Postnatal changes in the rheological properties of the aorta in Sprague-Dawley rats.

Postnatal changes in the rheological properties of the aortic wall were investigated in relation to morphological development of the wall in Sprague-Dawley (SD) rats at 3, 8 and 20 weeks old. The mechanical tensile characteristics of the longitudinal wall strip excised from the proximal thoracic aorta were assessed with stress-strain and stress-relaxation tests. Wall tension in the low and medium strain ranges was significantly lower in 3-week-old rats than in 8-week-old rats and in 8-week-old rats than in 20-week-old rats. Wall stress was significantly lower in 3-week-old rats than in 8- and 20-week-old rats mainly in the medium strain range, but was significantly greater in 3-week-old rats than in 8- and 20-week-old rats in the high strain range. The value of incremental elastic modulus at 3 weeks old was significantly smaller than that at 8 and 20 weeks old at a strain of 0.25 and significantly larger than that at 8 and 20 weeks old at a strain of 0.50. The value of relaxation strength at 5 min after the stretching was significantly greater at 3 weeks old than that at 8 and 20 weeks old. The wall was viscoelastic in the low and medium strain ranges at 3 weeks though large wall stress was generated in the high strain range. Histological investigation revealed that the smooth muscle layer, fine elastin fiber connecting thick elastin fibers and wall thickness were thin at 3 weeks old in comparison with those at 8 and 20 weeks old, though there was no significant difference in number of nuclei of the smooth muscle cells among the three age groups. Changes in the tensile characteristics of the wall reflected well those of the microstructure of the wall with growth. The rheological properties and microstructure of the aortic wall were close to maturation at 8 weeks in SD rats.     

Temporal and spatial variations of lipid droplets during adipocyte division and differentiation

By capturing time-lapse images of primary stromal-vascular cells (SVCs) derived from rat mesenteric adipose tissue, we revealed temporal and spatial variations of lipid droplets (LDs) in individual SVCs during adipocyte differentiation. Numerous small LDs (a few micrometers in diameter) appeared in the perinuclear region at an early stage of differentiation; subsequently, several LDs grew to more than 10 microm in diameter and occupied the cytoplasm. We have developed a method for the fluorescence staining of LDs in living adipocytes. Time-lapse observation of the stained cells at higher magnification showed that nascent LDs (several 100 nm in diameter) grew into small LDs while moving from lamellipodia to the perinuclear region. We also found that adipocytes are capable of division and that they evenly distribute the LDs between two daughter cells. Immunofluorescence observations of LD-associated proteins revealed that such cell divisions of SVCs occurred even after LDs were coated with perilipin, suggesting that the "final" cell division during adipocyte differentiation occurs considerably later than that characterized in 3T3-L1 cells. Our time-lapse observations have provided a detailed account of the morphological changes that SVCs undergo during adipocyte division and differentiation.     

Discovery and SAR of a novel series of Natriuretic Peptide Receptor-A (NPR-A) agonists

Novel thienopyrimidine compounds 2 and 3 were discovered from high-throughput screening as Natriuretic Peptide Receptor A (NPR-A) agonists. Scaffold hopping of a thienopyrimidine ring to a quinazoline ring, introduction of the basic functional group and optimization of the substituent on the 6-position of the benzene ring of quinazoline led to improved agonistic activity. We discovered compound 48, which showed potent agonistic activity for NPR-A with an EC₅₀ value of 0.073 μM, indicating 350-fold potency compared to the hit compound 3.     

Capsinoid is biosynthesized from phenylalanine and valine in a non-pungent pepper, Capsicum annuum L. cv. CH-19 sweet

The biosynthetic pathway of capsinoid in 'CH-19 Sweet' was investigated. [(3)H]Valine and [(14)C]phenylalanine were injected into the fruits of the intact plant. Both of radioactivities were detected in capsinoid fractions. (14)C radioactivity was observed in phenylpropanoid compounds, and in vanillin, vanillylamine, vanillyl alcohol, and vanillic acid. We confirmed that capsinoid is biosynthesized from phenylalanine and valine.     

Distribution and microhabitats of freshwater mussels in waterbodies in the terrestrialized floodplains of a lowland river

Even when anthropogenically altered, river floodplains continue to contribute to biodiversity. This study examined the distribution of freshwater mussels in relation to environmental factors in waterbodies in the terrestrialized floodplain of a lowland river. Mussels were captured, and environmental measurements were taken in November of 2013 and 2014 in quadrats established in three floodplain waterbodies (FWBs), which were isolated from the main river channel. Among the three FWBs, mussel abundance was highest in a shallow FWB (depth range 18–45 cm) that had intermediate conditions of mud depth and fine sediment rate. Mussel abundance showed a hump-shaped relationship with water depth (the peak 45–50 cm) and mud depth (the peak 8–12 cm). Mussel abundance was also negatively related to the abundance of benthic litter. Litter abundance was positively related to branch abundance and the presence of tree cover, and negatively related to the distance to tree cover, indicating that benthic litter was derived from riparian trees. Our results indicate that relatively shallow (≤ 50 cm) FWBs with moderately accumulated mud, which are not scoured even during flooding, appear to be suitable habitats for mussels. Moreover, it is possible that riparian trees negatively impact mussel distribution in FWBs. Possible short-term measures for improving mussel habitat in FWBs may include the elimination of riparian trees and benthic litter.     

Distribution Patterns of 14C in the Labelled Vitamin B6 Prepared with the Cell-suspension of Achromobacter cycloclastes

The biosynthetic pathway of vitamin B₆ (abbreviated as Be) has been studied with the cell-suspension of B₆-producing bacteria, Achromobacter cycloclastes A.M.S. 6201. The distribution of ¹⁴C in the Be molecule prepared with the cell-suspensions containing glycerol-1,3-¹⁴C, glycerol-2-¹⁴C or γ-aminobutyric acid-U-¹⁴C was investigated by using three novel degradation methods. The results showed that carbon skeletons of glycerol and γ-aminobutyric acid were used for the formation of the major part of B₆ carbon skeleton respectively. The implication of these compounds as precursors of B₆ was discussed.     

Local mechanical properties measured by atomic force microscopy for cultured bovine endothelial cells exposed to shear stress

Morphology and mechanical properties of cultured endothelial cells were measured, using a novel atomic force microscope (AFM) system, developed in our laboratory, in conjunction with an inverted confocal laser scanning microscope. We used this system to examine endothelial cell both in static cultures and exposed to a shear stress of 2 Pa. Initially, the three-dimensional topography of a cell was measured by the AFM and a location was selected for the subsequent measurement of the mechanical response of the cell. The surface of statically cultured cell was smooth. The cell height was not altered by the exposed duration of shear stress. A relationship between external force, F, and the indentation depth, delta, was obtained for several different locations on a cell. This force-indentation response was modelled using a quadratic equation, F = adelta2 + bdelta, indicating that two parameters, a and b, will be constants which are representative of the mechanical response. Endothelial cells cultured at static conditions demonstrated a polygonal shape and less stiff mechanical characteristics around the nucleus compared to those at peripheral regions. The stiffness of the endothelial cells exposed to shear stress increased with the duration time of exposure. At 6-h exposures, the stiffness was higher at upstream side of the cell than the downstream side. However, after 24-h exposure, the stiffness was similar on both sides of the cell. These changes in the stiffness of endothelial cells when exposed to shear stress were suggested to correspond with the distribution of stress fibers in the cell.     

Membrane-rigidifying effects of anti-cancer dietary factors

Since several anti-cancer drugs interact with cell membrane lipids, the effects of anti-cancer dietary factors on liposomal membranes with different lipid composition were comparatively studied by measuring fluorescence polarization. Fluidity was imparted on both hydrophobic and hydrophilic regions of lipid bilayers by decreasing cholesterol and increasing unsaturated phosphatidylcholine in membranes. At 0.625-10 microM, (-)-epigallocatechin gallate, genistein, apigenin, resveratrol and a reference anti-cancer drug, doxorubicin, rigidified the tumor cell model membranes consisting of 20 mol% cholesterol and 80 mol% phosphatidylcholine with the acyl chain 18:1/16:0 ratio of 1.0, but not daidzein. They were more effective on the membrane core than the membrane surface. Quercetin showed a biphasic effect on the hydrophobic regions of membrane lipid bilayers to rigidify above 5 microM and fluidize below 2.5 microM. In contrast, anti-cancer dietary factors and doxorubicin were not or much less effective in rigidifying the normal cell model membranes consisting of 40 mol% cholesterol and 60 mol% phosphatidylcholine with the acyl chain 18:1/16:0 ratio of 0.5. The membrane-rigidifying effects were greater depending on a decrease of the cholesterol/phosphatidylcholine ratio and an increase of the phosphatidylcholine unsaturation degree. Membrane-active dietary factors and doxorubicin inhibited the growth of mouse myeloma cells at 10-100 microM, while the growth inhibition by membrane-inactive daidzein was relatively weak. Anti-cancer dietary factors appear to act on more fluid membranes like tumor cells as well as doxorubicin to induce rigidification, especially in the hydrocarbon core of membrane lipids, which is determined by the composition of cholesterol and unsaturated phospholipids.     

A new structural model for the thyrotropin (TSH) receptor, as determined by covalent cross-linking of TSH to the recombinant receptor in intact cells: evidence for a single polypeptide chain.

The most widely held model for the human TSH receptor is of holoreceptor of 80 kDa with two subunits of approximately 50 and 30 kDa linked by disulfide bridges, with the former subunit containing the major hormone-binding site. We reexamined this model by covalently cross-linking radiolabeled TSH to the recombinant human TSH receptor stably expressed in Chinese hamster ovary (CHO) cells. When cross-linking was performed after the preparation of CHO membranes, analysis of hormone-receptor complexes under reducing and nonreducing conditions provided results supporting the two-subunit TSH receptor model. In contrast, however, cross-linking of TSH to the TSH receptor in intact CHO cells before membrane preparation revealed, even under reducing conditions, an approximately 100-kDa receptor as well as an approximately 54-kDa hormone-binding subunit. The approximately 100-kDa holoreceptor size is consistent with the size of the TSH receptor, as predicted from its derived amino acid sequence. The proportions of the approximately 100-kDa TSH receptor and the 54-kDa fragment varied in different experiments, suggesting the occurrence of proteolytic cleavage. Cross-linking of radiolabeled TSH to intact cells expressing a mutant TSH receptor (TSHR-D1) lacking amino acids 317-366 localized the proteolytic cleavage site to just up-stream of amino acid residue 317. In summary, the present data obtained by cross-linking TSH to recombinant human TSH receptors in intact cells provides evidence that the receptor exists in vivo as an approximately 100-kDa glycoprotein with a single polypeptide chain with intramolecular disulfide bridges.(ABSTRACT TRUNCATED AT 250 WORDS)