bluuebunny/crossref_metadata_2025 huggingface dataset:
DegU and YczE Positively Regulate the Synthesis of Bacillomycin D byBacillus amyloliquefaciensStrain FZB42
Authors: Koumoutsi Alexandra , Chen Xiao-Hua , Vater Joachim , Borriss Rainer
Published: September 2007

Environmental strainBacillus amyloliquefaciensFZB42 differs from the domesticated model organism of the same genus,Bacillus subtilis168, in its ability to promote plant growth and suppress plant-pathogenic organisms present in the rhizosphere. This behavior is exerted mainly through the production of several nonribosomal cyclic lipopeptides and polyketides, which exhibit a broad range of action against phytopathogenic bacteria, fungi, and nematodes. Here, we provide evidence that the synthesis of the main antifungal agent ofB. amyloliquefaciensFZB42, bacillomycin D, is regulated in multiple layers. Expression of the bacillomycin D operon (bmy) is dependent on a single σA-dependent promoter, Pbmyand is favored in its natural host by the small regulatory protein DegQ. The global regulators DegU and ComA are required for the full transcriptional activation ofbmy. DegU retains a key role since it binds directly to two sites located upstream of the bacillomycin D promoter. Moreover, both DegU and a transmembrane protein of unknown function, YczE, act on a later level of gene expression, exerting their posttranscriptional effects in a hitherto-unknown manner.
Impact of Substrate Glycoside Linkage and Elemental Sulfur on Bioenergetics of and Hydrogen Production by the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Chou Chung-Jung , Shockley Keith R. , Conners Shannon B. , Lewis Derrick L. , Comfort Donald A. , Adams Michael W. W. , Kelly Robert M.
Published: September 2007

Glycoside linkage (cellobiose versus maltose) dramatically influenced bioenergetics to different extents and by different mechanisms in the hyperthermophilic archaeon
Pyrococcus furiosus
when it was grown in continuous culture at a dilution rate of 0.45 h
−1
at 90°C. In the absence of S
0
, cellobiose-grown cells generated twice as much protein and had 50%-higher specific H
2
generation rates than maltose-grown cultures. Addition of S
0
to maltose-grown cultures boosted cell protein production fourfold and shifted gas production completely from H
2
to H
2
S. In contrast, the presence of S
0
in cellobiose-grown cells caused only a 1.3-fold increase in protein production and an incomplete shift from H
2
to H
2
S production, with 2.5 times more H
2
than H
2
S formed. Transcriptional response analysis revealed that many genes and operons known to be involved in α- or β-glucan uptake and processing were up-regulated in an S
0
-independent manner. Most differentially transcribed open reading frames (ORFs) responding to S
0
in cellobiose-grown cells also responded to S
0
in maltose-grown cells; these ORFs included ORFs encoding a membrane-bound oxidoreductase complex (MBX) and two hypothetical proteins (PF2025 and PF2026). However, additional genes (242 genes; 108 genes were up-regulated and 134 genes were down-regulated) were differentially transcribed when S
0
was present in the medium of maltose-grown cells, indicating that there were different cellular responses to the two sugars. These results indicate that carbohydrate characteristics (e.g., glycoside linkage) have a major impact on S
0
metabolism and hydrogen production in
P. furiosus
. Furthermore, such issues need to be considered in designing and implementing metabolic strategies for production of biofuel by fermentative anaerobes.
Quantification of Cell Proliferation and Alpha-Toxin Gene Expression ofClostridium perfringensin the Development of Necrotic Enteritis in Broiler Chickens
Authors: Si Weiduo , Gong Joshua , Han Yanming , Yu Hai , Brennan John , Zhou Huaijun , Chen Shu
Published: September 2007

Cell proliferation and alpha-toxin gene expression ofClostridium perfringensin relation to the development of necrotic enteritis (NE) were investigated. Unlike bacitracin-treated chickens, non-bacitracin-treated birds exhibited typical NE symptoms and reduced growth performance. They also demonstrated increasedC. perfringensproliferation and alpha-toxin gene expression that were positively correlated and progressed according to the regression modely=b0+b1X−b2X2. The averageC. perfringenscount of 5 log10CFU/g in the ileal digesta appears to be a threshold for developing NE with a lesion score of 2.
Coupling Immunomagnetic Separation on Magnetic Beads with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Detection of Staphylococcal Enterotoxin B
Authors: Schlosser Gitta , Kačer Petr , Kuzma Marek , Szilágyi Zoltán , Sorrentino Alida , Manzo Carla , Pizzano Rosa , Malorni Livia , Pocsfalvi Gabriella
Published: September 2007

The growing importance of mass spectrometry for the identification and characterization of bacterial protein toxins is a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, especially when these techniques are combined with affinity methods. Here we describe a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization-time of flight mass spectrometry for selective purification and detection of staphylococcal enterotoxin B (SEB). SEB is a potent bacterial protein toxin responsible for food poisoning, as well as a potential biological warfare agent. Unambiguous detection of SEB at low-nanogram levels in complex matrices is thus an important objective. In this work, an affinity molecular probe was prepared by immobilizing anti-SEB antibody on the surface of
para
-toluene-sulfonyl-functionalized monodisperse magnetic particles and used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB immunoglobulin G and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB could be detected directly “on beads” by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, “off beads” after its acidic elution. Application of this method to complex biological matrices was demonstrated by selective detection of SEB present in different matrices, such as cultivation media of S
taphylococcus aureus
strains and raw milk samples.
Structural and Mutational Analysis of tRNA Intron-Splicing Endonuclease fromThermoplasma acidophilumDSM 1728: Catalytic Mechanism of tRNA Intron-Splicing Endonucleases
Authors: Kim Young Kwan , Mizutani Kenji , Rhee Kyung-Hee , Nam Ki-Hyun , Lee Won Ho , Lee Eun Hye , Kim Eunice Eunkyeong , Park Sam-Yong , Hwang Kwang Yeon
Published: September 2007

In archaea, RNA endonucleases that act specifically on RNA with bulge-helix-bulge motifs play the main role in the recognition and excision of introns, while the eukaryal enzymes use a measuring mechanism to determine the positions of the universally positioned splice sites relative to the conserved domain of pre-tRNA. Two crystallographic structures of tRNA intron-splicing endonuclease fromThermoplasma acidophilumDSM 1728 (EndATa) have been solved to 2.5-Å and 2.7-Å resolution by molecular replacement, using the 2.7-Å resolution data as the initial model and the single-wavelength anomalous-dispersion phasing method using selenomethionine as anomalous signals, respectively. The models show that EndATais a homodimer and that it has overall folding similar to that of other archaeal tRNA endonucleases. From structural and mutational analyses of H236A, Y229F, and K265I in vitro, we have demonstrated that they play critical roles in recognizing the splice site and in cleaving the pre-tRNA substrate.
Requirement of the Zinc-Binding Domain of ClpX for Spx Proteolysis inBacillus subtilisand Effects of Disulfide Stress on ClpXP Activity
Authors: Zhang Ying , Zuber Peter
Published: September 2007

Spx, a transcriptional regulator of the disulfide stress response inBacillus subtilis, is under the proteolytic control of the ATP-dependent protease ClpXP. Previous studies suggested that ClpXP activity is down-regulated in response to disulfide stress, resulting in elevated concentrations of Spx. The effect of disulfide stress on ClpXP activity was examined using the thiol-specific oxidant diamide. ClpXP-catalyzed degradation of either Spx or a green fluorescent protein derivative bearing an SsrA tag recognized by ClpXP was inhibited by diamide treatment in vitro. Spx is also a substrate for MecA/ClpCP-catalyzed proteolysis in vitro, but diamide used at the concentrations that inhibited ClpXP had little observable effect on MecA/ClpCP activity. ClpX bears a Cys4 Zn-binding domain (ZBD), which in other Zn-binding proteins is vulnerable to thiol-reactive electrophiles. Diamide treatment caused partial release of Zn from ClpX and the formation of high-molecular-weight species, as observed by electrophoresis through nonreducing gels. Reduced Spx proteolysis in vitro and elevated Spx concentration in vivo resulted when two of the Zn-coordinating Cys residues of the ClpX ZBD were changed to Ser. This was reflected in enhanced Spx activity in both transcription activation and repression in cells expressing the Cys-to-Ser mutants. ClpXP activity in vivo is reduced when cells are exposed to diamide, as shown by the enhanced stability of an SsrA-tagged protein after treatment with the oxidant. The results are consistent with the hypothesis that inhibition of ClpXP by disulfide stress is due to structural changes to the N-terminal ZBD of ClpX.
Perturbation of Cell Wall Synthesis Suppresses Autolysis inStaphylococcus aureus: Evidence for Coregulation of Cell Wall Synthetic and Hydrolytic Enzymes
Authors: Antignac Aude , Sieradzki Krzysztof , Tomasz Alexander
Published: September 2007

Bacterial peptidoglycan hydrolases are considered to have destructive potential, which in the presence of inhibitory concentrations of cell wall synthesis inhibitors is involved in cell lysis. Therefore, the expression and activity of autolytic enzymes must be tightly regulated in growing cells. We describe here a series of experiments undertaken to examine further the coordination between cell wall synthesis and degradation. Cell growth in the presence of subinhibitory concentrations of β-lactam antibiotics was used to determine the effects of the partial inhibition of cell wall synthesis on the status of the autolytic system inStaphylococcus aureus. Our results revealed that, despite increased in vitro hydrolysis of cell walls by autolytic enzymes due to hypo-cross-linked peptidoglycans, cells grown in the presence of β-lactams were dramatically less prone to autolysis as a result of decreased transcription and enzymatic activities of several major autolytic enzymes. Similar repression of autolytic enzymatic activity and transcription was also observed when cell wall synthesis was disturbed by lowering the level of transcription ofpbpB, the gene encoding the major transpeptidase inS. aureus. Our data show that the perturbation of cell wall synthesis in growing cells ofS. aureusinduces strong repression of the autolytic system and provide evidence for transcriptional regulation between cell wall synthetic and hydrolytic enzymes.
Modulation of Glucose Transport Causes Preferential Utilization of Aromatic Compounds in
Pseudomonas putida
CSV86
Authors: Basu Aditya , Shrivastava Rahul , Basu Bhakti , Apte Shree K. , Phale Prashant S.
Published: September 2007

Pseudomonas putida
CSV86 utilizes aromatic compounds in preference to glucose and coutilizes aromatics and organic acids. Protein analysis of cells grown on different carbon sources, either alone or in combination, revealed that a 43-kDa periplasmic-space protein was induced by glucose and repressed by aromatics and succinate. Two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis identified this protein as closely resembling the sugar ABC transporter of
Pseudomonas putida
KT2440. A partially purified 43-kDa protein showed glucose binding activity and was specific for glucose. The results demonstrate that the aromatic- and organic acid-mediated repression of a periplasmic-space glucose binding protein and consequent inhibition of glucose transport are responsible for this strain's ability to utilize aromatics and organic acids in preference to glucose.
Isolation and Characterization of Unsaturated Fatty Acid Auxotrophs ofStreptococcus pneumoniaeandStreptococcus mutans
Authors: Altabe Silvia , Lopez Paloma , de Mendoza Diego
Published: September 2007

Unsaturated fatty acid (UFA) biosynthesis is essential for the maintenance of membrane structure and function in many groups of anaerobic bacteria. LikeEscherichia coli, the human pathogenStreptococcus pneumoniaeproduces straight-chain saturated fatty acids (SFA) and monounsaturated fatty acids. InE. coliUFA synthesis requires the action of two gene products, the essential isomerase/dehydratase encoded byfabAand an elongation condensing enzyme encoded byfabB. S. pneumoniaelacks both genes and instead employs a single enzyme with only an isomerase function encoded by thefabMgene. In this paper we report the construction and characterization of anS. pneumoniae708fabMmutant. This mutant failed to grow in complex medium, and the defect was overcome by addition of UFAs to the growth medium.S. pneumoniae fabMmutants did not produce detectable levels of monounsaturated fatty acids as determined by gas chromatography-mass spectrometry and thin-layer chromatography analysis of the radiolabeled phospholipids. We also demonstrate that afabMnull mutant of the cariogenic organismStreptococcus mutantsis a UFA auxotroph, indicating that FabM is the only enzyme involved in the control of membrane fluidity in streptococci. Finally we report that thefabNgene ofEnterococcus faecalis, coding for a dehydratase/isomerase, complements the growth ofS. pneumoniae fabMmutants. Taken together, these results suggest that FabM is a potential target for chemotherapeutic agents against streptococci and thatS. pneumoniaeUFA auxotrophs could help identify novel genes encoding enzymes involved in UFA biosynthesis.
Implicit Social Cognition and Law
Authors: Lane Kristin A. , Kang Jerry , Banaji Mahzarin R.
Published: September 2007

Experimental psychology has provided substantial evidence that the human mind can operate in automatic, uncontrollable fashion as well as without conscious awareness of its workings and the sources of influence on it. With methods available to measure implicit or less conscious aspects of social cognition, especially group-specific attitudes and stereotypes, several aspects of the nature of implicit social cognition are now regarded as well established. Such results primarily include the pervasive and robust implicit favoritism for one's own groups and socially dominant groups, the dissociation between implicit and explicit social cognition, the ability of both to predict behavior, the greater impact of the former on certain discriminatory behaviors, and the sensitivity of seemingly implicit thoughts, feelings, and behaviors to change in response to situational features and experience. Legal scholarship and judicial opinions are beginning to consider how the law can and should adapt to such findings, in particular how they call into question existing assumptions regarding the notion of intent, and their relevance for antidiscrimination law.
Flow patterns in three-dimensional porcine epicardial coronary arterial tree
Authors: Huo Yunlong , Wischgoll Thomas , Kassab Ghassan S.
Published: September 2007

The branching pattern of epicardial coronary arteries is clearly three-dimensional, with correspondingly complex flow patterns. The objective of the present study was to perform a detailed hemodynamic analysis using a three-dimensional finite element method in a left anterior descending (LAD) epicardial arterial tree, including main trunk and primary branches, based on computed tomography scans. The inlet LAD flow velocity was measured in an anesthetized pig, and the outlet pressure boundary condition was estimated based on scaling laws. The spatial and temporal wall shear stress (WSS), gradient of WSS (WSSG), and oscillatory shear index (OSI) were calculated and used to identify regions of flow disturbances in the vicinity of primary bifurcations. We found that low WSS and high OSI coincide with disturbed flows (stagnated, secondary, and reversed flows) opposite to the flow divider and lateral to the junction orifice of the main trunk and primary branches. High time-averaged WSSG occurs in regions of bifurcations, with the flow divider having maximum values. Low WSS and high OSI were found to be related through a power law relationship. Furthermore, zones of low time-averaged WSS and high OSI amplified for larger diameter ratio and high inlet flow rate. Hence, different focal atherosclerotic-prone regions may be explained by different physical mechanism associated with certain critical levels of low WSS, high OSI, and high WSSG, which are strongly affected by the diameter ratio. The implications of the flow patterns for atherogenesis are enumerated.
Maturational changes in the regulation of pulmonary vascular tone by nitric oxide in neonatal rats
Authors: Chicoine Louis G. , Paffett Michael L. , Girton Mark R. , Metropoulus Matthew J. , Joshi Mandar S. , Bauer John Anthony , Nelin Leif D. , Resta Thomas C. , Walker Benjimen R.
Published: September 2007

Nitric oxide (NO) is an important regulator of vasomotor tone in the pulmonary circulation. We tested the hypothesis that the role NO plays in regulating vascular tone changes during early postnatal development. Isolated, perfused lungs from 7- and 14-day-old Sprague-Dawley rats were studied. Baseline total pulmonary vascular resistance (PVR) was not different between age groups. The addition of KCl to the perfusate caused a concentration-dependent increase in PVR that did not differ between age groups. However, the nitric oxide synthase (NOS) inhibitor Nω-nitro-l-arginine augmented the K+-induced increase in PVR in both groups, and the effect was greater in lungs from 14-day-old rats vs. 7-day-old rats. Lung levels of total endothelial, inducible, and neuronal NOS proteins were not different between groups; however, the production rate of exhaled NO was greater in lungs from 14-day-old rats compared with those of 7-day-old rats. Vasodilation to 0.1 μM of the NO donor spermine NONOate was greater in 14-day lungs than in 7-day lungs, and lung levels of both soluble guanylyl cyclase and cGMP were greater at 14 days than at 7 days. Vasodilation to 100 μM of the cGMP analog 8-(4-chlorophenylthio)guanosine-3′,5′-cyclic monophosphate was greater in 7-day lungs than in 14-day lungs. Our results demonstrate that the pulmonary vascular bed depends more on NO production to modulate vascular tone at 14 days than at 7 days of age. The observed differences in NO sensitivity may be due to maturational increases in soluble guanylyl cyclase protein levels.
Insulin-like growth factor-1 potentiates platelet activation via the IRS/PI3Kα pathway
Authors: Hers Ingeborg
Published: September 2007

As insulin-like growth factor-1 (IGF-1) is present in the α granules of platelets and its receptor is expressed on the platelet surface, it may contribute to the amplification of platelet responses and pathogenesis of cardiovascular disease. The functional and signaling pathways that are involved in IGF-1 modulation of platelet function, however, are presently unknown. Here, I report that IGF-1 stimulation of platelets results in dose-dependent phosphorylation of the IGF receptor in the range of 1 to 100 nM. Phosphorylation of the IGF receptor is rapid and sustained, with maximal phosphorylation reached within 1 minute. Furthermore, IGF-1 stimulates tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-2 and their association with the p85 subunit of phosphoinositide-3 kinase (PI3K). IGF-1–stimulated tyrosine phosphorylation of IRS-1 and IRS-2 and subsequent p85 binding is transient and precedes phosphorylation of protein kinase B (PKB) on Ser473. PAR-1–mediated platelet aggregation is potentiated by IGF-1 and this potentiation, together with PKB phosphorylation, is abolished by the PI3Kα inhibitors PI-103 and PIK-75. Importantly, the IGF receptor inhibitor NVP-AEW541 and the neutralization antibody αIR3 inhibit SFLLRN-stimulated aggregation, implicating IGF-1 in autocrine regulation of platelet function. These results demonstrate that IGF-1 activates the IGF receptor/IRS/PI3K/PKB pathway, and that PI3Kα is essential for the potentiatory effect of IGF-1 on platelet responses.
Costimulatory blockade of CD154-CD40 in combination with T-cell lymphodepletion results in prevention of allogeneic sensitization
Authors: Xu Hong , Yan Jun , Huang Yiming , Chilton Paula M. , Ding Chuanlin , Schanie Carrie L. , Wang Li , Ildstad Suzanne T.
Published: September 2007

Sensitization is a critical unresolved challenge in transplantation. We show for the first time that blockade of CD154 alone or combined with T-cell depletion prevents sensitization. Allogeneic skin grafts were rejected by recipients treated with anti-αβ T-cell receptor (TCR), anti-CD154, anti-OX40L, or anti–inducible costimulatory pathway (ICOS) mAb alone with a kinetic similar to untreated recipients. However, the production of anti–donor MHC antibody was prevented in mice treated with anti-CD154 mAb only, suggesting a specific role for the CD154-CD40 pathway in B-cell activation. The impairment of T cell–dependent B-cell responses by blocking CD154 occurs through inhibiting activation of T and B cells and secretion of IFN-γ and IL-10. Combined treatment with both anti-CD154 and anti–αβ TCR abrogated antidonor antibody production and resulted in prolonged skin graft survival, suggesting the induction of both T- and B-cell tolerance with prevention of allogeneic sensitization. In addition, we show that the tolerance induced by combined treatment was nondeletional. Moreover, these sensitization-preventive strategies promote bone marrow engraftment in recipients previously exposed to donor alloantigen. These findings may be clinically relevant to prevent allosensitization with minimal toxicity and point to humoral immunity as playing a dominant role in alloreactivity in sensitized recipients.
CD44-mediated phagocytosis induces inside-out activation of complement receptor-3 in murine macrophages
Authors: Vachon Eric , Martin Raiza , Kwok Vivian , Cherepanov Vera , Chow Chung-Wai , Doerschuk Claire M. , Plumb Jonathan , Grinstein Sergio , Downey Gregory P.
Published: September 2007

Diverse receptors, including Fcγ receptors and β2 integrins (complement receptor-3 [CR3], CD11b/CD18), have been implicated in phagocytosis, but their distinct roles and interactions with other receptors in particle engulfment are not well defined. CD44, a transmembrane adhesion molecule involved in binding and metabolism of hyaluronan, may have additional functions in regulation of inflammation and phagocytosis. We have recently reported that CD44 is a fully competent phagocytic receptor that is able to trigger ingestion of large particles by macrophages. Here, we investigated the role of coreceptors and intracellular signaling pathways in modulation of CD44-mediated phagocytosis. Using biotinylated erythrocytes coated with specific antibodies (anti-CD44–coated erythrocytes [Ebabs]) as the phagocytic prey, we determined that CD44-mediated phagocytosis is reduced by 45% by a blocking CD11b antibody. Further, CD44-mediated phagocytosis was substantially (42%) reduced in CD18-null mice. Immunofluorescence microscopy revealed that CD11b is recruited to the phagocytic cup. The mechanism of integrin activation and mobilization involved activation of the GTPase Rap1. CD44-mediated phagocytosis was also sensitive to the extracellular concentration of the divalent cation Mg2+ but not Ca2+. In addition, buffering of intracellular Ca2+ did not affect CD44-mediated phagocytosis. Taken together, these data suggest that CD44 stimulation induces inside-out activation of CR3 through the GTPase Rap1.
Insulin-like growth factor-1 regulates platelet activation through PI3-Kα isoform
Authors: Kim Soochong , Garcia Analia , Jackson Shaun P. , Kunapuli Satya P.
Published: September 2007

Platelets release insulin-like growth factor-1 (IGF-1) from α granules upon activation. We have investigated the regulation of IGF-1 in Gi-dependent pathways leading to Akt activation and the role of IGF-1 in platelet activation. IGF-1 alone failed to induce platelet aggregation, but IGF-1 potentiated 2-MeSADP–induced platelet aggregation in a concentration-dependent manner. IGF-1 triggered platelet aggregation in combination with selective P2Y1 receptor activation. IGF-1 also caused platelet aggregation without shape change when combined with selective Gz stimulation by epinephrine, suggesting the role of IGF-1 in platelet aggregation by supplementing Gi pathways. The potentiating effect of IGF-1 was not affected by intracellular calcium chelation. Importantly, IGF-1 was unable to potentiate platelet aggregation by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, suggesting a critical regulation by PI3-K. Moreover, the potentiating effect of IGF-1 was abolished by the presence of PI3-K p110α inhibitor PIK-75. Stimulation of platelets with IGF-1 resulted in phosphorylation of Akt, a downstream effector of PI3-K, which was completely inhibited by wortmannin. IGF-1-induced Akt phosphorylation was abolished by PIK-75 suggesting the contribution of PI3-K p110α for activation of Akt by IGF-1. These results demonstrate that IGF-1 plays a role in potentiating platelet aggregation by complementing Gi- but not Gq-signaling pathways via PI3-K p110α.
Genetic variation in 1253 immune and inflammation genes and risk of non-Hodgkin lymphoma
Authors: Cerhan James R. , Ansell Stephen M. , Fredericksen Zachary S. , Kay Neil E. , Liebow Mark , Call Timothy G. , Dogan Ahmet , Cunningham Julie M. , Wang Alice H. , Liu-Mares Wen , Macon William R. , Jelinek Diane , Witzig Thomas E. , Habermann Thomas M. , Slager Susan L.
Published: September 2007

Smaller-scale evaluations suggest that common genetic variation in candidate genes related to immune function may predispose to the development of non-Hodgkin lymphoma (NHL). We report an analysis of variants within genes associated with immunity and inflammation and risk of NHL using a panel of 9412 single-nucleotide polymorphisms (SNPs) from 1253 genes in a study of 458 patients with NHL and 484 frequency-matched controls. We modeled haplotypes and risk of NHL, as well as the main effects for all independent SNPs from a gene in multivariate logistic regression models; we separately report results for nonsynonymous (ns) SNPs. In gene-level analyses, the strongest findings (P ≤ .001) were for CREB1, FGG, MAP3K5, RIPK3, LSP1, TRAF1, DUSP2, and ITGB3. In nsSNP analyses, the strongest findings (P ≤ .01) were for ITGB3 L59P (odds ratio [OR] = 0.66; 95% confidence interval [CI] 0.52-0.85), TLR6 V427A (OR = 5.20; CI 1.77-15.3), SELPLG M264V (OR = 3.20; CI 1.48-6.91), UNC84B G671S (OR = 1.50; CI 1.12-2.00), B3GNT3 H328R (OR = 0.74; CI 0.59-0.93), and BAT2 V1883L (OR = 0.64; CI 0.45-0.90). Our results suggest that genetic variation in genes associated with immune response (TRAF1, RIPK3, BAT2, and TLR6), mitogen-activated protein kinase (MAPK) signaling (MAP3K5, DUSP2, and CREB1), lymphocyte trafficking and migration (B3GNT3, SELPLG, and LSP1), and coagulation pathways (FGG and ITGB3) may be important in the etiology of NHL, and should be prioritized in replication studies.
A prospective study of G-CSF–primed bone marrow as a stem-cell source for allogeneic bone marrow transplantation in children: a Pediatric Blood and Marrow Transplant Consortium (PBMTC) study
Authors: Frangoul Haydar , Nemecek Eneida R. , Billheimer Dean , Pulsipher Michael A. , Khan Shakila , Woolfrey Ann , Manes Becky , Cole Catherine , Walters Mark C. , Ayas Mouhab , Ravindranath Yaddanapudi , Levine John E. , Grupp Stephan A.
Published: September 2007

A prospective multicenter trial was conducted to evaluate the safety and feasibility of granulocyte colony-stimulating factor (G-CSF)–primed bone marrow (G-BM) in children receiving allogeneic bone marrow transplantation (BMT). A total of 42 children with a median age of 9.8 years (range, 0.8-17 years) were enrolled. Donors with median age of 9.2 years (range, 1.1-22 years) received 5 μg/kg per day of subcutaneous G-CSF for 5 consecutive days. BM was harvested on the fifth day. No donor experienced complications related to G-CSF administration or marrow har-vest. Median nucleated (NC) and CD34 cells infused was 6.7 × 108/kg (range, 2.4-18.5 × 108/kg) and 7.4 × 106/kg (range, 2-27.6 × 106/kg), respectively. Neutrophil and platelet engraftment was at a median of 19 days (range, 13-28 days) and 20 days (range, 9-44 days), respectively. A total of 13 (32%) patients developed grade 2 graft-versus-host disease (GVHD), and 5 (13%) of 40 evaluable patients developed chronic GVHD (3 limited and 2 extensive). Higher cell dose was not associated with increased risk of acute or chronic GVHD. Overall survival and event-free survival at 2 years were 81% and 69%, respectively. Collection of G-BM from pediatric donors is safe, and can result in high NC and CD34 cell doses that facilitate engraftment after myeloablative BMT without a discernable increase in the risk of GVHD.
Giant panda BAC library construction and assembly of a 650-kb contig spanning major histocompatibility complex class II region
Authors: Zeng Chang-Jun , Pan Hui-Juan , Gong Shao-Bin , Yu Jian-Qiu , Wan Qiu-Hong , Fang Sheng-Guo
Published: September 2007

Background

Giant panda is rare and endangered species endemic to China. The low rates of reproductive success and infectious disease resistance have severely hampered the development of captive and wild populations of the giant panda. The major histocompatibility complex (MHC) plays important roles in immune response and reproductive system such as mate choice and mother-fetus bio-compatibility. It is thus essential to understand genetic details of the giant panda MHC. Construction of a bacterial artificial chromosome (BAC) library will provide a new tool for panda genome physical mapping and thus facilitate understanding of panda MHC genes.

Results

A giant panda BAC library consisting of 205,800 clones has been constructed. The average insert size was calculated to be 97 kb based on the examination of 174 randomly selected clones, indicating that the giant panda library contained 6.8-fold genome equivalents. Screening of the library with 16 giant panda PCR primer pairs revealed 6.4 positive clones per locus, in good agreement with an expected 6.8-fold genomic coverage of the library. Based on this BAC library, we constructed a contig map of the giant panda MHC class II region from BTNL2 to DAXX spanning about 650 kb by a three-step method: (1) PCR-based screening of the BAC library with primers from homologous MHC class II gene loci, end sequences and BAC clone shotgun sequences, (2) DNA sequencing validation of positive clones, and (3) restriction digest fingerprinting verification of inter-clone overlapping.

Conclusion

The identifications of genes and genomic regions of interest are greatly favored by the availability of this giant panda BAC library. The giant panda BAC library thus provides a useful platform for physical mapping, genome sequencing or complex analysis of targeted genomic regions. The 650 kb sequence-ready BAC contig map of the giant panda MHC class II region from BTNL2 to DAXX, verified by the three-step method, offers a powerful tool for further studies on the giant panda MHC class II genes.
Higher maturity and connective tissue association distinguish resident from ecently generated human tonsil plasma cells
Authors: Medina Francisco , Segundo Carmen , Jiménez-Gómez Gema , González-García Inés , Campos-Caro Antonio , Brieva José A
Published: September 2007

Human plasma cells (PC) are present in cell suspensions obtained from the tonsil by mechanical disaggregation (PCMECH). The present study shows that a collagenase treatment of tonsillar debris remaining after mechanical disaggregation yielded similar proportions of PC (PCCOLL). Moreover, PCMECH were present in suspensions highly enriched in germinal center cells whereas PCCOLL contained most of the IgA-secreting cells, suggesting their predominant location in follicular and parafollicular areas and connective tissue-rich zones such as tonsil subepithelium, respectively. Tonsil PCMECH and PCCOLL shared the phenotype CD38high CD19+ CD20low CD45high, expressed equivalent amounts of PRDI BF1/Blimp-1 transcription factor, and carried similarly mutated IgVH6 genes. However, they differed in several features. 1) PCMECH still expressed the early B cell transcription factor BSAP and were HLA-DRhigh; in contrast, PCCOLL were BSAP−and HLA-DRlow. 2) PCMECH were CD95+ and Bcl-2+/− whereas PCCOLL showed CD95+/− and Bcl-2+ expression; in addition, PCMECH exhibited increased spontaneous apoptosis. 3) The two PC subsets exhibited distinctive adhesion molecule profiles, since PCCOLL expressed higher levels of CD31, CD44, and CD49d, but a lower level of CD11a than PCMECH. These results suggest that PCMECH are recently generated, short-living PC, and PCCOLL constitutes a subset with higher maturity and survival, which resides in connective tissue-rich areas.
Stimulation of angiostatic platelet factor-4 variant (CXCL4L1/PF-4var) versus inhibition of angiogenic granulocyte chemotactic protein-2 (CXCL6/GCP-2) in normal and tumoral mesenchymal cells
Authors: Vandercappellen Jo , Noppen Samuel , Verbeke Hannelien , Put Willy , Conings René , Gouwy Mieke , Schutyser Evemie , Proost Paul , Sciot Raf , Geboes Karel , Opdenakker Ghislain , Van Damme Jo , Struyf Sofie
Published: September 2007

Chemokines affect inflammation and cancer through leukocyte attraction and angiogenesis. Here, we demonstrate that CXCL4L1/platelet factor-4 variant (PF-4var), a highly angiostatic chemokine, is poorly chemotactic for phagocytes and is inducible in monocytes by inflammatory mediators but remained undetectable in macrophages and neutrophils. In addition, CXCL4L1/PF-4var production by mesenchymal tumor cells was evidenced in vitro and in vivo by specific ELISA and immunohistochemistry. CXCL4L1/PF-4var, but not CXCL4/PF-4, was coinduced with the angiogenic chemokine CXCL6/granulocyte chemotactic protein-2 (GCP-2) by cytokines, e.g., IL-1β and IL-17, in sarcoma cells, but not in diploid fibroblasts. Furthermore, the induction of CXCL6/GCP-2 in endothelial cells by IL-1β was enhanced synergistically by TNF-α but inhibited by IFN-γ, which synergized with IL-1β to produce the angiostatic CXCL10/IFN-γ-induced protein-10. These findings indicate that the equilibrium between angiostatic and angiogenic factors during inflammation and tumor progression is rather complex and differs depending on the chemokine, cell type, and stimulus. Selective intervention in the chemokine network may drastically disturb this delicate balance of angiogenesis and tissue repair. Application of angiostatic CXCL4L1/PF-4var without attraction of protumoral phagocytes may be beneficial in cancer therapy.
Dap12 expression in activated microglia from retinoschisin-deficient retina and its PU.1-dependent promoter regulation
Authors: Weigelt Karin , Ernst Wolfgang , Walczak Yana , Ebert Stefanie , Loenhardt Thomas , Klug Maja , Rehli Michael , Weber Bernhard H F , Langmann Thomas
Published: September 2007

Several alterations in the expression of immune-related transcripts were identified recently in the degenerating retina of the retinoschisin knockout (Rs1h−/Y) mouse, including the strong expression of the adaptor protein Dap12. As Dap12 is found in leukocytes, we hypothesized that its disease-related expression may be confined to activated retinal microglia cells. To test this hypothesis, we established a procedure for isolation and culture of retinal microglia cells and performed genome-wide expression profiling from Rs1h−/Y and control microglia. While retaining their activated state in culture, ex vivo microglia expressed high levels of Dap12 and the transcription factor PU.1. The activation-dependent induction of Dap12 was also confirmed in the microglia cell line BV-2 following in vitro stimulation. To examine the transcriptional regulation of Dap12 further, macrophage cell lines were transfected with several Dap12 reporter constructs. Promoter deletion assays and site-directed mutagenesis experiments demonstrated an essential role of evolutionarily conserved PU.1 consensus sites in the proximal −104/+118 Dap12 promoter. In vitro and in vivo binding of PU.1 to this promoter region was demonstrated using EMSA and chromatin immunoprecipitation. Knockdown of PU.1 by RNA interference caused a significant reduction of endogenous Dap12 expression and re-expression, and activation of PU.1 in PU.1−/− progenitor cells induced Dap12 transcription. Taken together, our results indicate that activated microglia from degenerating retinae express high levels of Dap12 and PU.1, and PU.1 controls the myeloid-specific regulation of Dap12 directly and may also play a general role in microglia gene expression during retinal degeneration.
Nose-to-brain delivery of tacrine
Authors: Jogani Viral V , Shah Pranav J , Misra Ambikanandan R , Mishra Pushpa , Mishra Anil Kumar
Published: September 2007

In the treatment of Alzheimer's disease tacrine, a cholinesterase inhibitor, is not the drug of choice due to its low oral bioavailability, extensive hepatic first-pass effect, rapid clearance from the systemic circulation, pronounced hepatotoxicity, and the availability of drugs better than tacrine in the same pharmacological class. Hence, the aim of this investigation was to ascertain the possibility of direct nose-to-brain delivery of tacrine to improve bioavailability, to avoid the first-pass effect and to minimize hepatotoxicity. Tacrine solution (TS) in propylene glycol was radiolabelled with 99mTc (technetium) and administered in BALB/c mice intranasally (i.n.) and intravenously (i.v.). Drug concentrations in blood and brain were determined at predetermined time intervals post dosing. Drug targeting efficiency (DTE %) and the brain drug direct transport percentage (DTP %) were calculated to evaluate the brain targeting efficiency. Brain scintigraphy imaging in rabbits was performed to ascertain the uptake of the drug into the brain. Tacrine solution was effectively labelled with 99mTc and was found to be stable and suitable for in-vivo studies. Following intranasal administration tacrine was delivered quickly (Tmax 60 min) to the brain compared with intravenous administration (Tmax 120 min). The brain/blood ratios of the drug were found to be higher for [99mTc]TSi.n. compared with [99mTc]TSi.v. at all time points. The DTE (207.23%) and DTP (51.75%) following intranasal administration suggested that part of tacrine was directly transported to brain from the nasal cavity. Rabbit brain scintigraphy imaging showed higher uptake of the drug into the brain following intranasal administration compared with intravenous administration. The results showed that tacrine could be directly transported into the brain from the nasal cavity and intranasal administration resulted in higher bioavailability of drug with reduced distribution into non-targeted tissues. This selective localization of tacrine in the brain may be helpful in reducing dose, frequency of dosing and dose-dependent side effects, and may prove an interesting new approach in delivery of the drug to the brain for the treatment of Alzheimer's disease.
The protective role of Kangen-karyu against fructose-induced metabolic syndrome in a rat model
Authors: Yokozawa Takako , Kim Hyun Ju , Yamabe Noriko , Okamoto Takuya , Cho Eun Ju
Published: September 2007

The protective effect of Kangen-karyu extract and its mechanisms against fructose-induced metabolic syndrome have been investigated using a rat model. Male Wistar rats were fed a high fructose (65%) diet or standard chow for one week, and for two subsequent weeks were treated with 50 or 100 mg kg−1 body weight/day Kangen-karyu extract or vehicle. Serum glucose, glycosylated protein, triglyceride (TG), total cholesterol, and blood pressure levels of high-fructose-fed rats were increased compared with those of normal rats. However, Kangen-karyu extract ameliorated the high-fructose-induced metabolic syndrome including hyperglycaemia and hypertriglyceridaemia. In addition, the increase of hepatic TG content in rats given the high fructose diet was significantly inhibited with the regulation of sterol regulatory element-binding protein (SREBP)-1 expression by Kangen-karyu extract. On the other hand, peroxisome proliferator-activated receptor α and SREBP-2 protein levels were not affected by the feeding of the high fructose diet or Kangen-karyu extract. Moreover, Kangen-karyu extract administration to high-fructose-fed rats markedly reduced the thiobarbituric acid-reactive substance levels in serum, hepatic homogenate, and mitochondria. Furthermore, it inhibited the increase of cyclooxygenase (COX)-2 with the regulation of nuclear factor-kappa B (NF-κB) and bcl-2 proteins in the liver, suggesting that the protective potential of Kangenkaryu extract against metabolic syndrome would be attributed to the regulation of COX-2, NF-κB, and bcl-2 signalling pathways. This study indicated that Kangen-karyu extract significantly improved high-fructose-induced metabolic syndrome such as hyperglycaemia, hyperlipidaemia, and hypertension through the reductions of TG and cholesterol contents with the regulation of hepatic SREBP-1 protein and the NF-κB signalling pathway.
Evaluation of the estrogenic activity of the constituents in the fruits of Vitex rotundifolia L. for the potential treatment of premenstrual syndrome
Authors: Hu Yuan , Hou Ting-Ting , Zhang Qiao-Yan , Xin Hai-Liang , Zheng Han-Chen , Qin Lu-Ping , Rahman Khalid
Published: September 2007

The ethanol extract of the fruits of Vitex rotundifolia (VRE) and its four major compounds (casticin, luteolin, rotundifuran and agnuside) were tested for their estrogen-like activity by using the modified cell proliferation assay (E-SCREEN assessment system), as well as the estrogen receptor (ERα), estrogen receptor-regulated progesterone receptor and pS2 mRNA expression in MCF-7 cells. The results showed that only agnuside and rotundifuran could stimulate the proliferation of MCF-7 cells. These actions were dose dependent (range from 100 nm to 10 μm) and could be significantly inhibited by the specific estrogen receptor antagonist ICI 182,780. The estrogen receptor ERα and the estrogen receptor-regulated progesterone receptor and pS2 mRNA levels were increased by treatment with rotundifuran and agnuside within 24 h, and the effects could be reversed by ICI 182,780. The standardization of the extract and constituents were carried out by means of a high-performance liquid chromatography-fingerprint. It was concluded that VRE and its compounds showed estrogen-like activity and that the estrogenic effects of rotundifuran and agnuside were mediated by the estrogen inducible gene, which may be useful in regulating the hormone levels to treat related diseases. However, further studies are required to assess the physiological significance of VRE in animals and man.
A linear mixed-effects statistical model for in-vivo evaluation of recombinant human growth hormone implants in hypophysectomized rats
Authors: Santoveña A , García J T , Fariña J B , Llabrés M
Published: September 2007

We have used a linear mixed-effects statistical model to evaluate previously published results of body weight evolution in hypophysectomized (Hpx) rats after administration of two different controlled-release formulations of recombinant human growth hormone (rhGH). Using the linear mixed-effects model, it was possible to distinguish between maximal pharmacological response with time in different subjects and relate it to the structure of the different formulations, the release of the hormone from them and the time necessary to obtain a quantitative result as a consequence of the hormone activity, contrary to the multivariate variance analysis model (MANOVA) used in our earlier work. These results confirmed that the maximum body weight gain obtained with the controlled-release implants is similar to that with subcutaneous rhGH, but with the advantage that laminar biodegradable implants need to be administered only once every 2 weeks.
Adolescents’ Discussions About Contraception or STDs with Partners Before First Sex
Authors: Ryan Suzanne , Franzetta Kerry , Manlove Jennifer , Holcombe Emily
Published: September 2007

CONTEXT: Limited research has examined the individual, family and relationship factors that determine whether teenagers discuss contraception or STDs with their sexual partners before having sex for the first time.

METHODS: Logistic regression analyses of data from 1,426 teenagers who participated in Waves 1 (1995) and 2 (1996) of the National Longitudinal Study of Adolescent Health and who had first sex between waves examined factors influencing whether respondents discussed contraception or STDs with their partner before first sex, and whether these factors differ by gender.

RESULTS: Fifty‐three percent of females and 45% of males discussed contraception or STDs before having first sex. The greater respondents’ perceived condom knowledge and the greater their communication with their parents about everyday life, the higher their odds of discussing contraception or STDs before first sex (odds ratio, 1.2 for each). Being black was positively associated with sexual communication before first sex (1.9); as the number of dating activities and score on a test of verbal ability increased, so did the odds of such communication (1.6 and 1.02, respectively). The predictors of discussions about contraception or STDs did not differ by gender.

CONCLUSIONS: By increasing teenagers’ knowledge about condoms and other methods of contraception, pregnancy and STD prevention programs can help to encourage communication among teenage partners before the initiation of sexual intercourse. Programs should also encourage conversations between parents and teenagers, even when not about sex.
California Parents’ Preferences and Beliefs Regarding School‐Based Sex Education Policy
Authors: Constantine Norman A. , Jerman Petra , Huang Alice X.
Published: September 2007

CONTEXT:  Policy debates over the merits of abstinence‐only versus comprehensive approaches to sex education are ongoing, despite well‐documented public support for comprehensive sex education. Although parents are key stakeholders in the outcomes of these debates, their views have been less thoroughly considered.

METHODS:  A random digit dial survey of 1,284 California parents was conducted in 2006. Parents were asked about their sex education policy preferences, the importance of teaching selected topics at different grade levels and reasons for their preferences. Cross‐tabulations and odds ratios were used to assess regional and other subgroup differences.

RESULTS:  Overall, 89% of parents reported a preference for comprehensive sex education, and 11% for abstinence‐only education. Support for comprehensive sex education was high in all regions (87–93%) and across all subgroup characteristics: race or ethnicity (79–92%), age (86–94%), education (84–93%), household income (87–92%), religious affiliation (86–91%), religious service attendance (69–96%) and ideological leaning (71–96%). Four types of reasons for preferences emerged: those focused on the consequences of actions, on the importance of providing complete information, on the inevitability of adolescents’ engaging in sex and on religious or purity‐based morality concerns. While 64% of abstinence‐only supporters cited the last type (absolutist reasons), 94% of comprehensive sex education supporters cited one of the first three (pragmatic reasons).

CONCLUSIONS:  The high levels of support for comprehensive sex education across California’s diverse regions and demographic subgroups suggest that such support may be generalizable to communities and school districts both in California and around the country. Furthermore, ideological differences might be less important to the sex education debates than the distinction between pragmatic and absolutist perspectives.