bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparison of the minimum canal wall thickness remaining following preparation using two nickel–titanium rotary systems
Authors: Garala M. , Kuttler S. , Hardigan P. , Steiner‐Carmi R. , Dorn S.
Published: September 2003

Aim  To examine in vitro whether Profile or Hero 642 nickel–titanium rotary instrument systems substantially reduce the minimum canal wall thickness present following root‐canal preparation.

Methodology  The canal anatomy of 26 mesial roots from mandibular molars with separate buccal and lingual canals was evaluated before and after instrumentation using the Endodontic Cube. Following access, working lengths were determined and then samples invested within the cube using acrylic resin. Canal cross‐sections were subsequently prepared. Samples were then instrumented within the cube and pre‐ and post‐instrumentation images of the sections were compared to determine the minimum canal wall thickness remaining after preparation.

Results  Buccal and lingual canals within the sections from each level (coronal to apical) showed a reduction in minimum canal wall thickness after instrumentation. No statistically significant differences in canal wall thickness were found between the two systems at any level of the root.

Conclusions  Instrumentation with either of the two systems under investigation did not compromise canal wall thickness. Pre‐operative canal wall thickness was found to be the most significant factor determining the minimum canal wall thickness after preparation.
Examination on Candida spp. in refractory periapical granulomas
Authors: Waltimo T. , Kuusinen M. , Järvensivu A. , Nyberg P. , Väänänen A. , Richardson M. , Salo T. , Tjäderhane L.
Published: September 2003

Aim  To examine the occurrence of Candida spp. in refractory periapical granulomas.

Methodology  One hundred and three surgically removed periapical granulomas were subjected to molecular analysis for the occurrence of Candida albicans. DNA was extracted from the samples using a modified phenol/chloroform/isoamyl alcohol method and was subjected to polymerase chain reaction (PCR) with OPA‐03 and repetitive sequence (GACA)4 primers. The PCR products were separated in agarose gel electrophoresis, stained with ethidium bromide, visualized using UV light and the sequences were analysed. Samples indicating possible occurrence of Candida were further investigated by histological and immunohistological methods. Periodic acid–Shiff staining (PAS) was used to detect yeast cells and hyphae, and specific monoclonal antibodies to recognize high molecular mass mannoproteins present in the C. albicans cell wall. DNA extraction was controlled by running PCR using β‐actin primers (a housekeeping gene). C. albicans CCUG19915, C. tropicalis ATCC750, C. krusei ATCC6258, C. guilliermondii ATCC6260 and C. glabrata CCUG32725 served as positive controls in PCR. A tissue preparation of chronic atrophic candidosis in oral buccal mucosa served as a positive control for histological and immunohistological examinations.

Results  Polymerase chain reaction with β‐actin primers indicated successful DNA extraction in 68 out of 103 samples. The majority of the samples (50) were negative whereas 18 of the samples showed PCR products indicating possible occurrence of Candida spp. PAS‐staining and immunohistological examination of these samples were, however, negative. Further analysis of the PCR products revealed sequences not typical for Candida spp.

Conclusions  Candida spp. do not seem to occur in periapical granuloma.
Nursing Minimum Data Set development and implementation in Thailand
Authors: Volrathongchai Kanittha , Delaney Connie W. , Phuphaibul Rutja
Published: August 2003

Background. In 1997, the Thai Ministry of Public Health began planning to implement a national health information system. Development of the nursing component of this system is an ongoing process. The first step in developing a nursing information system is to identify an essential Nursing Minimum Data Set (NMDS).

Aim. To describe the development of a NMDS in Thailand and explore the challenges of implementing it, including the issue of the comparability with data sets in other countries, primarily the United States of America.

Methods. The process of developing a NMDS specific to Thailand is reviewed. Strategies for implementing this data set and important issues related to it are then discussed.

Findings. Although a preliminary Thai NMDS has been identified, challenges associated with its development and implementation within the Thai National health information system remain.

Conclusion. A Thai NMDS and its elements have been identified. The International Classification of Nursing Practice was translated and is to be used to implement the data set describing the nursing care of patients and their families. However, many issues, such as the need for conceptual translation and increasing nurses’ involvement in the process, still need to be addressed in order to implement the data set successfully.
Nurse practitioner practice and deployment: electronic mail Delphi study
Authors: Marsden Janet , Dolan Brian , Holt Lynda
Published: August 2003

Background. The nature of modern government and needs of policy‐makers demand accurate information that is delivered quickly. This study was part of a larger project for the Department of Health relating to nurse practitioner (NP) education and practice in UK.

Aims. The aim of the study was to identify the principal factors that help or hinder the development of NP roles in the National Health Service.

Methods. In order to facilitate a rapid response, a Delphi study was undertaken using electronic mail (e‐mail) and was completed within 4 weeks. Key stakeholders in NP practice, education and research and (non‐governmental) policy‐making were invited to participate. Key themes relating to the deployment and practice of NPs were generated. These were refined and collated by the research team and then rated by the respondents.

Results. This approach generated valuable expert consensus data around NP deployment and practice. Nurse practitioners’ practice is recognized as an integral part of health care that needs to be recognized by regulatory bodies to promote understanding of their potential at local levels. Nurse practitioners need freedom to innovate, adequate support and appropriate education to allow true autonomy. Limited prescribing and local restrictions on requesting investigations hinder practice.

Limitations. Although this was a small‐scale study, the expert panel was wide‐ranging and achieved substantial consensus.

Conclusion. The organisational and cultural changes that are required to foster the development of practice and deployment of NPs have yet to be instigated, according to the comments of the key stakeholders in this study. There appears to be a clear message that the government needs to take a more interventionist role in supporting nursing developments, rather than leaving this to local arrangements.
Families’ needs in emergency departments: instrument development
Authors: Redley Bernice , LeVasseur Sandra A. , Peters Gwenda , Bethune Elizabeth
Published: August 2003

Background. Families who accompany critically ill relatives in emergency departments (EDs) are an integral part of the care unit. However, there are few empirical data on their needs during this phase of care. In order to guide quality care, general and specific needs of families accompanying these critically ill relatives should be systematically examined.

Aim. The aim of this pilot project was to test the tool, methods and analysis plan for a study to examine the perceived needs of family members accompanying critically ill patients in EDs and their perceptions of ED staff's ability to meet these needs.

Method. Over a 6‐week period in 1996, 84 relatives who met the inclusion criteria were recruited to the study. A postal questionnaire, to uncover the needs of family members, was pilot tested. The questionnaire consisted of 40 need statements reflecting five major themes: meaning, proximity, communication, comfort and support. Of the 84 relatives selected for the study, 73% returned completed questionnaires.

Results. The findings of this pilot study suggest that the questionnaire is a valid and reliable tool for researchers wishing to examine and rank the needs of family members who accompany critically ill people in EDs. In addition, the analysis plan was found to be appropriate.

Conclusions. This pilot study provides both a method and a tool for further research into family needs. Examination of the pilot data supported the reliability and validity of the tool and produced findings that challenge nurses to move beyond traditional practice that has excluded families from being an integral part of caring for critically ill patients in EDs.
Nurses’ attitudes towards older residents with long‐term schizophrenia
Authors: Hellzén Ove , Kristiansen Lisbeth , Norbergh Karl Gustaf
Published: August 2003

Background. In institutional care, a symptom‐oriented approach is a frequently used but seldom‐discussed method for treating people with severe mental illness.

Aim. To investigate whether nurses’ approach could be explained with reference to a client's individual clinical picture or the fact that they had a diagnosis of schizophrenia.

Methods. An exploratory study of the staff's view of a caring approach for a fictitious older long‐term schizophrenic resident was conducted. All nurses working in the field of psychiatry at seven different units in one municipality in northern Sweden were an integral part of the study. The units were divided into two groups and classified as ‘dwelling’ or ‘support’. The ‘dwelling group’ was characterized by nurses working at traditional group dwellings, and the ‘support group’ by nurses working in small teams and visiting people with long‐term mental illness in their homes. Responses were received from 62 women and 23 men, of whom 14 were Registered Nurses and 69 were Enrolled Nurses. A questionnaire was used, developed from a case description of a 68‐year‐old woman with typical symptoms of severe cognitive decline, with problematic behaviour and a diagnosis of long‐term schizophrenia.

Findings. The main finding was that nurses with long experience became less sensitive in their relationship with the resident than less experienced nurses. There appeared to be a tendency for long work experience to have a negative effect on nurses’ attitudes towards the resident.

Conclusions. The nurses could be interpreted as being caught in a moral dilemma between ends and means. This dilemma could be represented on the one hand as the ‘conformist mode’, with an acceptance of ends and means, and on the other hand as the ‘innovation mode’, with acceptance of ends but with few legitimate means to achieve them.
Preterm infants’ behavioural indicators of oxygen decline during bottle feeding
Authors: Thoyre Suzanne M. , Carlson John R.
Published: August 2003

Background. During the time when preterm infants’ oral feeding skills are developing they often experience physiological instability and need assistance from caregivers to maintain adequate oxygenation. Assisting infants to maintain optimal oxygenation during oral feeding requires an understanding of how they express and aim to self‐regulate their oxygen status.

Aim. The purpose of this study was to identify potential behavioural indicators of declining oxygenation during preterm infant early bottle‐feeding.

Method. The design was explorative. Data were collected from a secondary analysis of 20 videotapes of preterm infant bottle feedings which included concurrent oxygen saturation data. In this analysis infant behaviours and quality of breathing were coded and compared across three periods: high oxygen saturation, immediately preceding an oxygen desaturation event, and during an oxygen desaturation event.

Findings. Infants gave limited behavioural indicators of declining oxygenation. Immediately prior to a desaturation event, they had an increase in eye flutter and were typically sucking and apnoeic. During a desaturation event, they typically relaxed their arms/hands and stopped sucking.

Conclusions. Reliance on preterm infant behavioural cues will be insufficient for detection of oxygen desaturation during oral feeding. Attention to changes in breath sounds and to the pattern of sucking are potentially important intervention strategies for the prevention of and appropriate response to oxygen decline during feeding. Sucking pauses may be a time when preterm infants aim to regulate their breathing pattern and thereby increase oxygenation. Interventions that focus on detection and minimization of apnoea during feeding, and which aim to protect infant sucking pauses, may reduce the number and severity of desaturation events preterm infants experience during bottle feeding.
The relationship between flesh quality and numbers of Kudoa thyrsites plasmodia and spores in farmed Atlantic salmon, Salmo salar L.
Authors: Dawson‐Coates J A , Chase J C , Funk V , Booy M H , Haines L R , Falkenberg C L , Whitaker D J , Olafson R W , Pearson T W
Published: September 2003

Atlantic salmon, Salmo salar L., were exposed to Kudoa thyrsites (Myxozoa, Myxosporea)‐containing sea water for 15 months, and then harvested and assessed for parasite burden and fillet quality. At harvest, parasites were enumerated in muscle samples from a variety of somatic and opercular sites, and mean counts were determined for each fish. After 6 days storage at 4 °C, fillet quality was determined by visual assessment and by analysis of muscle firmness using a texture analyzer. Fillet quality could best be predicted by determining mean parasite numbers and spore counts in all eight tissue samples (somatic and opercular) or in four fillet samples, as the counts from opercular samples alone showed greater variability and thus decreased reliability. The variability in both plasmodia and spore numbers between tissue samples taken from an individual fish indicated that the parasites were not uniformly distributed in the somatic musculature. Therefore, to best predict the probable level of fillet degradation caused by K. thyrsites infections, multiple samples must be taken from each fish. If this is performed, a mean plasmodia count of 0.3 mm−2 or a mean spore count of 4.0 × 105 g−1 of tissue are the levels where the probability of severe myoliquefaction becomes a significant risk.
Experimental production of gastric dilation and its association with osmoregulatory stress and biogenic amines in chinook salmon, Oncorhynchus tshawytscha (Walbaum)
Authors: Lumsden J S , Marshall S , Gillard M , Wybourne B , Minamikawa M
Published: September 2003

Chinook salmon smolt in fresh water fed a commercial diet known to produce minimal gastric dilation and air sacculitis (GDAS) were randomly assigned to four experimental tanks with flow‐through sea water. All four groups were acclimatized to sea water for 3 weeks and fed a diet of minced fresh seafood. After 3 weeks the groups were fed either; seafood as before, a different commercial pelleted diet associated with the development of GDAS on farms, or either diet supplemented with 500 mg L−1 putrescine, 300 mg L−1 cadaverine and 250 mg L−1 tyramine. Gastric dilation was produced in fish fed the commercial diet for 1 month but not by feeding a diet of minced seafood. The addition of putrescine, cadaverine and tyramine to either diet had no significant effect on the development of gastric dilation. Fish fed the commercial diet had significantly (P < 0.0001) wider weight‐adjusted stomach widths, less prominent longitudinal stomach folds (P < 0.0001) and lower (P < 0.0001) stomach‐width ratios than fish fed the fresh seafood diet. There was no significant difference in serum osmolality or sodium concentration between fish from groups with or without gastric dilation or fed biogenic amines.
Comparative challenge model of Flavobacterium columnare using abraded and unabraded channel catfish, Ictalurus punctatus (Rafinesque)
Authors: Bader J A , Nusbaum K E , Shoemaker C A
Published: September 2003

The early entry of the fish pathogen Flavobacterium columnare and enhancement by abrasion was studied in channel catfish, Ictalurus punctatus (Rafinesque), using the polymerase chain reaction and a species‐specific primer set for a bacterial 16S rRNA gene product. Evaluations were conducted following an abrasion bath immersion challenge with F. columnare. Abrasion, a practice which has historically been used prior to bacterial challenge, had significant effects on the early entry of the pathogen and on cumulative percent survival (CPS). The FvpF1–FvpR1 primer set was useful in detecting the early entry of F. columnare in mucus, skin, gill, blood, liver and trunk kidney tissues in both abraded and unabraded fish following immersion challenge at 29 ± 2 °C. Bacteria were detected earlier in all tissues in abraded fish, except in the trunk kidney. These differences were not significant, except in the case of blood. Mucus, skin and gill tissues were positive for F. columnare earliest regardless of treatment (after 5 min in abraded fish and after 15 min in unabraded fish). CPS following challenge with F. columnare was significantly affected by abrasion, which supports the use of abrasion for the F. columnare challenge model for channel catfish.
Synergistic effects of dietary iron and omega‐3 fatty acid levels on survival of farmed Atlantic salmon, Salmo salar L., during natural outbreaks of furunculosis and cold water vibriosis
Authors: Rørvik K‐A , Dehli A , Thomassen M , Ruyter B , Steien S H , Salte R
Published: September 2003

The present study demonstrates that farmed Atlantic salmon, Salmo salar, health is positively and significantly affected by synergistic effects between very long‐chain polyunsaturated fatty acids of the n‐3 family eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA) and iron, where positive effects of high dietary levels of EPA/DHA are enhanced when combined with low levels of iron. Based on cumulative mortalities in the different experimental groups, relative percentage of survival (RPS) for the high EPA/DHA‐low iron group was 70% during an outbreak of furunculosis and 96% during an outbreak of cold water vibriosis compared with the controls. A non‐additive effect between EPA/DHA and iron was confirmed by statistical analyses that revealed a significant effect of EPA/DHA alone and an interaction of iron with EPA/DHA. Liver cell cultures treated with EPA/DHA revealed that the synergistic effect could be related to an EPA/DHA dependent regulation of mRNA for proteins important for transport (transferrin) and storage (ferritin) of iron in the salmon. In keeping with this finding, the transcriptional down‐regulation of iron metabolism in vitro was reflected in decreased in vivo iron stores with increasing levels of dietary EPA/DHA. Hence, to avoid overloading of the iron transport/storage‐systems resulting in increased susceptibility to bacterial infections, high levels of dietary EPA/DHA should be accompanied by low levels of dietary iron.
White spot syndrome virus infection in cultured Penaeus vannamei (Boone) in Ecuador with emphasis on histopathology and ultrastructure
Authors: Rodríguez J , Bayot B , Amano Y , Panchana F , De Blas I , Alday V , Calderón J
Published: September 2003

Mortalities of cultured shrimp, Penaeus vannamei (Boone), induced by white spot syndrome virus (WSSV) have occurred in Ecuador since May 1999. Three epidemiological surveys in Ecuadorian farms were carried out and showed an apparent association between lower temperature and increased mortality rates in commercial ponds. Infected animals showed a reddish discolouration and lethargy and occasionally, white spots in the exoskeleton. Histopathological studies revealed that infected cells presented nuclear hypertrophy with eosinophilic to basophilic inclusions. In some cases, two other pathologies were observed: (a) lymphoid organ spheroids and (b) cells with pyknotic and karyorrhectic nuclei in the lymphoid organ, haematopoietic tissue, connective tissue, heart and antennal gland. Occasionally pyknotic cells were encapsulated without apparent injury to the adjacent tissue and without melanization. Transmission electron microscopy showed the presence of WSSV particles in the cytoplasm of cells with pyknotic nuclei in the stomach hypodermis. Viral structure and morphogenesis agreed with previous descriptions by other authors in WSSV‐infected shrimp. Occasionally, two nucleocapsids within one envelope were present amongst single enveloped nucleocapsids. A long rod‐shaped structure that could reach 2.4 μm in length was present in the nuclei of some infected cells.
Channel catfish virus gene expression in experimentally infected channel catfish, Ictalurus punctatus (Rafinesque)
Authors: Stingley R L , Griffin B R , Gray W L
Published: September 2003

Channel catfish virus (CCV) produces an acute haemorrhagic disease in fingerling channel catfish and establishes latent infection in fish that survive the primary infection. This study investigated CCV gene expression in tissues of experimentally infected fish. Reverse transcriptase polymerase chain reaction assays were developed for detection of transcripts expressed by each of the CCV direct repeat region genes in CCV‐infected channel catfish ovary cells and in tissues of infected fish. Immediate‐early, early and late gene transcripts were detected in the blood, brain, kidney and liver tissues of acutely infected catfish demonstrating active viral replication in multiple tissues during the early stages of CCV infection. However, there was no evidence for viral replication by 24 days post‐infection in tissues of fish that survived the acute disease. Viral latency‐associated transcripts encoded by CCV direct repeat genes were not detected in latently infected catfish. The results of this study provide a foundation for further studies to investigate the molecular basis of CCV pathogenesis and latency.
Valproate disrupts regulation of inositol responsive genes and alters regulation of phospholipid biosynthesis
Authors: Ju Shulin , Greenberg Miriam L.
Published: September 2003

Valproate (VPA) is one of the two drugs approved by the Food and Drug Administration (FDA) for the treatment of bipolar disorder. The therapeutic mechanism of VPA has not been established. We have shown previously that growth of the yeast Saccharomyces cerevisiae in the presence of VPA causes a decrease in intracellular inositol and inositol‐1‐P, and a dramatic increase in expression of INO1, which encodes the rate limiting enzyme for de novo inositol biosynthesis. To understand the underlying mechanism of action of VPA, INO1, CHO1 and INO2 expression, intracellular inositol and phospholipid biosynthesis were studied as a function of acute and chronic exposure of growing cells to the drug. A decrease in intracellular inositol was apparent immediately after addition of VPA. Surprisingly, expression of genes that are usually derepressed during inositol depletion, including INO1, CHO1 and INO2 (that contain inositol‐responsive UASINO sequences) decreased several fold during the first hour, after which expression began to increase. Incorporation of 32Pi into total phospholipids was significantly decreased. Pulse labelling of CDP‐DG and PG, shown previously to increase during inositol depletion, increased within 30 min. However, pulse labelling of PS, which normally increases during inositol depletion, was decreased within 30 min. PS synthase activity in cell extracts decreased with time, although VPA did not directly inhibit PS synthase enzyme activity. Thus, in contrast to the effect of chronic VPA treatment, short‐term exposure to VPA abrogated the normal response to inositol depletion of inositol responsive genes and led to aberrant synthesis of phospholipids.
RacA and the Soj‐Spo0J system combine to effect polar chromosome segregation in sporulating Bacillus subtilis
Authors: Wu Ling Juan , Errington Jeff
Published: September 2003

Sporulating cells of Bacillus subtilis undergo a highly polarized cell division and possess a specialized mechanism to move the oriC region of the chromosome close to the cell pole before septation. DivIVA protein, which localizes to the cell pole, and the Soj and Spo0J proteins, which associate with the chromosome, are part of the mechanism that delivers the chromosome to the cell pole. A sporulation‐specific protein, RacA, encodes a third DNA‐binding protein, which acts in conjunction with Soj and Spo0J to effect efficient polar chromosome segregation. divIVA mutants and soj racA double mutants have an unexpected phenotype in which specific markers to the left and right of oriC can be captured in the prespore compartment but the central oriC region is efficiently excluded. This ‘residual’ trapping requires Spo0J protein. We suggest that the Soj RacA DivIVA system is required to extract the oriC region from its position determined by the vegetative chromosome segregation machinery and anchor it to the cell pole.
Dynamic control of Dps protein levels by ClpXP and ClpAP proteases in Escherichia coli
Authors: Stephani Kunigunde , Weichart Dieter , Hengge Regine
Published: September 2003

The Escherichia coli starvation‐induced DNA protection protein Dps was observed to be degraded rapidly during exponential growth. This turnover is dependent on the clpP and clpX genes. The clpA gene is not required for Dps proteolysis, suggesting that Dps is a substrate for ClpXP protease but not for ClpAP protease. Dps proteolysis was found to be highly regulated. Upon carbon starvation, Dps is stabilized, which together with increased Dps synthesis allows strong accumulation of Dps in the stationary phase. The addition of glucose to starving cells results in rapid resumption of Dps proteolysis by ClpXP. Oxidative stress also leads to efficient stabilization of Dps. After hyperosmotic shift, however, proteolysis remains unaffected. Thus, regulated proteolysis of Dps strongly contributes to controlling Dps levels under very specific stress conditions. In contrast to the regulated degradation of RpoS by ClpXP, Dps proteolysis is independent of the recognition factor RssB. In addition, during starvation, clpP and, to a somewhat lesser extent, clpA are involved in maintaining ongoing Dps synthesis (acting at the level of Dps translation), which is required for strong Dps accumulation in long‐term stationary phase cells. In summary, both ClpXP and ClpAP exert significant control of Dps levels by affecting log phase stability and stationary phase synthesis of Dps respectively.
The global transcriptional response of Bacillus subtilis to manganese involves the MntR, Fur, TnrA and σB regulons
Authors: Guedon Emmanuel , Moore Charles M. , Que Qiang , Wang Tao , Ye Rick W. , Helmann John D.
Published: September 2003

We have used DNA microarrays to monitor the global transcriptional response of Bacillus subtilis to changes in manganese availability. Mn(II) leads to the MntR‐dependent repression of both the mntH and mntABCD operons encoding Mn(II) uptake systems. Mn(II) also represses the Fur regulon. This repression is unlikely to be a direct effect of Mn(II) on Fur as repression is sensitive to 2,2′‐dipyridyl, an iron‐selective chelator. We suggest that elevated Mn(II) displaces iron from cellular‐binding sites and the resulting rise in free iron levels leads to repression of the Fur regulon. Many of the genes induced by Mn(II) are activated by σB or TnrA. Both of these regulators are controlled by Mn(II)‐dependent enzymes. Induction of the σB‐dependent general stress response by Mn(II) is largely dependent on RsbU, a Mn(II)‐dependent phosphatase that dephosphorylates RsbV, ultimately leading to release of active σB from its antisigma, RsbW. The activity of TnrA is inhibited when it forms an inactive complex with feedback‐inhibited glutamine synthetase. Elevated Mn(II) reduces the sensitivity of glutamine synthetase to feedback inhibitors, and we suggest that this leads to the observed increase in TnrA activity. In sum, three distinct mechanisms can account for most of the transcriptional effects elicited by manganese: (i) direct binding of Mn(II) to metalloregulators such as MntR, (ii) perturbation of cellular iron pools leading to increased Fur activity and (iii) altered activity of Mn(II)‐dependent enzymes that regulate the activity of σB and TnrA.
Attenuated virulence of Streptococcus agalactiae deficient in D‐alanyl‐lipoteichoic acid is due to an increased susceptibility to defensins and phagocytic cells
Authors: Poyart Claire , Pellegrini Elisabeth , Marceau Michael , Baptista Marina , Jaubert Francis , Lamy Marie‐Cécile , Trieu‐Cuot Patrick
Published: September 2003

D‐alanylation of lipoteichoic acid (LTA), allows Gram‐positive bacteria to modulate their surface charge, regulate ligand binding and control the electromechanical properties of the cell wall. In this study, the role of D‐alanyl LTA in the virulence of the extracellular pathogen Streptococcus agalactiae was investigated. We demonstrate that a DltA– isogenic mutant displays an increased susceptibility to host defence peptides such as human defensins and animal‐derived cationic peptides. Accordingly, the mutant strain is more susceptible to killing by mice bone marrow‐derived macrophages and human neutrophils than the wild‐type strain. In addition, the virulence of the DltA– mutant is severely impaired in mouse and neonatal rat models. This mutant was eliminated more rapidly than the wild‐type strain from the lung of three‐week‐old mice inoculated intranasally and, consequently, is unable to induce a pneumonia. Finally, after intravenous injection of three‐week‐old mice, the survival of the DltA– mutant is markedly reduced in the blood in comparison to that of the wild‐type strain. We hypothesize that the decreased virulence of the DltA– mutant is a consequence of its increased susceptibility to cationic antimicrobial peptides and to killing by phagocytes. These results demonstrate that the D‐alanylation of LTA contributes to the virulence of S. agalactiae.
Tetracycline‐aptamer‐mediated translational regulation in yeast
Authors: Hanson Shane , Berthelot Karine , Fink Barbara , McCarthy John E. G. , Suess Beatrix
Published: September 2003

We describe post‐transcriptional gene regulation in yeast based on direct RNA–ligand interaction. Tetracycline‐dependent translational regulation could be imposed via specific aptamers inserted at two different positions in the 5′ untranslated region (5′UTR). Translation in vivo was suppressed up to ninefold upon addition of tetracycline. Repression via an aptamer located near the start codon (cap‐distal) in the 5′UTR was more effective than repression via a cap‐proximal position. On the other hand, suppression in a cell‐free system reached maximally 50‐fold and was most effective via a cap‐proximal aptamer. Examination of the kinetics of tetracycline‐dependent translational inhibition in vitro revealed that preincubation of tetracycline and mRNA before starting translation led not only to the fastest onset of inhibition but also the most effective repression. The differences between the behaviour of the regulatory system in vivo and in vitro are likely to be related to distinct properties of mRNP structure and mRNA accessibility in intact cells as opposed to cell‐extracts. Tetracycline‐dependent regulation was also observed after insertion of an uORF sequence upstream of the aptamer, indicating that our system also targets reinitiating ribosomes. Polysomal gradient analyses provided insight into the mechanism of regulation. Cap‐proximal insertion inhibits binding of the 43S complex to the cap structure whereas start‐codon‐proximal aptamers interfere with formation of the 80S ribosome, probably by blocking the scanning preinitiation complex.
PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP, the cognate phospho‐Ser/Thr phosphatase, in Mycobacterium tuberculosis
Authors: Boitel Brigitte , Ortiz‐Lombardía Miguel , Durán Rosario , Pompeo Fréderique , Cole Stewart T. , Cerveñansky Carlos , Alzari Pedro M.
Published: September 2003

Bacterial genomics revealed the widespread presence of eukaryotic‐like protein kinases and phosphatases in prokaryotes, but little is known on their biochemical properties, regulation mechanisms and physiological roles. Here we focus on the catalytic domains of two trans‐membrane enzymes, the Ser/Thr protein kinase PknB and the protein phosphatase PstP from Mycobacterium tuberculosis. PstP was found to specifically dephosphorylate model phospho‐Ser/Thr substrates in a Mn2+‐dependent manner. Autophosphorylated PknB was shown to be a substrate for Pstp and its kinase activity was affected by PstP‐mediated dephosphorylation. Two threonine residues in the PknB activation loop, found to be mostly disordered in the crystal structure of this kinase, namely Thr171 and Thr173, were identified as the target for PknB autophosphorylation and PstP dephosphorylation. Replacement of these threonine residues by alanine significantly decreased the kinase activity, confirming their direct regulatory role. These results indicate that, as for eukaryotic homologues, phosphorylation of the activation loop provides a regulation mechanism of mycobacterial kinases and strongly suggest that PknB and PstP could work as a functional pair in vivo to control mycobacterial cell growth.
TPR‐mediated interaction of RapC with ComA inhibits response regulator‐DNA binding for competence development in Bacillus subtilis
Authors: Core Leighton , Perego Marta
Published: September 2003

The Bacillus subtilis Rap family of proteins are characterized by protein–protein interaction modules containing the so‐called tetratricopeptide repeats (TPRs). The six TPR motifs of RapC mediate its interaction with the pentapeptide inhibitor PhrC (ERGMT) or with its target protein ComA, a phosphorylation‐dependent response regulator transcription factor for genetic competence. Our results show that RapC interaction with ComA inhibits the response regulator's ability to bind its target DNA promoter but does not affect its phosphorylation state. RapC binds equally well to ComA or to ComA∼P. The PhrC pentapeptide binds to RapC and inhibits its interaction with ComA. The D195 residue in TPR3 and the P263 residue in TPR5 of RapC are critical for the interaction with PhrC as their mutation to asparagine or leucine, respectively, prevents peptide inhibitory activity. The RapC mechanism of regulating ComA activity is a new example of how TPR motifs and their structural organization have been adapted for different specific functions within the B. subtilis Rap family.
Essential role of DivIVA in polar growth and morphogenesis in Streptomyces coelicolor A3(2)
Authors: Flärdh Klas
Published: September 2003

Streptomycetes grow by cell wall extension at hyphal tips. The molecular basis for such polar growth in prokaryotes is largely unknown. It is reported here that DivIVASC, the Streptomyces coelicolor homologue of the Bacillus subtilis protein DivIVA, is essential and directly involved in hyphal tip growth and morphogenesis. A DivIVASC‐EGFP hybrid was distinctively localized to hyphal tips and lateral branches. Reduction of divIVASC expression to about 10% of the normal level produced a phenotype strikingly similar to that of many tip growth mutants in fungi, including irregular curly hyphae and apical branching. Overexpression of the gene dramatically perturbed determination of cell shape at the growing tips. Furthermore, staining of nascent peptidoglycan with a fluorescent vancomycin conjugate revealed that induction of overexpression in normal hyphae disturbed tip growth, and gave rise to several new sites of cell wall assembly, effectively causing hyperbranching. The results show that DivIVASC is a novel bacterial morphogene, and it is localized at or very close to the apical sites of peptidoglycan assembly in Streptomyces hyphae.
Genes controlled by the essential YycG/YycF two‐component system of Bacillus subtilis revealed through a novel hybrid regulator approach
Authors: Howell Alistair , Dubrac Sarah , Andersen Kasper Krogh , Noone David , Fert Juliette , Msadek Tarek , Devine Kevin
Published: September 2003

The YycG/YycF two‐component system, originally identified in Bacillus subtilis, is very highly conserved and appears to be specific to low G + C Gram‐positive bacteria. This system is required for cell viability, although the basis for this and the nature of the YycF regulon remained elusive. Using a combined hybrid regulator/transcriptome approach involving the inducible expression of a PhoP′‐′YycF chimerical protein in B. subtilis, we have shown that expression of yocH, which encodes a potential autolysin, is specifically activated by YycF. Gel mobility shift and DNase I footprinting assays were used to show direct binding in vitro of purified YycF to the regulatory regions of yocH as well as ftsAZ, previously reported to be controlled by YycF. Nucleotide sequence analysis and site‐directed mutagenesis allowed us to define a potential consensus recognition sequence for the YycF response regulator, composed of two direct repeats: 5′‐TGT A/T A A/T/C‐N5‐TGT A/T A A/T/C‐3′. A DNA‐motif analysis indicates that there are potentially up to 10 genes within the B. subtilis YycG/YycF regulon, mainly involved in cell wall metabolism and membrane protein synthesis. Among these, YycF was shown to bind directly to the region upstream from the ykvT gene, encoding a potential cell wall hydrolase, and the intergenic region of the tagAB/tagDEF divergon, encoding essential components of teichoic acid biosynthesis. Definition of a potential YycF recognition sequence allowed us to identify likely members of the YycF regulon in other low G + C Gram‐positive bacteria, including several pathogens such as Listeria monocytogenes, Staphylococcus aureus and Streptococcus pneumoniae.
A supramolecular complex in the environmental stress signalling pathway ofBacillus subtilis
Authors: Chen Chien‐Cheng , Lewis Richard J. , Harris Robin , Yudkin Michael D. , Delumeau Olivier
Published: September 2003

σB, an alternative sigma‐factor ofBacillus subtilis,mediates the response of the cell to a variety of physical insults. Within the environmental stress signalling pathway RsbU, a protein phosphatase, is stimulated by its interaction with the protein kinase RsbT. In the absence of stress RsbT is expected to be trapped by an alternative binding partner, RsbS. Here, we have demonstrated that RsbS alone cannot act as an alternative partner for RsbT, but instead requires the presence of RsbR to create a high molecular mass RsbR:RsbS complex (∼ 1 MDa) able to capture RsbT. In this complex the phosphorylation state of RsbS, and not that of RsbR, controlled the binding to RsbT, whose kinase activity towards RsbS could be counterbalanced by the activity of RsbX, the phosphatase for RsbS‐P. The RsbR:RsbS complex recruited RsbT from a mixture of RsbT and RsbU. The phosphorylated form of RsbR in the complex enhanced the kinase activity of RsbT towards RsbS. This supramolecular complex thus has the functional properties of an alternative partner for RsbT. Electron micrographs of this complex are presented, and the purification of the RsbR:RsbS complex from cellular extracts provides evidence for the existence of such a complexin vivo.
The HfaB and HfaD adhesion proteins of Caulobacter crescentus are localized in the stalk
Authors: Cole Jennifer L. , Hardy Gail G. , Bodenmiller Diane , Toh Evelyn , Hinz Aaron , Brun Yves V.
Published: September 2003

The differentiating bacterium Caulobacter crescentus produces two different cell types at each cell division, a motile swarmer cell and an adhesive stalked cell. The stalked cell harbours a stalk, a thin cylindrical extension of the cell surface. The tip of the stalk is decorated with a holdfast, an adhesive organelle composed at least in part of polysaccharides. The synthesis of the stalk and holdfast occur at the same pole during swarmer cell differentiation. Mutations in the hfaABDC gene cluster had been shown to disrupt the attachment of the holdfast to the tip of the stalk, but the role of individual genes was unknown. We used lacZ fusions of various DNA fragments from the hfaABDC region to show that these genes form an operon. In order to analyse the relative contribution of the different genes to holdfast attachment, mutations were constructed for each gene. hfaC was not required for holdfast attachment or binding to surfaces. The hfaA and hfaD mutants shed some holdfast material into the surrounding medium and were partially deficient in binding to surfaces. Unlike hfaA and hfaB mutants, hfaD mutants were still able to form rosettes efficiently. Cells with insertions in hfaB were unable to bind to surfaces, and lectin binding studies indicated that the hfaB mutants had the strongest holdfast shedding phenotype. We determined that HfaB and HfaD are membrane‐associated proteins and that HfaB is a lipoprotein. Purification of stalks and cell bodies indicated that both HfaB and HfaD are enriched in the stalk as compared to the cell body. These results suggest that HfaB and HfaD, and probably HfaA, serve to anchor the holdfast to the tip of the stalk.
Binding of response regulator DegU to the aprE promoter is inhibited by RapG, which is counteracted by extracellular PhrG in Bacillus subtilis
Authors: Ogura Mitsuo , Shimane Kana , Asai Kei , Ogasawara Naotake , Tanaka Teruo
Published: September 2003

We screened the putative rap‐phr (response regulator aspartyl‐phosphate phosphatase‐phosphatase regulator) systems identified in the Bacillus subtilis genome for a rap gene that affects aprE (alkaline protease gene) expression by using a multicopy plasmid. We found that rapG was involved in the regulation of aprE, which belongs to the regulon of DegU, the response regulator of the DegS‐DegU two‐component system. Disruption of rapG and phrG resulted in enhancement and reduction of aprE‐lacZ expression, respectively, suggesting that PhrG inhibits RapG activity. Addition of 1–30 nM of a synthetic pentapeptide (PhrG; NH2‐EKMIG‐COOH) to the phrG disruptant completely rescued aprE‐lacZ expression, indicating that the PhrG peptide is indeed involved in aprE‐lacZ expression. Surprisingly, either introduction of multicopy phrG or addition of the PhrG peptide at high concentrations (100–300 nM) to the phrG cells decreased aprE‐lacZ expression. These results are reminiscent of the previous observation that at higher concentrations the PhrC peptide inhibits srfA‐lacZ expression directed by ComA, the regulator of the ComP‐ComA two‐component system. Because the Rap proteins belong to a family of aspartyl protein phosphatases, we tried to investigate the possible influence of RapG on dephosphorylation of DegU‐P (phosphorylated DegU) in vitro. RapG, however, did not affect dephosphorylation of DegU‐P under the adopted experimental conditions. Therefore, we hypothesized that RapG might inhibit the binding activity of DegU to the target promoters. We analysed the interaction of DegU and RapG using the aprE promoter and another target, a comK promoter. Gel shift analysis revealed that RapG served as the inhibitor of DegU binding to the promoter regions of aprE and comK and that this inhibition was counteracted by the PhrG peptide.
Genetic and molecular evidence that the Pseudomonas syringae type III effector protein AvrRpt2 is a cysteine protease
Authors: Axtell Michael J. , Chisholm Stephen T. , Dahlbeck Douglas , Staskawicz Brian J.
Published: September 2003

Upon delivery to the plant cell during infection, the Pseudomonas syringae effector protein AvrRpt2 undergoes proteolytic processing, enhances pathogen virulence and causes the elimination of the Arabidopsis RIN4 protein. A structure‐prediction method was employed in order to investigate possible biochemical functions of AvrRpt2. Results of a secondary structure prediction algorithm suggest that the functional C‐terminal portion of AvrRpt2 is a cysteine protease. Mutation of predicted catalytic residues within this portion of AvrRpt2 abolished in planta processing, elimination of Arabidopsis RIN4, and the ability to trigger an RPS2‐specific resistance response. These data indicate that AvrRpt2 is most likely a sequence divergent cysteine protease whose activity is required for elimination of RIN4 during infection.
Requirement for kasB in Mycobacterium mycolic acid biosynthesis, cell wall impermeability and intracellular survival: implications for therapy
Authors: Gao Lian‐Yong , Laval Francoise , Lawson Elise H. , Groger Richard K. , Woodruff Andy , Morisaki J. Hiroshi , Cox Jeffery S. , Daffe Mamadou , Brown Eric J.
Published: September 2003

Mycobacterium tuberculosis infects one‐third of the world's population and causes two million deaths annually. The unusually low permeability of its cell wall contributes to the ability of M. tuberculosis to grow within host macrophages, a property required for pathogenesis of infection. Mycobacterium marinum is an established model for discovering genes involved in mycobacterial infection. Mycobacterium marinum mutants with transposon insertions in the β‐ketoacyl‐acyl carrier protein synthase B gene (kasB) grew poorly in macrophages, although growth in vitro was unaffected. Detailed analyses by thin‐layer chromatography, nuclear magnetic resonance (NMR), matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, infrared spectroscopy, and chemical degradations showed that the kasB mutants synthesize mycolic acids that are 2–4 carbons shorter than wild type; the defect was localized to the proximal portion of the meromycolate chain. In addition, these mutants showed a significant (∼30%) reduction in the abundance of keto‐mycolates, with a slight compensatory increase of both α‐ and methoxy‐mycolates. Despite these small changes in mycolate length and composition, the kasB mutants exhibited strikingly altered cell wall permeability, leading to a marked increase in susceptibility to lipophilic antibiotics and the host antimicrobial molecules defensin and lysozyme. The abnormalities of the kasB mutants were fully complemented by expressing M. tuberculosis kasB, but not by the closely related gene kasA. These studies identify kasB as a novel target for therapeutic intervention in mycobacterial diseases.
The Salmonella SpiC protein targets the mammalian Hook3 protein function to alter cellular trafficking
Authors: Shotland Yoram , Krämer Helmut , Groisman Eduardo A.
Published: September 2003

The Salmonella SpiC protein is secreted into the cytosol of macrophages via a unique type III secretion system that functions intracellularly to translocate proteins across the phagosomal membrane. The SpiC protein is required for survival within macrophages and inhibition of phagosome‐lysosome fusion in vivo, and it is sufficient to inhibit endosome‐endosome fusion in vitro. Here, we establish that SpiC targets the function of Hook3, a mammalian protein implicated in cellular trafficking. Purified GST‐SpiC pulled down Hook3 from murine macrophages, and anti‐Hook3 antibodies precipitated SpiC from the cytosol of Salmonella‐infected macrophages. Expression of the spiC gene disrupted Golgi morphology in Vero cells and altered the distribution of lysosomes in macrophages, mimicking the phenotype of cells expressing a hook3 dominant‐negative mutant. By inactivating Hook3 function, the SpiC protein may alter the lysosome network and prevent phagosome‐lysosome fusion.