Inhibition of inducible nitric oxide synthase and cyclooxygenase II by Platycodon grandiflorum saponins via suppression of nuclear factor-kappaB activation in RAW 264.7 cells

Saponins are glycosidic compounds present in many edible and inedible plants. They exhibit potent biological activities in mammalian systems, including several beneficial effects such as anti-inflammation and immunomodulation. In this study, we investigated the effects of seven platycodin saponins on the activities of inducible nitric oxide synthase (iNOS) and cyclooxygenase II (COX-2) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. We found that 2"-O-acetyl polygalacin D (S1), platycodin A (S2), platycodin D (S3), and polygalacin D (S6) inhibited LPS-induced NO production in a concentration-dependent manner. Furthermore, these compounds inhibited the expression of LPS-induced iNOS and COX-2 protein and mRNA without an appreciable cytotoxic effect on RAW 264.7 macrophages, and could suppress induction by LPS of pro-inflammatory cytokines such as prostaglandin E2 (PGE2). Treatment with these compounds of RAW 264.7 cells transfected with a reporter construct indicated a reduced level of LPS-induced nuclear factor-kappaB (NF-kappaB) activity and effectively lowered NF-kappaB binding as measured by electrophoretic mobility shift assay (EMSA). The suppression of NF-kappaB activation appears to occur through the prevention of inhibitor kappaB (IkappaB) degradation. In vivo, platycodin saponin mixture (PS) and S3 protected mice from the lethal effects of LPS. The 89% lethality induced by LPS/galactosamine was reduced to 60% and 50% when PS and S3, respectively, were administered simultaneously with LPS. These results suggest that the main inhibitory mechanism of the platycodin saponins may be the reduction of iNOS and COX-2 gene expression through blocking of NF-kappaB activation.     

Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the Acipenseriformes

The shortnose sturgeon Acipenser brevirostrum was revealed to have a larger number of chromosomes than previously reported for other sturgeon species. Its chromosome number ranged from 362 to 372 (of ten specimens examined), showing intraindividual variation. The karyotype of metaphase with the highest chromosome number (372) consisted of 89 pairs of macrochromosomes and 97 pairs of microchromosomes (fundamental number; NF=550). Although the microchromosomes were relatively shorter than the macrochromosomes, most of them had discernible arms and centromeres. Silver-stained nucleolar organizer regions (Ag-NORs) were localized on the telomeric regions of 5 pairs of chromosomes (Ag-NORs=10): 4 were made up of small meta/submetacentrics and 1 of acrocentrics. Polyploidy of A. brevirostrum should be hexaploid based on the karyotype, numerous chromosomes, Ag-NORs, and previously reported large genome size (ca. 13pg DNA/cell).Supplementary material to this paper is available in electronic format at http://dx.doi.org/10.1007/s10228-004-0257-z     

Controlled transcriptional regulation in eukaryotes by a novel transcription factor derived from Escherichia coli purine repressor

Neb-LFamide or AYRKPPFNGSLFamide was originally purified from the grey flesh fly Neobellieria bullata as a myotropic neuropeptide. We studied the occurrence of this peptide and its isoforms in the central nervous system of different insect species by means of whole mount fluorescence immunohistochemistry, mass spectrometry, and data mining. We found that both sequence and immunoreactive distribution pattern are very conserved in the studied insects. In all species and stages we counted two pairs of immunoreactive cells in the pars intercerebralis. These cells projected axons throughout the ventral nerve cord. In the adult CNSs they formed a large number of immunoreactive varicosities as well. Mass spectrometry and data mining revealed that SIFamide exists in two isoforms: [G1]-SIFamide and [A1]-SIFamide. In addition, the SIFamide joining peptide is relatively well conserved throughout arthropod species. The conserved presence of two cysteine residues, separated by six amino acid residues, allows the formation of disulphide bridges.     

Eotaxin and monocyte chemotactic protein-3 use different modes of action

Eotaxin selectively binds CC chemokine receptor (CCR) 3, whereas monocyte chemotactic protein (MCP)-3 binds CCR1, CCR2, and CCR3. To identify the functional determinants of the chemokines, we generated four reciprocal chimeric chemokines-M10E9, M22E21, E8M11, and E20M23-by shuffling the N-terminus and N-loop of eotaxin and MCP-3. M22E21 and E8M11, which shared the N-loop from MCP-3, bound to monocytes with high affinity, and activated monocytes. In contrast, M10E9 and E20M23, which lacked the N-loop, failed to bind and transduce monocyte responses, identifying the N-loop of MCP-3 as the selectivity determinant for CCR1/CCR2. A BIAcore assay with an N-terminal peptide of CCR3 (residues 1-35) revealed that all chimeras except E20M23 exhibited varying degrees of binding affinity with commensurate chemotaxis activity of eosinophils. Surprisingly, E20M23 could neither bind the CCR3 peptide nor activate eosinophils, despite having both N-terminal motifs from eotaxin. These results suggest that the two N-terminal motifs of eotaxin must cooperate with other regions to successfully bind and activate CCR3.     

An Insect Multiligand Recognition Protein Functions as an Opsonin for the Phagocytosis of Microorganisms

We characterize a novel pathogen recognition protein obtained from the lepidopteran Galleria mellonella. This protein recognizes Escherichia coli, Micrococcus luteus, and Candida albicans via specific binding to lipopolysaccharides, lipoteichoic acid, and β-1,3-glucan, respectively. As a multiligand receptor capable of coping with a broad variety of invading pathogens, it is constitutively produced in the fat body, midgut, and integument but not in the hemocytes and is secreted into the hemolymph. The protein was confirmed to be relevant to cellular immune response and to further function as an opsonin that promotes the uptake of invading microorganisms into hemocytes. Our data reveal that the mechanism by which a multiligand receptor recognizes microorganisms contributes substantially to their phagocytosis by hemocytes. A better understanding of an opsonin with the required repertoire for detecting diverse invaders might provide us with critical insights into the mechanisms underlying insect phagocytosis.     

Population structure of the olive flounder (Paralichthys olivaceus) in Korea inferred from microsatellite marker analysis

The population structure of olive flounder Paralichthys olivaceus was estimated using nine polymorphic microsatellite (MS) loci in 459 individuals collected from eight populations, including five wild and three hatchery populations in Korea. Genetic variation in hatchery (mean number of alleles per locus, A = 10·2–12·1; allelic richness, A_R = 9·3–10·1; observed heterozygosity, H_O = 0·766–0·805) and wild (mean number of alleles per locus, A = 11·8–19·6; allelic richness, A_R = 10·9–16·1; observed heterozygosity, H_O = 0·820–0·888) samples did not differ significantly, suggesting a sufficient level of genetic variation in these well‐managed hatchery populations, which have not lost a substantial amount of genetic diversity. Neighbour‐joining tree and principal component analyses showed that genetic separation between eastern and pooled western and southern wild populations in Korea was probably influenced by restricted gene flow between regional populations due to the barrier effects of sea currents. The pooled western and southern populations are genetically close, perhaps because larval dispersal may depend on warm currents. One wild population (sample from Wando) was genetically divergent from the main distribution, but it was genetically close to hatchery populations, indicating that the genetic composition of the studied populations may be affected by hydrographic conditions and the release of fish stocks. The estimated genetic population structure and potential applications of MS markers may aid in the proper management of P. olivaceus populations.     

Acute lipoprotein lipase deletion in adult mice leads to dyslipidemia and cardiac dysfunction

The most energy-requiring organ in the body, the cardiac muscle, relies primarily on lipoprotein-derived fatty acids. Prenatal loss of cardiac lipoprotein lipase (LPL) leads to hypertriglyceridemia, but no cardiac dysfunction, in young mice. Cardiac specific loss of LPL in 8-wk-old mice was produced by a 2-wk tamoxifen treatment of MerCreMer (MCM)/Lpl(flox/flox) mice. LPL gene deletion was confirmed by PCR analysis, and LPL mRNA expression was reduced by approximately 70%. One week after tamoxifen was completed, triglyceride was increased with LPL deletion, 162 +/- 53 vs. 91 +/- 21 mg/dl, P < 0.01. Tamoxifen treatment of Lpl(flox/flox) mice did not cause a significant increase in triglyceride levels. Four weeks after tamoxifen, MCM/Lpl(flox/flox) mice had triglyceride levels of 190 +/- 27 mg/dl, similar to those of mice with prenatal LPL deletion. One week after the tamoxifen, MCM/Lpl(flox/flox), but not Lpl(flox/flox), mice had decreases in carnitine palmitoyl transferase I mRNA (18%) and pyruvate dehydrogenase kinase 4 mRNA (38%). These changes in gene expression became more robust with time. Acute loss of LPL decreased ejection fraction and increased mRNA levels for atrial natriuretic factor. Our studies show that acute loss of LPL can be produced and leads to rapid alteration in gene expression and cardiac dysfunction.