Unraveling Antimicrobial Resistance Genes and Phenotype Patterns among Enterococcus faecalis Isolated from Retail Chicken Products in Japan

Multidrug-resistant enterococci are considered crucial drivers for the dissemination of antimicrobial resistance determinants within and beyond a genus. These organisms may pass numerous resistance determinants to other harmful pathogens, whose multiple resistances would cause adverse consequences. Therefore, an understanding of the coexistence epidemiology of resistance genes is critical, but such information remains limited. In this study, our first objective was to determine the prevalence of principal resistance phenotypes and genes among Enterococcus faecalis isolated from retail chicken domestic products collected throughout Japan. Subsequent analysis of these data by using an additive Bayesian network (ABN) model revealed the co-appearance patterns of resistance genes and identified the associations between resistance genes and phenotypes. The common phenotypes observed among E. faecalis isolated from the domestic products were the resistances to oxytetracycline (58.4%), dihydrostreptomycin (50.4%), and erythromycin (37.2%), and the gene tet(L) was detected in 46.0% of the isolates. The ABN model identified statistically significant associations between tet(L) and erm(B), tet(L) and ant(6)-Ia, ant(6)-Ia and aph(3’)-IIIa, and aph(3’)-IIIa and erm(B), which indicated that a multiple-resistance profile of tetracycline, erythromycin, streptomycin, and kanamycin is systematic rather than random. Conversely, the presence of tet(O) was only negatively associated with that of erm(B) and tet(M), which suggested that in the presence of tet(O), the aforementioned multiple resistance is unlikely to be observed. Such heterogeneity in linkages among genes that confer the same phenotypic resistance highlights the importance of incorporating genetic information when investigating the risk factors for the spread of resistance. The epidemiological factors that underlie the persistence of systematic multiple-resistance patterns warrant further investigations with appropriate adjustments for ecological and bacteriological factors.     

Construction of bioengineered yeast platform for direct bioethanol production from alginate and mannitol

Brown macroalgae are a sustainable and promising source for bioethanol production because they are abundant in ocean ecosystems and contain negligible quantities of lignin. Brown macroalgae contain cellulose, hemicellulose, mannitol, laminarin, and alginate as major carbohydrates. Among these carbohydrates, brown macroalgae are characterized by high levels of alginate and mannitol. The direct bioconversion of alginate and mannitol into ethanol requires extensive bioengineering of assimilation processes in the standard industrial microbe Saccharomyces cerevisiae. Here, we constructed an alginate-assimilating S. cerevisiae recombinant strain by genome integration and overexpression of the genes encoding endo- and exo-type alginate lyases, DEH (4-deoxy-l-erythro-5-hexoseulose uronic acid) transporter, and components of the DEH metabolic pathway. Furthermore, the mannitol-metabolizing capacity of S. cerevisiae was enhanced by prolonged culture in a medium containing mannitol as the sole carbon source. When the constructed strain AM1 was anaerobically cultivated in a fermentation medium containing 6% (w/v) total sugars (approximately 1:2 ratio of alginate/mannitol), it directly produced ethanol from alginate and mannitol, giving 8.8 g/L ethanol and yields of up to 32% of the maximum theoretical yield from consumed sugars. These results indicate that all major carbohydrates of brown macroalgae can be directly converted into bioethanol by S. cerevisiae. This strain and system could provide a platform for the complete utilization of brown macroalgae.     

Measurements of cytoplasmic and vacuolar pH in Neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy

The nitrogen-15 chemical shift of the N1 (tau)-nitrogen of 15N-labeled histidine and the half-height line widths of proton-coupled resonances of the delta- and omega,omega'-nitrogens of 15N-labeled arginine and of the alpha-nitrogens of 15N-labeled alanine and proline were measured in intact mycelia of Neurospora crassa to obtain to estimates of intracellular pH. For intracellular 15N-labeled histidine, the N1 (tau)-nitrogen chemical shift was 200.2 ppm. In vitro measurements showed that the chemical shift was slightly affected by the presence of phosphate, with which the basic amino acids may be associated in vivo. These considerations indicate a pH of 5.7-6.0 for the environment of intracellular histidine. The half-height line widths of the delta- and omega,omega'-nitrogens of [15N]arginine were 15 and 26 Hz, respectively. In vitro studies showed that these line widths also are influenced by the presence of phosphate, and, after suitable allowance for this, the line widths indicate pH 6.1-6.5 for intracellular arginine. The half-height line widths for intracellular alanine and proline were 17 and 12 Hz, respectively, which are consistent with an intracellular pH of 7.1-7.2. Pools of histidine and arginine are found principally in the vacuole of Neurospora, most likely in association with polyphosphates. Proline and alanine are cytoplasmic. The results reported here are consistent with these localizations and indicate that the vacuolar pH is 6.1 +/- 0.4 while the cytoplasmic pH is 7.15 +/- 0.10. Comparisons of these estimates with those obtained by other techniques and their implications for vacuolar function are discussed.     

A quantitative estimation of sequential foliage development and fertility inDryopteris crassirhizoma

The foliage development ofDryopteris crassirhizoma Nakai was quantitatively estimated by measurements of shape and size of leaves from different developmental stages of sporophytes, to lead to an understanding of the life history characteristics of the species. The number of midrib branches (NV, number of veins) of the leaf corresponds to the leaf-shape complexity (DI, dissection index; shape complexity from a circle) and length of leafblade (BL). Some quantitative characters, such as leaf uniformity (decrease in NV variation), changes in shape and increase in number of leaves, vary progressively during foliage formation. The sequence of foliage development can be quantified using the parameter NV: for example, 15-NV for leaf uniformity, 30-NV for leaf-shape change from triangular to oblanceolate, 60-NV for increase in leaf number and leaf fertility in the course of sporophyte ontogeny. Contribution No. 3297 from the Institute of Low Temperature Science, Hokkaido University.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.     

Cancer Metastasis as Disrupted Developmental Phenotype

Cancer metastasis is a complex processes, associated with the invasion to tissues with extensive degradation of the surrounding normal components, penetration into vessels, circulation, and then invasion to normal tissues in body. It would be not surprising that tumor cells usurp pathways critical to the developing embryo during metastasis. For the better understanding of tumor metastasis, this review will highlight the recent progress and significance of the signal transduction pathways, relevant to developmental biology.     

(−)‐Epigallocatechin‐3‐gallate inhibits human angiotensin‐converting enzyme activity through an autoxidation‐dependent mechanism

We investigated the molecular mechanisms involved in the angiotensin‐converting enzyme (ACE) inhibition by (?)‐epigallocatechin‐3‐gallate (EGCg), a major tea catechin. EGCg inhibited both the ACE activity in the lysate of human colorectal cancer cells and human recombinant ACE (rh‐ACE) in a dose‐dependent manner. Co‐incubation with zinc sulfate showed no influence on the rh‐ACE inhibition by EGCg, whereas it completely counteracted the inhibitory effect of ethylenediaminetetraacetic acid, a chelating‐type ACE inhibitor. Although hydrogen peroxide was produced by the autoxidation of EGCg, hydrogen peroxide itself had little effect on the ACE activity. Conversely, the co‐incubation of EGCg with borate or ascorbic acid significantly diminished the EGCg inhibition. A redox‐cycling staining experiment revealed that rh‐ACE was covalently modified by EGCg. A Lineweaver–Burk plot analysis indicated that EGCg inhibited the ACE activity in a non‐competitive manner. These results suggested that EGCg might allosterically inhibit the ACE activity through oxidative conversion into an electrophilic quinone.