bluuebunny/crossref_metadata_2025 huggingface dataset:
Visualizing extracellular matrix and sensing fibroblasts metabolism in human dermis by nonlinear spectral imaging
Authors: Zhuo Shuangmu , Chen Jianxin , Jiang Xingshan , Cheng Xiaochun , Xie Shusen
Published: April 2007

Background/purpose: The purpose of this work was to demonstrate the use of the nonlinear spectral imaging technique with the image‐guided spectral analysis function and the extracting channels function in visualizing the extracellular maxtrix (ECM) structures and sensing fibroblasts metabolism within fresh and untreated human dermis.

Methods: A Zeiss LSM 510 META laser scanning microscopy and a femtosecond Ti: sapphire laser was used to obtain the nonlinear spectral images of human dermis.

Results: The nonlinear spectral imaging technique was useful in obtaining the biomorphology and biochemistry information from human dermis. By combining the image‐guided spectral analysis function, intrinsic components were identified and their corresponding emission spectra can be analyzed. By integrating the extracting channels function, the information of ultrastructure of the ECM can be quantitatively obtained.

Conclusion: The nonlinear spectral imaging technique has been demonstrated to be an effective technique for a detailed analysis and qualitative visualization of the ECM structure, and sensing of cell metabolism within fresh and untreated human dermis. Specifically, the ultrastructure of the ECM can be revealed, and collagen fibril spacing, elastic fiber diameters, the ration of collagen content over elastin can be quantitatively determined. It was found that the nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) signals excited at 830 nm provide the information for a good estimation of cellular redox ratio and a parameter (NF), the ration of NADH over FAD fluorescence signal, is used for sensing fibroblasts metabolism. With the advent of the clinical portability of typical multiphoton microscopy, the technique has the potential to be applied for in vivo studies and clinical examinations.
Plethodontid modulating factor, a hypervariable salamander courtship pheromone in the three‐finger protein superfamily
Authors: Palmer Catherine A. , Hollis David M. , Watts Richard A. , Houck Lynne D. , McCall Maureen A. , Gregg Ronald G. , Feldhoff Pamela W. , Feldhoff Richard C. , Arnold Stevan J.
Published: April 2007

The soluble members of the three‐finger protein superfamily all share a relatively simple ‘three‐finger’ structure, yet perform radically different functions. Plethodontid modulating factor (PMF), a pheromone protein produced by the lungless salamander, Plethodon shermani, is a new and unusual member of this group. It affects female receptivity when delivered to the female's nares during courtship. As with other plethodontid pheromone genes, PMF is hyperexpressed in a specialized male mental (chin) gland. Unlike other plethodontid pheromone genes, however, PMF is also expressed at low levels in the skin, liver, intestine and kidneys of both sexes. The PMF sequences obtained from all tissue types were highly variable, with 103 unique haplotypes identified which averaged 35% sequence dissimilarity (range 1–60%) at the protein level. Despite this variation, however, all PMF sequences contained a conserved ≈ 20‐amino‐acid secretion signal sequence and a pattern of eight cysteines that is also found in cytotoxins and short neurotoxins from snake venoms, as well as xenoxins from Xenopus. Although they share a common cysteine pattern, PMF isoforms differ from other three‐finger proteins in: (a) amino‐acid composition outside of the conserved motif; (b) length of the three distinguishing ‘fingers’; (c) net charge at neutral pH. Whereas most three‐finger proteins have a net positive charge at pH 7.0, PMF has a high net negative charge at neutral pH (pI range of most PMFs 3.5–4.0). Sequence comparisons suggest that PMF belongs to a distinct multigene subfamily within the three‐finger protein superfamily.
Discovery of a eugenol oxidase from Rhodococcus sp. strain RHA1
Authors: Jin Jianfeng , Mazon Hortense , van den Heuvel Robert H. H. , Janssen Dick B. , Fraaije Marco W.
Published: April 2007

A gene encoding a eugenol oxidase was identified in the genome from Rhodococcus sp. strain RHA1. The bacterial FAD‐containing oxidase shares 45% amino acid sequence identity with vanillyl alcohol oxidase from the fungus Penicillium simplicissimum. Eugenol oxidase could be expressed at high levels in Escherichia coli, which allowed purification of 160 mg of eugenol oxidase from 1 L of culture. Gel permeation experiments and macromolecular MS revealed that the enzyme forms homodimers. Eugenol oxidase is partly expressed in the apo form, but can be fully flavinylated by the addition of FAD. Cofactor incorporation involves the formation of a covalent protein–FAD linkage, which is formed autocatalytically. Modeling using the vanillyl alcohol oxidase structure indicates that the FAD cofactor is tethered to His390 in eugenol oxidase. The model also provides a structural explanation for the observation that eugenol oxidase is dimeric whereas vanillyl alcohol oxidase is octameric. The bacterial oxidase efficiently oxidizes eugenol into coniferyl alcohol (KM = 1.0 µm, kcat = 3.1 s−1). Vanillyl alcohol and 5‐indanol are also readily accepted as substrates, whereas other phenolic compounds (vanillylamine, 4‐ethylguaiacol) are converted with relatively poor catalytic efficiencies. The catalytic efficiencies with the identified substrates are strikingly different when compared with vanillyl alcohol oxidase. The ability to efficiently convert eugenol may facilitate biotechnological valorization of this natural aromatic compound.
Two conserved regions within the tissue‐type plasminogen activator gene promoter mediate regulation by brain‐derived neurotrophic factor
Authors: Daniel Philip B. , Lux Wolfram , Samson Andre L. , Schleuning Wolf‐Dieter , Niego Be'eri , Weiss Thomas W. , Tjärnlund‐Wolf Anna , Medcalf Robert L.
Published: April 2007

Tissue‐type plasminogen activator (t‐PA) has recently been identified as a modulator of neuronal plasticity and can initiate conversion of the pro‐form of brain‐derived neurotrophic factor (BDNF) into its mature form. BDNF also increases t‐PA gene expression implicating t‐PA as a downstream effector of BDNF function. Here we demonstrate that BDNF‐mediated induction of t‐PA mRNA requires an increase in t‐PA gene transcription. Reporter constructs harboring 9.5 kb of the human t‐PA promoter conferred BDNF‐responsiveness in transfected mouse primary cortical neurons. This regulation was recapitulated in HEK 293 cells coexpressing the TrkB neurotrophin receptor. t‐PA promoter‐deletion analysis revealed the presence of two BDNF‐responsive domains, one located between −3.07 and −2.5 kb and the other within the proximal promoter. The upstream region was shown to confer BDNF responsiveness in a TrkB‐dependent manner when attached to a heterologous promoter. We also identify homologous regions within the murine and bovine t‐PA gene promoters and demonstrate that the equivalent upstream murine sequence functions as a BDNF‐responsive enhancer when inserted 5′ of the human proximal t‐PA promoter. Hence, BDNF‐mediated induction of t‐PA transcription relies on conserved modular promoter elements including a novel upstream BDNF‐responsive domain and the proximal t‐PA gene promoter.
Randomised clinical trial of Hydrofiber dressing with silver versus povidone–iodine gauze in the management of open surgical and traumatic wounds
Authors: Jurczak Florent , Dugré Thierry , Johnstone Alison , Offori Theodor , Vujovic Zorica , Hollander Dirk ,   AQUACEL Ag Surgical/Trauma Wound Study Group
Published: April 2007

This prospective, randomised clinical trial compared pain, comfort, exudate management, wound healing and safety with Hydrofiber® dressing with ionic silver (Hydrofiber Ag dressing) and with povidone–iodine gauze for the treatment of open surgical and traumatic wounds. Patients were treated with Hydrofiber Ag dressing or povidone–iodine gauze for up to 2 weeks. Pain severity was measured with a 10‐cm visual analogue scale (VAS). Other parameters were assessed clinically with various scales. Pain VAS scores decreased during dressing removal in both groups, and decreased while the dressing was in place in the Hydrofiber Ag dressing group (n = 35) but not in the povidone–iodine gauze group (n = 32). Pain VAS scores were similar between treatment groups. At final evaluation, Hydrofiber Ag dressing was significantly better than povidone–iodine gauze for overall ability to manage pain (P < 0·001), overall comfort (P ≤ 0·001), wound trauma on dressing removal (P = 0·001), exudate handling (P < 0·001) and ease of use (P ≤ 0·001). Rates of complete healing at study completion were 23% for Hydrofiber Ag dressing and 9% for povidone–iodine gauze (P = ns). No adverse events were reported with Hydrofiber Ag dressing; one subject discontinued povidone–iodine gauze due to adverse skin reaction. Hydrofiber Ag dressing supported wound healing and reduced overall pain compared with povidone–iodine gauze in the treatment of open surgical wounds requiring an antimicrobial dressing.
Paediatric partial‐thickness scald burns – is Biobrane the best treatment available?
Authors: Mandal Anirban
Published: April 2007

The article critically analyses the role of Biobrane, one of the widely used modern biosynthetic dressings, in paediatric partial‐thickness scald burns. Most of the studies, available in the literature, confirmed that Biobrane has significantly reduced the hospital stay, wound healing time and requirements of pain medications. However, very few studies with long‐term follow up are available. Further randomised controlled trials are required to challenge the supremacy of Biobrane in paediatric partial‐thickness burns.
Wound bed preparation of difficult wounds: an evolution of the principles of TIME
Authors: Ligresti Claudio , Bo Filippo
Published: April 2007

In the last few years, considerable progress has been made in the treatment of chronic ulcers, thanks to new therapy methods. Wound bed preparation is a modern approach for the removal of local barriers to healing by optimising debridement, reduction of bioburden and exudate management through the TIME principles, which have been introduced by the International Advisory Board on Wound Bed Preparation. However, this protocol does not evaluate the state of the repair process and therefore does not suggest the ideal therapeutic choice for each single patient. The revised TIME‐H concept considers also the supposed healing time, H, and gives a score that correlates the wound condition with the incidental concomitance with medical pathologies related to the therapeutic measures, thus guiding the clinician towards a practical and systematic approach in the treatment. By applying this scheme to our situation, the average healing time was considerably reduced. The formulation of the new protocol TIME‐H for a critical assessment of treatment scheme, which also includes the general conditions of the patient, represents a more rational and adequate approach for an accurate prognosis and therefore for a more suitable therapeutic choice in the treatment of difficult wounds.
Diagnostic clues to megaloblastic anaemia without macrocytosis
Authors: CHAN C. W. J. , LIU S. Y. H. , KHO C. S. B. , LAU K. H. T. , LIANG Y. S. , CHU W. R. , MA S. K. E.
Published: April 2007

Masking of the macrocytic expression of megaloblastic anaemia (MA) by coexisting thalassaemia, iron deficiency and chronic illness has been widely reported. We described the haematological and clinical features of 20 Chinese patients with MA presenting with mean corpuscular volume (MCV) ≦99 fl, and analysed the steps leading to the final diagnosis of MA with concomitant thalassaemia trait (n = 11), thalassaemia trait and iron deficiency (n = 3), iron deficiency (n = 4) and chronic illness (n = 2). We also compared the haematological characteristics of this group of patients with a group of normocytic anaemic patients without vitamin B12/folate deficiency, and identified certain laboratory information useful for differentiating the two groups. Statistically significant parameters included the mean values of haemoglobin, MCV, red cell distribution width (RDW), reticulocyte index, platelet count and serum bilirubin. All provided clues to maturation disorders within the marrow. A decision flowchart for the diagnosis of MA without macrocytosis was proposed. In the studied population, by using the parameters of haemoglobin <10 g/dl, MCV 80–99 fl, RDW ≥ 16% and reticulocyte index ≦ 2% as indicators, there was a 58% chance that a patient had MA without macrocytosis if he/she had all the four indicators, and a 2.2% chance of having it if he/she did not have these indicators. We emphasized the importance of including peripheral blood smear examination in the diagnostic procedures for such patients, as well as the importance of paying attention to patients’ medical history, racial background and previous MCV value.
Antiphospholipid antibodies and hepatitis C virus infection in Iranian thalassemia major patients
Authors: KASHEF S. , KARIMI M. , AMIRGHOFRAN Z. , AYATOLLAHI M. , PASALAR M. , GHAEDIAN M. M. , KASHEF M. A.
Published: April 2007

Although the precise nature of Antiphospholipid antibodies is still not clearly defined, they are known to have association with thromboembolic events and have been found in hepatitis C virus (HCV) infection. Moreover, high prevalence of HCV infection and thrombotic risk is described in thalassemia. We aimed at investigating the prevalence of anticardiolipin antibodies (aCLAbs), lupus anticoagulant (LA), and their relation with HCV infection in Iranian thalassemic patients. Presence of anti‐HCV antibody, serum HCV‐RNA, aCLAbs, and LA activity was determined in 131 patients with thalassemia major (male/female: 63/68 aged 3–29 years) registered at thalassemia unit, Dastgheib Hospital, Shiraz, Iran. Sixty‐one healthy controls were also included. Anti‐HCV antibody was positive in 24 (18.3%), IgG aCLAbs in 56 (42.7%), and LA activity in 9 (6.9%) patients. 87.5% of patients positive for aCLAbs had a low titer of aCLAbs. Although none of the participants had a previous history of thrombosis, higher prevalence of aCLAbs was detected in thalassemic patients compared with controls. No significant difference in the prevalence of aCLAbs was found between HCV‐infected and noninfected patients. A high prevalence of aCLAbs, the majority in low titers, was detected in Iranian thalassemic patients irrespective of previous history of thrombosis and presence of HCV infection.
Placebo or Protector?
Authors: Ankeny Rachel A.
Published: April 2007

Dr. Golem
. How to Think About Medicine.
By Harry Collins and Trevor Pinch
. University of Chicago Press, Chicago, 2005. 258 pp. $25, £17.50. ISBN 9780226113661.

Collins and Pinch present eight case studies (including tonsillectomies, chronic fatigue syndrome, and vaccination) to explore the uncertainties and limitations of medical science.
Atlantic Meridional Overturning Circulation During the Last Glacial Maximum
Authors: Lynch-Stieglitz Jean , Adkins Jess F. , Curry William B. , Dokken Trond , Hall Ian R. , Herguera Juan Carlos , Hirschi Joël J.-M. , Ivanova Elena V. , Kissel Catherine , Marchal Olivier , Marchitto Thomas M. , McCave I. Nicholas , McManus Jerry F. , Mulitza Stefan , Ninnemann Ulysses , Peeters Frank , Yu Ein-Fen , Zahn Rainer
Published: April 2007

The circulation of the deep Atlantic Ocean during the height of the last ice age appears to have been quite different from today. We review observations implying that Atlantic meridional overturning circulation during the Last Glacial Maximum was neither extremely sluggish nor an enhanced version of present-day circulation. The distribution of the decay products of uranium in sediments is consistent with a residence time for deep waters in the Atlantic only slightly greater than today. However, evidence from multiple water-mass tracers supports a different distribution of deep-water properties, including density, which is dynamically linked to circulation.
Rapid and Recent Changes in Fungal Fruiting Patterns
Authors: Gange A. C. , Gange E. G. , Sparks T. H. , Boddy L.
Published: April 2007

Information on responses of higher organisms to climate change is dominated by events in spring. Far less is known about autumnal events and virtually nothing about communities of microorganisms. We analyzed autumnal fruiting patterns of macrofungi over the past 56 years and found that average first fruiting date of 315 species is earlier, while last fruiting date is later. Fruiting of mycorrhizal species that associate with both deciduous and coniferous trees is delayed in deciduous, but not in coniferous, forests. Many species are now fruiting twice a year, indicating increased mycelial activity and possibly greater decay rates in ecosystems.
Chimeras of Two Isoprenoid Synthases Catalyze All Four Coupling Reactions in Isoprenoid Biosynthesis
Authors: Thulasiram Hirekodathakallu V. , Erickson Hans K. , Poulter C. Dale
Published: April 2007

The carbon skeletons of over 55,000 naturally occurring isoprenoid compounds are constructed from four fundamental coupling reactions: chain elongation, cyclopropanation, branching, and cyclobutanation. Enzymes that catalyze chain elongation and cyclopropanation are well studied, whereas those that catalyze branching and cyclobutanation are unknown. We have catalyzed the four reactions with chimeric proteins generated by replacing segments of a chain-elongation enzyme with corresponding sequences from a cyclopropanation enzyme. Stereochemical and mechanistic considerations suggest that the four coupling enzymes could have evolved from a common ancestor through relatively small changes in the catalytic site.
Giant Fluctuations of Coulomb Drag in a Bilayer System
Authors: Price A. S. , Savchenko A. K. , Narozhny B. N. , Allison G. , Ritchie D. A.
Published: April 2007

The Coulomb drag in a system of two parallel layers is the result of electron-electron interaction between the layers. We have observed reproducible fluctuations of the drag, both as a function of magnetic field and electron concentration, which are a manifestation of quantum interference of electrons in the layers. At low temperatures the fluctuations exceed the average drag, giving rise to random changes of the sign of the drag. The fluctuations are found to be much larger than previously expected, and we propose a model that explains their enhancement by considering fluctuations of local electron properties.
Cloning, Functional Characterization, and Mode of Action of a Novel Insecticidal Pore-Forming Toxin, Sphaericolysin, Produced by
Bacillus sphaericus
Authors: Nishiwaki Hisashi , Nakashima Kenta , Ishida Chiharu , Kawamura Tadayuki , Matsuda Kazuhiko
Published: March 2007

An insecticidal protein produced by
Bacillus sphaericus
A3-2 was purified to elucidate its structure and mode of action. The active principle purified from the culture broth of A3-2 was a protein with a molecular mass of 53 kDa that rapidly intoxicated German cockroaches (
Blattela germanica
) at a dose of about 100 ng when injected. The insecticidal protein sphaericolysin possessed the undecapeptide motif of cholesterol-dependent cytolysins and had a unique N-terminal sequence. The recombinant protein expressed in
Escherichia coli
was equally as potent as the native protein. Sphaericolysin-induced hemolysis resulted from the protein's pore-forming action. This activity as well as the insecticidal activity was markedly reduced by a Y159A mutation. Also, coapplication of sphaericolysin with cholesterol abolished the insecticidal action, suggesting that cholesterol binding plays an important role in insecticidal activity. Sphaericolysin-lysed neurons dissociated from the thoracic ganglia of the German cockroaches. In addition, sphaericolysin's activity in ganglia was suppressed by the Y159A mutation. The sphaericolysin-induced damage to the cockroach ganglia was greater than the damage to the ganglia of common cutworms (
Spodoptera litura
), which accounts, at least in part, for the higher sensitivity to sphaericolysin displayed by the cockroaches than that displayed by cutworms.
A Homologue of the
Aspergillus velvet
Gene Regulates both Cephalosporin C Biosynthesis and Hyphal Fragmentation in
Acremonium chrysogenum
Authors: Dreyer Jacqueline , Eichhorn Heiko , Friedlin Ernst , Kürnsteiner Hubert , Kück Ulrich
Published: March 2007

The
Aspergillus nidulans velvet
(
veA
) gene encodes a global regulator of gene expression controlling sexual development as well as secondary metabolism. We have identified the
veA
homologue Ac
veA
from
Acremonium chrysogenum
, the major producer of the β-lactam antibiotic cephalosporin C. Two different disruption strains as well as the corresponding complements were generated as a prelude to detailed functional analysis. Northern hybridization and quantitative real-time PCR clearly indicate that the nucleus-localized AcVEA polypeptide controls the transcriptional expression of six cephalosporin C biosynthesis genes. The most drastic reduction in expression is seen for
cefEF
, encoding the deacetoxycephalosporine/deacetylcephalosporine synthetase. After 120 h of growth, the
cefEF
transcript level is below 15% in both disruption strains compared to the wild type. These transcriptional expression data are consistent with results from a comparative and time-dependent high-performance liquid chromatography analysis of cephalosporin C production. Compared to the recipient, both disruption strains have a cephalosporin C titer that is reduced by 80%. In addition to its role in cephalosporin C biosynthesis, Ac
veA
is involved in the developmentally dependent hyphal fragmentation. In both disruption strains, hyphal fragmentation is already observed after 48 h of growth, whereas in the recipient strain, arthrospores are not even detected before 96 h of growth. Finally, the two mutant strains show hyperbranching of hyphal tips on osmotically nonstabilized media. Our findings will be significant for biotechnical processes that require a defined stage of cellular differentiation for optimal production of secondary metabolites.
Sensitive Whole-Cell Biosensor Suitable for Detecting a Variety of
N
-Acyl Homoserine Lactones in Intact Rhizosphere Microbial Communities
Authors: DeAngelis Kristen M. , Firestone Mary K. , Lindow Steven E.
Published: March 2007

To investigate quorum sensing in rhizosphere soil, a whole-cell biosensor,
Agrobacterium tumefaciens
(pAHL-Ice), was constructed. The biosensor responded to all
N
-acyl homoserine lactones (AHLs) tested, except C
4
homoserine lactone, with a minimum detection limit of 10
−12
M, as well as to both exogenously added AHLs and AHL-producing bacterial strains in soil. This highly sensitive biosensor reveals for the first time the increased AHL availability in intact rhizosphere microbial communities compared to that in bulk soil.
Development of High-Throughput Phenotyping of Metagenomic Clones from the Human Gut Microbiome for Modulation of Eukaryotic Cell Growth
Authors: Gloux Karine , Leclerc Marion , Iliozer Harout , L'Haridon René , Manichanh Chaysavanh , Corthier Gérard , Nalin Renaud , Blottière Hervé M. , Doré Joël
Published: March 2007

Metagenomic libraries derived from human intestinal microbiota (20,725 clones) were screened for epithelial cell growth modulation. Modulatory clones belonging to the four phyla represented among the metagenomic libraries were identified (hit rate, 0.04 to 8.7% depending on the screening cutoff). Several candidate loci were identified by transposon mutagenesis and subcloning.
Differentiation of
Bacillus anthracis
,
B. cereus
, and
B. thuringiensis
by Using Pulsed-Field Gel Electrophoresis
Authors: Zhong Wenwan , Shou Yulin , Yoshida Thomas M. , Marrone Babetta L.
Published: March 2007

A pulsed-field gel electrophoresis (PFGE) method was developed for discriminating
Bacillus anthracis
from
B. cereus
and
B. thuringiensis
. A worldwide collection of 25
B. anthracis
isolates showed high-profile homology, and these isolates were unambiguously distinguished from
B. cereus
and
B. thuringiensis
isolates by cluster analysis of the whole-genome macrorestriction enzyme digestion patterns generated by NotI.
High-Resolution DNA Melt Curve Analysis of the Clustered, Regularly Interspaced Short-Palindromic-Repeat Locus of
Campylobacter jejuni
Authors: Price Erin P. , Smith Helen , Huygens Flavia , Giffard Philip M.
Published: March 2007

A novel method for genotyping the clustered, regularly interspaced short-palindromic-repeat (CRISPR) locus of
Campylobacter jejuni
is described. Following real-time PCR, CRISPR products were subjected to high-resolution melt (HRM) analysis, a new technology that allows precise melt profile determination of amplicons. This investigation shows that the CRISPR HRM assay provides a powerful addition to existing
C. jejuni
genotyping methods and emphasizes the potential of HRM for genotyping short sequence repeats in other species.
Phylogenetic Diversity of Gram-Positive Bacteria Cultured from Marine Sediments
Authors: Gontang Erin A. , Fenical William , Jensen Paul R.
Published: March 2007

Major advances in our understanding of marine bacterial diversity have been gained through studies of bacterioplankton, the vast majority of which appear to be gram negative. Less effort has been devoted to studies of bacteria inhabiting marine sediments, yet there is evidence to suggest that gram-positive bacteria comprise a relatively large proportion of these communities. To further expand our understanding of the aerobic gram-positive bacteria present in tropical marine sediments, a culture-dependent approach was applied to sediments collected in the Republic of Palau from the intertidal zone to depths of 500 m. This investigation resulted in the isolation of 1,624 diverse gram-positive bacteria spanning 22 families, including many that appear to represent new taxa. Phylogenetic analysis of 189 representative isolates, based on 16S rRNA gene sequence data, indicated that 124 (65.6%) belonged to the class
Actinobacteria
while the remaining 65 (34.4%) were members of the class
Bacilli
. Using a sequence identity value of ≥98%, the 189 isolates grouped into 78 operational taxonomic units, of which 29 (37.2%) are likely to represent new taxa. The high degree of phylogenetic novelty observed during this study highlights the fact that a great deal remains to be learned about the diversity of gram-positive bacteria in marine sediments.
Molecular Characterization of a Glucokinase with Broad Hexose Specificity from
Bacillus sphaericus
Strain C3-41
Authors: Han Bei , Liu Haizhou , Hu Xiaomin , Cai Yajun , Zheng Dasheng , Yuan Zhiming
Published: March 2007

Bacillus sphaericus
cannot metabolize sugar since it lacks several of the enzymes necessary for glycolysis. Our results confirmed the presence of a glucokinase-encoding gene,
glcK
, and a phosphofructokinase-encoding gene,
pfk
, on the bacterial chromosome and expression of glucokinase during vegetative growth of
B. sphaericus
strains. However, no phosphoglucose isomerase gene (
pgi
) or phosphoglucose isomerase enzyme activity was detected in these strains. Furthermore, one
glcK
open reading frame was cloned from
B. sphaericus
strain C3-41 and then expressed in
Escherichia coli
. Biochemical analysis revealed that this gene encoded a protein with a molecular mass of 33 kDa and that the purified recombinant glucokinase had
K

m

values of 0.52 and 0.31 mM for ATP and glucose, respectively. It has been proved that this ATP-dependent glucokinase can also phosphorylate fructose and mannose, and sequence alignment of the
glcK
gene indicated that it belongs to the ROK protein family. It is postulated that the absence of the phosphoglucose isomerase-encoding gene
pgi
in
B. sphaericus
might be one of the reasons for the inability of this bacterium to metabolize carbohydrates. Our findings provide additional data that further elucidate the specific metabolic pathway and could be used for genetic improvement of
B. sphaericus
.
Diversity and Abundance of Nitrate Reductase Genes (
narG
and
napA
), Nitrite Reductase Genes (
nirS
and
nrfA
), and Their Transcripts in Estuarine Sediments
Authors: Smith Cindy J. , Nedwell David B. , Dong Liang F. , Osborn A. Mark
Published: March 2007

Estuarine systems are the major conduits for the transfer of nitrate from agricultural and other terrestrial-anthropogenic sources into marine ecosystems. Within estuarine sediments some microbially driven processes (denitrification and anammox) result in the net removal of nitrogen from the environment, while others (dissimilatory nitrate reduction to ammonium) do not. In this study, molecular approaches have been used to investigate the diversity, abundance, and activity of the nitrate-reducing communities in sediments from the hypernutrified Colne estuary, United Kingdom, via analysis of nitrate and nitrite reductase genes and transcripts. Sequence analysis of cloned PCR-amplified
narG
,
napA
, and
nrfA
gene sequences showed the indigenous nitrate-reducing communities to be both phylogenetically diverse and also divergent from previously characterized nitrate reduction sequences in soils and offshore marine sediments and from cultured nitrate reducers. In both the
narG
and
nrfA
libraries, the majority of clones (48% and 50%, respectively) were related to corresponding sequences from delta-proteobacteria. A suite of quantitative PCR primers and TaqMan probes was then developed to quantify phylotype-specific nitrate (
narG
and
napA
) and nitrite reductase (
nirS
and
nrfA
) gene and transcript numbers in sediments from three sites along the estuarine nitrate gradient. In general, both nitrate and nitrite reductase gene copy numbers were found to decline significantly (
P
< 0.05) from the estuary head towards the estuary mouth. The development and application, for the first time, of quantitative reverse transcription-PCR assays to quantify mRNA sequences in sediments revealed that transcript numbers for three of the five phylotypes quantified were greatest at the estuary head.
Experimental Infection of Neonatal Foals withRhodococcus equiTriggers Adult-Like Gamma Interferon Induction
Authors: Jacks Stephanie , Giguère Steeve , Crawford P. Cynda , Castleman William L.
Published: April 2007

Rhodococcus equiis a facultative intracellular pathogen that causes pneumonia in young foals but does not induce disease in immunocompetent adult horses. Clearance ofR. equidepends mainly on gamma interferon (IFN-γ) production by T lymphocytes, whereas the predominance of interleukin 4 (IL-4) is detrimental. Young foals, like neonates of many other species, are generally deficient in the ability to produce IFN-γ. The objective of this study was to compare the cytokine profiles, as well as cell-mediated and antibody responses, of young foals to those of adult horses following intrabronchial challenge withR. equi. The lymphoproliferative responses of bronchial lymph node (BLN) cells to concanavalin A were significantly higher in foals than in adult horses. In contrast, adult horses had significantly higher lymphoproliferative responses toR. equiantigens than did foals. Infected foals had significantly lower IL-4 mRNA expression but significantly higher IFN-γ expression and IFN-γ/IL-4 ratio inR. equi-stimulated BLN lymphocytes than did infected adults. Infection withR. equiin foals resulted in a significant increase in the percentage of T lymphocytes and CD4+T lymphocytes in bronchoalveolar lavage fluid in association with a significant decrease in the percentage of these cell populations in BLNs. Infection of foals also resulted in a marked increase in serum immunoglobulin Ga (IgGa) and IgGb levels, resulting in concentrations in serum that were significantly higher than those of adult horses. This study demonstrates that the immune response toR. equiin foals is not biased toward IL-4 and is characterized by the predominant induction of IFN-γ.
Genome Sequence and Analysis of aPropionibacterium acnesBacteriophage
Authors: Farrar Mark D. , Howson Karen M. , Bojar Richard A. , West David , Towler James C. , Parry James , Pelton Katharine , Holland Keith T.
Published: March 2007

Cutaneous propionibacteria are important commensals of human skin and are implicated in a wide range of opportunistic infections.Propionibacterium acnesis also associated with inflammatory acne vulgaris. Bacteriophage PA6 is the first phage ofP. acnesto be sequenced and demonstrates a high degree of similarity to many mycobacteriophages both morphologically and genetically. PA6 possesses an icosahedreal head and long noncontractile tail characteristic of theSiphoviridae. The overall genome organization of PA6 resembled that of the temperate mycobacteriophages, although the genome was much smaller, 29,739 bp (48 predicted genes), compared to, for example, 50,550 bp (86 predicted genes) for the Bxb1 genome. PA6 infected onlyP. acnesand produced clear plaques with turbid centers, but it lacked any obvious genes for lysogeny. The host range of PA6 was restricted toP. acnes, but the phage was able to infect and lyse allP. acnesisolates tested. Sequencing of the PA6 genome makes an important contribution to the study of phage evolution and propionibacterial genetics.
Membrane-Bound Nitrate Reductase Is Required for Anaerobic Growth in Cystic Fibrosis Sputum
Authors: Palmer Kelli L. , Brown Stacie A. , Whiteley Marvin
Published: March 2007

The autosomal recessive disorder cystic fibrosis (CF) affects approximately 70,000 people worldwide and is characterized by chronic bacterial lung infections with the opportunistic pathogen
Pseudomonas aeruginosa
. To form a chronic CF lung infection,
P. aeruginosa
must grow and proliferate within the CF lung, and the highly viscous sputum within the CF lung provides a likely growth substrate. Recent evidence indicates that anaerobic microenvironments may be present in the CF lung sputum layer. Since anaerobic growth significantly enhances
P. aeruginosa
biofilm formation and antibiotic resistance, it is important to examine
P. aeruginosa
physiology and metabolism in anaerobic environments. Measurement of nitrate levels revealed that CF sputum contains sufficient nitrate to support significant
P. aeruginosa
growth anaerobically, and mutational analysis revealed that the membrane-bound nitrate reductase is essential for
P. aeruginosa
anaerobic growth in an in vitro CF sputum medium. In addition, expression of genes coding for the membrane-bound nitrate reductase complex is responsive to CF sputum nitrate levels. These findings suggest that the membrane-bound nitrate reductase is critical for
P. aeruginosa
anaerobic growth with nitrate in the CF lung.
An Extracytoplasmic Function Sigma Factor Acts as a General Stress Response Regulator inSinorhizobium meliloti
Authors: Sauviac Laurent , Philippe Heinui , Phok Kounthéa , Bruand Claude
Published: March 2007

Sinorhizobium melilotigenes transcriptionally up-regulated after heat stress, as well as upon entry into stationary phase, were identified by microarray analyses. Sixty stress response genes were thus found to be up-regulated under both conditions. One of them,rpoE2(smc01506), encodes a putative extracytoplasmic function (ECF) sigma factor. We showed that this sigma factor controls its own transcription and is activated by various stress conditions, including heat and salt, as well as entry into stationary phase after either carbon or nitrogen starvation. We also present evidence that the product of the gene cotranscribed withrpoE2negatively regulates RpoE2 activity, and we therefore propose that it plays the function of anti-sigma factor. By combining transcriptomic, bioinformatic, and quantitative reverse transcription-PCR analyses, we identified 44 RpoE2-controlled genes and predicted the number of RpoE2 targets to be higher. Strikingly, more than one-third of the 60 stress response genes identified in this study are RpoE2 targets. Interestingly, two genes encoding proteins with known functions in stress responses, namely,katCandrpoH2, as well as a second ECF-encoding gene,rpoE5, were found to be RpoE2 regulated. Altogether, these data suggest that RpoE2 is a major global regulator of the general stress response inS. meliloti. Despite these observations, and although this sigma factor is well conserved among alphaproteobacteria, no in vitro nor in planta phenotypic difference from the wild-type strain could be detected forrpoE2mutants. This therefore suggests that other important actors in the general stress response have still to be identified inS. meliloti.
Re
-Citrate Synthase from
Clostridium kluyveri
Is Phylogenetically Related to Homocitrate Synthase and Isopropylmalate Synthase Rather Than to
Si
-Citrate Synthase
Authors: Li Fuli , Hagemeier Christoph H. , Seedorf Henning , Gottschalk Gerhard , Thauer Rudolf K.
Published: March 2007

The synthesis of citrate from acetyl-coenzyme A and oxaloacetate is catalyzed in most organisms by a
Si
-citrate synthase, which is
Si
-face stereospecific with respect to C-2 of oxaloacetate. However, in
Clostridium kluyveri
and some other strictly anaerobic bacteria, the reaction is catalyzed by a
Re
-citrate synthase, whose primary structure has remained elusive. We report here that
Re
-citrate synthase from
C. kluyveri
is the product of a gene predicted to encode isopropylmalate synthase.
C. kluyveri
is also shown to contain a gene for
Si
-citrate synthase, which explains why cell extracts of the organism always exhibit some
Si
-citrate synthase activity.
ScrG, a GGDEF-EAL Protein, Participates in Regulating Swarming and Sticking inVibrio parahaemolyticus
Authors: Kim Yun-Kyeong , McCarter Linda L.
Published: March 2007

In this work, we describe a new gene controlling lateral flagellar gene expression. The gene encodes ScrG, a protein containing GGDEF and EAL domains. This is the second GGDEF-EAL-encoding locus determined to be involved in the regulation of swarming: the first was previously characterized and namedscrABC(for “swarming and capsular polysaccharide regulation”). GGDEF and EAL domain-containing proteins participate in the synthesis and degradation of the nucleotide signal cyclic di-GMP (c-di-GMP) in many bacteria. Overexpression ofscrGwas sufficient to induce lateral flagellar gene expression in liquid, decrease biofilm formation, decreasecpsgene expression, and suppress the ΔscrABCphenotype. Removal of its EAL domain reversed ScrG activity, converting ScrG to an inhibitor of swarming and activator ofcpsexpression. Overexpression ofscrGdecreased the intensity of a32P-labeled nucleotide spot comigrating with c-di-GMP standard, whereas overexpression ofscrGΔEALenhanced the intensity of the spot. Mutants with defects inscrGshowed altered swarming and lateral flagellin production and colony morphology (but not swimming motility); furthermore, mutation of two GGDEF-EAL-encoding loci (scrGandscrABC) produced cumulative effects on swarming, lateral flagellar gene expression, lateral flagellin production and colony morphology. Mutant analysis supports the assignment of the primary in vivo activity of ScrG to acting as a phosphodiesterase. The data are consistent with a model in which multiple GGDEF-EAL proteins can influence the cellular nucleotide pool: a low concentration of c-di-GMP favors surface mobility, whereas high levels of this nucleotide promote a more adhesiveVibrio parahaemolyticuscell type.
YhdJ, a Nonessential CcrM-Like DNA Methyltransferase of
Escherichia coli
and
Salmonella enterica
Authors: Broadbent Sarah E. , Balbontin Roberto , Casadesus Josep , Marinus Martin G. , van der Woude Marjan
Published: March 2007

The
Caulobacter crescentus
DNA adenine methyltransferase CcrM and its homologs in the α-
Proteobacteria
are essential for viability. CcrM is 34% identical to the
yhdJ
gene products of
Escherichia coli
and
Salmonella enterica
. This study provides evidence that the
E. coli yhdJ
gene encodes a DNA adenine methyltransferase. In contrast to an earlier report, however, we show that
yhdJ
is not an essential gene in either
E. coli
or
S. enterica
.
Zinc-Responsive Regulation of Alternative Ribosomal Protein Genes inStreptomyces coelicolorInvolves Zur and σR
Authors: Owen Gillian A. , Pascoe Ben , Kallifidas Dimitris , Paget Mark S. B.
Published: March 2007

Streptomyces coelicolorcontains paralogous versions of seven ribosomal proteins (S14, S18, L28, L31, L32, L33, and L36), which differ in their potential to bind structural zinc. The paralogues are termed C+or C−on the basis of the presence or absence of putative cysteine ligands. Here, mutational studies suggest that the C−version of L31 can functionally replace its C+paralogue only when expressed at an artificially elevated level. We show that the level of expression of four transcriptional units encoding C−proteins is elevated under conditions of zinc deprivation. Zur controls the expression of three transcriptional units (includingrpmG2,rpmE2,rpmB2,rpsN2,rpmF2, and possiblyrpsR2). Zur also controls the expression of theznuACBoperon, which is predicted to encode a high-affinity zinc transport system. Surprisingly, the zinc-responsive control of therpmG3-rpmJ2operon is dictated by σR, a sigma factor that was previously shown to control the response to disulfide stress inS. coelicolor. The induction of σRactivity during zinc limitation establishes an important link between thiol-disulfide metabolism and zinc homeostasis.
Evaluation of a Commercial Real-Time PCR Kit for Detection of Dengue Virus in Samples Collected during an Outbreak in Goiânia, Central Brazil, in 2005
Authors: Levi José Eduardo , Tateno Adriana Fumie , Machado Adriana Freire , Ramalho Débora Camillo , de Souza Vanda Akico Ueda Fick , Guilarde Adriana Oliveira , de Rezende Feres Valéria Christina , Martelli Celina Maria Turchi , Turchi Marília Dalva , Siqueira João Bosco , Pannuti Cláudio Sérgio
Published: April 2007

In the past 2 decades, dengue has reemerged in Brazil as a significant public health problem. Clinicians demand a diagnostic test with high sensitivity that is applicable during the early symptomatic phase. We aimed to test two distinct molecular methods on samples from suspected dengue cases during an outbreak in Central Brazil. Acute-phase serum specimens from 254 patients suspected of having dengue were collected during 2005 in the city of Goiânia, Central Brazil. Samples were blindly evaluated by real-time and multiplex PCR in addition to routine immunoglobulin M serology and virus culture. Overall, acute dengue was confirmed by serology, multiplex PCR, or virus isolation for 80% of patients (203/254). Another four patients presented real-time PCR-positive results as the unique marker of dengue. Higher real-time PCR positivity levels and viral loads were observed in the early symptomatic phase of disease (≤5 days) than after this period. Multiplex and real-time PCR assays presented a high kappa agreement (0.85). According to multiplex PCR, 60 samples harbored dengue virus type 3 (DEN-3), 4 samples harbored DEN-2, and 1 sample displayed a pattern compatible with a double infection with DEN-2 and -3. The dengue virus real-time kit was found to be practical and adjustable for high throughput, to display the best performance in the early symptomatic phase of dengue cases, and to be valuable for confirming dengue diagnosis in a timely manner.
Frequent Occult Infection with Cytomegalovirus in Cardiac Transplant Recipients despite Antiviral Prophylaxis
Authors: Potena Luciano , Holweg Cecile T. J. , Vana Marcy L. , Bashyam Leena , Rajamani Jaya , McCormick A. Louise , Cooke John P. , Valantine Hannah A. , Mocarski Edward S.
Published: April 2007

Despite antiviral prophylaxis, a high percentage (over 90%) of heart transplant patients experience active cytomegalovirus (CMV) infection, diagnosed by detection of viral DNA in peripheral blood polymorphonuclear leukocytes within the first few months posttransplantation. Viral DNA was detected in mononuclear cells prior to detection in granulocytes from CMV-seropositive recipients (R
+
) receiving a heart from a CMV-seropositive donor (D
+
). Based on assessment of systemic infection in leukocyte populations, both R
+
subgroups (R
+
/D
−
and R
+
/D
+
) experienced a greater infection burden than the R
−
/D
+
subgroup, which was aggressively treated because of a higher risk of acute CMV disease. Despite widespread systemic infection in all at-risk patient subgroups, CMV DNA was rarely (<3% of patients) detected in transplanted heart biopsy specimens. The R
+
patients more frequently exceeded the 75th percentile of the CMV DNA copy number distribution in leukocytes (110 copies/10
5
polymorphonuclear leukocytes) than the R
−
/D
+
subgroup. Therefore, active systemic CMV infection involving leukocytes is common in heart transplant recipients receiving prophylaxis to reduce acute disease. Infection of the transplanted organ is rare, suggesting that chronic vascular disease attributed to CMV may be driven by the consequences of systemic infection.
Multicenter Evaluation of the New Abbott RealTime Assays for Quantitative Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA
Authors: Schutten M. , Peters D. , Back N. K. T. , Beld M. , Beuselinck K. , Foulongne V. , Geretti A.-M. , Pandiani L. , Tiemann C. , Niesters H. G. M.
Published: April 2007

The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS TaqMan assays. Two different protocols used during the testing period with and without a pre-m1000 RNA isolation spin were compared. The difference proved to be nonsignificant. A uracil-N-glycosylase (UNG) contamination control option in the HCV test for previous Roche COBAS Amplicor users was evaluated. It proved to decrease amplicon carryover by 100-fold independent of the amplicon input concentration. The protocol including UNG proved to overcome problems with false-positive negative controls. Comparison with other assays revealed only minor differences. The largest difference was observed between the Abbott HCV RealTime assay and the Roche COBAS Amplicor HCV Monitor version 2.0 assay.
Influence of Disk Preparation on Detection of Metallo-β-Lactamase-Producing Isolates by the Combined Disk Assay
Authors: Andrade Soraya S. , Picão Renata C. , Campana Eloiza H. , Nicoletti Adriana G. , Pignatari Antônio C. C. , Gales Ana C.
Published: April 2007

The combined disk assay has been used for detection of metallo-β-lactamase-producing isolates. We have observed that the size of inhibition zones produced by many β-lactam/metallo-β-lactamase inhibitor (IMBL) combinations may differ depending on the way that the combined disks were prepared. Among the 10 β-lactam/IMBL combinations tested, only the imipenem/EDTA combination produced similar results.
Metallo-β-Lactamase or Extended-Spectrum β-Lactamase: a Wolf in Sheep's Clothing
Authors: Espedido Björn A. , Thomas Lee C. , Iredell Jonathan R.
Published: April 2007

Diagnostic algorithms in commonly used automated bacterial identification systems fail to reliably identify a metallo-β-lactamase in the
Enterobacteriaceae
. Misidentification as an extended-spectrum β-lactamase may result in inappropriate dismissal of drugs such as aztreonam in favor of carbapenems, which may in turn select for a highly carabapenem resistant phenotype.