bluuebunny/crossref_metadata_2025 huggingface dataset:
High‐pressure studies of aggregation of recombinant human interleukin‐1 receptor antagonist: Thermodynamics, kinetics, and application to accelerated formulation studies
Authors: Seefeldt Matthew B. , Kim Yong‐Sung , Tolley Kevin P. , Seely Jim , Carpenter John F. , Randolph Theodore W.
Published: August 2005

Recombinant human interleukin‐1 receptor antagonist (IL‐1ra) in aqueous solutions unfolds and aggregates when subjected to hydrostatic pressures greater than about 180 MPa. This study examined the mechanism and thermodynamics of pressure‐induced unfolding and aggregation of IL‐1ra. The activation free energy for growth of aggregates (ΔG∓aggregation) was found to be 37 ± 3 kJ/mol, whereas the activation volume (ΔV∓aggregation) was −120 ± 20 mL/mol. These values compare closely with equilibrium values for denaturation: The free energy for denaturation, ΔGdenaturation, was 20 ± 5 kJ/mol, whereas the partial specific volume change for denaturation, ΔVdenaturation, was −110 ± 30 mL/mol. When IL‐1ra begins to denature at pressures near 140 MPa, cysteines that are normally buried in the native state become exposed. Under oxidizing conditions, this results in the formation of covalently cross‐linked aggregates containing nonnative, intermolecular disulfide bonds. The apparent activation free energy for nucleation of aggregates, ΔG∓nuc, was 42 ± 4 kJ/mol, and the activation volume for nucleation, ΔV∓nuc,was −175 ± 37 mL/mol, suggesting that a highly solvent‐exposed conformation is needed for nucleation. We hypothesize that the large specific volume of IL‐1ra, 0.752 ± 0.004 mL/g, coupled with its relatively low conformational stability, leads to its susceptibility to denaturation at relatively low pressures. The positive partial specific adiabatic compressibility of IL‐1ra, 4.5 ± 0.7 ± 10−12cm2/dyn, suggests that a significant component of the ΔVdenaturationis attributable to the elimination of solvent‐free cavities. Lastly, we propose that hydrostatic pressure is a useful variable to conduct accelerated formulation studies of therapeutic proteins.
Redesigning symmetry‐related “mini‐core” regions of FGF‐1 to increase primary structure symmetry: Thermodynamic and functional consequences of structural symmetry
Authors: Dubey Vikash Kumar , Lee Jihun , Blaber Michael
Published: August 2005

Previous reports detailing mutational effects within the hydrophobic core of human acidic fibroblast growth factor (FGF‐1) have shown that a symmetric primary structure constraint is compatible with a stably folded protein. In the present report, we investigate symmetrically related pairs of buried hydrophobic residues in FGF‐1 (termed “mini‐cores”) that are not part of the central core. The effect upon the stability and function of FGF‐1 mutations designed to increase primary structure symmetry within these “mini‐core” regions was evaluated. At symmetry‐related positions 22, 64, and 108, the wild‐type protein contains either Tyr or Phe side chains. The results show that either residue can be readily accommodated at these positions. At symmetry‐related positions 42, 83, and 130, the wild‐type protein contains either Cys or Ile side chains. While positions 42 and 130 can readily accommodate either Cys or Ile side chains, position 83 is substantially destabilized by substitution by Ile. Tertiary structure asymmetry in the vicinity of position 83 appears responsible for the inability to accommodate an Ile side chain at this position, and is known to contribute to functional half‐life. A mutant form of FGF‐1 with enforced primary structure symmetry at positions 22, 64, and 108 (all Tyr) and 42, 83, and 130 (all Cys) is shown to be more stable than the reference FGF‐1 protein. The results support the hypothesis that a symmetric primary structure within a symmetric protein superfold represents a solution to achieving a foldable, stable polypeptide, and highlight the role that function may play in the evolution of asymmetry within symmetric superfolds.
Young gymnasts’ understanding of sport‐related pain: a contribution to prevention of injury
Authors: Nemeth R. L. , Von Baeyer C. L. , Rocha E. M.
Published: August 2005

Introduction  Pain may signal impending or actual injury, or the achievement of optimum workload to produce a physical conditioning effect. These different functions of pain present a challenge for athletes wanting to improve their skill and conditioning level in the most efficient manner without injury. As children may be particularly vulnerable to exacerbating pain and injury owing to limited knowledge, it is important to learn more about the development of their understanding of pain concepts (e.g. pain as a signal of impending injury vs. soreness from exertion).

Methods  A structured interview and scoring criteria were developed to measure children's understanding of the functions of pain in sport and the consequences of pain and injury. Competitive gymnasts (6–13 years; n = 68; 63% girls) were interviewed and their responses were scored for indices of understanding of different types of pain.

Results  Age differences were found in: number of different types of pain identified; understanding of pain causality; understanding the value of pain; distinguishing pain from exertion; and use of pain descriptors. Analyses revealed that gymnasts responded differently to different types of pain and were aware of the need to stop their sport in some cases and to continue in others. Most did not describe social pressure to continue gymnastics while in pain. Gymnasts demonstrated an understanding that there was little they could do about chronic pain, yet appreciated that pain or damage could worsen with continued practice.

Discussion  This study was a first step in elucidating young gymnasts’ understanding of sport‐related pain. Further research is needed with athletes from other sports, and comparisons should be made with non‐athletic children and those with pain from other sources. Within various sports, it will be important to determine the relative effects of age, sex, and number of hours spent training. Appreciation of individual differences in children's understanding of pain may contribute to prevention of injury in sport. For example, children who understand the difference between soreness from exertion and acute pain owing to injury may be able to make better decisions about pain management and continued practice.
Is the personal child health record used in secondary care?
Authors: Moss A. L. H.
Published: August 2005

Background  Hampshire et al. (2004) have recently reported on the variation in use of the Personal Child Health Record (PCHR) (The Red Book). This study aims to add further data to the discussion.

Objective  To estimate the proportion of parents bringing the Red Book to a hospital surgical outpatient appointment.

Methods  An audit was performed over 4 months ending 14 July 2004. This involved the author noting whether parents brought the Red Book to the Paediatric Plastic and Reconstructive Surgery clinics, on different sites and including specialist multidisciplinary clinics. 200 consecutive attendees were reviewed.
Comparison of a dedicated children's Seizure Clinic to mixed General Paediatric Clinics
Authors: Mar S. , Dunkley C. , Al‐Ansari I. , Whitehouse W. P.
Published: August 2005

Background  In the light of recent recommendations regarding the current management of children with possible epilepsy in the UK, different models of care are compared using an existing validated audit tool.

Methods  The initial clinical assessment process, investigation, management and communication regarding children referred with suspected epilepsy to general paediatric clinics or a paediatric seizure clinic was compared using the British Paediatric Neurology Association (BPNA) audit tool.

Results  Ninety‐three children were included in the comparison. The history and description of the episodes was better documented in the Seizure Clinic (SC). Children's early development (79% vs. 50%) and school performance (86% vs. 42%) were better documented in the SC. Documentation of possible side effects relating to newly prescribed anti‐epileptic drugs was poor in both groups (33% vs. 15%).

Conclusions  Differences between models of epilepsy care can be detected using audit tools although there are some methodological limitations of this particular study. Future similar studies as well as informing local practice can add to the debate on the appropriate way forward in improving the care for children with epilepsy.
Rates and social patterning of household smoking and breastfeeding in contrasting European settings
Authors: Papadimitriou G. , Kotzaeridou U. , Mouratidis C. , Goularas P. , Coe C. , Ganas A. , Spencer N. J.
Published: August 2005

Objective  To compare rates and social patterning of household smoking and breastfeeding in families with newborn infants in birth cohorts in Coventry, UK and Veria, North Greece.

Methods  Infants born in 1996 in Coventry, 1999 in Veria were recruited into birth cohort studies using similar methodologies. In Coventry recruitment was by family health visitor at the primary visit; in Veria, hospital‐based paediatricians enrolled infants at the neonatal examination. Data were collected at the initial contact on household smoking, type of feeding, and household socio‐demographic characteristics. Rates of initial breastfeeding and household smoking with 95% confidence intervals were estimated and breastfeeding and household smoking regressed on parental education and housing tenure in logistic regression models.

Results  Data were available on 2612 Coventry infants and 773 Veria infants. Rates of household smoking and breastfeeding were higher in Veria compared to Coventry. In Coventry, living in rented accommodation and lower maternal and paternal education were associated with household smoking and bottle feeding. Logistic regression models fitted on initiation of breastfeeding failed to show social patterning in Veria but more educated mothers showed a longer duration of breastfeeding. Only low paternal education was associated with household smoking after adjustment for maternal education and housing tenure.

Conclusions  Smoking and breastfeeding are more prevalent among households with young infants in Veria compared with Coventry. The social patterning of health‐related behaviours noted in Coventry is less marked in Veria. The relevance of these findings for public health interventions in the contrasting settings is discussed.
Mothers’ attitudes to the randomized controlled trial (RCT): the case of acute lymphoblastic leukaemia (ALL) in children
Authors: Eiser C. , Davies H. , Jenney M. , Glaser A.
Published: August 2005

Objectives  Survival rates for childhood cancer have improved substantially partly as a result of national and international randomized clinical trials (RCT). However, the decision for families is complex and emotional. Our aim was to describe the views of mothers of children newly diagnosed with ALL regarding consent to randomized controlled trials.

Design  Qualitative interview to explore mothers knowledge, and reasons for involving their child in RCTs. Interviews took place in mothers’ homes.

Participants  Fifty mothers of children with newly diagnosed ALL (age 4–16 years; mean = 7.4) recruited through research nurses at outpatient appointments.

Results  All but three families had consented for their child to be treated in the RCT, although there was wide variation in their understanding of the aims, costs and benefits. Most mothers reported the aim of the trial to compare ‘old’ and ‘new’ treatments.

Conclusion  Despite detailed verbal and written information, mothers were poorly informed about the purpose of the trial, and possibility of side effects. Individual preferences for either standard or new treatment were routinely reported. The data raise questions about the extent to which families give truly informed consent to recruitment of their child to an RCT.
Influence of family environment on language outcomes in children with myelomeningocele
Authors: Vachha B. , Adams R.
Published: August 2005

Background  Previously, our studies demonstrated language differences impacting academic performance among children with myelomeningocele and shunted hydrocephalus (MMSH). This follow‐up study considers the environmental facilitators within families (achievement orientation, intellectual–cultural orientation, active recreational orientation, independence) among a cohort of children with MMSH and their relationship to language performance.

Methods  Fifty‐eight monolingual, English‐speaking children (36 females; mean age: 10.1 years; age range: 7–16 years) with MMSH were evaluated. Exclusionary criteria were prior shunt infection; seizure or shunt malfunction within the previous 3 months; uncorrected visual or auditory impairments; prior diagnoses of mental retardation or attention deficit disorder. The Comprehensive Assessment of Spoken Language (CASL) and the Wechsler Abbreviated Scale of Intelligence (WASI) were administered individually to all participants. The CASL Measures four subsystems: lexical, syntactic, supralinguistic and pragmatic. Parents completed the Family Environment Scale (FES) questionnaire and provided background demographic information. Spearman correlation analyses and partial correlation analyses were performed.

Results  Mean intelligence scores for the MMSH group: full scale IQ 92.2 (SD = 11.9). The CASL revealed statistically significant difficulty for supralinguistic and pragmatic (or social) language tasks. FES scores fell within the average range for the group. Spearman correlation and partial correlation analyses revealed statistically significant positive relationships for the FES ‘intellectual–cultural orientation’ variable and performance within the four language subsystems. Socio‐economic status (SES) characteristics were analyzed and did not discriminate language performance when the intellectual–cultural orientation factor was taken into account.

Conclusion  The role of family facilitators on language skills in children with MMSH has not previously been described. The relationship between language performance and the families’ value on intellectual/cultural activities seems both statistically and intuitively sound. Focused interest in the integration of family values and practices should assist developmental specialists in supporting families and children within their most natural environment.
Overweight and obesity in Irish primary schools: retrospective cohort study
Authors: McMaster C. , Cullen L. , Raymond N.
Published: August 2005

Background  This retrospective cohort study investigates the feasibility of using established methods and routinely generated data from the statutory primary school health‐screening programme to estimate prevalence rates for childhood overweight and obesity in children from a rural area in the Republic of Ireland (ROI).

Method  Paper‐based records in the primary school health service for County Leitrim and parts of County Cavan in north‐west of ROI were hand searched to identify children attending senior infant classes during academic year 2001/2002. Electronic calculation of body mass index (BMI) and age at examination was carried out. Application of age‐ and sex‐specific cut‐off points from International Obesity Task Force (IOTF) and United Kingdom (UK) standard definitions for childhood overweight and obesity was used to determine age‐ and sex‐specific prevalence rates for childhood overweight and obesity.

Results  The eligible cohort was almost completely identified and consisted of 361 children. Weight and height measurements were available on 328 (91%) children aged between 4.22 and 7.92 years. IOTF standard application gave prevalence rates of 25% for obesity and overweight in boys and 26% in girls. With the UK growth standard, this increased to 34% in boys and reduced to 23% in girls.

Conclusion  It is feasible to generate prevalence rates for childhood overweight and obesity from data routinely obtained through the statutory school health‐screening programme in ROI. This study suggests levels of childhood overweight and obesity comparable to other Western societies. Further research on developing a universally accepted standard definition of childhood overweight and obesity is required.
Illness incidence and health seeking behaviour among street children in Rawalpindi and Islamabad, Pakistan – a qualitative study
Authors: Ali M. , De Muynck A.
Published: August 2005

Background  This is the first study in Pakistan to elicit street children's perceptions of health and the barriers to service utilization.

Methods  A descriptive, cross‐sectional study was carried out during September and October 2000. The data were collected in twin‐cities of Rawalpindi and Islamabad through individual, semi‐structured street‐based interviews; with 40 school age participants; in addition, three focus group discussions were also completed. The sampling was convenience based. This strategy was applied because of the non‐existence of a sampling frame for the street‐based children owing to the absence of any census or other reports, and also the difficulty of tracking very mobile street children.

Results  Results indicate that these youth were highly susceptible to many adverse health outcomes. The common ailments were injuries, respiratory and skin infections. Along with low self‐perceived severity of medical problems, self‐medication was preferred and medical pluralism existed. Their perceived constraints to services included long waiting time, monetary, negative attitude of service providers and their inferior status.

Conclusions  In developing user‐friendly services, it is important to be sensitive to street children's needs and requirements. Eliminating these barriers and the integration of health services among public and private resources are imperative for the regular and sustainable provision of health care to this vulnerable, under‐served group of children.
At HOME in Scotland: validation of the home observation for measurement of the environment inventory
Authors: Burston A. , Puckering C. , Kearney E.
Published: August 2005

Background  This study used the Home Observation for Measurement of the Environment (HOME) Inventory to measure aspects of the quality and quantity of psychological stimulation and cognitive support available in the home environment of a sample of Scottish children and to examine the effectiveness of this measure in a British context.

Methods  Forty‐seven families who had previously attended parent‐training programmes at family centres were assessed. The Middle Childhood (MC) and Early Adolescent (EA) version of the HOME were used with children aged between 8 and 13 years old. Measures of family adversity were also taken.

Results  There were no significant differences in the demographics of the MC‐HOME and EA‐HOME groups nor in their total HOME scores. Total HOME scores were compared with independent measures of family adversity. The results of the MC‐HOME and the EA‐HOME were similar to results reported for socially deprived American samples.

Discussion  This Scottish sample had similar MC‐HOME and EA‐HOME scores as previously reported in American samples and the HOME scores related strongly to family adversity, supplying a proximal link between social conditions and the environment of children. These results suggest that the HOME Inventory is reliable and has concurrent validity with measures of social adversity in a British sample.
The Tayside children's sleep questionnaire: a simple tool to evaluate sleep problems in young children
Authors: McGreavey J. A. , Donnan P. T. , Pagliari H. C. , Sullivan F. M.
Published: August 2005

Background  Behavioural sleep problems in young children are relatively common with between 20% and 40% of those aged 1 to 5 years being affected. This paper describes the development of a simple questionnaire to assess disorders of initiating and maintaining sleep (DIMS) in children aged between 1 and 5 years for use as a selection tool for research purposes or as a screening instrument in primary care.

Methods  A subsection of the Sleep Disturbance Scale for Children was adapted and piloted with a small sample of children in two inner city GP practices (n = 81). Face and content validity were initially established by expert review. Discriminant validity was assessed qualitatively using interviews with mothers of identified cases and non‐cases. The validity of the cut‐off score was assessed by blinded case note reviews off known cases; inter‐rater reliability was also calculated. Following modifications, the final questionnaire was posted to a representative sample of parents across the region with children in the appropriate age band (n = 1023). Internal reliability was assessed using Cronbach's alpha and factor analysis was undertaken to identify significant factors within the questionnaire.

Results  The response rate to the population questionnaire was 61.5% (n = 628) with 218 of the children having sleep scores that were indicative of DIMS (35%), echoing other figures reported in the literature. There was good internal consistency for the items (Cronbach's alpha = 0.85) with two main factors accounting for 58% of the variance.

Conclusion  The Tayside Children's Sleep Questionnaire (TCSQ) is an easy‐to‐read and reliable tool that could be used both as a clinical and research instrument to assess the severity and prevalence of DIMS in young children.
Low‐income Canadian Aboriginal and non‐Aboriginal parent–child interactions
Authors: Letourneau N. L. , Hungler K. M. , Fisher K.
Published: August 2005

Background  Limited research is available on the performance of North American Aboriginal1 people on the Nursing Child Assessment Teaching Scales (NCATS) and available research examines parent–child interactions within Aboriginal samples drawn from the USA. Most published normative data on low‐income people's performance on the NCATS are also limited to samples drawn from the USA. The purpose of this research study was to use the NCATS measure to: (1) begin to develop a knowledge base that describes the parent–child interactions observed in Canadian low‐income samples; and (2) explore the relationship between parent–infant interactions and ethnicity (Aboriginal or Non‐Aboriginal) within a low‐income Canadian sample.

Methods  Secondary analysis was completed on data collected as part of a larger study designed to examine the impact of low‐income situations on pre‐school children's health and development in Edmonton, Alberta, Canada. The NCATS measure of parent–child interaction was administered to all children (1–36 months old) and their parents in the low‐income non‐probability sample. The sample derived for secondary analysis consists of 12 Aboriginal parent–child pairs (11 mothers and 1 father) and 48 Non‐Aboriginal parent–child pairs (47 mothers and 1 father).

Results  The data analysis suggests that although low‐income Aboriginal parents may be less verbal with their children in interactions, the overall interaction quality is not different from that of other low‐income parents. However, both groups’ parent–child interaction scores were less than the published 10th percentile cut‐off score, indicating ‘worrisome scores’ and less than optimal interactions.

Conclusion  While the findings that compare the Aboriginal and Non‐Aboriginal samples are limited by the small sample size, the fact that these findings agree with those from heterogeneous Aboriginal samples drawn from the USA are encouraging. Finally, the findings provide needed information about parent–child interactions in Canadian low‐income urban samples including data from Aboriginal parents and children.
Long‐term outcomes of seriously injured children: a study using the child health questionnaire
Authors: Reading Richard
Published: August 2005

.
 
Davey T. M.,
Aitken L. M.,
Kassulke D.,
Bellamy N.,
Ambrose J.,
Gee T. &
Clark M.
(
2005
)

,
41
,
278
–
283
.

Objective  To assess the health‐related quality of life (HRQoL) in children 1–2 years after they had sustained an injury.

Methods  Parents of all children who were identified by the Queensland Trauma Registry during their admission to either of the two paediatric specialty hospitals in Brisbane, Australia, for the treatment of an injury, were invited to participate in this study. Parents who consented to participation received a copy of the Child Health Questionnaire (CHQ) that required them to provide information regarding their child's HRQoL following injury. The CHQ scores for the study respondents were compared with those of the Australian norms. This study was approved by the relevant ethics committees.

Results  Two hundred and forty‐one completed questionnaires were returned. The majority of cases were male (65%) and there was even representation across all age groups. The majority of injuries were considered to be minor (81%) and were predominantly the result of falls and cycling accidents causing mainly fractures and intracranial injury. On the majority of sub‐scales of the CHQ, study participants recorded scores that were statistically significantly below those of the Australian norms. None of the relevant variables collected by the Queensland Trauma Registry were found to predict scores on the CHQ in this study (for those children hospitalized for > 24 h).

Conclusion  Injured children are worse off than their Australian counterparts in terms of HRQoL even up to 2 years following an injury. Further research needs to be undertaken to identify factors that predict lower HRQoL in order to reduce the burden of injury on children and their families.
Association between congenital toxoplasmosis and parent‐reported developmental outcomes, concerns, and impairments, in 3‐year‐old children
Authors: Reading Richard
Published: August 2005

.
 
Freeman K.,
Salt A.,
Prusa A.,
Malm G.,
Ferret N.,
Buffalono W.,
Schmidt D.,
Tan H. K.,
Gilbert R. E. and
. (
2005
)

,
5
,
23
. Retrieved from http://www.biomedcentral.com/bmcpaediatr/ (accessed 14 July 2005).

Background  Information is lacking on the effects of congenital toxoplasmosis on development, behaviour, and impairment in later childhood, as well as on parental concerns and anxiety. This information is important for counselling parents about the prognosis for an infected child and for policy decisions on screening.

Methods  We prospectively studied a cohort of children identified by screening for toxoplasmosis in pregnant women or neonates between 1996 and 2000 in 10 European centres. At 3 years of age, parents of children with and without congenital toxoplasmosis were surveyed about their child's development, behaviour, and impairment, and about parental concerns and anxiety, using a postal questionnaire.

Results  Parents of 178/223 (80%) infected, and 527/821 (64%) uninfected children responded. We found no evidence that impaired development or behaviour were more common in infected children, or that any potential effect of congenital toxoplasmosis was masked by prenatal treatment. Parents of infected children were significantly more anxious and reported more visual problems in their children.

Conclusions  On average, children aged 3 to 4 years with congenital toxoplasmosis identified by screening and treated during infancy in this European setting had risks of abnormal development and behaviour similar to uninfected children. Parental anxiety about infected children needs to be addressed by clinicians. Future studies with longer follow‐up and clinician‐administered assessments may be better able to detect any subtle differences in child outcomes.
Evidence that WbpD is an N‐acetyltransferase belonging to the hexapeptide acyltransferase superfamily and an important protein for O‐antigen biosynthesis in Pseudomonas aeruginosa PAO1
Authors: Wenzel Cory Q. , Daniels Craig , Keates Robert A. B. , Brewer Dyanne , Lam Joseph S.
Published: August 2005

Di‐N‐acetylated uronic acid residues are unique sugar moieties observed in the lipopolysaccharides (LPS) of respiratory pathogens including several serotypes of Pseudomonas aeruginosa and several species of Bordetella. WbpD of P. aeruginosa PAO1 (serotype O5) is a putative 3‐N‐acetyltransferase that has been implicated in the biosynthesis of UDP‐2,3‐diacetamido‐2,3‐dideoxy‐d‐mannuronic acid [UDP‐d‐Man(2NAc3NAc)A], a precursor for the d‐Man(2NAc3NAc)A residues in the B‐band O antigen of this bacterium. A chromosomal knockout mutant of wbpD is incapable of producing either long‐chain B‐band O antigen (≥ 2 repeating units) or semi‐rough LPS (lipid A‐core + one repeat). Adding wbpD in trans restored LPS production to the wild‐type level; this indicates that wbpD is important for biosynthesis of individual B‐band O‐antigen repeating units. WbpD contains left‐handed beta‐helical (LβH) structure as observed by Conserved Domain analysis and in silico secondary and tertiary structure predictions. This feature suggested that WbpD belongs to the hexapeptide acyltransferase (HexAT) superfamily of enzymes. WbpD was overexpressed as an N‐terminally histidine‐tagged fusion protein (His6–WbpD) and purified to > 95% purity. The protein was subjected to Far‐UV circular dichroism spectroscopy, and the data revealed that WbpD contains left‐handed helical structure, which substantiated in silico predictions made earlier. Results from SDS‐PAGE, matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry (MS), and gel filtration analyses indicated that His6‐WbpD has trimeric organization, consistent with the quaternary structure of HexATs. The binding of acetyl‐CoA by WbpD was demonstrated by MALDI‐TOF MS, suggesting that WbpD is an acetyltransferase that utilizes a direct‐transfer reaction mechanism. Incubation of WbpD with acetyl‐CoA significantly enhanced the stability of the protein and prevented precipitation over a course of 14 days. As a substrate for studying the enzymatic activity of WbpD is unavailable at present, a structure‐based model for the LβH domain of WbpD was generated. Comparisons between this model and the LβH domains of known HexATs suggested that Lys136 plays a role in acetyl‐CoA binding. A K136A site‐directed mutant construct could only partially complement the wbpD knockout, and this mutation also reduced the stabilizing effects of acetyl‐CoA, while a K136R mutation showed no discernible effect on complementation of the wbpD mutant or the stabilizing effects of acetyl‐CoA on the purified mutant protein. A modified pathway was proposed for the biosynthesis of UDP‐d‐Man(2NAc3NAc)A, in which WbpD is involved in the catalysis of the fourth step by acting as a UDP‐2‐acetamido‐3‐amino‐2,3‐dideoxy‐d‐glucuronic acid 3‐N‐acetyltransferase.
Rhamnolipids mediate detachment of Pseudomonas aeruginosa from biofilms
Authors: Boles Blaise R. , Thoendel Matthew , Singh Pradeep K.
Published: August 2005

The process of detachment, through which bacteria use active mechanisms to leave biofilms and return to the planktonic (free‐living) state, is perhaps the least understood aspect of the biofilm life cycle. Like other stages of biofilm development, detachment is a dynamic, regulated process, controlled by specific genes, and induced by particular environmental cues. In previous work we discovered Pseudomonas aeruginosa variants that exhibit accelerated biofilm detachment. These hyper‐detaching variants arise spontaneously from biofilms at a high frequency, and they exhibit robust detachment under different biofilm growth conditions. Here we show that these variants detach by a mechanism requiring the biosurfactant rhamnolipid and that this detachment mechanism rapidly restores antibiotic sensitivity to separating bacteria. We also show that rhamnolipids can bring about detachment in wild‐type P. aeruginosa biofilms. These findings raise the possibility that this detachment mechanism may be useful as a treatment to disrupt established biofilms. Interestingly, the rhamnolipid‐mediated detachment mechanism involves the formation of cavities within the centre of biofilm structures. Our data suggest a model to explain detachment that occurs via this pattern.
MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization
Authors: Kim Soonok , Ahn Il‐Pyung , Rho Hee‐Sool , Lee Yong‐Hwan
Published: August 2005

Fungal hydrophobins are implicated in cell morphogenesis and pathogenicity in several plant pathogenic fungi including the rice blast fungus Magnaporthe grisea. A cDNA clone encoding a hydrophobin (magnaporin, MHP1) was isolated from a cDNA library constructed from rice leaves infected by M. grisea. The MHP1 codes for a typical fungal hydrophobin of 102 amino acids containing eight cysteine residues spaced in a conserved pattern. Hydropathy analysis of amino acids revealed that MHP1 belongs to the class II group of hydrophobins. The amino acid sequence of MHP1 exhibited about 20% similarity to MPG1, an M. grisea class I hydrophobin. Expression of MHP1 was highly induced during plant colonization and conidiation, but could hardly be detected during mycelial growth. Transformants in which MHP1 was inactivated by targeted gene replacement showed a detergent wettable phenotype, but were not altered in wettability with water. mhp1 mutants also exhibited pleiotropic effects on fungal morphogenesis, including reduction in conidiation, conidial germination, appressorium development and infectious growth in host cells. Furthermore, conidia of mhp1 mutants were defective in their cellular organelles and rapidly lose viability. As a result, mhp1 mutants exhibited a reduced ability to infect and colonize a susceptible rice cultivar. These phenotypes were recovered by re‐introduction of an intact copy of MHP1. Taken together, these results indicate that MHP1 has essential roles in surface hydrophobicity and infection‐related fungal development, and is required for pathogenicity of M. grisea.
Solving a sticky problem: new genetic approaches to host cell adhesion by the Lyme disease spirochete
Authors: Coburn Jenifer , Fischer Joshua R. , Leong John M.
Published: August 2005

The Lyme disease spirochetes, comprised of at least three closely related species, Borrelia burgdorferi, Borrelia garinii and Borrelia afzelii, are fascinating and enigmatic bacterial pathogens. They are maintained by tick‐mediated transmission between mammalian hosts, usually small rodents. The ability of these bacteria, which have relatively small genomes, to survive and disseminate in both an immunocompetent mammal and in an arthropod vector suggests that they have evolved elegant and indispensable strategies for interacting with their hosts. Recognition of specific mammalian and tick tissues is likely to be essential for successful completion of the enzootic life cycle but, given the historical difficulties in genetic manipulation of these organisms, characterization of factors promoting cell adhesion has until recently largely been confined to either the manipulation of host cells or the analysis of potential bacterial ligands in the form of recombinant proteins. These studies have led to the identification of several mammalian receptors for Lyme disease spirochetes, including glycosaminoglycans, decorin, fibronectin and integrins, as well as a tick receptor for the bacterium, and also candidate cognate bacterial ligands. Recent advances in our ability to genetically manipulate Lyme disease spirochetes, particularly B. burgdorferi, are now providing us with firm evidence that these ligands indeed do promote bacterial adherence to host cells, and with new insights into the roles of these multifacted Borrelia–host cell interactions during mammalian and arthropod infection.
Characterization of the molecular mechanisms involved in the differential production of erythrose‐4‐phosphate dehydrogenase, 3‐phosphoglycerate kinase and class II fructose‐1,6‐bisphosphate aldolase in Escherichia coli
Authors: Bardey Vincent , Vallet Corinne , Robas Nathalie , Charpentier Bruno , Thouvenot Benoit , Mougin Annie , Hajnsdorf Eliane , Régnier Philippe , Springer Mathias , Branlant Christiane
Published: August 2005

A gapA‐pgk gene tandem coding the glyceraldehyde 3‐phosphate dehydrogenase and 3‐phosphoglycerate kinase, is most frequently found in bacteria. However, in Enterobacteriaceae, gapA is replaced by an epd open reading frame (ORF) coding an erythrose‐4‐phosphate dehydrogenase and an fbaA ORF coding the class II fructose‐1,6‐bisphosphate aldolase follows pgk. Although epd expression is very low in Escherichia coli, we show that, in the presence of glucose, the 3 epd, pgk and fbaA ORFs are efficiently cotranscribed from promoter epd P0. Conservation of promoter epd P0 is likely due to its important role in modulation of the metabolic flux during glycolysis and gluconeogenesis. As a consequence, we found that the epd translation initiation region and ORF have been adapted in order to limit epd translation and to create an efficient RNase E entry site. We also show that fbaA is cotranscribed with pgk, from promoter epd P0 or an internal pgk P1 promoter of the extended ‐10 class. The differential expression of pgk and fbaA also depends upon an RNase E segmentation process, leading to individual mRNAs with different stabilities. The secondary structures of the RNA regions containing the RNase E sites were experimentally determined which brings important information on the structural features of RNase E ectopic sites.
NopP, a phosphorylated effector of Rhizobium sp. strain NGR234, is a major determinant of nodulation of the tropical legumes Flemingia congesta and Tephrosia vogelii
Authors: Skorpil Peter , Saad Maged M. , Boukli Nawal M. , Kobayashi Hajime , Ares‐Orpel Florencia , Broughton William J. , Deakin William J.
Published: August 2005

Rhizobium sp. NGR234 nodulates many plants, some of which react to proteins secreted via a type three secretion system (T3SS) in a positive‐ (Flemingia congesta, Tephrosia vogelii) or negative‐ (Crotalaria juncea, Pachyrhizus tuberosus) manner. T3SSs are devices that Gram‐negative bacteria use to inject effector proteins into the cytoplasm of eukaryotic cells. The only two rhizobial T3SS effector proteins characterized to date are NopL and NopP of NGR234. NopL can be phosphorylated by plant kinases and we show this to be true for NopP as well. Mutation of nopP leads to a dramatic reduction in nodule numbers on F. congesta and T. vogelii. Concomitant mutation of nopL and nopP further diminishes nodulation capacity to levels that, on T. vogelii, are lower than those produced by the T3SS null mutant NGRΩrhcN. We also show that the T3SS of NGR234 secretes at least one additional effector, which remains to be identified. In other words, NGR234 secretes a cocktail of effectors, some of which have positive effects on nodulation of certain plants while others are perceived negatively and block nodulation. NopL and NopP are two components of this mix that extend the ability of NGR234 to nodulate certain legumes.
Positioning and the specific sequence of each 13‐mer motif are critical for activity of the plasmid RK2 replication origin
Authors: Kowalczyk Lukasz , Rajewska Magdalena , Konieczny Igor
Published: August 2005

The minimal replication origin of the broad‐host‐range plasmid RK2, oriV, contains five iterons which are binding sites for the plasmid‐encoded replication initiation protein TrfA, four DnaA boxes, which bind the host DnaA protein, and an AT‐rich region containing four 13‐mer sequences. In this study, 26 mutants with altered sequence and/or spacing of 13‐mer motifs have been constructed and analysed for replication activity in vivo and in vitro. The data show that the replacement of oriV 13‐mers by similar but not identical 13‐mer sequences from Escherichia coli oriC inactivates the origin. In addition, interchanging the positions of the oriV 13‐mers results in greatly reduced activity. Mutants with T/A substitutions are also inactive. Furthermore, introduction of single‐nucleotide substitutions demonstrates very restricted sequence requirements depending on the 13‐mer position. Only two of the mutants are host specific, functional in Pseudomonas aeruginosa but not in E. coli. Our experiments demonstrate considerable complexity in the plasmid AT‐rich region architecture required for functionality. It is evident that low internal stability of this region is not the only feature contributing to origin activity. Our studies suggest a requirement for sequence‐specific protein interactions within the 13‐mers during assembly of replication complexes at the plasmid origin.
The moonlighting protein Tsa1p is implicated in oxidative stress response and in cell wall biogenesis in Candida albicans
Authors: Urban Constantin , Xiong Xin , Sohn Kai , Schröppel Klaus , Brunner Herwig , Rupp Steffen
Published: August 2005

Candida albicans is one of the most common fungal pathogens in humans. The cell wall is the first contact site between host and pathogen and thus is critical for colonization and infection of the host. We have identified Tsa1p, a protein that is differentially localized to the cell wall of C. albicans in hyphal cells but remains in the cytosol and nucleus in yeast‐form cells. This is different from Saccharomyces cerevisiae, where the homologous protein solely has been found in the cytoplasm. We report here that TSA1 confers resistance towards oxidative stress as well as is involved in the correct composition of hyphal cell walls. However, no significant change of the cell wall composition was observed in a TSA1 deletion strain in yeast‐form cells, which is in good agreement with the observation that Tsa1p is absent from the yeast‐form cell wall. This indicates that Tsa1p of C. albicans might represent a moonlighting protein with specific functions correlating to its respective localization. Furthermore, the translocation of Tsa1p to the hyphal cell wall of C. albicans depends on Efg1p, suggesting a contribution of the cAMP/PKA pathway to the localization of this protein. In a strain deleted for TUP1 that filaments constitutively Tsa1p can be found in the cell wall under all conditions tested, confirming the result that Tsa1p localization to the cell wall is correlated to the morphology of C. albicans.
Distinct mechanisms govern proteolytic shedding of a key invasion protein in apicomplexan pathogens
Authors: Howell Steven A. , Hackett Fiona , Jongco Artemio M. , Withers‐Martinez Chrislaine , Kim Kami , Carruthers Vern B. , Blackman Michael J.
Published: August 2005

Apical membrane antigen‐1 (AMA1) is a conserved apicomplexan protein that plays an important but undefined role in host cell invasion. We have studied the fate of Plasmodium falciparum AMA1 (PfAMA1) during erythrocyte invasion by the malaria merozoite, and compared it with that of the Toxoplasma gondii orthologue, TgAMA1. Shedding of the PfAMA1 ectodomain goes essentially to completion during invasion, and occurs predominantly or exclusively via juxtamembrane cleavage at the previously identified sheddase cleavage site, Thr517. Only the resulting juxtamembrane stub of the ectodomain is efficiently carried into the host cell, and this remains distributed around the plasma membrane of the intracellular ring‐stage parasite. Inhibition of normal shedding, however, results in proteolysis at an intramembrane, rhomboid‐like cleavage site, and PfAMA1 is susceptible to cleavage by Drosophila rhomboid‐1, showing that it can be a substrate for intramembrane cleavage but is not normally processed in this manner. In contrast, shedding of TgAMA1 from the surface of extracellular tachyzoites occurs exclusively via cleavage within the luminal half of its transmembrane domain by a rhomboid‐like protease. Also unlike PfAMA1, complete TgAMA1 shedding does not accompany Toxoplasma invasion as the intact protein was readily detected on the surface of newly invaded tachyzoites. This work reveals unexpected differences in the manner in which Plasmodium and Toxoplasma shed AMA1 from the surface of invasive zoites, and demonstrates the presence at the malaria merozoite surface of a rhomboid‐like protease.
The α‐toxin of Clostridium septicum is essential for virulence
Authors: Kennedy Catherine L. , Krejany Efrosinia O. , Young Lauren F. , O'Connor Jennifer R. , Awad Milena M. , Boyd Richard L. , Emmins John J. , Lyras Dena , Rood Julian I.
Published: August 2005

Clostridium septicum is the causative agent of spontaneous gas gangrene or atraumatic myonecrosis, a sudden and frequently fatal infection that is increasingly associated with malignancy of the colon. Little is known about the disease process although the focus of virulence studies has been the α‐toxin, a pore‐forming cytolysin that is encoded by the csa gene and secreted as an inactive protoxin. Until now a lack of techniques for the genetic manipulation of C. septicum has hindered the use of molecular approaches to understand pathogenesis. By introducing plasmids by conjugation from Escherichia coli, we have developed methods for the genetic manipulation of C. septicum and constructed a chromosomal csa mutant by allelic exchange. Virulence testing of an isogenic series of strains consisting of the wild type, the csa mutant, and a csa mutant complemented with the wild‐type csa gene revealed that the development of fulminant myonecrosis in mice was dependent on the ability to produce a functional haemolytic α‐toxin. Furthermore, the inhibition of leukocyte influx into the lesion, which is very typical of clostridial myonecrosis, was also dependent on the ability to produce α‐toxin. This study represents the first definitive identification of a virulence factor in this organism and opens the way for further studies that will delineate the role of other putative virulence factors in this significant pathogen.
VirR, a response regulator critical for Listeria monocytogenes virulence
Authors: Mandin Pierre , Fsihi Hafida , Dussurget Olivier , Vergassola Massimo , Milohanic Eliane , Toledo‐Arana Alejandro , Lasa Iñigo , Johansson Jörgen , Cossart Pascale
Published: August 2005

Signature‐tagged mutagenesis (STM) was used to identify new genes involved in the virulence of the Gram‐positive intracellular pathogen Listeria monocytogenes. One of the mutants isolated by this technique had the transposon inserted in virR, a gene encoding a putative response regulator of a two‐component system. Deletion of virR severely decreased virulence in mice as well as invasion in cell‐culture experiments. Using a transcriptomic approach, we identified 12 genes regulated by VirR, including the dlt‐operon, previously reported to be important for L. monocytogenes virulence. However, a strain lacking dltA, was not as impaired in virulence as the ΔvirR strain, suggesting a role in virulence for other members of the vir regulon. Another VirR‐regulated gene is homologous to mprF, which encodes a protein that modifies membrane phosphatidyl glycerol with l‐lysine and that is involved in resistance to human defensins in Staphylococcus aureus. VirR thus appears to control virulence by a global regulation of surface components modifications. These modifications may affect interactions with host cells, including components of the innate immune system. Surprisingly, although controlling the same set of genes as VirR, the putative cognate histidine kinase of VirR, VirS, encoded by a gene located three genes downstream of virR, was shown not to be essential for virulence. By monitoring the activity of VirR with a GFP reporter construct, we showed that VirR can be activated independently of VirS, for example through a mechanism involving variations in the level of intracellular acetyl phosphate. In silico analysis of the VirR‐regulated promoters revealed a VirR DNA‐binding consensus site and specific interaction between purified VirR protein and this consensus sequence was demonstrated by gel mobility shift assays. This study identifies a second key virulence regulon in L. monocytogenes, after the prfA regulon.