bluuebunny/crossref_metadata_2025_split huggingface dataset:
Communication among Oral Bacteria
Authors: Kolenbrander Paul E. , Andersen Roxanna N. , Blehert David S. , Egland Paul G. , Foster Jamie S. , Palmer Robert J. 
Published: January 2003

SUMMARYHuman oral bacteria interact with their environment by attaching to surfaces and establishing mixed-species communities. As each bacterial cell attaches, it forms a new surface to which other cells can adhere. Adherence and community development are spatiotemporal; such order requires communication. The discovery of soluble signals, such as autoinducer-2, that may be exchanged within multispecies communities to convey information between organisms has emerged as a new research direction. Direct-contact signals, such as adhesins and receptors, that elicit changes in gene expression after cell-cell contact and biofilm growth are also an active research area. Considering that the majority of oral bacteria are organized in dense three-dimensional biofilms on teeth, confocal microscopy and fluorescently labeled probes provide valuable approaches for investigating the architecture of these organized communities in situ. Oral biofilms are readily accessible to microbiologists and are excellent model systems for studies of microbial communication. One attractive model system is a saliva-coated flowcell with oral bacterial biofilms growing on saliva as the sole nutrient source; an intergeneric mutualism is discussed. Several oral bacterial species are amenable to genetic manipulation for molecular characterization of communication both among bacteria and between bacteria and the host. A successful search for genes critical for mixed-species community organization will be accomplished only when it is conducted with mixed-species communities.
Microbial Cellulose Utilization: Fundamentals and Biotechnology
Authors: Lynd Lee R. , Weimer Paul J. , van Zyl Willem H. , Pretorius Isak S. 
Published: January 2003

SUMMARYFundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for “consolidated bioprocessing” (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.
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

AbstractGlucocorticoids 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

AbstractAdvance 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

AbstractThe 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.
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

ABSTRACT

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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACT

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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACT

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

ABSTRACTThe 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.