Comparison of the morphology of the inner ear between newts and frogs in relation to their locomotory capability

The ultrastructural differences between the inner ears of Japanese red-bellied newts (Cynops pyrrhogaster) and black-spotted pond frogs (Rana nigromaculata) were investigated. Scanning electron microscopic observations showed apparent morphological differences in the shape of the ampulla cristae and the localization of the striola in the saccular macula. There were differences in the length of the kinocilia of the sensory hairs in each sensory region. In addition, the diameters of the bundles of stereocilia differed between the two species: the bundles of stereocilia in the semicircular cristae were thicker in frogs than in newts, while those of the utricular and lagenal maculae were thicker in newts than in frogs.     

Brain HSP70 mRNA expression is linked with plasma cortisol levels in goldfish (Carassius auratus) exposed to a potential predator

We previously found that when goldfish were exposed to a potential predator, bluegills, the goldfish experienced an increase in HSP70 mRNA expression in the brains and increased plasma cortisol levels. In the present study, we examined the potential causative relationship between HSP70 mRNA expression and plasma cortisol levels. Cortisol agonists (corticotropin releasing factor and cortisol) and antagonists (metyrapone and betamethasone) were used to modulate plasma cortisol levels. HSP70 mRNA expression and plasma cortisol levels were analyzed by Northern blotting and ELISA, respectively. Goldfish treated with the cortisol agonists showed marked increases in plasma cortisol levels and also in brain HSP70 mRNA expression. When goldfish were exposed to bluegills, plasma cortisol levels increased and HSP70 mRNA expression was enhanced after 6 hr. However, pre-treatment with the cortisol antagonists 24 hr prior to the exposure inhibited the enhancement as well as the increase in plasma cortisol levels. These results suggest that plasma cortisol plays a key role in the enhancement of brain HSP70 mRNA expression in goldfish stressed by exposure to bluegills.     

Rolling properties of rGPIbalpha-conjugated phospholipid vesicles with different membrane flexibilities on vWf surface under flow conditions

The recombinant fragment of the platelet membrane glycoprotein, rGPIbalpha, was conjugated to phospholipid vesicles with the average diameter of ca. 1 microm using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). We used five kinds of rGPIbalpha-vesicles with different fluorescent anisotropies of 1,6-diphenyl-1,3,5-hexatriene (DPH) to study the rolling properties of the vesicles on the von Willebrand factor (vWf)-immobilized surface. Under flow conditions, the rolling velocity of the rGPIbalpha-vesicles decreased with the increasing membrane flexibility. It is considered that the vesicles with a high membrane flexibility have a high deformability and can be flattened to a high degree during rolling on the vWf surface, thus resulting in the large contact area. We obtained a recipe to control the rolling velocity of artificial platelets by membrane flexibility.     

Single nucleotide polymorphisms of thrifty genes for energy metabolism: evolutionary origins and prospects for intervention to prevent obesity-related diseases

The "thrifty" genotype and phenotype that save energy are detrimental to the health of people living in affluent societies. Individual differences in energy metabolism are caused primarily by single nucleotide polymorphisms (SNPs), some of which promote the development of obesity/type 2 diabetes mellitus. In this review, four major questions are addressed: (1) Why did regional differences in energy metabolism develop during evolution? (2) How do genes respond to starvation and affluence? (3) Which SNPs correspond to the hypothetical "thrifty genes"? (4) How can we cope with disease susceptibility caused by the "thrifty" SNPs? We examined mtDNA and genes for energy metabolism in people who live in several parts of Asia and the Pacific islands. We included 14 genes, and the SNP frequencies of PPAR gamma 2, LEPR, and UCP3-p and some other genes differ significantly between Mongoloids and Caucasoids. These differences in SNPs may have been caused by natural selection depending on the types of agriculture practiced in different regions. Interventions to counteract the adverse effects of "thrifty" SNPs have been partially effective.     

cDNA cloning and characterization of Drb1, a new member of RRM-type neural RNA-binding protein

Neural RNA recognition motif (RRM)-type RNA-binding proteins play essential roles in neural development. To search for a new member of neural RRM-type RNA-binding protein, we screened rat cerebral expression library with polyclonal antibody against consensus RRM sequences. We have cloned and characterized a rat cDNA that belongs to RRM-type RNA-binding protein family, which we designate as drb1. Orthologs of drb1 exist in human and mouse. The predicted amino acid sequence reveals an open reading frame of 476 residues with a corresponding molecular mass of 53kDa and consists of four RNA-binding domains. drb1 gene is specifically expressed in fetal (E12, E16) rat brain and gradually reduced during development. In situ hybridization demonstrated neuron-specific signals in fetal rat brain. RNA-binding assay indicated that human Drb1 protein possesses binding preference on poly(C)RNA. These results indicate that Drb1 is a new member of neural RNA-binding proteins, which expresses under spatiotemporal control.     

Dominant-negative mutant of BIG2, an ARF-guanine nucleotide exchange factor,specifically affects membrane trafficking from the trans-Golgi network through inhibiting membrane association of AP-1 and GGA coat proteins

BIG2 is one of the guanine nucleotide exchange factors (GEFs) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and AP-1 coat protein complexes and GGA proteins. Brefeldin A (BFA), an ARF-GEF inhibitor, causes redistribution of the coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). We have recently shown that BIG2 overexpression blocks BFA-induced redistribution of the AP-1 complex but not TGN membrane tubulation. In the present study, we constructed a dominant-negative BIG2 mutant and found that when expressed in cells it induced redistribution of AP-1 and GGA1 and membrane tubulation of the TGN. By contrast, the mutant did not induce COPI redistribution or Golgi membrane tubulation. These observations indicate that BIG2 is involved in trafficking from the TGN by regulating membrane association of AP-1 and GGA through activating ARF.     

Structural requirements for selective binding of ISC1 to anionic phospholipids

Yeast ISC1 (Yer019w) encodes inositolphosphosphingolipid-phospholipase C and is activated by phosphatidylserine (PS) and cardiolipin (CL) (Sawai, H., Okamoto, Y., Lubert, C., Mao, C., Bielawska, A., Domae, M., and Hannun, Y. A. (2000) J. Biol. Chem. 275, 39793-39798). In this study, the structural requirements for anionic phospholipid-selective binding of ISC1 were determined using site-directed and deletion mutants. FLAG-tagged Isc1p was activated by PS, CL, and phosphatidylglycerol (PG) in a dose-dependent manner. Using lipid-protein overlay assays, Isc1p interacted specifically and directly with PS/CL/PG. Lipid-protein binding studies of a series of deletion mutants demonstrated that the second transmembrane domain (TMII) and the C terminus were required for PS binding. Moreover, the TMII and the C terminus domain were sufficient to impart PS binding to a heterologous protein, green fluorescence protein. In addition, mutations of positively charged amino acid residues at the C terminus of ISC1 reduced the activating effects of PS, suggesting involvement of these amino acids in interaction with PS/CL/PG and in the activation of the enzyme. Finally, when separate fragments containing the N terminus-TMI and TMII-C terminus were expressed heterologously, enzyme activity was reconstituted, demonstrating that the interaction of the N terminus and the C terminus is required for activity of Isc1p. These results raise the hypothesis that in the presence of PS/CL/PG, the catalytic domain in the N terminus of Isc1p is "pulled" to the membrane to interact with substrate. These studies provide unique insights into the properties of ISC1 and define a novel mechanism for activation of enzymes by lipids cofactors.     

Gene replacement in mice reveals that the heavily phosphorylated tail of neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport

The COOH-terminal tail of mammalian neurofilament heavy subunit (NF-H), the largest neurofilament subunit, contains 44-51 lysine-serine-proline repeats that are nearly stoichiometrically phosphorylated after assembly into neurofilaments in axons. Phosphorylation of these repeats has been implicated in promotion of radial growth of axons, control of nearest neighbor distances between neurofilaments or from neurofilaments to other structural components in axons, and as a determinant of slow axonal transport. These roles have now been tested through analysis of mice in which the NF-H gene was replaced by one deleted in the NF-H tail. Loss of the NF-H tail and all of its phosphorylation sites does not affect the number of neurofilaments, alter the ratios of the three neurofilament subunits, or affect the number of microtubules in axons. Additionally, it does not reduce interfilament spacing of most neurofilaments, the speed of action potential propagation, or mature cross-sectional areas of large motor or sensory axons, although its absence slows the speed of acquisition of normal diameters. Most surprisingly, at least in optic nerve axons, loss of the NF-H tail does not affect the rate of transport of neurofilament subunits.     

Heterologous production of flavanones in<Emphasis Type="Italic"> Escherichia coli</Emphasis>: potential for combinatorial biosynthesis of flavonoids in bacteria

Chalcones, the central precursor of flavonoids, are synthesized exclusively in plants from tyrosine and phenylalanine via the sequential reaction of phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL) and chalcone synthase (CHS). Chalcones are converted into the corresponding flavanones by the action of chalcone isomerase (CHI), or non-enzymatically under alkaline conditions. PAL from the yeast Rhodotorula rubra, 4CL from an actinomycete Streptomyces coelicolor A3(2), and CHS from a licorice plant Glycyrrhiza echinata, assembled as artificial gene clusters in different organizations, were used for fermentation production of flavanones in Escherichia coli. Because the bacterial 4CL enzyme attaches CoA to both cinnamic acid and 4-coumaric acid, the designed biosynthetic pathway bypassed the C4H step. E. coli carrying one of the designed gene clusters produced about 450 μg naringenin/l from tyrosine and 750 μg pinocembrin/l from phenylalanine. The successful production of plant-specific flavanones in bacteria demonstrates the usefulness of combinatorial biosynthesis approaches not only for the production of various compounds of plant and animal origin but also for the construction of libraries of "unnatural" natural compounds. Dedicated to Professor Sir David Hopwood.     

Disruption of the CAR1 gene encoding arginase enhances freeze tolerance of the commercial baker's yeast Saccharomyces cerevisiae

The effect of intracellular charged amino acids on freeze tolerance in dough was determined by constructing homozygous diploid arginase-deficient mutants of commercial baker's yeast. An arginase mutant accumulated higher levels of arginine and/or glutamate and showed increased leavening ability during the frozen-dough baking process, suggesting that disruption of the CAR1 gene enhances freeze tolerance.