bluuebunny/crossref_metadata_2025 huggingface dataset:
Recognition of DNA modified by trans‐[PtCl2NH3(4‐hydroxymethylpyridine)] by tumor suppressor protein p53 and character of DNA adducts of this cytotoxic complex
Authors: Stehlíková Kristýna , Kašpárková Jana , Nováková Olga , Martínez Alberto , Moreno Virtudes , Brabec Viktor
Published: December 2005

trans‐[PtCl2NH3(4‐Hydroxymethylpyridine)] (trans‐PtHMP) is an analogue of clinically ineffective transplatin, which is cytotoxic in the human leukemia cancer cell line. As DNA is a major pharmacological target of antitumor platinum compounds, modifications of DNA by trans‐PtHMP and recognition of these modifications by active tumor suppressor protein p53 were studied in cell‐free media using the methods of molecular biology and biophysics. Our results demonstrate that the replacement of the NH3 group in transplatin by the 4‐hydroxymethylpyridine ligand affects the character of DNA adducts of parent transplatin. The binding of trans‐PtHMP is slower, although equally sequence‐specific. This platinum complex also forms on double‐stranded DNA stable intrastrand and interstrand cross‐links, which distort DNA conformation in a unique way. The most pronounced conformational alterations are associated with a local DNA unwinding, which was considerably higher than those produced by other bifunctional platinum compounds. DNA adducts of trans‐PtHMP also reduce the affinity of the p53 protein to its consensus DNA sequence. Thus, downstream effects modulated by recognition and binding of p53 protein to DNA distorted by trans‐PtHMP and transplatin are not likely to be the same. It has been suggested that these different effects may contribute to different antitumor effects of these two transplatinum compounds.
The monopartite nuclear localization signal of autoimmune regulator mediates its nuclear import and interaction with multiple importin α molecules
Authors: Ilmarinen Tanja , Melén Krister , Kangas Hannele , Julkunen Ilkka , Ulmanen Ismo , Eskelin Petra
Published: December 2005

Autoimmune regulator (AIRE) is a transcriptional regulator involved in establishing immunological self‐tolerance. Mutations in the AIRE gene lead to the development of the autosomal, recessively inherited, organ‐specific autoimmune disease, autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED). The AIRE protein is mainly localized in the cell nucleus where it is associated with nuclear bodies. The N‐terminal part of AIRE has been previously shown to mediate nuclear localization of the protein. However, the functional nuclear localization signal (NLS) and nuclear import mechanisms of AIRE have not been identified. We show that, although the amino‐acid sequence of AIRE contains a potential bipartite NLS consisting of amino acids 110–114 and 131–133, only the latter part constitutes a functional NLS. Furthermore, we show by in vitro binding assays that AIRE interacts with multiple members of the nuclear transport receptor importin α family, mainly α1, α3, and α5, and that these interactions depend on the intactness of the Arg‐Lys‐rich NLS of AIRE. In addition, we found that AIRE binds to the ‘minor’ NLS‐binding site of importin α3 and α5 proteins consisting of the C‐terminal armadillo repeats 7–9. Our findings strongly suggest that the nuclear import of AIRE is mediated by the classical importin α/β pathway through binding to several importin α family members.
Systemic RNAi of the cockroach vitellogenin receptor results in a phenotype similar to that of the Drosophila yolkless mutant
Authors: Ciudad Laura , Piulachs Maria‐Dolors , Bellés Xavier
Published: December 2005

During vitellogenesis, one of the most tightly regulated processes in oviparous reproduction, vitellogenins are incorporated into the oocyte through vitellogenin receptor (VgR)‐mediated endocytosis. In this paper, we report the cloning of the VgR cDNA from Blattella germanica, as well as the first functional analysis of VgR following an RNA interference (RNAi) approach. We characterized the VgR, VgR mRNA and protein expression patterns in pre‐adult and adult stages of this cockroach, as well as VgR immunolocalization in ovarioles, belonging to the panoistic type. We then specifically disrupted VgR gene function using RNAi techniques. Knockdown of VgR expression led to a phenotype characterized by low yolk content in the ovary and high vitellogenin concentration in the haemolymph. This phenotype is equivalent to that of the yolkless mutant of Drosophila melanogaster, which have the yl (VgR) gene disrupted. The results additionally open the perspective that development genes can be functionally analyzed via systemic RNAi in this basal species.
The role of glucose 6‐phosphate in mediating the effects of glucokinase overexpression on hepatic glucose metabolism
Authors: Härndahl Linda , Schmoll Dieter , Herling Andreas W. , Agius Loranne
Published: December 2005

Pharmacological activation or overexpression of glucokinase in hepatocytes stimulates glucose phosphorylation, glycolysis and glycogen synthesis. We used an inhibitor of glucose 6‐phosphate (Glc6P) hydrolysis, namely the chlorogenic derivative, 1‐[2‐(4‐chloro‐phenyl)‐cyclopropylmethoxy]‐3, 4‐dihydroxy‐5‐(3‐imidazo[4,5‐b]pyridin‐1‐yl‐3‐phenyl‐acryloyloxy)‐cyclohexanecarboxylic acid (also known as S4048), to determine the contribution of Glc6P concentration, as distinct from glucokinase protein or activity, to the control of glycolysis and glycogen synthesis by glucokinase overexpression. The validity of S4048 for testing the role of Glc6P was supported by its lack of effect on glucokinase binding and its nuclear/cytoplasmic distribution. The stimulation of glycolysis by glucokinase overexpression correlated strongly with glucose phosphorylation, whereas glycogen synthesis correlated strongly with Glc6P concentration. Metabolic control analysis was used to determine the sensitivity of glycogenic flux to glucokinase or Glc6P at varying glucose concentrations (5–20 mm). The concentration control coefficient of glucokinase on Glc6P (1.4–1.7) was relatively independent of glucose concentration, whereas the flux control coefficients of Glc6P (2.4–1.0) and glucokinase (3.7–1.8) on glycogen synthesis decreased with glucose concentration. The high sensitivity of glycogenic flux to Glc6P at low glucose concentration is consistent with covalent modification by Glc6P of both phosphorylase and glycogen synthase. The high control strength of glucokinase on glycogenic flux is explained by its concentration control coefficient on Glc6P and the high control strength of Glc6P on glycogen synthesis. It is suggested that the regulatory strength of pharmacological glucokinase activators on glycogen metabolism can be predicted from their effect on the Glc6P content.
Factor VIIIa regulates substrate delivery to the intrinsic factor X‐activating complex
Authors: Panteleev Mikhail A. , Ananyeva Natalya M. , Greco Nicholas J. , Ataullakhanov Fazoil I. , Saenko Evgueni L.
Published: December 2005

Activation of coagulation factor X (fX) by activated factors IX (fIXa) and VIII (fVIIIa) requires the assembly of the enzyme–cofactor–substrate fIXa–fVIIIa–fX complex on negatively charged phospholipid membranes. Using flow cytometry, we explored formation of the intermediate membrane‐bound binary complexes of fIXa, fVIIIa, and fX. Studies of the coordinate binding of coagulation factors to 0.8‐µm phospholipid vesicles (25/75 phosphatidylserine/phosphatidylcholine) showed that fVIII (fVIIIa), fIXa, and fX bind to 32 700 ± 5000 (33 200 ± 14 100), 20 000 ± 4500, and 30 500 ± 1300 binding sites per vesicle with apparent Kd values of 76 ± 23 (71 ± 5), 1510 ± 430, and 223 ± 79 nm, respectively. FVIII at 10 nm induced the appearance of additional high‐affinity sites for fIXa (1810 ± 370, 20 ± 5 nm) and fX (12 630 ± 690, 14 ± 4 nm), whereas fX at 100 nm induced high‐affinity sites for fIXa (541 ± 67, 23 ± 5 nm). The effects of fVIII and fVIIIa on the binding of fIXa or fX were similar. The apparent Michaelis constant of the fX activation by fIXa was a linear function of the fVIIIa concentration with a slope of 1.00 ± 0.12 and an intrinsic Km value of 8.0 ± 1.5 nm, in agreement with the hypothesis that the reaction rate is limited by the fVIIIa–fX complex formation. In addition, direct correlation was observed between the fX activation rate and formation of the fVIIIa–fX complex. Titration of fX, fVIIIa, phospholipid concentration and phosphatidylserine content suggested that at high fVIIIa concentration the reaction rate is regulated by the concentration of free fX rather than of membrane‐bound fX. The obtained results reveal formation of high‐affinity fVIIIa–fX complexes on phospholipid membranes and suggest their role in regulating fX activation by anchoring and delivering fX to the enzymatic complex.
Structural characterization of the large soluble oligomers of the GTPase effector domain of dynamin
Authors: Chugh Jeetender , Chatterjee Amarnath , Kumar Ashutosh , Mishra Ram Kumar , Mittal Rohit , Hosur Ramakrishna V.
Published: December 2005

Dynamin, a protein playing crucial roles in endocytosis, oligomerizes to form spirals around the necks of incipient vesicles and helps their scission from membranes. This oligomerization is known to be mediated by the GTPase effector domain (GED). Here we have characterized the structural features of recombinant GED using a variety of biophysical methods. Gel filtration and dynamic light scattering experiments indicate that in solution, the GED has an intrinsic tendency to oligomerize. It forms large soluble oligomers (molecular mass > 600 kDa). Interestingly, they exist in equilibrium with the monomer, the equilibrium being largely in favour of the oligomers. This equilibrium, observed for the first time for GED, may have regulatory implications for dynamin function. From the circular dichroism measurements the multimers are seen to have a high helical content. From multidimensional NMR analysis we have determined that about 30 residues in the monomeric units constituting the oligomers are flexible, and these include a 17 residue stretch near the N‐terminal. This contains two short segments with helical propensities in an otherwise dynamic structure. Negatively charged SDS micelles cause dissociation of the oligomers into monomers, and interestingly, the helical characteristics of the oligomer are completely retained in the individual monomers. The segments along the chain that are likely to form helices have been predicted from five different algorithms, all of which identify two long stretches. Surface electrostatic potential calculation for these helices reveals that there is a distribution of neutral, positive and negative potentials, suggesting that both electrostatic and hydrophobic interactions could be playing important roles in the oligomer core formation. A single point mutation, I697A, in one of the helices inhibited oligomerization quite substantially, indicating firstly, a special role of this residue, and secondly, a decisive, though localized, contribution of hydrophobic interaction in the association process.
Human‐blind probes and primers for dengue virus identification
Authors: Putonti Catherine , Chumakov Sergei , Mitra Rahul , Fox George E. , Willson Richard C. , Fofanov Yuriy
Published: December 2005

Reliable detection and identification of pathogens in complex biological samples, in the presence of contaminating DNA from a variety of sources, is an important and challenging diagnostic problem for the development of field tests. The problem is compounded by the difficulty of finding a single, unique genomic sequence that is present simultaneously in all genomes of a species of closely related pathogens and absent in the genomes of the host or the organisms that contribute to the sample background. Here we describe ‘host‐blind probe design’– a novel strategy of designing probes based on highly frequent genomic signatures found in the pathogen genomes of interest but absent from the host genome. Upon hybridization, an array of such informative probes will produce a unique pattern that is a genetic fingerprint for each pathogen strain. This multiprobe approach was applied to 83 dengue virus genome sequences, available in public databases, to design and perform in silico microarray experiments. The resulting patterns allow one to unequivocally distinguish the four major serotypes, and within each serotype to identify the most similar strain among those that have been completely sequenced. In an environment where dengue is indigenous, this would allow investigators to determine if a particular isolate belongs to an ongoing outbreak or is a previously circulating version. Using our probe set, the probability that misdiagnosis at the serotype level would occur is ≈ 1 : 10150.
Stage‐specific expression of Caenorhabditis elegans ribonuclease H1 enzymes with different substrate specificities and bivalent cation requirements
Authors: Kochiwa Hiromi , Itaya Mitsuhiro , Tomita Masaru , Kanai Akio
Published: December 2005

Ribonuclease H1 (RNase H1) is a widespread enzyme found in a range of organisms from viruses to humans. It is capable of degrading the RNA moiety of DNA–RNA hybrids and requires a bivalent ion for activity. In contrast with most eukaryotes, which have one gene encoding RNase H1, the activity of which depends on Mg2+ ions, Caenorhabditis elegans has four RNase H1‐related genes, and one of them has an isoform produced by alternative splicing. However, little is known about the enzymatic features of the proteins encoded by these genes. To determine the differences between these enzymes, we compared the expression patterns of each RNase H1‐related gene throughout the development of the nematode and the RNase H activities of their recombinant proteins. We found gene‐specific expression patterns and different enzymatic features. In particular, besides the enzyme that displays the highest activity in the presence of Mg2+ ions, C. elegans has another enzyme that shows preference for Mn2+ ion as a cofactor. We characterized this Mn2+‐dependent RNase H1 for the first time in eukaryotes. These results suggest that there are at least two types of RNase H1 in C. elegans depending on the developmental stage of the organism.
Simulated beetle defoliation on willow genotypes in mixture and monotype plantations
Authors: Powers S.J. , Peacock L. , Yap M.‐L. , Brain P.
Published: December 2005

The effect of simulated beetle damage (0%, 25%, 50% and 75% mechanical defoliation) on 12 willow genotypes, grown in short‐rotation coppice, was studied in a modified criss‐cross experimental design. The design enabled the above‐ground effects of monoculture and mixed planting to be assessed. Repeated measurements were modelled to produce derived variables in terms of time or, more appropriately, in terms of accumulated day length (i.e. ‘developmental time’) units. These derived variables were then analysed using the REsidual Maximum Likelihood (REML) method implemented in GenStat™ (2001). No significant competition effect between the genotypes due to planting regime was detected. Genotypes Salix viminalis × Salix schwerinii‘Beagle’ and S. viminalis × S. schwerinii‘Torhild’ were found to have the greatest rate of increase in leaves regardless of defoliation and also the greatest height prior to defoliation. Genotype Salix dasyclados‘Loden’ showed the highest rate of growth under the stress of defoliation. When assessing height at the end of the growing season, S. viminalis × S. schwerinii‘Olof’ was the highest genotype for 25% and 75% levels of defoliation, but genotypes Salix aurita × Salix cinerea‘Delamere’, Loden and S. viminalis × Salix burjatica‘Ashton Parfitt’ appeared to be most tolerant by having consecutively lower base day lengths (i.e. increasing the accumulation of developmental units and the length of the growing season) for increasing defoliation. Shorter genotypes tended to be more tolerant, but of the higher genotypes reaching a control height of greater than 3 m by the end of the growing season, S. viminalis × S. schwerinii‘Tora’ and Beagle performed best to 50% defoliation.
Attractiveness of single and multiple species flower patches to beneficial insects in agroecosystems
Authors: Pontin D.R. , Wade M.R. , Kehrli P. , Wratten S.D.
Published: December 2005

The provision of floral resources for the enhancement of beneficial insect populations has shown promise as a strategy to enhance biological control and pollination in agroecosystems. One approach involves the provision of a single flower species while a second involves the multiple flower species, but the two have never been compared experimentally. Here we examine the influence of single and multiple species flower treatments on the abundance and foraging behaviour of key beneficial insects in two agricultural agroecosystems (broccoli and lucerne crops). The five flower treatments comprised buckwheat only, phacelia only, a simple mixture of buckwheat and phacelia, a complex mixture of buckwheat, phacelia and a commercial seed blend or the existing crop as a control. The abundance of bumble‐bees (Bombus hortorum) and honey bees (Apis mellifera) was highest in the three treatments that contained phacelia, while hoverfly (Melanostoma fasciatum) numbers were high in all four flower treatments. Bumble‐bees and honey bees probed almost exclusively phacelia flowers, even when provided with a choice of other flower species in the simple and complex mixture treatments. In contrast, hoverflies probed the flowers of all plant species in single and multiple species treatments, with no apparent difference in acceptance. However, in mixture treatments, the majority of individual bumble‐bees, honey bees and hoverflies probed the flowers from only one species, despite the presence of alternative flower species. Our results illustrate how an appreciation of insect floral attractiveness can be used to customise the species composition of floral patches to potentially maximise biological control and pollination in targeted agroecosystems.
Regulation of sexual behaviour in male macaques by sex steroid modulation of the serotonergic system
Authors: Barrett Gordon M. , Bardi Massimo , Guillén Ana Karina Zavala , Mori Akio , Shimizu Keiko
Published: December 2005

The view that androgen action is the primary impetus underlying male‐typical behaviour has been irrevocably altered by the profound perturbations in social and sexual behaviour observed in recent models of oestrogen insufficiency in male mice. Evidence is also accumulating for an involvement of oestrogens in the modulation of neural systems that are thought to play important roles in male reproductive functioning. Specifically, the serotonergic system is implicated in diverse autonomic functions, most or all of which are sensitive to oestradiol as well. Although their interaction domains have yet to be examined in male primates, roles have been established for both oestrogen and serotonin in the regulation of male sexual behaviour. We used a blinded, sham‐treated and self‐controlled, randomized, multitreatment cross‐over design to test the hypothesis that male sexual behaviour is regulated by oestrogen modulation of the serotonergic system in intact male Japanese macaques. Regression analysis revealed that oestradiol and whole blood tryptophan, but not testosterone or 5α‐dihydrotestosterone, had additive, independent effects on male potentia over a range of hormone concentrations, whereas androgens were confirmed to be the primary determinants of sexual motivation. We suggest that modulation of the serotonergic system by ‘female hormones’ may be fundamental to the regulation of male mating success in higher primates. This might also explain, at least in part, why significant correlations between steroid hormones and male copulatory behaviour have traditionally proven so elusive in this order, thereby warranting a re‐evaluation of the current notion that male sexual behaviour has been emancipated from activational hormonal control in higher primates.
The influence of gender on parasympathetic vasodilatation in the submandibular gland of the rat
Authors: Anderson Leigh C. , Martin David J. , Phillips Dallen L. , Killpack Kenneth J. , Bone Sven E. , Rahimian Roshanak
Published: December 2005

Parasympathetic vasodilatation in the rat submandibular gland is mediated by nitric oxide‐dependent and ‐independent mechanisms (prostacyclin and endothelium‐derived hyperpolarizing factor (EDHF)). The purpose of this study was to determine the influence of gender on the relative contributions of each pathway to nerve‐stimulated vasodilatation. Absolute increases in perfusion (laser Doppler flowmetry) were similar in male and female rats (in arbitrary perfusion units: 6159 ± 4530 and 5601 ± 3877 at 2 Hz; 15645 ± 6830 and 14848 ± 6118 at 5 Hz; and 22418 ± 7660 and 18878 ± 5864 at 10 Hz). However, expressed as a percentage increase above resting values, stimulated perfusion was higher in males than in females (P< 0.05). In males bothNω‐nitro‐l‐arginine methyl ester (l‐NAME) and indomethacin partly blocked parasympathetic vasodilatation at all frequencies tested (P< 0.05). In female rats significant reductions in nerve‐stimulated perfusion were observed only at 2 and 5 Hz, but the effects ofl‐NAME were greater than in males (−64 compared with −45% at 2 Hz and −45 compared with −33% at 5 Hz,P< 0.05). Indomethacin by itself had no apparent effect in females. The combined effects ofl‐NAME and indomethacin were dependent on the order of administration and on gender. Followingl‐NAME, indomethacin had no further effect in males or females.l‐NAME reduced indomethacin‐resistant vasodilatation in males and females, but the added effect of indomethacin was more pronounced in males. Finally, atropine‐resistant vasodilatation was partly blocked byl‐NAME, and the remaining vasodilatation was abolished by spantide I (substance P receptor antagonist). We conclude that NO, products of cyclo‐oxygenase activity and EDHF all play a role in parasympathetic vasodilatation, but that NO and EDHF are the major endothelium‐derived vasodilators in the rat submandibular gland. In addition, when other pathways are blocked EDHF makes a greater contribution in females. Lastly, both vasoactive intestinal peptide and substance P contribute to the atropine‐resistant vasodilatation.
The
Brucella suis
Type IV Secretion System Assembles in the Cell Envelope of the Heterologous Host
Agrobacterium tumefaciens
and Increases IncQ Plasmid pLS1 Recipient Competence
Authors: Carle Anna , Höppner Christoph , Ahmed Aly Khaled , Yuan Qing , den Dulk-Ras Amke , Vergunst Annette , O'Callaghan David , Baron Christian
Published: December 2005

Pathogenic
Brucella
species replicate within mammalian cells, and their type IV secretion system is essential for intracellular survival and replication. The options for biochemical studies on the
Brucella
secretion system are limited due to the rigidity of the cells and biosafety concerns, which preclude large-scale cell culture and fractionation. To overcome these problems, we heterologously expressed the
Brucella suis virB
operon in the closely related α
2
-proteobacterium
Agrobacterium tumefaciens
and showed that the VirB proteins assembled into a complex. Eight of the twelve VirB proteins were detected in the membranes of the heterologous host with specific antisera. Cross-linking indicated protein-protein interactions similar to those in other type IV secretion systems, and the results of immunofluorescence analysis supported the formation of VirB protein complexes in the cell envelope. Production of a subset of the
B. suis
VirB proteins (VirB3-VirB12) in
A. tumefaciens
strongly increased its ability to receive IncQ plasmid pLS1 in conjugation experiments, and production of VirB1 further enhanced the conjugation efficiency. Plasmid recipient competence correlated with periplasmic leakage and the detergent sensitivity of
A. tumefaciens
, suggesting a weakening of the cell envelope. Heterologous expression thus permits biochemical characterization of
B. suis
type IV secretion system assembly.
Sexual Dimorphism in the Control of Amebic Liver Abscess in a Mouse Model of Disease
Authors: Lotter Hannelore , Jacobs Thomas , Gaworski Iris , Tannich Egbert
Published: December 2005

Amebic liver abscess (ALA) is the most common extraintestinal manifestation of human infection by the enteric protozoan parasiteEntamoeba histolytica. In contrast to intestinal infection, ALA greatly predominates in males but is rare in females. Since humans are the only relevant host forE. histolytica, experimental studies concerning this sexual dimorphism have been hampered by the lack of a suitable animal model. By serial liver passage of culturedE. histolyticatrophozoites in gerbils and mice, we generated amebae which reproducibly induce ALA in C57BL/6 mice. Interestingly, all animals developed ALA, but the time courses of abscess formation differed significantly between the genders. Female mice were able to clear the infection within 3 days, whereas in male mice the parasite could be recovered for at least 14 days. Accordingly, male mice showed a prolonged time of recovery from ALA. Immunohistology of abscesses revealed that polymorphonuclear leukocytes and macrophages were the dominant infiltrates, but in addition, γ,δ-T cells, NK cells, and natural killer T (NKT) cells were also present at early times during abscess development, whereas conventional α,β-T cells appeared later, when female mice had already cleared the parasite. Interestingly, male and female mice differed in early cytokine production in response to ameba infection. Enzyme-linked immunospot assays performed with spleen cells of infected animals revealed significantly higher numbers of interleukin-4-producing cells in male mice but significantly higher numbers of gamma interferon (IFN-γ)-producing cells in female mice. Early IFN-γ production and the presence of functional NKT cells were found to be important for the control of hepatic amebiasis as application of an IFN-γ-neutralizing monoclonal antibody or the use of NKT knockout mice (Vα14iNKT, Jα 18−/−) dramatically increased the size of ALA in female mice. In addition,E. histolyticatrophozoites could be reisolated from liver abscesses of Jα18−/−mice on day 7 postinfection, when wild-type mice had already cleared the parasite. These data suggest that the sexual dimorphism in the control of ALA is due to gender-specific differences in early cytokine production mediated at least in part by NKT cells in response toE. histolyticainfection of the liver.
The Chemokines CXCL9 and CXCL10 Promote a Protective Immune Response but Do Not Contribute to Cardiac Inflammation following Infection withTrypanosoma cruzi
Authors: Hardison Jenny L. , Wrightsman Ruth A. , Carpenter Philip M. , Lane Thomas E. , Manning Jerry E.
Published: December 2005

The expression of chemokines within the heart during experimental infection of susceptible mice with the Colombiana strain ofTrypanosoma cruziwas characterized in an attempt to determine a functional role for these molecules in both host defense and disease. Analysis of chemokine transcripts revealed that CXC chemokine ligand 9 (CXCL9) and CXCL10, as well as CC chemokine ligand 2 (CCL2) and CCL5, were prominently expressed during acute disease, whereas transcripts for CXCL9, CXCL10, and CCL5 remained elevated during chronic infection. Inflammatory macrophages present within the heart were the primary cellular source of these chemokines followingT. cruziinfection. Peak chemokine expression levels coincided with increased gamma interferon expression and inflammation within the heart, suggesting a role for these molecules in both host defense and disease. Indeed, simultaneous treatment ofT. cruzi-infected mice with neutralizing antibodies specific for CXCL9 and CXCL10 resulted in an increased parasite burden that was sustained out to 50 days p.i. Antibody targeting either CXCL10 or CCL5 did not change eitherT. cruziburden within the heart nor attenuate the severity of cardiac inflammation at any time point examined, while targeting CXCL9 in combination with CXCL10 resulted in increased parasite burden. Collectively, these studies imply that CXCL9 and CXCL10 signaling enhances immune responses following parasite infection. However, antibody targeting of CXCL9 and CXCL10, or CXCL10 alone, or CCL5 alone does not directly modulate the inflammatory response within the heart, suggesting that other proinflammatory factors are able to regulate inflammation in this tissue in response toT. cruziinfection.
The CC Chemokine Receptor 5 Is Important in Control of Parasite Replication and Acute Cardiac Inflammation following Infection withTrypanosoma cruzi
Authors: Hardison Jenny L. , Wrightsman Ruth A. , Carpenter Philip M. , Kuziel William A. , Lane Thomas E. , Manning Jerry E.
Published: December 2005

Infection of susceptible mice with the Colombiana strain ofTrypanosoma cruziresults in an orchestrated expression of chemokines and chemokine receptors within the heart that coincides with parasite burden and cellular infiltration. CC chemokine receptor 5 (CCR5) is prominently expressed during both acute and chronic disease, suggesting a role in regulating leukocyte trafficking and accumulation within the heart followingT. cruziinfection. To better understand the functional role of CCR5 and its ligands with regard to both host defense and/or disease, CCR5−/−mice were infected withT. cruzi, and the disease severity was evaluated. Infected CCR5−/−mice develop significantly higher levels of parasitemia (P≤ 0.05) and cardiac parasitism (P ≤ 0.01) during acute infection that correlated with reduced survival. Further, we show that CCR5 is essential for directing the migration of macrophages and T cells to the heart early in acute infection withT. cruzi. In addition, data are provided demonstrating that CCR5 does not play an essential role in maintaining inflammation in the heart during chronic infection. Collectively, these studies clearly demonstrate that CCR5 contributes to the control of parasite replication and the development of a protective immune response during acute infection but does not ultimately participate in maintaining a chronic inflammatory response within the heart.
Binding and Internalization of Glucuronoxylomannan, the Major Capsular Polysaccharide ofCryptococcus neoformans, by Murine Peritoneal Macrophages
Authors: Chang Zong Liang , Netski Dale , Thorkildson Peter , Kozel Thomas R.
Published: December 2005

Glucuronoxylomannan (GXM), the major component of the capsular polysaccharide ofCryptococcus neoformans, is essential to virulence of the yeast. Previous studies found that the interaction between GXM and phagocytic cells has biological consequences that may contribute to the pathogenesis of cryptococcosis. We found that GXM binds to and is taken up by murine peritoneal macrophages. Uptake is dose and time dependent. Examination of the sites of GXM accumulation by immunofluorescence microscopy showed that the pattern was discontinuous and punctate both on the surfaces of macrophages and at intracellular depots. Although resident macrophages showed appreciable accumulation of GXM, uptake was greatest with thioglycolate-elicited macrophages. A modest stimulation of GXM binding followed treatment of resident macrophages with phorbol 12-myristate 13-acetate, but treatment with lipopolysaccharide or gamma interferon alone or in combination had no effect. Accumulation of GXM was critically dependent on cytoskeleton function; a near complete blockade of accumulation followed treatment with inhibitors of actin. GXM accumulation by elicited macrophages was blocked by treatment with inhibitors of tyrosine kinase, protein kinase C, and phospholipase C, but not by inhibitors of phosphatidylinositol 3-kinase, suggesting a critical role for one or more signaling pathways in the macrophage response to GXM. Taken together, the results demonstrate that it is possible to experimentally enhance or suppress binding of GXM to macrophages, raising the possibility for regulation of the interaction between this essential virulence factor and binding sites on cells that are central to host resistance.
Trypanosoma cruzi
Infection Induces Proliferation of Vascular Smooth Muscle Cells
Authors: Hassan Ghada S. , Mukherjee Shankar , Nagajyothi Fnu , Weiss Louis M. , Petkova Stefka B. , de Almeida Cecilia J. , Huang Huan , Desruisseaux Mahalia S. , Bouzahzah Boumediene , Pestell Richard G. , Albanese Chris , Christ George J. , Lisanti Michael P. , Tanowitz Herbert B.
Published: December 2005

Trypanosoma cruzi
infection causes cardiomyopathy and vasculopathy. Previous studies have demonstrated that infection of human umbilical vein endothelial and smooth muscle cells resulted in activation of extracellular signal-regulated kinase (ERK). In the present study, smooth muscle cells were infected with trypomastigotes, and immunoblot analysis revealed an increase in the expression of cyclin D1 and proliferating cell nuclear antigen (PCNA), important mediators of smooth muscle cell proliferation. Interestingly, after infection, the expression of caveolin-1 was reduced in both human umbilical vein endothelial cells and smooth muscle cells. Immunoblot and immunohistochemical analyses of lysates of carotid arteries obtained from infected mice revealed increased expression of PCNA, cyclin D1, its substrate, phospho-Rb (Ser780), and phospho-ERK1/2. The expression of the cyclin-dependent kinase inhibitor p21

Cip1/Waf1

, caveolin-1, and caveolin-3 was reduced in carotid arteries obtained from infected mice. There was an increase in the abundance of pre-pro-endothelin-1 mRNA in the carotid artery and aorta from infected mice. The ET
A
receptor was also elevated in infected arteries. ERK activates endothelin-1, which in turn exerts positive feedback activating ERK, and cyclin D1 is a downstream target of both endothelin-1 and ERK. There was significant incorporation of bromodeoxyuridine into smooth muscle cell DNA when treatment was with conditioned medium obtained from infected endothelial cells. Taken together, these data suggest that
T. cruzi
infection stimulates smooth muscle cell proliferation and is likely a result of the upregulation of the ERK-cyclin D1-endothelin-1 pathway.
Transcriptional Profiling of
Mycoplasma hyopneumoniae
during Heat Shock Using Microarrays
Authors: Madsen Melissa L. , Nettleton Dan , Thacker Eileen L. , Edwards Robert , Minion F. Chris
Published: December 2005

Bacterial pathogens undergo stress during host colonization and disease processes. These stresses result in changes in gene expression to compensate for potentially lethal environments developed in the host during disease.
Mycoplasma hyopneumoniae
colonizes the swine epithelium and causes a pneumonia that predisposes the host to enhanced disease from other pathogens. How
M. hyopneumoniae
responds to changing environments in the respiratory tract during disease progression is not known. In fact, little is known concerning the capabilities of mycoplasmas to respond to changing growth environments. With limited genes, mycoplasmas are thought to possess only a few mechanisms for gene regulation. A microarray consisting of 632 of the 698 open reading frames of
M. hyopneumoniae
was constructed and used to study gene expression differences during a temperature shift from 37°C to 42°C, a temperature swing that might be encountered during disease. To enhance sensitivity, a unique hexamer primer set was employed for generating cDNA from only mRNA species. Our analysis identified 91 genes that had significant transcriptional differences in response to heat shock conditions (
P
< 0.01) with an estimated false-discovery rate of 4 percent. Thirty-three genes had a change threshold of 1.5-fold or greater. Many of the heat shock proteins previously characterized in other bacteria were identified as significant in this study as well. A proportion of the identified genes (54 of 91) currently have no assigned function.
Antigen Discovery: a Postgenomic Approach to Leprosy Diagnosis
Authors: Aráoz Romulo , Honoré Nadine , Cho Sungae , Kim Jong-Pill , Cho Sang-Nae , Monot Marc , Demangel Caroline , Brennan Patrick J. , Cole Stewart T.
Published: December 2005

Leprosy is an infectious, neurodegenerative disease of humans caused by
Mycobacterium leprae
. Despite effective control programs, the incidence of leprosy remains stubbornly high, suggesting that transmission may be more common than expected. The rationale of this work was to use bioinformatics and comparative genomics to identify potentially antigenic proteins for diagnostic purposes. This approach defined three classes of proteins: those restricted to
M. leprae
(class I), those present in
M. leprae
with orthologues in other organisms besides mycobacteria (class II), and exported or surface-exposed proteins (class III). Twelve genes (two class I, four class II, and six class III proteins) were cloned in
Escherichia coli
, and their protein products were purified. Six of these proteins were detected in cell extracts of
M. leprae
by immunoblotting. The immunogenicity of each recombinant protein was then investigated in leprosy patients by measuring the reactivity of circulating antibody and gamma interferon (IFN-γ) responses in T-cell restimulation assays. Several class II and class III proteins were recognized by circulating antibodies. Importantly, most class II proteins elicited IFN-γ responses that were significantly stronger than those produced by previously identified antigens. Among them, two class II proteins, ML0308 and ML2498, showed marked humoral and cellular immunogenicity, therefore providing promising candidates for the diagnosis of both tuberculoid and lepromatous forms of leprosy.
Enterocyte Cytoskeleton Changes Are Crucial for Enhanced Translocation of NonpathogenicEscherichia coliacross Metabolically Stressed Gut Epithelia
Authors: Nazli Aisha , Wang Arthur , Steen Oren , Prescott David , Lu Jun , Perdue Mary H. , Söderholm Johan D. , Sherman Philip M. , McKay Derek M.
Published: December 2005

Substantial data implicate the commensal flora as triggers for the initiation of enteric inflammation or inflammatory disease relapse. We have shown that enteric epithelia under metabolic stress respond to nonpathogenic bacteria by increases in epithelial paracellular permeability and bacterial translocation. Here we assessed the structural basis of these findings. Confluent filter-grown monolayers of the human colonic T84 epithelial cell line were treated with 0.1 mM dinitrophenol (which uncouples oxidative phosphorylation) and noninvasive, nonpathogenicEscherichia coli(strain HB101, 106CFU) with or without pretreatment with various pharmacological agents. At 24 h later, apoptosis, tight-junction protein expression, transepithelial resistance (TER; a marker of paracellular permeability), and bacterial internalization and translocation were assessed. Treatment with stabilizers of microtubules (i.e., colchicine), microfilaments (i.e., jasplakinolide) and clathrin-coated pit endocytosis (i.e., phenylarsine oxide) all failed to block DNP+E. coliHB101-induced reductions in TER but effectively prevented bacterial internalization and translocation. Neither the TER defect nor the enhanced bacterial translocations were a consequence of increased apoptosis. These data show that epithelial paracellular and transcellular (i.e., bacterial internalization) permeation pathways are controlled by different mechanisms. Thus, epithelia under metabolic stress increase their endocytotic activity that can result in a microtubule-, microfilament-dependent internalization and transcytosis of bacteria. We speculate that similar events in vivo would allow excess unprocessed antigen and bacteria into the mucosa and could evoke an inflammatory response by, for example, the activation of resident or recruited immune cells.
Implications for Induction of Autoimmunity via Activation of B-1 Cells byHelicobacter pyloriUrease
Authors: Yamanishi Shingo , Iizumi Tadasu , Watanabe Eri , Shimizu Masumi , Kamiya Shigeru , Nagata Kumiko , Kumagai Yoshihiro , Fukunaga Yoshitaka , Takahashi Hidemi
Published: December 2005

Besides various gastroduodenal diseases,Helicobacter pyloriinfection may be involved in autoimmune disorders like rheumatoid arthritis (RA) or idiopathic thrombocytopenic purpura. Such autoimmune disorders are often associated with autoreactive antibodies produced by B-1 cells, a subpopulation of B lymphocytes. These B-1 cells are mainly located in the pleural cavity or mucosal compartment. The existence ofH. pyloriurease-specific immunoglobulin A (IgA)-producing B cells in the mucosal compartment and of their specific IgM in the sera of acutely infected volunteers suggests the possibility that urease stimulates mucosal innate immune responses. Here, we show for the first time that purifiedH. pyloriurease predominantly stimulates the B-1-cell population rather than B-2 cells, which produce antigen-specific conventional antibodies among splenic B220+B cells. The fact that such stimulation of B-1 cells was not affected by the addition of polymyxin B indicates that the effect of purifiedH. pyloriurease was not due to the contamination with bacterial lipopolysaccharide. Furthermore, the production of various B-1-cell-related autoreactive antibodies such as IgM-type rheumatoid factor, anti-single-stranded DNA antibody, and anti-phosphatidyl choline antibody was observed when the splenic B cells were stimulated with purifiedH. pyloriurease in vitro. These findings suggest thatH. pyloricomponents, urease in particular, may be among the environmental triggars that initiate various autoimmune diseases via producing autoreactive antibodies through the activation of B-1 cells. The findings shown here offer important new insights into the pathogenesis of autoimmune disorders related toH. pyloriinfection.
Target Antigen, Age, and Duration of Antigen Exposure Independently Regulate Immunoglobulin G Subclass Switching in Malaria
Authors: Tongren J. Eric , Drakeley Christopher J. , McDonald Suzanna L. R. , Reyburn Hugh G. , Manjurano Alphaxard , Nkya Watoky M. M. , Lemnge Martha M. , Gowda Channe D. , Todd Jim E. , Corran Patrick H. , Riley Eleanor M.
Published: December 2005

The isotype/subclass of immunoglobulin determines antibody function, but rather little is known about factors that direct class switching in vivo. To evaluate factors that might influence the maturation of the antibody response during infection, we conducted a seroepidemiological study of the immunoglobulin G (IgG) subclass response to four merozoite-associated antigens ofPlasmodium falciparumin a mountainous region of northeastern Tanzania, where malaria endemicity declines with increasing altitudes. We found that IgG1/IgG3 class switching is independently affected by the nature of the antigen, cumulative exposure to the antigen, and the maturity of the immune system (i.e., the age of the individual). These observations provide insights into the effects of immune system maturity, the duration and intensity of antigen exposure, and inherent characteristics of individual antigens on the process of class switching in human B cells. Our data also throw light on the consequences of class switch decisions on the gradual acquisition of antimalarial immunity.
Dietary Iron Content Mediates Hookworm Pathogenesis In Vivo
Authors: Held Melissa R. , Bungiro Richard D. , Harrison Lisa M. , Hamza Iqbal , Cappello Michael
Published: December 2005

Hookworm infection is associated with growth delay and iron deficiency anemia in developing countries. A series of experiments were designed in order to test the hypothesis that host dietary iron restriction mediates susceptibility to hookworm infection using the hamster model of
Ancylostoma ceylanicum
. Animals were maintained on diets containing either 10 ppm iron (iron restricted) or 200 ppm iron (standard/high iron), followed by infection with
A. ceylanicum
third-stage larvae. Infected animals fed the standard diet exhibited statistically significant growth delay and reduced blood hemoglobin levels compared to uninfected controls on day 20 postinfection. In contrast, no statistically significant differences in weight or hemoglobin concentration were observed between infected and uninfected animals fed the iron-restricted diet. Moreover, iron-restricted animals were observed to have reduced intestinal worm burdens on day 10 and day 20 postinfection compared to those of animals maintained on the standard/high-iron diet. In a subsequent study, animals equilibrated on diets containing a range of iron levels (10 ppm, 40 ppm, 100 ppm, or 200 ppm) were infected with
A. ceylanicum
and followed for evidence of hookworm disease. Infected animals from the intermediate-dietary iron (40- and 100-ppm) groups exhibited greater weight loss and anemia than those in the low (10-ppm)- or high (200-ppm)-iron diet groups. Mortality was also significantly higher in the intermediate-dietary-iron groups. These data suggest that severe dietary iron restriction impairs hookworm development in vivo but that moderate iron restriction enhances host susceptibility to severe disease.
Immunogenicity and Protection of a Recombinant Human Adenovirus Serotype 35-Based Malaria Vaccine againstPlasmodium yoeliiin Mice
Authors: Ophorst O. J. A. E. , Radošević K. , Havenga M. J. E. , Pau M. G. , Holterman L. , Berkhout B. , Goudsmit J. , Tsuji M.
Published: December 2005

Given the promise of recombinant adenovirus type 5 (rAd5) as a malaria vaccine carrier in preclinical models, we evaluated the potency of rAd35 coding forPlasmodium yoeliicircumsporozoite protein (rAd35PyCS). We chose rAd35 since a survey with serum samples from African subjects demonstrated that human Ad35 has a much lower seroprevalence of 20% and a much lower geometric mean neutralizing antibody titer (GMT) of 48 compared to Ad5 (seroprevalence, 85%; GMT, 1,261) in countries with a high malaria incidence. We also demonstrated that immunization with rAd35PyCS induced a dose-dependent and potent, CS-specific CD8+cellular and humoral immune response and conferred significant inhibition (92 to 94%) of liver infection upon high-dose sporozoite challenge. Furthermore, we showed that in mice carrying neutralizing antibody activity against Ad5, mimicking a human situation, CS-specific T- and B-cell responses were significantly dampened after rAd5PyCS vaccination, resulting in loss of inhibition of liver infection upon sporozoite challenge. In contrast, rAd35 vaccine was as potent in naive mice as in Ad5-preimmunized mice. Finally, we showed that heterologous rAd35-rAd5 prime-boost regimens were more potent than rAd35-rAd35 because of induction of anti-Ad35 antibodies after rAd35 priming. The latter data provide a further rationale for developing rAd prime-boost regimens but indicate that priming and boosting Ad vectors must be immunologically distinct and also should be distinct from Ad5. Collectively, the data presented warrant further development of rAd35-based vaccines against human malaria.