bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of Epstein-Barr Virus Kinases To Sensitize Tumor Cells to Nucleoside Analogues
Authors: Moore Stacy M. , Cannon Jennifer S. , Tanhehco Yvette C. , Hamzeh Fayez M. , Ambinder Richard F.
Published: July 2002

The presence of Epstein-Barr virus (EBV) in the tumor cells of some EBV-associated malignancies may facilitate selective killing of these tumor cells. We show that treatment of an EBV
+
Burkitt's lymphoma cell line with 5-azacytidine led to a dose-dependent induction of EBV lytic antigen expression, including expression of the viral thymidine kinase (TK) and phosphotransferase (PT). Azacytidine treatment for 24 h modestly sensitized the cell line to all nucleosides tested. To better characterize EBV TK with regard to various nucleoside analogues, we expressed EBV TK in stable cell clones. Two EBV TK-expressing clones were moderately sensitive to high doses of acyclovir and penciclovir (PCV) (62.5 to 500 μM) and to lower doses of ganciclovir (GCV) and bromovinyldeoxyuridine (BVdU) (10 to 100 μM) compared to a control clone and were shown to phosphorylate GCV. Similar experiments in a transient overexpression system showed more killing of cells transfected with the EBV TK expression vector than of cells transfected with the control mutant vector (50 μM GCV for 4 days). A putative PT was also studied in the transient transfection system and appeared similar to the TK in phosphorylating GCV and conferring sensitivity to GCV, but not in BVdU- or PCV-mediated cell killing. Induction of EBV kinases in combination with agents such as GCV merits further evaluation as an alternative strategy to gene therapy for selective killing of EBV-infected cells.
In Vitro Activity of Gemifloxacin (SB-265805, LB20304a) against
Legionella pneumophila
and Its Pharmacokinetics in Guinea Pigs with
L. pneumophila
Pneumonia
Authors: Edelstein Paul H. , Shinzato Takashi , Doyle Edward , Edelstein Martha A. C.
Published: July 2002

The activity of gemifloxacin against intracellular
Legionella pneumophila
and for the treatment of guinea pigs with
L. pneumophila
pneumonia was studied. Gemifloxacin, azithromycin, and levofloxacin (1 μg/ml) reduced bacterial counts of two
L. pneumophila
strains grown in guinea pig alveolar macrophages by 2 to 3 log
10
units. Gemifloxacin and levofloxacin had roughly equivalent intracellular activities. In contrast, erythromycin had static activity only. Therapy studies of gemifloxacin, azithromycin, and levofloxacin were performed in guinea pigs with
L. pneumophila
pneumonia. When gemifloxacin (10 mg/kg) was given by the intraperitoneal (i.p.) route to infected guinea pigs, mean peak levels in plasma were 1.3 μg/ml at 0.5 h and 1.2 μg/ml at 1 h postinjection. The terminal half-life phase of elimination from plasma was 1.3 h, and the area under the concentration-time curve from 0 to 24 h (AUC
0–24
) was 2.1 μg · h/ml. For the same drug dose, mean levels in lungs were 3.4 μg/g at both 0.5 and 1 h, with a half-life of 1.5 h and an AUC
0–24
of 6.0 μg · h/ml. All 15
L. pneumophila
-infected guinea pigs treated with gemifloxacin (10 mg/kg/dose given i.p. once daily) for 2 days survived for 9 days after antimicrobial therapy, as did 13 of 14 guinea pigs treated with the same dose of gemifloxacin given for 5 days. All 12 azithromycin-treated animals (15 mg/kg/dose given i.p. once daily for 2 days) survived, as did 11 of 12 animals treated with levofloxacin (10 mg/kg/dose given i.p. once daily for 5 days). None of 12 animals treated with saline survived. Gemifloxacin is effective against
L. pneumophila
in infected macrophages and in a guinea pig model of Legionnaires' disease, even with an abbreviated course of therapy. These data support studies of the clinical effectiveness of gemifloxacin for the treatment of Legionnaires' disease.
Intravenous Infusion of Cereport Increases Uptake and Efficacy of Acyclovir in Herpes Simplex Virus-Infected Rat Brains
Authors: Bidanset Deborah J. , Placidi Laurent , Rybak Rachel , Palmer Joyce , Sommadossi Jean-Pierre , Kern Earl R.
Published: July 2002

The outcome of herpes simplex virus (HSV) infections manifesting as encephalitis in healthy or immunocompromised individuals is generally very poor with mortality rates of about 8 to 28% with treatment. The long-term prognosis of survivors is often problematic, posing the need for alternative treatments that may decrease the mortality and morbidity associated with herpes encephalitis. This study addresses one such approach that includes a temporary permeabilization of the blood-brain barrier during treatment with acyclovir (ACV). In these studies we utilized a synthetic bradykinin analog, Cereport (RMP-7), in conjunction with ACV to treat HSV infection of the brain in a rat model. Cereport, infused intravenously via the jugular vein, was shown to increase [
14
C]ACV uptake in both the HSV-1-infected and -uninfected rat brain by approximately two- to threefold, correlating with enhanced efficacy of ACV in various brain compartments. In another series of experiments to determine efficacy, various doses of unlabeled ACV were administered during infusion with RMP-7. The decrease in viral titers in the temporal regions of the brain after 5 days of treatment suggested that this approach enhanced the efficacy of ACV treatment. These data indicated that Cereport infused with ACV enhances both the penetration and efficacy of this drug in the treatment of an experimental HSV-1 infection of the rat brain.
In Vitro Activities of ABT-773, a New Ketolide, against Aerobic and Anaerobic Pathogens Isolated from Antral Sinus Puncture Specimens from Patients with Sinusitis
Authors: Goldstein Ellie J. C. , Conrads Georg , Citron Diane M. , Merriam C. Vreni , Warren Yumi , Tyrrell Kerin
Published: July 2002

The comparative in vitro activities of ABT-773 against 207 aerobic and 162 anaerobic antral sinus puncture isolates showed that erythromycin-resistant pneumococcal strains were susceptible to ABT-773 (≤0.125 μg/ml); the MIC at which 90% of the isolates tested were inhibited for
Haemophilus influenzae
and other
Haemophilus
spp. was 4 μg/ml; and all
Moraxella
spp. and beta-lactamase-producing
Prevotella
species strains were inhibited by ≤0.125 μg/ml. Among the anaerobes tested, only fusobacteria (45%) required ≥4 μg of ABT-773/ml for inhibition. ABT-773 may offer a therapeutic alternative for sinus infections.
Antifungal Efficacy of Caspofungin (MK-0991) in Experimental Pulmonary Aspergillosis in Persistently Neutropenic Rabbits: Pharmacokinetics, Drug Disposition, and Relationship to Galactomannan Antigenemia
Authors: Petraitiene Ruta , Petraitis Vidmantas , Groll Andreas H. , Sein Tin , Schaufele Robert L. , Francesconi Andrea , Bacher John , Avila Nilo A. , Walsh Thomas J.
Published: July 2002

The antifungal efficacy, pharmacokinetics, and safety of caspofungin (CAS) were investigated in the treatment and prophylaxis of invasive pulmonary aspergillosis due to
Aspergillus fumigatus
in persistently neutropenic rabbits. Antifungal therapy consisted of 1, 3, or 6 mg of CAS/kg of body weight/day (CAS1, CAS3, and CAS6, respectively) or 1 mg of deoxycholate amphotericin B (AMB)/kg/day intravenously for 12 days starting 24 h after endotracheal inoculation. Prophylaxis (CAS1) was initiated 4 days before endotracheal inoculation. Rabbits treated with CAS had significant improvement in survival and reduction in organism-mediated pulmonary injury (OMPI) measured by pulmonary infarct score and total lung weight (
P
< 0.01). However, animals treated with CAS demonstrated a paradoxical trend toward increased residual fungal burden (log CFU per gram) and increased serum galactomannan antigen index (GMI) despite improved survival. Rabbits receiving prophylactic CAS1 also showed significant improvement in survival and reduction in OMPI (
P
< 0.01), but there was no effect on residual fungal burden. In vitro tetrazolium salt hyphal damage assays and histologic studies demonstrated that CAS had concentration- and dose-dependent effects on hyphal structural integrity. In parallel with a decline in GMI, AMB significantly reduced the pulmonary tissue burden of
A. fumigatus
(
P
≤ 0.01). The CAS1, CAS3, and CAS6 dose regimens demonstrated dose-proportional exposure and maintained drug levels in plasma above the MIC for the entire 24-h dosing interval at doses that were ≥3 mg/kg/day. As serial galactomannan antigen levels may be used for therapeutic monitoring, one should be aware that profoundly neutropenic patients receiving echinocandins for aspergillosis might have persistent galactomannan antigenemia despite clinical improvement. CAS improved survival, reduced pulmonary injury, and caused dose-dependent hyphal damage but with no reduction in residual fungal burden or galactomannan antigenemia in persistently neutropenic rabbits with invasive pulmonary aspergillosis.
Development of Enzymatic Assays for Quantification of Intracellular Lamivudine and Carbovir Triphosphate Levels in Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus-Infected Patients
Authors: Kewn Stephen , Hoggard Patrick G. , Sales Sean D. , Jones Kevin , Maher Bridget , Khoo Saye H. , Back David J.
Published: July 2002

In this paper, we describe the development and use of enzymatic assays to determine intracellular lamivudine triphosphate (3TCTP) and carbovir triphosphate (CBVTP) concentrations in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus (HIV)-infected patients. The assays involve inhibition of HIV reverse transcriptase (RT), which normally incorporates radiolabeled deoxynucleoside triphosphates into a synthetic template primer. For the 3TCTP assay, a preincubation procedure was added whereby 3TCTP becomes incorporated before [
3
H]dCTP. At a 1:400 template primer dilution, control product formation was reduced by 88.0% with 0.8 pmol of 3TCTP. Standard 3TCTP inhibition curves were performed using this procedure. For the CBVTP assay, 0.1 pmol of CBVTP inhibited control product formation with and without the use of a preincubation step, so inhibition curves were constructed using both procedures. However, reduced template primer stability with assays using preincubation steps led to a single-incubation procedure being adopted for future studies. The presence of PBMC extracts interfered with the 3TCTP assay. However, this was overcome by the addition of CuSO
4
. PBMC extracts did not interfere with the CBVTP assay. Intracellular 3TCTP and CBVTP concentrations were determined in PBMCs from HIV-infected patients over 24 h or greater. Peak concentrations were obtained 6 to 8 h after dosing, and the half-lives of the anabolites suggested the possibility of once-daily dosing. These assays are currently being used for determination of 3TCTP and CBVTP concentrations in clinical studies.
Combination Therapy with Indolylquinoline Derivative and Sodium Antimony Gluconate Cures Established Visceral Leishmaniasis in Hamsters
Authors: Pal Chiranjib , Raha Mousumi , Basu Anirban , Roy Keshab Chandra , Gupta Anasuya , Ghosh Monidipa , Sahu Niranjan Prasad , Banerjee Sukdeb , Mandal Nirup Bikash , Bandyopadhyay Santu
Published: July 2002

2-(2″-Dichloroacetamidobenzyl)-3-(3′-indolylquinoline), designated indolylquinoline derivative A, reduced the splenic and the liver parasite burdens by >93.0% in
Leishmania donovani
-infected hamsters, whereas sodium antimony gluconate (SAG) reduced the burdens approximately 80.0%. Complete clearance of parasitemia from the livers and spleens was noticed when infected animals received indolylquinoline derivative A plus SAG, suggesting that indolylquinoline derivative A has potential as a new agent for sole or conjunctive therapy for leishmaniasis.
TEM-80, a Novel Inhibitor-Resistant β-Lactamase in a Clinical Isolate of
Enterobacter cloacae
Authors: Arpin Corinne , Labia Roger , Dubois Véronique , Noury Patrick , Souquet Muriel , Quentin Claudine
Published: July 2002

Enterobacter cloacae
Ecl261 was isolated with
Escherichia coli
Ec257 from the urine of a patient living in a nursing home. Both isolates were resistant to ticarcillin (MICs, 1,024 μg/ml), without significant potentiation of its activity by 2 μg of clavulanate per ml (MICs, 512 μg/ml), and susceptible to naturally active cephalosporins. This inhibitor-resistant phenotype was conferred in both strains by similar conjugative plasmids of 40 kb (Ecl261) and 30 kb (Ec257), which also conveyed resistance to sulfonamides and trimethoprim. Clinical and transconjugant strains produced a β-lactamase with a pI of 5.2 which belonged to the TEM family, as indicated by specific PCR amplification. Compared with TEM-1, this enzyme exhibited lower catalytic efficiencies (14- and 120-fold less for amoxicillin and ticarcillin, respectively), and higher concentrations of β-lactamase inhibitors were required to yield a 50% reduction in benzylpenicillin hydrolysis (750-, 82-, and 50-fold higher concentrations for clavulanate, sulbactam, and tazobactam, respectively). Gene sequencing revealed four nucleotide differences with the nucleotide sequence of
bla
TEM-1A
. The first replacement (T32C), located in the promoter region, was described as being responsible for the increase in the level of β-lactamase production. The three other changes led to amino acid substitutions that define a new inhibitor-resistant TEM (IRT) β-lactamase, TEM-80 (alternate name, IRT-24). Two of them, Met69Leu and Asn276Asp, have previously been related to inhibitor resistance. The additional mutation, Ile127Val, was demonstrated by site-directed mutagenesis to have a very weak effect, at least alone, on the IRT phenotype. This is the first description of an IRT β-lactamase in
E. cloacae
. The horizontal transfer of
bla
TEM-80
may have occurred either from Ec257 to Ecl261 or in the reverse order.
Macrolide and Tetracycline Resistance and Molecular Relationships of Clinical Strains of
Streptococcus agalactiae
Authors: Culebras Esther , Rodriguez-Avial Iciar , Betriu Carmen , Redondo Montserrat , Picazo Juan J.
Published: July 2002

Mechanisms for tetracycline and macrolide resistance in 54 isolates of erythromycin-resistant
Streptococcus agalactiae
were analyzed by PCR. The
erm
(B),
erm
(A), and
mef
(A) genes, either alone or in combination, were detected in all the erythromycin-resistant isolates. The
tet
(M) and
tet
(O) genes were responsible for tetracycline resistance. Random amplification of polymorphic DNA indicated different clonal origins of the isolates.
Synergistic Effect of Ofloxacin and Magnesium Deficiency on Joint Cartilage in Immature Rats
Authors: Lozo Edith , Riecke Kai , Schwabe Rudolf , Vormann Jürgen , Stahlmann Ralf
Published: July 2002

Single high oral doses of fluoroquinolones (e.g., 1,200 mg of ofloxacin/kg of body weight) are chondrotoxic in juvenile rats. Characteristic cartilage lesions are detectable as early as 12 h after treatment. Since this dosing regimen does not reflect the therapeutic situation, we studied the effects of a 5- or 7-day treatment with ofloxacin at lower oral doses (10, 30, and 100 mg/kg twice a day [b.i.d.]) on joint cartilage in 4-week-old rats. We additionally investigated whether the effects of ofloxacin under these conditions are enhanced in animals kept on a magnesium-deficient diet during treatment. Knee joints were examined histologically. The concentrations of ofloxacin and magnesium were determined in plasma and cartilage. The lowest ofloxacin dose at which cartilage lesions occurred in animals on a standard diet was 100 mg/kg b.i.d. for 5 days. Peak plasma ofloxacin levels were approximately 10 mg/liter in these rats and thus were in the same range as the levels in the plasma of humans during therapy with high doses of ofloxacin. Treatment with 30 mg of ofloxacin/kg b.i.d. for 7 days caused no cartilage lesions in rats on a standard diet, but lesions did occur in 10 of 12 rats that were simultaneously fed a magnesium-deficient diet. Magnesium concentrations in bone, plasma, and cartilage from animals on an Mg
2+
-deficient diet were significantly lower than those in the controls. The concentration in plasma from animals on an Mg
2+
-deficient diet was 0.27 ± 0.03 mmol/liter, whereas it was 0.88 ± 0.08 mmol/liter in plasma from rats on a standard diet (means ± standard deviations). Ofloxacin treatment did not change the total magnesium concentrations in tissues, as determined with ashed samples. The incidence of ofloxacin-induced lesions was higher in the magnesium-deficient animals, suggesting a synergistic effect. These results must be taken into account for a benefit-risk evaluation if ofloxacin is considered for use in the pediatric population.
Reducing Mortality in
Salmonella enterica
Serovar Typhimurium-Infected Mice with a Tripeptidic Serum Fraction
Authors: Parker Todd A. , Willeford Kenneth O. , Parker Suzanne , Buddington Karyl
Published: July 2002

Salmonellosis-induced mortality in female Swiss Webster mice decreased significantly when tripeptidic immunostimulant (TPI) was administered prophylactically. Prophylactic benefits developed in a dose-dependent manner wherein 15 mg of TPI given 1 day before challenge reduced mortality by 70%.
Antisense Silencing of the
creA
Gene in
Aspergillus nidulans
Authors: Bautista L. Fernando , Aleksenko Alexei , Hentzer Morten , Santerre-Henriksen Anne , Nielsen Jens
Published: July 2002

Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in
Aspergillus nidulans
resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect was approximately one-half of that achieved in a null
creA
mutant. Unlike results for that mutant, however, growth parameters and colony morphology in the antisense transformants were not affected.
The Autoproteolysis of
Lactococcus lactis
Lactocepin III Affects Its Specificity towards β-Casein
Authors: Flambard Benedicte , Juillard Vincent
Published: July 2002

The effect of autoproteolysis of
Lactococcus lactis
lactocepin III on its specificity towards β-casein was investigated. β-Casein degradation was performed by using either an autolysin-defective derivative of
L. lactis
MG1363 carrying the proteinase genes of
L. lactis
SK11, which was unable to transport oligopeptides, or autoproteolyzed enzyme purified from
L. lactis
SK11. Comparison of the peptide pools by high-performance liquid chromatography analysis revealed significant differences. To analyze these differences in more detail, the peptides released by the cell-anchored proteinase were identified by on-line coupling of liquid chromatography to mass spectrometry. More than 100 oligopeptides were released from β-casein by the cell-anchored proteinase. Analysis of the cleavage sites indicated that the specificity of peptide bond cleavage by the cell-anchored proteinase differed significantly from that of the autoproteolyzed enzyme.
Quantification of Siderophore and Hemolysin from
Stachybotrys chartarum
Strains, Including a Strain Isolated from the Lung of a Child with Pulmonary Hemorrhage and Hemosiderosis
Authors: Vesper Stephen J. , Dearborn Dorr G. , Elidemir Okan , Haugland Richard A.
Published: July 2002

A strain of
Stachybotrys chartarum
was recently isolated from the lung of a pulmonary hemorrhage and hemosiderosis (PH) patient in Texas (designated the Houston strain). This is the first time that
S. chartarum
has been isolated from the lung of a PH patient. In this study, the Houston strain and 10 strains of
S. chartarum
isolated from case (
n
= 5) or control (
n
= 5) homes in Cleveland were analyzed for hemolytic activity, siderophore production, and relatedness as measured by random amplified polymorphic DNA analysis.
Reactions Involved in the Lower Pathway for Degradation of 4-Nitrotoluene by
Mycobacterium
Strain HL 4-NT-1
Authors: He Zhongqi , Spain Jim C.
Published: July 2002

In spite of the variety of initial reactions, the aerobic biodegradation of aromatic compounds generally yields dihydroxy intermediates for ring cleavage. Recent investigation of the degradation of nitroaromatic compounds revealed that some nitroaromatic compounds are initially converted to 2-aminophenol rather than dihydroxy intermediates by a number of microorganisms. The complete pathway for the metabolism of 2-aminophenol during the degradation of nitrobenzene by
Pseudomonas pseudoalcaligenes
JS45 has been elucidated previously. The pathway is parallel to the catechol extradiol ring cleavage pathway, except that 2-aminophenol is the ring cleavage substrate. Here we report the elucidation of the pathway of 2-amino-4-methylphenol (6-amino-
m
-cresol) metabolism during the degradation of 4-nitrotoluene by
Mycobacterium
strain HL 4-NT-1 and the comparison of the substrate specificities of the relevant enzymes in strains JS45 and HL 4-NT-1. The results indicate that the 2-aminophenol ring cleavage pathway in strain JS45 is not unique but is representative of the pathways of metabolism of other
o
-aminophenolic compounds.
Modification of Sorbitol MacConkey Medium Containing Cefixime and Tellurite for Isolation of
Escherichia coli
O157:H7 from Radish Sprouts
Authors: Fujisawa Tomohiko , Sata Shin , Aikawa Katsuhiro , Takahashi Takanori , Yamai Shiro , Shimada Toshio
Published: July 2002

A modified version of sorbitol MacConkey medium containing cefixime and tellurite (CT-SMAC medium) was produced by adding salicin and 4-methylumbelliferyl-β-
d
-galactopyranoside to CT-SMAC medium; this medium was designated CT-SSMAC medium and was used to isolate
Escherichia coli
O157:H7 from radish sprouts. Of 101 non-
E. coli
bacteria isolated from radish sprouts that produced colorless colonies similar to colonies of
E. coli
O157:H7 grown on CT-SMAC medium, 92 (91%) formed colonies that were red to pink or were β-galactosidase negative and colorless on CT-SSMAC medium. On the other hand, colonies of
E. coli
O157:H7 strains were colorless and β-galactosidase positive on CT-SSMAC medium. Our results suggest that CT-SSMAC medium is more selective than CT-SMAC medium for isolating
E. coli
O157:H7.
The α-Helix 4 Residue, Asn135, Is Involved in the Oligomerization of Cry1Ac1 and Cry1Ab5
Bacillus thuringiensis
Toxins
Authors: Tigue Natalie J. , Jacoby Juliette , Ellar David J.
Published: July 2002

The insecticidal Cry toxins produced by the bacterium
Bacillus thuringiensis
are comprised of three structural domains. Domain I, a seven-helix bundle, is thought to penetrate the insect epithelial cell plasma membrane through a hairpin composed of α-helices 4 and 5, followed by the oligomerization of four hairpin monomers. The α-helix 4 has been proposed to line the lumen of the pore, whereas some residues in α-helix 5 have been shown to be responsible for oligomerization. Mutation of the Cry1Ac1 α-helix 4 amino acid Asn135 to Gln resulted in the loss of toxicity to
Manduca sexta
, yet binding was still observed. In this study, the equivalent mutation was made in the Cry1Ab5 toxin, and the properties of both wild-type and mutant toxin counterparts were analyzed. Both mutants appeared to bind to
M. sexta
membrane vesicles, but they were not able to form pores. The ability of both N135Q mutants to oligomerize was also disrupted, providing the first evidence that a residue in α-helix 4 can contribute to toxin oligomerization.
The Effect of the
Agrobacterium tumefaciens attR
Mutation on Attachment and Root Colonization Differs between Legumes and Other Dicots
Authors: Matthysse Ann G. , McMahan Susan
Published: July 2002

Infections of wound sites on dicot plants by
Agrobacterium tumefaciens
result in the formation of crown gall tumors. An early step in tumor formation is bacterial attachment to the plant cells. AttR mutants failed to attach to wound sites of both legumes and nonlegumes and were avirulent on both groups of plants. AttR mutants also failed to attach to the root epidermis and root hairs of nonlegumes and had a markedly reduced ability to colonize the roots of these plants. However, AttR mutants were able to attach to the root epidermis and root hairs of alfalfa, garden bean, and pea. The mutant showed little reduction in its ability to colonize these roots. Thus,
A. tumefaciens
appears to possess two systems for binding to plant cells. One system is AttR dependent and is required for virulence on all of the plants tested and for colonization of the roots of all of the plants tested except legumes. Attachment to root hairs through this system can be blocked by the acetylated capsular polysaccharide. The second system is AttR independent, is not inhibited by the acetylated capsular polysaccharide, and allows the bacteria to bind to the roots of legumes.
Comparison of Statistical Methods for Identification of
Streptococcus thermophilus,
Enterococcus faecalis,
and
Enterococcus faecium
from Randomly Amplified Polymorphic DNA Patterns
Authors: Moschetti Giancarlo , Blaiotta Giuseppe , Villani Francesco , Coppola Salvatore , Parente Eugenio
Published: July 2002

Thermophilic streptococci play an important role in the manufacture of many European cheeses, and a rapid and reliable method for their identification is needed. Randomly amplified polymorphic DNA (RAPD) PCR (RAPD-PCR) with two different primers coupled to hierarchical cluster analysis has proven to be a powerful tool for the classification and typing of
Streptococcus thermophilus
,
Enterococcus faecium
, and
Enterococcus faecalis
(G. Moschetti, G. Blaiotta, M. Aponte, P. Catzeddu, F. Villani, P. Deiana, and S. Coppola, J. Appl. Microbiol. 85:25–36, 1998). In order to develop a fast and inexpensive method for the identification of thermophilic streptococci, RAPD-PCR patterns were generated with a single primer (XD9), and the results were analyzed using artificial neural networks (Multilayer Perceptron, Radial Basis Function network, and Bayesian network) and multivariate statistical techniques (cluster analysis, linear discriminant analysis, and classification trees). Cluster analysis allowed the identification of
S. thermophilus
but not of enterococci. A Bayesian network proved to be more effective than a Multilayer Perceptron or a Radial Basis Function network for the identification of
S. thermophilus
,
E. faecium
, and
E. faecalis
using simplified RAPD-PCR patterns (obtained by summing the bands in selected areas of the patterns). The Bayesian network also significantly outperformed two multivariate statistical techniques (linear discriminant analysis and classification trees) and proved to be less sensitive to the size of the training set and more robust in the response to patterns belonging to unknown species.
Characterization of a Heme-Dependent Catalase from
Methanobrevibacter arboriphilus
Authors: Shima Seigo , Sordel-Klippert Melanie , Brioukhanov Andrei , Netrusov Alexander , Linder Dietmar , Thauer Rudolf K.
Published: July 2002

Recently it was reported that methanogens of the genus
Methanobrevibacter
exhibit catalase activity. This was surprising, since
Methanobrevibacter
species belong to the order
Methanobacteriales
, which are known not to contain cytochromes and to lack the ability to synthesize heme. We report here that
Methanobrevibacter arboriphilus
strains AZ and DH1 contained catalase activity only when the growth medium was supplemented with hemin. The heme catalase was purified and characterized, and the encoding gene was cloned. The amino acid sequence of the catalase from the methanogens is most similar to that of
Methanosarcina barkeri
.
Decrease in
Cryptosporidium parvum
Oocyst Infectivity In Vitro by Using the Membrane Filter Dissolution Method for Recovering Oocysts from Water Samples
Authors: Carreno Ramon A. , Pokorny Nicholas J. , Weir Susan C. , Lee Hung , Trevors Jack T.
Published: July 2002

Exposure of
Cryptosporidium parvum
oocysts to solutions used for cellulose acetate membrane (CAM) dissolution filtration reduced their infectivity in HCT-8 cells. Ethanol (95% [vol/vol] and 70% [vol/vol]) alone and short exposure times to acetone decreased infectivity. These findings contrast with similar experiments using excystation assays and infectivity in mice.
Transformation of 2,2′-Bimorphine to the Novel Compounds 10-α-
S
-Monohydroxy-2,2′-Bimorphine and 10,10′-α,α′-
S
,
S
′-Dihydroxy-2,2′-Bimorphine by
Cylindrocarpon didymum
Authors: Stabler Peter J. , Holt Peter J. , Bruce Neil C.
Published: July 2002

Whole-cell suspensions of
Cylindrocarpon didymum
were observed to transform 2,2′-bimorphine to the compounds 10-α-
S
-monohydroxy-2,2′-bimorphine and 10,10′-α,α′-
S
,
S
′-dihydroxy-2,2′-bimorphine. Mass spectrometry and
1
H nuclear magnetic resonance spectroscopy confirmed the identities of these new morphine alkaloids.
Mapping Microbial Biodiversity
Authors: Stoner Daphne L. , Geary Micah C. , White Luke J. , Lee Randy D. , Brizzee Julie A. , Rodman Ann C. , Rope Ronald C.
Published: July 2002

We report the development of a prototype database that “maps” microbial diversity in the context of the geochemical and geological environment and geographic location. When it is fully implemented, scientists will be able to conduct database searches, construct maps containing the information of interest, download files, and enter data over the Internet.
Salmonella enterica
Serotype Bredeney: Antimicrobial Susceptibility and Molecular Diversity of Isolates from Ireland and Northern Ireland
Authors: Cormican Martin , DeLappe Niall , O’Hare Colette , Doran Geraldine , Morris Dearbhaile , Corbett-Feeney Geraldine , Fanning Séamus , Daly Mairead , Fitzgerald Margaret , Moore John
Published: July 2002

Salmonella enterica
serotype Bredeney has emerged as the third most commonly identified serotype among human clinical isolates referred to the Irish National Salmonella Reference Laboratory in the years 1998 to 2000. A collection of 112 isolates of
S. enterica
serotype Bredeney collected during the period 1995 to 1999 from animal, food, and human sources from both Ireland and Northern Ireland were studied. Antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and DNA amplification fingerprinting (DAF) were performed on all isolates. Plasmid profiles were examined on a subset of 33 isolates. A high proportion (74%) of isolates were susceptible to all antimicrobial agents tested. Resistance to both sulfonamide and trimethoprim was observed in 21% of isolates, and resistance to multiple (five) antimicrobial agents was observed in a single isolate (0.9%). Eight different PFGE patterns were obtained, with 87% of isolates grouping as PFGE type A. PFGE type A was predominant in animals, food, and humans. There was good overall concordance between the groups identified by PFGE and DAF. Overall results indicate that most
S. enterica
serotype Bredeney isolates in Ireland and Northern Ireland from animal and human sources are clonally related.
Electrochemical Regeneration of Fe(III) To Support Growth on Anaerobic Iron Respiration
Authors: Ohmura Naoya , Matsumoto Norio , Sasaki Kazuhiro , Saiki Hiroshi
Published: July 2002

Here we describe artificial help for the respiratory electron flow supporting anaerobic growth of
Thiobacillus ferrooxidans
through exogenous electrolysis. Flux between H
2
and a anode through cells was accomplished with electrochemical regeneration of iron. The electrochemical help resulted in a 12-fold increase in yield compared with the yield observed in its absence.
Effects of Temperature, Water Activity, and Syrup Film Composition on the Growth of
Wallemia sebi
: Development and Assessment of a Model Predicting Growth Lags in Syrup Agar and Crystalline Sugar
Authors: Vindeløv Jannik , Arneborg Nils
Published: July 2002

We investigated the effects of temperature, water activity (a
w
), and syrup film composition on the CFU growth of
Wallemia sebi
in crystalline sugar. At a high a
w
(0.82) at both high (20°C) and low (10°C) temperatures, the CFU growth of
W. sebi
in both white and extrawhite sugar could be described using a modified Gompertz model. At a low a
w
(0.76), however, the modified Gompertz model could not be fitted to the CFU data obtained with the two sugars due to long CFU growth lags and low maximum specific CFU growth rates of
W. sebi
at 20°C and due to the fact that growth did not occur at 10°C. At an a
w
of 0.82, regardless of the temperature, the carrying capacity (i.e., the cell concentration at
t
= ∞) of extrawhite sugar was lower than that of white sugar. Together with the fact that the syrup film of extrawhite sugar contained less amino-nitrogen relative to other macronutrients than the syrup film of white sugar, these results suggest that CFU growth of
W. sebi
in extrawhite sugar may be nitrogen limited. We developed a secondary growth model which is able to predict colony growth lags of
W. sebi
on syrup agar as a function of temperature and a
w
. The ability of this model to predict CFU growth lags of
W. sebi
in crystalline sugar was assessed.
Energy Generation by Extracellular Aldose Oxidation in N
2
-Fixing
Gluconacetobacter diazotrophicus
Authors: Luna María F. , Bernardelli Cecilia E. , Mignone Carlos F. , Boiardi José L.
Published: July 2002

Gluconacetobacter diazotrophicus
PAL3 was grown in a chemostat with N
2
and mixtures of xylose and gluconate. Xylose was oxidized to xylonate, which was accumulated in the culture supernatants. Biomass yields and carbon from gluconate incorporated into biomass increased with the rate of xylose oxidation. By using metabolic balances it is demonstrated that extracellular xylose oxidation led N
2
-fixing
G. diazotrophicus
cultures to increase the efficiency of energy generation.
Thermodynamic Analysis of Growth Temperature Dependence in the Adhesion of
Candida parapsilosis
to Polystyrene
Authors: Gallardo-Moreno Amparo M. , González-Martín M. Luisa , Pérez-Giraldo Ciro , Garduño Eugenio , Bruque José M. , Gómez-García Antonio C.
Published: July 2002

The purpose of this work was to study the adhesion to polystyrene of two
Candida parapsilosis
strains, grown at 22 and 37°C, in terms of hydrophobicity, surface charge, and interaction free energy. Growth temperature changed the surface properties of microorganisms, yielding a good correlation between thermodynamic predictions and adhesion behavior.
Hemoglobin in
Frankia
, a Nitrogen-Fixing Actinomycete
Authors: Tjepkema John D. , Cashon Robert E. , Beckwith Jason , Schwintzer Christa R.
Published: July 2002

Frankia
strain CcI3 grown in culture produced a hemoglobin which had optical absorption bands typical of a hemoglobin and a molecular mass of 14.1 kDa. Its equilibrium oxygen binding constant was 274 nM, the oxygen dissociation rate constant was 56 s
−1
, and the oxygen association rate constant was 206 μM
−1
s
−1
.
Comparison of Polymorphonuclear Cells from Healthy Donors and Differentiated HL-60 Cells as Phagocytes in an Opsonophagocytic Assay Using Antigen-Coated Fluorescent Beads
Authors: Guy Bruno , Testart Catherine , Gimenez Sophie , Sanchez Violette , Lheritier Philippe , Rossin Danielle , Mignon Michèle , Danve Bernard , Trannoy Emanuelle
Published: July 2002

Polymorphonuclear cells (PMNs) from healthy donors and differentiated HL-60 cells were compared in an opsonophagocytic assay using fluorescent latex beads coated with
Streptococcus pneumoniae
polysaccharide conjugates. Serum-specific phagocytosis was efficiently mediated by both sources of cells, as measured by flow cytometry, but the mean number of beads ingested per cell was three- to fivefold higher when PMNs were used than when HL-60 cells were used. Nevertheless, differentiated HL-60 cells could be a convenient and standardized source of cells to evaluate the functionality of specific antibodies to vaccine candidates as a coating on fluorescent beads.
Antifreeze Solution Improves DNA Recovery by Preserving the Integrity of Pathogen-Infected Blood and Other Tissues
Authors: Leal-Klevezas Diana Sara , Martı́nez-Vázquez Irma Olivia , Cuevas-Hernández Baltazar , Martı́nez-Soriano Juan Pablo
Published: July 2002

Preserving blood samples for shipping and later DNA extraction has been performed by cooling, freezing, drying, freeze-drying, and protease treatment, among other methods. Most methods to preserve field samples for further DNA extraction do not prevent cellular and DNA damage or are useful only in preserving them for short periods. This report introduces a novel method for blood and tissue that allows preservation in freezing temperatures for a prolonged period of time. The solution reported here (20% ethylene glycol-propylene glycol) preserves cells and tissues integrity, as judged by microscopic analysis, and improves DNA yield and quality.
A Point Mutation in a Domain of Gamma Interferon Receptor 1 Provokes Severe Immunodeficiency
Authors: Allende Luis M. , López-Goyanes Alberto , Paz-Artal Estela , Corell Alfredo , Garcı́a-Pérez M. Angel , Varela Pilar , Scarpellini Atilio , Negreira Sagrario , Palenque Elia , Arnaiz-Villena Antonio
Published: July 2002

Gamma interferon (IFN-γ) and the cellular responses induced by it are essential for controlling mycobacterial infections. Most patients bearing an IFN-γ receptor ligand-binding chain (IFN-γR1) deficiency present gross mutations that truncate the protein and prevent its expression, giving rise to severe mycobacterial infections and, frequently, a fatal outcome. In this report a new mutation that affects the IFN-γR1 ligand-binding domain in a Spanish patient with mycobacterial disseminated infection and multifocal osteomyelitis is characterized. The mutation generates an amino acid change that does not abrogate protein expression on the cellular surface but that severely impairs responses after the binding of IFN-γ (CD64 and HLA class II induction and tumor necrosis factor alpha and interleukin-12 production). A patient's younger brother, who was also probably homozygous for the mutation, died from meningitis due toMycobacterium bovis. These findings suggest that a point mutation may be fatal when it affects functionally important domains of the receptor and that the severity is not directly related to a lack of IFN-γ receptor expression. Future research on these nontruncating mutations will make it possible to develop new therapeutical alternatives in this group of patients.
Internal Transcribed Spacer Regions of rRNA Genes of
Pneumocystis carinii
from Monkeys
Authors: Hsueh John Y. C. , Bohm Rudolf P. , Didier Peter J. , Tang Xing , Lasbury Mark E. , Li Baozheng , Jin Shaoling , Bartlett Marilyn S. , Smith James W. , Lee Chao-Hung
Published: July 2002

Analysis of sequence variations among isolates of
Pneumocystis carinii
f. sp.
macacae
from 14 Indian rhesus monkeys (
Macaca mulatta
) at the internal transcribed spacer (ITS) regions of the nuclear rRNA gene was undertaken. Like those from
P. carinii
f. sp.
hominis
, the ITS sequences from various
P. carinii
f. sp.
macacae
isolates were not identical. Two major types of sequences were found. One type of sequence was shared by 13 isolates. These 13 sequences were homologous but not identical. Variations were found at 13 of the 180 positions in the ITS1 region and 28 of the 221 positions in the ITS2 region. These sequence variations were not random but exhibited definite patterns when the sequences were aligned. According to this sequence variation, ITS1 sequences were classified into three types and ITS2 sequences were classified into five types. The remaining specimen had ITS1 and ITS2 sequences substantially different from the others. Although some specimens had the same ITS1 or ITS2 sequence, all 14 samples exhibited a unique whole ITS sequence (ITS1 plus ITS2). The 5.8S rRNA gene sequences were also analyzed, and only two types of sequences that differ by only one base were found. Unlike
P. carinii
f. sp.
hominis
infections in humans, none of the monkey lung specimens examined in this study were found to be infected by more than one type of
P. carinii
f. sp.
macacae
. These results offer insights into the genetic differences between
P. carinii
organisms which infect distinct species.
Neutralizing Antibodies against Autologous Human Immunodeficiency Virus Type 1 Isolates in Patients with Increasing CD4 Cell Counts despite Incomplete Virus Suppression during Antiretroviral Treatment
Authors: Sarmati Loredana , d'Ettorre Gabriella , Nicastri Emanuele , Ercoli Lucia , Uccella Ilaria , Massetti Paola , Parisi Saverio Giuseppe , Vullo Vincenzo , Andreoni Massimo
Published: July 2002

Antiretroviral-treated human immunodeficiency virus (HIV) type 1-seropositive individuals can remain clinically stable for a long period of time with an increasing CD4 cell count irrespective of incomplete viral suppression. We evaluated the role of neutralizing antibody (NtAb) activity in the etiopathogenesis of this viro-immunological disconnection (defined as an increasing CD4
+
-cell count despite a persistent, detectable viral load during antiretroviral therapy) in 33 patients failing therapy with two analogue nucleoside reverse transcriptase inhibitors. An HIV NtAb titer of ≥1:25 was detected in specimens from 16 out of 33 (48%) patients. A significant correlation was found between NtAb titers and CD4
+
-cell counts (
P
= 0.001;
r
= 0.546) but not with HIV RNA levels in plasma. Five patients with a viro-immunological disconnection had an NtAb titer of >1:125, statistically higher than the NtAb titers for the remaining 28 patients with both virologic and immunologic failure (
P
< 0.0001). The HIV-specific humoral immune response could play a role during antiretroviral treatment to improve immunological function despite an incomplete suppression of viral load.
Spinal Cord Involvement in Uncomplicated Herpes Zoster
Authors: Steiner Israel , Steiner-Birmanns Bettina , Levin Netta , Hershko Klila , Korn-Lubetzki Isabelle , Biran Iftah
Published: July 2002

We prospectively evaluated herpes zoster patients during the acute phase of the disease for central nervous system involvement. Of 24 patients with spinal zoster, 13 (54%) had spinal cord abnormality, which was asymptomatic in 12 of the 13. Age but not lack of acyclovir treatment was associated with such involvement. In all but 2, neurological involvement resolved within 6 months. Although the mechanism responsible for the neurological abnormalities is unknown, findings may support the hypothesis that zoster is associated with spread of viral infection into the spinal cord and therefore support the possibility that zoster is due to active viral replication in the ganglion.
Biological Activity of a Mouse-Human Chimeric Immunoglobulin G2 Antibody toCryptococcus neoformansPolysaccharide
Authors: Westin Kwon Katherine , Lendvai Nikoletta , Morrison Sherie , Trinh K. Ryan , Casadevall Arturo
Published: July 2002

The variable regions of the heavy and light chains of the protective murine monoclonal antibody (MAb) 2H1 (m2H1) were expressed with the human constant region genes for immunoglobulin G2 (IgG2) and kappa, respectively, to construct a chimeric antibody (ch2H1). ch2H1 retains the specificity of the parent MAb, exhibits biological activity, and lacks the toxicity of the parent murine IgG1 in chronically infected mice.
Preconception Seroconversion and Maternal Seronegativity at Delivery Do Not Rule Out the Risk of Congenital Toxoplasmosis
Authors: Chemla C. , Villena I. , Aubert D. , Hornoy P. , Dupouy D. , Leroux B. , Bory J. P. , Pinon J. M.
Published: July 2002

We describe two unusual cases of congenital toxoplasmosis, one occurring after preconception maternal infection with cervical adenopathies and the other occurring after maternal infection at the very end of pregnancy with maternal seronegativity at delivery. These documented cases of congenital toxoplasmosis demonstrate the value of extending the serologic monitoring period during pregnancy, according to the individual clinical context.
Interaction of Fimbriae of
Haemophilus influenzae
Type B with Heparin-Binding Extracellular Matrix Proteins
Authors: Virkola Ritva , Brummer Mirko , Rauvala Heikki , van Alphen Loek , Korhonen Timo K.
Published: July 2002

The interaction of the fimbriae of
Haemophilus influenzae
type b (Hib) with two heparin-binding extracellular matrix proteins, human fibronectin (Fn) and heparin-binding growth-associated molecule (HB-GAM) from mouse, were studied. The fimbriated Hib strain 770235 fim+, as well as the recombinant strain
E. coli
HB101(pMH140), which expressed Hib fimbriae, adhered strongly to Fn and HB-GAM immobilized on glass. Purified Hib fimbriae bound to Fn and HB-GAM, and within the Fn molecule, the binding was localized to the N-terminal 30,000-molecular-weight (30K) and 40K fragments, which contain heparin-binding domains I and II, respectively. Fimbrial binding to Fn, HB-GAM, and the 30K and the 40K fragments was inhibited by high concentrations of heparin. The results show that fimbriae of Hib interact with heparin-binding extracellular matrix proteins. The nonfimbriated Hib strain 770235 fim− exhibited a low level of adherence to Fn but did not react with HB-GAM, indicating that Hib strains also possess a fimbria-independent mechanism to interact with Fn.
Protective Antigen-Mediated Antibody Response against a Heterologous Protein Produced In Vivo byBacillus anthracis
Authors: Brossier Fabien , Weber-Levy Martine , Mock Michèle , Sirard Jean-Claude
Published: July 2002

Bacillus anthracissecretes a lethal toxin composed of two proteins, the lethal factor (LF) and the protective antigen (PA), which interact within the host or in vitro at the surfaces of eukaryotic cells. Immunization with attenuatedB. anthracisstrains induces an antibody response against PA and LF. The LF-specific response is potentiated by the binding of LF to PA. In this study, we investigated the capacity of PA to increase the antibody response against a foreign antigen. We constructed a chimeric gene encoding the PA-binding part of LF (LF254) fused to the C fragment of tetanus toxin (ToxC). The construct was introduced by allelic exchange into the locus encoding LF. Two recombinantB. anthracisstrains secreting the hybrid protein LF254-ToxC were generated, one in a PA-producing background and the other in a PA-deficient background. Mice were immunized with spores of the strains, and the humoral response and protection against tetanus toxin were assessed. TheB. anthracisstrain producing both PA and LF254-ToxC induced significantly higher antibody titers and provided better protection against a lethal challenge with tetanus toxin than did its PA-deficient counterpart. Thus, PA is able to potentiate protective immunity against a heterologous antigen, demonstrating the potential ofB. anthracisrecombinant strains for use as live vaccine vehicles.
Stable Expression of a New Chimeric Fluorescent Reporter in the Human Malaria Parasite
Plasmodium falciparum
Authors: Kadekoppala Madhusudan , Kline Kimberly , Akompong Thomas , Haldar Kasturi
Published: July 2002

Stable transfection of a new, chimeric reporter in the human malaria parasite
Plasmodium falciparum
confers green fluorescence and methotrexate resistance that can be quantitated by Western blotting and flow cytometry. This provides a sensitive, live reporter for exploitation of genomic and high-throughput assays for the identification of new pathogenic determinants.