bluuebunny/crossref_metadata_2025 huggingface dataset:
Diversity of Methanotrophic Bacteria in Tropical Upland Soils under Different Land Uses
Authors: Knief Claudia , Vanitchung Supika , Harvey Narumon W. , Conrad Ralf , Dunfield Peter F. , Chidthaisong Amnat
Published: July 2005

Three upland soils from Thailand, a natural forest, a 16-year-old reforested site, and an agricultural field, were studied with regard to methane uptake and the community composition of methanotrophic bacteria (MB). The methane uptake rates were similar to rates described previously for forest and farmland soils of the temperate zone. The rates were lower at the agricultural site than at the native forest and reforested sites. The sites also differed in the MB community composition, which was characterized by denaturing gradient gel electrophoresis (DGGE) of
pmoA
gene fragments (coding for a subunit of particulate methane monooxygenase) that were PCR amplified from total soil DNA extracts. Cluster analysis based on the DGGE banding patterns indicated that the MB communities at the forested and reforested sites were similar to each other but different from that at the farmland site. Sequence analysis of excised DGGE bands indicated that
Methylobacter
spp. and
Methylocystis
spp. were present. Sequences of the “forest soil cluster” or “upland soil cluster α,” which is postulated to represent organisms involved in atmospheric methane consumption in diverse soils, were detected only in samples from the native forest and reforested sites. Additional sequences that may represent uncultivated groups of MB in the
Gammaproteobacteria
were also detected.
Definition of Genetically Distinct Attenuation Mechanisms in Naturally Virulence-Attenuated
Listeria monocytogenes
by Comparative Cell Culture and Molecular Characterization
Authors: Roberts Angela , Chan Yvonne , Wiedmann Martin
Published: July 2005

Listeria monocytogenes
is a foodborne pathogen able to cause serious disease in humans and animals. Not all isolates are equally pathogenic, however, and several isolates have been characterized as naturally virulence attenuated. We sought to identify the genetic basis of natural virulence attenuation using cell culture assays and molecular techniques. By comparing the phenotypes of naturally virulence-attenuated isolates to those of defined virulence gene mutants in plaque, cytotoxicity, and hemolysis assays and by characterizing selected virulence genes and their expression using DNA sequencing and TaqMan reverse transcriptase PCR, we classified virulence-attenuated isolates into four categories. Both group A and group B isolates were noncytotoxic and nonhemolytic; however, group A isolates underexpressed listeriolysin O (LLO, encoded by
hlyA
), while group B isolates produced LLO proteins that were inactive. The single isolate in group C was fully cytotoxic, had higher than wild-type hemolytic activity, and was, therefore, likely virulence attenuated due to overexpression of LLO. Group D isolates were characterized by normal cytotoxicity, hemolytic activity, and
hlyA
expression but had reduced intracellular growth. The genetic mechanisms causing virulence-attenuated phenotypes among the group D isolates could not be determined definitively but may involve defects in the expression of
actA
or the function of the ActA protein. Our results show (i) that the combination of cell culture assays and molecular techniques used in this study allows for identification and characterization of naturally virulence-attenuated isolates and (ii) that multiple distinct genetic mechanisms are responsible for natural virulence attenuation in
L. monocytogenes
.
Reduction of Perchlorate and Nitrate by Microbial Communities in Vadose Soil
Authors: Nozawa-Inoue Mamie , Scow Kate M. , Rolston Dennis E.
Published: July 2005

Perchlorate contamination is a concern because of the increasing frequency of its detection in soils and groundwater and its presumed inhibitory effect on human thyroid hormone production. Although significant perchlorate contamination occurs in the vadose (unsaturated) zone, little is known about perchlorate biodegradation potential by indigenous microorganisms in these soils. We measured the effects of electron donor (acetate and hydrogen) and nitrate addition on perchlorate reduction rates and microbial community composition in microcosm incubations of vadose soil. Acetate and hydrogen addition enhanced perchlorate reduction, and a longer lag period was observed for hydrogen (41 days) than for acetate (14 days). Initially, nitrate suppressed perchlorate reduction, but once perchlorate started to be degraded, the process was stimulated by nitrate. Changes in the bacterial community composition were observed in microcosms enriched with perchlorate and either acetate or hydrogen. Denaturing gradient gel electrophoresis analysis and partial sequencing of 16S rRNA genes recovered from these microcosms indicated that formerly reported perchlorate-reducing bacteria were present in the soil and that microbial community compositions were different between acetate- and hydrogen-amended microcosms. These results indicate that there is potential for perchlorate bioremediation by native microbial communities in vadose soil.
Evaluation of Lateral-Flow
Clostridium botulinum
Neurotoxin Detection Kits for Food Analysis
Authors: Sharma Shashi K. , Eblen Brian S. , Bull Robert L. , Burr Donald H. , Whiting Richard C.
Published: July 2005

The suitability and sensitivity of two in vitro lateral-flow assays for detecting
Clostridium botulinum
neurotoxins (BoNTs) in an assortment of foods were evaluated. Toxin extraction and preparation methods for various liquid, solid, and high-fat-content foods were developed. The lateral-flow assays, one developed by the Naval Medical Research Center (Silver Spring, MD) and the other by Alexeter Technologies (Gaithersburg, MD), are based on the immunodetection of BoNT types A, B, and E. The assays were found to be rapid and easy to perform with minimum requirements for laboratory equipment or skills. They can readily detect 10 ng/ml of BoNT types A and B and 20 ng/ml of BoNT type E. Compared to other in vitro detection methods, these assays are less sensitive, and the assessment of a result is strictly qualitative. However, the assay was found to be simple to use and to require minimal training. The assays successfully detected BoNT types A, B, and E in a wide variety of foods, suggesting their potential usefulness as a preliminary screening system for triaging food samples with elevated BoNT levels in the event of a
C. botulinum
contamination event.
Cloning and Expression of Islandisin, a New Thermostable Subtilisin from
Fervidobacterium islandicum
, in
Escherichia coli
Authors: Gödde Carolin , Sahm Kerstin , Brouns Stan J. J. , Kluskens Leon D. , van der Oost John , de Vos Willem M. , Antranikian Garabed
Published: July 2005

A gene encoding a subtilisin-like protease, designated islandisin, from the extremely thermophilic bacterium
Fervidobacterium islandicum
(DSMZ 5733) was cloned and actively expressed in
Escherichia coli
. The gene was identified by PCR using degenerated primers based on conserved regions around two of the three catalytic residues (Asp, His, and Ser) of subtilisin-like serine protease-encoding genes. Using inverse PCR regions flanking the catalytic residues, the gene could be cloned. Sequencing revealed an open reading frame of 2,106 bp. The deduced amino acid sequence indicated that the enzyme is synthesized as a proenzyme with a putative signal sequence of 33 amino acids (aa) in length. The mature protein contains the three catalytic residues (Asp177, His215, and Ser391) and has a length of 668 aa. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme could be classified as a subtilisin-like serine protease in the subgroup of thermitase. The whole gene was amplified by PCR, ligated into pET-15b, and successfully expressed in
E. coli
BL21(DE3)pLysS. The recombinant islandisin was purified by heat denaturation, followed by hydroxyapatite chromatography. The enzyme is active at a broad range of temperatures (60 to 80°C) and pHs (pH 6 to 8.5) and shows optimal proteolytic activity at 80°C and pH 8.0. Islandisin is resistant to a number of detergents and solvents and shows high thermostability over a long period of time (up to 32 h) at 80°C with a half-life of 4 h at 90°C and 1.5 h at 100°C.
Improvement of the Fungal Biocontrol Agent
Trichoderma atroviride
To Enhance both Antagonism and Induction of Plant Systemic Disease Resistance
Authors: Brunner Kurt , Zeilinger Susanne , Ciliento Rosalia , Woo Sheridian L. , Lorito Matteo , Kubicek Christian P. , Mach Robert L.
Published: July 2005

Biocontrol agents generally do not perform well enough under field conditions to compete with chemical fungicides. We determined whether transgenic strain SJ3-4 of
Trichoderma atroviride
, which expresses the
Aspergillus niger
glucose oxidase-encoding gene,
goxA
, under a homologous chitinase (
nag1
) promoter had increased capabilities as a fungal biocontrol agent. The transgenic strain differed only slightly from the wild-type in sporulation or the growth rate.
goxA
expression occurred immediately after contact with the plant pathogen, and the glucose oxidase formed was secreted. SJ3-4 had significantly less
N
-acetylglucosaminidase and endochitinase activities than its nontransformed parent. Glucose oxidase-containing culture filtrates exhibited threefold-greater inhibition of germination of spores of
Botrytis cinerea
. The transgenic strain also more quickly overgrew and lysed the plant pathogens
Rhizoctonia solani
and
Pythium ultimum
. In planta, SJ3-4 had no detectable improved effect against low inoculum levels of these pathogens. Beans planted in heavily infested soil and treated with conidia of the transgenic
Trichoderma
strain germinated, but beans treated with wild-type spores did not germinate. SJ3-4 also was more effective in inducing systemic resistance in plants. Beans with SJ3-4 root protection were highly resistant to leaf lesions caused by the foliar pathogen
B. cinerea
. This work demonstrates that heterologous genes driven by pathogen-inducible promoters can increase the biocontrol and systemic resistance-inducing properties of fungal biocontrol agents, such as
Trichoderma
spp., and that these microbes can be used as vectors to provide plants with useful molecules (e.g., glucose oxidase) that can increase their resistance to pathogens.
Fate of
14
C-Labeled Microbial Products Derived from Nitrifying Bacteria in Autotrophic Nitrifying Biofilms
Authors: Okabe Satoshi , Kindaichi Tomonori , Ito Tsukasa
Published: July 2005

The cross-feeding of microbial products derived from
14
C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [
14
C]bicarbonate, biofilm samples were incubated with and without NH
4
+
as a sole energy source for 10 days. The transfer of
14
C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the β-
Proteobacteria
showed significant uptake of
14
C-labeled microbial products. In particular, the members of the
Chloroflexi
were strongly MAR positive in the culture without NH
4
+
addition, in which nitrifying bacteria tended to decay. This indicated that the members of the
Chloroflexi
preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the
Cytophaga
-
Flavobacterium
cluster gradually utilized
14
C-labeled products in the culture with NH
4
+
addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of
14
C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.
Protein Engineering of Epoxide Hydrolase from
Agrobacterium radiobacter
AD1 for Enhanced Activity and Enantioselective Production of (
R
)-1-Phenylethane-1,2-Diol
Authors: Rui Lingyun , Cao Li , Chen Wilfred , Reardon Kenneth F. , Wood Thomas K.
Published: July 2005

DNA shuffling and saturation mutagenesis of positions F108, L190, I219, D235, and C248 were used to generate variants of the epoxide hydrolase of
Agrobacterium radiobacter
AD1 (EchA) with enhanced enantioselectivity and activity for styrene oxide and enhanced activity for 1,2-epoxyhexane and epoxypropane. EchA variant I219F has more than fivefold-enhanced enantioselectivity toward racemic styrene oxide, with the enantiomeric ratio value (E value) for the production of (
R
)-1-phenylethane-1,2-diol increased from 17 for the wild-type enzyme to 91, as well as twofold-improved activity for the production of (
R
)-1-phenylethane-1,2-diol (1.96 ± 0.09 versus 1.04 ± 0.07 μmol/min/mg for wild-type EchA). Computer modeling indicated that this mutation significantly alters (
R
)-styrene oxide binding in the active site. Another three variants from EchA active-site engineering, F108L/C248I, I219L/C248I, and F108L/I219L/C248I, also exhibited improved enantioselectivity toward racemic styrene oxide in favor of production of the corresponding diol in the (
R
) configuration (twofold enhancement in their E values). Variant F108L/I219L/C248I also demonstrated 10-fold- and 2-fold-increased activity on 5 mM epoxypropane (24 ± 2 versus 2.4 ± 0.3 μmol/min/mg for the wild-type enzyme) and 5 mM 1,2-epoxyhexane (5.2 ± 0.5 versus 2.6 ± 0.0 μmol/min/mg for the wild-type enzyme). Both variants L190F (isolated from a DNA shuffling library) and L190Y (created from subsequent saturation mutagenesis) showed significantly enhanced activity for racemic styrene oxide hydrolysis, with 4.8-fold (8.6 ± 0.3 versus 1.8 ± 0.2 μmol/min/mg for the wild-type enzyme) and 2.7-fold (4.8 ± 0.8 versus 1.8 ± 0.2 μmol/min/mg for the wild-type enzyme) improvements, respectively. L190Y also hydrolyzed 1,2-epoxyhexane 2.5 times faster than the wild-type enzyme.
Development and Application of an Insertional System for Gene Delivery and Expression in
Campylobacter jejuni
Authors: Karlyshev A. V. , Wren B. W.
Published: July 2005

The genetic investigation of
Campylobacter jejuni
, an important gastrointestinal pathogen, has been hampered by the lack of an efficient system for introduction of exogenous genetic information, as commonly used vectors designed for
Escherichia coli
and other bacteria cannot be maintained in
Campylobacter
cells. Additionally, gene expression in
Campylobacter
requires the presence of species-specific promoters. In this study we exploited the availability of several conserved copies of rRNA gene clusters for insertion of various genes into the chromosome by homologous recombination. The high conservation of the rRNA sequences means that the procedure can be applied to other
Campylobacter
strains. The presence of a
Campylobacter
-derived promoter in this vector ensures expression of exogenous genes in target cells. The efficiency of the procedure was demonstrated by complementation of mutations in two strains of
Campylobacter
. In addition, we applied the system for introduction and expression of a green fluorescent protein (GFP). GFP-expressing
Campylobacter
allowed visualization of sessile bacteria attached to a glass surface in stationary liquid culture. The study demonstrated that the attached bacteria contained an assemblage of coccoid and spiral forms with liquid channels preserving viable highly motile cells. We demonstrate a novel universal procedure for gene delivery and expression that can be used as an efficient tool to study this poorly understood pathogen. The principles developed in this study could be more widely applied for the manipulation of other bacteria that are refractory to genetic analysis.
Susceptibility of Biofilms to
Bdellovibrio bacteriovorus
Attack
Authors: Kadouri Daniel , O'Toole George A.
Published: July 2005

Biofilms are communities of microorganisms attached to a surface, and the growth of these surface attached communities is thought to provide microorganisms with protection against a range of biotic and abiotic agents. The capability of the gram-negative predatory bacterium
Bdellovibrio bacteriovorus
to control and reduce an existing
Escherichia coli
biofilm was evaluated in a static assay. A reduction in biofilm biomass was observed as early as 3 h after exposure to the predator, and an 87% reduction in crystal violet staining corresponding to a 4-log reduction in biofilm cell viability was seen after a 24-h exposure period. We observed that an initial titer of
Bdellovibrio
as low as 10
2
PFU/well or an exposure to the predator as short as 30 min is sufficient to reduce a preformed biofilm. The ability of
B. bacteriovorus
to reduce an existing biofilm was confirmed by scanning electron microscopy. The reduction in biofilm biomass obtained after the first 24 h of exposure to the predator remained unchanged even after longer exposure periods and reinoculation of the samples with fresh
Bdellovibrio
; however, no genetically stable resistant population of the host bacteria could be detected. Our data suggest that growth in a biofilm does not prevent predation by
Bdellovibrio
but allows a level of survival from attack greater than that observed for planktonic cells. In flow cell experiments
B. bacteriovorus
was able to decrease the biomass of both
E. coli
and
Pseudomonas fluorescens
biofilms as determined by phase-contrast and epifluorescence microscopy.
Mediterranean Fruit Fly as a Potential Vector of Bacterial Pathogens
Authors: Sela Shlomo , Nestel David , Pinto Riky , Nemny-Lavy Esther , Bar-Joseph Moshe
Published: July 2005

The Mediterranean fruit fly (
Ceratitis capitata
) is a cosmopolitan pest of hundreds of species of commercial and wild fruits. It is considered a major economic pest of commercial fruits in the world. Adult Mediterranean fruit flies feed on all sorts of protein sources, including animal excreta, in order to develop eggs. After reaching sexual maturity and copulating, female flies lay eggs in fruit by puncturing the skin with their ovipositors and injecting batches of eggs into the wounds. In view of the increase in food-borne illnesses associated with consumption of fresh produce and unpasteurized fruit juices, we investigated the potential of Mediterranean fruit fly to serve as a vector for transmission of human pathogens to fruits. Addition of green fluorescent protein (GFP)-tagged
Escherichia coli
to a Mediterranean fruit fly feeding solution resulted in a dose-dependent increase in the fly's bacterial load. Flies exposed to fecal material enriched with GFP-tagged
E. coli
were similarly contaminated and were capable of transmitting
E. coli
to intact apples in a cage model system. Washing contaminated apples with tap water did not eliminate the
E. coli
. Flies inoculated with
E. coli
harbored the bacteria for up to 7 days following contamination. Fluorescence microscopy demonstrated that the majority of fluorescent bacteria were confined along the pseudotrachea in the labelum edge of the fly proboscis. Wild flies captured at various geographic locations were found to carry coliforms, and in some cases presumptive identification of
E. coli
was made. These findings support the hypothesis that the common Mediterranean fruit fly is a potential vector of human pathogens to fruits.
Assessment of Soil Microbial Community Structure by Use of Taxon-Specific Quantitative PCR Assays
Authors: Fierer Noah , Jackson Jason A. , Vilgalys Rytas , Jackson Robert B.
Published: July 2005

Here we describe a quantitative PCR-based approach to estimating the relative abundances of major taxonomic groups of bacteria and fungi in soil. Primers were thoroughly tested for specificity, and the method was applied to three distinct soils. The technique provides a rapid and robust index of microbial community structure.
Impacts of Goethite Particles on UV Disinfection of Drinking Water
Authors: Wu Youxian , Clevenger Thomas , Deng Baolin
Published: July 2005

A unique association between bacterial cells and small goethite particles (∼0.2 by 2 μm) protected
Escherichia coli
and
Pseudomonas putida
from UV inactivation. The protection increased with the particle concentration in the turbidity range of 1 to 50 nephelometric turbidity units and with the bacterium-particle attachment time prior to UV irradiation. The lower degree of bacterial inactivation at longer attachment time was mostly attributed to the particle aggregation surrounding bacteria that provided shielding from UV radiation.
Colonic Microbiota Signatures across Five Northern European Countries
Authors: Lay Christophe , Rigottier-Gois Lionel , Holmstrøm Kim , Rajilic Mirjana , Vaughan Elaine E. , de Vos Willem M. , Collins Matthew D. , Thiel Ralph , Namsolleck Pawel , Blaut Michaël , Doré Joël
Published: July 2005

The composition of the colonic microbiota of 91 northern Europeans was characterized by fluorescent in situ hybridization using 18 phylogenetic probes. On average 75% of the bacteria were identified, and large interindividual variations were observed.
Clostridium coccoides
and
Clostridium leptum
were the dominant groups (28.0% and 25.2%), followed by the
Bacteroides
(8.5%). According to principal component analysis, no significant grouping with respect to geographic origin, age, or gender was observed.
Bov-tA Short Interspersed Nucleotide Element Sequences in Circulating Nucleic Acids from Sera of Cattle with Bovine Spongiform Encephalopathy (BSE) and Sera of Cattle Exposed to BSE
Authors: Schütz Ekkehard , Urnovitz Howard B. , Iakoubov Leonid , Schulz-Schaeffer Walter , Wemheuer Wilhelm , Brenig Bertram
Published: July 2005

Circulating nucleic acids (CNA) are known to be enriched in repetitive DNA sequences in humans. Here, bovine sera CNA were analyzed to determine if cell stress-related short interspersed nucleotide elements (SINEs) could be detected in sera from cattle associated with bovine spongiform encephalopathy (BSE). Nucleic acids were extracted, amplified, cloned, and sequenced from the sera of protease-resistant prion protein (PrPres)-positive cattle (n= 2) and sera from BSE-cohort cows (n= 6); 150 out of 163 clones revealed the presence of, on average, an 80-bp sequence from the 3′ region of Bov-tA SINE. A PCR protocol was developed that differentially identified SINE-associated CNA in BSE-exposed versus normal cattle. CNA were extracted from a serum vesicular fraction after controlled blood collection and processing procedures. Sera from four confirmed cases of BSE, 137 BSE-exposed cohort animals associated with eight confirmed BSE cases, and 845 healthy, PrPres-negative control cows were tested. All four sera from confirmed BSE cases were repeatedly reactive in the assay. BSE-exposed cohorts had a 100-fold higher occurrence of repeatedly reactive individuals per cohort (average = 63%; range = 33% to 91%), compared to healthy controls (average = 0.6%;P< 0.001). This study shows that BSE-confirmed and cohort animals possess a unique profile of SINE-associated serum CNA that can be utilized as a marker that highly correlates to BSE exposure.
Multivalent Group A Streptococcal Vaccine Elicits Bactericidal Antibodies against Variant M Subtypes
Authors: Dale James B. , Penfound Thomas , Chiang Edna Y. , Long Valerie , Shulman Stanford T. , Beall Bernard
Published: July 2005

Group A streptococci cause a wide spectrum of clinical illness. One of several strategies for vaccine prevention of these infections is based on the type-specific M protein epitopes. A multivalent M protein-based vaccine containing type-specific determinants from 26 different M serotypes is now in clinical trials. Recent epidemiologic studies have shown that, within some serotypes, the amino-terminal M protein sequence may show natural variation, giving rise to subtypes. This raises the possibility that vaccine-induced antibodies against the parent type may not be as effective in promoting bactericidal killing of variant subtypes. In the present study we used rabbit antisera against the 26-valent M protein-based vaccine in bactericidal tests against M1, M3, and M5 streptococci, which were represented by multiple subtypes. We show that the vaccine antibodies effectively promoted in vitro bactericidal activity despite the fact that the M proteins contained naturally occurring variant sequences in the regions corresponding to the vaccine sequence. Our results show that the variant M proteins generally do not result in significant differences in opsonization promoted by rabbit antisera raised against the 26-valent vaccine, suggesting that a multivalent M protein vaccine may not permit variant subtypes of group A streptococci to escape in a highly immunized population.
Evaluation of Inapparent Nosocomial Severe Acute Respiratory Syndrome Coronavirus Infection in Vietnam by Use of Highly Specific Recombinant Truncated Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay
Authors: Yu Fuxun , Le Mai Quynh , Inoue Shingo , Thai Hong Thi Cam , Hasebe Futoshi , del Carmen Parquet Maria , Morita Kouichi
Published: July 2005

Severe acute respiratory syndrome (SARS) is a recently emerged human disease associated with pneumonia. Inapparent infection with SARS coronavirus (CoV) is not well characterized. To develop a safe, simple, and reliable screening method for SARS diagnosis and epidemiological study, two recombinant SARS-CoV nucleocapsid proteins (N′ protein and NΔ
121
protein) were expressed in
Escherichia coli
, purified by affinity chromatography, and used as antigens for indirect, immunoglobulin G enzyme-linked immunosorbent assays (ELISA). Serum samples collected from healthy volunteers and SARS patients in Vietnam were used to evaluate the newly developed methods. The N′ protein-based ELISA showed a highly nonspecific reaction. The NΔ
121
protein-based ELISA, with a nonspecific reaction drastically reduced compared to that of the nearly-whole-length N′ protein-based ELISA, resulted in higher rates of positive reactions, higher titers, and earlier detection than the SARS-CoV-infected cell lysate-based ELISA. These results indicate that our newly developed SARS-CoV NΔ
121
protein-based ELISA is not only safe but also a more specific and more sensitive method to diagnose SARS-CoV infection and hence a useful tool for large-scale epidemiological studies. To identify inapparent SARS-CoV infections, serum samples collected from health care workers (HCWs) in Vietnam were screened by the NΔ
121
protein-based ELISA, and positive samples were confirmed by a virus neutralization test. Four out of 149 HCWs were identified to have inapparent SARS-CoV infection in Vietnam, indicating that subclinical SARS-CoV infection in Vietnam is rare but does exist.
Polyethylene Glycol-Conjugated Adenosine Deaminase (ADA) Therapy Provides Temporary Immune Reconstitution to a Child with Delayed-Onset ADA Deficiency
Authors: Lainka Elke , Hershfield Michael S. , Santisteban Ines , Bali Pawan , Seibt Annette , Neubert Jennifer , Friedrich Wilhelm , Niehues Tim
Published: July 2005

We describe the effects of polyethylene glycol-conjugated adenosine deaminase (ADA) replacement therapy on lymphocyte counts, activation, apoptosis, proliferation, and cytokine secretion in a 14-month-old girl with “delayed-onset” ADA deficiency and marked immunodysregulation. Pretreatment lymphopenia affected T cells (CD4, 150/μl; CD8, 459/μl), B cells (16/μl), and NK cells (55/μl). T cells were uniformly activated and largely apoptotic (CD4, 59%; CD8, 82%); and T-cell-dependent cytokine levels in plasma were elevated, including the levels of interleukin 2 (IL-2; 26 pg/ml), IL-4 (81 pg/ml), IL-5 (46 pg/ml), gamma interferon (1,430 pg/ml), tumor necrosis factor alpha (210 pg/ml), and IL-10 (168 pg/ml). Mitogen-stimulated peripheral blood mononuclear cells show reduced IL-2 secretion and proliferation. During the first 5 months of therapy there was clinical improvement and partial immune reconstitution, with nearly normal lymphocyte subset numbers, reduced T-cell activation and CD4-cell apoptosis, and decreased plasma cytokine levels. In parallel, IL-2 secretion and the lymphocyte mitogenic response improved. Between 4 and 7 months, immunoglobulin G antibodies to bovine ADA developed and resulted in the complete reversal of immune recovery.
Saccharomyces cerevisiae
Rab-GDI Displacement Factor Ortholog Yip3p Forms Distinct Complexes with the Ypt1 Rab GTPase and the Reticulon Rtn1p
Authors: Geng Jinming , Shin Marcus E. , Gilbert Penney M. , Collins Ruth N. , Burd Christopher G.
Published: July 2005

Rab GTPases are crucial regulators of organelle biogenesis, maintenance, and transport. Multiple Rabs are expressed in all cells, and each is localized to a distinct set of organelles, but little is known regarding the mechanisms by which Rabs are targeted to their resident organelles. Integral membrane proteins have been postulated to serve as receptors that recruit Rabs from the cytosol in a complex with the Rab chaperone, GDI, to facilitate the dissociation of Rab and GDI, hence facilitating loading of Rabs on membranes. We show here that the yeast (
Saccharomyces cerevisiae
) Golgi Rab GTPase Ypt1p can be copurified with the integral membrane protein Yip3p from detergent cell extracts. In addition, a member of the highly conserved reticulon protein family, Rtn1p, is also associated with Yip3p in vivo. However, Ypt1p did not copurify with Rtn1p, indicating that Yip3p is a component of at least two different protein complexes. Yip3p and Rtn1p are only partially colocalized in cells, with Yip3p localized predominantly to the Golgi and secondarily to the endoplasmic reticulum, whereas Rtn1p is localized predominantly to the endoplasmic reticulum and secondarily to the Golgi. Surprisingly, the intracellular localization of Rabs was not perturbed in
yip3
Δ or
rtn1
Δ mutants, suggesting that these proteins do not play a role in targeting Rabs to intracellular membranes. These data indicate that Yip3p may have multiple functions and that its interaction with Rabs is not critical for their recruitment to organelle membranes.
Release from Quorum-Sensing Molecules Triggers Hyphal Formation during
Candida albicans
Resumption of Growth
Authors: Enjalbert Brice , Whiteway Malcolm
Published: July 2005

Candida albicans
is a pathogenic fungus able to change morphology in response to variations in its growth environment. Simple inoculation of stationary cells into fresh medium at 37°C, without any other manipulations, appears to be a powerful but transient inducer of hyphal formation; this process also plays a significant role in classical serum induction of hyphal formation. The mechanism appears to involve the release of hyphal repression caused by quorum-sensing molecules in the growth medium of stationary-phase cells, and farnesol has a strong but incomplete role in this process. We used DNA microarray technology to study both the resumption of growth of
Candida albicans
cells and molecular regulation involving farnesol. Maintaining farnesol in the culture medium during the resumption of growth both delays and reduces the induction of hypha-related genes yet triggers expression of genes encoding drug efflux components. The persistence of farnesol also prevents the repression of histone genes during hyphal growth and affects the expression of putative or demonstrated morphogenesis-regulating cyclin genes, such as
HGC1
,
CLN3
, and
PCL2
. The results suggest a model explaining the triggering of hyphae in the host based on quorum-sensing molecules.
Functional Characterization of an Evolutionarily Distinct Phosphopantetheinyl Transferase in the Apicomplexan
Cryptosporidium parvum
Authors: Cai Xiaomin , Herschap Dustin , Zhu Guan
Published: July 2005

Recently, two types of fatty acid synthases (FASs) have been discovered from apicomplexan parasites. Although significant progress has been made in characterizing these apicomplexan FASs, virtually nothing was previously known about the activation and regulation of these enzymes. In this study, we report the discovery and characterization of two distinct types of phosphopantetheinyl transferase (PPTase) that are responsible for synthesizing holo-acyl carrier protein (ACP) from three apicomplexan parasites: surfactin production element (SFP) type in
Cryptosporidium parvum
(CpSFP-PPT), holo-ACP synthase (ACPS)-type in
Plasmodium falciparum
(PfACPS-PPT), and both SFP and ACPS types in
Toxoplasma gondii
(TgSFP-PPT and TgACPS-PPT). CpSFP-PPT and TgSFP-PPT are monofunctional, cytosolic, and phylogenetically related to animal PPTases. However, PfACPS-PPT and TgACPS-PPT are bifunctional (fused with a metal-dependent hydrolase), likely targeted to the apicoplast, and more closely related to proteobacterial PPTases. The function of apicomplexan PPTases has been confirmed by detailed functional analysis using recombinant CpSFP-PPT expressed from an artificially synthesized gene with codon usage optimized for
Escherichia coli
. The recombinant CpSFP-PPT was able to activate the ACP domains from the
C. parvum
type I FAS in vitro using either CoA or acetyl-CoA as a substrate, or in vivo when coexpressed in bacteria, with kinetic characteristics typical of PPTases. These observations suggest that the two types of fatty acid synthases in the Apicomplexa are activated and regulated by two evolutionarily distinct PPTases.
Autocrine, Mitogenic Pheromone Receptor Loop of the Ciliate
Euplotes raikovi
: Pheromone-Induced Receptor Internalization
Authors: Vallesi Adriana , Ballarini Patrizia , Di Pretoro Barbara , Alimenti Claudio , Miceli Cristina , Luporini Pierangelo
Published: July 2005

The ciliate
Euplotes raikovi
produces a family of diffusible signal proteins (pheromones) that function as prototypic growth factors. They may either promote cell growth, by binding to pheromone receptors synthesized by the same cells from which they are secreted (autocrine activity), or induce a temporary cell shift from the growth stage to a mating (sexual) one by binding to pheromone receptors of other, conspecific cells (paracrine activity). In cells constitutively secreting the pheromone E
r
-1, it was first observed that the expression of the E
r
-1 receptor “p15,” a type II membrane protein of 130 amino acids, is quantitatively correlated with the extracellular concentration of secreted pheromone. p15 expression on the cell surface rapidly and markedly increased after the removal of secreted E
r
-1 and gradually decreased in parallel with new E
r
-1 secretion. It was then shown that p15 is internalized through endocytic vesicles following E
r
-1 binding and that the internalization of p15/E
r
-1 complexes is specifically blocked by the paracrine p15 binding of E
r
-2, a pheromone structurally homologous to, and thus capable of fully antagonizing, E
r
-1. Based on previous findings that the p15 pheromone-binding site is structurally equivalent to E
r
-1 and that E
r
-1 molecules polymerize in crystals following a pattern of cooperative interaction, it was proposed that p15/E
r
-1 complexes are internalized as a consequence of their unique property (not shared by p15/E
r
-2 complexes) of undergoing clustering.
Deletion and Allelic Exchange of the
Aspergillus fumigatus veA
Locus via a Novel Recyclable Marker Module
Authors: Krappmann Sven , Bayram Özgür , Braus Gerhard H.
Published: July 2005

Detailed evaluation of gene functions in an asexual fungus requires advanced methods of molecular biology. For the generation of targeted gene deletions in the opportunistic pathogen
Aspergillus fumigatus
we designed a novel blaster module allowing dominant selection of transformants due to resistance to phleomycin as well as dominant (counter)selection of a Cre recombinase-mediated marker excision event. For validation purposes we have deleted the
A. fumigatus pabaA
gene in a wild-type isolate by making use of this cassette. The resulting
pabaA
::
loxP
strain served as the recipient for subsequent targeting of the
velvet
locus. Homologous reconstitution of the deleted gene was performed by an allele whose expression is driven in a nitrogen source-dependent manner, as validated by Northern analyses. Overexpression of the
veA
locus in
A. fumigatus
does not result in any obvious phenotype, whereas the sporulation capacities of the
veA
null mutant are reduced on nitrate-containing medium, a phenotype that is completely restored in the reconstituted strain.
WAVY WILSON LINE AND AdS/CFT
Authors: SEMENOFF GORDON W. , YOUNG DONOVAN
Published: July 2005

Wilson loops which are small deviations from straight, infinite lines, called wavy lines, are considered in the context of the AdS/CFT correspondence. A single wavy line and the connected correlation function of a straight and wavy line are considered. It is argued that, to leading order in "waviness," the functional form of the loop is universal and the coefficient, which is a function of the 't Hooft coupling, is found in weak coupling perturbation theory and the strong coupling limit using the AdS/CFT correspondence. Supersymmetric arguments are used to simplify the computation and to show that the straight line obeys the Migdal–Makeenko loop equation.
SYMMETRY OF MASSIVE RARITA–SCHWINGER FIELDS
Authors: PILLING TERRY
Published: July 2005

We derive the general Lagrangian and propagator for a vector-spinor field in d-dimensions and show that the physical observables are invariant under the so-called point transformation symmetry. Until now the symmetry has not been exploited in any nontrivial way, presumably because it is not an invariance of the classical action nor is it a gauge symmetry. Nevertheless, we develop a technique for exploring the consequences of the symmetry leading to a conserved vector current and charge. The current and charge are identically zero in the free field case and only contribute in a background such as a electromagnetic or gravitational field. The current can couple spin-[Formula: see text] fields to vector and scalar fields and may have important consequences in intermediate energy hadron physics as well as linearized supergravity. The consistency problem which plagues higher spin field theories is then discussed and some ideas regarding the possibility of solutions are presented.
MINIMIZING THE EFFECT OF SINUSOIDAL TRENDS IN DETRENDED FLUCTUATION ANALYSIS
Authors: NAGARAJAN RADHAKRISHNAN , KAVASSERI RAJESH G.
Published: July 2005

The detrended fluctuation analysis (DFA) [Peng et al., 1994] and its extensions (MF-DFA) [Kantelhardt et al., 2002] have been used extensively to determine possible long-range correlations in self-affine signals. While the DFA has been claimed to be a superior technique, recent reports have indicated its susceptibility to trends in the data. In this report, a smoothing filter is proposed to minimize the effect of sinusoidal trends and distortion in the log–log plots obtained by DFA and MF-DFA techniques.
BIFURCATIONS DUE TO SMALL TIME-LAG IN COUPLED EXCITABLE SYSTEMS
Authors: BURIĆ NIKOLA , TODOROVIĆ DRAGANA
Published: July 2005

A system of ODE's is used to attempt an approximation of the dynamics of two delayed coupled FitzHugh–Nagumo excitable units, described by delay-differential equations. It is shown that the codimension 2 generalized Hopf bifurcation acts as the organizing center for the dynamics of ODE's for small time-lags. Furthermore, this is used to explain important qualitative properties, like the phenomenon of oscillator death, of the exact dynamics for small time-delays.
BIFURCATIONS AND LIMIT DYNAMICS IN ADAPTIVE CONTROL SYSTEMS
Authors: LAMOOKI G. R. ROKNI , TOWNLEY S. , OSINGA H. M.
Published: July 2005

Adaptive controllers are used in systems where one or more parameters are unknown. Such controllers are designed to stabilize the system using an estimate for the unknown parameters that is adapted automatically as part of the stabilization. One drawback in adaptive control design is the possibility that the closed-loop limit system is not stable. The worst situation is the existence of a destabilized limit system attracting a large open subset of initial conditions.

These situations lie behind bad behavior of the closed-loop adaptive control system. The main issue in this paper is to identify and characterize the occurrence of such bad behavior in the adaptive stabilization of first- and second-order systems with one unknown parameter. We develop normal forms for all possible cases and find the conditions that lead to bad behavior. In this context, we discuss a number of bifurcation-like phenomena.
LOCAL ANALYSIS OF DISSIPATIVE DYNAMICAL SYSTEMS
Authors: NAGARAJAN RADHAKRISHNAN
Published: July 2005

Linear transformation techniques such as singular value decomposition (SVD) have been used widely to gain insight into the qualitative dynamics of data generated by dynamical systems. There have been several reports in the past that had pointed out the susceptibility of linear transformation approaches in the presence of nonlinear correlations. In this tutorial review, the local dispersion along with the surrogate testing is suggested to discriminate nonlinear correlations arising in deterministic and non-deterministic settings.
THE ROLE OF THE DIRECT TRANSFER MATRIX AS A CONNECTIVITY MATRIX AND APPLICATION TO THE HELMHOLTZ EQUATION IN 2D: RELATION TO NUMERICAL METHODS AND FREE FIELD RADIATION EXAMPLE
Authors: MAGRANS F. X. , GUASCH O.
Published: July 2005

The Direct Transfer Function (DTF) matrix was developed in the framework of the Global Transfer Direct Transfer (GTDT) method of transmission path analysis. This method aims at solving the problem of transmission paths among subsystems from a general N-dimensional linear network, representing a vibro-acoustical model under study. The DTF matrix can be calculated from the Global Transfer Functions (GTFs), which are measurable quantities, and it is built from all the Direct Transfer Functions (DTFs) between subsystem pairs. The DTFs allow to define transmission paths by relating the signals between two network subsystems when the remaining ones become somehow blocked.

In this paper, the role of the DTF matrix as a connectivity matrix is first shown by solving the Helmholtz equation in a two-dimensional grid. The results are compared with those arising from the analysis of the stencils of various numerical methods. Some finite difference and finite element methods have been considered. The connectivity role of the DTF matrix is also elucidated by means of a free field radiation example.
NUMERICAL SIMULATION OF EXTRACTING MODAL BACK-SCATTERING MATRIX FROM REVERBERATION IN SHALLOW-WATER WAVEGUIDE
Authors: PENG LINHUI , WANG NING , SHANG ER CHANG
Published: July 2005

Modal back-scattering matrix is the key component for reverberation predicting and also important for understanding the mechanism of bottom back-scattering. To extract the modal back-scattering matrix from reverberation data is a challening topic. So far, inversion of modal back-scattering matrix from reverberation data is based on some a priori assumptions. A new method for extracting modal back-scattering matrix from reverberation data in shallow-water waveguide is proposed recently [Shang et al., 2002]. In this paper numerical simulation of extracting the modal back-scattering matrix has been performed, it is shown that the inversion by changing the source depth works very well for lower frequency. Alternatively, the inversion by using the reverberation data at different ranges is proposed and it is shown that this approach is suitable on certain conditions.
DEPENDENCE OF THE AXIAL WAVE ON RANGE AND SOUND-SPEED PROFILE PROPERTIES IN A RANGE-INDEPENDENT OCEAN
Authors: GRIGORIEVA NATALIE S. , FRIDMAN GREGORY M.
Published: July 2005

For ducted propagation in a waveguide when the source and receiver are placed closely to the depth of the waveguide axis, there exist cusped caustics repeatedly along the axis. In neighborhoods of these cusped caustics, the interference of the wave fields that correspond to near-axial rays occurs. This results in the formation of a coherent structure (the axial wave) that propagates along the waveguide axis like a wave. In this paper the integral representation of the axial wave obtained before for an arbitrary waveguide in a two-dimensional range-independent medium is generalized to a three-dimensional range-independent medium. Through numerical simulation, the dependencies of the axial wave on range, sound-speed profile properties, and geometry of the experiment are studied for two sound-speed profiles: the average profile from the AET experiment and the Munk canonical profile. The sound source frequency is taken equal to 75 Hz; the propagation range is up to 3250 km. The strong difference between shapes of the axial wave for the average profile from the AET experiment and the Munk canonical profile is shown for all the examined models.
Spatiotemporal analysis of exocytosis in mouse parotid acinar cells
Authors: Chen Ying , Warner Jennifer D. , Yule David I. , Giovannucci David R.
Published: July 2005

Exocrine cells of the digestive system are specialized to secrete protein and fluid in response to neuronal and/or hormonal input. Although morphologically similar, parotid and pancreatic acinar cells exhibit important functional divergence in Ca2+ signaling properties. To address whether there are fundamental differences in exocytotic release of digestive enzyme from exocrine cells of salivary gland versus pancreas, we applied electrophysiological and optical methods to investigate spatial and temporal characteristics of zymogen-containing secretory granule fusion at the single-acinar cell level by direct or agonist-induced Ca2+ and cAMP elevation. Temporally resolved membrane capacitance measurements revealed that two apparent phases of exocytosis were induced by Ca2+ elevation: a rapidly activated initial phase that could not be resolved as individual fusion events and a second phase that was activated after a delay, increased in a staircaselike fashion, was augmented by cAMP elevation, and likely reflected both sequential compound and multivesicular fusion of zymogen-containing granules. Optical measurements of exocytosis with time-differential imaging analysis revealed that zymogen granule fusion was induced after a minimum delay of ∼200 ms, occurred initially at apical and basolateral borders of acinar cells, and under strong stimulation proceeded from apical pole to deeper regions of the cell interior. Zymogen granule fusions appeared to coordinate subsequent fusions and produced persistent structures that generally lasted several minutes. In addition, parotid gland slices were used to assess secretory dynamics in a more physiological context. Parotid acinar cells were shown to exhibit both similar and divergent properties compared with the better-studied pancreatic acinar cell regarding spatial organization and kinetics of exocytotic fusion of zymogen granules.