bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitation of Viral DNA by Real-Time PCR Applying Duplex Amplification, Internal Standardization, and Two-Color Fluorescence Detection
Authors: Gruber Franz , Falkner Falko G. , Dorner Friedrich , Hämmerle Thomas 
Published: July 2002

ABSTRACT

A real-time PCR method was developed to quantitate viral DNA that includes duplex amplification, internal standardization, and two-color fluorescence detection without the need to generate an external standardization curve. Applied to human parvovirus B19 DNA, the linear range was from 10
2
to at least 5 × 10
6
copies per ml of sample. The coefficient of variation was 0.29 using a run control of 2,876 copies per ml. The method reduces the risk of false-negative results, yields high precision, and is applicable for other DNA targets.
Improvement of Cellulolytic Properties of
Clostridium cellulolyticum
by Metabolic Engineering
Authors: Guedon Emmanuel , Desvaux Mickaël , Petitdemange Henri 
Published: July 2002

ABSTRACT

Cellulolytic clostridia have evolved to catabolize lignocellulosic materials at a seasonal biorhythm, so their biotechnological exploitation requires genetic improvements. As high carbon flux leads to pyruvate accumulation, which is responsible for the cessation of growth of
Clostridium cellulolyticum
, this accumulation is decreased by heterologous expression of pyruvate decarboxylase and alcohol dehydrogenase from
Zymomonas mobilis
. In comparison with that of the wild strain, growth of the recombinant strain at the same specific rate but for 145 h instead of 80 h led to a 150% increase in cellulose consumption and a 180% increase in cell dry weight. The fermentation pattern was shifted significantly: lactate production decreased by 48%, whereas the concentrations of acetate and ethanol increased by 93 and 53%, respectively. This study demonstrates that the fermentation of cellulose, the most abundant and renewable polymer on earth, can be greatly improved by using genetically engineered
C. cellulolyticum
.
Transcript Levels of the Eukaryotic Translation Initiation Factor 5A Gene Peak at Early G
1
Phase of the Cell Cycle in the Dinoflagellate
Crypthecodinium cohnii
Authors: Chan K. L. , New D. , Ghandhi S. , Wong F. , Lam C. M. C. , Wong J. T. Y. 
Published: July 2002

ABSTRACT

A cDNA encoding a eukaryotic translation initiation factor 5A (eIF-5A) homolog in heterotrophic dinoflagellate
Crypthecodinium cohnii
(CceIF-5A) was isolated through random sequencing of a cDNA library. The predicted amino acid sequence possesses the 12 strictly conserved amino acids around lysine 52 (equivalent to lysine 50 or 51 in other eukaryotes). A single 1.2-kb band was detected in Northern blot analysis. In synchronized
C. cohnii
cells, the transcript level peaked at early G
1
and decreased dramatically on the entry to S phase. Although this has not been previously reported, studies of budding yeast (
Saccharomyces cerevisiae
) and certain mammalian cell types suggest a role for eIF-5A in the G
1
/S transition of the eukaryotic cell cycle. Phylogenetic trees constructed with 26 other published eIF-5A sequences suggest that CceIF-5A, while falling within the eukaryotic branches, forms a lineage separate from those of the plants, animals, and archaebacteria. The posttranslational modification of eIF-5A by a transfer of a 4-aminobutyl moiety from spermidine to conserved lysine 50 or 51, forming amino acid hypusine, is the only demonstrated specific function of polyamines in cell proliferation. It has been suggested that polyamines stimulate population growth of bloom-forming dinoflagellates in the sea. We demonstrate here putrescine-stimulated cell proliferation. Furthermore, ornithine decarboxylase inhibitor
d
-difluoromethylornithine and the specific hypusination inhibitor
N
-guanyl-1,7-diaminoheptane exhibited inhibitory effects in two species of dinoflagellates. The possible links of polyamines and saxitoxin synthesis to the arginine cycle are also discussed.
Sequence Variation in the
porA
Gene of a Clone of
Neisseria meningitidis
during Epidemic Spread
Authors: Jelfs J. , Munro R. , Wedege E. , Caugant D. A. 
Published: July 2002

ABSTRACT

The ET-15 clone within the electrophoretic type (ET)-37 complex of
Neisseria meningitidis
was first detected in Canada in 1986 and has since been associated with outbreaks of meningococcal disease in many parts of the world. While the majority of the strains of the ET-37 complex are serosubtype P1.5,2, serosubtype determination of ET-15 strains may often be incomplete, with either only one or none of the two variable regions (VRs) of the serosubtype PorA outer membrane protein reacting with monoclonal antibodies. DNA sequence analysis of the
porA
gene from ET-15 strains with one or both unidentified serosubtype determinants was undertaken to identify the genetic basis of the lack of reaction with the monoclonal antibodies. Fourteen different
porA
alleles were identified among 38 ET-15 strains from various geographic origins. The sequences corresponding to subtypes P1.5a,10d, P1.5,2, P1.5,10d, P1.5a,10k, and P1.5a,10a were identified in 18, 11, 2, 2, and 1 isolate, respectively. Of the remaining four strains, which all were nonserosubtypeable, two had a stop codon within the VR1 and the VR2, respectively, while in the other two the
porA
gene was interrupted by the insertion element, IS
1301
. Of the strains with P1.5,2 sequence, one had a stop codon between the VR1 and VR2, one had a four-amino-acid deletion outside the VR2, and another showed no expression of PorA on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results reveal that numerous genetic events have occurred in the
porA
gene of the ET-15 clone in the short time of its epidemic spread. The magnitude of microevolutionary mechanisms available in meningococci and the remarkable genetic flexibility of these bacteria need to be considered in relation to PorA vaccine development.
Close Association between Pulmonary Disease Manifestation inMycoplasma pneumoniaeInfection and Enhanced Local Production of Interleukin-18 in the Lung, Independent of Gamma Interferon
Authors: Narita Mitsuo , Tanaka Hiroshi , Abe Shosaku , Yamada Satoshi , Kubota Mitsuru , Togashi Takehiro 
Published: July 2002

ABSTRACTTo investigate pathophysiologies ofMycoplasma pneumoniaeinfection from an immunological point of view, we measured the levels of interleukin-18 (IL-18) (originally designated gamma interferon [IFN-γ]-inducing factor) in 19 serum samples from 10 patients with pneumonia without pleural effusion (ages 1 to 16 years), 3 serum and 13 pleural fluid samples from 11 patients with pleural effusions (ages 11 months to 15 years), and 18 serum and 27 cerebrospinal fluid samples from 24 patients with central nervous system complications (ages 1 to 15 years). IL-18 was measured by a commercially available enzyme-linked immunosorbent assay kit (MBL, Nagoya, Japan). In addition, the levels of tumor necrosis factor alpha, IFN-γ, IL-6, IL-12, and KL-6 (a mucin-like glycoprotein expressed on type 2 pneumocytes) were measured in selected samples. The results concerning pleural effusions showed that elevated levels of IL-18 in pleural fluid, but not in serum, were solely associated with a sustained fibrotic change of the lung on chest roentgenography which might represent a pathological feature of intraluminal organization. All the pleural fluid samples with elevated levels of IL-18 were positive by PCR forM. pneumoniaeDNA. There was no association between IL-18 and IFN-γ levels in serum or in the pleural fluid. On the other hand, elevated levels of IL-18 in serum, but not in cerebrospinal fluid samples, were observed in the cases complicated by central nervous system involvement, including profound brain dysfunction with seizures. Our study demonstrated thatM. pneumoniaecan induce IL-18 and that the enhanced local production of IL-18 in the lung is closely associated with pulmonary disease manifestation.
Quantitative Analyses of Variations in the Injury of Endothelial Cells Elicited by 11 Isolates ofRickettsia rickettsii
Authors: Eremeeva Marina E. , Dasch Gregory A. , Silverman David J. 
Published: July 2002

ABSTRACTEleven isolates of spotted fever group rickettsiae from the blood of patients or ixodid ticks from North and South America were characterized. All isolates were identified asRickettsia rickettsiiusing restriction fragment length polymorphism analysis of a 532-bp rOmpA gene fragment obtained by PCR. The ability of theR. rickettsiiisolates to elicit cytopathic effects and parameters of oxidative injury were examined in cultured human EA.hy 926 endothelial cells. Cytopathic effects were determined by direct observation of infected cultures, by measuring the release of cytoplasmic lactate dehydrogenase (LDH), and by determination of intracellular pools of peroxide and reduced glutathione. Four biotypes ofR. rickettsiiwere defined. Group I included two highly cytopathic isolates from Montana, Bitterroot and Sheila Smith, and three isolates from Maryland, North Carolina, and Brazil. These isolates rapidly damaged cells, released large amounts of cytoplasmic LDH, caused accumulation of intracellular peroxide, and depleted intracellular pools of reduced glutathione. Group II contained three isolates, two from Montana, Hlp#2 and Lost Horse Canyon, and an isolate from Colombia, which were similar to group I but caused either lower responses in LDH release or smaller changes in intracellular peroxide levels. The group III isolates, Sawtooth from Montana and 84JG from North Carolina, caused lower cellular injury by all measures. Group IV isolate Price T from Montana was the least cytopathic and caused minimal alterations of all parameters measured. Understanding the molecular basis for the varied cellular injury caused by different isolates ofR. rickettsiimay contribute to improved treatment of Rocky Mountain spotted fever and to the rapid identification of those isolates which are more likely to cause fulminant disease.
Periodontal Disease as a Specific, albeit Chronic, Infection: Diagnosis and Treatment
Authors: Loesche Walter J. , Grossman Natalie S. 
Published: July 2002

SUMMARYPeriodontal disease is perhaps the most common chronic infection in adults. Evidence has been accumulating for the past 30 years which indicates that almost all forms of periodontal disease are chronic but specific bacterial infections due to the overgrowth in the dental plaque of a finite number of mostly anaerobic species such as Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola. The success of traditional debridement procedures and/or antimicrobial agents in improving periodontal health can be associated with the reduction in levels of these anaerobes in the dental plaque. These findings suggest that patients and clinicians have a choice in the treatment of this overgrowth, either a debridement and surgery approach or a debridement and antimicrobial treatment approach. However, the antimicrobial approach, while supported by a wealth of scientific evidence, goes contrary to centuries of dental teaching that states that periodontal disease results from a “dirty mouth.” If periodontal disease is demonstrated to be a risk factor for cardiovascular disease and stroke, it will be a modifiable risk factor since periodontal disease can be prevented and treated. Since the antimicrobial approach may be as effective as a surgical approach in the restoration and maintenance of a periodontally healthy dentition, this would give a cardiac or stroke patient and his or her physician a choice in the implementation of treatment seeking to improve the patient's periodontal condition so as to reduce and/or delay future cardiovascular events.
Clinical Aspects and Pathophysiology of Inflammatory Bowel Disease
Authors: Hendrickson Barbara A. , Gokhale Ranjana , Cho Judy H. 
Published: July 2002

SUMMARYThe chronic inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis, are recognized as important causes of gastrointestinal disease in children and adults. In this review we delineate the clinical manifestations and diagnostic features of IBD. In addition, we summarize important recent advances in our understanding of the immune mediators of intestinal inflammation. This information has led to new therapeutic approaches in IBD. Further, we discuss the considerable data that point to the significance of genetic factors in the development of IBD and the genetic loci which have been implicated through genomewide searches. The commensal bacterial flora also appears to be a critical element, particularly in regards to Crohn's disease, although the precise role of the bacteria in the disease manifestations remains unclear. Current investigations promise to yield fresh insights in these areas.
Molecular Variation among Type IV Pilin (
bfpA
) Genes from Diverse Enteropathogenic
Escherichia coli
Strains
Authors: Blank T. Eric , Zhong Hailang , Bell Alison L. , Whittam Thomas S. , Donnenberg Michael S. 
Published: July 2002

ABSTRACT

Typical enteropathogenic
Escherichia coli
(EPEC) strains produce bundle-forming pili (BFP), type IVB fimbriae that have been implicated in EPEC virulence, antigenicity, autoaggregation, and localized adherence to epithelial cells (LA). BFP are polymers of bundlin, a pilin protein that is encoded by the
bfpA
gene found on a large EPEC plasmid. Striking sequence variation has previously been observed among type IV pilin genes of other gram-negative bacterial pathogens (e.g.,
Pseudomonas
and
Neisseria
spp.). In contrast, the established sequences of
bfpA
genes from two distantly related prototype EPEC strains vary by only a single base pair. To determine whether bundlin sequences vary more extensively, we used PCR to amplify the
bfpA
genes from 19 EPEC strains chosen for their various serotypes and sites and years of isolation. Eight different
bfpA
alleles were identified by sequencing of the PCR products. These alleles can be classified into two major groups. The α group contains three alleles derived from strains carrying O55, O86, O111, O119, O127, or O128 somatic antigens. The β group contains five alleles derived from strains carrying O55, O110, O128ab, O142, or nontypeable antigens. Sequence comparisons show that bundlin has highly conserved and variable regions, with most of the variation occurring in the C-terminal two-thirds of the protein. The results of multilocus enzyme electrophoresis support the hypothesis that
bfpA
sequences have spread horizontally across distantly related clonal lineages. Strains with divergent bundlin sequences express bundlin protein, produce BFP, and carry out autoaggregation and LA. However, four strains lack most or all of these phenotypes despite having an intact
bfpA
gene. These results have important implications for our understanding of bundlin structure, transmission of the
bfp
gene cluster among EPEC strains, and the role of bundlin variation in the evasion of host immune system responses.
Disease Outcome Subsequent to Primary and Secondary Urogenital Infection with Murine or Human Biovars ofChlamydia trachomatis
Authors: Ramsey Kyle H. , DeWolfe Jennifer L. , Salyer Rena D. 
Published: July 2002

ABSTRACTA susceptible strain of mice infected intravaginally with the mouse pneumonitis biovar ofChlamydia trachomatisbecame infertile and sustained high rates of hydrosalpinx formation regardless of prior infection with a human serovar. Conversely, susceptible mice infected with human serovars remained fertile unless challenged with a homologous human serovar.
Distribution of Core Oligosaccharide Types in Lipopolysaccharides from
Escherichia coli
Authors: Amor Karen , Heinrichs David E. , Frirdich Emilisa , Ziebell Kim , Johnson Roger P. , Whitfield Chris 
Published: July 2002

ABSTRACT

In the lipopolysaccharides of
Escherichia coli
there are five distinct core oligosaccharide (core OS) structures, designated K-12 and R1 to R4. The objective of this work was to determine the prevalences of these core OS types within the species. Unique sequences in the
waa
(core OS biosynthesis) gene operon were used to develop a PCR-based system that facilitated unequivocal determination of the core OS types in isolates of
E. coli
. This system was applied to the 72 isolates in the
E. coli
ECOR collection, a compilation of isolates that is considered to be broadly representative of the genetic diversity of the species. Fifty (69.4%) of the ECOR isolates contained the R1 core OS, 8 (11.1%) were representatives of R2, 8 (11.1%) were R3, 2 (2.8%) were R4, and only 4 (5.6%) were K-12. R1 is the only core OS type found in all four major phylogenetic groups (A, B1, B2, and D) in the ECOR collection. Virulent extraintestinal pathogenic
E. coli
isolates tend to be closely related to group B2 and, to a lesser extent, group D isolates. All of the ECOR representatives from the B2 and D groups had the R1 core OS. In contrast, commensal
E. coli
isolates are more closely related to group A, which contains isolates representing each of the five core OS structures. R3 was the only core OS type found in 38 verotoxigenic
E. coli
(VTEC) isolates from humans and cattle belonging to the common enterohemorrhagic
E. coli
serogroups O157, O111, and O26. Although isolates from other VTEC serogroups showed more core OS diversity, the R3 type (83.1% of all VTEC isolates) was still predominant. When non-VTEC commensal isolates from cattle were analyzed, it was found that most possessed the R1 core OS type.
Regulation of Immunoglobulin G2 Production by Prostaglandin E2and Platelet-Activating Factor
Authors: Ishihara Yuichi , Zhang Ji-Bo , Quinn Steve M. , Schenkein Harvey A. , Best Al M. , Barbour Suzanne E. , Tew John G. 
Published: July 2002

ABSTRACTPatients with localized juvenile periodontitis (LJP) have elevated levels of immunoglobulin G2 (IgG2) in their sera. This is also observed in vitro when peripheral blood leukocytes from LJP patients are stimulated with pokeweed mitogen. In previous studies, we showed that lymphocytes from subjects with no periodontitis (NP subjects) produced substantial amounts of IgG2 when they were cultured with monocytes from LJP patients (LJP monocytes). These observations indicate that monocytes or monocyte-derived mediators are positive regulators of the production of IgG2. The present study was initiated to determine if secreted factors from LJP monocytes were capable of enhancing IgG2 production and to determine if prostaglandin E2 (PGE2), which LJP monocytes produce at elevated levels, enhances IgG2 production. Experiments in a transwell system and with monocyte-conditioned media indicated that cell-cell contact was not necessary for LJP monocytes to augment the production of IgG2 by T and B cells from NP subjects. Moreover, the production of IgG2 was selectively induced by the addition of PGE2or platelet-activating factor (PAF), another lipid cytokine, which can elevate PGE2synthesis. Furthermore, IgG2 production was abrogated when cells were treated with indomethacin, a cyclooxygenase inhibitor that blocks the synthesis of PGE2, or the PAF antagonists CV3988 and TEPC-15. The effects of indomethacin were completely reversed by PGE2, indicating that this is the only prostanoid that is essential for the production of IgG2. Similarly, PGE2reversed the effects of a PAF antagonist, suggesting that the effects of PAF are mediated through the induction of PGE2synthesis. Together, these data indicate that PGE2and PAF are essential for the production of IgG2.
Developmental Expression of a Tandemly Repeated, Glycine- and Serine-Rich Spore Wall Protein in the Microsporidian Pathogen
Encephalitozoon cuniculi
Authors: Bohne Wolfgang , Ferguson David J. P. , Kohler Karoline , Gross Uwe 
Published: July 2002

ABSTRACT

Microsporidia are intracellular organisms of increasing importance as opportunistic pathogens in immunocompromised patients. Host cells are infected by the extrusion and injection of polar tubes located within spores. The spore is surrounded by a rigid spore wall which, in addition to providing mechanical resistance, might be involved in host cell recognition and initiation of the infection process. A 51-kDa outer spore wall protein was identified in
Encephalitozoon cuniculi
with the aid of a monoclonal antibody, and the corresponding gene,
SWP1
, was cloned by immunoscreening of a cDNA expression library. The cDNA encodes a protein of 450 amino acids which displays no significant similarities to known proteins in databases. The carboxy-terminal region consists of five tandemly arranged glycine- and serine-rich repetitive elements.
SWP1
is a single-copy gene that is also present in the genomes of
Encephalitozoon intestinalis
and
Encephalitozoon hellem
as demonstrated by Southern analysis. Indirect immunofluorescence and immunoelectron microscopy revealed that SWP1 is differentially expressed during the infection cycle. The protein is absent in replicative meronts until 24 h postinfection, and its expression is first induced in early sporonts at a time when organisms translocate from the periphery to the center of the parasitophorous vacuole. Expression of SWP1 appears to be regulated at the mRNA level, as was shown by reverse transcriptase PCR analysis. Further identification and characterization of stage-specific genes might help to unravel the complex intracellular differentiation process of microsporidia.
Vervet Monkeys Vaccinated with KilledLeishmania majorParasites and Interleukin-12 Develop a Type 1 Immune Response but Are Not Protected against Challenge Infection
Authors: Gicheru Michael M. , Olobo Joseph O. , Anjili Christopher O. , Orago Alloys S. , Modabber Farrokh , Scott Phillip 
Published: July 2002

ABSTRACTLeishmania majoris a protozoan parasite that causes chronic cutaneous lesions that often leave disfiguring scars. Infections in mice have demonstrated that leishmanial vaccines that include interleukin-12 (IL-12) as an adjuvant are able to induce protective immunity. In this study, we assessed the safety, immunopotency, and adjuvant potential of two doses of IL-12 when used with a killedL. majorvaccine in vervet monkeys. The induction of cell-mediated immunity following vaccination was determined by measuring delayed-type hypersensitivity, in vitro lymphocyte proliferation, and gamma interferon (IFN-γ) production. Protection was assessed by challenging the animals withL. majorparasites and monitoring the course of infection. At low doses of IL-12 (10 μg), a small increase in the parameters of cell-mediated immunity was observed, relative to those in animals that received antigen without IL-12. However, none of these animals were protected against a challenge infection. At higher doses of IL-12 (30 μg), a substantial increase inLeishmania-specific immune responses was observed, and monkeys immunized with antigen and IL-12 exhibited an IFN-γ response that was as great as that in animals that had resolved a primary infection and were immune. Nevertheless, despite the presence of correlates of protection, the disease course was only slightly altered, and protection was low compared to that in self-cured monkeys. These data suggest that protection against leishmaniasis may require more than the activation ofLeishmania-specific IFN-γ-producing T cells, which has important implications for designing a vaccine against leishmaniasis.