bluuebunny/crossref_metadata_2025 huggingface dataset:
The Family Experience of “Sudden Health”: The Case of Intractable Epilepsy
Authors: SEABURN DAVID B. , ERBA GIUSEPPE
Published: August 2004

This article reports the experience of “sudden health” among six families who participated in an exploratory qualitative study of families with a member who elects to have corrective surgery for intractable epilepsy. Families were interviewed pre‐ and post surgery (6–8months) and the interviews were analyzed using a constant comparative methodology. Findings indicated that (1) families were organized in two primary ways (nesting and crisis) to deal with epilepsy and the aftermath of surgery and (2) “sudden health” had differing effects on these families depending on their organizational style, emotional communication process, and developmental dynamics.
Small Steps and Big Leaps in an Era of Cultural Transition: A Crisis in a Traditional Kosovar Albanian Family
Authors: SLUZKI CARLOS E. , AGANI FERID N.
Published: August 2004

This ethnographic vignette details the way a difficult crisis in an extended Kosovar family was managed by its own members within the mandates of that overtly patriarchal culture while creating important avenues for change. The cultural and historic context for these events is provided, and the nuances of this solution‐oriented, culturally congruent, “natural” (i.e., not in a therapeutic context) process are discussed.
The Adult Attachment Interview and Observed Couple Interaction: Implications for an Intergenerational Perspective on Couple Therapy
Authors: WAMPLER KAREN S. , SHI LIN , NELSON BRIANA S. , KIMBALL THOMAS G.
Published: August 2004

One of the assumptions of intergenerational family therapy is that how a person thinks and talks about family of origin experiences has important implications for current family relationships. The Adult Attachment Interview (AAI) is a measure of attachment security based on how coherently the person can discuss attachment experiences in childhood. This study examined the relationship between attachment security. As measured by the AAI, and couple interaction, as measured by the Georgia Marriage Q‐Sort (GMQ), in a sample of 28 couples in therapy for relationship problems. During a conflict resolution discussion, those individuals who wee less coherent in discussing their family of origin expressed more negative affect, less respect, less openness, more avoidance, and less willingness to negotiate when interacting with their partner. No evidence of intra‐couple effects or “buffering” was found The findings support a key assumption of intergenerational approaches to family therapy and suggest that applying attachment theory is a promising direction for refining and developing new interventions for couples.
Urban Youth Disruptive Behavioral Difficulties: Exploring Association with Parenting and Gender
Authors: SCHIFF MIRIAM , MCKAY MARY MCKERNAN
Published: August 2004

The current study will examine behavioral difficulties among a sample of African American urban youth who were exposed to violence. Possible gender differences in disruptive behavioral difficulties, as well as possible associations between parental practices, family relationships, and youth disruptive behavioral difficulties are examined. A secondary data analysis from baseline data for 125 African American urban mothers and their children collected as part of a large‐scale, urban, family‐based, HIV prevention research study was analyzed. Findings reveal that externalizing behavioral problems in youth are associated with exposure to violence. Girls displayed significantly higher levels of externalizing behavioral difficulties than boys. Mothers' parenting practices and family relationships were associated with youths' externalizing behavior problems. Implications for interventions to reduce youths' exposure to violence and to develop gender sensitive interventions for youth and supportive interventions for their parents are discussed.
An Outcome Evaluation of the Implementation of the Triple P‐Positive Parenting Program in Hong Kong
Authors: LEUNG CYNTHIA , SANDERS MATTHEW R , LEUNG SHIRLEY , MAK ROSE , LAU JOSEPH
Published: August 2004

The present study evaluated the effectiveness of the Positive Parenting Program (Triple P) with a sample of Chinese parents of children with early onset conduct‐related problems in Hong Kong. The participants consisted of 91 parents whose children attended maternal and child health centers and child assessment centers for service, and were between three to seven years old. Participants were randomly assigned to the intervention (TP) and a waitlist control group (WL). There was no significant difference in pre‐intervention measures between the two groups. However, at post intervention, participants in the TP group reported significantly lower levels of child behavior problems, lower dysfunctional parenting styles, and higher parent sense of competence, compared to the WL group. Implications of these findings for the use of Triple P with families of Chinese descent are discussed.
Thermoelastic Stresses in an Axisymmetric Thick-Walled Tube Under an Arbitrary Internal Transient
Authors: Segall A. E.
Published: August 2004

A closed-form axisymmetric solution was derived for the transient thermal-stress fields developed in thick-walled tubes subjected to an arbitrary thermal loading on the internal surface with convection to the surrounding external environment. Generalization of the temperature excitation was achieved by using a versatile polynomial composed of integral-and half-order terms. In order to avoid the difficult and potentially error prone evaluation of functions with complex arguments, Laplace transformation and a ten-term Gaver-Stehfest inversion formula were used to solve the resulting Volterra integral equation. The ensuing series representation of the temperature distribution as a function of time and radial position was then used to derive new relationships for the transient thermoelastic stress-states. Excellent agreement was seen between the derived temperature and stress relationships, existing series solutions, and a finite-element analysis when the thermophysical and thermoelastic properties were assumed to be independent of temperature. The use of a smoothed polynomial in the derived relationships allows the incorporation of empirical data not easily represented by standard functions. This in turn permits an easy analysis of the significance of the exponential boundary condition and convective coefficient in determining the magnitudes and distribution of the resulting stress states over time. Moreover, the resulting relationships are easily programmed and can be used to verify and calibrate numerical calculations.
Residual Stress of Thin-Wall Pipe Subjected to Axisymmetric Plastic Expansion
Authors: Chiba Norimasa , Ishida Yuji , Ogasawara Nagahisa , Ito Hiroshi , Enomoto Kunio , Kimoto Hiroshi
Published: August 2004

A straight pipe was expanded at one end by inserting a rigid disk. The residual stress measured at the inner surface shows a strong tensile peak beyond the region where the pipe was directly expanded by the insertion of the disk. The reason the residual stress reaches its peak at a location further into the pipe is discussed based on a FE analysis. The residual stress was found to reach its tensile peak at the plastic region front that was developed during the pipe expansion. A simple formula for identifying the tensile peak location is also proposed.
Fatigue Crack Propagation Rates for Notched 304 Stainless Steel Specimens In Elevated Temperature Water
Authors: Wire Gary L. , Mills William J.
Published: August 2004

Fatigue crack propagation (FCP) rates for 304 stainless steel (304 SS) were determined in 24°C and 288°C air and 288°C water with 20–60 cc H2/kg H2O using double-edged notch (DEN) specimens. Tests performed at matched loading conditions in air and water provided a direct comparison of the relative crack growth rates over a wide range of test conditions. Crack growth rates of 304 SS in water were about 12 times the air rate for both short cracks (0.03–0.25 mm) and long cracks up to 4.06 mm beyond the notch, which are consistent with conventional deep crack tests. The large environmental degradation for 304 SS crack growth is consistent with the strong reduction of fatigue life in high hydrogen water. Further, very similar environmental effects were reported in fatigue crack growth tests in hydrogen water chemistry (HWC). Prior to the recent tests reported by Wire and Mills [1] and Evans and Wire [2], most literature data in high hydrogen water showed only a mild environmental effect for 304 SS, of order 2.5 times air or less. However, the tests were predominantly performed at high cyclic stress intensities or high frequencies where environmental effects are small. The environmental effect in low oxygen environments at low stress intensity depends strongly on both the stress ratio, R, and the load rise time, Tr. Fractographic examinations were performed on specimens tested in both air and water to understand the operative cracking mechanisms associated with environmental effects. In 288°C water, the fracture surfaces were crisply faceted with a crystallographic appearance, and showed striations under high magnification. The cleavage-like facets suggest that hydrogen embrittlement is the primary cause of accelerated cracking.
Comparison of Fracture Response of Preflawed Tubes Under Internal Static and Detonation Loading
Authors: Chao Tong Wa , Shepherd Joseph E.
Published: August 2004

The role of loading rate and fluid properties in the fracture of piping under internal explosions is an important issue for designers and accident analysis. This was examined by fracture experiments on thin-walled and preflawed aluminum 6061-T6 tubes. The tubes were (1) statically loaded with oil, (2) statically loaded with nitrogen, and (3) dynamically loaded with gaseous detonations traveling at 2.4 km/s. The tubes were also instrumented with pressure transducers, crack detection gages, and strain gages. The experiments were controlled so that comparisons could be made on identical specimens. Fracture behavior was discussed along with fluid dynamics.
Failure Assessment on Tensile Cracked Specimens of Aluminum Alloys
Authors: Christopher T. , Sankaranarayanasamy K. , Rao B. Nageswara
Published: August 2004

A three-parameter fracture criterion is applied to correlate fracture data from tensile cracked plates made of aluminum alloys. Fracture parameters to generate the failure assessment diagram are determined for the materials considered in the present study. Failure load estimates were found to be in good agreement with test results. The failure assessment diagram of a material generated from tensile fracture plate configurations can also be applied for failure pressure estimations of flawed pressure vessels.
The Reaction Center H Subunit Is Not Required for High Levels of Light-Harvesting Complex 1 in
Rhodospirillum rubrum
Mutants
Authors: Lupo Domenico , Ghosh Robin
Published: August 2004

The gene (
puhA
) encoding the H subunit of the reaction center (RC) was deleted by site-directed interposon mutagenesis by using a kanamycin resistance cassette lacking transcriptional terminators to eliminate polar effects in both the wild-type strain
Rhodospirillum rubrum
S1 and the carotenoid-less strain
R. rubrum
G9. The
puhA
interposon mutants were incapable of photoheterotrophic growth but grew normally under aerobic chemoheterotrophic conditions. Absorption spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the RCs were absent. In minimal medium and also in modified medium containing succinate and fructose, the light-harvesting 1 complex (LH1) levels of the S1-derived mutants were about 70 to 100% of the wild-type levels in the same media. The correct assembly of LH1 in the membrane and the pigment-pigment interaction were confirmed by near-infrared circular dichroism spectroscopy. LH1 formation was almost absent when the carotenoid-less G9-derived
puhA
mutants were grown in standard minimal medium, suggesting that carotenoids may stabilize LH1. In the fructose-containing medium, however, the LH1 levels of the G9 mutants were 70 to 100% of the parental strain levels. Electron micrographs of thin sections of
R. rubrum
revealed photosynthetic membranes in all mutants grown in succinate-fructose medium. These studies indicate that the H subunit of the RC is necessary neither for maximal formation of LH1 nor for photosynthetic membrane formation but is essential for functional RC assembly.
Functional Characterization of an Aminotransferase Required for Pyoverdine Siderophore Biosynthesis in
Pseudomonas aeruginosa
PAO1
Authors: Vandenende Chris S. , Vlasschaert Matthew , Seah Stephen Y. K.
Published: August 2004

The fluorescent dihydroxyquinoline chromophore of the pyoverdine siderophore in
Pseudomonas
is a condensation product of
d
-tyrosine and
l
-2,4-diaminobutyrate. Both
pvdH
and
asd
(encoding aspartate β-semialdehyde dehydrogenase) knockout mutants of
Pseudomonas aeruginosa
PAO1 were unable to synthesize pyoverdine under iron-limiting conditions in the absence of
l
-2,4-diaminobutyrate in the culture media. The
pvdH
gene was subcloned, and the gene product was hyperexpressed and purified from
P. aeruginosa
PAO1. PvdH was found to catalyze an aminotransferase reaction, interconverting aspartate β-semialdehyde and
l
-2,4-diaminobutyrate. Steady-state kinetic analysis with a novel coupled assay established that the enzyme adopts a ping-pong kinetic mechanism and has the highest specificity for α-ketoglutarate. The specificity of the enzyme toward the smaller keto acid pyruvate is 41-fold lower. The enzyme has negligible activity toward other keto acids tested. Homologues of PvdH were present in the genomes of other
Pseudomonas
spp. These homologues were found in the DNA loci of the corresponding genomes that contain other pyoverdine synthesis genes. This suggests that there is a general mechanism of
l
-2,4-diaminobutyrate synthesis in
Pseudomonas
strains that produce the pyoverdine siderophore.
TheEnterococcus faecalis fsrTwo-Component System Controls Biofilm Development through Production of Gelatinase
Authors: Hancock Lynn E. , Perego Marta
Published: August 2004

Bacterial growth as a biofilm on solid surfaces is strongly associated with the development of human infections. Biofilms on native heart valves (infective endocarditis) is a life-threatening disease as a consequence of bacterial resistance to antimicrobials in such a state. Enterococci have emerged as a cause of endocarditis and nosocomial infections despite being normal commensals of the gastrointestinal and female genital tracts. We examined the role of two-component signal transduction systems in biofilm formation by theEnterococcus faecalisV583 clinical isolate and identified thefsrregulatory locus as the sole two-component system affecting this unique mode of bacterial growth. Insertion mutations in thefsroperon affected biofilm formation on two distinct abiotic surfaces. Inactivation of thefsr-controlled genegelEencoding the zinc-metalloprotease gelatinase was found to prevent biofilm formation, suggesting that this enzyme may present a unique target for therapeutic intervention in enterococcal endocarditis.
The Bvg Virulence Control System Regulates Biofilm Formation in
Bordetella bronchiseptica
Authors: Irie Yasuhiko , Mattoo Seema , Yuk Ming H.
Published: August 2004

Bordetella
species utilize the BvgAS (
Bordetella
virulence gene) two-component signal transduction system to sense the environment and regulate gene expression among at least three phases: a virulent Bvg
+
phase, a nonvirulent Bvg
−
phase, and an intermediate Bvg
i
phase. Genes expressed in the Bvg
+
phase encode known virulence factors, including adhesins such as filamentous hemagglutinin (FHA) and fimbriae, as well as toxins such as the bifunctional adenylate cyclase/hemolysin (ACY). Previous studies showed that in the Bvg
i
phase, FHA and fimbriae continue to be expressed, but ACY expression is significantly downregulated. In this report, we determine that
Bordetella bronchiseptica
can form biofilms in vitro and that the generation of biofilm is maximal in the Bvg
i
phase. We show that FHA is required for maximal biofilm formation and that fimbriae may also contribute to this phenotype. However, expression of ACY inhibits biofilm formation, most likely via interactions with FHA. Therefore, the coordinated regulation of adhesins and ACY expression leads to maximal biofilm formation in the Bvg
i
phase in
B. bronchiseptica
.
Transposon-Mediated Linker Insertion Scanning Mutagenesis of the
Escherichia coli
McrA Endonuclease
Authors: Anton Brian P. , Raleigh Elisabeth A.
Published: August 2004

McrA is one of three functions that restrict modified foreign DNA in
Escherichia coli
K-12, affecting both methylated and hydroxymethylated substrates. We present here the first systematic analysis of the functional organization of McrA by using the GPS-LS insertion scanning system. We collected in-frame insertions of five amino acids at 46 independent locations and C-terminal truncations at 20 independent locations in the McrA protein. Each mutant was assayed for in vivo restriction of both methylated and hydroxymethylated bacteriophage (M.HpaII-modified λ and T4
gt
, respectively) and for induction of the
E. coli
SOS response in the presence of M.HpaII methylation, indicative of DNA damage. Our findings suggest the presence of an N-terminal DNA-binding domain and a C-terminal catalytic nuclease domain connected by a linker region largely tolerant of amino acid insertions. DNA damage inflicted by a functional C-terminal domain is required for restriction of phage T4
gt
. Disruption of the N-terminal domain abolishes restriction of both substrates. Surprisingly, truncation mutations that spare the N-terminal domain do not mediate DNA damage, as measured by SOS induction, but nevertheless partially restrict M.HpaII-modified λ in vivo. We suggest a common explanation for this “restriction without damage” and a similar observation seen in vivo with McrB, a component of another of the modified-DNA restriction functions. Briefly, we propose that unproductive site-specific binding of the protein to a vulnerable position in the λ genome disrupts the phage development program at an early stage. We also identified a single mutant, carrying an insertion in the N-terminal domain, which could fully restrict λ but did not restrict T4
gt
at all. This mutant may have a selective impairment in substrate recognition, distinguishing methylated from hydroxymethylated substrates. The study shows that the technically easy insertion scanning method can provide a rich source of functional information when coupled with effective phenotype tests.
The
vir
Gene of Bacteriophage MAV1 Confers Resistance to Phage Infection on
Mycoplasma arthritidis
Authors: Clapper Brenda , Tu Anh-Hue T. , Elgavish Ada , Dybvig Kevin
Published: August 2004

Lysogenization of
Mycoplasma arthritidis
with the MAV1 bacteriophage increases the virulence of the mycoplasma in rats. The MAV1
vir
gene is one of only two constitutively transcribed phage genes in the lysogen. We show here that Vir is a lipoprotein and is located on the outer surface of the cell membrane. To investigate whether Vir is a virulence factor, the
vir
gene was cloned into the transposon vector Tn
4001T
and inserted in the genome of the nonlysogen strain 158. The virulence of the resulting transformants was no different from that of the parent strain. Interestingly, all
vir
-containing transformants were resistant to infection by MAV1. Vir had no effect on MAV1 adsorption. We conclude that Vir is not a virulence factor but functions to exclude superinfecting phage, possibly by blocking the injection of phage DNA into the bacterial cytoplasm.
Mutations Altering the N-Terminal Receiver Domain of NRI (NtrC) That Prevent Dephosphorylation by the NRII-PII Complex inEscherichia coli
Authors: Pioszak Augen A. , Ninfa Alexander J.
Published: August 2004

The phosphorylated form of NRI is the transcriptional activator of nitrogen-regulated genes inEscherichia coli. NRI∼P displays a slow autophosphatase activity and is rapidly dephosphorylated by the complex of the NRII and PII signal transduction proteins. Here we describe the isolation of two mutations, causing the alterations ΔD10 and K104Q in the receiver domain of NRI, that were selected as conferring resistance to dephosphorylation by the NRII-PII complex. The mutations, which alter highly conserved residues near the D54 site of phosphorylation in the NRI receiver domain, resulted in elevated expression of nitrogen-regulated genes under nitrogen-rich conditions. The altered NRI receiver domains were phosphorylated by NRII in vitro but were defective in dephosphorylation. The ΔD10 receiver domain retained normal autophosphatase activity but was resistant to dephosphorylation by the NRII-PII complex. The K104Q receiver domain lacked both the autophosphatase activity and the ability to be dephosphorylated by the NRII-PII complex. The properties of these altered proteins are consistent with the hypothesis that the NRII-PII complex is not a true phosphatase but rather collaborates with NRI≈P to bring about its dephosphorylation.
Assembly Dynamics of FtsZ Rings in
Bacillus subtilis
and
Escherichia coli
and Effects of FtsZ-Regulating Proteins
Authors: Anderson David E. , Gueiros-Filho Frederico J. , Erickson Harold P.
Published: August 2004

FtsZ is the major cytoskeletal component of the bacterial cell division machinery. It forms a ring-shaped structure (the Z ring) that constricts as the bacterium divides. Previous in vivo experiments with green fluorescent protein-labeled FtsZ and fluorescence recovery after photobleaching have shown that the
Escherichia coli
Z ring is extremely dynamic, continually remodeling itself with a half time of 30 s, similar to microtubules in the mitotic spindle. In the present work, under different experimental conditions, we have found that the half time for fluorescence recovery of
E. coli
Z rings is even shorter (∼9 s). As before, the turnover appears to be coupled to GTP hydrolysis, since the mutant FtsZ84 protein, with reduced GTPase in vitro, showed an ∼3-fold longer half time. We have also extended the studies to
Bacillus subtilis
and found that this species exhibits equally rapid dynamics of the Z ring (half time, ∼8 s). Interestingly, null mutations of the FtsZ-regulating proteins ZapA, EzrA, and MinCD had only modest effects on the assembly dynamics. This suggests that these proteins do not directly regulate FtsZ subunit exchange in and out of polymers. In
B. subtilis
, only 30 to 35% of the FtsZ protein was in the Z ring, from which we conclude that a Z ring only 2 or 3 protofilaments thick can function for cell division.
A Novel
Acetivibrio cellulolyticus
Anchoring Scaffoldin That Bears Divergent Cohesins
Authors: Xu Qi , Barak Yoav , Kenig Rina , Shoham Yuval , Bayer Edward A. , Lamed Raphael
Published: August 2004

Sequencing of a cellulosome-integrating gene cluster in
Acetivibrio cellulolyticus
was completed. The cluster contains four tandem scaffoldin genes (
scaA
,
scaB
,
scaC
, and
scaD
) bounded upstream and downstream, respectively, by a presumed cellobiose phosphorylase and a nucleotide methylase. The sequences and properties of
scaA
,
scaB
, and
scaC
were reported previously, and those of
scaD
are reported here. The
scaD
gene encodes an 852-residue polypeptide that includes a signal peptide, three cohesins, and a C-terminal S-layer homology (SLH) module. The calculated molecular weight of the mature ScaD is 88,960; a 67-residue linker segment separates cohesins 1 and 2, and two ∼30-residue linkers separate cohesin 2 from 3 and cohesin 3 from the SLH module. The presence of an SLH module in ScaD indicates its role as an anchoring protein. The first two ScaD cohesins can be classified as type II, similar to the four cohesins of ScaB. Surprisingly, the third ScaD cohesin belongs to the type I cohesins, like the seven ScaA cohesins. ScaD is the first scaffoldin to be described that contains divergent types of cohesins as integral parts of the polypeptide chain. The recognition properties among selected recombinant cohesins and dockerins from the different scaffoldins of the gene cluster were investigated by affinity blotting. The results indicated that the divergent types of ScaD cohesins also differ in their preference of dockerins. ScaD thus plays a dual role, both as a primary scaffoldin, capable of direct incorporation of a single dockerin-borne enzyme, and as a secondary scaffoldin that anchors the major primary scaffoldin, ScaA and its complement of enzymes to the cell surface.
Genetic Evidence Identifying the True Gluconeogenic Fructose-1,6-Bisphosphatase in
Thermococcus kodakaraensis
and Other Hyperthermophiles
Authors: Sato Takaaki , Imanaka Hiroyuki , Rashid Naeem , Fukui Toshiaki , Atomi Haruyuki , Imanaka Tadayuki
Published: August 2004

Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes in gluconeogenesis. Although FBPase activity has been detected in several hyperthermophiles, no orthologs corresponding to the classical FBPases from bacteria and eukaryotes have been identified in their genomes. An inositol monophosphatase (IMPase) from
Methanococcus jannaschii
which displayed both FBPase and IMPase activities and a structurally novel FBPase (Fbp

Tk

) from the hyperthermophilic archaeon
Thermococcus kodakaraensis
KOD1 have been proposed as the “missing” FBPase. For this study, using
T. kodakaraensis
, we took a genetic approach to elucidate which candidate is the major gluconeogenic enzyme in vivo. The IMPase/FBPase ortholog in
T. kodakaraensis
, Imp

Tk

, was confirmed to possess high FBPase activity along with IMPase activity, as in the case of other orthologs. We therefore constructed Δ
fbp
and Δ
imp
strains by applying a gene disruption system recently developed for
T. kodakaraensis
and investigated their phenotypes. The Δ
fbp
strain could not grow under gluconeogenic conditions while glycolytic growth was unimpaired, and the disruption resulted in the complete abolishment of intracellular FBPase activity. Evidently,

fbp
Tk

is an indispensable gene for gluconeogenesis and is responsible for almost all intracellular FBPase activity. In contrast, the endogenous

imp
Tk

gene could not complement the defect of the
fbp
deletion, and its disruption did not lead to any detectable phenotypic changes under the conditions examined. These facts indicated that

imp
Tk

is irrelevant to gluconeogenesis, despite the high FBPase activity of its protein product, probably due to insufficient transcription. Our results provide strong evidence that the true FBPase for gluconeogenesis in
T. kodakaraensis
is the Fbp

Tk

ortholog, not the IMPase/FBPase ortholog.
Thermus thermophilus
L11 Methyltransferase, PrmA, Is Dispensable for Growth and Preferentially Modifies Free Ribosomal Protein L11 Prior to Ribosome Assembly
Authors: Cameron Dale M. , Gregory Steven T. , Thompson Jill , Suh Moo-Jin , Limbach Patrick A. , Dahlberg Albert E.
Published: August 2004

The ribosomal protein L11 in bacteria is posttranslationally trimethylated at multiple amino acid positions by the L11 methyltransferase PrmA, the product of the
prmA
gene. The role of L11 methylation in ribosome function or assembly has yet to be determined, although the deletion of
Escherichia coli prmA
has no apparent phenotype. We have constructed a mutant of the extreme thermophile
Thermus thermophilus
in which the
prmA
gene has been disrupted with the
htk
gene encoding a heat-stable kanamycin adenyltransferase. This mutant shows no growth defects, indicating that
T. thermophilus
PrmA, like its
E. coli
homolog, is dispensable. Ribosomes prepared from this mutant contain unmethylated L11, as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and are effective substrates for in vitro methylation by cloned and purified
T. thermophilus
PrmA. MALDI-TOF MS also revealed that
T. thermophilus
L11 contains a total of 12 methyl groups, in contrast to the 9 methyl groups found in
E. coli
L11. Finally, we found that, as with the
E. coli
methyltransferase, the ribosomal protein L11 dissociated from ribosomes is a more efficient substrate for in vitro methylation by PrmA than intact 70S ribosomes, suggesting that methylation in vivo occurs on free L11 prior to its incorporation into ribosomes.
Linkage between Catecholate Siderophores and the Multicopper Oxidase CueO in
Escherichia coli
Authors: Grass Gregor , Thakali Keshari , Klebba Phillip E. , Thieme Daniel , Müller Axel , Wildner Günter F. , Rensing Christopher
Published: August 2004

The multicopper oxidase CueO had previously been demonstrated to exhibit phenoloxidase activity and was implicated in intrinsic copper resistance in
Escherichia coli
. Catecholates can potentially reduce Cu(II) to the prooxidant Cu(I). In this report we provide evidence that CueO protects
E. coli
cells by oxidizing enterobactin, the catechol iron siderophore of
E. coli
, in the presence of copper. In vitro, a mixture of enterobactin and copper was toxic for
E. coli
cells, but the addition of purified CueO led to their survival. Deletion of
fur
resulted in copper hypersensitivity that was alleviated by additional deletion of
entC
, preventing synthesis of enterobactin. In addition, copper added together with 2,3-dihydroxybenzoic acid or enterobactin was able to induce a Φ(
cueO-lacZ
) operon fusion more efficiently than copper alone. The reaction product of the 2,3-dihydroxybenzoic acid oxidation by CueO that can complex Cu(II) ions was determined by gas chromatography-mass spectroscopy and identified as 2-carboxymuconate.
A Novel Sortase, SrtC2, from
Streptococcus pyogenes
Anchors a Surface Protein Containing a QVPTGV Motif to the Cell Wall
Authors: Barnett Timothy C. , Patel Aman R. , Scott June R.
Published: August 2004

The important human pathogen
Streptococcus pyogenes
(group A streptococcus GAS), requires several surface proteins to interact with its human host. Many of these are covalently linked by a sortase enzyme to the cell wall via a C-terminal LPXTG motif. This motif is followed by a hydrophobic region and charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. Previously, we identified two sortase enzymes in GAS. SrtA is found in all GAS strains and anchors most proteins containing LPXTG, while SrtB is present only in some strains and anchors a subset of LPXTG-containing proteins. We now report the presence of a third sortase in most strains of GAS, SrtC. We show that SrtC mediates attachment of a protein with a QVPTGV motif preceding a hydrophobic region and charged tail. We also demonstrate that the QVPTGV sequence is a substrate for anchoring of this protein by SrtC. Furthermore, replacing this motif with LPSTGE, found in the SrtA-anchored M protein of GAS, leads to SrtA-dependent secretion of the protein but does not lead to its anchoring by SrtA. We conclude that
srtC
encodes a novel sortase that anchors a protein containing a QVPTGV motif to the surface of GAS.
Characterization of
Salmonella enterica
Subspecies I Genovars by Use of Microarrays
Authors: Porwollik S. , Boyd E. F. , Choy C. , Cheng P. , Florea L. , Proctor E. , McClelland M.
Published: August 2004

Subspecies 1 of
Salmonella enterica
is responsible for almost all
Salmonella
infections of warm-blooded animals. Within subspecies 1 there are over 2,300 known serovars that differ in their prevalence and the diseases that they cause in different hosts. Only a few of these serovars are responsible for most
Salmonella
infections in humans and domestic animals. The gene contents of 79 strains from the most prevalent serovars were profiled by microarray analysis. Strains within the same serovar often differed by the presence and absence of hundreds of genes. Gene contents sometimes differed more within a serovar than between serovars. Groups of strains that share a distinct profile of gene content can be referred to as “genovars” to distinguish them from serovars. Several misassignments within the
Salmonella
reference B collection were detected by genovar typing and were subsequently confirmed serologically. Just as serology has proved useful for understanding the host range and pathogenic manifestations of
Salmonella
, genovars are likely to further define previously unrecognized specific features of
Salmonella
infections.
High-Resolution Structure of the Histidine-Containing Phosphocarrier Protein (HPr) fromStaphylococcus aureusand Characterization of Its Interaction with the Bifunctional HPr Kinase/Phosphorylase
Authors: Maurer Till , Meier Sebastian , Kachel Norman , Munte Claudia Elisabeth , Hasenbein Sonja , Koch Brigitte , Hengstenberg Wolfgang , Kalbitzer Hans Robert
Published: August 2004

A high-resolution structure of the histidine-containing phosphocarrier protein (HPr) fromStaphylococcus aureuswas obtained by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy on the basis of 1,766 structural restraints. Twenty-three hydrogen bonds in HPr could be directly detected by polarization transfer from the amide nitrogen to the carbonyl carbon involved in the hydrogen bond. Differential line broadening was used to characterize the interaction of HPr with the HPr kinase/phosphorylase (HPrK/P) ofStaphylococcus xylosus, which is responsible for phosphorylation-dephosphorylation of the hydroxyl group of the regulatory serine residue at position 46. The dissociation constantKdwas determined to be 0.10 ± 0.02 mM at 303 K from the NMR data, assuming independent binding. The data are consistent with a stoichiometry of 1 HPr molecule per HPrK/P monomer in solution. Using transversal relaxation optimized spectroscopy-heteronuclear single quantum correlation, we mapped the interaction site of the two proteins in the 330-kDa complex. As expected, it covers the region around Ser46 and the small helix b following this residue. In addition, HPrK/P also binds to the second phosphorylation site of HPr at position 15. This interaction may be essential for the recognition of the phosphorylation state of His15 and the phosphorylation-dependent regulation of the kinase/phosphorylase activity. In accordance with this observation, the recently published X-ray structure of the HPr/HPrK core protein complex fromLactobacillus caseishows interactions with the two phosphorylation sites. However, the NMR data also suggest differences for the full-length protein fromS. xylosus: there are no indications for an interaction with the residues preceding the regulatory Ser46 residue (Thr41 to Lys45) in the protein ofS. xylosus. In contrast, it seems to interact with the C-terminal helix of HPr in solution, an interaction which is not observed for the complex of HPr with the core of HPrK/P ofL. caseiin crystals.
Substrate Specificity of the
Escherichia coli
Outer Membrane Protease OmpT
Authors: McCarter John D. , Stephens Daren , Shoemaker Kevin , Rosenberg Steve , Kirsch Jack F. , Georgiou George
Published: August 2004

OmpT is a surface protease of gram-negative bacteria that has been shown to cleave antimicrobial peptides, activate human plasminogen, and degrade some recombinant heterologous proteins. We have analyzed the substrate specificity of OmpT by two complementary substrate filamentous phage display methods: (i) in situ cleavage of phage that display protease-susceptible peptides by
Escherichia coli
expressing OmpT and (ii) in vitro cleavage of phage-displayed peptides using purified enzyme. Consistent with previous reports, OmpT was found to exhibit a virtual requirement for Arg in the P1 position and a slightly less stringent preference for this residue in the P1′ position (P1 and P1′ are the residues immediately prior to and following the scissile bond). Lys, Gly, and Val were also found in the P1′ position. The most common residues in the P2′ position were Val or Ala, and the P3 and P4 positions exhibited a preference for Trp or Arg. Synthetic peptides based upon sequences selected by bacteriophage display were cleaved very efficiently, with
k
cat
/
K

m

values up to 7.3 × 10
6
M
−1
s
−1
. In contrast, a peptide corresponding to the cleavage site of human plasminogen was hydrolyzed with a
k
cat
/
K

m

almost 10
6
-fold lower. Overall, the results presented in this work indicate that in addition to the P1 and P1′ positions, additional amino acids within a six-residue window (between P4 and P2′) contribute to the binding of substrate polypeptides to the OmpT binding site.
Transcription Regulation ofezrAand Its Effect on Cell Division ofBacillus subtilis
Authors: Chung Kuei-Min , Hsu Hsin-Hsien , Govindan Suresh , Chang Ban-Yang
Published: August 2004

The EzrA protein ofBacillus subtilisis a negative regulator for FtsZ (Z)-ring formation. It is able to modulate the frequency and position of Z-ring formation during cell division. The loss of this protein results in cells with multiple Z rings located at polar as well as medial sites; it also lowers the critical concentration of FtsZ required for ring formation (P. A. Levin, I. G. Kurster, and A. D. Grossman, Proc. Natl. Acad. Sci. USA96:9642-9647, 1999). We have studied the regulation ofezrAexpression during the growth ofB. subtilisand its effects on the intracellular level of EzrA as well as the cell length ofB. subtilis. With the aid of promoter probing, primer extension, in vitro transcription, and Western blotting analyses, two overlapping σA-type promoters, P1 and P2, located about 100 bp upstream of the initiation codon ofezrA, have been identified. P1, supposed to be an extended −10 promoter, was responsible for most of theezrAexpression during the growth ofB. subtilis. Disruption of this promoter reduced the intracellular level of EzrA very significantly compared with disruption of P2. Moreover, deletion of both promoters completely abolished EzrA inB. subtilis. More importantly, the cell length and percentage of filamentous cells ofB. subtiliswere significantly increased by disruption of the promoter(s). Thus, EzrA is required for efficient cell division during the growth ofB. subtilis, despite serving as a negative regulator for Z-ring formation.
FlhF, the Third Signal Recognition Particle-GTPase of
Bacillus subtilis
, Is Dispensable for Protein Secretion
Authors: Zanen Geeske , Antelmann Haike , Westers Helga , Hecker Michael , van Dijl Jan Maarten , Quax Wim J.
Published: August 2004

Bacillus subtilis
contains three proteins of the signal recognition particle-GTPase family known as Ffh, FtsY, and FlhF. Here we show that FlhF is dispensable for protein secretion, unlike Ffh and FtsY. Although
flhF
is located in the
fla
/
che
operon,
B. subtilis
168
flhF
mutant cells assemble flagella and are motile.
Overexpression of
gnsA
, a Multicopy Suppressor of the
secG
Null Mutation, Increases Acidic Phospholipid Contents by Inhibiting Phosphatidylethanolamine Synthesis at Low Temperatures
Authors: Sugai Rie , Shimizu Hisayo , Nishiyama Ken-ichi , Tokuda Hajime
Published: August 2004

GnsA overproduction was previously found to suppress both the
secG
null mutation and the
fabA6
mutation in
Escherichia coli
by increasing the unsaturated fatty acid contents. We report here that it also increased the acidic phospholipid contents at 20°C but not at 37°C. GnsA overproduction at 20°C specifically inhibited phosphatidylethanolamine synthesis and therefore caused the increase in the proportion of acidic phospholipids.
Burnout and work environments of public health nurses involved in mental health care
Authors: Imai H , Nakao H , Tsuchiya M , Kuroda Y , Katoh T
Published: August 2004

Aims:(1) To examine whether prevalence of burnout is higher among community psychiatric nurses working under recently introduced job specific work systems than among public health nurses (PHNs) engaged in other public health services. (2) To identify work environment factors potentially contributing to burnout.

Methods:Two groups were examined. The psychiatric group comprised 525 PHNs primarily engaged in public mental health services at public health centres (PHCs) that had adopted the job specific work system. The control group comprised 525 PHNs primarily engaged in other health services. Pines’ Burnout Scale was used to measure burnout. Respondents were classified by burnout score into three groups: A (mentally stable, no burnout); B (positive signs, risk of burnout); and C (burnout present, action required). Groups B and C were considered representative of “burnout”. A questionnaire was also prepared to investigate systems for supporting PHNs working at PHCs and to define emergency mental health service factors contributing to burnout.

Results:Final respondents comprised 785 PHNs. Prevalence of burnout was significantly higher in the psychiatric group (59.2%) than in the control group (51.5%). Responses indicating lack of job control and increased annual frequency of emergency overtime services were significantly correlated with prevalence of burnout in the psychiatric group, but not in the control group.

Conclusions:Prevalence of burnout is significantly higher for community psychiatric nurses than for PHNs engaged in other services. Overwork in emergency services and lack of job control appear to represent work environment factors contributing to burnout.
Acute childhood leukaemia and environmental exposure to potential sources of benzene and other hydrocarbons; a case-control study
Authors: Steffen C , Auclerc M F , Auvrignon A , Baruchel A , Kebaili K , Lambilliotte A , Leverger G , Sommelet D , Vilmer E , Hémon D , Clavel J
Published: August 2004

Aim:To analyse the association between potential environmental exposure to hydrocarbons and the risk of acute childhood leukaemia.

Methods:A hospital based multicentre case control study, stratified on centre, age, and sex, with 280 leukaemia cases and 285 controls was carried out. Data were collected by a standardised interview of the mothers.

Results:No clear association was seen between maternal occupational exposure to hydrocarbons during pregnancy and leukaemia, or between residential traffic density and leukaemia. There was an association between dwellings neighbouring a petrol station or a repair garage during childhood and the risk of childhood leukaemia (OR 4.0, 95% CI 1.5 to 10.3), with a duration trend. The association, which appeared particularly strong for acute non-lymphocytic leukaemia (OR 7.7, 95% CI 1.7 to 34.3), was not altered by adjustment for potential confounding factors.

Conclusions:Results showed an association between acute childhood leukaemia and dwellings neighbouring auto repair garages and petrol stations, which are benzene emitting sources. These findings could be due to chance, although the strength of the association and the duration trend are arguments for a causal association.
Occupational asthma caused by cellulase and lipase in the detergent industry
Authors: Brant A , Hole A , Cannon J , Helm J , Swales C , Welch J , Newman Taylor A , Cullinan P
Published: August 2004

Three employees from two different detergent companies were investigated for occupational asthma, using skin prick tests, serum specific IgE, and specific bronchial challenge. Two were challenged with lipase and one with cellulase. All three cases had immunological evidence of sensitisation to the detergent enzymes with which they worked. Bronchial challenge in each provoked a reproducible dual asthmatic response, which reproduced their work related symptoms. These are the first reported cases of occupational asthma attributable to cellulase and lipase in the detergent industry. Four of the most common enzymes used in this industry have now been reported to cause occupational asthma; continued vigilance and caution are needed when working with these or other enzymes.
Airway inflammation in aluminium potroom asthma
Authors: Sjåheim T , Halstensen T S , Lund M B , Bjørtuft Ø , Drabløs P A , Malterud D , Kongerud J
Published: August 2004

Aims:To examine whether asthma induced by exposure to aluminium potroom emissions (potroom asthma) is associated with inflammatory changes in the airways.

Methods:Bronchial biopsy specimens from 20 asthmatic workers (8 non-smokers and 12 smokers), 15 healthy workers (8 non-smokers and 7 smokers), and 10 non-exposed controls (all non-smokers) were analysed. Immunohistofluorescent staining was performed to identify mucosal total leucocytes (CD45+leucocytes), neutrophils, and mast cells.

Results:Median RBM thickness was significantly increased in both asthmatic workers (8.2 μm) and healthy workers (7.4 μm) compared to non-exposed controls (6.7 μm). Non-smoking asthmatic workers had significantly increased median density of lamina propria CD45+leucocytes (1519 cells/mm2v660 and 887 cells/mm2) and eosinophils (27 cells/mm2v10 and 3 cells/mm2) and significantly increased concentrations of exhaled NO (18.1 ppbv6.5 and 5.1 ppb) compared to non-smoking healthy workers and non-exposed controls. Leucocyte counts and exhaled NO concentrations varied with smoking habits and fewer leucocytes were observed in asthmatic smokers than in non-smokers Asthmatic smokers had significantly increased numbers of eosinophils in lamina propria compared to non-exposed controls (10v3 cells/mm2). Both eosinophilic and non-eosinophilic phenotypes of asthma were recognised in the potroom workers and signs of airway inflammation were also observed in healthy workers.

Conclusions:Airway inflammation is a central feature of potroom asthma and exposure to potroom emissions induces pathological alterations similar to those described in other types of asthma. Cigarette smoking seems to affect the underlying mechanisms involved in asthma, as the cellular composition of airway mucosa appears different in asthmatic smokers and non-smokers.
Output power levels from mobile phones in different geographical areas; implications for exposure assessment
Authors: Lönn S , Forssén U , Vecchia P , Ahlbom A , Feychting M
Published: August 2004

Background:The power level used by the mobile phone is one of the most important factors determining the intensity of the radiofrequency exposure during a call. Mobile phone calls made in areas where base stations are densely situated (normally urban areas) should theoretically on average use lower output power levels than mobile phone calls made in areas with larger distances between base stations (rural areas).

Aims:To analyse the distribution of power levels from mobile phones in four geographical areas with different population densities.

Methods:The output power for all mobile phone calls managed by the GSM operator Telia Mobile was recorded during one week in four defined areas (rural, small urban, suburban, and city area) in Sweden. The recording included output power for the 900 MHz and the 1800 MHz frequency band.

Results:In the rural area, the highest power level was used about 50% of the time, while the lowest power was used only 3% of the time. The corresponding numbers for the city area were approximately 25% and 22%. The output power distribution in all defined urban areas was similar.

Conclusion:In rural areas where base stations are sparse, the output power level used by mobile phones are on average considerably higher than in more densely populated areas. A quantitative assessment of individual exposure to radiofrequency fields is important for epidemiological studies of possible health effects for many reasons. Degree of urbanisation may be an important parameter to consider in the assessment of radiofrequency exposure from mobile phone use.