bluuebunny/crossref_metadata_2025 huggingface dataset:
Amphotericin B Resistance and Membrane Fluidity in
Kluyveromyces lactis
Strains
Authors: Younsi Mohamed , Ramanandraibe Elise , Bonaly Roger , Donner Mireille , Coulon Joël
Published: July 2002

The membrane fluidity of reduced-amphotericin B (AmB)-sensitivity
Kluyveromyces lactis
mutant strain is higher than that of the wild-type
K. lactis
strain. After culture of the
K. lactis
and
K. lactis
mutant cells in the presence of subinhibitory doses of AmB (10 and 125 mg/liter, respectively), the plasma membranes of both yeast strains also showed a higher fluidity than did those of control cells. High membrane fluidity was associated with changes in the structural properties of the membranes. Culture of the
K. lactis
and
K. lactis
mutant cells in the presence of AmB induced changes in membrane lipid contents. In particular, phospholipid contents were increased in both strains treated with AmB, compared with their corresponding counterparts. As a result, the sterol/phospholipid ratio decreased. The relative proportion of monounsaturated fatty acids also increased after AmB treatment. The saturated fatty acid/monounsaturated fatty acid ratio decreased in
K. lactis
and
K. lactis
mutant cells treated with AmB but also in
K. lactis
mutant control cells compared to that in the
K. lactis
wild strain. These changes in lipid composition explain the higher fluidity, which could represent a process of metabolic resistance of the yeasts to AmB.
Effects of Amphotericin B and Three Azole Derivatives on the Lipids of Yeast Cells of
Paracoccidioides brasiliensis
Authors: Hahn Rosane Christine , Hamdan Júnia Soares
Published: July 2002

Yeast cells of five different strains of
Paracoccidioides brasiliensis
were obtained for partial analysis of lipid composition, and sterol content was determined quantitatively and qualitatively. The determinations were conducted with cells cultured in the presence and absence of amphotericin B and azole derivatives at levels below the MIC.
A Single Oral Dose of Thalidomide Enhances the Capacity of Lymphocytes to Secrete Gamma Interferon in Healthy Humans
Authors: Verbon Annelies , Juffermans Nicole P. , Speelman Peter , van Deventer Sander J. H. , ten Berge Ineke J. M. , Guchelaar Henk-Jan , van der Poll Tom
Published: July 2002

Thalidomide is increasingly being used as adjuvant therapy for patients with mycobacterial and human immunodeficiency virus (HIV) infections. The T-helper (Th)1 cytokine–Th2 cytokine balance critically determines the outcomes of these diseases. To obtain insight into the effect of thalidomide on the capacity of lymphocytes to produce Th1 and Th2 cytokines, six healthy volunteers received an oral dose (400 mg) of thalidomide. Before and at 3, 6, and 24 h after ingestion of thalidomide, peripheral blood mononuclear cells (PBMCs) were isolated and stimulated for 24 h with the T-cell stimulant staphylococcal enterotoxin B (SEB) or anti-CD3/CD28. In all six volunteers ingestion of thalidomide was associated with enhanced SEB- and anti-CD3/CD28-induced production of the Th1 cytokine gamma interferon (P< 0.05) and a decrease in the level of anti-CD3/CD28-induced interleukin-5 (IL-5) production (P< 0.05). The levels of IL-2 (Th1) and IL-4 (Th2) released remained unchanged. These changes were accompanied by an increase in the amount of IL-12p40 released by the PBMCs 6 h after ingestion of thalidomide (P< 0.05). Thus, a single oral dose of thalidomide causes a Th1-type response in healthy humans. This finding offers a potential explanation for the positive effect of thalidomide in patients with mycobacterial and HIV infections.
In Vitro and In Vivo Antibacterial Activities of L-084, a Novel Oral Carbapenem, against Causative Organisms of Respiratory Tract Infections
Authors: Miyazaki Shuichi , Hosoyama Takayuki , Furuya Nobuhiko , Ishii Yoshikazu , Matsumoto Tetsuya , Ohno Akira , Tateda Kazuhiro , Yamaguchi Keizo
Published: July 2002

L-084 (a prodrug of LJC 11,036 [L-036]) is a new oral carbapenem. Here we compared the in vitro and in vivo antibacterial activities of L-036 with those of imipenem, faropenem, ceditoren-pivoxil, cefdinir, amoxicillin, and levofloxacin. The MICs at which 90% of the isolates were inhibited of L-036 against methicillin-susceptible staphylococci,Streptococcus pneumoniaeincluding penicillin-resistant organisms,Escherichia coli,Klebsiella pneumoniae,Haemophilus influenzaeincluding ampicillin-resistant organisms,Legionella pneumophila, andMoraxella catarrhaliswere equal to or less than 1 μg/ml. In pharmacokinetics studies of L-084 in lungs of mice, the maximum concentration in serum, half-life, and area under the concentration-time curve of this drug were 9.09 μg/g of tissue, 6.18 h, and 31.0 μg · h/ml, respectively. In murine respiratory infection models of penicillin-susceptible and -resistantS. pneumoniaeandH. influenzae, the efficacies of L-084 were better than those of reference drugs. Our results indicate that the in vitro high potency and good distribution in the lungs might be the underlying mechanisms of its efficacy in the murine model of pneumonia.
Evidence for Transfer of CMY-2 AmpC β-Lactamase Plasmids between
Escherichia coli
and
Salmonella
Isolates from Food Animals and Humans
Authors: Winokur P. L. , Vonstein D. L. , Hoffman L. J. , Uhlenhopp E. K. , Doern G. V.
Published: July 2002

Escherichia coli
is an important pathogen that shows increasing antimicrobial resistance in isolates from both animals and humans. Our laboratory recently described
Salmonella
isolates from food animals and humans that expressed an identical plasmid-mediated, AmpC-like β-lactamase, CMY-2. In the present study, 59 of 377
E. coli
isolates from cattle and swine (15.6%) and 6 of 1,017 (0.6%) isolates of human
E. coli
from the same geographic region were resistant to both cephamycins and extended-spectrum cephalosporins. An
ampC
gene could be amplified with CMY-2 primers in 94.8% of animal and 33% of human isolates. Molecular epidemiological studies of chromosomal DNA revealed little clonal relatedness among the animal and human
E. coli
isolates harboring the CMY-2 gene. The
ampC
genes from 10 animal and human
E. coli
isolates were sequenced, and all carried an identical CMY-2 gene. Additionally, all were able to transfer a plasmid containing the CMY-2 gene to a laboratory strain of
E. coli
. CMY-2 plasmids demonstrated two different plasmid patterns that each showed strong similarities to previously described
Salmonella
CMY-2 plasmids. Additionally, Southern blot analyses using a CMY-2 probe demonstrated conserved fragments among many of the CMY-2 plasmids identified in
Salmonella
and
E. coli
isolates from food animals and humans. These data demonstrate that common plasmids have been transferred between animal-associated
Salmonella
and
E. coli
, and identical CMY-2 genes carried by similar plasmids have been identified in humans, suggesting that the CMY-2 plasmid has undergone transfer between different bacterial species and may have been transmitted between food animals and humans.
Synergism of Voriconazole and Terbinafine against
Candida albicans
Isolates from Human Immunodeficiency Virus-Infected Patients with Oropharyngeal Candidiasis
Authors: Weig Michael , Müller Frank-Michael C.
Published: July 2002

The synergism of voriconazole (VRC) and terbinafine was studied by using 39 genotypically defined clinical
Candida albicans
isolates that were cross-resistant to fluconazole and VRC and serial isolates that gradually developed azole resistance. Synergy was noticed in 100% (eight of eight) of the strains that were resistant to VRC. Antagonism was not observed.
Kanamycin Susceptibility Testing of
Mycobacterium tuberculosis
Using Mycobacterium Growth Indicator Tube and a Colorimetric Method
Authors: Bastian Ivan , Rigouts Leen , Palomino Juan Carlos , Portaels Françoise
Published: July 2002

Two novel systems were evaluated for performing indirect kanamycin susceptibility tests on 72 strains of
Mycobacterium tuberculosis
. The microplate Alamar blue colorimetric method (breakpoint, 2.5 μg/ml) and the Mycobacterium Growth Indicator Tube (MGIT) system (breakpoint, 5.0 μg/ml) both produced 98.6% agreement when compared with the conventional proportion method performed on 7H10 agar using 5.0 μg of kanamycin/ml. Both systems provided results within an average of 1 week.
Activity of LY333328 in Experimental Meningitis Caused by a
Streptococcus pneumoniae
Strain Susceptible to Penicillin
Authors: Gerber Joachim , Smirnov Alexander , Wellmer Andreas , Ragheb Jasmin , Prange Juliane , Schütz Eckhardt , Wettich Klaus , Kalich Siegfried , Nau Roland
Published: July 2002

In a rabbit model of
Streptococcus pneumoniae
meningitis single doses of 10 and 2.5 mg of the glycopeptide LY333328 per kg of body weight reduced bacterial titers in cerebrospinal fluid (CSF) almost as rapidly as ceftriaxone at 10 mg/kg/h (changes in log CFU, −0.29 ± 0.21 and −0.26 ± 0.22 versus −0.34 ± 0.15/ml/h). A dose of 1 mg/kg was bacteriostatic (change in log CFU, 0.01 ± 0.11/ml/h). In two animals receiving LY333328 at a dose of 40 mg/kg the bacterial titers were reduced by 0.54 and 0.51 log CFU/ml/h. The penetration of CSF by LY333328 was 1 to 5%. The concentrations of lipoteichoic and teichoic acids in CSF and neuronal damage were similar in ceftriaxone- and LY333328-treated animals.
In Vitro Antimycobacterial Activities of 2′-Monosubstituted Isonicotinohydrazides and Their Cyanoborane Adducts
Authors: Maccari Rosanna , Ottanà Rosaria , Monforte Francesca , Vigorita Maria Gabriella
Published: July 2002

As a result of our search for new isoniazid derivatives with extended spectra of activity, we evaluated the in vitro antimycobacterial activities of isonicotinohydrazides (compounds 2) and their cyanoborane adducts (compounds 3), both obtained by the reaction of isonicotinoylhydrazones (compounds 1) with sodium cyanoborohydride. Most of the tested compounds displayed moderate to high activity against
Mycobacterium tuberculosis
H37Rv, with MICs ranging from 0.2 to 12.5 μg/ml. In particular, some hydrazides showed activity similar to that of rifampin (MIC = 0.2 μg/ml) and rather low cytotoxicity, so that they were generally shown to possess high safety indices. In contrast, the coordination to a cyanoborane (BH
2
CN) group (compounds 3) in general brought about a decrease in antimycobacterial activity, while cytotoxicity increased. Interestingly, selected compounds 1 to 3, mostly hydrazides (compounds 2), were effective in killing
M. tuberculosis
growing within macrophages at concentrations in culture medium which were much lower than the corresponding MICs. These compounds also displayed good activity against drug-resistant
M. tuberculosis
strains.
Effects of Amino Acid Alterations in Penicillin-Binding Proteins (PBPs) 1a, 2b, and 2x on PBP Affinities of Penicillin, Ampicillin, Amoxicillin, Cefditoren, Cefuroxime, Cefprozil, and Cefaclor in 18 Clinical Isolates of Penicillin-Susceptible, -Intermediate, and -Resistant Pneumococci
Authors: Nagai Kensuke , Davies Todd A. , Jacobs Michael R. , Appelbaum Peter C.
Published: July 2002

Amino acid alterations in or flanking conserved motifs making up the active binding sites of penicillin-binding proteins (PBPs) 1a, 2b, and 2x of pneumococci were correlated with changes in affinities of penicillin, ampicillin, amoxicillin, cefditoren, cefuroxime, cefprozil, and cefaclor for these PBPs. Four penicillin-susceptible (PSSP), eight penicillin-intermediate (PISP), and six penicillin-resistant (PRSP) pneumococci were studied by DNA sequencing of the penicillin-binding sites of the
pbp1a
, -
2x
, and -
2b
genes of strains and by determining 50% inhibitory concentrations of the seven agents for PBP1a, -2x, and -2b. Two PSSP strains had alterations in PBP2x (L
546
→V) (one strain) or PBP2b (T
445
→A) (one strain). All eight PISP strains had at least two alterations--T
338
→P or A or H
394
→Y in PBP2X and T
445
→A in BPB2b. All PRSP strains had the same changes seen in PISP strains, as well as T
371
→A or S substitutions in PBP1a. The two most resistant PRSP strains had a second change in PBP2x (M
339
→F) in a conserved motif. The affinities of penicillin and ampicillin for all three PBPs were decreased for PRSP and most PISP strains. The affinity of amoxicillin for PBP1a and -2x was decreased only for PRSP. Cefaclor and cefprozil showed decreased affinity of PRSP but not PISP for all three PBPs. Cefuroxime showed decreased affinity of PISP and PRSP for PBP1a and -2x but no change for PBP2b. Cefditoren showed no difference in PBP affinity based on penicillin or cefditoren MICs, indicating a different PBP target for this agent. Overall, the MICs for and PBP affinities of the strains correlated with the changes found in the PBP active binding sites.
Cloning of a Cation Efflux Pump Gene Associated with Chlorhexidine Resistance in
Klebsiella pneumoniae
Authors: Fang Chi-Tai , Chen Haur-Chuan , Chuang Yi-Ping , Chang Shan-Chwen , Wang Jin-Town
Published: July 2002

Expression libraries of a chlorhexidine-resistant
Klebsiella pneumoniae
strain were constructed and transformed into
Escherichia coli
XLOLR. Twenty chlorhexidine-resistant transformants were obtained after selection. All clones contained a novel 903-nucleotide locus. Its sequences were compatible with a cation efflux pump, and the locus was thus designated as
cepA
. Retransformation using
cepA
-containing plasmids conferred chlorhexidine resistance to both XLOLR and a chlorhexidine-sensitive
K. pneumoniae
strain. Therefore, CepA is associated with chlorhexidine resistance and may act as a cation efflux pump.
Growth Limits of
Listeria monocytogenes
as a Function of Temperature, pH, NaCl, and Lactic Acid
Authors: Tienungoon S. , Ratkowsky D. A. , McMeekin T. A. , Ross T.
Published: July 2002

Models describing the limits of growth of pathogens under multiple constraints will aid management of the safety of foods which are sporadically contaminated with pathogens and for which subsequent growth of the pathogen would significantly increase the risk of food-borne illness. We modeled the effects of temperature, water activity, pH, and lactic acid levels on the growth of two strains of
Listeria monocytogenes
in tryptone soya yeast extract broth. The results could be divided unambiguously into “growth is possible” or “growth is not possible” classes. We observed minor differences in growth characteristics of the two
L. monocytogenes
strains. The data follow a binomial probability distribution and may be modeled using logistic regression. The model used is derived from a growth rate model in a manner similar to that described in a previously published work (K. A. Presser, T. Ross, and D. A. Ratkowsky, Appl. Environ. Microbiol. 64:1773–1779, 1998). We used “nonlinear logistic regression” to estimate the model parameters and developed a relatively simple model that describes our experimental data well. The fitted equations also described well the growth limits of all strains of
L. monocytogenes
reported in the literature, except at temperatures beyond the limits of the experimental data used to develop the model (3 to 35°C). The models developed will improve the rigor of microbial food safety risk assessment and provide quantitative data in a concise form for the development of safer food products and processes.
Trichloroethene Reductive Dehalogenase from
Dehalococcoides ethenogenes
: Sequence of
tceA
and Substrate Range Characterization
Authors: Magnuson Jon K. , Romine Margaret F. , Burris David R. , Kingsley Mark T.
Published: July 2002

The anaerobic bacterium
Dehalococcoides ethenogenes
is the only known organism that can completely dechlorinate tetrachloroethene or trichloroethene (TCE) to ethene via dehalorespiration. One of two corrinoid-containing enzymes responsible for this pathway, TCE reductive dehalogenase (TCE-RDase) catalyzes the dechlorination of TCE to ethene. TCE-RDase dehalogenated 1,2-dichloroethane and 1,2-dibromoethane to ethene at rates of 7.5 and 30 μmol/min/mg, respectively, similar to the rates for TCE,
cis
-dichloroethene (DCE), and 1,1-DCE. A variety of other haloalkanes and haloalkenes containing three to five carbon atoms were dehalogenated at lower rates. The gene encoding TCE-RDase,
tceA
, was cloned and sequenced via an inverse PCR approach. Sequence comparisons of
tceA
to proteins in the public databases revealed weak sequence similarity confined to the C-terminal region, which contains the eight-iron ferredoxin cluster binding motif, (CXXCXXCXXXCP)
2
. Direct N-terminal sequencing of the mature enzyme indicated that the first 42 amino acids constitute a signal sequence containing the twin-arginine motif, RRXFXK, associated with the Sec-independent membrane translocation system. This information coupled with membrane localization studies indicated that TCE-RDase is located on the exterior of the cytoplasmic membrane. Like the case for the two other RDases that have been cloned and sequenced, a small open reading frame,
tceB
, is proposed to be involved with membrane association of TCE-RDase and is predicted to be cotranscribed with
tceA
.
Isolation of Bacteriophages Specific to a Fish Pathogen,
Pseudomonas plecoglossicida
, as a Candidate for Disease Control
Authors: Park Se Chang , Shimamura Ichiro , Fukunaga Minoru , Mori Koh-Ichiro , Nakai Toshihiro
Published: July 2002

Two types of bacteriophage specific to
Pseudomonas plecoglossicida
, the causative agent of bacterial hemorrhagic ascites disease in cultured ayu fish (
Plecoglossus altivelis
), were isolated from diseased ayu and the rearing pond water. One type of phage, which formed small plaques, was tentatively classified as a member of the family
Myoviridae
, and the other type, which formed large plaques, was classified as a member of the family
Podoviridae
. All 27 strains of
P. plecoglossicida
examined, which were isolated from diseased ayu from geographically different areas in 1991 to 1999, exhibited quite similar sensitivities to either type of phage. One strain of
P. plecoglossicida
was highly virulent for ayu, and the 50% lethal dose (LD
50
) when intramuscular injection was used was 10
1.2
CFU fish
−1
; in contrast, phage-resistant variants of this organism were less virulent (LD
50
, >10
4
CFU fish
−1
). Oral administration of phage-impregnated feed to ayu resulted in protection against experimental infection with
P. plecoglossicida
. After oral administration of
P. plecoglossicida
cells of this bacterium were always detected in the kidneys of control fish that did not receive the phage treatment, while the cells quickly disappeared from the phage-treated fish. Bacterial growth in freshwater was lower in the presence of phage, and the number of phage PFU increased rapidly. These results suggest that it may be possible to use phage to control the disease caused by
P. plecoglossicida
.
Method for Rapid Purification of Class IIa Bacteriocins and Comparison of Their Activities
Authors: Guyonnet D. , Fremaux C. , Cenatiempo Y. , Berjeaud J. M.
Published: July 2002

A three-step method was developed for the purification of mesentericin Y105 (60% yield) from the culture supernatant of
Leuconostoc mesenteroides
Y105. The same procedure was successfully applied to the purification of five other anti-
Listeria
bacteriocins identified by mass spectrometry. Specific activities of the purified bacteriocins were compared.
Microbial Life beneath a High Arctic Glacier
Authors: Skidmore Mark L. , Foght Julia M. , Sharp Martin J.
Published: July 2002

The debris-rich basal ice layers of a high Arctic glacier were shown to contain metabolically diverse microbes that could be cultured oligotrophically at low temperatures (0.3 to 4°C). These organisms included aerobic chemoheterotrophs and anaerobic nitrate reducers, sulfate reducers, and methanogens. Colonies purified from subglacial samples at 4°C appeared to be predominantly psychrophilic. Aerobic chemoheterotrophs were metabolically active in unfrozen basal sediments when they were cultured at 0.3°C in the dark (to simulate nearly in situ conditions), producing
14
CO
2
from radiolabeled sodium acetate with minimal organic amendment (≥38 μM C). In contrast, no activity was observed when samples were cultured at subfreezing temperatures (≤−1.8°C) for 66 days. Electron microscopy of thawed basal ice samples revealed various cell morphologies, including dividing cells. This suggests that the subglacial environment beneath a polythermal glacier provides a viable habitat for life and that microbes may be widespread where the basal ice is temperate and water is present at the base of the glacier and where organic carbon from glacially overridden soils is present. Our observations raise the possibility that in situ microbial production of CO
2
and CH
4
beneath ice masses (e.g., the Northern Hemisphere ice sheets) is an important factor in carbon cycling during glacial periods. Moreover, this terrestrial environment may provide a model for viable habitats for life on Mars, since similar conditions may exist or may have existed in the basal sediments beneath the Martian north polar ice cap.
Ethylene Removal at Low Temperatures under Biofilter and Batch Conditions
Authors: Elsgaard Lars
Published: July 2002

Removal of the plant hormone ethylene (C
2
H
4
) is often required by horticultural storage facilities, which are operated at temperatures below 10°C. The aim of this study was to demonstrate an efficient, biological C
2
H
4
removal under such low-temperature conditions. Peat-soil, acclimated to degradation of C
2
H
4
, was packed in a biofilter (687 cm
3
) and subjected to an airflow (∼73 ml min
−1
) with 2 ppm (μl liter
−1
) C
2
H
4
. The C
2
H
4
removal efficiencies achieved at 20, 10, and 5°C, respectively, were 99.0, 98.8, and 98.4%. This corresponded to C
2
H
4
levels of 0.022 to 0.032 ppm in the biofilter outlet air. At 2°C, the average C
2
H
4
removal efficiency dropped to 83%. The detailed temperature response of C
2
H
4
removal was tested under batch conditions by incubation of 1-g soil samples in a temperature gradient ranging from 0 to 29°C with increments of 1°C. The C
2
H
4
removal rate was highest at 26°C (0.85 μg of C
2
H
4
g [dry weight]
−1
h
−1
), but remained at levels of 0.14 to 0.28 μg of C
2
H
4
g (dry weight)
−1
h
−1
at 0 to 10°C. At 35 to 40°C, the C
2
H
4
removal rate was negligible (0.02 to 0.06 μg of C
2
H
4
g [dry weight]
−1
h
−1
). The
Q
10
(i.e., the ratio of rates 10°C apart) for C
2
H
4
removal was 1.9 for the interval 0 to 10°C. In conclusion, the present results demonstrated microbial C
2
H
4
removal, which proceeded at 0 to 2°C and produced a moderately psychrophilic temperature response.
Transmission to Eels, Portals of Entry, and Putative Reservoirs of
Vibrio vulnificus
Serovar E (Biotype 2)
Authors: Marco-Noales Ester , Milán Miguel , Fouz Belén , Sanjuán Eva , Amaro Carmen
Published: July 2002

Vibrio vulnificus
serovar E (formerly biotype 2) is the etiologic agent that is responsible for the main infectious disease affecting farmed eels. Although the pathogen can theoretically use water as a vehicle for disease transmission, it has not been isolated from tank water during epizootics to date. In this work, the mode of transmission of the disease to healthy eels, the portals of entry of the pathogen into fish, and their putative reservoirs have been investigated by means of laboratory and field experiments. Results of the experiments of direct and indirect host-to-host transmission, patch contact challenges, and oral-anal intubations suggest that water is the prime vehicle for disease transmission and that gills are the main portals of entry into the eel body. The pathogen mixed with food can also come into the fish through the gastrointestinal tract and develop the disease. These conclusions were supported by field data obtained during a natural outbreak in which we were able to isolate this microorganism from tank water for the first time. The examination of some survivors from experimental infections by indirect immunofluorescence and scanning electron microscopy showed that
V. vulnificus
serovar E formed a biofilm-like structure on the eel skin surface. In vitro assays demonstrated that the ability of the pathogen to colonize both hydrophilic and hydrophobic surfaces was inhibited by glucose. The capacity to form biofilms on eel surface could constitute a strategy for surviving between epizootics or outbreaks, and coated survivors could act as reservoirs for the disease.
The Glycogen-Bound Polyphosphate Kinase from
Sulfolobus acidocaldarius
Is Actually a Glycogen Synthase
Authors: Cardona Silvia , Remonsellez Francisco , Guiliani Nicolas , Jerez Carlos A.
Published: July 2002

Inorganic polyphosphate (polyP) is obtained by the polymerization of the terminal phosphate of ATP through the action of the enzyme polyphosphate kinase (PPK). Despite the presence of polyP in every living cell, a gene homologous to that of known PPKs is missing from the currently sequenced genomes of
Eukarya
,
Archaea
, and several bacteria. To further study the metabolism of polyP in
Archaea
, we followed the previously published purification procedure for a glycogen-bound protein of 57 kDa with PPK as well as glycosyl transferase (GT) activities from
Sulfolobus acidocaldarius
(R. Skórko, J. Osipiuk, and K. O. Stetter, J. Bacteriol. 171:5162–5164, 1989). In spite of using recently developed specific enzymatic methods to analyze polyP, we could not reproduce the reported PPK activity for the 57-kDa protein and the polyP presumed to be the product of the reaction most likely corresponded to glycogen-bound ATP under our experimental conditions. Furthermore, no PPK activity was found associated to any of the proteins bound to the glycogen-protein complex. We cloned the gene corresponding to the 57-kDa protein by using reverse genetics and functionally characterized it. The predicted product of the gene did not show similarity to any described PPK but to archaeal and bacterial glycogen synthases instead. In agreement with these results, the recombinant protein showed only GT activity. Interestingly, the GT from
S. acidocaldarius
was phosphorylated in vivo. In conclusion, our results convincingly demonstrate that the glycogen-protein complex of
S. acidocaldarius
does not contain a PPK activity and that what was previously reported as being glycogen-bound PPK is a bacterial enzyme-like thermostable glycogen synthase.
Detection and Quantification of Snow Algae with an Airborne Imaging Spectrometer
Authors: Painter Thomas H. , Duval Brian , Thomas William H. , Mendez Maria , Heintzelman Sara , Dozier Jeff
Published: July 2002

We describe spectral reflectance measurements of snow containing the snow algaChlamydomonas nivalisand a model to retrieve snow algal concentrations from airborne imaging spectrometer data. Because cells ofC. nivalisabsorb at specific wavelengths in regions indicative of carotenoids (astaxanthin esters, lutein, β-carotene) and chlorophyllsaandb, the spectral signature of snow containingC. nivalisis distinct from that of snow without algae. The spectral reflectance of snow containingC. nivalisis separable from that of snow without algae due to carotenoid absorption in the wavelength range from 0.4 to 0.58 μm and chlorophyllaandbabsorption in the wavelength range from 0.6 to 0.7 μm. The integral of the scaled chlorophyllaandbabsorption feature (I0.68) varies with algal concentration (Ca). Using the relationshipCa= 81019.2I0.68+ 845.2, we inverted Airborne Visible Infrared Imaging Spectrometer reflectance data collected in the Tioga Pass region of the Sierra Nevada in California to determine algal concentration. For the 5.5-km2region imaged, the mean algal concentration was 1,306 cells ml−1, the standard deviation was 1,740 cells ml−1, and the coefficient of variation was 1.33. The retrieved spatial distribution was consistent with observations made in the field. From the spatial estimates of algal concentration, we calculated a total imaged algal biomass of 16.55 kg for the 0.495-km2snow-covered area, which gave an areal biomass concentration of 0.033 g/m2.
Integration of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry and Molecular Cloning for the Identification and Functional Characterization of Mobile
ortho
-Halobenzoate Oxygenase Genes in
Pseudomonas aeruginosa
Strain JB2
Authors: Hickey W. J. , Sabat G.
Published: July 2002

Protein mass spectrometry and molecular cloning techniques were used to identify and characterize mobile
o
-halobenzoate oxygenase genes in
Pseudomonas aeruginosa
strain JB2 and
Pseudomonas huttiensis
strain D1. Proteins induced in strains JB2 and D1 by growth on 2-chlorobenzoate (2-CBa) were extracted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry. Two bands gave significant matches to OhbB and OhbA, which have been reported to be the α and β subunits, respectively, of an
ortho
-1,2-halobenzoate dioxygenase of
P. aeruginosa
strain 142 (T. V. Tsoi, E. G. Plotnikova, J. R. Cole, W. F. Guerin, M. Bagdasarian, and J. M. Tiedje, Appl. Environ. Microbiol. 65:2151–2162, 1999). PCR and Southern hybridization experiments confirmed that
ohbAB
were present in strain JB2 and were transferred from strain JB2 to strain D1. While the sequences of
ohbA
from strains JB2, D1, and 142 were identical, the sequences of
ohbB
from strains JB2 and D1 were identical to each other but differed slightly from that of strain 142. PCR analyses and Southern hybridization analyses indicated that
ohbAB
were conserved in strains JB2 and D1 and in strain 142 but that the regions adjoining these genes were divergent. Expression of
ohbAB
in
Escherichia coli
resulted in conversion of
o
-chlorobenzoates to the corresponding (chloro)catechols with the following apparent affinity: 2-CBa ≈ 2,5-dichlorobenzoate > 2,3,5-trichlorobenzoate > 2,4-dichlorobenzoate. The activity of OhbAB
JB2
appeared to differ from that reported for OhbAB
142
primarily in that a chlorine in the
para
position posed a greater impediment to catalysis with the former. Hybridization analysis of spontaneous 2-CBa
−
mutants of strains JB2 and D1 verified that
ohbAB
were lost along with the genes, suggesting that all of the genes may be contained in the same mobile element. Strains JB2 and 142 originated from California and Russia, respectively. Thus,
ohbAB
and/or the mobile element on which they are carried may have a global distribution.
pAM401-Based Shuttle Vectors That Enable Overexpression of Promoterless Genes and One-Step Purification of Tag Fusion Proteins Directly from
Enterococcus faecalis
Authors: Fujimoto Shuhei , Ike Yasuyoshi
Published: July 2002

Two novel
Enterococcus faecalis-Escherichia coli
shuttle vectors that utilize the promoter and ribosome binding site of
bacA
on the
E. faecalis
plasmid pPD1 were constructed. The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in
E. coli
and
E. faecalis
and encodes the
bacA
promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless β-galactosidase gene from
E. coli
and cloned the
traA
gene of the
E. faecalis
plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. β-Galactosidase was expressed in
E. coli
and
E. faecalis
at levels of 10
3
and 10 Miller units, respectively. By cloning the pAD1
traA
into pMGS101, the protein could be purified directly from a crude lysate of
E. faecalis
or
E. coli
with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both
E. coli
and
E. faecalis
that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and
cis/trans
analysis of regulatory genes, purification and copurification of proteins from
E. faecalis
, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from
E. faecalis
.
Comparison of Logistic Regression and Linear Regression in Modeling Percentage Data
Authors: Zhao Lihui , Chen Yuhuan , Schaffner Donald W.
Published: July 2002

Percentage is widely used to describe different results in food microbiology, e.g., probability of microbial growth, percent inactivated, and percent of positive samples. Four sets of percentage data, percent-growth-positive, germination extent, probability for one cell to grow, and maximum fraction of positive tubes, were obtained from our own experiments and the literature. These data were modeled using linear and logistic regression. Five methods were used to compare the goodness of fit of the two models: percentage of predictions closer to observations, range of the differences (predicted value minus observed value), deviation of the model, linear regression between the observed and predicted values, and bias and accuracy factors. Logistic regression was a better predictor of at least 78% of the observations in all four data sets. In all cases, the deviation of logistic models was much smaller. The linear correlation between observations and logistic predictions was always stronger. Validation (accomplished using part of one data set) also demonstrated that the logistic model was more accurate in predicting new data points. Bias and accuracy factors were found to be less informative when evaluating models developed for percentage data, since neither of these indices can compare predictions at zero. Model simplification for the logistic model was demonstrated with one data set. The simplified model was as powerful in making predictions as the full linear model, and it also gave clearer insight in determining the key experimental factors.
Enzymatic Manganese(II) Oxidation by a Marine α-Proteobacterium
Authors: Francis Chris A. , Co Edgie-Mark , Tebo Bradley M.
Published: July 2002

A yellow-pigmented marine bacterium, designated strain SD-21, was isolated from surface sediments of San Diego Bay, San Diego, Calif., based on its ability to oxidize soluble Mn(II) to insoluble Mn(III, IV) oxides. 16S rRNA analysis revealed that this organism was most closely related to members of the genus
Erythrobacter
, aerobic anoxygenic phototrophic bacteria within the α-4 subgroup of the
Proteobacteria
(α-4
Proteobacteria
). SD-21, however, has a number of distinguishing phenotypic features relative to
Erythrobacter
species, including the ability to oxidize Mn(II). During the logarithmic phase of growth, this organism produces Mn(II)-oxidizing factors of ≈250 and 150 kDa that are heat labile and inhibited by both azide and
o
-phenanthroline, suggesting the involvement of a metalloenzyme. Although the expression of the Mn(II) oxidase was not dependent on the presence of Mn(II), higher overall growth yields were reached in cultures incubated with Mn(II) in the culture medium. In addition, the rate of Mn(II) oxidation appeared to be slower in cultures grown in the light. This is the first report of Mn(II) oxidation within the α-4
Proteobacteria
as well as the first Mn(II)-oxidizing proteins identified in a marine gram-negative bacterium.
Detection of
Campylobacter jejuni
and
Campylobacter coli
in Environmental Waters by PCR Enzyme-Linked Immunosorbent Assay
Authors: Sails A. D. , Bolton F. J. , Fox A. J. , Wareing D. R. A. , Greenway D. L. A.
Published: July 2002

A PCR enzyme-linked immunosorbent assay (ELISA) assay was applied to the detection of
Campylobacter jejuni
and
Campylobacter coli
in environmental water samples after enrichment culture. Bacterial cells were concentrated from 69 environmental water samples by using filtration, and the filtrates were cultured in
Campylobacter
blood-free broth. After enrichment culture, DNA was extracted from the samples by using a rapid-boiling method, and the DNA extracts were used as a template in a PCR ELISA assay. A total of 51 samples were positive by either PCR ELISA or culture; of these, 43 were found to be positive by PCR ELISA and 43 were found to be positive by culture. Overall, including positive and negative results, 59 samples were concordant in both methods. Several samples were positive in the PCR ELISA assay but were culture negative; therefore, this assay may be able to detect sublethally damaged or viable nonculturable forms of campylobacters. The method is rapid and sensitive, and it significantly reduces the time needed for the detection of these important pathogens by 2 to 3 days.
Enhanced Accumulation and Changed Monomer Composition in Polyhydroxyalkanoate (PHA) Copolyester by In Vitro Evolution of
Aeromonas caviae
PHA Synthase
Authors: Kichise Tomoyasu , Taguchi Seiichi , Doi Yoshiharu
Published: July 2002

By in vitro evolution experiment, we have first succeeded in acquiring higher active mutants of a synthase that is a key enzyme essential for bacterial synthesis of biodegradable polyester, polyhydroxyalkanoate (PHA).
Aeromonas caviae
FA440 synthase, termed PhaC
Ac
, was chosen as a good target for evolution, since it can synthesize a PHA random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-
co
-3HHx)] that is a tough and flexible material compared to polyhydroxybutyrate (PHB) homopolyester. The in vitro enzyme evolution system consists of PCR-mediated random mutagenesis targeted to a limited region of the

phaC
Ac

gene and screening mutant enzymes with higher activities based on two types of polyester accumulation system by using
Escherichia coli
for the synthesis of PHB (by JM109 strain) (S. Taguchi, A. Maehara, K. Takase, M. Nakahara, H. Nakamura, and Y. Doi, FEMS Microbiol. Lett. 198:65-71, 2001) and of P(3HB-
co
-3HHx) {by LS5218 [
fadR601 atoC
(Con)] strain}. The expression vector for the

phaC
Ac

gene, together with monomer-supplying enzyme genes, was designed to synthesize PHB homopolyester from glucose and P(3HB-
co
-3HHx) copolyester from dodecanoate. Two evolved mutant enzymes, termed E2-50 and T3-11, screened through the evolution system exhibited 56 and 21% increases in activity toward 3HB-coenzyme A, respectively, and consequently led to enhanced accumulation (up to 6.5-fold content) of P(3HB-
co
-3HHx) in the recombinant LS5218 strains. Two single mutations in the mutants, N149S for E2-50 and D171G for T3-11, occurred at positions that are not highly conserved among the PHA synthase family. It should be noted that increases in the 3HHx fraction (up to 16 to 18 mol%) were observed for both mutants compared to the wild type (10 mol%).
Characterization of the Priming Effect by Pituitary Canine Growth Hormone on Canine Polymorphonuclear Neutrophil Granulocyte Function
Authors: Petersen Thomas K. , Smith C. Wayne , Jensen Asger L.
Published: July 2002

In this report, we demonstrate that canine growth hormone (cGH) is capable of priming canine polymorphonuclear neutrophil granulocytes (PMN) in a manner resembling that of human PMN. The cGH influences important functions that are involved in the process of recruitment of PMN, i.e., shape change, chemotaxis, CD11b/CD18 expression, adhesion, and subsequent transendothelial migration. Also, intracellular O2−production was evaluated. We investigated the priming effect by incubating PMN with purified pituitary cGH at various concentrations (10 to 800 μg/liter). The capacity for shape change was significantly (P< 0.05) enhanced, whereas the chemotactic response under agarose was significantly (P< 0.05) reduced. The chemotactic migration in Boyden chambers (10-μm-thick polycarbonate filter; lower surface count technique) was significantly (P< 0.05) enhanced, presumably due to cGH-induced hyperadhesiveness to the lower surface of the filters. The adhesion in albumin-coated microtiter plates and adherence to canine pulmonary fibroblasts were significantly (P< 0.05) increased, and the increased adhesion resulted in a significant (P< 0.01) increase in transendothelial migration using canine jugular vein endothelial cells. The increase in adhesion was associated with a significant increase in CD11b/CD18 expression. Furthermore, intracellular O2−production was significantly enhanced in response to both phorbol myristate acetate (P< 0.01) and opsonized zymosan (P< 0.05). In the absence of a PMN-stimulating agent, cGH did not influence the effector functions investigated except for an increased expression of CD11b/CD18.
High-Quality, Cost-Effective Strategy for Detection of Autoantibodies to Extractable Nuclear Antigens
Authors: Phan Tri G. , Ng Watson W. S. , Bird Dana , Smithers Kara , Wong Vicky , Gallagher Kerri , Williams Andrew , Adelstein Stephen
Published: July 2002

We evaluated methods for the detection of autoantibodies to extractable nuclear antigens (ENAs) to determine the strategy that yielded the most cost effective and clinically meaningful result. We prospectively compared counterimmunoelectrophoresis (CIEP) with and without serum prediffusion (SPD) and found that SPD significantly improved the quality of precipitation lines. This resulted in a decreased requirement for repeat testing and, consequently, was associated with a significant decrease in reagent costs and specimen turnaround time. We also retrospectively compared reactivity by CIEP, CIEP plus SPD, enzyme-linked immunosorbent assay (ELISA), and line immunoassay (LIA) of 52 serum samples that were previously determined to be positive for ENAs, and we correlated the results with clinical diagnoses. There was significant agreement among CIEP, CIEP plus SPD, ELISA, and LIA for the detection of anti-SS-A, anti-SS-B and anti-RNP. In general, CIEP, CIEP plus SPD, and LIA correlated better with the clinical diagnoses than ELISA, even though ELISA detected anti-ENAs more often than the other methods. CIEP plus SPD is therefore the most cost effective method for the identification of clinically meaningful ENAs. Based on our experience, we now screen for ENAs by CIEP, and positive samples are then typed by CIEP plus SPD. Samples that are difficult to interpret are then further assessed by an alternative method.
Viral Load in Breast Milk Correlates with Transmission of Human Cytomegalovirus to Preterm Neonates, but Lactoferrin Concentrations Do Not
Authors: van der Strate B. W. A. , Harmsen M. C. , Schäfer P. , Swart P. J. , The T. H. , Jahn G. , Speer C. P. , Meijer D. K. F. , Hamprecht K.
Published: July 2002

In vitro, lactoferrin (LF) strongly inhibits human cytomegalovirus (HCMV), which led us to hypothesize that in vivo HCMV might also be inhibited in secretions with high LF concentrations. In breast milk, high viral loads observed as high viral DNA titers tended to coincide with higher LF levels. However, the LF levels did not correlate to virus transmission to preterm infants. The viral load in the transmitting group was highest compared to the nontransmitting group. We conclude that viral load in breast milk is an important factor for transmission of the virus.
Antibody Responses toAcinetobacterspp. andPseudomonas aeruginosain Multiple Sclerosis: Prospects for Diagnosis Using the Myelin-Acinetobacter-Neurofilament Antibody Index
Authors: Hughes L. E. , Bonell S. , Natt R. S. , Wilson C. , Tiwana H. , Ebringer A. , Cunningham P. , Chamoun V. , Thompson E. J. , Croker J. , Vowles J.
Published: July 2002

Antibody responses toAcinetobacter(five strains),Pseudomonas aeruginosa,Escherichia coli, myelin basic protein (MBP), and neurofilaments were measured in sera from 26 multiple sclerosis (MS) patients, 20 patients with cerebrovascular accidents (CVA), 10 patients with viral encephalitis, and 25 healthy blood donors. In MS patients, elevated levels of antibodies against all strains ofAcinetobactertested were present, as well as antibodies againstP. aeruginosa, MBP, and neurofilaments, but not antibodies toE. coli, compared to the CVA group and controls. The myelin-Acinetobacter-neurofilament antibody index appears to distinguish MS patients from patients with CVAs or healthy controls. The relevance of such antibodies to the neuropathology of MS requires further evaluation.
Human Lactoferrin Interacts with Soluble CD14 and Inhibits Expression of Endothelial Adhesion Molecules, E-Selectin and ICAM-1, Induced by the CD14-Lipopolysaccharide Complex
Authors: Baveye S. , Elass E. , Fernig D. G. , Blanquart C. , Mazurier J. , Legrand D.
Published: July 2002

Lipopolysaccharides (LPS), either in the free form or complexed to CD14, a LPS receptor, are elicitors of the immune system. Lactoferrin (Lf), a LPS-chelating glycoprotein, protects animals against septic shock. Since optimal protection requires administration of Lf prior to lethal doses of LPS, we hypothesized that interactions between Lf and soluble CD14 (sCD14) exist. In a first step, human sCD14 and human Lf (hLf) were used to determine the kinetic binding parameters of hLf to free sCD14 in an optical biosensor. The results demonstrated that hLf bound specifically and with a high affinity (Kd= 16 ± 7 nM) to sCD14. Affinity chromatography studies showed that hLf interacted not only with free sCD14 but also, though with different binding properties, with sCD14 complexed to LPS or lipid A–2-keto-3-deoxyoctonic acid–heptose. In a second step, we have investigated whether the capacity of hLf to interact with sCD14 could modulate the expression of endothelial-leukocyte adhesion molecule 1 (E-selectin) or intercellular adhesion molecule 1 (ICAM-1) induced by the sCD14-LPS complex on human umbilical vein endothelial cells (HUVEC). Our experiments show that hLf significantly inhibited both E-selectin and ICAM-1 expressions at the surface of HUVEC. In conclusion, these observations suggest that the anti-inflammatory effects of hLf are due not only to the ability of the molecule to chelate LPS but also to its ability to interact with sCD14 and with the sCD14 complexed to LPS, thus modifying the activation of endothelial cells.
Toxoplasma gondii
Homologue of
Plasmodium
Apical Membrane Antigen 1 Is Involved in Invasion of Host Cells
Authors: Hehl Adrian B. , Lekutis Christine , Grigg Michael E. , Bradley Peter J. , Dubremetz Jean-François , Ortega-Barria Eduardo , Boothroyd John C.
Published: July 2002

Proteins with constitutive or transient localization on the surface of
Apicomplexa
parasites are of particular interest for their potential role in the invasion of host cells. We describe the identification and characterization of TgAMA1, the
Toxoplasma gondii
homolog of the
Plasmodium
apical membrane antigen 1 (AMA1), which has been shown to elicit a protective immune response against merozoites dependent on the correct pairing of its numerous disulfide bonds. TgAMA1 shows between 19% (
Plasmodium berghei
) and 26% (
Plasmodium yoelii
) overall identity to the different
Plasmodium
AMA1 homologs and has a conserved arrangement of 16 cysteine residues and a putative transmembrane domain, indicating a similar architecture. The single-copy
TgAMA1
gene is interrupted by seven introns and is transcribed into an mRNA of ∼3.3 kb. The TgAMA1 protein is produced during intracellular tachyzoite replication and initially localizes to the micronemes, as determined by immunofluorescence assay and immunoelectron microscopy. Upon release of mature tachyzoites, TgAMA1 is found distributed predominantly on the apical end of the parasite surface. A ∼54-kDa cleavage product of the large ectodomain is continuously released into the medium by extracellular parasites. Mouse antiserum against recombinant TgAMA1 blocked invasion of new host cells by approximately 40%. This and our inability to produce a viable TgAMA1 knock-out mutant indicate that this phylogenetically conserved protein fulfills a key function in the invasion of host cells by extracellular
T. gondii
tachyzoites.