we developed a breath test for mice using [1-13C]phenylalanine in order to examine the BH4 responsiveness of normal PAH in vivo. The reliability of the test was verified using BTBR mice and its mutant strain lacking PAH activity, Pahenu2. BH4 supplementation significantly enhanced 13CO2 production in C57BL/6 mice when phenylalanine was pre-loaded. Furthermore, BH4 apparently activated PAH in just 5 min. These observations suggest that submaximal PAH activity occurs at the physiological concentrations of BH4 in vivo, and that PAH activity can be rapidly enhanced by supplementation with BH4. Thus, we propose a possible hypothesis that the responsiveness to BH4 in patients with PAH deficiency is due to the fact that suboptimal physiological concentrations of BH4 are normally present in hepatocytes and the enhancement of the residual activity may be associated with a wide range of mutations.

In vitro and in vivo antibacterial activities of DK-507k, a novel fluoroquinolone.

The antibacterial activities of DK-507k, a novel quinolone, were compared with those of other quinolones: ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, sitafloxacin, and garenoxacin (BMS284756). DK-507k was as active as sitafloxacin and was as active as or up to eightfold more active than gatifloxacin, moxifloxacin, and garenoxacin against Streptococcus pneumoniae, methicillin-susceptible and methicillin-resistant Staphylococcus aureus, and coagulase-negative staphylococci. DK-507k was as active as or 4-fold more active than garenoxacin and 2- to 16-fold more active than gatifloxacin and moxifloxacin against ciprofloxacin-resistant strains of S. pneumoniae, including clinical isolates and in vitro-selected mutants with known mutations. DK-507k inhibited all ciprofloxacin-resistant strains of S. pneumoniae at 1 microg/ml. A time-kill assay with S. pneumoniae showed that DK-507k was more bactericidal than gatifloxacin and moxifloxacin. The activities of DK-507k against most members of the family Enterobacteriaceae were comparable to those of ciprofloxacin and equal to or up to 32-fold higher than those of gatifloxacin, levofloxacin, moxifloxacin, and garenoxacin. DK-507k was fourfold less active than sitafloxacin and ciprofloxacin against Pseudomonas aeruginosa, while it was two to four times more potent than levofloxacin, gatifloxacin, moxifloxacin, and garenoxacin against P. aeruginosa. In vivo, intravenous treatment with DK-507k was more effective than that with gatifloxacin and moxifloxacin against systemic infections caused by S. aureus, S. pneumoniae, and P. aeruginosa in mice. In a mouse model of pneumonia due to penicillin-resistant S. pneumoniae, DK-507k administered subcutaneously showed dose-dependent efficacy and eliminated the bacteria from the lungs, whereas gatifloxacin and moxifloxacin had no significant efficacy. Oral treatment with DK-507k was slightly more effective than that with ciprofloxacin in a rat model of foreign body-associated urinary tract infection caused by a P. aeruginosa isolate for which the MIC of DK-507k was fourfold higher than that of ciprofloxacin. Oral administration of DK-507k to rats achieved higher peak concentrations in serum and higher concentrations in cumulative urine than those achieved with ciprofloxacin. These data indicate the potential advantages of DK-507k over other quinolones for the treatment of a wide range of community-acquired infections.