bluuebunny/crossref_metadata_2025 huggingface dataset:
84 SUCCESSFUL CRYOPRESERVATION OF PORCINE EMBRYOS BY THE METAL MESH VITRIFICATION (MMV) METHOD
Authors: Fujino Y. , Nakamura Y.
Published: December 2004

The present study was designed to generate piglets from porcine embryos by the metal mesh vitrification (MMV) method. Prepuberal gilts were administered eCG and hCG and were artificially inseminated. Morulae and expanding blastocysts (diameter = approximately 200 μm) were collected at 144 h and 168 h after hCG injection, respectively. The metal mesh and the plastic plate (control) were used as sample containers for ultrarapid vitrification. The metal mesh (75 μm stainless steel mesh) was 1.5 mm wide and 10 mm long, and the 3 mm at the end of the mesh was bent at a right angle. Plastic plates, made from 0.25 mL plastic straws, were the same size and form as the metal mesh. Embryos were equilibrated with 7.5% ethylene glycol (EG) + 7.5% DMSO + 10% fetal bovine serum (FBS) in PBS for 5 min, followed by exposure to 15% EG + 15% DMSO + 0.6 M trehalose + 10% FBS in PBS for 1 min. Embryos were picked up on the metal mesh or loaded onto plastic plates with minimum volume of the solution, and then plunged into liquid nitrogen. Sample containers were placed in 1.8-mL cryotubes and stored in liquid nitrogen. Warming and dilution were performed by moving the container from liquid nitrogen into 0.5 M trehalose + 10% FBS in PBS at 37°C for 5 min. Embryos were rinsed twice in 4 mg/mL BSA + 10% FBS in NCSU37 (mNCSU37) for 5 min. Survival of the vitrified embryos was assessed after culture in mNCSU37 for 24 h (expanding blastocysts) or 48 h (morulae), and those that survived to fully expanded, hatching or hatched blastocysts were scored as viable. The vitrified warmed embryos were transferred surgically to recipient gilts. Experiment 1: The survival rates of expanding blastocysts vitrified by MMV or the control method were compared. The rate by MMV was significantly higher (84%; P < 0.01 by χ2 test) than that of the control (53%). Experiment 2: The developmental stage (morula or expanding blastocyst) suitable for vitrification was examined. Survival of expanding blastocysts was significantly higher (84%; P < 0.05) than that of morulae (55%). Experiment 3: Expanding blastocysts were vitrified by MMV, warmed, and transferred into recipients (20 embryos per recipient). Eight of 10 recipients were found to be pregnant. Seven recipients farrowed a total of 37 live and 1 stillborn piglets. The other recipient miscarried and farrowed 4 stillborn piglets. These results showed that the viability of vitrified warmed porcine embryos was affected by the material of the sample container (Experiment 1) and also by the developmental stage (Experiment 2). Since the transferred embryos showed an excellent ability to develope into piglets (Experiment 3), the MMV method developed in the present study seems to be an effective preservation method for porcine embryos.
90 PRESERVATION OF HERITAGE LIVESTOCK BREEDS: INTEGRATED PROGRAM TO CRYOPRESERVE GERMPLASM FROM TENNESSEE MYOTONIC GOATS
Authors: Matsas D. , Huntress V. , Levine H. , Ayres S. , Amini J. , Duby R. , Borden P. , Saperstein G. , Overstrom E.
Published: December 2004

The genetic diversity of common commercial livestock has diminished precipitously due to intensive selection by agribusiness. Rare and/or heritage breeds of domestic livestock are thought to be of low commercial value, yet such breeds contain unique genes that impart valuable traits including disease and parasite resistance, efficient feed conversion, high fecundity, and unique food and fiber qualities. These irreplaceable genetics can be preserved by the establishment of a comprehensive germplasm preservation program whereby the complete genetics of a given unimproved breed can be reestablished in a single generation as needed. We describe and demonstrate here the successful preservation of germplasm from the Tennessee Myotonic (TM) goat breed, and the resulting initiation of a comprehensive germplasm cryolibrary of heritage livestock breeds. Cycling mature TM does (n = 18) were synchronized (PGF2α, 7.5–10 mg i.m.), superovulated (FSH, 50–40–30 mg bid i.m., decreasing over 3 days), and bred by natural service. Embryos were collected surgically from the uterus on Day 7 and good-excellent grade embryos were cryopreserved using a conventional multi-step freezing protocol and stored in LN2. Semen from mature TM bucks (n = 15) was collected biweekly (AV and teaser) and processed using a conventional slow cooling/LN2 plunge protocol. For each animal, a primary cell culture, derived from skin biopsy, was propagated (3 passages) and frozen, and blood (serum, plasma) was collected and cryobanked. A total of 242 embryos, representing 25 sire/dam pedigrees, and 1140 straws of semen were cryobanked. Embryo viability was confirmed by surgical transfer of thawed embryos (2 embryos into each of 2 does) resulting in the birth of 1 healthy buck. Semen viability was assessed by microscopy (30–50% motility; 10–55% live by fluorescence staining) and by AI with frozen-thawed semen (pregnancies confirmed by ultrasound). This report documents and validates the utility of contemporaneous cryopreservation methods to successfully establish a cryolibrary of germplasm from rare breeds of livestock. This strategy affords long term safe storage of viable germplasm with the capability for rapid and accurate reestablishment of the breed genetics if/when needed. Moreover, such storage provides material for assessment of quantitative genetic diversity and for testing of breed disease susceptibility/resistance should that be warranted in the future.
This work was supported by and in partnership with SVF Foundation.
The diabetes-linked transcription factor PAX4 promotes β-cell proliferation and survival in rat and human islets
Authors: Brun Thierry , Franklin Isobel , St-Onge Luc , Biason-Lauber Anna , Schoenle Eugene J. , Wollheim Claes B. , Gauthier Benoit R.
Published: December 2004

The mechanism by which the β-cell transcription factor Pax4 influences cell function/mass was studied in rat and human islets of Langerhans. Pax4 transcripts were detected in adult rat islets, and levels were induced by the mitogens activin A and betacellulin. Wortmannin suppressed betacellulin-induced Pax4 expression, implicating the phosphatidylinositol 3-kinase signaling pathway. Adenoviral overexpression of Pax4 caused a 3.5-fold increase in β-cell proliferation with a concomitant 1.9-, 4-, and 5-fold increase in Bcl-xL (antiapoptotic), c-myc, and Id2 mRNA levels, respectively. Accordingly, Pax4 transactivated the Bcl-xL and c-myc promoters, whereas its diabetes-linked mutant was less efficient. Bcl-xL activity resulted in altered mitochondrial calcium levels and ATP production, explaining impaired glucose-induced insulin secretion in transduced islets. Infection of human islets with an inducible adenoviral Pax4 construct caused proliferation and protection against cytokine-evoked apoptosis, whereas the mutant was less effective. We propose that Pax4 is implicated in β-cell plasticity through the activation of c-myc and potentially protected from apoptosis through Bcl-xL gene expression.
Processive capping by formin suggests a force-driven mechanism of actin polymerization
Authors: Kozlov Michael M. , Bershadsky Alexander D.
Published: December 2004

Regulation of actin polymerization is essential for cell functioning. Here, we predict a novel phenomenon—the force-driven polymerization of actin filaments mediated by proteins of the formin family. Formins localize to the barbed ends of actin filaments, but, in contrast to the standard capping proteins, allow for actin polymerization in the barbed direction. First, we show that the mechanism of such “leaky capping” can be understood in terms of the elasticity of the formin molecules. Second, we demonstrate that if a pulling force acts on the filament end via the leaky cap, the elastic stresses can drive actin polymerization. We estimate that a moderate pulling force of ∼3.4 pN is sufficient to reduce the critical actin concentration required for barbed end polymerization by an order of magnitude. Furthermore, the pulling force increases the polymerization rate. The suggested mechanism of force-driven polymerization could be a key element in a variety of cellular mechanosensing devices.
Premarital Sex, Procreation, and HIV Risk in Nigeria
Authors: Smith Daniel Jordan
Published: December 2004

In Nigeria, research has documented a significant disparity between people's knowledge of HIV/AIDS and the extent to which they act to protect themselves. Data from a survey of 863 adolescent and unmarried young adults, in‐depth interviews, and participant observation are combined in this study to explain some of this discrepancy. Young migrants appear to make sexual and contraceptive decisions in relation to gender norms and attitudes concerning procreation at least as much as in relation to fear of disease. Assessments of current and potential partners, choices about whether or not to have sex, and decisions about whether or not to use condoms are influenced by shared cultural values regarding the importance of parenthood. These cultural conceptions of parenthood are gender‐specific and put men and women in different negotiating positions with regard to sex and contraception.
The Role of Conflict in the Rapid Fertility Decline in Eritrea and Prospects for the Future
Authors: Blanc Ann K.
Published: December 2004

The large fertility decline observed in Eritrea between the mid‐1990s and the early part of the new century is examined using data from two detailed national household surveys. Little evidence is found that this decrease infertility signals the beginning of a sustained decline. Rather, the results indicate that one of the outcomes of the military mobilization and displacement associated with the 1998–2000 border conflict with Ethiopia was a steep reduction in the proportion of women exposed to the risk of pregnancy. Part of this reduction was due to delayed age at marriage, but it came about largely because married women were less likely to be living with their husbands in 2002 than in 1995. Projections of the number of births that could occur if women implement their stated reproductive intentions and a postwar “baby boom” results show that the health system may be facing an increased demand for child health services over the next several years.
Mitoxantrone induces cell death in peripheral blood leucocytes of multiple sclerosis patients
Authors: Chan A , Weilbach F X , Toyka K V , Gold R
Published: December 2004

Mitoxantrone (MX) is a cytotoxic drug with proven clinical efficacy in active multiple sclerosis (MS). In this ex vivo study we investigated the immunological effects of MX on peripheral blood leucocytes (PBL) from MS patients. PBL were isolated from 46 patients with active MS (mean age 42 years, female : male 1·4 : 1) before and immediately after 1 h MX infusion. Isolated PBL were cultured and stimulated with phytohaemagglutinin (PHA), T cell receptor stimulating monoclonal antibody (MoAb) X35 or kept in culture medium alone. Proliferation was measured by [3H]-thymidine incorporation. MX-uptake and cell death in PBL subpopulations was analysed by flow cytometry using antibodies against cluster of differentiation (CD)-surface antigens, annexin V (AnnV) and propidium iodide (PI). MX was incorporated rapidly into PBL. After only a 1-h in vivo exposure, MX reduced proliferative responses in unstimulated and stimulated PBL (PHA: − 17%, MoAb X35: − 13%). MX-exposed PBL showed an increase of AnnV+/PI+ cells (unstimulated: 12%, PHA: 15%), which was even more pronounced 2 weeks after infusion. No difference was observed between de novo MX-treated patients and those on long-term MX treatment. In T cell receptor stimulated PBL, cell death was induced preferentially in CD19-positive B cells and to a lesser extent in CD8-positive T cells. MX is incorporated rapidly in circulating PBL of MS patients and induces a pronounced suppression of proliferative responses. This suppression appears to be mediated at least partly by the induction of late apoptotic/necrotic cell death with a preferential susceptibility of B cells.
Vaccination of neonatal calves withMycobacterium bovisBCG induces protection against intranasal challenge with virulentM. bovis
Authors: Hope J C , Thom M L , Villarreal-Ramos B , Vordermeier H M , Hewinson R G , Howard C J
Published: December 2004

Vaccination of neonates with Mycobacterium bovis bacillus Calmette–Guérin (BCG) may be a strategy that overcomes reduced vaccine efficacy associated with exposure to environmental mycobacteria in humans and cattle. Preliminary comparisons indicated that 2-week-old calves produced an immune response to vaccination at least as intense as that observed in adults. Subsequently, five gnotobiotic hysterotomy derived calves aged 1 day were inoculated with BCG and 3 months later were challenged intranasally with virulent M. bovis. The number of tissues with lesions and the pathological extent of these lesions was reduced significantly in vaccinates. Furthermore, lesions were evident in the lung or associated chest lymph nodes of four of five controls but none of five vaccinates. BCG vaccination reduced significantly the level of bacterial colonization. However, lesions in the head associated lymph nodes were observed in three of five BCG-vaccinated cattle. Levels of interferon gamma (IFN-γ) detected by enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot (ELISPOT) in individual vaccinated animals at challenge did not correlate with subsequent resistance and in general immune responses post-challenge were lower in vaccinated calves. Low IL-10 responses were evident but IL-4 was not detected. Responses to ESAT-6 and/or CFP-10 were evident in four of four control calves that had lesions. Two of the BCG vaccinates with lesions did not produce a response to ESAT-6 and CFP-10, indicating that these antigens did not distinguish vaccinated immune animals from vaccinated animals with lesions. Overall, vaccination of neonatal calves with BCG induced significant protection against disease and has potential as a strategy for the reduction of the incidence of bovine tuberculosis.
Effects of straw and silicon soil amendments on some foliar and stem‐base diseases in pot‐grown winter wheat
Authors: Rodgers‐Gray B. S. , Shaw M. W.
Published: December 2004

Four foliar and two stem‐base pathogens were inoculated onto wheat plants grown in different substrates in pot experiments. Soils from four different UK locations were each treated in three ways: (i) straw incorporated in the field at 10 t ha−1 several months previously; (ii) silicon fertilization at 100 mg L−1 during the experiment; and (iii) no amendments. A sand and vermiculite mix was used with and without silicon amendment. The silicon treatment increased plant silica concentrations in all experiments, but incorporating straw was not associated with raised plant silica concentrations. Blumeria graminis and Puccinia recondita were inoculated by shaking infected plants over the test plants, followed by suitable humid periods. The silicon treatment reduced powdery mildew (B. graminis) substantially in sand and vermiculite and in two of the soils, but there were no effects on the slight infection by brown rust (P. recondita). Phaeosphaeria nodorum and Mycosphaerella graminicola were inoculated as conidial suspensions. Leaf spot caused by P. nodorum was reduced in silicon‐amended sand and vermiculite; soil was not tested. Symptoms of septoria leaf blotch caused by M. graminicola were reduced by silicon amendment in a severely infected sand and vermiculite experiment but not in soil or a slightly infected sand and vermiculite experiment. Oculimacula yallundae (eyespot) and Fusarium culmorum (brown foot rot) were inoculated as agar plugs on the stem base. Severity of O. yallundae was reduced by silicon amendment of two of the soils but not sand and vermiculite; brown foot rot symptoms caused by F. culmorum were unaffected by silicon amendment. The straw treatment reduced severity of powdery mildew but did not detectably affect the other pathogens. Both straw and silicon treatments appeared to increase plant resistance to all diseases only under high disease pressure.
Markers, old and new, for examining Phytophthora infestans diversity
Authors: Cooke D. E. L. , Lees A. K.
Published: December 2004

Late blight, caused by Phytophthora infestans, is an ongoing threat to potato and tomato crop production worldwide and considerable fundamental and applied research is conducted with the long‐term aim of improved disease control. Understanding the mechanisms, processes and rates of P. infestans evolution is an important factor in predicting the effectiveness and durability of new management practices. A range of phenotypic and genotypic tests has been applied to achieve this goal, but each has limitations and new methods are sought. Recent progress in P. infestans genomics is providing the raw data for such methods and new high‐throughput codominant biomolecular markers are currently being developed that have tremendous potential in the study of P. infestans population biology, epidemiology, ecology, genetics and evolution. This paper reviews some key applications, recommends some changes in approach and reports on the status and potential of new and existing methods for probing P. infestans genetic diversity.
Nitrogen per unit leaf area affects the upper asymptote ofPuccinia striiformisf.sp.triticiepidemics in winter wheat
Authors: Neumann S. , Paveley N. D. , Beed F. D. , Sylvester‐Bradley R.
Published: December 2004

Field trials tested which components of epidemic development ofPuccinia striiformis, the cause of yellow rust, were affected by nitrogen (N) fertilizer applied to winter wheat. Both timing and amount of N were varied to affect canopy size and leaf N content, and to provide a supply of mobile N to the pathogen, by causing fresh N uptake after leaf expansion was complete. No N was applied to control plots. A logistic disease‐progress function was fitted to disease‐severity data, which were assessed in absolute units. Leaf area and specific leaf N (g N per m2leaf tissue) were quantified. Large and highly significant effects of N on the upper asymptotes, or ‘carrying capacities’ (c) were found. Effects on rates and points of inflection of the epidemics were not significant. Early N resulted in larger shoot numbers and leaf area, but disease was also more severe, so that by grain filling, the remaining green leaf areas were larger without N than with N. Later N treatments did not increase canopy size, but did increase symptom area compared with the control. These effects differ from the concept that N affects disease as a result of its effect on canopy growth, and therefore canopy microclimate, and suggest instead a substrate effect. Linear regression revealed that 51% of the observed differences incwere explained by variation in specific leaf N, suggesting that growth of the rust fungus may depend directly on particular components of total leaf N.
Regulation of the expression and subcellular localization of the taurine transporter TauT in mouse NIH3T3 fibroblasts
Authors: Voss Jesper W. , Pedersen Stine F. , Christensen Søren T. , Lambert Ian H.
Published: December 2004

The cellular level of the organic osmolyte taurine is a balance between active uptake and passive leak via a volume sensitive pathway. Here, we demonstrate that NIH3T3 mouse fibroblasts express a saturable, high affinity taurine transporter (TauT, Km = 18 µm), and that taurine uptake via TauT is a Na+‐ and Cl–‐dependent process with an apparent 2.5 : 1 : 1 Na+/Cl–/taurine stoichiometry. Transport activity is reduced following acute administration of H2O2 or activators of protein kinases A or C. TauT transport activity, expression and nuclear localization are significantly increased upon serum starvation (24 h), exposure to tumour necrosis factor alpha (TNFα; 16 h), or hyperosmotic medium (24 h); conditions that are also associated with increased localization of TauT to the cytosolic network of microtubules. Conversely, transport activity, expression and nuclear localization of TauT are reduced in a reversible manner following long‐term exposure (24 h) to high extracellular taurine concentration. In contrast to active taurine uptake, swelling‐induced taurine release is significantly reduced following preincubation with TNFα (16 h) but unaffected by high extracellular taurine concentration (24 h). Thus, in NIH3T3 cells, (a) active taurine uptake reflects TauT expression; (b) TauT activity is modulated by multiple stimuli, both acutely, and at the level of TauT expression; (c) the subcellular localization of TauT is regulated; and (d) volume‐sensitive taurine release is not mediated by TauT.
Stoichiometry of LHCI antenna polypeptides and characterization of gap and linker pigments in higher plants Photosystem I
Authors: Ballottari Matteo , Govoni Chiara , Caffarri Stefano , Morosinotto Tomas
Published: December 2004

We report on the results obtained by measuring the stoichiometry of antenna polypeptides in Photosystem I (PSI) from Arabidopsis thaliana. This analysis was performed by quantification of Coomassie blue binding to individual LHCI polypeptides, fractionation by SDS/PAGE, and by the use of recombinant light harvesting complex of Photosystem I (Lhca) holoproteins as a standard reference. Our results show that a single copy of each Lhca1–4 polypeptide is present in Photosystem I. This is in agreement with the recent structural data on PSI–LHCI complex [Ben Shem, A., Frolow, F. and Nelson, N. (2003) Nature, 426, 630–635]. The discrepancy from earlier estimations based on pigment binding and yielding two copies of each LHCI polypeptide per PSI, is explained by the presence of ‘gap’ and ‘linker’ chlorophylls bound at the interface between PSI core and LHCI. We showed that these chlorophylls are lost when LHCI is detached from the PSI core moiety by detergent treatment and that gap and linker chlorophylls are both Chl a and Chl b. Carotenoid molecules are also found at this interface between LHCI and PSI core. Similar experiments, performed on PSII supercomplexes, showed that dissociation into individual pigment‐proteins did not produce a significant loss of pigments, suggesting that gap and linker chlorophylls are a peculiar feature of Photosystem I.
Differential regulation of fatty acid amide hydrolase promoter in human immune cells and neuronal cells by leptin and progesterone
Authors: Maccarrone Mauro , Gasperi Valeria , Fezza Filomena , Finazzi‐Agrò Alessandro , Rossi Antonello
Published: December 2004

We have shown recently that in human T lymphocytes, leptin stimulates activity and expression of the endocannabinoid‐degrading enzyme fatty acid amide hydrolase (FAAH), through STAT3 (signal transducer and activator of transcription 3) and its CRE (cAMP response element)‐like transcriptional target in the FAAH promoter [Maccarrone, M., Di Rienzo, M., Finazzi‐Agrò, A., & Rossi, A. (2003) J. Biol. Chem. 278, 13318–13324]. We have also shown that progesterone, alone or additively with leptin, up‐regulates the FAAH gene in human T‐cells, through the Ikaros transcription factor [Maccarrone, M., Bari, M., Di Rienzo, M., Finazzi‐Agrò, A., & Rossi, A. (2003) J. Biol. Chem. 278, 32726–32732]. Here, we extend these observations to immortalized human lymphoma U937 cells, where stimulation of FAAH by leptin (up to ≈ 300% of the controls) involves binding to a leptin receptor (Kd = 2.0 ± 0.1 nm, Bmax = 382 ± 5 fmol·mg protein−1, apparent molecular mass of ≈ 110 kDa), and stimulation by progesterone involves an intracellular receptor of ≈ 120 kDa. Unlike FAAH, the other proteins of the endocannabinoid system are not modulated by the two hormones. Interestingly, human neuroblastoma CHP100 cells also have a leptin receptor (≈ 110 kDa, Kd = 2.2 ± 0.2 nm, Bmax = 339 ± 8 fmol·mg protein−1), a progesterone receptor (≈ 120 kDa), STAT3 and Ikaros, yet their FAAH is not activated by leptin or progesterone. These data, corroborated by transient expression and electrophoretic mobility‐shift assays, demonstrate an unprecedented cell‐specific regulation of the FAAH gene, which has important implications for the control of tone and activity of AEA along the neuroimmune axis.
A novel γ‐N‐methylaminobutyrate demethylating oxidase involved in catabolism of the tobacco alkaloid nicotine by Arthrobacter nicotinovorans pAO1
Authors: Chiribau Calin B. , Sandu Cristinel , Fraaije Marco , Schiltz Emile , Brandsch Roderich
Published: December 2004

Nicotine catabolism, linked in Arthrobacter nicotinovorans to the presence of the megaplasmid pAO1, leads to the formation of γ‐N‐methylaminobutyrate from the pyrrolidine ring of the alkaloid. Until now the metabolic fate of γ‐N‐methylaminobutyrate has been unknown. pAO1 carries a cluster of ORFs with similarity to sarcosine and dimethylglycine dehydrogenases and oxidases, to the bifunctional enzyme methylenetetrahydrofolate dehydrogenase/cyclohydrolase and to formyltetrahydrofolate deformylase. We cloned and expressed the gene carrying the sarcosine dehydrogenase‐like ORF and showed, by enzyme activity, spectrophotometric methods and identification of the reaction product as γ‐aminobutyrate, that the predicted 89 395 Da flavoprotein is a demethylating γ‐N‐methylaminobutyrate oxidase. Site‐directed mutagenesis identified His67 as the site of covalent attachment of FAD and confirmed Trp66 as essential for FAD binding, for enzyme activity and for the spectral properties of the wild‐type enzyme. A Km of 140 µm and a kcat of 800 s−1 was determined when γ‐N‐methylaminobutyrate was used as the substrate. Sarcosine was also turned over by the enzyme, but at a rate 200‐fold slower than γ‐N‐methylaminobutyrate. This novel enzyme activity revealed that the first step in channelling the γ‐N‐methylaminobutyrate generated from nicotine into the cell metabolism proceeds by its oxidative demethylation.
The structure and biological characteristics of the Spirochaeta aurantia outer membrane glycolipid LGLB
Authors: Vinogradov Evgeny , Paul Catherine J. , Li Jianjun , Zhou Yuchen , Lyle Elizabeth A. , Tapping Richard I. , Kropinski Andrew M. , Perry Malcolm B.
Published: December 2004

In an attempt to isolate lipopolysaccharide from Spirochaeta aurantia, Darveau‐Hancock extraction of the cell mass was performed. While no lipopolysaccharide was found, two carbohydrate‐containing compounds were detected. They were resolved by size‐exclusion chromatography into high molecular mass (LGLA) and low molecular mass (LGLB) fractions. Here we present the results of the analysis of the glycolipid LGLB. Deacylation of LGLB with hydrazine and separation of the products by using anion‐exchange chromatography gave two major products. Their structure was determined by using chemical methods, NMR and mass spectrometry. All monosaccharides had the d‐configuration, and aspartic acid had the l‐configuration. Intact LGLB contained two fatty groups at O‐2 and O‐3 of the glycerol residue. Nonhydroxylated C14 to C18 fatty acids were identified, which were predominantly unsaturated or branched. LGLB was able to gel Limulus amebocyte lysate, albeit at a lower level than that observed for Escherichia coli O113 lipopolysaccharide. However, even large amounts of LGLB were unable to stimulate any Toll‐like receptor (TLR) examined, including TLR4 and TLR2, previously shown to be sensitive to lipopolysaccharide and glycolipids from diverse bacterial origins, including other spirochetes.
1,5‐Diamino‐2‐pentyne is both a substrate and inactivator of plant copper amine oxidases
Authors: Lamplot Zbyněk , Šebela Marek , Maloň Michal , Lenobel René , Lemr Karel , Havliš Jan , Peč Pavel , Qiao Chunhua , Sayre Lawrence M.
Published: December 2004

1,5‐Diamino‐2‐pentyne (DAPY) was found to be a weak substrate of grass pea (Lathyrus sativus, GPAO) and sainfoin (Onobrychis viciifolia, OVAO) amine oxidases. Prolonged incubations, however, resulted in irreversible inhibition of both enzymes. For GPAO and OVAO, rates of inactivation of 0.1–0.3 min−1were determined, the apparentKIvalues (half‐maximal inactivation) were of the order of 10−5 m. DAPY was found to be a mechanism‐based inhibitor of the enzymes because the substrate cadaverine significantly prevented irreversible inhibition. TheN1‐methyl andN5‐methyl analogs of DAPY were tested with GPAO and were weaker inactivators (especially theN5‐methyl) than DAPY. Prolonged incubations of GPAO or OVAO with DAPY resulted in the appearance of a yellow–brown chromophore (λmax = 310–325 nm depending on the working buffer). Excitation at 310 nm was associated with emitted fluorescence with a maximum at 445 nm, suggestive of extended conjugation. After dialysis, the color intensity was substantially decreased, indicating the formation of a low molecular mass secondary product of turnover. The compound provided positive reactions with ninhydrin, 2‐aminobenzaldehyde and Kovacs' reagents, suggesting the presence of an amino group and a nitrogen‐containing heterocyclic structure. The secondary product was separated chromatographically and was found not to irreversibly inhibit GPAO. MS indicated an exact molecular mass (177.14 Da) and molecular formula (C10H15N3). Electrospray ionization‐ and MALDI‐MS/MS analyses yielded fragment mass patterns consistent with the structure of a dihydropyridine derivative of DAPY. Finally,N‐(2,3‐dihydropyridinyl)‐1,5‐diamino‐2‐pentyne was identified by means of1H‐ and13C‐NMR experiments. This structure suggests a lysine modification chemistry that could be responsible for the observed inactivation.
Transcriptional responses to glucose at different glycolytic rates in Saccharomyces cerevisiae
Authors: Elbing Karin , Ståhlberg Anders , Hohmann Stefan , Gustafsson Lena
Published: December 2004

The addition of glucose to Saccharomyces cerevisiae cells causes reprogramming of gene expression. Glucose is sensed by membrane receptors as well as (so far elusive) intracellular sensing mechanisms. The availability of four yeast strains that display different hexose uptake capacities allowed us to study glucose‐induced effects at different glycolytic rates. Rapid glucose responses were observed in all strains able to take up glucose, consistent with intracellular sensing. The degree of long‐term responses, however, clearly correlated with the glycolytic rate: glucose‐stimulated expression of genes encoding enzymes of the lower part of glycolysis showed an almost linear correlation with the glycolytic rate, while expression levels of genes encoding gluconeogenic enzymes and invertase (SUC2) showed an inverse correlation. Glucose control of SUC2 expression is mediated by the Snf1‐Mig1 pathway. Mig1 dephosphorylation upon glucose addition is known to lead to repression of target genes. Mig1 was initially dephosphorylated upon glucose addition in all strains able to take up glucose, but remained dephosphorylated only at high glycolytic rates. Remarkably, transient Mig1‐dephosphorylation was accompanied by the repression of SUC2 expression at high glycolytic rates, but stimulated SUC2 expression at low glycolytic rates. This suggests that Mig1‐mediated repression can be overruled by factors mediating induction via a low glucose signal. At low and moderate glycolytic rates, Mig1 was partly dephosphorylated both in the presence of phosphorylated, active Snf1, and unphosphorylated, inactive Snf1, indicating that Mig1 was actively phosphorylated and dephosphorylated simultaneously, suggesting independent control of both processes. Taken together, it appears that glucose addition affects the expression of SUC2 as well as Mig1 activity by both Snf1‐dependent and ‐independent mechanisms that can now be dissected and resolved as early and late/sustained responses.
Mechanistic studies on bovine cytosolic 5′‐nucleotidase II, an enzyme belonging to the HAD superfamily
Authors: Allegrini Simone , Scaloni Andrea , Careddu Maria Giovanna , Cuccu Giovanna , D'ambrosio Chiara , Pesi Rossana , Camici Marcella , Ferrara Lino , Tozzi Maria Grazia
Published: December 2004

Cytosolic 5′‐nucleotidase/phosphotransferase specific for 6‐hydroxypurine monophosphate derivatives (cN‐II), belongs to a class of phosphohydrolases that act through the formation of an enzyme–phosphate intermediate. Sequence alignment with members of the P‐type ATPases/L‐2‐haloacid dehalogenase superfamily identified three highly conserved motifs in cN‐II and other cytosolic nucleotidases. Mutagenesis studies at specific amino acids occurring in cN‐II conserved motifs were performed. The modification of the measured kinetic parameters, caused by conservative and nonconservative substitutions, suggested that motif I is involved in the formation and stabilization of the covalent enzyme–phosphate intermediate. Similarly, T249 in motif II as well as K292 in motif III also contribute to stabilize the phospho–enzyme adduct. Finally, D351 and D356 in motif III coordinate magnesium ion, which is required for catalysis. These findings were consistent with data already determined for P‐type ATPases, haloacid dehalogenases and phosphotransferases, thus suggesting that cN‐II and other mammalian 5′‐nucleotidases are characterized by a 3D arrangement related to the 2‐haloacid dehalogenase superfold. Structural determinants involved in differential regulation by nonprotein ligands and redox reagents of the two naturally occurring cN‐II forms generated by proteolysis were ascertained by combined biochemical and mass spectrometric investigations. These experiments indicated that the C‐terminal region of cN‐II contains a cysteine prone to form a disulfide bond, thereby inactivating the enzyme. Proteolysis events that generate the observed cN‐II forms, eliminating this C‐terminal portion, may prevent loss of enzymic activity and can be regarded as regulatory phenomena.
Brain succinic semialdehyde dehydrogenase
Authors: Lee Byung Ryong , Kim Dae Won , Hong Joung‐Woo , Eum Won Sik , Choi Hee Soon , Choi Soo Hyun , Kim So Young , An Jae Jin , Ahn Jee‐Yin , Kwon Oh‐Shin , Kang Tae‐Cheon , Won Moo Ho , Cho SUNG‐Woo , Lee Kil Soo , Park Jinseu , Choi Soo Young
Published: December 2004

Incubation of an NAD+‐dependent succinic semialdehyde dehydrogenase from bovine brain with 4‐dimethylaminoazobenzene‐4‐iodoacetamide (DABIA) resulted in a time‐dependent loss of enzymatic activity. This inactivation followed pseudo first‐order kinetics with a second‐order rate constant of 168 m−1·min−1. The spectrum of DABIA‐labeled enzyme showed a characteristic peak of the DABIA alkylated sulfhydryl group chromophore at 436 nm, which was absent from the spectrum of the native enzyme. A linear relationship was observed between DABIA binding and the loss of enzyme activity, which extrapolates to a stoichiometry of 8.0 mol DABIA derivatives per mol enzyme tetramer. This inactivation was prevented by preincubating the enzyme with substrate, succinic semialdehyde, but not by preincubating with coenzyme NAD+. After tryptic digestion of the enzyme modified with DABIA, two peptides absorbing at 436 nm were isolated by reverse‐phase HPLC. The amino acid sequences of the DABIA‐labeled peptides were VCSNQFLVQR and EVGEAICTDPLVSK, respectively. These sites are identical to the putative active site sequences of other brain succinic semialdehyde dehydrogenases. These results suggest that the catalytic function of succinic semialdehyde dehydrogenase is inhibited by the specific binding of DABIA to a cysteine residue at or near its active site.
Structure, expression and regulation of the cannabinoid receptor gene (CB1) in Huntington's disease transgenic mice
Authors: McCaw Elizabeth A. , Hu Haibei , Gomez Geraldine T. , Hebb Andrea L. O. , Kelly Melanie E. M. , Denovan‐Wright Eileen M.
Published: December 2004

Loss of cannabinoid receptors (CB1) occurs prior to neurodegeneration in Huntington's disease (HD). The levels and distribution of CB1 RNA were equivalent in 3‐week‐old mice regardless of genotype demonstrating that the specific factors and appropriate chromatin structure that lead to the transcription of CB1 were present in the striatum of young R6/2 and R6/1 transgenic HD mice. The expression of the mutant HD transgene led progressively to decreased steady‐state levels of CB1 mRNA in neurons of the lateral striatum, which was dependent on the size of the CAG repeat and relative expression of the gene encoding mutant huntingtin (HD). Although it is known that the coding region of CB1 is contained within a single exon in mice, rats and humans, the 5′‐untranslated region of the mouse gene remained to be defined. CB1 mRNA is encoded by two exons separated by an 18.4‐kb intron. Transcription of CB1 occurred at multiple sites within a GC‐rich promoter region upstream of exon 1 encoding the 5′‐UTR of CB1. There was no difference in the selection of specific transcription initiation sites associated with higher levels of CB1 expression in the striatum compared to the cortex or between the striata of wild‐type and HD transgenic mice. The progressive decline in CB1 mRNA levels in R6 compared to wild‐type mice was due to decreased transcription, which is consistent with the hypothesis that mutant huntingtin exerts its effects by altering transcription factor activity. The cell‐specific conditions that allow for increased transcription of CB1 in the lateral striatum compared to other forebrain regions from all transcription start sites were affected by the expression of mutant huntingtin in a time‐dependent manner.
Monitoring ligand‐mediated nuclear receptor–coregulator interactions by noncovalent mass spectrometry
Authors: Sanglier Sarah , Bourguet William , Germain Pierre , Chavant Virginie , Moras Dino , Gronemeyer Hinrich , Potier Noelle , Van Dorsselaer Alain
Published: December 2004

Retinoid receptors are ligand‐dependent transcription factors belonging to the nuclear receptor superfamily. Retinoic acid (RARα, β, γ) and retinoid X (RXRα, β, γ) receptors mediate the retinoid/rexinoid signal to the transcriptional machineries by interacting at the first level with coactivators or corepressors, which leads to the recruitment of enzymatically active noncovalent complexes at target gene promoters. It has been shown that the interaction of corepressors with nuclear receptors involves conserved LXXI/HIXXXI/L consensus sequences termed corepressor nuclear receptor (CoRNR) boxes. Here we describe the use of nondenaturing electrospray ionization mass spectrometry (ESI‐MS) to determine the characteristics of CoRNR box peptide binding to the ligand binding domains of the RARα–RXRα heterodimer. The stability of the RARα–RXRα–CoRNR ternary complexes was monitored in the presence of different types of agonists or antagonists for the two receptors, including inverse agonists. These results show unambiguously the differential impact of distinct retinoids on corepressor binding. We show that ESI‐MS is a powerful technique that complements classical methods and allows one to: (a) obtain direct evidence for the formation of noncovalent NR complexes; (b) determine ligand binding stoichiometries and (c) monitor ligand effects on these complexes.
An examination of land use controls in the Lake Biwa watershed from the perspective of environmental conservation and management
Authors: Yamamoto Kayoko , Nakamura Masahisa
Published: December 2004

In the outskirts of metropolitan areas of Japan, there is an increasing tendency for residential and commercial facilities to be located in close proximity to transportation networks, particularly around railway stations. The resulting sprawl, involving residential development and industrial estates, perpetuates land use distortions in the semi‐urban areas, as well as in agricultural and forestry lands. Lake Biwa, the largest lake in Japan, provides water to a population of 14 million people in Shiga, Osaka, Kyoto and Hyogo Prefectures. The Lake Biwa watershed has undergone dramatic land use transformations resulting from the migration of the downstream population and industries attracted by the provision of necessary, basic infrastructure via the Lake Biwa Comprehensive Development Plan (1972–1997). The purposes of this study are to provide a quantitative analysis of land use transformations within the Lake Biwa watershed, with particular reference to urbanization, examine watershed land use controls by analysis of deviation of actual land uses from the land use control targets, assess the flaws in the present land use controls and to discuss the implications of the results within the context of the Lake Biwa Comprehensive Conservation Plan (1999–), using a geographic information system to illustrate the results. The major findings of this study are that the continued existence of farmland and forests, in conjunction with urban sprawl, in the Urbanization Promotion Areas and the Use Districts, has resulted in serious land use problems which are seriously aggravated in the south‐eastern and north‐eastern parts of the watershed, and the expansion of urban areas in the Urbanization Control Areas and the White Areas has resulted in land use control becoming more problematic, especially in the eastern part of the watershed.
Endoscopic transanal resection of rectal tumours using a urological resectoscope – still has a role in selected patients
Authors: Beattie G. C. , Paul I. , Calvert C. H.
Published: December 2004

Introduction  Transanal resection of rectal villous adenomas or adenocarcinomas can be carried out using various modalities such as operative excision, fulguration, laser coagulation or cryotherapy. Transanal endoscopic microsurgery is currently not widely available. Transanal resection can provide effective palliation for locally advanced rectal tumours in patients unfit for abdomino‐perineal excision of rectum. A urological resectoscope can be safely and repeatedly used to resect advanced primary or locally recurrent rectal rumours by colorectal surgeons with urological expertise. This study reports our experience of treating rectal lesions with endoscopic transanal resection (ETAR) using the urological resectoscope.

Methods  Patients were identified from one surgeons' prospectively collected operating data. Charts were retrieved and reviewed.

Results  Over a 13‐year period a total of 43 ETAR procedures were carried out in 20 patients (11 males; mean age 74 years; range 54–92 years) using the urological resectoscope. Twelve (60%) patients had a single resection; 8 (40%) patients required more than one resection; the mean number of procedures per patient was 2.2 (range1–8). The median interval between resections for recurrent disease (excluding planned repeat resections) was 340 days (range 168–2337 days). Histopathology revealed rectal adenoma (with varying degrees of dysplasia) in 11 (55%) patients and adenocarcinoma in 9 (45%). The majority (30; 70%) of resections were carried out in patients with benign disease, with 13 (30%) in patients with rectal adenocarcinoma. Mean operating time per resection was 25 min. Thirteen (30%) resections were carried out under spinal anaesthetic. There was no procedure related mortality. There were no cases of haemorrhage, rectal perforation, ‘TUR syndrome’ or pelvic sepsis. No patients with benign disease subsequently developed an invasive carcinoma.

Conclusions  Accepting that this technique provides limited histopathological information regarding extent of resection and tumour clearance, our experience demonstrates that ETAR of rectal tumours using the urological resectoscope can provide a minimally invasive, effective and safe means of treating and palliating patients with benign and malignant rectal disease. There remains a place for this technique in selected patients.
Crohn's‐like changes in the colon due to mycophenolate?
Authors: Dalle I. J. , Maes B. D. , Geboes K. P. , Lemahieu W. , Geboes K.
Published: December 2004

Objective  Mycophenolate mofetil (MMF) is used for prevention of allograft rejection in kidney transplant patients. A subset of patients suffers from chronic diarrhoea of unknown origin. The aim of the study was to investigate the effect of MMF on the colonic mucosa.

Materials and methods  Colonic mucosal biopsies from 24 kidney transplant patients receiving MMF and presenting with chronic diarrhoea were analysed using routine stainings and immunohistochemistry for Ki67 and E‐cadherin. Results were compared with a control group of 19 kidney transplant patients not receiving MMF. In all patients routine clinical and laboratory investigations were performed in order to explain the diarrhoea.

Results  In 11 patients, the diarrhoea seemed to be of infectious origin. Furthermore, 19/24 of MMF‐patients showed characteristic histological alterations of the mucosa that were Crohn's disease‐like: discontinuous crypt architectural distortion, increased epithelial mucin secretion, mildly active inflammation and focal presence of dilated and inflamed crypts. Ki67 staining was abnormal in 6/24 MMF patients but also in 4/19 control patients. E‐Cadherin staining was normal in most MMF and control patients.

Conclusions  Diarrhoea following MMF treatment is frequently infectious in origin and associated with morphological changes with a Crohn's‐like pattern in the colonic mucosa in a subset of patients. MMF does not induce major alteration in the proliferative compartment of colonic epithelium. The diarrhoea is not associated with altered E‐cadherin expression in the colonic epithelium.
Long‐term follow‐up of retrograde colonic irrigation for defaecation disturbances
Authors: Gosselink M. P. , Darby M. , Zimmerman D. D. E. , Smits A. A. A. , Van Kessel I. , Hop W. C. , Briel J. W. , Schouten W. R.
Published: December 2004

Objective  Irrigation of the distal part of the large bowel is a nonsurgical alternative for patients with defaecation disturbances. In our institution, all patients with defaecation disturbances, not responding to medical treatment and biofeedback therapy, were offered retrograde colonic irrigation (RCI). This study is aimed at evaluating the long‐term feasibility and outcome of RCI.

Methods  Between 1989 and 2001, a consecutive series of 267 patients was offered RCI. All patients received instructions about RCI by one of our enterostomal therapists. Twenty‐eight patients were lost to follow‐up. A detailed questionnaire was sent by mail to 239 patients. The total response rate was 79% (190 patients). Based on the returned questionnaires it became clear that 21 (11%) patients never started RCI. The long‐term feasibility and outcome of RCI was therefore assessed in the remaining group of 169 patients. Thirty‐two patients were admitted with soiling, 71 patients with faecal incontinence, 37 patients with obstructed defaecation and 29 had defaecation disturbances after low anterior resection or pouch surgery.

Results  According to the returned questionnaires, RCI was considered effective by 91 (54%) patients. Among patients with soling and faecal incontinence, RCI was found to be effective in, respectively, 47 and 41% of the subjects. Despite of the reported effectiveness, 10 (67%) patients with soiling and 5 (17%) patients with faecal incontinence decided to stop. Among patients with obstructed defaecation and those with defaecation disturbances after low anterior resection or pouch surgery the effectiveness of RCI was found to be 65 and 79%, respectively. None of these patients ceased their therapy. The overall success‐rate of long‐term RCI was therefore 45%.

Conclusions  Long‐term RCI is beneficial for 45% of patients with defaecation disturbances. In the group of patients who considered RCI effective and beneficial, discontinuation of therapy was only observed among those with soiling and faecal incontinence.