bluuebunny/crossref_metadata_2025 huggingface dataset:
High Temperature Stress in Wild Wheats and Spring Wheats
Authors: Waines JG
Published: January 2006

The effect of high temperature stress on wild and spring wheats is reviewed. Wild wheats include species in the genera Aegilops L. and Triticum L. Species exist in a polyploid series, diploid, tetraploid and hexaploid, based on the genome formula, n = x = 7 chromosomes. Commercial durum wheat is tetraploid with the genome formula BBAA, while bread wheat is hexaploid (BBAADD). Wheats grown at Riverside, California, from June to October exhibit heat stress at the vegetative and reproductive stages. Under high temperatures (28/15�C day/night) during the vegetative stage, many diploid species do not grow well. Wild diploid T. urartu (AA) and T. monococcum ssp. boeoticum (AA) exhibited more effects of heat stress than the goat grasses A. speltoides (SS = BB?) or A. tauschii (DD). Wild tetraploid T. turgidum L. ssp. dicoccoides Korn (BBAA) exhibited more vegetative-phase stress tolerance than the diploid wheats. Modern Mexican cultivars of durum and bread wheats showed good establishment under high field temperatures, but often tiller number was reduced, and the developmental stages were reduced in time. All the spring durum and bread wheats tested flowered and set seed. They produced anthers with fertile pollen, and they had reproductive heat tolerance. Many wild Aegilops and Triticum accessions did not boot for lack of vernalisation, or they showed reproductive heat stress. Ten wild accessions, including A. speltoides, A. longissima and A. searsii, showed normal vegetative and reproductive development and were considered heat tolerant. They came from the same geographic area in Palestine which should be searched for landraces of wheats that show heat tolerance.
The Heat-Shock Response Relevant to Molecular and Structural Changes in Wheat Yield and Quality
Authors: Blumenthal C , Wrigley CW , Batey IL , Barlow EWR
Published: January 2006

When wheat coleoptiles or plants are subjected to a period of heat stress (e.g, at > 35�C for 1 h or more), there is a reduction in normal protein synthesis, accompanied by de novo synthesis of the classical range of heat-shock proteins (based on radioactive tracer experiments) in virtually all parts of the plant. Study of coleoptile elongation rates indicates that this synthesis is related to a protective effect, whereby a preliminary heat shock provides a degree of protection against a later lethal shock. This thermotolerance is also associated with the appearance in coleoptiles and roots of a small peptide (detected without radioactive labelling) whose amino acid sequence (12 residues) is the same as the N-terminal sequence of the alpha- and beta-gliadin proteins of the endosperm.
Heat stress during grain filling leads to important changes in the synthesis of gluten proteins with reduced synthesis of the high molecular weight (HMW) subunits of glutenin, and continuing synthesis of other gluten proteins, particularly various gliadin proteins. This latter group of polypeptides is thus presumed to be acting as heat-shock proteins, and indeed, multiple heat-shock elements are present in the published sequences of representative genes, up-stream of the coding regions.
HPLC analysis (with or without radioactive labelling) shows that there is a resulting change from the normal balance of gluten polypeptides immediately after the shock as well as in the mature grain. As a result, there is a lower proportion of large-sized aggregates of glutenin and weaker dough properties. This scenario indicates that it should be possible to identify genotypes that would be tolerant to stressrelated variations in quality by analysis of gluten composition and, at the gene level, by screening for heat-stress elements in the genes encoding HMW-glutenin subunits. In addition, heat stress modifies the particle size distribution of the starch fraction of mature grain, producing an increase in the proportion of large (A-type) starch granules. No change in chemical structure was detectable as a result of heat stress.
Modelling Nitrogen Uptake in Ingestad Units Using Michaelis-Menten Kinetics
Authors: Sands PJ , Smethurst PJ
Published: January 2006

The concept of nutrient flux density was developed to grow plants at a controlled and stable relative growth rate whilst maintaining a constant internal concentration of a limiting nutrient. The method requires frequent and exponentially increasing additions of nutrients to replenish uptake. In developing this approach there has been little reference to Michaelis-Menten-like nutrient uptake kinetics for characterising uptake by roots. This paper applies a simple model of nitrogen-limited plant growth using Michaelis-Menten uptake kinetics to data from previously published experiments based on the nutrient flux density approach. It is shown that the model can indeed reproduce key features of experiments: (1) plant relative growth rate equals nitrogen relative addition rate up to a limit; (2) when nitrogen uptake kinetic parameters are within the range reported in the literature, this limiting growth rate agrees with that observed; and (3) solution nitrogen concentrations are consistent with those published. We suggest that the understanding of nutrient uptake and utilisation by plants could be advanced by jointly considering these two approaches.
Elevated CO2 Effects on Water Use and Growth of Maize in Wet and Drying Soil
Authors: Samarakoon AB , Gifford RM
Published: January 2006

It is unclear from the literature as to whether growth of C4 species is responsive to elevated atmospheric CO2 concentration. Reports vary between no response to strong response. To explore the origin of this discrepancy, spaced plants of maize (Zea mays) were grown at atmospheric CO2 concentrations of 362 or 717 μL L-1 under continuously wet or drying soil regimes. The aims were to evaluate the comparative growth promotion from elevated CO2 in a C4 plant under the two contrasting water regimes and the causes of any such promotion, and also how water-use efficiency (WUE) is influenced by high CO2 under the two water regimes. In wet soil, transpiration rate was reduced on average by 29% at high CO2, but neither total dry matter nor plant height was significantly affected by CO2 level. Leaf area was not influenced significantly, so daily water use per plant was 25% lower and WUE was increased entirely due to reduced water use at high CO2. In soil that was drying from field capacity, plants in high CO2 used about 30% less water than those in ambient CO2 while the soil was still wet. This resulted in higher soil water content at high CO2. Plant growth showed a marked response, accumulating 35% more leaf area and 50% more dry matter. Young internodes elongated up to 170% more, giving taller plants. The growth enhancement was largely due to higher average net assimilation rate indicating that C4 photosynthesis responded to elevated CO2 during drought. In drying soil the increase in WUE was due to both increased dry matter and reduced water use, the contribution from each depending on the stage of soil drying. We hypothesise therefore that literature examples where maize growth responded to elevated CO2 may have involved (possibly unrecognised) minor water deficits.
Protein Composition of Wheat Apoplastic Fluid and Resistance to the Russian Wheat Aphid
Authors: Westhuizen AJVD , Pretorius Z
Published: January 2006

The protein composition of intercellular fluids from three different wheat cultivars (Triticum aestivum L. cvv. Tugela DN, Molopo DN and Betta DN) with resistance to the Russian wheat aphid, Diuraphis noxia (Mordvilko) and the corresponding near-isogenic susceptible cultivars (Tugela, Molopo and Betta) were studied under conditions of infestation and non-infestation. The aim was to gain information on the mechanism of resistance and the possible presence of molecular markers of resistance. Aphid infestation induced the accumulation of specific corresponding proteins in the intercellular fluids of resistant cultivars only. The infestation-related proteins were of different molecular mass ranges (28-33; 22-24; 18.5-19.5 and 15.5-17 kDa). Some were serologically related to PR proteins. We suspect that the infestation-related proteins are involved in resistance to the Russian wheat aphid. Although not particularly time saving, the intensified bands of infestation-related proteins on SDS-PAGE gels may be useful for determining resistance.
Generic concepts in Australian Gardenieae (Rubiaceae): a cladistic approach
Authors: Puttock Christopher F. , Quinn Christopher J.
Published: January 2006

Cladistic analyses of a morphological database for 37 Australian species of
Gardenia Ellis and Randia Houst.
ex L. demonstrate the polyphyly of both genera. Five clades are identified,
with Randia audasiiC. T. White placed in an isolated
position. Four of these clades are assigned to extant genera:
Aidia Lour., Atractocarpus Schltr.
et K. Krause, Gardenias.s. and
Kailarsenia Tirveng. The following genera are considered
as falling within Atractocarpus:
Neofranciella Guillaumin, Sukunia
A. C. Sm., and Trukia Kaneh.
Pelagodendron Seem. belongs with
Aidia. Whereas Randia audasii
falls within the current concept of Rothmannia Thunb.,
available data suggest that genus is paraphyletic, and should be confined to
the African taxa.
Infection, inflammation, height, and longevity
Authors: Crimmins Eileen M. , Finch Caleb E.
Published: December 2005

Using historical data from cohorts born before the 20th century in four northern European countries, we show that increasing longevity and declining mortality in the elderly occurred among the same birth cohorts that experienced a reduction in mortality at younger ages. Concurrently, these cohorts also experienced increasing adult height. We hypothesize that both the decline in old-age mortality and the increase in height were promoted by the reduced burden of infections and inflammation. Thus, early growth and cardiovascular diseases of old age may share infectious and inflammatory causes rooted in the external environment.
Division accuracy in a stochastic model of Min oscillations in
Escherichia coli
Authors: Kerr Rex A. , Levine Herbert , Sejnowski Terrence J. , Rappel Wouter-Jan
Published: December 2005

Accurate cell division in
Escherichia coli
requires the Min proteins MinC, MinD, and MinE as well as the presence of nucleoids. MinD and MinE exhibit spatial oscillations, moving from pole to pole of the bacterium, resulting in an average MinD concentration that is low at the center of the cell and high at the poles. This concentration minimum is thought to signal the site of cell division. Deterministic models of the Min oscillations reproduce many observed features of the system, including the concentration minimum of MinD. However, there are only a few thousand Min proteins in a bacterium, so stochastic effects are likely to play an important role. Here, we show that Monte Carlo simulations with a large number of proteins agree well with the results from a deterministic treatment of the equations. The location of minimum local MinD concentration is too variable to account for cell division accuracy in wild-type, but is consistent with the accuracy of cell division in cells without nucleoids. This finding confirms the need to include additional mechanisms, such as reciprocal interactions with the cell division ring or positioning of the nucleoids, to explain wild-type accuracy.
Demonstration of cue recruitment: Change in visual appearance by means of Pavlovian conditioning
Authors: Haijiang Qi , Saunders Jeffrey A. , Stone Rebecca W. , Backus Benjamin T.
Published: December 2005

Until half a century ago, associative learning played a fundamental role in theories of perceptual appearance [Berkeley, G. (1709)An Essay Towards a New Theory of Vision(Dublin), 1st Ed.]. But starting in 1955 [Gibson, J. J. & Gibson, E. J. (1955)Psychol. Rev.62, 32-41], most studies of perceptual learning have not been concerned with association or appearance but rather with improvements in discrimination ability. Here we describe a “cue recruitment” experiment, which is a straightforward adaptation of Pavlov's classical conditioning experiment, that we used to measure changes in visual appearance caused by exposure to novel pairings of signals in visual stimuli. Trainees viewed movies of a rotating wire-frame (Necker) cube. This stimulus is perceptually bistable. On training trials, depth cues (stereo and occlusion) were added to force the perceived direction of rotation. Critically, an additional signal was also added, contingent on rotation direction. Stimuli on test trials contained the new signal but not the depth cues. Over 45 min, two of the three new signals that we tested acquired the ability to bias perceived rotation direction on their own. Results were consistent across the eight trainees in each experiment, and the new cue's effectiveness was long lasting. Whereas most adaptation aftereffects on appearance are opposite in direction to the training stimuli, these effects were positive. An individual new signal can be recruited by the visual system as a cue for the construction of visual appearance. Cue recruitment experiments may prove useful for reexamining of the role of experience in perception.
Screening phage display libraries for organ-specific vascular immunotargeting
in vivo
Authors: Valadon Philippe , Garnett Jeff D. , Testa Jacqueline E. , Bauerle Marc , Oh Phil , Schnitzer Jan E.
Published: December 2005

The molecular diversity of the luminal endothelial cell surface arising
in vivo
from local variations in genetic expression and tissue microenvironment may create opportunities for achieving targeted molecular imaging and therapies. Here, we describe a strategy to identify probes and their cognate antigens for targeting vascular endothelia of specific organs
in vivo
. We differentially screen phage libraries to select organ-targeting antibodies by using luminal endothelial cell plasma membranes isolated directly from tissue and highly enriched in natively expressed proteins exposed to the bloodstream. To obviate liver uptake of intravenously injected phage, we convert the phage-displayed antibodies into scFv-Fc fusion proteins, which then are able to rapidly target select organ(s)
in vivo
as visualized directly by γ-scintigraphic whole-body imaging. Mass spectrometry helps identify the antigen targets. This comprehensive strategy provides new promise for harnessing the power of phage display for mapping vascular endothelia natively in tissue and for achieving vascular targeting of specific tissues
in vivo
.
Mechanism of bacterial cell-surface attachment revealed by the structure of cellulosomal type II cohesin-dockerin complex
Authors: Adams Jarrett J. , Pal Gour , Jia Zongchao , Smith Steven P.
Published: December 2005

Bacterial cell-surface attachment of macromolecular complexes maintains the microorganism in close proximity to extracellular substrates and allows for optimal uptake of hydrolytic byproducts. The cellulosome is a large multienzyme complex used by many anaerobic bacteria for the efficient degradation of plant cell-wall polysaccharides. The mechanism of cellulosome retention to the bacterial cell surface involves a calcium-mediated protein-protein interaction between the dockerin (Doc) module from the cellulosomal scaffold and a cohesin (Coh) module of cell-surface proteins located within the proteoglycan layer. Here, we report the structure of an ultra-high-affinity (
K
a
= 1.44 × 10
10
M
-1
) complex between type II Doc, together with its neighboring X module from the cellulosome scaffold of
Clostridium thermocellum
, and a type II Coh module associated with the bacterial cell surface. Identification of X module-Doc and X module-Coh contacts reveal roles for the X module in Doc stability and enhanced Coh recognition. This extremely tight interaction involves one face of the Coh and both helices of the Doc and comprises significant hydrophobic character and a complementary extensive hydrogen-bond network. This structure represents a unique mechanism for cell-surface attachment in anaerobic bacteria and provides a rationale for discriminating between type I and type II Coh modules.
Critical role of a thiolate-quinone charge transfer complex and its adduct form in
de novo
disulfide bond generation by DsbB
Authors: Inaba Kenji , Takahashi Yoh-hei , Ito Koreaki , Hayashi Shigehiko
Published: December 2005

Recent studies have revealed numerous examples in which oxidation and reduction of cysteines in proteins are integrated into specific cascades of biological regulatory systems. In general, these reactions proceed as thiol-disulfide exchange events. However, it is not exactly understood how a disulfide bond is created
de novo
. DsbB, an
Escherichia coli
plasma membrane protein, is one of the enzymes that create a new disulfide bond within itself and in DsbA, the direct catalyst of protein disulfide bond formation in the periplasmic space. DsbB is associated with a cofactor, either ubiquinone or menaquinone, as a source of an oxidizing equivalent. The DsbB-bound quinone undergoes transition to a pink (λ
max
, ≈500 nm, ubiquinone) or violet (λ
max
, ≈550 nm, menaquinone)-colored state during the course of the DsbB enzymatic reaction. Here we show that not only the thiolate form of Cys-44 previously suggested but also Arg-48 in the α-helical arrangement is essential for the quinone transition. Quantum chemical simulations indicate that proper positioning of thiolate anion and ubiquinone in conjunction with positively charged guanidinium moiety of arginine allows the formation of a thiolate-ubiquinone charge transfer complex with absorption peaks at ≈500 nm as well as a cysteinylquinone covalent adduct. We propose that the charge transfer state leads to the transition state adduct that accepts a nucleophilic attack from another cysteine to generate a disulfide bond
de novo
. A similar mechanism is conceivable for a class of eukaryotic dithiol oxidases having a FAD cofactor.
Constitutive activation of MKK6 in chondrocytes of transgenic mice inhibits proliferation and delays endochondral bone formation
Authors: Zhang Ren , Murakami Shunichi , Coustry Françoise , Wang Ying , de Crombrugghe Benoit
Published: December 2005

Accumulating
in vitro
evidence suggests that the p38 mitogen-activated protein kinase (MAPK) pathway is involved in endochondral ossification. To investigate the role of this pathway in endochondral ossification, we generated transgenic mice with expression in chondrocytes of a constitutively active mutant of MKK6, a MAPK kinase that specifically activates p38. These mice had a dwarf phenotype characterized by reduced chondrocyte proliferation, inhibition of hypertrophic chondrocyte differentiation, and a delay in the formation of primary and secondary ossification centers. Histological analysis with
in situ
hybridization showed reduced expression of
Indian hedgehog
,
PTH/PTH-related peptide receptor
(PTH, parathyroid hormone),
cyclin D1
, and increased expression of
p21
in chondrocytes. In addition, both
in vivo
and in transfected cells, p38 signaling increased the transcriptional activity of Sox9, a transcription factor essential for chondrocyte differentiation. In agreement with this observation, transgenic mice that express a constitutively active mutant of MKK6 in chondrocytes showed phenotypes similar to those of mice that overexpress
SOX9
in chondrocytes. These observations are consistent with the notion that increased activity of Sox9 accounts at least in part for the phenotype caused by constitutive activation of MKK6 in chondrocytes. Therefore, our study provides
in vivo
evidence for the role of p38 in endochondral ossification and suggests that Sox9 is a likely downstream target of the p38 MAPK pathway.
A nucleoporin is required for induction of Ca
2+
spiking in legume nodule development and essential for rhizobial and fungal symbiosis
Authors: Kanamori Norihito , Madsen Lene Heegaard , Radutoiu Simona , Frantescu Mirela , Quistgaard Esben M. H. , Miwa Hiroki , Downie J. Allan , James Euan K. , Felle Hubert H. , Haaning Line Lindegaard , Jensen Torben Heick , Sato Shusei , Nakamura Yasukazu , Tabata Satoshi , Sandal Niels , Stougaard Jens
Published: January 2006

Nuclear-cytoplasmic partitioning and traffic between cytoplasmic and nuclear compartments are fundamental processes in eukaryotic cells. Nuclear pore complexes mediate transport of proteins, RNAs and ribonucleoprotein particles in and out of the nucleus. Here we present positional cloning of a plant nucleoporin gene,
Nup133
, essential for a symbiotic signal transduction pathway shared by
Rhizobium
bacteria and mycorrhizal fungi. Mutation of
Nup133
results in a temperature sensitive nodulation deficient phenotype and absence of mycorrhizal colonization. Root nodules developing with reduced frequency at permissive temperatures are ineffective and electron microscopy show that
Rhizobium
bacteria are not released from infection threads. Measurement of ion fluxes using a calcium-sensitive dye show that
Nup133
is required for the Ca
2+
spiking normally detectable within minutes after application of purified rhizobial Nod-factor signal molecules to root hairs. Localization of NUP133 in the nuclear envelope of root cells and root hair cells shown with enhanced yellow fluorescent protein fusion proteins suggests a novel role for NUP133 nucleoporins in a rapid nuclear–cytoplasmic communication after host–plant recognition of symbiotic microbes. Our results identify a component of an intriguing signal process requiring interaction at the cell plasma membrane and at intracellular nuclear and plastid organelle-membranes to induce a second messenger.
RETRACTED: Packing defects as selectivity switches for drug-based protein inhibitors
Authors: Fernández Ariel , Scott Ridgway , Berry R. Stephen
Published: December 2005

The conservation of structure across homolog proteins often diffuses the impact of drug-based inhibition by promoting alternative protein-ligand associations that may lead to toxic side effects. However, sticky packing defects are typically not conserved across homologs, making them valuablea prioritargets to enhance specificity. By introducing a homology to quantify packing differences among proteins, we enable a previously undescribed strategy for the design of highly selective drug inhibitors involving ligands that wrap nonconserved packing defects. The selectivity of these ligands is validated by performing affinity assays on a cancer-related pharmacokinome. Minor reengineering of a powerful inhibitor guided by wrapping differences across its target kinome can selectively direct its impact toward a specific kinase. Thus, nonconserved packing defects may be used as selectivity switches across homolog targets, using spatial displacements of packing defects across aligned protein structures.
MAX1
, a regulator of the flavonoid pathway, controls vegetative axillary bud outgrowth in
Arabidopsis
Authors: Lazar Gabor , Goodman Howard M.
Published: December 2005

We show that
MAX1
, a specific repressor of vegetative axillary bud outgrowth in
Arabidopsis
, acts a positive regulator of the flavonoid pathway, including 11 structural genes and the transcription factor
An2
. Repression of bud outgrowth requires
MAX1
-dependent flavonoid gene expression. As the flavonoidless state leads to lateral outgrowth in
Arabidopsis
, our data suggest that a flavonoid-based mechanism regulates axillary bud outgrowth and that this mechanism is under the control of
MAX1
. Flavonoid gene expression results in the diminished expression of auxin transporters in the bud and stem, and this, in turn, decreases the rate of polar auxin transport. We speculate that
MAX1
could repress axillary bud outgrowth via regulating flavonoid-dependent auxin retention in the bud and underlying stem. Because
MAX1
is implicated in synthesis of the carotenoid-derived branch regulator(s) from the root, it likely links long-distance signaling with local control of bud outgrowth.
A simple physical model for scaling in protein-protein interaction networks
Authors: Deeds Eric J. , Ashenberg Orr , Shakhnovich Eugene I.
Published: December 2005

It has recently been demonstrated that many biological networks exhibit a “scale-free” topology, for which the probability of observing a node with a certain number of edges (
k
) follows a power law: i.e.,
p
(
k
) ∼
k
-γ
. This observation has been reproduced by evolutionary models. Here we consider the network of protein-protein interactions (PPIs) and demonstrate that two published independent measurements of these interactions produce graphs that are only weakly correlated with one another despite their strikingly similar topology. We then propose a physical model based on the fundamental principle that (de)solvation is a major physical factor in PPIs. This model reproduces not only the scale-free nature of such graphs but also a number of higher-order correlations in these networks. A key support of the model is provided by the discovery of a significant correlation between the number of interactions made by a protein and the fraction of hydrophobic residues on its surface. The model presented in this paper represents a physical model for experimentally determined PPIs that comprehensively reproduces the topological features of interaction networks. These results have profound implications for understanding not only PPIs but also other types of scale-free networks.
Regulation of tyrosinase trafficking and processing by presenilins: Partial loss of function by familial Alzheimer's disease mutation
Authors: Wang Runsheng , Tang Phuong , Wang Pei , Boissy Raymond E. , Zheng Hui
Published: December 2005

Presenilins (PS) are required for γ-secretase cleavage of multiple type I membrane proteins including the amyloid precursor protein and Notch and also have been implicated in regulating intracellular protein trafficking and turnover. Using genetic and pharmacological approaches, we reveal here a unique function of PS in the pigmentation of retinal pigment epithelium and epidermal melanocytes. PS deficiency leads to aberrant accumulation of tyrosinase (Tyr)-containing 50-nm post-Golgi vesicles that are normally destined to melanosomes. This trafficking is γ-secretase-dependent, and abnormal localization of Tyr in the absence of PS is accompanied by the simultaneous accumulation of its C-terminal fragment. Furthermore, we show that the PS1M146V familial Alzheimer's disease mutation exhibits a partial loss-of-function in pigment synthesis. Our results identify Tyr and related proteins as physiological substrates of PS and link γ-secretase activity with intracellular protein transport.
Autoantibodies to myelin basic protein catalyze site-specific degradation of their antigen
Authors: Ponomarenko Natalia A. , Durova Oxana M. , Vorobiev Ivan I. , Belogurov Alexey A. , Kurkova Inna N. , Petrenko Alexander G. , Telegin Georgy B. , Suchkov Sergey V. , Kiselev Sergey L. , Lagarkova Maria A. , Govorun Vadim M. , Serebryakova Marina V. , Avalle Bérangère , Tornatore Pete , Karavanov Alexander , Morse Herbert C. , Thomas Daniel , Friboulet Alain , Gabibov Alexander G.
Published: December 2005

Autoantibody-mediated tissue destruction is among the main features of organ-specific autoimmunity. This report describes “an antibody enzyme” (abzyme) contribution to the site-specific degradation of a neural antigen. We detected proteolytic activity toward myelin basic protein (MBP) in the fraction of antibodies purified from the sera of humans with multiple sclerosis (MS) and mice with induced experimental allergic encephalomyelitis. Chromatography and zymography data demonstrated that the proteolytic activity of this preparation was exclusively associated with the antibodies. No activity was found in the IgG fraction of healthy donors. The human and murine abzymes efficiently cleaved MBP but not other protein substrates tested. The sites of MBP cleavage determined by mass spectrometry were localized within immunodominant regions of MBP. The abzymes could also cleave recombinant substrates containing encephalytogenic MBP85-101peptide. An established MS therapeutic Copaxone appeared to be a specific abzyme inhibitor. Thus, the discovered epitope-specific antibody-mediated degradation of MBP suggests a mechanistic explanation of the slow development of neurodegeneration associated with MS.
Flow cytometric detection of specific RNAs in native human cells with quenched autoligating FRET probes
Authors: Abe Hiroshi , Kool Eric T.
Published: December 2005

We describe the use of modified fluorescent-labeled oligonucleotide probes in the sequence-specific detection of messenger RNAs in live human cells. To make this detection possible, we developed a previously undescribed probe design that combines earlier quenched autoligation chemistry with a previously undescribed fluorescence resonance energy transfer (FRET) strategy to lower background signals. The probe pairs consisted of a nucleophilic 3′-phosphorothioate probe carrying a Cy5 FRET acceptor, and an electrophilic probe containing the combination of a 5′ end electrophile/quencher and a fluorescein FRET donor. Probes were introduced to HL-60 cells by use of the streptolysin O pore-forming peptide. Signals from three different messenger RNAs, as well as 28S ribosomal RNA, could be detected and quantitated by flow cytometry. Probes targeted to ribosomal sequences and β-actin mRNA also could be detected over background by confocal fluorescence microscopy. Varying the target site and probe backbone chemistry were found to have large effects on signal. The data suggest that quenched autoligating probes may be of general utility as biological tools in following localization, transcription, and processing of eukaryotic cellular messages and may have applications in diagnostic or prognostic analysis of disease-related RNAs in human tissues.
Pneumocystis carinii pneumonia in Hong Kong: a 10 year retrospective study
Authors: Hui M. , Kwok W. T.
Published: December 2005

A retrospective review was performed of patients diagnosed with Pneumocystis carinii pneumonia (PCP) from 1994 to 2003 at the Prince of Wales Hospital in Hong Kong. Eighteen patients were identified. Six (33·3 %) were co-infected with human immunodeficiency virus (HIV). The remaining 12 non-HIV-infected patients had underlying diseases: three post-renal transplant recipients, three with haematological malignancies, two with auto-immune diseases, two with renal diseases, one with hepatocellular carcinoma and one with congenital cytomegalovirus disease. Cytomegalovirus co-infection was observed in four patients. All patients received cotrimoxazole therapy, with intolerance observed in four of them, including one with glucose-6-phosphate dehydrogenase deficiency, two with repeated vomiting and one with renal impairment. Overall crude mortality was 33·3 %. The results suggested that, apart from being a common infection for patients with HIV infection, PCP can occur during the course of many immunosuppressive diseases and therapies. The mortality of PCP was high despite appropriate treatment. Chemoprophylaxis should be considered in populations at risk.
Antimicrobial activities of silver dressings: an in vitro comparison
Authors: Ip Margaret , Lui Sau Lai , Poon Vincent K. M. , Lung Ivan , Burd Andrew
Published: December 2005

A range of silver-coated or -impregnated dressings are now commercially available for use but comparative data on their antimicrobial efficacies are limited. The antibacterial activities of five commercially available silver-coated/impregnated dressings were compared against nine common burn-wound pathogens, namely methicillin-sensitive and -resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloacae, Proteus vulgaris, Acinetobacter baumannii and a multi-drug-efflux-positive Acinetobacter baumannii (BM4454), using a broth culture method. The rapidity and extent of killing of these pathogens under in vitro conditions were evaluated. All five silver-impregnated dressings investigated exerted bactericidal activity, particularly against Gram-negative bacteria, including Enterobacter species, Proteus species and E. coli. The spectrum and rapidity of action, however, ranged widely for different dressings. Acticoat and Contreet had a broad spectrum of bactericidal activities against both Gram-positive and -negative bacteria. Contreet was characterized by a very rapid bactericidal action and achieved a reduction of ⩾10 000 c.f.u. ml−1 in the first 30 min for Enterobacter cloacae, Proteus vulgaris, Pseudomonas aeruginosa and Acinetobacter baumanii. Other dressings demonstrated a narrower range of bactericidal activities. Understanding the characteristics of these dressings may enable them to be targeted more appropriately according to the specific requirements for use of a particular dressing, as in for prophylaxis in skin grafting or for an infected wound with MRSA.
Identification of an RTX determinant of Burkholderia cenocepacia J2315 by subtractive hybridization
Authors: Whitby Paul W. , VanWagoner Timothy M. , Taylor Ashlee A. , Seale Thomas W. , Morton Daniel J. , LiPuma John J. , Stull Terrence L.
Published: December 2005

This study utilized suppressive subtractive hybridization between the clinical isolate Burkholderia cenocepacia J2315 and the closely related environmental isolate Burkholderia cepacia ATCC 25416T to isolate DNA fragments specific to B. cenocepacia J2315. Analysis of the resulting pools of B. cenocepacia-specific DNAs identified several fragments that may be part of putative virulence factors. Further in silico analysis of a single fragment indicated that it was internal to a gene of which the predicted product had characteristics of repeat in toxin (RTX)-like proteins and high similarity to proteins in other human or plant pathogens. In conjunction with this finding, phenotypic traits associated with known RTX proteins were assessed. A haemagglutinating activity of B. cenocepacia J2315 was identified that was absent in B. cepacia ATCC 25416T. The expression of this activity appeared to be growth phase-dependent. Analysis of the gene presence and haemagglutinating activity across the species of the B. cepacia complex showed that both were common to the ET12 lineage of B. cenocepacia, but were absent in the other species examined. Haemagglutinating activity was limited to isolates with the RTX-like gene. Expression studies utilizing quantitative PCR demonstrated an association between onset of haemagglutinating activity and increased expression of the gene, which suggests that the putative RTX determinant encodes a haemagglutinating activity.
Methicillin-resistant Staphylococcus aureus genotyping using a small set of polymorphisms
Authors: Stephens Alex J. , Huygens Flavia , Inman-Bamber John , Price Erin P. , Nimmo Graeme R. , Schooneveldt Jacqueline , Munckhof Wendy , Giffard Philip M.
Published: December 2005

The aim of this study was to identify a set of genetic polymorphisms that efficiently divides methicillin-resistant Staphylococcus aureus (MRSA) strains into groups consistent with the population structure. The rationale was that such polymorphisms could underpin rapid real-time PCR or low-density array-based methods for monitoring MRSA dissemination in a cost-effective manner. Previously, the authors devised a computerized method for identifying sets of single nucleotide polymorphisms (SNPs) with high resolving power that are defined by multilocus sequence typing (MLST) databases, and also developed a real-time PCR method for interrogating a seven-member SNP set for genotyping S. aureus. Here, it is shown that these seven SNPs efficiently resolve the major MRSA lineages and define 27 genotypes. The SNP-based genotypes are consistent with the MRSA population structure as defined by eburst analysis. The capacity of binary markers to improve resolution was tested using 107 diverse MRSA isolates of Australian origin that encompass nine SNP-based genotypes. The addition of the virulence-associated genes cna, pvl and bbp/sdrE, and the integrated plasmids pT181, pI258 and pUB110, resolved the nine SNP-based genotypes into 21 combinatorial genotypes. Subtyping of the SCCmec locus revealed new SCCmec types and increased the number of combinatorial genotypes to 24. It was concluded that these polymorphisms provide a facile means of assigning MRSA isolates into well-recognized lineages.
Distribution and genomic location of active insertion sequences in the Burkholderia cepacia complex
Authors: Kenna Dervla T. , Yesilkaya Hasan , Forbes Ken J. , Barcus Victoria A. , Vandamme Peter , Govan John R. W.
Published: December 2005

This study aimed firstly to establish the distribution and copy number within the Burkholderia cepacia complex of three insertion sequences (IS402, IS407 and IS1416) that possess the ability to activate transcription and hence influence gene expression. A second aim was to map the genomic insertion sites of one of the active insertion sequences (IS407) to establish putative links between insertion site and downstream gene activation. The resulting data revealed that all three insertion sequences were present in one-third of the 66 isolates tested. The three insertion sequences were prevalent across the nine B. cepacia complex species, although IS402 was absent from the 16 Burkholderia anthina strains tested and IS407 was absent from all 10 Burkholderia pyrrocinia strains. IS407 copies from six strains (two Burkholderia cenocepacia strains and one strain each of Burkholderia multivorans, Burkholderia stabilis, Burkholderia vietnamiensis and B. anthina) were mapped to the genome using hemi-nested inverse PCR. Insertions were found upstream of genes with wide-ranging functions. This study suggests that the abundance and distribution of these active insertion sequences is likely to affect genomic plasticity, and potentially gene transcription and pathogenicity.
PCR evaluation of false-positive signals from two automated blood-culture systems
Authors: Karahan Z. Ceren , Mumcuoglu Ipek , Guriz Haluk , Tamer Deniz , Balaban Neriman , Aysev Derya , Akar Nejat
Published: December 2005

Rapid detection of micro-organisms from blood is one of the most critical functions of a diagnostic microbiology laboratory. Automated blood-culture systems reduce the time needed to detect positive cultures, and reduce specimen handling. The false-positive rate of such systems is 1–10 %. In this study, the presence of pathogens in ‘false-positive’ bottles obtained from BACTEC 9050 (Becton Dickinson) and BacT/Alert (Biomérieux) systems was investigated by eubacterial and fungal PCR. A total of 169 subculture-negative aerobic blood-culture bottles (104 BacT/Alert and 65 BACTEC) were evaluated. Both fungal and eubacterial PCRs were negative for all BACTEC bottles. Fungal PCR was also negative for the BacT/Alert system, but 10 bottles (9·6 %) gave positive results by eubacterial PCR. Sequence analysis of the positive PCR amplicons indicated the presence of the following bacteria (number of isolates in parentheses): Pasteurella multocida (1), Staphylococcus epidermidis (2), Staphylococcus hominis (1), Micrococcus sp. (1), Streptococcus pneumoniae (1), Corynebacterium spp. (2), Brachibacterium sp. (1) and Arthrobacter/Rothia sp. (1). Antibiotic usage by the patients may be responsible for the inability of the laboratory to grow these bacteria on subcultures. For patients with more than one false-positive bottle, molecular methods can be used to evaluate the microbial DNA in these bottles. False positives from the BACTEC system may be due to elevated patient leukocyte counts or the high sensitivity of the system to background increases in CO2 concentration.
Peritonitis due to Cunninghamella bertholletiae in a patient undergoing continuous ambulatory peritoneal dialysis
Authors: Pimentel Jason D. , Dreyer Gavin , Lum Gary D.
Published: December 2005

Peritoneal dialysis-associated peritonitis due to fungi of the class Zygomycetes occurs very rarely. A case of fungal continuous ambulatory peritoneal dialysis peritonitis due to Cunninghamella bertholletiae is reported in a 39-year-old Aboriginal woman with end-stage renal failure and diabetes mellitus. This isolate was found to be resistant in vitro to amphotericin B, 5-fluorocytosine, fluconazole, itraconazole, ketoconazole and voriconazole. However, this patient was successfully treated with voriconazole and removal of the Tenckhoff dialysis catheter. Zygomycoses are an emerging threat among immunocompromised patients, including those with chronic renal failure. Zygomycosis due to C. bertholletiae is frequently fatal and is often non-responsive to systemic antifungal therapy. This is believed to be the first reported case of C. bertholletiae causing peritonitis in humans and one of the minority of cases involving this organism with a successful outcome.
Sequence and phylogenetic analysis of the gene for surface layer protein, slpA, from 14 PCR ribotypes of Clostridium difficile
Authors: Ní Eidhin Déirdre , Ryan Anthony W. , Doyle Rachael M. , Walsh J. Bernard , Kelleher Dermot
Published: December 2005

Clostridium difficile is the commonest cause of antibiotic-associated diarrhoea, with the hospitalized elderly being at particular risk. The organism makes a crystalline surface protein layer (S-layer), encoded by the slpA gene, the product of which is cleaved to give two mature peptides which associate to form the layer. The larger peptide (high molecular weight; HMW), derived from the C-terminal portion of the precursor, is relatively conserved, whereas the smaller peptide (low molecular weight; LMW), derived from the N-terminal portion of the precursor, is a dominant antigen which substantially forms the basis for serotyping of isolates. PCR ribotyping is a more discriminatory typing method, based on the intergenic rRNA. We obtained the sequence for slpA and some flanking DNA from a collection of C. difficile strains of 14 ribotypes isolated from elderly patients. Sequences from different ribotypes were compared with one another and with published sequences. Sequences from C. difficile ribotypes 046 and 092 were identical. Sequences from ribotype pairs 005 and 054, 012 and 046/092, 014 and 066 and 031 and 094 differed by 1–3 nt in the slpA gene. There were ultimately nine ribotypes or groups of ribotypes with very different slpA sequences, particularly in the region encoding the LMW peptide. The sequence from ribotype 002 was very different from previously published sequences. The DNA segment sequenced included the 5′ 315 bp of a secA homologue, encoding a putative transport protein required for peptide secretion across the plasma membrane. The amino acid sequences of the predicted HMW peptides were aligned and a neighbour-joining tree was produced using 10 000 bootstrap replicates. The predicted SecA N-terminal region was similarly analysed. For both SlpA and SecA, a strong association was found between ribotypes 012, 046/092, 017, 031 and 094. Ribotypes 001 and 078 formed part of this clade for SlpA but not SecA, indicating independent evolution for slpA and secA, presumably because they come under different selection pressures.
Antibacterial resistance in Helicobacter pylori strains isolated from Bulgarian children and adult patients over 9 years
Authors: Boyanova Lyudmila , Nikolov Rossen , Lazarova Elena , Gergova Galina , Katsarov Nikolai , Kamburov Victor , Spassova Zoya , Derejian Sirigan , Jelev Christo , Mitov Ivan , Krastev Zacharii
Published: December 2005

The aim of this study was to evaluate the primary, combined and post-treatment antibacterial resistance rates in 1205 Helicobacter pylori strains from non-treated (786 adults, 282 children) and treated (109 adults, 28 children) patients in Bulgaria. Susceptibility was tested by the limited agar dilution method. Overall primary resistance rates to metronidazole, clarithromycin, amoxicillin, tetracycline and both metronidazole and clarithromycin were respectively 15·0, 12·5, 1·5, 3·4 and 4·7 % in children and 25·6, 12·6, 0·8, 5·2 and 4·9 % in adults. Primary metronidazole resistance in adults was more common than in children, but the differences for other agents tested were not significant. Primary resistance rates were in the range of those reported worldwide. There was no significant increase in primary resistance rates from 1996/1999 to 2003/2004; however, clarithromycin resistance rates exhibited a slight tendency to increase. Post-treatment resistance to amoxicillin was detected in 1·6 % of 63 strains. Post-treatment resistance to metronidazole was common (81·6 %) and that to clarithromycin was considerable (36 %). Alarming emergence of strains with triple resistance to amoxicillin, metronidazole and clarithromycin was found in two non-treated and three treated patients. The results motivate a larger and continuing surveillance of H. pylori resistance in Bulgaria and worldwide.
Identification of bacteria in endodontic infections by sequence analysis of 16S rDNA clone libraries
Authors: Saito Daniel , de Toledo Leonardo Renato , Rodrigues Jorge Luiz Mazza , Tsai Siu Mui , Höfling José Francisco , Gonçalves Reginaldo Bruno
Published: December 2005

A significant proportion of oral bacteria are unable to undergo cultivation by existing techniques. In this regard, the microbiota from root canals still requires complementary characterization. The present study aimed at the identification of bacteria by sequence analysis of 16S rDNA clone libraries from seven endodontically infected teeth. Samples were collected from the root canals, subjected to the PCR with universal 16S rDNA primers, cloned and partially sequenced. Clones were clustered into groups of closely related sequences (phylotypes) and identification to the species level was performed by comparative analysis with the GenBank, EMBL and DDBJ databases, according to a 98 % minimum identity. All samples were positive for bacteria and the number of phylotypes detected per subject varied from two to 14. The majority of taxa (65·2 %) belonged to the phylum Firmicutes of the Gram-positive bacteria, followed by Proteobacteria (10·9 %), Spirochaetes (4·3 %), Bacteroidetes (6·5 %), Actinobacteria (2·2 %) and Deferribacteres (2·2 %). A total of 46 distinct taxonomic units was identified. Four clones with low similarity to sequences previously deposited in the databases were sequenced to nearly full extent and were classified taxonomically as novel representatives of the order Clostridiales, including a putative novel species of Mogibacterium. The identification of novel phylotypes associated with endodontic infections suggests that the endodontium may still harbour a relevant proportion of uncharacterized taxa.
Detection of mixed infections with Mycobacterium lentiflavum and Mycobacterium avium by molecular genotyping methods
Authors: Suffys Philip , da Silva Rocha Adalgiza , Brandão Adeilton , Vanderborght Bart , Mijs Wouter , Jannes Geert , Mello Fernanda C. Q. , Pedro Heloisa da Silveira Paro , de Souza Fonseca Leila , de Oliveira Rosângela Siqueira , Cardoso Leão Sylvia , Féres Saad Maria Helena
Published: December 2005

Three mycobacterial isolates, one from the blood of an HIV-infected patient and two consecutive isolates from a woman with unknown HIV status, had been identified as belonging to the Mycobacterium avium complex by conventional procedures. In both patients, using genetic analysis procedures such as PCR–restriction enzyme analysis (PRA) of the hsp65 gene, a commercially available reverse hybridization-based assay (INNO-LiPA mycobacteria) and/or sequencing analysis of the 16S–23S internal transcribed spacer (ITS), the presence of Mycobacterium lentiflavum was also demonstrated. At the time of detection, both cases were also infected with M. avium, suggesting an underestimation of infection with M. lentiflavum and co-infection with different Mycobacterium species.