bluuebunny/crossref_metadata_2025 huggingface dataset:
Fasting versus Gradual Initiation of the Ketogenic Diet: A Prospective, Randomized Clinical Trial of Efficacy
Authors: Bergqvist A. G. Christina , Schall Joan I. , Gallagher Paul R. , Cnaan Avital , Stallings Virginia A.
Published: November 2005

Summary: Purpose:The ketogenic diet (KD) is a 90% fat diet that is an effective treatment for intractable epilepsy. Rapid initiation of the KD requires hospital admission because of the complexity of the protocol and frequent mild and moderate adverse events. The purpose of the study was to compare the efficacy of a gradual KD initiation with the standard KD initiation preceded by a 24‐ to 48‐h fast.

Methods:Children ages 1 to 14 years with intractable epilepsy were randomized to a fasting initiation (FAST‐KD) or gradual initiation (GRAD‐KD). Baseline seizure activity was recorded daily for 28 days before admission and continued for the 3‐month duration of the study. Effectiveness was measured in two ways: (a) the proportion of subjects with >50% reduction in target seizure type from baseline to 3‐month evaluation, and (b) percentage reduction in the frequency of the target seizure type from baseline to 3‐month evaluation. Blood glucose was assessed q4 to 6h, and weights, electrolytes, hydration status, vomiting, acid balance, need for interventions (citric acid and sodium citrates (Bicitra) and IV fluids) were assessed daily. Fisher's exact tests were used to examine the association between protocol and occurrence of adverse events, and longitudinal mixed‐effects models were used to look for trends in tolerability data over time.

Results:Forty‐eight subjects, 24 in each arm, were randomized. In the FAST‐KD protocol, 58% of the children had >50% reduction in the target seizure type at 3 months, and 21% were seizure free. In the GRAD‐KD protocol, 67% had a >50% reduction at 3 months, and 21% were seizure free. The two protocols were equivalent in efficacy (p = 0.033). At 3 months, the FAST‐KD median percentage seizure reduction rate was 78% (ranging from 100% reduction to 73% increase in seizures per week) and was 94% (ranging from 100% reduction to 161% increase in seizures per week) for the GRAD‐KD protocol. By using a logarithmic transformed percentage reduction rate and an equivalence limit difference of 20%, the efficacy of the two protocols was equivalent (p = 0.0002). Children in the GRAD protocol lost significantly less weight (p = 0.006), and had fewer and less‐severe episodes of hypoglycemia (p < 0.001), fewer treatments for acidosis (citric acid and sodium citrates) (p < 0.04) and dehydration (IV fluids) (p < 0.04), but no difference in vomiting was noted.

Conclusions:These data suggest that in children with intractable epilepsy, a gradual initiation results in fewer adverse events and is tolerated better overall while maintaining the efficacy of the KD.
Vagus Nerve Stimulation Induces Concomitant Respiratory Alterations and a Decrease in SaO2 in Children
Authors: Zaaimi Boubker , Héberlé Claire , Berquin Patrick , Pruvost Mickael , Grebe Reinhard , Wallois Fabrice
Published: November 2005

Summary:  Purpose: To analyze respiratory alterations and effects on SaO2 caused by vagus nerve stimulation (VNS) in children with epilepsy.

Methods: Polysomnographic recordings, including electroencephalography, thoracoabdominal distention, nasal airflow, SaO2, and VNS artifact were evaluated in 10 children with pharmacoresistant epilepsy treated with VNS.

Results: Each VNS caused a significant increase in respiratory frequency (p < 0.05) throughout the stimulation period and a decrease in thoracoabdominal‐distention amplitude (p < 0.05), especially at the beginning of the stimulation. These respiratory alterations induced a decrease in SaO2 from 1 to 5%. The effects of VNS on respiration differed significantly between rapid‐eye‐movement (REM) and non‐REM (NREM) sleep states.

Conclusions: VNS caused a pronounced change in respiration in children with epilepsy, and this induced a decrease in SaO2. It is possible that VNS has a neuroprotective effect, and this possibility calls for further investigation.
In Vivo Epileptogenicity of Focal Cortical Dysplasia: A Direct Cortical Paired Stimulation Study
Authors: Matsumoto Riki , Kinoshita Masako , Taki Junya , Hitomi Takefumi , Mikuni Nobuhiro , Shibasaki Hiroshi , Fukuyama Hidenao , Hashimoto Nobuo , Ikeda Akio
Published: November 2005

Summary:  Purpose: Alternation of the intracortical inhibitory and excitatory mechanisms in focal cortical dysplasia (FCD) has not been well elucidated in vivo in humans. We investigated in vivo alternation of these mechanisms in epileptogenesis of FCD by means of paired‐pulse direct cortical electrical stimulation.

Methods: A 31‐year‐old man with FCD at the left foot primary somatosensory (SI) and motor areas who underwent invasive monitoring with subdural electrodes was studied. By means of subdural electrodes, paired‐pulse electrical stimulation was performed at the epileptic focus (foot SI) and control cortex (hand SI) with interstimulus interval (ISI) of 1–100 ms. Instead of using motor evoked potentials to investigate the degree of cortical excitability in response to motor cortex stimulation, we evaluated the size change of corticocortical evoked potentials (CCEPs), which are elicited at the adjacent cortex by direct cortical stimulation via fiber projection and thus reflect direct and indirect excitation of corticocortical projection neurons at the site of stimulation.

Results: During the interictal state, paired‐pulse stimulation of the focus revealed abnormally enhanced intracortical inhibition at ISI of 1–10 ms (maximum, 22%) compared with control stimulation of the hand SI (ISI of 1–2 ms; maximum, 18%) (p < 0.01). While the patient was having the somatosensory aura that later evolved into the left‐leg clonic seizure, single and paired stimulation at the focus showed increased cortical excitability (enlarged CCEP) and decreased intracortical inhibition, respectively.

Conclusions: During the aura, interictally enhanced intracortical inhibition at the focus was replaced by increased cortical excitability and decreased intracortical inhibition, suggesting increased net intrinsic epileptogenicity during seizure generation in this patient with FCD.
Victor Horsley's Contribution to Jacksonian Epileptology
Authors: Eadie Mervyn J.
Published: November 2005

Summary:  Purpose: To describe Victor Horsley's contribution to John Hughlings Jackson's understanding of the mechanisms involved in the generalization of convulsive epileptic seizures.

Methods: I reviewed Horsley's writings and other relevant late 19th century medical literature.

Results: Horsley's combination of strategically sited surgical lesions and cerebral cortex stimulation studies in experimental animals showed that, contrary to Hughlings Jackson's earlier belief, epileptic activity arising in one cerebral hemisphere had to spread to the contralateral hemisphere before bilateral convulsing could occur.

Conclusions: On the basis of well‐designed experiments, Horsley made a major contribution to the understanding of epileptic seizure propagation mechanisms.
Ipsilateral Head Deviation in Frontal Lobe Seizures
Authors: Rheims Sylvain , Demarquay Genevieve , Isnard Jean , Guenot Marc , Fischer Catherine , Sindou Marc , Mauguiere François , Ryvlin Philippe
Published: November 2005

Summary:  Purpose: The lateralizing value of ictal head deviation (HD) in frontal lobe epilepsy (FLE) is a matter of debate. Although FLE is typically associated with tonic or clonic HD contralateral to seizure onset, ipsilateral HD has been noted in numerous reports. Whether both types of HD can be distinguished according to their clinical patterns has not yet been specifically investigated.

Methods: We studied the clinical pattern and time of occurrence of HD of 129 seizures in 13 consecutive patients, who underwent successful surgery for FLE, including 12 investigated with an intracerebral stereotactic EEG procedure.

Results: Ictal HD was ipsilateral to the epileptogenic zone (EZ) in four (30%) patients and 27 (20.9%) seizures and contralateral in five (38%) patients and 15 (11.6%) seizures. Ipsilateral HD was rarely tonic and never associated with clonic manifestation. Contralateral HD was always tonic, unnatural, and associated with hemifacial clonic movements in 86% of seizures. Ipsilateral HD occurred earlier than contralateral HD (p < 0.03), with a mean delay of 1 ± 2 s after the first detectable ictal sign, as compared with a delay of 17 ± 11 s for contralateral HD. Moreover, ipsilateral HD always occurred before contralateral HD when both signs coexisted in the same seizure. Our patients with ipsilateral HD demonstrated either an anterior or dorsolateral frontal EZ.

Conclusions: Ipsilateral HD is a common ictal sign during FLE and can be distinguished from contralateral HD by its time of occurrence at or immediately after seizure onset and its lacking association with clonic movements.
Unilateral Intracarotid Amobarbital Procedure for Language Lateralization
Authors: Wellmer Jörg , Fernández Guillen , Linke Detlef B. , Urbach Horst , Elger Christian E. , Kurthen Martin
Published: November 2005

Summary:  Purpose: The determination of language dominance as part of the presurgical workup of patients with pharmacoresistant epilepsies has experienced fundamental changes. With the introduction of noninvasive functional magnetic resonance imaging (fMRI), the number of patients receiving intracarotid amobarbital procedures (IAPs) for assessment of language dominance has decreased considerably. However, recent studies show that because of methodologic limitations of fMRI, IAP remains an important tool for language lateralization. The current study examines whether unilateral instead of bilateral IAP is an adequate way to apply IAP with reduced invasiveness.

Methods: We retrospectively examine the predictive value of unilateral IAP for the results of bilateral IAP based on a sample of 75 patients with various types of language dominance. Target parameters are the prediction of the language‐dominant hemisphere and the identification of patients with atypical language dominance. For language assessment based on unilateral IAP, we introduce the measure hemispheric language capacity (HLC).

Results: Unilateral IAP performed on the side of intended surgery quantifies language capacity contralateral to the intended surgery. It detects atypical (bilateral or right) language dominance in the majority of patients. Experience with a separate series of 107 patients requiring presurgical language lateralization shows that in >80%, bilateral IAPs are redundant.

Conclusions: Unilateral IAP is principally sufficient for language lateralization in the presurgical evaluation of patients with pharmacoresistant epilepsies. Necessity of bilateral IAP is restricted to few indications (e.g., callosotomy). In times of noninvasive language lateralization, we propose unilateral IAP as the method of choice for the verification of doubtful (bilateral) fMRI activation patterns.
Three‐Dimensional Assemblies of Zirconia Nanocrystals Via Shape‐Preserving Reactive Conversion of Diatom Microshells
Authors: Shian Samuel , Cai Ye , Weatherspoon Michael R. , Allan Shawn M. , Sandhage Kenneth H.
Published: November 2005

The synthesis of three‐dimensional (3‐D) assemblies of zirconia nanocrystals via the shape‐preserving reactive conversion of biologically reproducible, silica‐based microtemplates (diatom microshells) is demonstrated for the first time. Silica diatom microshells were first converted into magnesia replicas via an oxidation–reduction displacement reaction with magnesium gas. The magnesia replicas were then converted into zirconia replicas via a metathetic displacement reaction with zirconium tetrachloride gas. By utilizing both types of shape‐preserving displacement reactions, bioclastic silica microtemplates may be converted into functional nanocrystalline assemblies with a variety of complex, but well‐controlled 3‐D shapes and chemistries for catalytic/chemical, biological, electrical, thermal, mechanical, and other applications.
Energy Release in Isothermally Stretched Silicate Glass fibers
Authors: Hornbøll Lasse , Lönnroth Nadja , Yue Yuanzheng
Published: November 2005

Three types of silicate glass fibers are annealed, simultaneously stretched in the glass transition region for certain time lengths, then slowly cooled to room temperature under load, and subsequently scanned by differential scanning calorimetry (DSC). During the DSC scanning, a broad exothermic peak (representing energy release) occurs in the stretched fibers well below the glass transition temperature, while it does not occur in the non‐stretched fibers. The peak indicates that mechanical stretching can result in an energy enhancement in the fibers. It also confirms that the energy released during reheating of the fibers formed using an industrial continuous fiber drawing process originates not only from thermal quenching but also from mechanical stretching. However, the mechanical stretching‐induced energy is much lower than the thermal hyperquenching‐induced energy in glass fibers. The effect of annealing temperature and time on the energy release behavior is discussed in terms of viscoelasticity.
Sulfanilic Acid: A Novel Consolidation Agent for Al2O3 in Aqueous Media
Authors: Zhang Jingxian , Xu Qiang , Iwasa Mikio , Ye Feng , Jiang Dongliang
Published: November 2005

A new solidifying agent, 4‐aminobenzene sulfonic acid (sulfanilic acid), is reported in this paper. The consolidation process and mechanism were followed using on viscoelastic, FTIR, SEM, and Hg porosimetry measurements. It was shown that the Al2O3 slurries with PAA‐Na as dispersant exhibited a high degree of particle stabilization. After the addition of sulfanilic acid, we observed an exponential increase in the storage modulus (G′) as a function of consolidation time. Correspondingly, Al2O3 slurries exhibited a transition from a viscous to an elastic state (in 62 min). FTIR analysis indicated that the consolidation process might follow two steps: first, the adsorption of sulfanilic acid on the Al2O3 particle surface; second, the acid–base interaction between the adsorbed PAA‐Na molecules and the sulfanilic acid molecules. This interaction could possibly induce the formation of three‐dimensional networks through a bridging or charge neutralization mechanism. The as‐consolidated Al2O3 green samples were homogeneous, with the relative green density being 54.69%. Results showed that it was feasible to introduce sulfanilic acid for the consolidation of Al2O3 slurries in aqueous media.
Methyl cellulose gel obstructed bone formation by GBR: an experimental study in rats
Authors: Lioubavina‐Hack Natalia , Karring Thorkild , Lynch Samuel E. , Lindhe Jan
Published: November 2005

Aim: To evaluate whether bone formation under Teflon capsules may be enhanced by concomitant implantation of recombinant human platelet‐derived growth factor‐BB/insulin‐like growth factor‐I (rhPDGF‐BB/IGF‐I) incorporated into a methyl cellulose gel.

Materials and Methods: Fifty‐five male 6‐month‐old albino rats of the Wistar strain were used in the study. The lateral aspect of the mandibular ramus was exposed on both sides of the jaw. In 70 sites, the periosteum was removed from the ramus, leaving the bone denuded, while in 35 sites, it was preserved. On 10 non‐periosteal (P−) sites and five periosteal (P+) sites, an empty rigid teflon capsule (d=7 mm), serving as control, was placed on the ramus. In the 40 test animals, the capsule placed on the one side of the jaw was filled at random with one of three different concentrations (1200, 600, 150 μg/ml) of rhPDGF‐BB/IGF‐I gel. The capsules placed on the contralateral side of the jaw contained a placebo methyl cellulose gel. Each growth factor group, defined according to the gel concentration, and the placebo group contained 10 capsules placed on the P− side and five capsules placed on the P+ side. Two months after surgery, all animals were sacrificed.

Results: Histologic analysis revealed that in the non‐filled control capsules, the amount of new bone including the bone marrow was 29.9% and 39.7% of the capsule area on the P− and P+ sides, respectively. In the test capsules with the growth factor gel and placed on the P−sides, the amounts of new bone ranged from 5.6% to 6.3%, which were similar (p>0.05) to that formed in the capsules filled with the methyl cellulose gel (5.5%). New bone formation was larger in the capsules on the P+ sides than in those on the P− sides but was similar in the capsules with different growth factor concentrations (range 17.9–19.6%) and in those with placebo gel (21.0%). In all groups, the carrier gel was poorly absorbed and occupied most of the capsules.

Conclusion: Local application of a methyl cellulose gel obstructed bone formation under Teflon capsules placed adjacent to uninjured cortical bone in the mandibular ramus of rats. These data suggest that another material should be utilized to deliver growth factors under Teflon membranes for guided bone regeneration.
Nicotine inhibits human gingival fibroblast migration via modulation of Rac signalling pathways
Authors: Fang Yiyu , Svoboda Kathy K. H.
Published: November 2005

Aim:Cigarette smoking is a risk factor in the development of periodontal diseases. In addition, a delayed healing process has been shown in smokers compared with non‐smokers after periodontal treatment. Cell migration is a key process of wound healing and it is highly regulated by a variety of signalling pathways. The small G protein, Rac, is necessary for cell migration. Our aim was to determine if nicotine disrupted Rac and its downstream signalling proteins, p21‐activated kinase 1/2 (PAK1/2), and p44/42 mitogen‐activated protein kinase (MAPK) (extracellular regulated kinase 1/2).

Material and Methods:Primary human fibroblasts from healthy gingival tissues were cultured and grown to confluence. Cells were serum starved for 24 h, and then treated with nicotine (0 or 0.5 μM) prior to in vitro wounding. Cell migration was analysed in live cell assays following in vitro wounds. Rac activity, phosphorylation levels of PAK1/2, and p44/42 MAPK were assessed in cultures treated with or without nicotine after multiple wounds.

Results:Nicotine decreased cell migration rates by 50% compared with controls. In addition, nicotine altered the activation patterns of Rac and PAK 1/2 and up‐regulated p44/42 MAPK.

Conclusion:Decreased cell migration in periodontal wounds exposed to nicotine may be mediated through the Rac and PAK1/2 signalling pathways.
Sonic‐powered toothbrushes and reversal of experimental gingivitis
Authors: Versteeg P. A. , Timmerman M. F. , Rosema N. A. M. , Warren P. R. , Van der Velden U. , Van der Weijden G. A.
Published: November 2005

Objective: To compare two sonic toothbrushes in relation to the reversal of experimental gingivitis.

Materials and Methods: Subjects refrained from brushing mandibular teeth for 21 days. During a 4‐week treatment phase, the right or left side of the mouth was brushed with either the Sonic Complete (SC) or Sonicare Elite (SE) toothbrush as randomly allocated. Plaque and gingivitis were assessed on day 0, after 21 days of no oral hygiene and after 1, 2 and 4 weeks of brushing twice daily.

Results: Thirty‐four subjects provided evaluable data. The experimentally induced gingivitis (EIG) resulted in higher bleeding and plaque scores compared with day 0. The mean plaque scores at day 21 changed from 3.09 to 1.30 for the SC, and from 3.02 to 1.21 for the SE. At the end of the treatment period, there was no significant difference between the two brushes. The mean bleeding scores changed from 1.87 (day 21) to 0.97 for the SC, and from 1.83 to 0.92 for the SE. For the assessments at 1, 2 and 4 weeks post‐EIG, both brushes showed a significant decrease in bleeding scores. There were no statistically significant differences between brushes.
Serum antibodies to periodontal pathogens and markers of systemic inflammation
Authors: Dye Bruce A. , Choudhary Khalid , Shea Steven , Papapanou Panos N.
Published: November 2005

Aim: We examined the relationship between serum antibodies against Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, and plasma fibrinogen and serum C‐reactive protein (CRP) in a nationally representative sample.

Methods: Data on 2973 participants aged 40 years and older from the third National Health and Nutrition Examination Survey, second phase (1991–1994) were used. Three logistic regression models adjusted for gender, race, educational attainment, diabetes, cigarette smoking, body mass index (BMI), and other inflammatory conditions were constructed, based on three different assumptions: (A) no access to dental/periodontal data; (B) knowledge of number of teeth present but not of clinical periodontal status; and (C) knowledge of both dental and clinical periodontal status.

Results: High fibrinogen (>400 mg/dl) was unrelated to P. gingivalis and A. actinomycetemcomitans antibodies in all models. High CRP (>0.4 mg/dl) was related to high antibody levels to P. gingivalis in models A [odds ratios (OR) 1.63, 95% confidence intervals (CI) 1.15–2.32], B (OR 1.69, 95% CI 1.18–2.41), and C (OR 1.58, 95% CI 1.12–2.23). In model C, high CRP was related to >30% extent of attachment loss of 3 mm (OR 1.58, 95% CI 1.19–2.08). Antibodies to A. actinomycetemcomitans were not associated with high CRP levels in any model.

Conclusions: High serum titre to P. gingivalis and the presence of periodontal disease are independently related to high CRP levels.
Accidental Presidents: Death, Assassination, Resignation, and Democratic Succession
Authors: ABBOTT PHILIP
Published: November 2005

One of the commonly stated virtues of modern constitutional democracies is their capacity to ensure reliable and accepted methods of political succession through election. This essay focuses on one particular, though not uncommon, complication in the democratic mode of political succession: American vice presidents who assume office as a result of the death, assassination, or resignation of a president. Three basic strategies by “accidental presidents” to establish and enhance their legitimacy are identified. The efforts of accidental presidents provide a framework to assess a democratic theory of succession, for a certain resiliency is necessary to respond to irregular modes of succession yet excessive plasticity might threaten election as the privileged method of succession.
Do Early Birds Get the Worm? Improving Timeliness of Presidential Nomination Forecasts
Authors: ADKINS RANDALL E. , DOWDLE ANDREW J.
Published: November 2005

This research explores whether data on polling, campaign expenditures, and cash reserves of campaigns collected in the year prior to the presidential election can produce accurate predictions of party nominees. Findings suggest that efforts to forecast nominations may begin as early as the first quarter of the year before the election for Republicans, where the trade‐off between timeliness and accuracy is negligible. In Democratic races where a sitting vice president does not run for president, exhibition season predictions are murky and the cost of trading timeliness for accuracy is higher.
History, Collective Memory, and the Appropriation of Martin Luther King, Jr.: Reagan's Rhetorical Legacy
Authors: BOSTDORFF DENISE M. , GOLDZWIG STEVEN R.
Published: November 2005

This article argues that President Ronald Reagan appropriated Dr. Martin Luther King, Jr.'s words and memory to suggest equal opportunity in the United States had been largely achieved. Individuals—rather than the government—now had to take responsibility for any additional progress. By arguing that the dismantling of federal civil rights laws and social programs was actually consistent with Dr. King's words, President Reagan advanced his own agenda for civil rights in direct violation of Dr. King's intentions, while narrowing the purview of civil rights to eliminate government intervention in employment, education, and other arenas.
White House Publicity Operations During the Korean War, June 1950–June 1951
Authors: CASEY STEVEN
Published: November 2005

Truman was the first modern president to face the challenge of selling a limited war. Based on a wide range of primary sources, this article explores the impact that the Korean War had on Truman's publicity operations. Whereas all wars place important new demands on presidents to speak out more frequently and forcefully, limited wars place significant constraints on what presidents can say and do. During the Korean War, Truman refused to go public at key moments, often employed rhetoric that was more restrained than at earlier moments of the Cold War, and shied away from creating new structures to coordinate the official message. Such actions also had important consequences. In 1950‐51, they hampered the task of effective presidential communication, and contributed to the war's growing unpopularity. For the longer term, they demonstrated the difficulties of selling a limited war, and hence place into sharper context the problems that beset Truman's successors during the subsequent conflict in Vietnam.
Presidential Signaling in a Market Economy
Authors: ESHBAUGH‐SOHA MATTHEW
Published: November 2005

Presidential leadership of the economy is vital to presidential success. Although research supports some presidential influence over the economy, consensus holds that presidents do not affect it consistently. Nevertheless, scholars have not examined the impact of the president's public statements on the economy. From signaling and anticipative reactions theories, I develop expectations for when presidential signals should affect the marketplace. I use impact assessment methodology to test this argument across three dependent variables: the daily price of oil, the daily yield on the thirty‐year Treasury bill, and monthly measures of the money supply. Signals tend to influence the market in the short term given the president's unitary authority and his credibility to affect the economy.
Grover Cleveland, William McKinley, and the Emergence of the President as Party Leader
Authors: KLINGHARD DANIEL P.
Published: November 2005

The modern concept of the president as party leader emerged during the late nineteenth century. Through independent leadership of the party‐in‐the‐electorate, presidents enhanced their capacities to be renominated, and thus their capacity to be reelected. This popular leadership also enhanced the president's ability to shape the content of national electoral campaigns in ways not available to traditional party organizations. There is good reason to suggest that this development, rather than the emergence of the rhetorical presidency or the formalization of national administrative capacities, marks the origins of the modern presidency.
Elections:Effects of the Stand by Your Ad Provision on Attitudes About Candidates and Campaigns
Authors: GALE KRISTINA , HAWKINS BETSEY GIMBEL , HAWKINS RICHARD , MAGLEBY DAVID B. , MONSON J. QUIN , PATTERSON KELLY D.
Published: November 2005

The “Stand by Your Ad” (SBYA) provision of the Bipartisan Campaign Reform Act requires federal candidates to claim responsibility for their advertising content. Using an experimental survey design to show respondents actual campaign ads with and without the SBYA language, we find that SBYA produces no effect on respondents’ levels of trust in candidates or the ads themselves. However, there is a statistically significant effect on respondents’ confidence in the campaign as well as on respondents’ support for the ad's sponsor. In ads sponsored by the presidential candidates, respondents’ reactions to the ads were further filtered through their predispositions toward the candidates.
The Activity Profile of the NhaD-Type Na
+
(Li
+
)/H
+
Antiporter from the Soda Lake Haloalkaliphile
Alkalimonas amylolytica
Is Adaptive for the Extreme Environment
Authors: Liu Jun , Xue Yanfen , Wang Quanhui , Wei Yi , Swartz Talia H. , Hicks David B. , Ito Masahiro , Ma Yanhe , Krulwich Terry A.
Published: November 2005

In extreme alkaliphiles, Na
+
/H
+
antiporters play a central role in the Na
+
cycle that supports pH homeostasis, Na
+
resistance, solute uptake, and motility. Properties of individual antiporters have only been examined in extremely alkaliphilic soil
Bacillus
spp., whereas the most alkaline natural habitats usually couple high pH with high salinity. Here, studies were conducted on a Na
+
(Li
+
)/H
+
antiporter, NhaD, from the soda lake haloalkaliphile
Alkalimonas amylolytica
. The activity profile of
A. amylolytica
NhaD at different pH values and Na
+
concentrations reflects its unique natural habitat. In membrane vesicles from antiporter-deficient
Escherichia coli
EP432 (Δ
nhaA
Δ
nhaB
), the pH optimum for NhaD-dependent Na
+
(Li
+
)/H
+
antiport was at least 9.5, the highest pH that could be tested; no activity was observed at pH ≤8.5. NhaD supported low Na
+
/H
+
antiport activity at pH 9.5 that was detectable over a range of Na
+
concentrations from 10 mM to at least 800 mM, with a 600 mM optimum. Although
A. amylolytica nhaD
was isolated by complementing the Li
+
sensitivity of the triple mutant
E. coli
strain KNabc (Δ
nhaA
Δ
nhaB
Δ
chaA
), sustained propagation of
nhaD
-bearing plasmids in this strain resulted in a glycine (Gly
327
)→serine mutation in a putative cytoplasmic loop of the mutant transporter. The altered activity profile of NhaD-G327S appears to be adaptive to the
E. coli
setting: a much higher activity than wild-type NhaD at Na
+
concentrations up to 200 mM but lower activity at 400 to 600 mM Na
+
, with a pH optimum and minimal pH for activity lower than those of wild-type NhaD.
High-Resolution Visualization ofPseudomonas aeruginosaPAO1 Biofilms by Freeze-Substitution Transmission Electron Microscopy
Authors: Hunter Ryan C. , Beveridge Terry J.
Published: November 2005

High-pressure freeze-substitution and transmission electron microscopy have been used for high-resolution imaging of the natural structure of a gram-negative biofilm. Unlike more conventional embedding techniques, this method confirms many of the observations seen by confocal microscopy but with finer structural detail. It further reveals that there is a structural complexity to biofilms at both the cellular and extracellular matrix levels that has not been seen before. Different domains of healthy and lysed cells exist randomly dispersed within a single biofilm as well as different structural organizations of exopolymers. Particulate matter is suspended within this network of fibers and appears to be an integral part of the exopolymeric substance (EPS). O-side chains extending from the outer membrane are integrated into EPS polymers so as to form a continuum. Together, the results support the concept of physical microenvironments within biofilms and show a complexity that was hitherto unknown.
Genetic Composition of theBacillus subtilisSOS System
Authors: Au Nora , Kuester-Schoeck Elke , Mandava Veena , Bothwell Laura E. , Canny Susan P. , Chachu Karen , Colavito Sierra A. , Fuller Shakierah N. , Groban Eli S. , Hensley Laura A. , O'Brien Theresa C. , Shah Amish , Tierney Jessica T. , Tomm Louise L. , O'Gara Thomas M. , Goranov Alexi I. , Grossman Alan D. , Lovett Charles M.
Published: November 2005

The SOS response in bacteria includes a global transcriptional response to DNA damage. DNA damage is sensed by the highly conserved recombination protein RecA, which facilitates inactivation of the transcriptional repressor LexA. Inactivation of LexA causes induction (derepression) of genes of the LexA regulon, many of which are involved in DNA repair and survival after DNA damage. To identify potential RecA-LexA-regulated genes inBacillus subtilis, we searched the genome for putative LexA binding sites within 300 bp upstream of the start codons of all annotated open reading frames. We found 62 genes that could be regulated by putative LexA binding sites. Using mobility shift assays, we found that LexA binds specifically to DNA in the regulatory regions of 54 of these genes, which are organized in 34 putative operons. Using DNA microarray analyses, we found that 33 of the genes with LexA binding sites exhibit RecA-dependent induction by both mitomycin C and UV radiation. Among these 33 SOS genes, there are 22 distinct LexA binding sites preceding 18 putative operons. Alignment of the distinct LexA binding sites reveals an expanded consensus sequence for theB. subtilisoperator: 5′-CGAACATATGTTCG-3′. Although the number of genes controlled by RecA and LexA inB. subtilisis similar to that ofEscherichia coli, only eightB. subtilisRecA-dependent SOS genes have homologous counterparts inE. coli.
Molecular and Biochemical Characterization of the
xlnD
-Encoded 3-Hydroxybenzoate 6-Hydroxylase Involved in the Degradation of 2,5-Xylenol via the Gentisate Pathway in
Pseudomonas alcaligenes
NCIMB 9867
Authors: Gao Xiaoli , Tan Chew Ling , Yeo Chew Chieng , Poh Chit Laa
Published: November 2005

The
xlnD
gene from
Pseudomonas alcaligenes
NCIMB 9867 (strain P25X) was shown to encode 3-hydroxybenzoate 6-hydroxylase I, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Active recombinant XlnD was purified as a hexahistidine fusion protein from
Escherichia coli
, had an estimated molecular mass of 130 kDa, and is probably a trimeric protein with a subunit mass of 43 kDa. This is in contrast to the monomeric nature of the few 3-hydroxybenzoate 6-hydroxylases that have been characterized thus far. Like other 3-hydroxybenzoate 6-hydroxylases, XlnD could utilize either NADH or NADPH as the electron donor. P25X harbors a second 3-hydroxybenzoate 6-hydroxylase II that was strictly inducible by specific aromatic substrates. However, the degradation of 2,5-xylenol and 3,5-xylenol in strain P25X was found to be dependent on the
xlnD
-encoded 6-hydroxylase I and not the second, strictly inducible 6-hydroxylase II.
In Vitro Analysis of Protein-Operator Interactions of the NikR and Fur Metal-Responsive Regulators of Coregulated Genes inHelicobacter pylori
Authors: Delany Isabel , Ieva Raffaele , Soragni Alice , Hilleringmann Markus , Rappuoli Rino , Scarlato Vincenzo
Published: November 2005

Two important metal-responsive regulators, NikR and Fur, are involved in nickel and iron homeostasis and controlling gene expression inHelicobacter pylori.To date, they have been implicated in the regulation of sets of overlapping genes. We have attempted here dissection of the molecular mechanisms involved in transcriptional regulation of the NikR and Fur proteins, and we investigated protein-promoter interactions of the regulators with known target genes. We show thatH. pyloriNikR is a tetrameric protein and, through DNase I footprinting analysis, we have identified operators for NikR to which it binds with different affinities in a metal-responsive way. Mapping of the NikR binding site upstream of the urease promoter established a direct role for NikR as a positive regulator of transcription and, through scanning mutagenesis of this binding site, we have determined two subsites that are important for the binding of the protein to its target sequence. Furthermore, by alignment of the operators for NikR, we have shown that theH. pyloriprotein recognizes a sequence that is distinct from its well-studied orthologue inEscherichia coli.Moreover, we show that NikR and Fur can bind independently at distinct operators and also compete for overlapping operators in some coregulated gene promoters, adding another dimension to the previous suggested link between iron and nickel regulation. Finally, the importance of an interconnection between metal-responsive gene networks for homeostasis is discussed.
Assembly and Function of a Spore Coat-Associated Transglutaminase of
Bacillus subtilis
Authors: Zilhão Rita , Isticato Rachele , Martins Lígia O. , Steil Leif , Völker Uwe , Ricca Ezio , Moran Charles P. , Henriques Adriano O.
Published: November 2005

The assembly of a multiprotein coat around the
Bacillus subtilis
spore confers resistance to lytic enzymes and noxious chemicals and ensures normal germination. Part of the coat is cross-linked and resistant to solubilization. The coat contains ε-(γ-glutamyl)lysyl cross-links, and the expression of the gene (
tgl
) for a spore-associated transglutaminase was shown before to be required for the cross-linking of coat protein GerQ. Here, we have investigated the assembly and function of Tgl. We found that Tgl associates, albeit at somewhat reduced levels, with the coats of mutants that are unable to assemble the outer coat (
cotE
), that are missing the inner coat and with a greatly altered outer coat (
gerE
), or that are lacking discernible inner and outer coat structures (
cotE gerE
double mutant). This suggests that Tgl is present at various levels within the coat lattice. The assembly of Tgl occurs independently of its own activity, as a single amino acid substitution of a cysteine to an alanine (C116A) at the active site of Tgl does not affect its accumulation or assembly. However, like a
tgl
insertional mutation, the
tglC116A
allele causes increased extractability of polypeptides of about 40, 28, and 16 kDa in addition to GerQ (20 kDa) and affects the structural integrity of the coat. We show that most Tgl is assembled onto the spore surface soon after its synthesis in the mother cell under σ
K
control but that the complete insolubilization of at least two of the Tgl-controlled polypeptides occurs several hours later. We also show that a multicopy allele of
tgl
causes increased assembly of Tgl and affects the assembly, structure, and functional properties of the coat.
Cell Division Defects inEscherichia coliDeficient in the Multidrug Efflux Transporter AcrEF-TolC
Authors: Lau Sze Yi , Zgurskaya Helen I.
Published: November 2005

TheEscherichia colichromosome contains several operons encoding confirmed and predicted multidrug transporters. Among these transporters only the inactivation of components of the AcrAB-TolC complex leads to substantial changes in susceptibility to multiple drugs. This observation prompted a conclusion that other transporters are silent or expressed at levels insufficient to contribute to multidrug resistance phenotype. We found that increased expression of AcrA, the periplasmic membrane fusion protein, is toxic only in cells lacking the multidrug efflux transporter AcrEF. AcrEF-deficient cells with increased expression of AcrA have a severe cell division defect that results in cell filamentation (>50 μm). Similar defects were obtained in cells lacking the outer membrane channel TolC, which acts with AcrEF, suggesting that cell filamentation is caused by the loss of AcrEF function. Green fluorescent protein-AcrA fusion studies showed that in normal and filamentous cells AcrA is associated with membranes in a confined manner and that this localization is not affected by the lack of AcrEF. Similarly, the structure and composition of membranes were normal in filamentous cells. Fluorescence microscopy showed that the filamentous AcrEF-deficientE. colicells are defective in chromosome condensation and segregation. Our results suggest that theE. coliAcrEF transporter is expressed under standard laboratory conditions and plays an important role in the normal maintenance of cell division.
Catabolite Repression and Activation in
Bacillus subtilis
: Dependency on CcpA, HPr, and HprK
Authors: Lorca Graciela L. , Chung Yong Joon , Barabote Ravi D. , Weyler Walter , Schilling Christophe H. , Saier Milton H.
Published: November 2005

Previous studies have suggested that the transcription factor CcpA, as well as the coeffectors HPr and Crh, both phosphorylated by the HprK kinase/phosphorylase, are primary mediators of catabolite repression and catabolite activation in
Bacillus subtilis
. We here report whole transcriptome analyses that characterize glucose-dependent gene expression in wild-type cells and in isogenic mutants lacking CcpA, HprK, or the HprK phosphorylatable serine in HPr. Binding site identification revealed which genes are likely to be primarily or secondarily regulated by CcpA. Most genes subject to CcpA-dependent regulation are regulated fully by HprK and partially by serine-phosphorylated HPr [HPr(Ser-P)]. A positive linear correlation was noted between the dependencies of catabolite-repressible gene expression on CcpA and HprK, but no such relationship was observed for catabolite-activated genes, suggesting that large numbers of the latter genes are not regulated by the CcpA-HPr(Ser-P) complex. Many genes that mediate nitrogen or phosphorus metabolism as well as those that function in stress responses proved to be subject to CcpA-dependent glucose control. While nitrogen-metabolic genes may be subject to either glucose repression or activation, depending on the gene, almost all glucose-responsive phosphorus-metabolic genes exhibit activation while almost all glucose-responsive stress genes show repression. These responses are discussed from physiological standpoints. These studies expand our appreciation of CcpA-mediated catabolite control and provide insight into potential interregulon control mechanisms in gram-positive bacteria.
The
Chlamydia pneumoniae
Type III Secretion-Related
lcrH
Gene Clusters Are Developmentally Expressed Operons
Authors: Ouellette Scot P. , AbdelRahman Yasser M. , Belland Robert J. , Byrne Gerald I.
Published: November 2005

Two chlamydial homologues of the
Yersinia lcrH
chaperone for type III secretion system structural components are present within separate gene clusters. Quantitative transcriptional analyses demonstrated that each cluster is differentially regulated and expressed as an operon using major sigma factor elements, suggesting the presence of more elaborate developmental regulation mechanisms in chlamydiae.
Actin-Binding Proteins from
Burkholderia mallei
and
Burkholderia thailandensis
Can Functionally Compensate for the Actin-Based Motility Defect of a
Burkholderia pseudomallei bimA
Mutant
Authors: Stevens Joanne M. , Ulrich Ricky L. , Taylor Lowrie A. , Wood Michael W. , DeShazer David , Stevens Mark P. , Galyov Edouard E.
Published: November 2005

Recently we identified a bacterial factor (BimA) required for actin-based motility of
Burkholderia pseudomallei
. Here we report that
Burkholderia mallei
and
Burkholderia thailandensis
are capable of actin-based motility in J774.2 cells and that BimA homologs of these bacteria can restore the actin-based motility defect of a
B. pseudomallei bimA
mutant. While the BimA homologs differ in their amino-terminal sequence, they interact directly with actin in vitro and vary in their ability to bind Arp3.
Identification of a Novel Essential Two-Component Signal Transduction System, YhcSR, inStaphylococcus aureus
Authors: Sun Junsong , Zheng Li , Landwehr Christina , Yang Junshu , Ji Yinduo
Published: November 2005

Two-component signal transduction systems play an important role in the ability of bacteria to adapt to various environments by sensing changes in their habitat and by altering gene expression. In this study, we report a novel two-component system, YhcSR, inStaphylococcus aureuswhich is required for bacterial growth in vitro. We found that the down-regulation ofyhcSRexpression by inducedyhcSantisense RNA can inhibit and terminate bacterial growth. Moreover, without complementaryyhcSoryhcR, no viableyhcSoryhcRgene replacement mutant was recoverable. Collectively, these results demonstrated that the YhcSR regulatory system is indispensable forS. aureusgrowth in culture. Moreover, inducedyhcSantisense RNA selectively increased bacterial susceptibility to phosphomycin. These data suggest that YhcSR probably modulates the expression of genes critical for bacterial survival and may be a potential target for the development of novel antibacterial agents.
Effect of intravenous amino acids on glutamine and protein kinetics in low-birth-weight preterm infants during the immediate neonatal period
Authors: Kadrofske Mark M. , Parimi Prabhu S. , Gruca Lourdes L. , Kalhan Satish C.
Published: November 2005

Glutamine may be a conditionally essential amino acid in low-birth-weight (LBW) preterm neonates. Exogenously administered amino acids, by providing anaplerotic carbon into the tricarboxylic acid cycle, could result in greater cataplerotic efflux and glutamine de novo synthesis. The effect of dose and duration of amino acid infusion on glutamine and nitrogen (N) kinetics was examined in LBW infants in the period immediately after birth. Preterm neonates (<32 weeks gestation, birth weights 809–1,755 g) were randomized to initially receive either 480 or 960 μmol·kg−1·h−1 of an intravenous amino acid solution for 19–24 hours, followed by a higher or lower amino acid load for either 5 h or 24 h. Glutamine de novo synthesis, leucine N, phenylalanine, and urea kinetics were determined using stable isotopic tracers. An increase in amino acid infusion from 480 to 960 μmol·kg−1·h−1 for 5 h resulted in decreased glutamine de novo synthesis in every neonate (384.4 ± 38.0 to 368.9 ± 38.2 μmol·kg−1·h−1, P < 0.01) and a lower whole body rate of proteolysis ( P < 0.001) and urea synthesis ( P < 0.001). However, when the increased amino acid infusion was extended for 24 h, glutamine de novo synthesis increased (369.7 ± 92.6 to 483.4 ± 97.5 μmol·kg−1·h−1, P < 0.001), whole body rate of proteolysis did not change, and urea production increased. Decreasing the amino acid load resulted in a decrease in glutamine rate of appearance (Ra) and leucine N Ra, but had no effect on phenylalanine Ra. Acutely stressed LBW infants responded to an increase in amino acid load by transiently suppressing whole body rate of glutamine synthesis, proteolysis, and oxidation of protein. The mechanisms of this transient effect on whole body protein/nitrogen metabolism remain unknown.
Oxandrolone enhances skeletal muscle myosin synthesis and alters global gene expression profile in Duchenne muscular dystrophy
Authors: Balagopal Prabhakaran , Olney Robert , Darmaun Dominique , Mougey Edward , Dokler Maryann , Sieck Gary , Hammond David
Published: November 2005

Earlier studies have shown that the progressive, unrelenting muscle loss associated with Duchenne muscular dystrophy (DMD) involves an imbalance between the rates of synthesis and degradation of muscle proteins. Although previous studies have suggested that oxandrolone may be beneficial in DMD, the mechanism of action of oxandrolone on muscle in DMD remains unclear. To address these issues, we combined stable isotope studies and gene expression analysis to measure the fractional synthesis rate of myosin heavy chain (MHC), the key muscle contractile protein, the transcript levels of the isoforms of MHC, and global gene expression profiles in four children with DMD before and after 3 mo of treatment with oxandrolone. Gastrocnemius muscle biopsies and blood samples were collected during the course of a primed 6-h continuous infusion of l-[U-13C]leucine on two separate occasions, before and after the 3-mo treatment with oxandrolone (0.1 mg·kg−1·day−1). Gene expression analysis was done with microarrays and RT-qPCR. In response to the treatment, MHC synthesis rate increased 42%, and this rise was accounted for, at least in part, by an upregulation of the transcript for MHC8 (perinatal MHC). Gene expression data suggested a decrease in muscle regeneration as a consequence of oxandrolone therapy, presumably because of a decrease in muscle degeneration. These findings suggest that 1) oxandrolone has a powerful protein anabolic effect on a key contractile protein and 2) larger and longer-term studies are warranted to determine whether these changes translate into meaningful therapy for these patients.