bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein kinase signalling requirements for metabotropic action of kainate receptors in rat CA1 pyramidal neurones
Authors: Grabauskas Gintautas , Lancaster Barrie , O'Connor Vincent , Wheal Howard V.
Published: December 2006

Hippocampal pyramidal neurones display a Ca2+‐dependent K+ current responsible for the slow afterhyperpolarization (IsAHP), a prominent regulator of excitability. There is considerable transmitter convergence onto IsAHP but little information about the interplay between the kinase‐based transduction mechanisms underlying transmitter action. We have added to existing information about the role of protein kinase C (PKC) in kainate receptor actions by demonstrating that direct postsynaptic activation of PKC with either 1‐oleoyl‐2‐acethylsn‐glycerol (OAG) or indolactam is sufficient to inhibit IsAHP. The physiological correlate of this action – activation of PKC by kainate receptors – requires Gαi/o proteins. The cAMP/PKA system is well documented to subserve the actions of monoamine transmitters. We have found an additional role for the cAMP/PKA system as a requirement for kainate receptor‐mediated inhibition of IsAHP. Inhibition of adenylyl cyclase with dideoxyadenosine or PKA with either H89 or RpcAMPs blocked kainate receptor‐mediated actions but did not prevent the actions of direct PKC activation with either OAG or indolactam. We therefore propose that the PKA requirement is upstream from the actions of PKC. We additionally report a downstream link in the form of increased mitogen‐activated protein (MAP) kinase activity, which may explain the long duration of metabotropic actions of kainate receptors on IsAHP.
TRPC‐like conductance mediates restoration of intracellular Ca2+in cochlear outer hair cells in the guinea pig and rat
Authors: Raybould Nicholas P. , Jagger Daniel J. , Kanjhan Refik , Greenwood Denise , Laslo Peter , Hoya Noriyuki , Soeller Christian , Cannell Mark B. , Housley Gary D.
Published: December 2006

Ca2+signalling is central to cochlear sensory hair cell physiology through its influence on sound transduction, membrane filter properties and neurotransmission. However, the mechanism for establishing Ca2+homeostasis in these cells remains unresolved. Canonical transient receptor potential (TRPC) Ca2+entry channels provide an important pathway for maintaining intracellular Ca2+levels. TRPC3 subunit expression was detected in guinea pig and rat organ of Corti by RT‐PCR, and localized to the sensory and neural poles of the inner and outer hair cells (OHCs) by confocal immunofluorescence imaging. A cation entry current with a TRPC‐like phenotype was identified in guinea pig and rat OHCs by whole‐cell voltage clamp. This slowly activating current was induced by the lowering of cytosolic Ca2+levels ([Ca2+]i) following a period in nominally Ca2+‐free solution. Activation was dependent upon the [Ca2+]oand was sustained until [Ca2+]iwas restored. Ca2+entry was confirmed by confocal fluorescence imaging, and rapidly recruited secondary charybdotoxin‐ and apamin‐sensitive KCacurrents. Dual activation by the G protein‐coupled receptor (GPCR)–phospholipase C–diacylglycerol (DAG) second messenger pathway was confirmed using the analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG). Ion substitution experiments showed that the putative TRPC Ca2+entry current was selective for Na+> K+with a ratio of 1: 0.6. The Ca2+entry current was inhibited by the TRPC channel blocker 2‐aminoethyl diphenylborate (2APB) and the tyrosine kinase inhibitor, erbstatin analogue. We conclude that TRPC Ca2+entry channels, most likely incorporating TRPC3 subunits, support cochlear hair cell Ca2+homeostasis and GPCR signalling.
Adenosine transporter antagonism in humans augments vasodilator responsiveness to adenosine, but not exercise, in both adenosine responders and non‐responders
Authors: Martin Elizabeth A. , Nicholson Wayne T. , Curry Timothy B. , Eisenach John H. , Charkoudian Nisha , Joyner Michael J.
Published: December 2006

We previously demonstrated a bimodal distribution of forearm vasodilator responsiveness to adenosine (ADO) infusion in the brachial arteries of human subjects. We also demonstrated that ADO receptor antagonism blunted exercise hyperaemia during heavy rhythmic handgripping, but vasodilator responses to exogenous ADO were only blunted in ADO responders. In this study, we continued investigating the contribution of ADO to exercise hyperaemia and possible differences between responders and non‐responders. We hypothesized that ADO transporter antagonism would increase vasodilatation in response to exogenous ADO in responders only, but not effect exercise‐mediated vasodilation. To test this hypothesis, we compared forearm vascular conductance (FVC) during infusion of ADO to FVC during handgripping before and after infusion of dipyridamole (DIP) in 20 subjects. In ADO responders, change in FVC above baseline (ml min−1 (100 mmHg)−1) for low, medium and high doses of ADO, respectively, was 58 ± 8, 121 ± 22 and 184 ± 38, and after DIP was 192 ± 32, 238 ± 50 and 310 ± 79. For non‐responders, these values were 23 ± 2, 43 ± 5 and 66 ± 9, respectively, before DIP (P < 0.01 versus responders). Contrary to our hypothesis, these values were increased by DIP in non‐responders (P < 0.001) and therefore not different from responders (P > 0.20). We found that ADO transporter blockade had no effect on exercise hyperaemia in either subgroup. We conclude that there may be increased ADO transporter activity in non‐responders resulting in reduced ADO‐mediated vasodilatation. The failure of DIP to augment exercise hyperemia under these conditions suggests that ADO concentrations may not rise enough during rhythmic handgripping to have a major impact on these responses.
Calcium sparklets regulate local and global calcium in murine arterial smooth muscle
Authors: Amberg Gregory C. , Navedo Manuel F. , Nieves‐Cintrón Madeline , Molkentin Jeffery D. , Santana Luis F.
Published: December 2006

In arterial smooth muscle, protein kinase Cα (PKCα) coerces discrete clusters of L‐type Ca2+ channels to operate in a high open probability mode, resulting in subcellular domains of nearly continual Ca2+ influx called ‘persistent Ca2+ sparklets’. Our previous work suggested that steady‐state Ca2+ entry into arterial myocytes, and thus global [Ca2+]i, is regulated by Ca2+ influx through clusters of L‐type Ca2+ channels operating in this persistently active mode in addition to openings of solitary channels functioning in a low‐activity mode. Here, we provide the first direct evidence supporting this ‘Ca2+ sparklet’ model of Ca2+ influx at a physiological membrane potential and external Ca2+ concentration. In support of this model, we found that persistent Ca2+ sparklets produced local and global elevations in [Ca2+]i. Membrane depolarization increased Ca2+ influx via low‐activity and high‐activity persistent Ca2+ sparklets. Our data indicate that Ca2+ entering arterial smooth muscle through persistent Ca2+ sparklets accounts for approximately 50% of the total dihydropyridine‐sensitive (i.e. L‐type Ca2+ channel) Ca2+ influx at a physiologically relevant membrane potential (−40 mV) and external Ca2+ concentration (2 mm). Consistent with this, inhibition of basal PKCα‐dependent persistent Ca2+ sparklets decreased [Ca2+]i by about 50% in isolated arterial myocytes and intact pressurized arteries. Taken together, these data support the conclusion that in arterial smooth muscle steady‐state Ca2+ entry and global [Ca2+]i are regulated by low‐activity and PKCα‐dependent high‐activity persistent Ca2+ sparklets.
Amplitude modulation of turbulence noise by voicing in fricatives
Authors: Pincas Jonathan , Jackson Philip J. B.
Published: December 2006

The two principal sources of sound in speech, voicing and frication, occur simultaneously in voiced fricatives as well as at the vowel-fricative boundary in phonologically voiceless fricatives. Instead of simply overlapping, the two sources interact. This paper is an acoustic study of one such interaction effect: the amplitude modulation of the frication component when voicing is present. Corpora of sustained and fluent-speech English fricatives were recorded and analyzed using a signal-processing technique designed to extract estimates of modulation depth. Results reveal a pattern, consistent across speaking style, speaker, and place of articulation, for modulation at f0 to rise at low voicing strengths and subsequently saturate. Voicing strength needed to produce saturation varied 60–66dB across subjects and experimental conditions. Modulation depths at saturation varied little across speakers but significantly for place of articulation (with [z] showing particularly strong modulation) clustering at approximately 0.4–0.5 (a 40%–50% fluctuation above and below unmodulated amplitude); spectral analysis of modulating signals revealed weak but detectable modulation at the second and third harmonics (i.e., 2f0 and 3f0)
Engineering Yeast Transcription Machinery for Improved Ethanol Tolerance and Production
Authors: Alper Hal , Moxley Joel , Nevoigt Elke , Fink Gerald R. , Stephanopoulos Gregory
Published: December 2006

Global transcription machinery engineering (gTME) is an approach for reprogramming gene transcription to elicit cellular phenotypes important for technological applications. Here we show the application of gTME to
Saccharomyces cerevisiae
for improved glucose/ethanol tolerance, a key trait for many biofuels programs. Mutagenesis of the transcription factor Spt15p and selection led to dominant mutations that conferred increased tolerance and more efficient glucose conversion to ethanol. The desired phenotype results from the combined effect of three separate mutations in the
SPT15
gene [serine substituted for phenylalanine (Phe
177
Ser) and, similarly, Tyr
195
His, and Lys
218
Arg]. Thus, gTME can provide a route to complex phenotypes that are not readily accessible by traditional methods.
Dual Infection with HIV and Malaria Fuels the Spread of Both Diseases in Sub-Saharan Africa
Authors: Abu-Raddad Laith J. , Patnaik Padmaja , Kublin James G.
Published: December 2006

Mounting evidence has revealed pathological interactions between HIV and malaria in dually infected patients, but the public health implications of the interplay have remained unclear. A transient almost one-log elevation in HIV viral load occurs during febrile malaria episodes; in addition, susceptibility to malaria is enhanced in HIV-infected patients. A mathematical model applied to a setting in Kenya with an adult population of roughly 200,000 estimated that, since 1980, the disease interaction may have been responsible for 8,500 excess HIV infections and 980,000 excess malaria episodes. Co-infection might also have facilitated the geographic expansion of malaria in areas where HIV prevalence is high. Hence, transient and repeated increases in HIV viral load resulting from recurrent co-infection with malaria may be an important factor in promoting the spread of HIV in sub-Saharan Africa.
Enzyme-Free Nucleic Acid Logic Circuits
Authors: Seelig Georg , Soloveichik David , Zhang David Yu , Winfree Erik
Published: December 2006

Biological organisms perform complex information processing and control tasks using sophisticated biochemical circuits, yet the engineering of such circuits remains ineffective compared with that of electronic circuits. To systematically create complex yet reliable circuits, electrical engineers use digital logic, wherein gates and subcircuits are composed modularly and signal restoration prevents signal degradation. We report the design and experimental implementation of DNA-based digital logic circuits. We demonstrate AND, OR, and NOT gates, signal restoration, amplification, feedback, and cascading. Gate design and circuit construction is modular. The gates use single-stranded nucleic acids as inputs and outputs, and the mechanism relies exclusively on sequence recognition and strand displacement. Biological nucleic acids such as microRNAs can serve as inputs, suggesting applications in biotechnology and bioengineering.
Ebola Outbreak Killed 5000 Gorillas
Authors: Bermejo Magdalena , Rodríguez-Teijeiro José Domingo , Illera Germán , Barroso Alex , Vilà Carles , Walsh Peter D.
Published: December 2006

Over the past decade, the Zaire strain of Ebola virus (ZEBOV) has repeatedly emerged in Gabon and Congo. Each human outbreak has been accompanied by reports of gorilla and chimpanzee carcasses in neighboring forests, but both the extent of ape mortality and the causal role of ZEBOV have been hotly debated. Here, we present data suggesting that in 2002 and 2003 ZEBOV killed about 5000 gorillas in our study area. The lag between neighboring gorilla groups in mortality onset was close to the ZEBOV disease cycle length, evidence that group-to-group transmission has amplified gorilla die-offs.
A Brown Dwarf Mass Donor in an Accreting Binary
Authors: Littlefair S. P. , Dhillon V. S. , Marsh T. R. , Gänsicke Boris T. , Southworth John , Watson C. A.
Published: December 2006

A long-standing and unverified prediction of binary star evolution theory is the existence of a population of white dwarfs accreting from substellar donor stars. Such systems ought to be common, but the difficulty of finding them, combined with the challenge of detecting the donor against the light from accretion, means that no donor star to date has a measured mass below the hydrogen burning limit. We applied a technique that allowed us to reliably measure the mass of the unseen donor star in eclipsing systems. We were able to identify a brown dwarf donor star, with a mass of 0.052 ± 0.002 solar mass. The relatively high mass of the donor star for its orbital period suggests that current evolutionary models may underestimate the radii of brown dwarfs.
An Inward-Facing Conformation of a Putative Metal-Chelate-Type ABC Transporter
Authors: Pinkett H. W. , Lee A. T. , Lum P. , Locher K. P. , Rees D. C.
Published: December 2006

The crystal structure of a putative metal-chelate–type adenosine triphosphate (ATP)–binding cassette (ABC) transporter encoded by genes
HI1470
and
HI1471
of
Haemophilus influenzae
has been solved at 2.4 angstrom resolution. The permeation pathway exhibits an inward-facing conformation, in contrast to the outward-facing state previously observed for the homologous vitamin B
12
importer BtuCD. Although the structures of both HI1470/1 and BtuCD have been solved in nucleotide-free states, the pairs of ABC subunits in these two structures differ by a translational shift in the plane of the membrane that coincides with a repositioning of the membrane-spanning subunits. The differences observed between these ABC transporters involve relatively modest rearrangements and may serve as structural models for inward- and outward-facing conformations relevant to the alternating access mechanism of substrate translocation.
Five Rules for the Evolution of Cooperation
Authors: Nowak Martin A.
Published: December 2006

Cooperation is needed for evolution to construct new levels of organization. Genomes, cells, multicellular organisms, social insects, and human society are all based on cooperation. Cooperation means that selfish replicators forgo some of their reproductive potential to help one another. But natural selection implies competition and therefore opposes cooperation unless a specific mechanism is at work. Here I discuss five mechanisms for the evolution of cooperation: kin selection, direct reciprocity, indirect reciprocity, network reciprocity, and group selection. For each mechanism, a simple rule is derived that specifies whether natural selection can lead to cooperation.
Armageddon in the Oceans
Authors: Worm Boris
Published: December 2006

The Swarm
. A Novel of the Deep.
By Frank Schätzing
. Translated from the German () by Sally-Ann Spencer. ReganBooks (HarperCollins), New York, 2006. 893 pp. $24.95, C$32.50. ISBN 0-06-081326-1. Paper, Hodder and Stoughton, London. £12.99. ISBN 0-340-89523-3.

A bestseller in Germany, this apocalyptic environmental thriller revolves around anthropogenic threats to the oceans.
Microwave-Induced Cooling of a Superconducting Qubit
Authors: Valenzuela Sergio O. , Oliver William D. , Berns David M. , Berggren Karl K. , Levitov Leonid S. , Orlando Terry P.
Published: December 2006

We demonstrated microwave-induced cooling in a superconducting flux qubit. The thermal population in the first-excited state of the qubit is driven to a higher-excited state by way of a sideband transition. Subsequent relaxation into the ground state results in cooling. Effective temperatures as low as ≈3 millikelvin are achieved for bath temperatures of 30 to 400 millikelvin, a cooling factor between 10 and 100. This demonstration provides an analog to optical cooling of trapped ions and atoms and is generalizable to other solid-state quantum systems. Active cooling of qubits, applied to quantum information science, provides a means for qubit-state preparation with improved fidelity and for suppressing decoherence in multi-qubit systems.
Sequential Interplay of Nicotinic and GABAergic Signaling Guides Neuronal Development
Authors: Liu Zhaoping , Neff Robert A. , Berg Darwin K.
Published: December 2006

GABA (γ-aminobutyric acid), the major inhibitory transmitter in the brain, goes through a transitory phase of excitation during development. The excitatory phase promotes neuronal growth and integration into circuits. We show here that spontaneous nicotinic cholinergic activity is responsible for terminating GABAergic excitation and initiating inhibition. It does so by changing chloride transporter levels, shifting the driving force on GABA-induced currents. The timing of the transition is critical, because the two phases of GABAergic signaling provide contrasting developmental instructions. Synergistic with nicotinic excitation, GABAergic inhibition constrains neuronal morphology and innervation. The results reveal a multitiered activity-dependent strategy controlling neuronal development.
RETRACTED: Secondary siRNAs Result from Unprimed RNA Synthesis and Form a Distinct Class
Authors: Sijen Titia , Steiner Florian A. , Thijssen Karen L. , Plasterk Ronald H. A.
Published: December 2006

In
Caenorhabditis elegans
, an effective RNA interference (RNAi) response requires the production of secondary short interfering RNAs (siRNAs) by RNA-directed RNA polymerases (RdRPs). We cloned secondary siRNAs from transgenic
C. elegans
lines expressing a single 22-nucleotide primary siRNA. Several secondary siRNAs start a few nucleotides downstream of the primary siRNA, indicating that non–RISC (RNA-induced silencing complex)–cleaved mRNAs are substrates for secondary siRNA production. In lines expressing primary siRNAs with single-nucleotide mismatches, secondary siRNAs do not carry the mismatch but contain the nucleotide complementary to the mRNA. We infer that RdRPs perform unprimed RNA synthesis. Secondary siRNAs are only of antisense polarity, carry 5′ di- or triphosphates, and are only in the minority associated with RDE-1, the RNAi-specific Argonaute protein. Therefore, secondary siRNAs represent a distinct class of small RNAs. Their biogenesis depends on RdRPs, and we propose that each secondary siRNA is an individual RdRP product.
Consequences of Telomere Shortening at an Active
VSG
Expression Site in Telomerase-Deficient
Trypanosoma brucei
Authors: Dreesen Oliver , Cross George A. M.
Published: December 2006

Trypanosoma brucei
evades the host immune response by sequential expression of a large family of variant surface glycoproteins (VSG) from one of ∼20 subtelomeric expression sites (ES).
VSG
transcription is monoallelic, and little is known about the regulation of antigenic switching. To explore whether telomere length could affect antigenic switching, we created a telomerase-deficient cell line, in which telomeres shortened at a rate of 3 to 6 bp at each cell division. Upon reaching a critical length, short silent ES telomeres were stabilized by a telomerase-independent mechanism. The active ES telomere progressively shortened and frequently broke. Upon reaching a critical length, the short active ES telomere stabilized, but the transcribed
VSG
was gradually lost from the population and replaced by a new
VSG
through duplicative gene conversion. We propose a model in which subtelomeric-break-induced replication-mediated repair at a short ES telomere leads to duplicative gene conversion and expression of a new
VSG
.
Candida albicans
Transcription Factor Ace2 Regulates Metabolism and Is Required for Filamentation in Hypoxic Conditions
Authors: Mulhern Siobhan M. , Logue Mary E. , Butler Geraldine
Published: December 2006

Ace2 transcription factor family genes are found in many fungal genomes and are required for regulation of expression of genes involved in cell separation. We used transcriptional profiling to identify the targets of Ace2 in
Candida albicans
, and we show that these include several cell wall components, such as glucanases and glycosylphosphatidylinositol-anchored proteins. Expression is downregulated in
ace2
deletion mutants in both yeast and hyphal cells. In addition, deleting
ace2
results in dramatic changes in expression of metabolic pathways. Expression of glycolytic enzymes is reduced, while expression of respiratory genes (including those involved in the tricarboxylic acid cycle, oxidative phosphorylation, and ATP synthesis) is increased. Similar changes occur in both yeast and hyphal cells. In contrast, genes required for acetyl-coenzyme A and lipid metabolism are upregulated in an
ace2
deletion mutant grown predominantly as yeast cells but are downregulated in hyphae. These results suggest that in wild-type strains, Ace2 acts to increase glycolysis and reduce respiration. This is supported by the observation that deleting
ace2
results in increased resistance to antimycin A, a drug that inhibits respiration. We also show that Ace2 is required for filamentation in response to low oxygen concentrations (hypoxia). We suggest that filamentation is induced in wild-type cells by reducing respiration (using low oxygen or respiratory drugs) and that mutants with increased respiratory activity fail to undergo filamentation under these conditions.
The
Tetrahymena thermophila
Phagosome Proteome
Authors: Jacobs Mary Ellen , DeSouza Leroi V. , Samaranayake Haresha , Pearlman Ronald E. , Siu K. W. Michael , Klobutcher Lawrence A.
Published: December 2006

In vertebrates, phagocytosis occurs mainly in specialized cells of the immune system and serves as a primary defense against invading pathogens, but it also plays a role in clearing apoptotic cells and in tissue remodeling during development. In contrast, unicellular eukaryotes, such as the ciliate
Tetrahymena thermophila
, employ phagocytosis to ingest and degrade other microorganisms to meet their nutritional needs. To learn more about the protein components of the multistep process of phagocytosis, we carried out an analysis of the
Tetrahymena
phagosome proteome.
Tetrahymena
cells were fed polystyrene beads, which allowed for the efficient purification of phagosomes. The protein composition of purified phagosomes was then analyzed by multidimensional separation coupled with tandem mass spectrometry. A total of 453 peptides were identified that resulted in the identification of 73 putative phagosome proteins. Twenty-eight of the proteins have been implicated in phagocytosis in other organisms, indicating that key aspects of phagocytosis were conserved during evolution. Other identified proteins have not previously been associated with phagocytosis, including some of unknown function. Live-cell confocal fluorescence imaging of
Tetrahymena
strains expressing green fluorescent protein-tagged versions of four of the identified phagosome proteins provided evidence that at least three of the proteins (including two with unknown functions) are associated with phagosomes, indicating that the bulk of the proteins identified in the analyses are indeed phagosome associated.
Proteins of the Glycine Decarboxylase Complex in the Hydrogenosome ofTrichomonas vaginalis
Authors: Mukherjee Mandira , Brown Mark T. , McArthur Andrew G. , Johnson Patricia J.
Published: December 2006

Trichomonas vaginalisis a unicellular eukaryote that lacks mitochondria and contains a specialized organelle, the hydrogenosome, involved in carbohydrate metabolism and iron-sulfur cluster assembly. We report the identification of two glycine cleavage H proteins and a dihydrolipoamide dehydrogenase (L protein) of the glycine decarboxylase complex inT. vaginaliswith predicted N-terminal hydrogenosomal presequences. Immunofluorescence analyses reveal that both H and L proteins are localized in hydrogenosomes, providing the first evidence for amino acid metabolism in this organelle. All three proteins were expressed inEscherichia coliand purified to homogeneity. The experimentalKmof L protein for the two H proteins were 2.6 μM and 3.7 μM, consistent with both H proteins serving as substrates of L protein. Analyses using purified hydrogenosomes showed that endogenous H proteins exist as monomers and endogenous L protein as a homodimer in their native states. Phylogenetic analyses of L proteins revealed that theT. vaginalishomologue shares a common ancestry with dihydrolipoamide dehydrogenases from the firmicute bacteria, indicating its acquisition via a horizontal gene transfer event independent of the origins of mitochondria and hydrogenosomes.
Analysis of
Euglena gracilis
Plastid-Targeted Proteins Reveals Different Classes of Transit Sequences
Authors: Durnford Dion G. , Gray Michael W.
Published: December 2006

The plastid of
Euglena gracilis
was acquired secondarily through an endosymbiotic event with a eukaryotic green alga, and as a result, it is surrounded by a third membrane. This membrane complexity raises the question of how the plastid proteins are targeted to and imported into the organelle. To further explore plastid protein targeting in
Euglena
, we screened a total of 9,461 expressed sequence tag (EST) clusters (derived from 19,013 individual ESTs) for full-length proteins that are plastid localized to characterize their targeting sequences and to infer potential modes of translocation. Of the 117 proteins identified as being potentially plastid localized whose N-terminal targeting sequences could be inferred, 83 were unique and could be classified into two major groups. Class I proteins have tripartite targeting sequences, comprising (in order) an N-terminal signal sequence, a plastid transit peptide domain, and a predicted stop-transfer sequence. Within this class of proteins are the lumen-targeted proteins (class IB), which have an additional hydrophobic domain similar to a signal sequence and required for further targeting across the thylakoid membrane. Class II proteins lack the putative stop-transfer sequence and possess only a signal sequence at the N terminus, followed by what, in amino acid composition, resembles a plastid transit peptide. Unexpectedly, a few unrelated plastid-targeted proteins exhibit highly similar transit sequences, implying either a recent swapping of these domains or a conserved function. This work represents the most comprehensive description to date of transit peptides in
Euglena
and hints at the complex routes of plastid targeting that must exist in this organism.
Binding Specificities and Potential Roles of Isoforms of Eukaryotic Initiation Factor 4E in
Leishmania
Authors: Yoffe Yael , Zuberek Joanna , Lerer Asaf , Lewdorowicz Magdalena , Stepinski Janusz , Altmann Michael , Darzynkiewicz Edward , Shapira Michal
Published: December 2006

The 5′ cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the
Leishmania
genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m
7
GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m
7
GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3′ untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m
7
GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the
Saccharomyces cerevisiae
eIF4E, indicating that despite their structural conservation, they are considerably diverged.
The Multifunctional β-Oxidation Enzyme Is Required for Full Symptom Development by the Biotrophic Maize Pathogen
Ustilago maydis
Authors: Klose Jana , Kronstad James W.
Published: December 2006

The transition from yeast-like to filamentous growth in the biotrophic fungal phytopathogen
Ustilago maydis
is a crucial event for pathogenesis. Previously, we showed that fatty acids induce filamentation in
U. maydis
and that the resulting hyphal cells resemble the infectious filaments observed in planta. To explore the potential metabolic role of lipids in the morphological transition and in pathogenic development in host tissue, we deleted the
mfe2
gene encoding the multifunctional enzyme that catalyzes the second and third reactions in β-oxidation of fatty acids in peroxisomes. The growth of the strains defective in
mfe2
was attenuated on long-chain fatty acids and abolished on very-long-chain fatty acids. The
mfe2
gene was not generally required for the production of filaments during mating in vitro, but loss of the gene blocked extensive proliferation of fungal filaments in planta. Consistent with this observation,
mfe2
mutants exhibited significantly reduced virulence in that only 27% of infected seedlings produced tumors compared to 88% tumor production upon infection by wild-type strains. Similarly, a defect in virulence was observed in developing ears upon infection of mature maize plants. Specifically, the absence of the
mfe2
gene delayed the development of teliospores within mature tumor tissue. Overall, these results indicate that the ability to utilize host lipids contributes to the pathogenic development of
U. maydis
.
VIB-1 Is Required for Expression of Genes Necessary for Programmed Cell Death in
Neurospora crassa
Authors: Dementhon Karine , Iyer Gopal , Glass N. Louise
Published: December 2006

Nonself recognition during somatic growth is an essential and ubiquitous phenomenon in both prokaryotic and eukaryotic species. In filamentous fungi, nonself recognition is also important during vegetative growth. Hyphal fusion between genetically dissimilar individuals results in rejection of heterokaryon formation and in programmed cell death of the fusion compartment. In filamentous fungi, such as
Neurospora crassa
, nonself recognition and heterokaryon incompatibility (HI) are regulated by genetic differences at
het
loci. In
N. crassa
, mutations at the
vib-1
locus suppress nonself recognition and HI mediated by genetic differences at
het-c/pin-c
,
mat
, and
un-24/het-6. vib-1
is a homolog of
Saccharomyces cerevisiae NDT80
, which is a transcriptional activator of genes during meiosis. For this study, we determined that
vib-1
encodes a nuclear protein and showed that VIB-1 localization varies during asexual reproduction and during HI.
vib-1
is required for the expression of genes involved in nonself recognition and HI, including
pin-c
,
tol
, and
het-6
; all of these genes encode proteins containing a HET domain.
vib-1
is also required for the production of downstream effectors associated with HI, including the production of extracellular proteases upon carbon and nitrogen starvation. Our data support a model in which mechanisms associated with starvation and nonself recognition/HI are interconnected. VIB-1 is a major regulator of responses to nitrogen and carbon starvation and is essential for the expression of genes involved in nonself recognition and death in
N. crassa
.
Reconstructing the Mosaic Glycolytic Pathway of the Anaerobic Eukaryote
Monocercomonoides
Authors: Liapounova Natalia A. , Hampl Vladimir , Gordon Paul M. K. , Sensen Christoph W. , Gedamu Lashitew , Dacks Joel B.
Published: December 2006

All eukaryotes carry out glycolysis, interestingly, not all using the same enzymes. Anaerobic eukaryotes face the challenge of fewer molecules of ATP extracted per molecule of glucose due to their lack of a complete tricarboxylic acid cycle. This may have pressured anaerobic eukaryotes to acquire the more ATP-efficient alternative glycolytic enzymes, such as pyrophosphate-fructose 6-phosphate phosphotransferase and pyruvate orthophosphate dikinase, through lateral gene transfers from bacteria and other eukaryotes. Most studies of these enzymes in eukaryotes involve pathogenic anaerobes;
Monocercomonoides
, an oxymonad belonging to the eukaryotic supergroup Excavata, is a nonpathogenic anaerobe representing an evolutionarily and ecologically distinct sampling of an anaerobic glycolytic pathway. We sequenced cDNA encoding glycolytic enzymes from a previously established cDNA library of
Monocercomonoides
and analyzed the relationships of these enzymes to those from other organisms spanning the major groups of
Eukaryota
,
Bacteria
, and
Archaea
. We established that, firstly,
Monocercomonoides
possesses alternative versions of glycolytic enzymes: fructose-6-phosphate phosphotransferase, both pyruvate kinase and pyruvate orthophosphate dikinase, cofactor-independent phosphoglycerate mutase, and fructose-bisphosphate aldolase (class II, type B). Secondly, we found evidence for the monophyly of oxymonads, kinetoplastids, diplomonads, and parabasalids, the major representatives of the Excavata. We also found several prokaryote-to-eukaryote as well as eukaryote-to-eukaryote lateral gene transfers involving glycolytic enzymes from anaerobic eukaryotes, further suggesting that lateral gene transfer was an important factor in the evolution of this pathway for denizens of this environment.
Pheromone-Induced Degradation of Ste12 Contributes to Signal Attenuation and the Specificity of Developmental Fate
Authors: Esch R. Keith , Wang Yuqi , Errede Beverly
Published: December 2006

The Ste12 transcription factor of
Saccharomyces cerevisiae
regulates transcription programs controlling two different developmental fates. One is differentiation into a mating-competent form that occurs in response to mating pheromone. The other is the transition to a filamentous-growth form that occurs in response to nutrient deprivation. These two distinct roles for Ste12 make it a focus for studies into regulatory mechanisms that impart biological specificity. The transient signal characteristic of mating differentiation led us to test the hypothesis that regulation of Ste12 turnover might contribute to attenuation of the mating-specific transcription program and restrict activation of the filamentation program. We show that prolonged pheromone induction leads to ubiquitin-mediated destabilization and decreased amounts of Ste12. This depletion in pheromone-stimulated cultures is dependent on the mating-pathway-dedicated mitogen-activated protein kinase Fus3 and its target Cdc28 inhibitor, Far1. Attenuation of pheromone-induced mating-specific gene transcription (
FUS1
) temporally correlates with Ste12 depletion. This attenuation is abrogated in the deletion backgrounds (
fus3Δ
or
far1Δ
) where Ste12 is found to persist. Additionally, pheromone induces haploid invasion and filamentous-like growth instead of mating differentiation when Ste12 levels remain high. These observations indicate that loss of Ste12 reinforces the adaptive response to pheromone and contributes to the curtailing of a filamentation response.
Reciprocal Nuclear Shuttling of Two Antagonizing Zn Finger Proteins Modulates Tup Family Corepressor Function To Repress Chromatin Remodeling
Authors: Hirota Kouji , Hoffman Charles S. , Ohta Kunihiro
Published: December 2006

The
Schizosaccharomyces pombe
global corepressors Tup11 and Tup12, which are orthologs of
Saccharomyces cerevisiae
Tup1, are involved in glucose-dependent transcriptional repression and chromatin alteration of the
fbp1
+
gene. The
fbp1
+
promoter contains two regulatory elements, UAS1 and UAS2, one of which (UAS2) serves as a binding site for two antagonizing C
2
H
2
Zn finger transcription factors, the Rst2 activator and the Scr1 repressor. In this study, we analyzed the role of Tup proteins and Scr1 in chromatin remodeling at
fbp1
+
during glucose repression. We found that Scr1, cooperating with Tup11 and Tup12, functions to maintain the chromatin of the
fbp1
+
promoter in a transcriptionally inactive state under glucose-rich conditions. Consistent with this notion, Scr1 is quickly exported from the nucleus to the cytoplasm at the initial stage of derepression, immediately after glucose starvation, at which time Rst2 is known to be imported into the nucleus. In addition, chromatin immunoprecipitation assays revealed a switching of Scr1 to Rst2 bound at UAS2 during glucose derepression. On the other hand, Tup11 and Tup12 persist in the nucleus and bind to the
fbp1
+
promoter under both derepressed and repressed conditions. These observations suggest that Tup1-like proteins recruited to the
fbp1
+
promoter are controlled by either of two antagonizing C
2
H
2
Zn finger proteins. We propose that the actions of Tup11 and Tup12 are regulated by reciprocal nuclear shuttling of the two antagonizing Zn finger proteins in response to the extracellular glucose concentration. This notion provides new insights into the molecular mechanisms of the Tup family corepressors in gene regulation.
Genetic Evidence that the Acyl Coenzyme A Binding Protein AcbA and the Serine Protease/ABC Transporter TagA Function Together in
Dictyostelium discoideum
Cell Differentiation
Authors: Cabral Matthew , Anjard Christophe , Loomis William F. , Kuspa Adam
Published: December 2006

The acyl coenzyme A (CoA) binding protein AcbA is cleaved to form a peptide (SDF-2) that coordinates spore encapsulation during the morphogenesis of
Dictyostelium discoideum
fruiting bodies. We present genetic evidence that the misspecification of cell types seen in mutants of the serine protease/ABC transporter TagA results from the loss of normal interactions with AcbA. Developmental phenotypes resulting from aberrant expression of the TagA protease domain, such as the formation of supernumerary tips on aggregates and the production of excess prestalk cells, are suppressed by null mutations in the
acbA
gene. Phenotypes resulting from the deletion of
tagA
, such as overexpression of the prestalk gene
ecmB
and the misexpression of the prespore gene
cotB
in stalk cells, are also observed in
acbA
mutants. Moreover,
tagA
−
mutants fail to produce SDF-2 during fruiting body morphogenesis but are able to do so if they are stimulated with exogenous SDF-2. These results indicate that the developmental program depends on TagA and AcbA working in concert with each other during cell type differentiation and suggest that TagA is required for normal SDF-2 signaling during spore encapsulation.
bla
VIM-2
and
bla
VIM-7
Carbapenemase-Producing
Pseudomonas aeruginosa
Isolates Detected in a Tertiary Care Medical Center in the United States: Report from the MYSTIC Program
Authors: Aboufaycal H. , Sader H. S. , Rolston K. , Deshpande L. M. , Toleman M. , Bodey G. , Raad I. , Jones R. N.
Published: December 2006

Two
Pseudomonas aeruginosa
strains resistant to beta-lactams, fluoroquinolones, aminoglycosides, tetracyclines, and carbapenems and susceptible only to polymyxin B (MIC ≤ 2 μg/ml) were identified as part of the Meropenem Yearly Susceptibility Test Information Collection program. Metallo-β-lactamase screening tests were positive, PCR yielded products with
bla
VIM
primers, and sequence analysis revealed
bla
VIM-7
and
bla
VIM-2
. The isolates had distinct ribotype and pulsed-field gel electorphoresis patterns and appeared independently, remote in time and location, at the same cancer center.
Improved Multilocus Sequence Typing Scheme for
Staphylococcus epidermidis
Authors: Thomas Jonathan C. , Vargas Miguel R. , Miragaia Maria , Peacock Sharon J. , Archer Gordon L. , Enright Mark C.
Published: December 2006

We evaluated three multilocus sequence typing (MLST) schemes for
Staphylococcus epidermidis
and selected the seven most discriminatory loci for the formation of a new, more powerful MLST scheme. This improved scheme gave 31 sequence types (STs) and 5 clonal complexes (CCs), whereas the other schemes delineate 16 to 24 STs and 1 to 3 CCs.
Stimulus Perception in Bacterial Signal-Transducing Histidine Kinases
Authors: Mascher Thorsten , Helmann John D. , Unden Gottfried
Published: December 2006

Two-component signal-transducing systems are ubiquitously distributed communication interfaces in bacteria. They consist of a histidine kinase that senses a specific environmental stimulus and a cognate response regulator that mediates the cellular response, mostly through differential expression of target genes. Histidine kinases are typically transmembrane proteins harboring at least two domains: an input (or sensor) domain and a cytoplasmic transmitter (or kinase) domain. They can be identified and classified by virtue of their conserved cytoplasmic kinase domains. In contrast, the sensor domains are highly variable, reflecting the plethora of different signals and modes of sensing. In order to gain insight into the mechanisms of stimulus perception by bacterial histidine kinases, we here survey sensor domain architecture and topology within the bacterial membrane, functional aspects related to this topology, and sequence and phylogenetic conservation. Based on these criteria, three groups of histidine kinases can be differentiated. (i) Periplasmic-sensing histidine kinases detect their stimuli (often small solutes) through an extracellular input domain. (ii) Histidine kinases with sensing mechanisms linked to the transmembrane regions detect stimuli (usually membrane-associated stimuli, such as ionic strength, osmolarity, turgor, or functional state of the cell envelope) via their membrane-spanning segments and sometimes via additional short extracellular loops. (iii) Cytoplasmic-sensing histidine kinases (either membrane anchored or soluble) detect cellular or diffusible signals reporting the metabolic or developmental state of the cell. This review provides an overview of mechanisms of stimulus perception for members of all three groups of bacterial signal-transducing histidine kinases.
How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria
Authors: Deutscher Josef , Francke Christof , Postma Pieter W.
Published: December 2006

The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.
Uses for JNK: the Many and Varied Substrates of the c-Jun N-Terminal Kinases
Authors: Bogoyevitch Marie A. , Kobe Bostjan
Published: December 2006

The c-Jun N-terminal kinases (JNKs) are members of a larger group of serine/threonine (Ser/Thr) protein kinases from the mitogen-activated protein kinase family. JNKs were originally identified as stress-activated protein kinases in the livers of cycloheximide-challenged rats. Their subsequent purification, cloning, and naming as JNKs have emphasized their ability to phosphorylate and activate the transcription factor c-Jun. Studies of c-Jun and related transcription factor substrates have provided clues about both the preferred substrate phosphorylation sequences and additional docking domains recognized by JNK. There are now more than 50 proteins shown to be substrates for JNK. These include a range of nuclear substrates, including transcription factors and nuclear hormone receptors, heterogeneous nuclear ribonucleoprotein K, and the Pol I-specific transcription factor TIF-IA, which regulates ribosome synthesis. Many nonnuclear substrates have also been characterized, and these are involved in protein degradation (e.g., the E3 ligase Itch), signal transduction (e.g., adaptor and scaffold proteins and protein kinases), apoptotic cell death (e.g., mitochondrial Bcl2 family members), and cell movement (e.g., paxillin, DCX, microtubule-associated proteins, the stathmin family member SCG10, and the intermediate filament protein keratin 8). The range of JNK actions in the cell is therefore likely to be complex. Further characterization of the substrates of JNK should provide clearer explanations of the intracellular actions of the JNKs and may allow new avenues for targeting the JNK pathways with therapeutic agents downstream of JNK itself.