Genetic evidence for multiple geographic populations of the giant mottled eel <Emphasis Type="Italic">Anguilla marmorata</Emphasis> in the Pacific and Indian oceans

Anguillid eels have been believed to have a unique catadromous life history completed within a particular ocean current system, and consequently to have extraordinary population structures, with each species comprising a single, virtually panmictic population with a species-specific spawning area far offshore. The giant mottled eel Anguilla marmorata is enigmatic in this respect because it is widely distributed from East Africa to the central South Pacific, where several different current systems exist, and apparently must have multiple spawning areas. To address this issue, the population structure was examined through the mitochondrial (mt) DNA analysis of 162 individuals collected from ten localities representing the overall distributional range. Nucleotide sequencing of a 626-bp segment in the control region of the mtDNA revealed 151 haplotypes, and these were clustered into several major groups supported by high bootstrap probabilities. Sequence differences among geographic samples revealed the existence of five geographic populations around North Pacific, Madagascar, Sumatra, Fiji, and Tahiti. Genetic differentiation among the populations in the Southern Hemisphere was supported by examination of nuclear DNA with amplified fragment length polymorphism (AFLP) analysis performed on 76 individuals from four sampling localities in the Southern Hemisphere. The distribution pattern of five populations was closely associated with the water-mass structure of oceans and major current systems. This observation suggests a hypothesis that present population differentiation in A. marmorata might have resulted from the establishment of new population-specific spawning sites in different oceanic current systems as the species colonized new areas, as its unique catadromous life history of anguillid eels.This revised version was published online in February 2005 with corrections to the repetition of the 1st authors name.     

Lamprey contractile protein genes mark different populations of skeletal muscles during development

Agnathan lampreys retain ancestral characteristics of vertebrates in the morphology of skeletal muscles derived from two mesodermal regions: trunk myotomes and unsegmented head mesoderm. During lamprey development, some populations of myoblasts migrate via pathways that differ from those of gnathostomes. To investigate the evolution of skeletal muscle differentiation in vertebrates, we characterize multiple contractile protein genes expressed in the muscle cells of the Japanese lamprey, Lethenteron japonicum. Lamprey actin gene LjMA2, and myosin heavy chain (MyHC) genes LjMyHC1 and LjMyHC2 are all expressed in the developing skeletal muscle cells of early embryos. However, LjMyHC1 and LjMyHC2 are expressed only in cells originating from myotomes, while LjMA2 is expressed in both myotomal and head musculature. Thus, in lampreys, myotomes and head mesoderm differ in the use of genes encoding contractile protein isoforms. Phylogenetic tree analyses including lamprey MyHCs suggest that the variety of muscle MyHC isoforms in different skeletal muscles may correspond to the morphological complexity of skeletal muscles of different vertebrate species. Another lamprey actin gene LjMA1 is likely to be the first smooth muscle actin gene isolated from non-tetrapods. We conclude that, in vertebrate evolution, the different regulatory systems for striated and smooth muscle-specific genes may have been established before the agnathan/gnathostome divergence.     

Distinct mechanisms by mutant presenilin 1 and 2 leading to increased intracellular levels of amyloid beta-protein 42 in Chinese hamster ovary cells

To characterize the properties of presenilin (PS) 1- and PS2-associated gamma-secretases, we established stable transfectants overexpressing amyloid precursor protein and wild-type (wt) or a number of mutant (mt) PS1 or PS2. Quantification of the intracellular amyloid beta-protein (Abeta) levels in mtPS1 and mtPS2 cell lines revealed the presence of two subtypes. One group consists of N141I, M239V, and T122P mutations of the PS2 gene and homologous mutations of PS1, N135D and M233T. These mutations led to an increase in the intracellular Abeta42 levels and a concomitant decrease in the intracellular Abeta40 levels. A cell-free assay for Abeta production using the membranes prepared from these transfectants exhibited predominant cleavage at position Abeta42 with marginal production of Abeta40. The other group consists of M146L, H163R, and G384A mutations of PS1, leading only to an increase in the intracellular Abeta42 levels. While the intracellular Abeta levels in M146L cells were consistent with the results from cell-free Abeta production, H163R and G384A cells showed significant discrepancies between the intracellular Abeta levels and cell-free Abeta production. Thus, all the mtPS1/2 examined here result in increases in the intracellular Abeta42 levels. This suggests that the underlying mechanisms for this shared phenotype may be diverse.     

Inhibition of lipopolysaccharide-induced pro-inflammatory cytokine expression via suppression of nuclear factor-kappaB activation by Mallotus japonicus phloroglucinol derivatives

An aqueous acetone extract obtained from the pericarps of Mallotus japonicus (MJE) was observed to inhibit pro-inflammatory cytokine (tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) production in a lipopolysaccharide (LPS)-activated murine macrophage-like cell line, RAW 264.7, or human blood monocytes. Several phloroglucinol derivatives were isolated from the pericarps as active compounds. Among these compounds, isomallotochromanol and isomallotochromene were the most potent in inhibiting cytokine production. MJE and the phloroglucinol derivatives significantly reduced these cytokine mRNA expressions. Gel shift analysis revealed that stimulation of macrophages with LPS caused an increase in the DNA binding activity of nuclear factor-kappaB (NF-kappaB), which was inhibited by isomallotochromanol and isomallotochromene. Western blot analysis showed that LPS reduced the IkappaB-alpha level in macrophages, while 10 microM isomallotochromanol and 10 microM isomallotochromene attenuated the LPS-induced decrease in IkappaB-alpha protein. We conclude that these phloroglucinol derivatives inhibit pro-inflammatory cytokine production and mRNA expression via suppression of NF-kappaB activation in activated macrophages.     

Sequence of the gene for Clostridium botulinum type B neurotoxin associated with infant botulism,expression of the C-terminal half of heavy chain and its binding activity

Previously, we demonstrated that the neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a foodborne botulism-related strain, Okra. In this study, the neurotoxin genes of 111/NT and Okra/NT were amplified and the sequences determined. The nucleotide sequences of the genes for both neurotoxins possessed an open reading frame of 3873 bp that encoded 1291 amino acids. The identities of nucleotide sequences and amino acid sequences were 97.6% and 95.7%, respectively. The ratio of nonsynonymous to synonymous substitutions was 0.47. The amino acid substitutions between 111/NT and Okra/NT occurred mainly in the domain of the C-terminal half of heavy chain (H(C)) responsible for binding to its receptor complex of protein and ganglioside. To characterize the binding capability of the H(C), recombinant genes for the H(C) and two hybrid H(C) in which one half of Okra/NT was replaced by the homologous half of 111/NT were constructed and expressed in Escherichia coli. The binding activity of the recombinant H(C) of 111/NT to the protein receptor synaptotagmin II, in the presence of ganglioside GT1b, was 4.2-fold less than Okra/NT, consistent with the corresponding two NTs. The use of hybrid H(C) revealed that mutation of 23 residues in carboxy terminal half of H(C) (1029-1291) of Okra/NT could be attributed to the lower binding activity of 111/NT and thus the differences in binding affinity between the two BoNT/B.     

Markedly increased secretion of VLDL triglycerides induced by gene transfer of apolipoprotein E isoforms in apoE-deficient mice

Apolipoprotein E (apoE) plays a key role in the receptor-mediated uptake of lipoproteins by the liver and therefore in regulating plasma levels of lipoproteins. ApoE may also facilitate hepatic secretion of very low density lipoprotein (VLDL) triglyceride (TG). We directly tested the hypothesis that reconstitution of hepatic apoE expression in adult apoE-deficient mice by gene transfer would acutely enhance VLDL-TG production and directly compared the three major human apoE isoforms using this approach. Second generation recombinant adenoviruses encoding the three major isoforms of human apoE (E2, E3, and E4) or a control virus were injected intravenously into apoE-deficient mice, resulting in acute expression of the apoE isoforms in the liver. Despite the expected decreases in total and VLDL cholesterol levels, apoE expression was associated with increased total and VLDL triglyceride levels (E2 > E4 > E3). The increase in TG levels significantly correlated with plasma apoE concentrations. In order to determine whether acute apoE expression influenced the rate of VLDL-TG production, additional experiments were performed. Three days after injection of adenoviruses, Triton WR1339 was injected to block lipolysis of TG-rich lipoproteins and VLDL-TG production rates were determined. Mice injected with control adenovirus had a mean VLDL-TG production rate of 74 +/- 7 micromol/h/kg. In contrast, VLDL-TG production rates in apoE-expressing mice were 363 +/- 162 micromol/h/kg, 286 +/- 175 micromol/h/kg, and 300 +/- 84 micromol/h/kg for apoE2, apoE3, and apoE4, respectively. The VLDL-TG production rates in apoE-expressing mice were all significantly greater than in control mice but were not significantly different from each other. In summary, acute expression of all three human apoE isoforms in livers of apoE-deficient mice markedly increased VLDL-TG production to a similar degree, consistent with the concept that apoE plays an important role in facilitating hepatic VLDL-TG production in an isoform-independent manner.     

Lysosomal accumulation of oxidized phosphatidylcholine-apolipoprotein B complex in macrophages: intracellular fate of oxidized low density lipoprotein

Oxidized phosphatidylcholine (OxPC) formed in oxidized low density lipoprotein (OxLDL) is thought to be involved in the development of atherosclerosis. OxPC has been found in foam cells in atherosclerotic lesions and suggested to be the epitope for OxLDL recognition by macrophages. OxPC is present as a complex with apolipoprotein B (apoB) in OxLDL, since some OxPC can bind with proteins. In the current study, the intracellular fate of OxPC-apoB complexes after internalization of OxLDL by macrophages was investigated. Murine macrophage cell line J774.1 was incubated with either OxLDL or acetylated LDL for 24 h, then the cells were further incubated for up to 24 h in new medium without lipoprotein. Modified apoB in the cells was quantitated by sandwich ELISA using monoclonal antibodies against OxPC and apoB. Intracellular OxLDL decreased rapidly for the first 4 h to approx. 20% of that before medium change, with the apparent metabolism of OxPC-apoB complex ceasing. OxPC-apoB complexes that remained in the cells after 24 h chasing increased as the period of OxLDL loading in macrophages prolongs. Acetylated LDL in the cells decreased quickly and disappeared after 4 h of chasing. Subcellular fractionation using sucrose density gradient ultracentrifugation of macrophages, which had already accumulated OxPC-apoB complexes by 24 h of incubation with OxLDL and further 24 h chasing, showed that the complex was co-localized with endosomal and lysosomal markers. Immunohistochemical double staining studies demonstrated that OxPC and apoB co-localize in foam cells in early atherosclerotic lesions obtained from human coronary artery. These results suggest that OxPC-apoB complexes originating from OxLDL accumulate in foam cells in human atherosclerotic lesions as well as in macrophages in vitro.