Dynamic transition of chemolithotrophic sulfur-oxidizing bacteria in response to amendment with nitrate in deposited marine sediments.

Although environmental stimuli are known to affect the structure and function of microbial communities, their impact on the metabolic network of microorganisms has not been well investigated. Here, geochemical analyses, high-throughput sequencing of 16S rRNA genes and transcripts, and isolation of potentially relevant bacteria were carried out to elucidate the anaerobic respiration processes stimulated by nitrate (20 mM) amendment of marine sediments. Marine sediments deposited by the Great East Japan Earthquake in 2011 were incubated anaerobically in the dark at 25∘C for 5 days. Nitrate in slurry water decreased gradually for 2 days, then more rapidly until its complete depletion at day 5; production of N2O followed the same pattern. From day 2 to 5, the sulfate concentration significantly increased and the sulfur content in solid-phase sediments significantly decreased. These results indicated that denitrification and sulfur oxidation occurred simultaneously. Illumina sequencing revealed the proliferation of known sulfur oxidizers, i.e., Sulfurimonas sp. and Chromatiales bacteria, which accounted for approximately 43.5% and 14.8% of the total population at day 5, respectively. These oxidizers also expressed 16S rRNA to a considerable extent, whereas the other microorganisms, e.g., iron(III) reducers and methanogens, became metabolically active at the end of the incubation. Extinction dilution culture in a basal-salts medium supplemented with sulfur compounds and nitrate successfully isolated the predominant sulfur oxidizers: Sulfurimonas sp. strain HDS01 and Thioalkalispira sp. strain HDS22. Their 16S rRNA genes showed 95.2-96.7% sequence similarity to the closest cultured relatives and they grew chemolithotrophically on nitrate and sulfur. Novel sulfur-oxidizing bacteria were thus directly involved in carbon fixation under nitrate-reducing conditions, activating anaerobic respiration processes and the reorganization of microbial communities in the deposited marine sediments.

Identification and characterization of an inhibitor of trichothecene 3-O-acetyltransferase, TRI101, by the chemical array approach.

Trichothecene 3-O-acetyltransferase (TRI101) is an indispensable enzyme for the biosynthesis of trichothecenes, a group of mycotoxins produced by Fusarium graminearum. In this study, an inhibitor of TRI101 was identified by chemical array analysis using compounds from the RIKEN Natural Products Depository (NPDepo) library. Although the addition of the identified enzyme inhibitor to the fungal culture did not inhibit trichothecene production, it can serve as a candidate lead compound in the development of a mycotoxin inhibitor that inactivates fungal defense mechanisms.

Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction.

Post-translational protein modification by tyrosine sulfation has an important role in extracellular protein-protein interactions. The protein tyrosine sulfation reaction is catalysed by the Golgi enzyme called the tyrosylprotein sulfotransferase. To date, no crystal structure is available for tyrosylprotein sulfotransferase. Detailed mechanism of protein tyrosine sulfation reaction has thus remained unclear. Here we present the first crystal structure of the human tyrosylprotein sulfotransferase isoform 2 complexed with a substrate peptide (C4P5Y3) derived from complement C4 and 3'-phosphoadenosine-5'-phosphate at 1.9 Å resolution. Structural and complementary mutational analyses revealed the molecular basis for catalysis being an SN2-like in-line displacement mechanism. Tyrosylprotein sulfotransferase isoform 2 appeared to recognize the C4 peptide in a deep cleft by using a short parallel ß-sheet type interaction, and the bound C4P5Y3 forms an L-shaped structure. Surprisingly, the mode of substrate peptide recognition observed in the tyrosylprotein sulfotransferase isoform 2 structure resembles that observed for the receptor type tyrosine kinases.

Identification of cargo proteins specific for the nucleocytoplasmic transport carrier transportin by combination of an in vitro transport system and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics.

The human importin-ß family consists of 21 nucleocytoplasmic transport carrier proteins that carry proteins and RNAs across the nuclear envelope through nuclear pores in specific directions. These transport carriers are responsible for the nucleocytoplasmic transport of thousands of proteins, but the cargo allocation of each carrier, which is necessary information if one wishes to understand the physiological context of transport, is poorly characterized. To address this issue, we developed a high-throughput method to identify the cargoes of transport carriers by applying stable isotope labeling by amino acids in cell culture to construct an in vitro transport system. Our method can be outlined in three steps. (1) Cells are cultured in a medium containing a stable isotope. (2) The cell membranes of the labeled cells are permeabilized, and proteins extracted from unlabeled cells are transported into the nuclei of the permeabilized cells. In this step, the reaction system is first depleted of all importin-ß family carriers and then supplemented with a particular importin-ß family carrier of interest. (3) Proteins in the nuclei are extracted and analyzed quantitatively via LC-MS/MS. As an important test case, we used this method to identify cargo proteins of transportin, a representative member of the importin-ß family. As expected, the identified candidate cargo proteins included previously reported transportin cargoes as well as new potential cargoes, which we corroborated via in vitro binding assays. The identified cargoes are predominately RNA-interacting proteins, affirming that cargoes allotted to the same carrier share functional characteristics. Finally, we found that the transportin cargoes possessed at least two classes of signal sequences: the well characterized PY-nuclear localization signals specific for transportin, and Lys/Arg-rich segments capable of binding to both transportin and importin-ß. Thus, our method will be useful for linking a carrier to features shared among its cargoes and to specific nuclear localization signals.

Efficacy and safety of moxifloxacin for community-acquired bacterial pneumonia based on pharmacokinetic analysis.

Moxifloxacin is a respiratory quinolone that is expected to be useful for treating community-acquired bacterial pneumonia, but few clinical studies and not a detailed evaluation of its pharmacokinetics have been conducted in Japan in patients with pneumonia. We assessed the efficacy and safety of moxifloxacin in 18 patients with community-acquired bacterial pneumonia using pharmacokinetic-pharmacodynamic analysis. There was significant improvement in body temperature, white blood cell count, C-reactive protein, and chest X-ray score on day 3 of moxifloxacin treatment, which persisted until the completion of treatment (all p < 0.05). Nine strains, including Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, and Enterobacter cloacae, were isolated from sputum cultures of nine patients. The isolated strains were eradicated by moxifloxacin. The mean area under the concentration-time curve from 0 to 24 hours [AUC(0-24 h) (AUC(0-24 h,ss))], maximum plasma concentration (C(max)), and trough plasma level (C(trough)) of moxifloxacin at steady state was 52.0 µg h/ml, 4.5, and 0.9 µg/ml, respectively. Mean AUC(0-24 h,ss)/mimimum inhibitory concentration (MIC), and C(max)/MIC ratios for patients in whom MICs of moxifloxacin were determined for pathogenic bacteria were 723 and 62, respectively. The median AUC(0-24 h,ss)/MIC and C(max)/MIC ratios (based on Monte Carlo simulation employing MICs for 257 strains of S. pneumoniae collected during a respiratory infection survey by the Japanese Society of Chemotherapy in 2007) were 209.56 and 17.88, respectively. Thus, when the target for the AUC/MIC ratio was set at ≥30 and that for the C(max)/MIC ratio at ≥5, the achievement rate for these two parameters was 97.36% and 96.71%, respectively. Two patients (11%) experienced three adverse effects [one nausea, another increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT)], but the events were not serious. Based on these results, moxifloxacin (400 mg once daily) was considered useful for treating community-acquired bacterial pneumonia and is expected to show excellent efficacy and safety as well as suppressing the emergence of resistance.

The development of a filter to enhance the efficacy and safety of excimer light (308 nm) therapy.

BACKGROUND: Excimer light (308 nm) therapy is a new ultraviolet (UV) B phototherapy for which the efficacy and resulting DNA damage are not well established. PURPOSE: To develop an effective and safe phototherapy using the excimer lamp, we studied the effects of different light cut-off filters, A and B. METHODS: Efficacy was evaluated by measuring apoptosis using fluorescence-activated cell sorting analysis. DNA damage was evaluated by measuring cyclobutane pyrimidine dimers (CPDs). Light sources, including normal wave and short wave (SW) excimer light, broad-band (BB) UVB, and narrow-band (NB) UVB, were examined using the filters. A human skin equivalent model was also examined. RESULTS: The ratio of positive apoptosis to CPD formation normalized to the mean induced by NB-UVB was 5.7 using the excimer lamp without a filter, 6.3 using the excimer lamp with the A filter, 6.4 using the SW excimer lamp without a filter, and 4.2 using the BB-UVB. The A filter reduced CPD formation induced by the normal wave and SW excimer lamp. In the human skin equivalent model, the use of filters significantly decreased the amount of CPD-positive cells. CONCLUSIONS: These findings suggest that using the A filter with the excimer lamp increases the efficacy and safety of excimer light therapy.

Melanin synthesis inhibitors from Lespedeza floribunda.

In the course of our search for new melanin synthesis inhibitors from plants, 40 new flavonoids and 11 known flavonoids were isolated from the roots of Lespedeza floribunda Bunge. The structures of the new compounds were determined by MS and NMR analyses, and the absolute configurations by CD spectra. Many of the compounds inhibited melanin synthesis in normal human epidermal melanocytes (NHEM), and compounds 3, 7, 8, 11, 16, 24, 27, 29, 33, 43, 45, and 51 were particularly inhibitory. Their activities were stronger than that of hydroquinone, which is known as a major skin-lightening drug.

A screening system for inhibitors of trichothecene biosynthesis: hydroxylation of trichodiene as a target.

Fusarium Tri4 encodes a key cytochrome P450 monooxygenase for hydroxylation of trichodiene early in the biosynthesis of trichothecenes. In this study, we established a system for screening for inhibitors of trichothecene biosynthesis using transgenic Saccharomyces cerevisiae expressing Tri4. For easy evaluation of the TRI4 activity, trichodiene-11-one was used as a substrate and the formation of 2alpha-hydroxytrichodiene-11-one was monitored by HPLC. Using this system, TRI4 proved to be inhibited by various flavones and furanocoumarins. We also found that a catechin-containing commercial beverage product, Catechin Supplement 300 (CS300), inhibited TRI4 activity, at a concentration which did not significantly affect the growth of the transgenic yeast. At an early stage of culture, both flavone and CS300 exhibited a toxin-inhibitory activity against Fusarium graminearum. However, inhibition of trichothecene production was not observed with longer incubation periods at minimum concentrations necessary to inhibit >50% of the TRI4 activity, presumably due to the metabolism by the fungus. The results suggest that this yeast screening system with TRI4 is useful for the rapid identification of lead compounds for the design of trichothecene biosynthesis inhibitors that are resistant to modification by the fungus.