HPLC Retention time prediction for metabolome analysi

Liquid Chromatography Time-of-Flight Mass Spectrometry (LC-TOF-MS) is widely used for profiling metabolite compounds. LC-TOF-MS is a chemical analysis technique that combines the physical separation capabilities of high-pressure liquid chromatography (HPLC) with the mass analysis capabilities of Time-of-Flight Mass Spectrometry (TOF-MS) which utilizes the difference in the flight time of ions due to difference in the mass-to-charge ratio. Since metabolite compounds have various chemical characteristics, their precise identification is a crucial problem of metabolomics research. Contemporaneously analyzed reference standards are commonly required for mass spectral matching and retention time matching, but there are far fewer reference standards than there are compounds in the organism. We therefore developed a retention time prediction method for HPLC to improve the accuracy of identification of metabolite compounds. This method uses a combination of Support Vector Regression and Multiple Linear Regression adaptively to the measured retention time. We achieved a strong correlation (correlation coefficient = 0.974) between measured and predicted retention times for our experimental data. We also demonstrated a successful identification of an E. coli metabolite compound that cannot be identified by precise mass alone.     

Diel and lunar spawning periodicity of the hawkfish Paracirrhites forsteri (Cirrhitidae) on the reefs of Kuchierabu-jima Island, southern Japan

We observed spawning behavior of the hawkfish Paracirrhites forsteri on reefs of southern Japan. Spawning generally occurred after sunset, prior to the full and new moon with semi-lunar spawning peak periodicity. No egg predation was observed in spawning after sunset, and high tide often occurred at dusk in the lunar phases. Thus, diel timing and lunar synchronicity may increase larval survival. We found that the mating activity tended to start earlier in the day during early mating season than during mid-to-late mating season. The advantages of the earlier start of mating activity were also examined in relation to adult biology contexts.     

Clinico-molecular study of dedifferentiation in well-differentiated liposarcoma

Well-differentiated liposarcoma (WD) acquires fully malignant potential when the histological progression named dedifferentiation occurs. This progression is supposed to occur in a time-dependent manner but this is still a debated issue. Clinically, the prediction of dedifferentiation for WD is very important from the therapeutic point of view. To identify genes that are predictive of dedifferentiation and to understand the mechanism of dedifferentiation, we investigated clinical information of 50 cases and studied the gene expression profiles of 36 lipomatous tumors using cDNA microarray. The clinical study showed that the dedifferentiation did not always seem to occur in a time-dependent manner. Interestingly, from the gene expression study, unsupervised hierarchical clustering analysis of well-differentiated lesions obtained from dedifferentiated liposarcoma (DD) cases that were indistinguishable from WD pathologically showed a clearly distinct gene expression pattern from WD. Using the pattern-matching program, 1687 genes including 487 known genes were identified, which discriminated WD cases from well-differentiated lipomatous lesions obtained from DD cases. These results suggest that the dedifferentiation may arise from different types of WD that could be distinguished from gene expression profiling but could hardly be classified by the pathological studies.     

Accumulation response of chloroplasts induced by mechanical stimulation in bryophyte cells

Chloroplast movement has been studied in many plants but mainly as a model system for light signaling. However, we recently showed that the avoidance response of chloroplasts is also induced by mechanical stimulation in fern protonemal cells. Here we report the discovery of a mechanically induced accumulation response of chloroplasts in bryophytes. When mechanical stimulation was directly applied with a capillary to a part of a cell, chloroplasts moved towards and accumulated at the pressed site within 30 min after the onset of stimulation in all species tested. The accumulation movement of chloroplasts was inhibited by Cremart but not by cytochalasin B in red-light-grown protonemata of Physcomitrella patens (Hedw.) B., S. & G. To determine the contribution of external Ca(2+) to the response, we examined the effects on the accumulation movement of gadolinium (Ga(3+)), an inhibitor of stretch-activated ion channels, and lanthanum (La(3+)), a potent inhibitor of calcium channels. Mechano-relocation of chloroplasts was abolished by these drugs, but no effects were observed on photo-relocation of chloroplasts, irrespective of light colors and intensity. These results suggest that influx of external Ca(2+) through the plasma membrane is essential for the early steps in signaling of mechano-relocation of chloroplasts whose motility system is dependent on microtubules.     

Secoisolariciresinol and isotaxiresinol inhibit tumor necrosis factor-alpha-dependent hepatic apoptosis in mice

The effects of secoisolariciresinol (1) and isotaxiresinol (2), two major lignans isolated from the wood of Taxus yunnanensis, on tumor necrosis factor-alpha (TNF-alpha)-dependent hepatic apoptosis induced by D-galactosamine (d-GalN)/lipopolysaccharide (LPS) were investigated in mice. Co-administration of d-GalN (700 mg/kg) and LPS (10 microg/kg) resulted in a typical hepatic apoptosis characterized by DNA fragmentation and the formation of apoptotic bodies. Serum glutamic pyruvic transaminase (sGPT) and glutamic oxaloacetic transaminase (sGOT) levels were also raised at 8 h after d-GalN/LPS intoxication due to a severe necrosis of hepatocytes. Pre-administration of 1 or 2 (50, 10 mg/kg, i.p.) 12 and 1 h before d-GalN/LPS significantly reduced DNA fragmentation and prevented chromatin condensation, apoptotic body formation and hepatitis. Pro-inflammatory cytokines such as TNF-alpha and interferon-gamma (IFN-gamma) secreted from LPS-activated macrophages are important mediators of hepatocyte apoptosis in this model. Pre-treatment with 1 or 2 significantly inhibited the elevation of serum TNF-alpha and IFN-gamma levels. In a separate experiment, both lignans had a significant dose-dependent protective effect on d-GalN/TNF-alpha-induced cell death in primary cultured mouse hepatocytes and TNF-alpha-mediated cell death in murine L929 fibrosarcoma cells. These results indicated that 1 and 2 prevent d-GalN/LPS-induced hepatic injury by inhibiting hepatocyte apoptosis through the blocking of TNF-alpha and IFN-gamma production by activated macrophages and direct inhibition of the apoptosis induced by TNF-alpha.     

Dopamine D1 and D2 Receptors and Their Signal System Present in Coated Vesicles Prepared from Bovine Striatal Tissue

Abstract: Coated vesicles (CVs) isolated from bovine striatal tissue were examined to determine whether they are associated with dopamine signal systems consisting of dopamine D₁ and D₂ receptors, G proteins, and adenylate cyclase. Dopamine receptors in CVs were characterized by a dopamine D₁ receptor antagonist, [³H]SCH 23390, and a dopamine D₂ receptor antagonist, [³H]-spiroperidol. The bindings of both ligands were specifically saturable and reversible with a dissociation constant ( K _D) of 0.65 and 0.5 n M , respectively. Dopaminergic antagonists and agonists inhibited the specific bindings of [³H]SCH 23390 and [³H]spiroperidol in a stereoselective and concentration-dependent manner with an appropriate rank order potency for dopamine D₁ or D₂ receptors. The regulations of the agonist binding by guanyl-5-ylimidodiphosphate were observed. ADP ribosylation of the CVs with [³²P]NAD demonstrated predominant labeling of bands of M_r 47,000–52,000, 42,000–45,000, and 40,000-39,000, which corresponded to the known molecular weights of the α subunits of G_s and G_i proteins. The presence of α and β subunits of G proteins in the CVs was also confirmed by immunoblotting assay. Adenylate cyclase activity, which was stimulated by SKF 38393 and inhibited by dopamine D₂ receptor agonists, was present in the CVs. These findings suggest that the dopamine D₁ and D₂ receptors in the CVs couple with adenylate cyclase via G_s/G_i protein.     

Molecular defect in human erythropoietic protoporphyria with fatal liver failure

We investigated the molecular basis of ferrochelatase in a Japanese patient with erythropoietic protoporphyria (EPP), complicated by fatal liver failure, and defined a novel point mutation in the ferrochelatase gene. cDNAs were synthesized using Epstein-Barr-virus-transformed lymphoblastoid cells from the proband. cDNA clones encoding ferrochelatase in the proband were isolated by amplification using the polymerase chain reaction. There were two sizes of ferrochelatase cDNAs; one was normal in size, the other being smaller. Sequence analysis of the abnormally sized cDNA clones revealed that they lacked exon 9 of the ferrochelatase gene. Genomic DNA analysis demonstrated that the proband had the abnormal allele and that it contained a G to A point mutation at the first position of the donor site of intron 9. An identical mutation was detected in the affected family members of the proband by allele-specific oligonucleotide hybridization analysis. EPP is inherited in an autosomal dominant manner in this family.     

Growth and uptake kinetics of a facultatively oligotrophic bacterium at low nutrient concentrations

In oligotrophic waters, not only community structure but also physiological properties of heterotrophic bacteria are influenced by the concentration of organic matter.The relationship between growth rate of two facultatively oligotrophic strains ofAeromonas sp. No. 6 andFlavobacterium sp. M1 was studied in comparison with that of two eutrophic strains ofEscherichia coli 7020 andFlavobacterium sp. M2. These strains had two or three different substrate constants (Ks values) depending on substrate concentrations: Ks values for the two former were remarkably lower than those for the two latter. For instance, Ks value forAeromonas sp. No. 6 was about 8.9M when substrate concentration was greater than 53M and about 1.1M when substrate concentration was less man 53M. InE. coli the Ks value was about 260M at greater than 5600M and about 47M at less than 5600M substrate concentration.Uptake kinetics ofAeromonas sp. grown in a medium containing 2.7 mM glutamate (H-cell) and 0.11M glutamate (L-cell) have been determined for the intact cells. H-cell had two distinct values of Km for glutamate assimilation and respiration, and L-cell had three distinct values of Km for glutamate assimilation and respiration: In H-cell Km of assimilation was 2.8×10^–7 M and 1.5×10^–4 M, and Km of respiration was 2.3×10^–7 M and 1.7×10^–4 M; in L-cell Km of assimilation was 7.4×10^–8 M, 8.3×10^–6 M, and 1.3×10^–4 M, and Km of respiration was 2.5×10^–7, 8.9×10^–6M, and 1.7×10^–4 M. More than 60% of glutamate taken up by the H- and L-cells was respired when the substrate concentration was less than 10^–6 M, although at greater than 10^–6 M, 50% and 30% of glutamate was respired by H-cells and L-cells, respectively. These results suggest that the facultatively oligotrophic bacteria grow with high efficiency in environments with extremely low nutrient concentration, such as oligotrophic waters of lakes and ocean, as compared with in their growth in conditions of high nutrient concentraton, such as nutrient broth.