bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of veterinarians in regional management plans
Authors: LAMBERSKI N. , WEST C.
Published: July 2007

Zoo and wildlife veterinarians play an evolving role in regional management plans. Veterinarians serve as advisors, collecting and collating information regarding the medical issues of a species or taxa and making recommendations. Veterinary involvement increases with participation in captive propagation programmes, reintroduction programmes andin situconservation efforts. With the increasing threat of emerging diseases, veterinarians have increasing responsibilities to these programmes. Many of the factors responsible for the emergence of infectious diseases also threaten the environment and wildlife populations. Understanding the potential impact a disease could have on a wildlife population begins by knowing the current health status of that population, identifying critical health factors and recognizing possible threats. Baseline health information is not always available for many wildlife populations. Veterinarians working with captive populations can build this missing database. This is a review of the ways in which veterinarians have contributed to the collection and collation of information that is essential when developing regional management plans. It includes current veterinary guidelines available to zoo veterinarians in the United States and Europe. Veterinarians can improve their contributions to conservation programmes by expanding their knowledge of epidemiology, wildlife biology, ecology and environmental science and by partnering with those who have this expertise.
Role of the zoo veterinarian in the conservation of captive and free‐ranging wildlife
Authors: DEEM S. L.
Published: July 2007

The challenges to execute wildlife conservation projects successfully have become more complex as anthropogenic changes continue to stress the planet, changing wild lands. As the wild becomes less so, more species are placed in captivity to improve their chances of long‐term survival, while concurrently management (and medicine) for free‐ranging wildlife has become increasingly important. A variety of disciplines, including veterinary medicine, is now recognized as key to wildlife conservation. Although veterinarians have been involved in conservation for decades, it is only recently that their role has become more appreciated in the larger conservation community. This realization of the contributions of veterinarians has occurred at a time when disease has been recognized as significantly impacting species' conservation bothin situandex situ. Today, veterinarians work with captive and free‐ranging animals to prevent and/or treat diseases that threaten species' survival. Most of these diseases are associated with the increasing human footprint. In this paper, I present wildlife health in today's world and the zoo veterinarian's role in wildlife conservation.
An integrated health approach to the conservation of Mountain gorillasGorilla beringei beringei
Authors: CRANFIELD M. , MINNIS R.
Published: July 2007

Conservation medicine, the medical practice that seeks to promote ecological health and well being of a defined habitat, functions at the intersection of animal, human and ecosystem health. It differs from classical public‐health epidemiology and medicine in that it aims to protect and improve animal health and related ecosystems, in addition to human health. Zoonotic diseases and emerging diseases are of primary concern and are particularly important when Endangered great‐ape populations are involved. The effective practice of conservation medicine demands an integrated cross‐disciplinary team approach involving wildlife and livestock veterinarians, local physicians, public‐health professionals, ecologists, government officials and communities. Common interests, improved data collection and economies of scale argue for combining health surveillance, data and delivery efforts. This team approach needs to be tailored to the infrastructure and sophistication of the host country's human and livestock/wildlife health systems. It is often, by default, the wildlife veterinarian(s) who coordinate(s) the integrated ‘one health’ approach because of their training in wildlife and livestock medicine as well as in zoonotic and emerging disease issues. This paper describes the Mountain Gorilla Veterinary Project's (MGVP Inc.) collaborative cross‐disciplinary strategy for Mountain gorillaGorilla beringei beringeiconservation and the database system developed during the process.
Magnetic resonance imaging and pathological findings in a case of canine idiopathic eosinophilic meningoencephalitis
Authors: Salvadori C , Baroni M , Arispici M , Cantile C
Published: July 2007

A case of idiopathic eosinophilic meningoencephalitis in a six‐month‐old male Maremma shepherd dog is reported. The dog was referred with a four month history of progressive weakness and depression with loss of trained habits. Tendency to recumbency, disorientation, visual impairment, bilaterally decreased menace response and hindlimb conscious proprioception deficits were detected. Magnetic resonance imaging showed a diffuse hypointense signal involving the cerebral grey matter with enlargement of the cerebral sulci on T1‐weighted and fast fluid‐attenuated inversion recovery (FLAIR) sequences consistent with a diffuse necrosis or atrophy of the cortical grey matter. Histological examination revealed severe inflammatory infiltration mainly composed of eosinophils and macrophages in the subarachnoid space and in the superficial layer of the cerebral cortex where parenchymal rarefaction and necrosis of neurones were also evident. No parasites, cysts or fungi were detected, and an immunologically mediated disorder was suspected. Magnetic resonance imaging may represent a useful diagnostic tool to differentiate idiopathic eosinophilic meningoencephalitis from other inflammatory brain diseases of young dogs.
Correlation of the MIC and Dose/MIC Ratio of Fluconazole to the Therapeutic Response of Patients with Mucosal Candidiasis and Candidemia
Authors: Rodríguez-Tudela Juan L. , Almirante Benito , Rodríguez-Pardo Dolors , Laguna Fernando , Donnelly J. Peter , Mouton Johan W. , Pahissa Albert , Cuenca-Estrella Manuel
Published: July 2007

We report on the correlation of the outcomes for two cohorts of patients who had been treated for candidemia (126 episodes) or oropharyngeal candidiasis (110 episodes) with various doses of fluconazole and the MIC of fluconazole obtained by using the EUCAST standard for fermentative yeasts. Of 145 episodes caused by an isolate with a fluconazole MIC ≤2 mg/liter, 93.7% (136 of 145) responded to fluconazole treatment. The response for those infected with a strain with a MIC of 4 mg/liter was 66% but reached 100% when the dose was greater than 100 mg/day, whereas the response for those infected with strains with MICs ≥8 mg/liter was only 12%. Hence, a MIC of 2 mg/liter or 4 mg/liter was able to predict successful treatment. A cure rate of 93.9% (140 of 149) was achieved when the dose/MIC ratio was ≥100 but fell to 14.6% (16 of 109) when the ratio was less. The dose/MIC required to achieve a response rate of 50% (the 50% effective concentration) was 43.7 for the cohort of patients with oropharyngeal candidiasis. Classification and regression analysis indicated that a dose/MIC of 35.5 was the threshold for the prediction of cure or failure. However, an increase in exposure above this threshold further increased the probability of cure, and all patients were cured when the dose/MIC exceeded 100. Monte Carlo simulations showed a probability of target attainment of 99% at MICs ≤2 mg/liter and a pharmacodynamic target of a dose/MIC ratio of 100, which was equivalent to an unbound fraction of the fluconazole area under the curve versus the MIC of 79.
Pharmacokinetics of Penicillin G in Infants with a Gestational Age of Less than 32 Weeks
Authors: Muller Anouk E. , DeJongh Joost , Bult Ymka , Goessens Wil H. F. , Mouton Johan W. , Danhof Meindert , van den Anker John N.
Published: July 2007

The pharmacokinetics of penicillin G were studied in 20 preterm neonates with a gestational age of less than 32 weeks on day 3 of life by using a population approach performed with the nonlinear mixed effects modeling program NONMEM. The derived population estimates and the correlation matrix of these estimates were used to perform Monte Carlo simulations and obtain the probability of target attainment (PTA). The pharmacokinetics of penicillin G were best described by a two-compartment pharmacokinetic model. The population estimates of the central volume of distribution, the peripheral volume of distribution, the intercompartmental clearance, and the total body clearance were 0.359 ± 0.06 liter, 0.152 ± 0.03 liter, 0.774 ± 0.28 liter/h, and 0.103 ± 0.01 liter/h (mean ± standard error), respectively. The terminal half-life was 3.9 h. Clearance increased significantly with increasing birth weight. Assuming the percentage of time that the concentration of unbound drug remained above the MIC of 50% for preterm neonates, the susceptibility breakpoint based on a 100% PTA was ≤4 mg/liter, simulating the current dosing regimen of 50,000 U/kg every 12 h. This regimen is therefore adequate for the treatment of common infections in neonates on the third day of life.
Reduction of
Streptococcus mutans
Adherence and Dental Biofilm Formation by Surface Treatment with Phosphorylated Polyethylene Glycol
Authors: Shimotoyodome Akira , Koudate Takashi , Kobayashi Hisataka , Nakamura Junji , Tokimitsu Ichiro , Hase Tadashi , Inoue Takashi , Matsukubo Takashi , Takaesu Yoshinori
Published: July 2007

Initial attachment of the cariogenic
Streptococcus mutans
onto dental enamel is largely promoted by the adsorption of specific salivary proteins on enamel surface. Some phosphorylated salivary proteins were found to reduce
S. mutans
adhesion by competitively inhibiting the adsorption of
S. mutans
-binding salivary glycoproteins to hydroxyapatite (HA). The aim of this study was to develop antiadherence compounds for preventing dental biofilm development. We synthesized phosphorylated polyethylene glycol (PEG) derivatives and examined the possibility of surface pretreatment with them for preventing
S. mutans
adhesion in vitro and dental biofilm formation in vivo. Pretreatment of the HA surface with methacryloyloxydecyl phosphate (MDP)-PEG prior to saliva incubation hydrophilized the surface and thereby reduced salivary protein adsorption and saliva-promoted bacterial attachment to HA. However, when MDP-PEG was added to the saliva-pretreated HA (S-HA) surface, its inhibitory effect on bacterial binding was completely diminished.
S. mutans
adhesion onto S-HA was successfully reduced by treatment of the surface with pyrophosphate (PP), which desorbs salivary components from S-HA. Treatment of S-HA surfaces with MDP-PEG plus PP completely inhibited saliva-promoted
S. mutans
adhesion even when followed by additional saliva treatment. Finally, mouthwash with MDP-PEG plus PP prevented de novo biofilm development after thorough teeth cleaning in humans compared to either water or PP alone. We conclude that MDP-PEG plus PP has the potential for use as an antiadherence agent that prevents dental biofilm development.
Steady-State Pharmacokinetic and Safety Profiles of Voriconazole and Ritonavir in Healthy Male Subjects
Authors: Liu Ping , Foster Grover , Gandelman Kuan , LaBadie Robert R. , Allison Mark J. , Gutierrez Maria J. , Sharma Amarnath
Published: July 2007

Since there is a likelihood of coadministration of voriconazole and ritonavir, two studies were conducted to evaluate the potential of drug interaction. Study A was a randomized, placebo-controlled, two-period, parallel-group trial (n= 34). Study B had the same design without the placebo group (n= 17). In period 1, subjects received 200 mg voriconazole or placebo twice daily (BID) for 3 days (400 mg BID on day 1). In period 2, following a 7-day washout, subjects received ritonavir alone at 400 mg BID (study A) or 100 mg BID (study B) for 10 days (days 11 to 20), and then ritonavir was coadministered with 200 mg BID voriconazole or placebo for the next 10 days (days 21 to 30). Serial plasma samples were collected on days 3, 20, and 30, and safety data were collected throughout the study. High-dose (400 mg BID) ritonavir substantially reduced the steady-state mean voriconazole exposure (area under the concentration-time curve from 0 to 12 h [AUC0-12], −82%; maximum concentration [Cmax], −66%). However, the effect of low-dose (100 mg BID) ritonavir was less pronounced (AUC0-12, −39%;Cmax, −24%). The decrease in voriconazole exposure was probably due to the induction of CYP2C19 and CYP2C9 by ritonavir. It is interesting that one subject in each study exhibited the opposite effect of ritonavir on voriconazole exposure (a 2.5- to 3-fold increase), probably due to lack of CYP2C19. Voriconazole had no apparent effect on the exposure of high-dose ritonavir but slightly decreased the exposure of low-dose ritonavir (AUC0-12, −14%;Cmax, −24%). The safety profile of combination therapy was not notably different from that of voriconazole or ritonavir alone. Due to the significant effect of ritonavir on voriconazole exposure, coadministration of voriconazole with 400 mg BID ritonavir is contraindicated; coadministration with 100 mg BID ritonavir should be avoided, unless an assessment of the benefit/risk to the patient justifies the use.
Comparative Evaluation of the VITEK 2, Disk Diffusion, Etest, Broth Microdilution, and Agar Dilution Susceptibility Testing Methods for Colistin in Clinical Isolates, Including Heteroresistant
Enterobacter cloacae
and
Acinetobacter baumannii
Strains
Authors: Lo-Ten-Foe Jerome R. , de Smet Anne Marie G. A. , Diederen Bram M. W. , Kluytmans Jan A. J. W. , van Keulen Peter H. J.
Published: July 2007

Increasing antibiotic resistance in gram-negative bacteria has recently renewed interest in colistin as a therapeutic option. The increasing use of colistin necessitates the availability of rapid and reliable methods for colistin susceptibility testing. We compared seven methods of colistin susceptibility testing (disk diffusion, agar dilution on Mueller-Hinton [MH] and Isosensitest agar, Etest on MH and Isosensitest agar, broth microdilution, and VITEK 2) on 102 clinical isolates collected from patient materials during a selective digestive decontamination or selective oral decontamination trial in an intensive-care unit. Disk diffusion is an unreliable method to measure susceptibility to colistin. High error rates and low levels of reproducibility were observed in the disk diffusion test. The colistin Etest, agar dilution, and the VITEK 2 showed a high level of agreement with the broth microdilution reference method. Heteroresistance for colistin was observed in six
Enterobacter cloacae
isolates and in one
Acinetobacter baumannii
isolate. This is the first report of heteroresistance to colistin in
E. cloacae
isolates. Resistance to colistin in these isolates seemed to be induced upon exposure to colistin rather than being caused by stable mutations. Heteroresistant isolates could be detected in the broth microdilution, agar dilution, Etest, or disk diffusion test. The VITEK 2 displayed low sensitivity in the detection of heteroresistant subpopulations of
E. cloacae
. The VITEK 2 colistin susceptibility test can therefore be considered to be a reliable tool to determine susceptibility to colistin in isolates of genera that are known not to exhibit resistant subpopulations. In isolates of genera known to (occasionally) exhibit heteroresistance, an alternative susceptibility testing method capable of detecting heteroresistance should be used.
Effect of Dosing and Dosing Frequency on the Efficacy of Ceftizoxime and the Emergence of Ceftizoxime Resistance during the Early Development of Murine Abscesses Caused by
Bacteroides fragilis
and
Enterobacter cloacae
Mixed Infection
Authors: Stearne Lorna E. T. , Goessens Wil H. F. , Mouton Johan W. , Gyssens Inge C.
Published: July 2007

The efficacy of β-lactams is thought to be dependent on the time that the unbound concentrations exceed the MIC (
fT
>MIC). However, the pharmacokinetic/pharmacodynamic index (PDI) that correlates best to the selection of resistance is not yet clear. The selection of ceftizoxime (CZX)-resistant
Enterobacter cloacae
mutant strains during the development of murine mixed-infection abscesses was studied to determine the PDI that is important for the emergence of resistance and the PDI value needed for the prevention of resistance. Studies were carried out 24 h after inoculation with
Bacteroides fragilis
ATCC 23745 and
E. cloacae
22491. Six to 1,536 mg of CZX/kg of body weight/day given every 2 h (q2h), q4h, q6h, or q8h was started 30 min before inoculation and continued for 24 h. Resistant mutants were isolated to determine mutant frequencies (MF). The
fT
>MIC varied from 9 to 98% for
E. cloacae
, the peak concentration (unbound fraction) was 0.6 to 578 mg/liter, and the area under the concentration-time curve (unbound fraction) (
f
AUC) was 1.9 to 553 mg·h/liter. The
f
AUC-to-MIC ratio best explained the in vivo efficacy. CZX-resistant
B. fragilis
and
E. cloacae
mutants were isolated from untreated controls at an MF of 10
−5
to 10
−7
. The MF of resistant
B. fragilis
did not increase during therapy. The selection of resistant
E. cloacae
strains at an MF of 10
−1
to 10
−2
was related to the
fT
>MIC and the ratio of
f
AUC to MIC following an inverse U shape. However, the ratio of
f
AUC to MIC was the stronger driver of resistance. The highest MFs were 0.7 to 0.9 at an
f
AUC-to-MIC ratio of approximately 250. We conclude that the ratio of
f
AUC to MIC is the PDI that correlated best to the in vivo efficacy of CZX and probably also to the emergence of resistant
E. cloacae
mutants. An
f
AUC-to-MIC ratio of 1,000 was needed to prevent the emergence of this resistance.
Endogenously Synthesized (−)-proto-Quercitol and Glycine Betaine Are Principal Compatible Solutes ofSchizochytriumsp. Strain S8 (ATCC 20889) and Three New Isolates of Phylogenetically Related Thraustochytrids
Authors: Jakobsen Anita N. , Aasen Inga M. , Strøm Arne R.
Published: July 2007

We report that endogenously synthesized (−)-proto-quercitol (1d-1,3,4/2,5-cyclohexanepentol) and glycine betaine were the principal compatible solutes ofSchizochytriumsp. strain S8 (ATCC 20889) and three new osmotolerant isolates of thraustochytrids (strains T65, T66, and T67). The compatible solutes were identified and quantified by use of nuclear magnetic resonance spectroscopy, and their identity was confirmed by mass spectroscopy and measurement of the specific optical rotation. The cellular content of compatible solutes increased with increasing NaCl concentration of a defined medium. (−)-proto-Quercitol was the dominating solute at all NaCl concentrations tested (0.25 to 1.0 M), e.g., cells of S8 and T66 stressed with 1.0 M NaCl accumulated about 500 μmol (−)-proto-quercitol and 100 μmol glycine betaine per g dry weight. To our knowledge, (−)-proto-quercitol has previously been found only in eucalyptus. The 18S rRNA gene sequences of the four (−)-proto-quercitol-producing strains showed 99% identity, and they displayed the same fatty acid profile. The only polyunsaturated fatty acids accumulated were docosahexaenoic acid (78%) and docosapentaenoic acid (22%). A less osmotolerant isolate (strain T29), which was closely phylogenetically related toThraustochytrium aureum(ATCC 34304), did not contain (−)-proto-quercitol or glycine betaine. Thus, the level of osmotolerance and the osmolyte systems vary among thraustochytrids.
Transcriptional Responses of
Enterococcus faecalis
V583 to Bovine Bile and Sodium Dodecyl Sulfate
Authors: Solheim Margrete , Aakra Ågot , Vebø Heidi , Snipen Lars , Nes Ingolf F.
Published: July 2007

Resistance to bile is a prerequisite property of the gastrointestinal bacterial flora. Bile acids are powerful detergents, and resistance to sodium dodecyl sulfate (SDS) has therefore often been considered relevant to studies of bile resistance. We have studied the effects of bovine bile (BB) and SDS on
Enterococcus faecalis
V583 by traditional growth studies and microarrays. Transcriptional responses were studied by time course experiments. In the presence of BB (V583-BB) or SDS (V583-SDS), 308 and 209 genes were identified as differentially expressed at one or more time points, respectively. In V583 treated with both BB and SDS (V583-BB-SDS), 254 genes showed differential expression. Detergents exert their toxic effects primarily on the microbial membrane. The enrichment of differentially transcribed genes that encode proteins with membrane-associated functions and/or locations indicates a major impact of all three treatments on the integrity and functionality of the cell membrane. Two gene clusters involved in fatty acid biosynthesis were repressed in V583-BB and V583-BB-SDS and partly induced in V583-SDS. Furthermore, two EmrB/QacA family drug resistance transporters and a vacuolar-type ATPase were induced in V583-BB and V583-BB-SDS. None of the putative bile salt hydrolase homologs in V583 showed differential expression during the bile treatments. The transcriptional profile of V583-BB-SDS was qualitatively more similar to the response in V583-BB than to that in V583-SDS, suggesting that the presence of bile suppresses the effects of SDS in V583-BB-SDS. The overall results presented here indicate that different mechanisms are involved in detergent resistance in
E. faecalis
.
No Evidence of an Impact on the Rhizosphere Diazotroph Community by the Expression of
Bacillus thuringiensis
Cry1Ab Toxin by Bt White Spruce
Authors: Lamarche Josyanne , Hamelin Richard C.
Published: July 2007

Nitrogen fixation is one of the most important roles played by soil bacterial communities, as fixation supplies nitrogen to many ecosystems which are often N limited. As impacts on this functional group of bacteria might harm the ecosystem's health and reduce productivity, monitoring that particular group is important. Recently, a field trial with Bt white spruce, which constitutively expresses the Cry1Ab insecticidal toxin of
Bacillus thuringiensis
, was established. The Bt white spruce was shown to be resistant to spruce budworm. We investigated the possible impact of these genetically modified trees on soil nitrogen-fixing bacterial communities. The trial consisted of untransformed controls, GUS white spruce (transformed with the β-glucuronidase gene), and Bt/GUS white spruce (which constitutively expresses both the Cry1Ab toxin and β-glucuronidase) in a random design. Four years after planting, soil samples from the control and the two treatments from plantation as well as from two natural stands of white spruce were collected. Diazotroph diversity was assessed by extracting soil genomic DNA and amplifying a region of the nitrogenase reductase (
nifH
) gene, followed by cloning and sequencing. Analysis revealed that nitrogen-fixing communities did not differ significantly among the untransformed control, GUS white spruce, and Bt/GUS white spruce. Nevertheless, differences in diazotroph diversity were observed between white spruce trees from the plantation site and those from two natural stands, one of which grew only a few meters away from the plantation. We therefore conclude, in the absence of evidence that the presence of the
B. thuringiensis cry1Ab
gene had an effect on diazotroph communities, that either site and/or field preparation prior to planting seems to be more important in determining diazotroph community structure than the presence of Bt white spruce.
Characteristics of
Gloeophyllum trabeum
Alcohol Oxidase, an Extracellular Source of H
2
O
2
in Brown Rot Decay of Wood
Authors: Daniel Geoffrey , Volc Jindřich , Filonova Lada , Plíhal Ondřej , Kubátová Elena , Halada Petr
Published: July 2007

A novel alcohol oxidase (AOX) has been purified from mycelial pellets of the wood-degrading basidiomycete
Gloeophyllum trabeum
and characterized as a homooctameric nonglycosylated protein with native and subunit molecular masses of 628 and 72.4 kDa, containing noncovalently bonded flavin adenine dinucleotide. The isolated
AOX
cDNA contained an open reading frame of 1,953 bp translating into a polypeptide of 651 amino acids displaying 51 to 53% identity with other published fungal AOX amino acid sequences. The enzyme catalyzed the oxidation of short-chain primary aliphatic alcohols with a preference for methanol (

K
m

= 2.3 mM,
k
cat
= 15.6 s
−1
). Using polyclonal antibodies and immunofluorescence staining, AOX was localized on liquid culture hyphae and extracellular slime in sections from degraded wood and on cotton fibers. Transmission electron microscopy immunogold labeling localized the enzyme in the hyphal periplasmic space and wall and on extracellular tripartite membranes and slime, while there was no labeling of hyphal peroxisomes. AOX was further shown to be associated with membranous or slime structures secreted by hyphae in wood fiber lumina and within the secondary cell walls of degraded wood fibers. The differences in AOX targeting compared to the known yeast peroxisomal localization were traced to a unique C-terminal sequence of the
G. trabeum
oxidase, which is apparently responsible for the protein's different translocation. The extracellular distribution and the enzyme's abundance and preference for methanol, potentially available from the demethylation of lignin, all point to a possible role for AOX as a major source of H
2
O
2
, a component of Fenton's reagent implicated in the generally accepted mechanisms for brown rot through the production of highly destructive hydroxyl radicals.
Characterization and Synergistic Interactions of
Fibrobacter succinogenes
Glycoside Hydrolases
Authors: Qi Meng , Jun Hyun-Sik , Forsberg Cecil W.
Published: July 2007

The objectives of this study were to characterize
Fibrobacter succinogenes
glycoside hydrolases from different glycoside hydrolase families and to study their synergistic interactions. The gene encoding a major endoglucanase (endoglucanase 1) of
F. succinogenes
S85 was identified as
cel9B
from the genome sequence by reference to internal amino acid sequences of the purified native enzyme. Cel9B and two other glucanases from different families, Cel5H and Cel8B, were cloned and overexpressed, and the proteins were purified and characterized. These proteins in conjunction with two predominant cellulases, Cel10A, a chloride-stimulated cellobiosidase, and Cel51A, formerly known as endoglucanase 2 (or CelF), were assayed in various combinations to assess their synergistic interactions using ball-milled cellulose. The degree of synergism ranged from 0.6 to 3.7. The two predominant endoglucanases produced by
F. succinogenes
, Cel9B and Cel51A, were shown to have a synergistic effect of up to 1.67. Cel10A showed little synergy in combination with Cel9B and Cel51A. Mixtures containing all the enzymes gave a higher degree of synergism than those containing two or three enzymes, which reflected the complementarity in their modes of action as well as substrate specificities.
Dissection and Modulation of the Four Distinct Activities of Nisin by Mutagenesis of Rings A and B and by C-Terminal Truncation
Authors: Rink Rick , Wierenga Jenny , Kuipers Anneke , Kluskens Leon D. , Driessen Arnold J. M. , Kuipers Oscar P. , Moll Gert N.
Published: July 2007

Nisin A is a pentacyclic antibiotic peptide produced by various
Lactococcus lactis
strains. Nisin displays four different activities: (i) it autoinduces its own synthesis; (ii) it inhibits the growth of target bacteria by membrane pore formation; (iii) it inhibits bacterial growth by interfering with cell wall synthesis; and, in addition, (iv) it inhibits the outgrowth of spores. Here we investigate the structural requirements and relevance of the N-terminal thioether rings of nisin by randomization of the ring A and B positions. The data demonstrate that: (i) mutation of ring A results in variants with enhanced activity and a modulated spectrum of target cells; (ii) for the cell growth-inhibiting activity of nisin, ring A is rather promiscuous with respect to its amino acid composition, whereas the bulky amino acid residues in ring B abolish antimicrobial activity; (iii) C-terminally truncated nisin A mutants lacking rings D and E retain significant antimicrobial activity but are unable to permeabilize the target membrane; (iv) the dehydroalanine in ring A is not essential for the inhibition of the outgrowth of
Bacillus
cells; (v) some ring A mutants have significant antimicrobial activities but have decreased autoinducing activities; (vi) the opening of ring B eliminates antimicrobial activity while retaining autoinducing activity; and (vii) some ring A mutants escape the nisin immune system(s) and are toxic to the nisin-producing strain NZ9700. These data demonstrate that the various activities of nisin can be engineered independently and provide a basis for the design and synthesis of tailor-made analogs with desired activities.
Differential Involvement of Histidine Kinase Receptors in Pseudohyphal Development, Stress Adaptation, and Drug Sensitivity of the Opportunistic Yeast
Candida lusitaniae
Authors: Chapeland-Leclerc Florence , Paccallet Paméla , Ruprich-Robert Gwenaël , Reboutier David , Chastin Christiane , Papon Nicolas
Published: July 2007

Fungal histidine kinase receptors (HKRs) sense and transduce many extracellular signals. We investigated the role of HKRs in morphogenetic transition, osmotolerance, oxidative stress response, and mating ability in the opportunistic yeast
Candida lusitaniae
. We isolated three genes,
SLN1
,
NIK1
, and
CHK1
, potentially encoding HKRs of classes VI, III, and X, respectively. These genes were disrupted by a transformation system based upon the “
URA3
blaster” strategy. Functional analysis of disruptants was undertaken, except for the
sln1 nik1
double mutant and the
sln1 nik1 chk1
triple mutant, which are not viable in
C. lusitaniae
. The
sln1
mutant revealed a high sensitivity to oxidative stress, whereas both the
nik1
and
chk1
mutants exhibited a more moderate sensitivity to peroxide. We also showed that the
NIK1
gene was implicated in phenylpyrrole and dicarboximide compound susceptibility while HKRs seem not to be involved in resistance toward antifungals of clinical relevance. Concerning mating ability, all disruptants were still able to reproduce sexually in vitro in unilateral or bilateral crosses. The most important result of this study was that the
sln1
mutant displayed a global defect of pseudohyphal differentiation, especially in high-osmolarity and oxidative-stress conditions. Thus, the
SLN1
gene could be crucial for the
C. lusitaniae
yeast-to-pseudohypha morphogenetic transition. This implication is strengthened by a high level of
SLN1
mRNAs revealed by semiquantitative reverse transcription-PCR when the yeast develops pseudohyphae. Our findings highlight a differential contribution of the three HKRs in osmotic and oxidant adaptation during the morphological transition in
C. lusitaniae
.
FEA1
,
FEA2
, and
FRE1
, Encoding Two Homologous Secreted Proteins and a Candidate Ferrireductase, Are Expressed Coordinately with
FOX1
and
FTR1
in Iron-Deficient
Chlamydomonas reinhardtii
Authors: Allen Michael D. , del Campo José A. , Kropat Janette , Merchant Sabeeha S.
Published: July 2007

Previously, we had identified FOX1 and FTR1 as iron deficiency-inducible components of a high-affinity copper-dependent iron uptake pathway in
Chlamydomonas
. In this work, we survey the version 3.0 draft genome to identify a ferrireductase, FRE1, and two ZIP family proteins, IRT1 and IRT2, as candidate ferrous transporters based on their increased expression in iron-deficient versus iron-replete cells. In a parallel proteomic approach, we identified FEA1 and FEA2 as the major proteins secreted by iron-deficient
Chlamydomonas reinhardtii
. The recovery of FEA1 and FEA2 from the medium of
Chlamydomonas
strain CC425 cultures is strictly correlated with iron nutrition status, and the accumulation of the corresponding mRNAs parallels that of the
Chlamydomonas FOX1
and
FTR1
mRNAs, although the magnitude of regulation is more dramatic for the
FEA
genes. Like the
FOX1
and
FTR1
genes, the
FEA
genes do not respond to copper, zinc, or manganese deficiency. The 5′ flanking untranscribed sequences from the
FEA1
,
FTR1
, and
FOX1
genes confer iron deficiency-dependent expression of
ARS2
, suggesting that the iron assimilation pathway is under transcriptional control by iron nutrition. Genetic analysis suggests that the secreted proteins FEA1 and FEA2 facilitate high-affinity iron uptake, perhaps by concentrating iron in the vicinity of the cell. Homologues of
FEA1
and
FRE1
were identified previously as high-CO
2
-responsive genes,
HCR1
and
HCR2
, in
Chlorococcum littorale
, suggesting that components of the iron assimilation pathway are responsive to carbon nutrition. These iron response components are placed in a proposed iron assimilation pathway for
Chlamydomonas
.
Human Monoclonal Antibodies against Anthrax Lethal Factor and Protective Antigen Act Independently To Protect againstBacillus anthracisInfection and Enhance Endogenous Immunity to Anthrax
Authors: Albrecht Mark T. , Li Han , Williamson E. Diane , LeButt Chris S. , Flick-Smith Helen C. , Quinn Conrad P. , Westra Hans , Galloway Darrell , Mateczun Alfred , Goldman Stanley , Groen Herman , Baillie Les W. J.
Published: July 2007

The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy forBacillus anthracisinfection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-μg dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] ofB. anthracisSterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected withB. anthracisSterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.
Binding of Serum Mannan Binding Lectin to a Cell Integrity-DefectiveCryptococcus neoformans ccr4Δ Mutant
Authors: Panepinto John C. , Komperda Kazimierz W. , Hacham Moshe , Shin Soowan , Liu Xiaoguang , Williamson Peter R.
Published: July 2007

Mannan binding lectin (MBL) is an innate immune mediator belonging to the collectin family known to bind to the surfaces of many viruses, bacteria, and fungi. However, pathogenic strains of the fungusCryptococcus neoformansare resistant to MBL binding. To dissect the mechanism of cryptococcal resistance to MBL, we compared MBL binding to an encapsulated wild-type strain, an encapsulatedccr4Δ mutant defective in cell integrity, and an acapsularcap60Δ strain. No MBL binding was detected on wild-typeC. neoformans. In contrast, theccr4Δ mutant bound MBL to the cell wall, predominantly at the ends of enlarged buds, whereas the acapsular strain bound MBL only at the bud neck and bud scars. In addition, theccr4Δ mutant was sensitive to the cell wall-active antifungal caspofungin and other cell wall stress inducers, and its virulence was reduced in a mouse model of cryptococcosis. Interestingly, treatment of wild-type cells with caspofungin also increased MBL binding toC. neoformans. These results suggest that both the presence of capsule and wild-type cell wall architecture preclude MBL binding toC. neoformans.
Cyclic Di-GMP Stimulates Protective Innate Immunity in Bacterial Pneumonia
Authors: Karaolis David K. R. , Newstead Michael W. , Zeng Xianying , Hyodo Mamoru , Hayakawa Yoshihiro , Bhan Urvhashi , Liang Hallie , Standiford Theodore J.
Published: July 2007

Innate immunity is the primary mechanism by which extracellular bacterial pathogens are effectively cleared from the lung. We have previously shown that cyclic di-GMP (c-di-GMP [c-diguanylate]) is a novel small molecule immunomodulator and immunostimulatory agent that triggers protective host innate immune responses. Using a murine model of bacterial pneumonia, we show that local intranasal (i.n.) or systemic subcutaneous (s.c.) administration of c-di-GMP prior to intratracheal (i.t.) challenge withKlebsiella pneumoniaestimulates protective immunity against infection. Specifically, i.n. or s.c. administration of c-di-GMP 48 and 24 h prior to i.t.K. pneumoniaechallenge resulted in significantly increased survival. Pretreatment with c-di-GMP resulted in a 5-fold reduction in bacterial CFU in the lung (P< 0.05) and an impressive >1,000-fold decrease in CFU in the blood (P< 0.01). c-di-GMP administration stimulated a robust innate response to bacterial challenge, characterized by enhanced accumulation of neutrophils and αβ T cells, as well as activated NK and αβ T lymphocytes, which was associated with earlier and more vigorous expression of chemokines and type I cytokines. Moreover, lung macrophages recovered fromKlebsiella-infected mice pretreated with c-di-GMP expressed greater quantities of inducible nitric oxide synthase and nitric oxide ex vivo than did macrophages isolated from infected mice pretreated with the control, c-GMP. These findings demonstrate that c-di-GMP delivered in either a compartmentalized or systemic fashion stimulates protective innate immunity in the lung and protects mice against bacterial invasion. We propose that the cyclic dinucleotide c-di-GMP may be used clinically as an effective immunomodulator, immune enhancer, and vaccine adjuvant to protect against respiratory infection and pneumonia in humans and animals.
Topology of AspT, the Aspartate:Alanine Antiporter of
Tetragenococcus halophilus
, Determined by Site-Directed Fluorescence Labeling
Authors: Nanatani Kei , Fujiki Takashi , Kanou Kazuhiko , Takeda-Shitaka Mayuko , Umeyama Hideaki , Ye Liwen , Wang Xicheng , Nakajima Tasuku , Uchida Takafumi , Maloney Peter C. , Abe Keietsu
Published: July 2007

The gram-positive lactic acid bacterium
Tetragenococcus halophilus
catalyzes the decarboxylation of
l
-aspartate (Asp) with release of
l
-alanine (Ala) and CO
2
. The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an
l
-aspartate-β-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity.
A Novel Filamentous Phage from the Deep-Sea Bacterium
Shewanella piezotolerans
WP3 Is Induced at Low Temperature
Authors: Wang Feng , Wang Fengping , Li Qiang , Xiao Xiang
Published: July 2007

Active filamentous phage particles were isolated from the deep-sea bacterium
Shewanella piezotolerans
WP3. A putative single-stranded DNA binding protein of the phage was found to be overexpressed at 4°C compared to its expression at 25°C by two-dimensional polyacrylamide gel electrophoresis. Reverse transcription quantitative PCR further revealed that the key genes of the SW1 phage were significantly induced at low temperature.
4-Sulfomuconolactone Hydrolases from
Hydrogenophaga intermedia
S1 and
Agrobacterium radiobacter
S2
Authors: Halak Sad , Basta Tamara , Bürger Sibylle , Contzen Matthias , Wray Victor , Pieper Dietmar Helmut , Stolz Andreas
Published: July 2007

The 4-carboxymethylen-4-sulfo-but-2-en-olide (4-sulfomuconolactone) hydrolases from
Hydrogenophaga intermedia
strain S1 and
Agrobacterium radiobacter
strain S2 are part of a modified protocatechuate pathway responsible for the degradation of 4-sulfocatechol. In both strains, the hydrolase-encoding genes occur downstream of those encoding the enzymes that catalyze the lactonization of 3-sulfomuconate. The deduced amino acid sequences of the 4-sulfomuconolactone hydrolases demonstrated the highest degree of sequence identity to 2-pyrone-4,6-dicarboxylate hydrolases, which take part in the
meta
cleavage pathway of protocatechuate. The 4-sulfomuconolactone hydrolases did not convert 2-pyrone-4,6-dicarboxylate, and the 2-pyrone-4,6-dicarboxylate hydrolase from
Sphingomonas paucimobilis
SYK-6 did not convert 4-sulfomuconolactone. Nevertheless, the presence of highly conserved histidine residues in the 4-sulfomuconolactone and the 2-pyrone-4,6-dicarboxylate hydrolases and some further sequence similarities suggested that both enzymes belong to the metallo-dependent hydrolases (the “amidohydrolase superfamily”). The 4-sulfomuconolactone hydrolases were heterologously expressed as His-tagged enzyme variants. Gel filtration experiments suggested that the enzymes are present as monomers in solution, with molecular weights of approximately 33,000 to 35,000. 4-Sulfomuconolactone was converted by sulfomuconolactone hydrolases to stoichiometric amounts of maleylacetate and sulfite. The 4-sulfomuconolactone hydrolases from both strains showed pH optima at pH 7 to 7.5 and rather similar catalytic constant (
k
cat
/
K

M

)values. The suggested 4-sulfocatechol pathway from 4-sulfocatechol to maleylacetate was confirmed by in situ nuclear magnetic resonance analysis using the recombinantly expressed enzymes.
The EssentialtacFGene Is Responsible for the Choline-Dependent Growth Phenotype ofStreptococcus pneumoniae
Authors: Damjanovic Marlen , Kharat Arun S. , Eberhardt Alice , Tomasz Alexander , Vollmer Waldemar
Published: July 2007

Streptococcus pneumoniaehas an absolute nutritional requirement for choline, and the choline molecules are known to incorporate exclusively into the cell wall and membrane teichoic acids of the bacterium. We describe here the isolation of a mutant of strain R6 in which a single G→T point mutation in the genetacF(formerly designatedspr1150) is responsible for generating a choline-independent phenotype. The choline-independent phenotype could be transferred to the laboratory strain R6 and to the encapsulated strain D39 by genetic transformation with a PCR product or with a plasmid carrying the mutatedtacFgene. ThetacFgene product belongs to the protein family of polysaccharide transmembrane transporters (flippases). A model is presented in which TacF is required for the transport of the teichoic acid subunits across the cytoplasmic membrane. According to this model, wild-type TacF has a strict specificity for choline-containing subunits, whereas the TacF present in the choline-independent mutant strain is able to transport both choline-containing and choline-free teichoic acid chains. The proposed transport specificity of parental-type TacF for choline-containing subunits would ensure the loading of the cell wall with teichoic acid chains decorated with choline residues, which appear to be essential for the virulence of this pathogen.
Methionine Sulfoxide Reductase from the Hyperthermophilic Archaeon
Thermococcus kodakaraensis
, an Enzyme Designed To Function at Suboptimal Growth Temperatures
Authors: Fukushima Eiji , Shinka Yasuhiro , Fukui Toshiaki , Atomi Haruyuki , Imanaka Tadayuki
Published: July 2007

Methionine sulfoxide reductase (Msr) catalyzes the thioredoxin-dependent reduction and repair of methionine sulfoxide (MetO). Although Msr genes are not present in most hyperthermophile genomes, an Msr homolog encoding an MsrA-MsrB fusion protein (MsrAB
Tk
) was present on the genome of the hyperthermophilic archaeon
Thermococcus kodakaraensis
. Recombinant proteins corresponding to MsrAB
Tk
and the individual domains (MsrA
Tk
and MsrB
Tk
) were produced, purified, and biochemically examined. MsrA
Tk
and MsrB
Tk
displayed strict substrate selectivity for Met-
S
-O and Met-
R
-O, respectively. MsrAB
Tk
, and in particular the MsrB domain of this protein, displayed an intriguing behavior for an enzyme from a hyperthermophile. While MsrAB
Tk
was relatively stable at temperatures up to 80°C (with a half-life of ∼30 min at 80°C), a 75% decrease in activity was observed after 2.5 min at 85°C, the optimal growth temperature of this archaeon. Moreover, maximal levels of MsrB activity of MsrAB
Tk
were observed at the strikingly low temperature of 30°C, which also was observed for MsrB
Tk
. Consistent with the low-temperature-specific biochemical properties of MsrAB
Tk
, the presence of the protein was greater in
T. kodakaraensis
cells grown at suboptimal temperatures (60 to 70°C) and could not be detected at 80 to 90°C. We found that the amount of intracellular MsrAB
Tk
protein increased with exposure to higher dissolved oxygen levels, but only at suboptimal growth temperatures. While measuring background rates of the Msr enzyme reactions, we observed significant levels of MetO reduction at high temperatures without enzyme. The occurrence of nonenzymatic MetO reduction at high temperatures may explain the specific absence of Msr homologs in most hyperthermophiles. Together with the fact that the presence of Msr in
T. kodakaraensis
is exceptional among the hyperthermophiles, the enzyme may represent a novel strategy for this organism to deal with low-temperature environments in which the dissolved oxygen concentrations increase.