Poly(ethylene oxide) (PEO) is a well-known biocompatible polymer and has widely been used for medical applications. Recently, we have investigated the dynamic behavior of hydration water in the vicinity of PEO chains at physiological temperature and shown the presence of slow water with diffusion coefficient one order of magnitude less than that of bulk water. This could be evidence for the intermediate water that is critical for biocompatibility; however, its detailed dynamical features were not established. In this article, we analyze the quasi-elastic neutron scattering from hydration water through mode distribution analysis and present a microscopic picture of hydration water as well as its relation to cold crystallization.

This chapter presents assignments of almost all of the polyethers together with their application to structural studies. One of the structural features of most polyether antibiotics is the repetition of similar partial structures in the molecules, which results in complicated C NMR spectra with many methylene and methine signals crowded in a narrow region. In addition to structural investigation, the methodology will further enable one to study important problems associated with the mechanisms of action of the polyether antibiotics, such as interaction with metal cations in solution. Subtle conformational changes of the antibiotics caused by complex formation will be amply reflected in their C NMR spectra. C NMR spectroscopy is well known to be a very useful tool for the structural elucidation of natural products. This is also true with the polyether antibiotics, especially when good crystals suitable for x-ray analysis are not obtainable, as evidenced in case of narasin.

Complexation of zwitterionic lipid, dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and protein, bovine serum albumin (BSA) at the air-water interface has been studied by surface pressure (π) - mean molecular area (A) isotherms and X-ray reflectivity. Although BSA has isoelectric point nearly at pH≈4.8, possibility of complex formation with lipid molecules has been investigated from low (≈4.0) to high (≈9.0) pH range in presence of divalent cation, Ca(2+) in the water subphase. Both the isotherm and reflectivity analysis show that the interaction of BSA with lipid monolayer takes place from that low to high subphase pH range, i.e., complexation occurs both below and above of the isoelectric point. Only one layer of BSA forms below the lipid monolayer and the probable reasons for such complex formation have been proposed.

Isoprenoids are a diverse group of molecules found in all organisms, where they perform such important biological functions as hormone signaling (e.g., steroids) in mammals, antioxidation (e.g., carotenoids) in plants, electron transport (e.g., ubiquinone), and cell wall biosynthesis intermediates in bacteria. All isoprenoids are synthesized by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer, dimethylallyl diphosphate (DMAPP). The biosynthetic pathway for the formation of IPP from acetyl-CoA (i.e., the mevalonate pathway) had been established mainly in mice and the budding yeast Saccharomyces cerevisiae. Curiously, most prokaryotic microorganisms lack homologs of the genes in the mevalonate pathway, even though IPP and DMAPP are essential for isoprenoid biosynthesis in bacteria. This observation provided an impetus to search for an alternative pathway to synthesize IPP and DMAPP, ultimately leading to the discovery of the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate pathway. This review article focuses on our significant contributions to a comprehensive understanding of the biosynthesis of IPP and DMAPP.

The first synthesis of (±)-cyclic dehypoxanthine futalosine (cyclic DHFL), a biosynthetic intermediate in the futalosine pathway for menaquinones operating in microorganisms, has been achieved. Efficient growth of the Streptomyces coelicolor mutant, which lacks the cyclic DHFL synthetase gene (mqnC gene) was observed in the presence of synthetic (±)-cyclic DHFL.

Efficient pulse sequences for measuring long‐range CH coupling constants (JCH) and proton‐proton spin coupling constants (JHH), named BIRD‐J‐resolved HMBC and BIRD‐high‐resolution HMBC, respectively, have been developed. In spin systems possessing a secondary methyl group positioned between protonated carbons (e.g.CH2CH(CH3)CH2), the methine proton splits in a complicated fashion, resulting in difficulty in the determination of its spin coupling constants. For easy and accurate measurements of the long‐range JCH and JHH in such a spin system, the BIRD pulse [90°x(H)− 180°x (H/C)− 90° (−x)(H)] or [90°x(H)− 180°x(H/C)− 90° (−x)(H)180°x(C)] is incorporated into the J‐resolved portion of the pulse sequence. As a result, the above secondary methyl group can be selectively decoupled, providing simplified cross‐peak patterns, which are suitable for the accurate measurements of the long‐range JCH and JHH. Copyright © 2010 John Wiley & Sons, Ltd.

New terpenoids named gifhornenolones A ( 1 ) and B ( 2 ) were isolated from the culture broth of Verrucosispora gifhornensis YM28-088, and their structures were established as hydroxylated isopimaradiene derivatives on the basis of extensive NMR and MS spectral analyses. In addition, a known sesquiterpene compound cyperusol C ( 3 ) was isolated. The absolute configuration of 1 was determined by nuclear Overhauser effect spectroscopy (NOESY) and CD spectra as 4 R , 5 S , 9 R , 10 S , 13 R , and that of 2 was determined by NOESY experiments as 3 R , 4 R , 5 R , 9 R , 10 S , 13 R . Labeling experiments with [1- 13 C]glucose and [ U - 13 C 6 ]glucose confirmed that the MEP (2- C -methyl- D -erythritol-4-phosphate) pathway was used for the biosynthesis of terpenoids in this organism. 1 showed potent inhibitory activity to the androgen receptor with an IC 50 of 2.8 μ g ml −1 .

Phase separation of giant vesicles composed of neutral saturated lipid, negatively charged unsaturated lipid, and cholesterol, is observed at different calcium concentrations. Confocal microscopy provides the information where the phase separation becomes distinct as the calcium concentration is increased. The charged domains tend to bud toward the interior of the vesicle. This budding is assumed to be due to an increase in the osmotic pressure, in cooperation with the spontaneous curvature change in the outer leaflet of the bilayer caused by the adsorption of calcium ions and charge screening effect. We interpret the effect of small cations on the phase separation based on the theoretical model with the Poisson–Boltzmann equation.

Menaquinone (MK) is an essential compound because it is an obligatory component of the electron transfer pathway in microorganisms. In Escherichia coli, MK was shown to be derived from chorismate by eight enzymes, designated MenA–H.1,2 However, we have revealed that an alternative pathway (we named it the futalosine pathway; Figure 1)3–5 was operating in some microorganisms including Helicobacter pylori, which causes gastric carcinoma. As humans and some useful intestinal bacteria, such as lactobacilli, possess the classical pathway, and MK biosynthesis is essential for survival of microorganisms,4 the futalosine pathway is an attractive target for the development of specific anti-H. pylori drugs. In this study, we tried to obtain such compounds from metabolites produced by actinomycetes and fungi. To identify compounds that specifically inhibit the futalosin pathway, we developed a screening method. We previously showed that the MqnA–D genes in the futalosine pathway were essential for survival, as these gene-disrupted Streptomyces coelicolor strains required exogenously added MK for their growth. Therefore, a compound that inhibits the growth of S. coelicolor but does not inhibit its growth in the presence of MK would become a candidate. However, this assay method is laborious and the growth of S. coelicolor is too slow to screen a mass of samples. Therefore, we employed a paper disk-agar diffusion assay, which is based on the phenomenon that antibiotics will diffuse from a paper disk into an agar medium containing test organisms and form a growth-inhibitory zone. We used two kinds of Bacillus strains as test organisms. One was Bacillus subtilis and the other is B. halodurans C-125. By genome sequencing, the latter strain was shown to be quite similar to the former strain in terms of genome size, G+C content of genomic DNA and the physiological properties used for taxonomical identification.6 Moreover, the phylogenetic placement of B. halodurans C-125 based on 16S rDNA sequence analysis indicated that this organism is more closely related to B. subtilis than to other members of the genus Bacillus.6 For example, both strains showed similar MIC values against representative antibiotics except for clarithromycin (Table 1). The resistance to clarithromycin was probably caused by the presence of an ermD gene that encodes the ribosome-methylation enzyme in B. halodurans.7 However, judging from the genome database of these strains, B. subtilis and B. halodurans C-125 use the classical pathway and the futalosine pathway, respectively, for the biosynthesis of MK.6 These facts suggested that a compound inhibiting the biosynthesis of MK in the futalosine pathway specifically represses the growth of only B. halodurans C-12. Therefore, we first screened candidate compounds for their ability to specifically inhibit B. halodurans C-125 using a paper disk assay. We tested approximately 1800 culture broths (1000 actinomycetes broths and 800 fungi broths). Of these, approximately 300 culture broths (17%) formed growth-inhibitory zone against both B. subtilis and B. halodurans C-125. However, we found that two actinomycetes culture broths specifically inhibited the growth of B. halodurans C-125 (hit ratio, 0.1%) (Figure 2). Then we examined whether B. halodurans C-125 could recover from this inhibition when MK (0.1 mg ml 1) was added into the culture broth during liquid cultivation. The growth of B. halodurans C-125 was clearly inhibited in the presence of sample no. AF50404, but this inhibition was reversed by adding MK, even in the presence of sample no. AF50404. This result strongly suggested that sample no. AF50404 contained a compound that specifically inhibited the futalosine pathway. The other candidate (AF50573) also showed the same inhibitory phenotype as that of no. AF50404, but it gradually lost its activity, probably because of its instability. Therefore, we used sample no. AF50404 in further analyses.

We recently demonstrated that the futalosine pathway was operating in some bacteria for the biosynthesis of menaquinone and that futalosine was converted into dehypoxanthinyl futalosine (DHFL) by an MqnB of Thermus thermophilus. In this study, we found that aminodeoxyfutalosine, which has adenine instead of hypoxanthine in futalosine, was directly converted into DHFL by an MqnB of Helicobacter pylori. Therefore, this step is potentially an attractive target for the development of specific anti-H. pylori drugs.

AbstractLabeling experiments confirm that the MEP (2‐C‐methyl‐D‐erythritol‐4‐phosphate) pathway is used for the biosynthesis of gifhornenolone A (I) and B (II) in the title microorganism.

Two peptides, tumescenamides A and B, were isolated from the fermentation broth of a marine bacterium, Streptomyces tumescens YM23-260. The structure of tumescenamide A was determined to be a cyclic depsipeptide consisting of α-amino-2-butenoic acid, tyrosine, valine, leucine and threonine, substituted with a 2,4-dimethylheptanoyl residue at the α-NH 2 position. Tumescenamide B possesses a 2,4,6-trimethylnonanoyl residue in place of the 2,4-dimethylheptanoyl substituent in tumescenamide A. Tumescenamide A induced reporter gene expression under the control of the insulin-degrading enzyme promoter.

AbstractTumescenamide A (Ia) induces gene expression under the control of the insulin‐degrading enzyme promoter.

A useful pulse sequence for measuring long-range C--H coupling constants (J(C-H)) named high resolution-HMBC (HR-HMBC) has been developed. In this pulse sequence, the J-scaling pulse [(nt(1))/2-180 degrees (H/C) - (nt(1))/2] is incorporated after the spin evolution period, and then followed by an (1)H 180 degrees pulse to reverse the magnetization of J(C-H) couplings. As a result, splittings of the cross peaks due to the long-range J(C-H) are realigned with separations of nJ(C-H) along the F(1) dimension, and thus even the small long-range J(C--H) values can easily be determined. The efficiency of measuring the long-range J(C--H) using the proposed pulse sequences has been demonstrated in application to the complicated natural product, portmicin.

The differences between the biosynthesis of sterols in higher plants and yeast/mammals are believed to originate at the cyclization step of oxidosqualene, which is cyclized to cycloartenol in higher plants and lanosterol in yeast/mammals. Recently, lanosterol synthase genes were identified from dicotyledonous plant species including Arabidopsis, suggesting that higher plants possess dual biosynthetic pathways to phytosterols via lanosterol, and through cycloartenol. To identify the biosynthetic pathway to phytosterol via lanosterol, and to reveal the contributions to phytosterol biosynthesis via each cycloartenol and lanosterol, we performed feeding experiments by using [6-(13)C(2)H(3)]mevalonate with Arabidopsis seedlings. Applying (13)C-{(1)H}{(2)H} nuclear magnetic resonance (NMR) techniques, the elucidation of deuterium on C-19 behavior of phytosterol provided evidence that small amounts of phytosterol were biosynthesized via lanosterol. The levels of phytosterol increased on overexpression of LAS1, and phytosterols derived from lanosterol were not observed in a LAS1-knockout plant. This is direct evidence to indicate that the biosynthetic pathway for phytosterol via lanosterol exists in plant cells. We designate the biosynthetic pathway to phytosterols via lanosterol "the lanosterol pathway." LAS1 expression is reported to be induced by the application of jasmonate and is thought to have evolved from an ancestral cycloartenol synthase to a triterpenoid synthase, such as beta-amyrin synthase and lupeol synthase. Considering this background, the lanosterol pathway may contribute to the biosynthesis of not only phytosterols, but also steroids as secondary metabolites. PMID: 19139393

In prokaryotes, menaquinone is used for respiration. In Escherichia coli, menaquinone is biosynthesized from chorismate by seven enzymes. However, very recently, we identified an alternative pathway (the futalosine pathway), which operates in some bacteria, including Streptomyces coelicolor, Helicobacter pylori, Campylobacter jejuni, and Thermus thermophilus. We describe the steps of this pathway, which branches at chorismate in a manner similar to the known pathway, but then follows a different route. This new pathway includes futalosine, an unusual nucleoside derivative consisting of inosine and o-substituted benzoate moieties, as a biosynthetic intermediate. In this study, a recombinant futalosine hydrolase (TTHA0556) of T. thermophilus, which participates in the second step of the pathway and catalyzes the reaction releasing hypoxanthine from futalosine, was prepared and used in functional analyses. Recombinant TTHA0556 formed a homotetramer and reacted only with futalosine; other structurally related nucleotides and nucleosides were not accepted. Recombinant TTHA0556 required no cofactors, and the optimum pH and temperature were 4.5 and 80 °C. The Km value was calculated to be 154.0±5.3 μM and the kcat value was 1.02/s. Recombinant TTHA0556 was slightly inhibited by hypoxanthine, with a Ki value of 1.1 mM.

Ammocidins B, C and D were isolated from the culture broth of Saccharothrix sp. AJ9571, an ammocidin A-producing strain. Their structures were determined by a detailed spectroscopic analysis and by a comparison of their NMR data with those of ammocidin A. Ammocidins A and B showed potent anti-proliferative activities against human cancer cell lines.

In microorganisms, menaquinone is an obligatory component of the electron- transfer pathway. It is derived from chorismate by seven enzymes in Escherichia coli. However, a bioinformatic analysis of whole genome sequences has suggested that some microorganisms, including pathogenic species such as Helicobacter pylori and Campylobacter jejuni, do not have orthologs of the men genes, even though they synthesize menaquinone. We deduced the outline of this alternative pathway in a nonpathogenic strain of Streptomyces by bioinformatic screening, gene knockouts, shotgun cloning with isolated mutants, and in vitro studies with recombinant enzymes. As humans and commensal intestinal bacteria, including lactobacilli, lack this pathway, it represents an attractive target for the development of chemotherapeutics.