bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship between astrocytic processes and “Perineuronal nets” in rat neocortex
Authors: Blümcke Ingmar , Eggli Peter , Celio Marco R.
Published: May 2005

“Perineuronal nets” (PNs) ensheath a subtype of inhibitory neurons in the mammalian neocortex. In the light of the proposal that PNs consist of glial processes, we have analyzed the relationship between intracellularly injected glial cells and PNs in the rat neocortex. Glial cells were injected iontophoretically with Lucifer Yellow in lightly fixed tissue slices and PNs were visualized with the lectin from Vicia villosa. Using confocal laser scanning microscopy, glial processes and PNs were identified as distinct structures. Lectin labeling was consistently associated with the extracellular space interposed between LY‐labeled astrocyte processes and neurons. Of the different types of glial cells injected, only the densely‐ramifying protoplasmic astrocytes extended processes which could be traced to contact PNs. These protoplasmic astrocytes also sent out processes to adjacent neurons not ensheathed by PNs, and to capillaries. The present data strongly suggests that PNs do not consist of glial processes but rather support the idea that PNs represent specialized extracellular material interposed between the surface of some inhibitory interneurons and astrocytic processes. © 1995 Wiley‐Liss, Inc.
Sodium‐dependent glutamate uptake as an activator of oxidative metabolism in primary astrocyte cultures from newborn rat
Authors: Eriksson Gun , Peterson Anders , Iverfeldt Kerstin , Walum Erik
Published: May 2005

In the present report we describe the effect of glutamate on respiratory activity in primary cultures of astrocytes, derived from cerebral cortex of newborn rat.

Glutamate (100 μM) caused an increased oxygen consumption. This effect could not be inhibited by antagonists to the NMDA or AMPA/kainate receptors. Neither trans‐ACPD (an agonist to the metabotropic glutamate receptor) nor the Krebs cycle intermediate α‐ketoglutarate had any effect on the respiratory rate. An uncontrolled influx of Na+, caused by gramicidin, could mimic the glutamate effect on respiratory activity. In addition, the glutamate effect was abolished by addition of ouabain or replacement of Na+ by Li+ in the perfusion buffer.

We conclude that the co‐transport of Na+, in the Na+ ‐dependent high‐affinity glutamate uptake system, mediated the glutamate‐induced increase in oxygen consumption through an increased activity of Na+/K+‐ATPases. © 1995 Wiley‐Liss, Inc.
Developmental regulation of Na+ and K+ conductances in glial cells of mouse hippocampal brain slices
Authors: Kressin Klaus , Kuprijanova Elena , Jabs Ronald , Seifert Gerald , Steinhäuser Christian
Published: May 2005

The relative contribution of voltage activated Na+ and K+ currents to the whole cell current pattern of hippocampal glial cells was analyzed and compared during different stages of postnatal maturation. The patch‐clamp technique was applied to identified cells in thin brain slices obtained from animals between postnatal day 5 and 35 (p5‐35). We focused on a subpopulation of glial cells in the CA1 stratum radiatum which most probably represents a pool of immature astrocytes, termed “complex” cells. These cells could not be labelled by 01/04 antibodies, but some of the older cells were positively stained for glial fibrillary acidic protein (GFAP). In the early postnatal days, the current pattern of the “complex” cells was dominated by two types of K+ outward currents: a delayed rectifier and a transient component. In addition, all cells expressed significant tetrodotoxin (TTX)‐sensitive Na+ currents. During maturation, the contribution of delayed rectifier and A‐type currents significantly decreased. Furthermore, almost all cells after p20 lacked Na+ currents. This down‐regulation of voltage gated Na+ and K+ outward currents was accompanied by a substantial increase in passive and inward rectifier K+ conductances. We found increasing evidence of electrical coupling between the “complex” cells with continued development. It is concluded that these developmental changes in the electrophysiological properties of “complex” glial cells could be jointly responsible for the well known impaired K+ homeostasis in the early postnatal hippocampus. © 1995 Wiley‐Liss, Inc.
Na+ channel aggregation in remyelinating mouse sciatic axons following transection
Authors: Tzoumaka Eleftheria‐Elda , Novaković Sanja D. , Rock Levinson S. , Shrager Peter
Published: May 2005

Mouse sciatic nerves from the degeneration‐resistant strain C57BL/6/Wld (Ola) were surgically injected with lysolecithin to induce focal demyelination. Three days later they were transected adjacent to the spinal cord to eliminate contact of the axons with their cell bodies. The Na+ channel distribution was assessed by immunocytochemistry and followed at several stages of remyelination. Control experiments were performed on nerves that were injected but not cut. At (3+4) days, namely, nerves cut 3 days post‐injection and examined 4 days after cutting, axons contained fully demyelinated regions. Na+ channel clusters appeared only at heminodes and at isolated sites that are likely to represent original nodes of Ranvier. During the next few days proliferating Schwann cells adhered to the axons and extended their processes. Clusters of Na+ channels appeared at their edges, and as the Schwann cells elongated the distance between these aggregates increased. A few clusters associated with neighboring Schwann cells approached each other and appeared to coalesce at sites where presumably new nodes of Ranvier would be formed. Beyond (3+6) days excessive degeneration of the transected axons precluded further observations. In the uncut controls, the spatio‐temporal sequence of Schwann cell proliferation and channel patch formation and movement was similar to that described above, although myelin formation was somewhat faster than in the cut axons. It is concluded that Na+ channel aggregation associated with the early stages of remyelination is not dependent upon continuous communication of the axon with its cell body and is under local control. © 1995 Wiley‐Liss, Inc.
Human epileptic astrocytes exhibit increased gap junction coupling
Authors: Lee S. H. , Magge S. , Spencer D. D. , Sontheimer H. , Cornell‐Bell A. H.
Published: May 2005

Fluorescence Recovery After Photobleach (FRAP) was used to quantify astrocyte gap junction coupling from tissues surgically resected from medically intractable epilepsy patients. Mesial temporal lobe cases provided hippocampus, surrounding hyperexcitable parahippocampus and normal cortex for culture. Cortical tumor cases yielded astrocytoma proper, cortex margins with normal EEG activity, and hyperexcitable cortex. Cells isolated from cortex surrounding astrocytomas and the parahippocampus surrounding the hippocampus showed an increase in glutamate‐induced Ca2+ oscillations and intercellular Ca2+ waves. Gap‐junction coupling was more pronounced in cells isolated from hyperexcitable tissue than from normal tissues as judged by their faster and more complete fluorescence recovery from laser bleach [FRAP]. This data suggests that intractable epilepsy may be associated with alterations in glial gap junction coupling. © 1995 Wiley‐Liss, Inc.
From rodent glial precursor cell to human glial neoplasia in the oligodendrocyte‐type‐2‐astrocyte lineage
Authors: Noble Mark , Gutowski Nick , Bevan Karen , Engel Ute , Linskey Mark , Urenjak Jutte , Bhakoo Kishore , Williams Steve
Published: May 2005

With only a few exceptions, the precursor cells representing the normal counterparts of human tumours are unknown. The comparative lack of information about the lineages involved in tissue development, and difficulties in growing many human tumors in a manner suitable for cellular biological analysis, together often make it difficult to study the differences between normal and tumor and tumor cells and to develop many of the model systems that would be useful in the study of human cancer. By applying techniques previously utilized to study glial progenitor cells, we have isolated a human glioblastoma multiforme (GBM)‐derived population that expresses many properties otherwise uniquely expressed by oligodendrocyte‐type‐2 astrocyte (O‐2A) progenitor cells. Hu‐O‐2A/Gb1 (for Human O‐2A lineage Glioblastoma number 1) cells responded to similar mitogens and differentiation modulators as rodent O‐2A progenitors, and generated cells with features of precursor cells, oligodendrocytes and astrocytes. Moreover, 1H‐NMR analysis of amino acid composition demonstrated a striking conservation of types and quantities of free amino acids between the human tumour cells and the rodent primary cells. Hu‐O‐2A/Gb1 cells represent the first human glioma‐derived population for which unambiguous lineage assignment has been possible, and our results indicate that the human O‐2A lineage can contribute to one of the most malignant of glial tumours. In addition, the highly diagnostic 1H‐NMR spectrum expressed by Hu‐O‐2A/Gb1 cells raises the possibility of eventual non‐invasive identification of tumors of this lineage. © 1995 Wiley‐Liss, Inc.
Physiology of transformed glial cells
Authors: Brismar Tom
Published: May 2005

Much of our present knowledge of glial cell function stems from studies of glioma cell lines, both rodent (C6, C6 polyploid, and TR33B) and human (1321N1, 138MG, D384, R‐111, T67, Tp‐301MG, Tp‐483MG, Tp‐378MG, U‐118MG, U‐251MG, U‐373MG, U‐787MG, U‐1242MG, and UC‐11MG). New methods such as patch clamp and Ca2+ imaging have lead to rapid progress the last few years in our knowledge about glial cells, where an unexpected presence and diversity of receptors and ion channels have emerged. Basic mechanisms related to membrane potential and K+ transport and the presence of voltage gated ion channels (Na+, inwardly rectifying K+, Ca2+ activated K+, Ca2+, and Cl− channels) have been identified. Receptor function and intracellular signaling for glutamate, acetylcholine, histamine, serotonin, cathecolamines, and a large number of neuropeptides (bradykinin, cholecystokinin, endothelin, opioids, and tachykinins) have been characterized. Such studies are facilitated in cell lines which offer a more homogenous material than primary cultures. Although the expression of ion channels and receptors vary considerably between different cell lines and comparative studies are rare, a few differences (compared to astrocytes in primary culture) have been identified which may turn out to be characteristic for glioma cells. Future identification of specific markers for receptors on glial and glioma cells related to cell type and growth properties may have great potential in clinical diagnosis and therapy. © 1995 Wiley‐Liss, Inc.
Tumor antigens in astrocytic gliomas
Authors: Kurpad Shekar N. , Zhao. X.‐G. , Wikstrand Carol J. , Batra Surinder K. , McLendon Roger E. , Bigner Darell D.
Published: May 2005

Gliomas affect 15,000 to 17,000 Americans every year and carry a dismal prognosis. The potential of immunologically mediated diagnosis and therapy, although greatly enhanced since the advent of monoclonal antibodies, has not been fully realized due to significant problems, most especially the challenge of identifying antigenic molecules specific to glial tumors. Other problematic issues include antigen‐associated factors such as heterogeneity, modulation, shedding, and cross‐reactivity with normal cells, and factors associated with therapeutic agent delivery, typically variable tumor perfusion and unfavorable diffusional forces in tumor microenvironment. An understanding of these problems called for the delineation of operationally specific antigens (tumor‐associated antigens not expressed by the normal central nervous system) combined with the use of compartmental therapeutic approaches to increase the specificity of therapy. Numerous antigens have been identified and are classified as extracellular/matrix‐associated, membrane‐associated, and intracellular antigens. Nevertheless, only a few have been demonstrated to be of significant therapeutic and diagnostic utility. These few include the extracellular matrix‐associated antigens tenascin and GP 240, defined by the antibody DMAb‐22. Recent identification of the overexpression of a deletion variant of the epidermal growth factor receptor (EGFRvIII) in up to 50% of the more malignant glial tumors and the subsequent creation of monoclonal antibodies that are specific to this molecule and do not recognize the wild‐type EGFR provide the most exciting development yet in the design of specific antiglioma immunoconjugates. In addition, the tumor‐specific nature of EGFRvIII combined with improved knowledge of immune mechanisms, especially in the context of the central nervous system, will facilitate the design of highly selective cell‐mediated therapeutic approaches with a view toward obtaining tumor‐specific immunity. © 1995 Wiley‐Liss, Inc.
Gene amplification in human gliomas
Authors: Collins V. P.
Published: May 2005

Gliomas represent the largest group of primary brain tumors in adults. The astrocytic variants are the most common and the adult forms are histologically stratified into three malignancy grades. Of these glioblastoma is the most common and the most malignant; it has also been best studied by molecular genetics and cytogenetics. Double‐minute chromosomes, known to represent amplified genes, are found in 50% of glioblastomas. Amplified genes are not detected in the most benign of the astrocytomas. Many genes have been shown to be amplified in more than single cases of gliomas and these include EGFR, CDK4, SAS, MDM2, GLI, PDGFAR, MYC, N MYC, MYCL1, MET, GADD153, and KIT. The most commonly amplified genes in glioblastomas are EGFR (in approximately 40%), CDK4, and SAS (in approximately 15%). The remainder of the genes are amplified at lower frequency. The best mapped amplicon in gliomas involves the 12q13–14 region. The amplicon is of undetermined size, encompasses a number of genes, and may be rearranged. It occurs in 15% of glioblastomas and almost always includes the CDK4 and SAS genes, in about 10% of tumors of MDM2 gene, and at lower frequency GLI, GADD153, and A2MR. All but A2MR are overexpressed if amplified. The amplified EGFR gene is frequently rearranged, resulting in changes in the regions of the transcript that codes for the extracellular domain. The resultant receptor is constitutively activated. These findings provide examples of the impact the use of modern molecular biological techniques has had on our understanding of oncogenic mechanisms in gliomas. © 1995 Wiley‐Liss, Inc.
The p53 gene and its role in human brain tumors
Authors: Bögler Oliver , Su Huang H.‐J. , Kleihues Paul , Cavenee Webster K.
Published: May 2005

Mutation of the p53 gene is among the most common lesions in a variety of human tumors, including those of the central nervous system. In most instances, mutation of one p53 allele is followed by loss of the remaining wild‐type allele, resulting in cells with a complete absence of functional wild‐type p53 protein. However, in some situations, such as at initiation of spontaneously arising gliomas or as the germline configuration of patients with the Li‐Fraumeni syndrome, cells clearly carry both wild‐type and mutant p53 alleles. These observations lead to the hypothesis that p53 mutations can give rise to loss of tumor suppressor functions as well as to gain of oncogenic transformation capabilities. In this review, we define the types of mutations that occur in the p53 gene in various glial tumors, contrast that with the spectra described in other human tumor types, and discuss the biochemistry and physiology of the p53 protein and its ability to regulate and be regulated by other gene products. We use this information to propose roles for p53 in the initiation and progression of human gliomas. © 1995 Wiley‐Liss, Inc.
A comparison of solute fluxes and sources from glacial and non‐glacial catchments over contrasting melt seasons
Authors: Lafrenière Melissa J. , Sharp Martin J.
Published: May 2005

Solute and runoff fluxes from two adjacent alpine streams (one glacial and one non‐glacial) were investigated to determine how the inorganic solute chemistry of runoff responded to seasonal and interannual changes in runoff sources and volume, and to differences in physical catchment properties. Intercatchment differences in solute composition were primarily controlled by differences in catchment geology and the presence of soils, whereas differences in total solute fluxes were largely dependent on specific discharge. The glacial stream catchment had higher chemical denudation rates due to the high rates of flushing (higher specific discharge). The non‐glacial Bow River had higher overall concentrations of solutes despite the greater prevalence of more resistant lithologies in this catchment. This is likely the result of both longer average water–rock contact times, and a greater supply of protons from organic soils and/or pyrite oxidation. Increases in snowpack depth/snowmelt runoff reduced the retention of nitrate in the Bow River catchment (i.e. increased nitrate export), probably by reducing net biological uptake, or by reducing the proportion of runoff that had contact with biologically active soil horizons that tend to remove nitrate.

The two streams exhibited opposite solute flux responses to climate perturbations over three melt seasons (1998, 1999, and 2000). The 1998 El Niño event resulted in an unusually thin winter snowpack, and increased runoff and solute fluxes from the glacial catchment, but decreased fluxes from the Bow River catchment. Solute fluxes in the Bow River increased proportionally to discharge, indicating that increased snowmelt runoff in this catchment resulted in a proportional increase in weathering rates. In contrast, the proportional variation in solute flux in the glacial stream was only ∼70–80% of the variation in water flux. This suggests that increased ablation of glacier ice and the development of subglacial channels during the 1998 El Niño year apparently reduced the average water–rock contact time in the glacial catchment relative to seasons when the subglacial drainage system was primarily distributed in character. Copyright © 2005 John Wiley & Sons, Ltd.
Fluxes of inorganic carbon from two forested catchments in the Appalachian mountains
Authors: Worrall Fred , Swank Wayne T. , Burt Tim
Published: May 2005

This study uses long‐term records of stream chemistry, discharge and air temperature from two neighbouring forested catchments in the southern Appalachians in order to calculate production of dissolved CO2 and dissolved inorganic carbon (DIC). One of the pair of catchments was clear‐felled during the period of the study. The study shows that: (1) areal production rates of both dissolved CO2 and DIC are similar between the two catchments even during and immediately after the period of clear‐felling; (2) flux of total inorganic carbon (dissolved CO2+ DIC) rises dramatically in response to a catchment‐wide acidification event; (3) DIC and dissolved CO2 are dominantly released on the old water portion of the discharge and concentrations peak in the early autumn when flows in the study catchments are at their lowest; (4) total fluvial carbon flux from the clear‐felled catchment is 11·6 t km−2 year−1 and for the control catchment is 11·4 t km−2 year−1. The total inorganic carbon flux represents 69% of the total fluvial carbon flux. The method presented in the study provides a useful way of estimating inorganic carbon flux from a catchment without detailed gas monitoring. The time series of dissolved CO2 at emergence to the stream can also be a proxy for the soil flux of CO2. Copyright © 2005 John Wiley & Sons, Ltd.
Analytical solutions of infiltration process under ponding irrigation
Authors: Chen Jiann‐Mou , Tan Yih‐Chi
Published: May 2005

The objective of this paper is to simulate the progress of the soil water content distribution in the soil profile with a water table at the bottom of the soil profile during ponding irrigation. This simulation can be done by solving the two‐dimensional Richards's equation for the assimilation of the advancing water jet, which uses the conditions of the two exponential functional forms k = ks eαψ and θ = θr + (θs − θr) eαψ to represent the hydraulic conductivity and volumetric water content, with ψ the pressure as the third variable. We assume that the ground surface becomes ponded and saturated as soon as the water flux passes the dry ground surface. By the technique of transformation, the analytical solution of these two‐dimensional Richards' equations has enabled figures of volumetric water content distribution to be obtained in successive time periods after irrigation. For the example of loam soil, it can simulate the variation of volumetric water content during and after irrigation in the soil profile. The analytical solutions of this paper reflect the real situation simulated, and can be applied to verify those complicated solutions from other analytical models. Copyright © 2005 John Wiley & Sons, Ltd.
A hybrid coating of biomimetic apatite and osteocalcin
Authors: Krout Ashley , Wen Hai Bo , Hippensteel Elizabeth , Li Panjian
Published: May 2005

A novel hybrid coating of biomimetic apatite (BAp) and osteocalcin (OC) was prepared by incubating BAp‐coated Ti6A14V coupons in an osteocalcin‐containing medium. A significant amount (up to 1.0 wt %) of OC was adsorbed by the BAp coating within 3 h of incubation as demonstrated by high‐performance liquid chromatography. Characterizations of the hybrid coating with environmental scanning microscopy and X‐ray diffraction indicated that protein adsorption does not alter the microstructure of the coating. The presence of OC in the hybrid coating was visualized with fluorescence microscopy using an immunolabeling procedure. The affinity of OC to the BAp coating was examined using a 20‐h elution test in phosphate‐buffered saline and only a minimal amount (<10%) of the loaded OC was eluted out. When the coating was fully dissolved in hydrochloric acid solution after elution, about 78% of the loaded OC could be recovered. Enzyme‐linked immunosorbent assay and peptide sodium dodecylsulfate–polyacrylamide gel electrophoresis confirmed the integrity and activity of OC molecules throughout the tests. The preliminary cell culture tests showed a significant effect of OC on the attachment and proliferation of osteoblasts. The quick loading profile and high affinity of OC to the BAp coating make it an ideal candidate for the hybrid coating preparation in clinical environment. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2005
In vitro and in vivo evaluation of Adacolumn ® cytapheresis in healthy subjects
Authors: Ramlow Wolfgang , Emmrich Jorg , Ahrenholz Peter , Sparmann Gisela , Kashiwagi Nobuhito , Franz Marita , Yokoyama Takashi , Yoshikawa Toshikazu
Published: May 2005

Adacolumn® is a medical device for adsorptive cytapheresis. It has been developed for selective adsorption of granulocytes and monocytes from peripheral blood of patients with immune disorders, such as autoimmune diseases and chronic inflammatory diseases. A double blind sham‐controlled crossover study design was used in order to evaluate in vivo biological responses of leukocytes as well as biocompatibility during and after Adacolumn® cytapheresis in healthy volunteers. In addition, experiments were undertaken to further evaluate leukocyte reactions to Adacolumn® carrier (G‐1: cellulose diacetate) beads in vitro. Six healthy volunteers, 4 males and 2 females, with a mean age of 26.7 years were randomly assigned to one of the two treatment arms in a crossover fashion. Three subjects received a single Adacolumn® treatment, followed by a single sham treatment at an interval of 7 days. The other three subjects received the two treatments in reverse order. All subjects were followed up 7 days after the last treatment. Additionally, in vitro investigations were carried out using blood from the healthy donors to examine the effect of G‐1 beads on granulocyte functions. In vitro exposure of human peripheral blood to G‐1 beads caused downregulation of L‐selectin expression and upregulation of Mac‐1 expression on granulocytes, leading to a marked reduction of adhesive capacity of granulocytes to endothelial cells. The exposure also led to decreased granulocyte chemotactic activity to IL‐8. The number of granulocytes and monocytes clearly decreased during Adacolumn® cytapheresis. Granulocytes showed marked phenotypic changes of L‐selectinLow and Mac‐1Hi after passing through Adacolumn® in vivo. Expression of TNF‐α and chemokine receptors was downregulated. In addition, TNF‐α and IL‐1β producing capacity of peripheral blood leukocytes was decreased after Adacolumn® cytapheresis and these changes lasted even one week after the cytapheresis. The level of complement fragments, C3a and C5a, increased, while bradykinin concentration did not change during Adacolumn® cytapheresis. Exposure of human peripheral blood to G‐1 beads, both in vitro and in vivo, caused a significant reduction of adhesive capacity and proinflammatory cytokine producing capacity of peripheral blood leukocytes. Such changes were not observed after sham apheresis. Despite complement activation, tolerability of Adacolum® cytapheresis was not influenced. These findings may at least partly explain the beneficial effect of Adacolumn® cytapheresis in the treatment of autoimmune diseases. J. Clin. Apheresis © 2005 Wiley‐Liss, Inc.
Quality systems in automated plateletpheresis in hospital‐based blood transfusion service in north India
Authors: Chaudhary Rajendra , Sekhar Das Sudipta , Agarwal Prashant , Shanker Shukla Jai
Published: May 2005

The issues of providing quality blood products and maintaining donor safety are primary aims of blood transfusion services. A comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (SOPs). The Drugs and Cosmetics Act of India, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. We, therefore, established our own SOP and operational flow chart for plateletpheresis that can be easily followed by other centers in India. A total of 100 plateletpheresis procedures performed using two cell separators (CS3000 Baxter Healthcare, Round Lake, IL; MCS3p, Haemonetics Corporation, Braintree, MA) were evaluated following our established SOP. The mean platelet yield in CS3000 was 2.9 ± 0.84 × 1011 and in MCS3p it was 2.88 ± 0.75 × 1011per unit. However, only 4–7% of SDPs showed WBC levels <5 × 106 due to lack of appropriate methods to quantitate residual WBC counts. Six of 100 donors complained of hypocalcemic symptoms. The operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. J. Clin. Apheresis © 2005 Wiley‐Liss, Inc.
HER‐2: A biomarker at the crossroads of breast cancer immunotherapy and molecular medicine
Authors: Pupa Serenella M. , Tagliabue Elda , Ménard Sylvie , Anichini Andrea
Published: May 2005

The oncoprotein encoded by the HER‐2 oncogene is a member of the HER family of receptor tyrosine kinases and is actually the first successfully exploited target molecule in new biomolecular therapies of solid tumors. The association of HER‐2 overexpression with human tumors, its extracellular accessibility, as well as its involvement in tumor aggressiveness are all factors that make this receptor an appropriate target for tumor‐specific therapy. In addition, HER‐2 overexpression fosters its immunogenicity, as shown by the frequency of B and T cell‐mediated responses against this oncoprotein in cancer patients, and it is being investigated as a promising molecule for either passive and active immunotherapy strategies. This review summarizes a number of immune intervention approaches that target HER‐2 in breast cancer. © 2005 Wiley‐Liss, Inc.
Arachidonic acid mediates non‐capacitative calcium entry evoked by CB1‐cannabinoid receptor activation in DDT1 MF‐2 smooth muscle cells
Authors: Demuth Dirk G. , Gkoumassi Effimia , Dröge Melloney J. , Dekkers Bart G.J. , Esselink Henk J. , van Ree Rutger M. , Parsons Mike E. , Zaagsma Johan , Molleman Areles , Nelemans S. Ad
Published: May 2005

Cannabinoid CB1‐receptor stimulation in DDT1 MF‐2 smooth muscle cells induces a rise in [Ca2+]i, which is dependent on extracellular Ca2+ and modulated by thapsigargin‐sensitive stores, suggesting capacitative Ca2+ entry (CCE), and by MAP kinase. Non‐capacitative Ca2+ entry (NCCE) stimulated by arachidonic acid (AA) partly mediates histamine H1‐receptor‐evoked increases in [Ca2+]i in DDT1 MF‐2 cells. In the current study, both Ca2+ entry mechanisms and a possible link between MAP kinase activation and increasing [Ca2+]i were investigated. In the whole‐cell patch clamp configuration, the CB‐receptor agonist CP 55, 940 evoked a transient, Ca2+‐dependent K+ current, which was not blocked by the inhibitors of CCE, 2‐APB, and SKF 96365. AA, but not its metabolites, evoked a transient outward current and inhibited the response to CP 55,940 in a concentration‐dependent manner. CP 55,940 induced a concentration‐dependent release of AA, which was inhibited by the CB1 antagonist SR 141716. The non‐selective Ca2+ channel blockers La3+ and Gd3+ inhibited the CP 55,940‐induced current at concentrations that had no effect on thapsigargin‐evoked CCE. La3+ also inhibited the AA‐induced current. CP 55,940‐induced AA release was abolished by Gd3+ and by phospholipase A2 inhibition using quinacrine; this compound also inhibited the outward current. The CP 55,940‐induced AA release was strongly reduced by the MAP kinase inhibitor PD 98059. The data suggest that in DDT1 MF‐2 cells, AA is an integral component of the CB1 receptor signaling pathway, upstream of NCCE and, via PLA2, downstream of MAP kinase. © 2005 Wiley‐Liss, Inc.
Nutrient removal performance of an anaerobic–anoxic–aerobic process as a function of influent C/P ratio
Authors: Ma Yong , Peng Yong‐zhen , Wang Xiao‐lian , Wang Shu‐ying
Published: May 2005

The laboratory scale anaerobic–anoxic–aerobic (A2O) process fed with synthetic brewage wastewater was designed to investigate the effects of changing feed C/P ratio on the performance of biological nutrient removal (BNR) processes. In the experiment, the influent chemical oxygen demand (COD) concentration was kept at approximately 300 mg L−1 while the total phosphorus concentration was varied to obtain the desired C/P ratio. Results showed that when the C/P ratio was lower than 32, phosphorus removal efficiency increased as C/P ratio increased linearly, while when the C/P ratio was higher than 32, the P removal efficiency was maintained at 90–98%, and effluent P concentration was lower than 0.5 mg L−1. However, regardless of the C/P ratio, excellent COD removal (90% or higher) and good total nitrogen removal (75–84%) were maintained throughout the experiments. It was also found that very good linear correlation was obtained between COD uptake per unit P released in the anaerobic zone and C/P ratio. In addition, the P content in the wasted activated sludge increased with the decrease in the C/P ratio. Based on the results, it was recommended that the wastewater C/P ratio and its effects be incorporated into BNR design and operational procedures, appropriate C/P ratios were used to achieve the effluent treatment goals. Copyright © 2005 Society of Chemical Industry
2,4,6‐Trichlorophenol and phenol removal in methanogenic and partially‐aerated methanogenic conditions in a fluidized bed bioreactor
Authors: Garibay‐Orijel Claudio , Ríos‐Leal Elvira , García‐Mena Jaime , Poggi‐Varaldo Héctor Mario
Published: May 2005

A fluidized bed bioreactor (FBBR) was operated for more than 575 days to remove 2,4,6‐trichlorophenol (TCP) and phenol (Phe) from a synthetic toxic wastewater containing 80 mg L−1 of TCP and 20 mg L−1 of Phe under two regimes: Methanogenic (M) and Partially‐Aerated Methanogenic (PAM). The mesophilic, laboratory‐scale FBBR consisted of a glass column (3 L capacity) loaded with 1 L of 1 mm diameter granular activated carbon colonized by an anaerobic consortium. Sucrose (1 g COD L−1) was used as co‐substrate in the two conditions. The hydraulic residence time was kept constant at 1 day. Both conditions showed similar TCP and Phe removal (99.9 + %); nevertheless, in the Methanogenic regime, the accumulation of 4‐chlorophenol (4CP) up to 16 mg L−1 and phenol up to 4 mg L−1 was observed, whereas in PAM conditions 4CP and other intermediates were not detected. The specific methanogenic activity of biomass decreased from 1.01 ± 0.14 in M conditions to 0.19 ± 0.06 mmolCH4 h−1 gTKN−1 in PAM conditions whereas the specific oxygen uptake rate increased from 0.039 ± 0.008 in M conditions to 0.054 ± 0.012 mmolO2 h−1 gTKN−1, which suggested the co‐existence of both methanogenic archaea and aerobic bacteria in the undefined consortium. The advantage of the PAM condition over the M regime is that it provides for the thorough removal of less‐substituted chlorophenols produced by the reductive dehalogenation of TCP rather than the removal of the parent compound itself. Copyright © 2005 Society of Chemical Industry
Tritiation of the cannabinoid receptor antagonist SR144528 involving lithium aluminum tritide reduction; assessment of the kinetic isotope effect by 3H‐NMR
Authors: Seltzman Herbert H. , Foster Matthew C. , Wyrick Christopher D. , Burgess Jason P. , Ivy Carroll F.
Published: May 2005

The cannabinoid receptor antagonist SR144528 was synthesized by an approach that enabled the incorporation of high specific activity tritium label while circumventing the lability of the target compound to catalytic hydrogenation. Lithium aluminum tritide of less than maximum specific activity was employed to introduce tritium, resulting in an H/T incorporation indicative of no kinetic isotope effect for the hydride/tritide reduction of a methyl benzoate. Copyright © 2005 John Wiley & Sons, Ltd.
Measurement of pulmonary surfactant disaturated‐phosphatidylcholine synthesis in human infants using deuterium incorporation from body water
Authors: Cogo Paola E. , Gucciardi Antonina , Traldi Umberto , Hilkert Andreas W. , Verlato Giovanna , Carnielli Virgilio
Published: May 2005

The aim of the study was to determine surfactant palmitate disaturated‐phosphatidylcholine (DSPC‐PA) synthesis in vivo in humans by the incorporation of deuterium from total body water into DSPC‐PA under steady state condition. We studied three newborns and one infant (body weight (BW) 4.6 ± 2.9 kg, gestational age 37.5 ± 2 weeks, age 9 ± 9 days) and four preterm newborns (BW 1.3 ± 0.6 kg, gestational age 30.3 ± 2.5 weeks, postnatal age 8.8 ± 9.2 h). All infants were mechanically ventilated during the study and the four preterm infants received exogenous surfactant at the start of the study.

We administered 0.44 g 2H2O/kg BW as a bolus intravenously, followed by 0.0125 g 2H2O/kg BW every 6 h to maintain deuterium enrichment at plateau over 2 days. Urine samples and tracheal aspirates (TA) were obtained prior to dosing and every 6 h thereafter. Isotopic enrichment curves of DSPC‐PA from sequential TA and urine deuterium enrichments were analyzed by Gas Chromatography‐Isotope Ratio—Mass Spectrometry (GC‐IRMS) and normalized for Vienna Standard Mean Ocean Water. Enrichment data were used to measure DSPC‐PA fractional synthesis rate (FSR) from the linear portion of the DSPC‐PA enrichment rise over time, relative to plateau enrichment of urine deuterium. Secretion time (ST) was defined as the time lag between the start of the study and the appearance of DSPC‐PA deuterium enrichment in TA. Data were given as mean ± SD.

All study infants reached deuterium‐steady state in urine. DSPC‐PA FSR was 6.5 ± 2.8%/day (range 2.6–10.2). FSR for infants who did not receive exogenous surfactant was 5.7 ± 3.5%/day (range 2.6–9.9%/day) and 7.3 ± 2.1%/day (range 5.1–10.2%/day) in the preterms, whereas DSPC‐PA ST was 10 ± 10 h and 31 ± 10 h respectively.

Surfactant DSPC‐PA synthesis can be measured in humans by the incorporation of deuterium from body water. This study is a simpler and less invasive method compared to previously published methods on surfactant kinetics by means of stable isotopes. Copyright © 2005 John Wiley & Sons, Ltd.
Structure elucidation of aplidine metabolites formed in vitro by human liver microsomes using triple quadrupole mass spectrometry
Authors: Brandon Esther F. A. , van Ooijen Ronald D. , Sparidans Rolf W. , Lázaro Luis López , Heck Albert J. R. , Beijnen Jos H. , Schellens Jan H. M.
Published: May 2005

The cyclic depsipeptide aplidine is a new anti‐cancer drug of marine origin. Four metabolites of this compound were found after incubation with pooled human microsomes using gradient high‐performance liquid chromatography with ultraviolet detection. After chromatographic isolation, the metabolites have been identified using nano‐electrospray triple quadrupole mass spectrometry. A highly specific sodium‐ion interaction with the cyclic structure opens the depsipeptide ring, and cleavage of the amino acid residues gives sequence information when activated by collision‐induced dissociation in the second quadrupole. The aplidine molecule could undergo the following metabolic reactions: hydroxylation at the isopropyl group (metabolites apli‐h 1 and apli‐h 2); C‐dealkylation at the N(Me)‐leucine group (metabolite apli‐da); hydroxylation at the isopropyl group and C‐dealkylation at the N(Me)‐leucine group (metabolite apli‐da/h), and C‐demethylation at the threonine group (metabolite apli‐dm). The identification of these metabolites formed in vitro may greatly aid the elucidation of the metabolic pathways of aplidine in humans. Copyright © 2005 John Wiley & Sons, Ltd.
Measurement of bile acid CoA esters by high‐performance liquid chromatography–electrospray ionisation tandem mass spectrometry (HPLC–ESI‐MS/MS)
Authors: Gan‐Schreier H. , Okun J. G. , Kohlmueller D. , Langhans C.‐D. , Peters V. , ten Brink H. J. , Verhoeven N. M. , Jakobs C. , Voelkl A. , Hoffmann G. F.
Published: May 2005

The novel and rapid assay presented here combines high‐performance liquid chromatography and electrospray ionisation tandem mass spectrometry (HPLC‐ESI‐MS/MS) to directly measure and quantify the CoA esters of 3α,7α,12α‐trihydroxy‐ and 3α,7α‐dihydroxy‐5β‐cholestan‐26‐oic acid (THCA and DHCA). The latter are converted inside peroxisomes to the primary bile acids, cholic and chenodeoxycholic acids, respectively. Prior to MS/MS, esters were separated by reversed‐phase HPLC on a C18 column using an isocratic mobile phase (acetonitrile/water/2‐propanol) and subsequently detected by multiple reaction monitoring. For quantification, the CoA ester of deuterium‐labelled 3α,7α,12α‐trihydroxy‐5β‐cholan‐24‐oic acid (d4‐CA) was used as internal standard. To complete an assay took less than 8 min.

To verify the validity of the assay, the effect of peroxisomal proteins on the efficacy of extraction of the CoA esters was tested. To this end, variable amounts of the CoA esters were spiked with a fixed amount of either intact peroxisomes or peroxisomal matrix proteins and then extracted using a solid‐phase extraction system. The CoA esters could be reproducibly recovered in the range of 0.1–4 µmol l−1 (linear correlation coefficient R2 > 0.99), with a detection limit of 0.1 µmol l−1.

In summary, electrospray ionization tandem mass spectrometry combined with HPLC as described here proved to be a rapid and versatile technique for the determination of bile acid CoA esters in a mixture with peroxisomal proteins. This suggests this technique to become a valuable tool in studies dealing with the multi‐step biosynthesis of bile acids and its disturbances in disorders like the Zellweger syndrome. Copyright © 2005 John Wiley & Sons, Ltd.
A new adaptive multi‐biasS‐parameter measurement algorithm for transistor characterization
Authors: van Niekerk Cornell , Schreurs Dominique
Published: May 2005

A new adaptive measurement algorithm is described for the control of an automatedS‐parameter measurement set‐up used to characterize transistors for non‐linear modelling. The procedure differs from previous algorithms in that is uses both the device DC‐ andS‐parameter data to identify DC bias regions where the device characteristics are changing rapidly. By placing more bias points in these areas and less data points in regions where the device response stays constant, the non‐linear behaviour of the device can be characterized more accurately while keeping the total volume of the experimental data and hence the measurement time to an acceptable level. Experimental results are presented that illustrates the operation of the adaptive algorithm as well as the influence that the selection procedure has on non‐linear modelling results. Copyright © 2005 John Wiley & Sons, Ltd.
Conformational Changes of the Amyloid β‐Peptide (1–40) Adsorbed on Solid Surfaces
Authors: Giacomelli Carla E. , Norde Willem
Published: May 2005

Summary: The conformational change of the 39–43 residues of the amyloid β‐peptide (Aβ) toward a β‐sheet enriched state promotes self‐aggregation of the peptide molecules and constitutes the major peptide component of the amyloid plaques in Alzheimer patients. The crucial question behind the self‐aggregation of Aβ is related to the different pathways the peptide may take after cleavage from the amyloid precursor proteins at cellular membranes. This work is aiming at determining the conformation of the Aβ (1–40) adsorbed on hydrophobic Teflon and hydrophilic silica particles, as model sorbent surfaces mimicking the apolar transmembrane environment and the polar, charged membrane surface, respectively. The mechanism by which the Aβ interacts with solid surfaces strongly depends on the hydrophobic/hydrophilic character of the particles. Hydrophobic and electrostatic interactions contribute differently in each case, causing a completely different conformational change of the adsorbed molecules on the two surfaces. When hydrophobic interactions between the peptide and the sorbent prevail, the adsorbed Aβ (1–40) mainly adopts an α‐helix conformation due to H‐bonding in the apolar part of the peptide that is oriented towards the surface. On the other hand, when the peptide adsorbs by electrostatic interactions β‐sheet formation is promoted due to intermolecular association between the apolar parts of the adsorbed peptide. Irrespective of the characteristics of the solid sorbent, crowding the surface results in intermolecular association between adsorbed molecules leading to a strong aggregation tendency of the Aβ (1–40).

CD spectra of Aβ (1–40) at pH 7: A) in solution ([Aβ] = 0.2 mg · ml−1) freshly prepared (line) and after overnight incubation (symbols); B) on Teflon (Γ = 0.5 mg · m−2).

magnified image

CD spectra of Aβ (1–40) at pH 7: A) in solution ([Aβ] = 0.2 mg · ml−1) freshly prepared (line) and after overnight incubation (symbols); B) on Teflon (Γ = 0.5 mg · m−2).
Recent advances in mycotoxin determination in food and feed by hyphenated chromatographic techniques/mass spectrometry
Authors: Sforza Stefano , Dall'Asta Chiara , Marchelli Rosangela
Published: May 2005

Mycotoxins are fungal toxins produced by molds, which occur universally in food and feed derivatives, and are produced under certain environmental conditions in the field before harvest, post‐harvest, during storage, processing, and feeding. Mycotoxin contamination is one of the most relevant and worrisome problem concerning food and feed safety because it can cause a variety of toxic acute and chronic effects in human and animals. In this review we report the use of mass spectrometry in connection with chromatographic techniques for mycotoxin determination by considering separately the most diffuse class of mycotoxins: patulin, aflatoxins, ochratoxin A, zearalenone, trichothecenes, and fumonisins. Although the selectivity of mass spectrometry is unchallenged if compared to common GC and LC detection methods, accuracy, precision, and sensitivity may be extremely variable concerning the different mycotoxins, matrices, and instruments. The sensitivity issue may be a real problem in the case of LC/MS, where the response can be very different for the different ionization techniques (ESI, APCI, APPI). Therefore, when other detection methods (such as fluorescence or UV absorbance) can be used for the quantitative determination, LC/MS appears to be only an outstanding confirmatory technique. In contrast, when the toxins are not volatile and do not bear suitable chromophores or fluorophores, LC/MS appears to be the unique method to perform quantitative and qualitative analyses without requiring any derivatization procedure. The problem of exact quantitative determination in GC/MS and LC/MS methods is particularly important for mycotoxin determination in food, given the high variability of the matrices, and can be solved only by the use of isotopically labeled internal standards or by the use of ionization interfaces able to lower matrix effects and ion suppressions. When the problems linked to inconstant ionization and matrix effects will be solved, only MS detectors will allow to simplify more and more the sample preparation procedures and to avoid clean‐up procedures, making feasible low‐cost, high‐throughput determination of mycotoxins in many different food matrices. © 2005 Wiley Periodicals, Inc.
Theoretical and experimental NMR study of protopine hydrochloride isomers
Authors: Toušek Jaromír , Maliňáková Kateřina , Dostál Jiří , Marek Radek
Published: May 2005

The 1H and 13C NMR chemical shifts of cis‐ and trans‐protopinium salts were measured and calculated. The calculations of the chemical shifts consisted of conformational analysis, geometry optimization (RHF/6–31G** method) and shielding constants calculations (B3LYP/6–31G** method). Based on the results of the quantum chemical calculations, two sets of experimental chemical shifts were assigned to the particular isomers. According to the experimental results, the trans‐isomer is more stable and its population is ∼68%. Copyright © 2005 John Wiley & Sons, Ltd.
Isolation and characterization of the major proteins of ram seminal plasma
Authors: Bergeron Annick , Villemure Michèle , Lazure Claude , Manjunath Puttaswamy
Published: May 2005

Mammalian seminal plasma contains among others, two major families of proteins, namely spermadhesins and those proteins that contain fibronectin type II domains. Spermadhesins are the major proteins of boar and stallion seminal plasma and homologous proteins have been identified in the bull. These proteins appear to be involved in capacitation and sperm–egg interaction. In bovine seminal plasma, proteins containing fibronectin type II domains are the major proteins and are designated BSP proteins. These proteins play a role in sperm capacitation. In this study, we present the isolation and characterization of the major proteins of ram seminal plasma. Precipitated proteins from Suffolk ram seminal plasma were loaded onto a gelatin–Agarose column. The unadsorbed (fraction A) and retarded proteins (fraction B) were removed by washing the column with phosphate buffered‐saline and the adsorbed proteins (fraction C) were eluted with 5M urea. SDS–PAGE of fraction B indicated the presence of a 15.5 kDa protein, which is the major protein of ram seminal plasma (∼45% of total protein by weight) and was identified as a spermadhesin by N‐terminal sequencing. SDS–PAGE analysis of fraction C revealed the presence of four proteins, which represented ∼20% of total ram seminal plasma proteins by weight, and were identified as proteins of the BSP family and named RSP proteins. These RSP proteins were designated RSP‐15 kDa, RSP‐16 kDa, RSP‐22 kDa, and RSP‐24 kDa. Only RSP‐15 kDa and ‐16 kDa proteins cross‐reacted with antibodies against BSP proteins. Ram spermadhesin and RSP proteins interact with heparin but only RSP proteins bind to hen's egg yolk low‐density lipoprotein. In conclusion, spermadhesin is the major protein of ram seminal plasma and other major proteins belong to the BSP protein family. Mol. Reprod. Dev. © 2005 Wiley‐Liss, Inc.