Effect of photoperiod on winter and summer diapause of the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Tortricidae)

To understand the geographical differences between diapause systems and synchronization of adult occurrence in the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Tortricidae), we examined the timing of winter diapause termination and intensity of summer diapause using univoltine and potentially bivoltine individuals in Iwate, Japan. In laboratory rearing experiments of mature larvae maintained at constant temperature (20 °C), winter diapause intensity weakened by January without photoperiodic responses. Meanwhile, summer diapause was maintained by the long day length and presumably terminated with the photoperiodic transition from long to short day length. The intensity of summer diapause was stronger for cocoons that transitioned from a 16 h light to 8 h dark (LD 16:8) to a LD 15:9 photoperiod than for those that transitioned from LD 15:9 to LD 14:10. These results suggest that populations distributed in relatively low-latitude areas, with partly or potentially bivoltine individuals, would have a weaker summer diapause or none at all. Moreover, sexual differences in the number of days to emergence were not detected when individuals experienced a photoperiodic transition from long to short day length, suggesting that the summer diapause system may function to synchronize the emergence of males and females in the population examined.     

Mass Isotopomer Analysis of Metabolically Labeled Nucleotide Sugars and N- and O-Glycans for Tracing Nucleotide Sugar Metabolisms

Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using ¹³C₆-glucose and ¹³C₂-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS.Metabolic labeling of cultured cells with ¹³C₆-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a ¹³C₆-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells.Using ¹³C₂-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism.Protein glycosylation, which is the most abundant post-translational modification, has important roles in many biological processes by modulating conformation and stability, whereas its dysregulation is associated with various diseases such as diabetes and cancer (1, 2). Glycosylation is regulated by various factors including glucose metabolism, the availability and localization of nucleotide sugars, and the expression and localization of glycosyltransferases (3, 4). Thus, ideally all of these components should be considered when detecting changes in a dynamic fashion; namely, it is necessary not only to take a snapshot but also to make movies of the dynamic changes in glycan metabolism.Glucose is used by living cells as an energy source via the glycolytic pathway as well as a carbon source for various metabolites including nucleotide sugars (e.g. UDP-GlcNAc and CMP-NeuAc). These nucleotide sugars are transported into the Golgi apparatus, and added to various glycans on proteins. UDP-GlcNAc is the donor substrate for N-acetylglucosaminyl (GlcNAc)¹ transferases; alternatively, it is used in the cytosol for O-GlcNAc modification (i.e. O-GlcNAcylation) of intracellular proteins (5). The UDP-GlcNAc synthetic pathway is complex as it is a converging point of glucose, nucleotide, fatty acid and amino acid metabolic pathways. Thus, the metabolic flow of glucose modulates the branching patterns of N-glycans via UDP-GlcNAc concentrations because many of the key GlcNAc transferases that determine the branching patterns have widely different K_m values for UDP-GlcNAc ranging from 0.04 mm to 11 mm (6, 7). Indeed, it was demonstrated that the branching formation of N-glycans in T cells is stimulated by the supply from the hexosamine pathway, whereby it regulates autoimmune reactions promoted by T cells (8).UDP-GlcNAc is also used for the synthesis of CMP-NeuAc, the donor substrate for sialyltransferases (9). The CMP-NeuAc concentration is controlled by the feedback inhibition of UDP-GlcNAc epimerase/ManNAc kinase by the final product CMP-NeuAc, and hence a high CMP-NeuAc level reduces metabolic flow in CMP-NeuAc de novo synthesis (10). However, there is still only limited information about how the levels of nucleotide sugars dynamically change in response to the environmental cues, and how such changes are reflected in the glycosylation of proteins.Stable isotope labeling is a promising approach to quantify metabolic changes in response to external cues (11, 12). For example, the use of nuclear magnetic resonance to obtain isotopomer signals of metabolically labeled molecules has been applied to trace the flux in glycolysis and fatty acid metabolism (13). An approach based on the mass isotopomers of labeled metabolites with ¹³C₆-glucose has been developed to monitor the UDP-GlcNAc synthetic pathway (13–15). The method based on the labeling ratio of each metabolite related to UDP-GlcNAc synthesis has clarified the contribution of each metabolic pathway (14). Moseley reported a novel deconvolution method for modeling UDP-GlcNAc mass isotopomers (15).Previous studies into the use of nucleotide sugars in glycosylation have relied on the specific detection of metabolically radiolabeled glycans (16). It is possible not only to deduce the glycan structures but also to trace their relative contributions to glycan synthesis without MS. On the other hand, mass isotopomer analysis of glycans labeled with stable isotope provides the ratios of labeled versus unlabeled molecules from MS spectra and structural details of the glycans. However, there are only a limited number of publications reporting the application of stable isotope labeling of glycans for monitoring the dynamics of glycans (17). To date, there have been no reports describing a systematic method for tracing cellular GlcNAc biosynthesis and use based on mass isotopomer analysis.The aim of this study was to extend our knowledge of the synthesis and metabolism of UDP-GlcNAc as well as its use in the synthesis of CMP-NeuAc, N- and O-glycans. We recently developed a conventional HPLC method for simultaneous determination of nucleotide sugars including unstable CMP-NeuAc (18). We first established an LC-MS method for isotopomer analysis of ¹³C₆-glucose labeled nucleotide sugars for tracing UDP-GlcNAc metabolism from synthesis to use, because previous methods were not suitable for estimating UDP-GlcNAc use in CMP-NeuAc de novo synthesis (15). We also established a method for isotopomer analysis of labeled N- and O-glycan to monitor the metabolic flow of hexosamine into glycans. Using these two methods, we demonstrated the differences in the use of hexosamines between hepatoma and pancreatic insulinoma cell lines. Our approach may be useful for identifying a metabolic “bottleneck” that governs the turnover speed and patterns of cellular glycosylation, which may be relevant for various applications including glycoprotein engineering and discovery of disease biomarkers.     

The short‐ and long‐term fitness consequences of natal dispersal in a wild bird population

Dispersal is a key process in population and evolutionary ecology. Individual decisions are affected by fitness consequences of dispersal, but these are difficult to measure in wild populations. A long‐term dataset on a geographically closed bird population, the Mauritius kestrel, offers a rare opportunity to explore fitness consequences. Females dispersed further when the availability of local breeding sites was limited, whereas male dispersal correlated with phenotypic traits. Female but not male fitness was lower when they dispersed longer distances compared to settling close to home. These results suggest a cost of dispersal in females. We found evidence of both short‐ and long‐term fitness consequences of natal dispersal in females, including reduced fecundity in early life and more rapid aging in later life. Taken together, our results indicate that dispersal in early life might shape life history strategies in wild populations.     

ERCC1 function in nuclear excision and interstrand crosslink repair pathways is mediated exclusively by the ERCC1-202 isoform

ERCC1 (excision repair cross-complementation group 1) plays essential roles in the removal of DNA intrastrand crosslinks by nucleotide excision repair, and that of DNA interstrand crosslinks by the Fanconi anemia (FA) pathway and homology-directed repair processes (HDR). The function of ERCC1 thus impacts on the DNA damage response (DDR), particularly in anticancer therapy when DNA damaging agents are employed. ERCC1 expression has been proposed as a predictive biomarker of the response to platinum-based therapy. However, the assessment of ERCC1 expression in clinical samples is complicated by the existence of 4 functionally distinct protein isoforms, which differently impact on DDR. Here, we explored the functional competence of each ERCC1 protein isoform and obtained evidence that the 202 isoform is the sole one endowed with ERCC1 activity in DNA repair pathways. The ERCC1 isoform 202 interacts with RPA, XPA, and XPF, and XPF stability requires expression of the ERCC1 202 isoform (but none of the 3 others). ERCC1-deficient non-small cell lung cancer cells show abnormal mitosis, a phenotype reminiscent of the FA phenotype that can be rescued by isoform 202 only. Finally, we could not observe any dominant-negative interaction between ERCC1 isoforms. These data suggest that the selective assessment of the ERCC1 isoform 202 in clinical samples should accurately reflect the DDR-related activity of the gene and hence constitute a useful biomarker for customizing anticancer therapies.     

Rhein,a Natural Anthraquinone Derivative,Attenuates the Activation of Pancreatic Stellate Cells and Ameliorates Pancreatic Fibrosis in Mice with Experimental Chronic Pancreatitis

Pancreatic fibrosis, a prominent histopathological feature of chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma, is essentially a dynamic process that leads to irreversible scarring of parenchymal tissues of the pancreas. Though the exact mechanisms of its initiation and development are poorly understood, recent studies suggested that the activation of pancreatic stellate cells (PSCs) plays a critical role in eliciting such active course of fibrogenesis. Anthraquinone compounds possess anti-inflammatory bioactivities whereas its natural derivative rhein has been shown to effectively reduce tissue edema and free-radical production in rat models of inflammatory conditions. Apart from its anti-inflammatory properties, rhein actually exerts strong anti-fibrotic effects in our current in-vivo and in-vitro experiments. In the mouse model of cerulein-induced CP, prolonged administration of rhein at 50 mg/kg/day significantly decreased immunoreactivities of the principal fibrotic activators alpha-smooth muscle actin (α-SMA) and transforming growth factor-beta (TGF-β) on pancreatic sections implicating the activation of PSCs, which is the central tread to fibrogenesis, was attenuated. Consequently, the overwhelmed deposition of extracellular matrix proteins fibronectin 1 (FN1) and type I collagen (COL I-α1) in exocrine parenchyma was found accordingly reduced. In addition, the expression levels of sonic hedgehog (SHH), which plays important roles in molecular modulation of various fibrotic processes, and its immediate effector GLI1 in pancreatic tissues were positively correlated to the degree of cerulein-induced fibrosis. Such up-regulation of SHH signaling was restrained in rhein-treated CP mice. In cultured PSCs, we demonstrated that the expression levels of TGF-β-stimulated fibrogenic markers including α-SMA, FN1 and COL I-α1 as well as SHH were all notably suppressed by the application of rhein at 10 μM. The present study firstly reported that rhein attenuates PSC activation and suppresses SHH/GLI1 signaling in pancreatic fibrosis. With strong anti-fibrotic effects provided, rhein can be a potential remedy for fibrotic and/or PSC-related pathologies in the pancreas.     

Simultaneous increases in the levels of compatible solutes by cost-effective cultivation of Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803, a cyanobacterium widely used for basic research, is often cultivated in a synthetic medium, BG-11, in the presence of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) or 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid buffer. Owing to the high cost of HEPES buffer (96.9% of the total cost of BG-11 medium), the biotechnological application of BG-11 is limited. In this study, we cultured Synechocystis sp. PCC 6803 cells in BG-11 medium without HEPES buffer and examined the effects on the primary metabolism. Synechocystis sp. PCC 6803 cells could grow in BG-11 medium without HEPES buffer after adjusting for nitrogen sources and light intensity; the production rate reached 0.54 g cell dry weight·L⁻¹·day⁻¹, exceeding that of commercial cyanobacteria and Synechocystis sp. PCC 6803 cells cultivated under other conditions. The exclusion of HEPES buffer markedly altered the metabolites in the central carbon metabolism; particularly, the levels of compatible solutes, such as sucrose, glucosylglycerol, and glutamate were increased. Although the accumulation of sucrose and glucosylglycerol under high salt conditions is antagonistic to each other, these metabolites accumulated simultaneously in cells grown in the cost-effective medium. Because these metabolites are used in industrial feedstocks, our results reveal the importance of medium composition for the production of metabolites using cyanobacteria.     

Fibronectin‐binding protein,FbpA, is the adhesin responsible for pathogenesis of Listeria monocytogenes infection

The role of fibronectin binding protein A (FbpA) in Listeria monocytogenes infection and its pathogenesis were studied in vivo and in vitro by constructing a fbpA‐deficient mutant of L. monocytogenes (ΔfbpA). In vivo, ΔfbpA was less pathogenic in mutant mice than was wild‐type L. monocytogenes. FbpA did not affect the amounts of various virulence‐determining factors, including internalin B and listeriolysin O. However, adherence to, and invasion of, mouse hepatocytes by the ΔfbpA mutant were reduced. In contrast, adherence to, but not invasion of, the ΔfbpA mutant to macrophages was attenuated. Fibronectin contributed to the efficient adherence and invasion of wild‐type L. monocytogenes, but not to those of the ΔfbpA mutant. Attenuation of adhesion and uptake of the ΔfbpA mutant were reversed by overexpression of FbpA in it. FbpA was not involved in intracellular growth, autophagy induction or actin tail formation. Thus, the present findings clearly show that FbpA acts as an important adhesion molecule of L. monocytogenes, especially regarding hepatocytes, without modulating the expression of other virulence factors that have been implicated in the pathogenesis of L. monocytogenes infection.