bluuebunny/crossref_metadata_2025 huggingface dataset:
The Presence of Eschars, but Not Greater Severity, in Portuguese Patients Infected with Israeli Spotted Fever
Authors: DE SOUSA RITA , ISMAIL NAHED , DÓRIA‐NÓBREGA SÓNIA , COSTA PEDRO , ABREU TIAGO , FRANÇA ANA , AMARO MÁRIO , PROENÇA PAULA , BRITO PAULA , POÇAS JOSÉ , RAMOS TERESA , CRISTINA GRAÇA , POMBO GRAÇA , VITORINO LILIANA , TORGAL JORGE , BACELLAR FÁTIMA , WALKER DAVID
Published: February 2006

Abstract: In Portugal, Mediterranean spotted fever (MSF) is caused by R. conorii Malish and Israeli spotted fever (ISF) strains. It has been suggested that the ISF strain isolated from patients with MSF causes different clinical manifestations compared to those caused by Malish strain, namely the absence of eschar and greater severity. The aim of this study was to analyze the presence or absence of eschar and of fatality in Portuguese patients infected with either Malish or ISF strain. Of 94 patients with a clinical diagnosis of MSF between 1994 to 2004, 47 were infected with Malish strain and 47 with ISF strain. Eschars were reported in 20 patients (49%) infected with Malish strain, and in 17 (39%) with ISF strain. The presence of eschar is not statistically associated to a greater extent with either R. conorii strain (P= 0.346). A total of 22 patients died, 9 infected with Malish strain and 13 infected with ISF strain, and no statistically significant difference was found (P= 0.330). This study showed that the concepts of absence of the eschar and greater severity in Israeli spotted fever infection should be revised.
Analysis of Immunoprotectivity of the Recombinant OmpA of Rickettsia heilongjiangensis
Authors: JIAO YANMEI , WEN BOHAI , CHEN MEILING , NIU DONGSHENG , ZHANG JUN , QIU LING
Published: February 2006

Abstract: A truncated gene ompA was amplified from Rickettsia heilongjiangensis isolated in China and a 56‐kDa truncated OmpA protein was expressed in E. coli cells transformed with the ompA‐recombined expression plasmid. High levels of serum antibodies to R. heilongjiangensis and proliferation of the splenic cells were found in mice immunized with the truncated OmpA. After challenge with R. heilongjiangensis or R. rickettsii, fever and pathological damages of the guinea pigs immunized with the truncated OmpA were significantly slighter as compared with those of nonimmunized guinea pigs. These results suggest that the truncated OmpA of R. heilongjiangii is immunogenic for effectively inducing humoral and cell‐mediated immune responses against homologous and heterologous species in the spotted fever group.
Production of Recombinant Protein Pap31 and Its Application for the Diagnosis of Bartonella bacilliformis Infection
Authors: TAYE A , CHEN H , DUNCAN K , ZHANG Z , HENDRIX L , GONZALEZ J , CHING W
Published: February 2006

Abstract: Tropical bartonellosis is a highly fatal epidemic and endemic infectious disease that occurs throughout the communities of the Andes Mountains in South America. The disease is caused by the facultative intracellular bacteria, Bartonella bacilliformis. The emergence of bartonellosis in new geographic areas and an increase in the number of reported cases suggest the need for a rapid test for epidemiologic study and investigation of the disease burden. The objective of this research is to develop a rapid serologic diagnostic test using recombinant antigens to overcome the limitations of the current standard IFA technique for laboratory diagnosis. Western blot analysis with patient sera of whole cell lysate separated on a 2D gel identified Pap31 as a dominant antigen. PCR primers were designed according to the sequence of ATCC strain 35685 to amplify the gene coding for Pap31 from a local isolate (HOSP 800–09, Peru). The amplicon was subsequently cloned into pET24a, adding the T7 tag, and expressed in E. coli. Patient sera with different IFA titers confirmed the diagnostic band of 31 kDa on a Western blot of SDS‐PAGE. The performance of affinity‐purified recombinant Pap31 (rPap31) was also evaluated in an ELISA format with 137 patient sera of known IFA titers. The range of ELISA reading from positive sera did not overlap with the range of those from negative sera, suggesting the potential application of rPap31 in both ELISA for high throughput regional hospital settings and in the construction of handheld rapid tests for rural clinical sites.
Bartonella bacilliformis GroEL: Effect on Growth of Human Vascular Endothelial Cells in Infected Cocultures
Authors: SMITHERMAN LAURA S. , MINNICK MICHAEL F.
Published: February 2006

Abstract: Bartonella are the only bacteria known to induce angioproliferative lesions of the human vasculature and liver during infection. Previous work from our lab suggests that GroEL participates in the mitogenic response observed in HUVEC cultures supplemented with the soluble fraction of Bartonella bacilliformis. Work in this study shows that exposure to high concentrations of the fraction is actually cytotoxic for HUVECs. To analyze this phenomenon, live B. bacilliformis‐HUVEC cocultures were employed to study the effect of excess bacterial GroEL on the host cell during active infection. Four B. bacilliformis strains were generated to produce varying levels of GroEL. HUVEC cocultures with LSS100, a strain that synthesizes markedly greater quantities of GroEL relative to others, significantly accelerates apoptosis of the cocultured HUVECs relative to other strains. Acceleration of apoptosis can be inhibited by Z‐VAD‐FMK, a pan‐caspase inhibitor. Time course data show that, at 18 h of infection, both LSS100 and control strains significantly inhibit spontaneous apoptosis of cocultured HUVECs, as previously reported for other Bartonella species. However, by 48 h, LSS100 significantly increases apoptosis of the host cell. We hypothesize that intracellular Bartonella GroEL functions as an Hsp60 analogue, a eukaryotic orthologue known to accelerate pro‐caspase 3 activation by enhancing its vulnerability to upstream activator caspases. These data suggest another strategy whereby Bartonella may regulate host cell growth.
Occurrence of Bartonella henselae and Bartonella quintana among Human Immunodeficiency Virus‐Infected Patients
Authors: PAPE M , KOLLARAS P , MANDRAVELI K , TSONA A , METALLIDIS S , NIKOLAIDIS P , ALEXIOU‐DANIEL S
Published: February 2006

Abstract: The purpose of this study was to determine the seroprevalence against Bartonella henselae and Bartonella quintana among a risk group, patients with HIV infection, and to identify the epidemiological factors involved. Our data indicate that the prevalence of Bartonella infection among HIV‐infected patients is much greater than that in the healthy population of the same area and that Bartonella infection should be considered in the differential diagnosis for patients with HIV disease.
Bacillary Angiomatosis Caused by Bartonella quintana
Authors: SALA MONTSERRAT , FONT BERNAT , SANFELIU ISABEL , QUESADA MARIELA , PONTS IMMA , SEGURA FERRAN
Published: February 2006

Abstract: Bacillary angiomatosis is a disorder of neovascular proliferation involving skin and lymph nodes of immunosuppressed patients. Bartonella henselae or Bartonella quintana have been inculpated as causative by direct culture or PCR amplification of DNA sequences. Here, we report the clinical evolution of a patient infected with human immunodeficiency virus (HIV) whose B. quintana infection was diagnosed by PCR.
Molecular Screening of Bartonella Species in Rodents from the Russian Far East
Authors: MEDIANNIKOV OLEG , IVANOV LEONID , ZDANOVSKAYA NINA , VYSOCHINA NELYA , FOURNIER PIERRE‐EDOUARD , TARASEVICH IRINA , RAOULT DIDIER
Published: February 2006

Abstract: A potentially broad‐range PCR system for gltA gene amplification has been designed. A nearly full (992 bp) sequence of the gltA gene was amplified in two steps. DNA was extracted from organs (spleen and liver) of four mostly spread species of rodents: Apodemus agrarius and Apodemus peninsulae mice; Clethrionomys rufocanus and Microtus fortis voles. All amplicons have been sequenced. The incidence of Bartonella species in rodents was 73%, 60%, 60%, and 83% for the above‐mentioned rodents, respectively. Several genotypes have been identified, including two gltA genotypes close to B. taylorii, four gltA genotypes close to B. grahamii, and one genotype of a potentially new species. Several Bartonella species were found in both mice species, although each vole species harbored a single species of Bartonella. High incidence of Bartonella infection was found in all studied species of small rodents in the Russian Far East. Several amplified genotypes probably belong to local strains of B. taylorii and B. grahamii, and one genotype represents a possible new species.
Rickettsia parkerias a Paradigm for Multiple Causes of Tick‐Borne Spotted Fever in the Western Hemisphere
Authors: PADDOCK CHRISTOPHER D.
Published: February 2006

Abstract:Among the many contributions made to rickettsiology by entomologist and rickettsiologist Ralph R. Parker was his discovery in 1937 of a novel rickettsia isolated from the Gulf Coast tick,Amblyomma maculatum. This bacterium was subsequently characterized as a unique rickettsial species in 1965 and namedRickettsia parkeriin honor of its discoverer. During the next several decadesR. parkeriwas generally considered as one of several “nonpathogenic” spotted fever group (SFG) rickettsiae that resided in ticks of the United States. The identification of novel rickettsioses on other continents during the last two decades of the twentieth century provided important evidence of the frequent coexistence of multiple and unique tick‐borne SFG rickettsiae sharing common geographic regions. Surprisingly, this paradigm, which was repeatedly demonstrated in Europe, Africa, and Australia during the last 10 years, had no confirmed correlate in the United States until 2002, whenR. parkeriwas isolated from a patient from the state of Virginia. Several pieces of epidemiologic, laboratory, and clinical evidence are compelling enough to suggest that this infection has occurred in other U.S. patients who reside within the range of the Gulf Coast tick. Just as important are new data indicating relatively high infection rates ofA. maculatumticks withR. parkeri, documenting the occurrence ofR. parkeriinAmblyomma tristeticks from Uruguay, and providing evidence that otherAmblyommaspecies might serve as efficient vectors ofR. parkeri. The recognition ofR. parkerias a cause of disease in humans will hopefully encourage a closer examination for specific etiologies of tick‐borne spotted fever rickettsioses in the United States and other countries of the Western Hemisphere.
Anaplasma and Ehrlichia Infection
Authors: DUMLER J STEPHEN
Published: February 2006

Abstract: Human and animal infections by Anaplasmataceae are increasingly recognized as important and potentially fatal arthropod‐transmitted diseases. Since the first recognition and implementation of diagnostic methods for human infection by Ehrlichia chaffeensis and Anaplasma phagocytophilum, the incidence of infections has linearly increased. Moreover, diagnostic and epidemiological testing indicates that “ehrlichia” infections are globally distributed and suggests that additional agents of human and veterinary “ehrlichiosis” will be identified. With increasing disease identification has come recognition that the infections can be severe, with approximately one‐half of patients requiring hospitalization for complications including respiratory distress, myocarditis, neurological complications, hepatitis, a septic or toxic shock‐like disease, opportunistic infections, and death in 0.5 to 3.0%. An understanding of the diseases, pathophysiology, pathogenesis, control, and management will best be developed through fundamental investigations. Advances in comprehension as to the separate contributions of bacteria and host are crucial since most data now suggest that alterations in host neutrophil function and protection from innate and adaptive immunity also contribute to disease manifestations. It is reasonable to operate from the hypothesis that these host cell functional changes ultimately benefit bacterial survival while inadvertently eliciting clinical disease in poorly adapted hosts. A firmer basis for the scientific understanding of Anaplasmataceae biology will allow logical and rational approaches toward infection control, prevention, and treatment.
Analysis of Ehrlichial p28 Gene Expression in a Murine Model of Persistent Infection
Authors: CROCQUET‐VALDES PATRICIA A. , McBRIDE JERE W. , FENG HUI‐MIN , ISMAIL NAHED , SMALL MELISSA A. , YU XUE‐JIE , WALKER DAVID H.
Published: February 2006

Abstract: Historically, ehrlichioses were tick‐borne diseases of veterinary medical importance and are now important emerging infectious diseases in humans. p28s are encoded by multigene families with ORFs tandemly arranged with intergenic spaces of variable lengths. We reported initial sequencing of the Ehrlichia muris p28 locus. A model of persistent infection was described and provided tools for study of persistent ehrlichial infection. We completed the sequence of the E. muris p28 locus and examined mRNA expression.
Cytokine Responses in Dogs Infected with Ehrlichia canis Oklahoma Strain
Authors: TAJIMA TOMOKO , RIKIHISA YASUKO
Published: February 2006

Abstract: Ehrlichia canis causes persistent infection of monocytes/macrophages and causes canine monocytic ehrlichiosis. Other than antibody development, very little is known how the dog immune system responds to this pathogen. In the present study, we examined the temporal expression of cytokine mRNA in peripheral blood mononuclear cells (PBMCs) from dogs inoculated with E. canis Oklahoma strain. We also examined production of interferon‐gamma (IFN‐γ) by canine PBMCs infected with E. canis in vitro.
Concentration of Procalcitonin and C‐Reactive Protein in Patients with Human Granulocytic Anaplasmosis and the Initial Phase of Tick‐Borne Encephalitis
Authors: LOTRIC‐FURLAN STANKA , ROJKO TEREZA , STRLE FRANC
Published: February 2006

Abstract: The aim of this prospective study was to assess the value of procalcitonin (PCT) level and serum concentration of C‐reactive protein (CRP) in differentiating viral from bacterial infection in selected groups of patients, including those with acute human granulocytic anaplasmosis (HGA), which is caused by an intracellular bacteria Anaplasma phagocytophilum, and patients with the initial phase of tick‐borne encephalitis (TBE), which is a viral disease caused by an RNA virus belonging to the Flavivirus genus of the family Flaviviridae.
Differential effects of experimental and cold-induced hyperthyroidism on factors inducing rat liver oxidative damage
Authors: Venditti P. , Pamplona R. , Ayala V. , De Rosa R. , Caldarone G. , Di Meo S.
Published: February 2006

Thyroid hormone-induced increase in metabolic rates is often associated with increased oxidative stress. The aim of the present study was to investigate the contribution of iodothyronines to liver oxidative stress in the functional hyperthyroidism elicited by cold, using as models cold-exposed and 3,5,3′-triiodothyronine (T3)- or thyroxine(T4)-treated rats. The hyperthyroid state was always associated with increases in both oxidative capacity and oxidative damage of the tissue. The most extensive damage to lipids and proteins was found in T3-treated and cold-exposed rats, respectively. Increase in oxygen reactive species released by mitochondria and microsomes was found to contribute to tissue oxidative damage, whereas the determination of single antioxidants did not provide information about the possible contribution of a reduced effectiveness of the antioxidant defence system. Indeed, liver oxidative damage in hyperthyroid rats was scarcely related to levels of the liposoluble antioxidants and activities of antioxidant enzymes. Conversely,other biochemical changes, such as the degree of fatty acid unsaturation and hemoprotein content, appeared to predispose hepatic tissue to oxidative damage associated with oxidative challenge elicited by hyperthyroid state.

As a whole, our results confirm the idea that T3 plays a key role in metabolic changes and oxidative damage found in cold liver. However,only data concerning changes in glutathione peroxidase activity and mitochondrial protein content favour the idea that dissimilarities in effects of cold exposure and T3 treatment could depend on differences in serum levels of T4.
The accumulation of methylamine counteracting solutes in elasmobranchs with differing levels of urea: a comparison of marine and freshwater species
Authors: Treberg Jason R. , Speers-Roesch Ben , Piermarini Peter M. , Ip Yuen K. , Ballantyne James S. , Driedzic William R.
Published: February 2006

We compared levels of the major organic osmolytes in the muscle of elasmobranchs, including the methylamines trimethylamine oxide (TMAO), betaine and sarcosine as well as the β-amino acids taurine and β-alanine,and the activities of enzymes of methylamine synthesis (betaine and TMAO) in species with a wide range of urea contents. Four marine, a euryhaline in freshwater (Dasyatis sabina), and two freshwater species, one that accumulates urea (Himantura signifer) and one that does not(Potamotrygon motoro), were analyzed. Urea contents in muscle ranged from 229–352 μmol g–1 in marine species to 2.0μmol g–1 in P. motoro. Marine elasmobranchs preferentially accumulate methylamines, possibly to counteract urea effects on macromolecules, whereas the freshwater species with lower urea levels accumulate the β-amino acid taurine as the major non-urea osmolyte. A strong correlation (r2=0.84, P<0.001) with a slope of 0.40 was found between muscle urea content and the combined total methylamines plus total β-amino acids, supporting the hypothesis that`non-urea' osmolytes are specifically maintained at an approximately 2:1 ratio with urea in the muscle of elasmobranchs. All species examined had measurable synthetic capacity for betaine in the liver but only one species had detectable TMAO synthetic capacity. We propose a phylogenetic explanation for the distribution of TMAO synthesis in elasmobranchs and suggest that activation of liver betaine aldehyde dehydrogenase, relative to choline dehydrogenase, coincides with betaine accumulation in elasmobranchs. The latter relationship may be important in maintaining methylamine levels during periods of low dietary TMAO intake for species lacking TMAO synthesis.
Pseudodiarrhoea in zebra mussels Dreissena polymorpha (Pallas)exposed to microcystins
Authors: Juhel Guillaume , Davenport John , O'Halloran John , Culloty Sarah , Ramsay Ruth , James Kevin , Furey Ambrose , Allis Orla
Published: February 2006

Microcystins are produced by bloom-forming cyanobacteria and pose significant health and ecological problems. In this study we show that zebra mussels respond differently to different strains of Microcystis aeruginosa, and that a highly toxic strain causes zebra mussels to produce large quantities of mucous pseudofaeces, `pseudodiarrhoea', that are periodically expelled hydraulically through the pedal gape by shell valve adductions rather than by the normal ciliary tracts. Analysis of the pseudofaecal ejecta showed that the proportion of Microcystis aeruginosa relative to Asterionella formosa was high in the pseudofaeces and even higher in the `pseudodiarrhoea' when a mixed diet was given to the mussels. This confirms that very toxic Microcystis aeruginosa were preferentially being rejected by comparison with the non-toxic diatom in the pseudofaeces and even more so in the`pseudodiarrhoea'. Such selective rejection was not observed with low or non-toxic strains and would therefore tend to enhance the presence of toxic Microcystis aeruginosa in mixed Microcystis aeruginosacyanobacterial blooms, as well as transferring toxins from the water column to the benthos.

The observed acute irritant response to the toxin represents the first demonstration of an adverse sublethal effect of microcystins on invertebrate ecophysiology. Our results also suggest that it could be a specific response to microcystin-LF, a little studied toxin variant.
Visual regulation of ground speed and headwind compensation in freely flying honey bees (Apis mellifera L.)
Authors: Barron Andrew , Srinivasan Mandyam V.
Published: February 2006

There is now increasing evidence that honey bees regulate their ground speed in flight by holding constant the speed at which the image of the environment moves across the eye (optic flow). We have investigated the extent to which ground speed is affected by headwinds. Honey bees were trained to enter a tunnel to forage at a sucrose feeder placed at its far end. Ground speeds in the tunnel were recorded while systematically varying the visual texture of the tunnel, and the strength of headwinds experienced by the flying bees. We found that in a flight tunnel bees used visual cues to maintain their ground speed, and adjusted their air speed to maintain a constant rate of optic flow, even against headwinds which were, at their strongest, 50% of a bee's maximum recorded forward velocity. Manipulation of the visual texture revealed that headwind is compensated almost fully even when the optic flow cues are very sparse and subtle, demonstrating the robustness of this visual flight control system. We discuss these findings in the context of field observations of flying bees.
Contractile recovery from acidosis in toad ventricle is independent of intracellular pH and relies upon Ca2+ influx
Authors: Salas Margarita A. , Vila-Petroff Martín G. , Venosa Roque A. , Mattiazzi Alicia
Published: February 2006

Hypercapnic acidosis produces a negative inotropic effect on myocardial contractility followed by a partial recovery that occurs in spite of the persistent extracellular acidosis. The underlying mechanisms of this recovery are far from understood, especially in those species in which excitation–contraction coupling differs from that of the mammalian heart. The main goal of the present experiments was to obtain a better understanding of these mechanisms in the toad heart. Hypercapnic acidosis,induced by switching from a bicarbonate-buffered solution equilibrated with 5%CO2 to the same solution equilibrated with 12% CO2,evoked a decrease in contractility followed by a recovery that reached values higher than controls after 30 min of continued acidosis. This contractile pattern was associated with an initial decrease in intracellular pH(pHi) that recovered to control values in spite of the persistent extracellular acidosis. Blockade of the Na+/H+ exchanger(NHE) with cariporide (5 μmol l–1) produced a complete inhibition of pHi restitution, without affecting the mechanical recovery. Hypercapnic acidosis also produced a gradual increase of diastolic and peak Ca2+i transient values, which occurred immediately after the acidosis was settled and persisted during the mechanical recovery phase. Inhibition of Ca2+ influx through the reverse mode of the Na+/Ca2+ exchanger (NCX) by KB-R (1 μmol l–1 for myocytes and 20 μmol l–1 for ventricular strips), or of L-type Ca2+ channels by nifedipine (0.5μmol l–1), completely abolished the mechanical recovery. Acidosis also produced an increase in the action potential duration. This prolongation persisted throughout the acidosis period. Our results show that in toad ventricular myocardium, acidosis produces a decrease in contractility,due to a decrease in Ca2+ myofilament responsiveness, followed by a contractile recovery, which is independent of pHi recovery and relies on an increase in the influx of Ca2+. The results further indicate that both the reverse mode NCX and the L-type Ca2+channels, appear to be involved in the increase in intracellular Ca2+ concentration that mediates the contractile recovery from acidosis.
A Spontaneous Mutation InvolvingKcnq2(Kv7.2) Reduces M-Current Density and Spike Frequency Adaptation in Mouse CA1 Neurons
Authors: Otto James F. , Yang Yan , Frankel Wayne N. , White H. Steve , Wilcox Karen S.
Published: February 2006

The M-type K+current [IK(M)] activates in response to membrane depolarization and regulates neuronal excitability. Mutations in two subunits (KCNQ2 and KCNQ3; Kv7.2 and Kv7.3) that underlie the M-channel cause the human seizure disorder benign familial neonatal convulsions (BFNC), presumably by reducingIK(M)function. In mice, theSzt1mutation, which deletes the genomic DNA encoding the KCNQ2 C terminus and all of CHRNA4 (nicotinic acetylcholine receptor α4 subunit) and ARFGAP-1 (GTPase-activating protein that inactivates ADP-ribosylation factor 1), reduces seizure threshold, and alters M-channel pharmacosensitivity. Genomic deletions affecting the C terminus of KCNQ2 have been identified in human families with BFNC, and truncation of the C terminus prevents proper KCNQ2/KCNQ3 channel assembly inXenopusoocytes. We showed previously thatSzt1mice have a reduced baseline seizure threshold and altered sensitivity to drugs that act at the M-channel. Specifically, the proconvulsant M-channel blocker linopirdine and anticonvulsant enhancer retigabine display increased and decreased potency, respectively, inSzt1mice. To investigate the effects of theSzt1mutation onIK(M)function explicitly, perforated-patch electrophysiology was performed in CA1 pyramidal neurons of the hippocampus in brain slices prepared from C57BL/6J-Szt1/+and control C57BL/6J+/+ mice. Our results show thatSzt1reduces bothIK(M)amplitude and current density, inhibits spike frequency adaptation, and alters many aspects of M-channel pharmacology. This is the first evidence that a naturally occurringKcnq2mutation diminishes the amplitude and function of the native neuronalIK(M), resulting in significantly increased neuronal excitability. Finally, the changes in single-cell biophysical properties likely underlie the altered seizure threshold and pharmacosensitivity reported previously inSzt1mice.
Forebrain Glucocorticoid Receptors Modulate Anxiety-Associated Locomotor Activation and Adrenal Responsiveness
Authors: Boyle Maureen P. , Kolber Benedict J. , Vogt Sherri K. , Wozniak David F. , Muglia Louis J.
Published: February 2006

Stress potently modulates anxiety- and depression-related behaviors. In response to stressors, the hypothalamic-pituitary-adrenal (HPA) axis is activated, resulting in the release of glucocorticoids from the adrenal cortex. These hormones act peripherally to restore homeostasis but also feed back to the CNS to control the intensity and duration of the stress response. Glucocorticoids act in limbic areas of the CNS to mediate the psychological and behavioral effects of stress. In this study, we investigate the effect of forebrain-specific disruption of the glucocorticoid receptor (GR) on stress- and anxiety-related behaviors. We demonstrate that mice with disruption of forebrain GR show alterations in stress-induced locomotor activation in a number of anxiety-related behavioral paradigms. These changes are associated with alterations in stress-induced HPA axis activation and, importantly, are not attenuated by chronic treatment with the tricyclic antidepressant imipramine. These data demonstrate the importance of forebrain GR in regulation of physiological and behavioral stress reactivity and suggest that distinct pathways regulate despair- and anxiety-related behaviors.
Presynaptic Control of Striatal Glutamatergic Neurotransmission by Adenosine A1–A2AReceptor Heteromers
Authors: Ciruela Francisco , Casadó Vicent , Rodrigues Ricardo J. , Luján Rafael , Burgueño Javier , Canals Meritxell , Borycz Janusz , Rebola Nelson , Goldberg Steven R. , Mallol Josefa , Cortés Antonio , Canela Enric I. , López-Giménez Juan F. , Milligan Graeme , Lluis Carme , Cunha Rodrigo A. , Ferré Sergi , Franco Rafael
Published: February 2006

The functional role of heteromers of G-protein-coupled receptors is a matter of debate. In the present study, we demonstrate that heteromerization of adenosine A1receptors (A1Rs) and A2Areceptors (A2ARs) allows adenosine to exert a fine-tuning modulation of glutamatergic neurotransmission. By means of coimmunoprecipitation, bioluminescence and time-resolved fluorescence resonance energy transfer techniques, we showed the existence of A1R–A2AR heteromers in the cell surface of cotransfected cells. Immunogold detection and coimmunoprecipitation experiments indicated that A1R and A2AR are colocalized in the same striatal glutamatergic nerve terminals. Radioligand-binding experiments in cotransfected cells and rat striatum showed that a main biochemical characteristic of the A1R–A2AR heteromer is the ability of A2AR activation to reduce the affinity of the A1R for agonists. This provides a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release. Furthermore, it is also shown that A1R–A2AR heteromers constitute a unique target for caffeine and that chronic caffeine treatment leads to modifications in the function of the A1R–A2AR heteromer that could underlie the strong tolerance to the psychomotor effects of caffeine.
Spatiotemporal Receptive Fields of Peripheral Afferents and Cortical Area 3b and 1 Neurons in the Primate Somatosensory System
Authors: Sripati Arun P. , Yoshioka Takashi , Denchev Peter , Hsiao Steven S. , Johnson Kenneth O.
Published: February 2006

Neurons in area 3b have been previously characterized using linear spatial receptive fields with spatially separated excitatory and inhibitory regions. Here, we expand on this work by examining the relationship between excitation and inhibition along both spatial and temporal dimensions and comparing these properties across anatomical areas. To that end, we characterized the spatiotemporal receptive fields (STRFs) of 32 slowly adapting type 1 (SA1) and 21 rapidly adapting peripheral afferents and of 138 neurons in cortical areas 3b and 1 using identical random probe stimuli. STRFs of peripheral afferents consist of a rapidly appearing excitatory region followed by an in-field (replacing) inhibitory region. STRFs of SA1 afferents also exhibit flanking (surround) inhibition that can be attributed to skin mechanics. Cortical STRFs had longer time courses and greater inhibition compared with peripheral afferent STRFs, with less replacing inhibition in area 1 neurons compared with area 3b neurons. The greater inhibition observed in cortical STRFs point to the existence of underlying intracortical mechanisms. In addition, the shapes of excitatory and inhibitory lobes of both peripheral and cortical STRFs remained mostly stable over time, suggesting that their feature selectivity remains constant throughout the time course of the neural response. Finally, the gradual increase in the proportion of surround inhibition from the periphery to area 3b to area 1, and the concomitant decrease in response linearity of these neurons indicate the emergence of increasingly feature-specific response properties along the somatosensory pathway.
Deficient Nonpeptidergic Epidermis Innervation and Reduced Inflammatory Pain in Glial Cell Line-Derived Neurotrophic Factor Family Receptor α2 Knock-Out Mice
Authors: Lindfors Päivi H. , Võikar Vootele , Rossi Jari , Airaksinen Matti S.
Published: February 2006

Most unmyelinated nociceptive neurons that mediate pain and temperature sensation from the skin bind isolectin B4 (IB4)-lectin and express Ret, the common signaling component of glial cell line-derived neurotrophic factor (GDNF) family. One of these factors, neurturin, is expressed in the epidermis, whereas its GDNF family receptor α2 (GFRα2) is expressed in the majority of unmyelinated Ret-positive sensory neurons. However, the physiological roles of endogenous neurturin signaling in primary sensory neurons are poorly understood. Here, we show that the vast majority (∼85%) of IB4 binding and P2X3purinoreceptor-positive neurons, but virtually none of the calcitonin gene-related peptide (CGRP) or vanilloid receptor transient receptor potential vanilloid 1-positive neurons in mouse dorsal root ganglion (DRG) express GFRα2. In GFRα2 knock-out (KO) mice, the IB4-binding and P2X3-positive DRG neurons were present but reduced in size, consistent with normal number but reduced caliber of unmyelinated axons in a cutaneous nerve. Strikingly, nonpeptidergic (CGRP-negative) free nerve endings in footpad epidermis were >70% fewer in GFRα2-KO mice than in their wild-type littermates. In contrast, the density of CGRP-positive epidermal innervation remained unaffected. In the formalin test, the KO mice showed a normal acute response but a markedly attenuated persistent phase, indicating a deficit in inflammatory pain response. Behavioral responses of GFRα2-KO mice to innocuous warm and noxious heat were not blunted; the mice were actually markedly hypersensitive to noxious cold in tail immersion test. Overall, our results indicate a critical role for endogenous GFRα2 signaling in maintaining the size and terminal innervation of the nonpeptidergic class of cutaneous nociceptorsin vivo.
State-Dependent Dendritic Computation in Hippocampal CA1 Pyramidal Neurons
Authors: Gasparini Sonia , Magee Jeffrey C.
Published: February 2006

Depending on the behavioral state, hippocampal CA1 pyramidal neurons receive very distinct patterns of synaptic input and likewise produce very different output patterns. We have used simultaneous dendritic and somatic recordings and multisite glutamate uncaging to investigate the relationship between synaptic input pattern, the form of dendritic integration, and action potential output in CA1 neurons. We found that when synaptic input arrives asynchronously or highly distributed in space, the dendritic arbor performs a linear integration that allows the action potential rate and timing to vary as a function of the quantity of the input. In contrast, when synaptic input arrives synchronously and spatially clustered, the dendritic compartment receiving the clustered input produces a highly nonlinear integration that leads to an action potential output that is extraordinarily precise and invariant. We also present evidence that both of these forms of information processing may be independently engaged during the two distinct behavioral states of the hippocampus such that individual CA1 pyramidal neurons could perform two different state-dependent computations: input strength encoding during theta states and feature detection during sharp waves.
Charged Residues in the α1and β2Pre-M1 Regions Involved in GABAAReceptor Activation
Authors: Mercado Jose , Czajkowski Cynthia
Published: February 2006

For Cys-loop ligand-gated ion channels (LGIC), the protein movements that couple neurotransmitter binding to channel gating are not well known. The pre-M1 region, which links the extracellular agonist-binding domain to the channel-containing transmembrane domain, is in an ideal position to transduce binding site movements to gating movements. A cluster of cationic residues in this region is observed in all LGIC subunits, and in particular, an arginine residue is absolutely conserved. We mutated charged pre-M1 residues in the GABAAreceptor α1(K219, R220, K221) and β2(K213, K215, R216) subunits to cysteine and expressed the mutant subunits with wild-type β2or α1inXenopusoocytes. Cysteine substitution of β2R216 abolished channel gating by GABA without altering the binding of the GABA agonist [3H]muscimol, indicating that this residue plays a key role in coupling GABA binding to gating. Tethering thiol-reactive methanethiosulfonate (MTS) reagents onto α1K219C, β2K213C, and β2K215C increased maximal GABA-activated currents, suggesting that structural perturbations of the pre-M1 regions affect channel gating. GABA altered the rates of sulfhydryl modification of α1K219C, β2K213C, and β2K215C, indicating that the pre-M1 regions move in response to channel activation. A positively charged MTS reagent modified β2K213C and β2K215C significantly faster than a negatively charged reagent, and GABA activation eliminated modification of β2K215C by the negatively charged reagent. Overall, the data indicate that the pre-M1 region is part of the structural machinery coupling GABA binding to gating and that the transduction of binding site movements to channel movements is mediated, in part, by electrostatic interactions.
Functional Dissection of Reelin Signaling by Site-Directed Disruption of Disabled-1 Adaptor Binding to Apolipoprotein E Receptor 2: Distinct Roles in Development and Synaptic Plasticity
Authors: Beffert Uwe , Durudas Andre , Weeber Edwin J. , Stolt Peggy C. , Giehl Klaus M. , Sweatt J. David , Hammer Robert E. , Herz Joachim
Published: February 2006

The Reelin signaling pathway controls neuronal positioning in human and mouse brain during development as well as modulation of long-term potentiation (LTP) and behavior in the adult. Reelin signals by binding to two transmembrane receptors, apolipoprotein E receptor 2 (Apoer2) and very-low-density lipoprotein receptor. After Reelin binds to the receptors, Disabled-1 (Dab1), an intracellular adaptor protein that binds to the cytoplasmic tails of the receptors, becomes phosphorylated on tyrosine residues, initiating a signaling cascade that includes activation of Src-family kinases and Akt. Here, we have created a line of mutant mice (Apoer2 EIG) in which the Apoer2 NFDNPVY motif has been altered to EIGNPVY to disrupt the Apoer2–Dab1 interaction to further study Reelin signaling in development and adult brain. Using primary neuronal cultures stimulated with recombinant Reelin, we find that normal Reelin signaling requires the wild-type NFDNPVY sequence and likely the interaction of Apoer2 with Dab1. Furthermore, examination of hippocampal, cortical, and cerebellar layering reveals that the NFDNPVY sequence of Apoer2 is indispensable for normal neuronal positioning during development of the brain. AdultApoer2 EIGmice display severe abnormalities in LTP and behavior that are distinct from those observed for mice lacking Apoer2. InApoer2 EIGslices, LTP degraded to baseline within 30 min, and this was prevented in the presence of Reelin. Together, these findings emphasize the complexity of Reelin signaling in the adult brain, which likely requires multiple adaptor protein interactions with the intracellular domain of Apoer2.
Relative Contributions of Axonal and Somatic Na Channels to Action Potential Initiation in Cerebellar Purkinje Neurons
Authors: Khaliq Zayd M. , Raman Indira M.
Published: February 2006

Neuronal excitability is likely to be regulated by the site of action potential initiation, the location on a neuron that crosses threshold first. Although initiation is axonal in many neurons, in Purkinje cells, somatic conductances can generate spontaneous action potentials, suggesting that the perisomatic region (soma and/or initial segment) contributes to spike initiation and may regulate firing. To identify directly the cellular regions at which Na channel modulation significantly influences firing, we measured spontaneous and evoked action potentials in Purkinje cells in cerebellar slices from postnatal day 14–28 mice while applying drugs locally to either the soma/initial segment or the first node of Ranvier. Na currents were decreased by tetrodotoxin (TTX) or increased by β-pompilidotoxin (β-PMTX). Dual somatic and axonal recordings indicated that spike thresholds and input–output curves were sensitive to TTX or β-PMTX at the perisomatic region but were unchanged by either drug at the first node. When perisomatic Na channel availability was reduced with subsaturating TTX, however, the input–output curve became shallower during additional TTX block of nodal channels, revealing a latent role for nodal Na channels in facilitating firing. In perisomatic TTX, axons failed to generate spontaneous or evoked spike trains. In contrast, choline block of the initial segment alone altered normal input–output curves. The data suggest that, although the first node reliably follows action potentials, spike initiation in Purkinje neurons occurs in the initial segment. Moreover, Purkinje cell output depends on the density, availability, and kinetics of perisomatic Na channels, a characteristic that may distinguish spontaneously firing from quiescent neurons.
Acceleration of Age-Related Hearing Loss by Early Noise Exposure: Evidence of a Misspent Youth
Authors: Kujawa Sharon G. , Liberman M. Charles
Published: February 2006

Age-related and noise-induced hearing losses in humans are multifactorial, with contributions from, and potential interactions among, numerous variables that can shape final outcome. A recent retrospective clinical study suggests an age–noise interaction that exacerbates age-related hearing loss in previously noise-damaged ears (Gates et al., 2000). Here, we address the issue in an animal model by comparing noise-induced and age-related hearing loss (NIHL; AHL) in groups of CBA/CaJ mice exposed identically (8–16 kHz noise band at 100 dB sound pressure level for 2 h) but at different ages (4–124 weeks) and held with unexposed cohorts for different postexposure times (2–96 weeks). When evaluated 2 weeks after exposure, maximum threshold shifts in young-exposed animals (4–8 weeks) were 40–50 dB; older-exposed animals (≥16 weeks) showed essentially no shift at the same postexposure time. However, when held for long postexposure times, animals with previous exposure demonstrated AHL and histopathology fundamentally unlike unexposed, aging animals or old-exposed animals held for 2 weeks only. Specifically, they showed substantial, ongoing deterioration of cochlear neural responses, without additional change in preneural responses, and corresponding histologic evidence of primary neural degeneration throughout the cochlea. This was true particularly for young-exposed animals; however, delayed neuropathy was observed in all noise-exposed animals held 96 weeks after exposure, even those that showed no NIHL 2 weeks after exposure. Data suggest that pathologic but sublethal changes initiated by early noise exposure render the inner ears significantly more vulnerable to aging.
Regulation of Cytoplasmic Dynein ATPase by Lis1
Authors: Mesngon Mariano T. , Tarricone Cataldo , Hebbar Sachin , Guillotte Aimee M. , Schmitt E. William , Lanier Lorene , Musacchio Andrea , King Stephen J. , Smith Deanna S.
Published: February 2006

Mutations in Lis1 cause classical lissencephaly, a developmental brain abnormality characterized by defects in neuronal positioning. Over the last decade, a clear link has been forged between Lis1 and the microtubule motor cytoplasmic dynein. Substantial evidence indicates that Lis1 functions in a highly conserved pathway with dynein to regulate neuronal migration and other motile events. Yeast two-hybrid studies predict that Lis1 binds directly to dynein heavy chains (Sasaki et al., 2000; Tai et al., 2002), but the mechanistic significance of this interaction is not well understood. We now report that recombinant Lis1 binds to native brain dynein and significantly increases the microtubule-stimulated enzymatic activity of dyneinin vitro. Lis1 does this without increasing the proportion of dynein that binds to microtubules, indicating that Lis1 influences enzymatic activity rather than microtubule association. Dynein stimulationin vitrois not a generic feature of microtubule-associated proteins, because tau did not stimulate dynein. To our knowledge, this is the first indication that Lis1 or any other factor directly modulates the enzymatic activity of cytoplasmic dynein. Lis1 must be able to homodimerize to stimulate dynein, because a C-terminal fragment (containing the dynein interaction site but missing the self-association domain) was unable to stimulate dynein. Binding and colocalization studies indicate that Lis1 does not interact with all dynein complexes found in the brain. We propose a model in which Lis1 stimulates the activity of a subset of motors, which could be particularly important during neuronal migration and long-distance axonal transport.
Combined Pharmacological and Electrophysiological Dissociation of Familiarity and Recollection
Authors: Curran Tim , DeBuse Casey , Woroch Brion , Hirshman Elliot
Published: February 2006

Dual-process theories of recognition memory hypothesize separate underlying familiarity and recollection processes, but the necessity of multiple processes is debated. Previous research has suggested that scalp-recorded event-related brain potentials (ERPs) may index the activity of separate familiarity and recollection processes. Other research indicates that the amnestic drug midazolam impairs recollection more than familiarity. Here, we used a convergent pharmacological and electrophysiological approach to manipulate and monitor human brain activity and provide evidence for separate processes. Midazolam selectively influenced the putative ERP-correlate of recollection but not the putative ERP-correlate of familiarity. Under control conditions (saline), subjects’ accuracy correlated with the recollection-related but not the familiarity-related ERP component, suggesting that recollection was dominant in driving memory. The opposite pattern was observed under midazolam administration, suggesting that when recollection fails, subjects may leverage familiarity to compensate. Thus, in contrast to perspectives holding that familiarity represents the default process, these results suggest that recollection was dominant until its impairment unveiled the influence of familiarity.
The Epidemic in Mental Illness: Clinical Fact or Survey Artifact?
Authors: Horwitz Allan V. , Wakefield Jerome C.
Published: February 2006

Do half of all Americans suffer from mental disorders at some point in their lives? Or do surveys misdiagnose the distress that is a normal part of every life?
Imaging Protein‐Protein Interactions by Fluorescence Resonance Energy Transfer (FRET) Microscopy
Authors: Wouters Fred S. , Bastiaens Philippe I.H.
Published: February 2006

Detection of specific protein‐protein interactions has long been restricted to bulk biochemical methods such as immunoprecipitation and immunoblotting. Even more sensitive methods using general immunofluorescence are limited, and it is difficult to infer protein‐protein interactions from the results of these tests. Fluoresence Resonance Energy Transfer (FRET) is a photophysical process that can be exploited to obtain highly sensitive information about such interactions. It can sense the presence of acceptor fluorophores in the vicinity of a donor fluorophore within a separation distance that is the size of a single protein molecule. This unit details FRET microscopy based on release of quenched donor fluorescence after acceptor photobleaching, microinjection of reagents into the nucleus or cytosol, and labeling of antibodies for these procedures.
Monitoring Protein‐Protein Interactions in Living Cells by Bioluminescence Resonance Energy Transfer (BRET)
Authors: Hamdan Fadi F. , Percherancier Yann , Breton Billy , Bouvier Michel
Published: February 2006

Bioluminescence resonance energy transfer (BRET) allows monitoring of protein‐protein interactions in real time in living cells. One candidate interacting protein is fused to a luminescent energy donor, such asRenillaluciferase, and the other to a fluorescent energy acceptor, such the green fluorescent protein (GFP), and the two are then coexpressed in the same cells. If the two proteins interact, their close proximity allows nonradiative energy transfer (BRET) between the luciferase and the GFP. BRET does not occur if the two proteins are separated by more than 100 Å, making the technique ideal for monitoring protein‐protein interactions in biological systems. This unit describes the use of BRET to study constitutive and agonist‐promoted interactions among signaling molecules, as illustrated by the homodimerization of the CXCR4 receptor and the recruitment of β‐arrestin2 to agonist‐activated G‐protein‐coupled receptors. This noninvasive and homogeneous assay provides a robust and sensitive proteomic platform with applications for basic science research and drug discovery.