bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure and kinetics of a transient antibody binding intermediate reveal a kinetic discrimination mechanism in antigen recognition
Authors: James Leo C. , Tawfik Dan S.
Published: August 2005

Induced fit is a predominant phenomenon in protein–ligand interactions, yet it is invariably attributed without establishing the existence, let alone the structure, of the initial, low-affinity encounter complex. We determined the crystal structure of the encounter complex on the pathway of ligand binding by IgE antibody SPE7. We show that this complex is formed by a wide range of ligands that initially bind with identical affinity. Nonspecific ligands rapidly dissociate, whereupon the antibody isomerizes to a nonbinding isomer. Specific ligand complexes, however, slowly isomerize to give a high-affinity complex. This isomerization involves backbone and side-chain rearrangements of up to 14 Å and the formation of specific hydrogen bonds. The postbinding conformational switch, combined with the prebinding isomerization to an energetically favorable nonbinding isomer, results in a “kinetic discrimination” mechanism that mediates selective binding, by a factor of >103, between highly related ligands that initially bind with the same affinity. This model may apply to proteins that bind multiple ligands in a specific manner or other proteins that, although capable of binding many ligands, are activated by only a few.
Structure of AMA1 from
Plasmodium falciparum
reveals a clustering of polymorphisms that surround a conserved hydrophobic pocket
Authors: Bai Tao , Becker Michael , Gupta Aditi , Strike Phillip , Murphy Vince J. , Anders Robin F. , Batchelor Adrian H.
Published: August 2005

Apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate that possesses polymorphisms that may pose a problem for a vaccine based on this antigen. Knowledge of the distribution of the polymorphic sites on the surface of AMA1 is necessary to obtain a detailed understanding of their significance for vaccine development. For this reason we have sought to determine the three-dimensional structure of AMA1 using x-ray crystallography. The central two-thirds of AMA1 is relatively conserved among
Plasmodium
species as well as more distantly related apicomplexan parasites, and contains two clusters of disulfide-bonded cysteines termed domains I and II. The crystal structure of this fragment of AMA1 reported here reveals that domains I+II consists of two intimately associated PAN domains. PAN domain I contains many long loops that extend from the domain core and form a scaffold for numerous polymorphic residues. This extreme adaptation of a PAN domain reveals how malaria parasites have introduced significant flexibility and variation into AMA1 to evade protective human antibody responses. The polymorphisms on the AMA1 surface are exclusively located on one side of the molecule, presumably because this region of AMA1 is most accessible to antibodies reacting with the parasite surface. Moreover, the most highly polymorphic residues surround a conserved hydrophobic trough that is ringed by domain I and domain II loops. Precedents set by viral receptor proteins would suggest that this is likely to be the AMA1 receptor binding pocket.
Covalent stabilization of coiled coils of the HIV gp41 N region yields extremely potent and broad inhibitors of viral infection
Authors: Bianchi Elisabetta , Finotto Marco , Ingallinella Paolo , Hrin Renee , Carella Anthony V. , Hou Xiaoli S. , Schleif William A. , Miller Michael D. , Geleziunas Romas , Pessi Antonello
Published: August 2005

Peptides from the N-heptad repeat region of the HIV gp41 protein can inhibit viral fusion, but their potency is limited by a low tendency to form a trimeric coiled-coil. Accordingly, stabilization of N peptides by fusion with the stable coiled-coil IZ yields nanomolar inhibitors [Eckert, D. M. & Kim, P. S. (2001)
Proc. Natl. Acad. Sci. USA
98, 11187-11192]. Because the antiviral potency of IZN17 is limited by self-association equilibrium, we covalently stabilized the peptide by using interchain disulfide bonds. The resulting covalent trimer, (CCIZN17)
3
, has an extraordinary thermodynamic stability that translates into unprecedented antiviral potency: (CCIZN17)
3
(
i
) inhibits fusion in a cell-cell fusion assay (IC
50
= 260 pM); (
ii
)is the most potent fusion inhibitor described to date (IC
50
= 40-380 pM) in a single-cycle infectivity assay against HIV
HXB2
, HIV
NL4-3
, and HIV
MN-1
; (
iii
) efficiently neutralizes acute viral infection in peripheral blood mononuclear cells; and (
iv
) displays a broad antiviral profile, being able to neutralize 100% of a large panel of HIV isolates, including R5, X4, and R5/X4 strains. In all of these assays, the potency of N-peptide inhibitor (CCIZN17)
3
was equal to or more than the C-peptide inhibitor in clinical use, DP178 (also known as Enfuvirtide and Fuzeon). More importantly, we show that the two inhibitors, which have different targets in gp41, synergize when used in combination. These features make (CCIZN17)
3
an attractive lead to develop as an antiviral drug, alone or in combination with DP178, as well as a promising immunogen to elicit a fusion-blocking neutralizing antibody response.
Amorphous layer around aragonite platelets in nacre
Authors: Nassif Nadine , Pinna Nicola , Gehrke Nicole , Antonietti Markus , Jäger Christian , Cölfen Helmut
Published: August 2005

We reveal that the aragonite CaCO3platelets in nacre ofHaliotis laevigataare covered with a continuous layer of disordered amorphous CaCO3and that there is no protein interaction with this layer. This finding contradicts classical paradigms of biomineralization, e.g., an epitaxial match between the structural organic matrix and the formed mineral. This finding also highlights the role of physicochemical effects in morphogenesis, complementing the previously assumed total control by biomolecules and bioprocesses, with many implications in nanotechnology and materials science.
MgcRacGAP controls the assembly of the contractile ring and the initiation of cytokinesis
Authors: Zhao Wei-meng , Fang Guowei
Published: August 2005

Initiation of cytokinesis requires the establishment of the cleavage plane, the assembly of the contractile ring, and the ingression of the cleavage furrow. MgcRacGAP, a GTPase-activating protein for RhoA, is required for cytokinesis, but the mechanism of its action remains unknown. We report here that MgcRacGAP is required for the assembly of anillin and myosin into the contractile ring. In addition, MgcRacGAP is required for the localized activation of myosin through the RhoA-mediated phosphorylation of the myosin regulatory light chain. Cells with MgcRacGAP RNA interference (RNAi) failed cytokinesis without any ingression of the cleavage furrow. Paradoxically, MgcRacGAP, a GTPase-activating protein, associates during cytokinesis with ECT2, a guanine nucleotide exchange factor for RhoA, and the localization of ECT2 to both the central spindle and the contractile ring depends on MgcRacGAP. Knockdown of ECT2 phenocopies that of MgcRacGAP. We conclude that MgcRacGAP controls the initiation of cytokinesis by regulating ECT2, which in turn induces the assembly of the contractile ring and triggers the ingression of the cleavage furrow.
Microbial regulation of intestinal radiosensitivity
Authors: Crawford Peter A. , Gordon Jeffrey I.
Published: August 2005

We describe a method for treating germ-free (GF) mice with γ-irradiation and transplanting them with normal or genetically manipulated bone marrow while maintaining their GF status. This approach revealed that GF mice are markedly resistant to lethal radiation enteritis. Furthermore, administering lethal doses of total body irradiation to GF mice produces markedly fewer apoptotic endothelial cells and lymphocytes in the mesenchymal cores of their small intestinal villi, compared with conventionally raised animals that have acquired a microbiota from birth. Analysis of GF and conventionally raised
Rag1
-/-
mice disclosed that mature lymphocytes are not required for the development of lethal radiation enteritis or the microbiota-associated enhancement of endothelial radiosensitivity. Studies of gnotobiotic knockout mice that lack fasting-induced adipose factor (Fiaf), a fibrinogen/angiopoietin-like protein normally secreted from the small intestinal villus epithelium and suppressed by the microbiota, showed that Fiaf deficiency results in loss of resistance of villus endothelial and lymphocyte populations to radiation-induced apoptosis. Together, these findings provide insights about the cellular and molecular targets involved in microbial regulation of intestinal radiosensitivity.
Gene targeting using a promoterless gene trap vector (“targeted trapping”) is an efficient method to mutate a large fraction of genes
Authors: Friedel Roland H. , Plump Andrew , Lu Xiaowei , Spilker Kerri , Jolicoeur Christine , Wong Karen , Venkatesh Tadmiri R. , Yaron Avraham , Hynes Mary , Chen Bin , Okada Ami , McConnell Susan K. , Rayburn Helen , Tessier-Lavigne Marc
Published: August 2005

A powerful tool for postgenomic analysis of mammalian gene function is gene targeting in mouse ES cells. We report that homologous recombination using a promoterless gene trap vector (“targeting trapping”) yields targeting frequencies averaging above 50%, a significant increase compared with current approaches. These high frequencies appear to be due to the stringency of selection with promoterless constructs, because most random insertions are silent and eliminated by drug selection. The promoterless design requires that the targeted gene be expressed in ES cells at levels exceeding a certain threshold (which we estimate to be ≈1% of the transferrin receptor gene expression level, for the secretory trap vector used here). Analysis of 127 genes that had been trapped by random (nontargeted) gene trapping with the same vector shows that virtually all are expressed in ES cells above this threshold, suggesting that targeted and random trapping share similar requirements for expression levels. In a random sampling of 130 genes encoding secretory proteins, about half were expressed above threshold, suggesting that about half of all secretory genes are accessible by either targeted or random gene trapping. The simplicity and high efficiency of the method facilitate systematic targeting of a large fraction of the genome by individual investigators and large-scale consortia alike.
Blocking protein farnesyltransferase improves nuclear shape in fibroblasts from humans with progeroid syndromes
Authors: Toth Julia I. , Yang Shao H. , Qiao Xin , Beigneux Anne P. , Gelb Michael H. , Moulson Casey L. , Miner Jeffrey H. , Young Stephen G. , Fong Loren G.
Published: August 2005

Defects in the biogenesis of lamin A from its farnesylated precursor, prelamin A, lead to the accumulation of prelamin A at the nuclear envelope, cause misshapen nuclei, and result in progeroid syndromes. A deficiency in ZMPSTE24, a protease involved in prelamin A processing, leads to prelamin A accumulation, an absence of mature lamin A, misshapen nuclei, and a lethal perinatal progeroid syndrome: restrictive dermopathy (RD). Hutchinson-Gilford progeria syndrome (HGPS) is caused by a mutant prelamin A that cannot be processed to lamin A. The hallmark cellular abnormality in RD and HGPS is misshapen nuclei. We hypothesized that the farnesylation of prelamin A is important for its targeting to the nuclear envelope in RD and HGPS and that blocking farnesylation would ameliorate the nuclear shape abnormalities. Indeed, when RD fibroblasts were treated with a farnesyltransferase inhibitor (FTI), prelamin A was partially mislocalized away from the nuclear envelope, and the frequency of nuclear shape abnormalities was reduced (
P
< 0.0001). A FTI also mislocalized prelamin A and improved nuclear shape in
Zmpste24
-deficient mouse embryonic fibroblasts (
P
< 0.0001) and improved nuclear shape in human HGPS fibroblasts (
P
< 0.0001). Most remarkably, a FTI significantly improved nuclear shape in two fibroblast cell lines from atypical progeria patients with lamin A missense mutations in the absence of prelamin A accumulation (
P
= 0.0003 and
P
< 0.0001). These findings establish a paradigm for ameliorating the most obvious cellular pathology in lamin-related progeroid syndromes and suggest a potential strategy for treating these diseases.
The evolution of lethal intergroup violence
Authors: Kelly Raymond C.
Published: August 2005

Recent findings and analyses in evolutionary biology, archaeology, and ethnology provide a favorable conjuncture for examining the evolution of lethal intergroup violence among hominids during the 2.9-million-year Paleolithic time span. Here, I seek to identify and investigate the main turning points in this evolutionary trajectory and to delineate the periodization that follows from this inquiry.
Three-dimensional structure of human monoamine oxidase A (MAO A): Relation to the structures of rat MAO A and human MAO B
Authors: De Colibus Luigi , Li Min , Binda Claudia , Lustig Ariel , Edmondson Dale E. , Mattevi Andrea
Published: August 2005

The three-dimensional structure of recombinant human monoamine oxidase A (hMAO A) as its clorgyline-inhibited adduct is described. Although the chain-fold of hMAO A is similar to that of rat MAO A and human MAO B (hMAO B), hMAO A is unique in that it crystallizes as a monomer and exhibits the solution hydrodynamic behavior of a monomeric form rather than the dimeric form of hMAO B and rat MAO A. hMAO A's active site consists of a single hydrophobic cavity of ≈550 Å3, which is smaller than that determined from the structure of deprenyl-inhibited hMAO B (≈700 Å3) but larger than that of rat MAO A (≈450 Å3). An important component of the active site structure of hMAO A is the loop conformation of residues 210–216, which differs from that of hMAO B and rat MAO A. The origin of this structural alteration is suggested to result from long-range interactions in the monomeric form of the enzyme. In addition to serving as a basis for the development of hMAO A specific inhibitors, these data support the proposal that hMAO A involves a change from the dimeric to the monomeric form through a Glu-151 → Lys mutation that is specific of hMAO A [Andrès, A. M., Soldevila, M., Navarro, A., Kidd, K. K., Oliva, B. & Bertranpetit, J. (2004)Hum. Genet.115, 377–386]. These considerations put into question the use of MAO A from nonhuman sources in drug development for use in humans.
Specificity versus stability in computational protein design
Authors: Bolon Daniel N. , Grant Robert A. , Baker Tania A. , Sauer Robert T.
Published: August 2005

Protein–protein interactions can be designed computationally by using positive strategies that maximize the stability of the desired structure and/or by negative strategies that seek to destabilize competing states. Here, we compare the efficacy of these methods in reengineering a protein homodimer into a heterodimer. The stability-design protein (positive design only) was experimentally more stable than the specificity-design heterodimer (positive and negative design). By contrast, only the specificity-design protein assembled as a homogenous heterodimer in solution, whereas the stability-design protein formed a mixture of homodimer and heterodimer species. The experimental stabilities of the engineered proteins correlated roughly with their calculated stabilities, and the crystal structure of the specificity-design heterodimer showed most of the predicted side-chain packing interactions and a main-chain conformation indistinguishable from the wild-type structure. These results indicate that the design simulations capture important features of both stability and structure and demonstrate that negative design can be critical for attaining specificity when competing states are close in structure space.
DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in
Arabidopsis thaliana
Authors: Xie Zhixin , Allen Edwards , Wilken April , Carrington James C.
Published: August 2005

Arabidopsis thaliana
contains four
DICER-LIKE
(
DCL
) genes with specialized functions in small RNA biogenesis for RNA interference-related processes. A mutant with defects in
DCL4
was identified and analyzed for microRNA- and endogenous, small interfering RNA (siRNA)-related functions. The
dcl4-2
mutant contained normal or near-normal levels of microRNAs (21 nt) and heterochromatin-associated siRNAs (24 nt). In contrast, this mutant lacked each of three families of 21-nt trans-acting siRNAs (ta-siRNAs) and possessed elevated levels of ta-siRNA target transcripts. The
dcl4-2
mutant resembled an
rna-dependent RNA polymerase 6
mutant in that both mutants lacked ta-siRNAs and displayed heterochronic defects in which vegetative phase change was accelerated. Double mutant analyses with
dcl2-1
,
dcl3-1
, and
dcl4-2
alleles revealed hierarchical redundancy among DCL activities, leading to alternative processing of ta-siRNA precursors in the absence of DCL4. These data support the concept that plants have specialized and compartmentalized DCL functions for biogenesis of distinct small RNA classes.
Ethnoveterinary plant preparations as livestock dewormers: practices, popular beliefs, pitfalls and prospects for the future
Authors: Githiori John B. , Höglund Johan , Waller Peter J.
Published: August 2005

Ethnomedicine is an integral part of traditional medical practices in many countries of the developing world. A large proportion of the population uses this form of treatment for primary health care and for the treatment of ailments in their livestock. Livestock is a major asset for resource-poor smallholder farmers and pastoralists throughout the world and internal parasites are recognized by these communities as having an impact on livestock health. Parasitic infections are among those infections that traditional healers confidently treat and against which an enormous variety of remedies exist. Many of these are based on the use of plant preparations. Although various methods have been used for the validation of traditional phytomedical preparations, there is a lack of standardization of these procedures. The present study is aimed at providing an overview of ethnoveterinary deworming preparations, the various methods that have been used in their validation and the future prospects for their use against helminth parasites of ruminant livestock in developing countries, with an emphasis on nematode parasites. Recommendations are made on the procedures that should be followed to conductin vivoandin vitroassays. Fostering better interaction between traditional healers and scientists is advocated to prevent harmful overexploitation, both of local knowledge and of plant species that may have effects against nematode parasites.
Biology and epidemiology ofToxoplasma gondiiin man and animals
Authors: Hill Dolores E. , Chirukandoth Sreekumar , Dubey J. P.
Published: August 2005

Toxoplasma gondiiis a coccidian parasite which utilizes felids as definitive hosts, and which has an unusually wide intermediate host range. The parasite was initially described by Nicolle and Manceaux in 1908 from the rodent,Ctenodactylus gundi. Infection withT. gondiiis one of the most common parasitic infections of man and other warm-blooded animals. It has been found worldwide from Alaska to Australia. Nearly one-third of humanity has been exposed to this parasite; serologic surveys indicate thatT. gondiiinfections are common in wild carnivores, including pigs, bears, felids, fox, raccoons, and skunks. Clinical and subclinical toxoplasmosis has been reported from wild cervids, ungulates, marsupials, monkeys, and marine mammals. Southern sea otter populations have been severely impacted byToxoplasmainfections.
Avian influenza virus: prospects for prevention and control by vaccination
Authors: Lee Chang-Won , Suarez David L.
Published: August 2005

Although vaccination does not always prevent infection of avian influenza (AI) virus, the clear benefit of vaccination is in its ability to prevent disease and to reduce the amount of virus in circulation. Thus, judicious use of vaccination can be an important component of an AI control program. However, the long-term use of vaccination without eradication may result in the selection of the antigenically divergent strains, which compromises the value of vaccination. In this review, the effectiveness of currently available and future AI vaccines is discussed with suggestions for the ideal use of vaccination even with antigenic drift of the virus.
Genetic diversity ofAnaplasmaspecies major surface proteins and implications for anaplasmosis serodiagnosis and vaccine development
Authors: de la Fuente José , Lew Ala , Lutz Hans , Meli Marina L. , Hofmann-Lehmann Regina , Shkap Varda , Molad Thea , Mangold Atilio J. , Almazán Consuelo , Naranjo Victoria , Gortázar Christian , Torina Alessandra , Caracappa Santo , García-Pérez Ana L. , Barral Marta , Oporto Beatriz , Ceci Luigi , Carelli Grazia , Blouin Edmour F. , Kocan Katherine M.
Published: August 2005

The genusAnaplasma(Rickettsiales: Anaplasmataceae) includes several pathogens of veterinary and human medical importance. An understanding of the diversity ofAnaplasmamajor surface proteins (MSPs), including those MSPs that modulate infection, development of persistent infections, and transmission of pathogens by ticks, is derived in part, by characterization and phylogenetic analyses of geographic strains. Information concerning the genetic diversity ofAnaplasmaspp. MSPs will likely influence the development of serodiagnostic assays and vaccine strategies for the control of anaplasmosis.
Escherichia coliin postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies
Authors: Fairbrother John M. , Nadeau Éric , Gyles Carlton L.
Published: August 2005

Escherichia coliis one of the most important causes of postweaning diarrhea in pigs. This diarrhea is responsible for economic losses due to mortality, morbidity, decreased growth rate, and cost of medication. TheE. colicausing postweaning diarrhea mostly carry the F4 (K88) or the F18 adhesin. Recently, an increase in incidence of outbreaks of severeE. coli-associated diarrhea has been observed worldwide. The factors contributing to the increased number of outbreaks of this more severe form ofE. coli-associated diarrhea are not yet fully understood. These could include the emergence of more virulentE. coliclones, such as the O149:LT:STa:STb:EAST1:F4ac, or recent changes in the management of pigs. Development of multiple bacterial resistance to a wide range of commonly used antibiotics and a recent increase in the prevalence and severity of the postweaning syndromes will necessitate the use of alternative measures for their control. New vaccination strategies include the oral immunization of piglets with live avirulentE. colistrains carrying the fimbrial adhesins or oral administration of purified F4 (K88) fimbriae. Other approaches to control this disease include supplementation of the feed with egg yolk antibodies from chickens immunized with F4 or F18 adhesins, breeding of F18- and F4-resistant animals, supplementation with zinc and/or spray-dried plasma, dietary acidification, phage therapy, or the use of probiotics. To date, not a single strategy has proved to be totally effective and it is probable that the most successful approach on a particular farm will involve a combination of diet modification and other preventive measures.
Improved manure management and utilization: A systems approach
Authors: McCann Laura , Abdalla Charles , Jenner Mark , Massey Ray
Published: August 2005

The manure issue is complex and inherently interdisciplinary but, more fundamentally, it requires systems thinking. Current policies, technologies, infrastructure, incentives and modes of thinking about the problem fail to consider the system-wide implications, and thus fail to foster the creation of new and innovative solutions. At the farm level, complexity, uncertainty and lack of compatibility with the current farming system need to be addressed in order to promote better manure management. Production facilities, feed management and waste treatment systems (including centralized treatment plants) need to be designed to allow for beneficial use of manure components. At the industry level, changes in the poultry, swine and beef industries have resulted in concentration, both in terms of decision-making and geography. This currently limits the ability of these farmers to take a systems approach to livestock production. Environmental policies thus need to take account and advantage of this new reality. At the economy-wide level, factors affecting the demand and supply for alternative manure products need to be considered. A number of innovative uses are being developed in the private sector, but there are constraints as far as technology, institutions and infrastructure are concerned. A systems perspective will allow the design of policies and technologies that reduce environmental problems associated with manure, while promoting efficient utilization of the resource.
Consumer perceptions and demand for organic food in Australia: Focus group discussions
Authors: Chang Hui-Shung , Zepeda Lydia
Published: August 2005

In Australia, the retail value of organic food production was estimated at A$250 million, with farm-gate value at around A$90 million and exports at around A$40 million, in 2002. The current share of organic sales in total food sales in Australia is about 1%. The growth rate in organic production was forecast to continue at 10–30% per annum. Despite the positive outlook, there are consumer concerns about product recognition and product integrity. To understand how demand for organic products is changing, it is important to investigate consumer attitudes and knowledge about these issues. The objective of this study was to identify issues that may hinder or promote demand. Given the qualitative nature of these issues, we used a focus group study to probe consumers. While focus group results are a qualitative approach and not intended to be generalizable, the results suggest directions for future research. The participants raised concerns about the use of chemicals, the treatment of farm animals and the environmental impact of food production. Organic food consumers were found to be more knowledgeable about organic foods, as well as being more tolerant of higher prices and inaccessibility. The results also suggest that increasing consumers' awareness of organic farming and certification, as well as the availability of organic foods, may be the most effective way of moving organic foods into mainstream.
Priority setting in agricultural land-use types for sustainable development
Authors: Akpinar Nevin , Talay Ilkden , Gun Sema
Published: August 2005

Even in developing countries that are faced with ecological and socio-economic problems, agricultural areas are constrained by land-use laws to be developed in particular ways. This being the case, the major issues in these areas are a better quality of life and sustainable use of the limited resources. This necessitates planning for sustainable development and evaluating various qualitative and quantitative data in a single framework. Multicriteria or multipurpose decision analysis methods are appropriate for this purpose. Using these methods, physical, economical and social data, as well as planning goals, can be combined and evaluated in the context of sustainable development. These multicriteria methods have been documented widely in a variety of problem areas, but two multicriteria methods, namely AHP (analytic hierarchy process) and ELECTRE II (elimination and choice translating reality), have not been used extensively in agricultural land-use decisions in developing countries. However, in situations where decision-making criteria are non-commensurable, non-comparable or non-countable, and when it is necessary to evaluate the criteria together, as in agricultural land-use decisions, AHP and ELECTRE II methods are warranted. This study reviews these methods briefly and suggests their potential application in the agricultural land-use decision process in a developing country. For this purpose, these methods were sampled in Ziyaret Stream Basin in Adiyaman, which is part of the Güneydoğu Anadolu Projesi (GAP) (South-eastern Anatolian Project) in the Republic of Turkey. The area could be characterized by its rural and agricultural features, although it is under the pressure of Adiyaman urban development. This study shows that both AHP and ELECTRE II methods can be applied successfully for the determination of agricultural land-use priorities, which are an essential part of the quality of life and of sustainable land-use studies.
Proposal for a more efficient subsidy system for organic farming: Potential use of the tax system within the European Union
Authors: Bahrs Enno
Published: August 2005

Agricultural enterprises that are run on ecological principles receive, in some countries, higher levels of financial support from the European Union (EU) than other agricultural businesses. The aim is to provide an incentive for businesses to adopt organic farming systems, a change desired by both politicians and society. However, with a fixed system of support payments based on land area, increasing the proportion of farms being run organically leads directly to higher costs of subsidies for the EU and the individual countries. This becomes especially true if enterprises with large land areas, high turnover and high income are converted to organic farming methods. Because financial resources within the member states are in very short supply, a modification of the current system of subsidies would seem to be necessary in the near future. The aim of any new system should be to reduce profit windfalls without making a drastic reduction in the incentive effect of the current system. At the same time, businesses that can run along these lines on a sustainable basis should be especially supported. This requires an increased level of individual assessment of the potential of the subsidized enterprises in relation to their cost–performance ratio for organic agricultural production. In this connection, a partial change to profit-based tax systems can be used as an effective instrument for co-financing. The method used to calculate profits for tax purposes, which varies from state to state, can be used to assess the performance and success of the organic farmer. Tax-free allowances or reductions in tax rates could be used to provide a selective subsidy method. The ability to relate subsidies to performance, thereby reducing windfall profits, is not the only advantage of using the tax system for this purpose. Another advantage is the low transaction costs. Using the example of the offsetting of profits between time periods, it will be shown here that the tax system can be used as an additional instrument for subsidization. The existing methods of subsidy can only be supported in this way, not completely substituted.
MCP-1 overexpressed in tuberous sclerosis lesions acts as a paracrine factor for tumor development
Authors: Li Shaowei , Takeuchi Fumiko , Wang Ji-an , Fuller Christopher , Pacheco-Rodriguez Gustavo , Moss Joel , Darling Thomas N.
Published: August 2005

Patients with tuberous sclerosis complex (TSC) develop hamartomatous tumors showing loss of function of the tumor suppressor TSC1 (hamartin) or TSC2 (tuberin) and increased angiogenesis, fibrosis, and abundant mononuclear phagocytes. To identify soluble factors with potential roles in TSC tumorigenesis, we screened TSC skin tumor–derived cells for altered gene and protein expression. Fibroblast-like cells from 10 angiofibromas and five periungual fibromas produced higher levels of monocyte chemoattractant protein-1 (MCP-1) mRNA and protein than did fibroblasts from the same patient's normal skin. Conditioned medium from angiofibroma cells stimulated chemotaxis of a human monocytic cell line to a greater extent than conditioned medium from TSC fibroblasts, an effect blocked by neutralizing MCP-1–specific antibody. Overexpression of MCP-1 seems to be caused by loss of tuberin function because Eker rat embryonic fibroblasts null for Tsc2 (EEF Tsc2−/−) produced 28 times as much MCP-1 protein as did EEF Tsc2+/+ cells; transient expression of WT but not mutant human TSC2 by EEF Tsc2−/− cells inhibited MCP-1 production; and pharmacological inhibition of the Rheb-mTOR pathway, which is hyperactivated after loss of TSC2, decreased MCP-1 production by EEF Tsc2−/− cells. Together these findings suggest that MCP-1 is an important paracrine factor for TSC tumorigenesis and may be a new therapeutic target.
Defective Fas expression exacerbates neurotoxicity in a model of Parkinson's disease
Authors: Landau Anne M. , Luk Kelvin C. , Jones Michelle-Lee , Siegrist-Johnstone Rosmarie , Young Yoon Kow , Kouassi Edouard , Rymar Vladimir V. , Dagher Alain , Sadikot Abbas F. , Desbarats Julie
Published: August 2005

Fas (CD95), a member of the tumor necrosis factor-receptor superfamily, has been studied extensively as a death-inducing receptor in the immune system. However, Fas is also widely expressed in a number of other tissues, including in neurons. Here, we report that defects in the Fas/Fas ligand system unexpectedly render mice highly susceptible to neural degeneration in a model of Parkinson's disease. We found that Fas-deficient lymphoproliferative mice develop a dramatic phenotype resembling clinical Parkinson's disease, characterized by extensive nigrostriatal degeneration accompanied by tremor, hypokinesia, and loss of motor coordination, when treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at a dose that causes no neural degeneration or behavioral impairment in WT mice. Mice with generalized lymphoproliferative disease, which express a mutated Fas ligand, display an intermediate phenotype between that of lymphoproliferative and WT mice. Moreover, Fas engagement directly protects neuronal cells from MPTP/1-methyl-4-phenylpyridinium ion toxicity in vitro. Our data show that decreased Fas expression renders dopaminergic neurons highly susceptible to degeneration in response to a Parkinson-causing neurotoxin. These findings constitute the first evidence for a neuroprotective role for Fas in vivo.
Repression of SPI2 transcription by nitric oxide-producing, IFNγ-activated macrophages promotes maturation ofSalmonellaphagosomes
Authors: McCollister Bruce D. , Bourret Travis J. , Gill Ronald , Jones-Carson Jessica , Vázquez-Torres Andrés
Published: August 2005

By remodeling the phagosomal membrane, the type III secretion system encoded within the Salmonella pathogenicity island-2 (SPI2) helps Salmonella thrive within professional phagocytes. We report here that nitric oxide (NO) generated by IFNγ-activated macrophages abrogates the intracellular survival advantage associated with a functional SPI2 type III secretion system. NO congeners inhibit overall expression of SPI2 effectors encoded both inside and outside the SPI2 gene cluster, reflecting a reduced transcript level of the sensor kinase SsrA that governs overall SPI2 transcription. Down-regulation of SPI2 expression in IFNγ-treated macrophages does not seem to be the result of global NO cytotoxicity, because transcription of the housekeeping rpoD sigma factor remains unchanged, whereas the expression of the hmpA-encoded, NO-metabolizing flavohemoprotein is stimulated. Because of the reduced SPI2 expression, Salmonella-containing vacuoles interact more efficiently with compartments of the late endosomal/lysosomal system in NO-producing, IFNγ-treated macrophages. These findings demonstrate that inhibition of intracellular SPI2 transcription by NO promotes the interaction of Salmonella phagosomes with the degradative compartments required for enhanced antimicrobial activity. Transcriptional repression of a type III secretion system that blocks phagolysosome biogenesis represents a novel mechanism by which NO mediates resistance of IFNγ-activated phagocytes to an intracellular pathogen.
Type I interferons act directly on CD8 T cells to allow clonal expansion and memory formation in response to viral infection
Authors: Kolumam Ganesh A. , Thomas Sunil , Thompson Lucas J. , Sprent Jonathan , Murali-Krishna Kaja
Published: August 2005

T cell expansion and memory formation are generally more effective when elicited by live organisms than by inactivated vaccines. Elucidation of the underlying mechanisms is important for vaccination and therapeutic strategies. We show that the massive expansion of antigen-specific CD8 T cells that occurs in response to viral infection is critically dependent on the direct action of type I interferons (IFN-Is) on CD8 T cells. By examining the response to infection with lymphocytic choriomeningitis virus using IFN-I receptor–deficient (IFN-IR0) and –sufficient CD8 T cells adoptively transferred into normal IFN-IR wild-type hosts, we show that the lack of direct CD8 T cell contact with IFN-I causes >99% reduction in their capacity to expand and generate memory cells. The diminished expansion of IFN-IR0 CD8 T cells was not caused by a defect in proliferation but by poor survival during the antigen-driven proliferation phase. Thus, IFN-IR signaling in CD8 T cells is critical for the generation of effector and memory cells in response to viral infection.
NKG2D function protects the host from tumor initiation
Authors: Smyth Mark J. , Swann Jeremy , Cretney Erika , Zerafa Nadeen , Yokoyama Wayne M. , Hayakawa Yoshihiro
Published: August 2005

The activation NKG2D receptor has been shown to play an important role in the control of experimental tumor growth and metastases expressing ligands for NKG2D; however, a function for this recognition pathway in host protection from de novo tumorigenesis has never been demonstrated. We show that neutralization of NKG2D enhances the sensitivity of wild-type (WT) C57BL/6 and BALB/c mice to methylcholanthrene (MCA)-induced fibrosarcoma. The importance of the NKG2D pathway was additionally illustrated in mice deficient for either IFN-γ or tumor necrosis factor–related apoptosis-inducing ligand, whereas mice depleted of natural killer cells, T cells, or deficient for perforin did not display any detectable NKG2D phenotype. Furthermore, IL-12 therapy preventing MCA-induced sarcoma formation was also largely dependent on the NKG2D pathway. Although NKG2D ligand expression was variable or absent on sarcomas emerging in WT mice, sarcomas derived from perforin-deficient mice were Rae-1+ and immunogenic when transferred into WT syngeneic mice. These findings suggest an important early role for the NKG2D in controlling and shaping tumor formation.
Comorbidities Associated with Constipation in Children Referred for Colon Manometry May Mask Functional Diagnoses
Authors: Gertken J T , Cocjin Jose , Pehlivanov Nonko , Danda Caroline , Hyman Paul E
Published: August 2005

Background:

In children with prolonged constipation of unclear pathogenesis or unresponsive to treatment, colon manometry can discriminate between functional fecal retention (FFR) and colon neuromuscular diseases.

Aim:

To identify the clinical features precipitating referral for colon manometry in children with functional constipation.

Method:

Retrospective medical record review of 173 constipated children (116 male, mean age 6.9 years, range 1‐17 years) referred for colon manometry.

Results:

Manometry was normal in 121 (70%). In those with normal manometry, FFR was identified in 96, irritable bowel syndrome (IBS) in 10, and functional constipation in 15. Of the 96 children FFR, 72 (76%) had comorbid conditions that might have interfered with the clinician's ability to diagnose FFR. Of 52 children with colon neuromuscular disease, only 12 (23%) had comorbid conditions (P < 0.001 compared with FFR). Of children more than 4 years, those with FFR were more likely to have fecal incontinence (44 of 62; 71%) than those with other functional disorders (2 of 19; 10%; P < 0.001) or neuromuscular disease (6 of 23; 26%; P < 0.001).

Conclusions:

Two thirds of children referred for colon manometry had normal studies and met criteria for a functional diagnosis. Three quarters of those with functional constipation had a comorbid condition that might alter the history sufficiently to obscure the diagnosis.
Diagnosing Functional Abdominal Pain with the Rome II Criteria: Parent, Child, and Clinician Agreement
Authors: Schurman Jennifer Verrill , Friesen Craig A , Danda Caroline Elder , Andre Linda , Welchert Elly , Lavenbarg Teri , Cocjin Jose T , Hyman Paul E
Published: August 2005

Objectives:

To compare the Rome II diagnoses made in children with recurrent abdominal pain by physicians and by parent and child responses on the Questionnaire on Pediatric Gastrointestinal Symptoms. Rates of diagnostic agreement and reasons for disagreement were examined to determine whether changes to the Rome II criteria are needed to improve diagnostic classification.

Methods:

One hundred and forty‐eight children and their parents or guardians completed the Questionnaire on Pediatric Gastrointestinal Symptoms during their first visit to a pediatric gastroenterology clinic. Parent‐ and child‐report Rome II diagnoses were based on Questionnaire on Pediatric Gastrointestinal Symptoms scoring criteria, whereas the physician's Rome II diagnosis was based on clinical impression from history and physical examination completed at this visit. Statistical comparisons involved Pearson χ2 tests and Fisher exact tests. Kappa and weighted kappa measured agreement rates.

Results:

Most children met the criteria for a functional gastrointestinal disorder based on the Rome II criteria. Functional dyspepsia was the most common diagnosis made by all three sources. The percentage of children classified as “no diagnosis” was small and was often a function of symptom duration (especially when diagnosis rested on the child self‐report). Diagnostic agreement was fair to moderate. Diagnoses based on parent and child questionnaires agreed more often on functional dyspepsia than irritable bowel syndrome. Diagnostic disagreement was most likely to result from parent and child disagreement on defecation symptoms.

Conclusions:

The Rome II classification system shows promise for improving diagnosis, study and treatment of children with recurrent abdominal pain. However, further refinement and clarification of the Rome II criteria for symptom duration and frequency may be needed to improve diagnostic agreement.
An extensive thermodynamic characterization of the dimerization domain of the HIV‐1 capsid protein
Authors: Lidón‐Moya María C. , Barrera Francisco N. , Bueno Marta , Pérez‐Jiménez Raúl , Sancho Javier , Mateu Mauricio G. , Neira José L.
Published: August 2005

The type 1 human immunodeficiency virus presents a conical capsid formed by several hundred units of the capsid protein, CA. Homodimerization of CA occurs via its C‐terminal domain, CA‐C. This self‐association process, which is thought to be pH‐dependent, seems to constitute a key step in virus assembly. CA‐C isolated in solution is able to dimerize. An extensive thermodynamic characterization of the dimeric and monomeric species of CA‐C at different pHs has been carried out by using fluorescence, circular dichroism (CD), absorbance, nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR), and size‐exclusion chromatography (SEC). Thermal and chemical denaturation allowed the determination of the thermodynamic parameters describing the unfolding of both CA‐C species. Three reversible thermal transitions were observed, depending on the technique employed. The first one was protein concentration‐dependent; it was observed by FTIR and NMR, and consisted of a broad transition occurring between 290 and 315 K; this transition involves dimer dissociation. The second transition (Tm∼ 325 K) was observed by ANS‐binding experiments, fluorescence anisotropy, and near‐UV CD; it involves partial unfolding of the monomeric species. Finally, absorbance, far‐UV CD, and NMR revealed a third transition occurring atTm∼ 333 K, which involves global unfolding of the monomeric species. Thus, dimer dissociation and monomer unfolding were not coupled. At low pH, CA‐C underwent a conformational transition, leading to a species displaying ANS binding, a low CD signal, a red‐shifted fluorescence spectrum, and a change in compactness. These features are characteristic of molten globule‐like conformations, and they resemble the properties of the second species observed in thermal unfolding.
Characterization of N‐linked oligosaccharides in chorion peroxidase of Aedes aegypti mosquito
Authors: Li Junsuo S. , Li Jianyong
Published: August 2005

A peroxidase is present in the chorion of Aedes aegypti eggs and catalyzes chorion protein cross‐linking during chorion hardening, which is critical for egg survival in the environment. The unique chorion peroxidase (CPO) is a glycoprotein. This study deals with the N‐glycosylation site, structures, and profile of CPO‐associated oligosaccharides using mass spectrometric techniques and enzymatic digestion. CPO was isolated from chorion by solubilization and several chromatographic methods. Mono‐saccharide composition was analyzed by HPLC with fluorescent detection. Our data revealed that carbohydrate (D‐mannose, N‐acetyl D‐glucosamine, D‐arabinose, N‐acetyl D‐galactosamine, and L‐fucose) accounted for 2.24% of the CPO molecular weight. A single N‐glycosylation site (Asn328‐Cys‐ Thr) was identified by tryptic peptide mapping and de novo sequencing of native and PNGase A‐deglycosylated CPO using matrix‐assisted laser/desorption/ionization time‐of‐flight mass spectrometry (MALDI/TOF/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The Asn328 was proven to be a major fully glycosylated site. Potential tryptic glycopeptides and profile were first assessed by MALDI/TOF/MS and then by precursor ion scanning during LC/MS/MS. The structures of N‐linked oligosaccharides were elucidated from the MS/MS spectra of glycopeptides and exoglycosidase sequencing of PNGase A‐released oligosaccharides. These CPO‐associated oligosaccharides had dominant Man3GlcNAc2 and Man3 (Fuc) GlcNAc2 and high mannose‐type structures (Man4–8GlcNAc2). The truncated structures, Man2GlcNAc2 and Man2 (Fuc) GlcNAc2, were also identified. Comparison of CPO activity and Stokes radius between native and deglycosylated CPO suggests that the N‐linked oligosaccharides influence the enzyme activity by stabilizing its folded state.
Oxidized and synchrotron cleaved structures of the disulfide redox center in the N‐terminal domain of Salmonella typhimurium AhpF
Authors: Roberts Blaine R. , Wood Zachary A. , Jönsson Thomas J. , Poole Leslie B. , Karplus P. Andrew
Published: August 2005

The flavoprotein component (AhpF) of Salmonella typhimurium alkyl hydroperoxide reductase contains an N‐terminal domain (NTD) with two contiguous thioredoxin folds but only one redox‐active disulfide (within the sequence ‐Cys129‐His‐Asn‐Cys132‐). This active site is responsible for mediating the transfer of electrons from the thioredoxin reductase‐like segment of AhpF to AhpC, the peroxiredoxin component of the two‐protein peroxidase system. The previously reported crystal structure of AhpF possessed a reduced NTD active site, although fully oxidized protein was used for crystallization. To further investigate this active site, we crystallized an isolated recombinant NTD (rNTD); using diffraction data sets collected first at our in‐house X‐ray source and subsequently at a synchrotron, we showed that the active site disulfide bond (Cys129–Cys132) is oxidized in the native crystals but becomes reduced during synchrotron data collection. The NTD disulfide bond is apparently particularly sensitive to radiation cleavage compared with other protein disulfides. The two data sets provide the first view of an oxidized (disulfide) form of NTD and show that the changes in conformation upon reduction of the disulfide are localized and small. Furthermore, we report the apparent pKa of the active site thiol to be ∼5.1, a relatively low pKa given its redox potential (∼265 mV) compared with most members of the thioredoxin family.