bluuebunny/crossref_metadata_2025 huggingface dataset:
A Patch of Surface-Exposed Residues Mediates Negative Regulation of Immune Signaling by Tomato Pto Kinase[W]
Authors: Wu Ai-Jiuan , Andriotis Vasilios M.E. , Durrant Marcus C. , Rathjen John P.
Published: October 2004

Tomato (Lycopersicon esculentum) Pto kinase specifically recognizes the Pseudomonas effector proteins AvrPto and AvrPtoB, leading to induction of defense responses and hypersensitive cell death. Structural modeling of Pto combined with site-directed mutagenesis identified a patch of surface-exposed residues required for native regulation of signaling. Mutations in this area resulted in constitutive gain-of-function (CGF) forms of Pto that activated AvrPto-independent cell death via the cognate signaling pathway. The patch overlaps the peptide binding region of the kinase catalytic cleft and is part of a broader region required for interaction with bacterial effectors. We propose that the negative regulatory patch is normally occupied by a peptide that represses Pto signaling. Furthermore, we found that Pto kinase activity was required for Avr-dependent activation but dispensable for signaling by CGF forms of Pto. This suggests that Pto signals by a conformational change rather than phosphorylation of downstream substrates in the defense signaling pathway.
Stimulus-Dependent, Promoter-Specific Binding of Transcription Factor WRKY1 to Its Native Promoter and the Defense-Related GenePcPR1-1in Parsley[W]
Authors: Turck Franziska , Zhou Aifen , Somssich Imre E.
Published: October 2004

WRKY transcription factors form a large family that plays a role in plant responses to biotic stress and during senescence. Defining in vivo relevant WRKY/promoter relationships has been hampered by the factors' indiscriminate binding to known W box DNA elements and their possible genetic redundance. Employing chromatin immunoprecipitations (ChIP) of cultured cells, we show that parsley (Petroselinum crispum) WRKY1 protein binds to the W boxes of its native promoter as well as to that of PcWRKY3 and the defense-related PR10-class marker gene Pathogenesis-Related1-1 (PcPR1-1). Although present at low concentrations in resting cells, WRKY1 does not appear to play a role in the immediate early gene response upon elicitation because it does not bind to the promoter at this time. Paradoxically, in vivo binding at the PcWRKY1 promoter correlates more with downregulation of gene expression, whereas previous overexpression studies suggested an activating function of WRKY1 on PcWRKY1 expression. By contrast, PcPR1-1 expression remains strong when its promoter is occupied in vivo by WRKY1. Unexpectedly, ChIP revealed that W boxes at promoter sites are constitutively occupied by other WRKY transcription factors, indicating that site recruitment does not seem to play a major role in their regulation. Rather, WRKY proteins very likely act in a network of mutually competing participants with temporal displacement occurring at defined preoccupied sites by other family members in a stimulus-dependent manner.
HANABA TARANU Is a GATA Transcription Factor That Regulates Shoot Apical Meristem and Flower Development in Arabidopsis[W]
Authors: Zhao Yuanxiang , Medrano Leonard , Ohashi Kazuaki , Fletcher Jennifer C. , Yu Hao , Sakai Hajime , Meyerowitz Elliot M.
Published: October 2004

We have isolated a new mutant, hanaba taranu (han), which affects both flower and shoot apical meristem (SAM) development in Arabidopsis thaliana. Mutants have fused sepals and reduced organ numbers in all four whorls, especially in the 2nd (petal) and 3rd (stamen) whorls. han meristems can become flatter or smaller than in the wild type. HAN encodes a GATA-3–like transcription factor with a single zinc finger domain. HAN is transcribed at the boundaries between the meristem and its newly initiated organ primordia and at the boundaries between different floral whorls. It is also expressed in vascular tissues, developing ovules and stamens, and in the embryo. han interacts strongly with clavata (clv) mutations (clv1, clv2, and clv3), resulting in highly fasciated SAMs, and we find that WUS expression is altered in han mutants from early embryogenesis. In addition, HAN is ectopically expressed both in clv1 and clv3 mutants. We propose that HAN is normally required for establishing organ boundaries in shoots and flowers and for controlling the number and position of WUS-expressing cells. Ectopic HAN expression causes growth retardation, aberrant cell division patterns, and loss of meristem activity, suggesting that HAN is involved in controlling cell proliferation and differentiation.
Spotted leaf11, a Negative Regulator of Plant Cell Death and Defense, Encodes a U-Box/Armadillo Repeat Protein Endowed with E3 Ubiquitin Ligase Activityw⃞
Authors: Zeng Li-Rong , Qu Shaohong , Bordeos Alicia , Yang Chengwei , Baraoidan Marietta , Yan Hongyan , Xie Qi , Nahm Baek Hie , Leung Hei , Wang Guo-Liang
Published: October 2004

The rice (Oryza sativa) spotted leaf11 (spl11) mutant was identified from an ethyl methanesulfonate–mutagenized indica cultivar IR68 population and was previously shown to display a spontaneous cell death phenotype and enhanced resistance to rice fungal and bacterial pathogens. Here, we have isolated Spl11 via a map-based cloning strategy. The isolation of the Spl11 gene was facilitated by the identification of three additional spl11 alleles from an IR64 mutant collection. The predicted SPL11 protein contains both a U-box domain and an armadillo (ARM) repeat domain, which were demonstrated in yeast and mammalian systems to be involved in ubiquitination and protein–protein interactions, respectively. Amino acid sequence comparison indicated that the similarity between SPL11 and other plant U-box-ARM proteins is mostly restricted to the U-box and ARM repeat regions. A single base substitution was detected in spl11, which results in a premature stop codon in the SPL11 protein. Expression analysis indicated that Spl11 is induced in both incompatible and compatible rice–blast interactions. In vitro ubiquitination assay indicated that the SPL11 protein possesses E3 ubiquitin ligase activity that is dependent on an intact U-box domain, suggesting a role of the ubiquitination system in the control of plant cell death and defense.
Biosynthesis of Very-Long-Chain Polyunsaturated Fatty Acids in Transgenic Oilseeds: Constraints on Their Accumulationw⃞
Authors: Abbadi Amine , Domergue Freéderic , Bauer Jörg , Napier Johnathan A. , Welti Ruth , Zähringer Ulrich , Cirpus Petra , Heinz Ernst
Published: October 2004

ω6- and ω3-polyunsaturated C20 fatty acids represent important components of the human diet. A more regular consumption and an accordingly sustainable source of these compounds are highly desirable. In contrast with the very high levels to which industrial fatty acids have to be enriched in plant oils for competitive use as chemical feedstocks, much lower percentages of very-long-chain polyunsaturated fatty acids (VLCPUFA) in edible plant oils would satisfy nutritional requirements. Seed-specific expression in transgenic tobacco (Nicotiana tabacum) and linseed (Linum usitatissimum) of cDNAs encoding fatty acyl-desaturases and elongases, absent from all agronomically important plants, resulted in the very high accumulation of Δ6-desaturated C18 fatty acids and up to 5% of C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acid. Detailed lipid analyses of developing seeds from transgenic plants were interpretated as indicating that, after desaturation on phosphatidylcholine, Δ6-desaturated products are immediately channeled to the triacylglycerols and effectively bypass the acyl-CoA pool. Thus, the lack of available Δ6-desaturated acyl-CoA substrates in the acyl-CoA pool limits the synthesis of elongated C20 fatty acids and disrupts the alternating sequence of lipid-linked desaturations and acyl-CoA dependent elongations. As well as the successful production of VLCPUFA in transgenic oilseeds and the identification of constraints on their accumulation, our results indicate alternative strategies to circumvent this bottleneck.
Crystal structures of possible lysine decarboxylases from Thermus thermophilus HB8
Authors: Kukimoto‐Niino Mutsuko , Murayama Kazutaka , Kato‐Murayama Miyuki , Idaka Miki , Bessho Yoshitaka , Tatsuguchi Ayako , Ushikoshi‐Nakayama Ryoko , Terada Takaho , Kuramitsu Seiki , Shirouzu Mikako , Yokoyama Shigeyuki
Published: October 2004

TT1887 and TT1465 from Thermus thermophilus HB8 are conserved hypothetical proteins, and are annotated as possible lysine decarboxylases in the Pfam database. Here we report the crystal structures of TT1887 and TT1465 at 1.8 Å and 2.2 Å resolutions, respectively, as determined by the multiwavelength anomalous dispersion (MAD) method. TT1887 is a homotetramer, while TT1465 is a homohexamer in the crystal and in solution. The structures of the TT1887 and TT1465 monomers contain single domains with the Rossmann fold, comprising six α helices and seven β strands, and are quite similar to each other. The major structural differences exist in the N terminus of TT1465, where there are two additional α helices. A comparison of the structures revealed the elements that are responsible for the different oligomerization modes. The distributions of the electrostatic potential on the solvent‐accessible surfaces suggested putative active sites.
Protein self‐association in crowded protein solutions: A time‐resolved fluorescence polarization study
Authors: Zorrilla Silvia , Rivas Germán , Acuña A. Ulises , Lillo M. Pilar
Published: October 2004

The self‐association equilibrium of a tracer protein, apomyoglobin (apoMb), in highly concentrated crowded solutions of ribonuclease A (RNase A) and human serum albumin (HSA), has been studied as a model system of protein interactions that occur in crowded macromolecular environments. The rotational diffusion of the tracer protein labeled with two different fluorescent dyes, 8‐anilinonaphthalene‐1‐sulfonate and fluorescein isothiocyanate, was successfully recorded as a function of the two crowder concentrations in the 50–200 mg/mL range, using picosecond‐resolved fluorescence anisotropy methods. It was found that apoMb molecules self‐associate at high RNase A concentration to yield a flexible dimer. The apparent dimerization constant, which increases with RNase A concentration, could also be estimated from the fractional contribution of monomeric and dimeric species to the total fluorescence anisotropy of the samples. In contrast, an equivalent mass concentration of HSA does not result in tracer dimerization. This different effect of RNase A and HSA is much larger than that predicted from simple models based only on the free volume available to apoMb, indicating that additional, nonspecific interactions between tracer and crowder should come into play. The time‐resolved fluorescence polarization methods described here are expected to be of general applicability to the detection and quantification of crowding effects in a variety of macromolecules of biological relevance.
Effect of sodium dodecyl sulfate on folding and thermal stability of acid‐denatured cytochrome c: A spectroscopic approach
Authors: Xu Qi , Keiderling Timothy A.
Published: October 2004

The molten globule (MG) state can be an intermediate in the protein folding pathway; thus, its detailed description can help understanding protein folding. Sodium dodecyl sulfate (SDS), an anionic surfactant that is commonly used to mimic hydrophobic binding environments such as cell membranes, is known to denature some native state proteins, including horse cytochrome c (cyt c). In this article, refolding of acid denatured cyt c is studied under the influence of SDS to form MG‐like states at both low concentration and above the critical micelle concentration using Fourier transform Infrared (FTIR) and ultraviolet and visible absorption as well as fluorescence and circular dichroism (CD). Thermal denaturation monitored with FTIR and CD shows distinct final high temperature states starting from MG‐like states formed with different SDS/protein ratios. The results suggest that the SDS/protein ratio as well as the actual SDS (or protein) concentration affects structure and its thermal stability. Thermal denaturation monitored with CD and FTIR for cyt c at neutral pH but denatured with SDS showed that at a high SDS/protein ratio, the thermal behavior of MG‐like states formed at low and neutral pH are quite similar. Based on the results obtained, the merits of two models of the protein–surfactant structure are discussed for different SDS concentrations.
Sonication of proteins causes formation of aggregates that resemble amyloid
Authors: Stathopulos Peter B. , Scholz Guenter A. , Hwang Young‐Mi , Rumfeldt Jessica A.O. , Lepock James R. , Meiering Elizabeth M.
Published: October 2004

Despite the widespread use of sonication in medicine, industry, and research, the effects of sonication on proteins remain poorly characterized. We report that sonication of a range of structurally diverse proteins results in the formation of aggregates that have similarities to amyloid aggregates. The formation of amyloid is associated with, and has been implicated in, causing of a wide range of protein conformational disorders including Alzheimer's disease, Huntington's disease, Parkinson's disease, and prion diseases. The aggregates cause large enhancements in fluorescence of the dye thioflavin T, exhibit green‐gold birefringence upon binding the dye Congo red, and cause a red‐shift in the absorbance spectrum of Congo red. In addition, circular dichroism reveals that sonication‐induced aggregates have high β‐content, and proteins with significant native α‐helical structure show increased β‐structure in the aggregates. Ultrastructural analysis by electron microscopy reveals a range of morphologies for the sonication‐induced aggregates, including fibrils with diameters of 5–20 nm. The addition of preformed aggregates to unsonicated protein solutions results in accelerated and enhanced formation of additional aggregates upon heating. The dye‐binding and structural characteristics, as well as the ability of the sonication‐induced aggregates to seed the formation of new aggregates are all similar to the properties of amyloid. These results have important implications for the use of sonication in food, biotechnological and medical applications, and for research on protein aggregation and conformational disorders.
Urea modulation of β‐amyloid fibril growth: Experimental studies and kinetic models
Authors: Kim Jin Ryoun , Muresan Adrian , Lee Ka Yee C. , Murphy Regina M.
Published: October 2004

Aggregation of β‐amyloid (Aβ) into fibrillar deposits is widely believed to initiate a cascade of adverse biological responses associated with Alzheimer's disease. Although it was once assumed that the mature fibril was the toxic form of Aβ, recent evidence supports the hypothesis that Aβ oligomers, intermediates in the fibrillogenic pathway, are the dominant toxic species. In this work we used urea to reduce the driving force for Aβ aggregation, in an effort to isolate stable intermediate species. The effect of urea on secondary structure, size distribution, aggregation kinetics, and aggregate morphology was examined. With increasing urea concentration, β‐sheet content and the fraction of aggregated peptide decreased, the average size of aggregates was reduced, and the morphology of aggregates changed from linear to a globular/linear mixture and then to globular. The data were analyzed using a previously published model of Aβ aggregation kinetics. The model and data were consistent with the hypothesis that the globular aggregates were intermediates in the amyloidogenesis pathway rather than alternatively aggregated species. Increasing the urea concentration from 0.4 M to 2 M decreased the rate of filament initiation the most; between 2 M and 4 M urea the largest change was in partitioning between the nonamyloid and amyloid pathways, and between 4 M and 6 M urea, the most significant change was a reduction in the rate of filament elongation.
Alteration of the tertiary structure of the major bee venom allergen Api m 1 by multiple mutations is concomitant with low IgE reactivity
Authors: Buhot Cécile , Chenal Alexandre , Sanson Alain , Pouvelle‐Moratille Sandra , Gelb Michael H. , Ménez André , Gillet Daniel , Maillère Bernard
Published: October 2004

We have engineered a recombinant form of the major bee venom allergen (Api m 1) with the final goal of reducing its IgE reactivity. This molecule (Api mut) contains 24 mutations and one deletion of 10 amino acids. The successive introduction of these sequence modifications led to a progressive loss of specific IgE and IgG reactivity and did not reveal any immunodominant epitopes. However, Api mut exhibited a clear loss of reactivity for Api m 1‐specific IgE and IgG. Injection of Api mut into mice induced specific antibody production. This humoral response was as high as that induced by the Api m 1 but the cross‐reactivity of the antibodies was weak. As inferred by far UV circular dichroism, this mutant was correctly folded. However, near UV circular dichroism and denaturation curves of Api mut showed that it exhibits a dynamic tertiary structure and that it is a highly flexible molecule. Finally, as all the sequence modifications have been introduced outside the human and murine T cell epitope regions, we investigated its T cell properties in mice. We showed that Api mut‐specific T lymphocytes induced in vivo were stimulated in vitro by both proteins. These data provide new insights in the design of hypoallergenic molecules.
Application of electrospray ionization mass spectrometry to study the hydrophobic interaction between the ε and θ subunits of DNA polymerase III
Authors: Gupta Rajesh , Hamdan Samir M. , Dixon Nicholas E. , Sheil Margaret M. , Beck Jennifer L.
Published: October 2004

The interactions between the N‐terminal domain of the ε (ε186) and θ subunits of DNA polymerase III of Escherichia coli were investigated using electrospray ionization mass spectrometry. The ε186–θ complex was stable in 9 M ammonium actetate (pH 8), suggesting that hydrophobic interactions have a predominant contribution to the stability of the complex. Addition of primary alkanols to ε186–θ in 0.1 M ammonium acetate (pH 8), led to dissociation of the complex, as observed in the mass spectrometer. The concentrations of methanol, ethanol, and 1‐propanol required to dissociate 50% of the complex were 8.9 M, 4.8 M, and 1.7 M, respectively. Closer scrutiny of the effect of alkanols on ε186, θ, and ε186–θ showed that ε186 formed soluble aggregates prior to precipitation, and that the association of ε186 with θ stabilized ε186. In‐source collision‐induced dissociation experiments and other results suggested that the ε186–θ complex dissociated in the mass spectrometer, and that the stability (with respect to dissociation) of the complex in vacuo was dependent on the solution from which it was sampled.
Insertion of the cytochrome b5 heme‐binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture
Authors: Knappenberger Jane A. , Kraemer‐Pecore Christina M. , Lecomte Juliette T.J.
Published: October 2004

Under native conditions, apocytochrome b5 exhibits a stable core and a disordered heme‐binding region that refolds upon association with the cofactor. The termini of this flexible region are in close proximity, suggesting that loop closure may contribute to the thermodynamic properties of the apocytochrome. A chimeric protein containing 43 residues encompassing the cytochrome loop was constructed using the cyanobacterial photosystem I accessory protein E (PsaE) from Synechococcus sp. PCC 7002 as a structured scaffold. PsaE has the topology of an SH3 domain, and the insertion was engineered to replace its 14‐residue CD loop. NMR and optical spectroscopies showed that the hybrid protein (named EbE1) was folded under native conditions and that it retained the characteristics of an SH3 domain. NMR spectroscopy revealed that structural and dynamic differences were confined near the site of loop insertion. Variable‐temperature 1D NMR spectra of EbE1 confirmed the presence of a kinetic unfolding barrier. Thermal and chemical denaturations of PsaE and EbE1 demonstrated cooperative, two‐state transitions; the stability of the PsaE scaffold was found only moderately compromised by the insertion, with a ΔTm of 8.3°C, a ΔCm of 1.5 M urea, and a ΔΔG° of 4.2 kJ/mole. The data implied that the penalty for constraining the ends of the inserted region was lower than the ∼6.4 kJ/mole calculated for a self‐avoiding chain. Extrapolation of these results to cytochrome b5 suggested that the intrinsic stability of the folded portion of the apoprotein reflected only a small detrimental contribution from the large heme‐binding domain.
Promiscuous protein biotinylation by Escherichia coli biotin protein ligase
Authors: Choi‐Rhee Eunjoo , Schulman Howard , Cronan John E.
Published: October 2004

Biotin protein ligases (BPLs) are enzymes of extraordinary specificity. BirA, the BPL of Escherichia coli biotinylates only a single cellular protein. We report a mutant BirA that attaches biotin to a large number of cellular proteins in vivo and to bovine serum albumin, chloramphenicol acetyltransferase, immunoglobin heavy and light chains, and RNAse A in vitro. The mutant BirA also self biotinylates in vivo and in vitro. The wild type BirA protein is much less active in these reactions. The biotinylation reaction is proximity‐dependent in that a greater extent of biotinylation was seen when the mutant ligase was coupled to the acceptor proteins than when the acceptors were free in solution. This approach may permit facile detection and recovery of interacting proteins by existing avidin/streptavidin technology.
Mimicry by asx‐ and ST‐turns of the four main types of β‐turn in proteins
Authors: Duddy William J. , Nissink J. Willem M. , Allen Frank H. , Milner‐White E. James
Published: October 2004

Hydrogen‐bonded β‐turns in proteins occur in four categories: type I (the most common), type II, type II', and type I'. Asx‐turns resemble β‐turns, in that both have an NH…OC hydrogen bond forming a ring of 10 atoms. Serine and threonine side chains also commonly form hydrogen‐bonded turns, here called ST‐turns. Asx‐turns and ST‐turns can be categorized into four classes, based on side chain rotamers and the conformation of the central turn residue, which are geometrically equivalent to the four types of β‐turns. We propose asx‐ and ST‐turns be named using the type I, II, I', and II' β‐turn nomenclature. Using this, the frequency of occurrence of both asx‐ and ST‐turns is: type II' > type I > type II > type I', whereas for β‐turns it is type I > type II > type I' > type II'. Almost all type II asx‐turns occur as a recently described three residue feature named an asx‐nest.
Crystal structure of human coactosin‐like protein at 1.9 Å resolution
Authors: Li Xuemei , Liu Xueqi , Lou Zhiyong , Duan Xin , Wu Hao , Liu Yiwei , Rao Zihe
Published: October 2004

Human coactosin‐like protein (CLP) shares high homology with coactosin, a filamentous (F)‐actin binding protein, and interacts with 5LO and F‐actin. As a tumor antigen, CLP is overexpressed in tumor tissue cells or cell lines, and the encoded epitopes can be recognized by cellular and humoral immune systems. To gain a better understanding of its various functions and interactions with related proteins, the crystal structure of CLP expressed in Escherichia coli has been determined to 1.9 Å resolution. The structure features a central β‐sheet surrounded by helices, with two very tight hydrophobic cores on each side of the sheet. CLP belongs to the actin depolymerizing protein superfamily, and is similar to yeast cofilin and actophilin. Based on our structural analysis, we observed that CLP forms a polymer along the crystallographic b axis with the exact same repeat distance as F‐actin. A model for the CLP polymer and F‐actin binding has therefore been proposed.
Peroxidative aggregation of α‐synuclein requires tyrosines
Authors: Olteanu Alina , Pielak Gary J.
Published: October 2004

α‐Synuclein is the main component of the intracellular protein aggregates in neurons of patients with Parkinson's disease. The occurrence of the disease is associated with oxidative damage. Although it is known that peroxidative chemistry leads to the aggregation of α‐synuclein in vitro, the specific amino acid types of α‐synuclein involved in this type of aggregation have not been identified. We show, using human cytochromecplus H2O2as the source oxidative stress, that the tyrosines of α‐synuclein are required for aggregation. The studies reveal the chemical basis for a crucial step in the aggregation process.
Crystal structure of a dodecameric FMN‐dependent UbiX‐like decarboxylase (Pad1) from Escherichia coli O157: H7
Authors: Rangarajan Erumbi S. , Li Yunge , Iannuzzi Pietro , Tocilj Ante , Hung Li‐Wei , Matte Allan , Cygler Miroslaw
Published: October 2004

The crystal structure of the flavoprotein Pad1 from Escherichia coli O157:H7 complexed with the cofactor FMN has been determined by the multiple anomalous diffraction method and refined at 2.0 Å resolution. This protein is a paralog of UbiX (3‐octaprenyl‐4‐hydroxybenzoate carboxylyase, 51% sequence identity) that catalyzes the third step in ubiquinone biosynthesis and to Saccharomyces cerevisiae Pad1 (54% identity), an enzyme that confers resistance to the antimicrobial compounds phenylacrylic acids through decarbox‐ylation of these compounds. Each Pad1 monomer consists of a typical Rossmann fold containing a non–covalently bound molecule of FMN. The fold of Pad1 is similar to MrsD, an enzyme associated with lantibiotic synthesis; EpiD, a peptidyl‐cysteine decarboxylase; and AtHAL3a, the enzyme, which decarboxylates 4′‐phosphopantothenoylcysteine to 4′‐phosphopantetheine during coenzyme A biosynthesis, all with a similar location of the FMN binding site at the interface between two monomers, yet each having little sequence similarity to one another. All of these proteins associate into oligomers, with a trimer forming the common structural unit in each case. In MrsD and EpiD, which belong to the homo‐dodecameric flavin‐containing cysteine decarboxylase (HFCD) family, these trimers associate further into dodecamers. Pad1 also forms dodecamers, although the association of the trimers is completely different, resulting in exposure of a different side of the trimer unit to the solvent. This exposure affects the location of the substrate binding site and, specifically, its access to the FMN cofactor. Therefore, Pad1 forms a separate family, distinguishable from the HFCD family.
Stabilization of membranes upon interaction of amphipathic polymers with membrane proteins
Authors: Picard Martin , Duval‐Terrié Caroline , Dé Emmanuelle , Champeil Philippe
Published: October 2004

Amphipathic polymers derived from polysaccharides, namely hydrophobically modified pullulans, were previously suggested to be useful as polymeric substitutes of ordinary surfactants for efficient and structure‐conserving solubilization of membrane proteins, and one such polymer, 18C10, was optimized for solubilization of proteins derived from bacterial outer membranes (Duval‐Terrié et al. 2003). We asked whether a similar ability to solubilize proteins could also be demonstrated in eukaryotic membranes, namely sarcoplasmic reticulum (SR) fragments, the major protein of which is SERCA1a, an integral membrane protein with Ca2+‐dependent ATPase and Ca2+‐pumping activity. We found that 18C10‐mediated solubilization of these SR membranes did not occur. Simultaneously, however, we found that low amounts of this hydrophobically modified pullulan were very efficient at preventing long‐term aggregation of these SR membranes. This presumably occurred because the negatively charged polymer coated the membranous vesicles with a hydrophilic corona (a property shared by many other amphipathic polymers), and thus minimized their flocculation. Reminiscent of the old Arabic gum, which stabilizes Indian ink by coating charcoal particles, the newly designed amphipathic polymers might therefore unintentionally prove useful also for stabilization of membrane suspensions.
Is Good Policy Unimplementable? Reflections on the Ethnography of Aid Policy and Practice
Authors: Mosse David
Published: October 2004

Despite the enormous energy devoted to generating the right policy models in development, strangely little attention is given to the relationship between these models and the practices and events that they are expected to generate or legitimize. Focusing on the unfolding activities of a development project over more than ten years as it falls under different policy regimes, this article challenges the assumption that development practice is driven by policy, suggesting that the things that make for ‘good policy’— policy which legitimizes and mobilizes political support — in reality make it rather unimplementable within its chosen institutions and regions. But although development practice is driven by a multi‐layered complex of relationships and the culture of organizations rather than policy, development actors work hardest of all to maintain coherent representations of their actions as instances of authorized policy, because it is always in their interest to do so. The article places these observations within the wider context of the anthropology of development and reflects on the place, method and contribution of development ethnography.
Resettlement, Rights to Development and the Ilisu Dam, Turkey
Authors: Morvaridi Behrooz
Published: October 2004

A cursory attempt to measure the extent of displacement over the past two decades indicates significant increases in conflict‐induced displacement and displacement resulting from development projects. At the same time a growing opposition to the latter form of displacement has raised questions over its legitimacy through a variety of media, including public campaigns and protests. This article focuses on some of the challenges that this presents to the displacement and resettlement discourse. In particular it considers the influences of the rights to development agenda on the spatial context of displacement and its associated economic and political changes. There appears to be a disjuncture between the practices of mainstream development, which tend to interpret development policy as it is defined and applied by a nation state and to assess inequalities within clear geographical definitions, and the universality of a rights based approach to development. This article examines these tensions in the context of displacement and resettlement management, drawing on evidence from a case study of the Ilisu dam in South East Anatolia, Turkey.
Morphometric and immunohistochemical study of the reticulum of red deer during prenatal development
Authors: Franco A. J. , Redondo E. , Masot A. J.
Published: October 2004

Iberian red deer need to be conserved for their economic role and for their genetic importance as an important component of the ecosystem. Modifications currently being made to traditional management systems require a better understanding of the structure, function and development of their alimentary system. Here we describe a histomorphometric and immunohistochemical analysis of the stomach of 25 red deer embryos and fetuses from 30 days of gestation until birth (235 days). Differentiation of the reticular compartment from the primitive gastric tube begins at 67 days, forming a three‐layered structure: epithelium, pluripotential blastemal tissue and serosa. The primitive reticular cells are initiated as small epithelial evaginations (primary ribs) at 117 days. At 142 days, lateral growths appear from the primary reticular ribs, forming the corneum papillae. The secondary reticular ribs form at 142 days as growths from the primary ribs. The uneven height of primary and secondary reticular ribs leads to the formation of cells of varying size. Growth of the reticular ribs involves the lamina propria but not the submucosa, so clear separation of these layers is maintained during histodifferentiation. Formation of the tunica muscularis from the pluripotential blastemal tissue begins at 67 days of intrauterine life, as two layers of longitudinally and circularly arranged myoblasts. Differentiation of the muscularis from the mucosa occurs at approximately 205 days, as longitudinal projections of the internal bundles of the tunica muscularis form the musculature of the primary ribs. The secretion of neutral and acid mucopolysaccharides by the reticular epithelial layer begins at 67 days, establishing the gradual adaptation of the mucosa to its protective function in postnatal life. Neuroendocrine (non‐neuron enolase) and glial cells (glial fibrillary acidic protein and vimentin) were detected by immunohistochemistry, in a similar localization and intensity to that reported in the rumen. The neuropeptides vasoactive intestinal peptide and neuropeptide Y showed a positive immunoreaction in the reticular epithelium from 142 days of prenatal life, again earlier than reported for the rumen. In comparison with domestic ruminants, deer were shown to be less precocious with regard to development of gastric tube, in their capacity to secrete neutral mucopolysaccharides, and in their neuroendocrine nature, as determined by the detection of positive neuroedocrine and/or glial cells.
Tissue‐specific expression of the tight junction proteins claudins and occludin in the rat salivary glands
Authors: Peppi M. , Ghabriel M. N.
Published: October 2004

Tight junctions (TJs) are essential features of endothelial barrier membranes and of fluid‐secreting epithelial cells, such as in the salivary glands. Novel integral membrane proteins have been identified as components of TJs, namely claudins and occludin. The aim of the present study was to determine the distribution of occludin and claudins in the large salivary glands of the rat. The parotid, submandibular and sublingual salivary glands were harvested from adult Sprague–Dawley rats and cryostat sections were stained using immunoperoxidase and immunofluorescence methods. Claudin‐1 was expressed in endothelial cells of microvessels and in short selected segments of the duct system. Claudin‐3 was expressed principally in the acinar cells and intercalated ducts, while claudin‐4 was principally expressed by the striated and interlobular ducts. Claudin‐5 was specific to endothelial cells of microvessels. Occludin was ubiquitously detected in the duct system. Double labelling and confocal microscopy showed some co‐localization of claudin‐3 with claudin‐4, and minimal co‐localization of occludin with claudin‐4, in the striated ducts. Claudin 2 was not detected in any of the salivary glands. The results indicate specificity of the chemical composition of tight junctions in the rat salivary glands, and may reflect different physiological roles for TJs in the glandular and duct epithelial cells, and in endothelial cells of salivary gland microvessels.
In vivo leptin expression in cartilage and bone cells of growing rats and adult humans
Authors: Morroni M. , De Matteis R. , Palumbo C. , Ferretti M. , Villa I. , Rubinacci A. , Cinti S. , Marotti G.
Published: October 2004

The present investigation was carried out to analyse, immunohistochemically, in vivo leptin expression in cartilage and bone cells, the latter restricted to the elements of the osteogenic system (stromal cells, osteoblasts, osteocytes, bone lining cells). Observations were performed on the first lumbar vertebra, tibia and femur of four rats and on the humerus, femur and acromion of four patients. Histological sections of paraffin‐embedded bone samples were immunostained using antibody to leptin. The results showed that, in growing rat bone, leptin is expressed in chondrocytes and stromal cells, but not in osteoblasts; bone lining cells were not found in the microscopic fields examined. In adult human bone, leptin is expressed in chondrocytes, stromal cells and bone lining cells; osteoblasts were not found in the microscopic fields examined. Osteocytes were found to be leptin positive only occasionally and focally in both rat and human bone. The in vivo findings reported show, for the first time, that leptin appears to be expressed only in the cells of the osteogenic lineage (stromal cells, bone lining cells, osteocytes) that, with respect to osteoblasts, are permanent and inactive, i.e. in those cells that according to our terminology constitute the bone basic cellular system (BBCS). Because the BBCS seems to be primarily involved in sensing and integrating mechanical strains and biochemical factors and then in triggering and driving bone formation and/or bone resorption, it appears that leptin seems to be mainly involved in modulating the initial phases of bone modelling and remodelling processes.
Interobserver error involved in independent attempts to measure cusp base areas of Pan M1s
Authors: Bailey Shara E. , Pilbrow Varsha C. , Wood Bernard A.
Published: October 2004

Cusp base areas measured from digitized images increase the amount of detailed quantitative information one can collect from post‐canine crown morphology. Although this method is gaining wide usage for taxonomic analyses of extant and extinct hominoids, the techniques for digitizing images and taking measurements differ between researchers. The aim of this study was to investigate interobserver error in order to help assess the reliability of cusp base area measurement within extant and extinct hominoid taxa. Two of the authors measured individual cusp base areas and total cusp base area of 23 maxillary first molars (M1) of Pan. From these, relative cusp base areas were calculated. No statistically significant interobserver differences were found for either absolute or relative cusp base areas. On average the hypocone and paracone showed the least interobserver error (< 1%) whereas the protocone and metacone showed the most (2.6–4.5%). We suggest that the larger measurement error in the metacone/protocone is due primarily to either weakly defined fissure patterns and/or the presence of accessory occlusal features. Overall, levels of interobserver error are similar to those found for intraobserver error. The results of our study suggest that if certain prescribed standards are employed then cusp and crown base areas measured by different individuals can be pooled into a single database.
A mesenchyme‐free culture system to elucidate the mechanism of otic vesicle morphogenesis
Authors: Miura Takashi , Shiota Kohei , Morriss‐Kay Gillian
Published: October 2004

The vertebrate inner ear has been extensively studied as a model system of morphogenesis and differentiation. The interactions between epithelium and surrounding mesenchyme have not previously been studied directly, because an appropriate experimental system had not been established. Here we describe a mesenchyme‐free culture system of E11.5 mouse otic vesicle which retains the ability for (1) formation of the cochlear loop, (2) emigration of ganglion cells from the epithelium and (3) invagination of semicircular canal epithelium. E10.5 otic vesicle was maintained using the same method, but morphogenesis was less successful. Culture of the E11.5 cochlear region alone resulted in regeneration of a structure with semicircular canal character from the cut end, indicating that region‐specific cell fate within the otic vesicle is not irreversibly determined at this stage. Co‐culturing otic vesicle with cochleovestibular ganglion (CVG) resulted in enhanced looping or ectopic diverticulum formation of the cochlear region, suggesting that the CVG provides a morphogenetic signal for cochlear looping. Cochlear looping was specifically blocked by inhibiting actin polymerization by cytochalasin D, while morphogenesis of the semicircular canal region remained intact. Hyaluronidase treatment inhibited semicircular canal morphogenesis, resulting in a cystic form of the otic vesicle. These data validate this culture system as a tool for elucidating the mechanism of morphogenesis of the otic vesicle.
Cytochemical and ultrastructural characterization of growing colonies of human embryonic stem cells
Authors: Johkura Kohei , Cui Li , Asanuma Kazuhiko , Okouchi Yasumitsu , Ogiwara Naoko , Sasaki Katsunori
Published: October 2004

The morphology of human embryonic stem (ES) cells changes with their colonial growth. For a better understanding of the growth of ES cell colonies in culture, we determined their cytochemical and ultrastructural characteristics focusing on images of living cells under a phase contrast microscope. During the initial growth stages, the colonies exhibited a mosaic appearance with discernible cell–cell borders. PAS staining coupled with amylase digestion demonstrated that the bright granules and dark deposits in the cytoplasm contained glycogen. Ultrastructurally they were glycogen accumulations, and clustered open spaces associated with various amounts of glycogen. Although intercellularly heterogeneous, these structures were detectable throughout colony growth. As the colonies grew, compaction towards the centre emerged and increased, accompanied by heterogeneous increases in coarse particles with or without a halo. TUNEL showed these particles to consist at least in part of apoptotic cells/bodies. Transmission electron microscopy indicated that most apoptotic cells had been phagocytosed by intact ES cells. Spontaneous differentiation was detected occasionally in the periphery of the colonies. The presence of PAS‐positive fibrous structures not susceptible to amylase digestion and laminin‐immunoreactivity indicated the accumulation of extracellular matrix in the peripheral differentiated areas. These findings made it possible to determine the growth stage of human ES cell colonies.
No change in neuron numbers in the dentate nucleus of patients with schizophrenia estimated with a new stereological method – the smooth fractionator
Authors: Andersen B. B. , Fabricius K. , Gundersen H. J. G. , Jelsing J. , Stark A. K.
Published: October 2004

The dentate nucleus is phylogenetically the most recent nucleus in the cerebellum. Owing to its connections to the thalamus and the prefrontal cortex it may be involved in the symptomathology in schizophrenia and other psychiatric illnesses. In this stereological study we implemented the smooth fractionator, which combines the unbiased principles of the optical fractionator with a new and more efficient sampling strategy to the dentate nucleus. The smooth fractionator represents the most efficient sampling strategy described so far in stereology, in terms of reducing the sampling variance and thus increasing the efficiency. It is the first application of the smooth fractionator to human brain tissue and presents estimations of total number of neurons in the dentate nuclei of eight patients with schizophrenia compared to eight control persons. The total number of neurons in the dentate nucleus was estimated to 3.36 × 106in subjects with schizophrenia, which was not statistically significant different from 3.65 × 106in control subjects (P = 0.63). The advantages and disadvantages of the smooth fractionator method are discussed and its precision in practical application is estimated.
A stereological evaluation of secretin and gastric inhibitory peptide‐containing mucosal cells of the perinatal small intestine of the pig
Authors: Van Ginneken C. , Weyns A.
Published: October 2004

Stereological methods were used to quantify secretin and gastric inhibitory peptide (GIP)‐immunoreactivity (GIP‐IR) in paraffin sections of the duodenum, jejunum and ileum of fetal and neonatal piglets. In addition, sections were processed for GLP‐1‐immunohistochemistry. The volume density of the tunica mucosa increased after birth, giving rise to a decreased volume density of the tela submucosa and tunica muscularis. Generally known region‐specific morphological distinctions were reflected in differing volume densities of the various layers. The highest volume density of GIP‐IR epithelial cells was observed in the jejunum of the neonate. In contrast, the volume density of secretin‐IR epithelial cells was highest in the duodenum of both fetal and neonatal piglets. The volume occupied by GIP‐IR and secretin‐IR epithelial cells increased in the jejunum after birth. Additionally, ileal secretin‐IR epithelial cells were more numerous in the neonatal piglet. In conclusion, the quantitative and qualitative presence of GIP‐IR and secretin‐IR epithelial cells agree with earlier reports of their presence and co‐localization between GIP‐IR and GLP‐1‐IR, in the pig small intestine.Furthermore, the differences suggest that age‐ and region‐related functional demands are temporally and probably causally related with the morphological diversification of the intestine and its endocrine cells.
Phylogeny of the leafhopper subfamily Evacanthinae with a review of Neotropical species and notes on related groups (Hemiptera: Membracoidea: Cicadellidae)
Authors: Dietrich C. H.
Published: October 2004

Abstract.  The phylogenetic analysis of ninety‐two adult morphological characters supports the treatment of Nirvaninae as a junior synonym of Evacanthinae and the redefinition of Evacanthinae to include the tribes Nirvanini, Balbillini, Evacanthini and Pagaroniini. The analysis indicates that Nirvaninae, as previously defined, is polyphyletic. A key to tribes and Neotropical genera is provided and diagnostic features for these taxa are reviewed. Jassoqualus Kramer, Neonirvana Oman, Synogonia Melichar (=Jassopronus Nielson & Godoy, syn.n.) and Tahura Melichar are retained within Nirvanini and two new Neotropical genera of this tribe are described and illustrated: Antillonirvana, gen.n., based on two new species from the Dominican Republic and one from Cuba; and Chibchanirvana, gen.n., based on two new species from Colombia. Pentoffia Kramer and Evanirvana Hill are treated as incertae sedis within Evacanthinae. Six new species of Pentoffia, a new species of Synogonia, a new species of Jassoqualus, two new species of Neonirvana and eleven new species of Tahura, all from South America, are also described and illustrated. The following taxa included previously in Nirvaninae are excluded from Evacanthinae, sensu lato: Tungurahuala Kramer to Cicadellinae; Columbonirvana Linnavuori to Typhlocybinae; Macroceratogoniini to Coelidiinae; Occinirvanini Evans to Deltocephalinae. Omaranus Distant, placed previously in Occinirvanini, is transferred to Doraturini (Deltocephalinae).
Treehopper trees: phylogeny of Membracidae (Hemiptera: Cicadomorpha: Membracoidea) based on molecules and morphology
Authors: Cryan Jason R. , Wiegmann Brian M. , Deitz Lewis L. , Dietrich Christopher H. , Whiting Michael F.
Published: October 2004

Abstract. Recent independent phylogenetic analyses of membracid relationships based on molecular and morphological data have identified monophyletic lineages within the family. However, the results of these studies have not fully resolved treehopper phylogeny, and relationships among some higher membracid lineages remain in doubt. Portions of three datasets (958 aligned nucleotides from elongation factor‐1α, 2363 aligned nucleotides from 28S ribosomal DNA, and eighty‐three morphological features of adults and nymphs) introduced in recent studies were reanalysed separately and in combination with two new molecular datasets (321 aligned nucleotides from wingless and 1829 aligned nucleotides from 18S ribosomal DNA). The results of the combined data analyses, contrary to previous analyses of morphological data alone, grouped membracids into two well‐supported lineages, one comprising Stegaspidinae and Centrotinae, the other comprising Membracinae, Darninae and Smiliinae. The analyses recovered Centrotinae, Membracinae and Darninae as monophyletic groups, but Stegaspidinae was paraphyletic with respect to Centrotinae, and Smiliinae was polyphyletic with Micrutalini placed as a sister group to the clade comprising Membracinae, Darninae and Smiliinae. These results are consistent with the following hypotheses, proposed previously based on an analysis of morphological data: (1) the posterior pronotal process was derived and lost multiple times during the evolution of Membracidae; (2) Membracidae originated in the New World and reached the Old World subsequently via dispersal; (3) maternal care evolved independently multiple times and may or may not have been preceded by the acquisition of ant mutualism.