Accumulation of anthranilic acid and N-glucosylanthranilic acid by a Corynebacterium glutamicum mutant resistant to DL-serine hydroxamate

During a study on the effect of DL-serine hydroxamate on Corynebacterium glutamicum (JCM1318, a wild strain), a mutant resistant to the drug, strain TO3002, was isolated. This mutant accumulated five Ehrlich's reagent positive fluorescent substances in the culture medium. Two major and one minor fluorescent products were isolated by preparative high-performance liquid chromatography following charcoal column chromatography from the culture supernatant. One major product was identified as anthranilic acid whose molecular ion was confirmed to be 137 by a measurement of liquid chromatography-mass spectrometry (LC-MS), and NMR spectrum coincided with that of anthranilic acid. LC-MS spectra of another major and the minor product showed that they had the same molecular weight of 299. This major product was supported to be N-glucosylanthranilic acid (N-o-carboxyphenyl-1-beta-glucosylamine) by two-dimensional (1)H and (13)C NMR analyses. The minor product was speculated to be an Amadori compound derived from N-glucosylanthranilic acid. N-Glucosylanthranilic acid accumulated in the early phase, then decreased in the late phase of the culture. In contrast, the accumulation of anthranilic acid increased remarkably in the late phase of the fermentation. Based on this phenomenon, it was assumed that N-glucosylanthranilic acid once accumulated was decomposed to form anthranilic acid, at least in large part, with the progress of fermentation. The strain TO3002 showed a leaky requirement for L-tryptophan or indole (but did not for anthranilic acid) and resistance to DL-serine hydroxamate.     

Effects of restricted feeding on daily fluctuations of hepatic functions including p450 monooxygenase activities in rats

Hepatic P450 monooxygenase activities, assessed by measurement of 7-alkoxycoumarin O-dealkylase (ACD) activities, show obvious daily fluctuations in male rats with high values during the dark period and low values during the light period. We have already confirmed that the ACD activities are controlled by the suprachiasmatic nucleus (SCN), which is well known as the oscillator of circadian rhythm. Recently, it is reported that circadian oscillators exist not only in the SCN but also in peripheral organs. To date, it is unclear which circadian oscillators predominantly drive the daily fluctuations of hepatic ACD activities. To address this question, we examined the effects of restricted feeding, which uncouples the circadian oscillators in the liver from the central pacemaker in the SCN, on the daily fluctuations in hepatic ACD activities in male rats. Here we show that restricted feeding inverts the oscillation phase of the daily fluctuations in hepatic ACD activities. Regarding the hepatic P450 content, there were no fluctuations between the light and dark periods under ad libitum and restricted feeding conditions. Therefore, it is considered that the daily fluctuations in hepatic ACD activities are predominantly driven by the circadian factors in peripheral organs rather than by the oscillator in the SCN directly.     

Inhibition of c-Jun NH2-terminal kinase activity improves ischemia/reperfusion injury in rat lungs

Although c-Jun NH(2)-terminal kinase (JNK) has been implicated in the pathogenesis of transplantation-induced ischemia/reperfusion (I/R) injury in various organs, its significance in lung transplantation has not been conclusively elucidated. We therefore attempted to measure the transitional changes in JNK and AP-1 activities in I/R-injured lungs. Subsequently, we assessed the effects of JNK inhibition by the three agents including SP600125 on the degree of lung injury assessed by means of various biological markers in bronchoalveolar lavage fluid and histological examination including detection of apoptosis. In addition, we evaluated the changes in p38, extracellular signal-regulated kinase, and NF-kappaB-DNA binding activity. I/R injury was established in the isolated rat lung preserved in modified Euro-Collins solution at 4 degrees C for 4 h followed by reperfusion at 37 degrees C for 3 h. We found that AP-1 was transiently activated during ischemia but showed sustained activation during reperfusion, leading to significant lung injury and apoptosis. The change in AP-1 was generally in parallel with that of JNK, which was activated in epithelial cells (bronchial and alveolar), alveolar macrophages, and smooth muscle cells (bronchial and vascular) on immunohistochemical examination. The change in NF-kappaB qualitatively differed from that of AP-1. Protein leakage, release of lactate dehydrogenase and TNF-alpha into bronchoalveolar lavage fluid, and lung injury were improved, and apoptosis was suppressed by JNK inhibition. In conclusion, JNK plays a pivotal role in mediating lung injury caused by I/R. Therefore, inhibition of JNK activity has potential as an effective therapeutic strategy for preventing I/R injury during lung transplantation.     

Distribution of the freshwater fishes of Japan: an historical overview

Japanese freshwater fishes, including lampreys, comprise 15 orders, 35 families, and 96 genera, with 211 species and subspecies. Most belong to the families Cyprinidae (29% of species and subspecies), Gobiidae (21%), Salmonidae (10%), and Cobitidae (8%). Cyprinids and cobitids presumably originated from east Asia, gobiids from southeast Asia, and cottids and salmonids from the north Pacific. Japanese freshwater fishes include 88 endemic species and subspecies, of which three have been extirpated. Fishes introduced into natural rivers and lakes for inland commercial fisheries and sport fishing, and by accident, include many exotic species, of which 23 now inhabit natural freshwaters. These often have destroyed the local fish fauna by predation, and caused genetic pollution by hybridization with local strains. Destruction of freshwater environments by land development also poses a threat to Japanese freshwater fish communities. In addition Japanese freshwater systems have been markedly altered by development of rice paddy fields which have caused some species to decline but others to flourish, and changed the distribution patterns of fishes between upstream and downstream areas. To conserve endangered species and declining communities of Japanese freshwater fishes, we need to clarify the characteristics of their original habitats and the effects of developing paddy fields, from both the ecological and historical points of view.     

Pyrazine Analogues Are Active Components of Wolf Urine That Induce Avoidance and Freezing Behaviours in Mice

Background

The common grey wolf (Canis lupus) is found throughout the entire Northern hemisphere and preys on many kinds of mammals. The urine of the wolf contains a number of volatile constituents that can potentially be used for predator–prey chemosignalling. Although wolf urine is put to practical use to keep rabbits, rodents, deer and so on at bay, we are unaware of any prior behavioural studies or chemical analyses regarding the fear-inducing impact of wolf urine on laboratory mice.

Methodology/Principal Findings

Three wolf urine samples harvested at different times were used in this study. All of them induced stereotypical fear-associated behaviors (i.e., avoidance and freezing) in female mice. The levels of certain urinary volatiles varied widely among the samples. To identify the volatiles that provoked avoidance and freezing, behavioural, chemical, and immunohistochemical analyses were performed. One of the urine samples (sample C) had higher levels of 2,6-dimethylpyrazine (DMP), trimethylpyrazine (TMP), and 3-ethyl-2,5-dimethyl pyrazine (EDMP) compared with the other two urine samples (samples A and B). In addition, sample C induced avoidance and freezing behaviours more effectively than samples A and B. Moreover, only sample C led to pronounced expression of Fos-immunoreactive cells in the accessory olfactory bulb (AOB) of female mice. Freezing behaviour and Fos immunoreactivity were markedly enhanced when the mice were confronted with a mixture of purified DMP, TMP, and EDMP vs. any one pyrazine alone.

Conclusions/Significance

The current results suggest that wolf urinary volatiles can engender aversive and fear-related responses in mice. Pyrazine analogues were identified as the predominant active components among these volatiles to induce avoidance and freezing behaviours via stimulation of the murine AOB.     

Involvement of meso-α,∊-Diamino-pimelate D-Dehydrogenase in Lysine Biosynthesis in Corynebacterium glutamicum

To characterize aspartyl aminopeptidase from Aspergillus oryzae, the recombinant enzyme was expressed in Escherichia coli. The enzyme cleaves N-terminal acidic amino acids. About 30% activity was retained in 20% NaCl. Digestion of defatted soybean by the enzyme resulted in an increase in the glutamic acid content, suggesting that the enzyme is potentially responsible for the release of glutamic acid in soy sauce mash.     

Truncation of N- and C-terminal regions of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> dextranase enhances catalytic activity

Multiple forms of native and recombinant endo-dextranases (Dexs) of the glycoside hydrolase family (GH) 66 exist. The GH 66 Dex gene from Streptococcus mutans ATCC 25175 (SmDex) was expressed in Escherichia coli. The recombinant full-size (95.4 kDa) SmDex protein was digested to form an 89.8 kDa isoform (SmDex90). The purified SmDex90 was proteolytically degraded to more than seven polypeptides (23–70 kDa) during long storage. The protease-insensitive protein was desirable for the biochemical analysis and utilization of SmDex. GH 66 Dex was predicted to comprise four regions from the N- to C-termini: N-terminal variable region (N-VR), conserved region (CR), glucan-binding site (GBS), and C-terminal variable region (C-VR). Five truncated SmDexs were generated by deleting N-VR, GBS, and/or C-VR. Two truncation-mutant enzymes devoid of C-VR (TM-NCGΔ) or N-VR/C-VR (TM-ΔCGΔ) were catalytically active, thereby indicating that N-VR and C-VR were not essential for the catalytic activity. TM-ΔCGΔ did not accept any further protease-degradation during long storage. TM-NCGΔ and TM-ΔCGΔ enhanced substrate hydrolysis, suggesting that N-VR and C-VR induce hindered substrate binding to the active site.     

The transition of mouse pluripotent stem cells from the naïve to the primed state requires Fas signaling through 3-O sulfated heparan sulfate structures recognized by the HS4C3 antibody

The characteristics of pluripotent embryonic stem cells of human and mouse are different. The properties of human embryonic stem cells (hESCs) are similar to those of mouse epiblast stem cells (mEpiSCs), which are in a later developmental pluripotency state, the so-called “primed state” compared to mouse embryonic stem cells (mESCs) which are in a naïve state. As a result of the properties of the primed state, hESCs proliferate slowly, cannot survive as single cells, and can only be transfected with genes at low efficiency. Generating hESCs in the naïve state is necessary to overcome these problems and allow their application in regenerative medicine. Therefore, clarifying the mechanism of the transition between the naïve and primed states in pluripotent stem cells is important for the establishment of stable methods of generating naïve state hESCs. However, the signaling pathways which contribute to the transition between the naïve and primed states are still unclear. In this study, we carried out induction from mESCs to mEpiSC-like cells (mEpiSCLCs), and observed an increase in the activation of Fas signaling during the induction. The expression of Fgf5, an epiblast marker, was diminished by inhibition of Fas signaling using the caspase-8 and -3 blocking peptides, IETD and DEVD, respectively. Furthermore, during the induction, we observed increased expression of 3-O sulfated heparan sulfate (HS) structures synthesized by HS 3-O-sulfotransferase (3OST), which are recognized by the HS4C3 antibody (HS4C3-binding epitope). Knockdown of 3OST-5 reduced Fas signaling and the potential for the transition to mEpiSCLCs. This indicates that the HS4C3-binding epitope is necessary for the transition to the primed state. We propose that Fas signaling through the HS4C3-binding epitope contributes to the transition from the naïve state to the primed state.     

Anaerobic degradation of benzene in BTX mixtures dependent on sulfate reduction

Abstract Enrichment cultures from marine sediments mineralized benzene while using sulfate as the terminal electron acceptor. Parallel cultures using river marsh sediment displayed no activity. Mineralization was confirmed by release of ¹⁴CO₂ from radiolabeled benzene. The dependence on sulfate reduction was demonstrated by stoichiometric balances and the use of specific inhibitors. This work supports recent observations that anaerobic benzene degradation takes place coupled to sulfate reduction.