bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dissolved Organic Nitrogen Hydrolysis Rates in Axenic Cultures of
Aureococcus anophagefferens
(Pelagophyceae): Comparison with Heterotrophic Bacteria
Authors: Berg Gry Mine , Repeta Daniel J. , Laroche Julie 
Published: July 2002

ABSTRACT

The marine autotroph
Aureococcus anophagefferens
(Pelagophyceae) was rendered axenic in order to investigate hydrolysis rates of peptides, chitobiose, acetamide, and urea as indicators of the ability to support growth on dissolved organic nitrogen. Specific rates of hydrolysis varied between 8 and 700% of rates observed in associated heterotrophic marine bacteria.
Simultaneous Extraction from Bacterioplankton of Total RNA and DNA Suitable for Quantitative Structure and Function Analyses
Authors: Weinbauer Markus G. , Fritz Ingo , Wenderoth Dirk F. , Höfle Manfred G. 
Published: July 2002

ABSTRACT

The aim of this study was to develop a protocol for the simultaneous extraction from bacterioplankton of RNA and DNA suitable for quantitative molecular analysis. By using a combined mechanical and chemical extraction method, the highest RNA and DNA yield was obtained with sodium lauryl sarcosinate-phenol or DivoLab-phenol as the extraction mix. The efficiency of extraction of nucleic acids was comparatively high and varied only moderately in gram-negative bacterial isolates and bacterioplankton (RNA, 52 to 66%; DNA, 43 to 61%); significant amounts of nucleic acids were also obtained for a gram-positive bacterial isolate (RNA, 20 to 30%; DNA, 20 to 25%). Reverse transcription-PCR and PCR amplification products of fragments of 16S rRNA and its genes were obtained from all isolates and communities, indicating that the extracted nucleic acids were intact and pure enough for community structure analyses. By using single-strand conformation polymorphism of fragments of 16S rRNA and its gene, community fingerprints were obtained from pond bacterioplankton. mRNA transcripts encoding fragments of the enzyme nitrite reductase gene (
nir
gene) could be detected in a pond water sample, indicating that the extraction method is also suitable for studying gene expression. The extraction method presented yields nucleic acids that can be used to perform structural and functional studies of bacterioplankton communities from a single sample.
Diversity, Dynamics, and Activity of Bacterial Communities during Production of an Artisanal Sicilian Cheese as Evaluated by 16S rRNA Analysis
Authors: Randazzo Cinzia L. , Torriani Sandra , Akkermans Antoon D. L. , de Vos Willem M. , Vaughan Elaine E. 
Published: July 2002

ABSTRACT

The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and culture-independent approaches. The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA). Bacterial and
Lactobacillus
group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene, respectively. DGGE profiles from samples taken during cheese production indicated dramatic shifts in the microbial community structure. Cloning and sequencing of rDNA amplicons revealed that mesophilic lactic acid bacteria (LAB), including species of
Leuconostoc
,
Lactococcus lactis
, and
Macrococcus caseolyticus
were dominant in the raw milk, while
Streptococcus thermophilus
prevailed during lactic fermentation. Other thermophilic LAB, especially
Lactobacillus delbrueckii
and
Lactobacillus fermentum
, also flourished during ripening. Comparison of the rRNA-derived patterns obtained by RT-PCR to the rDNA DGGE patterns indicated a substantially different degree of metabolic activity for the microbial groups detected. Identification of cultivated LAB isolates by phenotypic characterization and 16S rDNA analysis indicated a variety of species, reflecting to a large extent the results obtained from the 16S rDNA clone libraries, with the significant exception of the
Lactobacillus delbrueckii
species, which dominated in the ripening cheese but was not detected by cultivation. The present molecular approaches combined with culture can effectively describe the complex ecosystem of natural fermented dairy products, giving useful information for starter culture design and preservation of artisanal fermented food technology.
Development and Characterization of a Green Fluorescent Protein-Based Bacterial Biosensor for Bioavailable Toluene and Related Compounds
Authors: Stiner Lawrence , Halverson Larry J. 
Published: July 2002

ABSTRACT

A green fluorescent protein-based
Pseudomonas fluorescens
strain A506 biosensor was constructed and characterized for its potential to measure benzene, toluene, ethylbenzene, and related compounds in aqueous solutions. The biosensor is based on a plasmid carrying the toluene-benzene utilization (
tbu
) pathway transcriptional activator TbuT from
Ralstonia pickettii
PKO1 and a transcriptional fusion of its promoter
PtbuA1
with a promoterless
gfp
gene on a broad-host-range promoter probe vector. TbuT was not limiting, since it was constitutively expressed by being fused to the neomycin phosphotransferase (
nptII
) promoter. The biosensor cells were readily induced, and fluorescence emission after induction periods of 3 h correlated well with toluene, benzene, ethylbenzene, and trichloroethylene concentrations. Our experiments using flow cytometry show that intermediate levels of
gfp
expression in response to toluene reflect uniform induction of cells. As the toluene concentration increases, the level of
gfp
expression per cell increases until saturation kinetics of the TbuT-PtbuA1 system are observed. Each inducer had a unique minimum concentration that was necessary for induction, with
K
app
values that ranged from 3.3 ± 1.8 μM for toluene to 35.6 ± 16.6 μM for trichloroethylene (means ± standard errors of the means), and maximal fluorescence response. The fluorescence response was specific for alkyl-substituted benzene derivatives and branched alkenes (di- and trichloroethylene, 2-methyl-2-butene). The biosensor responded in an additive fashion to the presence of multiple inducers and was unaffected by the presence of compounds that were not inducers, such as those present in gasoline. Flow cytometry revealed that, in response to toxic concentrations of gasoline, there was a small uninduced population and another larger fully induced population whose levels of fluorescence corresponded to the amount of effectors present in the sample. These results demonstrate the potential for green fluorescent protein-based bacterial biosensors to measure environmental contaminants.
Fusarium Tri8
Encodes a Trichothecene C-3 Esterase
Authors: McCormick Susan P. , Alexander Nancy J. 
Published: July 2002

ABSTRACT

Mutant strains of
Fusarium graminearum
Z3639 produced by disruption of
Tri8
were altered in their ability to biosynthesize 15-acetyldeoxynivalenol and instead accumulated 3,15-diacetyldeoxynivalenol, 7,8-dihydroxycalonectrin, and calonectrin.
Fusarium sporotrichioides
NRRL3299
Tri8
mutant strains accumulated 3-acetyl T-2 toxin, 3-acetyl neosolaniol, and 3,4,15-triacetoxyscirpenol rather than T-2 toxin, neosolaniol, and 4,15-diacetoxyscirpenol. The accumulation of these C-3-acetylated compounds suggests that
Tri8
encodes an esterase responsible for deacetylation at C-3. This gene function was confirmed by cell-free enzyme assays and feeding experiments with yeast expressing
Tri8.
Previous studies have shown that
Tri101
encodes a C-3 transacetylase that acts as a self-protection or resistance factor during biosynthesis and that the presence of a free C-3 hydroxyl group is a key component of
Fusarium
trichothecene phytotoxicity. Since
Tri8
encodes the esterase that removes the C-3 protecting group, it may be considered a toxicity factor.
Distribution of Membrane Lipids of Planktonic
Crenarchaeota
in the Arabian Sea
Authors: Sinninghe Damsté Jaap S. , Rijpstra W. Irene C. , Hopmans Ellen C. , Prahl Fredrick G. , Wakeham Stuart G. , Schouten Stefan 
Published: July 2002

ABSTRACT

Intact core tetraether membrane lipids of marine planktonic
Crenarchaeota
were quantified in water column-suspended particulate matter obtained from four depth intervals (∼70, 500, 1,000 and 1,500 m) at seven stations in the northwestern Arabian Sea to investigate the distribution of the organisms at various depths. Maximum concentrations generally occurred at 500 m, near the top of the oxygen minimum zone, and the concentrations at this depth were, in most cases, slightly higher than those in surface waters. In contrast, lipids derived from eukaryotes (cholesterol) and from eukaryotes and bacteria (fatty acids) were at their highest concentrations in surface waters. This indicates that these crenarchaeotes are not restricted to the photic zone of the ocean, which is consistent with the results of recent molecular biological studies. Since the Arabian Sea has a strong oxygen minimum zone between 100 and 1,000 m, with minimum oxygen levels of <1 μM, the abundance of crenarchaeotal membrane lipids at 500 m suggests that planktonic
Crenarchaeota
are probably facultative anaerobes. The cell numbers we calculated from the concentrations of membrane lipids are similar to those reported for the Central Pacific Ocean, supporting the recent estimation of M. B. Karner, E. F. DeLong, and D. M. Karl (

Nature
409
:
507
-510,
2001

) that the world's oceans contain ca. 10
28
cells of planktonic
Crenarchaeota
.
Diversity of Hepatitis C Virus Quasispecies Evaluated by Denaturing Gradient Gel Electrophoresis
Authors: Harris K. A. , Teo C. G. 
Published: July 2002

ABSTRACT
Denaturing gradient gel electrophoresis (DGGE) was used to study the diversity of hepatitis C virus (HCV) quasispecies. Optimized DGGE running conditions were applied to screen for variations in sequences cloned from amplicons originating from the nonstructural 5b (NS5b) gene of HCV in blood of hemophilia patients, intravenous drug users, and blood donors (five specimens from each study group, ca. 40 clones studied per specimen). Clones identified by DGGE as unique were sequenced. NS5b sequence entropy and mean genetic distance in hemophiliacs did not differ significantly from those in the other groups, pointing to a lack of correlation between HCV diversity and the multiplicity of past HCV exposures. DGGE was also applied to investigate variation in the HCV envelope 2/hypervariable region 1 (E2/HVR-1) in serum samples serially taken from two patients during the seroconversion phase of HCV infection. E2/HVR-1 sequence entropy changes were small and not correlated with rising anti-HCV antibody levels, reflecting mutational changes not mediated by antibody selection.
Human T-Cell Responses to the Glucosyltransferases ofStreptococcus mutans
Authors: Chia Jean-San , You Chiou-Mien , Hu Chung-Yi , Chiang Bor-Luen , Chen Jen-Yang 
Published: July 2002

ABSTRACTWe previously reported differential humoral responses to glucosyltransferases (GTFs), with significantly higher saliva and serum antibody levels to GtfD than to GtfB or GtfC. To test the hypothesis that cellular immune responses to these molecules also may differ, peripheral blood mononuclear cell (PBMC) and T-cell proliferative responses in young adults and children with distinct genetic backgrounds were determined using purified recombinant GtfC and GtfD. PBMCs from all of the volunteers responded to GtfC and -D, but responses were directed predominantly towards GtfD and were major histocompatibility class II antigen dependent. A predominant T-cell response to GtfD, over GtfC, was detectable at various antigen concentrations ranging from 1 to 20 μg/ml and correlated with the differential serum immunoglobulin G (IgG) and salivary IgA antibody responses to the GTFs. Therefore, in naturally sensitized humans,Streptococcus mutansGTFs stimulate differential humoral and cellular immune responses, with the secreted form of GtfD eliciting a stronger response than the cell wall-associated form of GtfC.
Antigen-Driven T-Cell Selection in Patients with Cervical Cancer as Evidenced by T-Cell Receptor Analysis and Recognition of Autologous Tumor
Authors: Pilch H. , Höhn H. , Neukirch C. , Freitag K. , Knapstein P. G. , Tanner B. , Maeurer M. J. 
Published: July 2002

ABSTRACTWe characterized the T-cell receptor (TCR) repertoire in freshly harvested tumor lesions, in short-term-expanded CD4+tumor infiltrating lymphocytes (TIL) as well as in CD4+and CD8+peripheral blood lymphocytes (PBL) from three patients with cervical cancer. Skewing of the T-cell repertoire as defined by measuring the length of the complementarity-determining region 3 (CDR3) of the TCR VA and VB chains was observed in CD8+PBL, in freshly harvested tumor tissue, as well as in CD4+TIL. Comparative analysis of the TCR repertoire revealed unique monoclonal TCR transcripts within the tumor lesion which were not present in PBL, suggesting selection of TCR clonotypes due to antigenic stimulation. TCR repertoire analysis of the short-term (7-day) CD4+TIL lines revealed that the TCR composition is markedly different from that in CD4+PBL or in the freshly harvested tumor tissue. Only one-third of CD4+TIL lines showed HLA-DR-restricted recognition of autologous tumor cells as defined by cytolysis. These data provide support for the antigen-driven selection of T cells within cervical cancer lesions and suggest that analysis of the TCR repertoire may aid in obtaining an objective description of the immune response in patients with cervical cancer who are undergoing epitope-based immunotherapy.
Endotoxin-Induced Gamma Interferon Production: Contributing Cell Types and Key Regulatory Factors
Authors: Varma Tushar K. , Lin Cheng Y. , Toliver-Kinsky Tracy E. , Sherwood Edward R. 
Published: July 2002

ABSTRACTGamma interferon (IFN-γ) is an important mediator of endotoxin (lipopolysaccharide [LPS])-induced immune responses. However, the specific cell types that produce IFN-γ in response to LPS and the cellular factors that regulate LPS-induced IFN-γ production have not been fully determined. The present studies were undertaken to characterize the cell populations that produce IFN-γ after LPS challenge in the spleens of mice and to determine the regulatory factors that modulate LPS-induced production of IFN-γ. Our studies show that the levels of splenic IFN-γ mRNA and protein production peak at 6 and 8 h, respectively, after systemic LPS challenge. Approximately 60% of IFN-γ-producing cells are natural killer (NK) cells (CD3−DX5+) and 25% are NKT cells (CD3+DX5+). Most of the remaining IFN-γ-producing cells are T cells (CD3+DX5−), macrophages, and dendritic cells. Functionally, interleukin-12 (IL-12) is the major IFN-γ-stimulating factor after LPS challenge, with costimulation provided by IL-15, IL-18, and B7 proteins. IL-10 is a major inhibitor of LPS-induced IFN-γ production. Unlike intact heat-killed gram-negative and gram-positive bacteria, the class II major histocompatibility complex did not play a functional role in LPS-induced IFN-γ production. LPS is a potent stimulus for splenic IL-10, IL-12 p40, and IL-15 mRNA expression, whereas IL-12 p35 and IL-18 mRNAs, as well as B7 proteins, are constitutively expressed in the mouse spleen. Of the factors studied, IL-18 serves as the most potent costimulus with IL-12 for IFN-γ production, followed by IL-15 and B7 proteins. These data demonstrate that NK cells and NKT cells are the most abundant IFN-γ-producing cells in the mouse spleen after LPS challenge and that IL-10 and IL-12 are key functional regulators of LPS-induced IFN-γ production.
Comparison of O-Antigen Gene Clusters of
Escherichia coli
(
Shigella
) Sonnei and
Plesiomonas shigelloides
O17: Sonnei Gained Its Current Plasmid-Borne O-Antigen Genes from
P. shigelloides
in a Recent Event
Authors: Shepherd James G. , Wang Lei , Reeves Peter R. 
Published: July 2002

ABSTRACT

Escherichia coli
Sonnei has an O antigen identical to that of
Plesiomonas shigelloides
O17, and its O-antigen gene cluster is located on a plasmid. By sequencing the chromosomal O-antigen gene cluster of
P. shigelloides
O17 and comparing it with that of Sonnei, we showed that Sonnei gained its O-antigen genes recently.
Generation of a Recombinant 65-Kilodalton Mannoprotein, a Major Antigen Target of Cell-Mediated Immune Response toCandida albicans
Authors: La Valle Roberto , Sandini Silvia , Gomez Maria Jesus , Mondello Francesca , Romagnoli Giulia , Nisini Roberto , Cassone Antonio 
Published: July 2002

ABSTRACTA 65-kDa mannoprotein (CaMp65) has long been studied as a major, immunodominant antigen of the human opportunistic pathogenCandida albicans. An expression library ofC. albicanswas screened with serum from mice immunized with ScMp65 (ScW10), aSaccharomyces cerevisiaerecombinant protein of about 48 kDa. This serum recognized the CaMp65 from a cell wall extract ofC. albicans. After cloning and sequencing of the relevantC. albicanscDNA, an open reading frame encoding a protein of 379 amino acids was identified. Its deduced amino acid sequence showed regions of identity with all previously characterized tryptic fragments of CaMp65, as well as with the corresponding regions of ScMp65. A prepeptide of 32 amino acids with signal peptidase and Kex2 cleavage sites as well as a high number of potential O-glycosylation sites but no N-glycosylation sites or GPI anchor were observed in sequence studies of CaMp65. A putative adhesin RGD sequence was also present in the C-terminal region of the molecule. This triplet was absent in the ScMp65. The relevant gene (designatedCaMP65) was localized to chromosome R ofC. albicansas determined by pulse-field gel electrophoresis. Northern blot analysis demonstrated that gene transcription was heat inducible and associated with germ-tube formation by the fungus. A recombinant, His6-tagged protein (rCaMp65) was expressed inEscherichia coliunder an inducible promoter. After purification by nickel-chelate affinity chromatography, the recombinant product was detected as a 47-kDa protein band in immunoblots with the anti-ScMp65 serum, as well as with CaMp65-specific monoclonal antibodies. Both ScMp65 and CaMp65 were assayed for antigenic stimulation in cultures of peripheral blood mononuclear cells (PBMC) from 10 unselected human donors. While ScMp65 was substantially nonstimulatory, both rCaMp65 and the native CaMp65 were equally able to induce lymphoproliferation of the PBMC from all the donors. In addition, a number of CD4+T-cell clones were generated using aC. albicansmannoprotein fraction as an antigenic stimulant. Several of these clones specifically responded to both the native and the recombinantC. albicansMp65 but not to ScMp65. Thus, the recombinant Mp65 ofC. albicansretains antigenicity and, as such, could be a valid, standardized reagent for serodiagnostic and immunological studies.
Modulation of Cytokine Profiles by the Mycoplasma SuperantigenMycoplasma arthritidisMitogen Parallels Susceptibility to Arthritis Induced byM. arthritidis
Authors: Mu Hong-Hua , Sawitzke Allen D. , Cole Barry C. 
Published: July 2002

ABSTRACTMycoplasma arthritidismitogen (MAM) is a potent superantigen secreted byM. arthritidis, an agent of murine arthritis. Here we compare the abilities of MAM to induce a panel of cytokines in vitro and in vivo in BALB/c and C3H/HeJ mouse strains that differ in susceptibility to mycoplasmal arthritis. Splenocytes from both mouse strains produced high levels of all cytokines by 24 h following in vitro exposure to MAM. No differences in cytokine profiles were seen irrespective of the MAM dose. However, there were striking differences in cytokine profiles present in supernatants of splenocytes that had been collected from mice after intravenous (i.v.) injection of MAM and subsequently rechallenged with MAM in vitro. Splenocytes collected 24 and 72 h after i.v. injection of MAM and challenged in vitro with MAM showed the most marked divergence in the secreted cytokines. Type 1 cytokines were markedly elevated in C3H/HeJ cell supernatants, whereas they were depressed or remained low in BALB/c cell supernatants. In contrast, the levels of type 2 cytokines were all greatly increased in BALB/c cell cultures but were decreased or remained low in C3H/HeJ supernatants. Interleukin-12 mRNA and protein was also markedly elevated in C3H/HeJ mice, as were the levels of immunoglobulin G2a. The data indicate a major skewing in cytokine profiles to a type 1 inflammatory response in C3H/HeJ mice but to a protective type 2 response in BALB/c mice. These cytokine changes appear to be associated with the severe arthritis in C3H/HeJ mice following injection ofM. arthritidisin comparison to the mild disease seen in injected BALB/c mice.
Genetic Resistance to Experimental Infection with
Mycobacterium bovis
in Red Deer (
Cervus elaphus
)
Authors: Mackintosh Colin G. , Qureshi Tariq , Waldrup Ken , Labes Robert E. , Dodds Ken G. , Griffin J. Frank T. 
Published: July 2002

ABSTRACT

Tuberculosis (Tb) caused by
Mycobacterium bovis
is a worldwide threat to livestock and humans. One control strategy is to breed livestock that are more resistant to
Mycobacterium bovis
. In a 3-year heritability study 6 farmed red deer stags were selected from 39 on the basis of their differing responses to experimental challenge via the tonsillar sac with approximately 500 CFU of
M. bovis
. Two stags remained uninfected, two were moderately affected, and two developed serious spreading Tb. Seventy offspring, bred from these six stags by artificial insemination using stored semen, were similarly challenged with
M. bovis
. The offspring showed patterns of response to
M. bovis
challenge similar to those of their sires, providing evidence for a strong genetic basis to resistance to Tb, with an estimated heritability of 0.48 (standard error, 0.096;
P
< 0.01). This is the first time the heritability of Tb resistance in domestic livestock has been measured. The breeding of selection lines of resistant and susceptible deer will provide an ideal model to study the mechanisms of Tb resistance in a ruminant and could provide an additional strategy for reducing the number and severity of outbreaks of Tb in farmed deer herds. Laboratory studies to identify genetic and immunological markers for resistance to Tb are under way. Preliminary studies showed no associations between NRAMP or DRB genes and resistance to Tb in deer. Patterns of immune responses seen in resistant animals suggest that both innate and acquired pathways of immunity are necessary to produce the resistant phenotype.
Enhanced Hematopoietic Activity Accompanies Parasite Expansion in the Spleen and Bone Marrow of Mice Infected withLeishmania donovani
Authors: Cotterell Sara E. J. , Engwerda Christian R. , Kaye Paul M. 
Published: July 2002

ABSTRACTIn this study, we have analyzed hematopoietic activity in the spleen, bone marrow, and blood of BALB/c andscidmice infected withLeishmania donovani.Our analysis demonstrates that infection induces a rapid but transient mobilization of progenitor cells into the circulation, associated with elevated levels of granulocyte/macrophage colony-stimulating factor (GM-CSF) and MIP-1α. From 14 to 28 days postinfection, when parasite expansion begins in the spleen and bone marrow, both the frequency and cell cycle activity of hematopoietic progenitors, particulary CFU-granulocyte, monocyte, are dramatically increased in these organs. This is associated with increased accumulation of mRNA for GM-CSF, M-CSF, and G-CSF, but not interleukin-3. Our data also illustrate that hematopoietic activity, as assessed by changes in the frequency of progenitor cell populations and their levels of cell cycle activity, can be regulated in both a T-cell-independent and T-cell-dependent, as well as in an organ-specific, manner. Collectively, these data add to our knowledge of the long-term changes which occur in organs in whichL. donovaniis able to persist.