bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple Signaling Defects in the Absence of RIP140 Impair Both Cumulus Expansion and Follicle Rupture
Authors: Tullet Jennifer M. A. , Pocock Victoria , Steel Jennifer H. , White Roger , Milligan Stuart , Parker Malcolm G.
Published: May 2005

The nuclear receptor corepressor RIP140 is essential in the ovary for ovulation, but is not required for follicle growth and luteinization. To identify genes that may be subject to regulation by RIP140 or play a role in ovulation, we compared ovarian gene expression profiles in untreated immature wild-type and RIP140 null mice and after treatment with pregnant mare serum gonadotropin and human chorionic gonadotropin. Many genes involved in signaling, extracellular matrix formation, cell-cell attachment, and adhesion were aberrantly regulated in the absence of RIP140, varying according to the hormone status of the mice. Notable among these was the reduced expression of a number of genes that encode components of signaling pathways and matrix proteins required for cumulus expansion, a key remodeling process necessary for ovulation. Histological analysis confirmed that cumulus expansion in RIP140 null mice is reduced, oocyte detachment from the mural cell wall is impaired, and follicles fail to rupture in response to LH. Although the expression of many genes involved in cumulus cell expansion was reduced, there was a subset of genes involved in extracellular matrix formation and cell-cell interactions that was up-regulated and may interfere with ovarian tissue remodeling. We propose that widespread gene dysregulation in ovarian tissues in the absence of RIP140 leads to the anovulatory phenotype. This helps to define an important role for RIP140 in the regulation of multiple processes leading to ovulation.
Minireview: The Role of Oxidative Stress in Relation to Caloric Restriction and Longevity
Authors: Gredilla Ricardo , Barja Gustavo
Published: May 2005

Reduction of caloric intake without malnutrition is one of the most consistent experimental interventions that increases mean and maximum life spans in different species. For over 70 yr, caloric restriction has been studied, and during the last years the number of investigations on such nutritional intervention and aging has dramatically increased. Because caloric restriction decreases the aging rate, it constitutes an excellent approach to better understand the mechanisms underlying the aging process. Various investigations have reported reductions in steady-state oxidative damage to proteins, lipids, and DNA in animals subjected to restricted caloric intake. Most interestingly, several investigations have reported that these decreases in oxidative damage are related to a lowering of mitochondrial free radical generation rate in various tissues of the restricted animals. Thus, similar to what has been described for long-lived animals in comparative studies, a decrease in mitochondrial free radical generation has been suggested to be one of the main determinants of the extended life span observed in restricted animals. In this study we review recent reports of caloric restriction and longevity, focusing on mitochondrial oxidative stress and the proposed mechanisms leading to an extended longevity in calorie-restricted animals.
Minireview: Role of the Growth Hormone/Insulin-Like Growth Factor System in Mammalian Aging
Authors: Bartke Andrzej
Published: May 2005

The important role of IGF and insulin-related signaling pathways in the control of longevity of worms and insects is very well documented. In the mouse, several spontaneous or experimentally induced mutations that interfere with GH biosynthesis, GH actions, or sensitivity to IGF-I lead to extended longevity. Increases in the average life span in these mutants range from approximately 20–70% depending on the nature of the endocrine defect, gender, diet, and/or genetic background. Extended longevity of hypopituitary and GH-resistant mice appears to be due to multiple mechanisms including reduced insulin levels, enhanced insulin sensitivity, alterations in carbohydrate and lipid metabolism, reduced generation of reactive oxygen species, enhanced resistance to stress, reduced oxidative damage, and delayed onset of age-related disease. There is considerable evidence to suggest that the genetic and endocrine mechanisms that influence aging and longevity in mice may play a similar role in other mammalian species, including the human.
Domain rearrangement of SRP protein Ffh upon binding 4.5S RNA and the SRP receptor FtsY
Authors: BUSKIEWICZ IWONA , KUBARENKO ANDRIY , PESKE FRANK , RODNINA MARINA V. , WINTERMEYER WOLFGANG
Published: May 2005

The signal recognition particle (SRP) mediates membrane targeting of translating ribosomes displaying a signal-anchor sequence. In Escherichia coli, SRP consists of 4.5S RNA and a protein, Ffh, that recognizes the signal peptide emerging from the ribosome and the SRP receptor at the membrane, FtsY. In the present work, we studied the interactions between the NG and M domains in Ffh and their rearrangements upon complex formation with 4.5S RNA and/or FtsY. In free Ffh, the NG and M domains are facing one another in an orientation that allows cross-linking between positions 231 in the G domain and 377 in the M domain. There are binding interactions between the two domains, as the isolated domains form a strong complex. The interdomain contacts are disrupted upon binding of Ffh to 4.5S RNA, consuming a part of the total binding energy of 4.5S RNA-Ffh association that is roughly equivalent to the free energy of domain binding to each other. In the SRP particle, the NG domain binds to 4.5S RNA in a region adjacent to the binding site of the M domain. Ffh binding to FtsY also requires a reorientation of NG and M domains. These results suggest that in free Ffh, the binding sites for 4.5S RNA and FtsY are occluded by strong domain–domain interactions which must be disrupted for the formation of SRP or the Ffh-FtsY complex.
High-throughput RNAi screening in vitro: From cell lines to primary cells
Authors: OVCHARENKO DMITRIY , JARVIS RICHARD , HUNICKE-SMITH SCOTT , KELNAR KEVIN , BROWN DAVID
Published: May 2005

Small interfering RNAs (siRNAs) are being used to induce sequence-specific gene silencing in cultured cells to study mammalian gene function. Libraries of siRNAs targeting entire human gene classes can be used to identify genes with specific cellular functions. Here we describe high-throughput siRNA delivery methods to facilitate siRNA library screening experiments with both immortalized and primary cells. We adapted chemical reverse transfection for immortalized adherent cell lines in a 96-well format. The method is fast, robust, and exceptionally effective for many cell types. For primary cells and immortalized cells that are recalcitrant to lipofection-based methods, we developed electropermeabilization (electroporation) conditions that facilitate siRNA delivery to a broad range of cell types, including primary human T-cells, hMSC, NHA, NDHF-Neo, HUVEC, DI TNC1, RPTEC, PC12, and K562 cells. To enable high-throughput electropermeabilization of primary cells, we developed a novel 96-well electroporation device that provides highly efficient and reproducible delivery of siRNAs. The combination of high-throughput chemical reverse transfection and electroporation makes it possible to deliver libraries of siRNAs to virtually any cell type, enabling gene function analysis and discovery on a genome scale.
Automatisierung der Anästhesie: Ansätze zur Regelung der Analgesie (Automation of Anaesthesia: Approaches to Control Analgesia)
Authors: Stadler Konrad Shedden , Bänziger Bertram , Leibundgut Daniel , Schumacher Peter Matthias
Published: May 2005

Das Schmerzempfinden (Analgesie) eines anästhesierten Patienten ist schwer quantifizierbar und kann sich über die Zeit ändern. Der Blutdruck wird klinisch oft als Indikator verwendet. In den meisten Operationen wird dieser aber nicht kontinuierlich gemessen, sondern meist nur alle fünf Minuten (nichtinvasiv). Dieser Beitrag beschreibt Ansätze zur Regelung der Analgesie, die die nichtinvasive Messung mit Information aus der kontinuierlichen Herzfrequenzmessung ergänzt, um damit schneller auf Störungen reagieren zu können. Abschließend werden klinische Versuche vorgestellt.
HyGLEAM – eine neue Methode zur Leistungssteigerung Evolutionärer Algorithmen (HyGLEAM – A New Method for Increasing the Performance of Evolutionary Algorithms)
Authors: Jakob Wilfried
Published: May 2005

Nahezu alle praktischen Anwendungen Evolutionärer Algorithmen sind so genannte Hybride, die in irgendeiner Form anwendungsspezifisches Wissen in das evolutionäre Verfahren integrieren, wodurch eine problemspezifische Lösung entsteht. Dieser Beitrag konzentriert sich auf zwei Fragen: Wie hybridisiert man so, dass das resultierende Verfahren allgemein anwendbar bleibt und welches ist die beste Art der Hybridisierung? Die Ergebnisse der umfangreichen Untersuchungen sind viel versprechend: Die Anzahl der Berechnungen der Qualitätsfunktion konnte um einen Faktor von bis zu 100 reduziert werden.
Der mehrfädige Komodo-Mikrocontroller (The Multithreaded Komodo Microcontroller)
Authors: Brinkschulte Uwe , Uhrig Sascha , Ungerer Theo
Published: May 2005

Im Komodo-Projekt wurde ein mehrfädiger Java-Mikrocontroller mit hardwarebasiertem Echtzeit-Scheduling und hardwarebasiertem Energiemanagement als FPGA-Prototyp und als ASIC-Entwurf realisiert und bewertet. Dabei wurden die Scheduling-Algorithmen FPP, EDF, LLF und das neu entwickelte, nur bei mehrfädigen Prozessorkernen anwendbare Guaranteed-Percentage (GP) Scheduling in den Prozessorkern integriert. Auf der Grundlage des prozessorintegrierten GP- bzw. EDF-Scheduling wurden zwei neuartige hardwarebasierte Energiemanagement-Techniken für mehrfädige Prozessorarchitekturen entworfen, die den Energiebedarf des Prozessorkerns durch Frequenz- und Spannungsanpassung gemäß des benötigten Rechenzeitbedarfs reduzieren.
Parallel Processing in Network Processor Architectures (Parallelverarbeitung in Netzwerkprozessorarchitekturen)
Authors: Hagenau Rainer , Albrecht Carsten , Maehle Erik , Döring Andreas C.
Published: May 2005

Summury

Parallel processing is well established in high-performance computing. Currently, network processors as new emerging, special-purpose processors are targeted at the exploitation of parallelism to meet the requirements in data-plane processing with wire-speed. The achievable level of parallelism is determined by decisions in the architecture design and by the characteristics of the data-plane applications executed. We discuss two basic approaches in parallel processing, namely pipelining and concurrency, which establish basic models for parallel network processor organization. The features and constraints of these models are studied. Using this background some existing network processor architectures are reviewed and characterized regarding their potential in parallel data-plane processing.
Nichtstandardfehlermodelle für digitale Logikschaltkreise: Simulation, prüfgerechter Entwurf, industrielle Anwendungen (On Non-standard Fault Models for Logic Digital Circuits: Simulation, Design for Testability, Industrial Applications)
Authors: Polian Ilia
Published: May 2005

Konventionelle Testverfahren für integrierte Schaltungen sind zunehmend nicht mehr in der Lage, akzeptable Produktqualität zu gewährleisten. Ein möglicher Ausweg ist der Einsatz von verbesserten Modellen für Fertigungsdefekte (Nichtstandardfehlermodelle). Der erste Teil der Dissertation beschäftigt sich deshalb mit der Modellierung von Brückenfehlern. Insbesondere die Berücksichtigung des Einflusses des Brückenwiderstandes spiegelt die Gegebenheiten moderner Nanoscale-Technologien wider. Obwohl ein Kontinuum von Defekten unter Berücksichtigung nichttrivialer elektrischer Zusammenhänge modelliert wird, sind effiziente diskrete Simulationsalgorithmen möglich. Die einfachsten der vorgestellten Modelle wurden für den industriellen Einsatz optimiert; die Integration der komplexeren resistiven Modelle in die Werkzeuge eines führenden Entwurfsautomatisierungssoftware-Herstellers wird derzeit durchgeführt. Der zweite Teil der Dissertation befasst sich mit Entwurfsmethoden, welche die Testbarkeit des Schaltkreises auf dynamische Defekte erhöhen. Ein Ansatz zur Festlegung mehrerer Prüfpfade und eine Selbsttestarchitektur werden vorgestellt. Zwei Anhänge beschreiben den Zusammenhang zwischen den Nichtstandardfehlermodellen und dem konventionellen stuck-at-Modell und ihren Einsatz in der formalen Verifikation.
The effect of fetal pig size and stage of gestation on tissue fatty acid metabolism and profile
Authors: McNeil Christopher J , Finch Angela M , Page Kenneth R , Clarke Steve D , Ashworth Cheryl J , McArdle Harry J
Published: May 2005

The fetus requires an adequate supply of fatty acids for optimum growth and development. It has been hypothesized that reduced activity of enzymes of fatty acid metabolism could contribute to inadequate fetal growth. In a porcine model of differential fetal growth we examined heart and liver fatty acid synthase, Δ5-desaturase and Δ6-desaturase gene expression and measured hepatic fatty acid profile to assess long-chain polyunsaturated fatty acid status. On gestation days 45, 65 and 100 sows were killed and tissues extracted from an average-sized fetus and the smallest fetus from each litter. As early as day 45, considerable hepatic Δ5- and Δ6-desaturase was detected, and this expression significantly increased as gestation progressed. In contrast, cardiac desaturase expression remained stable with time. Fatty acid synthase expression was greatest at day 65 in the liver, but was not expressed in the heart. Overall, the smallest fetus did not exhibit reduced tissue Δ5- or Δ6-desaturase expression or compromised polyunsaturated fatty acid status at any stage. In fact, small fetuses expressed more cardiac Δ5-desaturase than their average-sized siblings, possibly in response to a stress to the heart. It is clear from this study that fatty acid metabolism changes markedly as gestation progresses, and reduced fatty acid supply does not cause inadequate growth in this porcine model of fetal development.
Follicular cysts occur after a normal estradiol-induced GnRH/LH surge if the corpus hemorrhagicum is removed
Authors: Gümen Ahmet , Wiltbank Milo C
Published: May 2005

The pathophysiology underlying follicular cysts appears to be lack of an estradiol (E2)-induced GnRH/LH surge due to hypothalamic insensitivity to E2. In addition, progesterone (P4) can cause animals with follicular cysts to resume normal cyclicity and normal hypothalamic responsiveness to E2. We postulated that follicular cysts may be a pathological manifestation of a physiological state that cows, and possibly other species, go through during the normal estrous cycle but the rise in P4following ovulation induces them back to normal hypothalamic responsiveness to E2. Based on this hypothesis, we expected that removal of the ovary containing the corpus hemorrhagicum would prevent the normal rise in P4following ovulation and induce development of follicular cysts. Cows (n= 24) on day 7 of the estrous cycle were treated with prostaglandin F2agr;(PGF2agr;) and time of ovulation was detected by ovarian ultrasonography every 8 h. Immediately following detection of ovulation, cows were randomly but unequally assigned to have the ovary containing the corpus hemorrhagicum removed (TRT;n= 16) or the ovary opposite to the corpus hemorrhagicum removed (CON;n= 8). Cows were subsequently evaluated by daily ultrasound and blood sampling to determine follicular dynamics. Ovulation was detected at 93.7 ± 4.5 h after PGF2agr;injection. All CON cows had a normal estrous cycle length (22.0 ± 0.6 days) after ovariectomy (OVX). Half of the TRT cows became anovular (TRT-ANO;n= 8) after OVX with large anovular follicles developing on the ovary (maximal size, 25.4 ± 1.4 mm; range, 20–32 mm). However, eight TRT cows ovulated (TRT-OV;n= 8) 7.3 ± 0.6 days after OVX. Control cows had a normal P4rise after ovulation. Removal of the newly formed corpus hemorrhagicum prevented the rise in circulating serum P4in TRT-ANO cows throughout the 25-day experimental period. The TRT-OV cows had a delayed increase in circulating P4but it was normal in relation to time of ovulation. Serum E2concentrations were similar among groups (TRT-OV, TRT-ANO and CON cows) until 7 days after OVX. Serum E2concentrations then decreased in TRT-OV and CON cows but remained elevated (>5 pg/ml) in TRT-ANO cows. Thus, the endogenous increase in circulating E2that induces the GnRH/LH surge and estrus is sufficient to induce cows into a physiological state that resembles follicular cysts if it is not followed by increased circulating P4.
Birth of piglets after transferring of in vitro-produced embryos pre-matured with R-roscovitine
Authors: Coy Pilar , Romar Raquel , Ruiz Salvador , Cánovas Sebastián , Gadea Joaquín , García Vázquez Francisco , Matás Carmen
Published: May 2005

The objectives of this study were to evaluate: (1) the nuclear maturation, (2) the intracellular glutathione (GSH) content, (3) the normality of fertilization and (4) full development after transplantation of embryos derived from porcine oocytes pre-cultured with 50 μmol/l roscovitine (an inhibitor of p34cdc2/cyclin B kinase) for 22 h. After treatment with roscovitine, the nuclear configuration of oocytes (Hoechst staining) was comparable with those examined just after collection: the majority of oocytes were arrested at the germinal vesicle (GV) 1 stage (63.2%). Roscovitine-treated oocytes progressed through meiosis to the metaphase II stage in a conventional step-wise in vitro maturation (IVM) program for 44 h in a proportion similar to control ones (>85.0%). When roscovitine-treated oocytes and non-treated oocytes were matured for 44 h and then co-cultured with fresh spermatozoa for 18 h, no differences were observed in oocyte penetrability, proportion of monospermic penetration and male pronuclear formation (>87%). Roscovitine increased the GSH synthesis in oocytes at 22 h, whereas, after 44 h, roscovitine-treated oocytes had similar amounts of GSH to non-treated oocytes. Finally, surgical transfer of zygotes at 22–24 h post-insemination, derived from roscovitine-treated oocytes, resulted in one pregnancy with 12 piglets born; control non-treated zygotes resulted in one pregnancy and 10 piglets born. The full-term developmental ability of mammalian oocytes pre-cultured with roscovitine prior to IVM is thereby demonstrated. This validation is important before the introduction of roscovitine into routine procedures.
Efficacy of intravenous phenytoin in the treatment of status epilepticus: Kinetics of central nervous system penetration
Authors: Wilder B. Joe , Ramsay R. Eugene , Willmore L. James , Feussner George F. , Perchalski Robert J. , Shumate Jack B.
Published: May 2005

Intravenous phenytoin (mean dose, 13.0 mg per kilogram of body weight) was administered to 14 patients at a rate not exceeding 100 mg per minute. Twelve hours after infusion the mean plasma phenytoin concentration was 10.2 μg per milliliter, and a therapeutic plasma concentration was found in 9 of the 14 patients. In 3 additional patients, during removal of a primary brain tumor, cortical samples were obtained after intravenous infusion of phenytoin (mean dose, 9.2 mg/kg). Rapid penetration of the brain was found. Concentrations at the end of infusion (16 μg/gm) and 60 minutes after infusion (23 μ/gm) were not significantly different. Six other patients received intravenous phenytoin (mean dose, 10.9 mg/kg) during pneumoencephalography. The cerebrospinal fluid phenytoin concentration gradually increased over 50 minutes. The plasma/cerebrospinal fluid ratio at 50 minutes was 11.0, which is approximately the ratio of total/free phenytoin in the plasma. An additional 10 patients in generalized convulsive status epilepticus were treated with intravenous phenytoin (mean dose, 12.2 mg/kg); status was controlled in 9 within 30 minutes. No major side‐effects were noted. Intravenous phenytoin rapidly enters the central nervous system and results in prompt and effective treatment of status epilepticus without suppressing higher cortical function or cardiorespiratory centers.
Morphological characteristics of central demyelination and remyelination: A single‐fiber study
Authors: Gledhill R. F. , McDonald W. I.
Published: May 2005

The characteristics of demyelination and remyelination in the central nervous system of the cat were examined using quantitative single‐fiber analysis. Internodal length, fiber diameter, and nodal gap length were measured in single fibers teased from the spinal cord of normal animals and of animals with transient experimental cord compression. Demyelination was primarily paranodal, but longer extents of myelin loss occurred. New myelin sheaths were formed by oligodendrocytes and organized into segments bounded by nodes. The internodal length remained inappropriately short for fiber diameter 6 months after compression. The findings demonstrate that the spinal cord is capable of remyelinating after injury, but whether this contributes to functional recovery remains unknown.
Visual evoked potentials in Leber's hereditary optic neuropathy
Authors: Dorfman L. J. , Nikoskelainen E. , Rosenthal A. R. , Sogg R. L.
Published: May 2005

Pattern‐reversal visual evoked potentials (VEPs) were measured serially in two brothers with Leber's hereditary optic neuropathy during the active phase of the disease. VEP latency and configuration were normal prior to the onset of visual symptoms. The earliest abnormalities consisted of either prolongation of VEP latency or unusual VEP morphology characterized by a double positive peak. As visual acuity declined progressively over a period of months, there was a parallel progressive prolongation of VEP latency accompanied by less consistent diminution of VEP amplitude. In two eyes, VEP ultimately could no longer be measured. VEP latency was normal in most of the asymptomatic family members, including the maternal presumptive carrier. These findings suggest that VEP measurements do not distinguish the presymptomatic or carrier states but do permit quantitative assessment of the activity and progression of the disease.
Acute failure of automatic respirations secondary to a unilateral brainstem infarct
Authors: Levin Barry E. , Margolis George
Published: May 2005

A case of failure of automatic respiration with sleep apnea is described in a patient with a unilateral lateral medullary tegmental lesion. This case is the first known with autopsy confirmation in which a unilateral lesion has caused this syndrome.
The serological diagnosis of St Louis encephalitis in a patient with the syndrome of opsoclonia, body tremulousness, and benign encephalitis
Authors: Estrin William J.
Published: May 2005

This report describes a 28‐year‐old man with serologically confirmed St Louis encephalitis who meets the criteria for the syndrome of opsoclonia, body tremulousness, and benign encephalitis.
Particle morphology and NMR structure of polymethylmethacrylate/polystyrene emulsifier‐free core–shell cationic latices in the presence of DBMEA
Authors: Zhang Jinzhi , Li Xiaoqin , Chai Shigan , Wang Shimin , Cheng Shiyuan
Published: May 2005

In the absence of emulsifier, we prepared stable emulsifier‐free polymethylmethacrylate/polystyrene (PMMA/PSt) copolymer latex by batch method with comonomer N,N‐dimethyl, N‐butyl, N‐methacryloloxylethyl ammonium bromide (DBMEA) by using A1BN as initiator. The size distribution of the latex particles was very narrow and the copolymer particles were spherical and very uniform. Under the same recipe and polymerization conditions, PMMA/PSt and PSt/PMMA composite polymer particle latices were prepared by a semicontinuous emulsifier‐free seeded emulsion polymerization method. The sizes and size distributions of composite latex particles were determined both by quasi‐elastic light scattering and transmission electron microscopy (TEM). The effects of feeding manner and staining agents on the morphologies of the composite particles were studied. The results were as follows: the latex particles were dyed with pH 2.0 phosphotungestic acid solution and with uranyl acetate solution, respectively, revealing that the morphologies of the composite latex particles were obviously core–shell structures. The core–shell polymer structure of PMMA/PSt was also studied by 1H, 13C, 2D NMR, and distortionless enhancement by polarization transfer, or DEPT, spectroscopy. Results showed that PMMA/PSt polymers are composed of PSt homopolymer, PMMA homopolymer, and PMMA‐g‐PSt graft copolymers; results by NMR are consistent with TEM results. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 1681–1687, 2005
Studies on engineering properties of PVC–PMMA semi‐interpenetrating polymer networks
Authors: Chakrabarti Rupa , Chakraborty Debabrata
Published: May 2005

Semi1 and semi2 interpenetrating polymer networks (IPNs) of PVC and in situ formed PMMA have been synthesized using diallyl phthalate and ethylene glycol dimethacrylate as the crosslinkers of PVC and PMMA, respectively. These two types of IPNs have been compared w.r.t their physical, mechanical, and thermal properties and an endeavor has been made to find a correlation of these properties with the morphology generated in these systems. The semi1 IPNs displayed a decrease in their mechanical parameters and the physical properties as well, while in contrast, the semi2 IPNs exhibited a marginal increase in the corresponding values after an initial drop upto about 15% of crosslinked PMMA incorporation when compared to the crosslinked PVC in the case of semi1 IPN and linear PVC in the case of semi2 IPN. The various samples of semi1 and semi2 IPNs showed a two‐stage degradation typical of PVC, while confirming the increased stability with the samples having higher percentages of PMMA. The influence of crosslinking of the major matrix in semi1 IPN was almost counterbalanced by the influence of crosslinking in the dispersed PMMA phase in the case of semi2 IPN. The softening characteristics as detected by the extent of penetration of the probe, as has been detected by thermomechanical analysis, are in conformity with their mechanicals. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 1725–1735, 2005
An indirect bioluminescence method for the quantitative measurement of polymorphonuclear cell chemotaxis
Authors: Partsch Gerald , Schwarzer Christine
Published: May 2005

A method is presented which allows the quantification of the effects of chemotactic factors on polymorphonuclear leukocytes on the basis of a sensitive ATP measurement using bioluminescence. The assay measures those cells which have migrated through a commercial 3 μm filter system (Transwell™). The assay was tested under standardized conditions with different chemotactic agents (leukotriene B4 [LTB4], N‐formyl‐methionyl‐leucyl‐phenylalanine [FMLP], N‐formyl‐methionyl‐leucyl‐phenylalanine‐methyl ester [M‐FMLP]). Under appropriate conditions the migration of PMN‐cells is time‐dependent and linear for 60 minutes. Spontaneous migration of PMN cells is simultaneously quantified in a simple way, and the value obtained allows a determination of the actual chemotactic stiuation of the PMN cells. In healthy humans the spontaneous migration varied between 4.2% and 14.4% of the total number of PMN cells. An optimal chemotactic activity was detected at 10−8/mol/I for FMLP and 10−7 mol/l for M‐FMLP in PMN leukocytes, which correlates with literature values. It was also found that in contrast to EDTA blood, heparinized blood lowers the ATP level of PMN cells (by about 50%) and therefore heparinized blood is not recommended for chemotactic experiments. This assay is a simple tool for quantification of the spontaneous migration, and the chemotactic response to specific factors and their inhibitors in particular for pharmacological experiments. In contrast to the ‘classical’ chemotactic assays this method also permits the simultaneous testing of the influence of chemotactic substances on cellular ATP levels.
Expression and quantification of firefly luciferase under control of Rhizobium meliloti symbiotic promoters
Authors: Cebolla Angel , Ruiz‐Berraquero Francisco , Palomares Antonio J.
Published: May 2005

We have tested the use of firefly luciferase for monitoring regulated symbiotic nitrogen fixation gene expression. Broad‐host‐range plasmids carrying translational fusions of Rhizobium meliloti nifH, fixA and nifA promoters were constructed. Despite low levels of promoter activity the absence of Escherichia coli endogenous luminescence and the high sensitivity of the bioluminescent assay for firefly luciferase allowed rapid screening for functional luciferase expression. Plasmids containing symbiotic promoter‐luc fusions were established in R. meliloti. Luciferase activity was detected and measured in both vegetative and symbiotic cells giving comparable results with those obtained by beta‐galactosidase assays. In addition, the luciferase assay was quicker, more sensitive and could be carried out with unrestricted cells. Furthermore, bioluminescence was high enough in alfalfa nodules containing nifH—luc fusion to be observed by a dark‐adapted eye and photographed.
Chemiluminescence intensities and spectra of luminol oxidation by sodium hypochlorite in the presence of hydrogen peroxide
Authors: Arnhold J. , Mueller S. , Arnold K. , Grimm E.
Published: May 2005

Hydrogen peroxide amplifies the chemiluminescence in the oxidation of luminol by sodium hypochlorite. A linear relationship between concentration of hydrogen peroxide and light intensity was found in the concentration range 5 × 10−8−7.5 × 10−6 mol/l. At 7.5 × 10−6 mol/l H2O2 the chemiluminescence is amplified 550—fold. The chemiluminescence spectra of these reactions have a wavelength maximum at 431 nm independent of the concentration of hydrogen peroxide. The results indicate that hydrogen peroxide is a necessary component in the chemiluminescent oxidation of the luminol by sodium hypochlorite.
Quantitative generation of singlet (1Δg) oxygen from acidified aqueous peroxynitrite produced by the reaction of nitric oxide and superoxide anion
Authors: Khan Ahsan Ullah
Published: May 2005

Simple acidification of aqueous alkaline peroxynitrite quantitatively generates singlet (1Δg) molecular oxygen, detected and quantitated spectroscopically (1270 nm). This observation provides a chemical basis for physiological cytotoxicity of ONOO− generated in the diffusion ‐ controlled reaction of cellular NO• and O. The experiments consist of (i) chemical generation of ONOO− from NO• gas and KO2 powder in alkaline aqueous solution; (ii) absorption spectral identification of ONOO− in the near‐UV with maximum at 302 nm; (iii) spectroscopic identification of 1O2 by its emission band at 1200–1340 nm with maximum at 1275 nm; and (iv) quantitation of 1O2 generated in ONOO−/H+ reaction by comparison of the chemiluminescence intensity at 1270 nm with that from H2O2/OCl− reaction that generates 1O2 with unit efficiency at alkaline pH. 1O2 was generated with unit efficiency with respect to ONOO− concentration by the ONOO−/H+ reaction.
Fluorescence quenching of β‐carboline alkaloids in micellar media. A study to select the adequate surfactant to use in analytical techniques
Authors: Martín Laura , Olives Ana I. , del Castillo Benito , Martín M. Antonia
Published: May 2005

The study of fluorescence quenching of the fluorophores allows the localization of the alkaloids (harmane and harmine) in the micelles (SDS, CTAB, Brij‐35) to be established. In aqueous micellar solutions (SDS and Brij‐35) at pH 13.0, emission corresponding to the neutral or zwitterionic forms can be observed. In the presence of CTAB (pH = 13.0) it was possible to observe the emission of anionic form. These species are not present in buffered aqueous solutions at these pH values. Bromide ion was added to the different surfactant solutions and the quenching effect was studied according to the Stern–Volmer equation. In the presence of SDS the quenching effect is considerably reduced compared to the aqueous solutions without surfactants, while for Brij‐35 micelles were similar to those observed in homogeneous aqueous solution. For CTAB micelles a notable fluorescence quenching was observed for the different pH values studied. The fluorescence quenching studies show that the neutral species are associated inside the micelles, instead of the ionic species (cationic, zwitterionic or anionic) remaining on the surface of the micelles. The anionic surface of SDS micelles prevents the quenching effect by anionic quenchers for both neutral and charged species. Copyright © 2005 John Wiley & Sons, Ltd.
Detection of synthetic corticosteroids in bovine urine by chemiluminescence high‐performance liquid chromatography
Authors: Vázquez B. I. , Feás X. , Lolo M. , Fente C. A. , Franco C. M. , Cepeda A.
Published: May 2005

The development of a black market of chemical cocktails for illegal growth promotion in food‐producing animals includes substances that are potentially dangerous for human health, such as synthetic corticosteroids. The potential presence of these residues in food makes it necessary to develop rapid and sensitive analytical methodologies to detect such substances, preferably in live animals before they arrive at the market. A chemiluminescence (CL) detection method for the determination of four synthetic corticosteroids (prednisolone, betamethasone, dexamethasone and flumethasone) in bovine urine has been developed. The proposed system, which does not need any derivatization procedure, offers an easy method well suited for routine research. Urine samples were homogenized with methanol:water (50:50, v[sol ]v) and centrifuged. The upper layer was collected and Strata™ X cartridges were used for cleaning up. The purified residues were evaporated to dryness and then redissolved in the mobile phase. Analysis of the extracts was performed using high‐performance liquid chromatography with chemiluminescence detection, employing luminol as the CL reagent. The recovery curves, obtained at four spiking levels (different for each corticosteroid), showed that recoveries of at least 70% could be obtained for urine. The chemiluminescence detection procedure afforded satisfactory results with respect to sensitivity and the LOD and LOQ, taken as the first point of the regression curve, ranged from 4 ppb to 65 ppb. The maximum mean RSD was below 13% and below 15% for intra‐ and inter‐day assay, respectively, in all cases. Copyright © 2005 John Wiley & Sons, Ltd.
Characteristics of endogenous flavin fluorescence of Photobacterium leiognathi luciferase and Vibrio fischeri NAD(P)H:FMN‐oxidoreductase
Authors: Vetrova E. V. , Kudryasheva N. S. , Visser A. J. W. G. , van Hoek A.
Published: May 2005

The bioluminescent bacterial enzyme system NAD(P)H:FMN‐oxidoreductase–luciferase has been used as a test system for ecological monitoring. One of the modes to quench bioluminescence is the interaction of xenobiotics with the enzymes, which inhibit their activity. The use of endogenous flavin fluorescence for investigation of the interactions of non‐fluorescent compounds with the bacterial luciferase from Photobacterium leiognathi and NAD(P)H:FMN‐oxidoreductase from Vibrio fischeri has been proposed. Fluorescence spectroscopy methods have been used to study characteristics of endogenous flavin fluorescence (fluorophore lifetime, the rotational correlation time). The fluorescence anisotropy behaviour of FMN has been analysed and compared to that of the enzyme‐bound flavin. The fluorescence characteristics of endogenous flavin of luciferase and NAD(P)H:FMN‐oxidoreductase have been shown to be applicable in studying enzymes' interactions with non‐fluorescent compounds. Copyright © 2005 John Wiley & Sons, Ltd.
Fluorescence analysis of photoinduced degradation of ecotoxicants in the presence of humic acids
Authors: Tchaikovskaya O. N. , Sokolova I. V. , Yudina N. V.
Published: May 2005

The photolysis of humic acids and phenols in water containing humic acids was investigated. Humic acids extracted from peat (Vasuygan Bog, Tomsk Region, Russian Federation) induce the phototransformation of 4‐chlorophenol at 365 and 222 nm. Humic acids were characterized by UV‐, fluorescence‐, IR‐ and EPR‐spectroscopy and laser‐induced fluorescence. The influence of humic acids on the phototransformation of phenols in different irradiation conditions was investigated. Comparison of the data on mercury lamp irradiation showed that the most effective phenols degradation was observed under exposure to KrCl* exilamp light (λ = 222 nm) in the presence of humic acids. Copyright © 2005 John Wiley & Sons, Ltd.
Bioluminescence determination of enzyme activity of firefly luciferase in the presence of pesticides
Authors: Trajkovska Snezana , Tosheska Katerina , Aaron Jean Jacques , Spirovski Filip , Zdravkovski Zoran
Published: May 2005

Firefly luciferase (EC 1.13.12.5) (FL) is the key enzyme in the firefly bioluminescence method (FB), which is widely used to determine the viability of living cells. The FB method can also be applied to monitoring the influence of different pollutants, such as pesticides. Firefly luciferase is a hydrophobic enzyme and its activity depends on the type of solvent, pH and substances present in the reaction mixture. The influence of three aromatic pesticides, including fenoxaprop‐p‐ethyl (I), diclofop‐methyl (II) and metsulfuron methyl (III), on the enzyme activity was indirectly evaluated through the measurement of emitted light in the bioluminescence reaction, expressed in relative luminescence units (RLU). The reaction mixture used in the bioluminescence measurements consisted of: Tris buffer (pH 7.75), adenosine triphosphate (ATP) and ATP monitoring reagent, where FL is present. Ethanol–water solutions of each pesticide were then added at concentrations of 2.4 × 10−4–2.4 × 10−8 mol[sol ]L. The FL activity inhibition factors (FL In%) were determined. The FL activity was maximally inhibited in the presence of all pesticides under study at a concentration of 2.4 × 10−4 mol[sol ]L and was lowered by about 15–26% for pesticide I at concentrations of 2.4 × 10−5–2.4 × 10−8 mol[sol ]L, whereas pesticides II and III, applied in the same concentration range, showed smaller FL inhibition values (5.3–20%). The pesticide degradation products (obtained after a 1 month period), measured in the same experimental conditions, in most cases exhibited a much less inhibitory effect on the enzyme activity than the corresponding initial pesticide. Copyright © 2005 John Wiley & Sons, Ltd.
Nanosized γ‐Al2O3 + Nd2O3‐based cataluminescence sensor for ethylene dichloride
Authors: Cao Xiaoan , Feng Guiming , Gao Haihua , Luo Xiaoqing , Lu Hailiang
Published: May 2005

A gas‐sensor utilizing cataluminescence (CTL) on nanosized γ‐Al2O3 + Nd2O3 for measuring low concentrations of gaseous ethylene dichloride (EDC) was developed. The results showed that γ‐Al2O3 nanoparticles as a catalyst offered high sensitivity and selectivity in the detection of EDC. The addition of a small quantity of Nd2O3 increased the intensity of CTL more than two‐fold. Quantitative analysis was performed at a wavelength of 400 nm and at an optimal temperature of 279°C, and the optimal flow rate of carrier gas was 320 mL[sol ]min. Under the optimized conditions, the linear range of CTL intensity vs. concentration is 6–5000 ppm (R = 0.9996; n = 7), with a detection limit of 2 ppm. The response time is <5 s. No interference or only very low levels of significant interference were observed when substances such as formaldehyde, n‐hexane, methyl benzene, carbon tetrachloride, chloroform and benzene were passed through the sensor. Copyright © 2005 John Wiley & Sons, Ltd.
Flow cytometric analysis of fine particles in a eutrophic lake
Authors: Guo Pei‐yong , Zhu Yin‐mei , Zhang Zhi‐jian
Published: May 2005

Fine particles play an important role, not only in aquatic biogeochemical processing but also in the distribution, transfer and transformation of pollutants in the aquatic environment. Flow cytometry, widely used in biomedical research, allows fast counting and optical analysis of individual particles. Organic autotrophic particles contain naturally fluorescing pigments, such as chlorophyll and phycoerythrin. Different populations have different sizes and pigments. They also have different ratios of pigments. In general, side angle scatter (SSC) is related to the size, shape and refractive index of particles. When a 488 nm wavelength was used to excite chlorophyll and phycoerythrin fluorescence, the pigments of organic autotrophic particles emitted red and orange light. Fine particles were detected by flow cytometry (FCM) in the southern part of a eutrophic lake in winter. We found that organic autotrophic particles belonged to three populations, which represented only 15.89% of total fine particles. Organic non‐living particles and inorganic particles represented the greater part (84.11%) of total fine particles. This study also demonstrated that flow cytometry is well suited to the dynamic monitoring and analysis of natural water aquatic particles that were difficult to study with traditional methods. Copyright © 2005 John Wiley & Sons, Ltd.
Proton relaxation in aqueous DNA solutions
Authors: Sprinz Helmut , Döllstädt Rosemarie , Hübner Gerhard
Published: May 2005

By use of D2O we found that the shortening of the longitudinal proton relaxation time which occurs in the investigated aqueous yeast DNA solutions (≦ 2.4% with 2% protein) was not based on a hydration effect, but was caused by magnetic impurities only. An estimate shows that the mobility of the hydrated water molecules is reduced by less than two orders of magnitude in comparison with the free water molecules.
Kinetics of denaturation of DNA
Authors: Massie Harold R. , Zimm Bruno H.
Published: May 2005

The kinetics of denaturation of DNA have been studied by relaxation techniques. Examination of the terminal relaxation times for a variety of DNA's under a variety of conditions has shown that DNA denaturation is principally a hydrodynamically limited process. Measurements within the helix–coil transition have demonstrated that the experimentally measured terminal relaxation times are a function of the following: (1) position in the helix–coil transition; (2) ionic strength of the solvent; (3) solvent viscosity; (4) DNA concentration; (5) molecular weight; (6) number and position of single‐strand breaks. The dependence of the terminal relaxation time on the above mentioned factors can be attributed to hydrodynamic effects. Thus a hydrodynamic model for DNA unwinding is required. The model which best fits the data involves the assumption of a rotational frictional coefficient independent of molecular weight. This assumption is suggested by the fact that the relaxation time is proportional to the first power of the molecular weight.
Counterion binding in partially neutralized poly(D‐glutamic acid) and poly(DL‐glutamic acid)
Authors: Sugai Shintaro , Nitta Katsutoshi
Published: May 2005

Sodium counterion association with partially neutralized poly(D‐glutamic acid) or poly(DL‐glutamic acid) was measured by use of Wall's transference method with radioactive sodium. In the region where both polyacids are in completely random coil form, fractions of association were considerably less than that with poly(acrylic acid) in the same region of degree of neutralization. Even in the region where poly (D‐glutamic acid) is in the helical form, the fraction of association was less than that with poly(acrylic acid) in the same region. No pronounced characteristics attributable to counterion association corresponding to the helix–coil transition could be found. The association phenomena were discussed on the basis of a rodlike model of polyelectrolyte.
Flow birefringence and intrinsic viscosity of a T2 bacteriophage DNA–methylated bovine serum albumin complex
Authors: Harrington Rodney E. , Martin Kathleen V.
Published: May 2005

The flow birefringence, extinction angles, and intrinsic viscosity have been determined at low velocity gradients for a complex of T2 bacteriophage DNA and methylated serum albumin prepared in dilute solution to a stoichiometry of approximately 90 proteins per DNA molecule. Comparative data upon equivalent solutions of pure uncomplexed T2 DNA are also presented, and these data are completely in accord with the results of previous study. The experimental data are interpreted in terms of current dynamical theory and indicate that the complex has an essentially linear chain structure, consisting of approximately two DNA molecules, which is hydrodynamically indistinguishable from the pure DNA and that extensive internal or intramolecular binding in the complex does not occur. Although interpretation of the results is hampered by an apparent moderate degree of polydispersity in the complex preparations and by relatively large shear extrapolations, the data for both DNA and the complex are substantially in accord with dynamical theory for a nondraining bead subchain model having high kinetic segmental rigidity.
Single crystals of poly(L‐glutamic acid)
Authors: Keith H. D. , Giannoni G. , Padden F. J.
Published: May 2005

Lamellar single crystals of alkaline earth salts of poly(L‐glutamic acid) have been grown by precipitation from dilute aqueous solution and studied by optical and electron microscopy and by x‐ray and electron diffraction. The calcium, strontium and barium salts were crystallized in the β form above room temperature and could be converted to crystals of β‐poly(L‐glutamic acid) by washing in dilute hydrochloric acid. The magnesium salt, on the other hand, was crystallized in the α form at or below room temperature but could not be converted into crystals of α‐poly(L‐glutamic acid) by washing in hydrochloric acid. The crystalline lamellae are very thin (thicknesses range from 25 to 60 Å in β crystals and are about 100 Å in α crystals) and the polypeptide chains are oriented normal to the planes of the lamellae. It is clear from the disparity between crystal thickness and molecular length that the molecules crystallize by folding at the upper and lower surfaces of the crystals. Conformations of the molecules at these folds are discussed briefly.
Single crystals of poly‐L‐lysine
Authors: Padden F. J. , Keith H. D. , Giannoni G.
Published: May 2005

Crystals of poly‐L‐lysine have been grown from aqueous solution in the presence of divalent anions. The most stable of these incorporate the HPO ion and are precipitated by the addition of sodium monohydrogen phosphate to solutions of poly‐L‐lysine HBr. Precipitation at or slightly above room temperature gives rise to single crystals of α‐poly‐L‐lysine HPO4 in the form of hexagonal lamellae about 150 Å thick. The axes of the helical polypeptide molecules are oriented normal to the planes of the lamellae, and since molecular length is about 1100 Å in the α‐helical conformation, these helices must be folded. The a parameter of the hexagonal unit cell is 19.55 Å for crystals immersed in mother liquor, and the lysine side chains are almost fully extended. Precipitation brought about by heating the same solutions to about 75°C produces micro‐crystals of β‐poly‐L‐lysine HPO4. A mode of packing of the anions in these crystals is proposed tentatively on the basis of an intersheet spacing determined from x‐ray powder diffraction patterns. In general, α crystals are transformed to β structures on drying; conditions under which the transition can either be forestalled or reversed are discussed.