Molecular studies on a Dictyostelium homolog of the tafazzin gene, the cause of Barth syndrome in humans

Der eukaryotische Mikroorganismus Dictyostelium discoideum lebt als einzellige Amöbe solange ausreichende Nahrungsressourcen zur Verfügung stehen. Sobald Nahrungsmangel eintritt, entwickeln sich die Zellen von einem einzelligen zu einem mehrzelligen Zustand, der mit einem multizellulären Fruchtkörper abschließt. Dieser Prozess wird durch eine Reihe aufeinanderfolgender Signale organisiert, die eine differentielle Genexpression regulieren. Die Gene der Discoidin I Familie gehören zu den Ersten, die im Laufe des Wachstums-Differenzierungs-Übergangs (engl. GDT) aktiviert werden. Sie eignen sich daher vorzüglich als Marker für den Beginn der Entwicklung. Mit Hilfe einer REMI-Mutagenese und Discoidin I als molekularem Marker sind verschiedene Komponenten des Wachstums-Differenzierungs-Übergangs in unserer Arbeitsgruppe identifiziert worden (Zeng et al., 2000 A und B; Riemann und Nellen, persönliche Mitteilung). Mit demselben Ansatz wurde in der vorliegenden Arbeit eine REMI-Mutante identifiziert, die eine Fehl-Expression von Discoidin zeigte und einen axenischen Wachstumsdefekt bei 15 °C aufwies. Das Gen wurde als Homolog zum humanen Tafazzin-Gen identifiziert. Dieses Gen wurde zur Rekonstruktion des Phänotyps über homologe Rekombination erneut disruptiert, was wie erwartet zu dem zuerst beschriebenen Phänotyp führte. Folgerichtig ergab eine Überexpression des Gens in den Mutanten eine Komplementation des Phänotyps. Immunfluoreszenz-Experimente zeigten eine mitochondriale Lokalisation des Dictyostelium discoideum Taffazzin Proteins. Dass ein mitochondriales Protein in Zusammenhang mit dem Wachstums-Differenzierungs-Übergang steht, ist ein unerwarteter Befund, der aber als Hinweis darauf gewertet werden kann, dass Mitochondrien einen direkten Einfluss auf die entwicklungsspezifische Signaltransduktion ausüben. Die Taffazzin Disruptions-Mutante in Dictyostelium führte zu einem abnormalen Cardiolipin Metabolismus. Dieses Phospholipid ist ein charakteristischer Bestandteil der inneren Mitochondrienmembran und für die Funktion verschiedener Enzyme erforderlich. Unsere vorläufigen Analysen des Phospholipid-Gehalts zeigten Übereinstimmung mit Daten von Patienten mit Barth-Syndrom, einer humanen Erkrankung, bei der das Taffazzin-Gen Mutationen aufweist, und mit Hefe-Mutanten dieses Gens. Dies zeigt den Wert von Dictyostelium discoideum als einen weiteren Modelorganismus zur Untersuchung des Barth-Syndroms und zur Erprobung möglicher Therapieansätze.

Solution properties of the de Branges differential recurrence equation

In this 1984 proof of the Bieberbach and Milin conjectures de Branges used a positivity result of special functions which follows from an identity about Jacobi polynomial sums thas was published by Askey and Gasper in 1976. The de Branges functions Tn/k(t) are defined as the solutions of a system of differential recurrence equations with suitably given initial values. The essential fact used in the proof of the Bieberbach and Milin conjectures is the statement Tn/k(t)<=0. In 1991 Weinstein presented another proof of the Bieberbach and Milin conjectures, also using a special function system Λn/k(t) which (by Todorov and Wilf) was realized to be directly connected with de Branges', Tn/k(t)=-kΛn/k(t), and the positivity results in both proofs Tn/k(t)<=0 are essentially the same. In this paper we study differential recurrence equations equivalent to de Branges' original ones and show that many solutions of these differential recurrence equations don't change sign so that the above inequality is not as surprising as expected. Furthermore, we present a multiparameterized hypergeometric family of solutions of the de Branges differential recurrence equations showing that solutions are not rare at all.

Systematics and evolution of the genus Deuterocohnia Mez (Bromeliaceae)

The present study investigates the systematics and evolution of the Neotropical genus Deuterocohnia Mez (Bromeliaceae). It provides a comprehensive taxonomic revision as well as phylogenetic analyses based on chloroplast and nuclear DNA sequences and presents a hypothesis on the evolution of the genus. A broad morphological, anatomical, biogeographical and ecological overview of the genus is given in the first part of the study. For morphological character assessment more than 700 herbarium specimens from 39 herbaria as well as living plant material in the field and in the living collections of botanical gardens were carefully examined. The arid habitats, in which the species of Deuterocohnia grow, are reflected by the morphological and anatomical characters of the species. Important characters for species delimitation were identified, like the length of the inflorescence, the branching order, the density of flowers on partial inflorescences, the relation of the length of the primary bracts to that of the partial inflorescence, the sizes of floral bracts, sepals and petals, flower colour, the presence or absence of a pedicel, the curvature of the stamina and the petals during anthesis. After scrutinizing the nomenclatural history of the taxa belonging to Deuterocohnia – including the 1992 syonymized genus Abromeitiella – 17 species, 4 subspecies and 4 varieties are accepted in the present revision. Taxonomic changes were made in the following cases: (I) New combinations: A. abstrusa (A. Cast.) N. Schütz is re-established – as defined by Castellanos (1931) – and transfered to D. abstrusa; D. brevifolia (Griseb.) M.A. Spencer & L.B. Sm. includes accessions of the former D. lorentziana (Mez) M.A. Spencer & L.B. Sm., which are not assigned to D. abstrusa; D. bracteosa W. Till is synonymized to D. strobilifera Mez; D. meziana Kuntze ex Mez var. carmineo-viridiflora Rauh is classified as a subspecies of D. meziana (ssp. carmineo-viridiflora (Rauh) N. Schütz); D. pedicellata W. Till is classified as a subspecies of D. meziana (ssp. pedicellata (W. Till) N. Schütz); D. scapigera (Rauh & L. Hrom.) M.A. Spencer & L.B. Sm ssp. sanctae-crucis R. Vásquez & Ibisch is classified as a species (D. sanctae-crucis (R. Vásquez & Ibisch) N. Schütz); (II) New taxa: a new subspecies of D. meziana Kuntze ex Mez is established; a new variety of D. scapigera is established; (the new taxa will be validly published elsewhere); (III) New type: an epitype for D. longipetala was chosen. All other species were kept according to Spencer and Smith (1992) or – in the case of more recently described species – according to the protologue. Beside the nomenclatural notes and the detailed descriptions, information on distribution, habitat and ecology, etymology and taxonomic delimitation is provided for the genus and for each of its species. An key was constructed for the identification of currently accepted species, subspecies and varieties. The key is based on easily detectable morphological characters. The former synonymization of the genus Abromeitiella into Deuterocohnia (Spencer and Smith 1992) is re-evalutated in the present study. Morphological as well as molecular investigations revealed Deuterocohnia incl. Abromeitiella as being monophyletic, with some indications that a monophyletic Abromeitiella lineage arose from within Deuterocohnia. Thus the union of both genera is confirmed. The second part of the present thesis describes and discusses the molecular phylogenies and networks. Molecular analyses of three chloroplast intergenic spacers (rpl32-trnL, rps16-trnK, trnS-ycf3) were conducted with a sample set of 119 taxa. This set included 103 Deuterocohnia accessions from all 17 described species of the genus and 16 outgroup taxa from the remainder of Pitcairnioideae s.str. (Dyckia (8 sp.), Encholirium (2 sp.), Fosterella (4 sp.) and Pitcairnia (2 sp.)). With its high sampling density, the present investigation by far represents the most comprehensive molecular study of Deuterocohnia up till now. All data sets were analyzed separately as well as in combination, and various optimality criteria for phylogenetic tree construction were applied (Maximum Parsimony, Maximum Likelihood, Bayesian inferences and the distance method Neighbour Joining). Congruent topologies were generally obtained with different algorithms and optimality criteria, but individual clades received different degrees of statistical support in some analyses. The rps16-trnK locus was the most informative among the three spacer regions examined. The results of the chloroplast DNA analyses revealed a highly supported paraphyly of Deuterocohnia. Thus, the cpDNA trees divide the genus into two subclades (A and B), of which Deuterocohnia subclade B is sister to the included Dyckia and Encholirium accessions, and both together are sister to Deuterocohnia subclade A. To further examine the relationship between Deuterocohnia and Dyckia/Encholirium at the generic level, two nuclear low copy markers (PRK exon2-5 and PHYC exon1) were analysed with a reduced taxon set. This set included 22 Deuterocohnia accessions (including members of both cpDNA subclades), 2 Dyckia, 2 Encholirium and 2 Fosterella species. Phylogenetic trees were constructed as described above, and for comparison the same reduced taxon set was also analysed at the three cpDNA data loci. In contrast to the cpDNA results, the nuclear DNA data strongly supported the monophyly of Deuterocohnia, which takes a sister position to a clade of Dyckia and Encholirium samples. As morphology as well as nuclear DNA data generated in the present study and in a former AFLP analysis (Horres 2003) all corroborate the monophyly of Deuterocohnia, the apparent paraphyly displayed in cpDNA analyses is interpreted to be the consequence of a chloroplast capture event. This involves the introgression of the chloroplast genome from the common ancestor of the Dyckia/ Encholirium lineage into the ancestor of Deuterocohnia subclade B species. The chloroplast haplotypes are not species-specific in Deuterocohnia. Thus, one haplotype was sometimes shared by several species, where the same species may harbour different haplotypes. The arrangement of haplotypes followed geographical patterns rather than taxonomic boundaries, which may indicate some residual gene flow among populations from different Deuteroccohnia species. Phenotypic species coherence on the background of ongoing gene flow may then be maintained by sets of co-adapted alleles, as was suggested by the porous genome concept (Wu 2001, Palma-Silva et al. 2011). The results of the present study suggest the following scenario for the evolution of Deuterocohnia and its species. Deuterocohnia longipetala may be envisaged as a representative of the ancestral state within the genus. This is supported by (1) the wide distribution of this species; (2) the overlap in distribution area with species of Dyckia; (3) the laxly flowered inflorescences, which are also typical for Dyckia; (4) the yellow petals with a greenish tip, present in most other Deuterocohnia species. The following six extant lineages within Deuterocohnia might have independently been derived from this ancestral state with a few changes each: (I) D. meziana, D. brevispicata and D. seramisiana (Bolivia, lowland to montane areas, mostly reddish-greenish coloured, very laxly to very densely flowered); (II) D. strobilifera (Bolivia, high Andean mountains, yellow flowers, densely flowered); (III) D. glandulosa (Bolivia, montane areas, yellow-greenish flowers, densely flowered); (IV) D. haumanii, D. schreiteri, D. digitata, and D. chrysantha (Argentina, Chile, E Andean mountains and Atacama desert, yellow-greenish flowers, densely flowered); (V) D. recurvipetala (Argentina, foothills of the Andes, recurved yellow flowers, laxly flowered); (VI) D. gableana, D. scapigera, D. sanctae-crucis, D. abstrusa, D. brevifolia, D. lotteae (former Abromeitiella species, Bolivia, Argentina, higher Andean mountains, greenish-yellow flowers, inflorescence usually simple). Originating from the lower montane Andean regions, at least four lineages of the genus (I, II, IV, VI) adapted in part to higher altitudes by developing densely flowered partial inflorescences, shorter flowers and – in at least three lineages (II, IV, VI) – smaller rosettes, whereas species spreading into the lowlands (I, V) developed larger plants, laxly flowered, amply branched inflorescences and in part larger flowers (I).

Time Discretization of the SST-generalized Navier-Stokes Equations: Positive and Negative Results

In the theory of the Navier-Stokes equations, the proofs of some basic known results, like for example the uniqueness of solutions to the stationary Navier-Stokes equations under smallness assumptions on the data or the stability of certain time discretization schemes, actually only use a small range of properties and are therefore valid in a more general context. This observation leads us to introduce the concept of SST spaces, a generalization of the functional setting for the Navier-Stokes equations. It allows us to prove (by means of counterexamples) that several uniqueness and stability conjectures that are still open in the case of the Navier-Stokes equations have a negative answer in the larger class of SST spaces, thereby showing that proof strategies used for a number of classical results are not sufficient to affirmatively answer these open questions. More precisely, in the larger class of SST spaces, non-uniqueness phenomena can be observed for the implicit Euler scheme, for two nonlinear versions of the Crank-Nicolson scheme, for the fractional step theta scheme, and for the SST-generalized stationary Navier-Stokes equations. As far as stability is concerned, a linear version of the Euler scheme, a nonlinear version of the Crank-Nicolson scheme, and the fractional step theta scheme turn out to be non-stable in the class of SST spaces. The positive results established in this thesis include the generalization of classical uniqueness and stability results to SST spaces, the uniqueness of solutions (under smallness assumptions) to two nonlinear versions of the Euler scheme, two nonlinear versions of the Crank-Nicolson scheme, and the fractional step theta scheme for general SST spaces, the second order convergence of a version of the Crank-Nicolson scheme, and a new proof of the first order convergence of the implicit Euler scheme for the Navier-Stokes equations. For each convergence result, we provide conditions on the data that guarantee the existence of nonstationary solutions satisfying the regularity assumptions needed for the corresponding convergence theorem. In the case of the Crank-Nicolson scheme, this involves a compatibility condition at the corner of the space-time cylinder, which can be satisfied via a suitable prescription of the initial acceleration.

Markov chain montecarlo method applied to rounding zeros of compositional data: first approach

As stated in Aitchison (1986), a proper study of relative variation in a compositional data set should be based on logratios, and dealing with logratios excludes dealing with zeros. Nevertheless, it is clear that zero observations might be present in real data sets, either because the corresponding part is completely absent –essential zeros– or because it is below detection limit –rounded zeros. Because the second kind of zeros is usually understood as “a trace too small to measure”, it seems reasonable to replace them by a suitable small value, and this has been the traditional approach. As stated, e.g. by Tauber (1999) and by Martín-Fernández, Barceló-Vidal, and Pawlowsky-Glahn (2000), the principal problem in compositional data analysis is related to rounded zeros. One should be careful to use a replacement strategy that does not seriously distort the general structure of the data. In particular, the covariance structure of the involved parts –and thus the metric properties– should be preserved, as otherwise further analysis on subpopulations could be misleading. Following this point of view, a non-parametric imputation method is introduced in Martín-Fernández, Barceló-Vidal, and Pawlowsky-Glahn (2000). This method is analyzed in depth by Martín-Fernández, Barceló-Vidal, and Pawlowsky-Glahn (2003) where it is shown that the theoretical drawbacks of the additive zero replacement method proposed in Aitchison (1986) can be overcome using a new multiplicative approach on the non-zero parts of a composition. The new approach has reasonable properties from a compositional point of view. In particular, it is “natural” in the sense that it recovers the “true” composition if replacement values are identical to the missing values, and it is coherent with the basic operations on the simplex. This coherence implies that the covariance structure of subcompositions with no zeros is preserved. As a generalization of the multiplicative replacement, in the same paper a substitution method for missing values on compositional data sets is introduced

An analysis of the anxiolytic effects of ethanol on consummatory successive negative contrast

The anxiolytic properties of ethanol (1 g/kg, 15% dose, i.p.) were studied in two experiments with rats involving incentive downshifts from a 32% to a 4% sucrose solution. In Experiment 1, alcohol administration before a downshift from 32% to 4% sucrose prevented the development of consummatory suppression (consummatory successive negative contrast, cSNC). In Experiment 2, ethanol prevented the attenuating effects of partial reinforcement (random sequence of 32% sucrose and nothing) on cSNC, causing a retardation of recovery from contrast. These effects of ethanol on cSNC are analogous to those described for the benzodiazepine anxiolytic chlordiazepoxide, suggesting that at least some of its anxiolytic effects are mediated by the same mechanisms. 

A cell by cell anisotropic adaptive mesh ALE scheme for the numerical solution of the Euler equations

In this paper a cell by cell anisotropic adaptive mesh technique is added to an existing staggered mesh Lagrange plus remap finite element ALE code for the solution of the Euler equations. The quadrilateral finite elements may be subdivided isotropically or anisotropically and a hierarchical data structure is employed. An efficient computational method is proposed, which only solves on the finest level of resolution that exists for each part of the domain with disjoint or hanging nodes being used at resolution transitions. The Lagrangian, equipotential mesh relaxation and advection (solution remapping) steps are generalised so that they may be applied on the dynamic mesh. It is shown that for a radial Sod problem and a two-dimensional Riemann problem the anisotropic adaptive mesh method runs over eight times faster.

On the convergence of the no response test

The no response test is a new scheme in inverse problems for partial differential equations which was recently proposed in [D. R. Luke and R. Potthast, SIAM J. Appl. Math., 63 (2003), pp. 1292–1312] in the framework of inverse acoustic scattering problems. The main idea of the scheme is to construct special probing waves which are small on some test domain. Then the response for these waves is constructed. If the response is small, the unknown object is assumed to be a subset of the test domain. The response is constructed from one, several, or many particular solutions of the problem under consideration. In this paper, we investigate the convergence of the no response test for the reconstruction information about inclusions D from the Cauchy values of solutions to the Helmholtz equation on an outer surface $\partial\Omega$ with $\overline{D} \subset \Omega$. We show that the one‐wave no response test provides a criterion to test the analytic extensibility of a field. In particular, we investigate the construction of approximations for the set of singular points $N(u)$ of the total fields u from one given pair of Cauchy data. Thus, the no response test solves a particular version of the classical Cauchy problem. Also, if an infinite number of fields is given, we prove that a multifield version of the no response test reconstructs the unknown inclusion D. This is the first convergence analysis which could be achieved for the no response test.

Investigation of the effects of the novel anticonvulsant compound carisbamate(RWJ-333369) on rat piriform cortical neurones in vitro

Background and purpose: Carisbamate is being developed for adjuvant treatment of partial onset epilepsy. Carisbamate produces anticonvulsant effects in primary generalized, complex partial and absence-type seizure models, and exhibits neuroprotective and antiepileptogenic properties in rodent epilepsy models. Phase IIb clinical trials of carisbamate demonstrated efficacy against partial onset seizures; however, its mechanisms of action remain unknown. Here, we report the effects of carisbamate on membrane properties, evoked and spontaneous synaptic transmission and induced epileptiform discharges in layer II-III neurones in piriform cortical brain slices. Experimental approach: Effects of carisbamate were investigated in rat piriform cortical neurones by using intracellular electrophysiological recordings. Key results: Carisbamate (50–400 mmol·L-1) reversibly decreased amplitude, duration and rise-time of evoked action potentials and inhibited repetitive firing, consistent with use-dependent Na+ channel block; 150–400 mmol·L-1 carisbamate reduced neuronal input resistance, without altering membrane potential. After microelectrode intracellular Cl- loading, carisbamate depolarized cells, an effect reversed by picrotoxin. Carisbamate (100–400 mmol·L-1) also selectively depressed lateral olfactory tract-afferent evoked excitatory synaptic transmission (opposed by picrotoxin), consistent with activation of a presynaptic Cl conductance. Lidocaine (40–320 mmol·L-1) mimicked carisbamate, implying similar modes of action. Carisbamate (300–600 mmol·L-1) had no effect on spontaneous GABAA miniature inhibitory postsynaptic currents and at lower concentrations (50–200 mmol·L-1) inhibited Mg2+-free or 4-aminopyridine-induced seizure-like discharges. Conclusions and implications: Carisbamate blocked evoked action potentials use-dependently, consistent with a primary action on Na+ channels and increased Cl- conductances presynaptically and, under certain conditions, postsynaptically to selectively depress excitatory neurotransmission in piriform cortical layer Ia-afferent terminals.

The relationship between diffuse spectral reflectance of the soil and its cation exchange capacity is scale-dependent

Diffuse reflectance spectroscopy (DRS) is increasingly being used to predict numerous soil physical, chemical and biochemical properties. However, soil properties and processes vary at different scales and, as a result, relationships between soil properties often depend on scale. In this paper we report on how the relationship between one such property, cation exchange capacity (CEC), and the DRS of the soil depends on spatial scale. We show this by means of a nested analysis of covariance of soils sampled on a balanced nested design in a 16 km × 16 km area in eastern England. We used principal components analysis on the DRS to obtain a reduced number of variables while retaining key variation. The first principal component accounted for 99.8% of the total variance, the second for 0.14%. Nested analysis of the variation in the CEC and the two principal components showed that the substantial variance components are at the > 2000-m scale. This is probably the result of differences in soil composition due to parent material. We then developed a model to predict CEC from the DRS and used partial least squares (PLS) regression do to so. Leave-one-out cross-validation results suggested a reasonable predictive capability (R2 = 0.71 and RMSE = 0.048 molc kg− 1). However, the results from the independent validation were not as good, with R2 = 0.27, RMSE = 0.056 molc kg− 1 and an overall correlation of 0.52. This would indicate that DRS may not be useful for predictions of CEC. When we applied the analysis of covariance between predicted and observed we found significant scale-dependent correlations at scales of 50 and 500 m (0.82 and 0.73 respectively). DRS measurements can therefore be useful to predict CEC if predictions are required, for example, at the field scale (50 m). This study illustrates that the relationship between DRS and soil properties is scale-dependent and that this scale dependency has important consequences for prediction of soil properties from DRS data

Ethnobiology and the evolution of the human mind

An evolutionary perspective on human thought and behaviour indicates that we should expect to find universal systems of perception, classification, and decision-making regarding the natural world. It is the interaction between these evolved aspects of the human mind, the biodiversity of the natural world, and unique historical, social, and economic contexts within which individuals develop and act that gives rise to cultural diversity. The palaeoanthropological record also indicates that language is a recently evolved phenomenon. This suggests that linguistic approaches in ethnobiology are likely to provide only a partial understanding of how humans perceive, classify, and engage with the natural world.

Unification of the probe and singular sources methods for the inverse boundary value problem by the no-response test

In this article, we use the no-response test idea, introduced in Luke and Potthast (2003) and Potthast (Preprint) and the inverse obstacle problem, to identify the interface of the discontinuity of the coefficient gamma of the equation del (.) gamma(x)del + c(x) with piecewise regular gamma and bounded function c(x). We use infinitely many Cauchy data as measurement and give a reconstructive method to localize the interface. We will base this multiwave version of the no-response test on two different proofs. The first one contains a pointwise estimate as used by the singular sources method. The second one is built on an energy (or an integral) estimate which is the basis of the probe method. As a conclusion of this, the probe and the singular sources methods are equivalent regarding their convergence and the no-response test can be seen as a unified framework for these methods. As a further contribution, we provide a formula to reconstruct the values of the jump of gamma(x), x is an element of partial derivative D at the boundary. A second consequence of this formula is that the blow-up rate of the indicator functions of the probe and singular sources methods at the interface is given by the order of the singularity of the fundamental solution.

Spectral analysis of the elliptic sine-Gordon equation in the quarter plane

We study the elliptic sine-Gordon equation in the quarter plane using a spectral transform approach. We determine the Riemann-Hilbert problem associated with well-posed boundary value problems in this domain and use it to derive a formal representation of the solution. Our analysis is based on a generalization of the usual inverse scattering transform recently introduced by Fokas for studying linear elliptic problems.

ASYM20: a program for force constant and normal coordinate calculations, with a critical review of the theory involved

The theory of harmonic force constant refinement calculations is reviewed, and a general-purpose program for force constant and normal coordinate calculations is described. The program, called ASYM20. is available through Quantum Chemistry Program Exchange. It will work on molecules of any symmetry containing up to 20 atoms and will produce results on a series of isotopomers as desired. The vibrational secular equations are solved in either nonredundant valence internal coordinates or symmetry coordinates. As well as calculating the (harmonic) vibrational wavenumbers and normal coordinates, the program will calculate centrifugal distortion constants, Coriolis zeta constants, harmonic contributions to the α′s. root-mean-square amplitudes of vibration, and other quantities related to gas electron-diffraction studies and thermodynamic properties. The program will work in either a predict mode, in which it calculates results from an input force field, or in a refine mode, in which it refines an input force field by least squares to fit observed data on the quantities mentioned above. Predicate values of the force constants may be included in the data set for a least-squares refinement. The program is written in FORTRAN for use on a PC or a mainframe computer. Operation is mainly controlled by steering indices in the input data file, but some interactive control is also implemented.

Reintroduction of the 2G12 epitope in an HIV-1 clade C gp120

Many clade C isolates of HIV-1 do not react with monoclonal antibody (MAb) 2G12, a broad-ranging human neutralizing MAb that recognizes high mannose carbohydrate groups attached to glycoprotein gp120. We reintroduced a partial and complete 2G12 epitope into a clade C background, HIV-1(CN54), and examined the antibody reactivity of the resulting recombinant molecules. Two glycosylation sites recovered 2G12 binding completely, but some binding was evident after the reintroduction of a single glycosylation site at Asn295.