bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic Modification of the Penicillin G Acylase Surface To Improve Its Reversible Immobilization on Ionic Exchangers
Authors: Montes Tamara , Grazú Valeria , López-Gallego Fernando , Hermoso Juan A. , García Jose L. , Manso Isabel , Galán Beatriz , González Ramón , Fernández-Lafuente Roberto , Guisán José M. 
Published: November 2006

ABSTRACTA new mutant of the industrial enzyme penicillin G acylase (PGA) fromEscherichia colihas been designed to improve its reversible immobilization on anionic exchangers (DEAE- or polyethyleneimine [PEI]-coated agarose) by assembling eight new glutamic residues distributed homogeneously through the enzyme surface via site-directed mutagenesis. The mutant PGA is produced and processed in vivo as is the native enzyme. Moreover, it has a similar specific activity to and shows the same pH activity profile as native PGA; however, its isoelectric point decreased from 6.4 to 4.3. Although the new enzyme is adsorbed on both supports, the adsorption was even stronger when supports were coated with PEI, allowing us to improve the enzyme stability in organic cosolvents. The use of restrictive conditions during the enzyme adsorption on anionic exchangers (pH 5 and high ionic strength) permitted us to still further increase the strength of adsorption and the enzyme stability in the presence of organic solvents, suggesting that these conditions allow the penetration of the enzyme inside the polymeric beds, thus becoming fully covered with the polymer. After the enzyme inactivation, it can be desorbed to reuse the support. The possibility to improve the immobilization properties on an enzyme by site-directed mutagenesis of its surface opens a promising new scenario for enzyme engineering.
Comparative Proteomics of
Dehalococcoides
spp. Reveals Strain-Specific Peptides Associated with Activity
Authors: Morris R. M. , Fung J. M. , Rahm B. G. , Zhang S. , Freedman D. L. , Zinder S. H. , Richardson R. E. 
Published: November 2006

ABSTRACT

Anaerobic reductive dehalogenation by
Dehalococcoides
spp. is an ideal system for studying functional diversity of closely related strains of bacteria. In
Dehalococcoides
spp., reductive dehalogenases (RDases) are key respiratory enzymes involved in the anaerobic detoxification of halogenated compounds at contaminated sites globally. Although housekeeping genes sequenced from
Dehalococcoides
spp. are >85% identical at the amino acid level, different strains are capable of dehalogenating diverse ranges of compounds, depending largely on the suite of RDase genes that each strain harbors and expresses. We identified RDase proteins that corresponded to known functions in four characterized cultures and predicted functions in an uncharacterized
Dehalococcoides
-containing mixed culture. Homologues within RDase subclusters containing PceA, TceA, and VcrA were among the most frequently identified proteins. Several additional proteins, including a formate dehydrogenase-like protein (Fdh), had high coverage in all strains and under all growth conditions.
High Viscosity and Anisotropy Characterize the Cytoplasm of Fungal Dormant Stress-Resistant Spores
Authors: Dijksterhuis J. , Nijsse J. , Hoekstra F. A. , Golovina E. A. 
Published: November 2006

ABSTRACTAscospores of the fungusTalaromyces macrosporusare dormant and extremely stress resistant, whereas fungal conidia—the main airborne vehicles of distribution—are not. Here, physical parameters of the cytoplasm of these types of spores were compared. Cytoplasmic viscosity and level of anisotropy as judged by spin probe studies (electron spin resonance) were extremely high in dormant ascospores and during early germination and decreased only partly after trehalose degradation and glucose efflux. Upon prosilition (ejection of the spore), these parameters fell sharply to values characteristic of vegetative cells. These changes occurred without major volume changes that suggest dramatic changes in cytoplasmic organization. Azide reversibly inhibited prosilition as well as the decline in cytoplasmic parameters. No organelle structures were observed in etched, cryoplaned specimens of ascospores by low-temperature scanning electron microscopy (LTSEM), confirming the high cytoplasmic viscosity. However, cell structures became visible upon prosilition, indicating reduced viscosity. The viscosity of fresh conidia of differentPenicilliumspecies was lower, namely, 3.5 to 4.8 cP, than that of ascospores, near 15 cP. In addition the level of anisotropic motion was markedly lower in these cells (h0/h+1= 1.16 versus 1.4). This was confirmed by LTSEM images showing cell structures. The decline of cytoplasmic viscosity in conidia during germination was linked with a gradual increase in cell volume. These data show that mechanisms of cytoplasm conservation during germination differ markedly between ascospores and conidia.
Chronic Exposure toHelicobacter pyloriImpairs Dendritic Cell Function and Inhibits Th1 Development
Authors: Mitchell Peter , Germain Conrad , Fiori Pier Luigi , Khamri Wafa , Foster Graham R. , Ghosh Subrata , Lechler Robert I. , Bamford Kathleen B. , Lombardi Giovanna 
Published: November 2006

ABSTRACTHelicobacter pyloricauses chronic gastric infection that affects the majority of the world's population. Despite generating an inflammatory response, the immune system usually fails to clear the infection. Since dendritic cells (DCs) play a pivotal role in shaping the immune response, we investigated the effects ofH. pylorion DC function. We have demonstrated thatH. pyloriincreased the expression of activation markers on DCs while upregulating the inhibitory B7 family molecule, PD-L1. Functionally,H. pylori-treated DCs resulted in the production of interleukin-10 (IL-10) and IL-23 but not of alpha interferon (IFN-α). While very little or no IL-12 was produced toH. pylorialone, simultaneous ligation of CD40 on DCs induced IL-12 release. We also demonstrated that DCs treated withH. pylori-induced IFN-γ production by allogeneic naive T cells. However, stimulation of DCs withH. pylorifor an extended period of time impaired their ability to produce cytokines after CD40 ligation and limited their ability to promote IFN-γ release, suggesting that the DCs had become exhausted by the prolonged stimulation. The effect of chronic infection withH. pylorion DC function was further investigated by focusing on DC development. Demonstrating that monocytes differentiated into DCs in the presence ofH. pyloriexhibited an exhausted phenotype with an impaired ability to produce IL-12 and a downregulation of CD1a. Our results raise the possibility that in chronicH. pyloriinfection DCs become exhausted after prolonged antigen exposure leading to suboptimal Th1 development. This effect may contribute to persistence ofH. pyloriinfection.
Murine Immune Responses to Liver-Stage Antigen 1 Protein FMP011, a Malaria Vaccine Candidate, Delivered with Adjuvant AS01B or AS02A
Authors: Brando Clara , Ware Lisa A. , Freyberger Helen , Kathcart April , Barbosa Arnoldo , Cayphas Sylvie , Demoitie Marie-Ange , Mettens Pascal , Heppner D. Gray , Lanar David E. 
Published: November 2006

ABSTRACTLiver-stage antigen 1 (LSA1) is expressed byPlasmodium falciparumonly during the intrahepatic cell stage of the parasite's development. Immunoepidemiological studies in regions where malaria is endemic suggested an association between the level of LSA1-specific humoral and cell-mediated immune responses and susceptibility to clinical malaria. A recombinant LSA1 protein, FMP011, has been manufactured as a preerythrocytic vaccine to induce an immune response that would have the effect of controlling parasitemia and disease in humans. To evaluate the immunogenicity of FMP011, we analyzed the immune response of three inbred strains of mice to antigen immunization using two different adjuvant formulations, AS01B and AS02A. We report here the ability of BALB/c and A/J mice, but not C57BL/6J mice, to mount FMP011-specific humoral (antibody titer) and cellular (gamma interferon [IFN-γ] production) responses following immunization with FMP011 formulated in AS01B or AS02A. Immunization of BALB/c and A/J mice with FMP011/AS01B induced more antigen-specific IFN-γ-producing splenocytes than immunization with FMP011/AS02A. A slightly higher titer of antibody was induced using AS02A than AS01B in both strains. C57BL/6J mice did not respond with any detectable FMP011-specific IFN-γ splenocytes or antibody when immunized with FMP011 in AS01B or AS02A. Intracellular staining of cells isolated from FMP011/AS01B-immunized BALB/c mice indicated that CD4+cells, but not CD8+cells, were the main IFN-γ-producing splenocyte. However, inclusion of blocking anti-CD4+antibody during the in vitro restimulation ELISpot analysis failed to completely abolish IFN-γ production, indicating that while CD4+T cells were the major source of IFN-γ, other cell types also were involved.
Iron-Responsive Repression of Urease Expression in
Helicobacter hepaticus
Is Mediated by the Transcriptional Regulator Fur
Authors: Belzer Clara , van Schendel Bart A. M. , Kuipers Ernst J. , Kusters Johannes G. , van Vliet Arnoud H. M. 
Published: November 2006

ABSTRACT

Persistent colonization of mucosal surfaces by bacteria in the mammalian host requires concerted expression of colonization factors, depending on the environmental conditions.
Helicobacter hepaticus
is a urease-positive pathogen that colonizes the intestinal and hepatobiliary tracts of rodents. Here it is reported that urease expression of
H. hepaticus
is iron repressed by the transcriptional regulator Fur. Iron restriction of growth medium resulted in a doubling of urease activity in wild-type
H. hepaticus
strain ATCC 51449 and was accompanied by increased levels of urease subunit proteins and
ureA
mRNA. Insertional inactivation of the
fur
gene abolished iron-responsive repression of urease activity, whereas inactivation of the
perR
gene did not affect iron-responsive regulation of urease activity. The iron-responsive promoter element was identified directly upstream of the
H. hepaticus ureA
gene. Recombinant
H. hepaticus
Fur protein bound to this
ureA
promoter region in a metal-dependent matter, and binding resulted in the protection of a 41-bp, Fur box-containing operator sequence located at positions −35 to −75 upstream of the transcription start site. In conclusion,
H. hepaticus
Fur controls urease expression at the transcriptional level in response to iron availability. This represents a novel type of urease regulation in ureolytic bacteria and extends the already diverse regulatory repertoire of the Fur protein.
Identification of
Helicobacter pylori
Genes That Contribute to Stomach Colonization
Authors: Baldwin David N. , Shepherd Benjamin , Kraemer Petra , Hall Michael K. , Sycuro Laura K. , Pinto-Santini Delia M. , Salama Nina R. 
Published: November 2006

ABSTRACT

Chronic infection of the human stomach by
Helicobacter pylori
leads to a variety of pathological sequelae, including peptic ulcer and gastric cancer, resulting in significant human morbidity and mortality. Several genes have been implicated in disease related to
H. pylori
infection, including the vacuolating cytotoxin and the
cag
pathogenicity island. Other factors important for the establishment and maintenance of infection include urease enzyme production, motility, iron uptake, and stress response. We utilized a C57BL/6 mouse infection model to query a collection of 2,400 transposon mutants in two different bacterial strain backgrounds for
H. pylori
genetic loci contributing to colonization of the stomach. Microarray-based tracking of transposon mutants allowed us to monitor the behavior of transposon insertions in 758 different gene loci. Of the loci measured, 223 (29%) had a predicted colonization defect. These included previously described
H. pylori
virulence genes, genes implicated in virulence in other pathogenic bacteria, and 81 hypothetical proteins. We have retested 10 previously uncharacterized candidate colonization gene loci by making independent null alleles and have confirmed their colonization phenotypes by using competition experiments and by determining the dose required for 50% infection. Of the genetic loci retested, 60% have strain-specific colonization defects, while 40% have phenotypes in both strain backgrounds for infection, highlighting the profound effect of
H. pylori
strain variation on the pathogenic potential of this organism.
The
Legionella pneumophila
Effector SidJ Is Required for Efficient Recruitment of Endoplasmic Reticulum Proteins to the Bacterial Phagosome
Authors: Liu Yancheng , Luo Zhao-Qing 
Published: November 2006

ABSTRACT

The virulence of
Legionella pneumophila
is dependent on the Dot/Icm type IV protein secretion system, which translocates effectors into infected cells. A large number of such translocated proteins have been identified, but few of these proteins are necessary for intracellular replication of the pathogen, making it difficult to correlate these genes with specific cell-biological events associated with
L. pneumophila
infection. We report here the identification and characterization of a family of two substrates, SidJ and SdjA, with distinctive phenotypes. In contrast to many Dot/Icm substrates, whose expression levels are elevated when bacteria are grown to postexponential phase, SidJ is produced at a constant rate during the entire bacterial growth cycle. Mutation in
sidJ
causes a significant growth defect in both macrophage and amoeba hosts, but an
sdjA
mutant is detectably defective only in protozoan hosts. However, in the amoeba host a mutant lacking both
sidJ
and
sdjA
does not display a more severe growth defect than the
sidJ
mutant. Despite its significant intracellular growth defect, the
sidJ
mutant is still able to effectively evade fusion with lysosomes. Importantly, recruitment of endoplasmic reticulum (ER) proteins by vacuoles containing the
sidJ
mutant was considerably delayed in both mammalian and amoeba cells. Our results suggest that SidJ modulates host cellular pathways, contributing to the trafficking or retention of ER-derived vesicles to
L. pneumophila
vacuoles.
Identification of the Gene Encoding the FhbB Protein ofTreponema denticola, a Highly Unique Factor H-Like Protein 1 Binding Protein
Authors: McDowell John V. , Frederick Jesse , Stamm Lola , Marconi Richard T. 
Published: November 2006

ABSTRACTThe gene encoding theTreponema denticolafactor H-like protein 1 (FHL-1) binding protein, FhbB, was recovered and characterized. Sequence conservation, expression, and properties of FhbB were analyzed. The identification of FhbB represents an important step in understanding the contribution of FHL-1 binding inT. denticolapathogenesis and in development of periodontal disease.
TccP2 of O157:H7 and Non-O157 Enterohemorrhagic
Escherichia coli
(EHEC): Challenging the Dogma of EHEC-Induced Actin Polymerization
Authors: Ogura Yoshitoshi , Ooka Tadasuke , Whale Andrew , Garmendia Junkal , Beutin Lothar , Tennant Sharon , Krause Gladys , Morabito Stefano , Chinen Isabel , Tobe Toru , Abe Hiroyuki , Tozzoli Rosangela , Caprioli Alfredo , Rivas Marta , Robins-Browne Roy , Hayashi Tetsuya , Frankel Gad 
Published: November 2006

ABSTRACT

Enterohemorrhagic
Escherichia coli
(EHEC) O157:H7 and enteropathogenic
E. coli
(EPEC) trigger actin polymerization at the site of bacterial adhesion by inducing different signaling pathways. Actin assembly by EPEC requires tyrosine phosphorylation of Tir, which subsequently binds the host adaptor protein Nck. In contrast, Tir
EHEC O157
is not tyrosine phosphorylated and instead of Nck utilizes the bacterially encoded Tir-cytoskeleton coupling protein (TccP)/EspF
U
, which mimics the function of Nck.
tccP
is carried on prophage CP-933U/Sp14 (TccP). Typical isolates of EHEC O157:H7 harbor a pseudo-
tccP
gene that is carried on prophage CP-933 M/Sp4 (
tccP2
). Here we report that atypical, β-glucuronidase-positive and sorbitol-fermenting, strains of EHEC O157 harbor intact
tccP
and
tccP2
genes, both of which are secreted by the LEE-encoded type III secretion system. Non-O157 EHEC strains, including O26, O103, O111, and O145, are typically
tccP
negative and translocate a Tir protein that encompasses an Nck binding site. Unexpectedly, we found that most clinical non-O157 EHEC isolates carry a functional
tccP2
gene that encodes a secreted protein that can complement an EHEC O157:H7 Δ
tccP
mutant. Using discriminatory, allele-specific PCR, we have demonstrated that over 90% of
tccP2
-positive non-O157 EHEC strains contain a Tir protein that can be tyrosine phosphorylated. These results suggest that the TccP pathway can be used by both O157 and non-O157 EHEC and that non-O157 EHEC can also trigger actin polymerization via the Nck pathway.
Endogenous CD4+CD25+Regulatory T Cells Have a Limited Role in the Control ofTrypanosoma cruziInfection in Mice
Authors: Kotner Joshua , Tarleton Rick 
Published: November 2006

ABSTRACTInfection with the protozoan parasiteTrypanosoma cruziresults in a robust and multifaceted immune response that controls parasite load but is unable to completely clear infection, resulting in parasite persistence and a chronic illness known as Chagas' disease in humans. The severity of Chagas' disease is correlated with persistent parasitism of muscle, neuronal, and gut tissues. The natural immunomodulatory function of endogenous CD4+CD25+regulatory T cells (Treg cells) to limit hyperactive immune responses may be exploited by microbes to persist despite host responses. In this study, we show that Treg cells are not necessary forT. cruzievasion of immune responses during acute or chronic infection. In vivo anti-CD25 monoclonal antibody-mediated depletion of Treg cells from mice prior to challenge with a lethal strain or prior to and during acute infection with a nonlethal strain of parasite neither improved nor worsened the outcome of immune responses: differences in parasitemia, kinetics of antigen-specific CD8+T-cell expansion, and CD8+T-cell effector function (both in vivo and ex vivo) were of similar magnitudes for both depleted and control groups. Furthermore, depletion of CD25+cells from chronically infected mice did not enhance immune responses of muscle-derived CD8+T cells, nor could FoxP3 mRNA/scurfin-expressing leukocytes be isolated from muscle tissue. Based on the results of this study, it is concluded that Treg cells do not appear to play a major role in regulating CD8+T-cell effector responses during the acute phase of infection or in the muscles of mice during chronicT. cruziinfection.
Streptococcus pneumoniaeDivIVA: Localization and Interactions in a MinCD-Free Context
Authors: Fadda Daniela , Santona Antonella , D'Ulisse Valeria , Ghelardini Patrizia , Ennas Maria Grazia , Whalen Michael B. , Massidda Orietta 
Published: November 2006

ABSTRACTTo clarify the function of DivIVA inStreptococcus pneumoniae, we localized this protein in exponentially growing cells by both immunofluorescence microscopy and immunoelectron microscopy and found thatS. pneumoniaeDivIVA (DivIVASPN) had a unique localization profile: it was present simultaneously both as a ring at the division septum and as dots at the cell poles. Double-immunofluorescence analysis suggested that DivIVA is recruited to the septum at a later stage than FtsZ and is retained at the poles after cell separation. All the other cell division proteins that we tested were localized in thedivIVAnull mutant, although the percentage of cells having constricted Z rings was significantly reduced. In agreement with its localization profile and consistent with its coiled-coil nature, DivIVA interacted with itself and with a number of known or putativeS. pneumoniaecell division proteins. Finally, a missensedivIVAmutant, obtained by allelic replacement, allowed us to correlate, at the molecular level, the specific interactions and some of the facets of thedivIVAmutant phenotype. Taken together, the results suggest that although the possibility of a direct role in chromosome segregation cannot be ruled out, DivIVA inS. pneumoniaeseems to be primarily involved in the formation and maturation of the cell poles. The localization and the interaction properties of DivIVASPNraise the intriguing possibility that a common, MinCD-independent function evolved differently in the various host backgrounds.
Functional Analysis ofluxSin the Probiotic StrainLactobacillus rhamnosusGG Reveals a Central Metabolic Role Important for Growth and Biofilm Formation
Authors: Lebeer Sarah , De Keersmaecker Sigrid C. J. , Verhoeven Tine L. A. , Fadda Abeer A. , Marchal Kathleen , Vanderleyden Jos 
Published: November 2006

ABSTRACTQuorum sensing is involved in the regulation of multicellular behavior through communication via small molecules. Given the high number and diversity of the gastrointestinal microbiota, it is postulated that members of this community communicate to coordinate a variety of adaptive processes. AI-2 is suggested to be a universal bacterial signaling molecule synthesized by the LuxS enzyme, which forms an integral part of the activated methyl cycle. We have previously reported that the well-documented probiotic strainLactobacillus rhamnosusGG, a human isolate, produces AI-2-like molecules. In this study, we identified theluxShomologue ofL. rhamnosusGG.luxSseems to be located in an operon with ayxjHgene encoding a putative cobalamin-independent methionine synthase. In silico analysis revealed a methionine-specific T box in the leader sequence of the putativeyxjH-luxSoperon. However, transcriptional analysis showed thatluxSis expressed mainly as a monocistronic transcript. Construction of aluxSknockout mutant confirmed that theluxSgene is responsible for AI-2 production inL. rhamnosusGG. However, this mutation also resulted in pleiotropic effects on the growth of this fastidious strain. Cysteine, pantothenate, folic acid, and biotin could partially complement growth, suggesting a central metabolic role forluxSinL. rhamnosusGG. Interestingly, theluxSmutant also showed a defect in monospecies biofilm formation. Experiments with chemically synthesized (S)-4,5-dihydroxy-2,3-pentanedione, coculture with the wild type, and nutritional complementation suggested that the main cause of this defect has a metabolic nature. Moreover, our data indicate that suppressor mutations are likely to occur inluxSmutants ofL. rhamnosusGG. Therefore, results ofluxS-related studies should be carefully interpreted.
Testing for thrombophilia: an evidence-based approach
Authors: Merriman L , Greaves M 
Published: November 2006

AbstractThrombophilia is a disorder of haemostasis in which there is a tendency for the occurrence of thrombosis. This tendency can be inherited or acquired. This review outlines common acquired and inherited thrombophilic conditions and discusses indications for testing. It is concluded that testing for acquired thrombophilic conditions should be considered in all cases of venous thrombosis, whereas testing for inherited thrombophilic conditions is unlikely to be helpful. If testing for inherited thrombophilia is to be carried out, the benefits, pitfalls and unwanted consequences of such testing should be taken into account.
Robotic technology in urology
Authors: Murphy D , Challacombe B , Khan M S , Dasgupta P 
Published: November 2006

Abstract
Urology has increasingly become a technology-driven specialty. The advent of robotic surgical systems in the past 10 years has led to urologists becoming the world leaders in the use of such technology. In this paper, we review the history and current status of robotic technology in urology. From the earliest uses of robots for transurethral resection of the prostate, to robotic devices for manipulating laparoscopes and to the current crop of master–slave devices for robotic-assisted laparoscopic surgery, the evolution of robotics in the urology operating theatre is presented. Future possibilities, including the prospects for nanotechnology in urology, are awaited.