Synthesis of cephalosporin C by a methionine analogue resistant mutant of Cephalosporium acremonium

Summary DL-seleno-methionine resistant mutants of Cephalosporium acremonium were isolated which have an enhanced capacity to utilized sulfate for the synthesis of cephalosporin C. Of these mutants, one designated as SMR-I3 produced three-fold more cephalosporin C from sulfate than its parent CW19. Mutant SMR-I3 required less dl-methionine for maximal synthesis of cephalosporin C, but an excess of dl-methionine inhibited the synthesis of the antibiotic. Furthermore, the mutant accumulated excessive methionine in the amino acid pool and possessed superior activity for sulfate uptake. These observations indicate that in the mutant SMR-I3, the biosynthesis of methionine from sulfate is very active and excess methionine becomes available for the synthesis of cephalosporin C.     

Optimum conditions for ursodeoxycholic acid production from lithocholic acid by Fusarium equiseti M41

Ursodeoxycholic acid dissolves cholesterol gallstones in humans. In the present study optimum conditions for ursodeoxycholic acid production by Fusarium equiseti M41 were studied. Resting mycelia of F. equiseti M41 showed maximum conversion at 28 degrees C, pH 8.0, and dissolved oxygen tension of higher than 60% saturation. Monovalent cations, such as Na+, K+, and Rb+, stimulated the conversion rate more than twofold. In the presence of 0.5 M KCl, the initial uptake rate and equilibrium concentration of lithocholic acid (substrate) were enhanced by 5.7- and 1.7-fold, respectively. We confirmed that enzyme activity catalyzing 7 beta-hydroxylation of lithocholic acid was induced by substrate lithocholic acid. The activity in the mycelium was controlled by dissolved oxygen tension during cultivation: with a dissolved oxygen tension of 15% and over, the activity peak appeared at 25 h of cultivation, whereas the peak was delayed to 34 and 50 h with 5 and 0% dissolved oxygen tension, respectively. After reaching the maximum, the 7 beta-hydroxylation activity in the mycelium declined rapidly at pH 7.0, but the decline was retarded by increasing the pH to 8.0. Several combinations of operations, such as pH shift (from pH 7 to 8), addition of 0.5 M KCl, and dissolved oxygen control, were applied to the production of ursodeoxycholic acid in a jar fermentor, and a much larger amount of ursodeoxycholic acid (1.2 g/liter) was produced within 96 h of cultivation.     

Elevated mitochondrial biogenesis in skeletal muscle is associated with testosterone-induced body weight loss in male mice

Androgen reduces fat mass, although the underlying mechanisms are unknown. Here, we examined the effect of testosterone on heat production and mitochondrial biogenesis. Testosterone-treated mice exhibited elevated heat production. Treatment with testosterone increased the expression level of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), ATP5B and Cox4 in skeletal muscle, but not that in brown adipose tissue and liver. mRNA levels of genes involved in mitochondrial biogenesis were elevated in skeletal muscle isolated from testosterone-treated male mice, but were down-regulated in androgen receptor deficient mice. These results demonstrated that the testosterone-induced increase in energy expenditure is derived from elevated mitochondrial biogenesis in skeletal muscle.     

Antibody-modified lipid nanoparticles for selective delivery of siRNA to tumors expressing membrane-anchored form of HB-EGF

An Fab’ antibody against heparin-binding epidermal growth factor-like growth factor (HB-EGF) was applied to achieve advanced tumor-targeted delivery of siRNA. Lipid nanoparticles (LNP) encapsulating siRNA (LNP-siRNA) were prepared, pegylated, and surface modified with Fab’ fragments of anti-HB-EGF antibody (αHB-EGF LNP-siRNA). αHB-EGF LNP-siRNA showed high-binding affinity to recombinant human HB-EGF in a Biacore assay. In addition, αHB-EGF LNP-siRNA selectively associated with cells expressing HB-EGF in vitro. Confocal microscopic images showed that siRNA formulated in αHB-EGF LNP-siRNA was efficiently internalized into MDA-MB-231 human breast cancer cells, on which HB-EGF is highly expressed. In addition, siRNA encapsulated in αHB-EGF LNP induced obvious suppression of both target mRNA and protein levels in MDA-MB-231 cells. These results indicate that αHB-EGF LNP have excellent potential to deliver siRNA to target cancer cells, resulting in effective gene silencing.     

Prognostic factors related to add-on dendritic cell vaccines on patients with inoperable pancreatic cancer receiving chemotherapy: a multicenter analysis

Objective

Dendritic cell (DC)-based cancer vaccines may have a significant benefit to patients with advanced pancreatic cancer. However, variations among clinical studies make it difficult to compare clinical outcomes. Here, we identified factors that determined the clinical benefits by analyzing data obtained at seven Japanese institutions that employed the same DC preparation and treatment regimens.

Methods

Of 354 patients who met the inclusion criteria, 255 patients who received standard chemotherapy combined with peptide-pulsed DC vaccines were analyzed.

Results

The mean survival time from diagnosis was 16.5 months (95 % CI 14.4–18.5) and that from the first vaccination was 9.9 months (95 % CI 8.0–12.9). Known prognostic baseline factors related to advanced pancreatic cancer, namely ECOG-PS, peritoneal metastasis, liver metastasis, and the prognostic nutrition index, were also representative. Importantly, we found that erythema reaction after vaccination was an independent and treatment-related prognostic factor for better survival and that OK-432 might be a good adjuvant enhancing the antitumor immunity during DC vaccination.

Conclusions

This is the first report of a multicenter clinical study suggesting the feasibility and possible clinical benefit of an add-on DC vaccine in patients with advanced pancreatic cancer who are undergoing chemotherapy. These findings need to be addressed in well-controlled prospective randomized trials.     

Photoprotective acclimation of micro- and nano-size phytoplankton assemblages in the Indian sector of the Southern Ocean

Phytoplankton in the mixed layer is exposed to increasing levels of light when transported to the surface layer of the ocean. The photoprotective response of natural assemblages of phytoplankton can differ among community structures. We investigated photoprotective acclimation and xanthophyll cycle pigments in size-fractionated natural phytoplankton assemblages during the austral summer in the Indian sector of the Southern Ocean. We estimated concentrations of phytoplankton pigments in the micro-size fractions (>20 μm) and nano-size fractions (2–20 μm) by subtracting concentrations in the <20 μm fractions from concentrations in the bulk samples, and by subtracting concentrations in the <2 μm fractions from concentrations in the <20 μm fractions, respectively. Changes in the ratios of the xanthophyll cycle pigments diadinoxanthin (DD) and diatoxanthin (DT) were determined at three optical depths in the mixed layer and during 48 h deck incubations under solar photosynthetically available radiation and ultraviolet radiation. Large variations in (DD + DT)/Chl a in the mixed layer (percent coefficient of variation >67 %) and in deck incubation bottles under variable light conditions (>75 % of the temporal variation) for the micro-size fractions suggest a higher potential for photoprotective acclimation than for the nano-size fractions. Decreases in DT/(DD + DT) with increases in the optical depth of the mixed layer (ζ _MLD) suggest that larger variations in light availability in the mixed layer might predict lower values of DT/(DD + DT) at the surface, regardless of cell size.     

The Core of Allosteric Motion in Thermus caldophilus l-Lactate Dehydrogenase

For Thermus caldophilus l-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S_0.5 value 10³-fold and increased the V_max value 4-fold at 30 °C and pH 7.0, indicating that TcLDH has a much more T state-sided allosteric equilibrium than Thermus thermophilus l-lactate dehydrogenase, which has only two amino acid replacements, A154G and H179Y. The inactive (T) and active (R) state structures of TcLDH were determined at 1.8 and 2.0 Å resolution, respectively. The structures indicated that two mobile regions, MR1 (positions 172–185) and MR2 (positions 211–221), form a compact core for allosteric motion, and His¹⁷⁹ of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased V_max 4-fold but reduced pyruvate S_0.5 only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase V_max, but 10²-reduced pyruvate S_0.5, and additively increased the FBP-independent activity of the Q4(R) enzyme. The two types of mutation consistently increased the thermal stability of the enzyme. The MR1-MR2 area is a positively charged cluster, and its center approaches another positively charged cluster (N domain cluster) across the Q-axis subunit interface by 5 Å, when the enzyme undergoes the T to R transition. Structural and kinetic analyses thus revealed the simple and unique allosteric machinery of TcLDH, where the MR1-MR2 area pivotally moves during the allosteric motion and mediates the allosteric equilibrium through electrostatic repulsion within the protein molecule.     

Phototropin Encoded by a Single-Copy Gene Mediates Chloroplast Photorelocation Movements in the Liverwort Marchantia polymorpha

Blue-light-induced chloroplast photorelocation movement is observed in most land plants. Chloroplasts move toward weak-light-irradiated areas to efficiently absorb light (the accumulation response) and escape from strong-light-irradiated areas to avoid photodamage (the avoidance response). The plant-specific kinase phototropin (phot) is the blue-light receptor for chloroplast movements. Although the molecular mechanisms for chloroplast photorelocation movement have been analyzed, the overall aspects of signal transduction common to land plants are still unknown. Here, we show that the liverwort Marchantia polymorpha exhibits the accumulation and avoidance responses exclusively induced by blue light as well as specific chloroplast positioning in the dark. Moreover, in silico and Southern-blot analyses revealed that the M. polymorpha genome encodes a single PHOT gene, MpPHOT, and its knockout line displayed none of the chloroplast photorelocation movements, indicating that the sole MpPHOT gene mediates all types of movement. Mpphot was localized on the plasma membrane and exhibited blue-light-dependent autophosphorylation both in vitro and in vivo. Heterologous expression of MpPHOT rescued the defects in chloroplast movement of phot mutants in the fern Adiantum capillus-veneris and the seed plant Arabidopsis (Arabidopsis thaliana). These results indicate that Mpphot possesses evolutionarily conserved regulatory activities for chloroplast photorelocation movement. M. polymorpha offers a simple and versatile platform for analyzing the fundamental processes of phototropin-mediated chloroplast photorelocation movement common to land plants.Light is not only an energy source for photosynthesis but it is also a signal that regulates numerous physiological responses for plants. Because chloroplasts are the important organelle for photosynthesis, most plant species possess a light-dependent mechanism to regulate the intracellular position of chloroplasts (chloroplast photorelocation movement). Intensive studies on chloroplast photorelocation movement have been performed since the 19th century (Böhm, 1856). Senn (1908) described the chloroplast distribution patterns under different light conditions in various plant species, including algae, liverworts, mosses, ferns, and seed plants, and revealed the general responses of chloroplasts to intensity and direction of light. Under low-light conditions, chloroplasts are positioned along the cell walls perpendicular to the direction of incident light (i.e. periclinal cell walls) to efficiently capture light for photosynthesis (the accumulation response). By contrast, under high-light conditions, chloroplasts are stacked along the cell walls parallel to the direction of incident light (i.e. anticlinal cell walls) to minimize total light absorption and to avoid photooxidative damage (the avoidance response). These chloroplast movements are induced primarily by blue light in most plant species (Suetsugu and Wada, 2007a). In some plant species, such as several ferns including Adiantum capillus-veneris, the moss Physcomitrella patens, and some charophycean green algae (Mougeotia scalaris and Mesotaenium caldariorum), red light is also effective to induce chloroplast movement (Suetsugu and Wada, 2007b). Analyses of chloroplast movement in response to irradiation with polarized light and/or a microbeam suggest that the photoreceptor for chloroplast movement is localized on or close to the plasma membrane (Haupt and Scheuerlein, 1990; Wada et al., 1993). In addition, chloroplasts assume their specific positions in the dark (dark positioning), although the patterns vary among plant species (Senn, 1908). For example, the chloroplasts are localized at the bottom of the cell in palisade cells of Arabidopsis (Arabidopsis thaliana; Suetsugu et al., 2005a) and on the anticlinal walls bordering neighboring cells in the prothallial cells of A. capillus-veneris (Kagawa and Wada, 1993; Tsuboi et al., 2007).Molecular mechanisms for chloroplast photorelocation movements have been revealed through molecular genetic analyses using Arabidopsis (Suetsugu and Wada, 2012). The light-activated kinase phototropin was identified as the blue-light receptor (Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001). Phototropin consists of two functional regions: a photosensory domain at the N terminus and a Ser/Thr kinase domain at the C terminus (Christie, 2007). The N-terminal photosensory domain contains two light, oxygen, or voltage (LOV) domains, which belong to the Per/ARNT/Sim domain superfamily. Each LOV domain binds to one FMN and functions as a blue-light sensor (Christie et al., 1999). The LOV2 domain is essential for blue-light-dependent regulation of the activation of the C-terminal kinase domain (Christie et al., 2002; Harper et al., 2003).Arabidopsis has two phototropins: phot1 and phot2 (Christie, 2007). Besides chloroplast photorelocation movement, phototropin controls other photoresponses to optimize the photosynthetic efficiency in plants and improves growth responses such as phototropism, stomatal opening, and leaf flattening (Christie, 2007). Both phot1 and phot2 redundantly regulate the chloroplast accumulation response (Sakai et al., 2001), hypocotyl phototropism (Huala et al., 1997; Sakai et al., 2001), stomatal opening (Kinoshita et al., 2001), and leaf flattening (Sakai et al., 2001; Sakamoto and Briggs, 2002). Rapid inhibition of hypocotyl elongation is specifically mediated by phot1 (Folta and Spalding, 2001), whereas the chloroplast avoidance response (Jarillo et al., 2001; Kagawa et al., 2001) and palisade cell development (Kozuka et al., 2011) are mediated primarily by phot2.It is thought that the phototropin-regulated photoresponses are mediated by mechanisms in which gene expression is not involved primarily. For example, chloroplast photorelocation movement can be observed even in enucleated fern cells (Wada, 1988), and phototropins show only a minor contribution to blue-light-induced gene expression in Arabidopsis (Jiao et al., 2003; Ohgishi et al., 2004; Lehmann et al., 2011). Furthermore, both phot1 and phot2 are localized on the plasma membrane despite the absence of a transmembrane domain (Sakamoto and Briggs, 2002; Kong et al., 2006). During chloroplast movement, phototropins, in particular phot2, associate not only with the plasma membrane but also with the chloroplast outer membrane (Kong et al., 2013b). In addition, phot1 shows blue-light-dependent internalization into the cytoplasm (Sakamoto and Briggs, 2002; Knieb et al., 2004; Wan et al., 2008; Kaiserli et al., 2009), whereas phot2 exhibits a blue-light-dependent association with the Golgi apparatus (Kong et al., 2006).PHOT genes have been identified from various green plants and are indicated to be duplicated in respective lineages such as seed plants, ferns, lycophytes, and mosses (Li et al., 2014). In the fern A. capillus-veneris, chloroplast accumulation and avoidance responses are induced by both blue and red light (Yatsuhashi et al., 1985). This fern has three phototropin family proteins, two phototropins (Acphot1 and Acphot2; Kagawa et al., 2004), and one neochrome that possesses the chromophore-binding domain of phytochrome and complete phototropin domains (Nozue et al., 1998). Neochrome is the red-light receptor that mediates chloroplast movement (Kawai et al., 2003) and possibly blue-light-induced chloroplast movement through its LOV domains (Kanegae et al., 2006). Because the Acphot2 mutant is defective in the chloroplast avoidance response and dark positioning (Kagawa et al., 2004; Tsuboi et al., 2007), similar to the phot2 mutant in Arabidopsis (Jarillo et al., 2001; Kagawa et al., 2001; Suetsugu et al., 2005a), the function of phot2 in the regulation of chloroplast movement is highly conserved in these vascular plants. In the moss P. patens, in which chloroplast accumulation and avoidance responses are induced by both blue and red light (Kadota et al., 2000), seven phototropin genes are present in the draft genome sequences (Rensing et al., 2008). The phototropins encoded by four of these genes (PpphotA1, PpphotA2, PpphotB1, and PpphotB2) function in the blue-light-induced chloroplast movement (Kasahara et al., 2004). Moreover, red-light-induced chloroplast movements are mediated by both conventional phytochromes (Mittmann et al., 2004; Uenaka and Kadota, 2007) and phototropins (Kasahara et al., 2004). Because the direct association between phytochromes and phototropins is suggested to be involved in red-light-induced chloroplast movement (Jaedicke et al., 2012), phototropins should be essential components in the chloroplast movement signaling pathway (Kasahara et al., 2004).A single PHOT gene was isolated in a unicellular green alga, Chlamydomonas reinhardtii (Huang et al., 2002; Kasahara et al., 2002). When expressed in Arabidopsis phot1 phot2 double-mutant plants, C. reinhardtii phototropin rescued the defects in chloroplast photorelocation movement in phot1 phot2 plants (Onodera et al., 2005), indicating that the initial step of the phototropin-mediated signal transduction mechanism for chloroplast movements is conserved in the green plant lineage. Although the existence of only one PHOT gene is ideal for elucidation of phototropin-mediated responses, C. reinhardtii cells contain a single chloroplast and show no chloroplast photorelocation movement.Liverworts represent the most basal lineage of extant land plants and offer a valuable experimental system for elucidation of various physiological responses commonly seen in land plants (Bowman et al., 2007). Marchantia polymorpha has emerged as a model liverwort because molecular biological techniques, such as genetic transformation and gene-targeting technologies, have been established for the species (Ishizaki et al., 2008, 2013a; Kubota et al., 2013; Sugano et al., 2014). Furthermore, an ongoing M. polymorpha genome sequencing project under the Community Sequencing Program at the Joint Genome Institute has indicated that many biological mechanisms found in other groups of land plants are conserved in a much less complex form. Blue-light-induced chloroplast movement was briefly reported in M. polymorpha (Senn, 1908; Nakazato et al., 1999). However, information on chloroplast photorelocation movement in liverworts, including M. polymorpha, is very limited.In this study, we investigated chloroplast photorelocation movement in detail in M. polymorpha and analyzed the molecular mechanism underlying the photoreceptor system through molecular genetic analysis of M. polymorpha phototropin.     

Very-long-chain polyunsaturated fatty acids accumulate in phosphatidylcholine of fibroblasts from patients with Zellweger syndrome and acyl-CoA oxidase1 deficiency

Peroxisomes are subcellular organelles that function in multiple anabolic and catabolic processes, including β-oxidation of very-long-chain fatty acids (VLCFA) and biosynthesis of ether phospholipids. Peroxisomal disorders caused by defects in peroxisome biogenesis or peroxisomal β-oxidation manifest as severe neural disorders of the central nervous system. Abnormal peroxisomal metabolism is thought to be responsible for the clinical symptoms of these diseases, but their molecular pathogenesis remains to be elucidated. We performed lipidomic analysis to identify aberrant metabolites in fibroblasts from patients with Zellweger syndrome (ZS), acyl-CoA oxidase1 (AOx) deficiency, D-bifunctional protein (D-BP) and X-linked adrenoleukodystrophy (X-ALD), as well as in peroxisome-deficient Chinese hamster ovary cell mutants. In cells deficient in peroxisomal biogenesis, plasmenylethanolamine was remarkably reduced and phosphatidylethanolamine was increased. Marked accumulation of very-long-chain saturated fatty acid and monounsaturated fatty acids in phosphatidylcholine was observed in all mutant cells. Very-long-chain polyunsaturated fatty acid (VLC-PUFA) levels were significantly elevated, whilst phospholipids containing docosahexaenoic acid (DHA, C22:6n-3) were reduced in fibroblasts from patients with ZS, AOx deficiency, and D-BP deficiency, but not in fibroblasts from an X-ALD patient. Because patients with AOx deficiency suffer from more severe symptoms than those with X-ALD, accumulation of VLC-PUFA and/or reduction of DHA may be associated with the severity of peroxisomal diseases.     

Peritoneal injection of fucoidan suppresses the increase of plasma IgE induced by OVA-sensitization

We previously reported that fucoidan, a dietary fiber purified from seaweed, inhibited IgE production by B cells in vitro. In this study, we examined the effect of fucoidan on IgE production in vivo. The OVA-induced increase of plasma IgE was significantly suppressed when fucoidan was intraperitoneally, but not orally, administered prior to the first immunization with OVA. The production of IL-4 and IFN-γ in response to OVA in spleen cells isolated from OVA-sensitized mice treated with fucoidan in vivo was lower than that from mice treated without fucoidan. Moreover, the flow cytometric analysis and ELISpot assay revealed that the administration of fucoidan suppressed a number of IgE-expressing and IgE-secreting B cells, respectively. These results indicate that fucoidan inhibits the increase of plasma IgE through the suppression of IgE-producing B cell population, and the effect of fucoidan in vivo is crucially dependent on the route and timing of its administration.