bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter
Authors: Kovacikova Gabriela , Skorupski Karen 
Published: March 2003

SummaryQuorum sensing negatively influences virulence gene expression in certain toxigenic Vibrio cholerae strains. At high cell densities, the response regulator LuxO fails to reduce the expression of HapR, which, in turn, represses the expression of the virulence cascade. A critical regulatory step in the cascade is activation of tcpPH expression by AphA and AphB. We show here that HapR influences the virulence cascade by directly repressing aphA expression. In strain C6706, aphA expression was increased in a ΔhapR mutant and decreased in a ΔluxO mutant, indicating a negative and positive influence, respectively, of these gene products on the promoter. Overexpression of HapR also reduced aphA expression in both C6706 and Escherichia coli. DNase I footprinting showed that purified HapR binds to the aphA promoter between −85 and −58. Although it appears that quorum sensing does not influence virulence gene expression in strain O395 solely because of a frameshift in hapR, overproduced HapR did not repress expression from the O395 aphA promoter in either Vibrio or E. coli, nor did the protein bind to the promoter. Two basepair differences from C6706 are present in the O395 HapR binding site at −85 and −77. Introducing the −77 change into C6706 prevented HapR binding and repression of aphA expression. This mutation also eliminated the repression of toxin‐co‐regulated pilus (TCP) and cholera toxin (CT) that occurs in a ΔluxO mutant, indicating that HapR function at aphA is critical for density‐dependent regulation of virulence genes.
Formation of the stoichiometric complex of EnvZ, a histidine kinase, with its response regulator, OmpR
Authors: Yoshida Takeshi , Qin Ling , Inouye Masayori 
Published: March 2003

SummaryEnvZ, a histidine kinase, and its cognate response regulator OmpR of Escherichia coli are responsible for adaptation to external osmotic changes by regulating the levels of the outer membrane porin proteins, OmpF and OmpC. The osmosensor, EnvZ, has dual enzymatic functions with OmpR kinase and OmpR‐P phosphatase. Here, we demonstrate that the cytoplasmic kinase domain of EnvZ (EnvZc) and OmpR are able to form a 1:1 complex detected by native PAGE. This indicates that two OmpR molecules can bind to one EnvZc dimer. As this 1:1 EnvZc/OmpR complex is formed even in the presence of a large excess of EnvZc, OmpR binding to EnvZc is co‐operative. The complex formation is also observed between EnvZc and phosphorylated OmpR for the phosphatase reaction. OmpR‐P bound to EnvZc was readily released upon the addition of OmpR, indicating that OmpR and OmpR‐P can compete for the binding to EnvZ. On the basis of these results, a model is discussed to explain how cellular OmpR‐P concentrations are regulated in response to medium osmolarity.
The expression level determines the surface distribution of the transferrin receptor in Trypanosoma brucei
Authors: Mußmann Rainer , Janssen Hans , Calafat Jero , Engstler Markus , Ansorge Iris , Clayton Christine , Borst Piet 
Published: March 2003

SummaryThe transferrin receptor (TfR) of Trypanosoma brucei is a heterodimer attached to the surface membrane by a glycosylphosphatidylinositol (GPI) anchor. The TfR is restricted to the flagellar pocket, a deep invagination of the plasma membrane. The membrane of the flagellar pocket and the rest of the cell surface are continuous, and the mechanism that selectively retains the TfR in the pocket is unknown. Here, we report that the TfR is retained in the flagellar pocket by a specific and saturable mechanism. In bloodstream‐form trypanosomes transfected with the TfR genes, TfR molecules escaped flagellar pocket retention and accumulated on the entire surface, even at modest (threefold) overproduction levels. Similar surface accumulation was observed when the TfR levels were physiologically upregulated threefold when trypanosomes were starved for transferrin. These results suggest that the TfR flagellar pocket retention mechanism is easily saturated and that control of the expression level is critical to maintain the restricted surface distribution of the receptor.
Modulation of phage Mu repressor DNA binding and degradation by distinct determinants in its C‐terminal domain
Authors: Mukhopadhyay Bani , Marshall‐Batty Kimberly R. , Kim Benjamin D. , O'Handley Diane , Nakai Hiroshi 
Published: March 2003

SummaryRapid degradation of the bacteriophage Mu immunity repressor can be induced in trans by mutant, protease‐hypersensitive repressors (Vir) with an altered C‐terminal domain (CTD). Genetic and biochemical analysis established that distinct yet overlapping determinants in the wild‐type repressor CTD modulate Vir‐induced degradation by Escherichia coli ClpXP protease and DNA binding by the N‐terminal DNA‐binding domain (DBD). Although deletions of the repressor C‐terminus resulted in both resistance to ClpXP protease and suppression of a temperature‐sensitive DBD mutation (cts62), some cysteine‐replacement mutations in the CTD elicited only one of the two phenotypes. Some CTD mutations prevented degradation induced by Vir and resulted in the loss of intrinsic ClpXP protease sensitivity, characteristic of wild‐type repressor, and at least two mutant repressors protected Vir from proteolysis. One protease‐resistant mutant became susceptible to Vir‐induced degradation when it also contained the cts62 mutation, which weakens DNA binding but apparently facilitates conversion to a protease‐sensitive conformation. Conversely, this CTD mutation was able to suppress temperature sensitivity of DNA binding by the cts62 repressor. The results suggest that determinants in the CTD not only provide a cryptic ClpX recognition motif but also direct CTD movement that exposes the motif and modulates DNA binding.
Specialized osmotic stress response systems involve multiple SigB‐like sigma factors in Streptomyces coelicolor
Authors: Viollier Patrick H. , Kelemen Gabriella H. , Dale Glenn E. , Nguyen Kien T. , Buttner Mark J. , Thompson Charles J. 
Published: March 2003

SummaryWhereas in Bacillus subtilis, a general stress response stimulon under the control of a single sigma factor (SigB) is induced by different physiological and environmental stresses (heat, salt or ethanol shock), in Streptomyces coelicolor, these environmental stresses induce independent sets of proteins, and its genome encodes nine SigB paralogues. To investigate possible functions of multiple sigB‐like genes in S. coelicolor, Pctc, a promoter routinely used to assay SigB activity in vivo, was analysed as a heterologous reporter. The fact that Pctc was activated by osmotic shock, but not by heat or ethanol, confirmed that stress response system(s) could operate independently in S. coelicolor. Pctc was also induced transiently during growth of liquid cultures, presumably by nutritional signals. We purified an RNA polymerase holoenzyme from crude extracts that catalysed specific transcription of Pctc in vitro. Its sigma subunit was identified as a product of the sigH gene, which is co‐transcribed downstream of a putative antisigma factor gene (prsH). Although the sigH function was not needed for normal colony morphology, prsH was conditionally required for both aerial hyphae formation and regulation of antibiotic biosynthesis. Levels of two different sigH‐encoded proteins were growth phase dependent but not significantly changed by osmotic stress, implying the important roles of post‐translational regulatory elements such as PrsH. In addition, synthesis of three other SigH‐like proteins was induced by osmotic stress, but not by ethanol or heat. Two of them were genetically assigned to sigH homologous loci sigI and sigJ and shown to be independently regulated. This family of SigH‐like proteins displayed different osmotic response kinetics. Thus, in contrast to many other bacteria, S. coelicolor uses an osmotic sensory system that can co‐ordinate the activity of multiple paralogues to control the relative activity of promoters within the same stress stimulon. Such specialized stress response systems may reflect adaptive functions needed for colonial differentiation.
The product of the UTH1 gene, required for Bax‐induced cell death in yeast, is involved in the response to rapamycin
Authors: Camougrand Nadine , Grelaud‐Coq Angela , Marza Esther , Priault Muriel , Bessoule Jean‐Jacques , Manon Stéphen 
Published: March 2003

SummaryA yeast mutant was isolated that was resistant to Bax‐induced cell death. It supports a mutation leading to decreased amounts of the protein Uth1p. A strain in which the UTH1 gene is disrupted also exhibits resistance to Bax expression. The absence of Uth1p does not change the mitochondrial localization of Bax, its insertion in the mitochondrial outer membrane or its cytochrome c‐releasing activity. On the other hand, the absence of Uth1p does prevent the appearance of other hallmarks related to Bax expression in yeast, such as oxidation of mitochondrial lipid, production of reactive oxygen species and maintenance of plasma membrane properties after ethanol stress. The absence of Uth1p was also found to induce resistance to rapamycin, a specific inducer of autophagy. This resistance only appears when cells are grown under respiratory conditions, but not under fermentative conditions, suggesting that Uth1p acts in an autophagic pathway involving mitochondria, in accordance with its main localization in the outer mitochondrial membrane. Taken together, these data show that Bax is able to activate a death pathway related to autophagy in yeast, which also exhibits typical hallmarks of apoptosis, revealing a possible dual function of Bax in both types of death. This hypothesis is discussed in the light of observations suggesting a co‐regulation of apoptosis and autophagy in mammalian cells.
Ssn6, an important factor of morphological conversion and virulence in Candida albicans
Authors: Hwang Cheol‐Sang , Oh Jang‐Hyun , Huh Won‐Ki , Yim Hyung‐Soon , Kang Sa‐Ouk 
Published: March 2003

Summary Candida albicans
, the major fungal pathogen in humans, undergoes morphological conversion from yeasts to filamentous growth forms depending upon various environmental conditions. Here, we have identified a
C. albicans
gene, namely
SSN6
, encoding a putative global transcriptional co‐repressor that is highly homologous to the
Saccharomyces cerevisiae
Ssn6. The isolated
C. albicans SSN6
complemented the pleiotropic phenotypes of
S. cerevisiae ssn6
mutation, and its expression levels declined significantly in response to a strong true hyphal inducer, serum. The mutant lacking
C. albicans
Ssn6 displayed a stubby pseudohyphal growth pattern, derepressed filament‐specific genes in response to elevated temperature 37°C and failed to develop true hyphae, extensive filamentation and virulence. Such morphological defects of
ssn6
/
ssn6
mutant were not rescued by overexpression of Tup1, Cph1 or Efg1. Moreover, epistatic analysis showed that, as far as cell morphology was concerned, Ssn6 was epistatic to Tup1 at the higher temperature but that, at the lower temperature, the
ssn6
/
ssn6 tup1
/
tup1
double mutant grew in a stubby form of pseudohyphae distinct from the phenotypes of either single mutant. Furthermore, overexpression of
SSN6
in
C. albicans led to enhanced filamentous growth and attenuated virulence. These findings suggest that Ssn6 may function as an activator as well as a repressor of filamentous growth and be a target for candidacidal drugs, as its excess or deficiency resulted in impaired virulence.
Iron and contact with host cells induce expression of adhesins on surface of Trichomonas vaginalis
Authors: Garcia Ana F. , Chang Te‐Hung , Benchimol Marlene , Klumpp David Jichael , Lehker Michael W. , Alderete John F. 
Published: March 2003

SummaryThe proteins AP65, AP51, AP33 and AP23 synthesized by Trichomonas vaginalis organisms in high iron play a role in adherence. Multigene families encode enzymes of the hydrogenosome organelles, which have identity to adhesins. This fact raises questions regarding the compartmentalization of the proteins outside the organelle and about the interactions of adhesins with host cells. Data here demonstrate the presence of the proteins outside the organelle under high‐iron conditions. Fluorescence and immuno‐cytochemical experiments show that high‐iron‐grown organisms coexpressed adhesins on the surface and intracellularly in contrast with low‐iron parasites. Furthermore, the AP65 epitopes seen by rabbit anti‐AP65 serum that blocks adherence and detects surface proteins were identified, and a mAb reacting to those epitopes recognized the trichomonal surface. Two‐dimensional electrophoresis and immunoblot of adhesins from surface‐labelled parasites provided evidence that all members of the multigene family were co‐ordinately expressed and placed on the trichomonal surface. Similar two‐dimensional analysis of proteins from purified hydrogenosomes obtained from iodinated trichomonads confirmed the specific surface labelling of proteins. Contact of trichomonads with vaginal epithelial cells increased the amount of surface‐expressed adhesins. Moreover, we found a direct relationship between the levels of adherence and amount of adhesins bound to immortalized vaginal and ureter epithelial cells, further reinforcing specific associations. Finally, trichomonads of MR100, a drug‐resistant isolate absent in hydrogenosome proteins and adhesins, were non‐adherent. Overall, the results confirm an important role for iron and contact in the surface expression of adhesins of T. vaginalis organisms.
Role of the GGDEF regulator PleD in polar development of Caulobacter crescentus
Authors: Aldridge Phillip , Paul Ralf , Goymer Patrick , Rainey Paul , Jenal Urs 
Published: March 2003

SummarySeveral members of the two‐component signal transduction family have been implicated in the control of  polar development in Caulobacter crescentus: PleC and DivJ, two polarly localized histidine sensor kinases; and the response regulators DivK and PleD. The PleD protein was shown previously to be required during the swarmer‐to‐stalked cell transition for flagellar ejection and efficient stalk biogenesis. Here, we present data indicating that PleD also controls the onset of motility and a cell density switch immediately preceding cell division. Constitutively active alleles of pleD or wspR, an orthologue from Pseudomonas fluorescens, almost completely suppressed C. crescentus motility and inhibited the increase in swarmer cell density during cell differentiation. The observation that these alleles also had a dominant‐negative effect on motility in a pleC divJ and a pleC divK mutant background indicated that PleD is located downstream of the other components in the signal transduction cascade, which controls the activity of the flagellar motor. In addition, the presence of a constitutive pleD or wspR allele resulted in a doubling of the average stalk length. Together, this is consistent with a model in which the active form of PleD, PleD∼P, negatively controls aspects of differentiation in the late predivisional cell, whereas it acts positively on polar development during the swarmer‐to‐stalked cell transition. In agreement with such a model, we found that DivJ, which localizes to the stalked pole during cell differentiation, positively controlled the in vivo phosphorylation status of PleD, and the swarmer pole‐specific PleC kinase modulated this status in a negative manner. Furthermore, domain switch experiments demonstrated that the WspR GGDEF output domain from P. fluorescens is active in C. crescentus, favouring a more general function for this novel signalling domain over a specific role such as DNA or protein interaction. Possible roles for PleD and its C‐terminal output domain in modulating the polar cell surface of C. crescentus are discussed.
Risk stratification and pathological mechanisms in preterm delivery
Authors: Lockwood Charles J. , Kuczynski Edward 
Published: March 2003

The delivery of infants before 37 weeks gestation is a leading cause of perinatal mortality and morbidity in the United States. Traditional methods of predicting women at risk relying on obstetric history or premonitory symptoms (detected clinically or by tocodynamometry) are neither sensitive nor specific. Recent approaches to predicting preterm delivery have included sonographic measurement of cervical length and various biochemical assays. Although more sensitive than traditional methods, none of these alone exhibits sufficient accuracy to warrant widespread use. We contend that the failure of current approaches to predicting preterm delivery reflects an inadequate understanding of the underlying pathogenesis. Clinical and experimental evidence support the concept that most cases of preterm delivery reflect four pathogenic processes, which share a common final biological pathway leading to uterine contractions and cervical changes with or without premature rupture of membranes. These pathogeneses are: (1) activation of the maternal or fetal hypothalamic‐pituitary‐adrenal axis; (2) decidual‐chorioamniotic or systemic inflammation; (3) decidual haemorrhage (i.e. abruption); and (4) pathological distention of the uterus. Our research seeks to combine the most useful biophysical and biochemical markers of such processes with optimal clinical and epidemiological predictors into a composite, easily applied risk tool. This integrated approach has the potential to identify at‐risk asymptomatic patients with high sensitivity, specificity, and positive and negative predictive values, and also to ascertain underlying pathogenic processes that can lead to targeted therapy. To accomplish these goals, we employ logistic regression and artificial neural network models to assess and apply the appropriate weight to markers associated with each of the above pathogenic pathways, in addition to markers of the final common pathway leading to fetal membrane rupture, cervical extracellular matrix degradation, and myometrial activation.By combining these markers, we expect ultimately to produce a predictive model that is more robust than any existing method, and that identifies the relative contribution of each pathogenic process. Further analysis of this model using a neural network will enable us to identify asymptomatic patients destined to deliver preterm with high sensitivity, specificity, positive and negative predictive values, and to assess the relative contribution of each of the four distinct pathogeneses to this preterm delivery risk.
Survey on maternal mortality in Swaziland using the sisterhood method
Authors: Lech Medard M. , Zwane Amos 
Published: March 2003

SummaryMonitoring and evaluating maternal mortality in African countries is impossible without specific and reliable data and indicators. This study of maternal mortality using the ‘Sisterhood Method’ was undertaken in Swaziland. The crude data on ‘sisterhood mortality’ were obtained from the 1993–94 Multi‐Purpose Household Survey carried out by the Central Statistics Office and Ministry of Health of Swaziland. A total fertility rate of 6.36, as given in the 1986 Swaziland census, was used in estimating these indicators. Prior to this study, the maternal mortality rate (MMR) in Swaziland (based only on health facility data) was considered to lie within the range of 107–125 maternal deaths per 100 000 live births. Use of the ‘Sisterhood Method’ allowed a more precise estimate of maternal mortality for the general population of Swaziland. The study revealed the estimated MMR to be 229 and the life‐time risk of maternal death to be 1 in 69. These values were most probably stable throughout the 6‐ to 7‐year period before the survey.
Murine schistosomiasis mansoni: coordinate cytokine regulation and differences in cellular immune responses of granuloma cells and splenocytes to endogenous and exogenous schistosome egg antigens
Authors: King Christopher L. , Xianli Jia , Stavitsky Abram B. 
Published: March 2003

To better understand the cellular immune mechanisms that regulate granulomatous inflammation to Schistosoma mansoni ova, we examined the dynamics of lymphocyte proliferation and cytokine expression by granuloma cells and splenocytes to endogenous and exogenous schistosome egg antigen (SEA) 6–19 weeks postinfection. Compared to splenocytes, granuloma cells (partially CD4+ cells) which are at the site of antigen release were highly activated by endogenous SEA and terminally differentiated as indicated by the more than 10‐fold greater frequency of ex vivo interleukin (IL)‐4, IL‐5 and interferon (IFN)‐γ ‐secreting cells, greater levels of constitutive cytokine production and failure to proliferate to either endogenous or exogenous SEA. Endogenous cytokine production by granuloma cells was coordinately regulated, enhanced little by exogenous SEA, and temporally correlated with granulomatous inflammation. By contrast, CD4+ splenocytes produced comparatively little cytokine release by endogenous antigen, whereas exogenous SEA strongly induced IL‐4, IL‐5, IL‐10 and IFN‐γ production and lymphocyte proliferation that correlated poorly with the dynamics of granulomatous inflammation. These results show that cytokine responses to endogenous SEA correlated better with granulomatous inflammation than responses to exogenous SEA. Furthermore, granuloma cells and splenocytes demonstrated strikingly different proliferative responses and dynamics of cytokine expression, suggesting that how SEA reactive lymphocytes traffic between lymphoid tissues and the granuloma is critical to a better understanding of the mechanisms of granulomatous inflammation and its modulation.
Polyspecific malaria antibodies present at the time of infection inhibit the development of immunity to malaria but antibodies specific for the malaria merozoite surface protein, MSP1, facilitate immunity
Authors: Zhang Wenbao , Liu Xue Qin , Xu Huji , Good Michael F. 
Published: March 2003

SummarySerum taken from mice immune to malaria as a result of infection and drug cure, or from mice immunized with a recombinant form of the merozoite surface protein, MSP1, can provide passive protection of recipient mice against the lethal parasite, Plasmodium yoelii YM. However, recipients of MSP1‐immune serum go on to develop long‐term immunity, whereas recipients of serum from mice naturally immune to malaria rapidly lose their resistance to infection. We demonstrate that ‘infection/cure’ serum suppresses the development of both antibody and cell‐mediated parasite‐specific responses in recipients, whereas these develop in recipients of MSP1‐specific antibodies. These data have profound implications for our understanding of the development of malaria immunity in babies who passively acquire antibodies from their mothers.