Molecular identity of uncoupling proteins in thermogenic skunk cabbage

Thermogenic skunk cabbage has been reported to have two types of uncoupling protein (UCP), a typical 6-transmembrane (TM) SrUCPA and an atypical 5-TM SrUCPB. To verify further the role of SrUCPs in thermogenic skunk cabbage, we examined the molecular identity of SrUCPs in more detail. Both mRNA and genomic analyses supported the presence of SrUCPA, but not SrUCPB. Furthermore, SrUCP protein purified from spadix mitochondria was identified as SrUCPA by mass spectrometry. These results clearly indicate that SrUCPA is the major expressed UCP in skunk cabbage, and the presence of atypical SrUCPB is unlikely to be associated with thermogenesis of skunk cabbage.     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.     

Comparative Genome Analysis of Lactobacillus reuteri and Lactobacillus fermentum Reveal a Genomic Island for Reuterin and Cobalamin Production.

Lactobacillus reuteri is a heterofermentative lactic acid bacterium that naturally inhabits the gut of humans and other animals. The probiotic effects of L. reuteri have been proposed to be largely associated with the production of the broad-spectrum antimicrobial compound reuterin during anaerobic metabolism of glycerol. We determined the complete genome sequences of the reuterin-producing L. reuteri JCM 1112(T) and its closely related species Lactobacillus fermentum IFO 3956. Both are in the same phylogenetic group within the genus Lactobacillus. Comparative genome analysis revealed that L. reuteri JCM 1112(T) has a unique cluster of 58 genes for the biosynthesis of reuterin and cobalamin (vitamin B(12)). The 58-gene cluster has a lower GC content and is apparently inserted into the conserved region, suggesting that the cluster represents a genomic island acquired from an anomalous source. Two-dimensional nuclear magnetic resonance (2D-NMR) with (13)C(3)-glycerol demonstrated that L. reuteri JCM 1112(T) could convert glycerol to reuterin in vivo, substantiating the potential of L. reuteri JCM 1112(T) to produce reuterin in the intestine. Given that glycerol is shown to be naturally present in feces, the acquired ability to produce reuterin and cobalamin is an adaptive evolutionary response that likely contributes to the probiotic properties of L. reuteri.     

A method for the design of 3D scaffolds for high-density cell attachment and determination of optimum perfusion culture conditions

The application of in vitro cultured cells in tissue engineering or drug screening, aimed at complex soft tissues such as liver, requires in vivo physiological function of the cultured cells. For this purpose, the scaffold in which cells are cultured should provide a microenvironment similar to an in vivo one with a three-dimensional extracellular matrix, a high supply capacity of O₂ and nutrients, and high cell density. In this paper, we propose a method to design (1) the geometry of the scaffold, with a surface/volume ratio optimized to allow high-density (5×10⁷ cells/mL) cell culture and (2) culture conditions that will supply optimal quantities of oxygen and nutrients. CFD modeling of mass transport was used to determine the shear stress as well as O₂ and glucose metabolism in the scaffold (20 mm width–35 mm length) for various flow rates. Validation of the model was done through comparison with flow resistance and micro-PIV experiments. CFD analysis showed the maximum metabolic rate densities for this scaffold are 6.04×10⁻³ mol/s/m³ for O₂ at 0.71 mL/min and 1.91×10⁻² mol/s/m³ for glucose at 0.35 mL/min.     

A method for detecting effect of beneficial mutations in natural populations of Drosophila melanogaster.

An experimental method is proposed for detecting the effects of positive natural selection on DNA polymorphisms. Since beneficial mutations are expected to increase in frequency faster than neutral mutations, variants which have reached high frequencies in a relatively short period could be linked to some beneficial mutation. D. melanogaster has a cosmopolitan polymorphic inversion -In(2L)t- whose age in some local populations has been estimated. Setting the age of In(2L)t as the upper limit for the age of variants, we searched for variants whose frequencies were possibly influenced by positive natural selection. We detected a single candidate whose frequency and distribution met the requirements imposed by our method.     

Continuous column culture system for adherent cells

A new continuous column culture system for adherent cells was developed using beads. The beads were packed in a column and an appropriate medium was continuously passed through. The whole system was kept under closed conditions. L cells and C6 cells were cultured by this new system. The number of cells increased linearly up to 16 days and reached a maximum at around 18 days. As the heat production remained constant for 16 days, it can be concluded that cells grown in this system had identical characteristics. The final concentration of cells reached was 1.0 × 10⁸ml^?1. The cells could grow both in the upward and the downward direction. Advantages of this system are: (1) Cells can be recovered in their adherent form on the beads; (2) cells can easily be collected from the column by trypsinization, and (3) cells remaining in the column after trypsinization can grow again.     

A stimulatory effect of somatostatin: enhancement of activin A-mediated FSH secretion in rat pituitary cells

The interaction between somatostatin and activin A was studied in terms of FSH secretion in rat pituitary cells in primary culture. Incubation of pituitary cells with 1 nM activin A for 48 hrs resulted in an increase in FSH release into incubation medium. The effect of activin A was dependent on cell-density and the higher the density, the smaller the stimulatory action of activin A. Somatostatin, by itself, did not affect the FSH secretion. When 100 nM somatostatin was included together with activin A or the cells were pretreated with somatostatin for 2 hrs, the activin A-induced FSH secretion was enhanced. This potentiation effect of somatostatin was inversely dependent on the cell-density. These results indicate that somatostatin enhances, rather than inhibits, the activin A action in pituitary cells.     

Enhancement of Newcastle Disease Virus-Induced Fusion of Mouse L Cells by Sodium Vanadate

Sodium vanadate enhanced Newcastle disease virus (NDV)-induced cell fusion in L cells, and there was a direct correlation between the degree of cell fusion and the dose of vanadate added. When anti-F protein of NDV monospecific antiserum was added to the culture fluid of L cells infected with NDV, the enhancement of cell fusion was suppressed. In contrast, neither anti-HN nor anti-M protein monospecific antiserum inhibited the enhancement. Incubation at low temperature (4 C) and addition of sodium azide to the culture fluid suppressed the enhancement. The suppression by azide was seen only when the drug was added within 5 min after the beginning of incubation of NDV-infected L cells with vanadate. On the other hand, incubation at low temperature inhibited the enhancement at any time during incubation with vanadate. Cytochalasin D also inhibited the enhancement if it was added at any time during incubation with vanadate.     

Production of hyperdorsal larvae by exposing uncleaved Xenopus eggs to a centrifugal force directed from the animal pole to the vegetal pole

Exposure of uncleaved Xenopus eggs to a centrifugal force directed from the animal pole to the vegetal pole produces larvae with enhanced dorsal structures, which resemble 'hyperdorso-anterior' larvae produced by D₂O-treatment at 0.3 normalized time (NT). Optimal conditions are 70 g for 6 min at 20% of the first cell cycle (0.2 NT). Exposure before removal of vegetal pole cortical cytoplasm, which we find has an effect of eliminating dorsal structures, protects eggs from losing their ability to form dorsal axial structures upon removal. In contrast, exposure after a slight ultraviolet (UV)-irradiation, which has virtually no effect on dorsal development, produces larvae with heavily reduced dorsal structures, which resemble 'ventralized' larvae produced by heavy UV-irradiation. Interestingly, none of these treatments prevents cortical rotation. Morphological and histological examinations reveal that exposure to the force causes displacement of both cortical and deep egg components from around the vegetal pole to subequatorial regions. We conclude that exposure to the centrifugal force enhances dorsal structures by displacing dorsal determinants from around the vegetal pole to subequatorial regions broader than normal. This is the first experiment in which displacement of egg components, by methods independent of the rotation, are shown to perturb larval body pattern.