bluuebunny/crossref_metadata_2025 huggingface dataset:
Gestão em um projeto de extensão universitária: uma abordagem multimeios
Authors: Lages Frederico Santos , Dias Danilo Rocha , Avelar Ewerton Alex
Published: February 2024

A extensão universitária é a atividade que se integra à matriz curricular e às atividades de pesquisa, constituindo-se em processo interdisciplinar, político-educacional, cultural, científico, tecnológico, que promove a interação transformadora entre as universidades e os outros setores da sociedade, por meio da produção e da aplicação do conhecimento. Na Odontologia, os cursos de graduação devem ser capazes de formar cirurgiões-dentistas que tenham habilidades e competências técnicas para execução dos procedimentos, mas que também sejam capazes de liderar equipes, tendo sempre em vista o bem-estar da população, e possuam conhecimentos relacionados a outras áreas como Contabilidade, Administração e Logística, áreas relacionadas, de forma geral, ao conceito de gestão. O objetivo deste trabalho é apresentar um relato de caso sobre práticas de gestão empregadas no projeto de extensão “Manutenção e controle em Implantodontia” da Faculdade de Odontologia da Universidade Federal de Minas Gerais. Este é um estudo descritivo do tipo relato de caso, nos quais apresentamos e discutimos sobre os métodos e ferramentas de gestão desenvolvidos e empregados em um projeto de extensão universitário na área da saúde. Todas as ferramentas desenvolvidas e apresentadas foram fundamentais para o desenvolvimento das diversas atividades, como ensino e acompanhamento dos alunos, análises estatísticas, pesquisa, e os procedimentos clínicos de manutenção e controle para os pacientes com reabilitações implantossuportadas.
To Examine the Effectiveness of a Hospital-Based Nurse-Led Secondary Prevention Clinic
Authors: Mainie Paula M. , Moore Gillian , Riddell John W. , Adgey A.A. Jennifer
Published: July 2005

Modification of cardiovascular risk factors can reduce the incidence of myocardial infarction (MI), effectively extend survival, decrease the need for interventional procedures, and improve quality of life in persons with known cardiovascular disease. Pharmacological treatments and important lifestyle changes reduce people's risks substantially (by 1/3 to 2/3) and can slow and perhaps reverse progression of established coronary disease. When used appropriately, these interventions are more cost-effective than many other treatments, currently provided by the National Health Service [Department of Health National Service Frameworks: coronary heart disease. Preventing coronary heart disease in high risk patients. 2000. HMSO.] Secondary prevention clinics are effective means by which to ensure targets are achieved and assist primary care in long-term maintenance of lifestyle change and drug optimisation.

A 2-year hospital-based pilot project was established at the Royal Hospitals, April 2001–April 2003. The aim of the project was to target patients with coronary heart disease, post-MI and/or coronary artery bypass grafting and/or percutaneous coronary intervention, 6 months following their cardiac event. The plan was to assess patient risk factors and medication a minimum of 6 months following their cardiac event to ascertain if government targets were being achieved; secondly, to examine the effectiveness of a hospital-based nurse-led secondary prevention clinic on modifying risk factors and optimising drug therapies.
Is grey literature essential for a better control of publication bias in psychiatry? An example from three meta-analyses of schizophrenia
Authors: Martin José Luis R. , Pérez Víctor , Sacristán Montse , Álvarez Enric
Published: July 2005

Systematic reviews in mental health have become useful tools for health professionals in view of the massive amount and heterogeneous nature of biomedical information available today. In order to determine the risk of bias in the studies evaluated and to avoid bias in generalizing conclusions from the reviews it is therefore important to use a very strict methodology in systematic reviews. One bias which may affect the generalization of results is publication bias, which is determined by the nature and direction of the study results. To control or minimize this type of bias, the authors of systematic reviews undertake comprehensive searches of medical databases and expand on the findings, often undertaking searches of grey literature (material which is not formally published). This paper attempts to show the consequences (and risk) of generalizing the implications of grey literature in the control of publication bias, as was proposed in a recent systematic work. By repeating the analyses for the same outcome from three different systematic reviews that included both published and grey literature our results showed that confusion between grey literature and publication bias may affect the results of a concrete meta-analysis.
Dose-related effect of methylphenidate on stopping and changing in children with attention-deficit/hyperactivity disorder
Authors: Lijffijt Marijn , Kenemans J. Leon , Wal Annemiek ter , Quik Elise H. , Kemner C. , Westenberg Herman , Verbaten Marinus N. , van Engeland Herman
Published: July 2005

Purpose

The effect of methylphenidate (MPH) on inhibitory control as assessed by the stop task in children with attention-deficit/hyperactivity disorder (ADHD) could be influenced by task difficulty and may be mediated by attention.

Subjects and methods

Fifteen children with ADHD performed the stop and the change task after placebo, 0.5 and 1.0 mg/kg MPH in a within-subject design.

Results

Linear-trend analysis showed a similar effect of MPH in both tasks and a stronger effect for inhibitory control than for attention. Furthermore, a correlation was found between blood serum metabolites of norepinephrine and dopamine for attentional measures and inhibitory control measures, respectively.

Discussion and conclusion

In children with ADHD MPH could act primarily on inhibitory control, and is not influenced by task difficulty. Also, attention and inhibitory control could have differential pharmacological profiles.
Risk of suicide after attempted suicide in the population of Slovenia from 1970 to 1996
Authors: Ziherl Slavko , Zalar Bojan
Published: July 2005

Objective:

All suicide attempts cannot predict suicide, therefore we examined those characteristics of suicide attempt which could most accurately predict completed suicide.

Subject and methods:

Subjects were all individuals registered as committed suicides (N= 16,522) or attempted suicides (N= 15,057) in the register of suicides of the Republic of Slovenia between 1970 and 1996. Log linear analysis of a frequency table was used to uncover relationship between categorical variables.

Results:

The model we found fit between variables: mode, number of repetitions and type, then between number of repetitions, type and gender, and between mode, type and gender.

Discussion:

The risk of suicide in those who previously attempted suicide is approximately 773 times higher than the risk of suicide without a previous suicide attempt. Those who attempt suicide by hanging (hanging being in Slovenia the most frequent mode of completed suicide) are at even greater risk to commit suicide.

Conclusion:

Our data suggests that clinicians should heighten their awareness that any suicide attempt can in some 20% predict suicide. Someone who has attempted suicide by hanging is at the highest risk of suicide.
Micromolar HgCl2 concentrations transitorily duplicate the ATP level in Saccharomyces cerevisiae cells
Authors: Silles Eduardo , Osorio Hugo , Maia Rita , Günther Sillero María A. , Sillero Antonio
Published: July 2005

Low concentrations of HgCl2 elicited, in Saccharomyces cerevisiae, a transitory increase in the ATP level followed by a decrease of its concentration, until almost disappearance. At 1 μM HgCl2, the increase in ATP lasted for about 30 min, while at 10 μM the increase was only observed in the first 5 min of treatment. The initial burst of ATP was accompanied by a decrease in the level of hexose phosphates, whereas during the decrease of ATP an increase in the inosine and hexose phosphates levels took place. The treatment with HgCl2 inhibited the plasma membrane proton ATPase but not the activities of hexokinase or 6‐phosphofructokinase.
Methylation of the guanidino group of arginine residues prevents citrullination by peptidylarginine deiminase IV
Authors: Hidaka Yuji , Hagiwara Teruki , Yamada Michiyuki
Published: July 2005

Peptidylarginine deiminase IV (PAD IV) catalyzes the citrullination of Arg residues of proteins, such as histones. Suzuki et al. recently reported that haplotypes of the PAD IV gene are associated with susceptibility to rheumatoid arthritis. To investigate the mechanism of substrate specificity and inhibitors of PAD IV, a series of the Arg derivatives were synthesized and their reactivity to PAD IV examined. The results suggest that both imino and carboxyl groups are important in the molecular recognition of PAD IV and that methylation of the guanidino group prevents citrullination. In addition, the findings herein show that Bz‐N
G‐monomethyl‐Arg and Bz‐N
G,N
G‐dimethyl‐Arg specifically inhibit citrullination.
Prevalence of feline infectious peritonitis in specific cat breeds
Authors: Pesteanu-Somogyi Loretta D. , Radzai Christina , Pressler Barrak M.
Published: July 2005

Although known that purebreed cats are more likely to develop feline infectious peritonitis (FIP), previous studies have not examined the prevalence of disease in individual breeds. All cats diagnosed with FIP at a veterinary teaching hospital over a 16-year period were identified. Breed, sex and reproductive status of affected cats were compared to the general cat population and to mixed breed cats evaluated during the same period. As with previous studies sexually intact cats and purebreed cats were significantly more likely to be diagnosed with FIP; males and young cats also had a higher prevalence of disease. Abyssinians, Bengals, Birmans, Himalayans, Ragdolls and Rexes had a significantly higher risk, whereas Burmese, Exotic Shorthairs, Manxes, Persians, Russian Blues and Siamese cats were not at increased risk for development of FIP. Although additional factors doubtlessly influence the relative prevalence of FIP, this study provides additional guidance when prioritizing differentials in ill purebreed cats.
Hypervitaminosis A in the cat: A case report and review of the literature
Authors: Polizopoulou Zoe S. , Kazakos George , Patsikas Michael N. , Roubies Nikolaos
Published: July 2005

A case of hypervitaminosis A with secondary entrapment and compression of the left brachial plexus nerve roots is described. A 9-year-old male castrated domestic shorthair, fed a home-made diet based on raw pork liver, was submitted for examination for a left forelimb lameness that evolved to paralysis over a 2-month period. Clinical examination revealed a flaccid paralysis and atrophy of all left forelimb muscles. An ipsilateral Horner's syndrome was also noted. Radiological examination of the cervical and thoracic spine showed massive new bone formation at the ventral aspect of the second cervical to sixth thoracic vertebra. The diagnosis of hypervitaminosis A was made, based on the clinical and radiographic findings, as well as the determination of serum vitamin A concentration, which was 630 μg/dl, three times above the upper normal limit for this species. Despite the unfavourable initial prognosis, the cat progressively regained function of the affected limb approximately 6 months after the diet was changed to a commercial canned food.
The Origins of the Thenar and Hypothenar Muscles
Authors: KUNG J. , BUDOFF J. E. , WEI M. L. , GHARBAOUI I. , LUO Z. P.
Published: July 2005

This paper presents an anatomical study of the origins of the thenar and hypothenar muscles and postulates the causes of weakness and pillar pain following carpal tunnel release.
Pin Arthrodesis of the Wrist – A Modified Technique
Authors: KUMAR K. , COX Q. G. N.
Published: July 2005

An intramedullary pin is commonly used for wrist arthrodesis in patients with rheumatoid arthritis. However, pin migration is a recognized complication of this technique. We report the results of wrist fusion in 15 patients using a modified technique with a transverse blocking screw inserted into the metacarpal distal to the intramedullary pin to prevent distal migration and backing out of the intramedullary pin. The procedure is simple to perform, does not add significantly to the operating time and has not been associated with any complications.
Eph kinases and ephrins support thrombus growth and stability by regulating integrin outside-in signaling in platelets
Authors: Prévost Nicolas , Woulfe Donna S. , Jiang Hong , Stalker Timothy J. , Marchese Patrizia , Ruggeri Zaverio M. , Brass Lawrence F.
Published: July 2005

The ability of activated platelets to adhere to each other at sites of vascular injury depends on the integrin α
IIb
β
3
. However, as aggregation continues, other signaling and adhesion molecules can contribute as well. We have previously shown that human platelets express on their surface the Eph receptor kinases EphA4 and EphB1 and the Eph kinase ligand ephrinB1. We now show that EphA4 is physically associated with α
IIb
β
3
in resting platelets, increases its surface expression when platelets are activated, and colocalizes with α
IIb
β
3
at sites of contact between platelets. We also show that Eph/ephrin interactions can support the stable accumulation of platelets on collagen under flow and contribute to postengagement “outside-in” signaling through α
IIb
β
3
by stabilizing platelet aggregates and facilitating tyrosine phosphorylation of the β
3
cytoplasmic domain. β
3
phosphorylation allows myosin to bind to α
IIb
β
3
and clot retraction to occur. The data support a model in which the onset of aggregation permits Eph/ephrin interactions to occur, after which signaling downstream from ephrinB1 and its receptors favors continued growth and stability of the thrombus by several mechanisms, including positive effects on outside-in signaling through α
IIb
β
3
.
The structural basis of androgen receptor activation: Intramolecular and intermolecular amino–carboxy interactions
Authors: Schaufele Fred , Carbonell Xavier , Guerbadot Martin , Borngraeber Sabine , Chapman Mark S. , Ma Aye Aye K. , Miner Jeffrey N. , Diamond Marc I.
Published: July 2005

Nuclear receptors (NRs) are ligand-regulated transcription factors important in human physiology and disease. In certain NRs, including the androgen receptor (AR), ligand binding to the carboxy-terminal domain (LBD) regulates transcriptional activation functions in the LBD and amino-terminal domain (NTD). The basis for NTD–LBD communication is unknown but may involve NTD–LBD interactions either within a single receptor or between different members of an AR dimer. Here, measurement of FRET between fluorophores attached to the NTD and LBD of the AR established that agonist binding initiated an intramolecular NTD–LBD interaction in the nucleus and cytoplasm. This intramolecular folding was followed by AR self-association, which occurred preferentially in the nucleus. Rapid, ligand-induced intramolecular folding and delayed association also were observed for estrogen receptor-α but not for peroxisome proliferator activated receptor-γ2. An antagonist ligand, hydroxyflutamide, blocked the NTD–LBD association within AR. NTD–LBD association also closely correlated with the transcriptional activation by heterologous ligands of AR mutants isolated from hormone-refractory prostate tumors. Intramolecular folding, but not AR–AR affinity, was disrupted by mutation of an α-helical (
23
FQNLF
27
) motif in the AR NTD previously described to interact with the AR LBD
in vitro
. This work establishes an intramolecular NTD–LBD conformational change as an initial component of ligand-regulated NR function.
RETRACTED:
HOS10
encodes an R2R3-type MYB transcription factor essential for cold acclimation in plants
Authors: Zhu Jianhua , Verslues Paul E. , Zheng Xianwu , Lee Byeong-ha , Zhan Xiangqiang , Manabe Yuzuki , Sokolchik Irina , Zhu Yanmei , Dong Chun-Hai , Zhu Jian-Kang , Hasegawa Paul M. , Bressan Ray A.
Published: July 2005

We report the identification and characterization of an
Arabidopsis
mutant,
hos10-1
(for high expression of osmotically responsive genes), in which the expression of
RD29A
and other stress-responsive genes is activated to higher levels or more rapidly activated than in wild-type by low temperature, exogenous abscisic acid (ABA), or salt stress (NaCl). The
hos10-1
plants are extremely sensitive to freezing temperatures, completely unable to acclimate to the cold, and are hypersensitive to NaCl. Induction of
NCED3
(the gene that encodes the rate-limiting enzyme in ABA biosynthesis) by polyethylene glycol-mediated dehydration and ABA accumulation are reduced by this mutation. Detached shoots from the mutant plants display an increased transpiration rate compared with wild-type plants. The
hos10-1
plants exhibit several developmental alterations, such as reduced size, early flowering, and reduced fertility. The
HOS10
gene encodes a putative R2R3-type MYB transcription factor that is localized to the nucleus. Together, these results indicate that HOS10 is an important coordinating factor for responses to abiotic stress and for growth and development.
Inducible costimulator is required for type 2 antibody isotype switching but not T helper cell type 2 responses in chronic nematode infection
Authors: Loke P'ng , Zang Xingxing , Hsuan Lisa , Waitz Rebecca , Locksley Richard M. , Allen Judith E. , Allison James P.
Published: July 2005

Inducible costimulator (ICOS) has been suggested to perform an important role in T helper cell type 2 (Th2) responses, germinal center formation, and isotype switching. The role of ICOS in chronic Th2 responses was studied in a nematode model with the filarial parasite,Brugia malayi. Contrary to expectations, we did not observe a significant defect in IL-4-producing Th2 cells in ICOS–/– mice or in eosinophil recruitment. We also found that ICOS was not required for the differentiation of alternatively activated macrophages (AAMΦ) that express Ym1 and Fizz1. Although the production of IgE was slightly reduced in ICOS–/– mice, this was not as significant as in CD28–/– mice. In contrast to live infection, the primary response of ICOS–/– mice immunized with solubleB. malayiantigen and complete Freund's adjuvant resulted in significantly fewer IL-4-producing cells in the lymph nodes. As previously reported, we observed a defect in antibody isotype switching toward the IgG1 isotype in ICOS–/– mice during live infection. Interestingly, there was a significant enhancement of parasite-specific IgG3 isotype antibodies. CD28–/– and MHC class II–/– mice also had enhanced parasite-specific IgG3 isotype antibodies. Our results suggest that ICOS is not required to maintain a chronic cellular Th2 response. The primary role of ICOS in a chronic helminth infection could be to drive antibodies toward type 2 isotypes. T-independent antibody response to the parasite could be enhanced in the absence of costimulation and T cell help.
Interferon prevents formation of replication-competent hepatitis B virus RNA-containing nucleocapsids
Authors: Wieland Stefan F. , Eustaquio Angelina , Whitten-Bauer Christina , Boyd Bryan , Chisari Francis V.
Published: July 2005

We have previously shown that IFN-β inhibits hepatitis B virus (HBV) replication by noncytolytic mechanisms that either destabilize pregenomic (pg)RNA-containing capsids or prevent their assembly. Using immortalized murine hepatocyte cell lines stably transfected with a doxycycline (dox)-inducible HBV replication system, we now show that replication-competent pgRNA-containing capsids are not produced when the cells are pretreated with IFN-β before HBV expression is induced with dox. Furthermore, the turnover rate of preformed HBV RNA-containing capsids is not changed in the presence of IFN-β or IFN-γ under conditions in which further pgRNA synthesis is inhibited by dox removal. In summary, these results demonstrate that types 1 and 2 IFN activate hepatocellular mechanism(s) that prevent the formation of replication-competent HBV capsids and, thereby, inhibit HBV replication.
Toll‐like receptor 4 mediates mitochondrial DNA damage and biogenic responses after heat‐inactivated E. coli
Authors: Suliman Hagir B. , Welty‐Wolf Karen E. , Carraway Martha Sue , Schwartz David A. , Hollingsworth John W. , Piantadosi Claude A.
Published: July 2005

An important site of cellular damage in bacterial sepsis is mitochondrial DNA (mtDNA), which we proposed is caused by reactive oxygen and nitrogen species generated by activation of signaling through specific toll‐like receptors (TLR). In wild‐type (Wt) mice injected with heat‐inactivated E. coli, hepatic TLR4 and TLR2 proteins were up‐regulated with TLR‐dependent increases in transcript levels for tumor necrosis factor (TNF‐α), interleukin 6, nitric oxide synthase‐II (iNOS), and NADPH oxidase 2 (Nox2). The accompanying stress significantly depleted hepatic mtDNA despite eight‐ and fourfold increases in manganese superoxide dismutase (MnSOD) and mitochondrial transcription factor A (Tfam) expression, respectively. The identical E. coli dose generated significantly less TNF‐α, NO, and Nox2 in TLR4−/− and TLR2/4−/− but not in TLR2−/− mice. TLR4−/− and TLR2/4−/− compared with Wt mice were protected from mtDNA oxidation but showed no Tfam up‐regulation and little copy number restoration. A critical role in the mtDNA damage was determined for TLR4‐mediated iNOS transcription through the MyD88 pathway. In Wt mice, mtDNA depletion was avoided by selective iNOS blockade, and residual mtDNA loss was linked to NF‐κB‐dependent TNF‐α expression. These data disclose the dual role of TLR4 in mtDNA damage and compensatory mitochondrial biogenic responses after innate immune activation.
Identification of a Gene in the Process of Being Lost from the Genus Agrostis
Authors: Li Huaijun Michael , Rotter David , Bonos Stacy A. , Meyer William A. , Belanger Faith C.
Published: July 2005

Lineage-specific gene loss is considered one of the processes contributing to speciation and genome diversity. Such gene loss has been inferred from interspecies comparisons of orthologous DNA segments. Examples of intraspecific gene loss are rare. Here we report identification of a gene, designated Crs-1 (creeping specific-1), that appears to be in the process of being lost from heterozygous populations of the species creeping bentgrass (Agrostis stolonifera). The Crs-1 gene encodes a protein with an N-terminal dirigent protein domain and a C-terminal lectin domain and is similar to the maize (Zea mays) β-glucosidase aggregating factor. Most individual creeping bentgrass plants examined are lacking Crs-1. Some individuals are hemizygous for the Crs-1 locus, indicating major haplotype noncolinearity at that locus. Crs-1 was not detected in several other Agrostis species, indicating it is being lost from the genus. The Crs-1 locus in creeping bentgrass provides a rare example of the evolutionary process of gene loss occurring within a plant species.
RAC GTPases in Tobacco and Arabidopsis Mediate Auxin-Induced Formation of Proteolytically Active Nuclear Protein Bodies That Contain AUX/IAA Proteins
Authors: Tao Li-zhen , Cheung Alice Y. , Nibau Candida , Wu Hen-ming
Published: July 2005

Auxin signaling relies on ubiquitin ligase SCFTIR1-mediated 26S proteasome-dependent proteolysis of a large family of short-lived transcription regulators, auxin/indole acetic acid (Aux/IAA), resulting in the derepression of auxin-responsive genes. We have shown previously that a subset of Rac GTPases is activated by auxin, and they in turn stimulate auxin-responsive gene expression. We show here that increasing Rac signaling activity promotes Aux/IAA degradation, whereas downregulating that activity results in the reduction of auxin-accelerated Aux/IAA proteolysis. Observations reported here reveal a novel function for these Rac GTPases as regulators for ubiquitin/26S proteasome-mediated proteolysis and further consolidate their role in auxin signaling. Moreover, our study reveals a cellular process whereby auxin induces and Rac GTPases mediate the recruitment of nucleoplasmic Aux/IAAs into proteolytically active nuclear protein bodies, into which components of the SCFTIR1, COP9 signalosome, and 26S proteasome are also recruited.
Pyruvate Decarboxylase Provides Growing Pollen Tubes with a Competitive Advantage in Petunia
Authors: Gass Nathalie , Glagotskaia Tatiana , Mellema Stefan , Stuurman Jeroen , Barone Mario , Mandel Therese , Roessner-Tunali Ute , Kuhlemeier Cris
Published: July 2005

Rapid pollen tube growth places unique demands on energy production and biosynthetic capacity. The aim of this work is to understand how primary metabolism meets the demands of such rapid growth. Aerobically grown pollen produce ethanol in large quantities. The ethanolic fermentation pathway consists of two committed enzymes: pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). Because adh mutations do not affect male gametophyte function, the obvious question is why pollen synthesize an abundant enzyme if they could do just as well without. Using transposon tagging in Petunia hybrida, we isolated a null mutant in pollen-specific Pdc2. Growth of the mutant pollen tubes through the style is reduced, and the mutant allele shows reduced transmission through the male, when in competition with wild-type pollen. We propose that not ADH but rather PDC is the critical enzyme in a novel, pollen-specific pathway. This pathway serves to bypass pyruvate dehydrogenase enzymes and thereby maintain biosynthetic capacity and energy production under the unique conditions prevailing during pollen–pistil interaction.
Nitric oxide synthase inhibition affects sarcoplasmic reticulum Ca2+ release in skeletal muscle fibres from mouse
Authors: Pouvreau Sandrine , Jacquemond Vincent
Published: July 2005

Nitric oxide (NO) generated by skeletal muscle is believed to regulate force production but how this is achieved remains poorly understood. In the present work we tested the effects of NO synthase (NOs) inhibitors on membrane current and intracellular calcium in isolated skeletal muscle fibres from mouse, under voltage‐clamp conditions. Resting [Ca2+] and [Ca2+] transients evoked by large depolarizations exhibited similar properties in control fibres and in fibres loaded with tenth millimolar levels of the NOs inhibitor N‐nitro‐l‐arginine (l‐NNA). Yet the voltage dependence of calcium release was found to be shifted by ∼15 mV towards negative values in the presence of l‐NNA. This effect could be reproduced by the other NOs inhibitor S‐methyl‐l‐thiocitrulline (l‐SMT). Separate experiments showed that the voltage dependence of charge movement and of the slow calcium current were unaffected by the presence of l‐NNA, ruling out an effect on the voltage sensor. A negative shift in the voltage dependence of calcium release with no concurrent alteration in the properties of charge movement was also observed in fibres exposed to the oxidant H2O2 (1 mm). Conversely the reducing agent dithiothreitol (10 mm) had no obvious effect on Ca2+ release. Overall, the results indicate that physiological levels of NO exert a tonic inhibitory control on the activation of the calcium release channels. Changes in the voltage dependence of Ca2+ release activation may be a ubiquitous physiological consequence of redox‐related modifications of the ryanodine receptor.
Beyond the brush border: NHERF4 blazes new NHERF turf
Authors: Thelin William R. , Hodson Caleb A. , Milgram Sharon L.
Published: July 2005

The Na exchanger regulatory factor (NHERF) family of epithelial‐enriched PDZ domain scaffolding proteins plays important roles in maintaining and regulating epithelial cell function. The NHERFs exhibit some overlap in tissue distribution and binding partners, suggesting redundant functions. Yet, it is clear that each NHERF protein exhibits distinct properties, translating into unique cellular functions. The work summarized in this review suggests the most recently identified family member, NHERF4, is the most divergent. Additional investigation is needed, however, to understand more completely the role of NHERF4 in the context of the NHERF family.
Structure of a Synaptic γδ Resolvase Tetramer Covalently Linked to Two Cleaved DNAs
Authors: Li Weikai , Kamtekar Satwik , Xiong Yong , Sarkis Gary J. , Grindley Nigel D. F. , Steitz Thomas A.
Published: July 2005

The structure of a synaptic intermediate of the site-specific recombinase γδ resolvase covalently linked through Ser
10
to two cleaved duplex DNAs has been determined at 3.4 angstrom resolution. This resolvase, activated for recombination by mutations, forms a tetramer whose structure is substantially changed from that of a presynaptic complex between dimeric resolvase and the cleavage site DNA. Because the two cleaved DNA duplexes that are to be recombined lie on opposite sides of the core tetramer, large movements of both protein and DNA are required to achieve strand exchange. The two dimers linked to the DNAs that are to be recombined are held together by a flat interface. This may allow a 180° rotation of one dimer relative to the other in order to reposition the DNA duplexes for strand exchange.
Understanding the Infrared Spectrum of Bare CH
5
+
Authors: Asvany Oskar , P Padma Kumar , Redlich Britta , Hegemann Ilka , Schlemmer Stephan , Marx Dominik
Published: July 2005

Protonated methane, CH
5
+
, continues to elude definitive structural assignment, as large-amplitude vibrations and hydrogen scrambling challenge both theory and experiment. Here, the infrared spectrum of bare CH
5
+
is presented, as detected by reaction with carbon dioxide gas after resonant excitation by the free electron laser at the FELIX facility in the Netherlands. Comparison of the experimental spectrum at ∼110 kelvin to finite-temperature infrared spectra, calculated by ab initio molecular dynamics, supports fluxionality of bare CH
5
+
under experimental conditions and provides a dynamical mechanism for exchange of hydrogens between CH
3
tripod positions and the three-center bonded H
2
moiety, which eventually leads to full hydrogen scrambling. The possibility of artificially freezing out scrambling and internal rotation in the simulations allowed assignment of the infrared spectrum despite this pronounced fluxionality.
Escherichia coli
HypA Is a Zinc Metalloprotein with a Weak Affinity for Nickel
Authors: Atanassova Anelia , Zamble Deborah B.
Published: July 2005

The
hyp
operon encodes accessory proteins that are required for the maturation of the [NiFe] hydrogenase enzymes and, in some organisms, for the production of urease enzymes as well. HypA or a homologous protein is required for nickel insertion into the hydrogenase precursor proteins. In this study, recombinant HypA from
Escherichia coli
was purified and characterized in vitro. Metal analysis was used to demonstrate that HypA simultaneously binds stoichiometric Zn
2+
and stoichiometric Ni
2+
. Competition experiments with a metallochromic indicator reveal that HypA binds zinc with nanomolar affinity. Spectroscopic analysis of cobalt-containing HypA provides evidence for a tetrathiolate coordination sphere, suggesting that the zinc site has a structural role. In addition, HypA can exist as several oligomeric complexes and the zinc content modulates the quaternary structure of the protein. Fluorescence titration experiments demonstrate that HypA binds nickel with micromolar affinity and that the presence of zinc does not dramatically affect the nickel-binding activity. Finally, complex formation between HypA and HypB, another accessory protein required for nickel insertion, was observed. These experiments suggest that HypA is an architectural component of the hydrogenase metallocenter assembly pathway and that it may also have a direct role in the delivery of nickel to the hydrogenase large subunit.
The
Burkholderia pseudomallei
BpeAB-OprB Efflux Pump: Expression and Impact on Quorum Sensing and Virulence
Authors: Chan Ying Ying , Chua Kim Lee
Published: July 2005

BpeAB-OprB is a multidrug efflux pump of the bacterial pathogen
Burkholderia pseudomallei
and is responsible for conferring antimicrobial resistance to aminoglycosides and macrolides. Expression of
bpeAB-oprB
is inducible by its substrate erythromycin and upon entry into stationary phase. BpeR, a member of the TetR family, functions as a repressor of the
bpeAB-oprB
operon.
bpeR
expression was similarly induced at stationary phase but lagged behind the induction of
bpeAB-oprB
expression. The induction of
bpeAB-oprB
expression could be advanced to the early exponential phase by exogenous addition of the
B. pseudomallei
autoinducers
N
-octanoyl-homoserine lactone (C8HSL) and
N
-decanoyl-homoserine lactone (C10HSL), suggesting that the
bpeAB-oprB
operon may be quorum regulated. On the other hand, acyl-homoserine lactone (acyl-HSL) production was undetectable in the
bpeAB
-null mutant and strains which overexpress
bpeR
. The failure of these strains to produce acyl-HSLs seemed to be at the level of synthesis of acyl-HSLs, as growth-phase-dependent expression of the autoinducer synthase BpsI was abolished in the
bpeAB
-null mutant.
bpsI
expression remained growth phase dependent in the
bpeR
mutant which had functional BpeAB-OprB. BpeAB-OprB function is likewise necessary for optimal production of quorum-sensing-controlled virulence factors such as siderophore and phospholipase C and for biofilm formation. Cell invasion and cytotoxicity towards human lung epithelial (A549) and human macrophage (THP-1) cells were also significantly attenuated in both the
bpeAB
mutant and
bpeR-
overexpressing strains, thus suggesting the possibility of attenuating
B. pseudomallei
virulence using inhibitors of the BpeAB-OprB efflux pump.
The Genome of
Salmonella enterica
Serovar Gallinarum: Distinct Insertions/Deletions and Rare Rearrangements
Authors: Wu Kai-Yu , Liu Gui-Rong , Liu Wei-Qiao , Wang Austin Q. , Zhan Sen , Sanderson Kenneth E. , Johnston Randal N. , Liu Shu-Lin
Published: July 2005

Salmonella enterica
serovar Gallinarum is a fowl-adapted pathogen, causing typhoid fever in chickens. It has the same antigenic formula (1,9,12:—:—) as
S. enterica
serovar Pullorum, which is also adapted to fowl but causes pullorum disease (diarrhea). The close relatedness but distinct pathogeneses make this pair of fowl pathogens good models for studies of bacterial genomic evolution and the way these organisms acquired pathogenicity. To locate and characterize the genomic differences between serovar Gallinarum and other salmonellae, we constructed a physical map of serovar Gallinarum strain SARB21 by using I-CeuI, XbaI, and AvrII with pulsed-field gel electrophoresis techniques. In the 4,740-kb genome, we located two insertions and six deletions relative to the genome of
S. enterica
serovar Typhimurium LT2, which we used as a reference
Salmonella
genome. Four of the genomic regions with reduced lengths corresponded to the four prophages in the genome of serovar Typhimurium LT2, and the others contained several smaller deletions relative to serovar Typhimurium LT2, including regions containing
srfJ
,
std
, and
stj
and gene clusters encoding a type I restriction system in serovar Typhimurium LT2. The map also revealed some rare rearrangements, including two inversions and several translocations. Further characterization of these insertions, deletions, and rearrangements will provide new insights into the molecular basis for the specific host-pathogen interactions and mechanisms of genomic evolution to create a new pathogen.
Analysis of theEscherichia coliAlp Phenotype: Heat Shock Induction inssrAMutants
Authors: Munavar Hussain , Zhou YanNing , Gottesman Susan
Published: July 2005

The major phenotypes oflonmutations, UV sensitivity and overproduction of capsule, are due to the stabilization of two substrates, SulA and RcsA. Inactivation of transfer mRNA (tmRNA) (encoded byssrA), coupled with a multicopy kanamycin resistance determinant, suppressed bothlonphenotypes and restored the rapid degradation of SulA. This novel protease activity was named Alp but was never identified further. We report here the identification, mapping, and characterization of a chromosomal mutation,faa(forfunctionaffectingAlp), that leads to full suppression of a Δlon ssrA::cathost and thus bypasses the requirement for multicopy Kanr;faaandssrAmutants are additive in their ability to suppresslonmutants. Thefaamutation was mapped to the C terminus ofdnaJ(G232);dnaJnull mutants have similar effects. The identification of alonsuppressor indnaJsuggested the possible involvement of heat shock. We find thatssrAmutants alone significantly induce the heat shock response. The suppression of UV sensitivity, both in the original Alp strain and infaamutants, is reversed by mutations inclpY, encoding a subunit of the heat shock-induced ClpYQ protease that is known to degrade SulA. However, capsule synthesis is not restored byclpYmutants, probably because less RcsA accumulates in the Alp strain and because the RcsA that does accumulate is inactive. BothssrAeffects are partially relieved byssrAderivatives encoding protease-resistant tags, implicating ribosome stalling as the primary defect. Thus,ssrAandfaaeach suppress twolonmutant phenotypes but by somewhat different mechanisms, with heat shock induction playing a major role.
Identification of Phthiodiolone Ketoreductase, an Enzyme Required for Production of Mycobacterial Diacyl Phthiocerol Virulence Factors
Authors: Onwueme Kenolisa C. , Vos Cheryl J. , Zurita Juan , Soll Clifford E. , Quadri Luis E. N.
Published: July 2005

Diacyl phthiocerol esters and their congeners are mycobacterial virulence factors. The biosynthesis of these complex lipids remains poorly understood. Insight into their biosynthesis will aid the development of rationally designed drugs that inhibit their production. In this study, we investigate a biosynthetic step required for diacyl (phenol)phthiocerol ester production, i.e., the reduction of the keto group of (phenol)phthiodiolones. We utilized comparative genomics to identify phthiodiolone ketoreductase gene candidates and provide a genetic analysis demonstrating gene function for two of these candidates. Moreover, we present data confirming the existence of a diacyl phthiotriol intermediate in diacyl phthiocerol biosynthesis. We also elucidate the mechanism underlying diacyl phthiocerol deficiency in some mycobacteria, such as
Mycobacterium ulcerans
and
Mycobacterium kansasii
. Overall, our findings shed additional light on the biosynthesis of an important group of mycobacterial lipids involved in virulence.
The Ubiquitous Protein Domain EAL Is a Cyclic Diguanylate-Specific Phosphodiesterase: Enzymatically Active and Inactive EAL Domains
Authors: Schmidt Andrew J. , Ryjenkov Dmitri A. , Gomelsky Mark
Published: July 2005

The EAL domain (also known as domain of unknown function 2 or DUF2) is a ubiquitous signal transduction protein domain in the
Bacteria
. Its involvement in hydrolysis of the novel second messenger cyclic dimeric GMP (c-di-GMP) was demonstrated in vivo but not in vitro. The EAL domain-containing protein Dos from
Escherichia coli
was reported to hydrolyze cyclic AMP (cAMP), implying that EAL domains have different substrate specificities. To investigate the biochemical activity of EAL, the
E. coli
EAL domain-containing protein YahA and its individual EAL domain were overexpressed, purified, and characterized in vitro. Both full-length YahA and the EAL domain hydrolyzed c-di-GMP into linear dimeric GMP, providing the first biochemical evidence that the EAL domain is sufficient for phosphodiesterase activity. This activity was c-di-GMP specific, optimal at alkaline pH, dependent on Mg
2+
or Mn
2+
, strongly inhibited by Ca
2+
, and independent of protein oligomerization. Linear dimeric GMP was shown to be 5′pGpG. The EAL domain from Dos was overexpressed, purified, and found to function as a c-di-GMP-specific phosphodiesterase, not as a cAMP-specific phosphodiesterase, in contrast to previous reports. The EAL domains can hydrolyze 5′pGpG into GMP, however, very slowly, thus implying that this activity is irrelevant in vivo. Therefore, c-di-GMP is the exclusive substrate of EAL. Multiple-sequence alignment revealed two groups of EAL domains hypothesized to correspond to enzymatically active and inactive domains. The domains in the latter group have mutations in residues conserved in the active domains. The enzymatic inactivity of EAL domains may explain their coexistence with GGDEF domains in proteins possessing c-di-GMP synthase (diguanulate cyclase) activity.
ExpR, a LuxR Homolog ofErwinia carotovorasubsp.carotovora, Activates Transcription ofrsmA, Which Specifies a Global Regulatory RNA-Binding Protein
Authors: Cui Yaya , Chatterjee Asita , Hasegawa Hiroaki , Dixit Vaishali , Leigh Nathan , Chatterjee Arun K.
Published: July 2005

N-acyl homoserine lactone (AHL) is required byErwinia carotovorasubspecies for the expression of various traits, including extracellular enzyme and protein production and pathogenicity. Previous studies withE. carotovorasubsp.carotovorahave shown that AHL deficiency causes the production of high levels of RsmA, an RNA binding protein that functions as a global negative regulator of extracellular enzymes and proteins and secondary metabolites (Rsm, regulator of secondary metabolites). We document here that ExpR, a putative AHL receptor belonging to the LuxR family of regulators, activates RsmA production. In the absence of AHL, an ExpR+E. carotovorasubsp.carotovorastrain compared to its ExpR−mutant, produces higher levels ofrsmARNA and better expresses anrsmA-lacZtranscriptional fusion. Moreover, the expression of thersmA-lacZfusion inEscherichia coliis much higher in the presence ofexpR71(theexpRgene ofE. carotovorasubsp.carotovorastrain Ecc71) than in its absence. We also show that purified preparation of MBP-ExpR71binds (MBP, maltose binding protein)rsmADNA. By contrast, MBP-ExpR71does not bindahlI(gene for AHL synthase),pel-1(gene for pectate lyase), orrsmB(gene for regulatory RNA that binds RsmA), nor does ExpR71activate expression of these genes. These observations strongly suggest transcriptional activation ofrsmAresulting from a direct and specific interaction between ExpR71and thersmApromoter. Several lines of evidence establish thatN-3-oxohexanoyl-l-homoserine lactone (3-oxo-C6-HL), the major AHL analog produced byE. carotovorasubsp.carotovorastrain Ecc71, inhibits ExpR71-mediated activation ofrsmAexpression. These findings for the first time establish that theexpReffect inE. carotovorasubsp.carotovorais channeled via RsmA, a posttranscriptional regulator ofE. carotovorasubspecies, and AHL neutralizes this ExpR effect.
Mutational Analysis of 16S and 23S rRNA Genes of
Thermus thermophilus
Authors: Gregory Steven T. , Carr Jennifer F. , Rodriguez-Correa Daniel , Dahlberg Albert E.
Published: July 2005

Structural studies of the ribosome have benefited greatly from the use of organisms adapted to extreme environments. However, little is known about the mechanisms by which ribosomes or other ribonucleoprotein complexes have adapted to functioning under extreme conditions, and it is unclear to what degree mutant phenotypes of extremophiles will resemble those of their counterparts adapted to more moderate environments. It is conceivable that phenotypes of mutations affecting thermophilic ribosomes, for instance, will be influenced by structural adaptations specific to a thermophilic existence. This consideration is particularly important when using crystal structures of thermophilic ribosomes to interpret genetic results from nonextremophilic species. To address this issue, we have conducted a survey of spontaneously arising antibiotic-resistant mutants of the extremely thermophilic bacterium
Thermus thermophilus
, a species which has featured prominently in ribosome structural studies. We have accumulated over 20 single-base substitutions in
T. thermophilus
16S and 23S rRNA, in the decoding site and in the peptidyltransferase active site of the ribosome. These mutations produce phenotypes that are largely identical to those of corresponding mutants of mesophilic organisms encompassing a broad phylogenetic range, suggesting that
T. thermophilus
may be an ideal model system for the study of ribosome structure and function.
Analysis of theospCRegulatory Element Controlled by the RpoN-RpoS Regulatory Pathway inBorrelia burgdorferi
Authors: Yang Xiaofeng F. , Lybecker Meghan C. , Pal Utpal , Alani Sophie M. , Blevins Jon , Revel Andrew T. , Samuels D. Scott , Norgard Michael V.
Published: July 2005

Outer surface lipoprotein C (OspC) is a key virulence factor ofBorrelia burgdorferi. ospCis differentially regulated during borrelial transmission from ticks to rodents, and such regulation is essential for maintaining the spirochete in its natural enzootic cycle. Recently, we showed that the expression ofospCinB. burgdorferiis governed by a novel alternative sigma factor regulatory network, the RpoN-RpoS pathway. However, the precise mechanism by which the RpoN-RpoS pathway controlsospCexpression has been unclear. In particular, there has been uncertainty regarding whetherospCis controlled directly by RpoS (σs) or indirectly through a transactivator (induced by RpoS). Using deletion analyses and genetic complementation in an OspC-deficient mutant ofB. burgdorferi, we analyzed theciselement(s) required for the expression ofospCin its native borrelial background. Two highly conserved upstream inverted repeat elements, previously implicated inospCregulation, were not required forospCexpression inB. burgdorferi. Using similar approaches, a minimal promoter that contained a canonical −35/−10 sequence necessary and sufficient for σs-dependent regulation ofospCwas identified. Further, targeted mutagenesis of a C at position −15 within the extended −10 region ofospC, which is postulated to function like the strategic C residue important for Eσsbinding inEscherichia coli, abolishedospCexpression. The minimalospCpromoter also was responsive to coumermycin A1, further supporting its σscharacter. The combined data constitute a body of evidence that the RpoN-RpoS regulatory network controlsospCexpression by direct binding of σsto a σs-dependent promoter ofospC. The implication of our findings to understanding howB. burgdorferidifferentially regulatesospCand otherospC-like genes via the RpoN-RpoS regulatory pathway is discussed.