bluuebunny/crossref_metadata_2025 huggingface dataset:
The usefulness of skin culture in the diagnosis of chronic meningococcaemia
Authors: TEXEREAU M. , ROBLOT P. , DUMARS A. , GRIGNON B. , BECQ‐GIRAUDON B.
Published: August 2010

Abstract. Texereau M, Roblot E', Dumars A, Grignon B, Becq‐Giraudon B (Hbpital la Miktrie, Centre Hospitalier Universitaire, Poitiers, Cedex, France). The usefulness of skin culture in the diagnosis of chronic meningococcaemia (Case Report)

We deal with the second reported case of chronic meningococcaemia in which the culture of skin biopmeningococcaemia in which the culture of skin biopa history of recurrent fever and rash. Laboratory studies revealed an inflammatory syndrome. Serologic tests as well as blood culture tests were negative. The histological examination of skin lesions revealed a perivascular infiltrate in the dermis without any picture of leukocytoclastic vasculitis. A culture of skin specimen tested positive for Neisseria meningitidis (N. meningitidis). After a week of antibiotic treatment, the patient recovered with no recurrence of either fever or rash over a two year period.
The influence of ketoacids on plasma creatinine assays in diabetic ketoacidosis
Authors: Kemperman F. A. W. , Weber J. A. , Gorgels J. , Van Zanten A. P. , Krediet R. T. , Arisz L.
Published: August 2010

Abstract. Kemperman FAW, Weber JA, Gorgels J, van Zanten AP, Krediet RT, Arisz L (University of Amsterdam and Slotervaart Hospital, Amsterdam, The Netherlands). The influence of ketoacids on plasma creatinine assays in diabetic ketoacidosis. J Intern Med 2000; 248: 513–519.

Objective. Analysis of the interference of ketoacids on various routine plasma creatinine assays during a clinical episode of diabetic ketoacidosis (DKA).

Design. Observational study. Blood samples were drawn before, during and after standard in‐hospital treatment. Plasma creatinine was measured with two dissimilar enzymatic assays (creatininase PAP + and creatinine iminohydrolase Serapak), a kinetic alkaline picrate method (Jaffé) and a high‐performance liquid chromatography (HPLC) procedure. Acetoacetate and β‐hydroxybutyrate were analysed by enzymatic methods.

Setting. Department of Medicine, University Hospital.

Subjects. Nine patients who experienced 10 episodes of DKA.

Main outcome measures. Agreement of the routine plasma creatinine assays with HPLC and analysis of possible interferents.

Results. At presentation, the Jaffé assay gave falsely high values of plasma creatinine (median 99 µmol L–1), in contrast to the PAP+ (median 60.5 µmol L–1) and HPLC assays (median 67.5 µmol L–1). This positive error decreased during treatment. This was due to a decrease in acetoacetate, as the positive error by the Jaffé method correlated with the acetoacetate concentration (r = 0.79, P < 0.0001). In the multiple regression analysis, β‐hydroxybutyrate caused no additional interference by the Jaffé assay, confirmed by in vitro experiments. Analysis of agreement showed that the difference between PAP+ and HPLC creatinine was – 4.6 ± 3.0 µmol L–1 (mean ± SD), and 2.0 ± 5.3 µmol L–1 between Serapak and HPLC. This was statistically significant, but clinically negligible.

Conclusion. Acetoacetate caused severe interference of the alkaline picrate (Jaffé) assay, which might influence therapeutic decisions at the start of diabetic ketoacidosis. Enzymatic assays lack this interference.
Increased mortality in diabetes during the first 10 years of the disease. A population‐based study (DISS) in Swedish adults 15–34 years old at diagnosis
Authors: Wibell L. , Nyström L. , Östman J. , Arnqvist H. , Blohmé G. , Lithner F. , Littorin B. , Sundkvist G.
Published: August 2010

Abstract. Wibell L, Nyström L, Östman J, Arnqvist H, Blohmé G, Lithner F, Littorin B, Sundkvist G (Uppsala University Hospital, Uppsala; Umeå University, Umeå; Huddinge Hospital, Stockholm; Linköping University Hospital, Linköping; Sahlgrenska Hospital, Gothenburg; University of Lund; and Malmö University Hospital, Malmö, Sweden). Increased mortality in diabetes during the first 10 years of the disease. A population‐based study (DISS) in Swedish adults 15–34 years old at diagnosis. J Intern Med 2001; 249: 263–270.

Objectives. To study, prospectively, in young adult patients, the mortality during the first years after the diagnosis of diabetes.

Design. The Diabetes Incidence Study in Sweden (DISS) aims to register all incident cases aged 15–34 years. During a 10‐year period all deaths were identified by record linkage to the national Cause of Death Registry.

Subjects. During the period, 4097 new cases were registered and classified as type 1 diabetes (73%), type 2 (16%), secondary (2%) and unclassified (9%). The median follow‐up was 5 years (21 001 person‐years).

Main outcome measures. Calculation of the standardized mortality ratio (SMR) and 95% confidence interval (CI). Evaluation of all deceased by scrutiny of clinical records, death certificates and autopsy protocols.

Results. Fifty‐eight patients died, corresponding to an SMR of 3.5 (CI=2.7–4.5), which increased from 1.5 at 15–19 years to 4.1 at 30–34 years. SMR was 2.7 in primary diabetes: 2.3 (1.6–3.3) in type 1 and 4.1 (2.6–6.7) in type 2. In secondary diabetes, alcohol‐associated pancreatitis a common cause, SMR was 32 (CI=24–45). Evidence of alcohol or drug misuse, mental dysfunction or suicide was found in 40 of all 58 deceased cases. Less often, hypoglycaemia (n=7) or hyperglycaemia‐ketoacidosis (n=11) was present at death. Unexplained ‘dead in bed’ was found once.

Conclusions. In the investigated population‐based cohort the early mortality was about threefold increased. Hypoglycaemia and ketoacidosis per se played a relatively small role compared with a heavy impact from social and mental dysfunction, and from careless use of alcohol or drugs.
Developing an optimal approach to global drug safety
Authors: Balkrishnan R. , Furberg C. D.
Published: August 2010

Abstract. Balkrishnan R, Furberg CD (Wake Forest University School of Medicine, Winston‐Salem, NC, USA). Developing an optimal approach to global drug safety (Review).J Intern Med2001:250; 271–279.

An increasing number of media reports on a number of marketed drugs withdrawn because of harmful effects, a scientific report on epidemic proportions of serious adverse drug reactions in hospitalized patients, and a disturbing report on medical mistakes that includes medication errors have recently all brought drug safety into intense focus and placed it under greater scrutiny. Concerted efforts are now being made to understand the causes of drug safety problems and to find ways to reduce their frequency. An international symposium, ‘Developing an Optimal Approach to Drug Safety’ was held at Wake Forest University in the Fall of 2000 to identify the issues and solutions to extant problems in this area. This report summarizes the resulting discussions of global postmarketing surveillance initiatives and describes efforts to reduce medication errors, and improve global communication about drug safety.
Fruit and vegetable consumption in the prevention of cancer: an update
Authors: Terry P. , Terry J. B. , Wolk A.
Published: August 2010

Abstract. Terry P, Terry JB, Wolk A (The Karolinska Institute, Stockholm, Sweden; Albert Einstein College of Medicine, Bronx, NY, USA; and National Food Administration, Uppsala, Sweden). Fruit and vegetable consumption in the prevention of cancer: an update (Review). J Intern Med 2001; 250: 280–290.

Fruit and vegetables are considered to be important means of preventing cancer through diet. However, several recent large prospective cohort studies and clinical trials have found no associations regarding several major cancers, including cancers of the colon, breast and stomach. Although these results are disconcerting, especially for health‐conscious individuals, there is also mounting evidence that people who avoid fruit and vegetables completely, or consume very little, are indeed at increased risk of these cancers. These individuals in particular should be identified, with the goal of helping them find ways to include more fruit and vegetables in their diets.
Chronic mild neutropenia in adults: relation to IgG3 deficiency and infection susceptibility
Authors: Karlström R. , Gustafson R. , Palmblad J.
Published: August 2010

Abstract. Karlström R, Gustafson R, Palmblad J (The Karolinska Institutet at Huddinge University Hospital, Stockholm, Sweden). Chronic mild neutropenia in adults: relation to IgG3 deficiency and infection susceptibility. J Intern Med 2001; 250: 342–347.

Objectives. Chronic mild neutropenias (NP, i.e. absolute neutrophil blood counts/ANC/0.5–1.5 × 109 L−1) are accompanied by a variable infection susceptibility, which may or may not be as a result of concomitant conditions. Here, we assessed whether such patients also displayed an immunoglobulin deficiency and if this condition contributed to infection proneness.

Design, setting and subjects. Thirty consecutive adult Caucasian patients with chronic mild NP were followed at one university hospital for up to 28 years. Comparisons were made with 49 IgG3 deficiency patients at an immunodeficiency clinic.

Main outcome measures. Recorded infections, ANC and serum immunoglobulin levels; flow cytometry assessments of blood lymphocyte subsets and tests for autoimmunity were run to determine neutropenia subtypes. Results. Forty per cent of the NP patients were treated for severe or recurrent infections. The mean IgG3 value for the NP patients was significantly lower than for healthy controls (P < 0.005) and 33% of the patients displayed IgG3 values below the reference values (i.e. below 0.21 g L−1), and an additional 13% had IgG3 values within the range others consider low (0.21–0.41 g L−1). Unexpectedly, neutropenic IgG3 deficiency patients exhibited less infection proneness than those with normal IgG3 values (P=0.03). Patients with autoimmune, large granular lymphocyte‐associated or idiopathic NP had IgG3 deficiency in 63, 44 and 38%, respectively. In addition, none of IgG3 deficiency patients followed at the immunodeficiency clinic displayed neutropenia.

Conclusion. IgG3 deficiency is common amongst chronic mild neutropenia patients, particularly in those with autoimmune background, but contributes not significantly to infection susceptibility.
Ultrasound imaging in diagnosis of superior mesenteric artery syndrome
Authors: NERI S. , SIGNORELLI S. S. , MONDATI E. , PULVIRENTI D. , CAMPANILE E. , DI PINO L. , SCUDERI M. , GIUSTOLISI N. , DI PRIMA P. , MAUCERI B. , ABATE G. , CILIO D. , MISSERI M. , SCUDERI R.
Published: August 2010

Objectives.  We screened a cohort of subjects affected by various degree of dyspepsia to reveal if they presented a reduction of the aorto‐mesenteric angle and to diagnose suspected cases of superior mesenteric artery (SMA) syndrome.

Design.  Controlled, prospective, study.

Setting. Subjects were studied as outpatients.

Subjects.  The study investigated a total of 3622 subjects referred to our department by their general practitioners for dyspepsia and/or abdominal pain.

Interventions.  Abdominal ultrasonography with power colour Doppler, gastroduodenoscopy, hypotonic duodenography, contrast‐enhanced spiral computerized tomography were performed.

Main outcome measurement and results.  Color Doppler revealed a significant reduction of the SMA angle in 29 of 950 subjects; gastroscopy showed duodenal compressive pulsation in 14 of 29 patients and X‐ray revealed compression of the third segment of the duodenum in 28 of 29 patients. CT confirmed the presence of a reduced angle and various degrees of duodenal compression in all patients. Ultrasonography and CT examinations gave overlapping results (P > 0.05) in diagnosing pathological aorto‐mesenteric angle.

Conclusion.  The authors believe that the incidence of reduced aorto‐mesenteric angle and SMA syndrome might be underrated.

Ultrasound power colour Doppler imaging is useful in epidemiological screening of reduced aorto‐mesenteric angle to diagnose suspected cases of SMA syndrome.
Weight change and mortality: the Nord‐Trøndelag Health Study
Authors: DRØYVOLD W. B. , LUND NILSEN T. I. , LYDERSEN S. , MIDTHJELL K. , NILSSON P. M. , NILSSON J.‐Å. , HOLMEN J.
Published: August 2010

Objectives.  The prevalence of obesity is increasing. Overweight and obese people have increased mortality compared with normal weight people. We investigated the effect of weight change on mortality.

Design.  Prospective population study.

Setting.  We utilized data from two large population‐based health studies conducted in 1984–86 and 1995–97 respectively. Cox proportional hazards models were used to calculate mortality rate ratios (RRs) with 95% confidence intervals (CIs) between people with a stable weight and people who lost or gained weight.

Subjects.  Totally 20 542 men and 23 712 women aged 20 years or more, without cardiovascular disease or diabetes at the first survey and without a history of cancer at the second survey were followed up on all‐cause mortality for 5 years after the second survey.

Results.  We found no association between weight gain and mortality. People who lost weight had a higher total mortality rate compared with those who were weight stable [RR was 1.6 (95% CI: 1.4–1.8) in men and 1.7 (95% CI: 1.5–2.0) in women]. Similar associations were found for cardiovascular and noncardiovascular mortality. Additional analysis showed a linear increase in mortality rates across categories of weight loss for both men and women (P < 0.001). There was a statistically significant interaction between weight change and initial BMI, but only amongst men (P = 0.001).

Conclusions.  Weight loss, but not weight gain, was associated with increased mortality amongst men and women. Although underlying undiagnosed disease is the most plausible explanation for this finding, the similar associations found for total mortality, cardiovascular mortality, and noncardiovascular mortality makes the causal pathway somewhat enigmatic.
Exhaled NO and iNOS expression in sputum cells of healthy, obese and OSA subjects
Authors: Depalo A. , Carpagnano G. E. , Spanevello A. , Sabato R. , Cagnazzo M. G. , Gramiccioni C. , Foschino‐Barbaro M. P.
Published: August 2010

Background.  Obstructive sleep apnoea (OSA) is associated with airways inflammation; a key role in this regard seems to be played by nitric oxide (NO). The aim of this study was to measure exhaled NO and expression of its enzyme, the inducible nitric oxide synthase (iNOS) in cells of induced sputum in OSA patients and in obese subjects without sleep apnoea and to correlate these inflammatory markers with severity of OSA.

Methods.  We enrolled 18 obese patients with OSA (10 men, age 48.2 ± 8.4 years), 15 obese patients without OSA (eight men, age 52.8 ± 11 years) and 10 healthy subjects (five men, age 42 ± 4 years). Exhaled NO was measured using a chemiluminescence analyser; iNOS expression was measured in the sputum cells by immunocytochemistry.

Results.  Exhaled NO resulted significantly increased in OSA and in obese patients (23.1 ± 2.1 and 17.9 ± 2.1 p.p.b.) than in healthy subjects (7.2 ± 0.6 p.p.b.; P < 0.001). OSA and obese patients showed a higher percentage of neutrophils and a lower percentage of macrophages in the induced sputum compared to healthy subjects. In addition, OSA and obese patients showed higher iNOS expression in neutrophils and in macrophages with respect to healthy subjects. A positive correlation between exhaled NO, iNOS expression and AHI was observed.

Conclusions.  These data confirm the presence of airway inflammation in OSA and in obese patients, and suggest the possible role for NO and iNOS expression in neutrophils of the induced sputum as noninvasive markers to identify and monitor the airway inflammation in these subjects.
T64A polymorphism in β3‐adrenergic receptor gene (ADRB3) and coronary heart disease: a case‐cohort study and meta‐analysis
Authors: Zafarmand M. H. , Van Der Schouw Y. T. , Grobbee D. E. , De Leeuw P. W. , Bots M. L.
Published: August 2010

Objectives.  A missense mutation of the human ADRB3 gene replacing tryptophan with arginine at codon 64 (Trp64Arg) has been related to obesity, insulin resistance, earlier onset of noninsulin‐dependent diabetes mellitus and hypertension. These findings may also suggest an increased risk of coronary heart disease (CHD). We therefore investigated the role of this polymorphism on the occurrence of acute myocardial infarction (AMI) and CHD in a population of healthy Dutch women.

Design.  We performed a case‐cohort study in a prospective cohort of 15 236 initially healthy Dutch women. We applied a Cox proportional hazards model with an estimation procedure adapted for case‐cohort designs to study the relationship between the polymorphism and AMI (n = 71) and CHD (n = 211). In addition, a meta‐analysis of published studies was performed using a random effect model.

Results.  Using the dominant model, carriers of the arginine allele (n = 222) compared to those with the more common genotype (n = 1508) were not at increased risk of AMI (hazard ratio = 1.60; 95% CI, 0.86–2.96) and for CHD (HR = 1.36; 95% CI, 0.92–2.02). We did not find any relationship using recessive and additive models, either. Our meta‐analysis corroborated these findings by showing no significant association between the polymorphism and risk of CHD using different genetic models.

Conclusions.  Our study in combination with a meta‐analysis of previous reports do not provide support for a role of missense mutation replacing tryptophan with arginine at codon 64 (Trp64Arg) at the human ADRB3 gene in CHD risk.
Efficacy and safety of anticoagulant prophylaxis to prevent venous thromboembolism in acutely ill medical inpatients: a meta‐analysis
Authors: Själander A. , Jansson J.‐H. , Bergqvist D. , Eriksson H. , Carlberg B. , Svensson P.
Published: August 2010

Objectives.  Venous thromboembolism (VTE) is a potentially serious complication of hospitalization and immobilization. The use of anticoagulant prophylaxis in acutely ill medical inpatients is still under debate. New data including a recent meta‐analysis have recently been published. We aim at studying the efficacy and safety of anticoagulant prophylaxis in acutely ill medical inpatients, and demonstrate differences between meta‐analyses due to different data extraction from the heterogeneous studies included.

Subjects.  The Cochrane Library, MEDLINE and EMBASE were searched from 1980 to present. Manual searches were performed regarding abstracts from major meetings. Seven blinded randomized controlled clinical trials assessing the prophylactic effect of heparin in acutely ill medical patients were identified and included in the meta‐analysis.

Results.  Low‐molecular weight heparin (LMWH) prophylaxis prevented 48% of symptomatic pulmonary embolism (PE), 48% of symptomatic deep vein thrombosis (DVT) (not significant) and 51% of asymptomatic DVT. A nonsignificant trend towards higher bleeding risk during LMWH prophylaxis was found. Death was not significantly affected. We compared our data with a recent meta‐analysis with different study selection and data extraction and found similar results.

Conclusions.  As DVT and PE are manifestations of the same illness, VTE, one can argue that anticoagulant prophylaxis prevents approximately half of the expected events. Most medical inpatients have short hospital stays, and a low risk of VTE. The important task for the clinician is to identify patients with a sufficiently high risk of symptomatic VTE to warrant LMWH prophylaxis. Despite differences in study selection and data extraction, our study shows results similar to a recent meta‐analysis.
SIRT6 in DNA repair, metabolism and ageing
Authors: Lombard D. B. , Schwer B. , Alt F. W. , Mostoslavsky R.
Published: August 2010

Ageing, or increased mortality with time, coupled with physiologic decline, is a nearly universal yet poorly understood biological phenomenon. Studies in model organisms suggest that two conserved pathways modulate longevity: DNA damage repair and Insulin/Igf1‐like signalling. In addition, homologs of yeast Sir2 – the sirtuins – regulate lifespan in diverse organisms. Here, we focus on one particular sirtuin, SIRT6. Mice lacking SIRT6 develop a degenerative disorder that in some respects mimics models of accelerated ageing [Cell (2006) 124:315]. We discuss how sirtuins in general and SIRT6 specifically relate to other evolutionarily conserved pathways affecting ageing, and how SIRT6 might function to ensure organismal homeostasis and normal lifespan.
The genetics of ageing: insight from genome‐wide approaches in invertebrate model organisms
Authors: Kennedy B. K.
Published: August 2010

Intense effort has been directed at understanding pathways modulating ageing in invertebrate model organisms. Prior to this decade, several longevity genes had been identified in flies, worms and yeast. More recently, with the development of RNAi libraries in worms and the yeast open reading frame (ORF) deletion collection, it has become routine to perform genome‐wide screens for phenotypes of interest. A number of worm screens have now been performed to identify genes whose reduced expression leads to longer lifespan, and two ORF deletion longevity screens have been performed in yeast. Interestingly, these screens have linked previously unidentified cellular pathways to invertebrate ageing. More surprising, however, is the sheer number of longevity genes in worms and yeast. In this review, I discuss data from genome‐wide screens in the context of evolutionary theories of ageing and raise issues regarding the increasing complexity associated with the genetics of longevity.
Stress‐induced proteolysis in yeast
Authors: Hilt Wolfgang , Wolf Dieter H.
Published: August 2010

Survival of cells in their natural environment is crucially dependent on their ability to adapt to constantly occurring changes. The ability of cells to respond to extremes of environmental influences is vital to survival. Proteolysis is a central cellular tool in stress response. Proteins of pathways necessary for normal growth, but harmful under stress conditions, as well as proteins damaged by stress have to be eliminated. The yeast Saccharomyces cerevisiae, a model eukaryote, has evolved two different proteolytic systems: (i) a membrane‐enveloped, vacuolar (lysosomal) system, which contains a variety of non‐specific peptidases and (ii) highly specific peptidases residing at different cellular locations. The best characterized peptidase of the specific system is proteinase yscE, the proteasome equivalent found in all eukaryotic cells. Both the vacuolar and the non‐vacuolar systems are vital components of the stress response in yeast.
The Crl protein activates cryptic genes for curli formation and fibronectin binding in Escherichia coli HB101
Authors: Arnqvist Anna , Olsén Arne , Pfeifer John , Russell David G. , Normark Staffan
Published: August 2010

Curli are thin, coiled, temperature‐regulated fibres on fibronectin‐binding Escherichia coli. The subunit protein of curli was highly homologous at its amino terminus to SEF‐17, the subunit protein of thin, aggregative fimbriae of Salmonella enteritidis 27655 strain 3b, suggesting that these fibres form a novel class of surface organelles on enterobacteria. E. coli HB101 is non‐curliated and unable to bind soluble, iodinated fibronectin. The phenotypically cryptic curlin subunit gene, csgA, in HB101 is transcriptionally activated by expressing the cytoplasmic Crl on a multicopy plasmid. Transcriptional activation of csgA by Crl was observed after growth at 26°C but not at 37°C, even though crl transcription was not thermoregulated. A deletion of the 39 carboxy‐terminal residues abolished Crl activity, whereas a deletion of 10 residues at the C‐terminus did not, implying that a region between residue 93 and 122 in the 132‐amino‐acid‐residue large Crl protein is required for activating curli expression in E. coli HB101. crl is a normal housekeeeping gene in E. coli and it is suggested that its gene product may either be a DNA‐binding protein affecting chromatin structure as has been suggested for histone‐like protein H1 or interact with specific regulatory protein(s) controlling transcription of genes required for curli formation and fibronectin binding.
Isolation and characterization of the aadA aminoglycoside‐resistance gene from Salmonella choleraesuis
Authors: Leung K. Y. , Ruschkowski S. R. , Finlay B. B.
Published: August 2010

The streptomycin‐ and spectinomycin‐resistance gene of Salmonella choleraesuis was cloned and its nucleotide sequence determined. The gene is 789 bases long, encoding a protein of a predicted size of 29353 Da. The gene product inactivated streptomycin and spectinomycin by an adenylation modification. It is homologous (c. 40% total identity) to streptomycin adenylyltransferase, a 3′ (9)‐O‐nucleotidyltransferase (AAD(3′)(9)), which is encoded by the aad A gene in Escherichia coli, Agrobacterium tumefaciens, Kiebsiella pneumonia, and Serratia marcescens. The AadA protein of S. choleraesuis differs significantly from the other AadA proteins, indicating that it may have diverged from the other members of this family earlier in evolution. Southern hybridization analysis revealed that homologous aad A sequences were also present in other streptomycin‐resistant Salmonella species.
A new aspect of transcriptional control of the Escherichia coli crp gene: positive autoregulation
Authors: Hanamura Akemi , Aiba Hiroji
Published: August 2010

Transcription of the Escherichia coli crp gene is negatively regulated by CRP–cAMP that binds to a specific site located downstream of the crp promoter. A second binding site for CRP–cAMP (CRP site II) exists upstream of the crp promoter. Using an in vitro transcription assay, we have demonstrated that CRP‐cAMP activates transcription of crp in certain conditions. A promoter which carries an altered CRP‐binding site II is no longer activated by CRP–cAMP, indicating that CRP site II mediates the activation of crp transcription. The concentrations of cAMP that are required for positive autoregulation are higher than those for negative autoregulation. Evidence for positive and negative autoregulation in vivo is presented by a quantitative S1 nuclease analysis.
Farnesol and dodecanol effects on the Candida albicans Ras1‐cAMP signalling pathway and the regulation of morphogenesis
Authors: Davis‐Hanna Amber , Piispanen Amy E. , Stateva Lubomira I. , Hogan Deborah A.
Published: August 2010

Candida albicans hypha formation which has been stimulated via the Ras1‐cAMP‐Efg1 signalling cascade is inhibited by farnesol, a C. albicans autoregulatory factor, and small molecules such as dodecanol. In cultures containing farnesol or dodecanol, hypha formation was restored upon addition of dibutyryl‐cAMP. The CAI4‐Ras1G13V strain, which carries a dominant‐active variant of Ras1 and forms hyphae in the absence of inducing stimuli, grew as yeast in medium with farnesol or dodecanol; the heat shock sensitivity of the CAI4‐Ras1G13V strain was also suppressed by these compounds. Neither Pde1 nor Pde2 was necessary for the repression of hyphal growth by farnesol or dodecanol. Two transcripts, CTA1 and HSP12, which are at higher levels upon mutation of Ras1 or Cdc35, were increased in abundance in cells grown with farnesol or dodecanol. Microscopic analysis of strains carrying CTA1 and HWP1 promoter fusions grown with intermediate concentrations of farnesol or dodecanol indicated a link between cells with the increased expression of cAMP‐repressed genes and cells repressed for hypha formation. Because several cAMP‐controlled outputs are affected by farnesol and dodecanol, our findings suggest that these compounds impact activity of the Ras1‐Cdc35 pathway, thus leading to an alteration of C. albicans morphology.
Co‐ordinated regulation of gluconate catabolism and glucose uptake in Corynebacterium glutamicum by two functionally equivalent transcriptional regulators, GntR1 and GntR2
Authors: Frunzke Julia , Engels Verena , Hasenbein Sonja , Gätgens Cornelia , Bott Michael
Published: August 2010

Corynebacterium glutamicum is a Gram‐positive soil bacterium that prefers the simultaneous catabolism of different carbon sources rather than their sequential utilization. This type of metabolism requires an adaptation of the utilization rates to the overall metabolic capacity. Here we show how two functionally redundant GntR‐type transcriptional regulators, designated GntR1 and GntR2, co‐ordinately regulate gluconate catabolism and glucose uptake. GntR1 and GntR2 strongly repress the genes encoding gluconate permease (gntP), gluconate kinase (gntK), and 6‐phosphogluconate dehydrogenase (gnd) and weakly the pentose phosphate pathway genes organized in the tkt‐tal‐zwf‐opcA‐devB cluster. In contrast, ptsG encoding the EIIGlc permease of the glucose phosphotransferase system (PTS) is activated by GntR1 and GntR2. Gluconate and glucono‐δ‐lactone interfere with binding of GntR1 and GntR2 to their target promoters, leading to a derepression of the genes involved in gluconate catabolism and reduced ptsG expression. To our knowledge, this is the first example for gluconate‐dependent transcriptional control of PTS genes. A mutant lacking both gntR1 and gntR2 shows a 60% lower glucose uptake rate and growth rate than the wild type when cultivated on glucose as sole carbon source. This growth defect can be complemented by plasmid‐encoded GntR1 or GntR2.
A stepwise model for double‐stranded RNA processing by ribonuclease III
Authors: Gan Jianhua , Shaw Gary , Tropea Joseph E. , Waugh David S. , Court Donald L. , Ji Xinhua
Published: August 2010

RNA interference is mediated by small interfering RNAs produced by members of the ribonuclease III (RNase III) family represented by bacterial RNase III and eukaryotic Rnt1p, Drosha and Dicer. For mechanistic studies, bacterial RNase III has been a valuable model system for the family. Previously, we have shown that RNase III uses two catalytic sites to create the 2‐nucleotide (nt) 3′ overhangs in its products. Here, we present three crystal structures of RNase III in complex with double‐stranded RNA, demonstrating how Mg2+is essential for the formation of a catalytically competent protein–RNA complex, how the use of two Mg2+ions can drive the hydrolysis of each phosphodiester bond, and how conformational changes in both the substrate and the protein are critical elements for assembling the catalytic complex. Moreover, we have modelled a protein–substrate complex and a protein–reaction intermediate (transition state) complex on the basis of the crystal structures. Together, the crystal structures and the models suggest a stepwise mechanism for RNase III to execute the phosphoryl transfer reaction.
Haloferax volcanii twin‐arginine translocation substates include secreted soluble, C‐terminally anchored and lipoproteins
Authors: Giménez María I. , Dilks Kieran , Pohlschröder Mechthild
Published: August 2010

Recent in silico and in vivo studies have suggested that the majority of proteins destined for secretion in the haloarchaea are trafficked through the twin‐arginine translocation (Tat) pathway. The presence of lipobox motifs in most haloarchaeal Tat signal sequences is intriguing as: (i) bioinformatic searches of archaeal genomes have not identified lipoprotein biogenesis enzymes and (ii) there are no known Tat substrates containing both a twin‐arginine and a bona fide lipobox. We have examined six computationally designated Tat substrates in the haloarchaeon Haloferax volcanii to verify previous computational predictions and to initiate studies of lipoprotein biogenesis via the Tat pathway. Our results confirmed that the six candidate proteins were not only Tat substrates, but also belonged to diverse classes of secretory proteins. Analysis of predicted lipoprotein Tat substrates revealed that they are anchored to the archaeal membrane in a cysteine‐dependent manner. Interestingly, despite the absence of an archaeal lipoprotein signal peptidase II (SPase II) homologue, the SPase II inhibitor globomycin impeded cell growth and specifically prevented maturation of lipoproteins. Together, this work not only represents the first experimental demonstration of a lipoprotein Tat substrate, but also indicates the presence of an unidentified lipoprotein biogenesis pathway in archaea.
Different regions of Mlc and NagC, homologous transcriptional repressors controlling expression of the glucose and N‐acetylglucosamine phosphotransferase systems in Escherichia coli, are required for inducer signal recognition
Authors: Pennetier Carole , Domínguez‐Ramírez Lenin , Plumbridge Jacqueline
Published: August 2010

Mlc and NagC are two homologous transcription factors which bind to similar DNA targets but for which the inducing signals and mechanisms of activation are very different. Displacing Mlc from its DNA binding sites necessitates its sequestration to the inner membrane via an interaction with PtsG (EIICBGlc), while NagC is displaced from its DNA targets by interacting with GlcNAc6P. We have isolated mutations in both proteins which prevent the inactivation of the repressors by growth on glucose or GlcNAc. These mutations are located in different and specific regions of each protein. For Mlc changes at the C‐terminal make it a constitutive repressor and also prevent it from binding to EIIBGlc. Mutations in NagC, at positions which form a structural motif resembling a glucose binding site in Mlc, produce permanently repressing forms of NagC, suggesting that this motif forms a GlcNAc6P binding site in NagC. The pattern of repression by chimeric proteins of NagC and Mlc confirms the importance of the C‐terminal region of Mlc for both repression and inducer binding and demonstrate that the helix–turn–helix DNA‐binding motif is not sufficient to determine the specificity of interaction of the repressor with DNA.
Periplasmic phosphorylation of lipid A is linked to the synthesis of undecaprenyl phosphate
Authors: Touzé Thierry , Tran An X. , Hankins Jessica V. , Mengin‐Lecreulx Dominique , Trent M. Stephen
Published: August 2010

One‐third of the lipid A found in the Escherichia coli outer membrane contains an unsubstituted diphosphate unit at position 1 (lipid A 1‐diphosphate). We now report an inner membrane enzyme, LpxT (YeiU), which specifically transfers a phosphate group to lipid A, forming the 1‐diphosphate species. 32P‐labelled lipid A obtained from lpxT mutants do not produce lipid A 1‐diphosphate. In vitro assays with Kdo2‐[4′‐32P]lipid A as the acceptor shows that LpxT uses undecaprenyl pyrophosphate as the substrate donor. Inhibition of lipid A 1‐diphosphate formation in wild‐type bacteria was demonstrated by sequestering undecaprenyl pyrophosphate with the cyclic polypeptide antibiotic bacitracin, providing evidence that undecaprenyl pyrophosphate serves as the donor substrate within whole bacteria. LpxT‐catalysed phosphorylation is dependent upon transport of lipid A across the inner membrane by MsbA, a lipid A flippase, indicating a periplasmic active site. In conclusion, we demonstrate a novel pathway in the periplasmic modification of lipid A that is directly linked to the synthesis of undecaprenyl phosphate, an essential carrier lipid required for the synthesis of various bacterial polymers, such as peptidoglycan.
Incorporation of a polypeptide segment into the β‐domain pore during the assembly of a bacterial autotransporter
Authors: Ieva Raffaele , Skillman Kristen M. , Bernstein Harris D.
Published: August 2010

Bacterial autotransporters consist of an N‐terminal ‘passenger domain’ that is transported into the extracellular space by an unknown mechanism and a C‐terminal ‘β‐domain’ that forms a β‐barrel in the outer membrane. Recent studies have revealed that fully assembled autotransporters have an unusual architecture in which a small passenger domain segment traverses the pore formed by the β‐domain. It is unclear, however, whether this configuration forms prior to passenger domain translocation or results from the translocation of the passenger domain through the β‐domain pore. By examining the accessibility of tobacco etch virus protease sites and single‐cysteine residues in the passenger domain of the Escherichia coli O157:H7 autotransporter EspP at different stages of protein biogenesis, we identified a novel pre‐translocation intermediate whose topology resembles that of the fully assembled protein. This intermediate was isolated in the periplasm in cell fractionation experiments. The data strongly suggest that the EspP β‐domain and an embedded polypeptide segment are integrated into the outer membrane as a single pre‐formed unit. The data also provide indirect evidence that at least some outer membrane proteins acquire considerable tertiary structure prior to their membrane integration.
The Chlamydia outer membrane protein OmcB is required for adhesion and exhibits biovar‐specific differences in glycosaminoglycan binding
Authors: Moelleken Katja , Hegemann Johannes H.
Published: August 2010

Chlamydia pneumoniae, an obligate intracellular human pathogen, causes a number of respiratory diseases. We explored the role of the conserved OmcB protein in C. pneumoniae infections, using yeast display technology. (i) Yeast cells presenting OmcB were found to adhere to human epithelial cells. (ii) Pre‐incubation of OmcB yeast cells with heparin, but not other glycosaminoglycans (GAGs), abrogated adhesion. (iii) Pre‐treatment of the target cells with heparinase inhibited adherence, and GAG‐deficient CHO cell lines failed to bind OmcB yeast. (iv) A heparin‐binding motif present near the N‐terminus of OmcB is required for host cell binding. (v) Pre‐treatment of chlamydial elementary bodies (EBs) with anti‐OmcB antibody or pre‐incubation of target cells with recombinant OmcB protein reduced infectivity upon challenge with C. pneumoniae. (vi) Adhesion of fluorescently labelled EBs to epithelial or endothelial cells was abrogated by prior addition of heparin or OmcB protein. Thus, C. pneumoniae OmcB is an adhesin that binds heparan sulphate‐like GAGs. OmcB from Chlamydia trachomatis serovar L1 also adheres to human cells in a heparin‐dependent way, unlike its counterpart from serovar E. We show that a single position in the OmcB sequence determines heparin dependence/independence, and variations there may reflect differences between the two serovars in cell tropism and disease pattern.
Undecaprenyl phosphate recycling comes out of age
Authors: Valvano Miguel A.
Published: August 2010

The recycling of the lipid carrier undecaprenyl‐phosphate (Und‐P) requires the dephosphorylation of Und‐PP, a reaction proposed to occur at the external or periplasmic side of the bacterial cell membrane. In this issue of Molecular Microbiology, experiments based on the analysis of lipopolysaccharide modifications in Escherichia coli demonstrate that the phosphorylation of lipid A at position 1 is catalysed by the membrane enzyme LpxT (formerly YeiU). This enzyme specifically transfers the distal phosphate group from Und‐PP to lipid A 1‐phosphate to produce lipid A 1‐diphosphate. Furthermore, this reaction requires a functionally intact MsbA protein, which catalyses the transfer of lipid A across the membrane, confirming that the LpxT‐mediated lipid A modification occurs on the periplasmic side of the membrane. These observations provide a novel and unexpected link between periplasmic lipid A modifications and the Und‐PP recycling pathway.
Division of labour during Bacillus subtilis biofilm formation
Authors: Kearns Daniel B.
Published: August 2010

Biofilms are multicellular aggregates of bacteria that are organized by an extracellular matrix. A key determinant of biofilm formation by a variety of bacteria is the proper regulation and assembly of the matrix components. In this issue of Molecular Microbiology, Chai et al. present the surprising finding that the genes encoding the components of the extracellular matrix in Bacillus subtilis are expressed in only a subpopulation of cells. Their results indicate that biofilms contain specialized cell types that are differentiated by transcriptional regulation. The population heterogeneity that they observe is another significant example in a growing list of phenotypes that arise owing to bistable gene expression.
Characterization of five novel Pseudomonas aeruginosa cell‐surface signalling systems
Authors: Llamas María A. , Mooij Marlies J. , Sparrius Marion , Vandenbroucke‐Grauls Christina M. J. E. , Ratledge Colin , Bitter Wilbert
Published: August 2010

Cell‐surface signalling is a sophisticated regulatory mechanism used by Gram‐negative bacteria to sense signals from outside the cell and transmit them into the cytoplasm. This regulatory system consists of an outer membrane‐localized TonB‐dependent receptor (TonB‐dependent transducer), a cytoplasmic membrane‐localized antisigma factor and an extracytoplasmic function (ECF) sigma factor. Pseudomonas aeruginosa contains 13 potential surface signalling systems of which only six have been studied in detail. In this work we have identified the regulons of five novel P. aeruginosa signalling systems. For that, the ECF sigmas PA0149, PA1912, PA2050, PA2093 and PA4896 have been overexpressed and their target gene candidates have been identified using DNA microarray, proteomic analysis, and/or lacZ reporter construct. All five ECF sigma factors control the production of one TonB‐dependent transducer. Interestingly, two sigma factors, PA2050 and PA2093, regulate the synthesis of a second transducer. Furthermore, we show that although all these sigma factors seem to control putative (metal) transport systems, one of them also regulates the expression of P. aeruginosa pyocins. Finally, we also show that the PA1912‐PA1911‐PA1910 (designated FemI‐FemR‐FemA in this work) signalling system responds to the presence of the Mycobacterium siderophores mycobactin and carboxymycobactin and is involved in the utilization of these heterologous siderophores.
A phage display system designed to detect and study protein–protein interactions
Authors: Bair Catherine L. , Oppenheim Amos , Trostel Andrei , Prag Gali , Adhya Sankar
Published: August 2010

Analysing protein–protein interactions is critical in proteomics and drug discovery. The usage of 2‐Hybrid (2λ) systems is limited to an in vivo environment. We describe a bacteriophage 2‐Hybrid system for studying protein interactions in vitro. Bait and prey are displayed as fusions to the surface of phage λ that are marked with different selectable drug‐resistant markers. An interaction of phages in vitro through displayed proteins allows bacterial infection by two phages resulting in double drug‐resistant bacterial colonies at very low multiplicity of infections. We demonstrate interaction of the protein sorting signal Ubiquitin with the Vps9‐CUE, a Ubiquitin binding domain, and by the interaction of (Gly‐Glu)4 and (Gly‐Arg)4 peptides. Interruptions of the phage interactions by non‐fused (free) bait or prey molecules show how robust and unique our approach is. We also demonstrate the use of Ubiquitin and CUE display phages to find binding partners in a λ‐display library. The unique usefulness to 2λ is also described.
Polymorphism and regulation of the spxB (pyruvate oxidase) virulence factor gene by a CBS‐HotDog domain protein (SpxR) in serotype 2 Streptococcus pneumoniae
Authors: Ramos‐Montañez Smirla , Tsui Ho‐Ching Tiffany , Wayne Kyle J. , Morris Jordan L. , Peters Lindsey E. , Zhang Faming , Kazmierczak Krystyna M. , Sham Lok‐To , Winkler Malcolm E.
Published: August 2010

spxB‐encoded pyruvate oxidase is a major virulence factor of Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes H2O2 and acetyl phosphate, which play roles in metabolism, signalling, and oxidative stress. We report here the first cis‐ and trans‐acting regulatory elements for spxB transcription. These elements were identified in a genetic screen for spontaneous mutations that caused colonies of strain D39 to change from a semitransparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency ≈ 3 × 10−5) were impaired for SpxB function or expression. Two mutations changed amino acids in SpxB likely required for cofactor or subunit binding. One mutation defined a cis‐acting adjacent direct repeat required for optimal spxB transcription. The other three spontaneous mutations created the same frameshift near the start of the trans‐acting spxR regulatory gene. The SpxR protein contains helix–turn–helix, CBS and HotDog domains implicated in binding DNA, adenosyl compounds, and CoA‐containing compounds respectively, and suggest that SpxR positively regulates spxB transcription in response to energy and metabolic state. Microarray analyses unexpectedly demonstrated that SpxR also positively regulates the strH exoglycosidase gene, which, like spxB, has been implicated in colonization. Finally, SpxR is required for full virulence in a murine model of infection.
Escherichia coli DnaA interacts with HU in initiation at the E. coli replication origin
Authors: Chodavarapu Sundari , Felczak Magdalena M. , Yaniv Josette Rouvière , Kaguni Jon M.
Published: August 2010

Escherichia coli HU protein is a dimer encoded by two closely related genes whose expression is growth phase‐dependent. As a major component of the bacterial nucleoid, HU binds to DNA non‐specifically, but acts at the chromosomal origin (oriC) during initiation by stimulating strand opening in vitro. We show that the α dimer of HU is more active than other forms of HU in initiation of an oriC‐containing plasmid because it more effectively promotes strand opening of oriC. Other results demonstrate that HU stabilizes the DnaA oligomer bound to oriC, and that the α subunit of HU interacts with the N‐terminal region of DnaA. These observations support a model whereby DnaA interacts with the α dimer or the αβ heterodimer, depending on their cellular abundance, to recruit the respective form of HU to oriC. The greater activity of the α dimer of HU at oriC may stimulate initiation during early log phase compared with the lesser activity of the αβ heterodimer or the β dimer.
The Plasmodium falciparum Maurer's clefts in 3D
Authors: Frischknecht Friedrich , Lanzer Michael
Published: August 2010

In 1902, the German physician Georg Maurer discovered a dotted staining pattern within the cytoplasm of Plasmodium falciparum infected erythrocytes that, according to the tradition at the time, was named in his honour. The significance of Georg Maurer's discovery remained unrecognized for almost a century. Only recently are Maurer's clefts appreciated as a novel type of secretory organelle. Established by the malaria parasite within its host cell, Maurer's clefts play an essential role in directing proteins from the parasite to the erythrocyte surface. In this issue of Molecular Microbiology, Hanssen et al. report on the three dimensional structure of Maurer's clefts, as determined by electron tomography. The data presented suggest that Maurer's clefts are connected to both the parasitophorous vacuolar and the erythrocyte plasma membrane, however, no continuum exists that would allow lipids or proteins to freely flow between these three compartments. This seminal work, which stands in the tradition of Georg Maurer's original discovery, represents a milestone in our understanding of the structure and function of this fascinating organelle.
Trans‐targeting of protease substrates by conformationally activating a regulable ClpX‐recognition motif
Authors: Marshall‐Batty Kimberly R. , Nakai Hiroshi
Published: August 2010

Conversion of bacteriophage Mu repressor to ClpXP‐sensitive form correlates with induced local flexibility at the ClpX recognition motif located at the C‐terminal end. Changing the C‐terminal valine to an alanine (RepV196A) caused the degradation tag to be constitutively active like that of mutant repressors called Vir, which have a dominant ClpXP‐sensitive conformation. However, unlike Vir, RepV196A was unable to convert wild‐type repressor (Rep) to the ClpXP‐sensitive form. In mixtures with Rep, only RepV196A was rapidly degraded by ClpXP. Unlike Rep, RepV196A was ClpXP sensitive without induced C‐terminal flexibility. And unlike adaptor proteins that tether and deliver substrates to ClpX for trans‐targeting, Vir promoted rapid degradation of Rep by ClpX deleted for the tethering site that binds adaptor proteins. Therefore, Rep's ClpX recognition motif has regulable properties, allowing an alternative trans‐targeting mechanism in which an inactive degradation tag is turned on by induced conformational changes.