bluuebunny/crossref_metadata_2025 huggingface dataset:
SHAPE OPTIMIZATION OF DENTAL IMPLANT DESIGNS UNDER OBLIQUE LOADING USING THE p-VERSION FINITE ELEMENT METHOD
Authors: PETRIE CYNTHIA S. , WILLIAMS JOHN L.
Published: January 2003

The aim of this study was to introduce design optimization of an endosseous dental implant in order to minimize cortical bone strains at the crest of the implant/bone interface. Linear elastic p-version finite element analysis was used to find the optimal design in four different two-dimensional finite element models of the mandibular bone: model 1 (medium density cancellous bone and 2 mm thickness of cortical bone), model 2 (low density cancellous bone and 2 mm thickness of cortical bone), model 3 (medium density cancellous bone and 1 mm thickness of cortical bone), model 4 (low density cancellous bone and 1 mm thickness of cortical bone). Shape of the implant was evaluated with respect to its length and diameter, as well as the presence or absence of taper in the neck. Implants with maximum length, maximum diameter, and no taper had the lowest crestal strains in models 1, 3, and 4. Taper in the neck of the implant increased the strains in models 1, 3, and 4. In cases of poor quality of bone, increasing the diameter of the implant appeared to be the most effective means of reducing crestal bone strains.
SKELETAL MUSCLE IMPEDANCE IN THE CONTROL OF MOTOR ACTIONS
Authors: HOGAN NEVILLE
Published: January 2003

Unlike ideal engineering actuators, skeletal muscle has neither zero nor infinite mechanical impedance. Non-zero neuro-motor mechanical impedance is required to stabilize posture; its possible contribution to movement will briefly be considered. The essential role of variable mechanical impedance to control physical interaction with objects will be described. Evidence that humans voluntarily control impedance to achieve task goals will be reviewed. Robot impedance control is a biologically-inspired approach to controlling robot interaction. How robust contact stability may be achieved by constraining mechanical impedance will be detailed. One robot application requiring contact and cooperation between robots and humans is robotic physiotherapy. Experience to date demonstrating the success of robotic neuro-rehabilitation of patients recovering from stroke will be summarized. Analysis of the geometry of musculo-skeletal attachment reveals that force control may require synergistic co-contraction of apparently antagonist muscles, rendering the conventional anatomical definition of synergist and antagonist muscles ambiguous. Analysis of workless constraint forces show that they may be used to economize muscle effort; exerting force components normal to a kinematic constraint never requires more muscle effort than exerting purely tangential forces.
MEDICAL IMAGE COMPRESSION USING JPEG-2000 AND JPEG: A COMPARISON STUDY
Authors: OH TICK HUI , BESAR ROSLI
Published: January 2003

Due to the constrained bandwidth and storage capacity, medical images must be compressed before transmission and storage. However, compression will reduce image fidelity, especially when the image is compressed of lower bit rate, which cannot be tolerated in medical field. In this paper, the compression performance of the new JPEG-2000 and the more conventional JPEG is studied. The parameters used for comparison include the compression efficiency, peak signal-to-noise ratio (PSNR), picture quality scale (PQS), and mean opinion score (MOS). Three types of medical images are used — X-ray, magnetic resonance imaging (MRI) and ultrasound. Overall, the study shows that JPEG-2000 compression is more acceptable and superior compare to JPEG for lossy compression.
Intramuscular versus Oral Dexamethasone for the Treatment of Moderate‐to‐severe Croup: A Randomized, Double‐blind Trial
Authors: Donaldson David , Poleski David , Knipple Eric , Filips Kurt , Reetz Linda , Pascual Rebecca G. , Jackson Raymond E.
Published: January 2003

Glucocorticoids are an effective treatment for croup, although the most beneficial route of administration remains unclear. Recent studies have concluded that both intramuscular dexamethasone and oral dexamethasone are effective treatments, but there are few data directly comparing the two for moderate‐to‐severe croup. Objectives: The authors' primary objective was to determine whether there is a difference in proportion of children with resolution of symptoms attributable to croup at 24 hours, when treated with oral or intramuscular dexamethasone. Secondarily, the authors sought to estimate whether there is a difference in proportion of children with resolution of symptoms attributable to croup at 10 days and to estimate the interval to complete resolution of symptoms between these two routes. Methods: The authors performed a prospective, randomized, double‐blind trial involving children aged 3‐84 months with moderate‐to‐severe croup, presenting to a suburban teaching emergency department (ED). Patients were eligible for enrollment if they had inspiratory stridor or a barky cough and a croup score of 2 or greater after 10‐15 minutes of cool mist therapy. The patients were randomized to one of two intervention groups. In both groups, the parents were not present in the treatment room during study drug administration. One group received 0.6 mg/kg of intramuscular dexamethasone and an oral placebo, while the other group received 0.6 mg/kg of oral dexamethasone and direct pressure on their thigh with the hub of a syringe. A nurse placed a Band‐Aid on the site of the real or mock injection. Parents were contacted by telephone approximately 1 and 10 days after the index visit to ask about their child's symptoms using a standardized questionnaire. Data were analyzed using an intention‐to‐treat approach. Results: Of 126 patients eligible, 96 were recruited, with complete follow‐up on 95. The groups were similar in all baseline characteristics, treatments received in the ED, and disposition. At 24 hours and 10 days after the visit, there were no statistical differences between the groups for the proportion with stridor, expiratory sounds, barky cough, sleep pattern, the degree of improvement, or the proportion with complete resolution of symptoms at one day. Conclusions: No statistical differences for any parameters were observed between intramuscular and oral dexamethasone treatments for children with moderate‐to‐severe croup at 24 hours or at any time the week after treatment. The durations of symptoms were similar between the treatment groups.
Ethics Seminars: Health Care Proxies and Suicidal Patients
Authors: Bania Theodore C. , Lee Richard , Clark Mark
Published: January 2003

Advance directives, health care proxies, and living wills are forms of advance planning that permit patients to make decisions regarding their health care and are used when the patient becomes incapacitated. The ethics of allowing these forms of advance planning are questioned when the patient has attempted suicide. The authors present an ethical analysis of a case of an elder patient who overdosed on sustained‐release diltiazem and had a health care proxy who wanted all treatments stopped.
Transcriptional Analysis of the Orphan Nuclear Receptor Constitutive Androstane Receptor (NR1I3) Gene Promoter: Identification of a Distal Glucocorticoid Response Element
Authors: Pascussi Jean Marc , Busson-Le Coniat Maryvonne , Maurel Patrick , Vilarem Marie-José
Published: January 2003

The constitutive androstane receptor (CAR, NR1I3) transcriptionally activates cytochrome P450 2B6, 2C9, and 3A4 when activated by xenobiotics, such as phenobarbital. Information on the human CAR promoter was obtained by searching the NCBI human genome database. A contig (NT026945) corresponding to a fragment of chromosome 1q21 was found to contain the complete CAR gene. These data were confirmed using chromosomal in situ hybridization. Both primer extension and 5′-rapid amplification of the cDNA end PCR analysis were carried out to determine the transcriptional start site of human CAR, which was found to be 32 nucleotides downstream of a potential TATA box (CATAAAA). In addition, we found that the 5′-untranslated region of CAR mRNA is 110 nucleotides shorter than previously reported. Using genomic PCR, we amplified and cloned approximately 4.9 kb (−4711/+144) of the CAR gene promoter. The activity of this promoter was measured by transient transfection. Deletion analysis suggested the presence of a glucocorticoid responsive element in its distal region (−4477/−4410). From cotransfection experiments, mutagenesis, and gel shift assays, we identified a glucocorticoid response element at −4447/−4432 that was recognized and transactivated by the human glucocorticoid receptor. Finally, using the chromatin immunoprecipitation assay, we demonstrated that the glucocorticoid receptor binds to the distal region of CAR promoter in cultured hepatocytes only in the presence of dexamethasone. Identification of this functional element provides a rational mechanistic basis for CAR induction by glucocorticoids. CAR appears to be a primary glucocorticoid receptor-response gene.
X-ray absorption spectroscopic studies of zinc in the N-terminal domain of HIV-2 integrase and model compounds
Authors: Feiters Martinus C. , Eijkelenboom Astrid P. A. M. , Nolting Hans-Friedrich , Krebs Bernt , van den Ent Fusinita M. I. , Plasterk Ronald H. A. , Kaptein Robert , Boelens Rolf
Published: January 2003

X-ray absorption spectroscopy (XAS), including extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) analysis, has been carried out at the ZnKedge of the N-terminal part of the integrase protein of the human immunodeficiency virus, type 2 (HIV-2), and of some zinc coordination compounds. In the presence of excess β-mercaptoethanol, which was present in the NMR structure elucidation of the protein [Eijkelenboomet al.(1997),Curr. Biol.7, 739–746; (2000),J. Biomol. NMR,18, 119–28], the protein spectrum was nearly identical to that recorded in its absence. Comparison of the XANES of the protein with that of model compounds and literature data permits the conclusion that the Zn ion is four-coordinated. The major shell of the EXAFS provides evidence for a mixed (N or O as well as S) coordination sphere, while the minor shells indicate imidazole coordination. Our approach to the analysis of the EXAFS, including quantification of the imidazole by multiple scattering simulations withEXCURV92, was validated on the model compounds. An important result is that with multiple scattering simulations using restraints on the parameters of the imidazole rings the number of imidazoles and their orientation could be determined. The integrase spectra can be fitted with two sulfur ligands at 2.26 Å (Debye–Waller-type factor 0.009 Å2) and two imidazole ligands with the N atoms at 1.99 Å (Debye–Waller-type factor 0.005 Å2). The XAS-derived geometry is fully consistent with that found in the NMR structure determination and, allowing for the volume contraction due to the temperature difference between the experiments, justifies the restraints applied in the structure calculation (Zn—S and Zn—N distances of 2.3 Å and 2.0 Å, respectively).
X-ray absorption spectroscopy to watch catalysis by metalloenzymes: status and perspectives discussed for the water-splitting manganese complex of photosynthesis
Authors: Dau Holger , Haumann Michael
Published: January 2003

Understanding structure–function relations is one of the main interests in the molecular biosciences. X-ray absorption spectroscopy of biological samples (BioXAS) has gained the status of a useful tool for characterization of the structure of protein-bound metal centers with respect to the electronic structure (oxidation states, orbital occupancies) and atomic structure (arrangement of ligand atoms). Owing to progress in the performance characteristics of synchrotron radiation sources and of experimental stations dedicated to the study of (ultra-dilute) biological samples, it is now possible to carry out new types of BioXAS experiments, which have been impracticable in the past. Of particular interest are approaches to follow biological catalysis at metal sites by characterization of functionally relevant structural changes. In this article, the first steps towards the use of BioXAS to `watch' biological catalysis are reviewed for the water-splitting reactions occurring at the manganese complex of photosynthesis. The following aspects are considered: the role of BioXAS in life sciences; methodological aspects of BioXAS; catalysis at the Mn complex of photosynthesis; combination of EXAFS and crystallographic information; the freeze-quench technique to capture semi-stable states; time-resolved BioXAS using a freeze-quench approach; room-temperature experiments and `real-time' BioXAS; tasks and perspectives.
TheMXANprocedure: a new method for analysing the XANES spectra of metalloproteins to obtain structural quantitative information
Authors: Benfatto M. , Della Longa S. , Natoli C. R.
Published: January 2003

The first quantitative analyses are reported of the FeK-edge polarized X-ray absorption near-edge structure (XANES) of a single crystal of the iron protein carbonmonoxy-myoglobin (MbCO) and of its cryogenic photoproduct Mb*CO. The CO—Fe–heme local structure has been determined using a novel fitting procedure, namedMXAN, which is able to fit the XANES part (from the edge to about 200 eV) of experimental X-ray absorption data. This method is based on the comparison between the experimental spectrum and several theoretical spectra that are generated by changing the relevant geometrical parameters of the site around the absorbing atom. The theoretical spectra are derived in the framework of the full multiple-scattering approach. TheMXANprocedure is able to recover information about the symmetry and atomic distances, and the solution is found to be independent of the starting conditions. The extracted local structure of Mb*CO includes an Fe—CO distance of 3.08 (7) Å, with a tilting angle between the heme normal and the Fe—C vector of 37 (7)° and a bending angle between the Fe—C vector and the C—O bond of 31 (5)°
Marriage of XAFS and crystallography for structure–function studies of metalloproteins
Authors: Hasnain S. Samar , Strange Richard W.
Published: January 2003

Metalloproteins constitute a significant proportion of all known genomes. In this article it is shown that the marriage of two synchrotron-radiation-based structural techniques, namely high-resolution crystallography and XAFS, can yield unprecedented levels of structural information that is directly relevant to the mechanism/function of these important macromolecules.
Phengite-Based Chronology of K- and Ba-Rich Fluid Flow in Two Paleosubduction Zones
Authors: Catlos E. J. , Sorensen S. S.
Published: January 2003

Subduction recycles aqueous fluids from slab and sediment to the mantle. Subduction zones are long-lived, but time scales for fluid-rock interaction within subduction complexes are uncertain. Large-ion lithophile elements (potassium and barium) were added to eclogite (subducted basalt) during high pressure/temperature metamorphism via phengite crystallization from subduction zone fluids. Phengite grains from eclogite blocks and their metasomatic selvages yielded
40
Ar/
39
Ar ages across grains and between samples that indicate 25 and 60 million years of fluid-rock interaction in the Samana Complex, Dominican Republic, and the Franciscan Complex, California, respectively.
Neuronal Activity in the Lateral Intraparietal Area and Spatial Attention
Authors: Bisley James W. , Goldberg Michael E.
Published: January 2003

Although the parietal cortex has been implicated in the neural processes underlying visual attention, the nature of its contribution is not well understood. We tracked attention in the monkey and correlated the activity of neurons in the lateral intraparietal area (LIP) with the monkey's attentional performance. The ensemble activity in LIP across the entire visual field describes the spatial and temporal dynamics of a monkey's attention. Activity subtending a single location in the visual field describes the attentional priority at that area but does not predict that the monkey will actually attend to or make an eye movement to that location.
Modulation of ATP-Dependent Chromatin-Remodeling Complexes by Inositol Polyphosphates
Authors: Shen Xuetong , Xiao Hua , Ranallo Ryan , Wu Wei-Hua , Wu Carl
Published: January 2003

Eukaryotes use adenosine triphosphate (ATP)–dependent chromatin-remodeling complexes to regulate gene expression. Here, we show that inositol polyphosphates can modulate the activities of several chromatin-remodeling complexes in vitro. Inositol hexakisphosphate (IP
6
) inhibits nucleosome mobilization by NURF, ISW2, and INO80 complexes. In contrast, nucleosome mobilization by the yeast SWI/SNF complex is stimulated by inositol tetrakisphosphate (IP
4
) and inositol pentakisphosphate (IP
5
). We demonstrate that mutations in genes encoding inositol polyphosphate kinases that produce IP
4
, IP
5
, and IP
6
impair transcription in vivo. These results provide a link between inositol polyphosphates, chromatin remodeling, and gene expression.
Thermonuclear Supernovae: Simulations of the Deflagration Stage and Their Implications
Authors: Gamezo Vadim N. , Khokhlov Alexei M. , Oran Elaine S. , Chtchelkanova Almadena Y. , Rosenberg Robert O.
Published: January 2003

Large-scale, three-dimensional numerical simulations of the deflagration stage of a thermonuclear supernova explosion show the formation and evolution of a highly convoluted turbulent flame in the gravitational field of an expanding carbon-oxygen white dwarf. The flame dynamics are dominated by the gravity-induced Rayleigh-Taylor instability that controls the burning rate. The thermonuclear deflagration releases enough energy to produce a healthy explosion. The turbulent flame, however, leaves large amounts of unburned and partially burned material near the star center, whereas observations that imply these materials are present only in outer layers. This disagreement could be resolved if the deflagration triggers a detonation.
MICROBICIDES:Raising New Barriers Against HIVInfection
Authors: Trager Rebecca Spieler
Published: January 2003

In January, the International Partnership for Microbicides (IPM), a nonprofit group dedicated to advancing a promising but undeveloped strategy for fighting HIV--using topical microbicides to block transmission--will open its doors. With $30 million in committed support, IPM intends to help evaluate an estimated 50 candidate compounds, clear barriers to testing, and facilitate access to microbicides that prove effective. IPM joins several other organizations supporting research in the area, but it will be the first to focus so intensely on coordinating microbicide R&D and reviewing products.
CelI, a Noncellulosomal Family 9 Enzyme from
Clostridium thermocellum
, Is a Processive Endoglucanase That Degrades Crystalline Cellulose
Authors: Gilad Rachel , Rabinovich Larisa , Yaron Sima , Bayer Edward A. , Lamed Raphael , Gilbert Harry J. , Shoham Yuval
Published: January 2003

The family 9 cellulase gene
celI
of
Clostridium thermocellum
, was previously cloned, expressed, and characterized (G. P. Hazlewood, K. Davidson, J. I. Laurie, N. S. Huskisson, and H. J. Gilbert, J. Gen. Microbiol. 139:307-316, 1993). We have recloned and sequenced the entire
celI
gene and found that the published sequence contained a 53-bp deletion that generated a frameshift mutation, resulting in a truncated and modified C-terminal segment of the protein. The enzymatic properties of the wild-type protein were characterized and found to conform to those of other family 9 glycoside hydrolases with a so-called theme B architecture, where the catalytic module is fused to a family 3c carbohydrate-binding module (CBM3c); CelI also contains a C-terminal CBM3b. The intact recombinant CelI exhibited high levels of activity on all cellulosic substrates tested, with pH and temperature optima of 5.5 and 70°C, respectively, using carboxymethylcellulose as a substrate. Native CelI was capable of solubilizing filter paper, and the distribution of reducing sugar between the soluble and insoluble fractions suggests that the enzyme acts as a processive cellulase. A truncated form of the enzyme, lacking the C terminal CBM3b, failed to bind to crystalline cellulose and displayed reduced activity toward insoluble substrates. A truncated form of the enzyme, in which both the cellulose-binding CBM3b and the fused CBM3c were removed, failed to exhibit significant levels of activity on any of the substrates examined. This study underscores the general nature of this type of enzymatic theme, whereby the fused CBM3c plays a critical accessory role for the family 9 catalytic domain and changes its character to facilitate processive cleavage of recalcitrant cellulose substrates.
Biochemical Characterization of a Mutationally Altered Protein Translocase: Proton Motive Force Stimulation of the Initiation Phase of Translocation
Authors: Mori Hiroyuki , Ito Koreaki
Published: January 2003

Protein translocation across the
Escherichia coli
plasma membrane is facilitated by concerted actions of the SecYEG integral membrane complex and the SecA ATPase. A
secY
mutation (
secY39
) affects Arg357, an evolutionarily conserved and functionally important residue, and impairs the translocation function in vivo and in vitro. In this study, we used the “superactive” mutant forms of SecA, which suppress the SecY39 deficiency, to characterize the mutationally altered SecY39EG translocase. It was found that SecY39-mediated preprotein translocation exhibited absolute dependence on the proton motive force. The proton motive force-dependent step proved to lie before signal peptide cleavage. We suggest that the proton motive force assists in the initiation phase of protein translocation.
Salmonella entericaSerovar TyphimuriumrdoAIs Growth Phase Regulated and Involved in Relaying Cpx-Induced Signals
Authors: Suntharalingam P. , Spencer H. , Gallant C. V. , Martin N. L.
Published: January 2003

The disulfide oxidoreductase, DsbA, mediates disulfide bond formation in proteins as they enter or pass through the periplasm of gram-negative bacteria. Although DsbA function has been well characterized, less is known about the factors that control its expression. Previous studies withEscherichia colidemonstrated thatdsbAis part of a two-gene operon that includes an uncharacterized, upstream gene,yihE, that is positively regulated via the Cpx stress response pathway. To clarify the role of theyihEhomologue ondsbAexpression inSalmonella entericaserovar Typhimurium, the effect of this gene (termedrdoA) on the regulation ofdsbAexpression was investigated. Transcriptional assays assessingrdoApromoter activity showed growth phase-dependent expression with maximal activity in stationary phase. Significant quantities ofrdoAanddsbAtranscripts exist in serovar Typhimurium, but only extremely low levels ofrdoA-dsbAcotranscript were detected. Activation of the Cpx system in serovar Typhimurium increased synthesis of bothrdoA-anddsbA-specific transcripts but did not significantly alter the levels of detectable cotranscript. These results indicate that Cpx-mediated induction ofdsbAtranscription in serovar Typhimurium does not occur through anrdoA-dsbAcotranscript. A deletion of therdoAcoding region was constructed to definitively test the relevance of therdoA-dsbAcotranscript todsbAexpression. The absence of RdoA affects DsbA expression levels when the Cpx system is activated, and providingrdoAintranscomplements this phenotype, supporting the hypothesis that a bicistronic mechanism is not involved in serovar TyphimuriumdsbAregulation. TherdoAnull strain was also shown to be altered in flagellar phase variation. First it was found that induction of the Cpx stress response pathway switched flagellar synthesis to primarily phase 2 flagellin, and this effect was then found to be abrogated in therdoAnull strain, suggesting the involvement of RdoA in mediating Cpx-related signaling.
Bioenergetic Properties of the Thermoalkaliphilic
Bacillus
sp. Strain TA2.A1
Authors: Olsson Karen , Keis Stefanie , Morgan Hugh W. , Dimroth Peter , Cook Gregory M.
Published: January 2003

The thermoalkaliphilic
Bacillus
sp. strain TA2.A1 was able to grow in pH-controlled batch culture containing a nonfermentable growth substrate from pH 7.5 to 10.0 with no significant change in its specific growth rate, demonstrating that this bacterium is a facultative alkaliphile. Growth at pH 10.0 was sensitive to the protonophore carbonyl cyanide
m
-chlorophenylhydrazone, suggesting that a proton motive force (Δp) generated via aerobic respiration was an obligate requirement for growth of strain TA2.A1. Strain TA2.A1 exhibited intracellular pH homeostasis as the external pH increased from 7.5 to 10.0; however, the maximum ΔpH generated over this pH range was only 1.1 units at an external pH of 9.5. The membrane potential (Δψ) was maintained between −114 mV and −150 mV, and little significant change was observed over the pH range for growth. In contrast, the Δp declined from −164 mV at pH 7.5 to approximately −78 mV at pH 10.0. An inwardly directed sodium motive force (ΔpNa
+
) of −100 mV at pH 10.0 indicated that cellular processes (i.e., solute transport) dependent on a sodium gradient would not be affected by the adverse Δp. The phosphorylation potential of strain TA2.A1 was maintained between −300 mV and −418 mV, and the calculated H
+
/ATP stoichiometry of the ATP synthase increased from 2.0 at pH 7.5 to 5.7 at pH 10.0. Based on these data, vigorous growth of strain TA2.A1 correlated well with the ΔpNa
+
, phosphorylation potential, and the ATP/ADP ratio, but not with Δp. This communication represents the first report on the bioenergetics of an extremely thermoalkaliphilic aerobic bacterium.
Combinatorial Redesign of the DNA Binding Specificity of a Prokaryotic Helix-Turn-Helix Repressor
Authors: Fromknecht Katja , Vogel Pia D. , Wise John G.
Published: January 2003

Redesign of the bacteriophage 434 Cro repressor was accomplished by using an in vivo genetic screening system to identify new variants that specifically bound previously unrecognized DNA sequences. Site-directed, combinatorial mutagenesis of the 434 Cro helix-turn-helix (HTH) motif generated libraries of new variants which were screened for binding to new target sequences. Multiple mutations of 434 Cro that functionally converted wild-type (wt) 434 Cro DNA binding-sequence specificity to that of a λ bacteriophage-specific repressor were identified. The libraries contained variations within the HTH sequence at only three positions. In vivo and in vitro analysis of several of the identified 434 Cro variants showed that the relatively few changes in the recognition helix of the HTH motif of 434 Cro resulted in specific and tight binding of the target DNA sequences. For the best 434 Cro variant identified, an apparent

K
d

for λ O
R
3 of 1 nM was observed. In competition experiments, this Cro variant was observed to be highly selective. We conclude that functional 434 Cro repressor variants with new DNA binding specificities can be generated from wt 434 Cro by mutating just the recognition helix. Important characteristics of the screening system responsible for the successful identifications are discussed. Application of the techniques presented here may allow the identification of DNA binding protein variants that functionally affect DNA regulatory sequences important in disease and industrial and biotechnological processes.
Identification of Secretion Determinants of theBordetella pertussisBrkA Autotransporter
Authors: Oliver David C. , Huang George , Fernandez Rachel C.
Published: January 2003

The autotransporters comprise a functionally diverse family of gram-negative proteins that mediate their own export across the bacterial outer membrane. They consist of an amino-terminal passenger region called the “α-domain” and the structural hallmark of the autotransporter family, a carboxy-terminal transporter region usually referred to as the “β-domain.” The passenger region can be quite diverse and constitutes the effector functions of these proteins, whereas the C-terminal region is conserved and is responsible for translocating the passenger moiety across the outer membrane. BrkA is the 103-kDa autotransporter protein inBordetella pertussisthat is cleaved to yield a 73-kDa N-terminal α-domain and a 30-kDa C-terminal β-domain. We have previously shown that a recombinant form of the β-domain of BrkA is capable of forming channels in artificial membranes. Here, we define two additional secretion determinants of BrkA. N-terminal sequencing of the 73-kDa BrkA passenger fromB. pertussisandEscherichia colirevealed that BrkA has a 42-amino-acid signal peptide. In addition, deletion analysis of BrkA identified a 31- to 39-amino-acid region found immediately upstream of the β-domain that was essential for surface expression. This 31- to 39-amino-acid linker region, together with the β-domain, defines the minimal BrkA translocation unit. The linker region may also serve to anchor the BrkA passenger to the bacterial surface.
Characterization of a Cellulase Containing a Family 30 Carbohydrate-Binding Module (CBM) Derived from
Clostridium thermocellum
CelJ: Importance of the CBM to Cellulose Hydrolysis
Authors: Arai Takamitsu , Araki Rie , Tanaka Akiyoshi , Karita Shuichi , Kimura Tetsuya , Sakka Kazuo , Ohmiya Kunio
Published: January 2003

Clostridium thermocellum
CelJ is a modular enzyme containing a family 30 carbohydrate-binding module (CBM) and a family 9 catalytic module at its N-terminal moiety. To investigate the functions of the CBM and the catalytic module, truncated derivatives of CelJ were constructed and characterized. Isothermal titration calorimetric studies showed that the association constants (

K
a

) of the CBM polypeptide (CBM30) for the binding of cellopentaose and cellohexaose were 1.2 × 10
4
and 6.4 × 10
4
M
−1
, respectively, and that the binding of CBM30 to these ligands is enthalpically driven. Qualitative analyses showed that CBM30 had strong affinity for cellulose and β-1,3-1,4-mixed glucan such as barley β-glucan and lichenan. Analyses of the hydrolytic action of the enzyme comprising the CBM and the catalytic module showed that the enzyme is a processive endoglucanse with strong activity towards carboxymethylcellulose, barley β-glucan and lichenan. By contrast, the catalytic module polypeptide devoid of the CBM showed negligible activity toward these substrates. These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.
FlhD/FlhC Is a Regulator of Anaerobic Respiration and the Entner-Doudoroff Pathway through Induction of the Methyl-Accepting Chemotaxis Protein Aer
Authors: Prüβ Birgit M. , Campbell John W. , Van Dyk Tina K. , Zhu Charles , Kogan Yakov , Matsumura Philip
Published: January 2003

The regulation by two transcriptional activators of flagellar expression (FlhD and FlhC) and the chemotaxis methyl-accepting protein Aer was studied with glass slide DNA microarrays. AnflhD::Kan insertion and anaerdeletion were independently introduced into twoEscherichia coliK-12 strains, and the effects upon gene regulation were investigated. Altogether, theflhD::Kan insertion altered the expression of 29 operons of known function. Among them was Aer, which in turn regulated a subset of these operons, namely, the ones involved in anaerobic respiration and the Entner-Doudoroff pathway. In addition, FlhD/FlhC repressed enzymes involved in aerobic respiration and regulated many other metabolic enzymes and transporters in an Aer-independent manner. Expression of 12 genes of uncharacterized function was also affected. FlhD increasedgltBD,gcvTHP, andompTexpression. The regulation of half of these genes was subsequently confirmed with reporter gene fusions, enzyme assays, and real-time PCR. Growth phenotypes offlhDandflhCmutants were determined with Phenotype MicroArrays and correlated with gene expression.
Global Regulation of
Staphylococcus aureus
Genes by Rot
Authors: Saïd-Salim B. , Dunman P. M. , McAleese F. M. , Macapagal D. , Murphy E. , McNamara P. J. , Arvidson S. , Foster T. J. , Projan S. J. , Kreiswirth B. N.
Published: January 2003

Staphylococcus aureus
produces a wide array of cell surface and extracellular proteins involved in virulence. Expression of these virulence factors is tightly controlled by numerous regulatory loci, including
agr
,
sar
,
sigB
,
sae
, and
arl
, as well as by a number of proteins with homology to SarA. Rot (repressor of toxins), a SarA homologue, was previously identified in a library of transposon-induced mutants created in an
agr-
negative strain by screening for restored protease and alpha-toxin. To date, all of the SarA homologues have been shown to act as global regulators of virulence genes. Therefore, we investigated the extent of transcriptional regulation of staphylococcal genes by Rot. We compared the transcriptional profile of a
rot agr
double mutant to that of its
agr
parental strain by using custom-made Affymetrix GeneChips. Our findings indicate that Rot is not only a repressor but a global regulator with both positive and negative effects on the expression of
S. aureus
genes. Our data also indicate that Rot and
agr
have opposing effects on select target genes. These results provide further insight into the role of Rot in the regulatory cascade of
S. aureus
virulence gene expression.
A
cka-gfp
Transcriptional Fusion Reveals that the Colicin K Activity Gene Is Induced in Only 3 Percent of the Population
Authors: Mulec Janez , Podlesek Zdravko , Mrak Peter , Kopitar Andreja , Ihan Alojz , Žgur-Bertok Darja
Published: January 2003

In prokaryotes, only a few examples of differential gene expression in cell populations have been described. Colicin production in natural populations of
Escherichia coli
, while providing a competitive advantage in the natural habitat, also leads to lysis of the toxin-producing cell. Colicin K synthesis has been found to be induced due to an increase in ppGpp (I. Kuhar, J. P. van Putten, D. Žgur-Bertok, W. Gaastra, and B. J. Jordi, Mol. Microbiol.
41:
207-216). Using two transcriptional fusions,
cka-gfp
and
cki-gfp
, we show that at the single-cell level, the colicin K activity gene
cka
is expressed in only 3% of the bacterial population upon induction by nutrient starvation. In contrast, the immunity gene
cki
is expressed in the large majority of the cells. Expression of the
cka-gfp
fusion in a
lexA
-defective strain and in a
relA spoT
mutant strain indicates that differential expression of
cka
is established primarily at the level of transcription.
Isolation of a Negative Control Mutant of the Nitrogen Assimilation Control Protein, NAC, in
Klebsiella aerogenes
Authors: Janes Brian K. , Rosario Christopher J. , Bender Robert A.
Published: January 2003

A negative control mutant of the nitrogen assimilation control protein, NAC, has been isolated. Mutants with the leucine at position 111 changed to a nonhydrophobic residue activate transcription from
hut
and
ure
promoters, but fail to repress
gdhA
expression. This failure does not result from failure to bind to either of the two sites required for
gdhA
repression, but the binding at those sites is altered in the mutant. It appears that the NAC negative control mutants fail to form the complex structures (probably tetramers) formed by wild-type NAC at the
gdhA
promoter.
PIISignal Transduction Proteins, Pivotal Players in Microbial Nitrogen Control
Authors: Arcondéguy Tania , Jack Rachael , Merrick Mike
Published: January 2003

The PIIfamily of signal transduction proteins are among the most widely distributed signal proteins in the bacterial world. First identified in 1969 as a component of the glutamine synthetase regulatory apparatus, PIIproteins have since been recognized as playing a pivotal role in control of prokaryotic nitrogen metabolism. More recently, members of the family have been found in higher plants, where they also potentially play a role in nitrogen control. The PIIproteins can function in the regulation of both gene transcription, by modulating the activity of regulatory proteins, and the catalytic activity of enzymes involved in nitrogen metabolism. There is also emerging evidence that they may regulate the activity of proteins required for transport of nitrogen compounds into the cell. In this review we discuss the history of the PIIproteins, their structures and biochemistry, and their distribution and functions in prokaryotes. We survey data emerging from bacterial genome sequences and consider other likely or potential targets for control by PIIproteins.
Pneumococcal Virulence Factors: Structure and Function
Authors: Jedrzejas Mark J.
Published: January 2003

The overall goal for this review is to summarize the current body of knowledge about the structure and function of major known antigens of Streptococcus pneumoniae, a major gram-positive bacterial pathogen of humans. This information is then related to the role of these proteins in pneumococcal pathogenesis and in the development of new vaccines and/or other antimicrobial agents. S. pneumoniae is the most common cause of fatal community-acquired pneumonia in the elderly and is also one of the most common causes of middle ear infections and meningitis in children. The present vaccine for the pneumococcus consists of a mixture of 23 different capsular polysaccharides. While this vaccine is very effective in young adults, who are normally at low risk of serious disease, it is only about 60% effective in the elderly. In children younger than 2 years the vaccine is ineffective and is not recommended due to the inability of this age group to mount an antibody response to the pneumococcal polysaccharides. Antimicrobial drugs such as penicillin have diminished the risk from pneumococcal disease. Several pneumococcal proteins including pneumococcal surface proteins A and C, hyaluronate lyase, pneumolysin, autolysin, pneumococcal surface antigen A, choline binding protein A, and two neuraminidase enzymes are being investigated as potential vaccine or drug targets. Essentially all of these antigens have been or are being investigated on a structural level in addition to being characterized biochemically. Recently, three-dimensional structures for hyaluronate lyase and pneumococcal surface antigen A became available from X-ray crystallography determinations. Also, modeling studies based on biophysical measurements provided more information about the structures of pneumolysin and pneumococcal surface protein A. Structural and biochemical studies of these pneumococcal virulence factors have facilitated the development of novel antibiotics or protein antigen-based vaccines as an alternative to polysaccharide-based vaccines for the treatment of pneumococcal disease.
Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance
Authors: Chopra Ian , Roberts Marilyn
Published: January 2003

Tetracyclines were discovered in the 1940s and exhibited activity against a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites. They are inexpensive antibiotics, which have been used extensively in the prophlylaxis and therapy of human and animal infections and also at subtherapeutic levels in animal feed as growth promoters. The first tetracycline-resistant bacterium, Shigella dysenteriae, was isolated in 1953. Tetracycline resistance now occurs in an increasing number of pathogenic, opportunistic, and commensal bacteria. The presence of tetracycline-resistant pathogens limits the use of these agents in treatment of disease. Tetracycline resistance is often due to the acquisition of new genes, which code for energy-dependent efflux of tetracyclines or for a protein that protects bacterial ribosomes from the action of tetracyclines. Many of these genes are associated with mobile plasmids or transposons and can be distinguished from each other using molecular methods including DNA-DNA hybridization with oligonucleotide probes and DNA sequencing. A limited number of bacteria acquire resistance by mutations, which alter the permeability of the outer membrane porins and/or lipopolysaccharides in the outer membrane, change the regulation of innate efflux systems, or alter the 16S rRNA. New tetracycline derivatives are being examined, although their role in treatment is not clear. Changing the use of tetracyclines in human and animal health as well as in food production is needed if we are to continue to use this class of broad-spectrum antimicrobials through the present century.
Autophagy in Yeast: Mechanistic Insights and Physiological Function
Authors: Abeliovich Hagai , Klionsky Daniel J.
Published: January 2003

Unicellular eukaryotic organisms must be capable of rapid adaptation to changing environments. While such changes do not normally occur in the tissues of multicellular organisms, developmental and pathological changes in the environment of cells often require adaptation mechanisms not dissimilar from those found in simpler cells. Autophagy is a catabolic membrane-trafficking phenomenon that occurs in response to dramatic changes in the nutrients available to yeast cells, for example during starvation or after challenge with rapamycin, a macrolide antibiotic whose effects mimic starvation. Autophagy also occurs in animal cells that are serum starved or challenged with specific hormonal stimuli. In macroautophagy, the form of autophagy commonly observed, cytoplasmic material is sequestered in double-membrane vesicles called autophagosomes and is then delivered to a lytic compartment such as the yeast vacuole or mammalian lysosome. In this fashion, autophagy allows the degradation and recycling of a wide spectrum of biological macromolecules. While autophagy is induced only under specific conditions, salient mechanistic aspects of autophagy are functional in a constitutive fashion. In Saccharomyces cerevisiae, induction of autophagy subverts a constitutive membrane-trafficking mechanism called the cytoplasm-to-vacuole targeting pathway from a specific mode, in which it carries the resident vacuolar hydrolase, aminopeptidase I, to a nonspecific bulk mode in which significant amounts of cytoplasmic material are also sequestered and recycled in the vacuole. The general aim of this review is to focus on insights gained into the mechanism of autophagy in yeast and also to review our understanding of the physiological significance of autophagy in both yeast and higher organisms.
Secretory Pathway of Trypanosomatid Parasites
Authors: McConville Malcolm J. , Mullin Kylie A. , Ilgoutz Steven C. , Teasdale Rohan D.
Published: January 2003

The Trypanosomatidae comprise a large group of parasitic protozoa, some of which cause important diseases in humans. These include Trypanosoma brucei (the causative agent of African sleeping sickness and nagana in cattle), Trypanosoma cruzi (the causative agent of Chagas' disease in Central and South America), and Leishmania spp. (the causative agent of visceral and [muco]cutaneous leishmaniasis throughout the tropics and subtropics). The cell surfaces of these parasites are covered in complex protein- or carbohydrate-rich coats that are required for parasite survival and infectivity in their respective insect vectors and mammalian hosts. These molecules are assembled in the secretory pathway. Recent advances in the genetic manipulation of these parasites as well as progress with the parasite genome projects has greatly advanced our understanding of processes that underlie secretory transport in trypanosomatids. This article provides an overview of the organization of the trypanosomatid secretory pathway and connections that exist with endocytic organelles and multiple lytic and storage vacuoles. A number of the molecular components that are required for vesicular transport have been identified, as have some of the sorting signals that direct proteins to the cell surface or organelles in the endosome-vacuole system. Finally, the subcellular organization of the major glycosylation pathways in these parasites is reviewed. Studies on these highly divergent eukaryotes provide important insights into the molecular processes underlying secretory transport that arose very early in eukaryotic evolution. They also reveal unusual or novel aspects of secretory transport and protein glycosylation that may be exploited in developing new antiparasite drugs.
Regulation of Cellular Differentiation in Filamentous Cyanobacteria in Free-Living and Plant-Associated Symbiotic Growth States
Authors: Meeks John C. , Elhai Jeff
Published: January 2003

Certain filamentous nitrogen-fixing cyanobacteria generate signals that direct their own multicellular development. They also respond to signals from plants that initiate or modulate differentiation, leading to the establishment of a symbiotic association. An objective of this review is to describe the mechanisms by which free-living cyanobacteria regulate their development and then to consider how plants may exploit cyanobacterial physiology to achieve stable symbioses. Cyanobacteria that are capable of forming plant symbioses can differentiate into motile filaments called hormogonia and into specialized nitrogen-fixing cells called heterocysts. Plant signals exert both positive and negative regulatory control on hormogonium differentiation. Heterocyst differentiation is a highly regulated process, resulting in a regularly spaced pattern of heterocysts in the filament. The evidence is most consistent with the pattern arising in two stages. First, nitrogen limitation triggers a nonrandomly spaced cluster of cells (perhaps at a critical stage of their cell cycle) to initiate differentiation. Interactions between an inhibitory peptide exported by the differentiating cells and an activator protein within them causes one cell within each cluster to fully differentiate, yielding a single mature heterocyst. In symbiosis with plants, heterocyst frequencies are increased 3- to 10-fold because, we propose, either differentation is initiated at an increased number of sites or resolution of differentiating clusters is incomplete. The physiology of symbiotically associated cyanobacteria raises the prospect that heterocyst differentiation proceeds independently of the nitrogen status of a cell and depends instead on signals produced by the plant partner.
T Antigens of Simian Virus 40: Molecular Chaperones for Viral Replication and Tumorigenesis
Authors: Sullivan Christopher S. , Pipas James M.
Published: January 2003

Simian virus 40 (SV40) is a small DNA tumor virus that has been extensively characterized due to its relatively simple genetic organization and the ease with which its genome is manipulated. The large and small tumor antigens (T antigens) are the major regulatory proteins encoded by SV40. Large T antigen is responsible for both viral and cellular transcriptional regulation, virion assembly, viral DNA replication, and alteration of the cell cycle. Deciphering how a single protein can perform such numerous and diverse functions has remained elusive. Recently it was established that the SV40 T antigens, including large T antigen, are molecular chaperones, each with a functioning DnaJ domain. The molecular chaperones were originally identified as bacterial genes essential for bacteriophage growth and have since been shown to be conserved in eukaryotes, participating in an array of both viral and cellular processes. This review discusses the mechanisms of DnaJ/Hsc70 interactions and how they are used by T antigen to control viral replication and tumorigenesis. The use of the DnaJ/Hsc70 system by SV40 and other viruses suggests an important role for these molecular chaperones in the regulation of the mammalian cell cycle and sheds light on the enigmatic SV40 T antigen—a most amazing molecule.