D- and l-amino acid concentrations in culture broth of Lactobacillus are highly dependent on the phylogenetic group of Lactobacillus

d-amino acids produced by Lactobacillus are thought to contribute to the taste quality and health functions; however, no studies have comprehensively evaluated the concentrations of the D- and L-forms of amino acids separately in individual Lactobacillus strains. To gain insight into amino acid concentrations in Lactobacillus, we evaluated amino acid concentrations in culture broth of Lactobacillus separately for the D- and L-forms. Lactobacillus strains were cultured in culture broth, and the amino acid concentrations in supernatant were assessed. The amino acid concentrations obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were subjected to cluster analysis based on Bray-Curtis distance with Ward's minimum variance method. In the analysis of amino acid concentrations under culture with different monosaccharides, the distances among strains cultured with the same monosaccharide were significantly greater than those among cultures of the same strain under different monosaccharides (p < 0.01). The cluster analysis of amino acid concentrations under culture with the same monosaccharide suggested that strains belonging to the same phylogenetic group of Lactobacillus exhibited similar concentrations of amino acids. Data analyses of 70 strains belonging to 17 Lactobacillus taxa indicated that the concentrations of amino acids were highly dependent on the phylogenetic group of Lactobacillus and that the group differences in amino acid concentration were strongly driven by differences in l-serine and d-alanine concentrations. Our results indicate that it is important to evaluate D- and l-amino acids separately when evaluating variations in amino acid concentrations. Because d-alanine has the potential to affect taste quality, the results of this study may provide insight into the taste quality of fermented food produced by Lactobacillus.     

Body‐color and behavioral responses by the mid‐instar nymphs of the desert locust,Schistocerca gregaria (Orthoptera: Acrididae) to crowding and visual stimuli

The effects of crowding and isolation on body color and behavior were observed for the mid‐instar nymphs of the desert locust, Schistocerca gregaria. Some of the solitarious (isolation‐reared) nymphs that were crowded for 1 or 4 h during the third instar developed black patterns at the fourth instar, but most individuals remained unaffected. Black patterns appeared in all individuals that were crowded for 1 day or longer, but even after 4 days of crowding the black patterning for some individuals was not as intense as that for the gregarious (crowd‐reared) controls. Isolation of gregarious nymphs caused the black patterns to recede or disappear at the last (fifth) nymphal instar, but it was necessary to isolate the nymphs from the beginning of the first instar to obtain body coloration looking like solitarious nymphs in most individuals. Solitarious nymphs that were allowed to see gregarious nymphs developed different intensities of black patterns depending on the body size and number of nymphs shown. The behavioral phase shift from one phase to another was observed when the nymphs were crowded or isolated for 2 days or longer, as previously reported for the last nymphal instars of the same strain. Behavioral gregarization was induced for isolated nymphs that were allowed to see a group of nymphs through a transparent double wall. These results suggested that body‐color phase shift occurred more rapidly for mid‐instar nymphs than for late instar nymphs but the rate of behavioral phase shift was similar for the two instars.     

Radical and superoxide scavenging activities of matairesinol and oxidized matairesinol

The radical and superoxide scavenging activities of oxidized matairesinols were examined. It could be assumed that the free benzylic position was important for higher radical scavenging activity. The different level of activity was observed between 7'-oxomatairesinol (Mat 2) and 7-oxomatairesinol (Mat 3). The activity of 8-hydroxymatairesinol was lower than that of matairesinol (Mat 1). The superoxide scavenging activity of the oxidized matairesinols was also demonstrated for the first time. It is assumed that the pKa value of phenol in the oxidized matairesinols affected this activity.     

Statistical Validation of Two Sample Comparison Methods for Oligonucleotide Microarray in Rat Ischemia Model

In gene expression analyses using a high-density oligonucleotide array in a rat ischemia model, two comparison methods, “pair-wise comparison” and “sample average comparison”, were evaluated based on statistical methods. The reliability of the elements screened with a 1.2 to 10-fold threshold was also evaluated. In pair-wise comparisons, most of the elements were significantly independent of the threshold value, with the percentage of significant elements remaining above 95%, when screened at 2.5-fold or higher threshold value. Pair-wise comparison structurally provided strict screening, which resulted in genes that were not selected despite significant alterations in expression. Screening by “sample average comparison” resulted in elements with low probability of significance, which suggested the necessity for increasing the reliability by additional statistical analyses after screening. When genes with altered expression were screened using an oligonucleotide array, marked differences in the numbers and reliability were proved to exist among elements screened by each sample comparison method.     

Synthesis of the glycosaminoglycan-protein linkage tetraosyl peptide moieties of betaglycan, which serve as a hexosamine acceptor for enzymatic glycosyl transfer

Betaglycan, also known as TGF-β type III receptor, is a membrane-anchored proteoglycan, which has two glycosaminoglycan (GAG) attachment sites (López-Casillas, F.; Payne, H. M.; Andres, J. L.; Massagué, J. J.Cell Biol.1994, 124, 557-568). Chondroitin sulfate (CS) or heparan sulfate (HS) can attach to the first site, Ser⁵³⁵, whereas only CS attaches to the second, Ser⁵⁴⁶. Although the mechanism behind the assembly of CS and HS is not fully understood, it has been reported that the assembly of HS requires not only a cluster of acidic residues but also hydrophobic residues located near the Ser-Gly attachment sites (Esko, J. D. Zhang, L. Curr. Opin. Struct. Biol.1996, 6, 663-670). To further understand the effects of amino acids close to the Ser residues of the GAG-attachment sites on the glycosyltransferases, two tetraosyl peptides derived from the CS attachment sites of betaglycan, GlcA-Gal-Gal-Xyl-SerGlyAspAsnGly (1) and GlcA-Gal-Gal-Xyl-SerGlyAspAsnGlyPheProGly (2), were synthesized, and used as donor substrates for β1,4-N-acetylgalactosaminyltransferase-I (β4GalNAcT-I) and α1,4-N-acetylglucosaminyltransferase-I (α4GlcNAcT-I). Both the chemically synthesized linkage region tetrasaccharides were far better acceptors for β4GalNAcT-I than for α4GlcNAcT-I in vitro, although they also showed appreciable acceptor activity for α4GlcNAcT-I.     

Evolution of oropharyngeal patterning mechanisms involving Dlx and endothelins in vertebrates

In jawed vertebrates, the Dlx code, or nested expression patterns of Dlx genes, specify the dorsoventral polarity of pharyngeal arches, downstream of endothelin-1 (Edn-1) and its effectors, Bapx1 (Nkx3.2) and dHand (Hand2). To elucidate the evolution of the specification mechanism of the oropharyngeal skeletal system, lamprey homologs of Dlx, Edn, endothelin receptor (Ednr), Bapx1, and dHand were identified. Our analysis suggested that the Edn gene family emerged at the advent of vertebrates, and that gene duplications leading to the different Edn gnathostome subtypes (Edn1-3) occurred before the cyclostome-gnathostome split. This timing of gene duplications, giving rise to multiple subtypes, was also implied for Dlx, Ednr, Hand, and Bapx. In lamprey embryos, nested expressions of Dlx genes were not observed in pharyngeal arches, nor was any focal expression of Bapx1, known in gnathostomes to specify the jaw joint. The dHand homolog, however, was expressed more intensively ventrally, as in gnathostomes. Lamprey homologs of Edn-1 and EdnrA were also shown to be expressed as described in mice, indicating involvement of this signaling pathway in the craniofacial patterning early in vertebrate evolution. These results suggest that the last common ancestor of all the extant vertebrates would have possessed basic gene repertoires involved in oropharyngeal patterning in gnathostomes, but the elaborate genetic program leading to the Dlx code is likely to have been acquired uniquely in gnathostomes.     

Regulation of hydroxyindole-O-methyltransferase gene expression in Japanese quail (Coturnix coturnix japonica).

Hydroxyindole-O-methyltrasferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. In this study, the expression of the HIOMT gene in Japanese quail was investigated with respect to tissue distribution and the effects of light and vitamin A deficiency. HIOMT mRNA in the pineal gland and eye had a clear daily rhythm with peak values in daytime. The testis also contained a detectable amount of HIOMT mRNA, which did not display a rhythmic change over a 24-h period. When birds were rendered vitamin A deficient through feeding with a vitamin A-free diet, the daily rhythm of the HIOMT gene almost disappeared in both the pineal gland and eye due to increases in the nighttime values. Our previous observations and these results suggest that vitamin A and a photo-signal are required to maintain the rhythmic expression of the HIOMT gene as well as the arylalkylamine N-acetyltransferase gene.     

Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin sulfate proteoglycans of whale cartilage.

From the carbohydrate-protein linkage region of whale cartilage proteoglycans, which bear predominantly chondroitin 4-sulfate, one nonsulfated, two monosulfated and one disulfated hexasaccharide alditols were isolated after exhaustive digestions with Actinase E and chondroitinase ABC, and subsequent beta-elimination. Their structures were analyzed by chondroitinase ACII digestion in conjunction with HPLC and by 500-MHz 1H-NMR spectroscopy. The nonsulfated compound (A) had the following conventional structure: delta GlcA(beta 1-3)-GalNAc(beta 1-4)GlcA(beta 1-3)Gal(beta 1-4)Xylol, where GlcA, delta GlcA and GalNAc are glucuronic acid; 4,5-unsaturated glucuronic acid and 2-deoxy-2-N-acetylamino-D-galactose, respectively. The other compounds were sulfated derivatives of compound A. Two monosulfated compounds (B and C) had an ester sulfate on C4 or C6 of the GalNAc residue, respectively and the disulfated compound (D) had two ester sulfate groups, namely, one on C4 of the GalNAc and the other on C4 of the Gal residue substituted by GlcA. The molar ratio of A/B/C/D was 0.21:0.16:0.36:0.27. The compound containing Gal-4-O-sulfate was previously isolated by us in the form of a sulfated glycoserine [delta GlcA(beta 1-3)GalNAc(4-O- sulfate)(beta 1-4)GlcA(beta 1-3)Gal(4-O-sulfate)(beta 1-3)-Gal(beta 1- 4)Xyl beta 1-O-Ser] from the carbohydrate-protein linkage region of rat chondrosarcoma chondroitin-4-sulfate proteoglycans [Sugahara K., Yamashina, I., DeWaard, P., Van Halbeek, H. & Vliegenthart, J.F.G. (1988) J. Biol. Chem. 263, 10,168-10,174]. The discovery of this structure in the carbohydrate-protein linkage region of chondroitin 4-sulfate proteoglycans from nontumorous cartilage indicates that it is not a tumor-associated product but rather a physiological biosynthetic product since it represents a significant proportion. The biological significance of this structure is discussed in relation to glycosaminoglycan biosynthesis.     

Dermatan sulfate domains defined by the novel antibody GD3A12, in normal tissues and ovarian adenocarcinomas

Dermatan sulfate (DS) expression in normal tissue and ovarian cancer was investigated using the novel, phage display-derived antibody GD3A12 that was selected against embryonic glycosaminoglycans (GAGs). Antibody GD3A12 was especially reactive with DS rich in IdoA-GalNAc4S disaccharide units. IdoA residues are important for antibody recognition as DS polymers with low numbers of IdoA residues were less reactive, and expression of the DS epimerase in ovarian carcinoma cells was associated with expression of the GD3A12 epitope. Moreover, staining of antibody GD3A12 was abolished by chondroitinase-B lyase digestion. Expression of DS domains defined by antibody GD3A12 was confined to connective tissue of most organs examined and presented as a typical fibrillar-type of staining. Differential expression of the DS epitopes recognized by antibodies GD3A12 and LKN1 (4/2,4 di-O-sulfated DS) was best seen in thymus and spleen, indicating differential expression of various DS domains in these organs. In ovarian carcinomas strong DS expression was found in the stromal parts, and occasionally on tumor cells. Partial co-localization in ovarian carcinomas was observed with decorin, versican and type I collagen suggesting a uniform distribution of this specific DS epitope. This unique anti-DS antibody may be instrumental to investigate the function, expression, and localization of specific DS domains in health and disease. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.