bluuebunny/crossref_metadata_2025 huggingface dataset:
Defects of β2‐adrenergic signal transduction in chronic lymphocytic leukaemia: relationship to disease progression
Authors: KAMP T. , LIEBL B. , HAEN E. , EMMERICH B. , HALLEK M.
Published: November 2003

The second messenger 3′:5′‐cyclic adenosine monophosphate (cAMP) inhibits the proliferation of human B lymphocytes. In lymphoid malignancies, cAMP levels or the number of β2‐adrenergic receptors seem to be decreased. In order to explore this phenomenon further, the function of the β2‐adrenergic receptor complex was examined in mononuclear leucocytes (MNLs) from patients with B‐cell chronic lymphocytic leukaemia (CLL). Peripheral blood MNLs from 25 CLL patients (16 male, nine female; aged 62 ± 9 years) and 10 healthy volunteers (seven male, three female; aged 47 ± 19 years) were used. The binding characteristics of β2‐adrenergic receptors (β2‐AR) on MNLs were determined by radioligand binding assays with [125I]‐cyanopindolol ([125I]‐CYP). The number of high‐affinity binding sites for [125I]‐CYP was significantly lower in CLL patients (313 ± 300 sites per cell; mean ± SD) than in control subjects (1479 ± 1268 sites per cell). Moreover, the density of β2‐AR decreased with disease progression, from Binet stage A (371 ± 236, n = 13) to B (236 ± 136, n = 7) and C (141 ± 59, n = 5) (P < 0.05; Kruskal–Wallis analysis). Functional analyses of the β2‐AR complex were performed by measuring the cellular cAMP content of MNLs in response to different stimulators. The cAMP production of MNLs upon isoprenaline stimulation (ISO; 10 min, 10−4 mol L−1) was slightly lower in CLL patients (12.5 ± 7.04 pmol 10−6 cells) than in control subjects (15.91 ± 10.08 pmol 10−6 cells), and decreased with CLL progression (stage A 14 ± 7; stage B 13.66 ± 3.91; stage C 3.07 ± 0.79 pmol 10−6 cells). In contrast, cAMP accumulation in response to cholera toxin (CHO; 10−4 g ml−1, 120 min) was not different in control subjects (70.07 ± 31.30 pmol 10−6 cells) and CLL patients (stage A 95.24 ± 123.07 , stage B 70.76 ± 57.37, stage C 33.21 ± 33.73 pmol 10−6 cells). When stimulated with forskolin (100 μmol L−1, 15 min), control MNLs produced about ten‐fold more cAMP than CLL MNLs (188.56 ± 92.53 vs. 17.88 ± 10.32 pmol 10−6 cells); this response was not stage dependent. Taken together, the results show that the β2‐AR transmembrane signalling is impaired in CLL patients. The correlation of some β2‐AR signalling defects with disease progression suggests that they may contribute to the disease progression of CLL patients.
Circulating immune complexes in human tuberculosis sera: demonstration of specific antibodies against Mycobacterium tuberculosis glycolipid (DAT, PGLTb1, LOS) antigens in isolated circulating immune complexes
Authors: SIMONNEY N. , MOLINA J. M. , MOLIMARD M. , OKSENHENDLER E. , LAGRANGE P. H.
Published: November 2003

An enzyme‐linked immunosorbent assay (ELISA) for IgG using three glycolipid antigens from Mycobacterium tuberculosis in 65 tuberculosis (TB) patients and 50 healthy control subjects was performed. The circulating immune complexes (CICs) were isolated by precipitation with polyethylene glycol 6000 (PEG). This method associated to ELISA measured the specific antibodies present in these CICs. PEG [optical density (OD) 280] was shown to be significantly elevated (P < 0.001) in tuberculous samples. The concentrations of IgG antibodies complexed to the three glycolipid antigens were shown to be higher in patient with tuberculosis than in normal control subjects (P < 0.001). No correlation was observed between levels of free and CIC‐bound antibodies. These antibodies isolated from CICs were responsible for almost all of the false‐negative serological results. However, great heterogeneity was noticed depending on the antigen used, showing a more positive ELISAs against DAT (77%) than against LOS (71%) or PGLTb1 (18.5%). No correlation was established between the presence of specific CIC‐complexed IgG and the bacteriological load or the tuberculosis localization (pulmonary vs. extrapulmonary). The sensitivity of ELISA for CIC‐complexed IgG to DAT and LOS was lower in HIV‐infected TB patients. From these results, we conclude that detection of complexed IgG DAT and LOS glycolipid antigen will be useful as a complementary technique for the serodiagnosis of tuberculosis.
An enzyme immunoassay for polymorphonuclear leucocyte‐mediated fibrinogenolysis
Authors: BOS R. , VAN LEUVEN C. J. M. , STOLK J. , HIEMSTRA P. S. , RONDAY H. K. , NIEUWENHUIZEN W.
Published: November 2003

Upon stimulation, polymorphonuclear leucocytes (PMNs) release potent serine proteases, i.e. elastase, cathepsin G and proteinase 3, which contribute to the degradation of tissue and plasma components. Here, we describe the development of a plasma test to assess PMN‐mediated fibrinogenolysis as a biochemical marker for actual PMN‐derived proteolysis in vivo, useful for monitoring therapeutic efficacy, i.e. of elastase inhibitors. We generated a monoclonal antibody (MAb), designated 1‐1/B3, with a high affinity for elastase‐degraded fibrinogen (EDF). The epitope for 1‐1/B3 becomes exposed in a time‐dependent manner during digestion of fibrinogen with purified PMN‐derived serine proteases and with isolated PMNs in vitro. However, 1‐1/B3 does not react with plasma fibrinogen or with fibrin(ogen) degradation products generated by plasmin or by other active proteases that may occur locally, i.e. metalloproteases and lysosomal cathepsins. On the basis of MAb 1‐1/B3, we developed a plasma test for the assessment of PMN‐mediated fibrin(ogen) degradation products (PMN‐FDP). In a panel of control plasmas, we observed concentrations of PMN‐FDP of 8.2 ± 0.9 ng mL−1 (n = 18). These values were increased twofold in patients with α1‐proteinase inhibitor deficiency (18.6 ± 3.3 ng mL−1; n = 12; P < 0.0001) and even more in patients with sepsis (365.7 ± 97.7 ng mL−1; n = 16; P < 0.0001). Furthermore, synovial tissue extracts from patients with rheumatoid arthritis contained increased levels of PMN‐FDP, compared with synovial tissue extracts (P < 0.005) from patients with osteoarthritis.
Neutrophil superoxide and hydrogen peroxide production in patients with acute liver failure
Authors: CLAPPERTON M. , ROLANDO N. , SANDOVAL L. , DAVIES E. , WILLIAMS R.
Published: November 2003

Defects in superoxide and hydrogen peroxide production may be implicated in the high incidence of bacterial infections in patients with acute liver failure (ALF). In the present study, oxygen radical production in patients with ALF due to paracetamol overdose was compared with that of healthy volunteers. Neutrophils from 14 ALF patients were stimulated via the complement receptors using zymosan opsonized with ALF or control serum. Superoxide and hydrogen peroxide production by ALF neutrophils stimulated with zymosan opsonized with ALF serum was significantly reduced compared with the control subjects (P < 0.01). This defect persisted when zymosan opsonized by control serum was used (P < 0.05). Superoxide and hydrogen peroxide production in neutrophils stimulated with formyl‐methionyl‐leucyl‐phenylalanine (fMLP) from a further 18 ALF patients was unaffected compared with control neutrophils. Serum C3 complement levels were significantly reduced in ALF patients compared with control subjects (P < 0.0005). These results demonstrate a neutrophil defect in ALF due to paracetamol overdose, that is complement dependent but independent of serum complement, possibly connected to the complement receptor.
Low‐density lipoprotein metabolism and its association to plasma lipoprotein(a) in the nephrotic syndrome
Authors: STENVINKEL P. , BERGLUND L. , ERICSSON S. , ALVESTRAND A. , ANGELIN B. , ERIKSSON M.
Published: November 2003

Patients with nephrotic syndrome have multiple abnormalities of lipoprotein metabolism, but the cause and exact nature of these abnormalities have not been established. In the present study we have determined the kinetics of plasma low‐density lipoprotein (LDL) apoB in seven nephrotic patients demonstrating an elevated LDL apoB production rate (25.7 ± 6.4 vs. 13.1 ± 0.3 mg kg–1 day–1; P < 0.001) but a normal LDL apoB fractional catabolic rate (FCR) (0.31 ± 0.04 vs. 0.33 ± 0.008 pools day–1; NS) compared with 41 healthy control subjects. However, two out of the seven patients had a markedly low LDL apoB‐FCR. Serum albumin was inversely correlated with the LDL apoB production rate (R = –0.82; P < 0.05). Plasma lipoprotien (a) [Lp(a)] levels were significantly (P < 0.001) increased in the nephrotic patients compared with control subjects. Significant correlations were observed between log Lp(a) and LDL apoB production rate (R = 0.90; P < 0.01), VLDL‐cholesterol (R = 0.95; P < 0.001) and VLDL‐triglycerides (R = 0.80; P < 0.05) respectively. In summary, the present study suggests that nephrotic hyperlipidaemia may be caused by at least two independent mechanisms. The elevated LDL apoB production rate is highly correlated with the prevailing levels of serum albumin, whereas some nephrotic patients seem to have a decreased LDL apoB clearance, suggesting impaired LDL receptor‐mediated clearance. The present results also suggest that the elevated plasma Lp(a) levels in nephrosis are related to an increased hepatic synthesis rather than a decreased catabolism of lipoproteins.
The diet of Chesapeake Bay striped bass in the late 1950s
Authors: Griffin J. C. , Margraf F. J.
Published: November 2003

The diet of Chesapeake Bay striped bass, (Morone saxatilis) Walbaum, based on unpublished stomach content data from 916 fish collected between 1955 and 1959 was described. The diet in the late 1950s, quantified using an index of relative importance (IRI), was dominated by Atlantic menhaden, Brevoortia tyrannus Latrobe. Atlantic menhaden (66%) and bay anchovy, Anchoa mitchilli Valenciennes, (19%) had the highest IRI value overall. Small striped bass (<600 mm total length) ate predominantly bay anchovy (IRI = 67%). Large striped bass (≥ 600 mm total length) ate predominantly Atlantic menhaden (IRI = 93%). Since 1990 small striped bass rely more on invertebrate prey and larger fish now rely more on small pelagic prey, such as bay anchovy and 0‐age clupeids. Analysis of historical data using current techniques provided a valuable tool for comparison to help in understanding the current striped bass predator–prey relationship in Chesapeake Bay.
Spatial and temporal patterns in the distribution and feeding habits of Morone saxatilis in marsh creeks of Delaware Bay, USA
Authors: Nemerson D. M. , Able K. W.
Published: November 2003

Delaware River and Bay Morone saxatilis (Walbaum), striped bass, have recently undergone a dramatic recovery. Here, we examine spatio‐temporal patterns in the distribution and food habits of striped bass in marsh creeks in the estuary (salinity range 1–20 psu), 1996–2000. Sampling occurred monthly, April through November, with otter trawls (4.9 m, 6 mm mesh, n = 6760) and weirs (intertidal block nets, 6 mm mesh, n = 647) at eight sites. Young‐of‐the‐year (YOY) fish were generally more than 10 times more abundant at oligohaline sites, although they were abundant at mesohaline sites in 1996, the lowest salinity year. Small striped bass (<100 mm) in creeks relied on mysids and other crustaceans and became more piscivorous with size. Overall, they consumed 46% by weight fish, of which 52% were mummichog, Fundulus heteroclitus (L.), and 18% were YOY Atlantic croaker, Micropogonias undulatus (L.), prey types which are not typical in most food habits studies and are indicative of feeding in marsh creeks. These data suggest that young striped bass are an abundant component of Delaware Bay marsh creek fauna and may play an important role in trophic relays within this system.
A retrospective evaluation of traumatized permanent teeth
Authors: Robertson A. , Robertson S. , NorÉn J. G.
Published: November 2003

Summary. The purpose of this retrospective study (15 years follow‐up) was to evaluate the long‐term results of treatment to injured teeth following acute trauma. A total of 198 patients with 488 injured teeth were available for analysis. Uncomplicated crown fractures were restored with composite restorations in 106 teeth. 19% of the restorations had been replaced more than 10 times and at the final examination approximately 25% were rated as unacceptable, i.e. needing clinical treatment. Fixed prosthetic therapy had been provided for 106 teeth and endodontic treatment had been provided in 114. Colour changes appearing at late stage suggested obliteration, necrosis or endodontically treated teeth. Anterior composite restorations have been considered acceptable as semi‐permanent restorations in patients of early school age (7–15 years), and may have been an advance on what was possible previously, but results of this clinical study suggest that they still do have major shortcomings. There remains a need for a simple, conservative method of restoring aesthetics to fractured and discoloured anterior teeth for clinical use in young patients of late school age.
Caries behaviour in Danish teenagers: a longitudinal radiographic study
Authors: Hintze H.
Published: November 2003

Summary. Coinciding with the decline in caries prevalence observed among children and adolescents, changes in the distribution and progression rates of the disease have been found. The aim of this study was to assess occlusal and approximal caries behaviour and disease distribution within a group of Danish teenagers, and to determine whether the presence of occlusal fillings at baseline could be used as predictor for caries behaviour. The material used was bitewing radiographs taken twice in 197 adolescents, at the age of 14·5 years (first examination) and approximately 3 years later (second examination). Of the occlusal surfaces assessed at both examinations, 93% remained unchanged, 1% developed new carious lesions, and 6% had been filled during the study period. All occlusal dentinal lesions found at the first examination and left unrestored during the study period did not appear to have progressed. Of the approximal surfaces, 86% remained unchanged, 9% developed caries, 2% showed caries progression, and 2·5% had been filled during the study period. 22% of the approximal enamel lesions found at the first examination progressed into the dentine during the study period. 18% of all the children were responsible for 50% of the total number of new lesions and fillings. The proportion of children exhibiting new lesions at the second examination was almost evenly distributed among children with and without occlusal fillings at the first examination. In the group of children with occlusal fillings at baseline, significantly more individuals than in the other group showed new fillings and lesions with and without progression. It is concluded that the progression percentage for approximal enamel lesions to penetrate into the dentine was low, that the distribution of new carious lesions and fillings among the children was polarized, and that the presence of occlusal fillings at baseline could not be used to identify children with a specific caries behaviour.
Motivation for dental hygiene in adolescents
Authors: Macgregor I. D. M. , Balding J. W. , Regis D.
Published: November 2003

Summary. The aim of this study was to investigate the relation between some ‘lifestyle’ factors and motivation for toothbrushing in young people. Data from a questionnaire survey of 7770 children aged 14–15 years were analysed to obtain information about their main reason for toothbrushing, their toothbrushing frequency, smoking behaviour and social efficacy. In those who brushed their teeth less frequently, toothbrushing was performed more for cosmetic reasons, that is, having teeth that look and feel good and breath that smells fresh, in contrast to the avoidance of toothache and false teeth, which are health reasons. The more socially advantaged individuals were more inclined to brush their teeth to avoid toothache, and less for the sake of good appearance, compared with the more disadvantaged. Motivation for toothbrushing varied significantly with smoking behaviour, committed smokers were more inclined to brush for cosmetic reasons whereas those who had never smoked were more concerned to avoid dental problems. Similarly, those who had a current friend of opposite sex, and in males, those expressing confidence when meeting members of the opposite sex, brushed more for cosmetic reasons. In conclusion, different people perform their dental hygiene for quite different reasons. These findings need to be taken into account when instructing patients in personal dental care and formulating dental health messages.
Reported infant feeding, oral hygiene and dental attendance patterns in children aged 5 years and under referred for extraction of teeth under general anaesthesia
Authors: HUNTER M. L. , HOOD C. A. , HUNTER B. , KINGDON A.
Published: November 2003

Summary. This study was designed to examine reported infant feeding, oral hygiene, and dental attendance practices in children who had established dental disease before attaining school age. Data were collected by structured interview from the mothers of 150 children aged 5 years and under who were referred to a hospital Paediatric Dentistry Unit for the extraction of teeth under general anaesthesia. Few children in this study had been exclusively breast‐fed, and, although unconfirmed by data derived from matched caries‐free controls, the development of dental caries would appear to be related to prolonged bottle‐feeding, including the use of a night‐time bottle until just before weaning, the early introduction and regular consumption of larger than average amounts of confectionery, coupled with reportedly regular but unsupervised toothbrushing. Although decay was already established in over a third of children before they visited a dentist for the first time, little evidence of restorative care was found. It would appear that the primary care services fail this youngest age‐group.
TRAUMATIC FRACTURES OF PRIMARY MOLARS: A CASE REPORT
Authors: SOVIERO Vera Mendes , GuimarÃes Laura , Miasato JosÉ MASSAO , RAMOS Maria Eliza , ALTO Luciane Almeida MONTE
Published: November 2003

Summary. Dental trauma occurs very often in children and its consequences vary from tiny fractures to the complete avulsion of the tooth. According to Andreasen, 86·5% of incidents of dental trauma suffered by preschool children cause injury to maxillary primary incisors, whereas only 0·5% cause injury to primary molars. This case report is of a male patient aged 7 years who had fallen down a month before, struck his chin against a step, and fractured the maxillary and mandibular left primary second molars (65 and 75) and the mandibular right primary second molar (85). The 65 required only smoothing of sharp edges, 75 received endodontic treatment and a stainless steel crown, and 85 was extracted and a space maintainer fitted. The early diagnosis of fractures of posterior teeth is very important; a thorough examination of all posterior teeth should be made, especially when child suffers trauma to the chin region.
Facial talon cusps
Authors: McNamara Triona , Haeussler Alice M. , Keane Jeanne
Published: November 2003

Summary. This is a report of two patients with isolated facial talon cusps. One occurred on a permanent mandibular central incisor; the other on a permanent maxillary canine. The locations of these talon cusps suggests that the definition of a talon cusp include teeth in addition to the incisor group and be extended to include the facial aspect of teeth.
U.K. National Clinical Guidelines in Paediatric Dentistry
Authors: GREGG TERRY A.
Published: November 2003

In 1994 the Department of Health requested the Royal College of Surgeons to produce National Clinical Guidelines. This task was delegated to the Paediatric Dentistry Clinical Audit Committee which is a subcommittee of the Consultants in Paediatric Dentistry Group of United Kingdom and Ireland.The purpose of Clinical Guidelines is to improve the effectiveness and efficiency of clinical care through the identification of good clinical practice and desired clinical outcomes. Each Guideline is intended to assist clinicians in making decisions about appropriate management of specific conditions. The aim has been to choose commonly encountered clinical situations and make recommendations on their management. In many areas of practice there is a shortage of reliable research data, so that although some recommendations are supported by robust data, others are made with a lesser degree of confidence, and may represent only ‘best current practice’. An anticipated benefit is that shortage of data will be highlighted, so stimulating research aimed at improving the scientific foundation of our clinical activity. It will be important to refine the existing Guidelines as further information becomes available, and also to add to the number of Guidelines in the future.The production of a guideline follows a consistent and thorough method. Draft authors were asked to review the scientific literature on selected topics and produce a draft guideline which was then circulated to an ‘Expert Panel’ for comment and opinion. Expert panels varied according to the subject of the guideline and consisted of all Consultants, the British Society of Paediatric Dentistry and individuals from other disciplines who were identfied as having a particular expertise in that subject. Where applicable each guideline consists of three broad sections. The first section is a series of recommendations for diagnosis and management, the second contains explanatory notes relating to the evolution of these recommendations, and the third section contains references and comments to assist further research into the subject. A final Guideline was eventually produced which, if appropriate, was assessed, according to the Scottish Intercollogiate Guideline Network (SIGN) classification, as to whether it was based on proven scientific evidence or currently accepted good clinical practice with limited scientific evidence. The SIGN classification is shown in Table 1 together with the Grading system used in the text of each document. The first guideline is published below.It should be understood that a Clinical Guideline is intended to assist the clinician in the management of patients in an effective and efficient way. It is not intended to restrict clinical freedom in the management of an individual case.
The effect of different methods of drinking on the pH of dental plaque in vivo
Authors: TAHMASSEBI J. F. , DUGGAL M. S.
Published: November 2003

Summary. The effect on the pH of human dental plaque of rinsing and drinking with or without a straw was investigated in eight volunteers using the plaque sampling method. A fruit‐based juice was used as the test drink with 10% sucrose and 10% sorbitol solutions serving as positive and negative controls, respectively. The maximum mean plaque pH drop (±SD) when a fruit juice was consumed with a straw (0·82±0·27) was significantly less (P<0·05) as compared to when it was consumed straight from a cup without a straw (1·14±0·25) and when it was used as a rinse (1·25±0·21). Also, the minimum plaque pH reached after challenge was significantly lower (P<0·0005) when the subjects rinsed before swallowing (5·22±0·13) as compared with drinking with a straw (5·92±0·39) or without (5·64±0·2). The area under the plaque pH curve was also found to be significantly less when a straw was used (1·81±0·71) as compared with drinking without (2·72±0·93). Drinking with a straw therefore resulted in a less pronounced pH drop and hence might reduce the demineralizing effect on the teeth of various potentially cariogenic drinks.
The influence of medical professionalism on scientific practice
Authors: Kirk‐Smith Michael D. , Stretch David D.
Published: November 2003

This paper examines how the practise of science in medicine may be subverted by the professionalism of medicine. The requirements of science as regards the axiom of the burden of proof and research design may be inevitably entangled with the influence of authority. This entanglement may be particularly strong in medical research because of the way the profession works and is organized. The nature and possible effects of this entanglement are explored by examining the cultural differences between scientists and professionals and their approach to authority. Then the nature of the axiom of burdens of proof and research design are described, followed by accompanying discussions of how aspects of authority and professional organization may influence them. Finally, concluding comments address ways forward.
A qualitative evaluation of information leaflets for gastroscopy procedure
Authors: Parahoo Kader , Ridley Tanya , Thompson Kate , Melby Vidar , Humphreys George
Published: November 2003

Written information sent to patients prior to diagnostic gastroscopy is an important part of the process of informing and preparing them for the procedure. Yet there is ample evidence in the literature that information leaflets do not measure up to the required standard. In this study, information leaflets from a random sample of seven hospitals in Northern Ireland that carried out gastroscopy as a day procedure were evaluated using a checklist of items recommended by the British Society of Gastroenterology (BSG) for inclusion in leaflets for patients undergoing diagnostic gastroscopy. The results showed that the number of written materials sent to patients prior to the procedure varied between units. There were inconsistencies in the information given by the same unit, and overall, there was a lack of vital information in most of the leaflets. Some of the information was confusing and ambiguous. The potential risk of the procedure was explained in only one of the leaflets. Patients’ right to choose to have a mild sedative was not made clear in most of the leaflets. More should be done to address these gaps and inconsistencies in the written information provided to patients prior to gastroscopy.
Improving blood gas control in mechanically ventilated, premature infants through monitoring and evaluation of clinical practice
Authors: Steinmetz Jacob , Greisen Gorm
Published: November 2003

Mechanical ventilation causing hypocapnia or hyperoxia carries a risk for the pre‐term infant. The aim was to improve blood gas control in our unit. A guideline was written, and all personnel were motivated concerning blood gas control. Case records of all mechanically ventilated premature infants were examined during two 3‐month periods, before and after intervention. The hours spent with hypocapnia (pCO2 < 4 kPa) or hyperoxia (pO2 > 12 kPa) were recorded. Case records of 31 infants were examined for a total of 1358 h of mechanical ventilation, 641 h before and 717 h after the intervention. The percentage time of hypocapnia before intervention (7.0%) was reduced significantly (P = 0.044) to less than half (2.9%) after intervention. Hyperoxia was reduced from 14.5% to 8.7% (P = 0.072). Blood gas control of mechanically ventilated premature infants could be improved with little effort, but hyperoxia is too frequent.
Applying a ‘stages of change’ model to enhance a traditional evaluation of a research transfer course
Authors: Buckley Leslie L. , Goering Paula , Parikh Sagar V. , Butterill Dale , Foo Emily K. H.
Published: November 2003

The aim of this study was to utilize an evaluation tool based on Prochaska's model of change in order to assess behaviour change as part of an evaluation process for a research transfer training programme (RTTP). The RTTP was a training programme offered to scientists in a psychiatry department and research institute to gain skills in research transfer. In addition to a traditional course evaluation framework evaluating overall satisfaction with the course and whether or not learning objectives were met, an additional ‘stages of change’ evaluation tool designed to assess change along a continuum was utilized. This instrument measured change in participants’ attitudes, intentions and actions with respect to research transfer practice and consisted of a 12‐question survey completed by participants prior to taking the course and 3 months post‐course. In two out of the three categories, attitudes and intention to practice, there was positive change from pre‐ to post‐course (P < 0.05). Although there was a trend of increased RT‐related action, this was less robust and did not reach significance. For the RTTP transfer course, a ‘stages of change’ model of evaluation provided an enhanced understanding by showing changes in participants that would otherwise have been overlooked if only changes in RT behaviour were measured. Additionally, evaluating along a change continuum specifically identifies areas for improvement in future courses. The instrument developed for this study could also be used as a pre‐course, participant needs assessment to tailor a course to the change needs of participants. Finally, this ‘stages of change’ approach provides insight into where barriers to change may exist for research transfer action.
Dynamics of experimental production of Thelohanellus hovorkai (Myxozoa: Myxosporea) in fish and oligochaete alternate hosts
Authors: Liyanage Y S , Yokoyama H , Wakabayashi H
Published: November 2003

The dynamics of development and production of Thelohanellus hovorkai (Myxozoa) were examined to investigate factors inducing haemorrhagic thelohanellosis in carp, Cyprinus carpio L. Fresh actinospores of T. hovorkai were harvested from the oligochaete alternate host, Branchiura sowerbyi, and used for infection experiments with myxosporean‐free carp. Visualization of actinospores by fluorescent labelling revealed that sporoplasms penetrated the gill filaments of carp immersed in an actinospore suspension as early as 30 min post‐exposure (PE). Plasmodia of T. hovorkai developed in the connective tissues of various organs and matured 3–5 weeks PE; dispersion of myxospores from degenerate plasmodia occurred 5–7 weeks PE. Challenges with a high dose of actinospores (4.5 × 106 spores per fish) resulted in the onset of disease, which was more easily achieved by the oral intubation of actinospores than by immersion in an actinospore suspension. Actinosporean‐free B. sowerbyi were exposed to different densities of myxospores (104–106 spores per oligochaete) and subsequently reared at different temperatures (15, 20, 25 °C). At 20 and 25 °C, actinospore releases were first detected 40–43 days PE, with multiple peaks of release (max. 7 × 105 actinospores day−1) during the next 60 days. We concluded that the developmental cycle of T. hovorkai was completed within 3–5 months at 20–25 °C, and that the ingestion of large numbers of actinospores orally, possibly by feeding on infected oligochaetes, resulted in a disease condition in carp.
Genome‐based detection methods of Macrobrachium rosenbergii nodavirus, a pathogen of the giant freshwater prawn, Macrobrachium rosenbergii: dot‐blot, in situ hybridization and RT‐PCR
Authors: Sri Widada J , Durand S , Cambournac I , Qian D , Shi Z , Dejonghe E , Richard V , Bonami J R
Published: November 2003

The availability of specific and rapid detection methods is essential for monitoring the health status of farmed species, particularly in viral diseases as in this case early diagnosis is a critical factor in containing disease outbreaks. Three complementary genome‐based methods were developed for the detection of Macrobrachium rosenbergii nodavirus (MrNV), i.e. dot‐blot hybridization, in situ hybridization and reverse transcriptase‐polymerase chain reaction (RT‐PCR). Detection limits were established for dot‐blot hybridization and RT‐PCR and are c. 7 fg and 8 pg of viral RNA, respectively. In situ hybridization indicated that infection was confined to the striated muscle tissue. As a result of its sensitivity, RT‐PCR can be used for in‐depth investigations to examine the extent of the viral infection and establish the onset of infection in hatcheries. The application of RT‐PCR on samples collected from prawn farms in China showed the possible use of this method in routine health monitoring.
Reduced total hardness of fresh water enhances the efficacy of bathing as a treatment for amoebic gill disease in Atlantic salmon, Salmo salar L.
Authors: Roberts S D , Powell M D
Published: November 2003

The current treatment for amoebic gill disease (AGD)‐affected Atlantic salmon involves bathing sea‐caged fish in fresh water, often sourced from local dams, for 3–4 h. In both a small‐scale laboratory and an on‐farm field experiment, the effects of water hardness on the efficacy of freshwater bathing were assessed. Results showed that soft fresh water (19.3–37.4 mg L−1 CaCO3), whether it be naturally soft city mains water or artificially softened dam water, was more efficacious at alleviating AGD in affected fish than hard fresh water (173–236.3 mg L−1 CaCO3). Soft freshwater bathing significantly reduced viable gill amoebae numbers (from 73.9 to 40.9% of total count) and significantly alleviated gill pathology, both gross and histological. Following bathing, gross gill pathological scores of soft freshwater bathed fish lagged 2 weeks behind hard freshwater bathed fish. Significant gill lesion fragmentation, and shedding of lesion‐associated hyperplastic tissue, was accompanied by a significant reduction in AGD‐affected gill filaments in soft freshwater bathed fish. Furthermore, soft freshwater bathing alleviated the blood plasma electrolyte imbalance seen in control (sea water) and hard freshwater bathed fish. This study showed that the use of soft fresh water for bathing AGD‐affected Atlantic salmon could be an improvement to the current method of treatment. Not only does it reduce gill amoeba numbers, but also, it is of a therapeutic advantage with the potential to reduce bathing frequency.
Amoebic gill disease: sequential pathology in cultured Atlantic salmon, Salmo salar L.
Authors: Adams M B , Nowak B F
Published: November 2003

Amoebic gill disease (AGD) affects the marine culture phase of Atlantic salmon, Salmo salar L., in Tasmania. Here, we describe histopathological observations of AGD from smolts, sampled weekly, following transfer to estuarine/marine sites. AGD was initially detected histologically at week 13 post‐transfer while gross signs were not observed for a further week post‐transfer. Significant increases (P < 0.001) in the proportion of affected gill filaments occurred at weeks 18 and 19 post‐transfer coinciding with the cessation of a halocline and increased water temperature at the cage sites. The progression of AGD histopathology, during the sampling period, was characterized by three phases. (1) Primary attachment/interaction associated with extremely localized host cellular alterations, juxtaposed to amoebae, including epithelial desquamation and oedema. (2) Innate immune response activation and initial focal hyperplasia of undifferentiated epithelial cells. (3) Finally, lesion expansion, squamation–stratification of epithelia at lesion surfaces and variable recruitment of mucous cells to these regions. A pattern of preferential colonization of amoebae at lesion margins was apparent during stage 3 of disease development. Together, these data suggest that AGD progression was linked to retraction of the estuarine halocline and increases in water temperature. The host response to gill infection with Neoparamoeba sp. is characterized by a focal fortification strategy concurrent with a migration of immunoregulatory cells to lesion‐affected regions.
Role of multiple ArcA recognition sites in anaerobic regulation of succinate dehydrogenase (sdhCDAB ) gene expression in Escherichia coli
Authors: Shen Joan , Gunsalus Robert P.
Published: November 2003

Succinate dehydrogenase (sdhCDAB ) gene expression in Escherichia coli is negatively regulated by the arcA and fnr gene products during anaerobic cell growth conditions. The controlled synthesis of this sole membrane‐bound enzyme of the tricarboxylic acid cycle allows optimal participation in the aerobic electron transport pathway for the generation of energy via oxidative phosphorylation reactions. To understand how ArcA participates in the anaerobic repression of sdhCDAB expression, a family of sdhC–lacZ fusions was constructed and analysed in vivo. DNase I footprint and gel shift assays using purified ArcA protein revealed the location of four distinct and independent ArcA binding sites in the sdhC promoter region. ArcA sites, designated sites 1 and 2, are centred at −205 bp and −119 bp upstream of the sdhC promoter, respectively, whereas ArcA site 3 overlaps the −35 and −10 regions of the sdhC promoter. A fourth ArcA site is centred at + 257 bp downstream of the sdhC promoter. They are bound with differing affinity by ArcA and ArcA phosphate. The in vivo studies, in combination with the in vitro studies, indicate that ArcA site 3 is necessary and sufficient for the ArcA‐dependent repression of sdhC gene expression, while the DNA region containing ArcA site 2 contributes to maximal gene expression. The DNA‐containing ArcA sites 1 and 4 provide minor roles in the ArcA regulation of sdhC expression. Lastly, the Fnr‐dependent control of sdhCDAB gene expression was shown to occur independently of the ArcA and to require DNA sequences near the start of sdhC transcription.
Cell‐surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S‐layer of Caulobacter crescentus
Authors: Bingle Wade H. , Nomellini John F. , Smit John
Published: November 2003

The paracrystalline surface (S)‐layer of Caulobacter crescentus is composed of a single secreted protein (RsaA) that interlocks in a hexagonal pattern to completely envelop the bacterium. Using a genetic approach, we inserted a 12 amino acid peptide from Pseudomonas aeruginosa strain K pilin at numerous semirandom positions in RsaA. We then used an immunological screen to identify those sites that presented the inserted pilin peptide on the C. crescentus cell surface as a part of the S‐layer. Eleven such sites (widely separated in the primary sequence) were identified, demonstrating for the first time that S‐layers can be readily exploited as carrier proteins to display ‘epitope‐size’ heterologous peptides on bacterial cell surfaces. Whereas intact RsaA molecules carrying a pilin peptide could always be found on the surface of C. crescentus regardless of the particular insertion site, introduction of the pilin peptide at 9 of the 11 sites resulted in some proteolytic cleavage of RsaA. Two types of proteolytic phenomena were observed. The first was characterized by a single cleavage within the pilin peptide insert with both fragments of the S‐layer protein remaining anchored to the outer membrane. The other proteolytic phenomenon was characterized by cleavage of the S‐layer protein at a point distant from the site of the pilin peptide insertion. This cleavage always occurred at the same location in RsaA regardless of the particular insertion site, yielding a surface‐anchored 26 kDa proteolytic fragment bearing the RsaA N‐terminus; the C‐terminal cleavage product carrying the pilin peptide was released into the growth medium. When the results of this work were combined with the results of a previous study, the RsaA primary sequence could be divided into three regions with respect to the location of a peptide insertion and its effect on S‐layer biogenesis: (i) insertions in the extreme N‐terminus of RsaA either produce no apparent effect on S‐layer biogenesis or disrupt surface‐anchoring of the protein; (ii) insertions in the extreme C‐terminus either produce no apparent effect on S‐layer biogenesis or disrupt protein secretion; and (iii) insertions more centrally located in the protein either have no apparent effect on S‐layer biogenesis or result in proteolytic cleavage of RsaA. These data are discussed in relation to our previous assignment of the RsaA N‐ and C‐terminus as regions that are important for surface anchoring and secretion respectively.
Virus‐like, double‐stranded RNAs in the parasitic protozoan Cryptosporidium parvum
Authors: Khramtsov Nikolai V. , Woods Keith M. , Nesterenko Michael V. , Dykstra Christine C. , Upton Steve J.
Published: November 2003

We have discovered and analysed two novel, linear extrachromosomal double‐stranded RNAs (dsRNAs) within oocysts of major north Amercian isolates of Cryptosporidium parvum, a parasitic protozoan that infects the gastrointestinal tract of a variety of mammals, including humans. These dsRNAs were found to reside within the cytoplasm of sporozoites, and were not detected in other species of the genus. cDNAs representing both dsRNA genomes were cloned and sequenced, 1786 and 1374 nt, and each encoded one large open reading frame (ORF). The deduced protein sequence of the larger dsRNA (L‐dsRNA) had homology with viral RNA‐dependent RNA polymerases (RDRP), with more similarity to polymerases from fungi than those from other protozoa. The deduced protein sequence from the smaller dsRNA (S‐dsRNA) had limited similarity with mitogen‐activated c‐June NH2 terminal protein kinases (JNK) from mammalian cells. Attempts to visually identify or purify virus‐like particles associated with the dsRNAs were unsuccessful. Sensitivity of the dsRNAs to RNase A also suggests that the dsRNAs may be unencapsidated. A RDRP activity was identified in crude extracts from C. parvum sporozoites and products of RNA polymerase activity derived in vitro were similar to the dsRNAs purified directly from the parasites.
Sok antisense RNA from plasmid R1 is functionally inactivated by RNase E and polyadenylated by poly(A) polymerase I
Authors: Dam Mikkelsen Nikolaj , Gerdes Kenn
Published: November 2003

The hok/sok system of plasmid R1, which mediates plasmid stabilization by the killing of plasmid‐free cells, codes for two RNA species, Sok antisense RNA and hok mRNA. Sok RNA, which is unstable, inhibits translation of the stable hok mRNA. The 64 nt Sok RNA folds into a single stem‐loop domain with an 11 nt unstructured 5′ domain. The initial recognition reaction between Sok RNA and hok mRNA takes place between the 5′ domain and the complementary region in hok mRNA. In this communication we examine the metabolism of Sok antisense RNA. We find that RNase E cleaves the RNA 6 nt from its 5′ end and that this cleavage initiates Sok RNA decay. The RNase E cleavage occurs in the part of Sok RNA that is responsible for the initial recognition of the target loop in hok mRNA and thus leads to functional inactivation of the antisense. The major RNase E cleavage product (denoted pSok‐6) is rapidly degraded by polynucleotide phosphorylase (PNPase). Thus, the RNase E cleavage tags pSok−6 for further rapid degradation by PNPase from its 3′ end. We also show that Sok RNA is polyadenylated by poly(A) polymerase I (PAP I), and that the poly(A)‐tailing is prerequisite for the rapid 3′‐exonucleolytic degradation by PNPase.
Deletion analysis of cspA of Escherichia coli: requirement of the AT‐rich UP element for cspA transcription and the downstream box in the coding region for its cold shock induction
Authors: Mitta Masanori , Fang Li , Inouye Masayori
Published: November 2003

In order to analyse the mechanism of cold shock induction of CspA, a major cold shock protein of Escherichia coli, deletion analysis of the cspA gene was carried out. It was found that (i) the AT‐rich sequence (−47 to −38) upstream of the cspA−35 region may act as the UP element playing a crucial role in cspA transcription at both 37°C and 15°C; (ii) the unusually long 5′‐UTR of the cspA mRNA has negative effects on cspA expression at 37°C; and (iii) in contrast, the 5′‐UTR exerts a positive effect on mRNA stabilization at low temperature. Furthermore, it was demonstrated that the 14 base downstream box (DB) locating 12 bases downstream of the initiation codon of the cspA mRNA and complementary to a region near the decoding region of 16S rRNA was essential for the mRNA translation during the growth lag acclimation phase immediately after cold shock. During this phase, translation of non‐cold shock gene mRNAs is blocked, since they require cold shock‐specific ribosomal factors for the formation of the translation initiation complex. It is proposed that DB in cold shock mRNAs allows the formation of a stable initiation complex at low temperature in the absence of the cold shock ribosomal factors.
Proline 21, a residue within the α‐helical domain of ΦX174 lysis protein E, is required for its function in Escherichia coli
Authors: Witte Angela , Schrot Gerald , Schön Petra , Lubitz Werner
Published: November 2003

ΦX174 lysis protein E‐mediated lysis of Escherichia coli is characterized by a protein E‐specific fusion of the inner and outer membrane and formation of a transmembrane tunnel structure. In order to understand the fusion process, the topology of protein E within the envelope complex of E. coli was investigated. Proteinase K protection studies showed that, during the time course of protein E‐mediated lysis process, more of the fusion protein E‐FXa‐streptavidin gradually became accessible to the protease at the cell surface. These observations postulate a conformational change in protein E during induction of the lysis process by movement of the C‐terminal end of the protein throughout the envelope complex from the inner side to the outer side spanning the entire pore and fusing the inner and outer membranes at distinct areas. The initiation mechanism for such a conformational change could be the cis–trans isomerization of proline residues within α‐helical membrane‐spanning segments. Conversion of proline 21, presumed to be in the membrane‐embedded α‐helix of protein E, to alanine, glycine, serine and valine, respectively, resulted in lysis‐negative E mutant proteins. Proteinase K accessibility studies using streptavidin as a reporter fused to the P21G mutant protein showed that the C‐terminal part of the fusion protein is not translocated to the outer side of the membrane, suggesting that this proline residue is essential for the correct folding of protein E within the cell wall complex of E. coli. Oligomerization of protein P21G‐StrpA was not disturbed.
Pheromone‐induced production of antimicrobial peptides in Lactobacillus
Authors: Brurberg May B. , Nes Ingolf F. , Eijsink Vincent G. H.
Published: November 2003

Production of the bacteriocin sakacin P by Lactobacillus sake LTH673 is dependent on a secreted 19‐residue peptide pheromone (IP‐673). The gene encoding IP‐673 (sppIP) was identified and sequenced. SppIP was shown to be co‐transcribed with genes encoding a histidine kinase (sppK ) and a response regulator (sppR) typical for signal transduction in bacteria. Further sequencing and transcription studies have shown that IP‐673 induces transcription of its own gene and of what are often considered to be all genes necessary for bacteriocin production and immunity. Studies with a reporter gene showed that the promoter in front of the sakacin P structural gene (sppA) is strictly regulated. The promoter in front of sppIP turned out to be less strictly regulated, and low basal promoter activity could be detected in uninduced cells. Bacteriocin production in Bac− isolates of L. plantarum C11 could be induced by the non‐cognate IP‐673 only after the introduction of sppK, indicating that sppK encodes the pheromone receptor. These results show that bacteriocin production in lactobacilli is regulated using a short, strain‐specific peptide pheromone. Growth conditions were shown to have considerable effects on the functionality of this regulatory mechanism.