bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Characterization ofOsMADS18, a Member of theAP1/SQUASubfamily of MADS Box Genes
Authors: Fornara Fabio , Pařenicová Lucie , Falasca Giuseppina , Pelucchi Nilla , Masiero Simona , Ciannamea Stefano , Lopez-Dee Zenaida , Altamura Maria Maddalena , Colombo Lucia , Kater Martin M.
Published: August 2004

MADS box transcription factors controlling flower development have been isolated and studied in a wide variety of organisms. These studies have shown that homologous MADS box genes from different species often have similar functions. OsMADS18 from rice (Oryza sativa) belongs to the phylogenetically defined AP1/SQUA group. The MADS box genes of this group have functions in plant development, like controlling the transition from vegetative to reproductive growth, determination of floral organ identity, and regulation of fruit maturation. In this paper we report the functional analysis of OsMADS18. This rice MADS box gene is widely expressed in rice with its transcripts accumulated to higher levels in meristems. Overexpression of OsMADS18 in rice induced early flowering, and detailed histological analysis revealed that the formation of axillary shoot meristems was accelerated. Silencing of OsMADS18 using an RNA interference approach did not result in any visible phenotypic alteration, indicating that OsMADS18 is probably redundant with other MADS box transcription factors. Surprisingly, overexpression of OsMADS18 in Arabidopsis caused a phenotype closely resembling the ap1 mutant. We show that the ap1 phenotype is not caused by down-regulation of AP1 expression. Yeast two-hybrid experiments showed that some of the natural partners of AP1 interact with OsMADS18, suggesting that the OsMADS18 overexpression phenotype in Arabidopsis is likely to be due to the subtraction of AP1 partners from active transcription complexes. Thus, when compared to AP1, OsMADS18 during evolution seems to have conserved the mechanistic properties of protein-protein interactions, although it cannot complement the AP1 function.
The lys5 Mutations of Barley Reveal the Nature and Importance of Plastidial ADP-Glc Transporters for Starch Synthesis in Cereal Endosperm
Authors: Patron Nicola J. , Greber Boris , Fahy Brendan F. , Laurie David A. , Parker Mary L. , Denyer Kay
Published: August 2004

Much of the ADP-Glc required for starch synthesis in the plastids of cereal endosperm is synthesized in the cytosol and transported across the plastid envelope. To provide information on the nature and role of the plastidial ADP-Glc transporter in barley (Hordeum vulgare), we screened a collection of low-starch mutants for lines with abnormally high levels of ADP-Glc in the developing endosperm. Three independent mutants were discovered, all of which carried mutations at the lys5 locus. Plastids isolated from the lys5 mutants were able to synthesize starch at normal rates from Glc-1-P but not from ADP-Glc, suggesting a specific lesion in the transport of ADP-Glc across the plastid envelope. The major plastidial envelope protein was purified, and its sequence showed it to be homologous to the maize (Zea mays) ADP-Glc transporter BRITTLE1. The gene encoding this protein in barley, Hv.Nst1, was cloned, sequenced, and mapped. Like lys5, Hv.Nst1 lies on chromosome 6(6H), and all three of the lys5 alleles that were examined were shown to carry lesions in Hv.Nst1. Two of the identified mutations in Hv.Nst1 lead to amino acid substitutions in a domain that is conserved in all members of the family of carrier proteins to which Hv.NST1 belongs. This strongly suggests that Hv.Nst1 lies at the Lys5 locus and encodes a plastidial ADP-Glc transporter. The low-starch phenotype of the lys5 mutants shows that the ADP-Glc transporter is required for normal rates of starch synthesis. This work on Hv.NST1, together with the earlier work on BRITTLE1, suggests that homologous transporters are probably present in the endosperm of all cereals.
Relocalization of Nuclear ALY Proteins to the Cytoplasm by the Tomato Bushy Stunt Virus P19 Pathogenicity Protein
Authors: Uhrig Joachim F. , Canto Tomas , Marshall David , MacFarlane Stuart A.
Published: August 2004

The P19 protein of tomato bushy stunt virus (TBSV) is a multifunctional pathogenicity determinant involved in suppression of posttranscriptional gene silencing, virus movement, and symptom induction. Here, we report that P19 interacts with the conserved RNA-binding domain of an as yet uncharacterized family of plant ALY proteins that, in animals, are involved in export of RNAs from the nucleus and transcriptional coactivation. We show that the four ALY proteins encoded by the Arabidopsis genome and two ALY proteins from Nicotiana benthamiana are localized to the nucleus. Moreover, and in contrast to animal ALY, all but one of the proteins are also in the nucleolus, with distinct subnuclear localizations. Infection of plants by TBSV or expression of P19 from Agrobacterium results in relocation of three of the six ALY proteins from the nucleus to the cytoplasm demonstrating specific targeting of the ALY proteins by P19. The differential effects on subcellular localization indicate that, in plants, the various ALY proteins may have different functions. Interaction with and relocalization of ALY is prevented by mutation of P19 at residues previously shown to be important for P19 function in plants. Down-regulation of expression of two N. benthamiana ALY genes by virus-induced gene silencing did not interfere with posttranscriptional gene silencing. Targeting of ALY proteins during TBSV infection may therefore be related to functions of P19 in addition to its silencing suppression activity.
Biological activity, membrane‐targeting modification, and crystallization of soluble human decay accelerating factor expressed inE. coli
Authors: White Jennifer , Lukacik Petra , Esser Dirk , Steward Michael , Giddings Naomi , Bright Jeremy R. , Fritchley Sarah J. , Morgan B. Paul , Lea Susan M. , Smith Geoffrey P. , Smith Richard A.G.
Published: August 2004

Decay‐accelerating factor (DAF, CD55) is a glycophosphatidyl inositol‐anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression inEscherichia coli, isolation and membrane‐targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C‐terminal cysteine residue to permit site‐specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed inPichia pastoris. Modification with a membrane‐localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF‐derived therapeutic. Crystals of theE. coli‐derived protein were obtained and diffracted to 2.2 Å, thus permitting the first detailed X‐ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.
Sensitivity of molecular dynamics simulations to the choice of the X‐ray structure used to model an enzymatic reaction
Authors: Garcia‐Viloca Mireia , Poulsen Tina D. , Truhlar Donald G. , Gao Jiali
Published: August 2004

A subject of great practical importance that has not received much attention is the question of the sensitivity of molecular dynamics simulations to the initial X‐ray structure used to set up the calculation. We have found two cases in which seemingly similar structures lead to quite different results, and in this article we present a detailed analysis of these cases. The first case is acyl‐CoA dehydrogenase, and the chief difference of the two structures is attributed to a slight shift in a backbone carbonyl that causes a key residue (the proton‐abstracting base) to be in a bad conformation for reaction. The second case is xylose isomerase, and the chief difference of the two structures appears to be the ligand sphere of a Mg2+ metal cofactor that plays an active role in catalysis.
Buried hydrophobic side‐chains essential for the folding of the parallel β‐helix domains of the P22 tailspike
Authors: Betts Scott , Haase‐Pettingell Cameron , Cook Kristen , King Jonathan
Published: August 2004

The processive β‐strands and turns of a polypeptide parallel β‐helix represent one of the topologically simplest β‐sheet folds. The three subunits of the tailspike adhesin of phage P22 each contain 13 rungs of a parallel β‐helix followed by an interdigitated section of triple‐stranded β‐helix. Long stacks of hydrophobic residues dominate the elongated buried core of these two β‐helix domains and extend into the core of the contiguous triple β‐prism domain. To test whether these side‐chain stacks represent essential residues for driving the chain into the correct fold, each of three stacked phenylalanine residues within the buried core were substituted with less bulky amino acids. The mutant chains with alanine in place of phenylalanine were defective in intracellular folding. The chains accumulated exclusively in the aggregated inclusion body state regardless of temperature of folding. These severe folding defects indicate that the stacked phenylalanine residues are essential for correct parallel β‐helix folding. Replacement of the same phenylalanine residues with valine or leucine also impaired folding in vivo, but with less severity. Mutants were also constructed in a second buried stack that extends into the intertwined triple‐stranded β‐helix and contiguous β‐prism regions of the protein. These mutants exhibited severe defects in later stages of chain folding or assembly, accumulating as misfolded but soluble multimeric species. The results indicate that the formation of the buried hydrophobic stacks is critical for the correct folding of the parallel β‐helix, triple‐stranded β‐helix, and β‐prism domains in the tailspike protein.
A directed evolution strategy for optimized export of recombinant proteins reveals critical determinants for preprotein discharge
Authors: Kaderbhai Mustak A. , Davey Hazel M. , Kaderbhai Naheed N.
Published: August 2004

A directed evolutionary approach is described that searches short, random peptide sequences for appendage at the secretory signal peptide–mature protein junction to seek ideal algorithms for both efficient and hyper export of recombinant proteins to the periplasm of Escherichia coli. The strategy employs simple, visual detection of positive clones using a PINK expression system that faithfully reports on export status of a mammalian hemoprotein in E. coli. With‐in “sequence spaces” ranging from 1 to 13 residues, a significant but highly variable secretory fitness was scored such that the rate of secretion reciprocally correlated with the membrane‐associated precursor pool of the evolved exportable hemoproteins. Three clusters of hyper, median, and hypo exporters were isolated. These had corresponding net charges of −1, 0, and +1 within the evolved sequence space, which in turn clearly correlated with the prevailing magnitude and polarity of the membrane energization states. The findings suggest that both the nature of the charged residue and the proximal sequence in the early mature region are the crucial determinants of the protonophore‐dependent electrophoretic discharge of the precursor across the inner membrane of E. coli. We conclude that the directed evolutionary approach will find ready application in engineering recombinant proteins for their efficient secretion via the sec export pathway in E. coli.
A fluorescence stopped‐flow kinetic study of the conformational activation of α‐chymotrypsin and several mutants
Authors: Verheyden Gert , Matrai Janka , Volckaert Guido , Engelborghs Yves
Published: August 2004

The kinetic activation parameters (activation free energy, activation free enthalpy, and activation free entropy change) of the conformational change of α‐chymotrypsin from an inactive to the active conformation were determined after a pH jump from pH 11.0 to pH 6.8 by the fluorescence stopped‐flow method. The conformational change was followed by measuring changes in the protein fluorescence. For the bovine wild‐type protein, the same kinetic parameters are obtained as in the study of proflavin binding. Several mutants were made with the goal to accelerate or decelerate this conformational transition. The inspiration for the choice of the mutants came from a previous modelling study done on the bovine wild‐type chymotrypsin. The results of the fluorescence stopped flow experiments show that several mutants behaved as was expected based on the information provided by the modeling study on the wild‐type variant. For some mutants our assumptions were not correct, and therefore additional modeling studies of the activation pathways of these mutant proteins are necessary to be able to explain the observed kinetic behavior.
On the modeling of snake venom serine proteinase interactions with benzamidine‐based thrombin inhibitors
Authors: Henriques Elsa S. , Fonseca Nelson , Ramos Maria João
Published: August 2004

Pit viper venoms contain a number of serine proteinases that exhibit one or more thrombin‐like activities on fibrinogen and platelets, this being the case for the kinin‐releasing and fibrinogen‐clotting KN‐BJ from the venom of Bothrops jararaca. A three‐dimensional structural model of the KN‐BJ2 serine proteinase was built by homology modeling using the snake venom plasminogen activator TSV‐PA as a major template and porcine kallikrein as additional structural support. A set of intrinsic buried waters was included in the model and its behavior under dynamic conditions was molecular dynamics simulated, revealing a most interesting similarity pattern to kallikrein. The benzamidine‐based thrombin inhibitors α‐NAPAP, 3‐TAPAP, and 4‐TAPAP were docked into the refined model, allowing for a more insightful functional characterization of the enzyme and a better understanding of the reported comparatively low affinity of KN‐BJ2 toward those inhibitors.
Solution structure of Escherichia coli Par10: The prototypic member of the Parvulin family of peptidyl‐prolyl cis/trans isomerases
Authors: Kühlewein Angelika , Voll Georg , Hernandez Alvarez Birte , Kessler Horst , Fischer Gunter , Rahfeld Jens‐Ulrich , Gemmecker Gerd
Published: August 2004

E. coli Par10 is a peptidyl‐prolyl cis/trans isomerase (PPIase) from Escherichia coli catalyzing the isomerization of Xaa‐Pro bonds in oligopeptides with a broad substrate specificity. The structure of E. coli Par10 has been determined by multidimensional solution‐state NMR spectroscopy based on 1207 conformational constraints (1067 NOE‐derived distances, 42 vicinal coupling‐constant restraints, 30 hydrogen‐bond restraints, and 68 ϕ/ψ restraints derived from the Chemical Shift Index). Simulated‐annealing calculations with the program ARIA and subsequent refinement with XPLOR yielded a set of 18 convergent structures with an average backbone RMSD from mean atomic coordinates of 0.50 Å within the well‐defined secondary structure elements. E. coli Par10 is the smallest known PPIase so far, with a high catalytic efficiency comparable to that of FKBPs and cyclophilins. The secondary structure of E. coli Par10 consists of four helical regions and a four‐stranded antiparallel β‐sheet. The N terminus forms a β‐strand, followed by a large stretch comprising three α‐helices. A loop region containing a short β‐strand separates these helices from a fourth α‐helix. The C terminus consists of two more β‐strands completing the four‐stranded anti‐parallel β‐sheet with strand order 2143. Interestingly, the third β‐strand includes a Gly‐Pro cis peptide bond. The curved β‐strand forms a hydrophobic binding pocket together with α‐helix 4, which also contains a number of highly conserved residues. The three‐dimensional structure of Par10 closely resembles that of the human proteins hPin1 and hPar14 and the plant protein Pin1At, belonging to the same family of highly homologous proteins.
Analysis of the stability of multimeric proteins by effective ΔG and effective m‐values
Authors: Park Chiwook , Marqusee Susan
Published: August 2004

Analyzing the stability of a multimeric protein is challenging because of the intrinsic difficulty in handling the mathematical model for the folded multimer‐unfolded monomer equilibrium. To circumvent this problem, we introduce the concept of effective stability, ΔGeff (= −RTlnKeff), where Keff is the equilibrium constant expressed in monomer units. Analysis of the denaturant effect on ΔGeff gives new insight into the stability of multimeric proteins. When a multimeric protein is mostly folded, the dependence of effective stability on denaturant concentration (effective m‐value) is simply the m‐value of its monomeric unit. However, when the protein is mostly unfolded, its stability depends on denaturant concentration with the m‐value of its multimeric form. We also find that the effective m‐value at the Cm is a good approximation of the apparent m‐value determined by fitting the equilibrium unfolding data from multimeric proteins with a two‐state monomer model. Moreover, when the m‐value of a monomeric unit is estimated from its size, the effective stability of a multimeric protein can be determined simply from Cm and this estimated m‐value. These simple and intuitive approaches will allow a facile analysis of the stability of multimeric proteins. These analyses are also applicable for high‐throughput analysis of protein stability on a proteomic scale.
Energetics of galactose– and glucose–aromatic amino acid interactions: Implications for binding in galactose‐specific proteins
Authors: Sujatha Mannargudi S. , Sasidhar Yellamraju U. , Balaji Petety V.
Published: August 2004

An aromatic amino acid is present in the binding site of a number of sugar binding proteins. The interaction of the saccharide with the aromatic residue is determined by their relative position as well as orientation. The position‐orientation of the saccharide relative to the aromatic residue was found to vary in different sugar‐binding proteins. In the present study, interaction energies of the complexes of galactose (Gal) and of glucose (Glc) with aromatic residue analogs have been calculated by ab initio density functional (U‐B3LYP/ 6‐31G**) theory. The position‐orientations of the saccharide with respect to the aromatic residue observed in various Gal‐, Glc‐, and mannose–protein complexes were chosen for the interaction energy calculations. The results of these calculations show that galactose can interact with the aromatic residue with similar interaction energies in a number of position‐orientations. The interaction energy of Gal–aromatic residue analog complex in position‐orientations observed for the bound saccharide in Glc/Man–protein complexes is comparable to the Glc–aromatic residue analog complex in the same position‐orientation. In contrast, there is a large variation in interaction energies of complexes of Glc‐ and of Gal‐ with the aromatic residue analog in position‐orientations observed in Gal–protein complexes. Furthermore, the conformation wherein the O6 atom is away from the aromatic residue is preferred for the exocyclic —CH2OH group in Gal–aromatic residue analog complexes. The implications of these results for saccharide binding in Gal‐specific proteins and the possible role of the aromatic amino acid to ensure proper positioning and orientation of galactose in the binding site have been discussed.
Interactions and chaperone function of αA‐crystallin with T5P γC‐crystallin mutant
Authors: Liang Jack J‐N
Published: August 2004

T5P γC‐crystallin mutation is associated with Coppock‐like cataract, one of the autosomal dominant congenital cataracts. It is not known why the abundant α‐crystallin cannot prevent the mutation‐related aggregation. Our previous studies indicate that the mutation changes conformation and reduces solubility and stability, but it is not known whether it is these events or the loss of interaction with other crystallins that causes the cataract. It is also not known whether the α‐crystallin can protect T5P mutant as effectively from heat‐induced aggregation as the wild‐type (WT) γC‐crystallin. To investigate the mechanism of interactions and chaperone function between αA‐ and γC‐crystallin, human αA‐crystallin and W9F mutant as well as WT γC‐crystallin and T5P mutant were cloned. Interactions between αA‐ and γC‐crystallin were studied with fluorescence resonance energy transfer (FRET), and chaperone activity was assessed by the suppression of heat‐induced aggregation of substrate proteins. Conformational changes of substrate proteins were studied by spectroscopic measurements. The results indicate that the T5P mutant showed a slightly greater FRET than WT γC‐crystallin with αA‐crystallin, and αA‐crystallin could effectively prevent both WT and T5P γC‐crystallin from heat‐induced aggregation. Spectroscopic measurements show that both αA‐crystallin and γC‐crystallin underwent only slight conformational change after chaperone binding. Together with previous results obtained with a two‐hybrid system assay of interactions between αA‐ and γC‐crystallin, the present FRET and chaperone results indicate that loss of interactions of T5P mutant with other crystallins may play a larger role than the protection afforded by chaperone‐like activity in Coppock‐like cataract.
Substrate‐induced asymmetry and channel closure revealed by the apoenzyme structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase
Authors: Morris Van K. , Izard Tina
Published: August 2004

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in prokaryotic coenzyme A (CoA) biosynthesis, directing the transfer of an adenylyl group from ATP to 4′‐phosphopantetheine (Ppant) to yield dephospho‐CoA (dPCoA). The crystal structures of Escherichia coli PPAT bound to its substrates, product, and inhibitor revealed an allosteric hexameric enzyme with half‐of‐sites reactivity, and established an in‐line displacement catalytic mechanism. To provide insight into the mechanism of ligand binding we solved the apoenzyme (Apo) crystal structure of PPAT from Mycobacterium tuberculosis. In its Apo form, PPAT is a symmetric hexamer with an open solvent channel. However, ligand binding provokes asymmetry and alters the structure of the solvent channel, so that ligand binding becomes restricted to one trimer.
Investigation of the nature of the methionine–π interaction in β‐hairpin peptide model systems
Authors: Tatko Chad D. , Waters Marcey L.
Published: August 2004

There are frequent contacts between aromatic rings and sulfur atoms in proteins. However, it is unclear to what degree this putative interaction is stabilizing and what the nature of the interaction is. We have investigated the aryl–sulfur interaction by placing a methionine residue diagonal to an aromatic ring on the same face of a β‐hairpin, which places the methionine side chain in close proximity to the aryl side chain. The methionine (Met)–aryl interaction was compared with an equivalent hydrophobic and cation–π interaction in the context of the β‐hairpin. The interaction between phenylalanine (Phe), tryptophan (Trp), or cyclohexylalanine (Cha) and Met stabilized the β‐hairpin by −0.3 to −0.5 kcal mole−1, as determined by double‐mutant cycles. The peptides were subjected to thermal denaturations that suggest a hydrophobic driving force for the interactions between Met and Trp or Cha. The observed interaction of Met or norleucine (Nle) with Trp or Cha are quite similar, implying a hydrophobic driving force for the Met–π interaction. However, the thermodynamic data suggest that there may be some differences between the interaction of Met with Trp and Phe and that there may be a small thermodynamic component to the Met•••Phe interaction.
Structure of the XPC binding domain of hHR23A reveals hydrophobic patches for protein interaction
Authors: Kamionka Mariusz , Feigon Juli
Published: August 2004

Rad23 proteins are involved both in the ubiquitin‐proteasome pathway and in nucleotide excision repair (NER), but the relationship between these two pathways is not yet understood. The two human homologs of Rad23, hHR23A and B, are functionally redundant in NER and interact with xeroderma pigmentosum complementation group C (XPC) protein. The XPC–hHR23 complex is responsible for the specific recognition of damaged DNA, which is an early step in NER. The interaction of the XPC binding domain (XPCB) of hHR23A/B with XPC protein has been shown to be important for its optimal function in NER. We have determined the solution structure of XPCB of hHR23A. The domain consists of five amphipathic helices and reveals hydrophobic patches on the otherwise highly hydrophilic domain surface. The patches are predicted to be involved in interaction with XPC. The XPCB domain has limited sequence homology with any proteins outside of the Rad23 family except for sacsin, a protein involved in spastic ataxia of Charlevoix‐Saguenay, which contains a domain with 35% sequence identity.
Genetic counseling in carriers of reciprocal chromosomal translocations involving long arm of chromosome 16
Authors: Stasiewicz‐Jarocka B , Haus O , Assche E Van , Kostyk E , Constantinou M , Rybałko A , Krzykwa B , Marcinkowska A , Barisič I , Kučinskas V , Katużewski B , Schwanitz G , Midro AT
Published: August 2004

Families with balanced chromosomal changes ascertained by unbalanced progeny, miscarriages, or by chance are interested in their probability for unbalanced offspring and other unfavorable pregnancy outcomes. This is usually done based on the original data published by Stengel‐Rutkowski et al. several decades ago. That data set has never been updated. It is particularly true for the subgroup with low number of observations, to which belong reciprocal chromosomal translocations (RCTs) with breakpoint in an interstitial segment of 16q. The 11 pedigrees from original data together with the new 18 pedigrees of RCT carriers at risk of single‐segment imbalance detected among 100 pedigrees of RCT carriers with breakpoint position at 16q were used for re‐evaluation of the probability estimation for unbalanced offspring at birth and at second trimester of prenatal diagnosis, published in 1988. The new probability rate for unbalanced offspring after 2 : 2 disjunction and adjacent‐1 segregation for the total group of pedigrees was 4 ± 3.9% (1/25). In addition, the probability estimate for unbalanced fetuses at second trimester of prenatal diagnosis was calculated as 2/11, i.e. 18.2 ± 11.6%. The probability rates for miscarriages and stillbirths/early deaths were about 16 ± 7.3% (4/25) and <2% (0/25), respectively. Considering different segment lengths of 16q, higher probability rate (0/8, i.e. <6.1%) for maternal RCT carriers at risk of distal 16q segment imbalance (shorter segment) was obtained in comparison with the rate (0/10, i.e. <4.8%) for RCT at risk of proximal segment imbalance (longer segment). It supports findings obtained from the original data for RCT with other chromosomes, where the probability for unbalanced offspring generally increased with decreasing length of the segments involved in RCT. Our results were applied for five new families with RCT involving 16q, namely three at risk of single‐segment imbalance [t(8;16)(q24.3;q22)GTG, ish(wcp8+,wcp16+;wcp8–,wcp16+), t(11;16)(q25;q22)GTG, and t(11;16)(q25;q13)GTG] and two with RCT at risk of double‐segment imbalance [t(16;19)(q13;q13.3)GTG, isht(16;19)(q13;q13.3) (D16Z3+,16QTEL013−D19S238E+,TEL19pR–; D16Z3−, D19S238E–,TEL19pR+), and t(16;20)(q11.1;q12)GTG, m ish,t(16;20)(wcp16+,wcp20+;wcp16+,wcp20+)]. They have been presented in details to illustrate how the available empiric data could be used in practice for genetic counseling.
New insights in congenital bowing of the femora
Authors: Cormier‐Daire V , Geneviève D , Munnich A , Le Merrer M
Published: August 2004

The aim of this study is to review the clinical, radiological and molecular findings of the bent bone dysplasia group including Stüve‐Wiedemann syndrome due to LIFR mutations, Compomelic dysplasia due to SOX9 mutations and Kyphomelic dysplasia with no known molecular bases.
Economic Restructuring and Income Inequality in Urban China
Authors: Meng Xin
Published: August 2004

Economic transition from a planned to a market oriented economy is often associated with a widening of income inequality. The nature of this change, however, may differ during different stages of the economic transition. This paper investigates the increase in income inequality in urban China during two phases of economic reform: a moderate reform era (1988–95) and a radical reform era (1995–99). It is found that although income inequality increased considerably during both stages, the nature and causes of the increase are different. In the moderate reform period, the increase in inequality was a result of some parts of society sharing more of the economic gain than others, and the main cause of this inequality is regional income dispersion. During the radical reform period income reductions at the lower end of the distribution is observed, and it is mainly due to the large‐scale unemployment generated by labor reallocation.
A ‘Continuous Meeting’ Type of Biomedical Education in Rijeka, Croatia
Authors: Volarević S.
Published: August 2004

During the second half of 2002 and the first half of 2003, several top international scientists gave guest courses, each in their own research field of expertise, as a part of a postgraduate (PhD) programme in biomedical education at the Medical school in Rijeka, Croatia. Due to its structure, the programme is a ‘continuous meeting’ type of learning. Courses attracted not only local medical doctors and dentists but also from the surrounding region as well as neighbouring countries in southeastern Europe. The highlights from presentations of several selected scientists were reported here as some might bear relevance to contemporary immunological research. I also describe the programme's structure and contents that could, if proved attractive, greatly stimulate an almost lacking interest in biomedical sciences in these areas of Europe.
Epitope DNA Vaccines Against Tuberculosis: Spacers and Ubiquitin Modulates Cellular Immune Responses Elicited by Epitope DNA Vaccine
Authors: Wang Q.‐M. , Sun S.‐H. , Hu Z.‐L. , Zhou F.‐J. , Yin M. , Xiao C.‐J. , Zhang J.‐C.
Published: August 2004

Cell‐mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed epitope DNA vaccines (p3‐M‐38) encoding cytotoxic T lymphocyte (CTL) epitopes of MPT64 and 38 kDa proteins of Mycobacterium tuberculosis. In order to observe the influence of spacer sequence (Ala‐Ala‐Tyr) or ubiquitin (UbGR) on the efficacy of the two CTL epitopes, we also constructed DNA vaccines, p3‐M‐S(spacer)‐38, p3‐Ub (UbGR)‐M‐S‐38 and p3‐Ub‐M‐38. The immune responses elicited by the four DNA vaccines were tested in C57BL/6 (H‐2b) mice. The cytotoxicity of T cells was detected by LDH‐release method and by enzyme‐linked immunospot assay for epitope‐specific cells secreting interferon‐γ. The results showed that DNA immunization with p3‐M‐38 vaccine could induce epitope‐specific CD8+ CTL response and that the spacer sequence (AAY) only enhanced M epitope presentation. The protein‐targeting sequence (UbGR) enhanced the immunogenicity of the two epitopes. The finding that defined spacer sequences at C‐terminus and protein‐targeting degradation modulated the immune response of epitope string DNA vaccines will be of importance for the further development of multi‐epitope DNA vaccines against tuberculosis.
The Receptor for Interleukin‐17E is Induced by Th2 Cytokines in Antigen‐Presenting Cells
Authors: Gratchev A. , Kzhyshkowska J. , Duperrier K. , Utikal J. , Velten F. W. , Goerdt S.
Published: August 2004

Interleukin‐17E (IL‐17E) (IL‐25) is a recently identified cytokine capable to induce Th2‐associated cytokine production (IL‐5 and IL‐13) and T helper 2 (Th2)‐type pathologies in animal models. The IL‐17E‐responsive cell population in vivo was described to be a further uncharacterized non‐T‐, non‐B‐splenic accessory cell. Despite the identification of IL‐17BR as the receptor for IL‐17E, the cell population expressing IL‐17BR has hitherto not been identified. Here, we show that human monocyte‐derived Th2‐skewed antigen‐presenting cells (APC2) express membrane‐bound and soluble forms of IL‐17BR on the mRNA and protein level upon stimulation with IL‐4, IL‐10, IL‐13 or transforming growth factor‐βin vitro. These results indicate that IL‐17BR‐expressing APC2s may mediate the development of the IL‐17E‐mediated immunological reaction patterns observed in vivo.
Induction of Systemic Tolerance in Normal but not in Transgenic Mice Through Continuous Feeding of Ovalbumin
Authors: Simioni P. U. , Fernandes L. G. R. , Gabriel D. L. , Tamashiro W. M. S. C.
Published: August 2004

The ingestion of most dietary protein can cause systemic tolerance, and such tolerance is easier to induce in younger than in older mice. In this study, we examined whether oral tolerance to ovalbumin (OVA) could be induced in OVA‐T‐cell receptor (OVA‐TCR)‐specific transgenic mice. Continuous feeding or gavage with OVA induced tolerance, measured as reduced antibody production, in young and aged BALB/c mice, in a dose‐dependent manner, but this effect was not observed in transgenic mice. Once BALB/c mice became tolerant, this state was maintained for over 44 weeks, although the tolerant state could be reversed by adoptive cell transfer. DO11.10 mice did not become tolerant upon continuous feeding with OVA, and the adoptive transfer of naïve cells increased the levels of specific antibodies in their sera after antigenic challenge. The immunization schedule used here leads to a Th2‐dependent antibody response in normal BALB/c mice. However, the same schedule induced both Th1‐ and Th2‐antibody responses in transgenic mice. Dendritic cells (DC) from tolerant BALB/c mice were less efficient in the induction of the proliferation of cocultured T cells from both BALB/c and DO11.10 mice, as well as Th1 [interleukin (IL)‐2 and interferon (IFN)‐γ] and Th2 (IL‐4 and IL‐10) cytokine production. The DC from DO11.10 transgenic mice were equally efficient in the induction of T‐cell proliferation in both normal and transgenic mice, as well as in the induction of Th1 and Th2 cytokines, whether or not the mice consumed OVA. Transforming growth factor (TGF)‐β secretion was significantly lower in the supernatants of T cells from both normal and transgenic mice cocultured with DC from DO11.10 mice that had consumed OVA, while it was significantly higher in the presence of DC from normal tolerant mice, thus implicating TGF‐β as a regulatory cytokine in oral tolerance in the murine model.
Single‐Base Oligodeoxyguanosine‐Binding Proteins on Nonspecific Cytotoxic Cells: Identification of a New Class of Pattern‐Recognition Receptors
Authors: Kaur H. , Jaso‐Friedmann L. , Leary J. H. , Evans D. L.
Published: August 2004

The present study was designed to identify a possible new class of pathogen‐recognition proteins that bind single‐base oligodeoxynucleotide (ODN) ligands. Binding by the teleost natural killer cell equivalent [referred to as nonspecific cytotoxic cells (NCC)] was compared with mammalian cells (mouse RAW264.7 cells and human THP‐1 cells). The ODN analysed were composed of 20‐mers of guanosine (dG20), adenosine (dA20), thymidine (dT20) or cytosine (dC20). Binding studies first determined the 50% saturation levels for NCC (1.25 µg/ml), RAW264.7 (0.2 µg/ml) and THP‐1 (0.8 µg/ml). Binding by dG20 to all the three cell types was saturable. Ligand blots of NCC membrane lysates with biotinylated dG20 revealed two different major molecular weight species (16–18 and 29 kDa) of binding proteins. The 29‐kDa protein was identified with the help of Western blot analysis using a polyclonal antibody specific to an NCC antimicrobial protein (ncamp‐1). The membrane expression of the 29‐kDa ncamp‐1 was determined by the binding of surface‐biotinylated NCC membrane proteins with digoxigenin dG20 followed by immunoprecipitation using anti‐digoxigenin agarose beads. The 29 and 14–18 kDa NCC membrane proteins were cross‐reactive using Western blot examination with a polyclonal anti‐histone 1 antibody. Function studies revealed that dG20 activated a twofold upregulation of membrane binding by homologous dG20‐biotin. dG20 also stimulated NCC‐increased membrane expression of NCC receptor protein 1. Additional experiments were performed to determine the DNase sensitivity of the different ODN. dG20 appeared to be more resistant to DNase treatment, compared to dC20, dA20 and dT20. The single‐base ODN‐binding proteins may represent a new class of pattern‐recognition receptors that are involved in innate anti‐bacterial resistance mediated by NCC.
Active Synthesis of Mouse Polymeric Immunoglobulin Receptor in the Epithelial Cells of the Distal Urinary Tubule in Kidney
Authors: Asano M. , Saito M. , Suguro H. , Nomura H. , Inage T. , Moro I.
Published: August 2004

The tissue distribution of mouse polymeric immunoglobulin receptor (pIgR) has been demonstrated. By Northern blot hybridization, pIgR mRNA expression was detected in liver, intestine, stomach, lung and kidney. A weak expression was also detected in thymus by reverse transcriptase‐polymerase chain reaction. The pIgR expression in kidney was further studied and confirmed that pIgR protein was actively synthesized in the epithelial cells of distal urinary tubule and of Henle's loop. Immunoelectron microscopical analysis showed the accumulation of pIgR‐containing vesicles in the apical portion of distal urinary tubule epithelial cells.
Tolerability and Anti‐Inflammatory Effects of Glucuronoxylomannan in Collagen‐Induced Arthritis
Authors: Mirshafiey A. , Ngoc Dzung D. T. , Murphy J. W. , Khorramizadeh M. R. , Saadat F. , Mehrabian F. , Larsson P.
Published: August 2004

This investigation was planned to assess the therapeutic efficacy of glucuronoxylomannan (GXM) in collagen‐induced arthritis (CIA). GXM was isolated from culture filtrate of Cryptococcus neoformans var. gattii, serotype C. CIA was induced by the immunization of Dark Agouti rats with bovine type II collagen in incomplete Freund's adjuvant. GXM solution at two doses, 25 and 50 mg/kg, was administered intraperitoneally. Onset of i.p. injections of GXM to prevention and treatment groups was days 0 and 10 postimmunization, respectively. The WEHI‐164 cell line was used for assaying tolerability, matrix metalloproteinase type 2 (MMP‐2) activity and apoptosis. MMP‐2 activity was assessed using zymography. For assessment of apoptosis, the terminal deoxyribonucleotidyl transferase‐mediated dUTP nick‐end labelling method was used. The results of this experiment showed that the treatment of CIA with GXM at a dose of 50 mg/kg could suppress disease development both prophylactically and therapeutically. This beneficial effect of GXM was associated with a significant decrease in the anti‐CII antibody response compared with untreated rats. Moreover, GXM therapy could diminish MMP‐2 activity, but it had no notable effect on apoptosis. GXM also showed a high tolerability compared with certain steroidal and non‐steroidal anti‐inflammatory drugs. We conclude that GXM suppresses the development of disease in CIA and it could be recommended as a new immunosuppressive and anti‐inflammatory agent for further investigations.
The Influence of Remaining Live BCG Organisms in Vaccinated Mice on the Maintenance of Immunity to Tuberculosis
Authors: Olsen A. W. , Brandt L. , Agger E. M. , Van Pinxteren L. A. H. , Andersen P.
Published: August 2004

The only available vaccine against Mycobacterium tuberculosis, the bacille Calmette‐Guérin (BCG) vaccine, is at present being used as a reference for the efficacy of novel vaccines. Herein, we demonstrate that viable BCG can be detected at late time points after vaccination in C57BL/6J mice. If BCG is cleared by antibiotic treatment, the number of mycobacteria‐reactive effector cells in the spleen gradually reverts to low levels and consequently immunity in this organ wanes, while resistance in the lung remains stable. The implications for comparing BCG vaccination with experimental vaccines including non‐replicating vaccines are discussed.
Upregulation of T‐Cell‐Stimulating Activity of Mycobacteria‐Infected Macrophages
Authors: Kimura H. , Maeda Y. , Takeshita F. , Takaoka L. E. , Matsuoka M. , Makino M.
Published: August 2004

Macrophages are one of the most abundant host cells to come in contact with mycobacteria. However, the infected macrophages less efficiently stimulate autologous T cells in vitro. We investigated the effect of the induction of phenotypic change of macrophages on the host cell activities by using Mycobacterium leprae as a pathogen. The treatment of macrophages with interferon‐γ (IFN‐γ), GM‐CSF and interleukin‐4 deprived macrophages of CD14 antigen expression but instead provided them with CD1a, CD83 and enhanced CD86 antigen expression. These phenotypic features resembled those of monocyte‐derived dendritic cells (DC). These macrophage‐derived DC‐like cells (MACDC) stimulated autologous CD4+ and CD8+ T cells when infected with M. leprae. Further enhancement of the antigen‐presenting function and CD1a expression of macrophages was observed when treated with IFN‐γ. The M. leprae‐infected and ‐treated macrophages expressed bacterial cell membrane‐derived antigens on the surface and were efficiently cytolysed by the cell membrane antigen‐specific CD8+ cytotoxic T lymphocytes (CTL). These results suggest that the induction of phenotypic changes in macrophages can lead to the upregulation of host defence activity against M. leprae.
Evidence for a Type 1 Diabetes Susceptibility Locus (IDDM10) on Chromosome 10p11‐q11 in a Russian Population
Authors: Chistiakov D. A. , Seryogin Y. , Savost'anov K. V. , Zilberman L. I. , Titovich E. V. , Kuraeva T. L. , Dedov I. I. , Nosikov V. V.
Published: August 2004

Around 20 susceptibility loci for type 1 diabetes mellitus (T1DM) have been mapped. One of these loci, IDDM10, was found on chromosome 10p11‐q11. Here, we investigated whether the IDDM10 locus contributes in the susceptibility to T1DM in a Russian family dataset. One hundred and fourteen simplex Russian families, each containing two siblings (one affected with T1DM diagnosed and one nondiabetic sibling), and 97 multiplex families, containing 106 affected full sibling pairs, were studied. Genomic DNA from the venous blood of the patients was genotyped by PCR using 12 microsatellites (D10S193, D10S548, D10S565, D10S586, D10S588, D10S675, D10S1243, D10S1426, D10S1733, D10S1772, D10S1780 and D10S1783) located on chromosome 10p11‐q11. Using the multipoint linkage analysis, the region of suggestive linkage, with a multipoint logarithm of odds (LOD) ratio (MLS) value of more than 2.2, was found between markers D10S1733 and D10S1780, an area of 9.0 cM on the genetic map. The maximum linkage peak (MLS = 2.85 and nonparametric logarithm = 2.68) was observed between markers D11S565 and D11S1243. Using the transmission disequilibrium test, an association of these markers, D10S565 (P overall = 0.0082) and D10S1243 (P overall = 0.017), with T1DM was shown. These results suggest the evidence for the IDDM10 susceptibility locus on chromosome 10p11‐q11.
A Simplified Cytokine Immunoassay Using Magnetic Polymer Particles
Authors: McNeill A. , Kastrup J. , Bäckström B. T.
Published: August 2004

Accurate detection of cytokines is essential for understanding their biological role in the immune system. Various methods to detect cytokines have been developed, including sandwich enzyme‐linked immunosorbent assay (ELISA) and flow cytometry‐based methods. All of the currently available methods have limitations, however. These limitations include time and extensive handling in standard sandwich ELISAs and the need for specialized equipment in flow cytometry‐based assays. We have developed a magnetic polymer cytokine immunoassay and demonstrate that this assay is rapid and simple, needs less handling and offers better dynamic range, compared to standard sandwich ELISA. Furthermore, it does not require flow cytometry equipment, which is often used in microparticle‐based polymer immunoassays. The magnetic polymer cytokine immunoassay described in this study is as sensitive as a standard sandwich ELISA. Because the method is not limited to the use of magnetic polymer particles, it is versatile and compatible with a number of different solid matrixes.
The Marinoan glaciation (Neoproterozoic) in northeast Svalbard
Authors: Halverson Galen P. , Maloof Adam C. , Hoffman Paul F.
Published: August 2004

Two separate and distinct diamictite‐rich units occur in the mixed carbonate‐siliciclastic Polarisbreen Group, which comprises the top kilometer of >7 km of Neoproterozoic strata in the northeast of the Svalbard archipelago. The platformal succession accumulated on the windward, tropical to subtropical margin of Laurentia. The older Petrovbreen Member is a thin glacimarine diamictite that lacks a cap carbonate. It contains locally derived clasts and overlies a regional karstic disconformity that was directly preceded by a large (>10‰) negativeδ13C anomaly in the underlying shallow‐marine carbonates. This anomaly is homologous to anomalies in Australia, Canada and Namibia that precede the Marinoan glaciation. The younger and thicker Wilsonbreen Formation comprises terrestrial ice‐contact deposits. It contains abundant extrabasinal clasts and is draped by a transgressive cap dolostone 3–18 m thick. The cap dolostone is replete with sedimentary features strongly associated with post‐Marinoan caps globally, and its isotopic profile is virtually identical to that of other Marinoan cap dolostones. From the inter‐regional perspective, the two diamictite‐rich units in the Polarisbreen Group should represent the first and final phases of the Marinoan glaciation. Above the Petrovbreen diamictite are ∼200 m of finely laminated, dark olive‐coloured rhythmites (MacDonaldryggen Member) interpreted here to represent suspension deposits beneath shorefast, multi‐annual sea ice (sikussak). Above the suspension deposits and below the Wilsonbreen diamictites is a <30‐m‐thick regressive sequence (Slangen Member) composed of dolomite grainstone and evaporitic supratidal microbialaminite. We interpret this sabkha‐like lagoonal sequence as an oasis deposit that precipitated when local marine ice melted away under greenhouse forcing, but while the tropical ocean remained covered due to inflow of sea glaciers from higher latitudes. It appears that the Polarisbreen Group presents an unusually complete record of the Marinoan snowball glaciation.