bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of protein kinase C in light adaptation of molluscan microvillar photoreceptors
Authors: Piccoli Giuseppe , Gomez Maria del Pilar , Nasi Enrico
Published: August 2002

The mechanisms by which Ca2+ regulates light adaptation in microvillar photoreceptors remain poorly understood. Protein kinase C (PKC) is a likely candidate, both because some sub‐types are activated by Ca2+ and because of its association with the macromolecular ‘light‐transduction complex’ in Drosophila. We investigated the possible role of PKC in the modulation of the light response in molluscan photoreceptors. Western blot analysis with isoform‐specific antibodies revealed the presence of PKCα in retinal homogenates. Immunocytochemistry in isolated cell preparations confirmed PKCα localization in microvillar photoreceptors, preferentially confined to the light‐sensing lobe. Light stimulation induced translocation of PKCα immunofluorescence to the photosensitive membrane, an effect that provides independent evidence for PKC activation by illumination; a similar outcome was observed after incubation with the phorbol ester PMA. Several chemically distinct activators of PKC, such as phorbol‐12‐myristate‐13‐acetate (PMA), (‐)indolactam V and 1,2,‐dioctanoyl‐sn‐glycerol (DOG) inhibited the light response of voltage‐clamped microvillar photoreceptors, but were ineffective in ciliary photoreceptors, in which light does not activate the Gq/PLC cascade, nor elevates intracellular Ca2+. Pharmacological inhibition of PKC antagonized the desensitization produced by adapting lights and also caused a small, but consistent enhancement of basal sensitivity. These results strongly support the involvement of PKC activation in the light‐dependent regulation of response sensitivity. However, unlike adapting background light or elevation of [Ca2+]i, PKC activators did not speed up the photoresponse, nor did PKC inhibitors antagonize the accelerating effects of background adaptation, suggesting that modulation of photoresponse time course may involve a separate Ca2+‐dependent signal.
Mechanisms of enhancement of human motor cortex excitability induced by interventional paired associative stimulation
Authors: Stefan Katja , Kunesch Erwin , Benecke Reiner , Cohen Leonardo G. , Classen Joseph
Published: August 2002

Associative stimulation has been shown to enhance excitability in the human motor cortex (Stefan et al. 2000); however, little is known about the underlying mechanisms. An interventional paired associative stimulation (IPAS) was employed consisting of repetitive application of single afferent electric stimuli, delivered to the right median nerve, paired with single pulse transcranial magnetic stimulation (TMS) over the optimal site for activation of the abductor pollicis brevis muscle (APB) to generate approximately synchronous events in the primary motor cortex. Compared to baseline, motor evoked potentials (MEPs) induced by unconditioned, single TMS pulses increased after IPAS. By contrast, intracortical inhibition, assessed using (i) a suprathreshold test TMS pulse conditioned by a subthreshold TMS pulse delivered 3 ms before the test pulse, and (ii) a suprathreshold test TMS pulse conditioned by afferent median nerve stimulation delivered 25 ms before the TMS pulse, remained unchanged when assessed with appropriately matching test stimulus intensities. The increase of single‐pulse TMS‐evoked MEP amplitudes was blocked when IPAS was performed under the influence of dextromethorphan, an N‐methyl‐d‐aspartate (NMDA) receptor antagonist known to block long‐term potentiation (LTP). Further experiments employing the double‐shock TMS protocol suggested that the afferent pulse, as one component of the IPAS protocol, induced disinhibition of the primary motor cortex at the time when the TMS pulse, as the other component of IPAS, was delivered. Together, these findings support the view that LTP‐like mechanisms may underlie the cortical plasticity induced by IPAS.
Allosteric modulation of the mouse kir6.2 channel by intracellular H+ and ATP
Authors: Wu Jianping , Cui Ningren , Piao Hailan , Wang Ying , Xu Haoxing , Mao Jinzhe , Jiang Chun
Published: August 2002

The ATP‐sensitive K+ (KATP) channels are regulated by intracellular H+ in addition to ATP, ADP, and phospholipids. Here we show evidence for the interaction of H+ with ATP in regulating a cloned KATP channel, i.e. Kir6.2 expressed with and without the SUR1 subunit. Channel sensitivity to ATP decreases at acidic pH, while the pH sensitivity also drops in the presence of ATP. These effects are more evident in the presence of the SUR1 subunit. In the Kir6.2 + SUR1, the pH sensitivity is reduced by about 0.4 pH units with 100 μM ATP and 0.6 pH units with 1 mm ATP, while a decrease in pH from 7.4 to 6.8 lowers the ATP sensitivity by about fourfold. The Kir6.2 + SUR1 currents are strongly activated at pH 5.9‐6.5 even in the presence of 1 mm ATP. The modulations appear to take place at His175 and Lys185 that are involved in proton and ATP sensing, respectively. Mutation of His175 completely eliminates the pH effect on the ATP sensitivity. Similarly, the K185E mutant‐channel loses the ATP‐dependent modulation of the pH sensitivity. Thus, allosteric modulations of the cloned KATP channel by ATP and H+ are demonstrated. Such a regulation allows protons to activate directly the KATP channels and release channel inhibition by intracellular ATP; the pH effect is further enhanced with a decrease in ATP concentration as seen in several pathophysiological conditions.
Environmental Change and Antarctic Seabird Populations
Authors: Croxall J. P. , Trathan P. N. , Murphy E. J.
Published: August 2002

Recent changes in Antarctic seabird populations may reflect direct and indirect responses to regional climate change. The best long-term data for high-latitude Antarctic seabirds (Adélie and Emperor penguins and snow petrels) indicate that winter sea-ice has a profound influence. However, some effects are inconsistent between species and areas, some in opposite directions at different stages of breeding and life cycles, and others remain paradoxical. The combination of recent harvest driven changes and those caused by global warming may produce rapid shifts rather than gradual changes.
SynCAM, a Synaptic Adhesion Molecule That Drives Synapse Assembly
Authors: Biederer Thomas , Sara Yildirim , Mozhayeva Marina , Atasoy Deniz , Liu Xinran , Kavalali Ege T. , Südhof Thomas C.
Published: August 2002

Synapses, the junctions between nerve cells through which they communicate, are formed by the coordinated assembly and tight attachment of pre- and postsynaptic specializations. We now show that SynCAM is a brain-specific, immunoglobulin domain–containing protein that binds to intracellular PDZ-domain proteins and functions as a homophilic cell adhesion molecule at the synapse. Expression of the isolated cytoplasmic tail of SynCAM in neurons inhibited synapse assembly. Conversely, expression of full-length SynCAM in nonneuronal cells induced synapse formation by cocultured hippocampal neurons with normal release properties. Glutamatergic synaptic transmission was reconstituted in these nonneuronal cells by coexpressing glutamate receptors with SynCAM, which suggests that a single type of adhesion molecule and glutamate receptor are sufficient for a functional postsynaptic response.
Global Biodiversity, Biochemical Kinetics, and the Energetic-Equivalence Rule
Authors: Allen Andrew P. , Brown James H. , Gillooly James F.
Published: August 2002

The latitudinal gradient of increasing biodiversity from poles to equator is one of the most prominent but least understood features of life on Earth. Here we show that species diversity can be predicted from the biochemical kinetics of metabolism. We first demonstrate that the average energy flux of populations is temperature invariant. We then derive a model that quantitatively predicts how species diversity increases with environmental temperature. Predictions are supported by data for terrestrial, freshwater, and marine taxa along latitudinal and elevational gradients. These results establish a thermodynamic basis for the regulation of species diversity and the organization of ecological communities.
Geographic Range Size and Determinants of Avian Species Richness
Authors: Jetz Walter , Rahbek Carsten
Published: August 2002

Geographic patterns in species richness are mainly based on wide-ranging species because their larger number of distribution records has a disproportionate contribution to the species richness counts. Here we demonstrate how this effect strongly influences our understanding of what determines species richness. Using both conventional and spatial regression models, we show that for sub-Saharan African birds, the apparent role of productivity diminishes with decreasing range size, whereas the significance of topographic heterogeneity increases. The relative importance of geometric constraints from the continental edge is moderate. Our findings highlight the failure of traditional species richness models to account for narrow-ranging species that frequently are also threatened.
Hierarchical Organization of Modularity in Metabolic Networks
Authors: Ravasz E. , Somera A. L. , Mongru D. A. , Oltvai Z. N. , Barabási A.-L.
Published: August 2002

Spatially or chemically isolated functional modules composed of several cellular components and carrying discrete functions are considered fundamental building blocks of cellular organization, but their presence in highly integrated biochemical networks lacks quantitative support. Here, we show that the metabolic networks of 43 distinct organisms are organized into many small, highly connected topologic modules that combine in a hierarchical manner into larger, less cohesive units, with their number and degree of clustering following a power law. Within
Escherichia coli
, the uncovered hierarchical modularity closely overlaps with known metabolic functions. The identified network architecture may be generic to system-level cellular organization.
Gated Visual Input to the Central Auditory System
Authors: Gutfreund Yoram , Zheng Weimin , Knudsen Eric I.
Published: August 2002

The central auditory system translates sound localization cues into a map of space guided, in part, by visual experience. In barn owls, this process takes place in the external nucleus of the inferior colliculus (ICX). However, to date, no trace of visual activity has been observed in this auditory nucleus. Here we show that strong visual responses, which are appropriate to guide auditory plasticity, appear in the ICX when inhibition is blocked in the optic tectum. Thus, visual spatial information is gated into the auditory system by an inhibitory mechanism that operates at a higher level in the brain.
Medulloblastoma Growth Inhibition by Hedgehog Pathway Blockade
Authors: Berman David M. , Karhadkar Sunil S. , Hallahan Andrew R. , Pritchard Joel I. , Eberhart Charles G. , Watkins D. Neil , Chen James K. , Cooper Michael K. , Taipale Jussi , Olson James M. , Beachy Philip A.
Published: August 2002

Constitutive Hedgehog (Hh) pathway activity is associated with initiation of neoplasia, but its role in the continued growth of established tumors is unclear. Here, we investigate the therapeutic efficacy of the Hh pathway antagonist cyclopamine in preclinical models of medulloblastoma, the most common malignant brain tumor in children. Cyclopamine treatment of murine medulloblastoma cells blocked proliferation in vitro and induced changes in gene expression consistent with initiation of neuronal differentiation and loss of neuronal stem cell–like character. This compound also caused regression of murine tumor allografts in vivo and induced rapid death of cells from freshly resected human medulloblastomas, but not from other brain tumors, thus establishing a specific role for Hh pathway activity in medulloblastoma growth.
Identification of a Potential Ejaculation Generator in the Spinal Cord
Authors: Truitt William A. , Coolen Lique M.
Published: August 2002

We tested the significance of a population of lumbar spinothalamic cells for male sexual behavior in rats. These cells are positioned to relay ejaculation-related signals from reproductive organs to the brain, and they express neurokinin-1 receptors. Ablation of these neurons by the selective toxin SSP-saporin resulted in a complete disruption of ejaculatory behavior. In contrast, other components of sexual behavior remained intact. These results suggest that this population of spinothalamic cells plays a pivotal role in generation of ejaculatory behavior and may be part of a spinal ejaculation generator.
Mass Balance of Polar Ice Sheets
Authors: Rignot Eric , Thomas Robert H.
Published: August 2002

Recent advances in the determination of the mass balance of polar ice sheets show that the Greenland Ice Sheet is losing mass by near-coastal thinning, and that the West Antarctic Ice Sheet, with thickening in the west and thinning in the north, is probably thinning overall. The mass imbalance of the East Antarctic Ice Sheet is likely to be small, but even its sign cannot yet be determined. Large sectors of ice in southeast Greenland, the Amundsen Sea Embayment of West Antarctica, and the Antarctic Peninsula are changing quite rapidly as a result of processes not yet understood.
Controlled Elimination of Clathrin Heavy-Chain Expression in DT40 Lymphocytes
Authors: Wettey Frank R. , Hawkins Steve F. C. , Stewart Abigail , Luzio J. Paul , Howard Jonathan C. , Jackson Antony P.
Published: August 2002

We exploited the high rate of homologous recombination shown by the chicken B cell line DT40 to inactivate the endogenous alleles for clathrin heavy chain and replace them with human clathrin complementary DNA under the control of a tetracycline-regulatable promoter. Clathrin repression perturbed the activities of Akt-mediated and mitogen-activated protein kinase–mediated signaling pathways and induced apoptosis; this finding suggests that in DT40 cells clathrin helps to maintain the integrity of antiapoptotic survival pathways. We also describe a variant cell line in which these signaling pathways were unaffected by clathrin down-regulation. This variant cell line did not undergo apoptosis in the absence of clathrin and was used to examine the effects of clathrin depletion on membrane-trafficking pathways. Receptor-mediated and fluid-phase endocytosis were both substantially inhibited, and transferrin-receptor recycling was modestly inhibited. Surprisingly, clathrin removal did not affect the morphology or biochemical composition of lysosomes.
Natural Product Terpenoids in Eocene and Miocene Conifer Fossils
Authors: Otto Angelika , White James D. , Simoneit Bernd R. T.
Published: August 2002

Numerous saturated and aromatic hydrocarbons, but not polar compounds, originating from plants and microorganisms (biomarkers) have been reported in sediments, coals, and petroleum. Here we describe natural product terpenoids found in two fossil conifers,
Taxodium balticum
(Eocene) and
Glyptostrobus oregonensis
(Miocene). A similar terpenoid pattern is also observed in extant
Taxodium distichum
. The preservation of characteristic terpenoids (unaltered natural products) in the fossil conifers supports their systematic assignment to the Cypress family (Cupressaceae sensu lato). The results also show that fossil conifers can contain polar terpenoids, which are valuable markers for (paleo)chemosystematics and phylogeny.
Fluidity of Bound Hydration Layers
Authors: Raviv Uri , Klein Jacob
Published: August 2002

We have measured the shear forces between solid surfaces sliding past each other across aqueous salt solutions, at pressures and concentrations typical of naturally occurring systems. In such systems the surface-attached hydration layers keep the compressed surfaces apart as a result of strongly repulsive hydration forces. We find, however, that the bound water molecules retain a shear fluidity characteristic of the bulk liquid, even when compressed down to films 1.0 ± 0.3 nanometer thick. We attribute this to the ready exchange (as opposed to loss) of water molecules within the hydration layers as they rub past each other under strong compression.
The Mechanism of Diamond Nucleation from Energetic Species
Authors: Lifshitz Y. , Köhler Th. , Frauenheim Th. , Guzmann I. , Hoffman A. , Zhang R. Q. , Zhou X. T. , Lee S. T.
Published: August 2002

A model for diamond nucleation by energetic species (for example, bias-enhanced nucleation) is proposed. It involves spontaneous bulk nucleation of a diamond embryo cluster in a dense, amorphous carbon hydrogenated matrix; stabilization of the cluster by favorable boundary conditions of nucleation sites and hydrogen termination; and ion bombardment–induced growth through a preferential displacement mechanism. The model is substantiated by density functional tight-binding molecular dynamics simulations and an experimental study of the structure of bias-enhanced and ion beam–nucleated films. The model is also applicable to the nucleation of other materials by energetic species, such as cubic boron nitride.
A Silver-Free, Single-Sheet Imaging Medium Based on Acid Amplification
Authors: Marshall John L. , Telfer Stephen J. , Young Michael A. , Lindholm Edward P. , Minns Richard A. , Takiff Larry
Published: August 2002

We describe a photographic medium that uses acid-amplified imaging (AAI) rather than silver halide development to amplify a latent image. The latent image is captured when small amounts of superacid are generated by the photolysis of iodonium salts sensitized by cationic dyes. During thermal processing, the quantity of acid is multiplied in a process catalyzed by strong acid, resulting in a much larger amount of a weaker acid. Acid-sensitive indicator dyes that diffuse into regions where acid has been produced form a colored image. The AAI system is not sufficiently sensitive for direct use in cameras but is suitable for many printing applications.
Air-Snow Interactions and Atmospheric Chemistry
Authors: Dominé Florent , Shepson Paul B.
Published: August 2002

The presence of snow greatly perturbs the composition of near-surface polar air, and the higher concentrations of hydroxyl radicals (OH) observed result in a greater oxidative capacity of the lower atmosphere. Emissions of nitrogen oxides, nitrous acid, light aldehydes, acetone, and molecular halogens have also been detected. Photolysis of nitrate ions contained in the snow appears to play an important role in creating these perturbations. OH formed in the snowpack can oxidize organic matter and halide ions in the snow, producing carbonyl compounds and halogens that are released to the atmosphere or incorporated into snow crystals. These reactions modify the composition of the snow, of the interstitial air, and of the overlying atmosphere. Reconstructing the composition of past atmospheres from ice-core analyses may therefore require complex corrections and modeling for reactive species.
Can Selection Explain the Presbyterians?
Authors: Ruse Michael
Published: August 2002

Darwin's Cathedral
Evolution, Religion, and the Nature of Society. David Sloan Wilson. University of Chicago Press, Chicago, 2002. 276 pp. $25, £16. ISBN 0-226-90134-3.

According to Wilson, human societies can be thought of as organisms and religion and morality are best understood as complex adaptations of these units.
What Hath Nature Wrought?
Authors: Dunlap Thomas R.
Published: August 2002

Down to Earth
Nature's Role in American History. Ted Steinberg. Oxford University Press, New York, 2002. 361 pp. $30. ISBN 0-19-514009-5.

Steinberg presents an account of the influence of the environment-and human interactions with it-on U.S. history, our current circumstances, and our future prospects.
Oxidation-Resistant Gold-55 Clusters
Authors: Boyen H.-G. , Kästle G. , Weigl F. , Koslowski B. , Dietrich C. , Ziemann P. , Spatz J. P. , Riethmüller S. , Hartmann C. , Möller M. , Schmid G. , Garnier M. G. , Oelhafen P.
Published: August 2002

Gold nanoparticles ranging in diameter from 1 to 8 nanometers were prepared on top of silicon wafers in order to study the size dependence of their oxidation behavior when exposed to atomic oxygen. X-ray photoelectron spectroscopy showed a maximum oxidation resistance for “magic-number” clusters containing 55 gold atoms. This inertness is not related to electron confinement leading to a size-induced metal-to-insulator transition, but rather seems to be linked to the closed-shell structure of such magic clusters. The result additionally suggests that gold-55 clusters may act as especially effective oxidation catalysts, such as for oxidizing carbon monoxide.
Structural Basis of Transcription Activation: The CAP-αCTD-DNA Complex
Authors: Benoff Brian , Yang Huanwang , Lawson Catherine L. , Parkinson Gary , Liu Jinsong , Blatter Erich , Ebright Yon W. , Berman Helen M. , Ebright Richard H.
Published: August 2002

The
Escherichia coli
catabolite activator protein (CAP) activates transcription at P

lac

, P

gal

, and other promoters through interactions with the RNA polymerase α subunit carboxyl-terminal domain (αCTD). We determined the crystal structure of the CAP-αCTD-DNA complex at a resolution of 3.1 angstroms. CAP makes direct protein-protein interactions with αCTD, and αCTD makes direct protein-DNA interactions with the DNA segment adjacent to the DNA site for CAP. There are no large-scale conformational changes in CAP and αCTD, and the interface between CAP and αCTD is small. These findings are consistent with the proposal that activation involves a simple “recruitment” mechanism.
Dynamics of Recent Climate Change in the Arctic
Authors: Moritz Richard E. , Bitz Cecilia M. , Steig Eric J.
Published: August 2002

The pattern of recent surface warming observed in the Arctic exhibits both polar amplification and a strong relation with trends in the Arctic Oscillation mode of atmospheric circulation. Paleoclimate analyses indicate that Arctic surface temperatures were higher during the 20th century than during the preceding few centuries and that polar amplification is a common feature of the past. Paleoclimate evidence for Holocene variations in the Arctic Oscillation is mixed. Current understanding of physical mechanisms controlling atmospheric dynamics suggests that anthropogenic influences could have forced the recent trend in the Arctic Oscillation, but simulations with global climate models do not agree. In most simulations, the trend in the Arctic Oscillation is much weaker than observed. In addition, the simulated warming tends to be largest in autumn over the Arctic Ocean, whereas observed warming appears to be largest in winter and spring over the continents.
Nanoparticles with Raman Spectroscopic Fingerprints for DNA and RNA Detection
Authors: Cao YunWei Charles , Jin Rongchao , Mirkin Chad A.
Published: August 2002

Multiplexed detection of oligonucleotide targets has been performed with gold nanoparticle probes labeled with oligonucleotides and Raman-active dyes. The gold nanoparticles facilitate the formation of a silver coating that acts as a surface-enhanced Raman scattering promoter for the dye-labeled particles that have been captured by target molecules and an underlying chip in microarray format. The strategy provides the high-sensitivity and high-selectivity attributes of gray-scale scanometric detection but adds multiplexing and ratioing capabilities because a very large number of probes can be designed based on the concept of using a Raman tag as a narrow-band spectroscopic fingerprint. Six dissimilar DNA targets with six Raman-labeled nanoparticle probes were distinguished, as well as two RNA targets with single nucleotide polymorphisms. The current unoptimized detection limit of this method is 20 femtomolar.
Biodegradation of the Polyketide Toxin Cercosporin
Authors: Mitchell Thomas K. , Chilton William Scott , Daub Margaret E.
Published: August 2002

Cercosporin is a non-host-specific polyketide toxin produced by many species of plant pathogens belonging to the genusCercospora. This red-pigmented, light-activated toxin is an important pathogenicity determinant forCercosporaspecies. In this study, we screened 244 bacterial isolates representing 12 different genera for the ability to degrade cercosporin. Cercosporin degradation was determined by screening for the presence of cleared zones surrounding colonies on cercosporin-containing culture medium and was confirmed by assaying the kinetics of degradation in liquid medium. Bacteria belonging to four different genera exhibited the cercosporin-degrading phenotype. The isolates with the greatest cercosporin-degrading activity belonged toXanthomonas campestrispv. zinniae andX. campestrispv. pruni. Isolates of these pathovars removed over 90% of the cercosporin from culture medium within 48 h. Bacterial degradation of red cercosporin was accompanied by a shift in the color of the growth medium to brown and then green. The disappearance of cercosporin was accompanied by the appearance of a transient green product, designated xanosporic acid. Xanosporic acid and its more stable lactone derivative, xanosporolactone, are nontoxic to cercosporin-sensitive fungi and to plant tissue and are labile in the presence of light. Detailed spectroscopic analysis (to be reported in a separate publication) of xanosporolactone revealed that cercosporin loses one methoxyl group and gains one oxygen atom in the bacterial conversion. The resulting chromophore (4,9-dihydroxy-3-oxaperlylen-10H-10-one) has never been reported before but is biosynthetically plausible via oxygen insertion by a cytochrome P-450 enzyme.
Correspondence of High Levels of Beta-Exotoxin I and the Presence of
cry1B
in
Bacillus thuringiensis
Authors: Espinasse Sylvain , Gohar Michel , Chaufaux Josette , Buisson Christophe , Perchat Stéphane , Sanchis Vincent
Published: August 2002

Examination of 640 natural isolates of
Bacillus thuringiensis
showed that the 58 strains (9%) whose supernatants were toxic to
Anthonomus grandis
(Coleoptera: Curculionidae) produced between 10 and 175 μg of β-exotoxin I per ml. We also found that 55 (46%) of a sample of 118 strains whose culture supernatants were not toxic to
A. grandis
nevertheless produced between 2 and 5 μg/ml. However, these amounts of β-exotoxin I were below the threshold for detectable toxicity against this insect species. Secretion of large amounts of β-exotoxin I was strongly associated with the presence of
cry1B
and
vip2
genes in the 640 natural
B. thuringiensis
isolates studied. We concluded that strains carrying
cry1B
and
vip2
genes also possess, on the same plasmid, genetic determinants necessary to promote high levels of production of β-exotoxin I.
Tissue Localization of the Endosymbiotic Bacterium “
Candidatus
Blochmannia floridanus” in Adults and Larvae of the Carpenter Ant
Camponotus floridanus
Authors: Sauer Christina , Dudaczek Dieter , Hölldobler Bert , Gross Roy
Published: August 2002

The distribution of endosymbiotic bacteria in different tissues of queens, males, and workers of the carpenter ant
Camponotus floridanus
was investigated by light and electron microscopy and by in situ hybridization. A large number of bacteria could be detected in bacteriocytes within the midguts of workers, young virgin queens, and males. Large amounts of bacteria were also found in the oocytes of workers and queens. In contrast, bacteria were not present in oocyte-associated cells or in the spermathecae of mature queens, although occasionally a small number of bacteria could be detected in the testis follicles of males. Interestingly, the number of bacteriocytes in mature queens was strongly reduced and the bacteriocytes contained only very few or no bacteria at all, although the endosymbionts were present in huge amounts in the ovaries of the same animals. During embryogenesis of the deposited egg, the bacteria were concentrated in a ring of endodermal tissue destined to become the midgut in later developmental stages. However, during larval development, bacteria could also be detected in other tissues although to a lesser extent. Only in the last-instar larvae were bacteria found exclusively in the midgut tissue within typical bacteriocytes. Tetracycline and rifampin efficiently cleansed
C. floridanus
workers of their symbionts and the bacteriocytes of these animals still remained empty several months after treatment had ceased. Despite the lack of their endosymbionts, these adult animals were able to survive without any obvious negative effect under normal cultivation conditions.
Development of a Standard Test To Assess the Resistance of
Staphylococcus aureus
Biofilm Cells to Disinfectants
Authors: Luppens Suzanne B. I. , Reij Martine W. , van der Heijden Rob W. L. , Rombouts Frank M. , Abee Tjakko
Published: August 2002

A standardized disinfectant test for
Staphylococcus aureus
cells in biofilms was developed. Two disinfectants, the membrane-active compound benzalkonium chloride (BAC) and the oxidizing agent sodium hypochlorite, were used to evaluate the biofilm test.
S. aureus
formed biofilms on glass, stainless steel, and polystyrene in a simple system with constant nutrient flow that mimicked as closely as possible the conditions used in the current standard European disinfectant test (EN 1040). The biofilm that was formed on glass contained cell clumps and extracellular polysaccharides. The average surface coverage was 60%, and most (92%) of the biofilm cells were viable. Biofilm formation and biofilm disinfection in different experiments were reproducible. For biofilms exposed to BAC and hypochlorite the concentrations needed to achieve 4-log killing were 50 and 600 times higher, respectively, than the concentrations needed to achieve this level of killing with the European phase 1 suspension test cells. Our results show that a standardized disinfectant test for biofilm cells is a useful addition to the current standard tests.
Effect of a
Sinorhizobium meliloti
Strain with a Modified
putA
Gene on the Rhizosphere Microbial Community of Alfalfa
Authors: van Dillewijn Pieter , Villadas Pablo J. , Toro Nicolás
Published: August 2002

The success of a rhizobial inoculant in the soil depends to a large extent on its capacity to compete against indigenous strains. M403, a
Sinorhizobium meliloti
strain with enhanced competitiveness for nodule occupancy, was recently constructed by introducing a plasmid containing an extra copy of a modified
putA
(proline dehydrogenase) gene. This strain and M401, a control strain carrying the same plasmid without the modified gene, were used as soil inoculants for alfalfa in a contained field release experiment at León, Spain. In this study, we determined the effects of these two strains on the indigenous microbial community. 16S rRNA genes were obtained from the rhizosphere of alfalfa inoculated with strain M403 or strain M401 or from noninoculated plants by amplification of DNA from soil with bacterial group-specific primers. These genes were analyzed and compared by restriction fragment length polymorphism and temperature gradient gel electrophoresis. The results allowed us to differentiate between alterations in the microbial community apparently caused by inoculation and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa plants and by the environment. Only moderate inoculation-dependent effects could be detected, while the alfalfa plants appeared to have a much stronger influence on the microbial community.
Environmental Regulation of
Campylobacter jejuni
Major Outer Membrane Protein Porin Expression in
Escherichia coli
Monitored by Using Green Fluorescent Protein
Authors: Dedieu Luc , Pagès Jean-Marie , Bolla Jean-Michel
Published: August 2002

Porins allow exchanges between bacteria and their environment. In the gram-negative food-borne pathogen
Campylobacter jejuni
two porins, major outer membrane protein (MOMP) and Omp50, have been identified. MOMP is synthesized at a very high level under laboratory culture conditions, suggesting that its promoter functions very efficiently under these conditions. In
Campylobacter
samples, we observed that MOMP porin expression increased at a high temperature (42°C) or a high pH (pH 8.5) compared to expression at a low temperature (31°C) or an acidic pH (pH 5.5). To study the regulation of MOMP expression at the transcriptional level, we constructed an
momp-gfp
fusion in which
gfp
expression was put under the control of the
momp
promoter. Interestingly, we observed the same pattern of regulation in
Escherichia coli
, as monitored by green fluorescent protein production, that was found in
Campylobacter
. The ranges of pH and temperature tested are physiologically relevant, because they can be found in the digestive tracts of both birds and humans, which are both colonized by
Campylobacter.
Our results suggest that a component of the regulatory mechanism is conserved in
C. jejuni
and
E. coli
. However, medium osmolarity and sodium salicylate did not have a significant effect on
C. jejuni momp
promoter activity in
E. coli
, suggesting that major regulatory elements of
E. coli
porin expression do not participate in MOMP regulation. In contrast, mechanisms involving DNA supercoiling may be involved, as shown by DNA gyrase inhibition assays. These findings are a step towards determining the role of outer membrane proteins in the adaptation of
C. jejuni
to its environment.
Genetic Heterogeneity in
Bacillus sporothermodurans
as Demonstrated by Ribotyping and Repetitive Extragenic Palindromic-PCR Fingerprinting
Authors: Guillaume-Gentil Olivier , Scheldeman Patsy , Marugg Joey , Herman Lieve , Joosten Han , Heyndrickx Marc
Published: August 2002

Thirty-eight strains of
Bacillus sporothermodurans
isolated from ultra-high-temperature (UHT)-treated milk or sterilized milk (UHT isolates) and from animal feed or raw milk (farm isolates) were characterized by automated ribotyping and by repetitive extragenic palindromic (REP)-PCR fingerprinting. By investigating the genetic relationships among isolates from these various sources, the relative importance of different contamination sources could be evaluated. The results of the separate clustering analyses of the
Pvu
II and
Eco
RI ribopatterns and the REP-PCR patterns were largely consistent with each other and revealed the existence of two main clusters; there was one homogeneous group containing all (REP-PCR) or most (ribotyping) of the UHT isolates, and there was a second more diverse group comprising the farm isolates. A combined three-dimensional analysis of all data showed that three German UHT isolates did not belong to the compact group containing the majority of the UHT isolates. These results demonstrate that
B. sporothermodurans
is more heterogeneous than previously assumed and that most of the UHT isolates form a genetically distinct subgroup and are capable of producing highly heat-resistant spores. The close genetic relationship of these UHT isolates suggests a clonal origin of a few predominant strains of
B. sporothermodurans
that can be found in UHT-treated or sterilized milk products.
Quantification of Uncultured
Ruminococcus obeum
-Like Bacteria in Human Fecal Samples by Fluorescent In Situ Hybridization and Flow Cytometry Using 16S rRNA-Targeted Probes
Authors: Zoetendal Erwin G. , Ben-Amor Kaouther , Harmsen Hermie J. M. , Schut Frits , Akkermans Antoon D. L. , de Vos Willem M.
Published: August 2002

A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured
Ruminococcus obeum
-like bacteria by fluorescent in situ hybridization (FISH). These bacteria have frequently been found in 16S ribosomal DNA clone libraries prepared from bacterial communities in the human intestine. Thirty-two reference strains from the human intestine, including a phylogenetically related strain and strains of some other
Ruminococcus
species, were used as negative controls and did not hybridize with the new probe. Microscopic and flow cytometric analyses revealed that a group of morphologically similar bacteria in feces did hybridize with this probe. Moreover, it was found that all hybridizing cells also hybridized with a probe specific for the
Clostridium coccoides-Eubacterium rectale
group, a group that includes the uncultured
R. obeum
-like bacteria. Quantification of the uncultured
R. obeum
-like bacteria and the
C. coccoides-E. rectale
group by flow cytometry and microscopy revealed that these groups comprised approximately 2.5 and 16% of the total community in fecal samples, respectively. The uncultured
R. obeum
-like bacteria comprise about 16% of the
C. coccoides-E. rectale
group. These results indicate that the uncultured
R. obeum
-like bacteria are numerically important in human feces. Statistical analysis revealed no significant difference between the microscopic and flow cytometric counts and the different feces sampling times, while a significant host-specific effect on the counts was observed. Our data demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of uncultured bacteria in the human gastrointestinal tract.
Cyclization Reaction Catalyzed by Glycogen Debranching Enzyme (EC 2.4.1.25/EC 3.2.1.33) and Its Potential for Cycloamylose Production
Authors: Yanase Michiyo , Takata Hiroki , Takaha Takeshi , Kuriki Takashi , Smith Steven M. , Okada Shigetaka
Published: August 2002

Glycogen debranching enzyme (GDE) has 4-α-glucanotransferase and amylo-1,6-glucosidase activities in the single polypeptide chain. We analyzed the detailed action profile of GDE from
Saccharomyces cerevisiae
on amylose and tested whether GDE catalyzes cyclization of amylose. GDE treatment resulted in a rapid reduction of absorbance of iodine-amylose complex and the accumulation of a product that was resistant to an exo-amylase (glucoamylase [GA]) but was degraded by an endo-type α-amylase to glucose and maltose. These results indicated that GDE catalyzed cyclization of amylose to produce cyclic α-1,4 glucan (cycloamylose). The formation of cycloamylose was confirmed by high-performance anion-exchange chromatography, and the size was shown to range from a degree of polymerization of 11 to a degree of polymerization around 50. The minimum size and the size distribution of cycloamylose were different from those of cycloamylose produced by other 4-α-glucanotransferases. GDE also efficiently produced cycloamylose even from the branched glucan substrate, starch, demonstrating its potential for industrial production of cycloamylose.
A Stationary-Phase Acyl-Coenzyme A Synthetase of
Streptomyces coelicolor
A3(2) Is Necessary for the Normal Onset of Antibiotic Production
Authors: Banchio C. , Gramajo H.
Published: August 2002

The
fadD1
and
macs1
genes of
Streptomyces coelicolor
are part of a two-gene operon. Both genes encode putative acyl coenzyme A synthetases (ACSs). The amino acid sequence of FadD1 has high homology with those of several ACSs, while MACS1 has the closest homology with medium-chain ACSs, broadly known as SA proteins. Like FadD of
Escherichia coli
, FadD1 also has a broad substrate specificity, although saturated long-chain fatty acids appears to be the preferred substrate.
fadD1
is a growth-phase-regulated gene, and its mRNA is detected only during the stationary phase of growth. Interestingly, a mutation in
fadD1
alters the levels of another ACS or ACSs, both at the stationary phase and at the exponential phase of growth, at least when glucose is used as a main carbon source. The mutant also shows a severe deficiency in antibiotic production, and at least for Act biosynthesis, this deficiency seems to be related to delayed expression of the Act biosynthetic genes. Antibiotic production is restored by the introduction of a wt
fadD1
allele into the cell, demonstrating a strict link between ACS activity and the biosynthesis of secondary metabolites. The results of this study indicate that the ACSs may be useful targets for the design of rational approaches to improving antibiotic production.
Biochemical Evidence for Formate Transfer in Syntrophic Propionate-Oxidizing Cocultures of
Syntrophobacter fumaroxidans
and
Methanospirillum hungatei
Authors: de Bok Frank A. M. , Luijten Maurice L. G. C. , Stams Alfons J. M.
Published: August 2002

The hydrogenase and formate dehydrogenase levels in
Syntrophobacter fumaroxidans
and
Methanospirillum hungatei
were studied in syntrophic propionate-oxidizing cultures and compared to the levels in axenic cultures of both organisms. Cells grown syntrophically were separated from each other by Percoll gradient centrifugation. In
S. fumaroxidans
both formate dehydrogenase and hydrogenase levels were highest in cells which were grown syntrophically, while the formate-H
2
lyase activities were comparable under the conditions tested. In
M. hungatei
the formate dehydrogenase and formate-H
2
lyase levels were highest in cells grown syntrophically, while the hydrogenase levels in syntrophically grown cells were comparable to those in cells grown on formate. Reconstituted syntrophic cultures from axenic cultures immediately resumed syntrophic growth, and the calculated growth rates of these cultures were highest for cells which were inoculated from the axenic
S. fumaroxidans
cultures that exhibited the highest formate dehydrogenase activities. The results suggest that formate is the preferred electron carrier in syntrophic propionate-oxidizing cocultures of
S. fumaroxidans
and
M. hungatei
.
Purification and Immunogenicity of a RecombinantBordetella pertussisS1S3FHA Fusion Protein Expressed byStreptococcus gordonii
Authors: Lee Song F. , Halperin Scott A. , Knight Jennifer B. , Tait Aaron
Published: August 2002

Acellular pertussis vaccines typically consist of antigens isolated fromBordetella pertussis, and pertussis toxin (PT) and filamentous hemagglutinin (FHA) are two prominent components. One of the disadvantages of a multiple-component vaccine is the cost associated with the production of the individual components. In this study, we constructed an in-frame fusion protein consisting of PT fragments (179 amino acids of PT subunit S1 and 180 amino acids of PT subunit S3) and a 456-amino-acid type I domain of FHA. The fusion protein was expressed by the commensal oral bacteriumStreptococcus gordonii.The fusion protein was secreted into the culture medium as an expected 155-kDa protein, which was recognized by a polyclonal anti-PT antibody, a monoclonal anti-S1 antibody, and a monoclonal anti-FHA antibody. The fusion protein was purified from the culture supernatant by affinity and gel permeation chromatography. The immunogenicity of the purified fusion protein was assessed in BALB/c mice by performing parenteral and mucosal immunization experiments. When given parenterally, the fusion protein elicited a very strong antibody titer against the FHA type I domain, a moderate titer against native FHA, and a weak titer against PT. When given mucosally, it elicited a systemic response and a mucosal response to FHA and PT. In Western blots, the immune sera recognized the S1, S3, and S2 subunits of PT. These data collectively indicate that fragments of the pertussis vaccine components can be expressed in a single fusion protein byS. gordoniiand that the fusion protein is immunogenic. This multivalent fusion protein approach may be used in designing a new generation of acellular pertussis vaccines.
Hxt-Carrier-Mediated Glucose Efflux upon Exposure of
Saccharomyces cerevisiae
to Excess Maltose
Authors: Jansen Mickel L. A. , De Winde Johannes H. , Pronk Jack T.
Published: August 2002

When wild-type
Saccharomyces cerevisiae
strains pregrown in maltose-limited chemostat cultures were exposed to excess maltose, release of glucose into the external medium was observed. Control experiments confirmed that glucose release was not caused by cell lysis or extracellular maltose hydrolysis. To test the hypothesis that glucose efflux involved plasma membrane glucose transporters, experiments were performed with an
S. cerevisiae
strain in which all members of the hexose transporter (
HXT
) gene family had been eliminated and with an isogenic reference strain. Glucose efflux was virtually eliminated in the hexose-transport-deficient strain. This constitutes experimental proof that Hxt transporters facilitate export of glucose from
S. cerevisiae
cells. After exposure of the hexose-transport-deficient strain to excess maltose, an increase in the intracellular glucose level was observed, while the concentrations of glucose 6-phosphate and ATP remained relatively low. These results demonstrate that glucose efflux can occur as a result of uncoordinated expression of the initial steps of maltose metabolism and the subsequent reactions in glucose dissimilation. This is a relevant phenomenon for selection of maltose-constitutive strains for baking and brewing.
Albidovulum inexpectatum
gen. nov., sp. nov., a Nonphotosynthetic and Slightly Thermophilic Bacterium from a Marine Hot Spring That Is Very Closely Related to Members of the Photosynthetic Genus
Rhodovulum
Authors: Albuquerque Luciana , Santos João , Travassos Pedro , Nobre M. Fernanda , Rainey Fred A. , Wait Robin , Empadinhas Nuno , Silva Manuel T. , da Costa Milton S.
Published: August 2002

Several bacterial isolates, with an optimum growth temperature of about 50°C, were recovered from the marine hot spring at Ferraria on the island of São Miguel in the Azores. The geothermal water emerged from a porous lava flow and rapidly cooled in contact with seawater except at low tide. The bacterial species represented by strains FRR-10
T
and FRR-11 was nonpigmented, strictly aerobic, and organotrophic. Several genes,
bchZ
,
pufB
,
pufA
,
pufL
, or
pufM
, encoding the photosynthetic reaction center proteins and the core light-harvesting complexes were not detected in these strains. The organism oxidized thiosulfate to sulfate with enhancement of growth. The organism did not require additional NaCl in the culture medium for growth, but NaCl at 1.0% enhanced growth. Phylogenetic analyses using the 16S rRNA gene sequence of strain FRR-10
T
indicated that the new organism represented a new species of the α-3 subclass of the
Proteobacteria
and that it branches within the species of the genus
Rhodovulum
. The contradiction of classifying an organism which branches within the radiation of the genus
Rhodovulum
but does not possess the hallmark characteristics of this genus is discussed. However, the absence of several of these characteristics, namely, the lack of photosynthesis and pigmentation, which could be related to colonization of dark environments, and growth at high temperatures, leads to our proposal that strains FRR-10
T
and FRR-11 should be classified as a new species of a novel genus,
Albidovulum inexpectatum
, representing, at present, the most thermophilic organism within the α-3 subclass of the
Proteobacteria
.
Expression of Genes Encoding F
1
-ATPase Results in Uncoupling of Glycolysis from Biomass Production in
Lactococcus lactis
Authors: Koebmann Brian J. , Solem Christian , Pedersen Martin B. , Nilsson Dan , Jensen Peter R.
Published: August 2002

We studied how the introduction of an additional ATP-consuming reaction affects the metabolic fluxes in
Lactococcus lactis
. Genes encoding the hydrolytic part of the F
1
domain of the membrane-bound (F
1
F
0
) H
+
-ATPase were expressed from a range of synthetic constitutive promoters. Expression of the genes encoding F
1
-ATPase was found to decrease the intracellular energy level and resulted in a decrease in the growth rate. The yield of biomass also decreased, which showed that the incorporated F
1
-ATPase activity caused glycolysis to be uncoupled from biomass production. The increase in ATPase activity did not shift metabolism from homolactic to mixed-acid fermentation, which indicated that a low energy state is not the signal for such a change. The effect of uncoupled ATPase activity on the glycolytic flux depended on the growth conditions. The uncoupling stimulated the glycolytic flux threefold in nongrowing cells resuspended in buffer, but in steadily growing cells no increase in flux was observed. The latter result shows that glycolysis occurs close to its maximal capacity and indicates that control of the glycolytic flux under these conditions resides in the glycolytic reactions or in sugar transport.