Oversulfated chondroitin sulfate-E binds to BMP-4 and enhances osteoblast differentiation

Small leucine-rich proteoglycans, such as biglycan, and their side chain sulfated glycosaminoglycans (GAGs), have been suggested to be involved in bone formation and mineralization processes. The present study was designed to investigate whether chondroitin sulfate (CS), one of the GAG, and its oversulfated structures coupled with bone morphogenetic protein-4 (BMP-4) alter the differentiation and subsequent mineralization of MC3T3-E1 osteoblastic cells. CS-E, one of the oversulfated CS structure, enhanced cell growth, alkaline phosphatase (ALP) activity, collagen deposition, and mineralization whereas heparin enhanced only ALP activity and mineralization. As well as CS-E, CS-H, and CPS also enhanced the mineralization of the cells. CS-E enhanced the mineralization of the cells by interacting with protein in the conditioned medium. CS-E induced mineralization was significantly inhibited by an antibody against BMP-4. The addition of exogenous BMP-4 further increased the capacity of CS-E to enhance mineralization. Fluorescence correlation spectroscopy method using fluoresceinamine-labeled GAG revealed that the oversulfated GAGs have a high affinity for BMP-4. The disaccharide analysis of the cells indicated that MC3T3-E1 cells are capable of producing oversulfated structures of CS by themselves. The lack of CS from the cells after chondroitinase treatment resulted in the inhibition of mineralization. These results in the present study indicate that oversulfated CS, which possesses 4,6-disulfates in N-acetyl-galactosamine, binds to BMP-4 and promotes osteoblast differentiation and subsequent mineralization.     

Activation of cytosolic phospholipase A2alpha in resident peritoneal macrophages by Listeria monocytogenes involves listeriolysin O and TLR2

Eicosanoid production by macrophages is an early response to microbial infection that promotes acute inflammation. The intracellular pathogen Listeria monocytogenes stimulates arachidonic acid release and eicosanoid production from resident mouse peritoneal macrophages through activation of group IVA cytosolic phospholipase A2 (cPLA2alpha). The ability of wild type L. monocytogenes (WTLM) to stimulate arachidonic acid release is partially dependent on the virulence factor listeriolysin O; however, WTLM and L. monocytogenes lacking listeriolysin O (DeltahlyLM) induce similar levels of cyclooxygenase 2. Arachidonic acid release requires activation of MAPKs by WTLM and DeltahlyLM. The attenuated release of arachidonic acid that is observed in TLR2-/- and MyD88-/- macrophages infected with WTLM and DeltahlyLM correlates with diminished MAPK activation. WTLM but not DeltahlyLM increases intracellular calcium, which is implicated in regulation of cPLA2alpha. Prostaglandin E2, prostaglandin I2, and leukotriene C4 are produced by cPLA2alpha+/+ but not cPLA2alpha-/- macrophages in response to WTLM and DeltahlyLM. Tumor necrosis factor (TNF)-alpha production is significantly lower in cPLA2alpha+/+ than in cPLA2alpha-/- macrophages infected with WTLM and DeltahlyLM. Treatment of infected cPLA2alpha+/+ macrophages with the cyclooxygenase inhibitor indomethacin increases TNFalpha production to the level produced by cPLA2alpha-/- macrophages implicating prostaglandins in TNFalpha down-regulation. Therefore activation of cPLA2alpha in macrophages may impact immune responses to L. monocytogenes.     

Roles of functional and structural domains of hepatocyte growth factor activator inhibitor type 1 in the inhibition of matriptase

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-bound, Kunitz-type serine protease inhibitor. HAI-1 inhibits serine proteases that have potent pro-hepatocyte growth factor-converting activity, such as the membrane-type serine protease, matriptase. HAI-1 comprises an N-terminal domain, followed by an internal domain, first protease inhibitory domain (Kunitz domain I), low-density lipoprotein receptor A module (LDLRA) domain, and a second Kunitz domain (Kunitz domain II) in the extracellular region. Our aim was to assess the roles of these domains in the inhibition of matriptase. Soluble forms of recombinant rat HAI-1 mutants made up with various combinations of domains were produced, and their inhibitory activities toward the hydrolysis of a chromogenic substrate were analyzed using a soluble recombinant rat matriptase. Kunitz domain I exhibited inhibitory activity against matriptase, but Kunitz domain II did not. The N-terminal domain and Kunitz domain II decreased the association rate between Kunitz domain I and matriptase, whereas the internal domain increased this rate. The LDLRA domain suppressed the dissociation of the Kunitz domain I-matriptase complex. Surprisingly, an HAI-1 mutant lacking the N-terminal domain and Kunitz domain II showed an inhibitor constant of 1.6 pm, and the inhibitory activity was 400 times higher in this HAI-1 mutant than in the mutant with all domains. These findings, together with the known occurrence of an HAI-1 species lacking the N-terminal domain and Kunitz domain II in vivo, suggest that the domain structure of HAI-1 is organized in a way that allows HAI-1 to flexibly control matriptase activity.     

Anaerobic ammonium oxidation (anammox) irreversibly inhibited by methanol

Methanol inhibition of anaerobic ammonium oxidation (anammox) activity was characterized. An enrichment culture entrapped in a polyethylene glycol gel carrier was designed for practical uses of wastewater treatment. Batch experiments demonstrated that anammox activity decreased with increases in methanol concentration, and relative activity reached to 29% of the maximum when 5 mM methanol was added. Also, batch experiments were conducted using anammox sludge without immobilization. Anammox activity was evaluated by quantifying ¹⁴N¹⁵N (²⁹N) emission by combined gas chromatography-quadrupole mass spectrometry, and the anammox activity was found to be almost as sensitive to methanol as in the earlier trials in which gel carriers were used. These results indicated that methanol inhibition was less severe than previous studies. When methanol was added in the influent of continuous feeding system, relative activity was decreased to 46% after 80 h. Although the addition was halted, afterwards the anammox activity was not resumed in another 19 days of cultivation, suggesting that methanol inhibition to anammox activity was irreversible. It is notable that methanol inhibition was not observed if anammox activity was quiescent when substrate for anammox was not supplied. These results suggest that methanol itself is not inhibitory and may not directly inhibit the anammox activity.     

Neoculin, a taste-modifying sweet protein, accumulates in ripening fruits of cultivated Curculigo latifolia

Neoculin is a sweet protein with a taste-modifying activity of converting sourness to sweetness. It occurs in the fruits of Curculigo latifolia, a wild plant found in tropical Asia. We successfully cultivated the plant and evaluated the production of neoculin. The neoculin content of the fruit was high for 10 weeks after flowering, following which the yield decreased gradually. The optimal period for harvesting the fruits with sensory activity coincided with this 10-week peak period during which the amount of neoculin was 1-3mg in the whole fruit and 1.3mg/g of pulp. Immunohistochemical staining showed that neoculin occurred in the whole fruit, especially at the basal portion. Although it is known that neoculin comprises an acidic subunit (NAS) with an N-glycosylated moiety and a basic subunit (NBS), protein gel blot analysis revealed the presence of a non-glycosylated NAS species. This suggests the presence of multiple NAS-NBS heterodimers in our cultivar.     

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.