bluuebunny/crossref_metadata_2025 huggingface dataset:
Different in vitro response to rIL‐1β of newborn and adult rat astroglia
Authors: Colasanti M. , Ramacci Maria T. , Foresta Piero , Lauro Giuliana M.
Published: April 2003

In recent literature, lymphokines have been reported to be able to promote both proliferation and maturation of some glial populations. In this paper, we compare the effect of rIL‐1 on newborn and adult rat astroglial cells in vitro. In newborn, but not in adult astrocytes, 100 U/ml of rIL‐1β increase [3H]thymidine incorporation with a maximal response by 3 days as compared to the control untreated culture. In contrast, rIL‐1β induces an increase of GFAP immunoreactivity both in newborn and in adult astrocytes, as compared to the control untreated cells. These data indicate that, while both newborn and adult astroglial cells are capable of responding to rIL‐1β, only newborn astrocytes can respond to this lymphokine with proliferation. Thus, it appears likely that different factors, other than rIL‐1β, are needed by adult astrocytes to proliferate.
Effect of a simple visual pattern on the early postnatal development oF GABA Receptor sites in the chick optic lobe
Authors: de Plazas Sara Fiszer , Conterjnic Daniela , Flores Vladimir
Published: April 2003

It is known that manipulation of the visual environment results in changes in the developmental pattern of several neurotransmitter receptors and that the GABA receptor shows a high degree of plasticity in differential illumination experiments. In the present paper we investigated whether exposure to a visual pattern has a developmental effect on GABA receptor expression during early postnatal life. Two groups of newly hatched chicks were used: one was exposed to a simple and specific visual pattern and the other was deprived of any visual pattern. GABA receptors at each developmental stage were determined by binding experiments performed in a crude membrane fraction. Saturation studies were carried out in a fraction enriched in synaptic membranes. The developmental pattern of both high and low affinity GABA binding sites was affected by the visual pattern. This effect displays its maximal expression by the end of the first postnatal week. The modification in receptor expression was due to a change in the receptor density while the affinity was not affected.

The change in receptor density induced by the presence of a visual pattern was highest at the end of the first postnatal week suggesting that at that time there is a brief period of higher plasticity for GABA receptor expression in the visual system than at other times. Our results also suggest that variations in GABA receptor density could be instrumental in adaptative changes in the visual system in response to variations in the environmental stimulation.
Developmental changes in B‐50 (GAP‐43) in primary cultures of cerebral cortex: Content and phosphorylation of B‐50
Authors: Ramakers G.J.A. , De Graan P.N.E. , Oestreicher A.B. , Boer G.J. , Corner M.A. , Gispen W.H.
Published: April 2003

The content and phosphorylation of the neuronal growth‐associated protein B‐50 (GAP‐43) were studied in cultured neocortex as a function of normal development and development in the presence of tetrodotoxin (TTX), a blocker of bioelectric activity (BEA). The observations were correlated with previous morphological findings on neurite outgrowth and B‐50 immunolocalization in the same cultures.

In control cultures, the concentration of B‐50 reached a maximum at 7 days in vitro (DIV) and decreased thereafter, whereas the concentration of neuron specific enolase (NSE), which was used as a neuronal reference marker, rose till 28 DIV and leveled off towards 42 DIV. The degree of basal phosphorylation of B‐50 (relative to that of total protein) decreased after the first week in vitro. Stimulation of B‐50 phosphorylation by phorbol ester also decreased with age in vitro, indicating that changes in B‐50 phosphorylation were mainly due to changes in protein kinase C (PKC) activity.

The chronic presence of TTX led to a reduced content of B‐50 and NSE after 14 DIV. The basal phosphorylation of B‐50 was neither affected by acute nor chronic TTX treatment. However, upon stimulation of PKC with phorbol esters, some alterations of B‐50 phosphorylation were revealed in cultures grown in TTX. These biochemical observations are in line with the absence of effects of TTX on neurite outgrowth during the first 2 weeks in culture, and later effects of TTX on neuronal survival.

The developmental changes in B‐50 concentration and phosphorylation largely correlate with previous morphological observations on axonal outgrowth and growth cone shape in the same cultures. We suggest that B‐50 phosphorylation plays an important role in transducing extracellular signals into directed neurite outgrowth.
Lack of retrograde axonal transport of the heparin‐binding growth factors by chick ciliary neurones
Authors: Hendry I.A. , Belford D.A.
Published: April 2003

In view of the likelihood that the heparin‐binding growth factors of fibroblast growth factors are neurotrophic for the neurones of the chick ciliary ganglion a study has been made of the retrograde axonal transport of the acidic and basic fibroblast growth factors by these neurones. No high capacity retrograde axonal transport of these molecules was seen. The amount of transport was equivalent to the low level seen with many proteins such as horse radish peroxidase or bovine serum albumin rather than the convincing transport seen for nerve growth factor in the sympathetic system. The iodinated proteins retained their ability to bind to neuronal receptors. Thus, if the fibroblast growth factors are neurotrophic in the ciliary ganglion, they may exert their action by mechanisms other than the retrograde axonal transport of the factor itself.
Action of the diabetogenic drug streptozotocin on glycolytic and glycogenolytic metabolism in adult rat brain cortex and hippocampus
Authors: Plaschke Konstanze , Hoyer Siegfried
Published: April 2003

In sporadic Alzheimer's disease (AD), a number of metabolic alterations to the brain have been observed soon after the onset of the initial clinical symptoms. In particular, impairments of glucose utilization and related metabolic pathways are prominent and well‐established findings in incipient AD, resembling metabolic abnormalities such as have been found in noninsulin‐dependent diabetes mellitus. To mimic these abnormalities, we administered an intracerebroventricular (icv) injection of streptozotocin (STZ) to rats and studied the effects on glucose and glycogen metabolism in the cerebral cortex and hippocampus compared with controls. The enzymatic activities studied dropped significantly by 10–30% in brain cortex (cort.) and hippocampus (hc) 3 and 6 weeks after icv STZ injection: hexokinase (15% 3 weeks cort.; 14% 6 weeks cort.; 12% 3 weeks hc; 28% 6 weeks hc), phosphofructokinase (15%; 15%; 24%; 15%), glyceraldehyde‐3‐phosphate dehydrogenase (10%; 12%; 30%; 19%), pyruvate kinase (22%; 13%; 22%; 28%), glucose‐6‐phosphatase (10%; 23%; 14%; 19%) and phosphorylase a (22%; 11%; 30%; 15%).

The content of glycogen was significantly higher in STZ‐treated rats than in control animals (7% 3 weeks and 15% 6 weeks in cortex).

In contrast to the reduced enzymatic activities, we observed no changes in the concentrations of the glycolytic intermediates glucose, glucose‐6‐phosphate, fructose‐6‐phosphate, fructose‐1,6‐diphosphate, pyruvate, lactate and glucose‐1‐phosphate. These data clearly indicate reduced glycolytic enzyme activity after icv administration of STZ and suggest gluconeogenesis consequent on abnormalities in glucose breakdown. This model may thus be assumed to be a useful tool to investigate pathogenetic factors involved in sporadic dementia of Alzheimer type.
Ontogenesis of type II spiral ganglion neurons during development: Peripherin immunohistochemistry
Authors: Hafidi A. , Després G. , Romand R.
Published: April 2003

In this study, we analysed the distribution of the intermediate filament peripherin in the developing cochlea of the rat. At gestational day 16, weak immunolabeling was observed in neuronal somas throughout the spiral ganglion. At gestational day 20, the peripherin labeling increased in intensity throughout the cochlea but became especially strong in some ganglion neurons of the basal turn. Homogeneous immunolabeling was observed throughout the spiral ganglion of the apical turn. Double immunofluorescence labeling of the prenatal cochlea with peripherin and neurofilament (NF) antibodies revealed colocalization on the same structures. By postnatal day 3, the peripherin labeling intensity had decreased in the majority of spiral ganglion neurons, but remained strong in some cells of the basal turn. Only a few neurons continued to be immunolabeled into adulthood that correspond to Type II spiral ganglion neurons expressing both NF protein and peripherin, two classes of intermediate filament proteins. In the organ of Corti, the first immunolabeling was observed on gestational day 20 as peripheral fibers reaching the receptor cells. Positive fibers were observed below both inner (IHCs) and outer (OHCs) hair cells. At birth and at postnatal day 3, peripherin immunolabeling was still observed below both IHCs and OHCs. By postnatal day 4, peripherin labeling became more dominant in fibers below OHCs, but some immunoreactivity was still present below IHCs. No immunoreactivity was present in the intraganglionic spiral bundle (IGSB) fibers containing the olivary complex efferent fibers before birth. A few days after birth some fibers of the IGSB started to be immunoreactive.
Laminin concentrations in serum and cerebrospinal fluid in aging and alzheimer's disease
Authors: Kittur S.D. , Adler W.H. , Martin G.R. , Schapiro M.B. , Rapoport S.I. , Gunzler V.
Published: April 2003

Laminin, a basement membrane protein, and a potent promoter of neurite outgrowth, surrounds all peripheral nerves. It appears in the central nervous system during development and reappears in response to injury. In Alzheimer's disease (AD), there is a progressive loss of neurons in specific areas of the brain along with the presence of an increased number of senile plaques and neurofibrillary tangles. Laminin levels have been shown to be increased in injury, so we undertook to examine levels of laminin by radioimmunoassay (RIA), in cerebrospinal fluid and serum of patients with AD and age‐matched controls. No difference in the CSF and serum laminin concentrations was found between Alzheimer's disease and age‐matched controls. We found a lack of correlation between severity of clinical dementia and laminin concentrations. Finally, we show that the CSF and serum laminin concentrations increase with age.
The migration and intermixing of donor and host glia on nitrocellulose polymers implanted into cortical lesion cavities in adult mice and rats
Authors: Harvey A.R. , Fan Y. , Connor A.M. , Grounds M.D. , Beilharz M.W.
Published: April 2003

The fate of neonatal glia (mostly glial fibrillary acidic protein‐positive astrocytes), cultured on nitrocellulose papers and implanted into cortical lesion cavities, was examined in adult mice and rats. In mice, a Y‐chromosome‐specific probe and in situ hybridization techniques were used to identify male cells. Male‐female grafts allowed visualization of donor glia and their behaviour after transplantation; female‐male grafts allowed an analysis of how host cells responded to the presence of the implants. There was substantial intermixing of cells, with many donor glia migrating away from the implants and host cells migrating onto both sides of the nitrocellulose paper. In rats, donor glia were labelled with fluorescein‐conjugated latex microspheres prior to transplantation on nitrocellulose polymers. The rat data were broadly consistent with those obtained from the mouse: moreover, immunohistochemical studies in rats suggested that the majority of host cells migrating onto the previously cell‐coated papers were astrocytes. In a number of studies, glia‐coated polymers have been used in an attempt to promote the regrowth of axons across lesion sites in the brain and spinal cord. The present work suggests that both transplanted and host glia may influence the regenerative growth seen in such implants.
Grafting of fast blue labeled glial cells into neonatal rat brain: Differential survival and migration among cell types
Authors: de los Monteros A. Espinosa , Bernard R. , Tiller B. , Rouget P. , de Vellis J.
Published: April 2003

Cultures of oligodendrocyte progenitor cells, ERD 1.1 cells, a nontransformed immortalized cell line of oligodendrocyte progenitors and C6 glioma cells were labeled with the fluorescent dye Fast Blue and transplanted into brains of 4 day postnatal Wistar rat pups. The localization of fluorescent cells within host brain was examined at various times post‐transplantation to determine patterns of cell migration as well as survival and integration among the host tissue. Oligodendrocyte progenitors migrated mainly along white matter tracks, integrating successfully into the host parenchyma. High survival rates were found between 5 and 27 days post grafting. ERD 1.1 cells survived and migrated between 1 and 5 days after transplantation. However, by 27 days survival had dropped from 60 to 20% of the initial cell population. The surviving cells were mainly localized to subventricular and subependymal regions at 27 days. C6 cells migrated extensively rostrally and caudally from the site of injection in the hippocampus and were tumorogenic. This finding confirmed previous reports on the survival and migration patterns of oligodendrocyte progenitors grafted into neonatal brain. However, they show that two cell lines that share phenotypic properties of oligodendrocyte progenitors markedly differ from these cells with respect to migration patterns and integration within host parenchyma. Fast Blue dye was still detectable after repeated cell division in grafted C6 cells, enabling us to track single cells as well as tumor formation. This dye should be useful not only to address issues of development, but also of tumor biology and therapeutic treatment.
Sequestering of immunoglobulins by astrocytes after cortical lesion and homografting of fetal cortex
Authors: Bernstein Jerald J. , Willingham L.A. , Goldberg William J.
Published: April 2003

The immunoglobulin (IgG, IgM, IgA) content of normal, reactive and migrated rat astrocytes was studied in lesioned adult rat cortex and after fetal cortex grafts. Implantation pockets were aspirated in the somatomotor cortex (under bregma) of adult Sprague‐Dawley rats. A fetal (E14) rat hemicortex incubated in the plant lectin Phaseolus vulgaris leucoagglutinin (graft premarker) was placed in the aspiration pocket or the pocket left empty (control). Sections of brain were immunohistochemically double labeled for immunoglobulins‐GFAP or Phaseolus vulgaris leucoagglutinin‐GFAP 3–60 days postoperative. Mature or fetal astrocytes in situ did not contain immunoglobulins. In pocket‐only animals, individual reactive astrocytes lining and subjacent to the pocket were positive for rat IgG, IgM and IgA (3–60 days). In grafted animals, graft derived astrocytes (Phaseolus vulgaris leucoagglutinin‐GFAP positive) were intermingled with host reactive astrocytes (Phaseolus vulgaris leucoagglutinin negative) lining and subjacent to the pocket. Both classes of astrocytes contained immunoglobulins IgG, IgM and IgA. Immunoglobulin positive graft derived astrocytes migrated into the corpus callosum, cingulum and habenula. These results demonstrate that astrocytes sequester immunoglobulins and probably other serum proteins as a critical function in restoring homeostasis to the injured or diseased nervous system.
Immunohistochemical staining for glial fibrillary acidic protein (GFAP) after deafferentation or ischemic infarction in rat visual system: Features of reactive and damaged astrocytes
Authors: Schmidt‐Kastner R. , Wietasch K. , Weigel H. , Eysel U.T.
Published: April 2003

Immunohistochemical staining for glial fibrillary acidic protein (GFAP) is standard for visualization of reactive astrocytes in tissue sections, whereas various forms of astrocytic damage remain to be described in detail. In this study we tested differences in GFAP labeling in reactive astrocytes and in glial cells damaged by ischemia and edema. Studies were performed in the anatomically well defined visual system of rat. Basic staining patterns for GFAP were established in subcortical visual nuclei and visual cortex. In the first model, deafferentation of visual centers was performed by unilateral optic nerve lesion, and characteristic changes of GFAP labeling in reactive astrocytes were studied at 0.5,1,1.5,2,4,8 and 21 days after lesion. Initial changes were seen in the deafferented superior colliculus at 1 day after deafferentation with a diffuse increase and stellate types of reactive cells formed at 2–8 days. In the second model, small ischemie infarcts were produced in the visual cortex of rats using the method of photochemically‐induced thrombosis. GFAP labeling with a polyclonal antiserum was massively enhanced in the infarct at 4 hr. Characteristic morphological changes in damaged astrocytes were seen which were also identified in experiments with simulated global ischemia. In the surround of the infarct, swelling of astrocytes also caused increased labeling. At 3–4 days infarction typical reactive astrocytes surrounded the lesioned area. In conclusion, these immunohistochemical studies on GFAP in rat visual system allow for the following classifications, (a) Normal astrocytes vary in labeling at different anatomical localizations, (b) Reactive astrocytes show enhanced labeling and larger cell‐size within an interval of 1–2 days after lesion (c) Astrocytes damaged by ischemia reveal increased labeling of disintegrating cellular elements within hours after a lesion (d) Swollen astrocytes undergo enhanced labeling in areas with vasogenic edema.
Quantitative analysis of exocytosis visualized by a video‐enhanced light/fluorescence microscope reveals two distinct components of exocytosis in RBL‐2H3 cells
Authors: Ozawa Koichiro , Kobayashi Hideyuki , Kawai Eriko , Suzaki Etsuko , Nonomura Yoshiaki , Masujima Tsutomu
Published: April 2003

Rat basophilic leukemia (RBL‐2H3) cells, which exhibit Ca2+‐dependent secretion of granules when stimulated with antigen or the Ca2+‐ionophore A23187, were observed under a video‐enhanced light/fluorescence microscope. Exocytotic events of individual granules were visualized in individual cells stimulated with antigen or A23187. Exocytosis of granules stimulated with A23187 showed two peaks in the time course. The earlier one was inhibited by selective inhibitors of protein kinase C (Ro31–8425, Ro31–8220, and chelerythrine) and the other was inhibited by an inhibitor of phosphatidate hydrolase, propranolol. Exocytosis by antigen stimulation, however, showed only one peak, which was inhibited by the selective inhibitors of protein kinase C, but not by propranolol. These results indicate that at least two distinct components of exocytosis exist in RBL2H3 cells.
14‐3‐3 proteins associate with the regulatory phosphorylation site of spinach leaf nitrate reductase in an isoform‐specific manner and reduce dephosphorylation of Ser‐543 by endogenous protein phosphatases
Authors: Bachmann Markus , Huber Joan L. , Athwal Gurdeep S. , Wu Ke , Ferl Robert J. , Huber Steven C.
Published: April 2003

Three lines of evidence indicate that the 14‐3‐3 proteins that inactivate the phosphorylated form of spinach leaf NADH:nitrate reductase (NR) bind to the enzyme at the regulatory phosphorylation site (Ser‐543). First, a phosphorylated synthetic peptide based on the regulatory site can prevent and also reverse the inactivation of phospho‐NR caused by 14‐3‐3 proteins. Second, sequence‐specific and phosphorylation‐dependent binding of the aforementioned synthetic peptide to the 14‐3‐3 proteins was demonstrated in vitro. Third, 14‐3‐3 proteins were required for the ATP‐dependent phosphorylation of NR (as assessed by activity measurements) in the presence of NR‐kinase and leaf protein phosphatases. Lastly, we demonstrate specificity of recombinant Arabidopsis 14‐3‐3 isoforms in the interaction with phospho‐NR: ω > χ > ν ⋙ gf, ψ.
Protein phosphorylation during phagosome maturation
Authors: Emans Neil , Nzala Nsumu N , Desjardins Michel
Published: April 2003

Phagolysosome biogenesis is driven by a series of interactions between phagosomes and organelles of the biosynthetic and endocytic pathways. The presence of endocytic markers on phagosomes suggests that phagosomes and endosomes share common structural and functional characteristics. In that line of thought, protein phosphorylation has been shown to be involved in regulatory aspects of the fusion properties of endosomes and other vacuolar organelles. To study further the mechanisms involved in phagolysosome biogenesis, we have investigated the presence of phagosome proteins that can be phosphorylated in vitro by endogenous phagosome‐associated kinases. The results obtained show that proteins phosphorylated on tyrosine residues are present on phagosomes. Moreover, complex phosphorylation/dephosphorylation cycles appear to occur during phagolysosome biogenesis. The addition of endosome fractions to phagosomes inhibit the phosphorylation of phagosome proteins. These results suggest that phosphorylation and dephosphorylation events could play roles in the biogenesis of phagolysosomes and regulate, in part, the complex in vivo interactions between phagosomes and endosomes.
Cell death after cartilage impact occurs around matrix cracks
Authors: Lewis Jack L. , Deloria Laurel B. , Oyen‐Tiesma Michelle , Thompson Roby C. , Ericson Marna , Oegema Theodore R.
Published: April 2003

The damage from rapid high energy impacts to cartilage may contribute to the development of osteoarthritis (OA). Understanding how and when cells are damaged during and after the impact may provide insight into how these lesions progress. Mature bovine articular cartilage on the intact patella was impacted with a flat impacter to 53 MPa in 250 ms. Cell viability was determined by culturing the cartilage with nitroblue tetrazolium for 18 h or for 4 days in medium containing 5% serum before labeling (5‐day sample) and compared to adjacent, non‐impacted tissue as viable cells per area. There was a decrease in viable cell density only in specimens with macroscopic cracks and the loss was localized primarily near matrix cracks, which were in the upper 25% of the tissue. This was confirmed using confocal microscopy with a fluorescent live/dead assay, using 5′‐chloromethylfluorescein diacetate and propidium iodide. Cell viability in the impacted regions distant from visible cracks was no different than the non‐impacted control. At 5 days, viable cell density decreased in the surface layer in both the control and impacted tissue, but there was no additional impact‐related change. In summary, cell death after the impaction of cartilage on bone occurred around impact induced cracks, but not in impacted areas without cracks. If true in vivo, early stabilization of the damaged area may prevent late sequelae that lead to OA. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Protection from inactivation of the adenine nucleotide translocator during hypoglycaemia-induced apoptosis by mitochondrial phospholipid hydroperoxide glutathione peroxidase
Authors: IMAI Hirotaka , KOUMURA Tomoko , NAKAJIMA Ryo , NOMURA Kazuhiro , NAKAGAWA Yasuhito
Published: April 2003

We demonstrated that mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx) first suppressed the dissociation of cytochrome c (cyt c) from cardiolipin (CL) in mitochondrial inner membranes and then apoptosis caused by the hypoglycaemia by the prevention of peroxidation of CL [Nomura, Imai, Koumura, Arai and Nakagawa (1999) J. Biol. Chem. 274, 29294–29302; Nomura, Imai, Koumura, Kobayashi and Nakagawa (2000) Biochem. J. 351, 183–193]. The present study shows the involvement of peroxidation of CL in the inactivation of adenine nucleotide translocator (ANT) and the opening of permeability transition pores by using the system of ANT-reconstituted liposome and isolated mitochondria. ANT activity appeared in dioleoyl phosphatidylcholine proteoliposome containing 10% (mol/mol) CL or phosphatidylglycerol (PG), but not other classes of phospholipids. ANT activity was competitively inhibited by the addition of cardiolipin hydroperoxide (CLOOH) in reconstituted liposomes containing CL. However, phosphatidylcholine hydroperoxide failed to inactivate the activity of ANT. The activity of ANT in reconstituted liposomes, including CLOOH, recovered when CLOOH in reconstituted liposome was reduced to hydroxycardiolipin by incubation with PHGPx. The activity of ANT was determined in rat basophil leukaemia RBL2H3 cells after their exposure to 2-deoxyglucose. ANT activity decreased to 50% of the control level by 4 h in response to apoptosis. In parallel, cyt c and apoptosis-inducing factor (AIF) were released from mitochondria. Suppression of the accumulation of CLOOH by overexpression of PHGPx in mitochondria effectively prevented the inactivation of ANT, the opening of permeability transition pores and the release of cyt c and AIF from mitochondria in hypoglycaemia-induced apoptotic cells. These findings suggest that mitochondrial PHGPx might be involved in the modulation of the activity of ANT and the opening of pores for the release of cyt c via the modulation of levels of CLOOH in the mitochondria.
Sarco/endoplasmic-reticulum calcium ATPase SERCA1 is maintained in the endoplasmic reticulum by a retrieval signal located between residues 1 and 211
Authors: NEWTON Thomas , BLACK John P. J. , BUTLER John , LEE Anthony G. , CHAD John , EAST J. Malcolm
Published: April 2003

The location of sarco/endoplasmic-reticulum calcium ATPase (SERCA) retention/retrieval motifs in the sequence of the SERCA1 has been investigated by examining the subcellular location in COS-7 cells of enhanced-green-fluorescent-protein-tagged calcium-pump chimaeras. These chimaeras have been constructed from the fast-twitch SERCA1 and the plasma-membrane calcium ATPase PMCA3. The N-terminal, central and C-terminal segments of these calcium pumps were exchanged between SERCA1 and PMCA3. The segments exchanged correspond to residues 1–211, 212–711 and 712–994 of SERCA1, and residues 1–264, 265–788 and 789–1159 of PMCA3 respectively. Only chimaeras containing the N-terminal segment of SERCA1 were located in the endoplasmic reticulum (ER), whereas chimaeras containing the N-terminal segment from PMCA3 were able to escape from the ER and enter the endomembrane pathway en route for the plasma membrane. Co-localization of SERCA1 in COS-7 cells with the ER/Golgi-intermediate compartment marker ERGIC53 indicates that SERCA1 is maintained in the ER by a process of retrieval. These results indicate that the N-terminal region of SERCA1, containing transmembrane helices M1 and M2, contains an ER-retrieval signal.
Identification and characterization of a prostate-specific androgen-independent protein-binding site in the probasin promoter
Authors: YEUNG Lillian H. Y. , READ Jason T. , SORENSON Pernille , NELSON Colleen C. , GLEAVE Martin , JIA William , RENNIE Paul S.
Published: April 2003

In this study we investigated the combination of transcription factors and proteins binding to the proximal part of the prostate-specific probasin (PB) promoter. Using DNaseI in vitro footprinting, several protected regions were identified on the proximal PB promoter (nucleotides −286 to +28 relative to the transcription start site) when nuclear extracts from LNCaP, a human prostate cancer cell line, were used. Four of the protected areas were observed only when LNCaP nuclear extracts treated with synthetic androgen (10 nM R1881) were used. Two other regions, referred to as FPI and FPII, showed protection regardless of the presence or absence of androgen. When DNaseI footprinting was done using other prostate and non-prostate nuclear extracts, protection of the FPII region was only seen in prostate cell lines. These androgen-independent regions were further tested for tissue and binding specificity using the electrophoretic mobility-shift assay. Eight complexes formed with the FPI probe while four complexes were observed with the FPII probe on incubation with the tested nuclear extracts. Methylation protection assays reveal that prostate cancer cell lines yield slightly different protection patterns for some of the protein complexes formed with non-prostate-derived cell lines, suggesting the presence of prostate-enriched or -exclusive proteins. Site-directed mutagenesis of the protected nucleotides within FPII resulted in a significant reduction in expression from the PB promoter. Identification of proteins binding to the FPII region revealed the participation of nuclear factor I (NF-I) or a closely related protein, although other unknown proteins are also involved. Defining the DNA and protein components that dictate prostate-specific expression of the PB promoter in an androgen-independent manner would provide a strong basis for the design and development of a gene therapy for systemic treatment of androgen-independent prostate cancer.
Structural requirements of human DNase IIalpha for formation of the active enzyme: the role of the signal peptide, N-glycosylation, and disulphide bridging
Authors: MacLEA Kyle S. , KRIESER Ronald J. , EASTMAN Alan
Published: April 2003

DNase IIα (EC 3.1.22.1) is an endonuclease, which is active at low pH, that cleaves double-stranded DNA to short 3′-phosphoryl oligonucleotides. Although its biochemistry is well understood, its structure–activity relationship has been largely unexamined. Recently, we demonstrated that active DNase IIα consists of one contiguous polypeptide, heavily glycosylated, and containing at least one intrachain disulphide linkage [MacLea, Krieser and Eastman (2002) Biochem. Biophys. Res. Commun. 292, 415–421]. The present paper describes further work to examine the elements of DNase IIα protein required for activity. Truncated forms and site-specific mutants were expressed in DNase IIα-null mouse cells. Results indicate that the signal-peptide leader sequence is required for correct glycosylation and that N-glycosylation is important for formation of the active enzyme. Despite this, enzymic deglycosylation of wild-type protein with peptide N-glycosidase F reveals that glycosylation is not intrinsically required for DNase activity. DNase IIα contains six evolutionarily conserved cysteine residues, and mutations in any one of these cysteines completely ablated enzymic activity, consistent with the importance of disulphide bridging in maintaining correct protein structure. We also demonstrate that a mutant form of DNase IIα that lacks the purported active-site His295 can still bind DNA, indicating that this histidine residue is not simply involved in DNA binding, but may have a direct role in catalysis. These results provide a more complete model of the DNase IIα protein structure, which is important for three-dimensional structural analysis and for production of DNase IIα as a potential protein therapeutic for cystic fibrosis or other disorders.
Characterization of Galpha13-dependent plasma membrane recruitment of p115RhoGEF
Authors: BHATTACHARYYA Raja , WEDEGAERTNER Philip B.
Published: April 2003

The Ras homology (Rho) guanine nucleotide exchange factor (GEF), p115RhoGEF, provides a direct link between the G-protein α subunit, α13, and the small GTPase Rho. In the present study, we demonstrate that activated mutants of α13 or α12, but not αq, promote the redistribution of p115RhoGEF from the cytoplasm to the plasma membrane (PM). We also show that the PM translocation of p115RhoGEF is promoted by stimulation of thromboxane A2 receptors. Furthermore, we define domains of p115RhoGEF required for its regulated PM recruitment. The RhoGEF RGS (regulators of G-protein signalling) domain of p115RhoGEF is required for PM recruitment, but it is not sufficient for strong α13-promoted PM recruitment, even though it strongly interacts with activated α13. We also identify the pleckstrin homology domain as essential for α13-mediated PM recruitment. An amino acid substitution of lysine to proline at position 677 in the pleckstrin homology domain of p115RhoGEF inhibits Rho-mediated gene transcription, but this mutation does not affect α13-mediated PM translocation of p115RhoGEF. The results suggest a mechanism whereby multiple signals contribute to regulated PM localization of p115RhoGEF.
Expression profile of the transient receptor potential (TRP) family in neutrophil granulocytes: evidence for currents through long TRP channel 2 induced by ADP-ribose and NAD
Authors: HEINER Inka , EISFELD Jörg , HALASZOVICH Christian R. , WEHAGE Edith , JÜNGLING Eberhard , ZITT Christof , LÜCKHOFF Andreas
Published: April 2003

An early key event in the activation of neutrophil granulocytes is Ca2+ influx. Members of the transient receptor potential (TRP) channel family may be held responsible for this. The aim of the present study is to analyse the expression pattern of TRP mRNA and identify characteristic currents unambiguously attributable to particular TRP channels. mRNA was extracted from human neutrophils, isolated by gradient centrifugation and also by magnetically labelled CD15 antibodies. The presence of mRNA was demonstrated using reverse transcriptase–PCR in neutrophils (controlled to be CD5-negative) as well as in human leukaemic cell line 60 (HL-60) cells, for the following TRP species: the long TRPC2 (LTRPC2), the vanilloid receptor 1, the vanilloid receptor-like protein 1 and epithelial Ca2+ channels 1 and 2. TRPC6 was specific for neutrophils, whereas only in HL-60 cells were TRPC1, TRPC2, TRPC3, melastatin 1 and melastatin-related 1 found. Patch-clamp measurements in neutrophils revealed non-selective cation currents evoked by intracellular ADP-ribose and by NAD+. Both these modes of activation have been found to be characteristic of LTRPC2. Furthermore, single-channel activity was resolved in neutrophils and it was indistinguishable from that in LTRPC2-transfected HEK-293 cells. The results provide evidence that LTRPC2 in neutrophil granulocytes forms an entry pathway for Na+ and Ca2+, which is regulated by ADP-ribose and the redox state.
Multimode seeded Richtmyer–Meshkov mixing in a convergent, compressible, miscible plasma system
Authors: Lanier N. E. , Barnes Cris W. , Batha S. H. , Day R. D. , Magelssen G. R. , Scott J. M. , Dunne A. M. , Parker K. W. , Rothman S. D.
Published: April 2003

Richtmyer–Meshkov (RM) mixing seeded by multimode initial surface perturbations in a convergent, compressible, miscible plasma system is measured on the OMEGA [T. R. Boehly et al., Opt. Commun. 133, 495 (1997)] laser system. A strong shock (Mach 12–20), created by 50 laser beams, is used to accelerate impulsively a thin aluminum shell into a lower density foam. As the system converges, both interfaces of the aluminum are RM unstable and undergo mixing. Standard x-ray radiographic techniques are employed to survey accurately the zero-order hydrodynamics, the average radius and overall width, of the marker. LASNEX [G. B. Zimmerman et al., Comments on Plasma Physics 2, 51 (1975)] simulations are consistent with the zero-order behavior of initially smooth markers. In experiments with smooth aluminum markers, the measured marker width shortly after shock passage behaves incompressibly and thickens due to Bell–Plesset effects. At high convergence (>4), the marker begins to compress as the rebounding shock passes back through the marker. When an initial multimode perturbation is introduced to the outer surface of the marker, the measured marker width is observed to increase by 10–15 μm, and is substantially smaller than as-shot simulations using RAGE [R. M. Baltrusaitis et al., Phys. Fluids 8, 2471 (1996)] would predict.
Surface excess in dilute polymer solutions and the adsorption transition versus wetting phenomena
Authors: Metzger Susanne , Müller Marcus , Binder Kurt , Baschnagel Jörg
Published: April 2003

The adsorption of long flexible chains from dilute solution is studied by Monte Carlo simulation of a coarse-grained bead-spring model, and the results are interpreted in terms of phenomenological theories, using both mean field approaches and scaling concepts. It is shown that the surface excess, i.e., the integral of the local density difference of the monomers close to the surface relative to the bulk changes its sign very close to the adsorption transition (that is a sharp transition in the limit where the chain length diverges to infinity) for long chains, and it can be described in terms of the standard scaling description that has previously been tested for polymers with one end anchored on the surface (“polymer mushrooms”). Attention is also paid to the question on how this description changes when the temperature T of the polymer solution approaches the theta temperature Θ. Since the theta point can also be considered as an end point of a line of critical points, where the polymer solution phase separates into a dilute solution of collapsed chains and a more concentrated solution in the bulk, the adsorbing wall for T<Θ causes the existence of wetting layers. Conjectures about relations between wetting transitions for T near Θ and the adsorption transition are also presented.
Representations of a symplectic type subalgebra of W∞
Authors: Boyallian Carina , Liberati José I.
Published: April 2003

We classify the irreducible quasifinite highest weight modules over the symplectic Lie subalgebra W∞, and realize them in terms of irreducible highest weight representations of classical Lie subalgebras of infinite matrices with finitely many nonzero diagonals.
Exploration of high harmonic fast wave heating on the National Spherical Torus Experiment
Authors: Wilson J. R. , Bell R. E. , Bernabei S. , Bitter M. , Bonoli P. , Gates D. , Hosea J. , LeBlanc B. , Mau T. K. , Medley S. , Menard J. , Mueller D. , Ono M. , Phillips C. K. , Pinsker R. I. , Raman R. , Rosenberg A. , Ryan P. , Sabbagh S. , Stutman D. , Swain D. , Takase Y. , Wilgen J. ,   NSTX Team
Published: April 2003

High harmonic fast wave (HHFW) heating has been proposed as a particularly attractive means for plasma heating and current drive in the high beta plasmas that are achievable in spherical torus (ST) devices. The National Spherical Torus Experiment (NSTX) [M. Ono, S. M. Kaye, S. Neumeyer et al., in Proceedings of the 18th IEEE/NPSS Symposium on Fusion Engineering, Albuquerque, 1999 (IEEE, Piscataway, NJ, 1999), p. 53] is such a device. An rf heating system has been installed on the NSTX to explore the physics of HHFW heating, current drive via rf waves and for use as a tool to demonstrate the attractiveness of the ST concept as a fusion device. To date, experiments have demonstrated many of the theoretical predictions for HHFW. In particular, strong wave absorption on electrons over a wide range of plasma parameters and wave parallel phase velocities, wave acceleration of energetic ions, and indications of current drive for directed wave spectra have been observed. In addition HHFW heating has been used to explore the energy transport properties of NSTX plasmas, to create H-mode discharges with a large fraction of bootstrap current and to control the plasma current profile during the early stages of the discharge.
Kinetic instabilities of thin current sheets: Results of two-and-one-half-dimensional Vlasov code simulations
Authors: Silin I. , Büchner J.
Published: April 2003

Nonlinear triggering of the instability of thin current sheets is investigated by two-and-one-half- dimensional Vlasov code simulations. A global drift-resonant instability (DRI) is found, which results from the lower-hybrid-drift waves penetrating from the current sheet edges to the center where they resonantly interact with unmagnetized ions. This resonant nonlinear instability grows faster than a Kelvin–Helmholtz instability obtained in previous studies. The DRI is either asymmetric or symmetric mode or a combination of the two, depending on the relative phase of the lower-hybrid-drift waves at the edges of the current sheet. With increasing particle mass ratio the wavenumber of the fastest-growing mode increases as kLz∼(mi/me)1/2/2 and the growth rate of the DRI saturates at a finite level.
Frequency dependence of asymmetry-induced transport in a non-neutral plasma trap
Authors: Eggleston D. L. , Carrillo B.
Published: April 2003

A key prediction of the theory of asymmetry-induced transport is that the particle flux will be dominated by particles that move in resonance with the asymmetry. For the case of a time-varying asymmetry, the resonance condition is ω−lωR−nπv/L=0, where v is the axial velocity, L is the plasma length, ωR is the E×B rotation frequency, and ω, l, and n are the asymmetry frequency, azimuthal wavenumber, and axial wavenumber, respectively. Data are presented from experiments on a low density trap in which ω, ωR, and n are varied and the resulting radial particle flux is measured. The experiments show a resonance in the flux similar to that predicted by theory. The peak frequency of this resonance increases with ωR and varies with n, in qualitative agreement with theory, but quantitative comparisons between experiment and theory show serious discrepancies.
Fractional operators and special functions. II. Legendre functions
Authors: Durand Loyal
Published: April 2003

Most of the special functions of mathematical physics are connected with the representation of Lie groups. The action of elements D of the associated Lie algebras as linear differential operators gives relations among the functions in a class, for example, their differential recurrence relations. In this paper, we apply the fractional generalizations Dμ of these operators developed in an earlier paper in the context of Lie theory to the group SO(2,1) and its conformal extension. The fractional relations give a variety of interesting relations for the associated Legendre functions. We show that the two-variable fractional operator relations lead directly to integral relations among the Legendre functions and to one- and two-variable integral representations for those functions. Some of the relations reduce to known fractional integrals for the Legendre functions when reduced to one variable. The results enlarge the understanding of many properties of the associated Legendre functions on the basis of the underlying group structure.
Next-step-targeted experiments on the Mega-Amp Spherical Tokamak
Authors: Gryaznevich M. , Akers R. J. , Counsell G. F. , Cunningham G. , Dnestrovskij A. , Field A. R. , Hender T. C. , Kirk A. , Lloyd B. , Meyer H. , Morris A. W. , Sykes A. , Tabasso A. , Valovic M. , Voss G. M. , Wilson H. R. ,   MAST and NBI teams
Published: April 2003

Since its first physics campaign, the principal parameters on MAST (Mega-Amp Spherical Tokamak) [A. Sykes et al., Nuclear Fusion 41, 1423 (2001)] have been brought up towards their design values. Considerable advances have been made in a range of physics areas of direct relevance to the International Thermonuclear Experimental Reactor (ITER) [ITER Physics Basis, Nuclear Fusion 39, 2175 (1999)]. In this paper, results on H-mode access, global confinement and pedestal studies are presented and compared with conventional aspect ratio tokamak scalings. Physics and engineering requirements relevant to next step spherical tokamak devices are discussed, in particular the plasma formation, current ramp-up and sustainment, and plasma exhaust. Results of first experiments directly targeting these issues are presented: Plasma current up to 0.5 MA has been produced without use of the central solenoid flux, and current ramp-up and sustainment without use of the central solenoid flux has been demonstrated. Experiments on neutral beam heating and current drive (CD) demonstrate up to 50% bootstrap current fraction and good CD efficiency, and divertor power loading has been found to be tolerable and have a favorable outboard asymmetry.
Ion dynamics in helicon sources
Authors: Kline J. L. , Balkey M. M. , Keiter P. A. , Scime E. E. , Keesee A. M. , Sun X. , Hardin R. , Compton C. , Boivin R. F. , Zintl M. W.
Published: April 2003

Recent experiments have demonstrated that phenomena associated with ion dynamics, such as the lower hybrid resonance, play an important role in helicon source operation. In this work, a review of recent ion heating measurements and the role of the slow wave in heating ions at the edge of helicon sources is presented. The relationship between parametrically driven waves and ion heating near the rf antenna in helicon sources is also discussed. Recent measurements of parallel and rotational ion flows in helicon sources are presented and the implications for particle confinement, instability growth, and helicon source operation are reviewed.
Structure of a salt–amphiphile–water solution and the mechanism of salting out
Authors: Bowron D. T. , Finney J. L.
Published: April 2003

Salting out of amphiphiles from aqueous solution is a process of central importance in aqueous solution chemistry and of major application in the concentration of macromolecules and the crystallization of proteins. Despite its importance, our understanding of the mechanism is poor, there being no direct experimental evidence to support the several competing explanations in the literature. Using neutron diffraction with isotope substitution, we report a structural study of the effect of adding a simple salting out agent (NaCl) to a dilute solution of an amphiphile (t-butanol). The results show clearly that the anion is central to driving the changes in association of the amphiphile through the formation of an anion bridge between the polar ends of neighboring alcohol molecules. This further exposes the nonpolar surface of the amphiphile and suggests that further association may then occur through hydrophobic interaction. The mechanism uncovered is both different from those currently in the literature and relatively simple, and offers a possible route forward to understanding the variation in salting out efficacy of different ions as indicated by the Hofmeister or lyotropic series.
Wave packet study of the Ar–HBr photolysis: Stereodynamical effects
Authors: Prosmiti R. , Garcı́a-Vela A.
Published: April 2003

The ultraviolet photolysis of Ar–HBr(v=1) is studied through wave packet dynamics simulations, focusing on the fragmentation pathway Ar–HBr +ℏω→H+Ar–Br. Photolysis starts from two initial states of Ar–HBr(v=1) with a different angular shape, namely the ground and the first excited van der Waals (vdW) states, corresponding to the Ar–H–Br and Ar–Br–H isomers, respectively. It is found that the yield of Ar–Br radical products is substantially higher for the initial excited vdW state of the cluster, where H dissociation is less hindered. In addition, the yield of radical formation is much higher in the Ar–HBr(v=1) photolysis than that previously found in the Ar–HCl(v=0) case, even for the ground vdW state, where the initial angular distribution of both clusters is similar. Another unexpected difference is that Ar–HCl(v=0) photolysis exhibits strong manifestations of quantum interference, while these effects are much weaker in Ar–HBr(v=1). A lower probability of the first collision between the recoiling hydrogen and the Ar atom in the case of Ar–HBr(v=1), due to geometrical differences between its initial state and that of Ar–HCl(v=0), is suggested to explain the different photolysis behavior of both clusters. The implications of the present findings in the photolysis of other related precursor clusters are discussed.