bluuebunny/crossref_metadata_2025 huggingface dataset:
Cholera—a Step Too Far
Authors: Chetwynd Amanda , Short Hilary
Published: June 2006

In the mid-19th century epidemics of cholera presented a new threat to the population. In the face of conflicting theories as to how the disease was spread, William Farr came up with an interesting theory. Hilary Short and Amanda Chetwynd have investigated.
Genetic polymorphisms of adhesion molecules in children with severe RSV‐associated diseases
Authors: Krueger M. , Puthothu B. , Heinze J. , Forster J. , Heinzmann A.
Published: June 2006

Adhesion molecules are involved in the pathophysiology of respiratory syncytial virus (RSV)‐associated diseases. By testing polymorphisms within ICAM‐1, VCAM‐1 and E‐selectin, we found no evidence for association of any polymorphism with severe RSV infections. Thus, we conclude that these genes do not predispose to severe RSV‐associated diseases.
Protein tyrosine phosphatase non‐receptor type 22 (PTPN22) gene R620W variant and sporadic idiopathic hypoparathyroidism in Asian Indians
Authors: Ray D. , Tomar N. , Gupta N. , Goswami R.
Published: June 2006

Recently, a gain of function variant C1858T of the lymphoid‐specific protein tyrosine phosphatase non‐receptor (LYP, PTPN22) gene has been reported to be associated with several autoimmune disorders including Graves’ disease, type 1 diabetes, rheumatoid arthritis and vitiligo. The present study was carried out in 80 patients with sporadic idiopathic hypoparathyroidism (SIH) [43 males and 37 females, mean ± SD age and duration of symptoms 32.5 ± 14.1 years and 6.7 ± 7.2 years (range 1 day to 35 years), respectively] and 193 healthy controls (male : female ratio 91:102, mean ± SD age, 43.1 ± 11.6 years) to assess association of 1858T allele with the disease. Polymerase chain reaction–restriction fragment length polymorphism analysis was performed to genotype C1858T variant. The frequency of occurrence of 1858T allele was 4/160 (2.5%) in SIH and 5/386 (1.3%) in the control alleles (odds ratio 1.95, 95% CI 0.51–7.37). Thus, the present study reveals that 1858T allele is rare (1.3%) in Asian Indians. The trend of higher prevalence of 1858T allele in patients with SIH needs to be studied further in other population with higher rate of the allele to support the autoimmune basis of the disease.
MICA‐STR, HLA‐B haplotypic diversity and linkage disequilibrium in the Hunan Han population of southern China
Authors: Tian W. , Li L‐X. , Wang F. , Luo Q‐Z. , Yan M‐Y. , Yu P. , Guo S‐S. , Cao Y.
Published: June 2006

Major histocompatibility complex (MHC) class I chain‐related gene A (MICA) is located 46 kb centromeric to HLA‐B and encodes a stress‐inducible protein. MICA allelic variation is thought to be associated with disease susceptibility and immune response to transplants. This study was aimed to investigate the haplotypic diversity and linkage disequilibrium between human leukocyte antigen (HLA)‐B and (GCT)n short tandem repeat in exon 5 of MICA gene (MICA‐STR) in a southern Chinese Han population. Fifty‐eight randomly selected nuclear families with 183 members including 85 unrelated parental samples were collected in Hunan province, southern China. HLA‐B generic typing was performed by polymerase chain reaction–sequence‐specific priming (PCR–SSP), and samples showing novel HLA‐B‐MICA‐STR linkage were further typed for HLA‐B allelic variation by high‐resolution PCR–SSP. MICA‐STR allelic variation and MICA gene deletion (MICA*Del) were detected by fluorescent PCR–size sequencing and PCR–SSP. Haplotype was determined through family segregation analysis. Statistical analysis was applied to the data of the 85 unrelated parental samples. Nineteen HLA‐B specificities and seven MICA‐STR allelic variants were observed in 85 unrelated parental samples, the most predominant of which were HLA‐B*46, ‐B60, ‐B*13, and ‐B*15, and MICA*A5, MICA*A5.1 and MICA*A4, respectively. Genotype distributions of HLA‐B, MICA‐STR loci were consistent with Hardy–Weinberg proportions. The HLA‐B‐MICA‐STR haplotypic phases of all 85 unrelated parental samples were unambiguously assigned, which contained 30 kinds of HLA‐B, MICA‐STR haplotypic combinations, nine of them have not been reported in the literature. Significant positive linkage disequilibria between certain HLA‐B and MICA‐STR alleles, including HLA‐B*13 and MICA*A4, HLA‐B*38 and MICA*A9, HLA‐B*58 and MICA*A9, HLA‐B*46 and MICA*A5, HLA‐B*51 and MICA*A6, HLA‐B*52 and MICA*A6, and HLA‐B60 and MICA*A5.1, were observed. HLA‐B*48 was linked to MICA*A5, MICA*A5.1 and MICA*Del. HLA‐B*5801‐MICA*A10 linkage was found in a family. Our data indicated a high degree of haplotypic diversity and strong linkage disequilibrium between MICA‐STR and HLA‐B in a southern Chinese Han population, the data will inform future studies on anthropology, donor–recipient HLA matching in clinical transplantation and HLA‐linked disease association.
Activated calcineurin ameliorates contraction‐induced injury to skeletal muscles of mdx dystrophic mice
Authors: Stupka Nicole , Plant David R. , Schertzer Jonathan D. , Emerson Tennent M. , Bassel‐Duby Rhonda , Olson Eric N. , Lynch Gordon S.
Published: June 2006

Utrophin expression is regulated by calcineurin and up‐regulating utrophin can decrease the susceptibility of dystrophic skeletal muscle to contraction‐induced injury. We overexpressed the constitutively active calcineurin‐A α in skeletal muscle of mdx dystrophic mice (mdx CnA*) and examined the tibialis anterior muscle to determine whether the presence of activated calcineurin promotes resistance to muscle damage after lengthening contractions. Two stretches (10 s apart) of 40% strain relative to muscle fibre length were initiated from the plateau of a maximal isometric tetanic contraction. Muscle damage was assessed 1, 5 and 15 min later by the deficit in maximum isometric force and by quantifying the proportion of muscle fibres staining positive for intracytoplasmic albumin. The force deficit at all time points after the lengthening contractions was approximately 80% in mdx muscles and 30% in mdxCnA* muscles. The proportion of albumin‐positive fibres was significantly less in control and injured muscles from mdxCnA* mice than from mdx mice. Compared with mdx mice, mean fibre cross‐sectional area was 50% less in muscles from mdxCnA* mice. Furthermore, muscles from mdxCnA* mice exhibited a higher proportion of fibres expressing the slow(er) myosin heavy chain (MyHC) I and IIa isoforms, prolonged contraction and relaxation times, lower absolute and normalized maximum forces, and a clear leftward shift of the frequency–force relationship with greater force production at lower stimulation frequencies. These are structural and functional markers of a slower muscle phenotype. Taken together, our findings show that muscles from mdxCnA* mice have a smaller mean fibre cross‐sectional area, a greater sarcolemmal to cytoplasmic volume ratio, and an increase in utrophin expression, promoting an attenuated susceptibility to contraction‐induced injury. We conclude that increased calcineurin activity may confer functional benefits to dystrophic skeletal muscles.
Barium ions inhibit the dynamic response of guinea‐pig corneal cold receptors to heating but not to cooling
Authors: Brock James , Acosta M. Carmen , Al Abed Amr , Pianova Svetlana , Belmonte Carlos
Published: June 2006

An in vitro preparation of the guinea‐pig cornea was used to study the effects of the K+ channel blockers 4‐aminopyridine (4‐AP), tetraethylammonium (TEA) and Ba2+ on nerve terminal impulses (NTIs) recorded extracellularly from cold sensory receptors. These receptors have an ongoing discharge of NTIs that is increased by cooling and decreased by heating. The K+ channel blocker 4‐AP reduced the negative amplitude of the diphasic (positive–negative) NTIs, whereas TEA and Ba2+ prolonged the duration of the negative component. As the shape of the NTI is determined by the first derivative (dV/dt) of the membrane voltage change, these changes in the negative component are consistent with the blockade of K+ channels that contribute to action potential repolarization. Only TEA changed the basal activity of the receptors, increasing the likelihood of burst discharges. Ba2+ selectively reduced the response of the receptors to heating, whereas neither 4‐AP nor TEA modified the response to heating or to cooling. The findings indicate that K+ channels blocked by 4‐AP, TEA and Ba2+ contribute to action potential repolarization in corneal cold receptors, and that ionic mechanisms that underlie the reduction in NTI frequency in response to heating differ from those that increase activity in response to cooling.
Protease‐activated receptor 2 sensitizes TRPV1 by protein kinase Cɛ‐ and A‐dependent mechanisms in rats and mice
Authors: Amadesi Silvia , Cottrell Graeme S. , Divino Lorna , Chapman Kevin , Grady Eileen F. , Bautista Francisco , Karanjia Rustum , Barajas‐Lopez Carlos , Vanner Stephen , Vergnolle Nathalie , Bunnett Nigel W.
Published: June 2006

Proteases that are released during inflammation and injury cleave protease‐activated receptor 2 (PAR2) on primary afferent neurons to cause neurogenic inflammation and hyperalgesia. PAR2‐induced thermal hyperalgesia depends on sensitization of transient receptor potential vanilloid receptor 1 (TRPV1), which is gated by capsaicin, protons and noxious heat. However, the signalling mechanisms by which PAR2 sensitizes TRPV1 are not fully characterized. Using immunofluorescence and confocal microscopy, we observed that PAR2 was colocalized with protein kinase (PK) Cɛ and PKA in a subset of dorsal root ganglia neurons in rats, and that PAR2 agonists promoted translocation of PKCɛ and PKA catalytic subunits from the cytosol to the plasma membrane of cultured neurons and HEK 293 cells. Subcellular fractionation and Western blotting confirmed this redistribution of kinases, which is indicative of activation. Although PAR2 couples to phospholipase Cβ, leading to stimulation of PKC, we also observed that PAR2 agonists increased cAMP generation in neurons and HEK 293 cells, which would activate PKA. PAR2 agonists enhanced capsaicin‐stimulated increases in [Ca2+]i and whole‐cell currents in HEK 293 cells, indicating TRPV1 sensitization. The combined intraplantar injection of non‐algesic doses of PAR2 agonist and capsaicin decreased the latency of paw withdrawal to radiant heat in mice, indicative of thermal hyperalgesia. Antagonists of PKCɛ and PKA prevented sensitization of TRPV1 Ca2+ signals and currents in HEK 293 cells, and suppressed thermal hyperalgesia in mice. Thus, PAR2 activates PKCɛ and PKA in sensory neurons, and thereby sensitizes TRPV1 to cause thermal hyperalgesia. These mechanisms may underlie inflammatory pain, where multiple proteases are generated and released.
Comment on "Heterogeneous Hadean Hafnium: Evidence of Continental Crust at 4.4 to 4.5 Ga"
Authors: Valley John W. , Cavosie Aaron J. , Fu Bin , Peck William H. , Wilde Simon A.
Published: June 2006

Harrison
et al
. (Reports, 23 December 2005, p. 1947) proposed that plate tectonics and granites existed 4.5 billion years ago (Ga), within 70 million years of Earth's formation, based on geochemistry of >4.0 Ga detrital zircons from Australia. We highlight the large uncertainties of this claim and make the more moderate proposal that some crust formed by 4.4 Ga and oceans formed by 4.2 Ga.
Enhanced Mid-Latitude Tropospheric Warming in Satellite Measurements
Authors: Fu Qiang , Johanson Celeste M. , Wallace John M. , Reichler Thomas
Published: June 2006

The spatial distribution of tropospheric and stratospheric temperature trends for 1979 to 2005 was examined, based on radiances from satellite-borne microwave sounding units that were processed with state-of-the-art retrieval algorithms. We found that relative to the global-mean trends of the respective layers, both hemispheres have experienced enhanced tropospheric warming and stratospheric cooling in the 15 to 45° latitude belt, which is a pattern indicative of a widening of the tropical circulation and a poleward shift of the tropospheric jet streams and their associated subtropical dry zones. This distinctive spatial pattern in the trends appears to be a robust feature of this 27-year record.
A Clamping Mechanism Involved in SNARE-Dependent Exocytosis
Authors: Giraudo Claudio G. , Eng William S. , Melia Thomas J. , Rothman James E.
Published: June 2006

During neurotransmitter release at the synapse, influx of calcium ions stimulates the release of neurotransmitter. However, the mechanism by which synaptic vesicle fusion is coupled to calcium has been unclear, despite the identification of both the core fusion machinery [soluble
N
-ethylmaleimide–sensitive factor attachment protein receptor (SNARE)] and the principal calcium sensor (synaptotagmin). Here, we describe what may represent a basic principle of the coupling mechanism: a reversible clamping protein (complexin) that can freeze the SNAREpin, an assembled fusion-competent intermediate en route to fusion. When calcium binds to the calcium sensor synaptotagmin, the clamp would then be released. SNARE proteins, and key regulators like synaptotagmin and complexin, can be ectopically expressed on the cell surface. Cells expressing such “flipped” synaptic SNAREs fuse constitutively, but when we coexpressed complexin, fusion was blocked. Adding back calcium triggered fusion from this intermediate in the presence of synaptotagmin.
α-Synuclein Blocks ER-Golgi Traffic and Rab1 Rescues Neuron Loss in Parkinson's Models
Authors: Cooper Antony A. , Gitler Aaron D. , Cashikar Anil , Haynes Cole M. , Hill Kathryn J. , Bhullar Bhupinder , Liu Kangning , Xu Kexiang , Strathearn Katherine E. , Liu Fang , Cao Songsong , Caldwell Kim A. , Caldwell Guy A. , Marsischky Gerald , Kolodner Richard D. , LaBaer Joshua , Rochet Jean-Christophe , Bonini Nancy M. , Lindquist Susan
Published: June 2006

Alpha-synuclein (αSyn) misfolding is associated with several devastating neurodegenerative disorders, including Parkinson's disease (PD). In yeast cells and in neurons αSyn accumulation is cytotoxic, but little is known about its normal function or pathobiology. The earliest defect following αSyn expression in yeast was a block in endoplasmic reticulum (ER)–to–Golgi vesicular trafficking. In a genomewide screen, the largest class of toxicity modifiers were proteins functioning at this same step, including the Rab guanosine triphosphatase Ypt1p, which associated with cytoplasmic αSyn inclusions. Elevated expression of Rab1, the mammalian
YPT1
homolog, protected against αSyn-induced dopaminergic neuron loss in animal models of PD. Thus, synucleinopathies may result from disruptions in basic cellular functions that interface with the unique biology of particular neurons to make them especially vulnerable.
Role of
Porphyromonas gingivalis
FeoB2 in Metal Uptake and Oxidative Stress Protection
Authors: He Jia , Miyazaki Hiroshi , Anaya Cecilia , Yu Fan , Yeudall W. Andrew , Lewis Janina P.
Published: June 2006

Porphyromonas gingivalis
, a gram-negative anaerobic bacterium, is a recognized periodontopathogen. It exhibits a high degree of aerotolerance and is able to survive in host cells, indicating that efficient oxidative stress protection mechanisms must be present in this organism. Manganese homeostasis plays a major role in oxidative stress protection in a variety of organisms; however, the transport and role of this metal in
P. gingivalis
is not well understood. Analysis of the genome of
P. gingivalis
W83 revealed the presence of two genes encoding homologs of a ferrous iron transport protein, FeoB1 and FeoB2. FeoB2 has been implicated in manganese accumulation in
P. gingivalis
. We sought to determine the role of the FeoB2 protein in metal transport as well as its contribution to resistance to oxygen radicals. Quantitative reverse transcriptase PCR analyses demonstrated that expression of
feoB2
is induced in the presence of oxygen. The role of FeoB2 was investigated using an isogenic mutant strain deficient in the putative transporter. We characterized the FeoB2-mediated metal transport using
55
Fe
2+
and
54
Mn
2+
. The FeoB2-deficient mutant had dramatically reduced rates of manganese uptake (0.028 pmol/min/10
7
bacteria) compared with the parental strain (0.33 pmol/min/10
7
bacteria) (after 20 min of uptake using 50 nM of
54
Mn
2+
). The iron uptake rates, however, were higher in the mutant strain (0.75 pmol/min/10
7
bacteria) than in the wild type (0.39 pmol/min/10
7
bacteria). Interestingly, reduced survival rates were also noted for the mutant strain after exposure to H
2
O
2
and to atmospheric oxygen compared to the parental strain cultured under the same conditions. In addition, in vitro infection of host cells with the wild type, the FeoB2-deficient mutant, and the same-site revertant revealed that the mutant had a significantly decreased capability for intracellular survival in the host cells compared to the wild-type strain. Our results demonstrate that
feoB2
encodes a major manganese transporter required for protection of the bacterium from oxidative stress generated by atmospheric oxygen and H
2
O
2
. Furthermore, we show that FeoB2 and acquisition of manganese are required for intracellular survival of
P. gingivalis
in host cells.
A Monoclonal Antibody to
Bacillus anthracis
Protective Antigen Defines a Neutralizing Epitope in Domain 1
Authors: Rivera Johanna , Nakouzi Antonio , Abboud Nareen , Revskaya Ekaterina , Goldman David , Collier R. John , Dadachova Ekaterina , Casadevall Arturo
Published: June 2006

Antibody (Ab) responses to
Bacillus anthracis
toxins are protective, but relatively few protective monoclonal antibodies (MAbs) have been reported. Protective antigen (PA) is essential for the action of
B. anthracis
lethal toxin (LeTx) and edema toxin. In this study, we generated two MAbs to PA, MAbs 7.5G and 10F4. These MAbs did not compete for binding to PA, consistent with specificities for different epitopes. The MAbs were tested for their ability to protect a monolayer of cultured macrophages against toxin-mediated cytotoxicity. MAb 7.5G, the most-neutralizing MAb, bound to domain 1 of PA and reduced LeTx toxicity in BALB/c mice. Remarkably, MAb 7.5G provided protection without blocking the binding of PA or lethal factor or the formation of the PA heptamer complex. However, MAb 7.5G slowed the proteolytic digestion of PA by furin in vitro, suggesting a potential mechanism for Ab-mediated protection. These observations indicate that some Abs to domain 1 can contribute to host protection.
Search for
Bacillus anthracis
Potential Vaccine Candidates by a Functional Genomic-Serologic Screen
Authors: Gat Orit , Grosfeld Haim , Ariel Naomi , Inbar Itzhak , Zaide Galia , Broder Yehoshua , Zvi Anat , Chitlaru Theodor , Altboum Zeev , Stein Dana , Cohen Sara , Shafferman Avigdor
Published: June 2006

Bacillus anthracis
proteins that possess antigenic properties and are able to evoke an immune response were identified by a reductive genomic-serologic screen of a set of in silico-preselected open reading frames (ORFs). The screen included in vitro expression of the selected ORFs by coupled transcription and translation of linear PCR-generated DNA fragments, followed by immunoprecipitation with antisera from
B. anthracis-
infected animals. Of the 197 selected ORFs, 161 were chromosomal and 36 were on plasmids pXO1 and pXO2, and 138 of the 197 ORFs had putative functional annotations (known ORFs) and 59 had no assigned functions (unknown ORFs). A total of 129 of the known ORFs (93%) could be expressed, whereas only 38 (64%) of the unknown ORFs were successfully expressed. All 167 expressed polypeptides were subjected to immunoprecipitation with the anti-
B. anthracis
antisera, which revealed 52 seroreactive immunogens, only 1 of which was encoded by an unknown ORF. The high percentage of seroreactive ORFs among the functionally annotated ORFs (37%; 51/129) attests to the predictive value of the bioinformatic strategy used for vaccine candidate selection. Furthermore, the experimental findings suggest that surface-anchored proteins and adhesins or transporters, such as cell wall hydrolases, proteins involved in iron acquisition, and amino acid and oligopeptide transporters, have great potential to be immunogenic. Most of the seroreactive ORFs that were tested as DNA vaccines indeed appeared to induce a humoral response in mice. We list more than 30 novel
B. anthracis
immunoreactive virulence-related proteins which could be useful in diagnosis, pathogenesis studies, and future anthrax vaccine development.
Role of Tumor Necrosis Factor Alpha in Development of Immunity againstCryptosporidium parvumInfection
Authors: Lean I-Sarah , Lacroix-Lamandé Sonia , Laurent Fabrice , McDonald Vincent
Published: June 2006

Tumor necrosis factor (TNF-α) significantly reducedCryptosporidium parvumdevelopment in a murine enterocyte cell line, and a key mechanism of action appeared to be inhibition of parasite invasion. However, TNF-α-deficient mice controlled infection as effectively as wild-type mice. This suggests that TNF-α might have only a redundant role for establishing immunity againstC. parvum.
Burkholderia cenocepacia
ZmpB Is a Broad-Specificity Zinc Metalloprotease Involved in Virulence
Authors: Kooi C. , Subsin B. , Chen R. , Pohorelic B. , Sokol P. A.
Published: June 2006

In previous studies we characterized the
Burkholderia cenocepacia
ZmpA zinc metalloprotease. In this study, we determined that
B. cenocepacia
has an additional metalloprotease, which we designated ZmpB. The
zmpB
gene is present in the same species as
zmpA
and was detected in
B. cepacia
,
B. cenocepacia
,
B. stabilis
,
B. ambifaria
, and
B. pyrrocinia
but was absent from
B. multivorans
,
B. vietnamiensis
,
B. dolosa
, and
B. anthina
. The
zmpB
gene was expressed, and ZmpB was purified from
Escherichia coli
by using the pPROEXHTa His
6
Tag expression system. ZmpB has a predicted preproenzyme structure typical of thermolysin-like proteases and is distantly related to
Bacillus cereus
bacillolysin. ZmpB was expressed as a 63-kDa preproenzyme precursor that was autocatalytically cleaved into mature ZmpB (35 kDa) and a 27-kDa prepropeptide. EDTA, 1,10-phenanthroline, and Zn
2+
cations inhibited ZmpB enzyme activity, indicating that it is a metalloprotease. ZmpB had proteolytic activity against α-1 proteinase inhibitor, α
2
-macrogobulin, type IV collagen, fibronectin, lactoferrin, transferrin, and immunoglobulins.
B. cenocepacia zmpB
and
zmpA zmpB
mutants had no proteolytic activity against casein and were less virulent in a rat agar bead chronic infection model, indicating that
zmpB
is involved in
B. cenocepacia
virulence. Expression of
zmpB
was regulated by both the CepIR and CciIR quorum-sensing systems.
Phenotypic and Functional Characterization of Vaginal Dendritic Cells in a Rat Model ofCandida albicansVaginitis
Authors: De Bernardis Flavia , Lucciarini Roberta , Boccanera Maria , Amantini Consuelo , Arancia Silvia , Morrone Stefania , Mosca Michela , Cassone Antonio , Santoni Giorgio
Published: June 2006

This study analyzes the phenotype of vaginal dendritic cells (VDCs), their antigenic presentation and activation of T-cell cytokine secretion, and their protective role in a rat model ofCandidavaginitis. Histological observation demonstrated a significant accumulation of OX62+VDCs in the mucosal epithelium ofCandida albicans-infected rats at the third round of infection. We identified two subsets of OX62+VDCs differing in the expression of CD4 molecule in both noninfected andCandida-infected rats. The OX62+CD4+subset of VDCs displayed a lymphoid cell-like morphology and expressed the T-cell antigen CD5, whereas the OX62+CD4−VDC subset exhibited a myeloid morphology and was CD5 negative.Candidainfection resulted in VDC maturation with enhanced expression of CD80 and CD134L on both CD4+and CD4−VDC subsets at 2 and 6 weeks afterCandidainfection. CD5−CD4−CD86−CD80−CD134L+VDCs from infected, but not noninfected, rats spontaneously released large amounts of interleukin-12 (IL-12) and tumor necrosis factor alpha, whereas all VDC subsets released comparable levels of IL-10 and IL-2 cytokines. Furthermore, OX62+VDCs from infected rats primed naïve CD4+T-cell proliferation and release of cytokines, including gamma interferon, IL-2, IL-6, and IL-10, in response to staphylococcal enterotoxin B stimulation in vitro. Adoptive transfer of highly purified OX62+VDCs from infected rats induced a significant acceleration of fungal clearance compared with that in rats receiving naive VDCs, suggesting a protective role of VDCs in the anti-Candidamucosal immunity. Finally, VDC-mediated protection was associated with their ability to rapidly migrate to the vaginal mucosa and lymph nodes, as assessed by adoptive transfer of OX62+VDCs labeled with 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester.
LuxS Involvement in the Regulation of Genes Coding for Hemin and Iron Acquisition Systems inPorphyromonas gingivalis
Authors: James Chloe E. , Hasegawa Yoshiaki , Park Yoonsuk , Yeung Vincent , Tribble Gena D. , Kuboniwa Masae , Demuth Donald R. , Lamont Richard J.
Published: June 2006

The periodontal pathogenPorphyromonas gingivalisemploys a variety of mechanisms for the uptake of hemin and inorganic iron. Previous work demonstrated that hemin uptake inP. gingivalismay be controlled by LuxS-mediated signaling. In the present study, the expression of genes involved in hemin and iron uptake was determined in parent andluxSmutant strains by quantitative real-time reverse transcription-PCR. Compared to the parental strain, theluxSmutant showed reduced levels of transcription of genes coding for the TonB-linked hemin binding protein Tlr and the lysine-specific protease Kgp, which can degrade host heme-containing proteins. In contrast, there was up-regulation of the genes for another TonB-linked hemin binding protein, HmuR; a hemin binding lipoprotein, FetB; a Fe2+ion transport protein, FeoB1; and the iron storage protein ferritin. Differential expression of these genes in theluxSmutant was maximal in early-exponential phase, which corresponded with peak expression ofluxSand AI-2 signal activity. Complementation of theluxSmutation with wild-typeluxSintransrescued expression ofhmuR. Mutation of the GppX two-component signal transduction pathway caused an increase in expression ofluxSalong withtlrand lower levels of message forhmuR. Moreover, expression ofhmuRwas repressed, and expression oftlrstimulated, when theluxSmutant was incubated with AI-2 partially purified from the culture supernatant of wild-type cells. A phenotypic outcome of the altered expression of genes involved in hemin uptake was impairment of growth of theluxSmutant in hemin-depleted medium. The results demonstrate a role of LuxS/AI-2 in the regulation of hemin and iron acquisition pathways inP. gingivalisand reveal a novel control pathway forluxSexpression.
Evaluation of Protection Afforded by
Brucella abortus
and
Brucella melitensis
Unmarked Deletion Mutants Exhibiting Different Rates of Clearance in BALB/c Mice
Authors: Kahl-McDonagh M. M. , Ficht T. A.
Published: June 2006

Research for novel
Brucella
vaccines has focused upon the development of live vaccine strains, which have proven more efficacious than killed or subunit vaccines. In an effort to develop improved vaccines, signature-tagged mutant banks were screened to identify mutants attenuated for survival. Mutants selected from these screens exhibited various degrees of attenuation characterized by the rate of clearance, ranging from a failure to grow in macrophages after 24 h of infection to a failure to persist in the mouse model beyond 8 weeks. Ideal vaccine candidates should be safe to the host, while evoking protective immunity. In the present work, we constructed unmarked deletion mutants of three gene candidates,
manBA
,
virB2
, and
asp24
, in both
Brucella abortus
and
Brucella melitensis
. The Δ
asp24
mutants, which persist for extended periods in vivo, are superior to current vaccine strains and to other deletion strains tested in the mouse model against homologous challenge infection after 12, 16, and 20 weeks postvaccination. The Δ
asp24
mutants also display superior protection compared to Δ
manBA
and Δ
virB2
mutants against heterologous challenge in mice. From this study, a direct association between protection against infection and cytokine response was not apparent between all vaccine groups and, therefore, correlates of protective immunity will need to be considered further. A distinct correlation between persistence of the vaccine strain and protection against infection was corroborated.
Effect of a CD4-Depleting Antibody on the Development ofCryptococcus neoformans-Induced Allergic Bronchopulmonary Mycosis in Mice
Authors: Arora Shikha , McDonald Roderick A. , Toews Galen B. , Huffnagle Gary B.
Published: June 2006

Allergic bronchopulmonary mycosis (ABPM) is a hypersensitivity lung disease in which fungal colonization is accompanied by an allergic response to the fungus. Using a mouse model of ABPM caused byCryptococcus neoformansinfection of C57BL/6 mice, the goal of the present studies was to determine the effect of the CD4-depleting monoclonal antibody GK1.5 on the development of the allergic responses seen during active fungal infection. These results would provide insight into the role of CD4+T cells in this disease. Our results show that GK1.5 treatment resulted in attenuation of pulmonary inflammation and eosinophilia in these animals. These mice also had reduced T2 cytokine production and no serum immunoglobulin E production. Absence of CD4+T cells did not affect recruitment of CD8+T cells to the site of infection; however, the numbers of CD19+B cells were severely reduced in the lungs of CD4+T-cell-depleted animals. We also examined changes in the pulmonary architecture and found that depletion of CD4+T cells was associated with a significant reduction in mucus production and goblet cell metaplasia in these mice. Interestingly, attenuation of Th2 responses in CD4+T-cell-depleted animals did not increase the fungal load in their lungs. We also compared development of ABPM in young and mature mice and did not find any differences at any of the time points. Overall, our results show that depletion of CD4+T cells prevents the development of Th2 responses seen during ABPM.
The Novel
Helicobacter pylori
CznABC Metal Efflux Pump Is Required for Cadmium, Zinc, and Nickel Resistance,Urease Modulation, and Gastric Colonization
Authors: Stähler Frank Nils , Odenbreit Stefan , Haas Rainer , Wilrich Julia , Vliet Arnoud H. M. Van , Kusters Johannes G. , Kist Manfred , Bereswill Stefan
Published: June 2006

Maintaining
metal homeostasis is crucial for the adaptation of
Helicobacter
pylori
to the gastric environment. Iron, copper, and nickel
homeostasis has recently been demonstrated to be required for the
establishment of
H. pylori
infection in animal models. Here we
demonstrate that the HP0969-0971 gene cluster encoding the Czc-type
metal export pump homologs HP0969, HP0970, and the
H.
pylori
-specific protein HP0971 forms part of a novel
H.
pylori
metal resistance determinant, which is required for gastric
colonization and for the modulation of urease activity. Insertional
mutagenesis of the HP0971, HP0970, or HP0969 genes in
H.
pylori
reference strain 26695 resulted in increased sensitivity to
cadmium, zinc, and nickel (czn), suggesting that the encoded proteins
constitute a metal-specific export pump. Accordingly, the genes were
designated
cznC
(HP0971),
cznB
(HP0970), and
cznA
(HP0969). The CznC and CznA proteins play a predominant
role in nickel homeostasis, since only the
cznC
and
cznA
mutants but not the
cznB
mutant displayed an 8-
to 10-fold increase in urease activity. Nickel-specific affinity
chromatography demonstrated that recombinant versions of CznC and CznB
can bind to nickel and that the purified CznB protein interacted with
cadmium and zinc, since both metals competitively inhibited nickel
binding. Finally, single
cznA
,
cznB
, and
cznC
mutants did not colonize the stomach in a Mongolian
gerbil-based animal model. This demonstrates that the metal export
functions of
H. pylori cznABC
are essential for gastric
colonization and underlines the extraordinary importance of metal ion
homeostasis for the survival of
H. pylori
in the gastric
environment.
T-Cell Expression Cloning ofPorphyromonas gingivalisGenes Coding for T Helper-Biased Immune Responses during Infection
Authors: Gonçalves Reginaldo B. , Leshem Onir , Bernards Karen , Webb John R. , Stashenko Philip P. , Campos-Neto Antonio
Published: June 2006

Exposure of the mouse oral cavity toPorphyromonas gingivalisresults in the development of gingivitis and periapical bone loss, which apparently are associated with a Th1 response to bacterial antigens. We have used this infection model in conjunction with direct T-cell expression cloning to identify bacterial antigens that induce a preferential or biased T helper response during the infectious process. AP. gingivalis-specific CD4 T-cell line derived from mice at 3 weeks postchallenge was used to directly screen aP. gingivalisgenomic expression library. This screen resulted in the identification of five genes coding for previously identified proteins and three other putative protein antigens. One of the identified proteins,P. gingivalisthiol peroxidase, was studied in detail because this molecule belongs to a protein family that is apparently involved in microbial pathogenesis. Infection of mice withP. gingivalis, either via the subcutaneous route or after exposure of the animal's oral cavity to viable bacteria, resulted in the induction of a strong thiol peroxidase-specific immune response characterized by the production of high titers of specific serum immunoglobulin G2a antibody and the production of gamma interferon by antigen-stimulated lymphoid cells, a typical Th1-biased response. Thus, the use of a proven T-cell expression cloning approach and a mouse model of periodontal disease resulted in the identification and characterization ofP. gingivalisproteins that might be involved in pathogenesis.
Transient Neonatal
Cryptosporidium parvum
Infection Triggers Long-Term Jejunal Hypersensitivity to Distension in Immunocompetent Rats
Authors: Marion Rachel , Baishanbo Asiya , Gargala Gilles , François Arnaud , Ducrotté Philippe , Duclos Celia , Fioramonti Jean , Ballet Jean Jacques , Favennec Loïc
Published: June 2006

In 5-day-old immunocompetent Sprague-Dawley rats infected with either 10
2
or 10
5
Cryptosporidium parvum
oocysts, transient infection resulted 120 days later in increased cardiovascular depressor response to jejunal distension and jejunal myeloperoxidase activity (
P
< 0.05). Nitazoxanide treatment normalized jejunal sensitivity (
P
< 0.001) but not myeloperoxidase levels (
P
> 0.05). Data warrant further evaluation of the role of early cryptosporidiosis in the development of chronic inflammatory gut conditions.
Sialic Acid-Binding Immunoglobulin-Like Lectin 7 Mediates Selective Recognition of Sialylated Glycans Expressed on
Campylobacter jejuni
Lipooligosaccharides
Authors: Avril Tony , Wagner Eric R. , Willison Hugh J. , Crocker Paul R.
Published: June 2006

siglecs are a family of sialic-acid binding immunoglobulin-like lectins mostly expressed by cells of the immune system that have the potential to interact with sialylated glycans expressed not only on host cells but also on certain pathogens.
Campylobacter jejuni
is a common pathogen of humans that expresses surface lipooligosaccharides (LOS) that can be modified with ganglioside-like terminal structures in the core oligosaccharides. In this study, we examined the interaction of 10 siglecs with LOS purified from four different
C. jejuni
isolates expressing GM1-like, GD1a-like, GD3-like, and GT1a-like oligosaccharides. Of all siglecs examined, only Siglec-7 exhibited specific, sialic acid-dependent interactions with
C. jejuni
LOS in solid-phase binding assays. Binding was especially prominent with LOS from the HS:19(GM1
+
GT1a
+
) isolate, with weaker binding with LOS from the HS:19(GD3
+
) isolate. Binding of Siglec-7 was also observed with intact bacteria expressing these LOS structures. Specific binding of HS:19(GM1
+
GT1a
+
) bacteria was demonstrated with Siglec-7 expressed on transfected Chinese hamster ovary cells and with peripheral blood leukocytes, among which HS:19(GM1
+
GT1a
+
) bacteria bound selectively to both natural killer cells and monocytes which naturally express Siglec-7. These results raise the possibility that, in addition to their role in generating autoimmune antibody responses,
C. jejuni
LOS could interact with Siglec-7 expressed by leukocytes, modulate the host-pathogen interaction, and contribute to the clinical outcome and the development of secondary complications such as Guillain-Barré syndrome.
Protein Expression Profiles of
Chlamydia pneumoniae
in Models of Persistence versus Those of Heat Shock Stress Response
Authors: Mukhopadhyay Sanghamitra , Miller Richard D. , Sullivan Erin D. , Theodoropoulos Christina , Mathews Sarah A. , Timms Peter , Summersgill James T.
Published: June 2006

Chlamydia pneumoniae
is an obligate intracellular pathogen that causes both acute and chronic human disease. Several in vitro models of chlamydial persistence have been established to mimic chlamydial persistence in vivo. We determined the expression patterns of 52
C. pneumoniae
proteins, representing nine functional subgroups, from the gamma interferon (IFN-γ) treatment (primarily tryptophan limitation) and iron limitation (IL) models of persistence compared to those following heat shock (HS) at 42°C. Protein expression patterns of
C. pneumoniae
persistence indicates a strong stress component, as evidenced by the upregulation of proteins involved in protein folding, assembly, and modification. However, it is clearly more than just a stress response. In IFN persistence, but not IL or HS, amino acid and/or nucleotide biosynthesis proteins were found to be significantly upregulated. In contrast, proteins involved in the biosynthesis of cofactors, cellular processes, energy metabolism, transcription, and translation showed an increased in expression in only the IL model of persistence. These data represent the most extensive protein expression study of
C. pneumoniae
comparing the chlamydial heat shock stress response to two models of persistence and identifying the common and unique protein level responses during persistence.
Synthesis of 2'-C-Methylcytidine and 2'-C-Methyluridine Derivatives Modified in the 3'-Position as Potential Antiviral Agents
Authors: Pierra Claire , Amador Agnès , Badaroux Eric , Storer Richard , Gosselin Gilles
Published: June 2006

As part of our anti-hepatitis C program, we recently discovered 2'-C-methylcytidine (1) and 2'-C-methyluridine (2), which are potent inhibitors in cell culture of several viruses (bovine viral diarrhea virus (BVDV), yellow fever virus (YFV)) closely related to HCV. In order to characterize structure-activity relationships, we introduced some structural and functional modifications into the 3'-position of 2'-C-methylcytidine and 2'-C-methyluridine. All these hitherto unknown compounds thus synthesized were tested for the activity against a wide range of viruses and found to be inactive.
Nucleic Acid Related Compounds. 136. Synthesis of 2-Amino- and 2,6-Diaminopurine Derivatives via Inverse-Electron-Demand Diels-Alder Reactions
Authors: Lin Xiaoyu , Robins Morris J.
Published: June 2006

Thermal inverse-electron-demand Diels-Alder reactions of 5-aminoimidazoles and 2,4,6-tris(ethoxycarbonyl)-1,3,5-triazine (2) with spontaneous retro-Diels-Alder loss of ethyl cyanoformate and elimination of ammonia give 2,6-bis(ethoxycarbonyl)purines. A report that selective alkaline hydrolysis followed by acid-catalyzed decarboxylation gave 6-(ethoxycarbonyl)purine products was not in harmony with known reactions in purine chemistry. Our reinvestigation has shown that the 6-(ethoxycarbonyl) group undergoes preferential base-promoted hydrolysis, as expected, but regioselectivity for attack of hydroxide at the carbonyl group at C6 is not high (relative to hydrolysis of both C2 and C6 esters). The structure of 9-benzyl-2-(ethoxycarbonyl)purine was determined by X-ray crystallography and confirmed by Curtius rearrangement of the azidocarbonyl analogue to give 2-amino-6-benzylpurine. Acid-catalyzed decarboxylation of the 2,6-dicarboxylate formed during hydrolysis gave 9-benzylpurine, and Curtius rearrangement of 2,6-bis(azidocarbonyl)-9-benzylpurine gave 2,6-diamino-9-benzylpurine. Attempted applications of inverse-electron-demand Diels-Alder reactions of 2 with nucleoside derivatives were problematic.
Strategies for the prevention of volleyball related injuries
Authors: Reeser J C , Verhagen E , Briner W W , Askeland T I , Bahr R
Published: June 2006

Although the overall injury rate in volleyball and beach volleyball is relatively low compared with other team sports, injuries do occur in a discipline specific pattern. Epidemiological research has revealed that volleyball athletes are, in general, at greatest risk of acute ankle injuries and overuse conditions of the knee and shoulder. This structured review discusses both the known and suspected risk factors and potential strategies for preventing the most common volleyball related injuries: ankle sprains, patellar tendinopathy, and shoulder overuse.
Calcific tendonitis of the quadriceps
Authors: Varghese B , Radcliffe G S , Groves C
Published: June 2006

The case reported is of a 46 year old male who presented with a history of acute on chronic knee pain. The clinical features and investigations suggested a tear of the quadriceps tendon, with pre-existing chronic calcific enthesopathy. The operative findings were of an acute collection of calcific material within the quadriceps tendon. This acute presentation and calcific collection have not previously been reported in the quadriceps tendon.
FIFA’s approach to doping in football
Authors: Dvorak J , Graf-Baumann T , D’Hooghe M , Kirkendall D , Taennler H , Saugy M
Published: June 2006

Background and objectives:FIFA’s anti-doping strategy relies on education and prevention. A worldwide network of physicians guarantees doping control procedures that are straightforward and leave no place for cheating. FIFA actively acknowledges its responsibility to protect players from harm and ensure equal chances for all competitors by stringent doping control regulations, data collection of positive samples, support of research, and collaboration with other organisations. This article aims to outline FIFA’s approach to doping in football.

Method:Description of FIFA’s doping control regulations and procedures, statistical analysis of FIFA database on doping control, and comparison with data obtained by WADA accredited laboratories as for 2004.

Results:Data on positive doping samples per substance and confederation/nation documented at the FIFA medical office from 1994 to 2005 are provided. According to the FIFA database, the incidence of positive cases over the past 11 years was 0.12%, with about 0.42% in 2004 (based on the assumption of 20 750 samples per year) and 0.37% in 2005. Especially important in this regard is the extremely low incidence of the true performance enhancing drugs such as anabolic steroids and stimulants. However, there is a need for more consistent data collection and cross checks among international anti-doping agencies as well as for further studies on specific substances, methods, and procedures. With regard to general health impairments in players, FIFA suggests that principles of occupational medicine should be considered and treatment with banned substances for purely medical reasons should be permitted to enable players to carry out their profession. At the same time, a firm stand has to be taken against suppression of symptoms by medication with the aim of meeting the ever increasing demands on football players.

Conclusion:Incidence of doping in football seems to be low, but much closer collaboration and further investigation is needed with regard to banned substances, detection methods, and data collection worldwide.
Testosterone and doping control
Authors: Saudan C , Baume N , Robinson N , Avois L , Mangin P , Saugy M
Published: June 2006

Background and objectives:Anabolic steroids are synthetic derivatives of testosterone, modified to enhance its anabolic actions (promotion of protein synthesis and muscle growth). They have numerous side effects, and are on the International Olympic Committee’s list of banned substances. Gas chromatography-mass spectrometry allows identification and characterisation of steroids and their metabolites in the urine but may not distinguish between pharmaceutical and natural testosterone. Indirect methods to detect doping include determination of the testosterone/epitestosterone glucuronide ratio with suitable cut-off values. Direct evidence may be obtained with a method based on the determination of the carbon isotope ratio of the urinary steroids. This paper aims to give an overview of the use of anabolic-androgenic steroids in sport and methods used in anti-doping laboratories for their detection in urine, with special emphasis on doping with testosterone.

Methods:Review of the recent literature of anabolic steroid testing, athletic use, and adverse effects of anabolic-androgenic steroids.

Results:Procedures used for detection of doping with endogenous steroids are outlined. The World Anti-Doping Agency provided a guide in August 2004 to ensure that laboratories can report, in a uniform way, the presence of abnormal profiles of urinary steroids resulting from the administration of testosterone or its precursors, androstenediol, androstenedione, dehydroepiandrosterone or a testosterone metabolite, dihydrotestosterone, or a masking agent, epitestosterone.

Conclusions:Technology developed for detection of testosterone in urine samples appears suitable when the substance has been administered intramuscularly. Oral administration leads to rapid pharmacokinetics, so urine samples need to be collected in the initial hours after intake. Thus there is a need to find specific biomarkers in urine or plasma to enable detection of long term oral administration of testosterone.
Therapeutic use exemption
Authors: Dvorak J , Kirkendall D , Vouillamoz M
Published: June 2006

Football players who have either physical symptoms or disease after injury may need to be treated with specific medicines that are on the list of prohibited substances. Therapeutic use exemption may be granted to such players, in accordance with strictly defined criteria—these are presented in this article. Procedures of how to request for an abbreviated or a standard therapeutic use exemption are explained, and data on therapeutic use exemptions (UEFA and FIFA, 2004 and 2005) are also presented.
Erythropoietin and blood doping
Authors: Robinson N , Giraud S , Saudan C , Baume N , Avois L , Mangin P , Saugy M
Published: June 2006

Objective and method:To outline the direct and indirect approaches in the fight against blood doping in sports, the different strategies that have been used and are currently being used to fight efficiently against blood doping are presented and discussed.

Results and conclusions:The paper outlines the different approaches and diagnostic tools that some federations have to identify and target sportspeople demonstrating abnormal blood profiles. Originally blood tests were introduced for medical reasons and for limiting misuse of recombinant human erythropoietin (rHuEPO). In this way it became possible to prevent athletes with haematocrit levels well above normal, and potentially dangerous for their health, competing in sport. Today, with nearly a decade of blood testing experience, sports authorities should be familiar with some of the limitations and specially the ability of blood tests performed prior to competitions to fight efficiently against the misuse of rHuEPO, blood transfusion, and artificial haemoglobin.