bluuebunny/crossref_metadata_2025 huggingface dataset:
Personal Narratives of Students With and Without Learning Disabilities
Authors: Celinska Dorota K.
Published: April 2004

This study investigated personal narratives produced by children with and without learning disabilities in the context of naturalistic conversation. The high‐point analysis was applied to compare the referential and evaluative aspects of children's personal narratives. Participants were 60 students in Grades 4 and 5 in public suburban schools, with an almost equal number of boys and girls. The participants with learning disabilities were matched individually with typically achieving peers for chronological age, grade, ethnicity, and gender. Despite significant differences in expressive semantic and syntactic skills, both groups produced narratives that were equivalent in terms of length, structural organization, and global coherence. Group differences centered around the decreased inclusion of high points in narratives produced by girls with learning disabilities, indicating that these girls may struggle with generating coherent personal narratives and communicating their own perspective on the recounted personal experiences. This specific narrative performance is accounted for by the unique profile of cognitive and academic characteristics demonstrated by girls with learning disabilities. Practical implications for the multifaceted assessment of referential and evaluative narrative functions are discussed, and interventions for enhancing narrative skills are suggested. The study's findings are also discussed in the context of previous research and directions for future inquiries.
Academic Self‐Perceptions, Effort, and Strategy Use in Students with Learning Disabilities: Changes Over Time
Authors: Meltzer Lynn , Katzir Tamar , Miller Lynne , Reddy Ranjini , Roditi Bethany
Published: April 2004

This study investigated changes in teachers' and students' perceptions of students' effort, strategy use, and academic difficulties when strategy instruction was infused into the classroom curriculum. The sample consisted of 201 students with learning disabilities, 210 average achievers, and 57 teachers from Grades 4–9 in two urban and suburban communities. After six months of classroom‐based strategy instruction, students with learning disabilities reported more consistent use of strategies with their schoolwork and perceived themselves as struggling less in reading, writing, and spelling. Teachers perceived the students with learning disabilities as more strategic and as applying more effort to their schoolwork. Teachers also perceived their students as showing significant improvements in spelling, regardless of whether they had learning disabilities. These findings extended the results of previous investigations and indicated the small, positive impact of classroom‐based strategy instruction. Further investigations are critical to evaluate the generalizability of these findings.
Benefits from peer teaching in the dental gross anatomy laboratory
Authors: Brueckner Jennifer K. , MacPherson Brian R.
Published: April 2004

This study examined the impact of implementing a rotating dissection schedule on the attitudes and performance of first‐year dental students in the gross anatomy laboratory at the University of Kentucky. In 2002–2003, half of the students assigned to each cadaver dissected the assigned objectives during the first 90 min of the laboratory session. During the last 30 min, the non‐dissecting group members came into the laboratory and had the day's dissection demonstrated and explained to them via peer instruction. Dissection responsibilities rotated with each laboratory session. Eighty‐eight percent of student participants were satisfied with the rotating dissection approach according to a mid‐term survey, and this satisfaction level remained unchanged at the end of the semester for most students. Students' perceptions of the quality of peer laboratory presentations varied, with only 44% rating them as good or better. Eighty percent of students perceived that rotating dissection did not impede their performance, and this was confirmed by analysis of grade data. Student satisfaction and the ability to devote additional weekly curriculum time to studying anatomy in a very compressed curriculum were the main student‐described benefits.
A student learning perspective on teaching and learning, with implications for problem‐based learning
Authors: Prosser Michael
Published: April 2004

Over the last 20 years or so, there has been a substantial development in our understanding of how and what university students learn in their courses (here meaning a component of a programme) and programmes of study. This research has shown that rather than there being a direct connection between the way teachers teach and design their courses, and the quality of their students' learning outcomes, the relationship is indirect. The way students perceive and understand their learning context and the way they approach their learning in relationship to these perceptions have been found to be major intervening factors between teachers' teaching and students' learning outcomes. Their perceptions are, in turn, constituted in relation to their prior experiences of teaching and learning and what is designed for them to learn. In this paper, some of the key findings of this research and how it can be related to a problem‐based learning (PBL) perspective in higher education are outlined. In particular, it is argued that the variation in students' perceptions and understanding of what PBL is about is fundamental to the way they approach their studies and to their learning outcomes.
Stress Provoking Factors in Dental Education
Authors: Divaris K. , Polychronopoulou A.
Published: April 2004

Aim  To identify stress provoking factors in dental education and to explore the role of study level and gender on perceived sources of stress.

Methods  Five hundred seventy‐one undergraduates, officially registered at the University of Athens Dental School, participated in the survey by filling a 30‐item modified version of the Dental Environment Stress questionnaire.

Results  Assigned workload, performance pressure and self‐efficacy beliefs constituted the most stress provoking factors. Females reported significantly higher distress, whereas stress intensity greatly differentiated by class year. Multivariate analysis revealed that, irrespective of gender, fourth level and graduating students were less concerned about class work overload, clinical training difficulties and course failing, but were significantly more insecure about their professional future. Entry‐level students appeared to be the most concerned about the lack of time for relaxation. However, they were the least stressed about completing graduation requirements compared with the rest of their peers.

Conclusion  The reconsideration of the existing educational system towards a more balanced, flexible and student‐centred orientation, as well as the implementation of continuous self‐assessment and study counselling, might be effective in reducing inherent stress sources in dental education.
Student State of Readiness for E‐Learning: University of Sheffield's Faculty of Medicine
Authors: Stokes C. W. , Basford L. , Cannavina G.
Published: April 2004

A study to assess the state of readiness of students for the implementation of new e‐technologies into the learning environment formed the first phase of the FDTL4‐funded WILeN Project. The project will collaborate with institutions across the UK to develop a Web‐Based Interprofessional Learning Network.

Current learning theories place the emphasis on student–teacher and student–student interaction. However, previous research and development on e‐learning has focused on the interaction of the students with the learning material.

The aim of the study was to evaluate the state of students’ readiness for e‐learning and their ability to participate in online discussions, contribute to message forums and interact via video‐conferencing.

The study was undertaken in two phases. A survey, conducted electronically, was designed to find a ’base level’ of student IT skills that could be used to design the next‐generation of interactive and inter‐professional e‐learning materials, and students produced denture designs via video‐conferencing seminars.

In result, 191 valid responses were received reporting that most students (95.8%) check their e‐mail every few days or more, with slightly less (82.8%) using the web frequently. There was a low incidence of students having used technologies recognized as providing the basis for future online learning environments: Internet Relay Chat (37.7%), Message Forums (49.7%) and Video‐conferencing (5.8%). The data revealed that the students generally lacked experience of the new interactive technologies. However, the successful video‐conferencing seminars demonstrated that the students had a willingness and capability to learn new skills.

In conclusion, there is a lack of educational readiness for new interactive learning media, but the students have the transferable skills and enthusiasm to enable their use in future learning scenarios.
Preliminary Testing of an ICT‐Based Learning Support System in Helsinki
Authors: Karaharju‐Suvanto T. , Noponen J. , Pakkanen A. L.
Published: April 2004

Background and aim:  During dental education, it would be beneficial for the students to be able to combine theoretical teaching with practical work in a logical manner. For this purpose, a Portfolio software has been developed to facilitate ICT‐based learning with DecaMedia Ltd. Test phase had several aims; test the functionality of the software and add necessary refinements, test the flow of information from teachers to students in different forms and to analyse the possible problems in the use of this software and in ICT‐based learning in general.

Material and methods:  A group of teachers and students were gathered to test the usefulness of the new software. Prior to this phase, there had been some testing and a questionnaire study on ICT skills and attitudes. During the first period of this project, the software was customized by DecaMedia to meet our demands. Different access levels were created. The students gathered interesting patient cases and took documents in several media forms. The teachers added theoretical material to combine scientific knowledge with practical work either directly or using links.

Results and conclusions:  In general, the following areas were noted: this media is good for supporting assessment . When patient files are added, the importance of privacy protection is high as the files are transferred via network. For theoretical material, the copyright has to be considered as well as the size of network‐delivered documents. This program has a user‐friendly interface. Attachment of different file types was straightforward. The operating language of the program is Finnish, which helps less‐experienced students to get started. There are still some limitations, mainly in stability. We found the program useful to support dental education, but more ICT training will be required for both students and teachers. ICT learning can be beneficial addition in dental education.
Do osmotic forces play a role in the uptake of water by human skin?
Authors: Van Kemenade Patricia M. , Houben Mark M. J. , Huyghe Jacques M. , Douven Lucien F. A.
Published: April 2004

Background/purpose: To describe the water and ion transport through the skin under different conditions, we developed a three‐component mixture model. This model has proven to describe the transient change in transepidermal water loss (TEWL) after a change in relative humidity and the result of damage to the skin. Osmotic forces arc present in the model. To assess the influence of osmotic forces on the water uptake of the skin, we investigated transient TEWL values after 1 h application of salt solutions of different molarities (0, 1, and 4 M NaCl).

Methods: Filters saturated with 0, 1, and 4 M NaCl solution were applied for 1 h under occlusion. TEWL was measured 50–90 min after removal of the solution. The transient water loss curves were fit with an exponential function. The area under the fitted curve was calculated and regarded as a measure for the amount of extra water absorbed in the skin.

Results: For all molarities, TEWL is increased immediately after removal of the solution. In time, this increase decays until pre‐application values are reached again. The rate of decrease differs significantly for all three molarities. Ninety‐five per cent of the increase has been reversed after 30, 19, and 6 min for the 0, 1, and 4 M case, respectively. The amount of water absorbed differs significantly between the three molarities 7.3±2.0; 3.9±1.0; 2.0±0.5 g/m2, respectively.

Conclusions: In all cases, there was an increase in TEWL immediately after removal of the solution. The significant differences in decay time and amount of water absorbed between the three molarities indicate that osmotic forces do play an important role in the water uptake.
In vivo epidermal thickness measurement: ultrasound vs. confocal imaging
Authors: Nouveau‐Richard S. , Monot M. , Bastien P. , De Lacharrière O.
Published: April 2004

Background/purpose: In this study, in vivo skin imaging methods, ultrasound (US) and confocal microscopy (CM) were compared with regards to their accuracy in measuring the epidermal thickness. In addition an attempt was made to clarify the biological significance of the second echo‐rich line observed on US skin images, i.e. whether it represents the dermal–epidermal junction or the papillar–reticular dermis limit.

Methods: US images were obtained with an in‐house device (22 MHz probe) and the CM images with the VivaScope 1000 (Lucid Inc., Rochester, NY, USA). Skin from the dorsal forearm, the back of hand and the palm skin of 11 subjects (25–40 years) were examined. Repeatability of the procedure and reproducibility of the results were evaluated on repeated measurements taken at 1‐month interval.

Results: Both techniques are correlated. When a CM measurement is performed from the stratum corneum (SC) surface to the bottom of the papillae, results obtained with US and CM are very similar. Thus, the second echo‐rich line on US skin imaging is likely to reflect a virtual line joining the bottom of the papillae. CM is limited to the measurement of a relative thin epidermis, due to the signal‐to‐noise ratio, which decreases with depth. US technique offers a better repeatability and reproducibility, particularly for SC measurement. This is mainly due to the small size of the investigated field of view in CM.

Conclusion: This study confirms the accuracy of US and the feasibility of CM imaging techniques for in vivo epidermal thickness measurement. Echography probably measures a maximal epidermal thickness since it encompasses the bottom of the papillae.
Metabolic Activity of
Staphylococcus epidermidis
Is High during Initial and Low during Late Experimental Foreign-Body Infection
Authors: Vandecasteele Stefaan Johan , Peetermans Willy Eduard , Carbonez An , Van Eldere Johan
Published: April 2004

Foreign-body infection (FBI) is notoriously resistant to eradication by antibiotic treatment. It is hypothesized that reduced bacterial metabolic activity contributes to this resistance. We examined the metabolic activity of
Staphylococcus epidermidis
in 204 samples recovered during in vitro foreign-body colonization and in 424 samples recovered during in vivo FBI in a rat model. Metabolic activity was measured by determining the amount of 16S rRNA per genome by quantitative PCR. The initial foreign-body-associated growth proved to be a metabolically active process, both in vitro and in vivo. The initial 16S rRNA content was similar to that observed during in vitro exponential-growth phase. However, during late in vivo FBI, a 114-fold (
P
≪ 0.0001) decrease in the 16S rRNA content was observed, indicating that there was markedly decreased metabolic activity. This decreased metabolic activity during late FBI can explain at least in part why such infections are so difficult to eradicate with conventional antibiotic treatment.
TheHelicobacter pylori flaA1andwbpBGenes Control Lipopolysaccharide and Flagellum Synthesis and Function
Authors: Merkx-Jacques A. , Obhi R. K. , Bethune G. , Creuzenet C.
Published: April 2004

flaA1andwbpBare conserved genes with unknown biological function inHelicobacter pylori.Since both genes are predicted to be involved in lipopolysaccharide (LPS) biosynthesis, flagellum assembly, or protein glycosylation, they could play an important role in the pathogenesis ofH. pylori.To determine their biological role, both genes were disrupted in strain NCTC 11637. Both mutants exhibited altered LPS, with loss of most O-antigen and core modification, and increased sensitivity to sodium dodecyl sulfate compared to wild-type bacteria. These defects could be complemented in a gene-specific manner. Also,flaA1could complement these defects in thewbpBmutant, suggesting a potential redundancy of the reductase activity encoded by both genes. Both mutants were nonmotile, although thewbpBmutant still produced flagella. The defect in the flagellum functionality of this mutant was not due to a defect in flagellin glycosylation since flagellins from wild-type strain NCTC 11637 were shown not to be glycosylated. TheflaA1mutant produced flagellins but no flagellum. Overall, the similar phenotypes observed for both mutants and the complementation of thewbpBmutant byflaA1suggest that both genes belong to the same biosynthesis pathway. The data also suggest thatflaA1andwbpBare at the interface between several pathways that govern the expression of different virulence factors. We propose that FlaA1 and WbpB synthesize sugar derivatives dedicated to the glycosylation of proteins which are involved in LPS and flagellum production and that glycosylation regulates the activity of these proteins.
Transgenic Expression of RecA of the SpirochetesBorrelia burgdorferiandBorrelia hermsiiinEscherichia coliRevealed Differences in DNA Repair and Recombination Phenotypes
Authors: Putteet-Driver Adrienne D. , Zhong Jianmin , Barbour Alan G.
Published: April 2004

After unsuccessful attempts to recover a viable RecA-deficient mutant of the Lyme borreliosis agentBorrelia burgdorferi, we characterized the functional activities of RecA ofB. burgdorferi, as well as RecA of the relapsing fever spirocheteBorrelia hermsiiand the free-living spirocheteLeptospira biflexa, in arecAmutant ofEscherichia coli. As a control,E. coliRecA was expressed from the same plasmid vector. DNA damage repair activity was assessed after exposure of the transgenic cells to UV light or the radiomimetic chemicals methyl methanesulfonate and mitomycin C. Recombination activity in the cells was assessed by using an assay for homologous recombination between repeats in the chromosome and by measuring the ability of the cells to foster lytic growth byred gammutant bacteriophage λ. Overall, we found that transgenic cells withrecAgenes ofB. burgdorferi,B. hermsii, andL. biflexahad approximately equivalent activities in promoting homologous recombination in thelacZduplication assay, but cells withB. burgdorferi recAand, most notably,B. hermsii recAwere significantly less capable than cells withL. biflexa recAorE. coli recAin responding to DNA damage or in facilitating plaque formation in the phage assay. The comparatively poor function ofBorrelia recAin the latter set of assays may be the consequence of impaired coordination in the loading of the transgenic RecA by RecBCD and/or RecFOR inE. coli.
Pseudomonas aeruginosa
Autoinducer Enters and Functions in Mammalian Cells
Authors: Williams Simon C. , Patterson Erin K. , Carty Nancy L. , Griswold John A. , Hamood Abdul N. , Rumbaugh Kendra P.
Published: April 2004

Quorum sensing (QS) is a cell density-dependent signaling mechanism used by many bacteria to control gene expression. Several recent reports indicate that the signaling molecules (autoinducers) that mediate QS in
Pseudomonas aeruginosa
may also modulate gene expression in host cells; however, the mechanisms are largely unknown. Here we show that two
P. aeruginosa
autoinducers,
N
-3-oxododecanoyl-homoserine lactone and
N
-butyryl-homoserine lactone, can both enter eukaryotic cells and activate artificial chimeric transcription factors based on their cognate transcriptional activators, LasR and RhlR, respectively. The autoinducers promoted nuclear localization of chimeric proteins containing the full LasR or RhlR coding region, and the LasR-based proteins were capable of activating transcription of a LasR-dependent luciferase gene. Responsiveness to autoinducer required the N-terminal autoinducer-binding domains of LasR and RhlR. Truncated proteins consisting of only the C-terminal helix-turn-helix DNA-binding domains of both proteins attached to a nuclear localization signal efficiently translocated to the nucleus in the absence of autoinducer, and truncated LasR-based proteins functioned as constitutively active transcription factors. Chimeric LasR proteins were only activated by their cognate autoinducer ligand and not by
N
-butyryl-
l
-homoserine lactone. These data provide evidence that autoinducer molecules from human pathogens can enter mammalian cells and suggest that autoinducers may influence gene expression in host cells by interacting with and activating as-yet-unidentified endogenous proteins.
The Tp38 (TpMglB-2) Lipoprotein Binds Glucose in a Manner Consistent with Receptor Function in
Treponema pallidum
Authors: Deka Ranjit K. , Goldberg Martin S. , Hagman Kayla E. , Norgard Michael V.
Published: April 2004

A 38-kDa lipoprotein of
Treponema pallidum
(Tp38) was predicted to be a periplasmic sugar-binding protein based on its sequence similarity to the glucose/galactose-binding (MglB) protein of
Escherichia coli
(P. S. Becker, D. R. Akins, J. D. Radolf, and M. V. Norgard, Infect. Immun.
62:
1381-1391, 1994). Inasmuch as glucose is believed to be the principal, if not sole, carbon and energy source for
T. pallidum
and is readily available to the spirochete during its obligate infection of humans, we hypothesized that Tp38 may serve as the organism's requisite glucose receptor. For the present study, a nonacylated recombinant form of Tp38 was coexpressed with GroES and GroEL in
E. coli
to facilitate the isolation of soluble, properly folded Tp38. The highly sensitive method of intrinsic fluorescence spectroscopy, predicated on the manner in which tryptophan residues reside and move within protein microenvironments, was then used to assess sugar binding to Tp38. The intrinsic fluorescence of Tp38 was essentially unaltered when it was exposed to
d
-mannose,
d
-fucose,
d
-ribose,
l
-glucose, or
l
-galactose, but it changed markedly in the presence of
d
-glucose, and to a lesser extent,
d
-galactose, indicating binding. The

K
d

values for
d
-glucose and
d
-galactose binding to Tp38 were 152.2 ± 20.73 nM and 251.2 ± 55.25 nM, respectively. Site-directed mutagenesis of Trp-145, a residue postulated to contribute to the sugar-binding pocket in a manner akin to the essential Trp-183 in
E. coli
MglB, abolished Tp38's conformational change in response to
d
-glucose. The combined data are consistent with Tp38 serving as a glucose receptor for
T. pallidum
. These findings potentially have important implications for syphilis pathogenesis, particularly as they may pertain to glucose-mediated chemotactic responses by
T. pallidum
.
Relationship of Critical Temperature to Macromolecular Synthesis and Growth Yield in
Psychrobacter cryopegella
Authors: Bakermans Corien , Nealson Kenneth H.
Published: April 2004

Most microorganisms isolated from low-temperature environments (below 4°C) are eury-, not steno-, psychrophiles. While psychrophiles maximize or maintain growth yield at low temperatures to compensate for low growth rate, the mechanisms involved remain unknown, as does the strategy used by eurypsychrophiles to survive wide ranges of temperatures that include subzero temperatures. Our studies involve the eurypsychrophilic bacterium
Psychrobacter cryopegella
, which was isolated from a briny water lens within Siberian permafrost, where the temperature is −12°C.
P. cryopegella
is capable of reproducing from −10 to 28°C, with its maximum growth rate at 22°C. We examined the temperature dependence of growth rate, growth yield, and macromolecular (DNA, RNA, and protein) synthesis rates for
P. cryopegella.
Below 22°C, the growth of
P. cryopegella
was separated into two domains at the critical temperature (
T
critical
= 4°C). RNA, protein, and DNA synthesis rates decreased exponentially with decreasing temperatures. Only the temperature dependence of the DNA synthesis rate changed at
T
critical
. When normalized to growth rate, RNA and protein synthesis reached a minimum at
T
critical
, while DNA synthesis remained constant over the entire temperature range. Growth yield peaked at about
T
critical
and declined rapidly as temperature decreased further. Similar to some stenopsychrophiles,
P. cryopegella
maximized growth yield at low temperatures and did so by streamlining growth processes at
T
critical
. Identifying the specific processes which result in
T
critical
will be vital to understanding both low-temperature growth and growth over a wide range of temperatures.
Characterization of
Streptococcus pneumoniae
TrmD, a tRNA Methyltransferase Essential for Growth
Authors: O'Dwyer Karen , Watts Joseph M. , Biswas Sanjoy , Ambrad Jennifer , Barber Michael , Brulé Hervé , Petit Chantal , Holmes David J. , Zalacain Magdalena , Holmes Walter M.
Published: April 2004

Down-regulation of expression of
trmD
, encoding the enzyme tRNA (guanosine-1)-methyltransferase, has shown that this gene is essential for growth of
Streptococcus pneumoniae
. The
S. pneumoniae trmD
gene has been isolated and expressed in
Escherichia coli
by using a His-tagged T7 expression vector. Recombinant protein has been purified, and its catalytic and physical properties have been characterized. The native enzyme displays a molecular mass of approximately 65,000 Da, suggesting that streptococcal TrmD is a dimer of two identical subunits. In fact, this characteristic can be extended to several other TrmD orthologs, including
E. coli
TrmD. Kinetic studies show that the streptococcal enzyme utilizes a sequential mechanism. Binding of tRNA by gel mobility shift assays gives a dissociation constant of 22 nM for one of its substrates,

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathit{tRNA}_{\mathit{CAG}}^{\mathit{Leu}}\) \end{document}

. Other heterologous nonsubstrate tRNA species, like

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathit{tRNA}_{\mathit{GGT}}^{\mathit{Thr}}\) \end{document}

, tRNA
Phe
, and

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathit{tRNA}_{\mathit{TGC}}^{\mathit{Ala}}\) \end{document}

, bind the enzyme with similar affinities, suggesting that tRNA specificity is achieved via a postbinding event(s).
Properties of
Bacillus subtilis
σ
A
Factors with Region 1.1 and the Conserved Arg-103 at the N Terminus of Region 1.2 Deleted
Authors: Hsu Hsin-Hsien , Huang Wei-Cheng , Chen Jia-Perng , Huang Liang-Yin , Wu Chai-Fong , Chang Ban-Yang
Published: April 2004

σ factors in the σ
70
family can be classified into the primary and alternative σ factors according to their physiological functions and amino acid sequence similarities. The primary σ factors are composed of four conserved regions, with the conserved region 1 being divided into two subregions. Region 1.1, which is absent from the alternative σ factor, is poor in conservation; however, region 1.2 is well conserved. We investigated the importance of these two subregions to the function of
Bacillus subtilis
σ
A
, which belongs to a subgroup of the primary σ factor lacking a 254-amino-acid spacer between regions 1 and 2. We found that deletion of not more than 100 amino acid residues from the N terminus of σ
A
, which removed part or all region 1.1, did not affect the overall transcription activity of the truncated σ
A
-RNA polymerase in vitro, indicating that region 1.1 is not required for the functioning of σ
A
in RNA polymerase holoenzyme. This finding is consistent with the complementation data obtained in vivo. However, region 1.1 is able to negatively modulate the promoter DNA-binding activity of the σ
A
-RNA polymerase. Further deletion of the conserved Arg-103 at the N terminus of region 1.2 increased the content of stable secondary structures of the truncated σ
A
and greatly reduced the transcription activity of the truncated σ
A
-RNA polymerase by lowering the efficiency of transcription initiation after core binding of σ
A
. More importantly, the conserved Arg-103 was also demonstrated to be critical for the functioning of the full-length σ
A
in RNA polymerase.
Characterization of a Major
Bacillus anthracis
Spore Coat Protein and Its Role in Spore Inactivation
Authors: Kim Ho-San , Sherman D. , Johnson F. , Aronson A. I.
Published: April 2004

A major
Bacillus anthracis
spore coat protein of 13.4 kDa, designated Cotα, was found only in the
Bacillus cereus
group. A stable ca. 30-kDa dimer of this protein was also present in spore coat extracts. Cotα, which is encoded by a monocistronic gene, was first detected late in sporulation, consistent with a σ
K
-regulated gene. On the basis of immunogold labeling, the protein is in the outer spore coat and absent from the exosporium. In addition, disruption of the gene encoding Cotα resulted in spores lacking a dark-staining outer spore coat in thin-section electron micrographs. The mutant spores were stable upon heating or storage, germinated at the same rate as the wild type, and were resistant to lysozyme. They were, however, more sensitive than the wild type to phenol, chloroform, and hypochlorite but more resistant to diethylpyrocarbonate. In all cases, resistance or sensitivity to these reagents was restored by introducing a clone of the
cot
α gene into the mutant. Since Cotα is an abundant outer spore coat protein of the
B. cereus
group with a prominent role in spore resistance and sensitivity, it is a promising target for the inactivation of
B. anthracis
spores.
Characterization of Virulence Factor Regulation by SrrAB, a Two-Component System in
Staphylococcus aureus
Authors: Pragman Alexa A. , Yarwood Jeremy M. , Tripp Timothy J. , Schlievert Patrick M.
Published: April 2004

Workers in our laboratory have previously identified the staphylococcal respiratory response AB (SrrAB), a
Staphylococcus aureus
two-component system that acts in the global regulation of virulence factors. This system down-regulates production of
agr
RNAIII, protein A, and toxic shock syndrome toxin 1 (TSST-1), particularly under low-oxygen conditions. In this study we investigated the localization and membrane orientation of SrrA and SrrB, transcription of the
srrAB
operon, the DNA-binding properties of SrrA, and the effect of SrrAB expression on
S. aureus
virulence. We found that SrrA is localized to the
S. aureus
cytoplasm, while SrrB is localized to the membrane and is properly oriented to function as a histidine kinase.
srrAB
has one transcriptional start site which results in either an
srrA
transcript or a full-length
srrAB
transcript;
srrB
must be cotranscribed with
srrA
. Gel shift assays of the
agr
P2,
agr
P3, protein A (
spa
), TSST-1 (
tst
), and
srr
promoters revealed SrrA binding at each of these promoters. Analysis of SrrAB-overexpressing strains by using the rabbit model of bacterial endocarditis demonstrated that overexpression of SrrAB decreased the virulence of the organisms compared to the virulence of isogenic strains that do not overexpress SrrAB. We concluded that SrrAB is properly localized and oriented to function as a two-component system. Overexpression of SrrAB, which represses
agr
RNAIII, TSST-1, and protein A in vitro, decreases virulence in the rabbit endocarditis model. Repression of these virulence factors is likely due to a direct interaction between SrrA and the
agr
,
tst
, and
spa
promoters.
Population-Based Genetic and Evolutionary Analysis ofChlamydia trachomatisUrogenital Strain Variation in the United States
Authors: Millman Kim , Black Carolyn M. , Johnson Robert E. , Stamm Walter E. , Jones Robert B. , Hook Edward W. , Martin David H. , Bolan Gail , Tavaré Simon , Dean Deborah
Published: April 2004

Chlamydia trachomatisis a major cause of ocular and sexually transmitted diseases worldwide. While much of our knowledge about its genetic diversity comes from serotyping orompAgenotyping, no quantitative assessment of genetic diversity within serotypes has been performed. To accomplish this, 507 urogenital samples from a multicenter U.S. study were analyzed by phylogenetic and statistical modeling. No B, Da, or I serotypes were represented. Based on our analyses, all but one previous urogenital B serotype was identified as Ba. This, coupled with the lack of B serotypes in our population, suggests that B has specific tropism for ocular mucosa. We identified a Ba/D recombinant (putative crossover nucleotide 477;P< 0.0001) similar to a B/D mosaic we described previously from an African trachoma patient. Computational analyses of the Ba/D recombinant indicated that upstream changes were less important for tissue tropism than downstream incorporation of the D sequence. Since most serotypes had nonsynonymous/synonymous ratios of <1.0, the major outer membrane protein, encoded byompA, has many functional constraints and is under purifying selection. Surprisingly, all serotype groups except for J had a unimodal population structure indicating rapid clonal expansion. Of the groups with a unimodal structure, E and Ia and, to a lesser extent, G and K were prevalent, had infrequent incorporation of mutations, and, compared to other groups, had a relatively greater degree of diversifying selection, consistent with a selective sweep of mutations within these groups. Collectively, these data suggest a diverse evolutionary strategy for different serogroups of the organism.
Unraveling the Function of the
Rhodospirillum rubrum
Activator of Polyhydroxybutyrate (PHB) Degradation: the Activator Is a PHB-Granule-Bound Protein (Phasin)
Authors: Handrick Rene , Reinhardt Simone , Schultheiss Daniel , Reichart Thomas , Schüler Dirk , Jendrossek Verena , Jendrossek Dieter
Published: April 2004

Efficient hydrolysis of native poly(3-hydroxybutyrate) (nPHB) granules in vitro by soluble PHB depolymerase of
Rhodospirillum rubrum
requires pretreatment of nPHB with an activator compound present in
R. rubrum
cells (J. M. Merrick and M. Doudoroff, J. Bacteriol.
88:
60-71, 1964). Edman sequencing of the purified activator (17.4 kDa; matrix-assisted laser desorption ionization—time of flight mass spectrometry) revealed identity to a hypothetical protein deduced from a partially sequenced
R. rubrum
genome. The complete activator gene,
apdA
(activator of polymer degradation), was cloned from genomic DNA, expressed as a six-His-tagged protein in recombinant
Escherichia coli
(
M
r
, 18.3 × 10
3
), and purified. The effect of ApdA on PHB metabolism was studied in vitro and in vivo. In vitro, the activity of the activator could be replaced by trypsin, but recombinant ApdA itself had no protease activity. Comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein patterns of trypsin- and ApdA-treated nPHB granules isolated from different PHB-accumulating bacteria showed that trypsin activated nPHB by removing proteins of the surface layer of nPHB regardless of the origin of nPHB, but ApdA bound to and interacted with the surface layer of nPHB in a nonproteolytic manner, thereby transforming nPHB into an activated form that was accessible to the depolymerase. In vivo, expression of ApdA in
E. coli
harboring the PHB biosynthetic genes,
phaCBA
, resulted in significant increases in the number and surface/volume ratio of accumulated PHB granules, which was comparable to the effect of phasin proteins, such as PhaP in
Ralstonia eutropha
. The amino acid sequence of ApdA was 55% identical to the amino acid sequence of Mms16, a magnetosome-associated protein in magnetotactic
Magnetospirillum
species. Mms16 was previously reported to be a GTPase with an essential function in magnetosome formation (Y. Okamura, H. Takeyama, and T. Matsunaga, J. Biol. Chem.
276:
48183-48188, 2001). However, no GTPase activity of ApdA could be demonstrated. We obtained evidence that Mms16 of
Magnetospirillum gryphiswaldense
can functionally replace ApdA in
R. rubrum
. Fusions of
apdA
and
mms16
to
gfp
or
yfp
were functionally expressed, and both fusions colocalized with PHB granules after conjugative transfer to
R. rubrum
. In conclusion, ApdA in vivo is a PHB-bound, phasin-like protein in
R. rubrum.
The function of Mms16 in magnetotactic bacteria requires further clarification.
PhoP Can Activate Its Target Genes in a PhoQ-Independent Manner
Authors: Lejona Sergio , Castelli María Eugenia , Cabeza María Laura , Kenney Linda J. , García Véscovi Eleonora , Soncini Fernando C.
Published: April 2004

The PhoP/PhoQ two-component system controls the extracellular magnesium depletion response in
Salmonella enterica
. Previous studies have shown that PhoP is unable to up-regulate its target genes in the absence of PhoQ function. In this work, we demonstrate that PhoP overexpression can substitute for PhoQ- and phosphorylation-dependent activation. Either a high concentration of PhoP or activation via phosphorylation stimulates PhoP self-association.
Novel
rpoB
Mutations Conferring Rifampin Resistance on
Bacillus subtilis
: Global Effects on Growth, Competence, Sporulation, and Germination
Authors: Maughan Heather , Galeano Belinda , Nicholson Wayne L.
Published: April 2004

Previously, spontaneous rifampin resistance mutations were isolated in cluster I of the
rpoB
gene, resulting in amino acid replacements (Q469R, H482R, H482Y, or S487L) in the
Bacillus subtilis
RNA polymerase β subunit (W. L. Nicholson and H. Maughan, J. Bacteriol.
184:
4936-4940, 2002). In this study, each amino acid change in the β subunit was observed to result in its own unique spectrum of effects on growth and various developmental events, including sporulation, germination, and competence for transformation. The results thus establish the important role played by the RNA polymerase β subunit, not only in the catalytic aspect of transcription, but also in the regulation of major developmental events in
B. subtilis
.
Analysis of an Engineered
Salmonella
Flagellar Fusion Protein, FliR-FlhB
Authors: Van Arnam John S. , McMurry Jonathan L. , Kihara May , Macnab Robert M.
Published: April 2004

Salmonella
FliR and FlhB are membrane proteins necessary for flagellar export. In
Clostridium
a
fliR-flhB
fusion gene exists. We constructed a similar
Salmonella
fusion gene which is able to complement
fliR
,
flhB
, and
fliR flhB
null strains. Western blotting revealed that the FliR-FlhB fusion protein retains the FlhB protein's cleavage properties. We conclude that the FliR and FlhB proteins are physically associated in the wild-type
Salmonella
basal body, probably in a 1:1 ratio.
Interplay between Plasmid Partition and Postsegregational Killing Systems
Authors: Brendler Therese , Reaves Lucretia , Austin Stuart
Published: April 2004

Active partition systems and postsegregational killing (PSK) systems are present together in naturally occurring low-copy-number plasmids. Theory suggests that PSK may act as the ultimate determinant of plasmid retention, whereas the partition system may minimize the growth penalty to the host, resulting in a near-ideal symbiosis when the systems combine. Here, we prove the validity of this principle for a specific case involving the P1
par
system and the
mvp
PSK system.
Control of Expression of the Arginine Deiminase Operon ofStreptococcus gordoniiby CcpA and Flp
Authors: Dong Yiqian , Chen Yi-Ywan M. , Burne R. A.
Published: April 2004

InStreptococcus gordoniiDL1, inactivation of theccpAgene and a gene encoding an Fnr-like protein (Flp) demonstrated that CcpA was essential for carbohydrate catabolite repression and that Flp was required for optimal expression and anaerobic induction of the arginine deiminase system.
Physical mapping of barley genes using an ultrasensitive fluorescence in situ hybridization technique
Authors: Stephens J L , Brown S E , Lapitan N L.V , Knudson D L
Published: April 2004

The primary objective of this study was to elucidate gene organization and to integrate the genetic linkage map for barley (Hordeum vulgare L.) with a physical map using ultrasensitive fluorescence in situ hybridization (FISH) techniques for detecting signals from restriction fragment length polymorphism (RFLP) clones. In the process, a single landmark plasmid, p18S5Shor, was constructed that identified and oriented all seven of the chromosome pairs. Plasmid p18S5Shor was used in all hybridizations. Fourteen cDNA probes selected from the linkage map for barley H. vulgare 'Steptoe' × H. vulgare 'Morex' (Kleinhofs et al. 1993) were mapped using an indirect tyramide signal amplification technique and assigned to a physical location on one or more chromosomes. The haploid barley genome is large and a complete physical map of the genome is not yet available; however, it was possible to integrate the linkage map and the physical locations of these cDNAs. An estimate of the ratio of base pairs to centimorgans was an average of 1.5 Mb/cM in the distal portions of the chromosome arms and 89 Mb/cM near the centromere. Furthermore, while it appears that the current linkage maps are well covered with markers along the length of each arm, the physical map showed that there are large areas of the genome that have yet to be mapped.Key words: Hordeum vulgare, barley, physical mapping, FISH, cDNA, genetics, linkage, chromosome, BACs.
Search for methylation-sensitive amplification polymorphisms associated with the "mantled" variant phenotype in oil palm (Elaeis guineensis Jacq.)
Authors: Jaligot E , Beulé T , Baurens F-C , Billotte N , Rival A
Published: April 2004

The methylation-sensitive amplification polymorphism (MSAP) technique has been employed on somatic embryo-derived oil palms (Elaeis guineensis Jacq.) to identify methylation polymorphisms correlated with the "mantled" somaclonal variation. The variant phenotype displays an unstable feminization of male organs in both male and female flowers. Using MSAP, the methylation status of CCGG sites was compared in three normal versus three mantled regenerants sampled in clonal populations obtained through somatic embryogenesis from four genotypically distinct mother palms. Overall, 64 selective primer combinations were used and they have amplified 23 markers exhibiting a differential methylation pattern between the two phenotypes. Our results indicate that CCGG sites are poorly affected by the considerable decrease in global DNA methylation that has been previously associated with the mantled phenotype. Each of the 23 markers isolated in the present study could discriminate between the two phenotypes only when they were from the same genetic origin. This result hampers at the moment the direct use of MSAP markers for the early detection of variants, even though valuable information on putative target sequences will be obtained from a further characterization of these polymorphic markers.Key words: DNA methylation, epigenetics, MSAP, oil palm, somaclonal variation.
Genetic diversity ofEchinaceaspecies based upon amplified fragment length polymorphism markers
Authors: Kim D -H , Heber D , Still D W
Published: April 2004

The taxonomy of Echinacea is based on morphological characters and has varied depending on the monographer. The genus consists of either nine species and four varieties or four species and eight varieties. We have used amplified fragment length polymorphisms (AFLP) to assess genetic diversity and phenetic relationships among nine species and three varieties of Echinacea (sensu McGregor). A total of 1086 fragments, of which approximately 90% were polymorphic among Echinacea taxa, were generated from six primer combinations. Nei and Li's genetic distance coefficient and the neighbor-joining algorithm were employed to construct a phenetic tree. Genetic distance results indicate that all Echinacea species are closely related, and the average pairwise distance between populations was approximately three times the intrapopulation distances. The topology of the neighbor-joining tree strongly supports two major clades, one containing Echinacea purpurea, Echinacea sanguinea, and Echinacea simulata and the other containing the remainder of the Echinacea taxa (sensu McGregor). The species composition within the clades differs between our AFLP data and the morphometric treatment offered by Binns and colleagues. We also discuss the suitability of AFLP in determining phylogenetic relationships.Key words: Echinacea, AFLP, genetic distance, phylogeny.
Genetic characterization of a novel Tib-derived variant of soybean Kunitz trypsin inhibitor detected in wild soybean (Glycine soja)
Authors: Wang Ke-Jing , Yamashita Tetsuro , Watanabe Masao , Takahata Yoshihito
Published: April 2004

A novel variant of soybean Kunitz trypsin inhibitor (SKTI) was detected in 530 lines of wild soybean (Glycine soja). This variant showed an intermediate electrophoretic mobility between the Tia and Tic types. In isoelectric focusing polyacrylamide gel electrophoresis gels containing urea, this variant had a similar isoelectric point as that of Tia. The genetic analysis of SKTI bands in F2 seeds from crosses of the new variant type with Tia or Tic type showed that this variant type is controlled by a codominant allele at the SKTI locus. We propose the genetic symbol Tif for this novel variant. When the nucleotide sequence of the Tif gene was compared with those of other types of SKTI genes (Tia, Tib, and Tic), the sequence of Tif was identical to that of Tib with the exception of one A[Formula: see text]G transitional mutation occurring at position 676 of Tif. This mutation resulted in an amino acid change from Lys to Glu at the 178 residue. These results suggest that this variant is derived from Tib through a point mutation. In addition, we settled an inconsistency in the number of amino acid differences between Tia and Tib (eight or nine). Analysis of nucleotide and amino acid sequences revealed that Tib was different from Tia by nine amino acids.Key words: soybean Kunitz trypsin inhibitor, polymorphism, gene sequence, soybean, wild soybean.