Cell autonomous requirement for TGF-beta signaling during odontoblast differentiation and dentin matrix formation

TGF-β subtypes are expressed in tissues derived from cranial neural crest cells during early mouse craniofacial development. TGF-β signaling is critical for mediating epithelial-mesenchymal interactions, including those vital for tooth morphogenesis. However, it remains unclear how TGF-β signaling contributes to the terminal differentiation of odontoblast and dentin formation during tooth morphogenesis. Towards this end, we generated mice with conditional inactivation of the Tgfbr2 gene in cranial neural crest derived cells. Odontoblast differentiation was substantially delayed in the Tgfbr2^fl/fl;Wnt1-Cre mutant mice at E18.5. Following kidney capsule transplantation, Tgfbr2 mutant tooth germs expressed a reduced level of Col1a1 and Dspp and exhibited defects including decreased dentin thickness and absent dentinal tubules. In addition, the expression of the intermediate filament nestin was decreased in the Tgfbr2 mutant samples. Significantly, exogenous TGF-β2 induced nestin and Dspp expression in dental pulp cells in the developing tooth organ. Our data suggest that TGF-β signaling controls odontoblast maturation and dentin formation during tooth morphogenesis.     

Progression of flagellar stages during artificially delayed motility initiation in sea urchin sperm

Transition from immotile to motile flagella may involve a series of states, in which some of regulatory mechanisms underlying normal flagellar movement are working with others being still suppressed. To address ourselves to the study of starting transients of flagella, we analyzed flagellar movement of sea urchin sperm whose motility initiation had been retarded in an experimental solution, so that we could capture the instance at which individual spermatozoa began their flagellar beating. Initially straight and immotile flagella began to shiver at low amplitude, then propagated exclusively the principal bend (P bend), and finally started stable flagellar beating. The site of generation of the P bend in the P-bend propagating stage varied in position in the basal region up to 10 microm from the base, indicating that the ability of autonomous bend generation is not exclusively possessed by the very basal region but can be unmasked throughout a wider region when the reverse bend (R bend) is suppressed. The rate of change in the shear angle, the curvature of the R bend and the frequency and regularity of beating substantially increased upon transition from P-bend propagating to full-beating, while the propagation velocity of bends remained unchanged. These findings indicate that artificially delayed motility initiation may accompany sequential modification of the motile system and that mechanisms underlying flagellar motility can be analyzed separately under experimentally retarded conditions.     

Accumulation of glycolipids in mutant Chinese hamster ovary cells (Z65) with defective peroxisomal assembly and comparison of the metabolic rate of glycosphingolipids between Z65 cells and wild-type CHO-K1 cells

The influence of peroxisomal dysfunction on glycosphingolipid metabolism was investigated using mutant Chinese hamster ovary (CHO) cells (Z65) with defective assembly of the peroxisomal membranes. In accordance with previous observations, the concentration of very long chain fatty acid (C24:0) was shown to be higher in Z65 cells than in control cells. We then compared the composition of glycolipids in Z65 cells with that in CHO-K1 cells, which are wild-type Chinese hamster ovary cells with intact peroxisomes, and found significantly increased concentrations of ceramide monohexoside (CMH) and ganglioside GM3 in Z65 cells. However, there were no differences in the concentrations of glycerophospholipids, triglycerides, free fatty acids and cholesterol between Z65 and CHO-K1 cells. Further, to investigate the metabolic rate of the major lipids, Z65 and CHO-K1 cells were pulse-labeled with [3-14C]serine. [3-14C]Serine was incorporated into phosphatidylserine, phosphatidylethanolamine and sphingomyelin more quickly in CHO-K1 than in Z65 cells. However, after 48 h, the radioactivity incorporated into those lipids, including CMH, was greater in Z65 cells than in CHO-K1 cells. Thus, the altered metabolism of glycosphingolipids, probably due to peroxisomal dysfunction, was thought to be responsible for the change in glycosphingolipid composition in Z65 cells.     

Characterization of Endoplasmic Reticulum-Localized UDP-d-Galactose: Hydroxyproline O-Galactosyltransferase Using Synthetic Peptide Substrates in Arabidopsis

We characterized peptidyl hydroxyproline (Hyp) O-galactosyltransferase (HGT), which is the initial enzyme in the arabinogalactan biosynthetic pathway. An in vitro assay of HGT activity was established using chemically synthesized fluorescent peptides as acceptor substrates and extracts from Arabidopsis (Arabidopsis thaliana) T87 cells as a source of crude enzyme. The galactose residue transferred to the peptide could be detected by high-performance liquid chromatography and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analyses. HGT required a divalent cation of manganese for maximal activity and consumed UDP-d-galactose as a sugar donor. HGT exhibited an optimal pH range of pH 7.0 to 8.0 and an optimal temperature of 35°C. The favorable substrates for the activity seemed to be peptides containing two alternating imino acid residues including at least one acceptor Hyp residue, although a peptide with single Hyp residue without any other imino acids also functioned as a substrate. The results of sucrose density gradient centrifugation revealed that the cellular localization of HGT activity is identical to those of endoplasmic reticulum markers such as Sec61 and Bip, indicating that HGT is predominantly localized to the endoplasmic reticulum. To our knowledge, this is the first characterization of HGT, and the data provide evidence that arabinogalactan biosynthesis occurs in the protein transport pathway.O-glycosylation is the addition of a sugar to hydroxy amino acids such as Thr, Ser, Hyp, Hyl, or Tyr (Lehle et al., 2006). This type of protein modification occurs in many organisms to modify a large variety of proteins. Several types of sugars can be linked to proteins via O-glycosylation, including Man, N-acetylgalactosamine, Glc, Xyl, N-acetylglucosamine, Fuc, Gal, and arabinofuranose (Araf). In addition, elongation of the added sugar residues yields a large variety of oligo- and polysaccharide extensions on the substrate proteins. These modifications are known to play important roles in various phenomena, including pathways required to maintain biological systems and basic cellular functions.Structural analysis of oligo- and polysaccharides in plant cell walls has revealed the presence of three types of O-linked structures, Gal-O-Hyp, Araf-O-Hyp, and Gal-O-Ser (Kieliszewski and Shpak, 2001; Seifert and Roberts, 2007). A part of these three structures has been found on proteins in the super family that includes arabinogalactan protein (AGP) and extensin, which are localized to the cell surface. AGPs contain O-linked arabinogalactan oligo- or polysaccharides attached to Hyp residues (Gal-O-Hyp). It is known that arabinogalactan polysaccharides mainly consist of β-1,3 linkages of Gal polymers (Seifert and Roberts, 2007). Extensin contains short arabino-oligosaccharide chains attached to Hyp residues (Araf-O-Hyp) and single Gal residues linked to Ser residues (Gal-O-Ser). It has been suggested that these O-linked structures play an important role in many stages of growth and development in plants, including signaling, embryogenesis, and programmed cell death (Knox, 2006; Seifert and Roberts, 2007). However, our understanding of the biosynthesis of these O-linked structures is limited at present.Shpak et al. described a novel strategy to elucidate O-glycosylation of AGPs via introduction of synthetic genes encoding a protein substrate of glycosyltransferases into plant cells (Shpak et al., 1999; Estevez et al., 2006). This strategy provided good evidence for the substrate specificities of Hyp O-galactosyltransferase (HGT). Hyp galactosylation occurs on clustered noncontiguous Hyp residues such as Xaa-Hyp-Xaa-Hyp repeats of AGPs (where Xaa is any amino acid except Hyp; Tan et al., 2003). However, the arabinogalactosylation site is not limited to clustered noncontiguous Hyp residues, as isolated Hyp residues with appropriate surrounding sequences can be modified with arabinogalactan (Matsuoka et al., 1995; Shimizu et al., 2005). Therefore, the mechanism of glycosylation to Hyp residues seems complex in plants, while we have little information about the glycosyltransferase(s) involved in arabinogalactan biosynthesis. To examine the enzymatic properties and to identify genes involved in arabinogalactan biosynthesis, we first attempted to establish an in vitro assay for HGT activity, which catalyzes the initial step in arabinogalactan biosynthesis in plants.Here, we report a novel assay for HGT activity based on the use of endoplasmic reticulum (ER)-enriched cell lysates extracted from Arabidopsis (Arabidopsis thaliana) T87 cells as a source of the enzyme and chemically synthesized fluorescent peptides as enzyme substrates. The method enabled us to characterize the enzymatic properties of HGT and to determine the localization of HGT in Arabidopsis cells. Properties of the enzyme and the usefulness of our assay for various studies are discussed.     

A high-potential nonheme iron protein (HiPIP)-linked,thiosulfate-oxidizing enzyme derived fromChromatium vinosum

A thiosulfate-oxidizing enzyme was partially purified fromChromatium vinosum, and some of its properties were studied. The enzyme rapidly reducede HiPIP (high-potential nonheme iron protein) in the presence of thiosulfate. Cytochromesc of yeast and tuna and ferricyanide also acted well as electron acceptors for the enzyme; horse cytochromec was a poor electron acceptor. Cytochromec-552, cytochromec′, and cytochromec-553 did not act as electron acceptors. The enzyme was inhibited by cyanide and sulfite. On the basis of the stoichiometry in reduction of ferricyanide catalyzed by the enzyme in the presence of thiosulfate, the oxidized product of thiosulfate was inferred to be tetrathionate.     

Comparison of stress responses between mental tasks and white noise exposure

The purpose of this study was to compare the cardiovascular responses to different types of mental stress. Ten healthy males performed a mental arithmetic task (MA) on one day and were exposed to white noise (WN, 80dB) on another day. Both the MA and the WN were composed of four 5-min consecutive periods with a 3-min rest between them. On each day, the systolic and diastolic blood pressure (SBP and DBP), mean arterial pressure (MAP), cardiac output (CO), and total peripheral resistance (TPR) were measured continually during the entire experimental period. The changes from the baseline (Delta) in all periods were calculated for both mental stresses. As for the results, the DeltaMAP, DeltaCO, DeltaHR, and DeltaTPR in the MA did not significantly change during the task periods. However, in the WN, the DeltaMAP and DeltaTPR showed significant increases over the time of the consecutive periods. In addition, we discuss the response patterns for the two mental stresses. We examine three hemodynamic reactivity patterns: a cardiac pattern characterized by increased CO and decreased TPR, a mixed pattern characterized by a moderate increase in both CO and TPR, and a vascular pattern characterized by increased TPR and decreased CO. The results show that throughout all task/exposure periods, the response pattern remained the same for six subjects in each stress. Furthermore, of these six subjects, half showed the same response pattern in both the MA and the WN. In conclusion, compared to the MA task, consecutive WN exposure showed an accumulation of stress responses. A change in TPR contributed to a gradual increase in MAP in the WN. It is also possible that among the subjects there were different types of response to the MA and WN.     

Effect of Coccolith Polysaccharides Isolated from the Coccolithophorid, <Emphasis Type="Italic">Emiliania huxleyi</Emphasis>, on Calcite Crystal Formation in In Vitro CaCO<Subscript>3</Subscript> Crystallization

Marine coccolithophorids (Haptophyceae) produce calcified scales “coccoliths” which are composed of CaCO₃ and coccolith polysaccharides (CP) in the coccolith vesicles. CP was previously reported to be composed of uronic acids and sulfated residues, etc. attached to the polymannose main chain. Although anionic polymers are generally known to play key roles in biomineralization process, there is no experimental data how CP contributes to calcite crystal formation in the coccolithophorids. CP used was isolated from the most abundant coccolithophorid, Emiliania huxleyi. CaCO₃ crystallization experiment was performed on agar template layered onto a plastic plate that was dipped in the CaCO₃ crystallization solution. The typical rhombohedral calcite crystals were formed in the absence of CP. CaCO₃ crystals formed on the naked plastic plate were obviously changed to stick-like shapes when CP was present in the solution. EBSD analysis proved that the crystal is calcite of which c-axis was elongated. CP in the solution stimulated the formation of tabular crystals with flat edge in the agarose gel. SEM and FIB-TEM observations showed that the calcite crystals were formed in the gel. The formation of crystals without flat edge was stimulated when CP was preliminarily added in the gel. These observations suggest that CP has two functions: namely, one is to elongate the calcite crystal along c-axis and another is to induce tabular calcite crystal formation in the agarose gel. Thus, CP may function for the formation of highly elaborate species-specific structures of coccoliths in coccolithophorids.