Development of a chemical strategy to produce rare aldohexoses from ketohexoses using 2-aminopyridine

Rare sugars are monosaccharides that are found in relatively low abundance in nature. Herein, we describe a strategy for producing rare aldohexoses from ketohexoses using the classical Lobry de Bruyn–Alberda van Ekenstein transformation. Upon Schiff-base formation of keto sugars, a fluorescence-labeling reagent, 2-aminopyridine (2-AP), was used. While acting as a base catalyst, 2-AP efficiently promoted the ketose-to-aldose transformation, and acting as a Schiff-base reagent, it effectively froze the ketose–aldose equilibrium. We could also separate a mixture of Sor, Gul, and Ido in their Schiff-base forms using a normal-phase HPLC separation system. Although Gul and Ido represent the most unstable aldohexoses, our method provides a practical way to rapidly obtain these rare aldohexoses as needed.     

Vasotocin- and mesotocin-induced increases in short-circuit current across tree frog skin

In adult amphibian skin, Na⁺ crosses from outside to inside. This Na⁺ transport can be measured as the amiloride-blockable short-circuit current (SCC) across the skin. We investigated the effects of arginine vasotocin (AVT) and mesotocin (MT), and those of antagonists of the vasopressin and oxytocin receptors, on the SCC across Hyla japonica skin. (1) Both AVT (100 pmol/L or more) and MT (1 nmol/L or more) increased the SCC. (2) The AVT- and MT-induced increases in SCC recovered with time (downregulation). (3) These AVT/MT-induced effects were blocked by application of OPC-31260 (vasopressin V₂-receptor antagonist). (4) The OPC-31260 concentration needed to block the AVT-induced response was lower upon post-application (after application of agonist) than upon pre-application (before application of agonist), suggesting the number of receptors may have decreased after AVT application. (5) Upon repeated application of AVT (100 pmol/L), the induced SCC increase did not differ significantly between the 1st and 2nd applications. (6) The time to reach the half-maximum value of the AVT-induced or MT-induced increase in SCC was not significantly different between washout and post-application of OPC-31260, suggesting that post-application of OPC-31260 cleared AVT and MT from their receptors. The effects of AVT, MT, and their antagonists in H. japonica, which is adapted to a terrestrial habitat, are compared with our previously published data on Rana catesbeiana (=Lithobates catesbeianus), which is adapted to a semiaquatic habitat.     

Overtime Work and Prevalence of Diabetes in Japanese Employees: Japan Epidemiology Collaboration on Occupational Health Study

Objective

Epidemiologic evidence on long working hour and diabetes has been conflicting. We examined the association between overtime work and prevalence of diabetes among Japanese workers.

Methods

The subjects were 40,861 employees (35,170 men and 5,691 women), aged 16 to 83 years, of 4 companies in Japan. Hours of overtime were assessed using self-reported questionnaires. Diabetes was defined as a fasting plasma glucose ≥126 mg/dl (7.0 mmol/l), hemoglobin A1c ≥6.5% (48 mmol/mol), or current use of anti-diabetic drug. Multiple logistic regression analysis was used to calculate odds ratio of diabetes for each category of overtime.

Results

After adjustment for age, sex, company, smoking, and BMI, there was a suggestion of U-shaped relationship between overtime work and prevalence of diabetes (P for quadratic trend = 0.07). Compared with those who worked <45 hours of overtime per month, the adjusted odds ratios (95% confidence interval) of diabetes were 0.86 (0.77–0.94), 0.69 (0.53–0.89), and 1.03 (0.72–1.46) for those who worked 45–79, 80–99, and ≥100 hours of overtime per month, respectively. In one company (n = 33,807), where other potential confounders including shift work, job position, type of department, alcohol consumption, sleep duration, leisure time physical activity, and family history of diabetes was additionally adjusted for, similar result was obtained (P for quadratic trend = 0.05).

Conclusions

Long hours of overtime work may not be associated with increased prevalence of diabetes among Japanese workers.     

Increase in Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) Had a Strong Impact on the Development of Type 2 Diabetes in Japanese Individuals with Impaired Insulin Secretion: The Saku Study

Our aim was to assess the impact of increase in homeostasis model assessment of insulin resistance (HOMA-IR) on the development of type 2 diabetes in Japanese individuals with impaired insulin secretion (IIS). This study included 2,209 participants aged 30–69 without diabetes at baseline who underwent comprehensive medical check-ups between April 2006 and March 2007 at Saku Central Hospital. Participants were classified into eight groups according to the combination of baseline IIS status (non-IIS and IIS) and category of HOMA-IR change between the baseline and follow-up examinations (decrease, no change/small increase, moderate increase, and large increase). Type 2 diabetes was determined from fasting and 2 h post-load plasma glucose concentrations at the follow-up examination between April 2009 and March 2011. At baseline, 669 individuals (30.3%) were classified as having IIS. At follow-up, 74 individuals developed type 2 diabetes. After adjusting for confounding factors including baseline HOMA-IR values, the multivariable-adjusted odds ratios (95% confidence intervals) for type 2 diabetes in the non-IIS with a decrease (mean change in HOMA-IR: −0.47), non-IIS with a moderate increase (mean change in HOMA-IR: 0.28), non-IIS with a large increase (mean change in HOMA-IR: 0.83), IIS with a decrease (mean change in HOMA-IR: −0.36), IIS with no change/small increase (mean change in HOMA-IR: 0.08), IIS with a moderate increase (mean change in HOMA-IR: 0.27), and IIS with a large increase (mean change in HOMA-IR: 0.73) groups, relative to the non-IIS with no change/small increase (mean change in HOMA-IR: 0.08) group were 0.23 (0.04, 1.11), 1.22 (0.26, 5.72), 2.01 (0.70, 6.46), 1.37 (0.32, 4.28), 3.60 (0.83, 15.57), 5.24 (1.34, 20.52), and 7.01 (1.75, 24.18), respectively. Moderate and large increases in HOMA-IR had a strong impact on the development of type 2 diabetes among individuals with IIS in this Japanese population.     

Prolonged nuclear retention of activated extracellular signal-regulated kinase 1/2 is required for hepatocyte growth factor-induced cell motility

We examined the signaling pathway by which hepatocyte growth factor (HGF) induces cell motility, with special focus on the role of extracellular signal-regulated kinase (ERK) in the nucleus. We used Madin-Darby canine kidney cells overexpressing ERK2 because of their prominent motility response to HGF. HGF stimulation of the cells induces not only a rapid, marked, and sustained activation and rapid nuclear accumulation of ERK1/2, but also a prolonged nuclear retention of the activated ERK1/2. Interruption of the ERK1/2 activation by PD98059 treatment of the cells 30 min after HGF stimulation abolishes the HGF-induced cell motility. Enforced cytoplasmic retention of the activated ERK1/2 by the expression of an inactive form of MKP-3 cytoplasmic phosphatase inhibits the cell motility response. Although epidermal growth factor stimulation of the cells induces the activation and nuclear accumulation of ERK1/2, it does not induce the prolonged nuclear retention of the activated ERK1/2, and fails to induce cell motility. In the nucleus, activated ERK1/2 continuously phosphorylate Elk-1, leading to the prolonged expression of c-fos, which results in the expression of several genes such as matrix metalloproteinase (mmp)-9; MMP-9 activity is required for the induction of the cell motility response. Our results indicate that the sustained activity of ERK1/2 in the nucleus is required for the induction of HGF-induced cell motility.     

RNA interference during spermatogenesis in mice

Spermatogenesis consists of complex cellular and developmental processes, such as the mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and morphogenesis of haploid spermatids. In this study, we show that RNA interference (RNAi) functions throughout spermatogenesis in mice. We first carried out in vivo DNA electroporation of the testis during the first wave of spermatogenesis to enable foreign gene expression in spermatogenic cells at different stages of differentiation. Using prepubertal testes at different ages and differentiation stage-specific promoters, reporter gene expression was predominantly observed in spermatogonia, spermatocytes, and round spermatids. This method was next applied to introduce DNA vectors that express small hairpin RNAs, and the sequence-specific reduction in the reporter gene products was confirmed at each stage of spermatogenesis. RNAi against endogenous Dmc1, which encodes a DNA recombinase that is expressed and functionally required in spermatocytes, led to the same phenotypes observed in null mutant mice. Thus, RNAi is effective in male germ cells during mitosis and meiosis as well as in haploid cells. This experimental system provides a novel tool for the rapid, first-pass assessment of the physiological functions of spermatogenic genes in vivo.     

Traits of transgenic Atropa belladonna doubly transformed with different Agrobacterium rhizogenes strains

Hairy root cultures of Atropa belladonna L. were established by infection either with Agrobacterium rhizogenes ATCC 15834 or MAFF 03-01724, and transgenic plants were obtained from both hairy root cultures. Doubly transformed roots were induced by re-infection of the leaf segments of transgenic Atropa belladonna plants (A. rhizogenes 15834) with MAFF 03-01724. Shoots and viviparous leaves were regenerated from the doubly transformed roots. The genetic transformation was determined by the opine assay (agropine, mannopine and/or mikimopine) and polymerase chain reaction. Physiological changes and tropane alkaloid biosynthesis in the hairy roots (singly and doubly transformed) were investigated. The alkaloid content in the doubly transformed root strain was intermediate as compared to the root strains which were singly transformed. On the other hand endogenous IAA levels in doubly transformed roots were significantly decreased compared to both singly transformed roots.Abbreviations BA benzyladenine - IAA indoleacetic acid - NAA naphthaleneacetic acid - PCR polymerase chain reaction - t-ZR trans-zeatin     

Wide distribution of O157-antigen biosynthesis gene clusters in Escherichia coli

Most Escherichia coli O157-serogroup strains are classified as enterohemorrhagic E. coli (EHEC), which is known as an important food-borne pathogen for humans. They usually produce Shiga toxin (Stx) 1 and/or Stx2, and express H7-flagella antigen (or nonmotile). However, O157 strains that do not produce Stxs and express H antigens different from H7 are sometimes isolated from clinical and other sources. Multilocus sequence analysis revealed that these 21 O157:non-H7 strains tested in this study belong to multiple evolutionary lineages different from that of EHEC O157:H7 strains, suggesting a wide distribution of the gene set encoding the O157-antigen biosynthesis in multiple lineages. To gain insight into the gene organization and the sequence similarity of the O157-antigen biosynthesis gene clusters, we conducted genomic comparisons of the chromosomal regions (about 59 kb in each strain) covering the O-antigen gene cluster and its flanking regions between six O157:H7/non-H7 strains. Gene organization of the O157-antigen gene cluster was identical among O157:H7/non-H7 strains, but was divided into two distinct types at the nucleotide sequence level. Interestingly, distribution of the two types did not clearly follow the evolutionary lineages of the strains, suggesting that horizontal gene transfer of both types of O157-antigen gene clusters has occurred independently among E. coli strains. Additionally, detailed sequence comparison revealed that some positions of the repetitive extragenic palindromic (REP) sequences in the regions flanking the O-antigen gene clusters were coincident with possible recombination points. From these results, we conclude that the horizontal transfer of the O157-antigen gene clusters induced the emergence of multiple O157 lineages within E. coli and speculate that REP sequences may involve one of the driving forces for exchange and evolution of O-antigen loci.     

Construction of a virtual Mycobacterium tuberculosis consensus genome and its application to data from a next generation sequencer

Background

Although Mycobacterium tuberculosis isolates are consisted of several different lineages and the epidemiology analyses are usually assessed relative to a particular reference genome, M. tuberculosis H37Rv, which might introduce some biased results. Those analyses are essentially based genome sequence information of M. tuberculosis and could be performed in sillico in theory, with whole genome sequence (WGS) data available in the databases and obtained by next generation sequencers (NGSs). As an approach to establish higher resolution methods for such analyses, whole genome sequences of the M. tuberculosis complexes (MTBCs) strains available on databases were aligned to construct virtual reference genome sequences called the consensus sequence (CS), and evaluated its feasibility in in sillico epidemiological analyses.

Results

The consensus sequence (CS) was successfully constructed and utilized to perform phylogenetic analysis, evaluation of read mapping efficacy, which is crucial for detecting single nucleotide polymorphisms (SNPs), and various MTBC typing methods virtually including spoligotyping, VNTR, Long sequence polymorphism and Beijing typing. SNPs detected based on CS, in comparison with H37Rv, were utilized in concatemer-based phylogenetic analysis to determine their reliability relative to a phylogenetic tree based on whole genome alignment as the gold standard. Statistical comparison of phylogenic trees based on CS with that of H37Rv indicated the former showed always better results that that of later. SNP detection and concatenation with CS was advantageous because the frequency of crucial SNPs distinguishing among strain lineages was higher than those of H37Rv. The number of SNPs detected was lower with the consensus than with the H37Rv sequence, resulting in a significant reduction in computational time. Performance of each virtual typing was satisfactory and accorded with those published when those are available.

Conclusions

These results indicated that virtual CS constructed from genome sequence data is an ideal approach as a reference for MTBC studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1368-9) contains supplementary material, which is available to authorized users.     

Different impacts of pMP19 on the virulence of Melissococcus plutonius strains with different genetic backgrounds

Virulence factors responsible for bacterial pathogenicity are often encoded by plasmids. In Melissococcus plutonius, the causative agent of European foulbrood of honey bees, a putative virulence plasmid (pMP19) possessing mtxA, which encodes a putative insecticidal toxin, was found by comparative genome analyses. However, as the role of pMP19 in the pathogenesis of European foulbrood remains to be elucidated, we generated pMP19 cured-M. plutonius from representative strains of the three genetically distinct groups (CC3, CC12 and CC13) and compared their virulence against Apis mellifera larvae using our in vitro infection model. Under the conditions tested, the loss of pMP19 abrogated the pathogenicity in CC3 strains, and > 94% of pMP19-cured CC3 strain-infected larvae became adult bees, suggesting that pMP19 is a virulence determinant of CC3 strains. However, introduction of mtxA on its own did not increase the virulence of pMP19-cured strains. In contrast to CC3 strains, the representative CC12 strain remained virulent even in the absence of pMP19, whereas the representative CC13 strain was avirulent even in the presence of the plasmid. Thus, pMP19 plays a role in the virulence of M. plutonius; however, its impact on the virulence varies among strains with different genetic backgrounds.