Comparative studies on the conformational change and aggregation behavior of irradiated carrageenans and agar by dynamic light scattering

The conformational associative properties of kappa-, iota-, and lambda-carrageenan and agar with irradiation dose were studied by dynamic light scattering. The random scission of the carrageenans and agar by gamma irradiation resulted in the formation of polydispersed lower molecular weight fragments. At high doses, the system moves towards uniformity. Conformational change from coil to helix was observed in all carrageenans and agar at doses up to 100 kGy. The conformational change in lambda-carrageenan may be due to the irregular and hybrid structure of this polysaccharide. Only agar and lambda-carrageenan still undergo conformational transition at a high dose of 200 kGy. Gelation is observed for kappa-, iota-carrageenan up to a dose of 50 kGy while gelation is still observed at 100 kGy for agar. Increase in the hydrodynamic radius with decreasing temperatures for the non-irradiated carrageenans follows this order: lambda-carrageenan>kappa-carrageenan>iota-carrageenan. Slight increases in hydrodynamic radius were observed with irradiation.     

Magnesium Supplementation did not Affect the Increasing Bone Zinc Concentration in Rats Given Excess Calcium as Carbonate

We investigated the effect of magnesium supplementation on zinc distribution in rats given excess calcium as carbonate. Rats were given a control diet (5 g/kg calcium and 0.5 g/kg magnesium), a high calcium diet (HC, 25 g/kg calcium and 0.5 g/kg magnesium) or the high calcium diet supplied with magnesium (HCM, 25 g/kg calcium and 2.5 g/kg magnesium) for 4 weeks. Calcium carbonate and magnesium oxide were used for increasing these mineral concentrations in diets. Although feed intake did not differ among the groups, the excess calcium suppressed feed efficiency, irrespective of dietary magnesium concentration. Femoral magnesium concentration was lower in the HC group than in the control and the HCM groups. Femoral zinc concentration was higher in the HC group and the HCM group than in the control group. The zinc concentration in the kidney was lower in the HC group and the HCM group than in the control group. The excess calcium did not affect zinc concentration in plasma and other tissues such as the liver, testis, and spleen, irrespective of dietary magnesium. These results suggest that the increasing bone zinc and the decreasing renal zinc do not result from magnesium insufficiency in rats given excess calcium as carbonate.     

Analysis of cyclic-stretching responses using cell-adhesion-patterned cells

Human vascular endothelial cells form the interface between the bloodstream and vessel walls and are continuously subjected to mechanical stimulation. When endothelial cells are stretched cyclically, along one axis, they align perpendicular to the axis of stretch. We previously reported that applying a cyclic, uni-axial strain to cells induced tyrosine phosphorylation of focal adhesion kinase and stimulated mitogen-activated protein kinase. However, it is difficult to quantify and analyze the spatial distribution of tyrosine phosphorylation in these cells, as they form focal adhesions randomly. In this study, we developed a system to overcome this problem by preparing individual, uniform, patterned cells that could be stretched cyclically and uni-axially. We constructed polydimethylsiloxane stretch chambers and used microcontact printing technology to imprint a pattern of 2 microm fibronectin dots (10 lines x 10 columns in a 38 microm square) before seeding them with human umbilical vein endothelial cells (HUVEC). We found that most HUVEC attached to the patterned dots after 2h and were similar in size and morphology, based on phase-contrast microscopy. In this system we were able to statistically analyze tyrosine phosphorylation and actin polymerization in these patterned cells, when subjected to a cyclic, uni-axial strain, using fluorescent microscopy.     

Affixin activates Rac1 via betaPIX in C2C12 myoblast

Affixin/beta-parvin is an integrin-linked kinase (ILK)-binding focal adhesion protein highly expressed in skeletal muscle and heart. To elucidate the possible role of affixin in skeletal muscle, we established stable C2C12 cell line expressing T7-tagged human affixin (C2C12-affixin cells). Exogenous expression of affixin promotes lamellipodium formation where affixin, ILK alphap21-activated kinase (PAK)-interactive exchange factor (PIX) and betaPIX accumulate. The association of affixin and betaPIX was confirmed by immunoprecipitation and pull down assay. In C2C12-affixin cells, an increased level of activated Rac1 but not Cdc42 was observed, and mutant betaPIX lacking guanine nucleotide exchange factor activity inhibited lamellipodium formation. These results suggest that affixin is involved in reorganization of subsarcolemmal cytoskeletal actin by activation of Rac1 through alpha and betaPIXs in skeletal muscle.     

Involvement of Sema4A in the progression of experimental autoimmune myocarditis

Dilated cardiomyopathy often results from autoimmunity triggered by microbial infections during myocarditis. However, it remains unclear how immunological disorders are implicated in pathogenesis of autoimmune myocarditis. Here, we demonstrated that Sema4A, a class IV semaphorin, plays key roles in experimental autoimmune myocarditis (EAM). Dendritic cells pulsed with myosin heavy chain-α peptides induced severe myocarditis in wild-type mice, but not in Sema4A-deficient mice. In adoptive transfer experiments, CD4⁺ T-cells from wild-type mice induced severe myocarditis, while CD4⁺ T-cells from Sema4A-deficient mice exhibited considerably attenuated myocarditis. Our results indicated that Sema4A is critically involved in EAM by regulating differentiation of T-cells.     

Cloning, expression, purification and characterization of an isotype of clytin, a calcium-binding photoprotein from the luminous hydromedusa Clytia gregarium

The cDNA for an isotype of clytin, a calcium-binding photoprotein from the luminous jellyfish Clytia gregarium, was identified and named clytin-II. The histidine-tagged apoprotein of clytin-II expressed into the periplasmic space of Escherichia coli cells was isolated by nickel chelate affinity chromatography. Recombinant clytin-II regenerated from apoprotein by incubation with coelenterazine was purified. The yield of purified clytin-II was 13 mg from 2 l of cultured cells with purity >95%. The luminescence properties of clytin-II were characterized by comparison with clytin-I and aequorin, and semi-synthetic clytin-II was also prepared. The initial luminescence intensity of clytin-II triggered by Ca(2+) was 4.5 times higher than that of clytin-I and aequorin, but the luminescence capacity was close to clytin-I and aequorin. Thus, clytin-II is a useful protein, showing high sensitivity in the signal-to-noise ratio of luminescence intensity.     

Multipotent cells from the human third molar: feasibility of cell-based therapy for liver disease

Abstract Adult stem cells have been reported to exist in various tissues. The isolation of high-quality human stem cells that can be used for regeneration of fatal deseases from accessible resources is an important advance in stem cell research. In the present study, we identified a novel stem cell, which we named tooth germ progenitor cells (TGPCs), from discarded third molar, commonly called as wisdom teeth. We demonstrated the characterization and distinctiveness of the TGPCs, and found that TGPCs showed high proliferation activity and capability to differentiate in vitro into cells of three germ layers including osteoblasts, neural cells, and hepatocytes. TGPCs were examined by the transplantation into a carbon tetrachloride (CCl₄)-treated liver injured rat to determine whether this novel cell source might be useful for cell-based therapy to treat liver diseases. The successful engraftment of the TGPCs was demonstrated by PKH26 fluorescence in the recipient's rat as to liver at 4 weeks after transplantation. The TGPCs prevented the progression of liver fibrosis in the liver of CCl₄-treated rats and contributed to the restoration of liver function, as assessed by the measurement of hepatic serum markers aspartate aminotransferase and alanine aminotransferase. Furthermore, the liver functions, observed by the levels of serum bilirubin and albumin, appeared to be improved following transplantation of TGPCs. These findings suggest that multipotent TGPCs are one of the candidates for cell-based therapy to treat liver diseases and offer unprecedented opportunities for developing therapies in treating tissue repair and regeneration.     

Developmental anatomy of the reproductive shoot in <Emphasis Type="Italic">Hydrobryum japonicum</Emphasis> (Podostemaceae)

Podostemaceae are unusual aquatic angiosperms adapting to extreme habitats, i.e., rapids and waterfalls, and have unique morphologies. We investigated the developmental anatomy of reproductive shoots scattered on crustose roots of Hydrobryum japonicum by scanning electron microscopy and using semi-thin serial sections. Two developmental patterns were observed: bracts arise either continuously from an area of meristematic cells that has produced leaves, or within differentiated root ground tissue beneath, and internal to, leaf base scars after an interruption. In both patterns, the bract primordia arise endogenously at the base of youngest bracts in the absence of shoot apical meristem, involving vacuolated-cell detachment to each bract separately. The different transition patterns of reproductive shoot development may be caused by different stages of parental vegetative shoots. The floral meristem arises between the two youngest bracts, and is similarly accompanied by cell degeneration. In contrast, the floral organs, including the spathella, arise exogenously from the meristem. Bract development, like vegetative leaf development, is unique to this podostemad, while floral-organ development is conserved.     

Phylogeography of the genus <Emphasis Type="Italic">Ulva</Emphasis> (Ulvophyceae,Chlorophyta), with special reference to the Japanese freshwater and brackish taxa

The nuclear-encoded ITS and associated 5.8S rDNA regions were sequenced for 72 specimens of Ulva collected from 44 rivers across Japan, including U. prolifera Müller from the Shimanto River, Kochi Prefecture, as well as 26 samples originally identified as U. linza L. from 20 coastal marine areas. Sequence data revealed that the samples fall into six distinct clades: the U. flexuosa Wulfen clade (2 samples), the Ulva linza-procera-prolifera (LPP) complex clade (75 samples), Ulva sp. 1 clade (3 samples), Ulva sp. 2 clade (7 samples), Ulva sp. 3 clade (4 samples) and Ulva sp. 4 clade (7 samples). The LPP complex contained a mixture of 26 samples collected from seashores and 49 samples obtained from rivers, including U. prolifera from the Shimanto River, and GenBank data for U. linza and U. procera Ahlner. The samples of the LPP complex differed by only 0–7 substitutions (0–1.149%). Subsequent phylogeographic analyses of the LPP complex based on the 5S rDNA spacer region revealed the presence of two further groupings: a group including 22 strictly marine littoral U. linza samples and a U. prolifera group composed of a mixture of 4 marine samples and all 49 river samples. The monophyly of all river samples indicates that adaptation to low salinity might have occurred only once in the evolutionary history of the LPP complex.