bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Small-Molecule Inhibitors of Nucleoside Triphosphate Hydrolase in
Toxoplasma gondii
Authors: Asai Takashi , Takeuchi Tsutomu , Diffenderfer Jeff , Sibley L. David
Published: July 2002

Approximately 150,000 small-molecule compounds were tested by a robotic screening assay for their ability to inhibit nucleoside triphosphate hydrolase (NTPase), a novel enzyme of the tachyzoite form of
Toxoplasma gondii
. Five unrelated species of compounds were found to inhibit the activities of both NTPase isoforms (NTPase isoform I [NTPase-I] and NTPase-II). The 50% inhibitory concentrations (IC
50
s) ranged from 0.1 to 20 μM, and in general, the IC
50
s were similar for both NTPase isoforms. However, the activity of NTPase-I was 20 times more sensitive than the activity of NTPase-II to one of the inhibitors: 9-hydroxy-10-(pentachlorophenoxy)stearic acid. The five compounds identified also prevented tachyzoite replication in vitro, with IC
50
s ranging from ∼7 to ≥50 μM. The most effective of these initial compounds, 2-phenylthio-indole, was used to identify six additional, structurally related compounds, which were tested for their inhibitory effects on enzyme activities and tachyzoite replication. Surprisingly, these compounds were competitive inhibitors of NTPase-I but noncompetitive inhibitors of NTPase-II. Modifications to the indole and phenol rings resulted in alterations of activity, thus providing insight into the structural features that are important for inhibition of
T. gondii
NTPases.
Mutations in Cytochrome
b
Resulting in Atovaquone Resistance Are Associated with Loss of Fitness in
Plasmodium falciparum
Authors: Peters Jennifer M. , Chen Nanhua , Gatton Michelle , Korsinczky Michael , Fowler Elizabeth V. , Manzetti Sergio , Saul Allan , Cheng Qin
Published: July 2002

Drug resistance in malarial parasites has become a major obstacle in the control of the disease. Strategies are urgently needed to control the development of resistance and to possibly reverse existing resistance. One key element required to reverse malaria drug resistance is for the parasites to “pay” a biological “cost” or suffer a loss of fitness when acquiring resistance to antimalarial drugs. Such a situation would be a disadvantage to the resistant parasites in the absence of drug pressure. We compared here the relative fitness of atovaquone-resistant
Plasmodium falciparum
K1 clones with single and double base mutations in their cytochrome
b
genes to their parent clones during erythrocytic stages in the absence of drug pressure. We found that the double amino acid mutation (M133I and G280D) is associated with a 5 to 9% loss of fitness and that the single amino acid change of M133I did not result in any detectable loss of fitness. Molecular modeling of the interaction of
P. falciparum
cytochrome
b
with ubiquinone led to the prediction that a loss of fitness of the malaria parasites would result from the G280D mutation due to its close proximity to the putative ubiquinone-binding site. This appears to have resulted in a weakening of the cytochrome
b
-ubiquinone complex, thereby causing the electron transport chain to become less efficient. Our results suggest that the prevalence of resistant parasites may decrease after the drug usage is discontinued.
Immunomodulatory and Protective Effects of Moxifloxacin againstCandida albicans-Induced Bronchopneumonia in Mice Injected with Cyclophosphamide
Authors: Shalit Itamar , Horev-Azaria Limor , Fabian Ina , Blau Hannah , Kariv Naam , Shechtman Itsak , Alteraz Hannah , Kletter Yehudith
Published: July 2002

In a previous study, moxifloxacin was shown to ameliorate immunosuppression and enhance cytokine production in several tissues, including the lungs of cyclophosphamide-injected mice. We examined here the effects of moxifloxacin onCandida albicanslung infection in cyclophosphamide-injected mice. Mice were injected on day 0 with 250 mg of cyclophosphamide/kg, and on days 1 to 4 they were given moxifloxacin at 22.5 mg/kg/day compared to controls given ceftazidime at 75 mg/kg/day or saline. On day 6,C. albicans(10 7 CFU/mouse) was inoculated intratracheally, and animals were observed for the development of bronchopneumonia, weight loss, mortality, the presence ofC. albicans, and lung cytokine production. Histopathology on day 10 postinoculation revealed bronchopneumonia in 50, 67, and 0% of saline-, ceftazidime-, and moxifloxacin-treated mice, respectively (P < 0.05). The mortality rates were 28, 17, and 5%, respectively (P < 0.05), and weight loss occurred at 20, 32, and 0%, respectively (P < 0.05). By day 15,C. albicanswas eliminated from all moxifloxacin-treated mice but was still isolated from lung homogenates of 50 to 60% of the saline- and ceftazidime-treated groups. Among the cytokines tested on days 0 to 15, we found an increased production of tumor necrosis factor alpha, KC (functional interleukin-8), and gamma interferon in the lungs of ceftazidime- and saline-treated controls compared to the moxifloxacin pretreatment that abolished their secretion. In conclusion, moxifloxacin protected cyclophosphamide-injected mice fromC. albicans-induced lung infection and significantly reduced pneumonia, weight loss, and mortality despite the lack of direct antifungal activity. This is most likely due to an immunomodulating activity conferred by moxifloxacin, as shown in this model and in our previous studies. Its potential protective role should be studied in patients undergoing chemotherapy and immune suppression.
Overexpression of Sbe2p, a Golgi Protein, Results in Resistance to Caspofungin in
Saccharomyces cerevisiae
Authors: Osherov Nir , May Gregory S. , Albert Nathaniel D. , Kontoyiannis D. P.
Published: July 2002

Caspofungin inhibits the synthesis of 1, 3-β-
d
-glucan, an essential cell wall target in fungi. Genetic studies in the model yeast
Saccharomyces cerevisiae
have shown that mutations in
FKS1
and
FKS2
genes result in caspofungin resistance. However, direct demonstration of the role of gene overexpression in caspofungin resistance has been lacking. We transformed wild-type
S. cerevisiae
with an
S. cerevisiae URA3
-based
GAL1
cDNA library and selected transformants in glucose synthetic complete plates lacking uracil (glucose SC minus uracil plates). We then moved the transformants to galactose SC minus uracil plates containing caspofungin (1 μg/ml) and looked for caspofungin-resistant colonies. We retested the candidates (true positives were sensitive on glucose caspofungin and resistant on galactose caspofungin media, respectively). We identified 16 caspofungin-resistant candidates. Restriction analysis and hybridization confirmed that 15 of the 16 clones were identical. We sequenced one of the cDNA clones and found that it contained the cDNA for
SBE2. SBE2
has been described in
S. cerevisiae
to encode a Golgi protein involved in the transport of cell wall components (B. Santos and M. Snyder, Mol. Biol. Cell,
11:
435-452, 2000). The
SBE2
cDNA plasmid conferred again galactose-dependent caspofungin resistance when transformed back into the wild-type
S. cerevisiae
. Finally, the
SBE2
deletion mutant was hypersensitive to caspofungin. In conclusion, overexpression of Sbe2p under the regulated control of the
GAL1
promoter results in caspofungin resistance in
S. cerevisiae
. This transport pathway may provide insight into the tolerance or lack of sensitivity to caspofungin of some pathogenic fungi.
Efficacies of Entecavir against Lamivudine-Resistant Hepatitis B Virus Replication and Recombinant Polymerases In Vitro
Authors: Levine S. , Hernandez D. , Yamanaka G. , Zhang S. , Rose R. , Weinheimer S. , Colonno R. J.
Published: July 2002

Entecavir (ETV) is a potent and selective inhibitor of hepatitis B virus (HBV) replication in vitro and in vivo that is currently in clinical trials for the treatment of chronic HBV infections. A major limitation of the current HBV antiviral therapy, lamivudine (3TC), is the emergence of drug-resistant HBV in a majority of treated patients due to specific mutations in the nucleotide binding site of HBV DNA polymerase (HBV Pol). To determine the effects of 3TC resistance mutations on inhibition by ETV triphosphate (ETV-TP), a series of in vitro studies were performed. The inhibition of wild-type and 3TC-resistant HBV Pol by ETV-TP was measured using recombinant HBV nucleocapsids, and compared to that of 3TC-TP. These enzyme inhibition studies demonstrated that ETV-TP is a highly potent inhibitor of wild-type HBV Pol and is 100- to 300-fold more potent than 3TC-TP against 3TC-resistant HBV Pol. Cell culture assays were used to gauge the potential for antiviral cross-resistance of 3TC-resistant mutants to ETV. Results demonstrated that ETV inhibited the replication of 3TC-resistant HBV, but 20- to 30-fold higher concentrations were required. To gain further perspective regarding the potential therapeutic use of ETV, its phosphorylation was examined in hepatoma cells treated with extracellular concentrations representative of drug levels in plasma in ETV-treated patients. At these concentrations, intracellular ETV-TP accumulated to levels expected to inhibit the enzyme activity of both wild-type and 3TC-resistant HBV Pol. These findings are predictive of potent antiviral activity of ETV against both wild-type and 3TC-resistant HBV.
Antileishmanial Activity of the Antiulcer Agent Omeprazole
Authors: Jiang Suping , Meadows Juliana , Anderson Steven A. , Mukkada Antony J.
Published: July 2002

The benzimidazole compound omeprazole, used widely for the treatment of peptic ulcer disease, inhibits the growth of
Leishmania donovani
, the causative agent of visceral leishmaniasis. Promastigotes cultured at acidic pH and amastigotes within infected macrophages are reduced 90% or more with 150 μM omeprazole. Antiparasitic action of the drug is due to its inhibition of the P-type K
+
,H
+
-ATPase on the surface membrane. This enzyme is important for pH homeostasis and the maintenance of proton motive force across the membrane in
Leishmania.
The drug is effective only at acidic pH, a condition that mimics the in vivo environment within the phagolysosomal vesicles where the amastigote form of the parasite resides. Omeprazole deserves consideration as an alternative to currently available chemotherapeutics, which have severe toxic side effects.
Ciprofloxacin in Polyethylene Glycol-Coated Liposomes: Efficacy in Rat Models of Acute or Chronic
Pseudomonas aeruginosa
Infection
Authors: Bakker-Woudenberg Irma A. J. M. , ten Kate Marian T. , Guo Luke , Working Peter , Mouton Johan W.
Published: July 2002

In a previous study in experimental
Klebsiella pneumoniae
pneumonia, the therapeutic potential of ciprofloxacin was significantly improved by encapsulation in polyethylene glycol-coated (“pegylated”) long-circulating (STEALTH) liposomes. Pegylated liposomal ciprofloxacin in high doses was nontoxic and resulted in relatively high and sustained ciprofloxacin concentrations in blood and tissues, and hence an increase in the area under the plasma concentration-time curve (AUC). These data correspond to data from animal and clinical studies showing that for fluoroquinolones the AUC/MIC ratio is associated with favorable outcome in serious infections. Clinical failures and the development of resistance are observed for marginally susceptible organisms like
Pseudomonas aeruginosa
and for which sufficient AUC/MIC ratios cannot be achieved. In the present study the therapeutic efficacy of pegylated liposomal ciprofloxacin was investigated in two rat models of
Pseudomonas aeruginosa
pneumonia. In the acute model pneumonia developed progressively, resulting in a rapid onset of septicemia and a high mortality rate. Ciprofloxacin twice daily for 7 days was not effective at doses at or below the maximum tolerated dose (MTD). However, pegylated liposomal ciprofloxacin either at high dosage or given at low dosage in combination with free ciprofloxacin on the first day of treatment was fully effective (100% survival). Obviously, prolonged concentrations of ciprofloxacin in blood prevented death of the animals due to early-stage septicemia in this acute infection. However, bacterial eradication from the left lung was not effected. In the chronic model, pneumonia was characterized by bacterial persistence in the lung without bacteremia, and no signs of morbidity or mortality were observed. Ciprofloxacin administered for 7 days at the MTD twice daily resulted in killing of more than 99% of bacteria in the lung; this result can also be achieved with pegylated liposomal ciprofloxacin given once daily. Complete bacterial eradication is never observed.
Three-Dimensional Quantitative Structure-Activity Relationship and Comparative Molecular Field Analysis of Dipeptide Hydroxamic Acid
Helicobacter pylori
Urease Inhibitors
Authors: Mishra Hetal , Parrill Abby L. , Williamson John S.
Published: July 2002

A homology model of
Helicobacter pylori
urease was developed by using the crystal structure of urease from
Klebsiella aerogenes
(EC 3.5.1.5) as a template. The acetohydroxamic acid moiety was docked into the active pocket of the enzyme model, followed by relaxation of the complex by use of molecular dynamics. The resulting conformation was used as a template to construct 24 potential dipeptide hydroxamic acid inhibitors with which comparative molecular field analysis (CoMFA) was performed. The resulting model provided a cross-validation correlation coefficient (
q
2
L00
) of 0.610, a conventional
r
2
value of 0.988, and an
F
(Fisher indication of statistical significance) value of 294.88. We were able to validate the CoMFA model by using the 50% inhibitory concentrations of six compounds that were not included in the construction of the model. A very good structural correlation was observed between the amino acids in the model urease's active pocket and the contour maps derived from the CoMFA model. This correlation, accompanied by the validation supplied by use of the CoMFA data, illustrates that the model can aid in the prediction and design of novel
H. pylori
urease inhibitors.
Salicylate Reduces Susceptibility of
Mycobacterium tuberculosis
to Multiple Antituberculosis Drugs
Authors: Schaller Alain , Sun Zhonghe , Yang Yongping , Somoskovi Akos , Zhang Ying
Published: July 2002

Salicylate induces multiple antibiotic resistance in various bacterial species. Here we investigated the effect of salicylate on the susceptibility of
Mycobacterium tuberculosis
to a range of antituberculosis (anti-TB) drugs. In the presence of salicylate, the killing effects of isoniazid (INH), rifampin (RMP), ethambutol (EMB), streptomycin (STR), and
p
-aminosalicylate (PAS) were reduced, as shown with a tetrazolium redox dye viability assay and a bacterial survival assay. Salicylate-induced resistance was more pronounced for PAS, STR, and EMB but was not apparent for INH and RMP when salicylate and the anti-TB agents were incorporated into 7H11 plates. The significance of these findings for TB treatment needs to be further evaluated in vivo.
Resazurin Microtiter Assay Plate: Simple and Inexpensive Method for Detection of Drug Resistance in
Mycobacterium tuberculosis
Authors: Palomino Juan-Carlos , Martin Anandi , Camacho Mirtha , Guerra Humberto , Swings Jean , Portaels Françoise
Published: July 2002

A method for detecting multidrug-resistant
Mycobacterium tuberculosis
by using a reduction of resazurin is described. Eighty clinical isolates were evaluated against isoniazid and rifampin; results at 7 days were compared with those of the proportion method. Specificity and sensitivity were excellent. The method is simple, inexpensive, and rapid and might be used with other antituberculosis drugs.
Airborne Microbial Flora in a Cattle Feedlot
Authors: Wilson S. C. , Morrow-Tesch J. , Straus D. C. , Cooley J. D. , Wong W. C. , Mitlöhner F. M. , McGlone J. J.
Published: July 2002

A total of 1,408 cattle held in eight commercial feedlot pens were used to examine the quantity and diversity of microorganisms in cattle feedlot air. The effect of two feeding patterns on the generation of airborne dust and the total numbers of microorganisms was also examined (four feedlot pens/treatment). Microbial samples were collected, and dust particles that were 2.5 μm or less in diameter were measured with a Dustrak monitor during the evening dust peak for 4 days at sites both upwind and downwind of the feedlot pens. An Andersen biological cascade sampler was employed with different medium and incubation combinations for the capture and identification of bacteria and fungi. The results showed that when bacteria were considered, only nonpathogenic gram-positive organisms were recovered. However, gram-negative bacteria may have been present in a viable but nonculturable state. Fungi were recovered in smaller numbers than bacteria, and none of the fungi were pathogenic. The Dustrak results showed that one feeding pattern resulted in cattle behavior that generated levels of downwind dust lower (
P
= 0.04) than the levels generated by the behavior resulting from the other feeding pattern. However, the Andersen sampler results showed that there were no differences between feeding patterns with regard to the total number or diversity of microorganisms. The disparity may have been due to the different operating principles of the two systems. The overall numbers of microorganisms recovered were lower than those reported in studies of intensively housed farm animals in which similar recovery techniques were used.
Functional Production and Characterization of a Fibrin-Specific Single-Chain Antibody Fragment fromBacillus subtilis: Effects of Molecular Chaperones and a Wall-Bound Protease on Antibody Fragment Production
Authors: Wu Sau-Ching , Yeung Jonathan C. , Duan Yanjun , Ye Ruiqiong , Szarka Steven J. , Habibi Hamid R. , Wong Sui-Lam
Published: July 2002

To develop an ideal blood clot imaging and targeting agent, a single-chain antibody (SCA) fragment based on a fibrin-specific monoclonal antibody, MH-1, was constructed and produced via secretion fromBacillus subtilis. Through a systematic study involving a series ofB. subtilisstrains, insufficient intracellular and extracytoplasmic molecular chaperones and high sensitivity to wall-bound protease (WprA) were believed to be the major factors that lead to poor production of MH-1 SCA. Intracellular and extracytoplasmic molecular chaperones apparently act in a sequential manner. The combination of enhanced coproduction of both molecular chaperones andwprAinactivation leads to the development of an engineeredB. subtilisstrain, WB800HM[pEPP]. This strain allows secretory production of MH-1 SCA at a level of 10 to 15 mg/liter. In contrast, with WB700N (a seven-extracellular-protease-deficient strain) as the host, no MH-1 SCA could be detected in both secreted and cellular fractions. Secreted MH-1 SCA from WB800HM[pMH1, pEPP] could be affinity purified using a protein L matrix. It retains comparable affinity and specificity as the parental MH-1 monoclonal antibody. This expression system can potentially be applied to produce other single-chain antibody fragments, especially those with folding and protease sensitivity problems.
Survival of GacS/GacA Mutants of the Biological Control Bacterium
Pseudomonas aureofaciens
30-84 in the Wheat Rhizosphere
Authors: Chancey Scott T. , Wood Derek W. , Pierson Elizabeth A. , Pierson Leland S.
Published: July 2002

GacS/GacA comprises a two-component regulatory system that controls the expression of secondary metabolites required for the control of plant diseases in many pseudomonads. High mutation frequencies of
gacS
and
gacA
have been observed in liquid culture. We examined whether
gacS/gacA
mutants could competitively displace the wild-type populations on roots and thus pose a threat to the efficacy of biological control. The survival of a
gac
mutant alone and in competition with the wild type on roots was examined in the biological control strain
Pseudomonas aureofaciens
30-84. In this bacterium, GacS/GacA controls the expression of phenazine antibiotics that are inhibitory to plant pathogenic fungi and enhance the competitive survival of the bacterium. Wheat seedlings were inoculated with strain 30-84, and bacteria were recovered from roots after 21 days in sterile or nonsterile soil to check for the presence of
gacS
or
gacA
mutants. Although no mutants were detected in the inoculum,
gacS/gacA
mutants were recovered from 29 out of 31 roots and comprised up to 36% of the total bacterial populations. Southern hybridization analysis of the recovered
gacA
mutants did not indicate a conserved mutational mechanism. Replacement series analysis on roots utilizing strain 30-84 and a
gacA
mutant (30-84.gacA) or a
gacS
mutant (30-84.A2) demonstrated that although the mutant population partially displaced the wild type in sterile soil, it did not do so in natural soil. In fact, in natural soil final rhizosphere populations of wild-type strain 30-84 starting from mixtures were at least 1.5 times larger than would be predicted from their inoculation ratio and generally were greater than or equal to the population of wild type alone despite lower inoculation rates. These results indicate that although
gacS/gacA
mutants survive in natural rhizosphere populations, they do not displace wild-type populations. Better survival of wild-type populations in mixtures with mutants suggests that mutants arising de novo or introduced within the inoculum may be beneficial for the survival of wild-type populations in the rhizosphere.
Plant-Dependent Genotypic and Phenotypic Diversity of Antagonistic Rhizobacteria Isolated from Different
Verticillium
Host Plants
Authors: Berg Gabriele , Roskot Nicolle , Steidle Anette , Eberl Leo , Zock Angela , Smalla Kornelia
Published: July 2002

To study the effect of plant species on the abundance and diversity of bacterial antagonists, the abundance, the phenotypic diversity, and the genotypic diversity of rhizobacteria isolated from potato, oilseed rape, and strawberry and from bulk soil which showed antagonistic activity towards the soilborne pathogen
Verticillium dahliae
Kleb. were analyzed. Rhizosphere and soil samples were taken five times over two growing seasons in 1998 and 1999 from a randomized field trial. Bacterial isolates were obtained after plating on R2A (Difco, Detroit, Mich.) or enrichment in microtiter plates containing high-molecular-weight substrates followed by plating on R2A. A total of 5,854 bacteria isolated from the rhizosphere of strawberry, potato, or oilseed rape or bulk soil from fallow were screened by dual testing for in vitro antagonism towards
Verticillium
. The proportion of isolates with antagonistic activity was highest for strawberry rhizosphere (9.5%), followed by oilseed rape (6.3%), potato (3.7%), and soil (3.3%). The 331
Verticillium
antagonists were identified by their fatty acid methyl ester profiles. They were characterized by testing their in vitro antagonism against other pathogenic fungi; their glucanolytic, chitinolytic, and proteolytic activities; and their BOX-PCR fingerprints. The abundance and composition of
Verticillium
antagonists was plant species dependent. A rather high proportion of antagonists from the strawberry rhizosphere was identified as
Pseudomonas putida
B (69%), while antagonists belonging to the
Enterobacteriaceae
(
Serratia
spp.,
Pantoea agglomerans
) were mainly isolated from the rhizosphere of oilseed rape. For
P. putida
A and B plant-specific genotypes were observed, suggesting that these bacteria were specifically enriched in each rhizosphere.
Wine Yeast Strains Engineered for Glycogen Overproduction Display Enhanced Viability under Glucose Deprivation Conditions
Authors: Pérez-Torrado R. , Gimeno-Alcañiz J. V. , Matallana E.
Published: July 2002

We used metabolic engineering to produce wine yeasts with enhanced resistance to glucose deprivation conditions. Glycogen metabolism was genetically modified to overproduce glycogen by increasing the glycogen synthase activity and eliminating glycogen phosphorylase activity. All of the modified strains had a higher glycogen content at the stationary phase, but accumulation was still regulated during growth. Strains lacking
GPH1,
which encodes glycogen phosphorylase, are unable to mobilize glycogen. Enhanced viability under glucose deprivation conditions occurs when glycogen accumulates in the strain that overexpresses
GSY2
, which encodes glycogen synthase and maintains normal glycogen phosphorylase activity. This enhanced viability is observed under laboratory growth conditions and under vinification conditions in synthetic and natural musts. Wines obtained from this modified strain and from the parental wild-type strain don't differ significantly in the analyzed enological parameters. The engineered strain might better resist some stages of nutrient depletion during industrial use.
In Situ Transfer of Antibiotic Resistance Genes from Transgenic (Transplastomic) Tobacco Plants to Bacteria
Authors: Kay Elisabeth , Vogel Timothy M. , Bertolla Frank , Nalin Renaud , Simonet Pascal
Published: July 2002

Interkingdom gene transfer is limited by a combination of physical, biological, and genetic barriers. The results of greenhouse experiments involving transplastomic plants (genetically engineered chloroplast genomes) cocolonized by pathogenic and opportunistic soil bacteria demonstrated that these barriers could be eliminated. The
Acinetobacter
sp. strain BD413, which is outfitted with homologous sequences to chloroplastic genes, coinfected a transplastomic tobacco plant with
Ralstonia solanacearum
and was transformed by the plant's transgene (
aadA
) containing resistance to spectinomycin and streptomycin. However, no transformants were observed when the homologous sequences were omitted from the
Acinetobacter
sp. strain. Detectable gene transfer from these transgenic plants to bacteria were dependent on gene copy number, bacterial competence, and the presence of homologous sequences. Our data suggest that by selecting plant transgene sequences that are nonhomologous to bacterial sequences, plant biotechnologists could restore the genetic barrier to transgene transfer to bacteria.
Distribution of Genes Encoding the Trypsin-Dependent Lantibiotic Ruminococcin A among Bacteria Isolated from Human Fecal Microbiota
Authors: Marcille F. , Gomez A. , Joubert P. , Ladiré M. , Veau G. , Clara A. , Gavini F. , Willems A. , Fons M.
Published: July 2002

Fourteen bacterial strains capable of producing a trypsin-dependent antimicrobial substance active against
Clostridium perfringens
were isolated from human fecal samples of various origins (from healthy adults and children, as well as from adults with chronic pouchitis). Identification of these strains showed that they belonged to
Ruminococcus gnavus
,
Clostridium nexile
, and
Ruminococcus hansenii
species or to new operational taxonomic units, all from the
Clostridium coccoides
phylogenetic group. In hybridization experiments with a probe specific for the structural gene encoding the trypsin-dependent lantibiotic ruminococcin A (RumA) produced by
R. gnavus
, seven strains gave a positive response. All of them harbored three highly conserved copies of
rumA-
like genes. The deduced peptide sequence was identical to or showed one amino acid difference from the hypothetical precursor of RumA. Our results indicate that the
rumA
-like genes have been disseminated among
R. gnavus
and phylogenetically related strains that can make up a significant part of the human fecal microbiota.
Source of Enterococci in a Farmhouse Raw-Milk Cheese
Authors: Gelsomino R. , Vancanneyt M. , Cogan T. M. , Condon S. , Swings J.
Published: July 2002

Enterococci are widely distributed in raw-milk cheeses and are generally thought to positively affect flavor development. Their natural habitats are the human and animal intestinal tracts, but they are also found in soil, on plants, and in the intestines of insects and birds. The source of enterococci in raw-milk cheese is unknown. In the present study, an epidemiological approach with pulsed-field gel electrophoresis (PFGE) was used to type 646
Enterococcus
strains which were isolated from a Cheddar-type cheese, the milk it was made from, the feces of cows and humans associated with the cheese-making unit, and the environment, including the milking equipment, the water used on the farm, and the cows' teats. Nine different PFGE patterns, three of
Enterococcus casseliflavus
, five of
Enterococcus faecalis
, and one of
Enterococcus durans
, were found. The same three clones, one of
E. faecalis
and two of
E. casseliflavus
, dominated almost all of the milk, cheese, and human fecal samples. The two
E. casseliflavus
clones were also found in the bulk tank and the milking machine even after chlorination, suggesting that a niche where enterococci could grow was present and that contamination with enterococci begins with the milking equipment. It is likely but unproven that the enterococci present in the human feces are due to consumption of the cheese. Cow feces were not considered the source of enterococci in the cheese, as
Enterococcus faecium
and
Streptococcus bovis
, which largely dominated the cows' intestinal tracts, were not found in either the milk or the cheese.
Lactobacillus caseiIs Able To Survive and Initiate Protein Synthesis during Its Transit in the Digestive Tract of Human Flora-Associated Mice
Authors: Oozeer R. , Goupil-Feuillerat N. , Alpert C. A. , van de Guchte M. , Anba J. , Mengaud J. , Corthier G.
Published: July 2002

LiveLactobacillus caseiis present in fermented dairy products and has beneficial properties for human health. In the human digestive tract, the resident flora generally prevents the establishment of ingested lactic acid bacteria, the presence of which is therefore transient. The aim of this work was to determine ifL. caseiDN-114 001 survives during transit and how this bacterium behaves in the digestive environment. We used the human flora-associated (HFA) mouse model.L. caseiDN-114 001 was genetically modified by the introduction ofermandluxgenes, encoding erythromycin resistance and luciferase, respectively. For this modified strain (DN-240 041), light emission related to luciferase expression could easily be detected in the contents of the digestive tract. When inoculated into the digestive tract of HFA mice,L. casei(DN-240 041) survives but is eliminated with the same kinetics as an inert transit marker, indicating that it does not establish itself. In pure culture ofL. casei, luciferase activities were high in the exponential and early stationary growth phases but decreased to become undetectable 1 day after inoculation. Viability was only slightly reduced even after more than 5 days. After transit in HFA mice, luciferase activity was detected even when 5-day-oldL. caseicultures were given to the mice. In culture, the luciferase activity could be restored after 0.5 to 7 h of incubation in fresh medium or milk containing glucose, unless protein synthesis was inhibited by the addition of chloramphenicol or rifampin. These results suggest that in HFA miceL. caseiDN-240 041, and thus probablyL. caseiDN-114 001, is able to initiate new protein synthesis during its transit with the diet. The beneficial properties ofL. casei-fermented milk for human health might be related to this protein synthesis in the digestive tract.
Phylogenetic Relationships and Coaggregation Ability of Freshwater Biofilm Bacteria
Authors: Rickard Alex H. , Leach Stephen A. , Hall Laurence S. , Buswell Clive M. , High Nicola J. , Handley Pauline S.
Published: July 2002

Nineteen numerically dominant heterotrophic bacteria from a freshwater biofilm were identified by 16S ribosomal DNA gene sequencing, and their coaggregation partnerships were determined. Phylogenetic trees showed that both distantly related and closely related strains coaggregated at intergeneric, intrageneric, and intraspecies levels. One strain,
Blastomonas natatoria
2.1, coaggregated with all 18 other strains and may function as a bridging organism in biofilm development.
Cloning of a Gene Encoding Raw-Starch-Digesting Amylase from a
Cytophaga
sp. and Its Expression in
Escherichia coli
Authors: Jeang Chii-Ling , Chen Li-Shien , Chen Ming-Yu , Shiau Rong-Jen
Published: July 2002

A raw-starch-digesting amylase (RSDA) gene from a
Cytophaga
sp. was cloned and sequenced. The predicted protein product contained 519 amino acids and had high amino acid identity to α-amylases from three
Bacillus
species. Only one of the
Bacillus
α-amylases has raw-starch-digesting capability, however. The RSDA, expressed in
Escherichia coli
, had properties similar to those of the enzyme purified from the
Cytophaga
sp.
Responses of Adipose and Muscle Lipoprotein Lipase to Chronic Infection and Subsequent Acute Lipopolysaccharide Challenge
Authors: Picard Frédéric , Arsenijevic Denis , Richard Denis , Deshaies Yves
Published: July 2002

Infection of male Swiss Webster mice with
Toxoplasma gondii
or
Neospora caninum
leads to long-term alterations in energy balance. Following an initial 20 to 30% weight loss in all
T. gondii
-infected mice, half of the animals regain most of the lost weight (gainers), whereas the others maintain their low body weight (nongainers). Infection with
N. caninum
does not elicit weight loss. Lipoprotein lipase (LPL), the enzyme responsible for plasma triglyceride (TG) clearance and partitioning among tissues, is under tissue-specific modulation associated with energy balance. It is also a major determinant of infection-induced hypertriglyceridemia. This study aimed to assess the long-term modulation of adipose and muscle LPL activity in mice infected with
T. gondii
or
N. caninum
, to evaluate the effects of subsequent acute lipopolysaccharide (LPS) administration, and to relate LPL modulation in these conditions with infection-related changes in body weight gain. Twenty-eight days after infection, LPL activity in muscle of both gainer and nongainer
T. gondii
-infected mice was reduced by 40 to 50% compared with the levels in controls and
N. caninum
-infected mice, whereas LPL activity in adipose depots remained unchanged in all infected groups compared to the level in controls. LPS (from
Escherichia coli
, 100 ng/kg) injection induced a global reduction in adipose LPL in all groups, as assessed 90 min later. In both
T. gondii
-infected subgroups, muscle LPL was not further reduced by LPS treatment, whereas it was decreased by 40 to 50% in muscles of control and
N. caninum
-infected mice. Pre-LPS TG levels in plasma were similar in all groups. LPS greatly increased TG levels in plasma in both control and
N. caninum
-infected animals, whereas it did not alter those of
T. gondii
-infected gainer or nongainer animals. These results show that (i) independently of the extent of postinfection weight gain, long-term infection with
T. gondii
chronically reduces muscle LPL, which becomes unresponsive to acute endotoxemia; (ii) modulation of tissue LPL activity during chronic
T. gondii
infection favors TG partitioning towards adipose tissue; and (iii) skeletal muscle LPL is a key determinant of the acute response of triglyceridemia to LPS.
Interleukin-18 Induces Acute Biphasic Reduction in the Levels of Circulating Leukocytes in Mice
Authors: Hosohara Katsushi , Ueda Haruyasu , Kashiwamura Shin-Ichiro , Yano Takako , Ogura Takeharu , Marukawa Seishiro , Okamura Haruki
Published: July 2002

We investigated the acute hematological changes caused by interleukin-18 (IL-18) in mice. Intraperitoneal administration of IL-18 (2 μg/mouse) resulted in biphasic decreases in the number of leukocytes in the blood. The first phase of decrease occurred within 2 h of IL-18 administration and was followed by a transient increase at 5 h. The second phase of decrease occurred at around 6 h, reaching a nadir which lasted for more than 24 h. In mice deficient in inducible nitric oxide (NO) synthase, the first phase of reduction of leukocytes did not occur although the second phase of decrease was observed. In mice deficient in gamma interferon (IFN-γ) or in mice depleted of natural killer cells and incapable of producing IFN-γ, IL-18 had no effect on the number of circulating leukocytes. Levels of nitrite and/or nitrate in the serum were elevated within 2 h after administration of IL-18, reaching a peak at 4 h and then decreasing gradually to the basal level over a 24-h period of time. On the other hand, serum IFN-γ levels changed in a biphasic manner, reaching a peak at 2 h after IL-18 administration, followed by a decrease in the basal level and a second increase at 6 h. Levels of IL-18 receptor mRNAs also showed biphasic changes in correlation with the changes in serum IFN-γ levels. These results suggest that the changes in the leukocyte number following IL-18 administration are mediated by NO and IFN-γ, with NO being involved in the first phase of reduction and IFN-γ being involved in both phases.
Regulation of Interleukin-8 by Interleukin-10 and Transforming Growth Factor β in Human Monocytes Infected withMycobacterium bovis
Authors: Méndez-Samperio Patricia , García Elizabeth , Vázquez Abraham , Palma Janet
Published: July 2002

Recent studies indicate that interleukin 8 (IL-8) production contributes to the host immune responses against mycobacterial infection. In this study, we were interested to determine whether induction of IL-8 in human monocytes infected withMycobacterium boviswas regulated by other monocyte-derived cytokines important in antimycobacterial immunity: IL-10 and transforming growth factor β (TGF-β). Here, we report that IL-10 reduced, in a graded and significant manner, IL-8 production byM. bovis-infected human monocytes. Additionally, the specificity of the observed inhibition was further confirmed, since the addition of an anti-IL-10 neutralizing antibody completely reversed the inhibitory effect. In contrast, addition or neutralization of TGF-β appeared to have no significant effect onM. bovis-induced IL-8 secretion by human monocytes, whereas CD40 expression onM. bovis-infected monocytes was significantly inhibited by this cytokine. This was consistent with the finding by the reverse transcription-PCR method that pretreatment with IL-10, but not TGF-β, potently inhibited IL-8 mRNA levels. Interestingly, neutralization of endogenous IL-10 did not significantly alter IL-8 secretion, suggesting that induction of IL-8 was not significantly affected by coexpression of IL-10 during infection of human monocytes withM. bovis.Collectively, these data indicate that IL-8 production may be regulated when human monocytes are exposed to IL-10 prior to activation withM. bovisBCG. These data will aid in our understanding of the mechanisms involved in regulating the protective immune response to stimulation withM. bovisBCG.
Performance of Three Microimmunofluorescence Assays for Detection of
Chlamydia pneumoniae
Immunoglobulin M, G, and A Antibodies
Authors: Bennedsen Mette , Berthelsen Lene , Lind Inga
Published: July 2002

The microimmunofluorescence (MIF) test is considered the “gold standard” for laboratory diagnosis of acute and chronic
Chlamydia pneumoniae
infection. The performance of a MIF test based on
C. pneumoniae
antigen from Washington Research Foundation (WRF) was compared with those of assays from Labsystems (LAB) and MRL Diagnostics (MRL) by investigation of sera from three groups of patients: group I, 83 sera from 28 patients with atypical pneumonia; group II, 37 sera from 16 patients with acute
C. pneumoniae
or
Chlamydia psittaci
respiratory tract infection confirmed by PCR or culture; group III, 100 sera from 100 persons enrolled in the Copenhagen City Heart Study. The accordance among the results of the WRF assay and the two commercial assays was excellent for the immunoglobulin M (IgM) antibody detection rate (98%). The accordance in detection rates for IgG and IgA antibodies in sera from patients with acute infections was acceptable (87 and 88%), and in sera from group III, it was excellent (95 and 97%). The determinations of endpoint titers were reproducible with <1 dilution step difference for all three methods, except that the mean IgM antibody titer found by the LAB assay was almost 2 dilution steps higher than that found by the other two methods. Although the three assays use different
C. pneumoniae
strains as antigens, the detection rates and IgG and IgA endpoint titers were similar. The difference in endpoint titers of IgM antibodies is of no major concern, as the diagnosis of acute
C. pneumoniae
infection rests on the presence of IgM antibodies, not on their level.
Bactericidal Activity in Whole Blood as a Potential Surrogate Marker of Immunity after Vaccination against Tuberculosis
Authors: Cheon Seon-Hee , Kampmann Beate , Hise Amy G. , Phillips Manijeh , Song Ho-Yeon , Landen Katherine , Li Qing , Larkin Rhonda , Ellner Jerrold J. , Silver Richard F. , Hoft Daniel F. , Wallis Robert S.
Published: July 2002

The development of new tuberculosis (TB) vaccines will require the identification of correlates of human protection. This study examined the balance between immunity and virulence in a whole blood infection model in which intracellular mycobacterial survival was measured using BACTEC. In the blood of tuberculin-negative donors, counts ofMycobacterium tuberculosisH37Ra organisms fell by 0.14 log10CFU during 96 h of whole blood culture, whereas counts ofMycobacterium bovisBCG,M. tuberculosisH37Rv, and a clinical TB isolate's organisms increased by 0.13, 0.43, and 1.04 log10CFU, respectively (P< 0.001), consistent with their relative virulence. Inhibition of tumor necrosis factor alpha by the addition of methylprednisolone or pentoxifylline or removal of CD4+or CD8+T cells by magnetic beads had deleterious effects on immune control of intracellular growth only in the blood of tuberculin-positive donors. Repeated vaccination of eight tuberculin-negative volunteers withM. bovisBCG resulted in a 0.3 log (50%) reduction in BCG CFU counts in the model compared to baseline values (P< 0.05). Three of the volunteers responded only after the second vaccination. These experiments indicate that whole blood culture may be used to measure immunity toM. tuberculosisand that further studies of repeated BCG vaccination are warranted.
Cultivation of
Plasmodium
spp
Authors: Schuster Frederick L.
Published: July 2002

Cultivation of both human and non-human species of
Plasmodium
spp., the causal agent of malaria, has been a major research success, leading to a greater understanding of the parasite. Efforts at cultivating the organisms in vitro are complicated by the parasites' alternating between a human host and an arthropod vector, each having its own set of physiological, metabolic, and nutritional parameters. Life cycle stages of the four species that infect humans have been established in vitro. Of these four,
P. falciparum
remains the only species for which all stages have been cultured in vitro; different degrees of success have been achieved with the other human
Plasmodium
spp. The life cycle includes the exoerythrocytic stage (within liver cells), the erythrocytic stage (within erythrocytes or precursor reticulocytes), and the sporogonic stage (within the vector). Culture media generally consist of a basic tissue culture medium (e.g., minimal essential medium or RPMI 1640) to which serum and erythrocytes are added. Most of the efforts have been directed toward the stage found in the erythrocyte. This stage has been cultivated in petri plates or other growth vessels in a candle jar to generate elevated CO
2
levels or in a more controlled CO
2
atmosphere. Later developments have employed continuous-flow systems to reduce the labor-intensive nature of medium changing. The exoerythrocytic and sporogonic life cycle stages have also been cultivated in vitro. A number of avian, rodent, and simian malarial parasites have also been established in vitro. Although cultivation is of great help in understanding the biology of
Plasmodium
, it does not lend itself to use for diagnostic purposes.
In Vitro Cultivation of Microsporidia of Clinical Importance
Authors: Visvesvara Govinda S.
Published: July 2002

Although attempts to develop methods for the in vitro cultivation of microsporidia began as early as 1937, the interest in the culture of these organisms was confined mostly to microsporidia that infect insects. The successful cultivation in 1969 of
Encephalitozoon cuniculi
, a microsporidium of mammalian origin, and the subsequent identification of these organisms as agents of human disease heightened interest in the cultivation of microsporidia. I describe the methodology as well as the cell lines, the culture media, and culture conditions used in the in vitro culture of microsporidia such as
Brachiola (Nosema) algerae, Encephalitozoon cuniculi, E. hellem, E. intestinalis, Enterocytozoon bieneusi, Trachipleistophora hominis,
and
Vittaforma corneae
that cause human disease.
Roles of p38 Mitogen-Activated Protein Kinase, NF-κB, and Protein Kinase C in Proinflammatory Cytokine mRNA Expression by Human Peripheral Blood Leukocytes, Monocytes, and Neutrophils in Response toAnaplasma phagocytophila
Authors: Kim Hyung-Yong , Rikihisa Yasuko
Published: July 2002

Anaplasma phagocytophila, an obligately intracellular bacterium of granulocytes, causes human granulocytic ehrlichiosis. Within 2 h after addition ofA. phagocytophila, interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and IL-6 mRNAs are induced in human peripheral blood leukocytes (PBLs) or monocytes in vitro. However, neutrophils generate only IL-1β mRNA. In the present study, signaling pathways for induction of these three cytokines were examined. TNF-α and IL-6 mRNA expression by PBLs was inhibited with SB 203580 (a p38 mitogen-activated protein kinase [MAPK] inhibitor), MG-132 (a proteasome inhibitor), and SN-50 (an NF-κB inhibitor). Activation of p38 MAPK and NF-κB mRNAs in monocytes was detectable within 15 to 30 min after addition ofA. phagocytophila. Expression of these two cytokine mRNAs in PBLs and monocytes was also dependent on protein kinase C (PKC), protein kinase A (PKA), and protein tyrosine kinase (PTK). IL-1β mRNA expression by neutrophils was not dependent on p38 MAPK, and p38 MAPK was not activated in neutrophils incubated withA. phagocytophila. IL-1β mRNA induction by PBLs, monocytes, and neutrophils was dependent on PKC and PKA. Neutrophil expression of IL-1β mRNA was dependent on transglutaminase, phospholipase C, and PTK, all of which are also required for internalization ofA. phagocytophila. However, monocyte expression of IL-1β mRNA was less dependent on these enzymes. These results suggest thatA. phagocytophilatransduces different signals between its host neutrophils and monocytes for proinflammatory cytokine generation.