Structural analysis of the sugar chains of human urinary thrombomodulin

The sugar chains of human urinary thrombomodulin were studied. N- and O-linked sugar chains were simultaneously liberated by hydrazinolysis followed by N-acetylation and were tagged with 2-aminopyridine. Then the structures of the N- and O-linked pyridylamino (PA-) sugar chains were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion. The major N-linked sugar chains of human urinary thrombomodulin were found to be monosialo- and disialofucosylbiantennary chains, while the major O-linked sugar chain was +/-Siaalpha2-3Galbeta1-3(+/-Siaalpha2-6)GalNAc. Thrombomodulin also contained the reported structure SO4-3GlcAbeta1-3Galbeta1-3(+/-Siaalpha2-6)Galbeta1-4Xyl [H. Wakabayashi, S. Natsuka, T. Mega, N. Otsuki, M. Isaji, M. Naotsuka, S. Koyama, T. Kanamori, K. Sakai, and S. Hase (1999) J. Biol. Chem. 274, 5436-5442]. In addition to these sugar chains, a single Glc was linked to Ser 287.     

Precise region and the character of the pathogenicity island in clinical Vibrio parahaemolyticus strains

In this study, we determined the borders of the pathogenicity island in V. parahaemolyticus RIMD2210633 (Vp-PAI). Vp-PAI has features in common with Tn7 and other related elements at both terminal ends. Our findings indicate that the mobile element with a transposase which contains the DDE motif may have been involved in Vp-PAI formation.     

Interneurite affinity is regulated by heterophilic nectin interactions in concert with the cadherin machinery

Neurites recognize their specific partners during the formation of interneuronal connections. In hippocampal pyramidal neurons, axons attach to dendrites for their synaptogenesis, but the dendrites do not form stable contacts with each other, suggesting the presence of a mechanism to allow their selective associations. Nectin-1 (N1), an immunoglobulin domain adhesive protein, is preferentially localized in axons, and its heterophilic partner, N3, is present in both axons and dendrites; we tested their potential roles in interneurite recognition. The overexpression of N1, causing its mislocalization to dendrites, induced atypical dendrodendritic as well as excessive axodendritic associations. On the contrary, the genetic deletion of N1 loosened the contacts between axons and dendritic spines. Those actions of nectins required cadherin-catenin activities, but the overexpression of cadherin itself could not accelerate neurite attachment. These results suggest that the axon-biased localization of N1 and its trans-interaction with N3 in cooperation with the cadherin machinery is critical for the ordered association of axons and dendrites.     

Evolutionary Consequence of a Trade-Off between Growth and Maintenance along with Ribosomal Damages

Microorganisms in nature are constantly subjected to a limited availability of resources and experience repeated starvation and nutrition. Therefore, microbial life may evolve for both growth fitness and sustainability. By contrast, experimental evolution, as a powerful approach to investigate microbial evolutionary strategies, often targets the increased growth fitness in controlled, steady-state conditions. Here, we address evolutionary changes balanced between growth and maintenance while taking nutritional fluctuations into account. We performed a 290-day-long evolution experiment with a histidine-requiring Escherichia coli strain that encountered repeated histidine-rich and histidine-starved conditions. The cells that experienced seven rounds of starvation and re-feed grew more sustainably under prolonged starvation but dramatically lost growth fitness under rich conditions. The improved sustainability arose from the evolved capability to use a trace amount of histidine for cell propagation. The reduced growth rate was attributed to mutations genetically disturbing the translation machinery, that is, the ribosome, ultimately slowing protein translation. This study provides the experimental demonstration of slow growth accompanied by an enhanced affinity to resources as an evolutionary adaptation to oscillated environments and verifies that it is possible to evolve for reduced growth fitness. Growth economics favored for population increase under extreme resource limitations is most likely a common survival strategy adopted by natural microbes.     

Correlation between cell-associated mannose-sensitive hemagglutination by Vibrio parahaemolyticus and adherence to a human colonic cell line Caco-2

Abstract Cell-associated hemagglutination (cHA) activity with human erythrocytes was examined for 468 clinical and 71 environmental strains of Vibrio parahaemolyticus . Approximately 95% of the strains tested were cHA positive irrespective of source or Kanagawa phenomenon. 75% of clinical strains showed relatively strong mannose-sensitive hemagglutination (MSHA), whereas 88% of the environmental strains showed relatively weak mannose-resistant hemagglutination (MRHA). Adherence of V. parahaemolyticus to Caco-2 cells was also determined. A clear positive correlation between cell-associated MSHA and adherence to Caco-2 cells was observed.     

Enhancement of antioxidant activity of p-alkylaminophenols by alkyl chain elongation

The initial finding that the p-methylaminophenol (6) exhibited antioxidant activity led us to investigate whether the length of alkyl chains linked to the aminophenol residue might affect antioxidative activity. Therefore, we synthesized p-butylaminophenol (5), p-hexylaminophenol (4), p-octylaminophenol (3), and p-methoxybenzylaminophenol (7). All p-alkylaminophenols quenched alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radicals, with 7 being the most potent DPPH radical scavenger. Lipid peroxidation by rat liver microsomes was reduced by p-alkylaminophenols in dose- and aminophenol alkyl chain length-dependent fashion (3>4>5>6), with 3 being the most potent lipid peroxidation inhibitor, at approximately 350-fold higher potency than 6. These results indicate that elongation of alkyl chains in p-alkylaminophenols may increase antioxidative activity, and that p-alkylaminophenols may potentially be useful in the development of antioxidants.     

Essential Loci in Centromeric Heterochromatin of Drosophila melanogaster. I: The Right Arm of Chromosome 2

With the most recent releases of the Drosophila melanogaster genome sequences, much of the previously absent heterochromatic sequences have now been annotated. We undertook an extensive genetic analysis of existing lethal mutations, as well as molecular mapping and sequence analysis (using a candidate gene approach) to identify as many essential genes as possible in the centromeric heterochromatin on the right arm of the second chromosome (2Rh) of D. melanogaster. We also utilized available RNA interference lines to knock down the expression of genes in 2Rh as another approach to identifying essential genes. In total, we verified the existence of eight novel essential loci in 2Rh: CG17665, CG17683, CG17684, CG17883, CG40127, CG41265, CG42595, and Atf6. Two of these essential loci, CG41265 and CG42595, are synonymous with the previously characterized loci l(2)41Ab and unextended, respectively. The genetic and molecular analysis of the previously reported locus, l(2)41Ae, revealed that this is not a single locus, but rather it is a large region of 2Rh that extends from unextended (CG42595) to CG17665 and includes four of the novel loci uncovered here.THE term “heterochromatin” was introduced by Heitz (1928) to describe regions of mitotic chromosomes that remain condensed throughout the cell cycle, in contrast to regions of euchromatin, which condense only during cell division. Heterochromatin was later divided into two classes: constitutive and facultative heterochromatin (Brown 1966). Constitutive heterochromatin is found in large blocks near centromeres and telomeres, while facultative heterochromatin can be described as silenced euchromatin that undergoes heterochromatization at specific developmental stages. Other properties of constitutive heterochromatin include late replication in S phase, low gene density, strikingly reduced level of meiotic recombination, enrichment in transposable element sequences and highly repetitive satellite DNA sequences, and the ability to silence euchromatic gene expression in a phenomenon called position effect variegation.Approximately 30% of the Drosophila melanogaster genome consists of constitutive heterochromatin (Gatti and Pimpinelli 1992). Centromeric heterochromatin in D. melanogaster is composed of mainly middle-repeat satellite DNA sequences and clusters of transposable element sequences (Lohe et al. 1993; Pimpinelli et al. 1995). Genes that reside in the heterochromatin are scattered like islands between the satellites and clusters of transposable elements. On average, heterochromatic genes are larger than euchromatic genes, primarily due to the prevalent accumulation of transposable element sequences in their introns (Devlin et al. 1990; Biggs et al. 1994; Dimitri et al. 2003a,b; Hoskins et al. 2007). Heterochromatic genes also tend to be AT-rich compared to their euchromatic counterparts; there is some evidence suggesting that the coding sequences of heterochromatic genes evolve toward AT richness in response to being located in heterochromatin (Yasuhara et al. 2005; Díaz-Castillo and Golic 2007).Drosophila heterochromatin is vastly under-replicated in polytene chromosomes, so heterochromatic genes cannot easily be mapped through polytene analysis. However, by using Hoechst 33258 and N-chromosome banding techniques, Dimitri (1991) was successful in dividing heterochromatin in mitotic chromosomes into distinct cytological bands; this was an important step in mapping the precise location of heterochromatic genes because before this time heterochromatic genes could be mapped only relative to one another. Here we focus on further refining the previous mapping work on essential genes in the proximal heterochromatin of the right arm of the second chromosome (2Rh) in cytological region h41–h46 of D. melanogaster (Hilliker 1976; Hilliker et al. 1980; Coulthard et al. 2003; Myster et al. 2004).Early mapping studies in D. melanogaster putatively placed the light (lt) and rolled (rl) genes in, or near, chromosome 2 heterochromatin (Schultz 1936; Hannah 1951; Hessler 1958). The first large-scale mutagenesis specifically directed at finding vital loci in second chromosome heterochromatin was conducted by Hilliker (1976). Using heterochromatic deletions created by Hilliker and Holm (1975), Hilliker (1976) set out to map vital loci using the mutagen ethyl methanesulfonate (EMS). He identified seven individual lethal complementation groups in 2Rh that were interpreted as representing seven vital loci. One of these heterochromatic loci was identified as the previously described rl gene. Two of the remaining vital loci have since been identified: Nipped-A is synonymous with the l(2) 41Ah complementation group (Rollins et al. 1999) and RpL38 is synonymous with Minute(2)41A and Hilliker''s (1976) l(2)41Af complementation group (Marygold et al. 2005; also referred to as l(2)Ag in FlyBase). In addition, Rollins et al. (1999) found the Nipped-B gene to be located in 2Rh, but how this locus fit into the data from Hilliker (1976) was unclear.With the limited release of some of the more distal heterochromatic sequences (Hoskins et al. 2002), a more recent mutagenesis screen focusing on distal 2Rh was conducted by Myster et al. (2004). In the region defined by the overlap between Df(2R)41A8 and Df(2R)41A10 (the latter was previously shown to be deficient for most of 2Rh; Hilliker and Holm 1975), Myster et al. (2004) reported the existence of 15 vital loci, considerably more than the 4 essential loci predicted by Hilliker (1976). The discrepancy between these two studies was the catalyst for this current work. Each group used the same mutagen, EMS, yet each group came up with very different interpretations of the number of vital loci.Hilliker''s interpretation relied on earlier evidence that EMS preferentially produced point mutations and not large-scale aberrations (Lim and Snyder 1974). Assuming that the mutants isolated in his study were point mutations, or small aberrations limited to one locus, Hilliker found that some of the loci that he identified exhibited complex interallelic complementation; the most complex complementation pattern was observed with locus l(2)41Ae. On the other hand, the interpretation of Myster et al. (2004) was that heterochromatin was more sensitive to EMS and that EMS could produce large heterochromatic deletions; they proposed that the complex interallelic complementation in l(2)41Ae was due to the presence of deletions and that l(2)41Ae represented a region of 2Rh containing many genes, rather than being a single locus.To resolve these different interpretations of the genomic segment containing l(2)41Ae (i.e., is it a single locus or a region of 2Rh), we set out to map l(2)41Ae and the region surrounding the presumed location of l(2)41Ae (as in Myster et al. 2004) by performing a large-scale inter se complementation analysis between all available mutant lines that were previously mapped to l(2)41Ae (including Nipped-B). In addition, we undertook a molecular mapping and sequence analysis, using a candidate gene approach with the most recent annotation of 2Rh (Hoskins et al. 2007), to characterize the region and identify as many essential genes as possible. We also used these approaches to map l(2)41Ab and unextended [two of the more proximal complementation groups identified by Hilliker (1976)]. Finally, we also utilized available RNA interference (RNAi) lines to knock down the expression of 12 genes in 2Rh in an attempt to identify essential genes.