A newly identified lipoprotein lipase (LPL) gene mutation (F270L) in a Japanese patient with familial LPL deficiency

We have systematically investigated the molecular defects resulting in a primary lipoprotein lipase (LPL) deficiency in a Japanese male infant (proband SH) with fasting hyperchylomicronemia. Neither LPL activity nor immunoreactive LPL mass was detected in pre- or postheparin plasma from proband SH. DNA sequence analysis of the LPL gene of proband SH revealed homozygosity for a novel missense mutation of F270L (Phe(270)-->Leu/TTT(1065)-->TTG) in exon 6. The function of the mutant F270L LPL was determined by both biochemical and immunocytochemical studies. In vitro expression experiments on the mutant F270L LPL cDNA in COS-1 cells demonstrated that the mutant LPL protein was synthesized as a catalytically inactive form and its total amount was almost equal to that of the normal LPL. Moreover, the synthesized mutant LPL was non-releasable by heparin because the intracellular transport of the mutant LPL to the cell surface - by which normal LPL becomes heparin-releasable - was impaired due to the abnormal structure of the mutant LPL protein. These findings explain the failure to detect LPL activities and masses in pre- and postheparin plasma of the proband. The mutant F270L allele generated an XcmI restriction enzyme site in exon 6 of the LPL gene. The carrier status of F270L in the proband's family members was examined by digestion with XcmI. The proband was ascertained to be homozygous for the F270L mutation and his parents and sister were all heterozygous. The LPL activities and masses of the parents and the sister (carriers) were half or less than half of the control values. Regarding the phenotype of the carriers, the mother with a sign of hyperinsulinemia manifested hypertriglyceridemia (type IV hyperlipoproteinemia), whereas the healthy father and the sister were normolipidemic. Hyperinsulinemia may be a strong determinant of hypertriglyceridemia in subjects with heterozygous LPL deficiency.     

MCPIP1 ribonuclease antagonizes dicer and terminates microRNA biogenesis through precursor microRNA degradation

MicroRNAs (miRNAs) are versatile regulators of gene expression and undergo complex maturation processes. However, the mechanism(s) stabilizing or reducing these small RNAs remains poorly understood. Here we identify mammalian immune regulator MCPIP1 (Zc3h12a) ribonuclease as a broad suppressor of miRNA activity and biogenesis, which counteracts Dicer, a central ribonuclease in miRNA processing. MCPIP1 suppresses miRNA biosynthesis via cleavage of the terminal loops of precursor miRNAs (pre-miRNAs). MCPIP1 also carries a vertebrate-specific oligomerization domain important for pre-miRNA recognition, indicating its recent evolution. Furthermore, we observed potential antagonism between MCPIP1 and Dicer function in human cancer and found a regulatory role of MCPIP1 in the signaling axis comprising miR-155 and its target c-Maf. These results collectively suggest that the balance between processing and destroying ribonucleases modulates miRNA biogenesis and potentially affects pathological miRNA dysregulation. The presence of this abortive processing machinery and diversity of MCPIP1-related genes may imply a dynamic evolutional transition of the RNA silencing system.     

Studies on the Utilization of Hydrocarbons by Microorganisms

Taxonomical studies on ten strains of hydrocarbon-utilizing bacteria reported in previous paper, which produced various kinds of amino acid, were carried out. They were Achromo-bacter cycloclastes, Achromobacter delmarvae, Bacillus species, Corynebacterium species, Micrococcus species. Many of them were not identical with the species which are described in Bergey’s Manual of 7th Edition.     

Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction

Mesenchymal stem cells are multipotent cells that can differentiate into cardiomyocytes and vascular endothelial cells. Here we show, using cell sheet technology, that monolayered mesenchymal stem cells have multipotent and self-propagating properties after transplantation into infarcted rat hearts. We cultured adipose tissue-derived mesenchymal stem cells characterized by flow cytometry using temperature-responsive culture dishes. Four weeks after coronary ligation, we transplanted the monolayered mesenchymal stem cells onto the scarred myocardium. After transplantation, the engrafted sheet gradually grew to form a thick stratum that included newly formed vessels, undifferentiated cells and few cardiomyocytes. The mesenchymal stem cell sheet also acted through paracrine pathways to trigger angiogenesis. Unlike a fibroblast cell sheet, the monolayered mesenchymal stem cells reversed wall thinning in the scar area and improved cardiac function in rats with myocardial infarction. Thus, transplantation of monolayered mesenchymal stem cells may be a new therapeutic strategy for cardiac tissue regeneration.     

Calcium-activated neutral protease and its endogenous inhibitor Activation at the cell membrane and biological function

The structures of calcium-activated neutral protease (CANP) and its endogenous inhibitor elucidated recently have revealed novel features with respect to their structure-function relationship and enzyme activity regulation. The protease is regarded as a proenzyme which can be activated at the cell membrane in the presence of Ca²⁺ and phospholipid, and presumably regulates the functions of proteins, especially membrane-associated proteins, by limited proteolysis. Protein kinase C is hydrolysed and activated by CANP at the cell membrane to a cofactor-independent form. These results are reviewed and the possible involvement of CANP in signal transduction is discussed.     

A putative met-enkephalin releaser, kyotorphin enhances intracellular Ca2+ in the synaptosomes

Kyotorphin (Tyr-Arg) at 1 to 100 microM increased the intracellular [Ca2+]i, determined with Quin-II in the slice and the entry of 45Ca2+ entry into synaptosomes of the lower brain stem of the rat. These effects were not antagonized by nifedipine nor verapamil. However, since this dipeptide caused no changes on the membrane potentials of the synaptosomes, measured with Rhodamine 6G, it is suggested that the kyotorphin-induced increase in the [Ca2+]i may be due not to effects on the voltage dependent Ca2+ channels and Na+-Ca2+ exchange mechanisms caused by the changes of the membrane potentials, but to the specific receptor (kyotorphin receptor)-mediated mechanisms.     

The association between the phytoplankton, Rhopalosolen species (Chlorophyta; Chlorophyceae), and Anopheles gambiae sensu lato (Diptera: Culicidae) larval abundance in western Kenya

Algae are important food resources of the larvae of the African malaria vectors, Anopheles gambiae Giles and Anopheles arabiensis Patton (Anopheles gambiae sensu lato), and other zooplankton, but empirical evidence remains meager about the agal flora in ephemeral water bodies. The animals present in natural aquatic habitats in western Kenya were sampled from July to November 2002 to study abiotic and biotic environmental factors determining A. gambiae sl larval abundance. The five highest concentrations of third and fourth instars and pupae (hereafter referred to as old-stage larvae) were sampled in conjunction with the unicellular epizoic green algae, Rhopalosolen species (Chlorophyta; Chlorophyceae). Canonical correspondence analysis revealed that the presence of Rhopalosolen species was the most important determinant of the animal assemblage. The density of old-stage A. gambiae sl larvae was positively correlated with the presence of Rhopalosolen species, but the density of first and second instars of A. gambiae sl was not. The water bodies with Rhopalosolen sp. yielded larger mosquitoes in spite of the higher density of larvae. We demonstrated that the productivity of water bodies in terms of the larvae of malaria vectors can differ in magnitude depending on the agal flora. We discuss phytoplankton as a regulator of mosquito larval populations.     

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.     

Toll-like Receptors as a Target of Food-derived Anti-inflammatory Compounds

Toll-like receptors (TLRs) play a key role in linking pathogen recognition with the induction of innate immunity. They have been implicated in the pathogenesis of chronic inflammatory diseases, representing potential targets for prevention/treatment. Vegetable-rich diets are associated with the reduced risk of several inflammatory disorders. In the present study, based on an extensive screening of vegetable extracts for TLR-inhibiting activity in HEK293 cells co-expressing TLR with the NF-κB reporter gene, we found cabbage and onion extracts to be the richest sources of a TLR signaling inhibitor. To identify the active substances, we performed activity-guiding separation of the principal inhibitors and identified 3-methylsulfinylpropyl isothiocyanate (iberin) from the cabbage and quercetin and quercetin 4′-O-β-glucoside from the onion, among which iberin showed the most potent inhibitory effect. It was revealed that iberin specifically acted on the dimerization step of TLRs in the TLR signaling pathway. To gain insight into the inhibitory mechanism of TLR dimerization, we developed a novel probe combining an isothiocyanate-reactive group and an alkyne functionality for click chemistry and detected the probe bound to the TLRs in living cells, suggesting that iberin disrupts dimerization of the TLRs via covalent binding. Furthermore, we designed a variety of iberin analogues and found that the inhibition potency was influenced by the oxidation state of the sulfur. Modeling studies of the iberin analogues showed that the oxidation state of sulfur might influence the global shape of the isothiocyanates. These findings establish the TLR dimerization step as a target of food-derived anti-inflammatory compounds.