bluuebunny/crossref_metadata_2025 huggingface dataset:
Fatty Acid Ethyl Ester Effects on Interleukin‐2 Production, Cyclic AMP Synthesis, and Calcium Influx in Human Mononuclear Cells
Authors: Alhomsi Khaled , Laposata Michael
Published: June 2006

Background: Fatty acid ethyl esters (FAEE), nonoxidative ethanol metabolites, are produced by the esterification of fatty acids and ethanol. Plasma and serum FAEE correlate linearly with blood ethanol levels and are present in organs most commonly damaged by ethanol abuse. Our previous studies have shown that there is significant synthesis of FAEE by human mononuclear cells within seconds of exposure to physiologic doses of ethanol.

Objectives: We studied the effects of FAEE on selected early events of mononuclear cell activation that follow stimulation with phytohemagglutinin (PHA). Fatty acid ethyl esters induced changes in cytosolic free calcium ([Ca2+]) levels, the production and secretion of interleukin‐2 (IL‐2) by the cells, and intracellular cAMP concentrations.

Methods: A mononuclear fraction of human white blood cells (WBC) was incubated with physiologic doses of FAEE. For experiments involving IL‐2 production and calcium influx, PHA‐stimulated cells were incubated with 10, 25, 50, or 100 μM ethyl oleate, a representative FAEE species, for 60 minutes. Interleukin‐2 was measured by enzyme immunometeric assay and maximum levels of Ca2+ were monitored by spectrofluorimetry. In other experiments, mononuclear cells were incubated with 10, 25, and 50 μM ethyl oleate for 0.08 to 120 minutes, and then the concentration of cAMP was determined by a cAMP competitive enzyme immunoassay system.

Results: Fatty acid ethyl esters inhibited the PHA‐induced IL‐2 production and secretion in activated human mononuclear cells. Fatty acid ethyl esters also inhibited PHA‐induced [Ca2+] influx into cells in a dose‐dependent fashion. There was a rapid increase in the intracellular cAMP concentration of mononuclear cells induced by FAEE, with FAEE dose dependence. The cAMP concentration decreased as the incubation time with FAEE was increased.

Conclusions: Fatty acid ethyl esters inhibited PHA‐stimulated IL‐2 production and Ca2+ influx into human mononuclear cells and elevated intracellular cAMP concentration. These changes in mononuclear cell signaling may be associated with the immunosuppression associated with alcohol abuse.
Novelty Seeking as a Moderator of Familial Risk for Alcohol Dependence
Authors: Grucza Richard A. , Robert Cloninger C. , Bucholz Kathleen K. , Constantino John N. , Schuckit Marc I. , Dick Danielle M. , Bierut Laura J.
Published: June 2006

Background: Disinhibitory personality traits such as high novelty seeking (NS) are moderately heritable, and individuals with substance use disorders (SUDs) frequently exhibit such traits. However, recent studies have cast doubt on the supposition that such traits are true familial risk factors for SUD and particularly for alcohol dependence. Another possibility is that familial risk interacts with personality‐associated risk, in which case the association between personality and familial risk might depend on sample composition, accounting for the lack of consensus among studies to date. We examined this possibility by analyzing the association between NS and alcohol dependence in individuals at intermediate and high levels of familial risk for alcohol dependence.

Methods: Data from the Collaborative Study on the Genetics of Alcoholism, a multisite family study, were examined. Subjects were 1,111 adult siblings of alcohol‐dependent index cases. Parental diagnoses of alcohol dependence and personality scores of NS from the Tridimensional Personality Questionnaire were used to predict alcohol dependence.

Results: A significant interaction between NS and familial risk for alcoholism was seen, such that NS was a significantly stronger predictor of alcohol dependence in subjects with one or more parents with alcohol dependence than in subjects without alcohol‐dependent parents.

Conclusions: Novelty seeking and familial risk interact so that the risk associated with high NS is magnified in families with parental alcohol dependence and NS is a moderator of familial risk. Accordingly, high NS is strongly associated with alcohol dependence in subjects with a parent diagnosed with alcohol dependence, but low NS may protect against the risk associated with familial alcoholism. This interaction may account for conflicting findings from studies that have examined this question previously.
Applying Conceptual Grammar to Advanced‐Level Language Teaching: The Case of Two Completive Constructions in Korean
Authors: STRAUSS SUSAN , LEE JIHYE , AHN KYUNGJA
Published: June 2006

This article introduces conceptual grammar as an approach to the analysis and teaching of grammar in foreign and second language contexts through a combination of paradigms: corpus, discourse analysis, and cognitive linguistics. Although the approach is applicable to virtually any language and any construction within that language at various levels of study, we provide a detailed demonstration using Korean as a model. In particular, we focus on constructions expressing the completive aspect. The Korean system of marking aspect can be quite complex; what renders the Korean completive even more perplexing is the fact that it is expressed through two seemingly similar auxiliary forms, each of which signals different elements in the speaker's or writer's stance vis‐à‐vis the described event. By combining the paradigms of corpus, discourse analysis, and cognitive linguistics, the article demonstrates how a conceptual grammatical approach can render salient the particular discursive and conceptual patterns underlying the target forms. It is designed as a pedagogical tool to guide users to discern both inductively and deductively how native speakers conceptualize these differences and express them morphosyntactically—a perspective that is absent from most existing reference grammars and textbooks. In this article, we present samples of pedagogical materials developed using this model in addition to results of an experiment in which a version of those materials was administered to teachers and students of advanced Korean.
Effect of Liquid Phase Chemistry on Single‐Crystal Growth in PMN–35PT
Authors: Gorzkowski Edward P. , Watanabe Masashi , Chan Helen M. , Harmer Martin P.
Published: June 2006

This study investigated the liquid phase chemistry in Pb(Mg1/3Nb2/3)O3–35 mol% PbTiO3with excess PbO as previous studies suggested that the chemistry of the grain boundary phase may change {001} single‐crystal growth. It has been shown that the chemistry of the liquid phase was dynamic as characterized in an analytical electron microscope. In fact, MgO was found to precipitate out of the system due to the saturation of MgO in the liquid, which in turn altered the liquid/solid surface energy. By changing the surface energy of the system, the matrix grains became more faceted, i.e. the frequency of {001} surfaces increased. This reduced the driving force for growth of the single crystal, which appears to be responsible for reducing the growth rate during annealing. Additional supporting evidence came from observing the effect of varying the PbTiO3content.
Consolidation of Nano‐Sized TiN Powders by Spark Plasma Sintering
Authors: Wang Lianjun , Jiang Wan , Chen Lidong , Yang Mei , Zhu Hongmin
Published: June 2006

Nano‐sized TiN powders with an average particle size of 19 nm were synthesized via a new method, reduction–nitridation reaction in liquid ammonia. A consolidation procedure using spark plasma sintering (SPS) was used, and a dense TiN ceramic (>98% of theoretical) with mean grain size of 100–150 nm was obtained at 1380°C. The influence of sintering temperature on grain growth and microstructural evolution was also discussed.
Healing of intrabony peri‐implantitis defects following application of a nanocrystalline hydroxyapatite (Ostim™) or a bovine‐derived xenograft (Bio‐Oss™) in combination with a collagen membrane (Bio‐Gide™). A case series
Authors: Schwarz F. , Bieling K. , Latz T. , Nuesry E. , Becker J.
Published: June 2006

Objectives: The aim of the present case series was to evaluate the healing of intrabony peri‐implantitis defects following application of a nanocrystalline hydroxyapatite (NHA) or a bovine‐derived xenograft in combination with a collagen membrane (BDX+BG).

Material and Methods: Twenty‐two patients having moderate peri‐implantitis (n=22 intrabony defects) were randomly treated with (i) access flap surgery (AFS) and the application of NHA, or with AFS and the application of BDX+BG. Clinical parameters were recorded at baseline and after 6 months of non‐submerged healing.

Results: Post‐operative wound healing revealed that NHA compromized initial adhesion of the mucoperiosteal flaps in all patients. At 6 months after therapy, NHA showed a reduction in the mean PD from 7.0 ± 0.6 to 4.9 ± 0.6 mm and a change in the mean clinical attachment loss (CAL) from 7.5 ± 0.8 to 5.7 ± 1.0 mm. In the BDX+BC group, the mean PD was reduced from 7.1 ± 0.8 to 4.5 ± 0.7 mm and the mean CAL changed from 7.5 ± 1.0 to 5.2 ± 0.8 mm.

Conclusion: Within the limits of the present case series, it can be concluded that at 6 months after surgery both therapies resulted in clinically important PD reductions and CAL gains.
Linking habitat selection, emigration and population dynamics of freshwater fishes: a synthesis of ideas and approaches
Authors: McMahon T. E. , Matter W. J.
Published: June 2006

Abstract – The consequences of individual behaviour to dynamics of populations has been a critical question in fish ecology, but linking the two has proven difficult. A modification of Sale's habitat selection model provides a conceptual linkage for relating resource availability and individual habitat selection to exploratory behaviour, emigration and population‐level responses. Whole‐population experiments with pupfishCyprinodon maculariusthat linked all factors along this resource to population continuum lend support to this conceptual model, and illustrate that emigration may be much more common in fish populations than considered in most individual‐ or population‐based models. Accommodating emigration can enhance the ecological appropriateness of behavioural experiments and increase confidence in extrapolation of experimental observations to population‐level effects. New experimental designs and advancing technologies offer avenues for assessing population consequences of habitat selection and emigration by individual fish. Emigration often is the key linkage between individual behaviour and population responses, and greater understanding of the underlying factors affecting this often‐overlooked demographic parameter could offer new approaches for management and conservation of fishes.
Annual and seasonal dynamics of fish in the brackish‐water Matsalu Bay, Estonia
Authors: Vetemaa M. , Eschbaum R. , Verliin A. , Albert A. , Eero M. , Lillemägi R. , Pihlak M. , Saat T.
Published: June 2006

Abstract –  Matsalu Bay in the Väinameri (West‐Estonian Archipelago Sea) is a relatively shallow but large bay. It is the only real delta estuary in the northern Baltic Sea. The fish fauna is strongly dominated by freshwater species such as perch Perca fluviatilis, roach Rutilus rutilus, white bream Blicca bjoerkna and rudd Scardinius erythrophthalmus. The study presents CPUE data describing annual and seasonal variations in the species composition, abundance and spatial distribution, based on 1 year of monthly samplings from August 2001 to August 2002 and routine coastal monitoring (in late July–early August) between 1994 and 2003. The bay can be divided into three rather distinct parts. The fish fauna of the inner part (salinity 0–2 p.p.t.) is different from two open parts and resembles that of freshwater lakes. In the openmost part (salinity usually 4–6 p.p.t.) some marine species, such as herring Clupea harengus membras, flounder Platichthys flesus and eelpout Zoarces viviparus may seasonally be abundant. Species composition and abundance of most species varies seasonally and interannually. Only the abundance of few species such as pike Esox lucius and pikeperch Stizostedion lucioperca was not impacted by seasonality. The most dynamic period in the bay is spring, when several species (some of them very rare in summer, such as herring and smelt Osmerus eperlanus) enter the bay for spawning.
Modification of 0+ perch foraging behaviour by indirect cues of predation risk
Authors: Wanzenböck J. , Mikheev V. N. , Pasternak A. F.
Published: June 2006

Abstract – Intake rate and prey size selection of 0+ perch,Perca fluviatilis, from Lake Wallersee (Austria) was studied at different illumination (day light: 400 lx, twilight: 2 lx) during 5 days of habituation to novel surroundings in aquaria. The hypothesis was tested that high illumination and novelty of surroundings (transfer from holding to test aquaria) as indirect cues of predation risk influences 0+ perch foraging behaviour. Significantly lower total intake and lower proportion of largeDaphnia magna(two prey size groups, 2.9 and 1.2 mm, were used) were observed at higher illumination and under novel surroundings. Habituation to novelty caused an increase in consumption of large prey and decrease in consumption of small prey. During the whole period of habituation, more large prey was ingested at twilight than at daylight; no light‐induced difference in ingestion of small prey was found. Foraging 0+ perch responded to indirect nonspecific cues of predation risk by reduction of intake of large prey, which are costly in terms of handling time. This allows fish to be more vigilant without ceasing their foraging activity even in potentially dangerous situations. In the lake, young perch are most vulnerable to abundant piscivorous fish and birds during the day; in twilight perch can afford a more time‐consuming foraging activity.
Breastfeeding and family life
Authors: Lavender Tina , McFadden Catherine , Baker Lisa
Published: June 2006

The problems faced by breastfeeding mothers are well documented. However, the influence of social networks has mainly received attention solely through the eyes of the women. Therefore, we explored the views of the family as a whole. This exploratory study utilizes semi‐structured interviews, diaries and questionnaires. A purposive sample of 24 women and their families, from a hospital in the north‐west of England were invited to participate. Questionnaire data were analysed descriptively. Diaries and interviews were analysed using an open coding mechanism to identify emergent themes. Twenty‐three women and 27 of their family members participated. Questionnaire data showed that the majority of women (n = 17) expected to breastfeed for more than 3 months; 12 actually did this. Women anticipated that family members would provide the main source of breastfeeding support. Three main themes emerged from the interviews and diaries: ‘moving with the times’, ‘marketable commodity’ and ‘disparate communications’. The authors conclude that multi‐layered approach to breastfeeding promotion and support should be considered. Society needs to proactively encourage a positive breastfeeding culture, family members need direction on how to support a woman to breastfeed and women need to be able to articulate their individual requirements. Midwives could be instrumental in supporting such needs and facilitating change.
Effects of food type, feeding frequency, and temperature on juvenile survival and growth of Marisa cornuarietis (Mollusca: Gastropoda)
Authors: Selck Henriette , Aufderheide John , Pounds Nadine , Staples Charles , Caspers Norbert , Forbes Valery
Published: June 2006

Abstract. The present experiments are part of a larger study designed to investigate the influence of husbandry parameters on the life history of the ramshorn snail, Marisa cornuarietis, in order to identify suitable husbandry conditions for maintaining multi‐generation populations in the laboratory for use in ecotoxicological testing. In this paper we focus on the effects of a combination of food types and feeding frequencies (i.e., the frequency with which the snails were offered food) on juvenile growth and survival at different temperatures. Offspring produced in the laboratory by wild specimens of M. cornuarietis, from Puerto Rico, were used to test the effects of three types of food (lettuce, alginate with fish food, alginate with snail mix) fed at three frequencies (given ad libitum on 4/4, 2/4, or 1/4 d) on juvenile survival and growth. The 4‐d feeding regimens were repeated four times, giving a total of 16 d for the experiments. The experiments were conducted at two temperatures (22° and 25°C) under a 12 h light:12 h dark photoperiod. Juvenile growth rates increased with increasing feeding frequency for all food types. The most rapid growth rates occurred in the high‐frequency lettuce treatments and the slowest growth rates in the low‐frequency lettuce and alginate with snail mix treatments. Juvenile snails grew faster at 25° than at 22°C, and mortality was about twice as high at the lower temperature. Growth rates were used to provide a rough estimate of time to maturity, which was determined to take about twice as long at 22° than at 25°C. The results showed that lettuce is the best food if supplied in abundance, but effects on growth are very dependent on feeding frequency and temperature. We conclude that 25°C is a more appropriate temperature for maintaining populations than 22°C, that lettuce provides a suitable food source, and that food should be supplied continuously for husbandry and toxicity testing of populations of M. cornuarietis.
Direct development of the brittle star Amphiodia occidentalis (Ophiuroidea, Amphiuridae) from the northeastern Pacific Ocean
Authors: Emlet Richard B.
Published: June 2006

Abstract. The highly modified development of the brittle star Amphiodia occidentalis is described from post‐fertilization to the juvenile stage. Fertilized eggs are negatively buoyant, ∼190 μm in diameter, surrounded by a thick hyaline layer and a tough fertilization envelope. After gastrulation, embryos flatten into a bilaterally symmetrical disk with a U‐shaped ridge surrounding an indented stomodeum on the oral surface. Internally, a ring of ∼22 calcitic ossicles grows at the edge of the disk. Vestigial ophiopluteal structures such as a ciliated band, paired larval spicules, or larval arms are not expressed during development. Although the fertilization envelope disintegrates on day 3, developmental stages remain immotile for five more days until they move with podia. At hatching, five hydrocoelic lobes are evident on the left side of the post‐gastrula, and these migrate clockwise around the stomodeum, establishing pentamerous radial symmetry. Central and radial plates originate on the right side and migrate to a dorsocentral location as pentamerous symmetry is established. Development of the juvenile oral skeletal frame follows closely that described by Hendler (1978) for Amphioplus abditus except that A. occidentalis did not form buccal scales. The juvenile mouth opened by day 12. Fifty‐five days after fertilization, juveniles had not added their first arm segments, although the first lateral arm plates had appeared. Developmental stages identical to those described here have been found in plankton tows taken in Oregon usually after storms that bring high waves. The unusual development of this species probably occurs in both benthic and pelagic environments.
Immunohistochemical localization of vitellogenin in the hepatic diverticulum of the amphioxus Branchiostoma belcheri tsingtauense, with implications for the origin of the liver
Authors: Han Li , Zhang Shicui , Wang Yongjun , Sun Xutong
Published: June 2006

Abstract. The localization of vitellogenin (Vg) remains untested in amphioxus. Western blotting analysis showed that mouse anti‐amphioxus Vg sera cross‐reacted with the crude extracts of the hepatic diverticulum and ovary, and humoral fluids, including blood and coelomic fluid, from specimens of Branchiostoma belcheri. Similarly, immunohistochemical staining also revealed that Vg is localized in the hepatic diverticulum, sub‐intestinal vessels, and ovary in amphioxus. The hepatic diverticulum of amphioxus, like the vertebrate liver, synthesizes Vg, suggesting that amphioxus hepatic diverticulum is functionally equivalent to the vertebrate liver in respect of the synthesis of this compound. This agrees with the idea that amphioxus hepatic diverticulum is the precursor of vertebrate liver.
Effect of Repeat Copy Number on Variable-Number Tandem Repeat Mutations in
Escherichia coli
O157:H7
Authors: Vogler Amy J. , Keys Christine , Nemoto Yoshimi , Colman Rebecca E. , Jay Zack , Keim Paul
Published: June 2006

Variable-number tandem repeat (VNTR) loci have shown a remarkable ability to discriminate among isolates of the recently emerged clonal pathogen
Escherichia coli
O157:H7, making them a very useful molecular epidemiological tool. However, little is known about the rates at which these sequences mutate, the factors that affect mutation rates, or the mechanisms by which mutations occur at these loci. Here, we measure mutation rates for 28 VNTR loci and investigate the effects of repeat copy number and mismatch repair on mutation rate using in vitro-generated populations for 10
E. coli
O157:H7 strains. We find single-locus rates as high as 7.0 × 10
−4
mutations/generation and a combined 28-locus rate of 6.4 × 10
−4
mutations/generation. We observed single- and multirepeat mutations that were consistent with a slipped-strand mispairing mutation model, as well as a smaller number of large repeat copy number mutations that were consistent with recombination-mediated events. Repeat copy number within an array was strongly correlated with mutation rate both at the most mutable locus, O157-10 (
r
2
= 0.565,
P
= 0.0196), and across all mutating loci. The combined locus model was significant whether locus O157-10 was included (
r
2
= 0.833,
P
< 0.0001) or excluded (
r
2
= 0.452,
P
< 0.0001) from the analysis. Deficient mismatch repair did not affect mutation rate at any of the 28 VNTRs with repeat unit sizes of >5 bp, although a poly(G) homomeric tract was destabilized in the
mutS
strain. Finally, we describe a general model for VNTR mutations that encompasses insertions and deletions, single- and multiple-repeat mutations, and their relative frequencies based upon our empirical mutation rate data.
Mutations and Rearrangements in the Genome ofSulfolobus solfataricusP2
Authors: Redder Peter , Garrett Roger A.
Published: June 2006

The genome ofSulfolobus solfataricusP2 carries a larger number of transposable elements than any other sequenced genome from an archaeon or bacterium and, as a consequence, may be particularly susceptible to rearrangement and change. In order to gain more insight into the natures and frequencies of different types of mutation and possible rearrangements that can occur in the genome, thepyrEFlocus was examined for mutations that were isolated after selection with 5-fluoroorotic acid. About two-thirds of the 130 mutations resulted from insertions of mobile elements, including insertion sequence (IS) elements and a single nonautonomous mobile element, SM2. For each of these, the element was identified and shown to be present at its original genomic position, consistent with a progressive increase in the copy numbers of the mobile elements. In addition, several base pair substitutions, as well as small deletions, insertions, and a duplication, were observed, and about one-fifth of the mutations occurred elsewhere in the genome, possibly in an orotate transporter gene. One mutant exhibited a 5-kb genomic rearrangement at thepyrEFlocus involving a two-step IS element-dependent reaction, and its boundaries were defined using a specially developed “in vitro library” strategy. Moreover, while searching for the donor mobile elements, evidence was found for two major changes that had occurred in the genome of strain P2, one constituting a single deletion of about 4% of the total genome (124 kb), while the other involved the inversion of a 25-kb region. Both were bordered by IS elements and were inferred to have arisen through recombination events. The results underline the caution required in working experimentally with an organism such asS. solfataricuswith a continually changing genome.
Transcriptional and Translational Control of the
Salmonella fliC
Gene
Authors: Aldridge Phillip , Gnerer Joshua , Karlinsey Joyce E. , Hughes Kelly T.
Published: June 2006

The flagellin gene
fliC
encodes the major component of the flagellum in
Salmonella enterica
serovar Typhimurium. This study reports the identification of a signal within the 5′ untranslated region (5′UTR) of the
fliC
transcript required for the efficient expression and assembly of FliC into the growing flagellar structure. Primer extension mapping determined the transcription start site of the
fliC
flagellin gene to be 62 bases upstream of the AUG start codon. Using
tetA-fliC
operon fusions, we show that the entire 62-base 5′UTR region of
fliC
was required for sufficient
fliC
mRNA translation to allow normal FliC flagellin assembly, suggesting that translation might be coupled to assembly. To identify sequence that might couple
fliC
mRNA translation to FliC secretion, the 5′ end of the chromosomal
fliC
gene was mutagenized by PCR-directed mutagenesis. Single base sequences important for
fliC
-dependent transcription, translation, and motility were identified by using
fliC-lacZ
transcriptional and translational reporter constructs. Transcription-specific mutants identified the −10 and −35 regions of the consensus flagellar class 3 gene promoter. Single base changes defective in translation were located in three regions: the AUG start codon, the presumed ribosomal binding site region, and a region near the very 5′ end of the
fliC
mRNA that corresponded to a potential stem-loop structure in the 5′UTR. Motility-specific mutants resulted from base substitutions only in the
fliC
-coding region. The results suggest that
fliC
mRNA translation is not coupled to FliC secretion by the flagellar type III secretion system.
Characterization of the Ends and Target Site of a Novel Tetracycline Resistance-Encoding Conjugative Transposon from
Enterococcus faecium
664.1H1
Authors: Roberts Adam P. , Davis Ian J. , Seville Lorna , Villedieu Aurelie , Mullany Peter
Published: June 2006

Enterococcus faecium
664.1H1 is multiply antibiotic resistant and mercury resistant. In this study, the genetic support for the tetracycline resistance of
E. faecium
664.1H1 was characterized. The
tet
(S) gene is responsible for tetracycline resistance, and this gene is located on the chromosome of
E. faecium
664.1H1, on a novel conjugative transposon. The element is transferable to
Enterococcus faecalis
, where it integrates into a specific site. The element was designated EfcTn
1
. The integrase of EfcTn
1
is related to the integrase proteins found on staphylococcal pathogenicity islands. We show that the transposon is flanked by an 18-bp direct repeat, a copy of which is also present at the target site and at the joint of a circular form, and we propose a mechanism of insertion and excision.
Citric Acid Cycle in the Hyperthermophilic Archaeon
Pyrobaculum islandicum
Grown Autotrophically, Heterotrophically, and Mixotrophically with Acetate
Authors: Hu Yajing , Holden James F.
Published: June 2006

The hyperthermophilic archaeon
Pyrobaculum islandicum
uses the citric acid cycle in the oxidative and reductive directions for heterotrophic and autotrophic growth, respectively, but the control of carbon flow is poorly understood.
P. islandicum
was grown at 95°C autotrophically, heterotrophically, and mixotrophically with acetate, H
2
, and small amounts of yeast extract and with thiosulfate as the terminal electron acceptor. The autotrophic growth rates and maximum concentrations of cells were significantly lower than those in other media. The growth rates on H
2
and 0.001% yeast extract with and without 0.05% acetate were the same, but the maximum concentration of cells was fourfold higher with acetate. There was no growth with acetate if 0.001% yeast extract was not present, and addition of H
2
to acetate-containing medium greatly increased the growth rates and maximum concentrations of cells.
P. islandicum
cultures assimilated
14
C-labeled acetate in the presence of H
2
and yeast extract with an efficiency of 55%. The activities of 11 of 19 enzymes involved in the central metabolism of
P. islandicum
were regulated under the three different growth conditions. Pyruvate synthase and acetate:coenzyme A (CoA) ligase (ADP-forming) activities were detected only in heterotrophically grown cultures. Citrate synthase activity decreased in autotrophic and acetate-containing cultures compared to the activity in heterotrophic cultures. Acetylated citrate lyase, acetate:CoA ligase (AMP forming), and phosphoenolpyruvate carboxylase activities increased in autotrophic and acetate-containing cultures. Citrate lyase activity was higher than ATP citrate synthase activity in autotrophic cultures. These data suggest that citrate lyase and AMP-forming acetate:CoA ligase, but not ATP citrate synthase, work opposite citrate synthase to control the direction of carbon flow in the citric acid cycle.
N
-Acyl-
l
-Homoserine Lactone Quorum Sensing Controls Butanediol Fermentation in
Serratia plymuthica
RVH1 and
Serratia marcescens
MG1
Authors: Van Houdt Rob , Moons Pieter , Hueso Buj Maria , Michiels Chris W.
Published: June 2006

Butanediol fermentation in two
Serratia
species is shown to be affected by
N
-acyl-
l
-homoserine lactone-dependent quorum sensing. Knockout of quorum-sensing signal production caused a shift towards enhanced acid production, resulting in early growth arrest, which was reversible by the addition of synthetic signal molecules.
The Lipid A 1-Phosphatase ofHelicobacter pyloriIs Required for Resistance to the Antimicrobial Peptide Polymyxin
Authors: Tran An X. , Whittimore Judy D. , Wyrick Priscilla B. , McGrath Sara C. , Cotter Robert J. , Trent M. Stephen
Published: June 2006

Modification of the phosphate groups of lipid A with amine-containing substituents, such as phosphoethanolamine, reduces the overall net negative charge of gram-negative bacterial lipopolysaccharide, thereby lowering its affinity to cationic antimicrobial peptides. Modification of the 1 position ofHelicobacter pylorilipid A is a two-step process involving the removal of the 1-phosphate group by a lipid A phosphatase, LpxEHP(Hp0021), followed by the addition of a phosphoethanolamine residue catalyzed by EptAHP(Hp0022). To demonstrate the importance of modifying the 1 position ofH. pylorilipid A, we generated LpxEHP-deficient mutants in variousH. pyloristrains by insertion of a chloramphenicol resistance cassette intolpxEHPand examined the significance of LpxE with respect to cationic antimicrobial peptide resistance. Using both mass spectrometry analysis and an in vitro assay system, we showed that the loss of LpxEHPactivity in variousH. pyloristrains resulted in the loss of modification of the 1 position ofH. pylorilipid A, thus confirming the function of LpxEHP. Due to its unique lipid A structure,H. pyloriis highly resistant to the antimicrobial peptide polymyxin (MIC > 250 μg/ml). However, disruption oflpxEHPinH. pyloriresults in a dramatic decrease in polymyxin resistance (MIC, 10 μg/ml). In conclusion, we have characterized the first gram-negative LpxE-deficient mutant and have shown the importance of modifying the 1 position ofH. pylorilipid A for resistance to polymyxin.
A LuxS-Dependent Cell-to-Cell Language Regulates Social Behavior and Development inBacillus subtilis
Authors: Lombardía Esteban , Rovetto Adrián J. , Arabolaza Ana L. , Grau Roberto R.
Published: June 2006

Cell-to-cell communication in bacteria is mediated by quorum-sensing systems (QSS) that produce chemical signal molecules called autoinducers (AI). In particular, LuxS/AI-2-dependent QSS has been proposed to act as a universal lexicon that mediates intra- and interspecific bacterial behavior. Here we report that the model organismBacillus subtilisoperates aluxS-dependent QSS that regulates its morphogenesis and social behavior. We demonstrated thatB. subtilis luxSis a growth-phase-regulated gene that produces active AI-2 able to mediate the interspecific activation of light production inVibrio harveyi. We demonstrated that inB. subtilis,luxSexpression was under the control of a novel AI-2-dependent negative regulatory feedback loop that indicated an important role for AI-2 as a signaling molecule. Even thoughluxSdid not affect spore development, AI-2 production was negatively regulated by the master regulatory proteins of pluricellular behavior, SinR and Spo0A. Interestingly, wildB. subtiliscells, from the undomesticated and probioticB. subtilisnatto strain, required the LuxS-dependent QSS to form robust and differentiated biofilms and also to swarm on solid surfaces. Furthermore, LuxS activity was required for the formation of sophisticated aerial colonies that behaved as giant fruiting bodies where AI-2 production and spore morphogenesis were spatially regulated at different sites of the developing colony. We proposed that LuxS/AI-2 constitutes a novel form of quorum-sensing regulation where AI-2 behaves as a morphogen-like molecule that coordinates the social and pluricellular behavior ofB. subtilis.
Proteins Exported via the PrsD-PrsE Type I Secretion System and the Acidic Exopolysaccharide Are Involved in Biofilm Formation byRhizobium leguminosarum
Authors: Russo Daniela M. , Williams Alan , Edwards Anne , Posadas Diana M. , Finnie Christine , Dankert Marcelo , Downie J. Allan , Zorreguieta Angeles
Published: June 2006

The type I protein secretion system ofRhizobium leguminosarumbv. viciae encoded by theprsDandprsEgenes is responsible for secretion of the exopolysaccharide (EPS)-glycanases PlyA and PlyB. The formation of a ring of biofilm on the surface of the glass in shaken cultures by both theprsDandprsEsecretion mutants was greatly affected. Confocal laser scanning microscopy analysis of green-fluorescent-protein-labeled bacteria showed that during growth in minimal medium,R. leguminosarumwild type developed microcolonies, which progress to a characteristic three-dimensional biofilm structure. However, theprsDandprsEsecretion mutants were able to form only an immature biofilm structure. A mutant disrupted in the EPS-glycanaseplyBgene showed altered timing of biofilm formation, and its structure was atypical. A mutation in an essential gene for EPS synthesis (pssA) or deletion of several otherpssgenes involved in EPS synthesis completely abolished the ability ofR. leguminosarumto develop a biofilm. Extracellular complementation studies of mixed bacterial cultures confirmed the role of the EPS and the modulation of the biofilm structure by the PrsD-PrsE secreted proteins. Protein analysis identified several additional proteins secreted by the PrsD-PrsE secretion system, and N-terminal sequencing revealed peptides homologous to the N termini of proteins from the Rap family (Rhizobiumadheringproteins), which could have roles in cellular adhesion inR. leguminosarum. We propose a model forR. leguminosarumin which synthesis of the EPS leads the formation of a biofilm and several PrsD-PrsE secreted proteins are involved in different aspects of biofilm maturation, such as modulation of the EPS length or mediating attachment between bacteria.
Pseudomonas aeruginosa
1244 Pilin Glycosylation: Glycan Substrate Recognition
Authors: Horzempa Joseph , Dean Charles R. , Goldberg Joanna B. , Castric Peter
Published: June 2006

The pilin of
Pseudomonas aeruginosa
1244 is glycosylated with an oligosaccharide that is structurally identical to the O-antigen repeating unit of this organism. Concordantly, the metabolic source of the pilin glycan is the O-antigen biosynthetic pathway. The present study was conducted to investigate glycan substrate recognition in the 1244 pilin glycosylation reaction. Comparative structural analysis of O subunits that had been previously shown to be compatible with the 1244 glycosylation machinery revealed similarities among sugars at the presumed reducing termini of these oligosaccharides. We therefore hypothesized that the glycosylation substrate was within the sugar at the reducing end of the glycan precursor. Since much is known of PA103 O-antigen genetics and because the sugars at the reducing termini of the O7 (strain 1244) and O11 (strain PA103) are identical (β-
N
-acetyl fucosamine), we utilized PA103 and strains that express lipopolysaccharide (LPS) with a truncated O-antigen subunit to test our hypothesis. LPS from a strain mutated in the
wbjE
gene produced an incomplete O subunit, consisting only of the monosaccharide at the reducing end (β-
d
-
N
-acetyl fucosamine), indicating that this moiety contained substrate recognition elements for WaaL. Expression of
pilAO
1244
in PA103
wbjE
::
aacC1
, followed by Western blotting of extracts of these cells, indicated that pilin produced has been modified by the addition of material consistent with a single
N
-acetyl fucosamine. This was confirmed by analyzing endopeptidase-treated pilin by mass spectrometry. These data suggest that the pilin glycosylation substrate recognition features lie within the reducing-end moiety of the O repeat and that structures of the remaining sugars are irrelevant.
Translation Inhibition of the
Salmonella fliC
Gene by the
fliC
5′ Untranslated Region,
fliC
Coding Sequences, and FlgM
Authors: Rosu Valentina , Chevance Fabienne F. V. , Karlinsey Joyce E. , Hirano Takanori , Hughes Kelly T.
Published: June 2006

The 5′-untranslated region (5′UTR) of the
fliC
flagellin gene of
Salmonella
contains sequences critical for efficient
fliC
mRNA translation coupled to assembly. In a previous study we used targeted mutagenesis of the 5′ end of the
fliC
gene to isolate single base changes defective in
fliC
gene translation. This identified a predicted stem-loop structure, SL2, as an effector of normal
fliC
mRNA translation. A single base change (−38C:U) in the
fliC
5′UTR resulted in a mutant that is defective in
fliC
mRNA translation and was chosen for this study. Motile (Mot
+
) revertants of the −38C:T mutant were isolated and characterized, yielding several unexpected results. Second-site suppressors that restored
fliC
translation and motility included mutations that disrupt a RNA duplex stem formed between RNA sequences in the
fliC
5′UTR SL2 region (including a precise deletion of SL2) and bases early within the
fliC
-coding region. A stop codon mutation at position 80 of
flgM
also suppressed the −38C:T motility defect, while
flgM
mutants defective in anti-σ
28
activity had no effect on
fliC
translation. One remarkable mutation in the
fliC
5′UTR (−15G:A) results in a translation defect by itself but, in combination with the −38C:U mutation, restores normal translation. These results suggests signals intrinsic to the
fliC
mRNA that have both positive and negative effects on
fliC
translation involving both RNA structure and interacting proteins.
A Glutathione
S
-Transferase Catalyzes the Dehalogenation of Inhibitory Metabolites of Polychlorinated Biphenyls
Authors: Fortin Pascal D. , Horsman Geoff P. , Yang Hao M. , Eltis Lindsay D.
Published: June 2006

BphK is a glutathione
S
-transferase of unclear physiological function that occurs in some bacterial biphenyl catabolic (
bph
) pathways. We demonstrated that BphK of
Burkholderia xenovorans
strain LB400 catalyzes the dehalogenation of 3-chloro 2-hydroxy-6-oxo-6-phenyl-2,4-dienoates (HOPDAs), compounds that are produced by the cometabolism of polychlorinated biphenyls (PCBs) by the
bph
pathway and that inhibit the pathway's hydrolase. A one-column protocol was developed to purify heterologously produced BphK. The purified enzyme had the greatest specificity for 3-Cl HOPDA (
k
cat
/

K
m

, ∼10
4
M
−1
s
−1
), which it dechlorinated approximately 3 orders of magnitude more efficiently than 4-chlorobenzoate, a previously proposed substrate of BphK. The enzyme also catalyzed the dechlorination of 5-Cl HOPDA and 3,9,11-triCl HOPDA. By contrast, BphK did not detectably transform HOPDA, 4-Cl HOPDA, or chlorinated 2,3-dihydroxybiphenyls. The BphK-catalyzed dehalogenation proceeded via a ternary-complex mechanism and consumed 2 equivalents of glutathione (GSH) (

K
m

for GSH in the presence of 3-Cl HOPDA, ∼0.1 mM). A reaction mechanism consistent with the enzyme's specificity is proposed. The ability of BphK to dehalogenate inhibitory PCB metabolites supports the hypothesis that this enzyme was recruited to facilitate PCB degradation by the
bph
pathway.
Structural Classification of Bacterial Response Regulators: Diversity of Output Domains and Domain Combinations
Authors: Galperin Michael Y.
Published: June 2006

CheY-like phosphoacceptor (or receiver [REC]) domain is a common module in a variety of response regulators of the bacterial signal transduction systems. In this work, 4,610 response regulators, encoded in complete genomes of 200 bacterial and archaeal species, were identified and classified by their domain architectures. Previously uncharacterized output domains were analyzed and, in some cases, assigned to known domain families. Transcriptional regulators of the OmpR, NarL, and NtrC families were found to comprise almost 60% of all response regulators; transcriptional regulators with other DNA-binding domains (LytTR, AraC, Spo0A, Fis, YcbB, RpoE, and MerR) account for an additional 6%. The remaining one-third is represented by the stand-alone REC domain (∼14%) and its combinations with a variety of enzymatic (GGDEF, EAL, HD-GYP, CheB, CheC, PP2C, and HisK), RNA-binding (ANTAR and CsrA), protein- or ligand-binding (PAS, GAF, TPR, CAP_ED, and HPt) domains, or newly described domains of unknown function. The diversity of domain architectures and the abundance of alternative domain combinations suggest that fusions between the REC domain and various output domains is a widespread evolutionary mechanism that allows bacterial cells to regulate transcription, enzyme activity, and/or protein-protein interactions in response to environmental challenges. The complete list of response regulators encoded in each of the 200 analyzed genomes is available online athttp://www.ncbi.nlm.nih.gov/Complete_Genomes/RRcensus.html.
Proteomic Analysis ofCampylobacter jejuni11168 Biofilms Reveals a Role for the Motility Complex in Biofilm Formation
Authors: Kalmokoff Martin , Lanthier Patricia , Tremblay Tammy-Lynn , Foss Mary , Lau Peter C. , Sanders Greg , Austin John , Kelly John , Szymanski Christine M.
Published: June 2006

Campylobacter jejuniremains the leading cause of bacterial gastroenteritis in developed countries, and yet little is known concerning the mechanisms by which this fastidious organism survives within its environment. We have demonstrated thatC. jejuni11168 can form biofilms on a variety of surfaces. Proteomic analyses of planktonic and biofilm-grown cells demonstrated differences in protein expression profiles between the two growth modes. Proteins involved in the motility complex, including the flagellins (FlaA, FlaB), the filament cap (FliD), the basal body (FlgG, FlgG2), and the chemotactic protein (CheA), all exhibited higher levels of expression in biofilms than found in stationary-phase planktonic cells. Additional proteins with enhanced expression included those involved in the general (GroEL, GroES) and oxidative (Tpx, Ahp) stress responses, two known adhesins (Peb1, FlaC), and proteins involved in biosynthesis, energy generation, and catabolic functions. An aflagellateflhAmutant not only lost the ability to attach to a solid matrix and form a biofilm but could no longer form a pellicle at the air-liquid interface of a liquid culture. Insertional inactivation of genes that affect the flagellar filament (fliA,flaA,flaB,flaG) or the expression of the cell adhesin (flaC) also resulted in a delay in pellicle formation. These findings demonstrate that the flagellar motility complex plays a crucial role in the initial attachment ofC. jejuni11168 to solid surfaces during biofilm formation as well as in the cell-to-cell interactions required for pellicle formation. Continued expression of the motility complex in mature biofilms is unusual and suggests a role for the flagellar apparatus in the biofilm phenotype.
DARK-MATTER PARTICLES AND BARYONS FROM INFLATION AND SPONTANEOUS CP VIOLATION IN THE EARLY UNIVERSE
Authors: BARSHAY SAUL , KREYERHOFF GEORG
Published: June 2006

We present aspects of a model which attempts to unify the creation of cold dark matter, a CP-violating baryon asymmetry, and also a small, residual vacuum energy density, in the early universe. The model contains a primary scalar (inflaton) field and a primary pseudoscalar field, which are initially related by a cosmological, chiral symmetry. The nonzero vacuum expectation value of the pseudoscalar field spontaneously breaks CP invariance.
Formation of actin-ADF/cofilin rods transiently retards decline of mitochondrial potential and ATP in stressed neurons
Authors: Bernstein Barbara W. , Chen Hui , Boyle Judith A. , Bamburg James R.
Published: June 2006

When neurons in culture are transiently stressed by inhibition of ATP synthesis, they rapidly form within their neurites rodlike actin inclusions that disappear when the insult is removed. Oxidative stress, excitotoxic insults, and amyloid β-peptide oligomers also induce rods. Immunostaining of neurites indicates that these rods also contain the majority of the actin filament dynamizing proteins, actin-depolymerizing factor (ADF) and cofilin (AC). If the rods reappear within 24 h after the stress is removed, the neurite degenerates distal to the rod but with no increase in neuronal death. Here, rods were generated in cultured rat E18 hippocampal cells by overexpression of a green fluorescent protein chimera of AC. Surprisingly, we have found that, for a short period (∼60 min) immediately after initial rod formation, the loss of mitochondrial membrane potential (ΔΨm) and ATP in neurites with rods is slower than in neurites without them. The ΔΨm was monitored with the fluorescent dye tetramethylrhodamine methyl ester, and ATP was monitored with the fluorescent ion indicator mag-fura 2. Actin in rods is less dynamic than is filamentous actin in other cytoskeletal structures. Because ΔΨm depends on cellular ATP and because ATP hydrolysis associated with actin filament turnover is responsible for a large fraction of neuronal energy consumption (∼50%), the formation of rods transiently protects neurites by slowing filament turnover and its associated ATP hydrolysis.
In vivo oxygen imaging using green fluorescent protein
Authors: Takahashi Eiji , Takano Tomohiro , Nomura Yasutomo , Okano Satoshi , Nakajima Osamu , Sato Michihiko
Published: June 2006

In vivo oxygen measurement is the key to understanding how biological systems dynamically adapt to reductions in oxygen supply. High spatial resolution oxygen imaging is of particular importance because recent studies address the significance of within-tissue and within-cell heterogeneities in oxygen concentration in health and disease. Here, we report a new technique for in vivo molecular imaging of oxygen in organs using green fluorescent protein (GFP). GFP-expressing COS-7 cells were briefly photoactivated with a strong blue light while lowering the oxygen concentration from 10% to <0.001%. Red fluorescence (excitation 520–550 nm, emission >580 nm) appeared after photoactivation at <2% oxygen (the red shift of GFP fluorescence). The red shift disappeared after reoxygenation of the cell, indicating that the red shift is stable as long as the cell is hypoxic. The red shift of GFP fluorescence was also demonstrated in single cardiomyocytes isolated from the GFP knock-in mouse (green mouse) heart. Then, we tried in vivo molecular imaging of hypoxia in organs. The red shift could be imaged in the ischemic liver and kidney in the green mouse using macroscopic optics provided that oxygen diffusion from the atmospheric air was prevented. In crystalloid-perfused beating heart isolated from the green mouse, significant spatial heterogeneities in the red shift were demonstrated in the epicardium distal to the coronary artery ligation. We conclude that the present technique using GFP as an oxygen indicator may allow in vivo molecular imaging of oxygen in organs.