bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin‐1 in the Brain
Authors: PATEL H. C. , BOUTIN H. , ALLAN S. M.
Published: July 2009

Abstract: Interleukin‐1 (IL‐1) exerts a number of diverse actions in the brain, and it is currently well accepted that it contributes to experimentally induced neurodegeneration. Much of this is based on studies using the IL‐1 receptor antagonist, which inhibits cell death caused by ischemia, brain injury, or excitotoxins. Our aim is to determine how and where in the brain IL‐1 acts to produce these effects. Most of the neurodegenerative effects of IL‐1 are thought to be through IL‐1β. However, we have data implicating IL‐1α in excitotoxic cell death. Furthermore mice lacking both IL‐1α and IL‐1β show dramatically reduced ischemic cell death, whereas deletion of IL‐1α or IL‐1β alone fails to modify damage. It has also been demonstrated that IL‐1 exacerbates ischemic injury in mice in the absence of the type I IL‐1 receptor, suggesting the existence of novel IL‐1 receptors in the brain. IL‐1 also dramatically exacerbates neuronal loss in response to intrastriatal administration of the excitotoxin AMPA in the rat brain, an effect accompanied by marked increases in cytokine expression in the frontoparietal cortex, which precedes subsequent cell death in this region. Intrastriatal AMPA also results in limbic seizures that are exacerbated by IL‐1, and we hypothesize, therefore, that IL‐1 exacerbates cell death through increased seizure activity. Therefore, IL‐1 appears to induce acute neurodegeneration through a number of mechanisms.
Exploring the Stereostructural Requirements of Peptide Ligands for the Melanocortin Receptors
Authors: HRUBY VICTOR J. , CAI MINYING , GRIECO PAOLO , HAN GUOXIA , KAVARANA MALCOLM , TRIVEDI DEV
Published: July 2009

Abstract: The melanotropin peptides α‐MSH, γ‐MSH, and β‐MSH are believed to be the natural ligands for the four melanocortin receptors, MC1R, MC3R, MC4R, and MC5R. However, these peptides generally have low selectivity for these receptors. We report on some approaches to the development of selective agonists and antagonists peptide ligands for these receptors.
PT‐141: A Melanocortin Agonist for the Treatment of Sexual Dysfunction
Authors: MOLINOFF P. B. , SHADIACK A. M. , EARLE D. , DIAMOND L. E. , QUON C. Y.
Published: July 2009

Abstract: PT‐141, a synthetic peptide analogue of α‐MSH, is an agonist at melanocortin receptors including the MC3R and MC4R, which are expressed primarily in the central nervous system. Administration of PT‐141 to rats and nonhuman primates results in penile erections. Systemic administration of PT‐141 to rats activates neurons in the hypothalamus as shown by an increase in c‐Fos immunoreactivity. Neurons in the same region of the central nervous system take up pseudorabies virus injected into the corpus cavernosum of the rat penis. Administration of PT‐141 to normal men and to patients with erectile dysfunction resulted in a rapid dose‐dependent increase in erectile activity. The results suggest that PT‐141 holds promise as a new treatment for sexual dysfunction.
Avian Melanocortin System: α‐MSH May Act as an Autocrine/Paracrine Hormone
Authors: TAKEUCHI SAKAE , TAKAHASHI SUMIO , OKIMOTO RONALD , SCHIÖTH HELGI B. , BOSWELL TIMOTHY
Published: July 2009

Abstract: The interest in the physiological role of α‐MSH in birds has been limited because they lack the intermediate lobe of the pituitary, the main source of circulating α‐MSH in most vertebrates. Recent studies have improved our understanding of the avian melanocortin system. We have cloned and characterized all five MC‐R subtypes, POMC, and AGRP in chicken. Analyses of the tissue distribution of expression of these genes revealed widespread expression throughout the body, corresponding to the situation in mammals in which α‐MSH exerts a multiplicity of effects in different tissues by acting as a local mediator. We showed that the extended black locus controlling feather pigmentation in the chicken encodes MC1‐R. Moreover, black chickens carrying the dominant allele, the extended black, express the MC1‐R with ligand‐independent activity as the somber‐3J black mice. α‐MSH and AGRP were expressed in the infundibular nucleus of POMC and NPY neurons, respectively, in the brain of Japanese quail. Furthermore, fasting stimulated AGRP expression and lowered POMC expression. These data indicate that at least two of the major melanocortin systems reported in mammals, that is, regulation of pigmentation and energy homeostasis, was developed in a common ancestor to chicken and mammals at least 300 million years ago. Furthermore, α‐MSH peptide was identified in developing chicken eye, suggesting a possible involvement of α‐MSH in regulation of ocular development. Collectively, the data reviewed here indicate that α‐MSH is produced locally and acts as an autocrine/paracrine hormone in birds.
Cell Migration and Programmed Cell Death of Drosophila Germ Cells
Authors: COFFMAN CLARK R.
Published: July 2009

Abstract: Cell migration and programmed cell death are essential components of animal development and homeostasis, and the germ cells of Drosophila provide a simple genetic system to study the molecular mechanisms that govern these important cellular processes. Detailed descriptions of germ cell migration in Drosophila were accomplished long ago, but most genetic and molecular analyses of the process have occurred within the past 10 years. A few of the genes required for germ cell migration have been identified, and a very interesting picture is emerging. However, a process as complex as cell migration must involve the functions of many more molecules. In addition, cell migration and cell death mechanisms are often linked, as it is important to eliminate cells that are misplaced and could present a danger to the organism. In Drosophila, genes involved in germ cell migration can also affect programmed cell death. Currently, very little is known about how germ cells ectopic to the gonads are eliminated. To date, only four genes have been reported with roles in germ cell death, and three of these have additional functions in germ cell pathfinding. The nature of the cell death program has not been elucidated. Here, I provide a brief review of Drosophila germ cell migration and programmed cell death at both the descriptive and molecular levels. Many questions remain to be answered, but advances made in recent years are providing useful insights into these critical biological phenomena.
Epigenetic Control during Lymphoid Development and Immune Responses
Authors: MUEGGE KATHRIN , YOUNG HOWARD , RUSCETTI FRANCIS , MIKOVITS JUDY
Published: July 2009

Abstract: Methylation of cytosines controls a number of biologic processes such as imprinting and X chromosomal inactivation. DNA hypermethylation is closely associated with transcriptional silencing, while DNA hypomethylation is associated with transcriptional activation. Hypoacetylation of histones leads to compact chromatin with reduced accessibility to the transcriptional machinery. Methyl‐CpG binding proteins can recruit corepressors and histone deacetylases; thus, the interplay between these epigenetic mechanisms regulates gene activation. Methylation has been implicated as an important mechanism during immune development, controlling VDJ recombination, lineage‐specific expression of cell surface antigens, and transcriptional regulation of cytokine genes during immune responses. Aberrations in epigenetic machinery, either by genetic mutations or by somatic changes such as viral infections, are associated with early alterations in chronic diseases such as immunodeficiency and cancer.
Epidemiologic Considerations to Assess Altered DNA Methylation from Environmental Exposures in Cancer
Authors: MOORE LEE E. , HUANG WEN‐YI , CHUNG JOYCE , HAYES RICHARD B.
Published: July 2009

Abstract:Epidemiologic studies in human populations have identified a broad spectrum of risk factors for cancer. Gene‐damaging agents have been a primary focus of cancer epidemiology; however, all xenobiotics do not interact with DNA directly. Some exogenous agents induce epigenetic changes. In view of this, markers that measure changes to the epigenome must also be incorporated into molecular epidemiologic studies. We review the current understanding of the impact of exogenous agents including: micronutrients, chemotherapeutic agents, metals, and others, on DNA methylation. Two categories of genes are described: (1) genes that can alter susceptibility to aberrant DNA methylation and (2) genes that increase susceptibility to cancer when they are silenced through DNA methylation. Methods for incorporating markers of DNA methylation status into etiologic investigations of the impact of environmental exposures on disease (e.g., cancer) are discussed.
Promoter Hypermethylation as an Epigenetic Component in Type I and Type II Endometrial Cancers
Authors: RISINGER JOHN I. , MAXWELL G. LARRY , BERCHUCK ANDREW , BARRETT J. CARL
Published: July 2009

Abstract: Epigenetic mechanisms that result in aberrant gene expression are a prominent feature of many cancer types. One main epigenetic mechanism for gene silencing involves promoter hypermethylation. Type I and type II endometrial cancers exhibit differing clinical, histologic, and molecular genetic characteristics. We hypothesize that these differences also extend to epigenetic phenomena. Promoter methylation analysis of 11 genes in a panel of endometrial cancers supports this hypothesis. These initial data indicate that promoter hypermethylation events occur frequently in type 1 cancer and were not detected in type II cancers using this panel of loci. These data tend to support the hypothesis that type I and type II endometrial cancers will exhibit distinct patterns of gene silencing based on promoter hypermethylation events.
Closing Pulp and Paper Mill Water Circuits with Membrane Filtration
Authors: NUORTILA‐JOKINEN JUTTA , HUUHILO TIINA , NYSTRÖM MARIANNE
Published: July 2009

Abstract: In this study membrane filtration, ultrafiltration, and nanofiltration alone and as part of hybrid processes are considered as means to purify pulp and paper mill process waters suitable for reuse. Thermophilic ærobic biological treatment, pH adjustment, flocculation, and ozonation were tested as pretreatment methods on pilot or on laboratory scale. The aim was to increase flux and reduce fouling by various pretreatment steps and, thus, increase the competitiveness of the membrane process. The results were also evaluated by comparing the benefits obtained against the costs. It was discovered that benefits could be obtained with all the pretreatments tried. Thermophilic ærobic biology assisted in the removal of organic material and increased flux significantly, but the costs were the highest. The most cost‐effective processes, however, seem to be pH‐adjusted nanofiltration and flocculation nanofiltration hybrid processes, which is understandable because of their significantly lower investment costs compared to, for example, those of biological process. The pH adjustment increased the electrostatic repulsion between negatively charged solutes and membrane, thereby increasing the flux. Flocculation removed the foulants effectively from the feed and it both increased flux and reduced fouling. Yet, many noteworthy benefits were obtained also with ultrafiltration and ozonation. All of the hybrid processes tested could be applied at various points of the water circuit of an integrated pulp and paper mill for purification purposes. The eventual superiority and cost‐effectiveness of the applied process remains to be determined case by case.
Novel Charge‐Mosaic Membranes
Authors: SUN BENHUI , CHENG SHUYING
Published: July 2009

Abstract: Novel charge‐mosaic composite membranes composed of cation and anion permeable domains were prepared. Poly(4‐methylstyrene‐isoprene‐styrene‐isoprene‐4‐methylstyrene) pentablock copolymer (MsISIMs), poly(isoprene‐4‐methylstyrene) diblock copolymer (IMs), and poly(isoprene‐styrene‐isoprene) triblock copolymer (ISI), used as the precursor of the charge‐mosaic membranes, were polymerized from styrene (S), isoprene (I), and 4‐methylstyrene (Ms). MsISIMs and a blend of ISI and IMs were then dissolved in benzene and cast on a polypropylene microporous supporting membrane. After chemical modification, the MsISIMs and IMs/ISI charge‐mosaic composite membranes were obtained. Our experimental results show that the IMs/ISI charge‐mosaic composite membrane is similar in membrane structure and properties to the MsISIMs charge‐mosaic composite membrane.
Natural Gas Cleanup by Means of Membranes
Authors: OHLROGGE KLAUS , BRINKMANN TORSTEN
Published: July 2009

Abstract: This paper deals with the use of membranes for hydrocarbon dewpointing and dehydration of natural gas. Based on experience gained from membrane applications in separating organic vapors from off‐gas and process streams, as well as the dehydration of compressed air, membranes have been developed and tested for use in high pressure applications. Membranes and membrane modules have been modified to withstand the high operating pressure. Calculation programs were developed to understand the separation performance and to provide the necessary information for optimizing membrane design. A real challenge was the introduction of the vacuum mode dehydration operation in order to achieve the highest possible dewpoint reduction with minimum methane loss.
Coordination of T Cell Activation and Migration through Formation of the Immunological Synapse
Authors: DUSTIN MICHAEL L.
Published: July 2009

Abstract: T cell activation is based on interactions of T cell antigen receptors with MHC‐peptide complexes in a specialized cell‐cell junction between the T cell and antigen‐presenting cell—the immunological synapse. The immunological synapse coordinates naïve T cell activation and migration by stopping T cell migration with antigen‐presenting cells bearing appropriate major histocompatibility complex (MHC) peptide complexes. At the same time, the immunological synapse allows full T cell activation through sustained signaling over a period of several hours. The immunological synapse supports activation in the absence of continued T cell migration, which is required for T cell activation through serial encounters. Src and Syk family kinases are activated early in immunological synapse formation, but this signaling process returns to the basal level after 30 min; at the same time, the interactions between T cell receptors (TCRs) and MHC peptides are stabilized within the immunological synapse. The molecular pattern of the mature synapse in helper T cells is a self‐stabilized structure that is correlated with cytokine production and proliferation. I propose that this molecular pattern and its specific biochemical constituents are necessary to amplify signals from the partially desensitized TCR.
Major Peptide Autoepitopes for Nucleosome‐Centered T and B Cell Interaction in Human and Murine Lupus
Authors: DATTA SYAMAL K.
Published: July 2009

Abstract: The potential cross‐reactivity of normal T and B cells to nuclear antigens is vast, probably due to their ‘education’ by apoptotic cell antigens in generative lymphoid organs. Despite this ‘nucleocentric repertoire,’ as we call it, the peripheral immune system normally remains tolerant or ignorant of the products of apoptosis. However, the T helper (Th) cells, and also B cells of lupus, have a regulatory defect in the expression of CD40 ligand (CD40L). A sustained hyper‐expression of CD40L by lupus T cells can be triggered by sub‐threshold stimuli, and is associated with impaired phosphorylation of Cbl‐b, a critical downregulatory molecule in T cell signal transduction. This CD40L hyper‐expression abnormally prolongs co‐stimulatory signals to autoimmune B cells, and it probably instigates APC (dendritic cells, resting anti‐DNA B cells, and macrophages) to present apoptotic cell autoantigens in an immunogenic fashion. We have identified the dominant nucleosomal epitopes that are critical for cognate interactions between autoimmune Th cells and anti‐DNA B cells in lupus. By scanning of overlapping synthetic peptides, and by mass spectrometry of naturally processed peptides, five major epitopes in nucleosomal histones were localized, namely H1′22–42, H2B10‐33, H385‐105, H416‐39, and H471‐94. The autoimmune T cells as well as B cells of lupus recognize these epitopes, and with age, autoantibodies against the peptide epitopes cross‐react with nuclear autoantigens. Moreover, the peptide autoepitopes can be promiscuously presented and recognized by lupus T cells in the context of diverse MHC alleles. This cross‐reactivity opens up the possibility of developing ‘universally’ tolerogenic peptides for therapy of lupus in humans despite their MHC diversity. Indeed, tolerogenic therapy with a single histone peptide epitope can halt the progression of established glomerulonephritis in lupus‐prone mice by ‘tolerance spreading’ that inactivates a broad spectrum of autoimmune T and B cells in concert.
Novel Method to Control Pathogenic Bacteria on Human Mucous Membranes
Authors: FISCHETTI VINCENT A.
Published: July 2009

Abstract: Nearly all infections begin at a mucous membrane site. Also, the human mucous membranes are a reservoir for many pathogenic bacteria found in the environment (that is, pneumococci, staphylococci, streptococci), some of which are resistant to antibiotics. Clearly, if this human reservoir can be reduced or eliminated, the incidence of disease will be markedly reduced. However, compounds designed to eliminate this reservoir are not available. Towards this goal, we have exploited the highly lethal effects of bacteriophage lytic enzymes (lysins) to specifically destroy disease bacteria on mucous membranes. Such lysins are used by the phage to release their progeny at the end of their replicative cycle. We have identified and purified these enzymes and found that when applied externally to gram‐positive bacteria, they are killed seconds after contact. For example, 107S. pyogenes are reduced to undetectable levels 10 s after enzyme addition. A feature of these enzymes is their high specificity; that is, streptococcal lysins kill streptococci and pneumococcal lysins kill pneumococci without effects on the normal flora organisms. In vivo, an oral colonization model for S. pyogenes and a nasal colonization model for S. pneumoniae were developed to test the capacity of the lysins to kill organisms on these surfaces. In both cases, when the animals were pre‐colonized with their respective bacteria then treated with a small amount of lysin, specific for the colonizing organism, all the animals were found to be free of colonizing bacteria shortly after lysin treatment. Thus, lysins may be added to our armamentarium to control antibiotic‐resistant bacteria.
Gauge Theory and Chemical Structure
Authors: MATTINGLY JAMES
Published: July 2009

Abstract:The possibility of chemical structure in the context of quantized matter is examined by way of Richard Bader'sAtoms in Molecules. I critically examine his notion of ‘electronic charge density’—showing that he cannot really mean ‘density of charge’—and I argue that the appropriate concept is expectation value of charge. This still allows him to define chemical structure, but it makes problematic his appeals to the explanatory power of structure. This is because, as Rosenfeld and Bohr showed, the expectation value of charge cannot be taken as the electronic field experienced by other charges. I suggest that we can recover the efficacy of structure by thinking of chemistry as a gauge theory. Current consensus in the study of gauge theories indicates that gauge potentials represent a new type of property; while no member of the family of functions comprising the gauge potential is real, the potential itself is causally potent. I illustrate this in the case of electrodynamics, where the vector potential can causally influence charges in the absence of electric or magnetic fields. I show how chemical structure can be considered to be a gauge field. Following Bader, I take it to be a family of geometric configurations, no one of which is possessed by a given molecule. I claim that current research in gauge theory licenses the attribution of causal potency to this notion of structure, despite its lack of reality. I thus begin the process of freeing the explanatory resources of gauge theory from physics alone.
Site‐Directed Mutagenesis of Amino Acids in the Cytoplasmic Loop 6/7 of Na,K‐ATPase
Authors: XU G. , FARLEY R. A. , KANE D. J. , FALLER L. D.
Published: July 2009

Abstract: The loop between transmembrane helices 6 and 7 (L6/7) of P‐type ATPases has been suggested to be important for the functional linkage of ion binding and enzyme phosphorylation or to be a site of initial cation binding. To investigate the role of L6/7 in Na,K‐ATPase, alanine substitutions were made for charged and conserved residues in L6/7 of the human α1 subunit and the proteins were expressed in yeast for analysis. All mutants except the triple mutant E825A/E828A/D830A bound ouabain. Although the equilibrium dissociation constant for ouabain binding by most mutants was similar to the wild‐type value, the Kd of R837A for ouabain binding was ∼15‐fold higher than the wild‐type Kd. 18O exchange measurements indicated that the apparent affinity of this mutant for Pi was reduced about 3‐fold. The concentration dependence of KCl inhibition of ouabain binding or of NaCl inhibition of ouabain binding revealed 2–4‐fold changes in the apparent affinity for cations in the E825A, E828A, and R837A mutants. The E825A and E828A mutants lost the ability to bind ouabain after extraction with 0.1% SDS or after brief heating, indicating that these mutations affected the stability of the enzyme. The ATPase activity of the other mutants was measured after extraction of crude yeast membranes with 0.1% SDS. For all mutants except R834A, R837A, and R848A, the activity was at least 50% of wild‐type activity.
P‐Type ATPase Superfamily
Authors: OKAMURA HIDEYUKI , DENAWA MASATSUGU , OHNIWA RYOSUKE , TAKEYASU KUNIO
Published: July 2009

Abstract: The P‐type ATPase has become a protein superfamily. On the basis of sequence similarities, the phylogenetic analyses, and substrate specificities, this superfamily can be classified into 5 families and 11 subfamilies. A comparative phylogenetic analysis demonstrates the relationship between the molecular evolution of these subfamilies and the establishment of the kingdoms of living things.
Themes in Ion Pump Regulation
Authors: CORNELIUS F. , MAHMMOUD Y. A.
Published: July 2009

Abstract: The Na,K‐ATPase is an ion pump present in the plasma membrane. It is of vital importance for cell and body homeostasis and as such is under strict hormonal control. The molecular basis for the acute regulation of Na,K‐ATPase is multisite phosphorylation by protein kinases that can alter its behavior. This includes direct effects on the Na,K‐ATPase activity, regulation by membrane trafficking, and even dynamic regulation of interaction with regulatory proteins. In shark Na,K‐ATPase, the latter includes functional interaction with a small hydrophobic protein of the FXYD protein family, phospholemman‐like protein from shark, PLMS. This article summarizes our recent work on the mechanisms involved in the acute regulation of the Na,K‐ATPase studied using a plasma membrane preparation from shark salt glands as an epithelial transport model.
11‐Hydroxylation in the Biosynthesis of Endogenous Ouabain: Multiple Implications
Authors: HAMLYN JOHN M. , LAREDO JAMES , SHAH JUI R. , LU ZHUO REN , HAMILTON BRUCE P.
Published: July 2009

Abstract:Accumulating evidence indicates that mammals use steroidal glycosides with ‘digitalis‐like’ activity. An endogenous ouabain (EO) has been described and is linked with long‐term changes in sodium balance and cardiovascular structure and function. In the adrenal gland, the biosynthesis of EO and similar compounds appears to involve cholesterol side‐chain cleavage with sequential metabolism of pregnenolone and progesterone. The more distal events in the biosynthesis have not been elucidated. Preliminary work using primary cell cultures from the bovine adrenal cortex suggests that the biosynthesis of EO is affected by inhibitors of 11β‐hydroxylase. Direct participation of 11‐hydoxylase in EO synthesis would lead to an 11β isomer of ouabain in mammals and,in vivo, an 11β‐oriented hydroxyl group would spontaneously form a mixture of two 11–19 hemiketal isomers. The latter isomers would likely be converted back to a single 11β isomer of ouabain during isolation. The existence of an additional ring in the hemiketals, along with reduced flexion of the steroidal A, B, and C rings, raises the possibility that theirin vivophysiological targets and actions differ from the isolated form of EO.
Ehrlichia‐like Organism Gene Found in Small Mammals in the Suburban District of Guangzhou of China
Authors: PAN HUA , LIU SHIZHONG , MA YUHAI , TONG SHIDE , SUN YANG
Published: July 2009

Abstract: The 16S rRNA gene of Ehrlichia genus was examined from the blood and spleen of 26 wild small mammals that were captured at a canine feed lot where the first case of canine ehrlichiosis was found in south of China in 1998. Samples (3 blood and 1 spleen) from Rattus norvegicus were positive by polymerase chain reaction. The amplimers were then cloned and sequenced. All of the 16S rRNA gene sequence of these samples were found to be same and unique when compared with the other members of genus Ehrlichia. The most closely related species was Ehrlichia‐like sp. Schotti variant (99.03% related), which was detected and identified in Dutch Ixodes ricinus ticks by Schouls in 1999. The organism found on these animals was obviously not the species of E. muris, which was isolated and identified from a wild mouse in Japan in 1995, since the relatedness of this gene between the former and the latter was 93.28%. It is unclear whether this organism can cause disease in other mammals, including human beings.
Genetic Diversity of Bacterial Agents Detected in Ticks Removed from Asymptomatic Patients in Northeastern Italy
Authors: SANOGO Y. O. , PAROLA P. , SHPYNOV S. , CAMICAS J. L. , BROUQUI P. , CARUSO G. , RAOULT D.
Published: July 2009

Abstract: A total of 360 ticks were removed from 353 asymptomatic subjects in Belluno Province, Italy and surrounding areas, from 1998 to 2001. Ticks were identified as Ixodes ricinus (357), Ixodes hexagonus (1), Rhipicephalus sanguineus (1), and Ixodes ventalloi (1). Tick DNA was investigated by PCR and subsequent sequencing of amplified products to identity associated bacterial agents. Primers targeting different genes of Rickettsia (gltA and OmpA), Borrelia (16S rDNA, rpoB), Francisella (16S rDNA), and all genera members of the Anaplasmataceae (16S rDNA), were used. DNA of bacterial agents was identified in 28 Ixodes ricinus specimens (7.8%). Rickettsia helvetica was detected in 7 ticks. Rickettsia sp. IRS4 and Borrelia afzelii was detected in 4 ticks each. B. garinii and B. valaisiana were identified in one tick each. Anaplasma phagocytophilum, the agent of human granulocytic ehrlichiosis, was identified in 1 specimen of I. ricinus. A new Ehrlichia sp. (‘Candidatus Ehrlichia walkerii’, sp. nov.) was identified in 10 I. ricinus specimens.
Molecular Evidence of Coxiella‐like Microorganism Harbored by Haemaphysalis concinnae Ticks in the Russian Far East
Authors: MEDIANNIKOV OLEG , IVANOV LEONID , NISHIKAWA MAKOTO , SAITO REIKO , SIDELNIKOV YURY N. , ZDANOVSKAYA NINA I. , TARASEVICH IRINA V. , SUZUKI HIROSHI
Published: July 2009

Abstract: Twenty female imago of Haemaphysalis concinnae ticks collected from wild vegetation in May of 2001 in the Obor region near Khabarovsk in the Russian Far East were studied. DNA was extracted with commercially available kits following manufacturer's instructions. We used broad range primers SFG3 and SFG6 proposed for amplification of 16S ribosomal RNA gene's portion of spotted fever group Rickettsia. Initial amplification resulted in positive results in all 20 ticks. Direct sequencing of five randomly chosen amplicons showed that obtained sequences belong to a new species. Closest homology was found in Coxiella burnetii 16S rDNA. Using the obtained sequence as a basis, we designed internal primers highly specific for that sequence. Then we confirmed our results by nested PCR amplification with newly designed primers Cox1 and Cox2—all 20 samples also were positive. Having combined these primers with universal eubacterial primers fD1 and rP2 we retrieved a 1,043 bp portion of the 16S rRNA gene. Consecutive homology search demonstrated that the closest sequence similarity belongs to new Coxiella sp. recently found in Rhipicephalus sanguineus ticks (95.2% of homology) and Coxiella burnetii (94% of homology). So, our results suggest that a novel Coxiella‐like microorganism, provisionally called here ‘Cenerentola,’ is harbored by Haemaphysalis concinnae ticks in the Russian Far East.
Evaluation of Clinical Diagnosis Scores for Boutonneuse Fever
Authors: LETAÏEF A. , SOUISSI J. , TRABELSI H. , GHANNEM H. , JEMNI L.
Published: July 2009

Abstract: Mediterranean spotted fever (MSF) is endemic in Tunisia. Diagnosis is confirmed retrospectively based on serology. Clinical features are suggestive of diagnosis if the triad (fever, rash, and eschar) is present. Otherwise clinical diagnosis could be difficult. A diagnosis score was already proposed by Raoult et al. The aim of this study was to evaluate this score, considering only clinical parameters, in order to help clinicians in diagnosing MSF before confirmation. We retrospectively analyzed 62 consecutive charts of patients with diagnosis of suspected MSF. Diagnosis confirmation was made by serology (IgG anti‐R. conorii exceeding 128 or seroconversion). Epidemiological and clinical features and total score for each patient were reviewed. To validate the clinical score, we calculated sensitivity, specificity and the Youden index for each score in order to establish the ROC curve. SPSS was used for these statistical tests. Area under curve was 0.70 (P= 0.02). The cutoff score with the most predictive value of MSF diagnosis was 18, according to our study (sensitivity 60%, specificity 84,6% and Youden index 0.44). The MSF diagnosis score is a helpful tool to aid the clinician with presumptive management before confirmation. In our study we demonstrated that a score based only on epidemiological and clinical features could be sufficient, but we have to indicate that these results and cutoff score of 18 were from a retrospective study and should be confirmed by a well‐designed prospective one.
Report of Eight Cases of Fatal and Severe Mediterranean Spotted Fever in Portugal
Authors: AMARO M. , BACELLAR F. , FRANÇA A.
Published: July 2009

Abstract: Mediterranean spotted fever (MSF), endemically present, is associated with a low mortality and morbidity in Portugal. Etiological agents are Malish and Israeli tick typhus strains of Rickettsia conorii. In the last few years severe forms of MSF have emerged, with patients presenting atypical symptoms, major neurological manifestations, and multiorgan involvement, who have required intensive care facilities. Advanced age, underlying chronic disease, and delay of appropriate treatment are bad prognostic factors. In the acute phase of diagnosis, serological studies are delayed, inconclusive, and often unhelpful. A definitive diagnosis can only be made using isolation or molecular biology which can establish and clearly identify agents. Using evidence‐based case reports, clinical and laboratory data were evaluated from patients with severe or fatal MSF observed in Garcia da Orta Hospital‐Almada. Of the eight reference cases, four died, three in an acute fulminant stage. Of the survivors, four presented atypical involvement: ocular inoculation, massive gastric hemorrhage, acute respiratory disease (ARDS), and necrotizing vasculitis. Diagnosis by isolation of the agent was made in two cases, by immunohistochemistry in three, and by the indirect fluorescent antibody test (IFA) in three others. Israeli tick typhus and Malish R. conorii strains were isolated once each in fatal cases. In early stages, diagnosis continues to be clinical and patients should start appropriate therapy without delay if clinical suspicion of rickettsiosis arises to prevent poor outcome. Patients ranged in age from 39 to 71 years (mean 60), APACHE II ranged from 15 to 38 points and TISS 28 was between 24 and 46 points. In reported cases severity of disease was not obviously related to the usual comorbidities. Accelerated clinical course may not suggest classical MSF. Another relevant factor was prior prescription of an inappropriate antibiotic that contributed to misleading clinical features. The reported complications and atypical manifestations illustrate well the diversity of this disease.
Identification and Cloning Potentially Protective Antigens ofCoxiella burnetiiUsing Sera from Mice Experimentally Infected with Nine Mile Phase I
Authors: ZHANG G. Q. , SAMUEL J. E.
Published: July 2009

Abstract:Coxiella burnetiiis an obligate intracellular bacterium that causes acute Q fever and occasional chronic infections in humans. To determine the immunodominant antigens during infection withC. burnetii, sera from mice experimentally infected with Nine Mile phase I were tested by immunoblotting. The mouse sera recognized antigens with a variety of molecular weights, including proteins of 14, 22, 28, 34, and 60 kDa as immunodominant antigens. In order to clone potential protective antigens, a genomic DNA library of Nine Mile phase I was constructed in the expression vector Lambda ZAP Express and screened with sera from mice that recovered fromC. burnetiiinfection. A total of 102 immunoreactive clones with various signal intensities were identified from about 8,000 plaques. These clones were purified and expressed in the excised plasmid pBK‐CMV. The proteins expressed by these recombinant plasmids were analyzed by SDS‐PAGE and immunoblotting. Fifty‐four clones expressed immunoreactive proteins of molecular masses ranging from approximately 14 to 60 kDa. Sequence analysis and BLAST search of the recently completed genome sequence identified a variety of novel immunoreactive proteins. These proteins are logical vaccine candidates for testing protective activity againstC. burnetiichallenge. We established a sublethal challenge model in BALB/c mice with protection from the development of severe splenomegaly as an indicator of vaccinogenic activity. Further characterization of these proteins will provide essential information for developing novel, specific diagnostic reagents and potential subunit vaccine candidates againstC. burnetiiinfection.
Adherence to and Invasion of Vero Cells by Recombinant Escherichia coli Expressing the Outer Membrane Protein rOmpB of Rickettsia japonica
Authors: UCHIYAMA TSUNEO
Published: July 2009

Abstract: Recombinant Escherichia coli expressing the outer membrane protein rOmpB of rickettsiae on the surface were generated. The DNA corresponding to the open reading frame of the ompB gene of a spotted fever group rickettsia, Rickettsia japonica, was amplified by polymerase chain reaction. The amplified fragment was inserted between the Sal I and the Xho I sites of the expression vector pET‐22b(+). E. coli BL21(DE3) was transformed by the constructed plasmid. The recombinant bacteria expressed a recombinant protein with a molecular size of 165 kilodaltons on the surface. The size was consistent with that of the precursor of rOmpB. The protein was reactive with monoclonal antibodies to heat‐labile epitopes of the rOmpB. This result suggested a rather native conformation of the recombinant protein. Immunofluorescence of the recombinant bacteria demonstrated the surface expression of the protein. The recombinant bacteria acquired properties to enter Vero cells. The morphological change was observed by means of transmission electron microscopy and scanning electron microscopy. Adherence triggered the generation of abundant microvilli and membrane ruffling for the cells to engulf the bacteria The manner of entry of the recombinant bacteria was similar to that of rickettsiae. Thus it is suggested that the rOmpB plays an important role in the adherence to and invasion of host cells by rickettsiae. Moreover, since even the recombinant rOmpB precursor protein expressed on the surface of the bacteria promotes adherence and invasion, the conformation of the functional domain may be similar to that of the processed mature rOmpB.
Parachlamydia acanthamoeba Is Endosymbiotic or Lytic for Acanthamoeba polyphaga Depending on the Incubation Temperature
Authors: GREUB GILBERT , LA SCOLA BERNARD , RAOULT DIDIER
Published: July 2009

Abstract: Parachlamydiaceae are potential emerging pathogens that naturally infect free‐living amoebae. We investigated the affects of incubation temperature on the growth and cytopathic effect of P. acanthamoeba in Acanthamoeba polyphaga. A. polyphaga were infected with P. acanthamoeba and incubated at different temperatures for ten days. Bacterial growth was quantified by real‐time PCR. Cytopathic effects were determined by counting the number of cysts and viable amoebae (unstained with trypan blue) in Nageotte counting chambers. Uninfected amoebae cultures were used as negative control. At 32, 35, and 37°C, we observed a significant decrease in the number of viable A. polyphaga that contrasted with the delayed and smaller decrease in the number of living A. polyphaga observed at 25, 28, and 30°C. Higher incubation temperature, which is associated with amoebal lysis, surprisingly was not associated with increased growth rate. P. acanthamoeba is lytic for A. polyphaga at 32–37°C but endosymbiotic at 25–30°C. This suggests that A. polyphaga may be a reservoir of endosymbionts at the lower temperature of the nasal mucosa, which may be liberated by lysis at higher temperature, for instance, when the amoeba is inhaled and reaches the lower respiratory tract.
Pathogenic Rickettsiae as Bioterrorism Agents
Authors: AZAD ABDU F. , RADULOVIC SUZANA
Published: July 2009

Abstract: The diseases caused by rickettsiae vary from mild to severe clinical presentations, with case fatality ranging from none to over 30%. The severity of rickettsial diseases has been associated with age, delayed diagnosis, hepatic and renal dysfunction, central nervous system abnormalities, and pulmonary compromise. Despite the variability in clinical presentations many pathogenic rickettsiae cause debilitating diseases, any one of which could be used as a potential biological weapon. While Rickettsia prowazekii, R. rickettsii, and Coxiella burnetii pose serious problems and are currently considered bioterrorism agents, several other species could cause havoc once intentionally released into human populations. The complicating factors include misdiagnosis due to the similarity of rickettsial‐induced clinical signs to many commonly occurring infections and subsequent delayed treatment. Vigilance, preparedness, and the availability of efficacious vaccines and antibiotics are required to avert the morbidity and mortality and disturbances generated by the intentional release of pathogenic rickettsiae into large and immunologically naive human populations. This presentation reviews the rickettsial attributes that make them potential bioterrorism agents, as well as issues related to signs that would alert the responsible authorities, and the preventive measures that could reduce impact of these agents.
The Molecular Effects of Alcohol
Authors: KUMARI MEENA , ANJI ANTJE , WOODS HENRI , TICKU MAHARAJ K.
Published: July 2009

Abstract:Chronic ethanol treatment (50 mM, five days) induces stabilization of NR1 receptor subunit mRNA in cultured fetal cortical neurons. In this paper, we investigate the mechanism(s) by which ethanol mediates its effects on NR1 mRNA. Specifically, we have determined if cellular localization of NR1 mRNA in cortical neurons and/orde novoprotein synthesis is essential for ethanol‐mediated stabilization of NR1 mRNA. Subcellular fractionation studies show that all detectable NR1 mRNA is associated with rough endoplasmic reticulum, indicating that subcellular distribution of NR1 mRNA in fetal cortical neurons does not play a role in ethanol‐mediated NR1 mRNA stabilization. However, inhibition of protein synthesis by cycloheximide abolished the mRNA stabilizing effect of ethanol on NR1 mRNA, thus suggestingde novoprotein synthesis is crucial for the action of ethanol on NR1 mRNA stabilization.
Poly(ADP‐Ribose) Polymerase‐1 in Acute Neuronal Death and Inflammation
Authors: SKAPER STEPHEN D.
Published: July 2009

Abstract: Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is an abundant nuclear enzyme that is activated primarily by DNA damage. Upon activation, the enzyme hydrolyzes NAD+ to nicotinamide and transfers ADP ribose units to a variety of nuclear proteins, including histones and PARP‐1 itself. This process is important in facilitating DNA repair. However, excessive activation of PARP‐1 can lead to significant decrements in NAD+, and ATP depletion, and cell death (suicide hypothesis). In response to cellular damage by oxygen radicals or excitotoxicity, a rapid and strong activation of PARP‐1 occurs in neurons. Excessive PARP‐1 activation is implicated in a variety of insults, including cerebral and cardiac ischemia, 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐induced Parkinsonism, traumatic spinal cord injury, and streptozotocin‐induced diabetes. The use of PARP inhibitors has, therefore, been proposed as a protective therapy in decreasing excitotoxic neuronal cell death, as well as ischemic and other tissue damage. Excitotoxic brain lesions initially result in the primary destruction of brain parenchyma and subsequently in secondary damage of neighboring neurons hours after the insult. This secondary damage of initially surviving neurons accounts for most of the volume of the infarcted area and the loss of brain function after a stroke. One major component of secondary neuronal damage is the migration of macrophages and microglial cells toward the sites of injury, where they produce large quantities of toxic cytokines and oxygen radicals. Recent evidence indicates that this microglial migration is strongly controlled in living brain tissue by expression of the integrin CD11a, which is regulated in turn by PARP‐1, proposing that PARP‐1 downregulation may, therefore, be a promising strategy in protecting neurons from this secondary damage, as well. Studies demonstrating an important role for PARP‐1 in the regulation of gene transcription have further increased the intricacy of poly(ADP‐ribosyl)ation in the control of cell homeostasis and challenge the notion that energy collapse is the sole mechanism by which poly(ADP‐ribose) formation contributes to cell death. The hypothesis that PARPs might regulate cell fate as essential modulators of death and survival transcriptional programs is discussed with relation to nuclear factor κB and p53.
Systemic Vitamin D3 Attenuated Oxidative Injuries in the Locus Coeruleus of Rat Brain
Authors: CHEN KUEN‐BOR , LIN ANYA MAAN‐YUH , CHIU TSAI‐HSIEN
Published: July 2009

Abstract:Iron‐induced oxidative injuries in locus coeruleus (LC), a major source of noradrenergic projections in the central nervous system (CNS), were investigated in chloral‐hydrate anesthetized rats. Local infusion of iron dose‐dependently elevated lipid peroxidation of iron‐infused LC seven days after infusion. At the same time, norepinephrine content in the hippocampus ipsilateral to the iron‐infused LC was decreased in a concentration‐dependent manner. Our immunostaining study demonstrated reduced tyrosine hydroxylase‐positive neurons in the iron‐infused LC, indicating a reduction of neuron number by iron infusion. The involvement of apoptosis in iron‐induced oxidative injuries was studied. An abrupt increase in cytosolic cytochrome c content was demonstrated in the infused LC 48 hours after iron infusion. TUNEL‐positive cells, an indication of apoptosis, were detected in the iron‐infused LC. In an attempt to prevent iron‐induced neurotoxicity, vitamin D3, an active metabolite of vitamin D, was systemically administered. Iron‐induced increases in cytosolic cytochrome c and TUNEL‐positive cells were reduced by this treatment. Furthermore, systemic administration of vitamin D3 attenuated iron‐induced oxidative injuries in the infused LC. Our data suggest that local infusion of iron in LC induced oxidative stress and resulted in programmed cell death in the LC‐hippocampal noradrenergic system. Furthermore, vitamin D3 may be neuroprotective and therapeutic in attenuating iron‐induced neurotoxicity in CNS.
Amyloid Processing and Signal Transduction Properties of Antiparkinson‐Antialzheimer Neuroprotective Drugs Rasagiline and TV3326
Authors: YOUDIM MOUSSA B.H. , AMIT TAMAR , BAR‐AM ORIT , WEINSTOCK MARTA , YOGEV‐FALACH MERAV
Published: July 2009

Abstract: Two novel neuroprotective cholinesterase (ChE) inhibitors, TV3326 and TV3279 [(N‐propargyl‐(3R) and (3S) aminoindan‐5‐yl)‐ethyl methyl carbamate], respectively were derived from rasagiline, for the treatment of Alzheimer's disease (AD). TV3326 also inhibits monoamine oxidase (MAO)‐A and B, while its S‐isomer, TV3279, lacks MAO‐inhibitory activity. The actions of these drugs in the regulation of the amyloid precursor protein (APP) processing using rat PC12 and human SH‐SY5Y neuroblastoma cells were examined. Both isomers stimulated the release of the non‐amyloidogenic α‐secretase form of soluble APP (sAPPα) from these cell lines. The increases in sAPPα, induced by TV3326 and TV3279, were dose‐dependent (0.1‐100 μM) and blocked by the hydroxamic acid‐based metalloprotease inhibitor, Ro31‐9790, suggesting mediation via α‐secretase activity. Using several signal transduction inhibitors, the involvement of protein kinase C (PKC), mitogen‐activated protein (MAP) kinase, and tyrosine kinase‐dependent pathways in the enhancement of sAPPα release by TV3326 and TV3279 was identified. In addition, both drugs directly induced the phosphorylation of p44 and p42 MAP kinase, which was abolished by the specific inhibitors of MAP kinase activation, PD98059 and U0126. These data suggest a novel pharmacological mechanism, whereby these ChE inhibitors regulate the secretary processes of APP via activation of the MAP kinase pathway.
A miniaturized assay for influenza neuraminidase‐inhibiting antibodies utilizing reverse genetics‐derived antigens
Authors: Sandbulte Matthew R. , Gao Jin , Straight Timothy M. , Eichelberger Maryna C.
Published: July 2009

Background  Antibodies to neuraminidase (NA) contribute to protection during influenza virus infection, but NA inhibition (NI) titers are not routinely analyzed in vaccine trials. One reason is the cumbersome nature of the conventional thiobarbituric acid (TBA) NI assay, which uses chemical methods to quantify free sialic acid following incubation of NA with substrate in the presence of serum. In addition, the assay is complicated by the need to use virus of a hemagglutinin (HA) subtype novel to the host to detect NA‐specific antibodies only.

Objectives  Our primary objectives were to miniaturize the colorimetric NI assay to a format suitable for quantitative analysis of large numbers of samples, and validate the specificity and sensitivity of the miniaturized format with ferret and human sera. An additional aim was to use reverse genetics to construct HA‐mismatched viral reagents bearing NA of recent influenza A vaccine strains and H6 HA.

Results  Analysis of ferret antisera by the miniaturized assay demonstrated sensitivity and specificity comparable with the conventional assay. Similar increases in the NI titers in sera from vaccinated human volunteers were measured in miniaturized and conventional assays. Inactivated and live‐attenuated vaccines increased NI titers against a given subtype at approximately the same rate.

Conclusions  The reagents and miniaturized format of the TBA method described here provide a platform for practical serological monitoring of functional antibodies against NA.