bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Genomic Analysis Supports Conservation of Function Among Cellulose Synthase-Like A Gene Family Members and Suggests Diverse Roles of Mannans in Plants
Authors: Liepman Aaron H. , Nairn C. Joseph , Willats William G.T. , Sørensen Iben , Roberts Alison W. , Keegstra Kenneth
Published: February 2007

Mannan polysaccharides are widespread among plants, where they serve as structural elements in cell walls, as carbohydrate reserves, and potentially perform other important functions. Previous work has demonstrated that members of the cellulose synthase-like A (CslA) family of glycosyltransferases from Arabidopsis (Arabidopsis thaliana), guar (Cyamopsis tetragonolobus), and Populus trichocarpa catalyze β-1,4-mannan and glucomannan synthase reactions in vitro. Mannan polysaccharides and homologs of CslA genes appear to be present in all lineages of land plants analyzed to date. In many plants, the CslA genes are members of extended multigene families; however, it is not known whether all CslA proteins are glucomannan synthases. CslA proteins from diverse land plant species, including representatives of the mono- and dicotyledonous angiosperms, gymnosperms, and bryophytes, were produced in insect cells, and each CslA protein catalyzed mannan and glucomannan synthase reactions in vitro. Microarray mining and quantitative real-time reverse transcription-polymerase chain reaction analysis demonstrated that transcripts of Arabidopsis and loblolly pine (Pinus taeda) CslA genes display tissue-specific expression patterns in vegetative and floral tissues. Glycan microarray analysis of Arabidopsis indicated that mannans are present throughout the plant and are especially abundant in flowers, siliques, and stems. Mannans are also present in chloronemal and caulonemal filaments of Physcomitrella patens, where they are prevalent at cell junctions and in buds. Taken together, these results demonstrate that members of the CslA gene family from diverse plant species encode glucomannan synthases and support the hypothesis that mannans function in metabolic networks devoted to other cellular processes in addition to cell wall structure and carbohydrate storage.
Assessing the Efficiency of RNA Interference for Maize Functional Genomics
Authors: McGinnis Karen , Murphy Nick , Carlson Alvar R. , Akula Anisha , Akula Chakradhar , Basinger Heather , Carlson Michelle , Hermanson Peter , Kovacevic Nives , McGill M. Annie , Seshadri Vishwas , Yoyokie Jessica , Cone Karen , Kaeppler Heidi F. , Kaeppler Shawn M. , Springer Nathan M.
Published: February 2007

A large-scale functional genomics project was initiated to study the function of chromatin-related genes in maize (Zea mays). Transgenic lines containing short gene segments in inverted repeat orientation designed to reduce expression of target genes by RNA interference (RNAi) were isolated, propagated, and analyzed in a variety of assays. Analysis of the selectable marker expression over multiple generations revealed that most transgenes were transmitted faithfully, whereas some displayed reduced transmission or transgene silencing. A range of target-gene silencing efficiencies, from nondetectable silencing to nearly complete silencing, was revealed by semiquantitative reverse transcription-PCR analysis of transcript abundance for the target gene. In some cases, the RNAi construct was able to cause a reduction in the steady-state RNA levels of not only the target gene, but also another closely related gene. Correlation of silencing efficiency with expression level of the target gene and sequence features of the inverted repeat did not reveal any factors capable of predicting the silencing success of a particular RNAi-inducing construct. The frequencies of success of this large-scale project in maize, together with parameters for optimization at various steps, should serve as a useful framework for designing future RNAi-based functional genomics projects in crop plants.
Multiple Roles of ADP-Ribosylation Factor 1 in Plant Cells Include Spatially Regulated Recruitment of Coatomer and Elements of the Golgi Matrix
Authors: Matheson Loren A. , Hanton Sally L. , Rossi Marika , Latijnhouwers Maita , Stefano Giovanni , Renna Luciana , Brandizzi Federica
Published: February 2007

Recent evidence indicates that ADP-ribosylation factor 1 (ARF1) carries out multiple roles in plant cells that may be independent from the established effector complex COPI. To investigate potential COPI-independent functions, we have followed the dynamics of ARF1 and a novel putative effector, the plant golgin GRIP-related ARF-binding domain-containing Arabidopsis (Arabidopsis thaliana) protein 1 (GDAP1) in living plant cells. We present data that ascribe a new role to ARF1 in plant cell membrane traffic by showing that the GTPase functions to recruit GDAP1 to membranes. In addition, although ARF1 appears to be central to the recruitment of both COPI components and the golgin, we have established a different subcellular distribution of these ARF1 effectors. Live cell imaging demonstrates that GDAP1 and COPI are distributed on Golgi membranes. However, GDAP1 is also found on ARF1-labeled structures that lack coatomer, suggesting that the membrane environment, rather than ARF1 alone, influences the differential recruitment of ARF1 effectors. In support of this hypothesis, fluorescence recovery after photobleaching analyses demonstrated that GDAP1 and COPI have different kinetics on membranes during the cycle of activation and inactivation of ARF1. Therefore, our data support a model where modulation of the cellular functions of ARF1 in plant cells encompasses not only the intrinsic activities of the effectors, but also differential recruitment onto membranes that is spatially regulated.
TCP Transcription Factors Control the Morphology of Shoot Lateral Organs via Negative Regulation of the Expression of Boundary-Specific Genes inArabidopsis
Authors: Koyama Tomotsugu , Furutani Masahiko , Tasaka Masao , Ohme-Takagi Masaru
Published: February 2007

Plants form shoot meristems in the so-called boundary region, and these meristems are necessary for normal morphogenesis of aerial parts of plants. However, the molecular mechanisms that regulate the formation of shoot meristems are not fully understood. We report here that expression of a chimeric repressor from TCP3 (TCP3SRDX), a member of TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) transcription factors in Arabidopsis thaliana, resulted in the formation of ectopic shoots on cotyledons and various defects in organ development. Expression of TCP3SRDX induced ectopic expression of boundary-specific genes, namely the CUP-SHAPED COTYLEDON (CUC) genes, and suppressed the expression of miR164, whose product cleaves the transcripts of CUC genes. This abnormal phenotype was substantially reversed on the cuc1 mutant background. By contrast, gain of function of TCP3 suppressed the expression of CUC genes and resulted in the fusion of cotyledons and defects in formation of shoots. The pattern of expression of TCP3 did not overlap with that of the CUC genes. In addition, we found that eight TCPs had functions similar to that of TCP3. Our results demonstrate that the TCP transcription factors play a pivotal role in the control of morphogenesis of shoot organs by negatively regulating the expression of boundary-specific genes.
Light-Induced Energy Dissipation in Iron-Starved Cyanobacteria: Roles of OCP and IsiA Proteins
Authors: Wilson Adjélé , Boulay Clémence , Wilde Annegret , Kerfeld Cheryl A. , Kirilovsky Diana
Published: February 2007

In response to iron deficiency, cyanobacteria synthesize the iron stress–induced chlorophyll binding protein IsiA. This protein protects cyanobacterial cells against iron stress. It has been proposed that the protective role of IsiA is related to a blue light–induced nonphotochemical fluorescence quenching (NPQ) mechanism. In iron-replete cyanobacterial cell cultures, strong blue light is known to induce a mechanism that dissipates excess absorbed energy in the phycobilisome, the extramembranal antenna of cyanobacteria. In this photoprotective mechanism, the soluble Orange Carotenoid Protein (OCP) plays an essential role. Here, we demonstrate that in iron-starved cells, blue light is unable to quench fluorescence in the absence of the phycobilisomes or the OCP. By contrast, the absence of IsiA does not affect the induction of fluorescence quenching or its recovery. We conclude that in cyanobacteria grown under iron starvation conditions, the blue light–induced nonphotochemical quenching involves the phycobilisome OCP–related energy dissipation mechanism and not IsiA. IsiA, however, does seem to protect the cells from the stress generated by iron starvation, initially by increasing the size of the photosystem I antenna. Subsequently, the IsiA converts the excess energy absorbed by the phycobilisomes into heat through a mechanism different from the dynamic and reversible light-induced NPQ processes.
KANADI and Class III HD-Zip Gene Families Regulate Embryo Patterning and Modulate Auxin Flow during Embryogenesis inArabidopsis
Authors: Izhaki Anat , Bowman John L.
Published: February 2007

Embryo patterning in Arabidopsis thaliana is highly affected when KANADI or Class III HD-Zip genes are compromised. Triple loss-of-function kan1 kan2 kan4 embryos exhibit striking defects in the peripheral–central axis, developing lateral leaf-like organs from the hypocotyls, whereas loss of Class III HD-Zip gene activity results in a loss of bilateral symmetry. Loss of KANADI activity in a Class III HD-Zip mutant background mitigates the defects in bilateral symmetry, implying that the two gene families act antagonistically during embryonic pattern formation. Dynamic patterns of auxin concentration and flux contribute to embryo patterning. Polar cellular distribution of PIN-FORMED1 (PIN1) mediates auxin flow throughout embryogenesis and is required for establishment of the apical–basal axis and bilateral symmetry. Defects in the pattern of PIN1 expression are evident when members of either the KANADI or Class III HD-Zip gene families are compromised. Abnormal expression patterns of PIN1 in KANADI or Class III HD-Zip multiple mutants and the phenotype of plants in which members of both gene families are mutated suggest that pattern formation along the central–peripheral axis results from interplay between auxin and the KANADI and Class III HD-Zip transcription factors, whose defined spatial and temporal expression patterns may also be influenced by auxin.
Role of the MPN Subunits in COP9 Signalosome Assembly and Activity, and Their Regulatory Interaction withArabidopsisCullin3-Based E3 Ligases
Authors: Gusmaroli Giuliana , Figueroa Pablo , Serino Giovanna , Deng Xing Wang
Published: February 2007

The COP9 signalosome (CSN) is an evolutionarily conserved multisubunit protein complex that regulates a variety of biological processes. Among its eight subunits, CSN5 and CSN6 contain a characteristic MPN (for Mpr1p and Pad1p N-terminal) domain and, in Arabidopsis thaliana, are each encoded by two genes: CSN5A, CSN5B and CSN6A, CSN6B, respectively. We characterized both MPN subunits using a series of single and double mutants within each gene family. Our results indicate that although CSN6A and CSN6B retain mostly redundant functions, CSN5A and CSN5B play unequal roles in the regulation of plant development. Complete depletion of either of the two MPN members results in CSN instability and the decay of various CSN components, along with the complete loss of CUL1, CUL3, and CUL4 derubylation. Furthermore, we demonstrate that CSN interacts with CUL3, in addition to CUL1 and CUL4, and that the lack of CSN activity differentially affects the stability of those three cullins. Interestingly, we also show that optimal CUL3 activity is required to maintain the cellular pool of CSN5, through a posttranscriptional mechanism. Our data suggest the existence of reciprocal regulation between CUL3 and CSN5 accumulation. This study thus completes the genetic analysis of all CSN subunits and confirms the structural interdependence between PCI and MPN subunits in functional CSN complex formation.
The Arabidopsis EIN3 Binding F-Box Proteins EBF1 and EBF2 Have Distinct but Overlapping Roles in Ethylene Signaling
Authors: Binder Brad M. , Walker Joseph M. , Gagne Jennifer M. , Emborg Thomas J. , Hemmann Georg , Bleecker Anthony B. , Vierstra Richard D.
Published: February 2007

Ethylene signaling in Arabidopsis thaliana converges on the ETHYLENE-INSENSITIVE3 (EIN3)/EIN3-Like (EIL) transcription factors to induce various responses. EIN3 BINDING F-BOX1 (EBF1) and EBF2 were recently shown to function in ethylene perception by regulating EIN3/EIL turnover. In the absence of ethylene, EIN3 and possibly other EIL proteins are targeted for ubiquitination and subsequent degradation by Cullin 1–based E3 complexes containing EBF1 and 2. Ethylene appears to block this ubiquitination, allowing EIN3/EIL levels to rise and mediate ethylene signaling. Through analysis of mutant combinations affecting accumulation of EBF1, EBF2, EIN3, and EIL1, we show that EIN3 and EIL1 are the main targets of EBF1/2. Kinetic analyses of hypocotyl growth inhibition in response to ethylene and growth recovery after removal of the hormone revealed that EBF1 and 2 have temporally distinct but overlapping roles in modulating ethylene perception. Whereas EBF1 plays the main role in air and during the initial phase of signaling, EBF2 plays a more prominent role during the latter stages of the response and the resumption of growth following ethylene removal. Through their coordinated control of EIN3/EIL1 levels, EBF1 and EBF2 fine-tune ethylene responses by repressing signaling in the absence of the hormone, dampening signaling at high hormone concentrations, and promoting a more rapid recovery after ethylene levels dissipate.
Identification of Two Protein Kinases Required for Abscisic Acid Regulation of Seed Germination, Root Growth, and Gene Expression in Arabidopsis
Authors: Fujii Hiroaki , Verslues Paul E. , Zhu Jian-Kang
Published: February 2007

Abscisic acid (ABA) is an important phytohormone regulating various plant processes, including seed germination. Although phosphorylation has been suggested to be important, the protein kinases required for ABA signaling during seed germination and seedling growth remain elusive. Here, we show that two protein kinases, SNF1-RELATED PROTEIN KINASE2.2 (SnRK2.2) and SnRK2.3, control responses to ABA in seed germination, dormancy, and seedling growth in Arabidopsis thaliana. A snrk2.2 snrk2.3 double mutant, but not snrk2.2 or snrk2.3 single mutants, showed strong ABA-insensitive phenotypes in seed germination and root growth inhibition. Changes in seed dormancy and ABA-induced Pro accumulation consistent with ABA insensitivity were also observed. The snrk2.2 snrk2.3 double mutant had a greatly reduced level of a 42-kD kinase activity capable of phosphorylating peptides from ABF (for ABA Response Element Binding Factor) transcription factors. ABA-induced expression of several genes whose promoters contain an ABA response element (ABRE) was reduced in snrk2.2 snrk2.3, suggesting that the mechanism of SnRK2.2 and SnRK2.3 action in ABA signaling involves the activation of ABRE-driven gene expression through the phosphorylation of ABFs. Together, these results demonstrate that SnRK2.2 and SnRK2.3 are redundant but key protein kinases that mediate a major part of ABA signaling in Arabidopsis.
Arabidopsis BRANCHED1Acts as an Integrator of Branching Signals within Axillary Buds
Authors: Aguilar-Martínez José Antonio , Poza-Carrión César , Cubas Pilar
Published: February 2007

Shoot branching patterns depend on a key developmental decision: whether axillary buds grow out to give a branch or whether they remain dormant in the axils of leaves. This decision is controlled by endogenous and environmental stimuli mediated by hormonal signals. Although genes involved in the long-distance signaling of this process have been identified, the genes responding inside the buds to cause growth arrest remained unknown in Arabidopsis thaliana. Here, we describe an Arabidopsis gene encoding a TCP transcription factor closely related to teosinte branched1 (tb1) from maize (Zea mays), BRANCHED1 (BRC1), which represents a key point at which signals controlling branching are integrated within axillary buds. BRC1 is expressed in developing buds, where it arrests bud development. BRC1 downregulation leads to branch outgrowth. BRC1 responds to developmental and environmental stimuli controlling branching and mediates the response to these stimuli. Mutant and expression analyses suggest that BRC1 is downstream of the MORE AXILLARY GROWTH pathway and that it is required for auxin-induced apical dominance. Therefore, BRC1 acts inside the buds as an integrator of signals controlling bud outgrowth and translates them into a response of cell growth arrest. The conservation of BRC1/tb1 function among distantly related angiosperm species suggests that a single ancestral mechanism of branching control integration evolved before the radiation of flowering plants.
Tapetosomes in Brassica Tapetum Accumulate Endoplasmic Reticulum–Derived Flavonoids and Alkanes for Delivery to the Pollen Surface
Authors: Hsieh Kai , Huang Anthony H.C.
Published: February 2007

Tapetosomes are abundant organelles in tapetum cells during the active stage of pollen maturation in Brassicaceae species. They possess endoplasmic reticulum (ER)–derived vesicles and oleosin-coated lipid droplets, but their overall composition and function have not been established. In situ localization analyses of developing Brassica napus anthers revealed flavonoids present exclusively in tapetum cells, first in an ER network along with flavonoid-3′-hydroxylase and then in ER-derived tapetosomes. Flavonoids were absent in the cytosol, elaioplasts, vacuoles, and nuclei. Subcellular fractionation of developing anthers localized both flavonoids and alkanes in tapetosomes. Subtapetosome fractionation localized flavonoids in ER-derived vesicles, and alkanes and oleosins in lipid droplets. After tapetum cell death, flavonoids, alkanes, and oleosins were located on mature pollen. In the Arabidopsis thaliana mutants tt12 and tt19 devoid of a flavonoid transporter, flavonoids were present in the cytosol in reduced amounts but absent in tapetosomes and were subsequently located on mature pollen. tt4, tt12, and tt19 pollen was more susceptible than wild-type pollen to UV-B irradiation on subsequent germination. Thus, tapetosomes accumulate ER-derived flavonoids, alkanes, and oleosins for discharge to the pollen surface upon cell death. This tapetosome-originated pollen coat protects the haploidic pollen from UV light damage and water loss and aids water uptake.
In Vivo Efficacy of Anidulafungin and Caspofungin against
Candida glabrata
and Association with In Vitro Potency in the Presence of Sera
Authors: Wiederhold Nathan P. , Najvar Laura K. , Bocanegra Rosie , Molina Destiny , Olivo Marcos , Graybill John R.
Published: February 2007

In vitro studies have demonstrated that anidulafungin has greater potency than caspofungin against
Candida glabrata
. However, data from in vivo studies demonstrating that it has superior efficacy are lacking. The objective of this study was to compare the activities of anidulafungin and caspofungin against
C. glabrata
in a murine model of disseminated candidiasis. Two clinical
C. glabrata
isolates were used, including one with reduced caspofungin susceptibility. MICs were determined by broth microdilution in the presence and absence of sera. For the animal studies, mice were immunosuppressed with 5-fluorouracil one day prior to intravenous inoculation. Treatment with anidulafungin and caspofungin (0, 0.5, 1, 5, and 10 mg/kg of body weight per day) was begun 24 h later and was continued through day 7 postinoculation. The CFU were enumerated from kidney tissue. According to the standard microdilution methodology, anidulafungin had superior in vitro activity. However, this enhanced potency was attenuated by the addition of mouse and human sera. Caspofungin reduced the kidney fungal burden at lower doses compared to that achieved with anidulafungin in mice infected with the isolate with the lower MIC. Against the strain with the elevated caspofungin MIC, both anidulafungin and caspofungin were effective in reducing the kidney fungal burden at the higher doses studied. Despite the greater in vitro activity of anidulafungin in the absence of sera, both echinocandins were similarly effective in reducing the fungal burden in kidney tissue. The superior in vitro activity of anidulafungin did not confer enhanced in vivo efficacy against
C. glabrata
.
Clinical Pharmacodynamics of Meropenem in Patients with Lower Respiratory Tract Infections
Authors: Li Chonghua , Du Xiaoli , Kuti Joseph L. , Nicolau David P.
Published: February 2007

Studies of β-lactam pharmacodynamics in infected patients are sparse. In this study, classification and regression tree (CART) and logistic regression analyses were used to identify which pharmacodynamic indices and magnitudes were significant predictors of meropenem efficacy for 101 adult patients with lower respiratory tract infections (LRTI). Using demographic data, a validated population pharmacokinetic model was employed to predict pharmacokinetic parameters and free serum concentrations in the studied patients. Pharmacodynamic indices [percentage of the dosing interval that free drug concentrations remain above the MIC (%fT> MIC),f(maximum concentration of drug in serum) (fCmax)/MIC,fCmin/MIC, andf(area under the concentration-time curve) (fAUC)/MIC] were calculated based on the baseline pathogen with the highest drug MIC for each patient. The median (range) of percentfT> MIC,fCmax/MIC,fCmin/MIC, andfAUC/MIC were 100% (0 to 100%), 728.8 (0.8 to 15,777), 19.9 (0.01 to 278), and 3,605.4 (2.7 to 60,865.9), respectively. CART identified the following breakpoints as significant predictors for microbiological response: >54%fT> MIC, afCmax/MIC > 383, and afCmin/MIC > 5;fCmin/MIC > 5 was the only significant predictor of clinical response. Due to 100%fT> MIC achieved in the majority of LRTI patients,fCmin/MIC was the statistically significant parameter associated with meropenem clinical and microbiological response in the adults with LRTI. The findings for LRTI patients can be applied to optimize meropenem dose regimens to achieve clinical success and microbiological eradication in clinical practice.
Rapid Bactericidal Activity of Daptomycin against Methicillin-Resistant and Methicillin-Susceptible
Staphylococcus aureus
Peritonitis in Mice as Measured with Bioluminescent Bacteria
Authors: Mortin Lawrence I. , Li Tongchuan , Van Praagh Andrew D. G. , Zhang Shuxin , Zhang Xi-Xian , Alder Jeff D.
Published: February 2007

The rising rates of antibiotic resistance accentuate the critical need for new antibiotics. Daptomycin is a new antibiotic with a unique mode of action and a rapid in vitro bactericidal effect against gram-positive organisms. This study examined the kinetics of daptomycin's bactericidal action against peritonitis caused by methicillin-susceptible
Staphylococcus aureus
(MSSA) and methicillin-resistant
S. aureus
(MRSA) in healthy and neutropenic mice and compared this activity with those of other commonly used antibiotics. CD-1 mice were inoculated intraperitoneally with lethal doses of MSSA (Xen-29) or MRSA (Xen-1), laboratory strains transformed with a plasmid containing the
lux
operon, which confers bioluminescence. One hour later, the animals were given a single dose of daptomycin at 50 mg/kg of body weight subcutaneously (s.c.), nafcillin at 100 mg/kg s.c., vancomycin at 100 mg/kg s.c., linezolid at 100 mg/kg via gavage (orally), or saline (10 ml/kg s.c.). The mice were anesthetized hourly, and photon emissions from living bioluminescent bacteria were imaged and quantified. The luminescence in saline-treated control mice either increased (neutropenic mice) or remained relatively unchanged (healthy mice). In contrast, by 2 to 3 h postdosing, daptomycin effected a 90% reduction of luminescence of MSSA or MRSA in both healthy and neutropenic mice. The activity of daptomycin against both MSSA and MRSA strains was superior to those of nafcillin, vancomycin, and linezolid. Against MSSA peritonitis, daptomycin showed greater and more rapid bactericidal activity than nafcillin or linezolid. Against MRSA peritonitis, daptomycin showed greater and more rapid bactericidal activity than vancomycin or linezolid. The rapid decrease in the luminescent signal in the daptomycin-treated neutropenic mice underscores the potency of this antibiotic against
S. aureus
in the immune-suppressed host.
Competitive Inhibitors of the CphA Metallo-β-Lactamase from
Aeromonas hydrophila
Authors: Horsfall L. E. , Garau G. , Liénard B. M. R. , Dideberg O. , Schofield C. J. , Frère J. M. , Galleni M.
Published: February 2007

Various inhibitors of metallo-β-lactamases have been reported; however, none are effective for all subgroups. Those that have been found to inhibit the enzymes of subclass B2 (catalytically active with one zinc) either contain a thiol (and show less inhibition towards this subgroup than towards the dizinc members of B1 and B3) or are inactivators behaving as substrates for the dizinc family members. The present work reveals that certain pyridine carboxylates are competitive inhibitors of CphA, a subclass B2 enzyme. X-ray crystallographic analyses demonstrate that pyridine-2,4-dicarboxylic acid chelates the zinc ion in a bidentate manner within the active site. Salts of these compounds are already available and undergoing biomedical testing for various nonrelated purposes. Pyridine carboxylates appear to be useful templates for the development of more-complex, selective, nontoxic inhibitors of subclass B2 metallo-β-lactamases.
Multiple Staphylococcal Cassette Chromosomes and Allelic Variants of Cassette Chromosome Recombinases in
Staphylococcus aureus
and Coagulase-Negative Staphylococci from Norway
Authors: Hanssen Anne-Merethe , Sollid Johanna U. Ericson
Published: February 2007

We investigated the nature of the staphylococcal cassette chromosome
mec
(SCC
mec
) elements and cognate insertion sites in a collection of 42 clinical staphylococcal isolates of various species from Norway. The
ccr
and
mec
genes and the attachment sites (
attL
/
attR
) were identified by PCR, Southern blot hybridization, and DNA sequencing. We found 10 possibly new SCC
mec
types and one previously unreported variant of SCC
mec
type III (
mec
complex A,
ccrAB3
, and
ccrC7
) in
Staphylococcus epidermidis, Staphylococcus haemolyticus
, and
Staphylococcus hominis
. Eleven of 42 strains contained multiple copies of
ccr
, suggesting the presence of mosaic structures composed of multiple SCC elements.
S. haemolyticus
contained
ccrAB2
genes identical to those in
S. aureus
SCC
mec
type IV but lacked IS
1272
and
mec
regulators. Two new allelic
ccr
variants,
ccrC6
and
ccrC7
, were identified. Also, the presumed functional version of
ccrB1
was found in a
mecA
-positive
S. hominis
strain and in
mecA
-negative
S. epidermidis
and
S. hominis
strains. Only minor differences in direct repeats in the left and right boundaries (
attR
/
attL
) were observed, while there was more variation in the inverted repeats. Coagulase-negative staphylococci (CoNS) contained several representatives of different
ccr
complexes and thus seemed to harbor multiple or composite new types of SCC
mec
. The enormous diversity observed in the SCC
mec
elements implies a large SCC
mec
reservoir in CoNS.
Direct Inactivation of Human Immunodeficiency Virus Type 1 by a Novel Small-Molecule Entry Inhibitor, DCM205
Authors: Duong Yen T. , Meadows D. Christopher , Srivastava Indresh K. , Gervay-Hague Jacquelyn , North Thomas W.
Published: February 2007

With more than 40 million people living with human immunodeficiency virus (HIV), there is an urgent need to develop drugs that can be used in the form of a topical microbicide to prevent infection through sexual transmission. DCM205 is a recently discovered small-molecule inhibitor of HIV type 1 (HIV-1) that is able to directly inactivate HIV-1 in the absence of a cellular target. DCM205 is active against CXCR4-, CCR5-, and dual-tropic laboratory-adapted and primary strains of HIV-1. DCM205 binds to the HIV-1 envelope glycoprotein, and competition studies map the DCM205 binding at or near the V3 loop of gp120. Binding to this site interferes with the soluble CD4 interaction. With its ability to disable the virus particle, DCM205 represents a promising new class of HIV entry inhibitor that can be used as a strategy in the prevention of HIV-1/AIDS.
In Vitro Activities of Various Antimicrobials Alone and in Combination with Tigecycline against Carbapenem-Intermediate or -Resistant
Acinetobacter baumannii
Authors: Scheetz Marc H. , Qi Chao , Warren John R. , Postelnick Michael J. , Zembower Teresa , Obias Arlene , Noskin Gary A.
Published: February 2007

The activities of tigecycline alone and in combination with other antimicrobials are not well defined for carbapenem-intermediate or -resistant
Acinetobacter baumannii
(CIRA). Pharmacodynamic activity is even less well defined when clinically achievable serum concentrations are considered. Antimicrobial susceptibility testing of clinical CIRA isolates from 2001 to 2005 was performed by broth or agar dilution, as appropriate. Tigecycline concentrations were serially increased in time-kill studies with a representative of the most prevalent carbapenem-resistant clone (strain AA557; imipenem MIC, 64 mg/liter). The in vitro susceptibility of the strain was tested by time-kill studies in duplicate against the average free serum steady-state concentrations of tigecycline alone and in combination with various antimicrobials. Ninety-three CIRA isolates were tested and were found to have the following antimicrobial susceptibility profiles: tigecycline, MIC
50
of 1 mg/liter and MIC
90
of 2 mg/liter; minocycline, MIC
50
of 0.5 mg/liter and MIC
90
of 8 mg/liter; doxycycline, MIC
50
of 2 mg/liter and MIC
90
of ≥32 mg/liter; ampicillin-sulbactam, MIC
50
of 48 mg/liter and MIC
90
of 96 mg/liter; ciprofloxacin, MIC
50
of ≥16 mg/liter and MIC
90
of ≥16 mg/liter; rifampin, MIC
50
of 4 mg/liter and MIC
90
of 8 mg/liter; polymyxin B, MIC
50
of 1 mg/liter and MIC
90
of 1 mg/liter; amikacin, MIC
50
of 32 mg/liter and MIC
90
of ≥32 mg/liter; meropenem, MIC
50
of 16 mg/liter and MIC
90
of ≥128 mg/liter; and imipenem, MIC
50
of 4 mg/liter and MIC
90
of 64 mg/liter. Among the tetracyclines, the isolates were more susceptible to tigecycline than minocycline and doxycycline, according to FDA breakpoints (95%, 88%, and 71% of the isolates were susceptible to tigecycline, minocycline, and doxycycline, respectively). Concentration escalation studies with tigecycline revealed a maximal killing effect near the MIC, with no additional extent or rate of killing at concentrations 2× to 4× the MIC for tigecycline. Time-kill studies demonstrated indifference for tigecycline in combination with the antimicrobials tested. Polymyxin B, minocycline, and tigecycline are the most active antimicrobials in vitro against CIRA. Concentration escalation studies demonstrate that tigecycline may need to approach concentrations higher than those currently achieved in the bloodstream to adequately treat CIRA bloodstream infections. Future studies should evaluate these findings in vivo.
Role of Acidic pH in the Susceptibility of Intraphagocytic Methicillin-ResistantStaphylococcus aureusStrains to Meropenem and Cloxacillin
Authors: Lemaire Sandrine , Van Bambeke Françoise , Mingeot-Leclercq Marie-Paule , Glupczynski Youri , Tulkens Paul M.
Published: February 2007

Early studies showed that methicillin-resistantStaphylococcus aureus(MRSA) strains are susceptible to β-lactams when they are exposed to pH ≤ 5.5 in broth. BecauseS. aureussurvives in the phagolysosomes of macrophages, where the pH may be acidic, we have examined the susceptibility of MRSA ATCC 33591 phagocytized by human THP-1 macrophages to meropenem (MEM) and cloxacillin (CLX). Using a pharmacodynamic model assessing key pharmacological (50% effective concentration and maximal efficacy) and microbiological (static concentration) descriptors of antibiotic activity, we show that intraphagocytic MRSA strains are as sensitive to MEM and CLX as methicillin-susceptibleS. aureus(MSSA; ATCC 25923). This observation was replicated in broth if the pH was brought to 5.5 and was confirmed with clinical strains. Electron microscopy showed that both the MRSA and the MSSA strains localized and multiplied in membrane-bounded structures (phagolysosomes) in the absence of β-lactams. Incubation of the infected macrophages with ammonium chloride (to raise the phagolysosomal pH) made MRSA insensitive to MEM and CLX. No difference was seen inmec,mecA,mecI,mecR1,femA, andfemBexpression (reversed transcription-PCR) or in PBP 2a content (immunodetection) in MRSA grown in broth at pH 5.5 compared with that in MRSA grown in broth at 7.4. The level of [14C]benzylpenicillin binding to cell walls prepared from a non-β-lactamase-producing MRSA clinical isolate was two times lower than that to cell walls prepared from MSSA ATCC 25923 at pH 7.4, but the levels increased to similar values for both strains at pH 5.5. These data suggest that the restoration of susceptibility of intraphagocytic of MRSA to MEM and CLX is due to the acidic pH prevailing in phagolysosomes and is mediated by an enhanced binding to penicillin-binding proteins.
In Vitro Activities of Isavuconazole and Other Antifungal Agents against
Candida
Bloodstream Isolates
Authors: Seifert H. , Aurbach U. , Stefanik D. , Cornely O.
Published: February 2007

Isavuconazole is the active component of the new azole antifungal agent BAL8557, which is entering phase III clinical development. This study was conducted to compare the in vitro activities of isavuconazole and five other antifungal agents against 296
Candida
isolates that were recovered consecutively from blood cultures between 1995 and 2004 at a tertiary care university hospital. Microdilution testing was done in accordance with CLSI (formerly NCCLS) guideline M27-A2 in RPMI-1640 MOPS (morpholinepropanesulfonic acid) broth. The antifungal agents tested were amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, and isavuconazole.
C. albicans
was the most common species, representing 57.1% of all isolates. There was no trend found in favor of non-
Candida albicans
species over time. In terms of MIC
50
s, isavuconazole was more active (0.004 mg/liter) than amphotericin B (0.5 mg/liter), itraconazole (0.008 mg/liter), voriconazole (0.03 mg/liter), flucytosine (0.125 mg/liter), and fluconazole (8 mg/liter). For isavuconazole, MIC
50
s/MIC
90
s ranged from 000.2/0.004 mg/liter for
C. albicans
to 0.25/0.5 mg/liter for
C. glabrata
. Two percent of isolates (
C. glabrata
and
C. krusei
) were resistant to fluconazole;
C. albicans
strains resistant to fluconazole were not detected. There were only two isolates with MICs for isavuconazole that were >0.5 mg/liter: both were
C. glabrata
isolates, and the MICs were 2 and 4 mg/liter, respectively. In conclusion, isavuconazole is highly active against
Candida
bloodstream isolates, including fluconazole-resistant strains. It was more active than itraconazole and voriconazole against
C. albicans
and
C. glabrata
and appears to be a promising agent against systemic
Candida
infections.
In Vivo Pharmacodynamic Activity of the Glycopeptide Dalbavancin
Authors: Andes David , Craig William A.
Published: February 2007

Dalbavancin is a lipoglycopeptide antibiotic with broad-spectrum activity against gram-positive cocci and a markedly prolonged serum elimination half-life. We used the neutropenic murine thigh and lung infection models to characterize the pharmacodynamics of dalbavancin. Single-dose pharmacokinetic studies demonstrated linear kinetics and a prolonged elimination half-life which ranged from 7.6 to 13.1 h over the dose range of 2.5 to 80 mg/kg of body weight. The level of protein binding in mouse serum was 98.4%. The time course of in vivo activity of dalbavancin over the same dose range was examined in neutropenic ICR Swiss mice infected with a strain of either
Streptococcus pneumoniae
or
Staphylococcus aureus
by using the thigh infection model. The burden of organisms for
S. pneumoniae
was markedly reduced over the initial 24 h of study, and organism regrowth was suppressed in a dose-dependent fashion for up to the entire 96 h of study following dalbavancin doses of 2.5 mg/kg or greater. Dalbavancin doses of 20 mg/kg or greater resulted in less killing of
S. aureus
but were still followed by a prolonged suppression of regrowth. Multiple-dosing-regimen studies with the same organisms were used to determined which of the pharmacodynamic indices (maximum concentration in serum [
C
max
]/MIC, area under the concentration-versus-time curve [AUC]/MIC, or the duration of time that levels in serum exceed the MIC) best correlated with treatment efficacy. These studies used a dose range of 3.8 to 480 mg/kg/6 days fractionated into 2, 4, 6, or 12 doses over the 144-h dosing period. Nonlinear regression analysis was used to examine the data fit with each pharmacodynamic index. Dalbavancin administration by the use of large, widely spaced doses was the most efficacious for both organisms. Both the 24-h AUC/MIC and the
C
max
/MIC parameters correlated well with the in vivo efficacy of treatment against
S. pneumoniae
and
S. aureus
(for 24-h AUC/MIC,
R
2
= 78 and 77%, respectively; for
C
max
/MIC,
R
2
= 90 and 57%, respectively). The free-drug 24-h AUC/MICs required for a bacteriostatic effect were 17 ± 7 for five
S. pneumoniae
isolates. A similar treatment endpoint for the treatment against five strains of
S. aureus
required a larger dalbavancin exposure, with a mean free-drug 24-h AUC/MIC of 265 ± 143. Beta-lactam resistance did not affect the pharmacodynamic target. The dose-response curves were relatively steep for both species; thus, the pharmacodynamic target needed to achieve organism reductions of 1 or 2 log
10
in the mice were not appreciably larger (1.3- to 1.6-fold). Treatment was similarly efficacious in neutropenic mice and in the lung infection model. The dose-dependent efficacy and prolonged elimination half-life of dalbavancin support the widely spaced regimens used in clinical trials. The free-drug 24-h AUC/MIC targets identified in these studies should be helpful for discerning rational susceptibility breakpoints. The current MIC
90
for the target gram-positive organisms would fall within this value.
Two New
dfr
Genes in Trimethoprim-Resistant Integron-Negative
Escherichia coli
Isolates
Authors: Grape M. , Sundström L. , Kronvall G.
Published: February 2007

Two
Escherichia coli
isolates resistant to trimethoprim but negative for integrons carried two new resistance genes,
dfrA24
and
dfrA26
, remotely similar to one another and to the cassette-independent genes
dfrA8
and
dfrA9
. The
dfrA24
gene was not associated with known mobile elements, while
dfrA26
was associated with the CR1 common region.
Mutations in
fusA
Associated with Posttherapy Fusidic Acid Resistance in
Clostridium difficile
Authors: Norén T. , Åkerlund T. , Wullt M. , Burman L. G. , Unemo M.
Published: February 2007

In silico, we identified
fusA
(2,067 bp) in
Clostridium difficile
630. Sequencing of
fusA
in posttherapy fusidic acid-resistant
C. difficile
isolates from 12 patients with
C. difficile
-associated diarrhea (CDAD) identified
fusA
mutations, one or two nonsynonymous substitutions, or in one case a deletion of one codon associated with resistance. Five of these mutations have previously been described in
fusA
of fusidic acid-resistant
Staphylococcus aureus
, but seven were novel
fusA
mutations. Fusidic acid monotherapy for CDAD seemed to rapidly select conserved resistant mutants.
Antiplasmodial Activity of Lauryl-Lysine Oligomers
Authors: Radzishevsky I. , Krugliak M. , Ginsburg H. , Mor A.
Published: February 2007

The ever evolving resistance of the most virulent malaria parasite,
Plasmodium falciparum
, to antimalarials necessitates the continuous development of new drugs. Our previous analysis of the antimalarial activities of the hemolytic antimicrobial peptides dermaseptins and their acylated derivatives implicated the importance of hydrophobicity and charge for drug action. Following these findings, an oligoacyllysine (OAK) tetramer designed to mimic the characteristics of dermaseptin was synthesized and assessed for its antimalarial activity in cultures of
P. falciparum
. The tetramer inhibited the growth of different plasmodial strains at low micromolar concentrations (mean 50% inhibitory concentration [IC
50
], 1.8 μM). A structure-activity relationship study involving eight derivatives unraveled smaller, more potent OAK analogs (IC
50
s, 0.08 to 0.14 μM). The most potent analogs were the most selective, with selectivity ratios of 3 orders of magnitude. Selectivity was strongly influenced by the self-assembly properties resulting from interactions between hydrophobic OAKs, as has been observed with conventional antimicrobial peptides. Further investigations performed with a representative OAK revealed that the ring and trophozoite stages of the parasite developmental cycle were equally sensitive to the compound. A shortcoming of the tested compound was the need for long incubation times in order for it to exert its full effect. Nevertheless, the encouraging results obtained in this study regarding the efficiency and selectivity of some compounds establish them as leads for further development.
Tetracycline Resistance in Group A Streptococci: Emergence on a Global Scale and Influence on Multiple-Drug Resistance
Authors: Ayer Vanessa , Tewodros Wezenet , Manoharan Anand , Skariah Sini , Luo Feng , Bessen Debra E.
Published: February 2007

A global sample of group A streptococci (GAS) revealed ≥80 separate acquisitions of tetracycline resistance. Of 244 clones, 38 and 25% displayed resistance to tetracycline and erythromycin, respectively; a relatively high proportion (15%) were resistant to both classes of drugs.
tet
(M) displayed a highly significant association with
erm
(B).
Candida albicans
and
Candida dubliniensis
Respond Differently to Echinocandin Antifungal Agents In Vitro
Authors: Jacobsen Mette D. , Whyte Julie A. , Odds Frank C.
Published: February 2007

Candida dubliniensis
isolates tested for susceptibility to anidulafungin, caspofungin, and micafungin commonly showed artifactual regrowth and/or trailing effects with MIC tests done under conditions involving a high initial yeast concentration. The artifacts were less common with
Candida albicans
and seldom seen for either species under Clinical and Laboratory Standards Institute method M27-A test conditions.
A Complex Ergovaline Gene Cluster in Epichloë Endophytes of Grasses
Authors: Fleetwood Damien J. , Scott Barry , Lane Geoffrey A. , Tanaka Aiko , Johnson Richard D.
Published: February 2007

Clavicipitaceous fungal endophytes of the genera
Epichloë
and
Neotyphodium
form symbioses with grasses of the subfamily Pooideae, in which they can synthesize an array of bioprotective alkaloids. Some strains produce the ergopeptine alkaloid ergovaline, which is implicated in livestock toxicoses caused by ingestion of endophyte-infected grasses. Cloning and analysis of a nonribosomal peptide synthetase (NRPS) gene from
Neotyphodium lolii
revealed a putative gene cluster for ergovaline biosynthesis containing a single-module NRPS gene,
lpsB
, and other genes orthologous to genes in the ergopeptine gene cluster of
Claviceps purpurea
and the clavine cluster of
Aspergillus fumigatus
. Despite conservation of gene sequence, gene order is substantially different between the
N. lolii
,
C. purpurea
, and
A. fumigatus
ergot alkaloid gene clusters. Southern analysis indicated that the
N. lolii
cluster was linked with previously identified ergovaline biosynthetic genes
dmaW
and
lpsA
. The ergovaline genes are closely associated with transposon relics, including retrotransposons and autonomous and nonautonomous DNA transposons. All genes in the cluster were highly expressed in planta, but expression was very low or undetectable in mycelia from axenic culture. This work provides a genetic foundation for elucidating biochemical steps in the ergovaline pathway, the ecological role of individual ergot alkaloid compounds, and the regulation of their synthesis in planta.
Evidence of Horizontal Transfer of Symbiotic Genes from a
Bradyrhizobium japonicum
Inoculant Strain to Indigenous Diazotrophs
Sinorhizobium
(
Ensifer
)
fredii
and
Bradyrhizobium elkanii
in a Brazilian Savannah Soil
Authors: Barcellos Fernando Gomes , Menna Pâmela , da Silva Batista Jesiane Stefânia , Hungria Mariangela
Published: February 2007

The importance of horizontal gene transfer (HGT) in the evolution and speciation of bacteria has been emphasized; however, most studies have focused on genes clustered in pathogenesis and very few on symbiosis islands. Both soybean (
Glycine max
[L.] Merrill) and compatible
Bradyrhizobium japonicum
and
Bradyrhizobium elkanii
strains are exotic to Brazil and have been massively introduced in the country since the early 1960s, occupying today about 45% of the cropped land. For the past 10 years, our group has obtained several isolates showing high diversity in morphological, physiological, genetic, and symbiotic properties in relation to the putative parental inoculant strains. In this study, parental strains and putative natural variants isolated from field-grown soybean nodules were genetically characterized in relation to conserved genes (by repetitive extragenic palindromic PCR using REP and BOX A1R primers, PCR-restriction fragment length polymorphism, and sequencing of the 16SrRNA genes), nodulation, and N
2
-fixation genes (PCR-RFLP and sequencing of
nodY-nodA
,
nodC
, and
nifH
genes). Both genetic variability due to adaptation to the stressful environmental conditions of the Brazilian Cerrados and HGT events were confirmed. One strain (S 127) was identified as an indigenous
B. elkanii
strain that acquired a
nodC
gene from the inoculant
B. japonicum
. Another one (CPAC 402) was identified as an indigenous
Sinorhizobium
(
Ensifer
)
fredii
strain that received the whole symbiotic island from the
B. japonicum
inoculant strain and maintained an extra copy of the original
nifH
gene. The results highlight the strategies that bacteria may commonly use to obtain ecological advantages, such as the acquisition of genes to establish effective symbioses with an exotic host legume.