bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuroprotective effects of melatonin against anoxia/aglycemia stress, as assessed by synaptic potentials and superoxide production in rat hippocampal slices
Authors: Uchida Katsuhisa , Samejima Michikazu , Okabe Akihito , Fukuda Atsuo
Published: October 2004

Abstract:  Melatonin, which plays an important role in circadian rhythm regulation, is highly potent endogenous free radical scavenger and antioxidant. To clarify the neuroprotective effects of melatonin as a free radical scavenger, we recorded changes in synaptic potentials and monitored the generation of superoxide (O) (using hydroethidine) in the CA1 pyramidal layers of rat hippocampal slices exposed to anoxia/aglycemia (‘ischemic’) stress. Synaptic responses evoked by stimulation of Schaffer collateral/commissural afferents were suppressed by ischemic stress. When the duration of the stress was 8 min, the suppression was reversible, irrespective of the presence or absence of melatonin treatment, while the amount of O generated was reduced by the presence of melatonin. When stress duration was 12 min, the suppression of synaptic responses lasted more than 90 min, but melatonin significantly improved the recovery. The amount of O generated in the ‘recirculation’ phase after a 12 min ischemic stress was less in the ischemic alone group than in the melatonin‐treated group. This probably reflects that the number of viable cells with the ability to generate O had been reduced by the more severe ischemic stress. Other radical scavengers (ascorbic acid and α‐tocopherol) had similar effects. These results show that melatonin has the potential to protect the functions of neurons against an ischemic insult by reducing O generation.
Melatonin reduces amyloid β‐induced apoptosis in pheochromocytoma (PC12) cells
Authors: Feng Zheng , Zhang Jun‐tian
Published: October 2004

Abstract:  The finding that the amyloid β protein (Aβ) has neurotoxic properties and that such effects are partly mediated by free radicals has provided insights into the mechanisms of cell death in Alzheimer's disease (AD) and an avenue to explore new therapeutic approaches. Melatonin is a potent antioxidant and free radical scavenger. Previously, we showed that long‐term application of melatonin alleviated the learning and memory deficits in the APP695 transgenic mouse model of AD. In this study, the importance of melatonin in the management of Aβ‐induced apoptosis was investigated. Rat pheochromocytoma (PC12) cells treated with either Aβ25–35 or Aβ1–42 underwent apoptosis. Melatonin pretreatment at 10−5, 10−6 and 10−7 m significantly attenuated Aβ25–35‐ or Aβ1–42‐induced apoptosis in PC12 cells. The anti‐apoptotic effects of melatonin were highly reproducible and corroborated by multiple quantitative methods, including MTT cell viability assay, Hoechst 33342 nuclei staining, DNA fragmentation analysis, and flow cytometric analysis. In addition, melatonin effectively suppressed Aβ1–42‐induced nitric oxide formation, potently prevented Aβ1–40‐induced intracellular calcium overload and significantly alleviated Aβ1–40‐induced membrane rigidity. These results suggest that the mechanism of Aβ neurotoxicity involves oxidative stress, and the neuroprotective effects of melatonin are, at least in part, associated with its antioxidant properties. The use of melatonin or its derived analogs should be explored as a therapeutic approach in AD.
Maternally administered melatonin protects against ischemia and reperfusion‐induced oxidative mitochondrial damage in premature fetal rat brain
Authors: Watanabe Kazushi , Wakatsuki Akihiko , Shinohara Koichi , Ikenoue Nobuo , Yokota Kana , Fukaya Takao
Published: October 2004

Abstract:  We investigated the oxidative susceptibility of the brain and the effect of maternally administered melatonin on ischemia/reperfusion‐induced cerebral damage in premature fetal rat. Fetal brain mitochondria was separated on the 16th and 19th days of pregnant rats and the respiratory control index (RCI) was measured as an indicator of mitochondrial respiratory activity in the presence or absence of xanthine and xanthine oxidase. The utero–ovarian arteries were occluded bilaterally for 20 min in female rats on day 16 of pregnancy to induce fetal ischemia. Reperfusion was achieved by releasing the occlusion and restoring circulation for 30 min. A sham operation was performed in control rats. Melatonin (10 mg/kg) or vehicle was injected intraperitoneally into the dams 60 min prior to occlusion. The RCI and concentration of thiobarbituric acid‐reactive substances (TBARS) in fetal brain mitochondria were measured. The addition of xanthine and xanthine oxidase significantly decreased mitochondrial RCI at both the 16‐ and 19‐day‐old fetal brain. Xanthine and xanthine oxidase‐induced reduction in RCI was significantly greater in the 16‐day‐old fetal brain than that in the fetal brain from the 19th day of pregnancy. Ischemia/reperfusion significantly reduced RCI and elevated TBARS concentrations in the 16‐day‐old fetal brain mitochondria. Melatonin treatment reversed ischemia/reperfusion‐induced reduction in RCI (2.22 ± 0.10 to 2.53 ± 0.08, P < 0.01) and elevation in TBARS concentrations (13.50 ± 1.82 nmol/mg protein to 8.80 ± 0.78 nmol/mg protein, P < 0.01), resulting in values similar to those in untreated, sham‐treated animals. Results indicate that brain mitochondria in the premature fetal rats appear to be more susceptible to oxidative damage. Melatonin administration to pregnant rats may prevent ischemia/reperfusion‐induced oxidative mitochondrial damage in premature fetal brain.
Charged residue alterations in the inner‐core domain and carboxy‐terminus of α‐tropomyosin differentially affect mouse cardiac muscle contractility
Authors: Gaffin Robert D. , Tong Carl W. , Zawieja David C. , Hewett Timothy E. , Klevitsky Raisa , Robbins Jeffrey , Muthuchamy Mariappan
Published: October 2004

Two important charge differences between the α‐ and β‐tropomyosin (TM) isoforms are the exchange of a serine residue in the inner‐core region at position 229, and a histidine residue at the carboxy‐terminal end at position 276, with glutamic acid and asparagine, respectively. We have recently shown that altering these two residues in α‐TM to their β‐TM counterparts in transgenic (TG) mouse hearts causes a depression in both +dP/dt and −dP/dt and a decrease in calcium sensitivity. In this study, we address whether independent charge changes at these two residues in α‐TM modulate cardiac function differentially. To test this hypothesis we generated two TG lines: α‐TMSer229Glu and α‐TMHis276Asn. Molecular analyses show that 98% of native α‐TM is replaced by mutated protein in α‐TM229 hearts whereas α‐TM276 hearts show 82% replacement with the mutated protein. Isolated working heart data show that α‐TM229 TG hearts exhibit a significant decrease in both +dP/dt (7%) and −dP/dt (8%) compared with nontransgenics (NTGs) and time to peak pressure (TPP) is also reduced in α‐TM229 hearts. α‐TM276 hearts show a decrease only in −dP/dt (14%) and TPP is increased. pCa2+–tension relationships in skinned fibre preparations indicate decreased calcium sensitivity in α‐TM229 but no change in α‐TM276 preparations. Force–[Ca2+]IC measurements from intact papillary fibres indicate that α‐TM276 fibres produce more force per given [Ca2+]IC when compared to NTG fibres, while α‐TM229 fibres produce less force per given [Ca2+]IC. These data demonstrate that changing charged residues at either the inner‐core domain or the carboxyl end of TM alters sarcomeric performance differently, suggesting that the function of TM is compartmentalized along its length.
Advanced vaginal opening and precocious activation of the reproductive axis by KiSS‐1 peptide, the endogenous ligand of GPR54
Authors: Navarro V. M. , Fernández‐Fernández R. , Castellano J. M. , Roa J. , Mayen A. , Barreiro M. L. , Gaytan F. , Aguilar E. , Pinilla L. , Dieguez C. , Tena‐Sempere M.
Published: October 2004

The awakening of the gonadotrophic axis at puberty is the end‐point of a complex cascade of sex developmental events that leads to the attainment of reproductive capacity. Recently, loss‐of‐function mutations of the gene encoding GPR54, the putative receptor for the KiSS‐1‐derived peptide metastin, have been linked to hypogonadotrophic hypogonadism, both in rodents and humans. However, the actual role of the KiSS‐1/GPR54 system in the timing of puberty onset remains unexplored. We report herein that chronic central administration of KiSS‐1 peptide to immature female rats induced the precocious activation of the gonadotrophic axis, as estimated by advanced vaginal opening, elevated uterus weight, and increased serum levels of luteinizing hormone (LH) and oestrogen. The central effect of KiSS‐1 upon LH release appeared to be mediated via the hypothalamic LH‐releasing hormone. In contrast, despite the well‐documented permissive role of body fat stores and the adipocyte‐derived hormone leptin in puberty maturation, acute activation of the gonadotrophic axis by KiSS‐1 was persistently observed in pubertal animals under food deprivation, after central immunoneutralization of leptin, and in a model of leptin resistance. Overall, the present results, together with our recent data on maximum expression of KiSS‐1 and GPR54 genes in the hypothalamus at puberty, provide novel evidence for a role of the KiSS‐1 system as a downstream element in the hypothalamic network triggering the onset of puberty.
Differential involvement of oriens/pyramidale interneurones in hippocampal network oscillations in vitro
Authors: Gloveli Tengis , Dugladze Tamar , Saha Sikha , Monyer Hannah , Heinemann Uwe , Traub Roger D. , Whittington Miles A. , Buhl Eberhard H.
Published: October 2004

Using whole‐cell patch‐clamp recordings in conjunction with post hoc anatomy we investigated the physiological properties of hippocampal stratum oriens and stratum pyramidale inhibitory interneurones, before and following the induction of pharmacologically evoked gamma frequency network oscillations. Prior to kainate‐induced transient epochs of gamma activity, two distinct classes of oriens interneurones, oriens lacunosum‐moleculare (O‐LM) and trilaminar cells, showed prominent differences in their membrane and firing properties, as well as in the amplitude and kinetics of their excitatory postsynaptic events. In the active network both types of neurone received a phasic barrage of gamma frequency excitatory inputs but, due to their differential functional integration, showed clear differences in their output patterns. While O‐LM cells fired intermittently at theta frequency, trilaminar interneurones discharged on every gamma cycle and showed a propensity to fire spike doublets. Two other classes of fast spiking interneurones, perisomatic targeting basket and bistratified cells, in the active network discharged predominantly single action potentials on every gamma cycle. Thus, within a locally excited network, O‐LM cells are likely to provide a theta‐frequency patterned output to distal dendritic segments, whereas basket and bistratified cells are involved in the generation of locally synchronous gamma band oscillations. The anatomy and output profile of trilaminar cells suggest they are involved in the projection of locally generated gamma rhythms to distal sites. Therefore a division of labour appears to exist whereby different frequencies and spatiotemporal properties of hippocampal rhythms are mediated by different interneurone subtypes.
The 1.2-Megabase Genome Sequence of Mimivirus
Authors: Raoult Didier , Audic Stéphane , Robert Catherine , Abergel Chantal , Renesto Patricia , Ogata Hiroyuki , La Scola Bernard , Suzan Marie , Claverie Jean-Michel
Published: October 2004

We recently reported the discovery and preliminary characterization of Mimivirus, the largest known virus, with a 400-nanometer particle size comparable to mycoplasma. Mimivirus is a double-stranded DNA virus growing in amoebae. We now present its 1,181,404–base pair genome sequence, consisting of 1262 putative open reading frames, 10% of which exhibit a similarity to proteins of known functions. In addition to exceptional genome size, Mimivirus exhibits many features that distinguish it from other nucleocytoplasmic large DNA viruses. The most unexpected is the presence of numerous genes encoding central protein-translation components, including four amino-acyl transfer RNA synthetases, peptide release factor 1, translation elongation factor EF-TU, and translation initiation factor 1. The genome also exhibits six tRNAs. Other notable features include the presence of both type I and type II topoisomerases, components of all DNA repair pathways, many polysaccharide synthesis enzymes, and one intein-containing gene. The size and complexity of the Mimivirus genome challenge the established frontier between viruses and parasitic cellular organisms. This new sequence data might help shed a new light on the origin of DNA viruses and their role in the early evolution of eukaryotes.
Hysteretic Adsorption and Desorption of Hydrogen by Nanoporous Metal-Organic Frameworks
Authors: Zhao Xuebo , Xiao Bo , Fletcher Ashleigh J. , Thomas K. Mark , Bradshaw Darren , Rosseinsky Matthew J.
Published: October 2004

Adsorption and desorption of hydrogen from nanoporous materials, such as activated carbon, is usually fully reversible. We have prepared nanoporous metal-organic framework materials with flexible linkers in which the pore openings, as characterized in the static structures, appear to be too small to allow H
2
to pass. We observe hysteresis in their adsorption and desorption kinetics above the supercritical temperature of H
2
that reflects the dynamical opening of the “windows” between pores. This behavior would allow H
2
to be adsorbed at high pressures but stored at lower pressures.
Status and Trends of Amphibian Declines and Extinctions Worldwide
Authors: Stuart Simon N. , Chanson Janice S. , Cox Neil A. , Young Bruce E. , Rodrigues Ana S. L. , Fischman Debra L. , Waller Robert W.
Published: October 2004

The first global assessment of amphibians provides new context for the well-publicized phenomenon of amphibian declines. Amphibians are more threatened and are declining more rapidly than either birds or mammals. Although many declines are due to habitat loss and overutilization, other, unidentified processes threaten 48% of rapidly declining species and are driving species most quickly to extinction. Declines are nonrandom in terms of species' ecological preferences, geographic ranges, and taxonomic associations and are most prevalent among Neotropical montane, stream-associated species. The lack of conservation remedies for these poorly understood declines means that hundreds of amphibian species now face extinction.
Geochemical Precursors to Volcanic Activity at Mount St. Helens, USA
Authors: Berlo Kim , Blundy Jon , Turner Simon , Cashman Kathy , Hawkesworth Chris , Black Stuart
Published: October 2004

The importance of the interplay between degassing and crystallization before and after the eruption of Mount St. Helens (Washington, USA) in 1980 is well established. Here, we show that degassing occurred over a period of decades to days before eruptions and that the manner of degassing, as deduced from geochemical signatures within the magma, was characteristic of the eruptive style. Trace element (lithium) and short-lived radioactive isotope (lead-210 and radium-226) data show that ascending magma stalled within the conduit, leading to the accumulation of volatiles and the formation of lead-210 excesses, which signals the presence of degassing magma at depth.
Mycoplasma pneumoniaeand Its Role as a Human Pathogen
Authors: Waites Ken B. , Talkington Deborah F.
Published: October 2004

Mycoplasma pneumoniaeis a unique bacterium that does not always receive the attention it merits considering the number of illnesses it causes and the degree of morbidity associated with it in both children and adults. Serious infections requiring hospitalization, while rare, occur in both adults and children and may involve multiple organ systems. The severity of disease appears to be related to the degree to which the host immune response reacts to the infection. Extrapulmonary complications involving all of the major organ systems can occur in association withM. pneumoniaeinfection as a result of direct invasion and/or autoimmune response. The extrapulmonary manifestations are sometimes of greater severity and clinical importance than the primary respiratory infection. Evidence for this organism's contributory role in chronic lung conditions such as asthma is accumulating. Effective management ofM. pneumoniaeinfections can usually be achieved with macrolides, tetracyclines, or fluoroquinolones. As more is learned about the pathogenesis and immune response elicited byM. pneumoniae, improvement in methods for diagnosis and prevention of disease due to this organism may occur.
Impact of Malaria during Pregnancy on Low Birth Weight in Sub-Saharan Africa
Authors: Guyatt Helen L. , Snow Robert W.
Published: October 2004

Malaria during pregnancy can result in low birth weight (LBW), an important risk factor for infant mortality. This article reviews the pathological effects of malaria during pregnancy and the implications for the newborn's development and survival. Empirical data from throughout Africa on associations between placental malaria and birth weight outcome, birth weight outcome and infant mortality, and the rates of LBW in areas with various levels of malaria transmission are evaluated to assess the increased risks of LBW and infant mortality associated with malaria. It is estimated that in areas where malaria is endemic, around 19% of infant LBWs are due to malaria and 6% of infant deaths are due to LBW caused by malaria. These estimates imply that around 100,000 infant deaths each year could be due to LBW caused by malaria during pregnancy in areas of malaria endemicity in Africa.
Prevention of Infection Due toPneumocystisspp. in Human Immunodeficiency Virus-Negative Immunocompromised Patients
Authors: Rodriguez Martin , Fishman Jay A.
Published: October 2004

Pneumocystisinfection in humans was originally described in 1942. The organism was initially thought to be a protozoan, but more recent data suggest that it is more closely related to the fungi. Patients with cellular immune deficiencies are at risk for the development of symptomaticPneumocystisinfection. Populations at risk also include patients with hematologic and nonhematologic malignancies, hematopoietic stem cell transplant recipients, solid-organ recipients, and patients receiving immunosuppressive therapies for connective tissue disorders and vasculitides. Trimethoprim-sulfamethoxazole is the agent of choice for prophylaxis againstPneumocystisunless a clear contraindication is identified. Other options include pentamidine, dapsone, dapsone-pyrimethamine, and atovaquone. The risk for PCP varies based on individual immune defects, regional differences, and immunosuppressive regimens. Prophylactic strategies must be linked to an ongoing assessment of the patient's risk for disease.
Coccidioidomycosis: Host Response and Vaccine Development
Authors: Cox Rebecca A. , Magee D. Mitchell
Published: October 2004

Coccidioidomycosis is caused by the dimorphic fungi in the genusCoccidioides. These fungi live as mycelia in the soil of desert areas of the American Southwest, and when the infectious spores, the arthroconidia, are inhaled, they convert into the parasitic spherule/endospore phase. Most infections are mild, but these organisms are frank pathogens and can cause severe lethal disease in fully immunocompetent individuals. While there is increased risk of disseminated disease in certain racial groups and immunocompromised persons, the fact that there are hosts who contain the initial infection and exhibit long-term immunity to reinfection supports the hypothesis that a vaccine against these pathogens is feasible. Multiple studies have shown that protective immunity against primary disease is associated with T-helper 1 (Th-1)-associated immune responses. The single best vaccine in animal models, formalin-killed spherules (FKS), was tested in a human trial but was not found to be significantly protective. This result has prompted studies to better define immunodominantCoccidioidesantigen with the thought that a subunit vaccine would be protective. These efforts have defined multiple candidates, but the single best individual immunogen is the protein termed antigen 2/proline-rich antigen (Ag2/PRA). Studies in multiple laboratories have shown that Ag2/PRA as both protein and genetic vaccines provides significant protection against mice challenged systemically withCoccidioides. Unfortunately, compared to the FKS vaccine, it is significantly less protective as measured by both assays of reduction in fungal CFU and assays of survival. The capacity of Ag2/PRA to induce only partial protection was emphasized when animals were challenged intranasally. Thus, there is a need to define new candidates to create a multivalent vaccine to increase the effectiveness of Ag2/PRA. Efforts of genomic screening using expression library immunization or bioinformatic approaches to identify new candidates have revealed at least two new protective proteins, expression library immunization antigen 1 (ELI-Ag1) and a β-1,3-glucanosyltransferase (GEL-1). In addition, previously discovered antigens such asCoccidioides-specific antigen (CSA) should be evaluated in assays of protection. While studies have yet to be completed with combinations of the current candidates, the hypothesis is that with increased numbers of candidates in a multivalent vaccine, there will be increased protection. As the genome sequences of the twoCoccidioidesstrains which are under way are completed and annotated, the effort to find new candidates can increase to provide a complete genomic scan for immunodominant proteins. Thus, much progress has been made in the discovery of subunit vaccine candidates againstCoccidioidesand there are several candidates showing modest levels of protection, but for complete protection against pulmonary challenge we need to continue the search for additional candidates.
Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases
Authors: Clarridge Jill E.
Published: October 2004

The traditional identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic methods. Comparison of the bacterial 16S rRNA gene sequence has emerged as a preferred genetic technique. 16S rRNA gene sequence analysis can better identify poorly described, rarely isolated, or phenotypically aberrant strains, can be routinely used for identification of mycobacteria, and can lead to the recognition of novel pathogens and noncultured bacteria. Problems remain in that the sequences in some databases are not accurate, there is no consensus quantitative definition of genus or species based on 16S rRNA gene sequence data, the proliferation of species names based on minimal genetic and phenotypic differences raises communication difficulties, and microheterogeneity in 16S rRNA gene sequence within a species is common. Despite its accuracy, 16S rRNA gene sequence analysis lacks widespread use beyond the large and reference laboratories because of technical and cost considerations. Thus, a future challenge is to translate information from 16S rRNA gene sequencing into convenient biochemical testing schemes, making the accuracy of the genotypic identification available to the smaller and routine clinical microbiology laboratories.
Epidemiologic Background of Hand Hygiene and Evaluation of the Most Important Agents for Scrubs and Rubs
Authors: Kampf Günter , Kramer Axel
Published: October 2004

The etiology of nosocomial infections, the frequency of contaminated hands with the different nosocomial pathogens, and the role of health care workers' hands during outbreaks suggest that a hand hygiene preparation should at least have activity against bacteria, yeasts, and coated viruses. The importance of efficacy in choosing the right hand hygiene product is reflected in the new Centers for Disease Control and Prevention guideline on hand hygiene (J. M. Boyce and D. Pittet, Morb. Mortal. Wkly. Rep.51:1-45, 2002). The best antimicrobial efficacy can be achieved with ethanol (60 to 85%), isopropanol (60 to 80%), and n-propanol (60 to 80%). The activity is broad and immediate. Ethanol at high concentrations (e.g., 95%) is the most effective treatment against naked viruses, whereas n-propanol seems to be more effective against the resident bacterial flora. The combination of alcohols may have a synergistic effect. The antimicrobial efficacy of chlorhexidine (2 to 4%) and triclosan (1 to 2%) is both lower and slower. Additionally, both agents have a risk of bacterial resistance, which is higher for chlorhexidine than triclosan. Their activity is often supported by the mechanical removal of pathogens during hand washing. Taking the antimicrobial efficacy and the mechanical removal together, they are still less effective than the alcohols. Plain soap and water has the lowest efficacy of all. In the new Centers for Disease Control and Prevention guideline, promotion of alcohol-based hand rubs containing various emollients instead of irritating soaps and detergents is one strategy to reduce skin damage, dryness, and irritation. Irritant contact dermatitis is highest with preparations containing 4% chlorhexidine gluconate, less frequent with nonantimicrobial soaps and preparations containing lower concentrations of chlorhexidine gluconate, and lowest with well-formulated alcohol-based hand rubs containing emollients and other skin conditioners. Too few published data from comparative trials are available to reliably rank triclosan. Personnel should be reminded that it is neither necessary nor recommended to routinely wash hands after each application of an alcohol-based hand rub. Long-lasting improvement of compliance with hand hygiene protocols can be successful if an effective and accessible alcohol-based hand rub with a proven dermal tolerance and an excellent user acceptability is supplied, accompanied by education of health care workers and promotion of the use of the product.
Molecular Methods for Diagnosis of Viral Encephalitis
Authors: DeBiasi Roberta L. , Tyler Kenneth L.
Published: October 2004

Hundreds of viruses cause central nervous system (CNS) disease, including meningoencephalitis and postinfectious encephalomyelitis, in humans. The cerebrospinal fluid (CSF) is abnormal in >90% of cases; however, routine CSF studies only rarely lead to identification of a specific etiologic agent. Diagnosis of viral infections of the CNS has been revolutionized by the advent of new molecular diagnostic technologies to amplify viral nucleic acid from CSF, including PCR, nucleic acid sequence-based amplification, and branched-DNA assay. PCR is ideally suited for identifying fastidious organisms that may be difficult or impossible to culture and has been widely applied for detection of both DNA and RNA viruses in CSF. The technique can be performed rapidly and inexpensively and has become an integral component of diagnostic medical practice in the United States and other developed countries. In addition to its use for identification of etiologic agents of CNS disease in the clinical setting, PCR has also been used to quantitate viral load and monitor duration and adequacy of antiviral drug therapy. PCR has also been applied in the research setting to help discriminate active versus postinfectious immune-mediate disease, identify determinants of drug resistance, and investigate the etiology of neurologic disease of uncertain cause. This review discusses general principles of PCR and reverse transcription-PCR, including qualitative, quantitative, and multiplex techniques, with comment on issues of sensitivity, specificity, and positive and negative predictive values. The application of molecular diagnostic methods for diagnosis of specific infectious entities is reviewed in detail, including viruses for which PCR is of proven efficacy and is widely available, viruses for which PCR is less widely available or for which PCR has unproven sensitivity and specificity, and nonviral entities which can mimic viral CNS disease.
Antibody Therapy in the Management of Shiga Toxin-Induced Hemolytic Uremic Syndrome
Authors: Tzipori Saul , Sheoran Abhineet , Akiyoshi Donna , Donohue-Rolfe Arthur , Trachtman Howard
Published: October 2004

Hemolytic uremic syndrome (HUS) is a disease that can lead to acute renal failure and often to other serious sequelae, including death. The majority of cases are attributed to infections withEscherichia coli, serotype O157:H7 strains in particular, which cause bloody diarrhea and liberate one or two toxins known as Shiga toxins 1 and 2. These toxins are thought to directly be responsible for the manifestations of HUS. Currently, supportive nonspecific treatment is the only available option for the management of individuals presenting with HUS. The benefit of antimicrobial therapy remains uncertain because of several reports which claim that such intervention can in fact exacerbate the syndrome. There have been only a few specific therapies directed against neutralizing the activities of these toxins, but none so far has been shown to be effective. This article reviews the literature on the mechanism of action of these toxins and the clinical manifestations and current management and treatment of HUS. The major focus of the article, however, is the development and rationale for using neutralizing human antibodies to combat this toxin-induced disease. Several groups are currently pursuing this approach with either humanized, chimeric, or human antitoxin antibodies produced in transgenic mice. They are at different phases of development, ranging from preclinical evaluation to human clinical trials. The information available from preclinical studies indicates that neutralizing specific antibodies directed against the A subunit of the toxin can be highly protective. Such antibodies, even when administered well after exposure to bacterial infection and onset of diarrhea, can prevent the occurrence of systemic complications.
Role of Microglia in Central Nervous System Infections
Authors: Rock R. Bryan , Gekker Genya , Hu Shuxian , Sheng Wen S. , Cheeran Maxim , Lokensgard James R. , Peterson Phillip K.
Published: October 2004

The nature of microglia fascinated many prominent researchers in the 19th and early 20th centuries, and in a classic treatise in 1932, Pio del Rio-Hortega formulated a number of concepts regarding the function of these resident macrophages of the brain parenchyma that remain relevant to this day. However, a renaissance of interest in microglia occurred toward the end of the 20th century, fueled by the recognition of their role in neuropathogenesis of infectious agents, such as human immunodeficiency virus type 1, and by what appears to be their participation in other neurodegenerative and neuroinflammatory disorders. During the same period, insights into the physiological and pathological properties of microglia were gained from in vivo and in vitro studies of neurotropic viruses, bacteria, fungi, parasites, and prions, which are reviewed in this article. New concepts that have emerged from these studies include the importance of cytokines and chemokines produced by activated microglia in neurodegenerative and neuroprotective processes and the elegant but astonishingly complex interactions between microglia, astrocytes, lymphocytes, and neurons that underlie these processes. It is proposed that an enhanced understanding of microglia will yield improved therapies of central nervous system infections, since such therapies are, by and large, sorely needed.
The Genome Sequence ofMycoplasma hyopneumoniaeStrain 232, the Agent of Swine Mycoplasmosis
Authors: Minion F. Chris , Lefkowitz Elliot J. , Madsen Melissa L. , Cleary Barbara J. , Swartzell Steven M. , Mahairas Gregory G.
Published: October 2004

We present the complete genome sequence ofMycoplasma hyopneumoniae, an important member of the porcine respiratory disease complex. The genome is composed of 892,758 bp and has an average G+C content of 28.6 mol%. There are 692 predicted protein coding sequences, the average protein size is 388 amino acids, and the mean coding density is 91%. Functions have been assigned to 304 (44%) of the predicted protein coding sequences, while 261 (38%) of the proteins are conserved hypothetical proteins and 127 (18%) are unique hypothetical proteins. There is a single 16S-23S rRNA operon, and there are 30 tRNA coding sequences. The cilium adhesin gene has six paralogs in the genome, only one of which contains the cilium binding site. The companion gene,P102, also has six paralogs. Gene families constitute 26.3% of the total coding sequences, and the largest family is the 34-member ABC transporter family. Protein secretion occurs through a truncated pathway consisting of SecA, SecY, SecD, PrsA, DnaK, Tig, and LepA. Some highly conserved eubacterial proteins, such as GroEL and GroES, are notably absent. The DnaK-DnaJ-GrpR complex is intact, providing the only control over protein folding. There are several proteases that might serve as virulence factors, and there are 53 coding sequences with prokaryotic lipoprotein lipid attachment sites. Unlike other mycoplasmas,M. hyopneumoniaecontains few genes with tandem repeat sequences that could be involved in phase switching or antigenic variation. Thus, it is not clear howM. hyopneumoniaeevades the immune response and establishes a chronic infection.
Crystal Structure of the YgfZ Protein from
Escherichia coli
Suggests a Folate-Dependent Regulatory Role in One-Carbon Metabolism
Authors: Teplyakov Alexey , Obmolova Galina , Sarikaya Elif , Pullalarevu Sadhana , Krajewski Wojciech , Galkin Andrey , Howard Andrew J. , Herzberg Osnat , Gilliland Gary L.
Published: October 2004

The
ygfZ
gene product of
Escherichia coli
represents a large protein family conserved in bacteria to eukaryotes. The members of this family are uncharacterized proteins with marginal sequence similarity to the T-protein (aminomethyltransferase) of the glycine cleavage system. To assist with the functional assignment of the YgfZ family, the crystal structure of the
E. coli
protein was determined by multiwavelength anomalous diffraction. The protein molecule has a three-domain architecture with a central hydrophobic channel. The structure is very similar to that of bacterial dimethylglycine oxidase, an enzyme of the glycine betaine pathway and a homolog of the T-protein. Based on structural superposition, a folate-binding site was identified in the central channel of YgfZ, and the ability of YgfZ to bind folate derivatives was confirmed experimentally. However, in contrast to dimethylglycine oxidase and T-protein, the YgfZ family lacks amino acid conservation at the folate site, which implies that YgfZ is not an aminomethyltransferase but is likely a folate-dependent regulatory protein involved in one-carbon metabolism.
Tertiary Structure of
Staphylococcus aureus
Cell Wall Murein
Authors: Dmitriev Boris A. , Toukach Filip V. , Holst O. , Rietschel E. T. , Ehlers S.
Published: October 2004

The recently described scaffold model of murein architecture depicts the gram-negative bacterial cell wall as a gel-like matrix composed of cross-linked glycan strands oriented perpendicularly to the plasma membrane while peptide bridges adopt a parallel orientation (B. A. Dmitriev, F. V. Toukach, K. J. Schaper, O. Holst, E. T. Rietschel, and S. Ehlers, J. Bacteriol. 185:3458-3468, 2003). Based on the scaffold model, we now present computer simulation studies on the peptidoglycan arrangement of the gram-positive organism
Staphylococcus aureus
, which show that the orientation of peptide bridges is critical for the highly cross-linked murein architecture of this microorganism. According to the proposed refined model, staphylococcal murein is composed of glycan and oligopeptide chains, both running in a plane that is perpendicular to the plasma membrane, with oligopeptide chains adopting a zigzag conformation and zippering adjacent glycan strands along their lengths. In contrast to previous models of murein in gram-positive bacteria, this model reflects the high degree of cross-linking that is the hallmark of the staphylococcal cell wall and is compatible with distinguishing features of
S. aureus
cytokinesis such as the triple consecutive alteration of the division plane orientation and the strictly centripetal mode of septum closure.
The Completely Sequenced Plasmid pEST4011 Contains a Novel IncP1 Backbone and a Catabolic Transposon Harboring
tfd
Genes for 2,4-Dichlorophenoxyacetic Acid Degradation
Authors: Vedler Eve , Vahter Merle , Heinaru Ain
Published: October 2004

The
herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium
Achromobacter xylosoxidans
subsp.
denitrificans
strain EST4002 contains plasmid pEST4011. This plasmid ensures its host
a stable 2,4-D
+
phenotype. We determined the
complete 76,958-bp nucleotide sequence of pEST4011. This plasmid is a
deletion and duplication derivative of pD2M4, the 95-kb highly unstable
laboratory ancestor of pEST4011, and was self-generated during
different laboratory manipulations performed to increase the stability
of the 2,4-D
+
phenotype of the original strain,
strain D2M4(pD2M4). The 47,935-bp catabolic region of pEST4011 forms a
transposon-like structure with identical copies of the hybrid insertion
element IS
1071
::IS
1471
at the two
ends. The catabolic regions of pEST4011 and pJP4, the best-studied
2,4-D-degradative plasmid, both contain homologous,
tfd
-like
genes for complete 2,4-D degradation, but they have little sequence
similarity other than that. The backbone genes of pEST4011 are most
similar to the corresponding genes of broad-host-range
self-transmissible IncP1 plasmids. The backbones of the other three
IncP1 catabolic plasmids that have been sequenced (the
2,4-D-degradative plasmid pJP4, the haloacetate-catabolic plasmid pUO1,
and the atrazine-catabolic plasmid pADP-1) are nearly identical to the
backbone of R751, the archetype plasmid of the IncP1 β
subgroup. We show that despite the overall similarity in plasmid
organization, the pEST4011 backbone is sufficiently different (51 to
86% amino acid sequence identity between individual backbone
genes) from the backbones of members of the three IncP1 subgroups (theα
, β, and γ subgroups) that it belongs to a new
IncP1subgroup, the δ subgroup. This conclusion was also
supported by a phylogenetic analysis of the
trfA2
,
korA
, and
traG
gene products of different IncP1
plasmids.
Characterization of the
Escherichia coli
AaeAB Efflux Pump: a Metabolic Relief Valve?
Authors: Van Dyk Tina K. , Templeton Lori J. , Cantera Keith A. , Sharpe Pamela L. , Sariaslani F. Sima
Published: October 2004

Treatment of
Escherichia coli
with
p
-hydroxybenzoic acid (pHBA) resulted in upregulation of
yhcP
, encoding a protein of the putative efflux protein family. Also upregulated were the adjacent genes
yhcQ
, encoding a protein of the membrane fusion protein family, and
yhcR
, encoding a small protein without a known or suggested function. The function of the upstream, divergently transcribed gene
yhcS
, encoding a regulatory protein of the LysR family, in regulating expression of
yhcRQP
was shown. Furthermore, it was demonstrated that several aromatic carboxylic acid compounds serve as inducers of
yhcRQP
expression. The efflux function encoded by
yhcP
was proven by the hypersensitivity to pHBA of a
yhcP
mutant strain. A
yhcS
mutant strain was also hypersensitive to pHBA. Expression of
yhcQ
and
yhcP
was necessary and sufficient for suppression of the pHBA hypersensitivity of the
yhcS
mutant. Only a few aromatic carboxylic acids of hundreds of diverse compounds tested were defined as substrates of the YhcQP efflux pump. Thus, we propose renaming
yhcS
,
yhcR
,
yhcQ
, and
yhcP
, to reflect their role in aromatic carboxylic acid efflux, to
aaeR
,
aaeX
,
aaeA
, and
aaeB
, respectively. The role of pHBA in normal
E. coli
metabolism and the highly regulated expression of the AaeAB efflux system suggests that the physiological role may be as a “metabolic relief valve” to alleviate toxic effects of imbalanced metabolism.
Membrane Topology Analysis of Cyclic Glucan Synthase, a Virulence Determinant of
Brucella abortus
Authors: Ciocchini Andrés E. , Roset Mara S. , Iñón de Iannino Nora , Ugalde Rodolfo A.
Published: October 2004

Brucella abortus
cyclic glucan synthase (Cgs) is a 316-kDa (2,831-amino-acid) integral inner membrane protein that is responsible for the synthesis of cyclic β-1,2-glucan by a novel mechanism in which the enzyme itself acts as a protein intermediate.
B. abortus
Cgs uses UDP-glucose as a sugar donor and has the three enzymatic activities necessary for synthesis of the cyclic polysaccharide (i.e., initiation, elongation, and cyclization). Cyclic glucan is required in
B. abortus
for effective host interaction and complete expression of virulence. To gain further insight into the structure and mechanism of action of
B. abortus
Cgs, we studied the membrane topology of the protein using a combination of in silico predictions, a genetic approach involving the construction of fusions between the
cgs
gene and the genes encoding alkaline phosphatase (
phoA
) and β-galactosidase (
lacZ
), and site-directed chemical labeling of lysine residues. We found that
B. abortus
Cgs is a polytopic membrane protein with the amino and carboxyl termini located in the cytoplasm and with six transmembrane segments, transmembrane segments I (residues 419 to 441), II (residues 452 to 474), III (residues 819 to 841), IV (residues 847 to 869), V (residues 939 to 961), and VI (residues 968 to 990). The six transmembrane segments determine four large cytoplasmic domains and three very small periplasmic regions.
Ribose Utilization with an Excess of Mutarotase Causes Cell Death Due to Accumulation of Methylglyoxal
Authors: Kim Insook , Kim Eunjung , Yoo Seokho , Shin Daesung , Min Bumchan , Song Jeeyeon , Park Chankyu
Published: October 2004

Methylglyoxal (MG) is a highly reactive metabolic intermediate, presumably accumulated during uncontrolled carbohydrate metabolism. The major source of MG is dihydroxyacetone phosphate, which is catalyzed by MG synthase (the
mgs
product) in bacteria. We observed
Escherichia coli
cell death when the ribose transport system, consisting of the RbsDACBK proteins, was overproduced on multicopy plasmids. Almost 100% of cell death occurs a few hours after ribose addition (>10 mM), due to an accumulation of extracellular MG as detected by
1
H-nuclear magnetic resonance (
1
H-NMR). Under lethal conditions, the concentration of MG produced in the medium reached approximately 1 mM after 4 h of ribose addition as measured by high-performance liquid chromatography. An excess of the protein RbsD, recently characterized as a mutarotase that catalyzes the conversion between the β-pyran and β-furan forms of ribose, was critical in accumulating the lethal level of MG, which was also shown to require ribokinase (RbsK). The intracellular level of ribose 5-phosphate increased with the presence of the protein RbsD, as determined by
31
P-NMR. As expected, a mutation in the methylglyoxal synthase gene (
mgs
) abolished the production of MG. These results indicate that the enhanced ribose uptake and incorporation lead to an accumulation of MG, perhaps occurring via the pentose-phosphate pathway and via glycolysis with the intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate. It was also demonstrated that a small amount of MG is synthesized by monoamine oxidase.
The “Intracellular” Poly(3-Hydroxybutyrate) (PHB) Depolymerase of
Rhodospirillum rubrum
Is a Periplasm-Located Protein with Specificity for Native PHB and with Structural Similarity to Extracellular PHB Depolymerases
Authors: Handrick René , Reinhardt Simone , Kimmig Philipp , Jendrossek Dieter
Published: October 2004

Rhodospirillum rubrum
possesses a putative intracellular poly(3-hydroxybutyrate) (PHB) depolymerase system consisting of a soluble PHB depolymerase, a heat-stable activator, and a 3-hydroxybutyrate dimer hydrolase (J. M. Merrick and M. Doudoroff, J. Bacteriol.
88:
60-71, 1964). In this study we reinvestigated the soluble
R. rubrum
PHB depolymerase (PhaZ1). It turned out that PhaZ1 is a novel type of PHB depolymerase with unique properties. Purified PhaZ1 was specific for amorphous short-chain-length polyhydroxyalkanoates (PHA) such as native PHB, artificial PHB, and oligomer esters of (
R
)-3-hydroxybutyrate with 3 or more 3-hydroxybutyrate units. Atactic PHB, (
S
)-3-hydroxybutyrate oligomers, medium-chain-length PHA, and lipase substrates (triolein, tributyrin) were not hydrolyzed. The PHB depolymerase structural gene (
phaZ1
) was cloned. Its deduced amino acid sequence (37,704 Da) had no significant similarity to those of intracellular PHB depolymerases of
Wautersia eutropha
or of other PHB-accumulating bacteria. PhaZ1 was found to have strong amino acid homology with type-II catalytic domains of extracellular PHB depolymerases, and Ser
42
, Asp
138
, and His
178
were identified as catalytic-triad amino acids, with Ser
42
as the putative active site. Surprisingly, the first 23 amino acids of the PHB depolymerase previously assumed to be intracellular revealed features of classical signal peptides, and Edman sequencing of purified PhaZ1 confirmed the functionality of the predicted cleavage site. Extracellular PHB depolymerase activity was absent, and analysis of cell fractions unequivocally showed that PhaZ1 is a periplasm-located enzyme. The previously assumed intracellular activator/depolymerase system is unlikely to have a physiological function in PHB mobilization in vivo. A second gene, encoding the putative true intracellular PHB depolymerase (PhaZ2), was identified in the genome sequence of
R. rubrum
.
Osa Protein Constitutes a Strong Oncogenic Suppression System That Can Block
vir
-Dependent Transfer of IncQ Plasmids between
Agrobacterium
Cells and the Establishment of IncQ Plasmids in Plant Cells
Authors: Lee Lan-Ying , Gelvin Stanton B.
Published: October 2004

The
osa
(oncogenic suppressive activity) gene of the IncW group plasmid pSa is sufficient to suppress tumorigenesis by
Agrobacterium tumefaciens. osa
confers oncogenic suppression by inhibiting VirE2 protein export. This result is similar, but not identical, to that of oncogenic suppression by the IncQ plasmid RSF1010. We conducted a series of experiments to compare oncogenic suppression by these two systems.
Agrobacterium
strains harboring plasmids containing
osa
are more able to effect oncogenic suppression than are similar strains containing various RSF1010 derivatives. When
osa
is present within a donor
Agrobacterium
strain that also carries a derivative of RSF1010, the transfer of RSF1010 derivatives to recipient bacteria and their establishment in plants are blocked. Oncogenic suppression is still effected when the
osa
gene is integrated into the
Agrobacterium
chromosome, suggesting that it is the
osa
gene product that is active in suppression and that suppression does not require a protein-nucleic acid intermediate like that described for IncQ plasmids. Extracellular complementation experiments with tobacco leaf disks indicated that Osa blocks stable transfer of RSF1010 to plant cells by inhibiting transfer of VirE2, which is essential for the transfer of RSF1010 into plant cells, and not by inhibiting the actual transfer of RSF1010 itself. Our results suggest that Osa and RSF1010 cause oncogenic suppression by using different mechanisms.