Highly sensitive time-resolved fluorometric determination of estrogens by high-performance liquid chromatography using a beta-diketonate europium chelate

A new fluorescent europium chelate labeling reagent, 5-(4"-chlorosulfo-1',1"-diphenyl-4'-yl)-1,1,1,2,2-pentafluoro-3,5-pentanedione (CDPP), was synthesized for the time-resolved fluorometric detection of HPLC. The label can be directly bound to amino or phenolic hydroxyl groups of analytes with its chlorosulfonyl group, and the labeled analytes are separated on a HPLC column. After separation, EuCl(3), TOPO (tri-n-octylphosphine oxide), and Triton X-100 were added by post-column introduction to the eluent, and the fluorescence of the europium chelate was measured with the time-resolved fluorometric detector. Estrone (E1), 17beta-estradiol (E2), ethynylestradiol (EE2) and estriol (E3) were measured with the detection limits of 0.65, 0.65, 0.65 and 0.60 ng/ml, respectively. The recovery for river water samples was in the range of 86.0-105.1% with the RSD of 1.9-5.8%. The method was applied to the analysis of a river water sample and estrone (E1) was determined to be 2.1 ng/l. The results and processing have been compared with those of a GC-MS method and a high degree of correlation (r> or =0.98) was observed.     

Mouse model of Prinzmetal angina by disruption of the inward rectifier Kir6.1

The inwardly rectifying K(+) channel Kir6.1 forms K(+) channels by coupling with a sulfonylurea receptor in reconstituted systems, but the physiological roles of Kir6.1-containing K(+) channels have not been determined. We report here that mice lacking the gene encoding Kir6.1 (known as Kcnj8) have a high rate of sudden death associated with spontaneous ST elevation followed by atrioventricular block as seen on an electrocardiogram. The K(+) channel opener pinacidil did not induce K(+) currents in vascular smooth-muscle cells of Kir6.1-null mice, and there was no vasodilation response to pinacidil. The administration of methylergometrine, a vasoconstrictive agent, elicited ST elevation followed by cardiac death in Kir6.1-null mice but not in wild-type mice, indicating a phenotype characterized by hypercontractility of coronary arteries and resembling Prinzmetal (or variant) angina in humans. The Kir6.1-containing K(+) channel is critical in the regulation of vascular tonus, especially in the coronary arteries, and its disruption may cause Prinzmetal angina.     

Identification of eight members of the Argonaute family in the human genome

A number of genes have been identified as members of the Argonaute family in various nonhuman organisms and these genes are considered to play important roles in the development and maintenance of germ-line stem cells. In this study, we identified the human Argonaute family, consisting of eight members. Proteins to be produced from these family members retain a common architecture with the PAZ motif in the middle and Piwi motif in the C-terminal region. Based on the sequence comparison, eight members of the Argonaute family were classified into two subfamilies: the PIWI subfamily (PIWIL1/HIWI, PIWIL2/HILI, PIWIL3, and PIWIL4/HIWI2) and the eIF2C/AGO subfamily (EIF2C1/hAGO1, EIF2C2/hAGO2, EIF2C3/hAGO3, and EIF2C4/hAGO4). PCR analysis using human multitissue cDNA panels indicated that all four members of the PIWI subfamily are expressed mainly in the testis, whereas all four members of the eIF2C/AGO subfamily are expressed in a variety of adult tissues. Immunoprecipitation and affinity binding experiments using human HEK293 cells cotransfected with cDNAs for FLAG-tagged DICER, a member of the ribonuclease III family, and the His-tagged members of the Argonaute family suggested that the proteins from members of both subfamilies are associated with DICER. We postulate that at least some members of the human Argonaute family may be involved in the development and maintenance of stem cells through the RNA-mediated gene-quelling mechanisms associated with DICER.     

Freezing sensitivity in the sfr4 mutant of Arabidopsis is due to low sugar content and is manifested by loss of osmotic responsiveness

Protoplasts were tested to determine whether the freezing sensitivity of the sfr4 (sensitive to freezing) mutant of Arabidopsis was due to the mutant's deficiency in soluble sugars after cold acclimation. When grown under nonacclimated conditions, sfr4 protoplasts possessed freezing tolerance similar to that of wild type, with the temperature at which 50% of protoplasts are injured (LT(50)) of -4.5 degrees C. In both wild-type and sfr4 protoplasts, expansion-induced lysis was the predominant lesion between -2 degrees C and -4 degrees C, but its incidence was low (approximately 10%); below -5 degrees C, loss of osmotic responsiveness (LOR) was the predominant lesion. After cold acclimation, the LT(50) was decreased to only -5.6 degrees C for sfr4 protoplasts, compared with -9.1 degrees C for wild-type protoplasts. Although expansion-induced lysis was precluded in both types of protoplasts, the sfr4 protoplasts remained susceptible to LOR. After incubation of seedlings in Suc solution in the dark at 2 degrees C, freezing tolerance and the incidence of freeze-induced lesions in sfr4 protoplasts were examined. The freezing tolerance of isolated protoplasts (LT(50) of -9 degrees C) and the incidence of LOR were now similar for wild type and sfr4. These results indicate that the freezing sensitivity of cold-acclimated sfr4 is due to its continued susceptibility to LOR (associated with lyotropic formation of the hexagonal II phase) and associated with the low sugar content of its cells.     

Loading direction regulates the affinity of ADP for kinesin

Kinesin is an ATP-driven molecular motor that moves processively along a microtubule. Processivity has been explained as a mechanism that involves alternating single- and double-headed binding of kinesin to microtubules coupled to the ATPase cycle of the motor. The internal load imposed between the two bound heads has been proposed to be a key factor regulating the ATPase cycle in each head. Here we show that external load imposed along the direction of motility on a single kinesin molecule enhances the binding affinity of ADP for kinesin, whereas an external load imposed against the direction of motility decreases it. This coupling between loading direction and enzymatic activity is in accord with the idea that the internal load plays a key role in the unidirectional and cooperative movement of processive motors.     

Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme

DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus sp. KOD1) is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially available DNA polymerase [M. Takagi et al. (1997) Appl. Environ. Microbiol. 63, 4504-4510]. However, when long distance PCR (>5 kbp) was performed with KOD DNA polymerase, amplification efficiency (product yield) becomes lower because of its strong 3'-5' exonuclease activity for proof-reading. In order to improve a target length limitation in PCR, mutant DNA polymerases with decreased 3'-5' exonuclease activity were designed by substituting amino acid residues in conserved exonuclease motifs, Exo I (Asp141-Xaa-Glu), Exo II (Asn210-Xaa-Xaa-Xaa-Phe-Asp), and Exo III (Tyr311-Xaa-Xaa-Xaa-Asp). Exonuclease activity and amplification fidelity (error rate) of the DNA polymerases were altered by mutagenesis. However, long and accurate PCR by a single-type of mutant DNA polymerase was very difficult. The wild-type DNA polymerase (WT) and its exonuclease deficient mutant (N210D) were mixed in different ratio and their characteristics in PCR were examined. When the mixed enzyme (WT and N210D) was made at the ratio of 1:40, long PCR (15 kbp) at lower mutation frequency could be efficiently achieved.     

Catalytic diastereoselective sulfimidation of diaryl sulfides and application of chiral sulfimides to asymmetric allylic alkylation

The copper-catalyzed diastereoselective imidation of diaryl sulfides bearing a chiral oxazolinyl moiety at the ortho-position with [N-(p-toluenesulfonyl) imino]phenyliodinane (TsN=IPh) or Chloramine-T trihydrate [TsN(Cl)Na.3H2O] was successfully carried out to give the corresponding optically active N-tosylsulfimides in good yields. For example, the imidation of diphenyl sulfide bearing a methoxymethyl moiety at the 4-position of the oxazoline ring with TsN(Cl)Na.3H2O in acetonitrile in the presence of 10 mol% Cu(OTf)2 at 25 degrees C for 24 h affords the corresponding optically active N-tosylsulfimide in 52% isolated yield with a high diastereoselectivity of 99%. Hydrolysis of the optically active N-p-tosylsulfimides converts them into the corresponding optically active sulfimides in high yields without loss of diastereoselectivity. These novel optically active sulfimides and N-tosylsulfimides work as efficient chiral ligands for palladium(II)-catalyzed allylic alkylation of 1, 3-diphenyl-3-acetoxy-1-propene with dimethyl malonate to give the corresponding alkylation product quantitatively and with a high stereoselectivity (up to 90% ee).     

Inhibition of angiotensin-converting enzyme protects endothelial cell against hypoxia/reoxygenation injury

Cardiovascular tissue injury in ischemia/reperfusion has been shown to be prevented by angiotensin-converting enzyme (ACE) inhibitors. However, the mechanism on endothelial cells has not been assessed in detail. Cultured human aortic endothelial cells (HAEC) were exposed to hypoxia with or without reoxygenation. Hypoxia enhanced apoptosis along with the activation of caspase-3. Reoxygenation increased lactate dehydrogenase release time-dependently, along with an increase of intracellular oxygen radicals. ACE inhibitor quinaprilat and bradykinin significantly lessened apoptosis and lactate dehydrogenase release with these effects being diminished by a kinin B2 receptor antagonist and a nitric oxide synthase inhibitor. In conclusion, hypoxia activated the suicide pathway leading to apoptosis of HAEC by enhancing caspase-3 activity, while subsequent reoxygenation induced necrosis by enhancing oxygen radical production. Quinaprilat could ameliorate both apoptosis and necrosis through the upregulation of constitutive endothelial nitric oxide synthase via an increase of bradykinin, with the resulting increase of nitric oxide.     

Temporal and mosaic distribution of large ganglion cells in the retina of a daggertooth aulopiform deep-sea fish (Anotopterus pharao)

The daggertooth Anotopterus pharao (Aulopiformes: Anotopteridae) is a large, piscivorous predator that lives within the epipelagic zone at night. In this species, the distribution of retinal ganglion cells has been examined. An isodensity contour map of ganglion cells shows that the cells concentrate in a slightly ventral region of the temporal retina. The region of high ganglion cell density contains 4.07 x 10(3) cells mm(-2), and the resulting visual acuity is 3.5 cycles deg(-1). Outside the area centralis, conspicuously large ganglion cells (LGCs) are observed in the temporal margin of the retina. The LGCs are regularly arrayed, and displaced into the inner plexiform layer. Thick dendrites extend into the outer part (sublamina a) of the inner plexiform layer. In the retinal whole mount, the total number of LGCs is 1590 (90.7 cm specimen), and the mean size of the LGCs is about four times larger than that of the ordinary ganglion cells. The morphological appearance of the LGCs was similar to the off-type alpha cells of the cat retina. The function of these distinctive LGCs is discussed in relation to specific head-up feeding behaviour.