Electrotransformation of Thiobacillus ferrooxidans with plasmids containing a mer determinant.

The mer operon from a strain of Thiobacillus ferrooxidans (C. Inoue, K. Sugawara, and T. Kusano, Mol. Microbiol. 5:2707-2718, 1991) consists of the regulatory gene merR and an operator-promoter region followed by merC and merA structural genes and differs from other known gram-negative mer operons. We have constructed four potential shuttle plasmids composed of a T. ferrooxidans-borne cryptic plasmid, a pUC18 plasmid, and the above-mentioned mer determinant as a selectable marker. Mercury ion-sensitive T. ferrooxidans strains were electroporated with constructed plasmids, and one strain, Y4-3 (of 30 independent strains tested), was found to have a transformation efficiency of 120 to 200 mercury-resistant colonies per microgram of plasmid DNA. This recipient strain was confirmed to be T. ferrooxidans by physiological, morphological, and chemotaxonomical data. The transformants carried a plasmid with no physical rearrangements through 25 passages under no selective pressure. Cell extracts showed mercury ion-dependent NADPH oxidation activity.     

Ontogeny of surfactant apoprotein D, SP-D, in the rat lung

Surfactant protein D (SP-D) is a collagenous surfactant-associated glycoprotein synthesized by alveolar type II cells. Antiserum against rat SP-D was raised in rabbits and an enzyme-linked immunosorbant assay (ELISA) has been developed using anti-rat SP-D IgG. In the present study we examined the developmental profile of SP-D in the rat lung compared with that of surfactant protein A (SP-A). SP-A content in the lungs increased during late gestation and reached its maximum on day 1 of neonate, and then gradually decreased until at least day 5. SP-D content during early gestation was less than 10 ng/mg protein until day 18, but on day 19 there was a 4-fold increase in SP-D (compared to that on day 18). It increased twice between day 21 and the day of birth, when it reached the adult level of 250 ng/mg protein, which is about one fourth that of the adult level of SP-A. Unlike SP-A there seemed to be no decrease in SP-D content after birth. These results demonstrate that SP-D is regulated developmentally as are the other components of surfactant, but the inconsistency in the developmental profiles of SP-A and SP-D suggests that these proteins may play different roles in lung maturation.     

Physiological polyspermy: Selection of a sperm nucleus for the development of diploid genomes in amphibians

The union between a sperm and an egg nucleus in egg fertilization is necessary to mix genetic materials to create a new diploid genome for the next generation. In most animals, only one sperm is incorporated into the egg (monospermy), but several animals exhibit physiological polyspermy in which several sperms enter the egg during normal fertilization. However, only one sperm nucleus forms the zygote nucleus with the egg nucleus, even in a polyspermic egg. The cellular and molecular mechanisms involved in the selection of sperm nuclei in the egg cytoplasm have been well investigated in urodele amphibians. The principal sperm nucleus develops a larger sperm aster and contacts the egg nucleus to form a zygote nucleus, whereas other accessory sperm nuclei are unable to approach the egg nucleus. The diploid zygote nucleus induces cleavage and participates in embryonic development, whereas the accessory sperm nuclei undergo pyknosis and degenerate. We propose several models to account for the mechanisms of the selection of one sperm nucleus and the degeneration of accessory sperm nuclei. The roles of physiological polyspermy in animal reproduction are discussed by comparison with other polyspermic species.     

Tori lines mitigate seabird bycatch in a pelagic longline fishery

Reviews in Fish Biology and Fisheries - Albatross bycatch has been increasing over the past decade in the US tuna longline fishery of the central North Pacific. A controlled field...     

A Highlights from MBoC Selection: Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein–coupled receptor kinase–dependent manner

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.     

Functions of beta2-adrenergic receptor in human periodontal ligament cells

Adrenergic receptors (ARs) are receptors of noradrenalin and adrenalin, of which there are nine different subtypes. In particular, β2 adrenergic receptor (β2-AR) is known to be related to the restoration and maintenance of homeostasis in bone and cardiac tissues; however, the functional role of signaling through β2-AR in periodontal ligament (PDL) tissue has not been fully examined. In this report, we investigated that β2-AR expression in PDL tissues and their features in PDL cells. β2-AR expressed in rat PDL tissues and human PDL cells (HPDLCs) derived from two different patients (HPDLCs-2G and -3S). Rat PDL tissue with occlusal loading showed high β2-AR expression, while its expression was downregulated in that without loading. In HPDLCs, β2-AR expression was increased exposed to stretch loading. The gene expression of PDL-related molecules was investigated in PDL clone cells (2-23 cells) overexpressing β2-AR. Their gene expression and intracellular cyclic adenosine monophosphate (cAMP) levels were also investigated in HPDLCs treated with a specific β2-AR agonist, fenoterol (FEN). Overexpression of β2-AR significantly promoted the gene expression of PDL-related molecules in 2 to 23 cells. FEN led to an upregulation in the expression of PDL-related molecules and increased intracellular cAMP levels in HPDLCs. In both HPDLCs, inhibition of cAMP signaling by using protein kinase A inhibitor suppressed the FEN-induced gene expression of α-smooth muscle actin. Our findings suggest that the occlusal force is important for β2-AR expression in PDL tissue and β2-AR is involved in fibroblastic differentiation and collagen synthesis of PDL cells. The signaling through β2-AR might be important for restoration and homeostasis of PDL tissue.     

Mitochondria‐type GPAT is required for mitochondrial fusion

Glycerol‐3‐phosphate acyltransferase (GPAT) is involved in the first step in glycerolipid synthesis and is localized in both the endoplasmic reticulum (ER) and mitochondria. To clarify the functional differences between ER‐GPAT and mitochondrial (Mt)‐GPAT, we generated both GPAT mutants in C. elegans and demonstrated that Mt‐GPAT is essential for mitochondrial fusion. Mutation of Mt‐GPAT caused excessive mitochondrial fragmentation. The defect was rescued by injection of lysophosphatidic acid (LPA), a direct product of GPAT, and by inhibition of LPA acyltransferase, both of which lead to accumulation of LPA in the cells. Mitochondrial fragmentation in Mt‐GPAT mutants was also rescued by inhibition of mitochondrial fission protein DRP‐1 and by overexpression of mitochondrial fusion protein FZO‐1/mitofusin, suggesting that the fusion/fission balance is affected by Mt‐GPAT depletion. Mitochondrial fragmentation was also observed in Mt‐GPAT‐depleted HeLa cells. A mitochondrial fusion assay using HeLa cells revealed that Mt‐GPAT depletion impaired mitochondrial fusion process. We postulate from these results that LPA produced by Mt‐GPAT functions not only as a precursor for glycerolipid synthesis but also as an essential factor of mitochondrial fusion.     

Phosphorothioate analogs of sn-2 radyl lysophosphatidic acid (LPA): Metabolically stabilized LPA receptor agonists

We describe an efficient synthesis of metabolically stabilized sn-2 radyl phosphorothioate analogs of lysophosphatidic acid (LPA), and the determination of the agonist activity of each analog for the six LPA receptors (LPA_1–6) using a recently developed TGFα shedding assay. In general, the sn-2 radyl OMPT analogs showed similar agonist activities to the previous 1-oleoyl-2-O-methyl-glycerophosphothioate (sn-1 OMPT) analogs for LPA_1–6 receptors. In most cases, the sn-2 radyl-OMPT analogs were more potent agonists than LPA itself. Most importantly, sn-2 alkyl OMPT analogs were very potent LPA₅ and LPA₆ agonists. The availability of sn-2 radyl OPMT analogs further refines the structure–activity relationships for ligand–receptor interactions for this class of GPCRs.     

Administration of a maple syrup extract to mitigate their hepatic inflammation induced by a high-fat diet: a transcriptome analysis

Effects of the administration of maple syrup extract (MSX) on hepatic gene expression were investigated in mice fed a high-fat diet. Gene annotation enrichment analysis based on gene ontology revealed some changes in the expression of genes related to lipid metabolism and the immune response in MSX-fed mice. Detailed analysis of these data indicated that MSX ingestion mitigates hepatic inflammation.