bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vitro Development of Resistance to Six Quinolones in
Streptococcus pneumoniae, Streptococcus pyogenes
, and
Staphylococcus aureus
Authors: Boos Mechthild , Mayer Susanne , Fischer Ansgar , Köhrer Karl , Scheuring Sibylle , Heisig Peter , Verhoef Jan , Fluit Ad C. , Schmitz F.-J.
Published: July 2002

Streptococcus pneumoniae, Streptococcus pyogenes
, and
Staphylococcus aureus
isolates were exposed to subinhibitory MICs of ciprofloxacin, sparfloxacin, gatifloxacin, moxifloxacin, clinafloxacin, and gemifloxacin during a 10-day period. Subculturing led to resistance development, regardless of the initial potencies of the quinolones. None of the quinolones was associated with a significantly slower rate of resistance development.
Development of a Whole-Cell Assay for Peptidoglycan Biosynthesis Inhibitors
Authors: Barbosa Maria D. F. S. , Yang Gaoyun , Fang Jie , Kurilla Michael G. , Pompliano David L.
Published: July 2002

Osmotically stabilized
Escherichia coli
cells subjected to freezing and thawing were utilized as the source of enzymes for a peptidoglycan pathway assay that can be used to simultaneously test all targets of the committed steps of cell wall biosynthesis. The use of
14
C-labeled UDP-
N
-acetylglucosamine (UDP-GlcNAc) as a substrate allows the direct detection of cross-linked peptidoglycan formed. The assay was validated with known antibiotics. Fosfomycin was the strongest inhibitor of the pathway assay, with a 50% inhibitory concentration of 1 μM. Flavomycin, bacitracin, vancomycin,
d
-cycloserine, penicillin G, and ampicillin also inhibited formation of radiolabeled peptidoglycan by the
E. coli
cells. Screening of compounds identified two inhibitors of the pathway, Cpd1 and Cpd2. Subsequent tests with a biochemical assay utilizing purified enzyme implicated UDP-GlcNAc enolpyruvyl transferase (MurA) as the target of Cpd1. This compound inhibits the first enzyme of the pathway in a time-dependent manner. Moreover, enzyme inactivation is dependent on preincubation in the presence of UDP-GlcNAc, which forms a complex with MurA, exposing its active site. Cpd1 also displayed antimicrobial activity against a panel of microorganisms. The pathway assay used in conjunction with assays for individual enzymes provides an efficient means of detecting and characterizing novel antimicrobial agents.
Characterization of Imidazo[4,5-
d
]Pyridazine Nucleosides as Modulators of Unwinding Reaction Mediated by West Nile Virus Nucleoside Triphosphatase/Helicase: Evidence for Activity on the Level of Substrate and/or Enzyme
Authors: Borowski Peter , Lang Melanie , Haag Annemarie , Schmitz Herbert , Choe Joonho , Chen Huan-Ming , Hosmane Ramachandra S.
Published: July 2002

Compounds that interact with DNA or RNA generally act as inhibitors of enzymes that unwind DNA or RNA. In the present study we describe the synthesis and properties of some nucleoside analogues that interact with double-stranded DNA but that, in contrast, facilitate the unwinding reaction mediated by West Nile (WN) virus nucleoside triphosphatase (NTPase)/helicase. The nucleoside analogues described, 1-(2′-
O
-methyl-β-
d
-ribofuranosyl)imidazo[4,5-
d
]pyridazine-4,7(5
H
,6
H
)-dione (HMC-HO4), 1-(β-
d
-ribofuranosyl)imidazo[4,5-
d
]pyridazine-4,7(5
H
,6
H
)-dione, and 1-(2′-deoxy-α-
d
-ribofuranosyl)imidazo[4,5-
d
]pyridazine-4,7(5
H
,6
H
)dione, all contain the imidazo[4,5-
d
]pyridazine ring system. The extent of the enhancing effect on helicase activity was found to be dependent on the time of exposure of the DNA substrate to the compounds and their concentrations. The nucleoside analogues were nevertheless found to be capable of uncoupling the ATPase and helicase activities of the enzyme by a mechanism operating on the level of the enzyme. Thus, in the case of HMC-HO4, the direct interaction with the enzyme caused inhibition of its helicase activity, with a half-maximal inhibitory concentration of 30 μM. The similar potency of the compound against replication of WN virus in cell culture suggests that inhibition of the helicase activity of the viral enzyme is responsible for the observed antiviral activity of HMC-HO4 and may indeed represent an important mode of action of antiviral drugs in general. Comparative studies performed with the related NTPase/helicase from hepatitis C virus revealed that the extent of the effects mediated by imidazo[4,5-
d
]pyridazine nucleosides is enzyme specific. The substances described may represent a starting point for the development of a new class of helicase-specific antivirals.
Purification and Characterization of
Streptomyces griseus
Catechol
O
-Methyltransferase
Authors: Dhar Kajari , Rosazza John P. N.
Published: July 2002

A soluble (100,000 ×
g
supernatant) methyltransferase catalyzing the transfer of the methyl group of
S
-adenosyl-
l
-methionine to catechols was present in cell extracts of
Streptomyces griseus
. A simple, general, and rapid catechol-based assay method was devised for enzyme purification and characterization. The enzyme was purified 141-fold by precipitation with ammonium sulfate and successive chromatography over columns of DEAE-cellulose, DEAE-Sepharose, and Sephacryl S-200. The purified cytoplasmic enzyme required 10 mM magnesium for maximal activity and was catalytically optimal at pH 7.5 and 35°C. The methyltransferase had an apparent molecular mass of 36 kDa for both the native and denatured protein, with a pI of 4.4. Novel N-terminal and internal amino acid sequences were determined as DFVLDNEGNPLENNGGYXYI and RPDFXLEPPYTGPXKARIIRYFY, respectively. For this enzyme, the

K
m

for 6,7-dihydroxycoumarin was 500 ± 21.5 μM, and that for
S
-adenosyl-
l
-methionine was 600 ± 32.5 μM. Catechol, caffeic acid, and 4-nitrocatechol were methyltransferase substrates. Homocysteine was a competitive inhibitor of
S
-adenosyl-
l
-methionine, with a

K
i

of 224 ± 20.6 μM. Sinefungin and
S
-adenosylhomocysteine inhibited methylation, and the enzyme was inactivated by Hg
2+
,
p
-chloromercuribenzoic acid, and
N
-ethylmaleimide.
In Situ Determination of Sulfide Turnover Rates in a Meromictic Alpine Lake
Authors: Lüthy Lucas , Fritz Markus , Bachofen Reinhard
Published: July 2002

A push-pull method, previously used in groundwater analyses, was successfully adapted for measuring sulfide turnover rates in situ at different depths in the meromictic Lake Cadagno. In the layer of phototrophic bacteria at about 12 m in depth net sulfide consumption was observed during the day, indicating active bacterial photosynthesis. During the night the sulfide turnover rates were positive, indicating a net sulfide production from the reduction of more-oxidized sulfur compounds. Because of lack of light, no photosynthesis takes place in the monimolimnion; thus, only sulfide formation is observed both during the day and the night. Sulfide turnover rates in the oxic mixolimnion were always positive as sulfide is spontaneously oxidized by oxygen and as the rates of sulfide oxidation depend on the oxygen concentrations present. Sulfide oxidation by chemolithotrophic bacteria may occur at the oxicline, but this cannot be distinguished from spontaneous chemical oxidation.
Diversity of Infectious Pancreatic Necrosis Virus Strains Isolated from Fish, Shellfish, and Other Reservoirs in Northwestern Spain
Authors: Cutrin J. M. , Olveira J. G. , Barja J. L. , Dopazo C. P.
Published: July 2002

A comparison was done of 231 strains of birnavirus isolated from fish, shellfish, and other reservoirs in a survey study that began in 1986 in Galicia (northwestern Spain). Reference strains from all of the infectious pancreatic necrosis virus serotypes were included in the comparison, which was done by neutralization tests and agarose and polyacrylamide gel electrophoresis of the viral genome. The neutralization tests with antisera against the West Buxton, Spajarup (Sp), and Abild (Ab) strains showed that most of the Galician isolates were European types Sp and Ab; however, many isolates (30%) could not be typed. Results from agarose gels did not provided information for grouping of the strains, since all were found to have genomic segments of similar sizes. Analysis of polyacrylamide gels, however, allowed six electropherogroups (EGs) to be differentiated on the basis of genome mobility and separation among segments, and a certain relationship between EGs and serotypes was observed. A wide diversity of electropherotypes was observed among the Galician isolates, and as neutralization tests showed, most of the isolates were included in EGs corresponding to European types Ab and Sp. Only 6.5% of the isolates had the electropherotype characteristic of American strains.
Production of Pectate Lyases and Cellulases by
Chryseomonas luteola
Strain MFCL0 Depends on the Growth Temperature and the Nature of the Culture Medium: Evidence for Two Critical Temperatures
Authors: Laurent P. , Buchon L. , Guespin-Michel J. F. , Orange N.
Published: July 2002

Several extracellular enzymes that are responsible for plant tissue maceration were detected in culture supernatant of the psychrotrophic bacterium
Chryseomonas luteola
MFCL0. Isoelectrofocusing experiments showed that pectate lyase (PL) activity resulted from the cumulative action of three major isoenzymes, designated PLI, PLII, and PLIII. Cellulolytic activity was also detected in culture supernatants. These enzymes exhibited different behaviors with respect to growth temperature. PLII was not regulated by temperature, whereas PLI and PLIII were regulated similarly by growth temperature. Maximal levels of PLI and PLIII were produced at 14°C when cells were grown in polygalacturonate-containing synthetic medium and at around 20 to 24°C in nutrient broth. In contrast, thermoregulation of cellulolytic activity production differed from thermoregulation of PL. The level of cellulolytic activity was low in all media at temperatures up to 20°C, and then it increased dramatically until the temperature was 28°C, which is the optimal temperature for growth of
C. luteola
. Previously, we defined the critical temperature by using the modified Arrhenius equation to characterize bacterial behavior. This approach consists of monitoring changes in the maximal specific growth rate as a function of temperature. Our most striking result was the finding that the temperature at which maximum levels of PLI and PLIII were produced in two different media was the same as the critical temperature for growth observed in these two media.
Localization of Symbiotic Clostridia in the Mixed Segment of the Termite
Nasutitermes takasagoensis
(Shiraki)
Authors: Tokuda Gaku , Yamaoka Ikuo , Noda Hiroaki
Published: July 2002

Phylogeny and the distribution of symbiotic bacteria in the mixed segment of the wood-eating termite
Nasutitermes takasagoensis
(Shiraki) were studied. Bacterial 16S rRNA genes (rDNA) were amplified from the mixed segment of the gut by PCR, and two kinds of sequences were identified. The phylogenetic tree was constructed by neighbor-joining and maximum parsimony methods to identify symbionts harbored in the mixed segment. They are classified as low-G+C-content gram-positive bacteria and are most closely related to the genus
Clostridium
. The distribution of these bacteria throughout the whole gut was examined by PCR using specific primers, which suggested that they are confined to the mixed segment despite the presence of bacteria throughout the gut. In situ hybridization indicated that the symbiotic bacteria were localized to the ectoperitrophic space between the midgut wall and the peritrophic membrane in the mixed segment. Electron microscopy revealed the close association between these bacteria and the mesenteric epithelium, suggesting that they have some interactions with the gut tissue of termites.
Deletion of Various Carboxy-Terminal Domains of
Lactococcus lactis
SK11 Proteinase: Effects on Activity, Specificity, and Stability of the Truncated Enzyme
Authors: Bruinenberg Paul G. , De Vos Willem M. , Siezen Roland J.
Published: July 2002

The
Lactococcus lactis
SK11 cell envelope proteinase is an extracellular, multidomain protein of nearly 2,000 residues consisting of an N-terminal serine protease domain, followed by various other domains of largely unknown function. Using a strategy of deletion mutagenesis, we have analyzed the function of several C-terminal domains of the SK11 proteinase which are absent in cell envelope proteinases of other lactic acid bacteria. The various deletion mutants were functionally expressed in
L. lactis
and analyzed for enzyme stability, activity, (auto)processing, and specificity toward several substrates. C-terminal deletions of first the cell envelope W (wall) and AN (anchor) domains and then the H (helix) domain leads to fully active, secreted proteinases of unaltered specificity. Gradually increasing the C-terminal deletion into the so-called B domain leads to increasing instability and autoproteolysis and progressively less proteolytic activity. However, the mutant with the largest deletion (838 residues) from the C terminus and lacking the entire B domain still retains proteolytic activity. All truncated enzymes show unaltered proteolytic specificity toward various substrates. This suggests that the main role played by these domains is providing stability or protection from autoproteolysis (B domain), spacing away from the cell (H domain), and anchoring to the cell envelope (W and AN domains). In addition, this study allowed us to more precisely map the main C-terminal autoprocessing site of the SK11 proteinase and the epitope for binding of group IV monoclonal antibodies.
Diversity of Thiosulfate-Oxidizing Bacteria from Marine Sediments and Hydrothermal Vents
Authors: Teske A. , Brinkhoff T. , Muyzer G. , Moser D. P. , Rethmeier J. , Jannasch H. W.
Published: July 2002

Species diversity, phylogenetic affiliations, and environmental occurrence patterns of thiosulfate-oxidizing marine bacteria were investigated by using new isolates from serially diluted continental slope and deep-sea abyssal plain sediments collected off the coast of New England and strains cultured previously from Galapagos hydrothermal vent samples. The most frequently obtained new isolates, mostly from 10
3
- and 10
4
-fold dilutions of the continental slope sediment, oxidized thiosulfate to sulfate and fell into a distinct phylogenetic cluster of marine alpha-
Proteobacteria
. Phylogenetically and physiologically, these sediment strains resembled the sulfate-producing thiosulfate oxidizers from the Galapagos hydrothermal vents while showing habitat-related differences in growth temperature, rate and extent of thiosulfate utilization, and carbon substrate patterns. The abyssal deep-sea sediments yielded predominantly base-producing thiosulfate-oxidizing isolates related to Antarctic marine
Psychroflexus
species and other cold-water marine strains of the
Cytophaga-Flavobacterium-Bacteroides
phylum, in addition to gamma-proteobacterial isolates of the genera
Pseudoalteromonas
and
Halomonas-Deleya
. Bacterial thiosulfate oxidation is found in a wide phylogenetic spectrum of
Flavobacteria
and
Proteobacteria
.
marY1
, a Member of the
gypsy
Group of Long Terminal Repeat Retroelements from the Ectomycorrhizal Basidiomycete
Tricholoma matsutake
Authors: Murata Hitoshi , Yamada Akiyoshi
Published: July 2002

We cloned an intact copy of a long terminal repeat retroelement designated
marY1
from the ectomycorrhizal basidiomycete
Tricholoma matsutake
. The reverse transcriptase domain is found in
T. matsutake
and
Tricholoma magnivelare
worldwide. This finding suggests that retroelements associate with ectomycorrhizal basidiomycetes and may be useful as genetic markers for identification, phylogenetic analysis, and mutagenesis of this fungal group.
Pyrosequencing as a Method for Grouping of
Listeria monocytogenes
Strains on the Basis of Single-Nucleotide Polymorphisms in the
inlB
Gene
Authors: Unnerstad Helle , Ericsson Henrik , Alderborn Anders , Tham Wilhelm , Danielsson-Tham Marie-Louise , Mattsson Jens G.
Published: July 2002

By using pyrosequencing (i.e., sequencing by synthesis) 106 strains of different serovars of
Listeria monocytogenes
were rapidly grouped into four categories based on nucleotide variations at positions 1575 and 1578 of the
inlB
gene. Strains of serovars 1/2a and 1/2c constituted one group, and strains of serovars 1/2b and 3b constituted another group, whereas serovar 4b strains were separated into two groups.
Development of a Reverse Transcription-PCR–DNA Enzyme Immunoassay for Detection of “Norwalk-Like” Viruses and Hepatitis A Virus in Stool and Shellfish
Authors: Schwab Kellogg J. , Neill Frederick H. , Le Guyader Françoise , Estes Mary K. , Atmar Robert L.
Published: July 2002

Outbreaks of food- and waterborne gastroenteritis are being increasingly reported throughout the world. The analysis of environmental samples by newer diagnostic techniques such as reverse transcription-PCR (RT-PCR) amplification of nucleic acid has begun to identify human enteric viruses (predominantly “Norwalk-like” viruses [NLVs]) as the cause of many of these outbreaks. To streamline NLV detection from environmental samples such as shellfish, we have developed an RT-PCR–oligoprobe amplification and detection method using several new procedures that enable confirmed RT-PCR amplification and product detection in 1 day. The new steps include replacing reverse transcriptase and
Taq
polymerase with r
Tth
polymerase, a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase, in a single-tube, single-buffer, elevated temperature reaction. An internal standard Norwalk virus (NV) RNA control is added to each RT-PCR to identify sample inhibition, and thermolabile uracil
N
-glycosylase is incorporated into the reaction to prevent PCR product carryover contamination. Finally, RT-PCR-generated amplicons are detected in microtiter wells using virus-specific biotinylated oligoprobes in an enzyme-linked immunosorbent assay-based format. The DNA enzyme immunoassay is based on the capture of PCR product by biotinylated probes fixed onto individual streptavidin-coated wells. Using this method, low levels of NV were detected in stool and both NLV and hepatitis A virus were detected in bivalve mollusks following bioaccumulation. The method also successfully detected NLV in oysters implicated in an outbreak of NLV gastroenteritis. This method dramatically decreases the time needed for analysis and is amenable to automation.
Transposon Mutagenesis in Purple Sulfur Photosynthetic Bacteria: Identification of
hypF
, Encoding a Protein Capable of Processing [NiFe] Hydrogenases in α, β, and γ Subdivisions of the Proteobacteria
Authors: Fodor Barna , Rákhely Gábor , Kovács Ákos T. , Kovács Kornél L.
Published: July 2002

A random transposon-based mutagenesis system was optimized for the purple sulfur phototrophic bacterium
Thiocapsa roseopersicina
BBS. Screening for hydrogenase-deficient phenotypes resulted in the isolation of six independent mutants in a mini-Tn
5
library. One of the mutations was in a gene showing high amino acid sequence similarity to HypF proteins in other organisms. Inactivation of hydrogen uptake activity in the
hypF
-deficient mutant resulted in a dramatic increase in the hydrogen evolution capacity of
T. roseopersicina
under nitrogen-fixing conditions. This mutant is therefore a promising candidate for use in practical biohydrogen-producing systems. The reconstructed
hypF
gene was able to complement the
hypF
-deficient mutant of
T. roseopersicina
BBS. Heterologous complementation experiments, using
hypF
mutant strains of
T. roseopersicina
,
Escherichia coli
, and
Ralstonia eutropha
and various
hypF
genes, were performed. They were successful in all of the cases tested, although for
E. coli
, the regulatory region of the foreign gene had to be replaced in order to achieve partial complementation. RT-PCR data suggested that HypF has no effect on the transcriptional regulation of the structural genes of hydrogenases in this organism.
Degradation of Chlorinated Dibenzofurans and Dibenzo-
p
-Dioxins by Two Types of Bacteria Having Angular Dioxygenases with Different Features
Authors: Habe Hiroshi , Chung Jin-Sung , Lee Jong-Hoon , Kasuga Kano , Yoshida Takako , Nojiri Hideaki , Omori Toshio
Published: July 2002

Two kinds of bacteria having different-structured angular dioxygenases—a dibenzofuran (DF)-utilizing bacterium,
Terrabacter
sp. strain DBF63, and a carbazole (CAR)-utilizing bacterium,
Pseudomonas
sp. strain CA10—were investigated for their ability to degrade some chlorinated dibenzofurans (CDFs) and chlorinated dibenzo-
p
-dioxins (CDDs) (or, together, CDF/Ds) using either wild-type strains or recombinant
Escherichia coli
strains. First, it was shown that CAR 1,9a-dioxygenase (CARDO) catalyzed angular dioxygenation of all mono- to triCDF/Ds investigated in this study, but DF 4,4a-dioxygenase (DFDO) did not degrade 2,7-diCDD. Secondly, degradation of CDF/Ds by the sets of three enzymes (angular dioxygenase, extradiol dioxygenase, and
meta
-cleavage compound hydrolase) was examined, showing that these enzymes in both strains were able to convert 2-CDF to 5-chlorosalicylic acid but not other tested substrates to the corresponding chlorosalicylic acid (CSA) or chlorocatechol (CC). Finally, we tested the potential of both wild-type strains for cooxidation of CDF/Ds and demonstrated that both strains degraded 2-CDF, 2-CDD, and 2,3-diCDD to the corresponding CSA and CC. We investigated the sites for the attack of angular dioxygenases in each CDF/D congener, suggesting the possibility that the angular dioxygenation of 2-CDF, 2-CDD, 2,3-diCDD, and 1,2,3-triCDD (10 ppm each) by both DFDO and CARDO occurred mainly on the nonsubstituted aromatic nuclei.
Induction of a Mitosis Delay and Cell Lysis by High-Level Secretion of Mouse α-Amylase from
Saccharomyces cerevisiae
Authors: Wang Bi-Dar , Kuo Tsong-Teh
Published: July 2002

Some foreign proteins are produced in yeast in a cell cycle-dependent manner, but the cause of the cell cycle dependency is unknown. In this study, we found that
Saccharomyces cerevisiae
cells secreting high levels of mouse α-amylase have elongated buds and are delayed in cell cycle completion in mitosis. The delayed cell mitosis suggests that critical events during exit from mitosis might be disturbed. We found that the activities of PP2A (protein phosphatase 2A) and MPF (maturation-promoting factor) were reduced in α-amylase-oversecreting cells and that these cells showed a reduced level of assembly checkpoint protein Cdc55, compared to the accumulation in wild-type cells. MPF inactivation is due to inhibitory phosphorylation on Cdc28, as a
cdc28
mutant which lacks an inhibitory phosphorylation site on Cdc28 prevents MPF inactivation and prevents the defective bud morphology induced by overproduction of α-amylase. Our data also suggest that high levels of α-amylase may downregulate
PPH22,
leading to cell lysis. In conclusion, overproduction of heterologous α-amylase in
S. cerevisiae
results in a negative regulation of PP2A, which causes mitotic delay and leads to cell lysis.
New Immunofluorescence Assays for Detection of
Human Herpesvirus 8
-Specific Antibodies
Authors: Inoue Naoki , Mar Eng-Chun , Dollard Sheila C. , Pau Chou-Pong , Zheng Qi , Pellett Philip E.
Published: July 2002

Several assays have been developed for detection of immunoglobulin G antibodies to
Human herpesvirus 8
(HHV-8), including immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). However, the specificity and sensitivity of these assays are not completely defined due to the lack of a “gold standard.” Although IFAs based on primary effusion lymphoma (PEL) cell lines are used widely, the assays can be confounded by nonspecific reactions against cellular components and potential cross-reaction with antibodies against other herpesviruses. To provide more reliable IFAs, we established recombinant Semliki Forest viruses (rSFVs) expressing the HHV-8-specific proteins ORF73 and K8.1 and used BHK-21 cells infected with these rSFVs for IFA (ORF73-IFA and K8.1-IFA). Expression of the HHV-8-specific proteins at very high levels by the rSFV system allowed easy scoring for IFA and thereby increased specificity. The rSFV system also allowed detection of antibodies against glycosylation-dependent epitopes of K8.1. Titers measured by rSFV-based IFAs and PEL-based IFAs correlated well (correlation coefficients of >0.9), and concordances of seroreactivities between rSFV-based and PEL-based IFAs were >97% (κ > 0.93). K8.1-IFA was more sensitive than either ORF73-IFA or peptide ELISAs. Using PEL-based lytic IFA as a reference assay, the sensitivity and specificity of K8.1-IFA were estimated to be 94 and 100%, respectively. HHV-8 prevalences determined by K8.1-IFA among the human immunodeficiency virus (HIV)-positive (HIV
+
) Kaposi's sarcoma (KS) patients, HIV
+
KS
−
patients, and healthy controls were 100, 65, and 6.7%, respectively, which were consistent with prior reports. Therefore, our rSFV-based IFAs may provide a specific and sensitive method for use in epidemiology studies. In addition, they will provide a basis for further development of diagnostic tests for HHV-8 infection.
Effects of Androgen Treatment on Expression of Macrophage Fcγ Receptors
Authors: Gomez F. , Ruiz P. , Lopez R. , Rivera C. , Romero S. , Bernal J. A.
Published: July 2002

Macrophage Fcγ receptors (FcγRs) play an important role in the host defense against infection and in the pathophysiology of immune cytopenias. Modulation of macrophage FcγR expression is a potential therapeutic approach to immune disorders. Glucocorticoids and progesterones decrease macrophage FcγR expression. We assessed the effect of treatment with androgens and antiandrogens on the expression of macrophage FcγRs using an experimental guinea pig model. Four androgens (testosterone, dihydrotestosterone, mesterolone, and danazol) and five antiandrogens (flutamide, nilutamide, cyproterone acetate, spironolactone, and finasteride) were studied. Following in vivo treatment of guinea pigs, we determined the clearance of immunoglobulin G (IgG)-sensitized erythrocytes in vivo, the binding of IgG-sensitized erythrocytes by isolated splenic macrophages, and splenic macrophage FcγR cell surface expression. All of the androgens impaired the clearance of IgG-sensitized erythrocytes by decreasing splenic macrophage FcγR expression. Dihydrotestosterone and mesterolone were more effective than testosterone or dihydrotestosterone. Flow cytometry and fluorescence microscopy with monoclonal antibodies demonstrated that the androgens decreased the cell surface expression of FcγR1,2 more than that of FcγR2. Antiandrogens did not significantly alter macrophage FcγR expression. Nevertheless, antiandrogens counteracted the effects of androgens on macrophage FcγR expression. These data indicate that androgens impair the clearance of IgG-coated cells by decreasing splenic macrophage FcγR expression. Thus, androgens other than danazol are candidate drugs for the treatment of immune disorders.
Decreased Levels of CD54 (ICAM-1)-Positive Lymphocytes in the Peripheral Blood in Untreated Patients with Active Juvenile Dermatomyositis
Authors: O'Gorman Maurice R. G. , Bianchi Laura , Zaas David , Corrochano Virginia , Pachman Lauren M.
Published: July 2002

Significant abnormalities are observed in the peripheral blood of juvenile dermatomyositis (JDM) patients with active disease. In this study, we confirm that there is a significant increase in the relative percentage of B lymphocytes in the peripheral blood of a group of untreated children with newly diagnosed active JDM compared to healthy children (P< 0.0001). In order to investigate if properties intrinsic to B cells contributed to their relative increase in JDM, the percentage of B cells expressing activation markers (CD23, CD25, CD54, and CD69) was measured and compared to pediatric controls. Compared to healthy children less than 10 years of age (not significantly different from the JDM group), the JDM patients had an increase in the proportion of lymphocytes expressing CD19 (B cells;P= 0.0017) and decreases in the percentage of lymphocytes that were CD3−CD16+and/or CD56+(NK cells;P= 0.01) and CD3+CD8+(T suppressor/cytotoxic cells;P= 0.02). There were no significant differences in any of the B-cell activation markers assessed. Of note, the percentage of CD54+non-B lymphocytes (i.e., T cells and NK cells expressing CD54) was significantly lower in the JDM patients (25% ± 5%) than in the “age-related” healthy control group (43% ± 4%;P= 0.013). These results suggest the following for untreated children with active JDM: (i) the increase in the percentage of peripheral blood B cells is not due to intrinsic B-cell activation, and (ii) CD54/ICAM-1+non-B cells, CD8+T cells, and NK cells are being removed from circulation and may be participating in the pathophysiology of the disease.
Up-Regulation of CD40 Ligand and Induction of a Th2 Response in Children Immunized with Pneumococcal Polysaccharide Vaccines
Authors: Leiva Lily E. , Butler Boyd , Hempe James , Ortigas Alejandro P. , Sorensen Ricardo U.
Published: July 2002

We wished to determine whether pneumococcal polysaccharide antigens induce mRNA expression of CD40 ligand (CD40L) and Th1 or Th2 cytokines in unimmunized individuals in vitro and whether immunization with the 23-valent pneumococcal polysaccharide vaccine induces changes in CD40L and cytokine mRNA expression. Children with recurrent respiratory infections were studied before and 4 to 6 weeks after receiving the pneumococcal vaccine. One patient who failed to respond to the polysacharide vaccine subsequently received a single dose of the experimental 7-valent pneumococcal conjugate vaccine. Unimmunized healthy adults were included as controls. Quantification of mRNA expression of CD40L, interleukin-4 (IL-4), IL-12p40, and gamma interferon (IFN-γ) was performed by reverse transcription-PCR and enzyme-linked immunosorbent assay (ELISA)-PCR with resting and stimulated peripheral blood mononuclear cells. Serum immunoglobulin G (IgG) anti pneumococcal antibody levels were measured by ELISA. The results showed a significant increase in the expression of mRNAs for CD40L and IL-4, but not IL-12p40 or IFN-γ, in stimulated cultures from unimmunized individuals. CD40L and IL-4 mRNA expression was significantly higher in postimmunization than in preimmunization samples stimulated with the individual pneumococcal serotypes. These results suggest that pneumococcal polysaccharide antigens specifically up-regulate CD40L expression and induce a Th2 response in vitro which parallels the increase in IgG antipneumococcal antibody levels in serum.
Immunity to MurineChlamydia trachomatisGenital Tract Reinfection Involves B Cells and CD4+T Cells but Not CD8+T Cells
Authors: Morrison Sandra G. , Su Hua , Caldwell Harlan D. , Morrison Richard P.
Published: July 2002

CD4+T-helper type 1 (Th1) responses are essential for the resolution of a primaryChlamydia trachomatisgenital tract infection; however, elements of the immune response that function in resistance to reinfection are poorly understood. Defining the mechanisms of immune resistance to reinfection is important because the elements of protective adaptive immunity are distinguished by immunological memory and high-affinity antigen recognition, both of which are crucial to the development of efficacious vaccines. Using in vivo antibody depletion of CD4+and CD8+T cells prior to secondary intravaginal challenge, we identified lymphocyte populations that functioned in resistance to secondary chlamydial infection of the genital tract. Depletion of either CD4+or CD8+T cells in immune wild-type C57BL/6 mice had a limited effect on resistance to reinfection. However, depletion of CD4+T cells, but not CD8+T cells, in immune B-cell-deficient mice profoundly altered the course of secondary infection. CD4-depleted B-cell-deficient mice were unable to resolve a secondary infection, shed high levels of infectious chlamydiae, and did not resolve the infection until 3 to 4 weeks following the discontinuation of anti-CD4 treatment. These findings substantiated a predominant role for CD4+T cells in host resistance to chlamydial reinfection of the female genital tract and demonstrated that CD8+T cells are unnecessary for adaptive immune resistance. More importantly, however, this study establishes a previously unrecognized but very significant role for B cells in resistance to chlamydial reinfection and suggests that B cells and CD4+T cells may function synergistically in providing immunity in this model of chlamydial infection. Whether CD4+T cells and B cells function independently or dependently is unknown, but definition of those mechanisms is fundamental to understanding optimum protective immunity and to the development of highly efficacious immunotherapies against chlamydial urogenital infections.
Complement Activation inMycoplasma fermentans-Induced Mycoplasma Clearance from Infected Cells: Probing of the Organism with Monoclonal Antibodies against M161Ag
Authors: Kikkawa Satomi , Matsumoto Misako , Sasaki Tsuguo , Nishiguchi Miyuki , Tanaka Kazuhiko , Toyoshima Kumao , Seya Tsukasa
Published: July 2002

Mycoplasma fermentans, a cell wall-less prokaryote, is capable of infecting humans and has been suggested to serve as a cofactor in AIDS development. Recently, we discovered a novel lipoprotein with a molecular mass of 43 kDa originating fromM. fermentans. This protein, named M161Ag, activated human complement via the alternative pathway and efficiently induced the proinflammatory cytokines interleukin 1β (IL-1β), tumor necrosis factor alpha, IL-6, IL-10, and IL-12 in human peripheral blood monocytes. It is likely that M161Ag ofM. fermentansaffects the host immune system upon mycoplasma infection. In this study, we developed monoclonal antibodies (MAbs) against M161Ag and examined the direct role of complement inM. fermentansinfection using these MAbs as probes.M. fermentanswas rapidly cleared from the surfaces of infected cells by human complement, but a low-grade infection persisted in human tumor cell lines. Mycoplasma particles remaining alive in host cells may cause recurrent infection, and liberated M161Ag may serve as a biological response modifier affecting both innate and acquired immunity.
Eradication ofCryptosporidium parvumInfection by Mice with Ovalbumin-Specific T Cells
Authors: Lukin Kara , Cosyns Mary , Mitchell Tom , Saffry Milton , Hayward Anthony
Published: July 2002

CD154 is necessary for mice to clear aCryptosporidium parvuminfection, but whether this ligand has to be expressed on T cells with specificity forC. parvumhas not been determined. We infected DO11.10 (ovalbumin specific) T-cell receptor transgenic mice that had been bred to a RAG−/−background withC. parvumand found that the infection was cleared within 6 weeks, while RAG−/−controls were unable to clearC. parvuminfection. Recovery was accompanied by an increase in the number of splenic T cells with the CD44highphenotype that characterizes memory cells. To determine whether aC. parvum-infected environment sufficed to activate transgenic T cells, we reconstitutedC. parvum-infected BALB/c SCID mice with DO11.10 RAG−/−splenocytes. Fecal excretion ofC. parvumantigen ceased in the 12 weeks following the adoptive transfer, unless the mice were also injected with tolerizing doses of ovalbumin. DO11.10 T cells were found in the submucosa ofC. parvum-infected, but not uninfected, BALB/c SCID hosts within 48 h of injection. The transferred DO11.10 T cells divided and acquired a CD44highmemory phenotype inC. parvum-infected, but not uninfected, recipients. DO11.10 splenocytes from CD154 knockout donors failed to clear aC. parvuminfection, confirming a requirement for CD154 in recovery. In vitro, the DO11.10 cells did not proliferate in response toC. parvumantigen, and a tBlast GenBank search revealed no matches between the ovalbumin peptide andC. parvumDNA sequences.C. parvum-infected SCID mice given RAG−/−CD8+T cells with aListeria-specific transgene did not recover fromC. parvuminfection. Our data suggest that antigen-nonspecific CD4+T-cell effector mechanisms in combination with the innate arm of the immune system are sufficient for the eradication ofC. parvuminfection.
Investigation into the Mechanism of Gamma Interferon-Mediated Inhibition ofToxoplasma gondiiin Murine Astrocytes
Authors: Halonen Sandra K. , Weiss Louis M.
Published: July 2002

Toxoplasma gondiiis an obligate intracellular parasite that is a common opportunistic pathogen of the central nervous system in AIDS patients. Gamma interferon (IFN-γ) alone or in combination with interleukin-1 (IL-1), IL-6, or tumor necrosis factor alpha significantly inhibits the growth ofT. gondiiin murine astrocytes, suggesting these are important nonimmune effector cells in the brain. Inhibition was found to be independent of a nitric oxide-mediated or tryptophan starvation mechanism. Both reactive oxygen intermediates and iron deprivation are IFN-γ-mediated mechanisms known to operate against intracellular parasites in other cell types. Astrocytes generated from mice genetically deficient in the production of reactive oxygen intermediates (phox−/−mice) were found to inhibit growth ofT. gondiiwhen stimulated with IFN-γ alone or in combination with other cytokines. The reactive oxygen inhibitor catalase and the reactive oxygen scavengers mannitol and thiourea failed to reverse the IFN-γ-induced inhibition ofT. gondiiin astrocytes. These data indicate that IFN-γ-induced inhibition in astrocytes is independent of reactive oxygen intermediates. IFN-γ-induced inhibition could not be reversed by the addition of iron salts, ferric citrate, ferric nitrate, or ferric transferrin. Pretreatment of astrocytes with desferrioxamine also did not induce the inhibition ofT. gondii. These data indicate that the mechanism of IFN-γ inhibition was not due to iron deprivation. IFN-γ had no effect onT. gondiiinvasion of astrocytes, but inhibition of growth and loss of tachyzoite vacuoles were evident in IFN-γ-treated astrocytes by 24 h after invasion. Overall, these data suggest that IFN-γ-activated astrocytes inhibitT. gondiiby an as-yet-unknown mechanism.
The
opdB
Locus Encodes the Trypsin-Like Peptidase Activity of
Treponema denticola
Authors: Fenno J. Christopher , Lee Si Young , Bayer Christopher H. , Ning Yu
Published: July 2002

High levels of
Treponema denticola
in subgingival dental plaque are associated with severe periodontal disease.
T. denticola
, along with
Porphyromonas gingivalis
and
Bacteroides forsythus
, are the only cultivatable oral microorganisms that produce significant amounts of “trypsin-like” peptidase activity. The ability of subgingival plaque to hydrolyze
N
-α-benzoyl-
dl
-arginine-2-naphthylamide (BANA) is associated with high levels of one or more of these organisms. The purpose of this study was to identify the gene encoding trypsin-like activity in
T. denticola
and thus facilitate molecular-level studies of its potential role in disease. Using published peptide sequences of a
T. denticola
surface-associated oligopeptidase with BANA-hydrolyzing activity, we identified the gene, designated
opdB
, in an apparently noncoding region of the
T. denticola
genome unannotated contigs (11/2000;
http://www.tigr.org
). The
opdB
gene begins with a TTG start codon and encodes a 685-residue peptide with high homology to the oligopeptidase B family in prokaryotes and eukaryotes. An isogenic
T. denticola opdB
mutant was constructed by allelic replacement mutagenesis using an
ermF/AM
gene cassette. The mutant lacked BANA-hydrolyzing activity and had a slightly slower growth rate than the parent strain. This mutant will be used in future studies of interactions of
T. denticola
with host cells and tissue.
Attenuation of Virulence and Changes in Morphology in
Candida albicans
by Disruption of the
N
-Acetylglucosamine Catabolic Pathway
Authors: Singh Praveen , Ghosh Sharmistha , Datta Asis
Published: July 2002

A
Candida albicans
mutant with mutations in the
N
-acetylglucosamine (GlcNAc) catabolic pathway gene cluster, including the GlcNAc-6-phosphate deacetylase (
DAC1
), glucosamine-6-phosphate deaminase (
NAG1
), and GlcNAc kinase (
HXK1
) genes, was not able to grow on amino sugars, exhibited highly attenuated virulence in a murine systemic candidiasis model, and was less adherent to human buccal epithelial cells in vitro. No germ tubes were formed by the mutant after induction with GlcNAc, but the mutant exhibited hyperfilamentation under stress-induced filamentation conditions. In addition, the GlcNAc catabolic pathway played a vital role in determining the colony phenotype. Our results imply that this pathway is very important because of its diverse links with pathways involved in virulence and morphogenesis of the organism.
Novel Molecular Variants of Allele I of the
Escherichia coli
P Fimbrial Adhesin Gene
papG
Authors: Johnson James R. , Stell Adam L. , Kaster Nicholas , Fasching Claudine , O'Bryan Timothy T.
Published: July 2002

P fimbriae of extraintestinal pathogenic
Escherichia coli
mediate digalactoside-specific adherence via the tip adhesin molecule PapG, which occurs in three known variants (I to III), which are encoded by the corresponding three alleles of
papG
. In the present study, newly discovered variants of
papG
allele I and the respective wild-type source strains were characterized. One of the new
papG
allele I variants conferred a unique agglutination phenotype that combined the phenotypes associated with
papG
alleles I, II, and III. Comparative hydrophilicity analysis of predicted PapG peptides revealed regions that might explain the observed phenotypic similarities and differences between the PapG variants. The new
papG
allele I variants occurred either as the sole
papG
allele or together with both
papG
alleles II and III, rather than with only
papG
allele III, as in archetypal strains J96 and CP9. They also occurred in the absence of the usual F13
papA
allele. One of the new
papG
allele I variants occurred in a serogroup O6 strain that, according to random amplified polymorphic DNA analysis, was phylogenetically distant from the “J96-like” clonal group of
E. coli
O4:H5, which includes all previously identified examples of
papG
allele I. Cluster analysis of nucleotide and predicted peptide sequences suggested that
papG
allele I represents the earliest evolutionary branch from a common
papG
ancestor. These results demonstrate unexpected diversity within
papG
allele I and, together with previous findings, suggest that the J96-like clonal group of
E. coli
O4:H5 may represent the original source of
papG
within the species.
CD14 Is Expressed and Released as Soluble CD14 by Human Intestinal Epithelial Cells In Vitro: Lipopolysaccharide Activation of Epithelial Cells Revisited
Authors: Funda David P. , Tučková Ludmila , Farré Maria A. , Iwase Takashi , Moro Itaru , Tlaskalová-Hogenová Helena
Published: July 2002

Human endothelial as well as epithelial cells were shown to respond to lipopolysaccharides (LPSs). However, the expression and release of CD14 by these so-called CD14-negative cells have not been studied in detail. We investigated three human intestinal epithelial cell lines (ECLs), SW-480, HT-29, and Caco-2, for their expression of CD14 and CD11c/CD18 as well as their responsiveness to endotoxins. Fluorescence-activated cell sorter analysis revealed no expression of CD11c/CD18, but there was low expression of membrane-bound CD14 on HT-29, Caco-2, and SW-480 ECLs. Both Western blotting and reverse transcription-PCR confirmed the CD14 positivity of all three intestinal ECLs. No substantial modulation of CD14 expression was achieved after 6, 8, 18, 24, and 48 h of cultivation with 10-fold serial dilutions of LPS ranging from 0.01 ng/ml to 100 μg/ml. Interestingly, soluble CD14 was found in the tissue culture supernatants of all three ECLs. Finally, only HT-29 and SW-480, and not Caco-2, cells responded to LPS exposure (range, 0.01 ng/ml to 100 μg/ml) by interleukin 8 release. Thus, we show that HT-29, SW-480, and Caco-2 human intestinal ECLs express membrane-bound CD14. As Caco-2 cells did not respond to LPS, these cell lines might be an interesting model for studying the receptor complex for LPS. The fact that human intestinal epithelial cells are capable not only of expression but also of release of soluble CD14 may have important implications in vivo, e.g., in shaping the interaction between the mucosal immune system and bacteria in the gut and/or in the pathogenesis of endotoxin shock.
Potent Stimulation of the Innate Immune System by aLeishmania brasiliensisRecombinant Protein
Authors: Borges Monamaris M. , Campos-Neto Antonio , Sleath Paul , Grabstein Keneth H. , Morrissey Philip J. , Skeiky Yasir A. W. , Reed Steven G.
Published: July 2002

The interaction of the innate immune system with the microbial world involves primarily two sets of molecules generally known as microbial pattern recognition receptors and microbial pattern recognition molecules, respectively. Examples of the former are the Toll receptors present particularly in macrophages and dendritic cells. Conversely, the microbial pattern recognition molecules are conserved protist homopolymers, such as bacterial lipopolysaccharides, lipoteichoic acids, peptidoglycans, glucans, mannans, unmethylated bacterial DNA, and double-strand viral RNA. However, for protists that lack most of these molecules, such as protozoans, the innate immune system must have evolved receptors that recognize other groups of microbial molecules. Here we present evidence that a highly purified protein encoded by aLeishmania brasiliensisgene may be one such molecule. This recombinant leishmanial molecule, a homologue of eukaryotic ribosomal elongation and initiation factor 4a (LeIF), strongly stimulates spleen cells from severe combined immunodeficient (SCID) mice to produce interleukin-12 (IL-12), IL-18, and high levels of gamma interferon. In addition, LeIF potentiates the cytotoxic activity of the NK cells of these animals. Because LeIF is a conserved molecule and because SCID mice lack T and B lymphocytes but have a normal innate immune system (normal reticuloendothelial system and NK cells), these results suggest that proteins may also be included as microbial pattern recognition molecules. The nature of the receptor involved in this innate recognition is unknown. However, it is possible to exclude the Toll receptor Tlr4 as a putative LeIF receptor because the gene encoding this receptor is defective in C3H/HeJ mice, the mouse strain used in the present studies.
Ontogeny of Th1 Memory Responses against aBrucella abortusConjugate
Authors: Scharf Orit , Agranovich Inna , Lee Katherine , Eller Nancy L. , Levy Lily , Inman John , Scott Dorothy E. , Golding Basil
Published: July 2002

Protective immune responses to intracellular pathogens such asBrucella abortusare characteristically Th1-like. Recently we demonstrated that heat-killedB. abortus(HKBa), a strong Th1 stimulus, conjugated to ovalbumin (HKBA-OVA), but notB. abortusalone, can alter the antigen-specific cytokine profile from Th2- to Th1-like. In this report we study the ability of a single injection ofB. abortusto switch a Th2 to a Th1 response in immature mice. One-day- and 1-week-old mice were given a single injection ofB. abortusin the absence or presence of OVA, and at maturity mice were challenged with an allergenic preparation, OVA with alum (OVA-A).B. abortusgiven without OVA did not diminish the subsequent Th2 response in either age group. In contrast, mice receiving a single injection ofB. abortus-OVA at the age of 1 week, but not those injected at the age of 1 day, had reversal of the ratio of OVA-specific Th1 to Th2 cells and decreased immunoglobulin E levels after allergen challenge as adults. Within 6 h both 1-day- and 1-week-old mice expressed interleukin-12 p40 mRNA following eitherB. abortusorB. abortus-OVA administration. However, only the 1-week-old mice exhibited increased expression of gamma interferon (IFN-γ) mRNA. The absence of the early IFN-γ response in 1-day-old mice may explain their inability to generate a Th1 memory response. These results suggest that at early stages of immune development, responses to intracellular bacteria may be Th2- rather than Th1-like. Furthermore, they suggest that the first encounter with antigen evokes either a Th1- or a Th2-like response which becomes imprinted, so that subsequent memory responses conform to the original Th bias. This has implications for protection against infectious agents and development of allergic responses.