bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural basis for stereo‐specific catalysis in NAD+‐dependent (R)‐2‐hydroxyglutarate dehydrogenase from Acidaminococcus fermentans
Authors: Martins Berta M. , Macedo‐Ribeiro Sandra , Bresser Julia , Buckel Wolfgang , Messerschmidt Albrecht
Published: December 2004

NAD+‐dependent (R)‐2‐hydroxyglutarate dehydrogenase (HGDH) catalyses the reduction of 2‐oxoglutarate to (R)‐2‐hydroxyglutarate and belongs to the d‐2‐hydroxyacid NAD+‐dependent dehydrogenase (d‐2‐hydroxyacid dehydrogenase) protein family. Its crystal structure was determined by phase combination to 1.98 Å resolution. Structure–function relationships obtained by the comparison of HGDH with other members of the d‐2‐hydroxyacid dehydrogenase family give a chemically satisfying view of the substrate stereoselectivity and catalytic requirements for the hydride transfer reaction. A model for substrate recognition and turnover is discussed. The HGDH active site architecture is structurally optimized to recognize and bind the negatively charged substrate 2‐oxoglutarate. The structural position of the side chain of Arg52, and its counterparts in other family members, strongly correlates with substrate specificity towards substitutions at the C3 atom (linear or branched substrates). Arg235 interacts with the substrate's α‐carboxylate and carbonyl groups, having a dual role in both substrate binding and activation, and the γ‐carboxylate group can dock at an arginine cluster. The proton‐relay system built up by Glu264 and His297 permits His297 to act as acid–base catalyst and the 4Re‐hydrogen from NADH is transferred as hydride to the carbonyl group Si‐face leading to the formation of the correct enantiomer (R)‐2‐hydroxyglutarate.
Changes in lung function and tidal airflow patterns after increasing extrathoracic airway resistance
Authors: MORRIS M. Jocelyn , WILLIAMS E. Mark , MADGWICK Richard , BANERJEE Rajarshi , PHILLIPS Emma
Published: December 2004

Objective:  The aim of this study was to determine what changes occur in previously described tidal expiratory flow patterns when extrathoracic resistance is added.

Methodology:  A total of 16 subjects with normal lung function and 15 patients with chronic COPD, were studied. The following measurements were made before and after the addition of an extrathoracic expiratory resistance (EER; 0.89 kPa/L s at a flow of 0.5 L/s) during uncoached tidal breathing: expiratory time (tE), frequency (f), expiratory time to maximum flow/total expiratory time (tPTEF/tE), tidal maximum expiratory flow (PEFTIDAL), and time constant of the respiratory system calculated from the passive portion of tidal expiration (trsTIDAL). Post peak profiles were categorized as convex, linear or concave and time for flow to fall to 80% and 20% of maximum (t80 and t20) were measured.

Results:  Tidal indices tE, f and PEFTIDAL, were similar in the two groups, but tPTEF/tE was greater and trsTIDAL was less in the normal subjects than in the COPD patients. Addition of EER in both groups caused PEFTIDAL to decrease and trsTIDAL, t80 and the percentage of convex profiles to increase. tPTEF/tE increased in the COPD patients but was unchanged in the control group.

Conclusion:  After addition of EER during tidal breathing, expiratory flow in COPD patients resembled that seen in normal subjects.
Acute mountain sickness is associated with sleep desaturation at high altitude
Authors: BURGESS Keith R. , JOHNSON Pamela , EDWARDS Natalie , COOPER Jackie
Published: December 2004

Objective:  This study was intended to demonstrate a biologically important association between acute mountain sickness (AMS) and sleep disordered breathing.

Methodology:  A total of 14 subjects (eight males, six females aged 36 ± 10 years) were studied at six different altitudes from sea level to 5050 m over 12 days on a trekking route in the Nepal Himalaya. AMS was quantified by Lake Louise (LL) score. At each altitude, sleep was studied by 13 channel polysomnography (PSG). Resting arterial blood gases (ABG) and exercise SaO2 were measured. Ventilatory responses (VR) were measured at sea level. Individual data were analysed for association at several altitudes and mean data were analysed for association over all altitudes.

Results:  ABG showed partial acclimatization. For the mean data, there were strong positive correlations between LL score and altitude, and periodic breathing, as expected. Strong negative correlations existed between LL score and PaO2, PaCO2, sleep SaO2 and exercise SaO2, but there was no correlation with sea level VR. There were equally tight correlations between LLs/PaO2 and LL score/sleep SaO2.

The individual data showed no significant correlations with LL score at any altitude, probably reflecting the non‐steady state nature of the experiment. In addition, mean SaO2 during sleep was similar to minimum exercise SaO2 at each altitude and minimum sleep SaO2 was lower, suggesting that the hypoxic insult during sleep was equivalent to or greater than walking at high altitude.

Conclusions:  It is concluded that desaturation during sleep has a biologically important association with AMS, and it is speculated that under similar conditions (trekking) it is an important cause of AMS.
COPD in Japan: the Nippon COPD Epidemiology study
Authors: FUKUCHI Yoshinosuke , NISHIMURA Masaharu , ICHINOSE Masakazu , ADACHI Mitsuru , NAGAI Atsushi , KURIYAMA Takayuki , TAKAHASHI Keiji , NISHIMURA Koichi , ISHIOKA Shinichi , AIZAWA Hisamichi , ZAHER Carol
Published: December 2004

Objectives:  Despite high smoking rates, few prevalence studies of COPD have been performed in Asia. The Nippon COPD Epidemiology (NICE) Study used spirometry to measure prevalence of airflow limitation in Japanese adults.

Methodology:  Clinical, spirometric, and risk factor exposure data were collected on 2343 subjects aged ≥ 40 years who were demographically similar to the Japanese population. Airflow limitation was defined according to Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria (FEV1/FVC < 70%).

Results:  Prevalence of airflow limitation was 10.9%. Based upon GOLD severity criteria, 56% of these cases were found to be mild, 38% moderate, 5% severe, and 1% very severe. Airflow limitation was significantly more prevalent in males than females (16.4% vs. 5.0%; P < 0.001), in male ever‐smokers than female ever‐smokers (17.1% vs. 7.5%; P < 0.001), and in older subjects (3.5% in 40–49 years olds vs. 24.4% in those > 70 years; P < 0.001). Of note, airflow limitation was also found in 5.8% of non‐smokers and 4.6% of those younger than age 60 years. Only 9.4% of cases with airflow limitation reported a previous diagnosis of COPD.

Conclusions:  Prevalence of airflow limitation in Japan is higher than previously reported, suggesting a high degree of under‐recognition of COPD. The high prevalence of smoking coupled with an aging population threatens to further increase the burden of COPD, highlighting the need for enhanced screening efforts and interventions of prevention and treatment.
Confronting a biome crisis: global disparities of habitat loss and protection
Authors: Hoekstra Jonathan M. , Boucher Timothy M. , Ricketts Taylor H. , Roberts Carter
Published: December 2004

Human impacts on the natural environment have reached such proportions that in addition to an ‘extinction crisis’, we now also face a broader ‘biome crisis’. Here we identify the world's terrestrial biomes and, at a finer spatial scale, ecoregions in which biodiversity and ecological function are at greatest risk because of extensive habitat conversion and limited habitat protection. Habitat conversion exceeds habitat protection by a ratio of 8 : 1 in temperate grasslands and Mediterranean biomes, and 10 : 1 in more than 140 ecoregions. These regions include some of the most biologically distinctive, species rich ecosystems on Earth, as well as the last home of many threatened and endangered species. Confronting the biome crisis requires a concerted and comprehensive response aimed at protecting not only species, but the variety of landscapes, ecological interactions, and evolutionary pressures that sustain biodiversity, generate ecosystem services, and evolve new species in the future.
Generalized Linear Mixed Models for Ordered Responses in Complex Multilevel Structures: Effects Beneath the School or College in Education
Authors: Fielding Antony , Yang Min
Published: December 2004

The complexities of educational processes and structure and the need for disentangling effects beneath the level of the school or college are discussed. Ordinal response multilevel crossed random-effects models for educational grades are introduced. Weighted random effects for teacher contributions are then added. Estimation methodology is reviewed. Specially written macros for quasi-likelihood with second-order terms are described. The application discusses General Certificate of Education at advanced level grades cross-classified by student and teaching group within a number of institutions. The methods handle teacher effects where several teachers contribute to provision and where each teacher deals with several groups. Some methodological lessons are drawn for sparse data and the use of extra-multinomial variation. Developments of the analysis yield conclusions about the sources of variation in educational progress, and particularly the effect of teachers.
Releasing Multiply Imputed, Synthetic Public use Microdata: An Illustration and Empirical Study
Authors: Reiter Jerome P.
Published: December 2004

The paper presents an illustration and empirical study of releasing multiply imputed, fully synthetic public use microdata. Simulations based on data from the US Current Population Survey are used to evaluate the potential validity of inferences based on fully synthetic data for a variety of descriptive and analytic estimands, to assess the degree of protection of confidentiality that is afforded by fully synthetic data and to illustrate the specification of synthetic data imputation models. Benefits and limitations of releasing fully synthetic data sets are discussed.
Multiple‐Choice Tests and Student Understanding: What Is the Connection?
Authors: Simkin Mark G. , Kuechler William L.
Published: December 2004

Instructors can use both “multiple‐choice” (MC) and “constructed response” (CR) questions (such as short answer, essay, or problem‐solving questions) to evaluate student understanding of course materials and principles. This article begins by discussing the advantages and concerns of using these alternate test formats and reviews the studies conducted to test the hypothesis (or perhaps better described as the hope) that MC tests, by themselves, perform an adequate job of evaluating student understanding of course materials. Despite research from educational psychology demonstrating the potential for MC tests to measure the same levels of student mastery as CR tests, recent studies in specific educational domains find imperfect relationships between these two performance measures. We suggest that a significant confound in prior experiments has been the treatment of MC questions as homogeneous entities when in fact MC questions may test widely varying levels of student understanding. The primary contribution of the article is a modified research model for CR/MC research based on knowledge‐level analyses of MC test banks and CR question sets from basic computer language programming. The analyses are based on an operationalization of Bloom's Taxonomy of Learning Goals for the domain, which is used to develop a skills‐focused taxonomy of MC questions. However, we propose that their analyses readily generalize to similar teaching domains of interest to decision sciences educators such as modeling and simulation programming.
Within‐Class Variability in Student–Teacher Evaluations: Examples and Problems
Authors: Clayson Dennis E.
Published: December 2004

Although student evaluation of instruction has been shown to produce reliable results over class averages, considerable within‐class variability exists that has not been investigated. This study looked at examples of student evaluations in which students diametrically differed in their evaluation of the same instructor. Patterns were noted. A survey was conducted to determine the self‐reported incident of students changing evaluations based on noninstructional factors. About 60% of students admitted that they had changed evaluations for reasons related to the instructor's personality, while 30% admitted to changing evaluations based on grades and rigor. Possible reasons and effects of such changes are discussed.
An experimental model to demonstrate the carcinogenic action of oral chronic traumatic ulcer
Authors: Pérez Miguel Angel , Raimondi Ana Rosa , Itoiz María Elina
Published: December 2004

Background:  Oral chronic traumatic ulcer (CTU) is caused by constant irritation by dental edges or restorations and could behave as a pre‐malignant lesion in a field initiated by carcinogens such as tobacco or alcohol.

Methods:  We developed an experimental model in the hamster cheek pouch, combining the chemical carcinogen 7,12‐dimethyl‐benzanthracene (DMBA) with CTU.

Results:  The successive or simultaneous action of both agents induced a significantly larger number of endophytic carcinomas than larger doses of DMBA alone. CTU alone failed to induce tumor development. Ploidy analysis revealed significantly higher malignancy indices in endophytic than in exophytic carcinomas. 5‐Bromo‐2‐deoxyuridine labeling evidenced greater proliferation around the ulcers in chemically cancerized epithelium than around ulcers in healthy epithelium.

Conclusions:  The results show that CTU acts as a tumor promoter in this model. This finding is clinically relevant in that CTU may increase the risk of malignant transformation in patients with subclinical tumor initiation.
MMP‐3 and ‐8 expression is found in the condylar surface of temporomandibular joints with internal derangement
Authors: Tiilikainen P. , Pirttiniemi P. , Kainulainen T. , Pernu H. , Raustia A.
Published: December 2004

Background:  Internal derangement is one of the most common disorder of the temporomandibular joint (TMJ). The aim of this study was to investigate the associations of matrix metalloproteinase (MMP)‐3 and ‐8 expression in articular condylar surface with different stages of TMJ internal derangement according to Wilkes (Minn Med, 1978; 61: 645–52) and osteoarthrosis (OA) according to Dijkgraaf et al. (J Oral Maxillofac Surg, 1995; 53: 1182–92).

Methods:  The study was based on 54 condylar specimens obtained during TMJ surgery. Immunohistochemistry using antibodies specific to MMP‐3 and ‐8, represented in cartilage destruction, was carried out.

Results:  In all tissue specimens, MMP‐3 expression was intense in the surface layer but showed less intensive staining in the deeper layers. Some MMP‐8 expression was also seen. The severity of TMJ internal derangement, however, did not seem to have a statistically significant correlation (P < 0.05) with the expression of these enzymes.

Conclusion:  The study confirms that distinct expression of MMP‐3 and ‐8 is found in the condylar surface of TMJs with internal derangement.
Clinical and immunological effects of azithromycin in Behçet's disease
Authors: Mumcu G. , Ergun T. , Elbir Y. , Eksioglu‐Demiralp E. , Yavuz S. , Atalay T. , Direskeneli H.
Published: December 2004

Background:  The aim of this study was to evaluate the effects of azithromycin on mucocutaneous manifestations, oral health and immune response in Behçet's disease (BD).

Methods:  Eight BD patients with active mucocutaneous symptoms were treated with azithromycin for 4 weeks. Oral health, clinical manifestations and in vitro interleukin (IL)‐12, interferon (IFN)‐γ, IL‐10 and monocyte chemotactic protein (MCP)‐1 responses were evaluated before and after treatment.

Results:  The number of folliculitic lesions, healing time of oral ulcers and scores of plaque indexes (PLIs) were lower after azithromycin treatment (P < 0.05). Scores of PLIs correlated positively with the healing time of oral ulcers (P = 0.02). Although a trend towards increased stimulated IL‐10 responses with azithromycin was observed, no statistically significant difference was found. Stimulated and unstimulated MCP‐1, IFN‐γ and IL‐12 responses were similar before and after treatment (P > 0.05).

Conclusion:  Azithromycin was observed to be effective in decreasing folliculitic lesions and fastening the healing time of oral ulcers in BD.
The Economic Effects of Immigration into the United Kingdom
Authors: Coleman David , Rowthorn Robert
Published: December 2004

This article is concerned with the economic effects of immigration. The emphasis is on Britain, but extensive material is also provided on other countries. Since 1997 a new British immigration policy has displaced previous policy aims, which were focused on minimizing settlement. Large‐scale immigration is now seen as essential for Britain's economic well‐being, and measures have been introduced to increase inflows. The benefits claimed include fiscal advantages, increased prosperity, a ready supply of labor, and improvements to the age structure. Fears that large‐scale immigration might damage the interests of unskilled workers are discounted. This article examines these claims. It concludes that the economic consequences of large‐scale immigration are mostly minor, negative, or transient, that the interests of more vulnerable sections of the domestic population may well be damaged, and that any economic benefits are unlikely to bear comparison with immigration's probable substantial and permanent demographic and environmental impact. Our claims are in line with those from other developed countries.
Navigating the AIDS Epidemic in Rural Malawi
Authors: Watkins Susan Cotts
Published: December 2004

This article provides a perspective on rural Malawi during the unsettled times following the recognition by village residents that AIDS is a profound danger. The primary data are observational field journals in which local ethnographers wrote their recollections of conversations about AIDS that they overheard or participated in during the course of their daily lives. In their networks of friends, relatives, and neighbors, rural Malawians are publicly discussing a range of strategies of prevention. These strategies range from the abstinence, fidelity, and consistent condom use prescribed by international and national experts to innovative strategies of partner selection, divorce, and renewed religious commitment. The article also considers the effectiveness of these strategies and speculates about the applicability of the findings to other populations.
Religion as a Determinant of Economic and Demographic Behavior in the United States
Authors: Lehrer Evelyn L.
Published: December 2004

This note reviews and synthesizes research on the effects of religion on various economic and demographic behaviors of individuals and families in the United States, including the choice of marital partner, union formation and dissolution, fertility, female time allocation, education, wages, and wealth. Using a theoretical framework based on Gary Becker's contributions to the economics of the family, it demonstrates that religious affiliation affects economic and demographic behavior because of its impact on the costs and benefits of many interrelated decisions that people make over the life cycle. In addition, for behaviors that pertain to married‐couple households, religious affiliation matters because it is a complementary trait within the context of marriage. Religiosity, another dimension of religion, also affects economic and demographic outcomes, partly because it accentuates differences by religious affiliation, partly because religious involvement has generally beneficial effects on health and well‐being.
2004 Japanese Society for Dialysis Therapy Guidelines for Renal Anemia in Chronic Hemodialysis Patients*
Authors: Gejyo Fumitake , Saito Akira , Akizawa Tadao , Takashi Akiba  , Sakai Tatsuya , Suzuki Masashi , Nishi Shinichi , Tsubakihara Yoshiharu , Hirakata Hideki , Bessho Masami
Published: December 2004

Abstract:  The guideline committee of Japanese Society for Dialysis Therapy (JSDT), chaired by Professor F. Gejyo of Niigata University, now publishes an original Japanese guideline entitled ‘Guidelines for Renal Anemia in Chronic Hemodialysis Patients’. It includes the re‐evaluation of the usage of recombinant human erythropoietin (rHuEPO) with the medical and economical arguments regarding the prognosis and the quality of life of Japanese hemodialysis patients. This guideline consists of 7 sections. The first section comprises the general definition and the differential diagnosis of anemia. The hemoglobin (Hb) level of the Japanese population seemed to be low when compared with that of the European and American populations. The second section describes the target Hb level in hemodialysis patients. Multivariate analysis of the data that were collected from dialysis institutions throughout the country showed that an Hb level of 10–11 g/dL (Ht level 30–33%) at the first dialysis session in a week is the ideal range for chronic hemodialysis patients in terms of the 3–5 year survival rate. The supine position at blood sampling and the sampling timing at the first dialysis session in a week might affect the lower setting of target Hb hematocrit (Ht), compared to that of European and American guidelines. However, we particularly recommended that an Hb level of 11–12 g/dL (Ht level from 33 to 36%) at the first dialysis session in a week is desirable in relatively young patients. In the third section, the markers of iron deficiency are discussed. The Transferin saturation test (TSAT) and serum ferritin were emphasized as the standard markers. The routes of administration of rHuEPO and its dosages are written in the fourth section. The subcutaneous route was associated with the occurrence of secondary red cell aplasia due to anti‐rHuEPO antibodies; however, secondary red cell aplasia was seldom observed in the venous injection. From this fact we recommend venous injection for chronic hemodialysis patients. We advocate an initial dosage of 1500 U three times per week. The fifth section deals with the factors refractory to treatment with rHuEPO. If the patient shows an inadequate response to the usage of 9000 U per week, this condition defines the inadequate response to rHuEPO in Japan. Blood transfusion must be avoided where possible. The reasons for this and the adverse effects are interpreted in section six. In the final section, the adverse effects of rHuEPO are listed. Among them, hypertension, thrombotic events and secondary red cell aplasia were emphasized as the major complications.
Lenz’s law and dimensional analysis
Authors: Pelesko John A. , Cesky Michael , Huertas Sharon
Published: December 2004

We show that the time it takes a magnet to fall through a nonmagnetic metallic tube may be found via dimensional analysis. The simple analysis makes this classic demonstration of Lenz’s law accessible qualitatively and quantitatively to students with little knowledge of electromagnetism and only elementary knowledge of calculus. The analysis provides a new example of the power and limitations of dimensional analysis.
Determining dielectric constants using a parallel plate capacitor
Authors: Grove T. T. , Masters M. F. , Miers R. E.
Published: December 2004

The availability of low cost digital multimeters capable of measuring capacitance has made parallel plate capacitor investigations common in the introductory laboratory. Typically, students add multiple thin sheets of dielectric material between conducting plates. The capacitance is measured and then plotted versus the reciprocal of the dielectric thickness (the nominal plate separation). We explain why the experiment fails for small dielectric thickness and discuss an improved version of the experiment using dielectric sheets of various thicknesses rather than multiple thin sheets of a dielectric.
Strong-field approximation in laser-assisted dynamics
Authors: Madsen Lars Bojer
Published: December 2004

When electrons, atoms, and molecules are in a strong laser field, the wave function of the continuum electron plays an important role. Nonperturbative results for the cross-section for laser-assisted scattering and the laser-assisted photoelectric effect are obtained using the Volkov wave function of a free electron in a laser field. Also derived is the rate for strong-field electron detachment from negative ions.
Discovery of a Major D-Loop Replication Origin Reveals Two Modes of Human mtDNA Synthesis
Authors: Fish Jennifer , Raule Nicola , Attardi Giuseppe
Published: December 2004

Mammalian mitochondrial DNA (mtDNA) replication has long been considered to occur by asymmetric synthesis of the two strands, starting at the multiple origins of the strand-displacement loop (D-loop). We report the discovery of a major replication origin at position 57 in the D-loop of several human cell lines (HeLa, A549, and 143B.TK
–
) and immortalized lymphocytes. The nascent chains starting at this origin, in contrast to those initiated at the previously described origins, do not terminate prematurely at the 3′ end of the D-loop but proceed well beyond this control point, behaving as “true” replicating strands. This origin is mainly responsible for mtDNA maintenance under steady-state conditions, whereas mtDNA synthesis from the formerly identified D-loop origins may be more important for recovery after mtDNA depletion and for accelerating mtDNA replication in response to physiological demands.
Mammalian Tissue Oxygen Levels Modulate Iron-Regulatory Protein Activities in Vivo
Authors: Meyron-Holtz Esther G. , Ghosh Manik C. , Rouault Tracey A.
Published: December 2004

The iron-regulatory proteins (IRPs) posttranscriptionally regulate expression of transferrin receptor, ferritin, and other iron metabolism proteins. Although both IRPs can regulate expression of the same target genes, IRP2
–/–
mice significantly misregulate iron metabolism and develop neurodegeneration, whereas IRP1
–/–
mice are spared. We found that IRP2
–/–
cells misregulated iron metabolism when cultured in 3 to 6% oxygen, which is comparable to physiological tissue concentrations, but not in 21% oxygen, a concentration that activated IRP1 and allowed it to substitute for IRP2. Thus, IRP2 dominates regulation of mammalian iron homeostasis because it alone registers iron concentrations and modulates its RNA-binding activity at physiological oxygen tensions.
Building Programmable Jigsaw Puzzles with RNA
Authors: Chworos Arkadiusz , Severcan Isil , Koyfman Alexey Y. , Weinkam Patrick , Oroudjev Emin , Hansma Helen G. , Jaeger Luc
Published: December 2004

One challenge in supramolecular chemistry is the design of versatile, self-assembling building blocks to attain total control of arrangement of matter at a molecular level. We have achieved reliable prediction and design of the three-dimensional structure of artificial RNA building blocks to generate molecular jigsaw puzzle units called tectosquares. They can be programmed with control over their geometry, topology, directionality, and addressability to algorithmically self-assemble into a variety of complex nanoscopic fabrics with predefined periodic and aperiodic patterns and finite dimensions. This work emphasizes the modular and hierarchical characteristics of RNA by showing that small RNA structural motifs can code the precise topology of large molecular architectures. It demonstrates that fully addressable materials based on RNA can be synthesized and provides insights into self-assembly processes involving large populations of RNA molecules.
Autolysis of
Lactococcus lactis
Is Increased upon
d
-Alanine Depletion of Peptidoglycan and Lipoteichoic Acids
Authors: Steen Anton , Palumbo Emmanuelle , Deghorain Marie , Cocconcelli Pier Sandro , Delcour Jean , Kuipers Oscar P. , Kok Jan , Buist Girbe , Hols Pascal
Published: December 2004

Mutations in the genes encoding enzymes responsible for the incorporation of
d
-Ala into the cell wall of
Lactococcus lactis
affect autolysis. An
L. lactis
alanine racemase (
alr
) mutant is strictly dependent on an external supply of
d
-Ala to be able to synthesize peptidoglycan and to incorporate
d
-Ala in the lipoteichoic acids (LTA). The mutant lyses rapidly when
d
-Ala is removed at mid-exponential growth. AcmA, the major lactococcal autolysin, is partially involved in the increased lysis since an
alr acmA
double mutant still lyses, albeit to a lesser extent. To investigate the role of
d
-Ala on LTA in the increased cell lysis, a
dltD
mutant of
L. lactis
was investigated, since this mutant is only affected in the
d
-alanylation of LTA and not the synthesis of peptidoglycan. Mutation of
dltD
results in increased lysis, showing that
d
-alanylation of LTA also influences autolysis. Since a
dltD acmA
double mutant does not lyse, the lysis of the
dltD
mutant is totally AcmA dependent. Zymographic analysis shows that no degradation of AcmA takes place in the
dltD
mutant, whereas AcmA is degraded by the extracellular protease HtrA in the wild-type strain. In
L. lactis
, LTA has been shown to be involved in controlled (directed) binding of AcmA. LTA lacking
d
-Ala has been reported in other bacterial species to have an improved capacity for autolysin binding. Mutation of
dltD
in
L. lactis
, however, does not affect peptidoglycan binding of AcmA; neither the amount of AcmA binding to the cells nor the binding to specific loci is altered. In conclusion,
d
-Ala depletion of the cell wall causes lysis by two distinct mechanisms. First, it results in an altered peptidoglycan that is more susceptible to lysis by AcmA and also by other factors, e.g., one or more of the other (putative) cell wall hydrolases expressed by
L. lactis
. Second, reduced amounts of
d
-Ala on LTA result in decreased degradation of AcmA by HtrA, which results in increased lytic activity.
Regulation of thePseudomonassp. Strain ADP Cyanuric Acid Degradation Operon
Authors: García-González Vicente , Govantes Fernando , Porrúa Odil , Santero Eduardo
Published: December 2004

Pseudomonassp. strain ADP is the model strain for studying bacterial degradation of thes-triazine herbicide atrazine. In this work, we focused on the expression of theatzDEFoperon, involved in mineralization of the central intermediate of the pathway, cyanuric acid. Expression analysis ofatzD-lacZfusions inPseudomonassp. strain ADP andPseudomonas putidashowed thatatzDEFis subjected to dual regulation in response to nitrogen limitation and cyanuric acid. The gene adjacent toatzD,orf99(renamed hereatzR), encoding a LysR-like regulator, was found to be required for both responses. Expression ofatzR-lacZwas induced by nitrogen limitation and repressed by AtzR. Nitrogen regulation ofatzD-lacZandatzR-lacZexpression was dependent on the alternative σ factor σNand NtrC, suggesting that the cyanuric acid degradation operon may be subject to general nitrogen control. However, whileatzRis transcribed from a σN-dependent promoter,atzDEFtranscription appears to be driven from a σ70-type promoter. Expression ofatzRfrom a heterologous promoter revealed that although NtrC regulation ofatzD-lacZrequires the AtzR protein, it is not the indirect result of NtrC-activated AtzR synthesis. We propose that expression of the cyanuric acid degradation operonatzDEFis controlled by means of a complex regulatory circuit in which AtzR is the main activator. AtzR activity is in turn modulated by the presence of cyanuric acid and by a nitrogen limitation signal transduced by the Ntr system.
Molecular Diversity of a Putative Virulence Factor: Purification and Characterization of Isoforms of an Extracellular Serine Glutamyl Endopeptidase of
Enterococcus faecalis
with Different Enzymatic Activities
Authors: Kawalec Magdalena , Potempa Jan , Moon Jonathan L. , Travis James , Murray Barbara E.
Published: December 2004

A previously identified gene
sprE
of
Enterococcus faecalis
strain OG1 was shown to encode an extracellular serine protease that appears to belong to the glutamyl endopeptidase I staphylococcal group. A single form of SprE with a molecular mass of 25 kDa and a pH optimum between 7.0 and 7.5 was isolated from culture supernatant of wild-type
E. faecalis
strain OG1RF (TX4002); this form was apparently generated by cleavage of the Ser
−1
-Leu
1
and Arg
230
-Leu
231
peptide bonds of the secreted zymogen. In contrast, the culture supernatant of the gelatinase-null mutant, TX5264, with a nonpolar deletion of
gelE
which encodes the
E. faecalis
gelatinase, was found to contain several forms of SprE proteolytically processed on both the N and C termini; in addition to a full-length zymogen and a truncated zymogen, three mature forms of the SprE proteinase, Leu
1
-Ala
237
, Ser
−1
-Glu
227
, and Leu
1
-Glu
227
, were identified. As with the V8 proteinase of
Staphylococcus aureus
, the closest homologue of SprE, all of the active forms cleaved specifically Glu-Xaa peptide bonds but with substantially different efficiencies, while none was able to hydrolyze peptide bonds with Asp in the P1 position. The most active of all these enzyme forms against several substrates, including human fibrinogen and β-chain insulin, was the Ser
−1
-Glu
227
(
−1
S-SprE) isolated from TX5264;
−1
S-SprE, in contrast to other forms of SprE, was unstable at 37°C, apparently due to autodegradation. In conclusion, our results demonstrate that
sprE
encodes a highly specific serine-type glutamyl endopeptidase, the maturation of which is dependent on the presence of gelatinase. In the absence of gelatinase activity, the aberrant processing of pro-SprE results in the appearance of a “superactive” form of the enzyme,
−1
S-SprE.
The
katA
Catalase Gene Is Regulated by OxyR in both Free-Living and Symbiotic
Sinorhizobium meliloti
Authors: Jamet Alexandre , Kiss Ernö , Batut Jacques , Puppo Alain , Hérouart Didier
Published: December 2004

The characterization of an
oxyR
insertion mutant provides evidences that
katA
, which encodes the unique H
2
O
2
-inducible HPII catalase, is regulated by OxyR not only in free-living
Sinorhizobium meliloti
but also in symbiotic
S. meliloti
. Moreover,
oxyR
is expressed independently of exogenous H
2
O
2
and downregulates its own expression in
S. meliloti
.
Simulated Diffusion of Phosphorylated CheY through the Cytoplasm of
Escherichia coli
Authors: Lipkow Karen , Andrews Steven S. , Bray Dennis
Published: December 2004

We describe the use of a computational model to study the effects of cellular architecture and macromolecular crowding on signal transduction in
Escherichia coli
chemotaxis. A newly developed program,
Smoldyn
, allows the movement and interaction of a large number of individual molecules in a structured environment to be simulated (S. S. Andrews and D. Bray, Phys. Biol., in press). With
Smoldyn
, we constructed a three-dimensional model of an
E. coli
cell and examined the diffusion of CheYp from the cluster of receptors to the flagellar motors under control conditions and in response to attractant and repellent stimuli. Our simulations agree well with experimental observations of cell swimming responses and are consistent with the diffusive behavior expected in wild-type and mutant cells. The high resolution available to us in the new program allows us to calculate the loci of individual CheYp molecules in a cell and the distribution of their lifetimes under different cellular conditions. We find that the time delay between stimulus and response differs for flagellar motors located at different positions in the cell. We explore different possible locations for the phosphatase CheZ and show conditions under which a gradient of CheYp exists in the cell. The introduction of inert blocks into the cytoplasm, representing impenetrable structures such as the nucleoid and large protein complexes, produces a fall in the apparent diffusion coefficient of CheYp and enhances the differences between motors. These and other results are left as predictions for future experiments.
Identification of a Region in the Herpes Simplex Virus Scaffolding Protein Required for Interaction with the Portal
Authors: Singer Gregory P. , Newcomb William W. , Thomsen Darrel R. , Homa Fred L. , Brown Jay C.
Published: December 2004

The herpes simplex virus type 1 capsid is a protective shell that acts as a container for the genetic material of the virus. After assembly of the capsid, the viral DNA is translocated into the capsid interior through a channel formed by the portal. The portal is composed of a dodecamer of U
L
6 molecules which form a ring-like structure found at a single vertex within the icosahedron. Formation of portal-containing capsids minimally requires the four structural proteins (VP5, VP19C, VP23, and U
L
6) and a scaffolding protein (U
L
26.5). Recently, an interaction between U
L
26.5 and the portal has been identified, suggesting the scaffold functions by delivering the portal to the growing capsid shell. The aim of this study was to identify regions within U
L
26.5 required for its interaction with the portal. A specific region was identified by mutational analysis. Deletion of scaffold amino acids (aa) 143 to 151 was found to be sufficient to inhibit formation of the scaffold-portal complex as assayed in vitro. The aa 143 to 151 contain the sequence YYPGE, which is highly conserved among alphaherpesviruses. Although it did not bind to the portal, the Δ143-151 mutant was found to retain the ability to support assembly of morphologically normal capsids in vitro. Such capsids, however, did not contain the portal. The results suggest assembly of portal-containing capsids requires formation of a scaffold-portal complex in which intermolecular contact is dependent on scaffold aa 143 to 151.
Unrestricted Hepatocyte Transduction with Adeno-Associated Virus Serotype 8 Vectors in Mice
Authors: Nakai Hiroyuki , Fuess Sally , Storm Theresa A. , Muramatsu Shin-ichi , Nara Yuko , Kay Mark A.
Published: December 2004

Recombinant adeno-associated virus (rAAV) vectors can mediate long-term stable transduction in various target tissues. However, with rAAV serotype 2 (rAAV2) vectors, liver transduction is confined to only a small portion of hepatocytes even after administration of extremely high vector doses. In order to investigate whether rAAV vectors of other serotypes exhibit similar restricted liver transduction, we performed a dose-response study by injecting mice with β-galactosidase-expressing rAAV1 and rAAV8 vectors via the portal vein. The rAAV1 vector showed a blunted dose-response similar to that of rAAV2 at high doses, while the rAAV8 vector dose-response remained unchanged at any dose and ultimately could transduce all the hepatocytes at a dose of 7.2 × 10
12
vector genomes/mouse without toxicity. This indicates that all hepatocytes have the ability to process incoming single-stranded vector genomes into duplex DNA. A single tail vein injection of the rAAV8 vector was as efficient as portal vein injection at any dose. In addition, intravascular administration of the rAAV8 vector at a high dose transduced all the skeletal muscles throughout the body, including the diaphragm, the entire cardiac muscle, and substantial numbers of cells in the pancreas, smooth muscles, and brain. Thus, rAAV8 is a robust vector for gene transfer to the liver and provides a promising research tool for delivering genes to various target organs. In addition, the rAAV8 vector may offer a potential therapeutic agent for various diseases affecting nonhepatic tissues, but great caution is required for vector spillover and tight control of tissue-specific gene expression.
Crystal Structure of Complete Rhinovirus RNA Polymerase Suggests Front Loading of Protein Primer
Authors: Appleby Todd C. , Luecke Hartmut , Shim Jae Hoon , Wu Jim Z. , Cheney I. Wayne , Zhong Weidong , Vogeley Lutz , Hong Zhi , Yao Nanhua
Published: December 2004

Picornaviruses utilize virally encoded RNA polymerase and a uridylylated protein primer to ensure replication of the entire viral genome. The molecular details of this mechanism are not well understood due to the lack of structural information. We report the crystal structure of human rhinovirus 16 3D RNA-dependent RNA polymerase (HRV16 3D
pol
) at a 2.4-Å resolution, representing the first complete polymerase structure from the
Picornaviridae
family. HRV16 3D
pol
shares the canonical features of other known polymerase structures and contains an N-terminal region that tethers the
fingers
and
thumb
subdomains, forming a completely encircled active site cavity which is accessible through a small tunnel on the backside of the molecule. The small
thumb
subdomain contributes to the formation of a large cleft on the front face of the polymerase which also leads to the active site. The cleft appears large enough to accommodate a template:primer duplex during RNA elongation or a protein primer during the uridylylation stage of replication initiation. Based on the structural features of HRV16 3D
po1
and the catalytic mechanism known for all polymerases, a front-loading model for uridylylation is proposed.
Prevention of Virus Persistence and Protection against Immunopathology after Borna Disease Virus Infection of the Brain by a Novel Orf Virus Recombinant
Authors: Henkel Marco , Planz Oliver , Fischer Timo , Stitz Lothar , Rziha Hanns-Joachim
Published: December 2004

TheParapoxvirus Orf virusrepresents a promising candidate for novel vector vaccines due to its immune modulating properties even in nonpermissive hosts such as mouse or rat. The highly attenuated Orf virus strain D1701 was used to generate a recombinant virus (D1701-VrVp40) expressing nucleoprotein p40 of Borna disease virus, which represents a major antigen for the induction of a Borna disease virus-specific humoral and cellular immune response. Infection with Borna disease virus leads to distinct neurological symptoms mediated by the invasion of activated specific CD8+T cells into the infected brain. Usually, Borna disease virus is not cleared from the brain but rather persists in neural cells. In the present study we show for the first time that intramuscular application of the D1701-VrVp40 recombinant protected rats against Borna disease, and importantly, virus clearance from the infected brain was demonstrated in immunized animals. Even 4 and 8 months after the last immunization, all immunized animals were still protected against the disease. Initial characterization of the immune cells attracted to the infected brain areas suggested that D1701-VrVp40 mediated induction of B cells and antibody-producing plasma cells as well as T cells. These findings suggest the induction of various defense mechanisms against Borna disease virus. First studies on the role of antiviral cytokines indicated that D1701-VrVp40 immunization did not lead to an enhanced early response of gamma or alpha interferon or tumor necrosis factor alpha. Collectively, this study describes the potential of the Orf virus vector system in mediating long-lasting, protective antiviral immunity and eliminating this persistent virus infection without provoking massive neuronal damage.
Inverted Terminal Repeat Sequences Are Important for Intermolecular Recombination and Circularization of Adeno-Associated Virus Genomes
Authors: Yan Ziying , Zak Roman , Zhang Yulong , Engelhardt John F.
Published: December 2004

The relatively small package capacity (less than 5 kb) of adeno-associated virus (AAV) vectors has been effectively doubled with the development of dual-vector heterodimerization approaches. However, the efficiency of such dual-vector systems is limited not only by the extent to which intermolecular recombination occurs between two independent vector genomes, but also by the directional bias required for successful transgene reconstitution following concatemerization. In the present study, we sought to evaluate the mechanisms by which inverted terminal repeat (ITR) sequences mediate intermolecular recombination of AAV genomes, with the goal of engineering more efficient vectors for dual-vector trans-splicing approaches. To this end, we generated a novel AAV hybrid-ITR vector characterized by an AAV-2 and an AAV-5 ITR at opposite ends of the viral genome. This hybrid genome was efficiently packaged into either AAV-2 or AAV-5 capsids to generate infectious virions. Hybrid AV2:5 ITR viruses had a significantly lower capacity to form circular intermediates in infected cells than homologous AV2:2 and AV5:5 ITR vectors despite their similar capacity to express an encoded enhanced green fluorescent protein (EGFP) transgene. To examine whether the divergent ITR sequences contained within hybrid AV2:5 ITR vectors could direct intermolecular recombination in a tail-to-head fashion, we generated two hybrid ITR trans-splicing vectors (AV5:2LacZdonor and AV2:5LacZacceptor). Each delivered one exon of a β-galactosidase minigene flanked by donor or acceptor splice sequences. These hybrid trans-splicing vectors were compared to homologous AV5:5 and AV2:2 trans-splicing vector sets for their ability to reconstitute β-galactosidase gene expression. Results from this comparison demonstrated that hybrid ITR dual-vector sets had a significantly enhanced trans-splicing efficiency (6- to 10-fold, depending on the capsid serotype) compared to homologous ITR vectors. Molecular studies of viral genome structures suggest that hybrid ITR vectors provide more efficient directional recombination due to an increased abundance of linear-form genomes. These studies provide direct evidence for the importance of ITR sequences in directing intermolecular and intramolecular homologous recombination of AAV genomes. The use of hybrid ITR AAV vector genomes provides new strategies to manipulate viral genome conversion products and to direct intermolecular recombination events required for efficient dual-AAV vector reconstitution of the transgene.
Dendritic Cells and Macrophages Are Productively Infected by Poliovirus
Authors: Wahid Rahnuma , Cannon Martin J. , Chow Marie
Published: December 2004

Expression of the poliovirus receptor (PVR) on cells is a major host determinant of infection by poliovirus. Previously, the only immune cell type known to express PVR was the blood-derived monocyte, which is susceptible to infection at very low frequency. We demonstrate that professional antigen-presenting cells—macrophages and dendritic cells, generated upon differentiation of monocytes—retain expression of PVR and are highly susceptible to infection by type 1 Mahoney strain of poliovirus. Maximal cell-associated titers of virus are obtained within 6 to 8 h postinfection, and cell death and lysis occurs within 24 h postinfection. Similar kinetics are observed in cells infected with the Sabin 1 vaccine strain. Although protein synthesis and receptor-mediated endocytosis are inhibited upon poliovirus infection of these critical antigen-presenting cells, we demonstrate for the first time that functional presentation of antigen occurs in these infected cells via the HLA class II pathway.