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Vacuum-ultraviolet circular dichroism (VUVCD) spectra of malto-, laminari-, isomalto-, and cello-oligosaccharide series and their corresponding polysaccharides (laminarin and dextran) were measured from 200 to 168 nm in aqueous solution at 25°C using a synchrotron-radiation VUVCD spectrophotometer. Disaccharides exhibited markedly different CD spectra depending on the types of glycosidic linkages, and the CD spectra of each oligosaccharide series (with the exception of the isomalto-oligosaccharide series) varied with the chain length below 190 nm while retaining the spectral shape of the constituent disaccharide. These results indicate that the basic structures of oligosaccharides were greatly affected by the configurations of their constituent disaccharides, which had unique torsion angles restricted by the intramolecular hydrogen bonds between glucose units. Based on comparisons between the experimental and theoretical data, we suggest that the chain-length dependence of CD above 180 nm reflects the backbone structure of oligosaccharides (e.g., helical structures), while those below 180 nm are influenced by other factors associated with higher-energy chromophores such as the hydroxyl groups. The reported comprehensive VUVCD spectra provide basic information for understanding the complicated structures of oligosaccharides in aqueous solution that can be used in their theoretical assignments. [ABSTRACT FROM AUTHOR]

Endochondral ossification is a developmental process by which cartilage is replaced by bone. Terminally differentiated hypertrophic chondrocytes are calcified, vascularized, and removed by chondroclasts before bone matrix is laid down by osteoblasts. In mammals, the malleus is one of three auditory ossicles that transmit vibrations of the tympanic membrane to the inner ear. The malleus is formed from a cartilaginous precursor without growth plate involvement, but little is known about how bones of this type undergo endochondral ossification. Here, we demonstrate that in the processus brevis of the malleus, clusters of osteoblasts surrounding the capillary loop produce bone matrix, causing the volume of the capillary lumen to decrease rapidly in post-weaning mice. Synchrotron X-ray tomographic microscopy revealed a concentric, cylindrical arrangement of osteocyte lacunae along capillaries, indicative of pericapillary bone formation. Moreover, we report that overexpression of Fosl1, which encodes a component of the AP-1 transcription factor complex, in osteoblasts significantly blocked malleal capillary narrowing. These data suggest that osteoblast/endothelial cell interactions control growth plate-free endochondral ossification through 'osteogenic capillaries' in a Fosl1-regulated manner. [ABSTRACT FROM AUTHOR]

Vacuum-ultraviolet (VUV) electronic circular-dichroism (ECD) spectra of methyl β-D-glucopyranoside (methyl β-D-Glc), methyl α- and β-D-galactopyranosides (methyl α- and β-D-Gal), and methyl α- and β-D-xylopyranosides (methyl α- and β-D-Xyl) were measured down to 163 nm in aqueous solution using a synchrotron-radiation VUV-ECD spectrophotometer. Five methyl aldopyranosides exhibited characteristic ECD spectra depending on the gauche (G) and trans (T) conformations of the hydroxymethyl group at C-5 and the α-/β-anomer configurations of the methoxy group at C-1. To elucidate the contributions of these structures to the spectrum, the ECD spectra of three rotamers (GT, GG and TG) of methyl β-D-Glc, methyl α-D-Gal and methyl β-D-Gal were calculated using a time-dependent density functional theory and molecular dynamics simulations. These theoretical spectra were very similar to those observed experimentally, indicating that the GT and GG rotamers show negative and positive ECD around 170 nm, respectively, and that the α- and β-anomers exhibit negative and positive ECD around 160 nm, respectively. These spectral differences between the two rotamers and between the two anomers were attributable to changes in steric configurations, including the intramolecular hydrogen bond around the ring oxygen and the methoxy oxygen, respectively. These relationships were supported by the ECD spectra experimentally observed for methyl α-D-Xyl and methyl β-D-Xyl, demonstrating that the VUV-ECD spectroscopy is a powerful technique for characterizing the structures of saccharides in aqueous solution. [ABSTRACT FROM AUTHOR]

Developing long bones alter their shape while maintaining uniform cortical thickness via coordinated activity of bone)forming osteoblasts and bone-resorbing osteoclasts at periosteal and endosteal surfaces, a process we designate trans-pairing. Two types of trans-pairing shift cortical bone in opposite orientations: peri-forming trans-pairing (peri-t-p) increases bone marrow space and endo-forming trans-pairing (endo-t-p) decreases it, via paired activity of bone resorption and formation across the cortex. Here, we focused on endo-t-p in growing bones. Analysis of endo-t-p activity in the cortex of mouse fibulae revealed osteoclasts under the periosteum compressed by muscles, and expression of RANKL in periosteal cells of the cambium layer. Furthermore, mature osteoblasts were localized on the endosteum, while preosteoblasts were at the periosteum and within cortical canals. X-ray tomographic microscopy revealed the presence of cortical canals more closely associated with endo- than with peri-t-p. Sciatic nerve transection followed by muscle atrophy and unloading induced circumferential endo-t-p with concomitant spread of cortical canals. Such canals likely supply the endosteum with preosteoblasts from the periosteum under endo-t-p, allowing bone shape to change in response to mechanical stress or nerve injury. [ABSTRACT FROM AUTHOR]

Bacteria have two routes for the L-methionine biosynthesis. In one route called the direct sulfuration pathway, acetylated L-homoserine is directly converted into L-homocysteine. The reaction using H2S as the second substrate is catalyzed by a pyridoxal 5'-phosphate-dependent enzyme, O-acetylhomoserine sulfhydrylase (OAHS). In the present study, we determined the enzymatic functions and the structures of OAHS from Lactobacillus plantarum (LpOAHS). The LpOAHS enzyme exhibited the highest catalytic activity under the weak acidic pH condition. In addition, crystallographic analysis revealed that the enzyme takes two distinct structures, open and closed forms. In the closed form, two acidic residues are sterically clustered. The proximity may cause the electrostatic repulsion, inhibiting the formation of the closed form under the neutral to the basic pH conditions. We concluded that the pH-dependent regulation mechanism using the two acidic residues contributes to the acidophilic feature of the enzyme. [ABSTRACT FROM AUTHOR]

In the endoplasmic reticulum (ER), ER oxidoreductin 1 (ERO1) catalyzes intramolecular disulfide-bond formation within its substrates in coordination with protein-disulfide isomerase (PDI) and related enzymes. However, the molecular mechanisms that regulate the ERO1–PDI system in plants are unknown. Reduction of the regulatory disulfide bonds of the ERO1 from soybean, GmERO1a, is catalyzed by enzymes in five classes of PDI family proteins. Here, using recombinant proteins, vacuum-ultraviolet circular dichroism spectroscopy, biochemical and protein refolding assays, and quantitative immunoblotting, we found that GmERO1a activity is regulated by reduction of intramolecular disulfide bonds involving Cys-121 and Cys-146, which are located in a disordered region, similarly to their locations in human ERO1. Moreover, a GmERO1a variant in which Cys-121 and Cys-146 were replaced with Ala residues exhibited hyperactive oxidation. Soybean PDI family proteins differed in their ability to regulate GmERO1a. Unlike yeast and human ERO1s, for which PDI is the preferred substrate, GmERO1a directly transferred disulfide bonds to the specific active center of members of five classes of PDI family proteins. Of these proteins, GmPDIS-1, GmPDIS-2, GmPDIM, and GmPDIL7 (which are group II PDI family proteins) failed to catalyze effective oxidative folding of substrate RNase A when there was an unregulated supply of disulfide bonds from the C121A/C146A hyperactive mutant GmERO1a, because of its low disulfide-bond isomerization activity. We conclude that regulation of plant ERO1 activity is particularly important for effective oxidative protein folding by group II PDI family proteins. [ABSTRACT FROM AUTHOR]

Toxin-antitoxin (TA) systems consist of a toxin inhibiting essential cellular functions (such as DNA, RNA and protein synthesis), and its cognate antitoxin neutralizing the toxicity. Recently, we identified a TA system termed TsbA/TsbT in the Staphylococcus aureus genome. The induction of the tsbT gene in Escherichia coli halted both DNA and RNA synthesis, reduced supercoiled plasmid and resulted in increasingly relaxed DNA. These results suggested that DNA gyrase was the target of TsbT. In addition, TsbT inhibited both E. coli and S. aureus DNA gyrase activity and induced linearization of plasmid DNA in vitro. Taken together, these results demonstrate that the TsbT toxin targets DNA gyrase in vivo. Sitedirected mutagenesis experiments showed that the E27 and D37 residues in TsbT are critical for toxicity. Secondary structure prediction combining the analysis of vacuum-ultraviolet circular-dichroism spectroscopy and neural network method demonstrated that the 22nd-32nd residues of TsbT form an a-helix structure, and that the E27 residue is located around the centre of the a-helix segment. These findings give new insights not only into S. aureus TA systems, but also into bacterial toxins targeting DNA topoisomerases. [ABSTRACT FROM AUTHOR]

Vacuum-ultraviolet (VUV) circular dichroism (CD) spectroscopy has recently been used for secondary structure analysis of proteins; however, the contribution of aromatic side chains to protein VUV CD spectra is unresolved. In this report, VUV CD spectra of 10 Escherichia coli dihydrofolate reductase (DHFR) mutants, in which each phenylalanine or tyrosine residue was mutated to leucine, were measured down to 175 nm at 25 °C and pH 8.0 to elucidate the contributions of these aromatic side chains to the high-energy transitions of peptide bonds. The VUV CD spectra of these mutants were different from the spectrum of the wild-type protein, indicating that the contribution of the phenylalanine and tyrosine side chains of DHFR extends to the VUV region. Furthermore, the VUV CD spectrum and the folate- or NADP+-induced spectral change of F103L mutant DHFR indicated a modification and regeneration of exciton coupling between the Trp47 and Trp74 side chains, respectively, suggesting that exciton coupling may also contribute to the CD spectrum of DHFR in the VUV region. These results should be useful for theoretically characterizing the contribution of aromatic side chains to protein CD spectra, leading to the improvement of protein secondary-structure analysis by VUV CD spectroscopy. [ABSTRACT FROM AUTHOR]

The article studies the purification and characterization of cell surface-expressed chitinase ChiW in species Paenibacillus. It reports that active ChiW that lacked the anchoring domains in the N-terminus was successfully overproduced in Escherichia coli and was purified to homogenity. It informs that two catalytic domains at the C-terminal region were classified from glycoside hydrolase family 18 chitinase.

NFATc1 is a transcription factor that regulates T-cell development, osteoclastogenesis, and macrophage function. Given that T cells, osteoclasts, and macrophages in the tumor microenvironment are thought to modulate tumor progression, tumor cells may acquire NFATc1 expression through fusion with these NFATc1-expressing normal cells. We here revealed that a small proportion of tumor cells in human carcinoma specimens expressed NFATc1. To investigate the consequences of NFATc1 acquisition by tumor cells, we established A549 and MCF7 cell lines expressing a constitutively active form of NFATc1 (NFATc1CA) in an inducible manner. The expression of NFATc1CA promoted cancer cell invasion in association with changes in cell morphology. Analysis of gene expression and RNA interference experiments revealed that NFATc1CA suppressed E-cadherin expression by upregulating the transcriptional repressors Snail and Zeb1 in a manner independent of TGF-β signaling. Induced expression of NFATc1CA also downregulated E-cadherin expression and increased invasive activity in tumor xenografts in vivo. Our results thus suggest that the acquisition of NFATc1 expression contributes to tumor progression. [ABSTRACT FROM AUTHOR]