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Site Occupancy (site + occupancy)
Selected AbstractsA Quantitative Conservation Approach for the Endangered Butterfly Maculinea alconCONSERVATION BIOLOGY, Issue 2 2004MICHIEL F. WallisDeVries The quality and size of habitat patches and their isolation from other patches are the main parameters for an assessment of population persistence, but translating persistence probabilities into practical measures is still a weak link in conservation management. I provide a quantitative conservation approach for the endangered myrmecophilous butterfly Maculinea alcon in the Netherlands. All 127 colonies known on heathland since 1990 were investigated for patch quality, size, and isolation. I assessed habitat quality in three 10 × 10 m plots for most colonies. Site occupancy in 1998,1999 was only 56%. Occupancy was best explained by a logistic regression including patch area, host ant presence, host plant abundance, overall heathland area, and connectivity between sites ( R2= 0.410, p < 0.0001); it correctly classified the occupied or vacant status for 82% of the sites. Connectivity contributed only 3.6% to the total explained variation of site occupancy, indicating that habitat characteristics were more important than isolation in determining population persistence at the examined scale level (>500 m). Grazing and sod cutting had a beneficial impact, but in combination these practices proved detrimental. Hydrological measures to prevent drainage were also associated with lowered occupancy. I used the different components in the logistic regression to formulate objective management recommendations. These consisted of sod cutting, reduction of management intensity, enlargement of habitat, or combinations of these recommendations. The results highlight the importance of careful management when site quality is determined by multiple factors. The quantitative conservation approach followed here can be fruitfully extended to other endangered species, provided enough is known about their ecological requirements and how management actions affect them. Resumen:,La preservación de fragmentos individuales es extremadamente importante para especies en peligro con capacidad de dispersión limitada. La calidad y tamaño de los fragmentos de hábitat y su aislamiento de otros fragmentos son los parámetros principales para la evaluación de la persistencia de la población, pero la traducción de probabilidades de persistencia en medidas prácticas aun es un eslabón débil en la gestión de conservación. Proporciono un método cuantitativo de conservación para la mariposa mirmecófila Maculinea alcon en peligro en Holanda. Se investigó a las 127 colonias conocidas en brezales desde 1990 para calidad, tamaño y aislamiento del fragmento. Evalué la calidad del hábitat en tres parcelas de 10 × 10 m en la mayoría de las colonias. La ocupación de sitios en 1998-1999 fue sólo 56%. La ocupación fue mejor explicada por regresión logística incluyendo la superficie del fragmento, presencia de hormigas huésped, abundancia de plantas huésped, superficie total del brezal y conectividad entre sitios ( R2= 0.410, p < 0.0001); clasificó el estatus de ocupado o vacante en 82% de los sitios. La conectividad contribuyó con solo 36% de la variación total de sitio de ocupación, lo que indica que las características de hábitat fueron más importantes que el aislamiento en la determinación de la persistencia de la población en el nivel de escala examinado (>500 m). El pastoreo y el corte de pasto tuvieron un impacto benéfico pero combinadas, estas prácticas fueron perjudiciales. Obras hidrológicas para prevenir la desecación también se asociaron con una disminución en la ocupación. Utilicé los diferentes componentes de la regresión logística para formular recomendaciones objetivas de gestión. Estas incluyeron el corte de pasto, reducción en la intensidad de manejo, aumento de hábitat o combinaciones de estas recomendaciones. Los resultados resaltan la importancia de la gestión cuidadosa cuando la calidad del sitio está determinada por múltiples factores. El método cuantitativo de preservación utilizado puede ser extendido exitosamente a otras especies en peligro, siempre que sean suficientemente conocidos sus requerimientos ecológicos y la forma en que le afectan las acciones de manejo. [source] Spectral reflectance-compositional properties of spinels and chromites: Implications for planetary remote sensing and geothermometryMETEORITICS & PLANETARY SCIENCE, Issue 4 2004Edward A. Cloutis These two groups of minerals are spectrally distinct, which relates largely to differences in the types of major cations present. Both exhibit a number of absorption features in the 0.3,26 ,m region that show systematic variations with composition and can be used to quantify or constrain certain compositional parameters, such as cation abundances, and site occupancies. For spinels, the best correlations exist between Fe2+ content and wavelength positions of the 0.46, 0.93, 2.8, Restrahelen, 12.3, 16.2, and 17.5 ,m absorption features, Al and Fe3+ content with the wavelength position of the 0.93 ,m absorption feature, and Cr content from the depth of the absorption band near 0.55 ,m. For chromites, the best correlations exist between Cr content and wavelength positions of the 0.49, 0.59, 2, 17.5, and 23 ,m absorption features, Fe2+ and Mg contents with the wavelength position of the 1.3 ,m absorption feature, and Al content with the wavelength position of the 2 ,m absorption feature. At shorter wavelengths, spinels and chromites are most readily distinguished by the wavelength position of the absorption band in the 2 ,m region (<2.1 ,m for spinels, >2.1 ,m for chromite), while at longer wavelengths, spectral differences are more pronounced. The importance of being able to derive compositional information for spinels and chromites from spectral analysis stems from the relationship between composition and petrogenetic conditions (pressure, temperature, oxygen fugacity) and the widespread presence of spinels and chromites in the inner solar system. When coupled with the ability to derive compositional information for mafic silicates from spectral analysis, this opens up the possibility of deriving petrogenetic information for remote spinel- and chromite-bearing targets from analysis of their reflectance spectra. [source] Order,disorder transition in monoclinic sulfur: a precise structural study by high-resolution neutron powder diffractionACTA CRYSTALLOGRAPHICA SECTION B, Issue 6 2006W. I. F. David High-resolution neutron powder diffraction has been used in order to characterize the order,disorder transition in monoclinic cyclo-octasulphur. Rapid data collection and the novel use of geometrically constrained refinements has enabled a direct and precise determination of the order parameter, based on molecular site occupancies, to be made. The transition is critical and continuous; with a transition temperature, Tc = 198.4,(3),K, and a critical exponent, , = 0.28,(3), which is indicative of three-dimensional ordering. Difficulties encountered as a consequence of the low thermal conductivity of the sample are discussed. [source] A structural systematic study of four isomers of difluoro- N -(3-pyridyl)benzamideACTA CRYSTALLOGRAPHICA SECTION C, Issue 7 2009Joyce McMahon The four isomers 2,4-, (I), 2,5-, (II), 3,4-, (III), and 3,5-difluoro- N -(3-pyridyl)benzamide, (IV), all with formula C12H8F2N2O, display molecular similarity, with interplanar angles between the C6/C5N rings ranging from 2.94,(11)° in (IV) to 4.48,(18)° in (I), although the amide group is twisted from either plane by 18.0,(2),27.3,(3)°. Compounds (I) and (II) are isostructural but are not isomorphous. Intermolecular N,H...O=C interactions form one-dimensional C(4) chains along [010]. The only other significant interaction is C,H...F. The pyridyl (py) N atom does not participate in hydrogen bonding; the closest H...Npy contact is 2.71,Å in (I) and 2.69,Å in (II). Packing of pairs of one-dimensional chains in a herring-bone fashion occurs via,-stacking interactions. Compounds (III) and (IV) are essentially isomorphous (their a and b unit-cell lengths differ by 9%, due mainly to 3,4-F2 and 3,5-F2 substitution patterns in the arene ring) and are quasi-isostructural. In (III), benzene rotational disorder is present, with the meta F atom occupying both 3- and 5-F positions with site occupancies of 0.809,(4) and 0.191,(4), respectively. The N,H...Npy intermolecular interactions dominate as C(5) chains in tandem with C,H...Npy interactions. C,H...O=C interactions form R22(8) rings about inversion centres, and there are ,,, stacks about inversion centres, all combining to form a three-dimensional network. By contrast, (IV) has no strong hydrogen bonds; the N,H...Npy interaction is 0.3,Å longer than in (III). The carbonyl O atom participates only in weak interactions and is surrounded in a square-pyramidal contact geometry with two intramolecular and three intermolecular C,H...O=C interactions. Compounds (III) and (IV) are interesting examples of two isomers with similar unit-cell parameters and gross packing but which display quite different intermolecular interactions at the primary level due to subtle packing differences at the atom/group/ring level arising from differences in the peripheral ring-substitution patterns. [source] Twinning and structure of Eu0.6Sr0.4MnO3ACTA CRYSTALLOGRAPHICA SECTION C, Issue 1 2006Nicola Rotiroti The crystal structure of europium strontium manganese trioxide, Eu0.6Sr0.4MnO3, has been refined using a multiply twinned single crystal containing six twin components. The MnO6 octahedra show Jahn,Teller distortions with nearly fourfold symmetry, but the octahedral tilting scheme reduces the crystal symmetry to orthorhombic (space group Pbnm). The refinement of site occupancies and the analysis of difference Fourier maps show that the Eu3+ and Sr2+ cations occupy different crystallographic positions with eightfold and twelvefold coordination, respectively. [source] A Quantitative Conservation Approach for the Endangered Butterfly Maculinea alconCONSERVATION BIOLOGY, Issue 2 2004MICHIEL F. WallisDeVries The quality and size of habitat patches and their isolation from other patches are the main parameters for an assessment of population persistence, but translating persistence probabilities into practical measures is still a weak link in conservation management. I provide a quantitative conservation approach for the endangered myrmecophilous butterfly Maculinea alcon in the Netherlands. All 127 colonies known on heathland since 1990 were investigated for patch quality, size, and isolation. I assessed habitat quality in three 10 × 10 m plots for most colonies. Site occupancy in 1998,1999 was only 56%. Occupancy was best explained by a logistic regression including patch area, host ant presence, host plant abundance, overall heathland area, and connectivity between sites ( R2= 0.410, p < 0.0001); it correctly classified the occupied or vacant status for 82% of the sites. Connectivity contributed only 3.6% to the total explained variation of site occupancy, indicating that habitat characteristics were more important than isolation in determining population persistence at the examined scale level (>500 m). Grazing and sod cutting had a beneficial impact, but in combination these practices proved detrimental. Hydrological measures to prevent drainage were also associated with lowered occupancy. I used the different components in the logistic regression to formulate objective management recommendations. These consisted of sod cutting, reduction of management intensity, enlargement of habitat, or combinations of these recommendations. The results highlight the importance of careful management when site quality is determined by multiple factors. The quantitative conservation approach followed here can be fruitfully extended to other endangered species, provided enough is known about their ecological requirements and how management actions affect them. Resumen:,La preservación de fragmentos individuales es extremadamente importante para especies en peligro con capacidad de dispersión limitada. La calidad y tamaño de los fragmentos de hábitat y su aislamiento de otros fragmentos son los parámetros principales para la evaluación de la persistencia de la población, pero la traducción de probabilidades de persistencia en medidas prácticas aun es un eslabón débil en la gestión de conservación. Proporciono un método cuantitativo de conservación para la mariposa mirmecófila Maculinea alcon en peligro en Holanda. Se investigó a las 127 colonias conocidas en brezales desde 1990 para calidad, tamaño y aislamiento del fragmento. Evalué la calidad del hábitat en tres parcelas de 10 × 10 m en la mayoría de las colonias. La ocupación de sitios en 1998-1999 fue sólo 56%. La ocupación fue mejor explicada por regresión logística incluyendo la superficie del fragmento, presencia de hormigas huésped, abundancia de plantas huésped, superficie total del brezal y conectividad entre sitios ( R2= 0.410, p < 0.0001); clasificó el estatus de ocupado o vacante en 82% de los sitios. La conectividad contribuyó con solo 36% de la variación total de sitio de ocupación, lo que indica que las características de hábitat fueron más importantes que el aislamiento en la determinación de la persistencia de la población en el nivel de escala examinado (>500 m). El pastoreo y el corte de pasto tuvieron un impacto benéfico pero combinadas, estas prácticas fueron perjudiciales. Obras hidrológicas para prevenir la desecación también se asociaron con una disminución en la ocupación. Utilicé los diferentes componentes de la regresión logística para formular recomendaciones objetivas de gestión. Estas incluyeron el corte de pasto, reducción en la intensidad de manejo, aumento de hábitat o combinaciones de estas recomendaciones. Los resultados resaltan la importancia de la gestión cuidadosa cuando la calidad del sitio está determinada por múltiples factores. El método cuantitativo de preservación utilizado puede ser extendido exitosamente a otras especies en peligro, siempre que sean suficientemente conocidos sus requerimientos ecológicos y la forma en que le afectan las acciones de manejo. [source] The Role of Functionalisation, Asymmetry and Shape of a New Macrocyclic Compartmental Ligand in the Formation of Mononuclear, Homo- and Heterodinuclear Lanthanide(III) ComplexesEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 1 2009Sergio Tamburini Abstract The compartmental [1+1] macrocycle H3L, obtained by self-condensation of the formyl precursor 3,3,-(3,6-dioxaoctane-1,8-diyldioxy)bis(2-hydroxybenzaldehyde) with the amine precursor N,N -bis(2-aminoethyl)-2-hydroxybenzylamine, contains one inner ON3O2 Schiff base and one outer O2O4 crown-like chamber. According to the experimental conditions it forms, by a template process, the stable mononuclear complexes Ln(H3L)(Cl)2(CH3COO)·nS·mHCl or [Ln(L)]·nS (Ln = La, Lu, Y, Yb, Er, Dy, Tb, Gd, Eu, Ce) with the lanthanide(III) ion encapsulated in the crown-ether-like and in the Schiff base site. The mononuclear complexes Ln(H3L)(Cl)2(CH3COO)·nS·mHCl, by further complexation with a different lanthanide(III) ion, give rise to the related heterodinuclear complexes [LnLn,(L)(Cl)2(CH3COO)]·nS while the homodinuclear and the heterodinuclear complexes [Ln2(L)](Cl)3·nH2O and [LnLn,(L)](Cl)3·nS could be prepared by a template reaction using the appropriate molar ratio of reactants. Their properties have been studied by using SEM-EDS microscopy, IR and NMR spectroscopy and their compositions confirmed by thermal and ESI-Mass spectrometric analyses. In the heterodinuclear complexes, the site occupancy of the different lanthanide(III) ions was determined by 1H and 13C NMR spectroscopy in CD3OD or (CD3)2SO , it was found that heterodinuclear complexation occurs in methanol with the smaller lanthanide(III) ion mainly coordinating to the Schiff base site and the larger lanthanide(III) ion to the crown site whereas, in dimethyl sulfoxide, demetalation of the weaker coordinated lanthanide(III) ion into the crown ether chamber occurs with the subsequent formation of mononuclear species in solution. The thermal decomposition of the heterodinuclear complexes forms the related mixed oxides, the stoichiometries and properties of which were determined by SEM-EDS microscopy and X-ray powder diffraction studies (XRD). (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source] Factors influencing Willow Tit Poecile montanus site occupancy: a comparison of abandoned and occupied woodsIBIS, Issue 2007ALEX J. G. LEWIS The British Willow Tit Poecile montanus kleinschmidti underwent a decline of 85% between 1970 and 2003. The cause of this decline is unknown. However, several hypotheses have been put forward to account for it: competition from other tit species, predation by Great Spotted Woodpeckers Dendrocopos major and habitat change. In order to test these, woods that are currently occupied by Willow Tits were paired with woods (within 50 km) that had been abandoned by Willow Tits five or more years previously. Point counts for other tit species (potential competitors) and woodpecker species (potential predators) were carried out at ten evenly spaced points throughout each wood. Habitat variables were collected within a 50-m radius of where a Willow Tit was located (in the occupied woods) or where maps showed a Willow Tit had been located (for abandoned woods). No evidence was found for differences in numbers of potential competitor or potential predator species in abandoned and occupied woods. Soil water content was found to be higher at occupied sites. No other habitat features differed between the two categories of site. The drying up of British woods could therefore be implicated in the Willow Tit decline and this warrants further investigation. [source] Abundance and co-occurrence patterns of core and satellite species of ground beetles on small lake islandsOIKOS, Issue 2 2006Werner Ulrich 15 lake islands and two mainland sites of Mamry lake in Poland were sampled to investigate community structures and patterns of co-occurrences of ground beetles (Carabidae). The total ground beetle metacommunity of 71 species was divided into a group of core species occupying at least half of all study sites and of satellite species, which occurred at two sites or less. This division is mirrored by reduced dispersal abilities and non-random patterns of site occupancy. Core and satellite species also differed in patterns of relative abundance. The core group followed a lognormal distribution, the satellite group a power function as predicted by the self-similarity model of occurrence. We conclude that the division into core and satellite species is not a sample artefact but reflects different life history strategies. We also conclude that current models of niche division and co-occurrence might miss important aspects of community structure if they do not refer to patterns of dispersal. From these findings we infer that the regional distribution of core species might be shaped by species interactions and processes of niche divisions whereas the spatial distribution of satellite species are best interpreted as stemming from random dispersal. [source] Synthesis and Seebeck coefficient of nanostructured phosphorus-alloyed bismuth telluride thick filmsPHYSICA STATUS SOLIDI (C) - CURRENT TOPICS IN SOLID STATE PHYSICS, Issue 11 2008Jian Zhou Abstract Phosphorous-alloyed Bi2Te3 thick films have been prepared by electrochemical deposition. The average grain size of the films was calculated to be 14-26 nm based on Scherrer's equation. The effect of P on the Seebeck coefficient of thermoelectric P-alloyed Bi2Te3 thick film was investigated. The results show that P-alloyed thick film has n-type conductivity with the Seebeck coefficient of -35 ,V/K. The correlation between P site occupancy in the crystal and the Seebeck coefficient was discussed. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Neutron powder diffraction study of orthorhombic and monoclinic defective silicaliteACTA CRYSTALLOGRAPHICA SECTION B, Issue 1 2000G. Artioli The crystal structure of silicalite (SiO2) with a substantial amount of structural hydroxyl groups [Si(1,x)O(2,4x)(OH)4x, with 0.08 < x < 0.10] has been refined from neutron powder diffraction data measured using the HRPD instrument at the ISIS pulsed neutron source. Powder data were collected on the as-synthesized orthorhombic sample at 298,K, and on the deuterated monoclinic sample at 100,K. Preferential location of Si-atom vacancies was found on four out of 12 independent T sites in the orthorhombic silicalite [Si(6), Si(7), Si(10) and Si(11)], although the H atoms of the substituting hydroxyl groups could not be located because of the low statistical site occupancy on multiple sites. No significant population of D atoms or of Si vacancies was found in the tetrahedral sites of the monoclinic sample. The detected long-range order of adjacent Si atoms in defective orthorhombic [MFI] structures is compatible with a mechanism of Si vacancy clustering and with the model of hydroxyl nests assumed in the literature on the basis of IR spectroscopic evidence. Crystal data: orthorhombic, Pnma, Z = 8, a = 20.0511,(1), b = 19.8757,(1), c = 13.36823,(9),Å, V = 5327.62,(5),Å3, Dx = 1.798,g,cm,3, Mr = 721.01; monoclinic, P21/n, Z = 4, a = 19.8352,(2), b = 20.0903,(2), c = 13.3588,(1),Å, , = 90.892,(1)°, V = 5322.78,(6),Å3, Dx = 1.799,g,cm,3, Mr = 1442.02. [source] Old growth and secondary forest site occupancy by nocturnal birds in a neotropical landscapeANIMAL CONSERVATION, Issue 1 2010M. Sberze Abstract High rates of old growth (OG) forest destruction and difficult farming conditions result in increasing cover of secondary forests (SF) in the Amazon. In this setting, it is opportune to ask which animals use newly available SF and which stay restricted to OG. This study presents a comparison of SF and OG site occupancy by nocturnal birds in terra firme forests of the Amazon Guianan shield, north of Manaus, Brazil. We tested species-specific occupancy predictions for two owls (Lophostrix cristata/Glaucidium hardyi), two potoos (Nyctibius leucopterus/Nyctibius griseus) and two nightjars (Caprimulgus nigrescens/Nyctidromus albicollis). For each pair, we predicted that one species would have higher occupancy in OG while the other would either be indifferent to forest type or favor SF sites. Data were collected in 30 OG and 24 SF sites with monthly samples from December 2007 to December 2008. Our analytic approach accounts for the possibility of detection failure and for spatial autocorrelation in occupancy, thus leading to strong inferences about changes in occupancy between forest types and between species. Nocturnal bird richness and community composition were indistinguishable between OG and SF sites. Owls were relatively indifferent to forest type. Potoos followed the a priori predictions, and one of the nightjars (C. nigrescens) favored SF instead of OG as predicted. Only one species, Nyctib. leucopterus, clearly favored OG. The landscape context of our SF study sites, surrounded by a vast expanse of continuous OG forest, partially explains the resemblance between SF and OG fauna but leaves unexplained the higher occupancy for SF than OG sites for several study species. The causal explanation of high SF occupancy remains an open question, but the result itself motivates further comparisons for other groups, as well as recognition of the conservation potential of SF. [source] Di-,-oxido-bis{bis[N,N,-bis(2-pyridylmethyl)ethane-1,2-diamine]manganese(III,IV)} tris(perchlorate) hexahydrate: clarification of an order,disorder phase transitionACTA CRYSTALLOGRAPHICA SECTION C, Issue 11 2009Anne Nielsen The title compound, [Mn2O2(C14H18N4)2](ClO4)3·6H2O, contains a mixed-valent MnIII/MnIV complex. In accordance with a previous report [Collins, Hodgson, Michelsen & Towle (1987). J. Chem. Soc. Chem. Commun. pp. 1659,1660], the structure at 295,K is best described in the space group C2/c, with the complex exhibiting twofold rotational symmetry, and with half site occupancy for one perchlorate anion and several solvent water molecules. At 180,K, the structure is ordered in the subgroup P21/n and is clearly shown to be a hexahydrate, rather than the previously reported trihydrate. The origin of the order,disorder phase transition lies in the thermal motion of the perchlorate anions. [source] Non-linear conductivity in Coulomb glassesANNALEN DER PHYSIK, Issue 12 2009M. Caravaca Abstract We have studied the nonlinear conductivity of two-dimensional Coulomb glasses. We have used a Monte Carlo algorithm to simulate the dynamic of the system under an applied electric field E. We have compared results for two different models: a regular square lattice with only diagonal disorder and a random array of sites with diagonal and off-diagonal disorder. We have found that for moderate fields the logarithm of the conductivity is proportional to , reproducing experimental results. We have also found that in the nonlinear regime the site occupancy in the Coulomb gap follows a Fermi-Dirac distribution with an effective temperature Teff higher than the phonon bath temperature T. [source] Site Occupancy Models with Heterogeneous Detection ProbabilitiesBIOMETRICS, Issue 1 2006J. Andrew Royle Summary Models for estimating the probability of occurrence of a species in the presence of imperfect detection are important in many ecological disciplines. In these "site occupancy" models, the possibility of heterogeneity in detection probabilities among sites must be considered because variation in abundance (and other factors) among sampled sites induces variation in detection probability (p). In this article, I develop occurrence probability models that allow for heterogeneous detection probabilities by considering several common classes of mixture distributions for p. For any mixing distribution, the likelihood has the general form of a zero-inflated binomial mixture for which inference based upon integrated likelihood is straightforward. A recent paper by Link (2003, Biometrics59, 1123,1130) demonstrates that in closed population models used for estimating population size, different classes of mixture distributions are indistinguishable from data, yet can produce very different inferences about population size. I demonstrate that this problem can also arise in models for estimating site occupancy in the presence of heterogeneous detection probabilities. The implications of this are discussed in the context of an application to avian survey data and the development of animal monitoring programs. [source] Amino acid and manganese supplementation modulates the glycosylation state of erythropoietin in a CHO culture systemBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2007Christopher K. Crowell Abstract The manufacture of secreted proteins is complicated by the need for both high levels of expression and appropriate processing of the nascent polypeptide. For glycoproteins, such as erythropoietin (EPO), posttranslational processing involves the addition of oligosaccharide chains. We initially noted that a subset of the amino acids present in the cell culture media had become depleted by cellular metabolism during the last harvest cycle in our batch fed system and hypothesized that by supplementing these nutrients we would improve EPO yields. By increasing the concentration of these amino acids we increased recombinant human erythropoietin (rHuEPO) biosynthesis in the last harvest cycle as expected but, surprisingly, we also observed a large increase in the amount of rHuEPO with a relatively low sialic acid content. To understand the nature of this process we isolated and characterized the lower sialylated rHuEPO pool. Decreased sialylation correlated with an increase in N-linked carbohydrates missing terminal galactose moieties, suggesting that ,-1,4-galactosyltransferase may be rate limiting in our system. To test this hypothesis we supplemented our cultures with varying concentrations of manganese (Mn2+), a cofactor for ,-1,4-galactosyltransferase. Consistent with our hypothesis we found that Mn2+ addition improved galactosylation and greatly reduced the amount of rHuEPO in the lower sialylated fraction. Additionally, we found that Mn2+ addition increased carbohydrate site occupancy and narrowed carbohydrate branching to bi-antennary structures in these lower sialylated pools. Surprisingly Mn2+ only had this effect late in the culture process. These data indicate that the addition of Mn2+ has complex effects on stressed batch fed cultures. Biotechnol. Bioeng. 2007;96: 538,549. © 2006 Wiley Periodicals, Inc. [source] RNA interference of sialidase improves glycoprotein sialic acid content consistencyBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Frederyk A. Ngantung Abstract An important challenge facing therapeutic protein production in mammalian cell culture is the cleavage of terminal sialic acids on recombinant protein glycans by the glycosidase enzymes released by lysed cells into the supernatant. This undesired phenomenon results in a protein product which is rapidly cleared from the plasma by asialoglycoprotein receptors in the liver. In this study, RNA interference was utilized as a genetic approach to silence the activity of sialidase, a glycosidase responsible for cleaving terminal sialic acids on IFN-, produced by Chinese Hamster Ovary (CHO) cells. We first identified a 21-nt double stranded siRNA that reduced endogenous sialidase mRNA and protein activity levels. Potency of each siRNA sequences was compared using real time RT-PCR and a sialidase activity assay. We next integrated the siRNA sequence into CHO cells, allowing production and selection of stable cell lines. We isolated stable clones with sialidase activity reduced by over 60% as compared to the control cell line. Micellar electrokinetic chromatography (MEKC), thiobarbituric acid assay (TAA), and high performance anion exchange chromatography (HPAEC) coupled to amperometric detection were performed to analyze glycan site occupancy, sialic acid content, and distribution of asialo-/sialylated-glycan structures, respectively. Two of the stable clones successfully retained the full sialic acid content of the recombinant IFN-,, even upon cells' death. This was comparable to the case where a chemically synthesized sialidase inhibitor was used. These results demonstrated that RNA interference of sialidase can prevent the desialylation problem in glycoprotein production, resulting improved protein quality during the entire cell culture process. © 2006 Wiley Periodicals, Inc. [source] |