bluuebunny/crossref_metadata_2025 huggingface dataset:
Verbal and Visuospatial Short‐Term and Working Memory in Children: Are They Separable?
Authors: Alloway Tracy Packiam , Gathercole Susan Elizabeth , Pickering Susan J.
Published: November 2006

This study explored the structure of verbal and visuospatial short‐term and working memory in children between ages 4 and 11 years. Multiple tasks measuring 4 different memory components were used to capture the cognitive processes underlying working memory. Confirmatory factor analyses indicated that the processing component of working memory tasks was supported by a common resource pool, while storage aspects depend on domain‐specific verbal and visuospatial resources. This model is largely stable across this developmental period, although some evidence exists that the links between the domain‐specific visuospatial construct and the domain‐general processing construct were higher in the 4‐ to‐ 6‐year age group. The data also suggest that all working memory components are in place by 4 years of age.
Sexual Offences in Police Reports and Court Dossiers: A Case‐File Study
Authors: Van Den Bergh Peter M. , Hoekman Joop
Published: November 2006

Background  In many cases, sexual abuse involving a person with an intellectual disability (ID; as a victim and/or as a perpetrator) is not reported to the police. There are very few studies addressing police procedures in these cases, and even fewer addressing procedures at the Public Prosecutor level. The present study had two goals: (i) to describe relevant characteristics of cases of sexual abuse, involving people with ID (as a victim or perpetrator), that were either reported to the police or sent to the Public Prosecutor; (ii) to describe the dynamics of the process from making the report to passing the sentence.

Method  In this case‐file research, 75 police reports and dossiers at the Public Prosecutor, regarding sexual abuse were followed from the initial report to the police, up to the conviction.

Results  Even if a case was reported to the police, it did not always go forward to the Public Prosecutor and when a person with ID was involved as the perpetrator, few were actually convicted.

Conclusions  Whether the perpetrator had an ID did not appear to be a decisive factor in furnishing proof or in the conviction. There seemed to be no evidence for any specific disability‐related influence on the judicial process in cases against perpetrators of sexual abuse. Differentiating police data from court data, however, revealed an important finding. Relatives appeared in court dossiers far more than in police files (34% versus 14%), while on the contrary staff made up 24% in police files and only 13% in court dossiers. This suggested that different sensitivities, protecting staff compared with relatives, may be present in the judicial process, rather than in services.
Parents’ Use of Physical Interventions in the Management of Their Children's Severe Challenging Behaviour
Authors: Allen David , Hawkins Sarah , Cooper Viv
Published: November 2006

Background  Although training staff supporting people with challenging behaviour in physical interventions has become accepted practice, parents are often left to fend for themselves while managing equivalent behaviours. The study explores parents’ experience of managing severe challenging behaviours, their use of physical interventions and access to training in reactive strategies.

Method  A postal survey methodology was employed that utilized a novel 20‐item questionnaire. The questionnaire yielded both quantitative and qualitative data concerning the types of challenging behaviour displayed by the children, frequency of use and nature of physical interventions, types of training received and any difficulties experienced in obtaining training. The sample was drawn from the membership of a national charitable organization and comprised of 72 respondents.

Results  The majority of respondents had experienced major episodes of challenging behaviour. The emotional impact of living with such behaviours was apparent. Although the vast majority of respondents had employed restrictive physical interventions, few had received any formal training in such strategies. There was evidence that a number of high‐risk physical interventions were sometimes being employed. Accessing training appeared more problematic for parents when compared with paid care staff.

Conclusions  In this specific sample, parental use of physical intervention was high while parent training in physical intervention was uncommon. Impediments to parent training in physical intervention are discussed, as are wider issues concerning parental support.
The Prevalence of Psychiatric Disorders among People with Intellectual Disabilities: An Analysis of the Literature
Authors: Whitaker Simon , Read Stephen
Published: November 2006

Background  It has often been stated that the prevalence of psychiatric disorders in people with intellectual disabilities is greater than it is in the population as a whole.

Method  The epidemiological studies on psychiatric disorders in people with intellectual disabilities were reviewed.

Results  There is evidence that the prevalence of psychiatric disorder is greater in children with intellectual disabilities, compared with children with normal IQs, that it is higher in both adults and children with severe intellectual disabilities compared with people with mild or no intellectual disability and that the rate of problematic behaviour is higher in both adults and children having intellectual disabilities, than in their non‐disabled peers.

Conclusions  There is no sound evidence that the prevalence of psychiatric disorders in adults with mild intellectual disability is greater than in the population as a whole.
The Tissue Fixation System provides a new structural method for cystocoele repair: A preliminary report
Authors: PETROS Peter E. P. , RICHARDSON Peter A. , GOESCHEN Klaus , ABENDSTEIN Burghard
Published: November 2006

Background:  A new reconstruction principle that uses tensioned tapes instead of large mesh is described for cystocoele repair.

Aim:  To apply this method to patients with central, paravaginal and cervical ring defects.

Methods:  Ninety patients, mean age 63 years (29–83) and mean weight 73 kg (52–117 kg), underwent cystocoele repair using the Tissue Fixation System (TFS). Tapes were applied as a retro‐obturator U‐sling (n = 29), transversely between both arcus tendineus fascia pelvis (ATFP) ligaments (n = 45), along the path of the cardinal ligament in patients with cervical ring defects (n = 12), and longitudinally along the ATFP ligament (n = 4).

Results:  At mean eight months review (three to 15 months), two failures were reported. There was one haematoma that drained spontaneously at seven days, and there were no erosions. Mean hospital stay was one a half days for the Australian group (one to seven days) and five days (four to eight days) for the European group. After using single U‐sling, one patient required intermittent catheterisation for seven days before she could pass urine freely. In one patient the bladder was perforated during dissection laterally towards the ATFP; the perforation was successfully repaired.

Conclusions:  The tensioned tape operation is simple and accurate, and appears to work well in the short term. Longer‐term studies are required.
Serum advanced oxidation protein products, myeloperoxidase and ascorbic acid in pre‐eclampsia and eclampsia
Authors: NOYAN Tevfik , GÜLER Ayşe , ŞEKEROĞLU Mehmet Ramazan , KAMACI Mansur
Published: November 2006

Background:  Activation products from neutrophils and the complement system might cause endothelial dysfunction, which is central to the aetiology of pre‐eclampsia. This study aimed to investigate the activity of myeloperoxidase (MPO), and its association with advanced oxidation protein products (AOPP), in women with pre‐eclampsia and eclampsia.

Materials and method:  Twenty‐one pregnant women with pre‐eclampsia, 11 pregnant women with eclampsia and 19 healthy pregnant women were studied. Serum levels of malondialdehyde (MDA), AOPP, ascorbic acid (AA) and activities of MPO and catalase (CAT) were measured using a colorimetric method.

Results:  The MDA level was significantly higher in the pre‐eclampsia (3.15 ± 0.28 nmol/mL) and eclampsia (4.01 ± 0.66 nmol/mL) groups than in controls (1.85 ± 0.18 nmol/mL); the difference between MDA levels in the pre‐eclampsia and eclampsia groups was not statistically significant. MPO activity was significantly higher in the eclampsia (347.59 ± 88.06 U/L) group than in the pre‐eclampsia (196.17 ± 30.8) and control (93.22 ± 9.52) groups, and there was also no significant difference in these levels between the pre‐eclampsia and control groups. CAT activity was significantly higher in the pre‐eclampsia (166.35 ± 31.75 U/L) and eclampsia (166.98 ± 40.31 U/L) groups than in controls (81.28 ± 7.41 U/L), and AA level was significantly higher in the pre‐eclampsia (0.54 ± 0.15 mg/dL) group than in controls (0.18 ± 0.01 mg/dL); the differences in AA and CAT activity between the pre‐eclampsia and eclampsia groups were not statistically significant. AOPP levels did not change significantly among the control, pre‐eclampsia and eclampsia groups (106.88 ± 5.62, 98.89 ± 6.47, 111.89 ± 6.8 µmol/L, respectively).

Conclusions:  We suggest that increased oxidative stress might contribute to the pathophysiological mechanisms of pre‐eclampsia and eclampsia, and that AA and CAT might have a protective role via free radical‐scavenging properties. However, further study is needed.
Perinatal outcomes and macrosomia in a multi‐ethnic population of women with type 2 diabetes
Authors: HUGHES Ruth , ROWAN Janet
Published: November 2006

We reviewed pregnancy outcomes of women with type 2 diabetes giving birth over a six‐year period, comparing the main ethnic groups. Asian women had significantly smaller babies and lower rates of macrosomia as defined by standard growth charts. Other outcomes were similar between the Asian, European and Polynesian women.
Dynamic Organization of the Actin Cytoskeleton During Meiosis and Spore Formation in Budding Yeast
Authors: Taxis Christof , Maeder Celine , Reber Simone , Rathfelder Nicole , Miura Kota , Greger Klaus , Stelzer Ernst H. K. , Knop Michael
Published: November 2006

During sporulation in Saccharomyces cerevisiae, the four daughter cells (spores) are formed inside the boundaries of the mother cell. Here, we investigated the dynamics of spore assembly and the actin cytoskeleton during this process, as well as the requirements for filamentous actin during the different steps of spore formation. We found no evidence for a polarized actin cytoskeleton during sporulation. Instead, a highly dynamic network of non‐polarized actin cables is present underneath the plasma membrane of the mother cell. We found that a fraction of prospore membrane (PSM) precursors are transported along the actin cables. The velocity of PSM precursors is diminished if Myo2p or Tpm1/2p function is impaired. Filamentous actin is not essential for meiotic progression, for shaping of the PSMs or for post‐meiotic cytokinesis. However, actin is essential for spore wall formation. This requires the function of the Arp2/3p complex and involves large carbohydrate‐rich compartments, which may be chitosome analogous structures.
The stellar mass spectrum in warm and dusty gas: deviations from Salpeter in the Galactic centre and in circumnuclear starburst regions
Authors: Klessen Ralf S. , Spaans Marco , Jappsen Anne-Katharina
Published: November 2006

Understanding the origin of stellar masses is a key problem in astrophysics. In the solar neighbourhood, the mass distribution of stars follows a seemingly universal pattern. In the centre of the Milky Way, however, there are indications for strong deviations and the same may be true for the nuclei of distant starburst galaxies. Here we present the first numerical hydrodynamical calculations of stars formed in a molecular region with chemical and thermodynamic properties similar to those of warm and dusty circumnuclear starburst regions. The resulting initial mass function is top-heavy with a peak at ∼15 M⊙, a sharp turn-down below ∼7 M⊙ and a power-law decline at high masses. We find a natural explanation for our results in terms of the temperature dependence of the Jeans mass, with collapse occurring at a temperature of ∼100 K and an H2 density of a few ×105 cm−3, and discuss possible implications for galaxy formation and evolution.
New anticoagulants and the management of their bleeding complications
Authors: NG HENG JOO , CROWTHER MARK
Published: November 2006

Limitations of the currently available anticoagulants have fanned the continuing search for new anticoagulants with improved pharmacological and biosafety profile, and equal, if not superior efficacy. Targets of inhibition include the factor VIIa/tissue factor pathway (recombinant nematode anticoagulant peptide c2, tissue factor pathway inhibitor), factor Xa (fondaparinux, idraparinux, razaxaban), factor Va and VIIIa pathway (recombinant activated protein C, soluble thrombomodulin) and thrombin (hirudin, bivalirudin, argatroban, ximelagatran, dabigatran). Irrespective of their mode of action, bleeding complications are invariable with all anticoagulants. Conventional assessment and measures should remain as first‐line responses to bleeding complicating the use of these anticoagulants. Antidotes do not exist for the overwhelming majority of these agents. The role of recombinant activated factor VIIa in controlling bleeding is still investigational. Definitive haemostatic strategies for bleeding complications can only evolve with accumulating experience with these new agents.
The airway‐related parasympathetic motoneurones in the ventrolateral medulla of newborn rats were dissociated anatomically and in functional control
Authors: Chen Yonghua , Li Min , Liu Han , Wang Jijiang
Published: November 2006

The respiratory‐related synaptic control of the airway‐related preganglionic parasympathetic motoneurones (APPMs) has not been investigated, and whether differently targeted APPMs receive differential respiratory‐related synaptic modulation is unknown. In this study, putative APPMs in the ventrolateral medulla of newborn rats were retrogradely traced with fluorescent tracer and were examined using the patch‐clamp method in brainstem slices with respiratory rhythm. The results indicate that tracer application directly to the recurrent laryngeal nerve only labelled the putative APPMs within the compact portion of nucleus ambiguus (cNA), while tracer injection into the trachea wall labelled the putative APPMs both in cNA and in the area ventral/ventrolateral to cNA (vNA). The putative APPMs within cNA received mainly inhibitory inputs, which in some (9 of 20) neurones showed an inspiratory‐related attenuation and in others (7 of 20) showed an inspiratory‐related augmentation. At least some putative APPMs within cNA, of which the inhibitory synaptic inputs showed inspiratory‐related changes, might be related to the control of laryngeal muscles. The putative APPMs in vNA receive both excitatory and inhibitory inputs, and central inspiratory activity excited some (11 of 19) neurones via augmentation of their excitatory inputs and inhibited others (8 of 19) via augmentation of their inhibitory inputs. At least some putative APPMs in vNA might be trachea‐related motoneurones. These results provide evidence that APPMs controlling different segments of the airway might be dissociated in the ventrolateral medulla both anatomically and in functional control.
Mechanism of Activation of β-
d
-2′-Deoxy-2′-Fluoro-2′-
C
-Methylcytidine and Inhibition of Hepatitis C Virus NS5B RNA Polymerase
Authors: Murakami Eisuke , Bao Haiying , Ramesh Mangala , McBrayer Tamara R. , Whitaker Tony , Micolochick Steuer Holly M. , Schinazi Raymond F. , Stuyver Lieven J. , Obikhod Aleksandr , Otto Michael J. , Furman Phillip A.
Published: November 2006

β-
d
-2′-Deoxy-2′-fluoro-2′-
C
-methylcytidine (PSI-6130) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5′-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2′-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a

K
m

of 81 μM and a
k
cat
of 0.007 s
−1
, but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (

K
i

) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 μM. Similar results were obtained with 2′-
C
-methyladenosine triphosphate (

K
i

= 1.5 μM) and 2′-
C
-methylcytidine triphosphate (

K
i

= 1.6 μM). NS5B with the S282T mutation, which is known to confer resistance to 2′-
C
-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.
Novel Macrolide Resistance Module Carried by the IncP-1β Resistance Plasmid pRSB111, Isolated from a Wastewater Treatment Plant
Authors: Szczepanowski Rafael , Krahn Irene , Bohn Nadine , Pühler Alfred , Schlüter Andreas
Published: November 2006

The macrolide resistance plasmid pRSB111 was isolated from bacteria residing in the final effluents of a wastewater treatment plant. The 47-kb plasmid confers resistance to azithromycin, clarithromycin, erythromycin, roxithromycin, and tylosin when it is carried by
Pseudomonas
sp. strain B13 and is very similar to prototype IncP-1β plasmid pB3, which was previously isolated from an activated-sludge bacterial community of a wastewater treatment plant. The two plasmids differ in their accessory regions, located downstream of the conjugative transfer module gene
traC
. Nucleotide sequence analysis of the pRSB111 accessory region revealed that it contains a new macrolide resistance module composed of the genes
mphR
(E),
mph
(E), and
mrx
(E), which putatively encode a transcriptional regulator, a macrolide phosphotransferase, and a transmembrane transport protein, respectively. Analysis of the contributions of the individual genes of the macrolide resistance module revealed that
mph
(E) and
mrx
(E) are required for high-level macrolide resistance. The resistance genes are flanked by two insertion sequences, namely, IS
Pa15
and IS
RSB111
. Two truncated transposable elements, IS
6100
and remnants of a Tn
3
-like transposon, were identified in the vicinity of IS
RSB111
. The accessory element of pRSB111 apparently replaced the Tn
402-
like element present on the sister plasmid, pB3, as suggested by the conservation of Tn
402
-specific terminal inverted repeats on pRSB111.
Molecular Dynamics of
Emiliania huxleyi
and Cooccurring Viruses during Two Separate Mesocosm Studies
Authors: Martínez Joaquín Martínez , Schroeder Declan C. , Larsen Aud , Bratbak Gunnar , Wilson William H.
Published: November 2006

In this study we used denaturing gradient gel electrophoresis, sequencing analysis, and analytical flow cytometry to monitor the dynamics and genetic richness of
Emiliania huxleyi
isolates and cooccurring viruses during two mesocosm experiments in a Norwegian fjord in 2000 and 2003. We exploited variations in a gene encoding a protein with calcium-binding motifs (GPA) and in the major capsid protein (MCP) gene to assess allelic and genotypic richness within
E. huxleyi
and
E. huxleyi
-specific viruses (EhVs), respectively. To our knowledge, this is the first report that shows the effectiveness of the GPA gene for analysis of natural communities of
E. huxleyi
. Our results revealed the existence of a genetically rich, yet stable
E. huxleyi
and EhV community in the fjordic environment. Incredibly, the same virus and host genotypes dominated in separate studies conducted 3 years apart. Both
E. huxleyi
-dominated blooms contained the same six
E. huxleyi
alleles. In addition, despite the presence of at least six and four EhV genotypes at the start of the blooms in 2000 and 2003, respectively, the same two virus genotypes dominated the naturally occurring infections during the exponential and termination phases of the blooms in both years.
Identification and Characterization of a Fructose Phosphotransferase System in
Bifidobacterium breve
UCC2003
Authors: Mazé Alain , O'Connell-Motherway Mary , Fitzgerald Gerald F. , Deutscher Josef , van Sinderen Douwe
Published: November 2006

In silico analysis of the
Bifidobacterium breve
UCC2003 genome allowed identification of four genetic loci, each of which specifies a putative enzyme II (EII) protein of a phosphoenolpyruvate:sugar phosphotransferase system. The EII encoded by
fruA
, a clear homologue of the unique EIIBCA enzyme encoded by the
Bifidobacterium longum
NCC2705 genome, was studied in more detail. The
fruA
gene is part of an operon which contains
fruT
, which is predicted to encode a homologue of the
Bacillus subtilis
antiterminator LicT. Transcriptional analysis showed that the
fru
operon is induced by fructose. The genetic structure, complementation studies, and the observed transcription pattern of the
fru
operon suggest that the EII encoded in
B. breve
is involved in fructose transport and that its expression is controlled by an antiterminator mechanism. Biochemical studies unequivocally demonstrated that FruA phosphorylates fructose at the C-6 position.
Antimicrobial-Resistant Enteric Bacteria from Dairy Cattle
Authors: Sawant Ashish A. , Hegde Narasimha V. , Straley Beth A. , Donaldson Sarah C. , Love Brenda C. , Knabel Stephen J. , Jayarao Bhushan M.
Published: November 2006

A study was conducted to understand the descriptive and molecular epidemiology of antimicrobial-resistant gram-negative enteric bacteria in the feces of healthy lactating dairy cattle. Gram-negative enteric bacteria resistant to ampicillin, florfenicol, spectinomycin, and tetracycline were isolated from the feces of 35, 8, 5, and 42% of 213 lactating cattle on 74, 39, 9, 26, and 82% of 23 farms surveyed, respectively. Antimicrobial-resistant gram-negative bacteria accounted for 5 (florfenicol) to 14% (tetracycline) of total gram-negative enteric microflora. Nine bacterial species were isolated, of which
Escherichia coli
(87%) was the most predominant species. MICs showing reduced susceptibility to ampicillin, ceftiofur, chloramphenicol, florfenicol, spectinomycin, streptomycin, and tetracycline were observed in
E. coli
isolates. Isolates exhibited resistance to ampicillin (48%), ceftiofur (11%), chloramphenicol (20%), florfenicol (78%), spectinomycin (18%), and tetracycline (93%). Multidrug resistance (≥3 to 6 antimicrobials) was seen in 40% of
E. coli
isolates from healthy lactating cattle. Of 113 tetracycline-resistant
E. coli
isolates,
tet
(B) was the predominant resistance determinant and was detected in 93% of isolates, while the remaining 7% isolates carried the
tet
(A) determinant. DNA-DNA hybridization assays revealed that
tet
determinants were located on the chromosome. Pulsed-field gel electrophoresis revealed that tetracycline-resistant
E. coli
isolates (
n
= 99 isolates) belonged to 60 subtypes, which is suggestive of a highly diverse population of tetracycline-resistant organisms. On most occasions,
E. coli
subtypes, although shared between cows within the herd, were confined mostly to a dairy herd. The findings of this study suggest that commensal enteric
E. coli
from healthy lactating cattle can be an important reservoir for tetracycline and perhaps other antimicrobial resistance determinants.
Analysis of Diversity and Activity of Sulfate-Reducing Bacterial Communities in Sulfidogenic Bioreactors Using 16S rRNA and
dsrB
Genes as Molecular Markers
Authors: Dar Shabir A. , Yao Li , van Dongen Udo , Kuenen J. Gijs , Muyzer Gerard
Published: November 2006

Here
we describe the diversity and activity of sulfate-reducing bacteria
(SRB) in sulfidogenic bioreactors by using the simultaneous analysis of
PCR products obtained from DNA and RNA of the 16S rRNA and
dissimilatory sulfite reductase (
dsrAB
) genes. We subsequently
analyzed the amplified gene fragments by using denaturing gradient gel
electrophoresis (DGGE). We observed fewer bands in the RNA-based DGGE
profiles than in the DNA-based profiles, indicating marked differences
in the populations present and in those that were metabolically active
at the time of sampling. Comparative sequence analyses of the bands
obtained from rRNA and
dsrB
DGGE profiles were congruent,
revealing the same SRB populations. Bioreactors that received either
ethanol or isopropanol as an energy source showed the presence of SRB
affiliated with
Desulfobulbus rhabdoformis
and/or
Desulfovibrio sulfodismutans
, as well as SRB related to the
acetate-oxidizing
Desulfobacca acetoxidans
. The reactor that
received wastewater containing a diverse mixture of organic compounds
showed the presence of nutritionally versatile SRB affiliated with
Desulfosarcina variabilis
and another acetate-oxidizing SRB,
affiliated with
Desulfoarculus baarsii
. In addition to DGGE
analysis, we performed whole-cell hybridization with fluorescently
labeled oligonucleotide probes to estimate the relative abundances of
the dominant sulfate-reducing bacterial populations.
Desulfobacca
acetoxidans
-like populations were most dominant (50 to 60%)
relative to the total SRB communities, followed by
Desulfovibrio-
like populations (30 to 40%), and
Desulfobulbus
-like populations (15 to 20%). This study is the
first to identify metabolically active SRB in sulfidogenic bioreactors
by using the functional gene
dsrAB
as a molecular marker. The
same approach can also be used to infer the ecological role of
coexisting SRB in other
habitats.
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

A 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

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

Ascospores 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

Helicobacter 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

Liver-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

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

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

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

The 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

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

Infection 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

To 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

Quorum 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.