Substrate selectivity of imidazole-appended dimethyl-β-cyclodextrin

The substrate selectivity of the hydrolysis reaction by imidazole-appended dimethyl-β-cyclodextrin (2) was studied using different kinds of p-nitrophenyl esters of amino acids. The tendency for the hydrolysis reaction of the amino acid ester by 2 reflected a difference in K_m rather than in k_cat. 2 had the ability to undergo a stereo-selective hydrolysis reaction.     

Phosphoprotein synthesis and secretion by odontoblasts in rat incisors as revealed by electron microscopic radioautography

The secretory pathway of dentin phosphoproteins in rat incisors was studied by electron microscopic radioautography after the injection of 3H-serine, and the results were compared with those using 3H-proline as a tracer. Five min after injection of 3H-serine, radioactivity was found in the rough endoplasmic reticulum. At 10 min, silver grains were observed over the spherical portions of the cisface of the Golgi apparatus. At 20 min after injection, silver grains were seen over the cylindrical portions of the transface of the Golgi apparatus. The secretory granules showed the strongest reaction from 20 min to 1 hr. At 45 min, a significant labeled band appeared at the mineralization front. At 1 hr, the labeling at the mineralization front began to appear in the mineralized dentin, and after 12 hr this labeled band was located within the mineralized dentin. The pathway of 3H-proline was essentially the same as that of 3H-serine, but 3H-proline moved more slowly than 3H-serine, especially in transit from the rough endoplasmic reticulum to the Golgi apparatus. Secretory granules were heavily labeled from 30 min to 1 hr after injection of 3H-proline; no labeling was found at the mineralization front at 45 min. The labeling seen initially over the predentin was over the mineralized dentin no earlier than 6 hr after injection. The labeling pattern with 3H-serine is closely related to the localization of phosphoproteins, whereas the pattern with 3H-proline reflects the production of collagen rather than of phosphoproteins. The present radioautographic results indicate that dentin phosphoproteins are related to secretory granules and are secreted by odontoblasts at the mineralization front and also that phosphoproteins are involved in the process of mineralization of the circumpulpal dentin.     

The effect of conformity and plastic thickness on contact stresses in metal-backed plastic implants

Surface damage in polyethylene components for total joint replacement is associated with large contact stresses. An elasticity solution and finite element analyses were used to determine the influence of design parameters on the stresses due to contact in metal-backed components. For nearly conforming contact surfaces, it was found that the stresses in the plastic are very sensitive to clearance, that minimum plastic thickness of 4-6 mm should be maintained for metal-backed components, and that bonding the plastic to the metal backing reduces tensile stresses in the plastic at the edge of the contact zone.     

Invertase biosynthesis by Saccharomyces carlsbergensis in batch and continuous cultures

The biosynthesis of invertase by Saccharomyces carlsbergensis LAM 1068 was studied in relation to its glucose effect at both unsteady and steady states of growth. Experimental correlations between the dilution rate and invertase specific activity (E/X) in chemostat, cultures led to an optimum for the enzyme synthesis at a particular intermediate growth rate. The value of E/X increased from 1.1 (U/mg biomass) in batch cultures to 13 (U/mg biomass) in chemostat cultures. A mutant strain A3 showed the highest value for E/X = 25 (U/mg biomass) at high dilution rates where glucose repression was observed with the wild strain.     

Deletion of tumor necrosis factor-α receptor type 1 exacerbates insulin resistance and hepatic steatosis in aromatase knockout mice

The relevance of estrogen functions in lipid metabolism has been suggested in patients with estrogen-signaling deficiencies. Their importance was further implied by studies in estrogen-deficient mice (ArKO mice), which progressively developed hepatic steatosis. As circulating tumor necrosis factor (TNF)-α levels are known to positively correlate with disturbances in lipid metabolism, we investigated the impact of the loss of TNF-α signaling on carbohydrate and lipid metabolism in ArKO mice. Histological examinations of the livers of mice at 5 months of age revealed that ArKO male mice lacking the TNF-α receptor type 1 (TNFR1) gene (ArKO/TNFR1KO) or both the TNFR 1 and 2 genes (ArKO/TNFR1&2KO) developed more severe hepatic steatosis than ArKO or ArKO/TNFR2KO mice. Serum analyses demonstrated a clear increase in cholesterol and insulin levels in the ArKO/TNFR1KO mice compared with the ArKO mice. Glucose- and insulin-tolerance tests further revealed exacerbation of the systemic insulin resistant phenotype in the ArKO/TNFR1KO mice. Hepatic expression of lipogenic genes including fatty-acid synthase and stearoyl-Coenzyme A desaturase 1 were more markedly upregulated in the ArKO/TNFR1KO mice than the ArKO mice. These findings indicate that under estrogen-deficient physiological conditions, hepatic lipid metabolism would benefit from TNF-α mediated signaling via TNFR1.     

Relict distribution of Microhyla (Amphibia: Microhylidae) in the Ryukyu Archipelago: High diversity in East Asia maintained by insularization

The Ryukyu Archipelago, located at the southwestern part of Japan, is known as a group of continental islands and harbours many endemic taxa, supposedly reflecting its fairly long isolation from the Eurasian continent, Taiwan and the Japanese main islands. Microhyla okinavensis has been known as an endemic member of the terrestrial fauna of this archipelago. Molecular phylogenetic analyses using samples from nearly all island populations of the species and representative samples of other east Asian congeneric species revealed that M. okinavensis consists of four distinct subclades, of which the Amami, Okinawa and Miyako subclades, though exhibiting distinct genetic differentiations from each other, formed a monophyletic group (clade A). The remaining Yaeyama subclade was exclusively sister to M. mixtura from inland China, forming another monophyletic group (clade B), rendering M. okinavensis in the current definition paraphyletic. These results, as well as estimated dates of divergence from related taxa, indicate that M. okinavensis actually includes more than one distinct species. The results indicate that M. okinavensis and M. mixtura are relict species with disjunct distributions which had been most probably caused by invasion of M. fissipes in intervening areas.     

Suppression of BCR-ABL mRNA by various ribozymes in HeLa cells.

Ribozymes are RNA molecules with enzymatic activity that can cleave target RNA molecules in a sequence specific manner. To date, various types of ribozyme have been constructed to cleave other RNAs and such trans-acting ribozymes include hammerhead, hairpin and HDV ribozymes. External guide sequence (EGS) can also induce the suppression of a gene-expression by taking advantage of cellular RNase P. Here we compared the activities of various functional RNA cleavers both in vitro and in vivo. The first purpose of this comparison was intended to determine the best ribozyme motif with the highest activity in cells. The second purpose is to know the correlation between the activities of ribozymes in vitro and in vivo. Our results indicated that the intrinsic cleavage activity of ribozymes is not the sole determinant that is responsible for the activity of a ribozyme in cultured cells.     

Expression profiles and functional characterization of common carp (Cyprinus carpio) T2Rs

Bitter taste perception is mediated by a family of G protein-coupled receptors (T2Rs) in vertebrates. Common carp (Cyprinus carpio), which has experienced an additional round of whole genome duplication during the course of evolution, has a small number of T2R genes similar to zebrafish, a closely related cyprinid fish species, and their expression pattern at the cellular level or their cognate ligands have not been elucidated yet. Here, we showed through in situ hybridization experiments, that three common carp T2R (ccT2R) genes encoding ccT2R200-1, ccT2R202-1, and ccT2R202-2, were specifically expressed in the subsets of taste receptor cells in the lips and gill rakers. ccT2R200-1 was co-expressed with genes encoding downstream signal transduction molecules, such as PLC-β2 and Gαia. Heterologous expression system revealed that each ccT2R showed narrowly, intermediately, or broadly tuned ligand specificity, as in the case of zebrafish T2Rs. However, ccT2Rs showed different ligand profiles from their orthologous zebrafish T2Rs previously reported. Finally, we identified three ccT2Rs, namely ccT2R200-1, ccT2R200-2, and ccT2R203-1, to be activated by natural bitter compounds, andrographolide and/or picrotoxinin, which elicited no response to zebrafish T2Rs, in a dose-dependent manner. These results suggest that some ccT2Rs may have evolved to function in the oral cavity as taste receptors for natural bitter compounds found in the habitats in a species-specific manner.     

Temperature Changes in Brown Adipocytes Detected with a Bimaterial Microcantilever

Mammalian cells must produce heat to maintain body temperature and support other biological activities. Methods to measure a cell’s thermogenic ability by inserting a thermometer into the cell or measuring the rate of oxygen consumption in a closed vessel can disturb its natural state. Here, we developed a noninvasive system for measuring a cell’s heat production with a bimaterial microcantilever. This method is suitable for investigating the heat-generating properties of cells in their native state, because changes in cell temperature can be measured from the bending of the microcantilever, without damaging the cell and restricting its supply of dissolved oxygen. Thus, we were able to measure increases in cell temperature of <1 K in a small number of murine brown adipocytes (n = 4–7 cells) stimulated with norepinephrine, and observed a slow increase in temperature over several hours. This long-term heat production suggests that, in addition to converting fatty acids into heat energy, brown adipocytes may also adjust protein expression to raise their own temperature, to generate more heat. We expect this bimaterial microcantilever system to prove useful for determining a cell’s state by measuring thermal characteristics.