Sporulation and Antibiotic Production by Bacillus subtilis Mutants Deficient in Intracellular Proteases

Erythropoietin (Ep) was isolated from the urine of patients with aplastic anemia [Yanagawa et al., J. Biol. Chem., 259, 2707 (1984)] and burst-promoting activity (BPA) was extensively purified from the residue obtained after removal of Ep. These erythropoietic factors were studied for their effcects on erythroid burst-colony formation of human peripheral blood mononuclear cells in methylcellulose cultures. Reddish bursts were formed with the addition of Ep alone. Addition of BPA not only elevated the number of bursts but also greatly reduced the amount of Ep required for burst formation. The presence of BPA alone in cultures did not permit bursts to form but did permit the growth of small colonies that did not contain hemoglobin (Hb). Addition of Ep to these small colonies led to the formation of erythroid bursts. Administration of Ep to the cultures could be delayed for 6 days without decreasing the number of bursts if the cultures were initiated in the presence of BPA; in the absence of BPA, the erythroid precursors (BUF-E) were rapidly lost if Ep was not provided at the start of the cultures. BPA produced larger bursts than those formed in the presence of Ep alone. Microassays of Hb in the bursts indicated that BPA increased the amonut of Hb per burst. This increase could not be entirely explained by the augumentation in cell number per burst but was partly ascribable to the increased amount of Hb per cell.     

In isolated human centrosomes,the associated kinases phosphorylate a specific subset of centrosomal proteins

Summary— Several studies have shown that kinases and phosphatases can interact with the centrosome during interphase and mitosis suggesting that centrosomal components might be the targets of these enzymes. The association of the cAMP-dependent protein kinase type II and the mitotic kinase p34^cdc2 with centrosomes from human lymphoblast cells has previously been shown (Keryer et al, 1993, Exp Cell Res 204, 230–240; Bailly et al, 1989, EMBO J 8, 3985–3995). In this paper we demonstrate that isolated centrosomes are able to phosphorylate a few number of centrosomal proteins (M_r 230–220000; 135000 and 50000) and also H1 histone. The phosphorylation of H1-histone is cell cycle dependent and modulated by phosphatases. The use of kinase and phosphatase inhibitors and the addition of the catalytic subunit of cAMP-dependent kinase or of cyclinB-p34^cdc2 kinase showed that both kinases phosphorylate the same centrosomal substrates. In addition two centrosomal proteins (M_r 100000 and 37000) were phosphorylated only by p34^cdc2 kinase. Although the low amount of centrosomal proteins precluded a full characterization of these substrates we discuss the identity of the major centrosomal phosphoproteins by comparison with proteins known to associate with microtubule-organizing centres or mitotic spindles. Our results raise also the intriguing possibility that the cAMP-dependent protein kinase could be regulated by the mitotic kinase at the entry of mitosis.     

铬黄圆痕叶蝉属的订正及圆痕叶蝉族巴西二新属二新种描记（半翅目：叶蝉科）

基于比较形态学对曾归入圆痕叶蝉亚科铬黄圆痕叶蝉属Chromagallia的8个有效种（C. saucia (St?l), C. flavofasciata (St?l), C. longistilata (Coelho & Dutra), C. carvalhoi Gon?alves et al., C. lamasi Gon?alves et al., C. lanceolata Gon?alves et al., C. paraguayensis Gon?alves et al.和C. zanolae Gon?alves et al.）进行了订正,明确了该属的范围仅限于具有黄斑的3个种（C. flavofasciata (St?l), C. longistilata (Coelho & Dutra) 和C. rodriguesoi sp. nov.）,对该属进行了重新描述,并修订了鉴别特征。此外,对模式种C. flavofasciata 进行了重新描记,首次提供了C. longistilata的雌性生殖器图,并作了描记。把曾归入铬黄圆痕叶蝉属Chromagallia具有红斑的6个种移出并新建了2个新属：Rubragallia 和Neorubragallia,其中Rubragallia 包括R. saucia (St?l) n. comb.和R. paraguayensis (Gon?alves et al.) n. comb., Neorubragallia包括N. lamasi (Gon?alves et al.) n. comb., N. lanceolata (Gon?alves et al.) n. comb., N. zanolae (Gon?alves et al.) n. comb., N. carvalhoi (Gon?alves et al.) n. comb. 和N. mervini sp. nov.。文中提供了3个属的分种检索表,并对不同种的分类地位及3个属的划分进行了讨论。     

Open and shut: Crystal structures of the dodecylmaltoside solubilized mechanosensitive channel of small conductance from Escherichia coli and Helicobacter pylori at 4.4 Å and 4.1 Å resolutions

The mechanosensitive channel of small conductance (MscS) contributes to the survival of bacteria during osmotic downshock by transiently opening large diameter pores for the efflux of cellular contents before the membrane ruptures. Two crystal structures of the Escherichia coli MscS are currently available, the wild type protein in a nonconducting state at 3.7 Å resolution (Bass et al., Science 2002; 298:1582–1587) and the Ala106Val variant in an open state at 3.45 Å resolution (Wang et al., Science 2008; 321:1179–1183). Both structures used protein solubilized in the detergent fos‐choline‐14. We report here crystal structures of MscS from E. coli and Helicobacter pylori solubilized in the detergent β‐dodecylmaltoside at resolutions of 4.4 and 4.2 Å, respectively. While the cytoplasmic domains are unchanged in these structures, distinct conformations of the transmembrane domains are observed. Intriguingly, β‐dodecylmaltoside solubilized wild type E. coli MscS adopts the open state structure of A106V E. coli MscS, while H. pylori MscS resembles the nonconducting state structure observed for fos‐choline‐14 solubilized E. coli MscS. These results highlight the sensitivity of membrane protein conformational equilibria to variations in detergent, crystallization conditions, and protein sequence.     

Improved Bile Acid-binding Ability of Soybean Glycinin A1a Polypeptide by the Introduction of a Bile Acid-binding Peptide (VAWWMY)

We have previously identified a potential bile acid-binding peptide sequence (VAWWMY) in acidic polypeptide A1a of the soybean glycinin A1aB1b subunit (Choi, S. K., et al., Biosci. Biotechnol. Biochem., 66, 2395–2401 (2002)). In this study, we introduced the nucleotide sequence encoding this peptide in the coding DNA which corresponds to amino acids between 251 and 256, and 282 and 287 into the A1a polypeptide by replacement to respectively give modified versions A1aM1 and A1aM2. A fluorescence analysis demonstrates that their bile acid-binding ability was improved compared to A1a. Moreover, modified proglycinin A1aB1b with the VAWWMY sequence at the same sites as those of A1aM1 and A1aM2 was judged to assume the correct conformation. These results suggest the possibility of developing transgenic crops to accumulate the modified glycinin.     

The pattern of neuropeptide F and RF-amide in two tapeworms

Two years ago the first platyhelminth regulatory peptide, neuropeptide F (NPF), was isolated from the tapeworm Moniezia expansa by Maule et al. (1991). NPF is a 39 amino acid peptide with a C terminal phenylalaninamide. NPF is the first platyhelminth neuropeptide to be sequenced fully. Preabsorption with NPF quenches the immunostaining with anti-FMRF-amide and anti-bovine PP (Halton et al. 1992). As the first authentic flatworm neuropeptide, the occurence and distribution of NPF along the whole flatworm line are under investigation. Both free-living and parasitic flatworms are being studied. So far NPF-immunoreactivity has been reported from three free- living flatworms (see Grahn et al., 1995) and from four parasitic flatworms (Marks et al., 1993).FMRF- and RF-amide immunoreactive (IR) nerve cells and fibres are common in the gull-tapeworm Diphyllobothrium dendriticum. In order to test whether the patterns for NPF- and RF-immunoreactivity co- localize in the gull-tapeworm, immunostaining with anti-NPF and anti-RF were performed. To broaden the study, adult Proteochepalus exiguus from the intestine of whitefish were included in the experiment.The study was performed on whole mounts of skinned worms (Gustafsson, 1991). Anti-NPF was used in concentrations 1:500 and 1:1000. Controls included liquid phase absorption with the homologous antigen (1000 ng ml^–1).In D. dendriticum NPF-immunoreactivity occurs in nerve cells and varicose nerve fibres of larval and adult worms. The NPF-IR cell bodies are more common in the peripheral nerve cords than along the main nerve cords, which contain nerve fibres with large varicosities. The cell bodies in the PNS are often triangular in shape. Immediately beneath the tegumental surface a thin NPF-IR nerve fibre is observed. As to the co-localization of NPF and RF nothing definite can be said but the general pattern seems tobe the same. In the brain commissure of D. dendriticum one large ganglion cell stains with both antisera, indicating coexistence.In P. exiguus NPF- and RF-immunoreactivity was observed in the two main nerve cords situated laterally and in the pairs of thin dorsal and ventral longitudinal nerve cords. Numerous transverse commissures connect the longitudinal cords forming an orthogonal pattern. The cell bodies along the nerve cords are multipolar. Thin projections extend from the main nerve cords to the surface of the worm. The main nerve cords are lined with NPF-and RF-IR cell bodies. The general staining patterns of NPF and RF are very similar.     

Synthesis and Some Properties of UDP-(1)fructose

UDP-(1)fructose was synthesized essentially by the method of Michelson or Roseman et al. The product obtained was much more stable to acid than UDP-fructose isolated from Jerusalem artichoke tubers by Umemura et al.⁷⁾ and UDP-glucose. Hydrolysis time curves of UDP-(1)fructose and fructose-1-phosphate in 0.01N HGl and 0.1N HCl both at 100°C are presented. It was concluded from these curves that UDP-(1)fructose was first hydrolyzed into UMP and fructose-1-phosphate, and then fructose-1-phosphate was hydrolyzed more slowly into free fructose and inorganic phosphate.     

Influence of the last amino acid in the nascent peptide on EF-Tu during decoding

The last two amino acids of the nascent peptide at the ribosomal P-site influence the efficiency of termination readthrough at the stop codon UGA (Mottagui-Tabar et al (1994) EMBO J 13, 249–257; Björnsson et al (1996) EMBO J 15, 1696–1704). Here we analyze this effect on readthrough by wild type or a UGA suppressor form (Su9) of tRNA^Trp by varying the codons at positions −1 and −2 at the 5′ side of UGA. Strains with wild-type or mutant (ArBr) forms of elongation factor Tu (EF-Tu) were analyzed (Vijgenboom et al (1985) EMBO J 4, 1049–1052). The effect on readthrough by changing these −1 and −2 codons is different on the two forms of tRNA^Trp and is also dependent on the structure of EF-Tu. Readthrough by the tRNA^Trp-derived suppressor, but not wild-type tRNA^Trp, is sensitive to the van der Waals volume of the last amino acid in the nascent peptide. Together with mutant EF-Tu, both forms of tRNA^Trp are sensitive. The data suggest that the C-terminal amino acid in the nascent peptide is in a functional interaction with the EF-Tu ternary complex. This interaction is changed by mutation in tRNA^Trp at position 24 or in EF-Tu at position 375. No indication of a changed interaction between the mutant EF-Tu and the penultimate amino acid could be found. Mutant forms of RF2 (Mikuni et al (1991) Biochimie 73, 1509–1516) and ribosomal proteins S4 and S12 (Fáxen et al (1988) J Bacteriol 170, 3756–3760) were found not to be altered in sensitivity to the last two amino acids in the nascent peptide.     

Structural studies on the tri- and tetrasaccharides isolated from porcine intestinal heparin and characterization of heparinase/heparitinases using them as substrates

We prepared a series of oligosaccharides from porcine intestinalheparin after extensive digestion with a mixture of Flavobacteriumheparinase as well as heparitinases I and II. Previously, wereported the structures of the two glycoserines derived fromthe carbohydrate-protein linkage region [Sugahara et al., J.Biol. Chem., 267, 1528–1533 (1992)] and three tetrasaccharidesderived from the antithrombin III-binding site [Yamada et al.,J. Biol. Chem., 268, 4780–4787 (1993)]. In this study,we determined the structures of 10 other tetrasaccharides anda trisaccharide by enzymatic digestion, fast atom bombardmentmass spectrometry and 500-MHz ¹H NMR spectroscopy. These tetrasaccharidesshare the common disulphated structure,     

Seeing the forest beyond the trees

In a recent paper (Mitchard et al. 2014, Global Ecology and Biogeography, 23 , 935–946) a new map of forest biomass based on a geostatistical model of field data for the Amazon (and surrounding forests) was presented and contrasted with two earlier maps based on remote‐sensing data Saatchi et al. (2011; RS1) and Baccini et al. (2012; RS2). Mitchard et al. concluded that both the earlier remote‐sensing based maps were incorrect because they did not conform to Mitchard et al. interpretation of the field‐based results. In making their case, however, they misrepresented the fundamental nature of primary field and remote‐sensing data and committed critical errors in their assumptions about the accuracy of research plots, the interpolation methodology and the statistical analysis. By ignoring the large uncertainty associated with ground estimates of biomass and the significant under‐sampling and spatial bias of research plots, Mitchard et al. reported erroneous trends and artificial patterns of biomass over Amazonia. Because of these misrepresentations and methodological flaws, we find their critique of the satellite‐derived maps to be invalid.     

Control of renal and extrarenal salt and water excretion by plasma angiotensin II in the kelp gull (Larus dominicanus)

Summary The osmoregulatory effects of intravenously (i.v.) administered angiotensin II (AII) at dose rates of 5, 15 and 45 ng · kg^–1 · min^–1 were examined in kelp gulls utilizing salt glands and/or kidneys as excretory organs.In birds given i.v. infusion of 1200 mOsmolal NaCl at 0.3 ml · min^–1 and utilizing only the salt glands to excrete the load, infusion of AII for 30 min consistently inhibited salt gland function in a dose-dependent manner.In birds given i.v. infusion of 500 mOsmolal NaCl at 0.72 ml · min^–1 and utilizing both salt glands and kidneys to excrete the load, each dose of AII given for 2 h inhibited salt gland function but stimulated the kidney, so that the overall outputs of salt and water were enhanced and showed significant (2P<0.01) positive correlations with plasma AII.In birds given i.v. infusion of 200 mOsmolal glucose at 0.5 ml · min^–1 and utilizing only the kidneys to excrete the load, low doses of AII (5 and 15 ng · kg^–1 · min^–1) caused renal salt and water retention, whereas a high dose (45 ng · kg^–1 · min^–1) stimulated salt and water output.The actions of plasma AII in kelp gulls support the concept that this hormone plays a vital role in avian osmoregulation, having effects on both salt gland and kidney function. Elevation of plasma AII consistently inhibits actively secreting salt glands, but its effects upon renal excretion depend primarily on the osmotic status as well as on the plasma AII concentration. In conditions of salt and volume loading doses of AII stimulate sodium and water excretion. With salt and volume depletion, the action of AII is bi-phasic with low doses promoting renal sodium and water retention but high circulating levels causing natriuresis and diuresis.     

Macrosiagon deuvei n. sp. (Coleoptera: Ripiphoridae) from the French Eocene amber

Macrosiagon deuvei n. sp., the second fossil representative of this extant genus of Ripiphoridae: Ripiphorinae: Macrosiagonini is described from the lowermost Eocene amber of Oise (France). The new species is compared with the extant species of the genus. Taxonomic position of other two fossil representatives of the family described from France by Perrichot et al. (2004) is discussed. The genus Paleoripiphorus Perrichot et al. 2004 is tentatively transferred from Ripiphorinae to Ripidiinae.     

Genomic Characterization of the First Parechovirus in Bats

<正>Dear Editor,Parechoviruses (PeVs) are non-enveloped, spherical viruses of genus Parechovirus and family Picornaviridae.Within the capsid is a naked monopartite, linear, singlestranded positive-sense RNA genome of 7.3 kb, comprising a single long open reading frame (ORF) encoding a     

Identification of the yolk receptor protein in oocytes of Nereis virens (Annelida,Polychaeta) and comparison with the locust vitellogenin receptor

Summary In oviparous animals large amounts of yolk proteins of extraovarian origin are accumulated by developing oocytes during vitellogenesis. The yolk protein precursors, the vitellogenins (VTG), are transported into the oocytes by receptor-mediated endocytosis. In oocytes of the polychaetous annelid, Nereis virens, the receptor protein for VTG was visualized by ligand blotting studies as a protein with an apparent molecular mass of 190 kDa under non-reducing conditions. Anti-Locusta VTG receptor antibodies recognize the Nereis VTG receptor protein. The Nereis VTG receptor protein binds Locusta and Schistocerca VTG; the VTG receptor proteins of both locust species bind the Nereis vitellin. These results indicate the conservation of structural elements important for internalization of VTG.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane-sulphonic acid - HBS HEPES-buffered saline - PAP peroxidase-anti-peroxidase - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - TRIS, TBS TRIS-buffered saline - VT vitellin - VTG vitellogenin     

Randomization inference with general interference and censoring

Interference occurs between individuals when the treatment (or exposure) of one individual affects the outcome of another individual. Previous work on causal inference methods in the presence of interference has focused on the setting where it is a priori assumed that there is “partial interference,” in the sense that individuals can be partitioned into groups wherein there is no interference between individuals in different groups. Bowers et al. (2012, Political Anal, 21, 97–124) and Bowers et al. (2016, Political Anal, 24, 395–403) consider randomization-based inferential methods that allow for more general interference structures in the context of randomized experiments. In this paper, extensions of Bowers et al. that allow for failure time outcomes subject to right censoring are proposed. Permitting right-censored outcomes is challenging because standard randomization-based tests of the null hypothesis of no treatment effect assume that whether an individual is censored does not depend on treatment. The proposed extension of Bowers et al. to allow for censoring entails adapting the method of Wang et al. (2010, Biostatistics, 11, 676–692) for two-sample survival comparisons in the presence of unequal censoring. The methods are examined via simulation studies and utilized to assess the effects of cholera vaccination in an individually randomized trial of 73 000 children and women in Matlab, Bangladesh.     

Reverse-phase protein microarray highlights HER2 signaling activation in immunohistochemistry/FISH/HER2-negative breast cancers

Evaluation of: Wulfkuhle JD, Berg D, Wolff C et al. Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping. Clin. Cancer Res. 18(23), 6426–6435 (2012).

Exhaustive characterization and mapping of pivotal molecules and downstream effectors deregulated in breast cancer is of fundamental clinical value to define the most effective therapy. Wulfkuhle et al. applied reverse-phase protein microarray, a highly sensitive immunoassay able to perform quantitative and multiplexed analysis of total and/or modified cellular proteins, to assess protein levels and activation/phosphorylation status of the HER family (EGFR, HER2, HER3) and downstream signaling molecules in HER2⁺ and HER2^- breast cancers. The research was performed using laser capture microdissected tumor epithelial cells from frozen samples and formalin-fixed paraffin embedded specimens, which were also analyzed by immunohistochemistry (IHC) and FISH. This study identified a subgroup of IHC/FISH/HER2^- patients with HER2 activation/phosphorylation levels comparable with those obtained from IHC/FISH/HER2⁺ tumors. HER2 signaling activation was independent from total HER2 expression and involved HER3 and EGFR activation. These findings indicate that molecular characterization by reverse-phase protein microarray of HER2 and its partners/effectors in the signaling cascade enables the identification of a subgroup of IHC/FISH/HER2^- patients showing HER2 signaling activation. These patients, currently excluded from targeted therapy administration, could potentially benefit from this and it could improve prognosis and survival.     

Bioturbation by Nereis sp. and its effects on the phosphate flux across the sediment-water interface in the Palmones River estuary

The effects of Nereis sp. on the flux of dissolved phosphate across the sediment-water interface has been studied in Palmones River estuary using benthic flux-chambers and intact cores. Diffusive fluxes of phosphate were calculated from pore water gradient concentration and compared with those obtained from benthic chambers experiments. The high abundance of Nereis in the upper sediment layers appears to play an important part in the dissolved oxygen profiles in the overlying water, but had no effect on the redox potential. A negative relationship was found between the Nereis abundance and the phosphate gradient; this gradient ranged between 40 µmol 1^–1 cm^–1 with 340 Nereis individuals m^–2 and 20 µmol 1^–1 cm^–1 with 900 Nereis individuals m^–2. The ratio of the in situ flux to the flux gradient concentration for dissolved phosphate increased with the abundance of Nereis (from 1.7 at low abundance to 5.8 at high abundance).     

Prediction studies on number of teratocytes and eggs of Microplitis rufiventris Kok. (Hym., Braconidae) parasitoid in superparasitized host larvae

Microplitis rufiventris Kok. (Hym., Braconidae) is an internal solitary parasitoid of many noctuid caterpillars including the cotton leafworm Spodoptera littoralis (Boisd.) ( Kokujev 1914 ; Hammad et al. 1965 ; Gerling 1969 ), the lesser cotton worm S. exigua Hbn. ( Meier 1929 ; Thompson 1946 ; El-Minshawy 1963 ); S. latebrosa Lederer ( Hammad et al. 1965 ) and American bollworm Heliothis armigera Hbn. ( Meier 1929 ; Ibrahim & Tawfik 1975 ). When the egg of M. rufiventris hatches in its host, S. littoralis, spherical cells from the serosa that envelope the parasitoid embryo are released into the host’s haemolymph. Approximately 400 cells are liberated from an egg of the parasitoid. These cells increase in size, reaching a maximum average diameter of 137 μm at the completion of parasitoid development ( Khafagi 1997 ). These cells are most frequently called ‘teratocytes’ ( — 1968 , 1971; Vinson 1970 ). It is reported that the presence of large numbers of the teratocytes is indicative of superparasitism but their number does not give an indication to the exact number of parasitoid eggs from which the cells have been derived ( —, — Khafagi et al. 1998 ) . Therefore, it was of interest to initiate prediction studies on egg and teratocyte numbers in superparasitized host larvae.     

Does host plant richness explain diversity of ectomycorrhizal fungi? Re‐evaluation of Gao et al. (2013) data sets reveals sampling effects

The generally positive relationship between biodiversity of groups of directly or indirectly interacting organisms is one of the most important ecological concepts (Gaston, 2000 Nature, 405 , 220–227; Scherber C, Eisenhauer N, Weisser WW et al., 2010 Nature, 468 , 553–556). In a recent issue of Molecular Ecology, Gao C, Shi N‐N, Liu Y‐X et al. (2013: 22 , 3403–3414) reported that the richness of plants and ectomycorrhizal fungi is positively correlated both at local and at global scales. Here, we challenge these findings by re‐analysis of data and ascribe the reported results to sampling effect and poor data compilation.     

Trifluoroethanol‐containing RP‐HPLC mobile phases for the separation of transmembrane peptides human glycophorin‐A,integrin alpha‐1, and p24: analysis and prevention of potential side reactions due to formic acid

Reversed‐phase high‐pressure liquid chromatography analysis and purification of three hydrophobic, aggregation‐prone peptides, composed mainly of the transmembrane (TM) sequence, were performed using elution systems containing 2,2,2‐trifluoroethanol (TFE). The addition of 10–16% TFE to a common mobile phase, such as a water/acetonitrile/propanol (PrOH) or a water/PrOH/formic acid system, markedly improved the chromatographic separation of these peptides. The superior performance of TFE‐containing systems in separating peptides over water/PrOH/formic acid systems [Bollhagen R. et al., J. Chromatogr. A, 1995; 711 : 181–186.] clearly demonstrated that adding TFE to the mobile phase is one of best methods for TM‐peptide purification. Characterization of the potential side reactions using MALDI and ESI‐LIT/Orbitrap mass spectrometry indicated that prolonged incubation of peptides in a mixture of TFE–formic acid possibly induces O‐formylation of the Ser residue and N‐formylation of the N‐terminus of peptides. The conditions for selective removal of the formyl groups from TM peptides were also screened. We believe that these results will expand our ability to analyze and prepare hydrophobic, aggregation‐prone TM peptides and proteins. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.