bluuebunny/crossref_metadata_2025_split huggingface dataset:
Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence
Authors: Kannan Yuji , Koga Yuichi , Inoue Yohei , Haruki Mitsuru , Takagi Masahiro , Imanaka Tadayuki , Morikawa Masaaki , Kanaya Shigenori 
Published: July 2002

ABSTRACT

The gene encoding subtilisin-like protease
T. kodakaraensis
subtilisin was cloned from a hyperthermophilic archaeon
Thermococcus kodakaraensis
KOD1.
T. kodakaraensis
subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins,
T. kodakaraensis
subtilisin was overproduced in
Escherichia coli
in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding,
T. kodakaraensis
subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant
T. kodakaraensis
subtilisin which is composed of 398 amino acid residues (Gly
−82
to Gly
316
), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that
T. kodakaraensis
subtilisin exists in a monomeric form.
T. kodakaraensis
subtilisin hydrolyzed the synthetic substrate
N
-succinyl-Ala-Ala-Pro-Phe-
p
-nitroanilide only in the presence of the Ca
2+
ion with an optimal pH and temperature of pH 9.5 and 80°C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80°C, 20 min at 90°C, and 7 min at 100°C.
Detection of
Lactobacillus, Pediococcus, Leuconostoc
, and
Weissella
Species in Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel Electrophoresis
Authors: Walter Jens , Hertel Christian , Tannock Gerald W. , Lis Claudia M. , Munro Karen , Hammes Walter P. 
Published: July 2002

ABSTRACT

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera
Lactobacillus, Pediococcus, Leuconostoc
, and
Weissella
in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as
Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides
, and
Pediococcus pentosaceus
. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain
Lactobacillus rhamnosus
DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that
L. rhamnosus
was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects.
The Functional
ccpA
Gene Is Required for Carbon Catabolite Repression in
Lactobacillus plantarum
Authors: Muscariello Lidia , Marasco Rosangela , De Felice Maurilio , Sacco Margherita 
Published: July 2002

ABSTRACT

We report the characterization of the
ccpA
gene of
Lactobacillus plantarum
, coding for catabolite control protein A. The gene is linked to the
pepQ
gene, encoding a proline peptidase, in the order
ccpA-pepQ
, with the two genes transcribed in tandem from the same strand as distinct transcriptional units. Two
ccpA
transcription start sites corresponding to two functional promoters were found, expression from the upstream promoter being autogenously regulated through a catabolite-responsive element (
cre
) sequence overlapping the upstream +1 site. During growth on ribose, the upstream promoter showed maximal expression, while growth on glucose led to transcription from the downstream promoter. In a
ccpA
mutant strain, the gene was transcribed mainly from the upstream promoter in both repressing and non repressing conditions. Expression of two enzyme activities, β-glucosidase and β-galactosidase, was relieved from carbon catabolite repression in the
ccpA
mutant strain. In vivo footprinting analysis of the catabolite-controlled
bglH
gene regulatory region in the
ccpA
mutant strain showed loss of protection of the
cre
under repressing conditions.
Effect of Turbulent-Flow Pasteurization on Survival ofMycobacterium aviumsubsp.paratuberculosisAdded to Raw Milk
Authors: Pearce Lindsay E. , Truong H. Tuan , Crawford Robert A. , Yates Gary F. , Cavaignac Sonia , de Lisle Geoffrey W. 
Published: July 2002

ABSTRACTA pilot-scale pasteurizer operating under validated turbulent flow (Reynolds number, 11,050) was used to study the heat sensitivity ofMycobacterium aviumsubsp.paratuberculosisadded to raw milk. The ATCC 19698 type strain, ATCC 43015 (Linda, human isolate), and three bovine isolates were heated in raw whole milk for 15 s at 63, 66, 69, and 72°C in duplicate trials. No strains survived at 72°C for 15 s; and only one strain survived at 69°C. Means of pooledDvalues (decimal reduction times) at 63 and 66°C were 15.0 ± 2.8 s (95% confidence interval) and 5.9 ± 0.7 s (95% confidence interval), respectively. The mean extrapolatedD72°Cwas <2.03 s. This was equivalent to a >7 log10kill at 72°C for 15 s (95% confidence interval). The meanZvalue (degrees required for the decimal reduction time to traverse one log cycle) was 8.6°C. These five strains showed similar survival whether recovery was on Herrold's egg yolk medium containing mycobactin or by a radiometric culture method (BACTEC). Milk was inoculated with fresh fecal material from a high-level fecal shedder with clinical Johne's disease. After heating at 72°C for 15 s, the minimumM. aviumsubsp.paratuberculosiskill was >4 log10. Properly maintained and operated equipment should ensure the absence of viableM. aviumsubsp.paratuberculosisin retail milk and other pasteurized dairy products. An additional safeguard is the widespread commercial practice of pasteurizing 1.5 to 2° above 72°C.
Two-Dimensional Electrophoresis Study of
Lactobacillus delbrueckii
subsp.
bulgaricus
Thermotolerance
Authors: Gouesbet Gwenola , Jan Gwenael , Boyaval Patrick 
Published: July 2002

ABSTRACT

The response of
Lactobacillus delbrueckii
subsp.
bulgaricus
cells to heat stress was studied by use of a chemically defined medium. Two-dimensional electrophoresis (2-DE) analysis was used to correlate the kinetics of heat shock protein (HSP) induction with cell recovery from heat injury. We demonstrated that enhanced viability, observed after 10 min at 65°C, resulted from the overexpression of HSP and from mechanisms not linked to protein synthesis. In order to analyze the thermoadaptation mechanisms involved, thermoresistant variants were selected. These variants showed enhanced constitutive tolerance toward heat shock. However, contrary to the wild-type strain, these variants were poorly protected after osmotic or heat pretreatments. This result suggests that above a certain threshold, cells reach a maximum level of protection that cannot be easily exceeded. A comparison of protein patterns showed that the variants were able to induce more rapidly their adaptive mechanisms than the original strain. In particular, the variants were able to express constitutively more HSP, leading to the higher level of thermoprotection observed. This is the first report of the study by 2-DE of the heat stress response in
L. delbrueckii
subsp.
bulgaricus
.
Temporal Changes in Archaeal Diversity and Chemistry in a Mid-Ocean Ridge Subseafloor Habitat
Authors: Huber Julie A. , Butterfield David A. , Baross John A. 
Published: July 2002

ABSTRACT

The temporal variation in archaeal diversity in vent fluids from a midocean ridge subseafloor habitat was examined using PCR-amplified 16S rRNA gene sequence analysis and most-probable-number (MPN) cultivation techniques targeting hyperthermophiles. To determine how variations in temperature and chemical characteristics of subseafloor fluids affect the microbial communities, we performed molecular phylogenetic and chemical analyses on diffuse-flow vent fluids from one site shortly after a volcanic eruption in 1998 and again in 1999 and 2000. The archaeal population was divided into particle-attached (>3-μm-diameter cells) and free-living fractions to test the hypothesis that subseafloor microorganisms associated with active hydrothermal systems are adapted for a lifestyle that involves attachment to solid surfaces and formation of biofilms. To delineate between entrained seawater archaea and the indigenous subseafloor microbial community, a background seawater sample was also examined and found to consist only of Group I
Crenarchaeota
and Group II
Euryarchaeota
, both of which were also present in vent fluids. The indigenous subseafloor archaeal community consisted of clones related to both mesophilic and hyperthermophilic
Methanococcales
, as well as many uncultured
Euryarchaeota
, some of which have been identified in other vent environments. The particle-attached fraction consistently showed greater diversity than the free-living fraction. The fluid and MPN counts indicate that while culturable hyperthermophiles represent less than 1% of the total microbial community, the subseafloor at new eruption sites does support a hyperthermophilic microbial community. The temperature and chemical indicators of the degree of subseafloor mixing appear to be the most important environmental parameters affecting community diversity, and it is apparent that decreasing fluid temperatures correlated with increased entrainment of seawater, decreased concentrations of hydrothermal chemical species, and increased incidence of seawater archaeal sequences.
Effect of Growth Conditions and Staining Procedure upon the Subsurface Transport and Attachment Behaviors of a Groundwater Protist
Authors: Harvey Ronald W. , Mayberry Naleen , Kinner Nancy E. , Metge David W. , Novarino Franco 
Published: July 2002

ABSTRACT

The transport and attachment behaviors of
Spumella guttula
(Kent), a nanoflagellate (protist) found in contaminated and uncontaminated aquifer sediments in Cape Cod, Mass., were assessed in flowthrough and static columns and in a field injection-and-recovery transport experiment involving an array of multilevel samplers. Transport of
S. guttula
harvested from low-nutrient (10 mg of dissolved organic carbon per liter), slightly acidic, granular (porous) growth media was compared to earlier observations involving nanoflagellates grown in a traditional high-nutrient liquid broth. In contrast to the highly retarded (retardation factor of ∼3) subsurface transport previously reported for
S. guttula
, the peak concentration of porous-medium-grown
S. guttula
traveled concomitantly with that of a conservative (bromide) tracer. About one-third of the porous-medium-grown nanoflagellates added to the aquifer were transported at least 2.8 m downgradient, compared to only ∼2% of the broth-grown nanoflagellates. Flowthrough column studies revealed that a vital (hydroethidine [HE]) staining procedure resulted in considerably less attachment (more transport) of
S. guttula
in aquifer sediments than did a staining-and-fixation procedure involving 4′,6′-diamidino-2-phenylindole (DAPI) and glutaraldehyde. The calculated collision efficiency (∼10
−2
for porous-medium-grown, DAPI-stained nanoflagellates) was comparable to that observed earlier for the indigenous community of unattached groundwater bacteria that serve as prey. The attachment of HE-labeled
S. guttula
onto aquifer sediment grains was independent of pH (over the range from pH 3 to 9) suggesting a primary attachment mechanism that may be fundamentally different from that of their prey bacteria, which exhibit sharp decreases in fractional attachment with increasing pH. The high degree of mobility of
S. guttula
in the aquifer sediments has important ecological implications for the protistan community within the temporally changing plume of organic contaminants in the Cape Cod aquifer.
Identification of NovelHelicobacterspp. from a Beluga Whale
Authors: Harper Claudia M. G. , Xu Shilu , Feng Yan , Dunn J. Lawrence , Taylor Nancy S. , Dewhirst Floyd E. , Fox James G. 
Published: July 2002

ABSTRACTThe gastric fluid and feces of three belugas from the Mystic Aquarium were assessed for the presence ofHelicobacterspp. Gastric fluid and feces from the two clinically healthy belugas were negative for helicobacter, and endoscopy performed on these animals revealed no lesions. However, a helicobacter isolate and PCR product similar to helicobacter strains previously recovered from dolphins were identified, respectively, from the feces and gastric fluid of a beluga manifesting intermittent inappetence and lethargy. Esophageal and forestomach ulcers were noted on endoscopy. This is the first report of novelHelicobacterspp. being identified from whales.
Novel Plant-Microbe Rhizosphere Interaction Involving
Streptomyces lydicus
WYEC108 and the Pea Plant (
Pisum sativum
)
Authors: Tokala Ranjeet K. , Strap Janice L. , Jung Carina M. , Crawford Don L. , Salove Michelle Hamby , Deobald Lee A. , Bailey J. Franklin , Morra M. J. 
Published: July 2002

ABSTRACT

A previously undescribed plant-microbe interaction between a root-colonizing
Streptomyces
species,
S. lydicus
WYEC108, and the legume
Pisum sativum
is described. The interaction is potentially of great importance to the health and growth in nature of this nodulating legume. The root-colonizing soil actinomycete
S. lydicus
WYEC108 influences pea root nodulation by increasing root nodulation frequency, possibly at the level of infection by
Rhizobium
spp.
S. lydicus
also colonizes and then sporulates within the surface cell layers of the nodules. Colonization leads to an increase in the average size of the nodules that form and improves the vigor of bacteroids within the nodules by enhancing nodular assimilation of iron and possibly other soil nutrients. Bacteroid accumulation of the carbon storage polymer, poly-β-hydroxybutyrate, is reduced in colonized nodules. Root nodules of peas taken from agricultural fields in the Palouse hills of northern Idaho were also found to be colonized by actinomycete hyphae. We hypothesize that root and nodule colonization is one of several mechanisms by which
Streptomyces
acts as a naturally occurring plant growth-promoting bacterium in pea and possibly other leguminous plants.
Salmonella entericaInfections in Market Swine with and without Transport and Holding
Authors: Hurd H. S. , McKean J. D. , Griffith R. W. , Wesley I. V. , Rostagno M. H. 
Published: July 2002

ABSTRACTThe objective of this study was to compare, by using identical sample types, theSalmonella entericaprevalences and serovar diversities between pigs necropsied on the farm and those necropsied at the abattoir after transport and holding. We necropsied 567 market weight pigs (>70 kg) from six herds. Pigs were alternately assigned to be necropsied on the farm or at the abattoir. One-half of the group was sent in clean, disinfected trailers to slaughter at a commercial abattoir. After transport (mean distance, 169 km) and 2 to 3 h of holding in antemortem pens, these pigs were necropsied. The 50 pigs remaining on the farm were necropsied the following day. The same sample types and amounts were collected forS. entericaculture at both locations. Results show a sevenfold-higher (P< 0.001)S. entericaisolation rate from pigs necropsied at the abattoir (39.9%; 114 of 286) than from those necropsied on the farm (5.3%; 15 of 281). This difference was also observed for each individual herd. All sample types showed a significantly higher prevalence when comparing abattoir to on-farm collection, respectively: lymph nodes, 9.15 versus 3.6%; cecal contents, 13.6 versus 1.8%; 1 g of fecal matter, 25.2 versus 0.7%. Recovery of additional serovars at the abattoir suggests the pigs are receivingS. entericafrom extra-farm sources. This study demonstrates that rapid infection during transport, and particularly during holding, is a major reason for increasedS. entericaprevalence in swine. This finding identifies the holding pen as an importantS. entericacontrol point in the pork production chain.
Apoptosis in T-Lymphocyte Subsets in Human Immunodeficiency Virus-Infected Children Measured Immediately Ex Vivo and following In Vitro Activation
Authors: Niehues Tim , McCloskey Thomas W. , Ndagijimana Jennifer , Horneff Gerd , Wahn Volker , Pahwa Savita 
Published: July 2002

ABSTRACTPhosphatidylserine molecules are translocated to the outer plasma membrane of lymphocytes undergoing apoptosis and can be detected by the binding of fluorochrome-conjugated annexin V. Using the annexin V assay, we examined CD4 and CD8 T cells from human immunodeficiency virus (HIV)-infected children for apoptosis upon isolation or following in vitro culture. Immediate ex vivo analysis or overnight culture showed significantly higher levels of apoptosis in CD8 cells than in CD4 cells. Following culture with the activating stimulus phytohemagglutinin or anti-CD3 monoclonal antibody, we observed an increase in the percentage of apoptotic CD4 cells, whereas there was no change in the rate of CD8 cell death. These results demonstrate that in HIV-infected children, CD8 apoptosis may occur at a greater rate than CD4 apoptosis in vivo; greater CD4 depletion may be observed due to more efficient mechanisms for peripheral lymphocyte replacement in the CD8 compartment. Furthermore, our data suggest that CD8 lymphocytes may be maximally activated in vivo, a condition which may lead to the exhaustion of CD8-mediated immunity. These findings clarify the differences between the CD4 and CD8 apoptotic responses to HIV.
Expression of Urokinase Plasminogen Activator Receptor on Monocytes from Patients with Relapsing-Remitting Multiple Sclerosis: Effect of Glatiramer Acetate (Copolymer 1)
Authors: Balabanov Roumen , Lisak Deena , Beaumont Thomas , Lisak Robert P. , Dore-Duffy Paula 
Published: July 2002

ABSTRACTMultiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system in which peripheral blood monocytes play an important role. We have previously reported that patients with chronic progressive MS (CPMS) have significantly increased numbers of circulating monocytes which express the urokinase plasminogen activator receptor (uPAR). In the present study, we examined the expression of uPAR on monocytes in patients with relapsing-remitting multiple sclerosis (RRMS) not currently participating in a clinical trial and in patients with RRMS who were enrolled in a double-blind multicenter clinical trial designed to examine the effect of glatiramer acetate (copolymer 1; Copaxone) on relapsing disease. Patients with CPMS have sustained high levels of circulating uPAR-positive (uPAR+) monocytes. In comparison, patients with RRMS displayed variable levels of circulating uPAR+monocytes. Mean values for uPAR in patients with RRMS were above those seen for controls but were not as high as those observed for patients with secondary progressive MS. Patients with RRMS in the clinical trial also had variable levels of monocyte uPAR. However, patients in the treatment group displayed lower levels following 2 years of treatment. In both placebo-treated and glatiramer acetate-treated patients, the percentage of circulating uPAR+monocytes, as well as the density of uPAR expressed per cell (mean linear fluorescence intensity), increased just prior to the onset of a clinically documented exacerbation. Values fell dramatically with the development of clinical symptoms. uPAR levels in all groups correlated with both clinical activity and severity. Results indicate that monocyte activation is impatient in MS and that glatiramer acetate may have a significant effect on monocyte activation in patients with RRMS.
Interpretation of the Gamma Interferon Test for Diagnosis of Subclinical Paratuberculosis in Cattle
Authors: Jungersen G. , Huda A. , Hansen J. J. , Lind P. 
Published: July 2002

ABSTRACTA group of 252 cattle without clinical signs of paratuberculosis (paraTB) in 10 herds infected with paraTB and a group of 117 cattle in 5 herds without paraTB were selected. Whole-blood samples were stimulated with bovine, avian, and johnin purified protein derivative (PPD) and examined for gamma interferon (IFN-γ) release. For diagnosis of paraTB, satisfactory estimated specificities (95 to 99%) could be obtained by johnin PPD stimulation irrespective of interpretation relative to bovine PPD or no-antigen stimulation alone, but numbers of test positives in the infected herds varied from 64 to 112 with different interpretation criteria. For a limited number of test-positive animals, no change in the test results could be observed with increasing antigen concentrations but IFN-γ responses were significantly reduced (P< 0.0001) and four out of seven reactors tested negative when stimulation was performed on day-old samples. Denmark is free of bovine tuberculosis, but cross-reactivity with paraTB could be documented for cattle more than 14 months old in paraTB-infected herds compared with those in non-paraTB-infected herds. In both paraTB-free and paraTB-infected herds, false positives were observed when the test was applied to calves less than 15 months of age. Until novel antigen formulations more specific for these diseases are available, interpretation of the IFN-γ test must be individually adjusted to fit specific needs and the context within which the test is applied and, for paraTB, the test seems most appropriate for use as a supportive tool for evaluation of disease-preventive measures in young stock.
Cytokine Induction byStreptococcus mutansand Pulpal Pathogenesis
Authors: Hahn Chin-Lo , Best Al M. , Tew John G. 
Published: July 2002

ABSTRACTChronic pulpal inflammation under caries appears to be elicited by bacterial antigens that diffuse into the pulp through dentinal tubules. This prompted the hypothesis that cytokines elicited by antigens fromStreptococcus mutans, which frequently dominates shallow lesions, could play a major role in eliciting the initial T-cell response in the pulp. To test this, we examined the ability ofS. mutansto stimulate T cells and elicit cytokines and usedLactobacillus casei, which often predominates in deep carious lesions where B cells and plasma cells predominate, as a control. In addition, the presence of cytokines in the pulp was analyzed at the mRNA level.S. mutanselicited potent gamma interferon (IFN-γ) responses in peripheral blood mononuclear cell cultures and reduced the CD4/CD8 ratio by promoting CD8+T cells. Multiple inflammatory cytokine mRNAs (IFN-γ, interleukin 4 [IL-4], and IL-10) were detected in human dental pulp. A higher prevalence of IFN-γ (67%) than IL-4 (19%) or IL-10 (29%) was obtained in shallow caries, suggesting a type 1 cytokine mechanism in early pulpitis whereS. mutanspredominates. In contrast, in deep caries no differences in cytokine frequency were observed. Furthermore, the presence of IFN-γ in the pulp correlated with the presence ofS. mutans. The extraordinary induction of type 1 cytokines and the preferential activation of CD8+T cells byS. mutansoffers an explanation for the etiology of the CD8+T-cell-dominant lesion in early pulpitis and suggests thatS. mutansmay have a major impact on the initial lesion and pulpal pathology.