bluuebunny/crossref_metadata_2025 huggingface dataset:
FLASH is required for histone transcription and S-phase progression
Authors: Barcaroli D. , Bongiorno-Borbone L. , Terrinoni A. , Hofmann T. G. , Rossi M. , Knight R. A. , Matera A. G. , Melino G. , De Laurenzi V.
Published: September 2006

Cajal bodies are nuclear subdomains that are involved in maturation of small ribonucleoproteins and frequently associate with small nuclear RNA and histone gene clusters in interphase cells. We have recently identified FADD-like IL-1β-converting enzyme (FLICE) associated huge protein (FLASH) as an essential component of Cajal bodies. Here we show that FLASH associates with nuclear protein, ataxia-telangiectasia, a component of the cell-cycle-dependent histone gene transcription machinery. Reduction of FLASH expression by RNA interference results in disruption of the normal Cajal body architecture and relocalization of nuclear protein, ataxia-telangiectasia. Furthermore, FLASH down-regulation results in a clear reduction of histone transcription and a dramatic S-phase arrest of the cell cycle. Chromatin immunoprecipitation reveals that FLASH interacts with histone gene promoter sequences. These results identify FLASH as an important component of the machinery required for histone precursor mRNA expression and cell-cycle progression.
Two ligand-binding sites in the O
2
-sensing signal transducer HemAT: Implications for ligand recognition/discrimination and signaling
Authors: Pinakoulaki Eftychia , Yoshimura Hideaki , Daskalakis Vangelis , Yoshioka Shiro , Aono Shigetoshi , Varotsis Constantinos
Published: September 2006

We have identified a ligand (CO) accommodation cavity in the signal transducer sensor protein HemAT (heme-based aerotactic transducer) that allows us to gain single-molecule insights into the mechanism of gas sensor proteins. Specific mutations that are distal and proximal to the heme were designed to perturb the electrostatic field near the ligand that is bound to the heme and near the accommodated ligand in the cavity. We report the detection of a second site in heme proteins in which the exogenous ligand is accommodated in an internal cavity. The conformational gate that directs the ligand-migration pathway from the distal to the proximal site of the heme, where the ligand is trapped, has been identified. The data provide evidence that the heme pocket is the specific ligand trap and suggest that the regulatory mechanism may be tackled starting from more than one position in the protein. Based on the results, we propose a dynamic coupling between the two distinct binding sites as the underlying allosteric mechanism for gas recognition/discrimination that triggers a conformational switch for signaling by the oxygen sensor protein HemAT.
Nonhomologous chromosomal integration of foreign DNA is completely dependent on MUS-53 (human Lig4 homolog) in
Neurospora
Authors: Ishibashi Kazuma , Suzuki Keiichiro , Ando Yoshinori , Takakura Chihiro , Inoue Hirokazu
Published: September 2006

Homologous integration of a foreign DNA segment into a chromosomal target sequence enables precise disruption or replacement of genes of interest and provides an effective means to analyze gene function. However, integration after transformation is predominantly nonhomologous in most species other than yeast. Here, we show that homologous integration in the filamentous fungus
Neurospora
requires the homologous-recombination proteins MEI-3 (yeast Rad51 homolog) and MUS-25 (yeast Rad54 homolog), whereas nonhomologous integration requires nonhomologous end-joining protein MUS-52 (yeast Ku80 homolog). Two additional minor integration pathways are present, one MEI-3-independent and homologous, the other MUS-52-independent and nonhomologous. Homologous and nonhomologous mechanisms compete when external DNA is integrated. In
Neurospora
, both nonhomologous integration pathways, MUS-52-dependent and MUS-52-independent, require MUS-53 (a homolog of human Lig4), which functions in the final step of nonhomologous end-joining. Because nonhomologous integration is eliminated in a
LIG4
-disrupted strain, integration occurs only at the targeted site in
mus-53
mutants, making them an extremely efficient and safe host for gene targeting.
A class of iron chelators with a wide spectrum of potent antitumor activity that overcomes resistance to chemotherapeutics
Authors: Whitnall Megan , Howard Jonathan , Ponka Prem , Richardson Des R.
Published: September 2006

Novel chemotherapeutics with marked and selective antitumor activity are essential to develop, particularly those that can overcome resistance to established therapies. Iron (Fe) is critical for cell-cycle progression and DNA synthesis and potentially represents a novel molecular target for the design of new anticancer agents. The aim of this study was to evaluate the antitumor activity and Fe chelation efficacy of a new class of Fe chelators using human tumors. In this investigation, the ligands showed broad antitumor activity and could overcome resistance to established antitumor agents. The
in vivo
efficacy of the most effective chelator identified, di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT), was assessed by using a panel of human xenografts in nude mice. After 7 weeks, net growth of a melanoma xenograft in Dp44mT-treated mice was only 8% of that in mice treated with vehicle. In addition, no differences in these latter animals were found in hematological indices between Dp44mT-treated mice and controls. No marked systemic Fe depletion was observed comparing Dp44mT- and vehicle-treated mice, probably because of the very low doses required to induce anticancer activity. Dp44mT caused up-regulation of the Fe-responsive tumor growth and metastasis suppressor
Ndrg1
in the tumor but not in the liver, indicating a potential mechanism of selective anticancer activity. These results indicate that the novel Fe chelators have potent and broad antitumor activity and can overcome resistance to established chemotherapeutics because of their unique mechanism of action.
Role of the Menkes copper-transporting ATPase in NMDA receptor-mediated neuronal toxicity
Authors: Schlief Michelle L. , West Tim , Craig Ann Marie , Holtzman David M. , Gitlin Jonathan D.
Published: September 2006

Menkes disease, a fatal neurodegenerative disorder resulting in seizures, hypotonia, and failure to thrive, is due to inherited loss-of-function mutations in the gene encoding a copper-transporting ATPase (Atp7a) on the X chromosome. Although affected patients exhibit signs and symptoms of copper deficiency, the mechanisms resulting in neurologic disease remain unknown. We recently discovered that Atp7a is required for the production of an NMDA receptor-dependent releasable copper pool within hippocampal neurons, a finding that suggests a role for copper in activity-dependent modulation of synaptic activity. In support of this hypothesis, we now demonstrate that copper chelation exacerbates NMDA-mediated excitotoxic cell death in primary hippocampal neurons, whereas the addition of copper is specifically protective and results in a significant decrease in cytoplasmic Ca
2+
levels after NMDA receptor activation. Consistent with the known neuroprotective effect of NMDA receptor nitrosylation, we show here that this protective effect of copper depends on endogenous nitric oxide production in hippocampal neurons, demonstrating
in vivo
links among neuroprotection, copper metabolism, and nitrosylation. Atp7a is required for these copper-dependent effects: Hippocampal neurons isolated from newborn

Mo
br

mice reveal a marked sensitivity to endogenous glutamate-mediated NMDA receptor-dependent excitotoxicity
in vitro
, and mild hypoxic/ischemic insult to these mice
in vivo
results in significantly increased caspase 3 activation and neuronal injury. Taken together, these data reveal a unique connection between copper homeostasis and NMDA receptor activity that is of broad relevance to the processes of synaptic plasticity and excitotoxic cell death.
Cationic amino acid transporter 2 regulates inflammatory homeostasis in the lung
Authors: Rothenberg Marc E. , Doepker Matthew P. , Lewkowich Ian P. , Chiaramonte Monica G. , Stringer Keith F. , Finkelman Fred D. , MacLeod Carol L. , Ellies Lesley G. , Zimmermann Nives
Published: September 2006

Arginine is an amino acid that serves as a substrate for nitric oxide synthase and arginase. As such, arginine has the potential to influence diverse fundamental processes in the lung. Here we report that the arginine transport protein, cationic amino acid transporter (CAT)2, has a critical role in regulating lung inflammatory responses. Analysis of CAT2-deficient mice revealed spontaneous inflammation in the lung. Marked eosinophilia, associated with up-regulation of eotaxin-1, was present in the bronchoalveolar lavage fluid of 3-week-old CAT2-deficient mice. The eosinophilia was gradually replaced by neutrophilia in adult mice, while eotaxin-1 levels decreased and GRO-α levels increased. Despite the presence of activated alveolar macrophages in CAT2-deficient mice, NO production was compromised in these cells. Examination of dendritic cell activation, which can be affected by NO release, indicated increased dendritic cell activation in the lungs of CAT2-deficient mice. This process was accompanied by an increase in the number of memory T cells. Thus, our data suggest that CAT2 regulates anti-inflammatory processes in the lungs via regulation of dendritic cell activation and subsequent T cell responses.
Reconstruction of New Orleans after Hurricane Katrina: A research perspective
Authors: Kates R. W. , Colten C. E. , Laska S. , Leatherman S. P.
Published: September 2006

Four propositions drawn from 60 years of natural hazard and reconstruction research provide a comparative and historical perspective on the reconstruction of New Orleans after Hurricane Katrina. Decisions taken over its 288-year history that have made New Orleans so vulnerable to Katrina reflect a long-term pattern of societal response to hazard events—reducing consequences to relatively frequent events, and increasing vulnerability to very large and rare events. Thus Katrina's consequences for New Orleans were truly catastrophic—accounting for most of the estimated 1,570 deaths of Louisiana residents and $40–50 billion in monetary losses. A comparative sequence and timing of recovery provides a calendar of historical experience against which to gauge progress in reconstruction. Using this calendar, the emergency postdisaster period appears to be longer in duration than that of any other studied disaster. The restoration period, the time taken to restore urban services for the smaller population, is in keeping with or ahead of historical experience. The effort to reconstruct the physical environment and urban infrastructure is likely to take 8–11 years. Conflicting policy goals for reconstruction of rapid recovery, safety, betterment, and equity are already evident. Actions taken demonstrate the rush to rebuild the familiar in contrast to planning efforts that emphasize betterment. Because disasters tend to accelerate existing economic, social, and political trends, the large losses in housing, population, and employment after Katrina are likely to persist and, at best, only partly recover. However, the possibility of breaking free of this gloomy trajectory is feasible and has some historical precedent.
A hypothesis for vulnerable plaque rupture due to stress-induced debonding around cellular microcalcifications in thin fibrous caps
Authors: Vengrenyuk Yuliya , Carlier Stéphane , Xanthos Savvas , Cardoso Luis , Ganatos Peter , Virmani Renu , Einav Shmuel , Gilchrist Lane , Weinbaum Sheldon
Published: September 2006

In this article, we advance a hypothesis for the rupture of thin fibrous cap atheroma, namely that minute (10-μm-diameter) cellular-level microcalcifications in the cap, which heretofore have gone undetected because they lie below the visibility of current
in vivo
imaging techniques, cause local stress concentrations that lead to interfacial debonding. New theoretical solutions are presented for the local stress concentration around these minute spherical inclusions that predict a nearly 2-fold increase in interfacial stress that is relatively insensitive to the location of the hypothesized microinclusions in the cap. To experimentally confirm the existence of the hypothesized cellular-level microcalcifications, we examined autopsy specimens of coronary atheromatous lesions using
in vitro
imaging techniques whose resolution far exceeds conventional magnetic resonance imaging, intravascular ultrasound, and optical coherence tomography approaches. These high-resolution imaging modalities, which include confocal microscopy with calcium-specific staining and micro-computed tomography imaging, provide images of cellular-level calcifications within the cap proper. As anticipated, the minute inclusions in the cap are very rare compared with the numerous calcified macrophages observed in the necrotic core. Our mathematical model predicts that inclusions located in an area of high circumferential stress (>300 kPa) in the cap can intensify this stress to nearly 600 kPa when the cap thickness is <65 μm. The most likely candidates for the inclusions are either calcified macrophages or smooth muscle cells that have undergone apoptosis.
When motion appears stopped: Stereo motion standstill
Authors: Tseng Chia-huei , Gobell Joetta L. , Lu Zhong-Lin , Sperling George
Published: September 2006

Motion standstill is different from the usual perceptual experiences associated with objects in motion. In motion standstill, a pattern that is moving quite rapidly is perceived as being motionless, and yet its details are not blurred but clearly visible. We revisited motion standstill in dynamic random-dot stereograms similar to those first used by Julesz and Payne [Julesz B, Payne R (1968)
Vision Res
8:433–444]. Three improvements were made to their paradigm to avoid possible confounds: The temporal frequency of the motion stimuli was manipulated independently from that of individual stereo gratings so that the failure of motion perception is not due to inability to compute stereo. The motion of the stereo gratings was continuous across the visual field so that the perceived pattern in motion standstill was not a simple average of a back-and-forth display wobble over time. Observers discriminated three spatial frequencies to demonstrate pattern recognition. Three objective psychophysical methods, instead of merely self-report, were used to objectively demonstrate motion standstill. Our results confirm that motion standstill occurs in dynamic random-dot stereogram motion displays at 4–6 Hz. Motion standstill occurs when the stimulus spatiotemporal frequency combination exceeds that of the salience-based third-order motion system in a spatiotemporal frequency range in which the shape and depth systems still function. The ability of shape systems to extract a representative image from a series of moving samples is a significant component of a biological system's ability to derive a stable perceptual world from a constantly changing visual environment.
Follicle-stimulating hormone stimulates TNF production from immune cells to enhance osteoblast and osteoclast formation
Authors: Iqbal Jameel , Sun Li , Kumar T. Rajendra , Blair Harry C. , Zaidi Mone
Published: September 2006

Declining estrogen production after menopause causes osteoporosis in which the resorption of bone exceeds the increase in bone formation. We recently found that mice deficient in the β-subunit of follicle-stimulating hormone (FSHβ) are protected from bone loss despite severe estrogen deficiency. Here we show that FSHβ-deficient mice have lowered TNFα levels. However, TNFα-deficient mice are resistant to hypogonadal bone loss despite having elevated FSH, suggesting that TNFα is critical to the effect of FSH on bone mass. We find that FSH directly stimulates TNFα production from bone marrow granulocytes and macrophages. We also explore how TNFα up-regulation induces bone loss. By modeling the known actions of TNFα, we attribute the high-turnover bone loss to an expanded osteoclast precursor pool, together with enhanced osteoblast formation. TNFα inhibits osteoblastogenesis in the presence of ascorbic acid in culture medium, but in its absence this effect becomes stimulatory; thus, ascorbic acid reverses the true action of TNFα. Likewise, ascorbic acid blunts the effects of TNFα in stimulating osteoclast formation. We propose that hypogonadal bone loss is caused, at least in part, by enhanced FSH secretion, which in turn increases TNFα production to expand the number of bone marrow osteoclast precursors. Ascorbic acid may prevent FSH-induced hypogonadal bone loss by modulating the catabolic actions of TNFα.
Posttranslational hydroxylation of ankyrin repeats in IκB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH)
Authors: Cockman Matthew E. , Lancaster David E. , Stolze Ineke P. , Hewitson Kirsty S. , McDonough Michael A. , Coleman Mathew L. , Coles Charlotte H. , Yu Xiaohong , Hay Ronald T. , Ley Steven C. , Pugh Christopher W. , Oldham Neil J. , Masson Norma , Schofield Christopher J. , Ratcliffe Peter J.
Published: September 2006

Studies on hypoxia-sensitive pathways have revealed a series of Fe(II)-dependent dioxygenases that regulate hypoxia-inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The recognition of these unprecedented signaling processes has led to a search for other substrates of the HIF hydroxylases. Here we show that the human HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also efficiently hydroxylates specific asparaginyl (Asn)-residues within proteins of the IκB family. After the identification of a series of ankyrin repeat domain (ARD)-containing proteins in a screen for proteins interacting with FIH, the ARDs of p105 (
NFKB1
) and IκBα were shown to be efficiently hydroxylated by FIH at specific Asn residues in the hairpin loops linking particular ankyrin repeats. The target Asn residue is highly conserved as part of the ankyrin consensus, and peptides derived from a diverse range of ARD-containing proteins supported FIH enzyme activity. These findings demonstrate that this type of protein hydroxylation is not restricted to HIF and strongly suggest that FIH-dependent ARD hydroxylation is a common occurrence, potentially providing an oxygen-sensitive signal to a diverse range of processes.
An open study of the comparative efficacy and safety of caspofungin and liposomal amphotericin B in treating invasive fungal infections or febrile neutropenia in patients with haematological malignancy
Authors: Ellis Michael , Frampton Chris , Joseph Jose , Alizadeh Hussain , Kristensen Jorgen , Hauggaard Anders , Shammas Fuad
Published: September 2006

In a clinical non-trial setting, the efficacy and safety of caspofungin was compared with liposomal amphotericin B for the management of febrile neutropenia or invasive fungal infections in 73 episodes in patients with haematological malignancy. There were fewer episodes of drug toxicity with caspofungin than liposomal amphotericin B (58.3 vs 83.7 %, P=0.02). The favourable response rate for episodes of febrile neutropenia treated with caspofungin or liposomal amphotericin B was similar at 37.5 and 53.8 %, respectively, but more breakthrough fungal infections occurred with caspofungin than with liposomal amphotericin B (33.3 vs 0 %, P<0.05) in these patients who did not receive antifungal prophylaxis. None of four episodes of candidaemia or hepatosplenic candidiasis responded to caspofungin compared with three of four episodes treated with liposomal amphotericin B. Mortality was significantly higher with caspofungin treatment compared with liposomal amphotericin B (6/24 vs 2/49, P=0.01), mainly due to an excess of fungal infections (P=0.04). Caspofungin treatment was a significant independent predictor of mortality [odds ratio=7.6 (95 % confidence interval 1.2–45.5)] when sepsis severity, prolonged neutropenia and length of antifungal therapy were considered in a multiple logistic regression model. In clinical practice, there is a suggestion that caspofungin may not be as effective as liposomal amphotericin B in preventing breakthrough invasive fungal infections in febrile neutropenia or in preventing fungus-related deaths. Because of the potential biases in this observational study, these preliminary findings should be interpreted with caution and clarified with a larger cohort of patients.
In vitro activity of tea-tree oil against clinical skin isolates of meticillin-resistant and -sensitive Staphylococcus aureus and coagulase-negative staphylococci growing planktonically and as biofilms
Authors: Brady Aaron , Loughlin Ryan , Gilpin Deirdre , Kearney Paddy , Tunney Michael
Published: September 2006

The susceptibility of Staphylococcus aureus [meticillin-resistant (MRSA) and meticillin-sensitive (MSSA)] and coagulase-negative staphylococci (CoNS), which respectively form part of the transient and commensal skin flora, to tea-tree oil (TTO) was compared using broth microdilution and quantitative in vitro time–kill test methods. MRSA and MSSA isolates were significantly less susceptible than CoNS isolates, as measured by both MIC and minimum bactericidal concentration. A significant decrease in the mean viable count of all isolates in comparison with the control was seen at each time interval in time–kill assays. However, the only significant difference in the overall mean log10 reduction in viable count between the groups of isolates was between CoNS and MSSA at 3 h, with CoNS isolates demonstrating a significantly lower mean reduction. To provide a better simulation of in vivo conditions on the skin, where bacteria are reported to grow as microcolonies encased in glycocalyx, the bactericidal activity of TTO against isolates grown as biofilms was also compared. Biofilms formed by MSSA and MRSA isolates were completely eradicated following exposure to 5 % TTO for 1 h. In contrast, of the biofilms formed by the nine CoNS isolates tested, only five were completely killed, although a reduction in viable count was apparent for the other four isolates. These results suggest that TTO exerts a greater bactericidal activity against biofilm-grown MRSA and MSSA isolates than against some biofilm-grown CoNS isolates.
Predominance of multi-drug-resistant LAM and Beijing family strains among Mycobacterium tuberculosis isolates recovered from prison inmates in Tula Region, Russia
Authors: Ignatova Anna , Dubiley Svetlana , Stepanshina Valentina , Shemyakin Igor
Published: September 2006

The genotypic characteristics and drug susceptibility profiles of clinical isolates of Mycobacterium tuberculosis recovered from prison hospital patients in the Tula region (central Russia) during 2001 and 2002 are reported. The emergence of multi-drug-resistant tuberculosis (TB) poses a major health risk to the population, with economic implications for TB control. Prisons serve as a continuous source of TB transmission. The results showed that members of the LAM and Beijing families are major contributors to the epidemiological picture of TB in the population studied. The two families of strains accounted for most of the drug-resistant TB in the population. The genotypic characteristics of the M. tuberculosis predominant LAM strain that was responsible for 31 % of TB cases in this setting are presented.
Optimal sampling site for mucosal candidosis in oral cancer patients is the labial sulcus
Authors: Rautemaa Riina , Rusanen Peter , Richardson Malcolm , Meurman Jukka H.
Published: September 2006

Traditional sampling methods for the diagnosis of oral candidosis in head and neck cancer patients, i.e. saliva collection or tongue scrapings, are often impossible to perform. The aim was to determine the optimal sampling method. Eighteen oral cancer patients and five control subjects were sampled semi-quantitatively from the labial sulcus, dorsum of the tongue, dental plaque and saliva for cultivation of yeasts. The patients were examined prior to all cancer treatment (n=5), or 2–4 weeks (n=5) or 8–12 weeks (n=8) post-operatively. The incidence of Candida was found to increase from 40 % at the control and pre-operative level up to 73 % 8–12 weeks post-operatively. Candida albicans was found to be the only species until 4 weeks post-operatively. Thereafter, the incidence of species other than C. albicans was 38 %. The most sensitive sampling site was found to be the vestibular sulcus, from which all culture-positive cases could be confirmed. Tongue surface scraping was found to be more sensitive than saliva collection in detecting Candida. All sampling methods and sites were equally sensitive in detecting the different Candida species. Dental plaque was found to have the highest density of Candida colonization, and was thus found to be the most significant source of Candida infection, which emphasizes the role of dental care in these patients.
Use of voriconazole in a patient with aspergilloma caused by an itraconazole-resistant strain of Aspergillus fumigatus
Authors: Dannaoui E. , Garcia-Hermoso D. , Naccache J. M. , Meneau I. , Sanglard D. , Bouges-Michel C. , Valeyre D. , Lortholary O.
Published: September 2006

The case is reported of a patient with cavitary sarcoidosis complicated by an aspergilloma caused by an itraconazole-resistant strain of Aspergillus fumigatus, who was treated with voriconazole. The authors suggest that susceptibility testing of A. fumigatus strains is of value during long-term therapy with itraconazole, and that voriconazole may be a good option for treatment of patients infected with itraconazole-resistant strains of A. fumigatus.
Immunization with 3-oxododecanoyl-l-homoserine lactone–protein conjugate protects mice from lethal Pseudomonas aeruginosa lung infection
Authors: Miyairi Shinichi , Tateda Kazuhiro , Fuse Etsu T. , Ueda Chihiro , Saito Hiroaki , Takabatake Tohru , Ishii Yoshikazu , Horikawa Manabu , Ishiguro Masaji , Standiford Theodore J. , Yamaguchi Keizo
Published: September 2006

Quorum-sensing systems have been reported to play a critical role in the pathogenesis of several bacterial infections. Recent data have demonstrated thatPseudomonas N-3-oxododecanoyl-l-homoserine lactone (3-oxo-C12-homoserine lactone, 3-oxo-C12-HSL), but notN-butanoyl-l-homoserine lactone (C4-HSL), induces apoptosis in macrophages and neutrophils. In the present study, the effects of active immunization with 3-oxo-C12-HSL–carrier protein conjugate on acuteP. aeruginosalung infection in mice were investigated. Immunization with 3-oxo-C12-HSL–BSA conjugate (subcutaneous, four times, at 2-week intervals) elaborated significant amounts of specific antibody in serum. Control and immunized mice were intranasally challenged with approximately 3×106 c.f.u.P. aeruginosaPAO1, and survival was then compared. All control mice died by day 2 post bacterial challenge, while 36 % of immunized mice survived to day 4 (P<0.05). Interestingly, bacterial numbers in the lungs did not differ between control and immunized groups, whereas the levels of pulmonary tumour necrosis factor (TNF)-αin the immunized mice were significantly lower than those of control mice (P<0.05). Furthermore, the extractable 3-oxo-C12-HSL levels in serum and lung homogenate were also significantly diminished in the immunized mice. Immune serum completely rescued reduction of cell viability by 3-oxo-C12-HSL-mediated apoptosis in macrophagesin vitro. These results demonstrated that specific antibody to 3-oxo-C12-HSL plays a protective role in acuteP. aeruginosainfection, probably through blocking of host inflammatory responses, without altering lung bacterial burden. The present data identify a promising potential vaccine strategy targeting bacterial quorum-sensing molecules, including autoinducers.
IgG and IgG subclass antibodies in patients with active cutaneous leishmaniasis
Authors: Ozbılge Hatice , Aksoy Nurten , Gurel Mehmet S. , Yazar Suleyman
Published: September 2006

This research was planned to detect IgG and IgG subclasses in sera of patients with active cutaneous leishmaniasis (CL). Sera from 30 patients with active CL aged between 10 and 50 years and from 30 healthy controls aged between 8 and 50 years were included in the study. Levels of IgG and its subclasses were measured by a nephelometer. Levels of IgG, IgG1 and IgG3 in the CL patients were higher than in the controls. In addition, IgG and IgG1, and IgG and IgG3 levels showed a significant positive correlation. These results showed that IgG subclasses could possibly be used as a helpful diagnostic marker in CL.
A review of an emerging enteric pathogen: enteroaggregative Escherichia coli
Authors: Huang David B. , Mohanty Alakananda , DuPont Herbert L. , Okhuysen Pablo C. , Chiang Tom
Published: September 2006

Enteroaggregative Escherichia coli (EAEC) is an increasingly recognized enteric pathogen. It is a cause of both acute and persistent diarrhoea among children, adults and HIV-infected persons, in both developing and developed countries. In challenge studies, EAEC has caused diarrhoeal illness with the ingestion of 1010 c.f.u. Outbreaks of diarrhoeal illness due to EAEC have been reported, and linked to the ingestion of contaminated food. Diarrhoeal illness due to EAEC is the result of a complex pathogen–host interaction. Some infections due to EAEC result in diarrhoeal illness and elicit an inflammatory response, whereas other infections do not result in a symptomatic infection. Many putative virulence genes and EAEC strains that produce biofilm have been identified; however, the clinical significance of these genes and of biofilm production has yet to be defined. A −251 AA single nucleotide polymorphism (SNP) in the interleukin (IL)-8 promoter region is reported to increase host susceptibility to EAEC diarrhoea. Ciprofloxacin and rifaximin continue to be an effective treatment in persons infected with EAEC. This review is intended to provide an updated review for healthcare workers on EAEC, an emerging enteric pathogen.
Synthesis and characterization of Pseudomonas aeruginosa alginate–tetanus toxoid conjugate
Authors: Kashef Nasim , Behzadian-Nejad Qorban , Najar-Peerayeh Shahin , Mousavi-Hosseini Kamran , Moazzeni Mohammad , Djavid Gholamreza Esmaeeli
Published: September 2006

Chronic infection withPseudomonas aeruginosais the main proven perpetrator of lung function decline and ultimate mortality in cystic fibrosis (CF) patients. Mucoid strains of this bacterium elaborate mucoid exopolysaccharide, also referred to as alginate. Alginate-based immunization of naïve animals elicits opsonic antibodies and leads to clearance of mucoidP. aeruginosafrom the lungs. Alginate was isolated from mucoidP. aeruginosastrain 8821M by repeated ethanol precipitation, dialysis, proteinase and nuclease digestion, and chromatography. To improve immunogenicity, the purified antigen was coupled to tetanus toxoid (TT) with adipic acid dihydrazide (ADH) as a spacer and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) as a linker. The reaction mixture was passed through a Sepharose CL-4B column. The resulting conjugate was composed of TT and large-size alginate polymer at a ratio of about 3 : 1; it was non-toxic and non-pyrogenic, and elicited high titres of alginate-specific IgG. Antisera raised against the conjugate had high opsonic activity against the vaccine strain. The alginate conjugate was also able to protect mice against a lethal dose of mucoidP. aeruginosa. These data indicate that an alginate-based vaccine has significant potential to protect against chronic infection with mucoidP. aeruginosain the CF host.
Evaluation of antigenic variations between two virulent toxoplasma strains
Authors: Delibas Songul Bayram , Ertabaklar Hatice , Ertug Sema
Published: September 2006

Toxoplasma gondii infection in humans is routinely assessed by serological means. Here, the authors attempted to compare the response of different Toxoplasma strains to serological tests and to evaluate the antigenic profiles of the RH and RH Ankara (TRH) strains with Western blotting. Anti-Toxoplasma IgG antibodies of 72 patients were examined with the indirect immunofluorescence antibody (IFA) test, ELISA and Western blotting (WB) by using antigen from both strains. Antigenic variations between strains did not affect IFA and ELISA test results, but qualitative and quantitative differences between the WB patterns were observed. A number of bands with molecular masses varying between 17 and 105 kDa were detected in WB. Fourteen different bands were obtained with the assay performed with RH strain antigen. An additional four bands were observed with TRH strain antigen. Also, an 80 kDa band was observed to stain darker in the blot with TRH strain antigen, whereas with RH strain antigen 30 and 38 kDa bands were darker. The results showed that strain-specific polymorphism in tachyzoite antigens of different Toxoplasma strains is important in the evaluation of WB but not in conventional serological analyses such as ELISA and IFA.
The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to enhance survival in lethal acid conditions
Authors: Reeve Wayne G. , Bräu Lambert , Castelli Joanne , Garau Giovanni , Sohlenkamp Christian , Geiger Otto , Dilworth Michael J. , Glenn Andrew R. , Howieson John G. , Tiwari Ravi P.
Published: September 2006

Sinorhizobium medicae WR101 was identified as a mutant of WSM419 that contained a minitransposon-induced transcriptional gusA fusion activated at least 20-fold at pH 5.7. The expression of this fusion in moderately acid conditions was dependent on the calcium concentration; increasing the calcium concentration to enhance cell growth and survival in acid conditions decreased the expression of the fusion. A gene region containing the gusA fusion was sequenced, revealing five S. medicae genes: tcsA, tcrA, fsrR, lpiA and acvB. The gusA reporter in WR101 was fused to lpiA, which encodes a putative transmembrane protein also found in other Alphaproteobacteria such as Sinorhizobium meliloti, Rhizobium tropici and Agrobacterium tumefaciens. As LpiA has partial sequence similarity to the lysyl-phosphatidylglycerol (LPG) synthetase FmtC/MprF from Staphylococcus aureus, membrane lipid compositions of S. medicae strains were analysed. Cells cultured under neutral or acidic growth conditions did not induce any detectable LPG and therefore this lipid cannot be a major constituent of S. medicae membranes. Expression studies in S. medicae localized the acid-activated lpiA promoter within a 372 bp region upstream of the start codon. The acid-activated transcription of lpiA required the fused sensor–regulator product of the fsrR gene, because expression of lpiA was severely reduced in an S. medicae fsrR mutant. S. meliloti strain 1021 does not contain fsrR and acid-activated expression of the lpiA-gusA fusion did not occur in this species. Although acid-activated lpiA transcription was not required for cell growth, its expression was crucial in enhancing the viability of cells subsequently exposed to lethal acid (pH 4.5) conditions.
Interaction of the transmembrane domain of lysis protein E from bacteriophage ϕX174 with bacterial translocase MraY and peptidyl-prolyl isomerase SlyD
Authors: Mendel Sharon , Holbourn Joanne M. , Schouten James A. , Bugg Timothy D. H.
Published: September 2006

The molecular target for the bacteriolytic E protein from bacteriophageϕX174, responsible for host cell lysis, is known to be the enzyme phospho-MurNAc-pentapeptide translocase (MraY), an integral membrane protein involved in bacterial cell wall peptidoglycan biosynthesis, with an essential role being played by peptidyl-prolyl isomerase SlyD. A synthetic 37 aa peptide Epep, containing the N-terminal transmembraneα-helix of E, was found to be bacteriolytic againstBacillus licheniformis, and inhibited membrane-bound MraY. The solution conformation of Epepwas found by circular dichroism (CD) spectroscopy to be 100 %α-helical. No change in the CD spectrum was observed upon addition of purifiedEscherichia coliSlyD, implying that SlyD does not catalyse prolyl isomerization upon E. However, Epepwas found to be a potent inhibitor of SlyD-catalysed peptidyl-prolyl isomerization (IC500.15 μM), implying a strong interaction between E and SlyD. Epepwas found to inhibitE. coliMraY activity when assayed in membranes (IC500.8 μM); however, no inhibition of solubilized MraY was observed, unlike nucleoside natural product inhibitor tunicamycin. These results imply that the interaction of E with MraY is not at the MraY active site, and suggest that a protein–protein interaction is formed between E and MraY at a site within the transmembrane region.
Glycerol, ethylene glycol and propanediol elicit pimaricin biosynthesis in the PI-factor-defective strain Streptomyces natalensis npi287 and increase polyene production in several wild-type actinomycetes
Authors: Recio Eliseo , Aparicio Jesús F. , Rumbero Ángel , Martín Juan F.
Published: September 2006

Production of pimaricin by Streptomyces natalensis ATCC 27448 is elicited by the PI-factor, an autoinducer secreted by the producer strain during the rapid growth phase. Exogenous PI-factor restored pimaricin production in a mutant strain npi287 defective in PI-factor biosynthesis. During purification of the PI-factor, a second pimaricin-inducing fraction different from PI-factor was isolated from the culture broth of wild-type S. natalensis ATCC 27448. After purification by HPLC and analysis by MS and NMR, this active fraction was shown to contain glycerol and lactic acid. Pure glycerol restored pimaricin production in liquid cultures of the autoinducer-defective npi287 mutant. A similar effect was exerted by ethylene glycol, 1,2-propanediol and 1,3-propanediol but not by higher polyalcohols or by glycerol acetate or glycerol lactate esters. Glycerol stimulated (30–270 %) the production of six different polyene macrolide antibiotics by their respective producer strains. Addition of glycerol to the inducer-defective npi287 strain restored pimaricin production but did not result in extracellular or intracellular accumulation of PI-factor. Exogenously added PI-factor was internalized by the cells in the presence of glycerol, and a mixture of both PI-factor and glycerol produced a slightly higher inducing effect on pimaricin production than PI-factor alone. In summary, glycerol, ethylene glycol and propanediol exert a bypass of the PI-factor inducing effect on pimaricin biosynthesis.
Inhibitors of Helicobacter pylori ATPase Cagα block CagA transport and cag virulence
Authors: Hilleringmann Markus , Pansegrau Werner , Doyle Michael , Kaufman Susan , MacKichan Mary Lee , Gianfaldoni Claudia , Ruggiero Paolo , Covacci Antonello
Published: September 2006

With the steadily increasing occurrence of antibiotic resistance in bacteria, there is a great need for new antibacterial compounds. The approach described here involves targeting virulence-related bacterial type IV secretion systems (TFSSs) with small-molecule inhibitors. The cag TFSS of Helicobacter pylori was chosen as a model, and novel inhibitors directed against the cag VirB11-type ATPase Cagα were identified. The cag genes encode proteins that are components of a contact-dependent secretion system used by the bacterium to translocate the effector molecule CagA into host cells. Translocated CagA is associated with severe gastritis, and carcinoma. Furthermore, functional TFSSs and immunodominant CagA play a role in interleukin (IL)-8 induction, which is an important factor for chronic inflammation. Inhibitors of Cagα were identified by high-throughput screening of chemical libraries that comprised 524 400 small molecules. The ATPase activity of Cagα was inhibited by the selected compounds in an in vitro enzymic assay using the purified enzyme. The most active compound, CHIR-1, reduced TFSS function to an extent that cellular effects on AGS cells mediated by CagA were virtually undetectable, while reduced levels of IL-8 induction were observed. Gastric colonization by CHIR-1-pre-treated bacteria was found to be impaired in a dose-dependent manner using a mouse model of infection. Small-molecule Cagα inhibitors, the first described inhibitors of a TFSS, are potential candidates for the development of new antibacterial compounds that may lead to alternative medical treatments. The compounds are expected to impose weak selective pressure, since they target virulence functions. Moreover, the targeted virulence protein is conserved in a variety of bacterial pathogens. Additionally, TFSS inhibitors are potent tools to study the biology of TFSSs.
A truncated Bacillus subtilis dal gene with a 3′ ssrA gene tag regulates the growth and virulence of racemase-deficient Listeria monocytogenes
Authors: Li Zhongxia , Zhao Xinyan , Zhou Chenghui , Gu Baiyan , Frankel Fred R.
Published: September 2006

Listeria monocytogenes(Lm) is a Gram-positive intracellular pathogen that can elicit strong cellular immunity. An attenuated strain (Lmdd) with deletions in two genes (dalanddat) required ford-alanine synthesis and viability has been shown to induce long-lived protective systemic and mucosal immune responses in mice when administered in the presence of the required amino acid. To bypass the necessity for exogenousd-alanine without compromising the safety of the original strain, the defect of Lmdd was complemented with a heterologousBacillus subtilis dalgene, and the effects of truncating the upstream region of the gene on its transcription efficiency and of modifying its protein product with anssrAtag at the 3′-terminus were examined. The strains with 551 bp and 80 bp upstream regions showed high levels of transcription and grew withoutd-alanine. The strains with the shortest upstream regions, 48 bp and 18 bp, showed greatly decreased levels of transcription and failed to grow in the absence ofd-alanine. Addition of anssrAtag to the longer genes resulted in a somewhat altered growth pattern in media and a reduced plaque size on L2 fibroblasts. These bacteria contained low levels of racemase protein and reduced free pools ofd-alanine. One of the strains tested further, Lmdd/pA80S, was rapidly cleared from the spleens of infected mice but nevertheless induced a strong immune response that protected mice against challenge by wild-typeL. monocytogenes. These bacteria can thus induce immune responses in mice comparable to the original Lmdd strain, but without the need for exogenousd-alanine, and may have use as a live vaccine vector against infectious diseases and cancers.
Transcriptional profiling of a Staphylococcus aureus clinical isolate and its isogenic agr and sarA mutants reveals global differences in comparison to the laboratory strain RN6390
Authors: Cassat James , Dunman Paul M. , Murphy Ellen , Projan Steven J. , Beenken Karen E. , Palm Katherine J. , Yang Soo-Jin , Rice Kelly C. , Bayles Kenneth W. , Smeltzer Mark S.
Published: September 2006

The production of Staphylococcus aureus virulence factors is under the control of complex regulatory circuits. Most studies aimed at defining these regulatory networks have focused on derivatives of the strain NCTC 8325, most notably RN6390. However, all NCTC 8325 derivatives, including RN6390, possess an 11 bp deletion in rsbU. This deletion renders NCTC 8325 derivatives naturally sigma-factor-B deficient. Recent studies have shown that RN6390 is also deficient, in comparison to clinical isolates, with respect to biofilm formation, a process which is important for both pathogenesis and antimicrobial resistance. Based on these considerations, the authors carried out genome-scale transcriptional profiling, comparing RN6390 with the virulent rsbU-positive clinical isolate UAMS-1. The results revealed significant genome-wide differences in expression patterns between RN6390 and UAMS-1, and suggested that the overall transcriptional profile of UAMS-1 is geared toward expression of factors that promote colonization and biofilm formation. In contrast, the transcriptional profile of RN6390 was heavily influenced by RNAIII expression, resulting in a phenotype characterized by increased production of exoproteins, and decreased capacity to form a biofilm. The greater influence of agr in RN6390 relative to UAMS-1 was also evident when the transcriptional profile of UAMS-1 was compared with that of its isogenic sarA and agr mutants. Specifically, the results indicate that, in contrast to NCTC 8325 derivatives, agr plays a limited role in overall regulation of gene expression in UAMS-1, when compared with sarA. Furthermore, by defining the sarA regulon in a biofilm-positive clinical isolate, and comparing the results with transcriptional profiling experiments defining biofilm-associated gene expression patterns in the same strain, the authors identified a sarA-regulated operon (alsSD) that is also induced in biofilms, and demonstrated that mutation of alsSD results in reduced capacity to form a biofilm.
The enduracidin biosynthetic gene cluster from Streptomyces fungicidicus
Authors: Yin Xihou , Zabriskie T. Mark
Published: September 2006

The biosynthetic gene cluster for the 17 aa peptide antibiotic enduracidin has been cloned and sequenced from Streptomyces fungicidicus ATCC 21013. The 84 kb gene cluster contains 25 ORFs and is located within a 116 kb genetic locus that was fully sequenced. Targeted disruption of non-ribosomal peptide synthetase (NRPS) genes in the cluster abolished enduracidin production and confirmed function. The cluster includes four genes, endA-D, encoding two-, seven-, eight- and one-module NRPSs, respectively, and includes unique modules for the incorporation of citrulline and enduracididine. The NRPS organization generally follows the collinearity principle, and starts with a condensation domain (C domain) similar to those found in other lipopeptide systems for the coupling of an acyl group to the starting amino acid. The sixth module of EndB, corresponding to Thr8, is missing an adenylation domain (A domain) and this module is presumed to be loaded in trans by the single module protein EndD. The most striking feature of the NRPS organization is the lack of epimerization domains (E domains) in light of the fact that the product has seven d-amino acid residues. Sequence analysis reveals that C domains following modules corresponding to d-amino acids belong to a unique subset of C domains able to catalyse both epimerization and condensation reactions. Other genes directing lipid modification and activation, and formation of the non-proteinogenic amino acids 4-hydroxyphenylglycine and enduracididine are readily identified, as are genes possibly involved in regulation of antibiotic biosynthesis and export. These findings provide the basis to further genetically manipulate and improve lipodepsipeptide antibiotics via combinatorial and chemical methods.