bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological evidence for a high‐affinity cadmium transporter highly expressed in a Thlaspi caerulescens ecotype
Authors: Lombi E. , Zhao F. J. , McGrath S. P. , Young S. D. , Sacchi G. A.
Published: March 2003

• Uptake kinetics and translocation characteristics of cadmium and zinc are presented for two contrasting ecotypes of the Cd/Zn hyperaccumulator Thlaspi caerulescens, Ganges (southern France) and Prayon (Belgium).

• Experiments using radioactive isotopes were designed to investigate the physiology of Cd and Zn uptake, and a pressure‐chamber system was employed to collect xylem sap.

• In contrast to similar Zn uptake and translocation, measurements of concentration‐dependent influx of Cd revealed marked differences between ecotypes. Ganges alone showed a clear saturable component in the low Cd concentration range; maximum influx Vmax for Cd was fivefold higher in Ganges; and there was a fivefold difference in the Cd concentration in xylem sap. Addition of Zn to the uptake solution at equimolar concentration to Cd did not decrease Cd uptake by Ganges, but caused a 35% decrease in Prayon.

• There is strong physiological evidence for a high‐affinity, highly expressed Cd transporter in the root cell plasma membranes of the Ganges ecotype of T. caerulescens. This raises evolutionary questions about specific transporters for non‐essential metals. The results also show the considerable scope for selecting hyperaccumulator ecotypes to achieve higher phytoextraction efficiencies.
Ancestry of KNOX genes revealed by bryophyte (Physcomitrella patens) homologs
Authors: Champagne Connie E. M. , Ashton Neil W.
Published: March 2003

Structural and phylogenetic studies of KNOX
genes identified in the bryophyte
Physcomitrella patens
are reported here, to provide insights into the evolution of class 1 and class 2
KNOX genes.

Three KNOTTED1
‐like homeobox (
KNOX
) genomic clones were isolated and sequenced from
P. patens . Corresponding cDNAs from a library, prepared from mRNA transcripts isolated from gametophytic tissues, were also sequenced.

Conceptual translation and analysis of the bryophyte coding sequences revealed a domain pattern and secondary structures typical of higher plant KNOX proteins. Intron number and positions within the genes were also highly conserved between moss and angiosperm loci, providing further support for their homology. Structural and phylogenetic analyses indicated that moss clones ( MKN2
and
MKN4
) represent class 1
KNOX
genes and the remaining clone (
MKN1–3
) is a class 2
KNOX gene.

We conclude that the observed protein domain pattern is encoded by homeobox genes that evolved after separation of the plant lineage from that of fungi and animals, and must have been present in the common ancestor to mosses and seed plants. It is proposed that gene duplication and diversification, which created class 1 and 2 KNOX
gene subfamilies, occurred after separation of this common ancestor from its algal progenitor (since a characterized algal
KNOX gene cannot be assigned to class 1 or 2), but before the moss and higher plant lineages diverged.
Phosphorus uptake by arbuscular mycorrhizal hyphae does not increase when the host plant grows under atmospheric CO2 enrichment
Authors: Gavito Mayra E. , Bruhn Dan , Jakobsen Iver
Published: March 2003

• We conducted an experiment to test whether phosphorus (P) uptake by mycorrhizal hyphae could be enhanced by growing the host plant under [CO2] enrichment and whether any response to [CO2] was dependent on C source‐sink relationships.

• Plant C assimilation, mass allocation, growth and P uptake were measured in pea (Pisum sativum) plants inoculated with 0, 1 or 5% of a mixture of three Glomus spp. Intra‐ and extra‐radical mycorrhizal development was followed and hyphal 33P uptake from a root‐exclusion compartment was measured.

• Total P and 33P content measurements indicated that root, not hyphal, P uptake was increased by elevated [CO2] in the mycorrhizal treatments and that hyphal P uptake was actually reduced by elevated [CO2] after 57 d. Neither intra‐ nor extraradical mycorrhizal development was related to this response.

• Plant and fungal measurements suggested positive interactions in plant growth and P uptake only when C source‐sink relationships were balanced; high C source (enhanced assimilation at elevated [CO2]) and high C sink (increasing mycorrhizal development). The results also indicated that enhanced plant C supply does not alter growth or function of arbuscular mycorrhizal fungi.
Insect‐mediated reproduction of systemic infections by Puccinia arrhenatheri on Berberis vulgaris
Authors: Naef Andreas , Roy Bitty A. , Kaiser Roman , Honegger Rosmarie
Published: March 2003

• Witches’ brooms on Berberis vulgaris are induced by a systemically infecting rust fungus, Puccinia arrhenatheri. These witches’ brooms bear yellow discolored leaves on which the fungus exposes its gametes in a sugary nectar. During the spermatial stage of the fungus the infected leaves emit a strong, flowery scent.

• An exclusion‐experiment was used to evaluate whether fungal reproductive success, defined by the ability of the fungus to produce aeciospores, depended on gamete transfer by insects. To determine whether insects were attracted to the infected leaves, and if so, why, visitation to infected and uninfected leaves was quantified and volatiles produced by leaves, infected leaves and flowers were analyzed.

• The production of aeciospores was significantly higher on witches’ brooms with insect visitation. Visitation rates were higher and visits were longer on witches’ brooms than on uninfected branches. A wide diversity of visitors, mainly Diptera and Hymenoptera, was observed. The volatiles emitted by infected leaves were composed of sweet floral fragrances and insect pheromones.

• Our results suggest that sexual reproduction of the pathogen requires out‐crossing by insects and that infected leaves attract insects by floral mimicry (bright yellow color and the production of sugary nectar and volatiles).
The cell biology of phytochrome signalling
Authors: Møller Simon G. , Ingles Patricia J. , Whitelam Garry C.
Published: March 2003

Phytochrome signal transduction has in the past often been viewed as being a nonspatially separated linear chain of events. However, through a combination of molecular, genetic and cell biological approaches, it is becoming increasingly evident that phytochrome signalling constitutes a highly ordered multidimensional network of events. The discovery that some phytochromes and signalling intermediates show light‐dependent nucleo‐cytoplasmic partitioning has not only led to the suggestion that early signalling events take place in the nucleus, but also that subcellular localization patterns most probably represent an important signalling control point. Moreover, detailed characterization of signalling intermediates has demonstrated that various branches of the signalling network are spatially separated and take place in different cellular compartments including the nucleus, cytosol, and chloroplasts. In addition, proteasome‐mediated degradation of signalling intermediates most probably act in concert with subcellular partitioning events as an integrated checkpoint. An emerging view from this is that phytochrome signalling is separated into several subcellular organelles and that these are interconnected in order to execute accurate responses to changes in the light environment. By integrating the available data, both at the cellular and subcellular level, we should be able to construct a solid foundation for further dissection of phytochrome signal transduction in plants.

Contents

Summary
553

I.
Introduction
554

II.
Nucleus vs cytoplasm
556

III.
The nucleus
562

IV.
The cytoplasm
571

V.
Interactions with other signalling pathways
577

VI.
Conclusions and the future
582

Acknowledgements
583

References
583
Implications of leaf nitrogen recycling on the nitrogen isotope composition of deciduous plant tissues
Authors: Kolb K. J. , Evans R. D.
Published: March 2003

The effect of nitrogen (N) recycling on tissue δ15N was studied using drought‐deciduous shrubs (Encelia farinosaandE. frutescens) and deciduous trees (Quercus rubraandQ. alba) to determine if isotope discrimination occurs with N reabsorption or reallocation.

There was substantial reabsorption of leaf nitrogen before abscission in all species, yet there was no difference in the δ15N of living vs abscised leaves, suggesting no isotope discrimination with reabsorption. However, the contribution of stored N for leaf production resulted in a difference in foliar and absorbed nitrogen δ15N, suggesting that isotope discrimination occurs with reallocation.

The contribution of stored nitrogen to leaf production varied with timing of leaf development: leaves produced first had a δ15N that reflected utilization of both absorbed and stored N, yet the contribution of stored N decreased over time.

The results from this study as well as previous research on intraplant variation in isotopic composition suggest that caution needs to be exercised when interpreting foliar δ15N because it is not always a reliable indicator of whole‐plant δ15N.
An approach to the detection of synthetic milk in dairy milk: 3. Detection of vegetable oil and sodium bicarbonate
Authors: Paradkar Manish M , Singhal Rekha S , Kulkarni Pushpa R
Published: March 2003

A high‐performance thin layer chromatography (HPTLC) method for the detection of groundnut oil and palm oil, the main constituents of synthetic milk, was developed. The minimum detectable level of the oils was 80 mg/100 ml. The rosolic acid qualitative test was found to be sensitive enough to detect levels as low as 20 mg/100 ml sodium bicarbonate in milk.
Changes in the microbiological and chemical characteristics of an artisanal, low‐fat cheese made from raw ovine milk during ripening
Authors: Nikolaou E , Tzanetakis N , Litopoulou‐Tzanetaki E , Robinson R K
Published: March 2003

Changes in the microbial flora of batzos cheese made from raw ovine milk were studied during ripening. Lactic acid bacteria and Enterobacteriaceae were the predominant groups of micro‐organisms. Cheeses manufactured in summer had higher microbial counts than those made in spring, with the exception of staphylococci. Nevertheless, Enterobacteriaceae and coliforms decreased more rapidly in cheese made in summer and counts at the end of storage were lower than those in spring cheese.

Enterococci predominated in the ripened curd of cheese made in spring, whereas lactobacilli were the most abundant lactic acid bacteria in cheese made in summer. Enterococcus faecium was the predominant species in spring, and Lactobacillus paracasei ssp. paracasei predominated in cheese made in summer. The pH of the cheeses was > 5.0 throughout ripening, and NaCl‐in‐moisture content (> 8.0%) permitted the growth and survival of salt‐tolerant micro‐organisms. αs1‐Casein degraded at a faster rate than β‐casein; both caseins were hydrolysed more rapidly in spring than in summer. The free amino acid content became higher in summer cheese (566.24–3460.25 µg/g of glycine equivalent) than in spring cheese because of the progress of ripening. Moreover, the milk fat of the cheese was degraded more in the summer than in the spring. The results suggest that there could be advantages to using starter cultures and improving the level of hygiene during milk and cheese production in order to eliminate undesirable micro‐organisms and standardize cheese quality.
The effects of varying sugar content and fruit concentration on the physical properties of vanilla and fruit ice‐cream‐type frozen yogurts
Authors: Güven M , Karaca O B
Published: March 2003

In this study, the physical properties of samples of ice cream produced from yogurt with varying levels of sugar (18, 20 or 22%) and fruit (strawberry) concentration (15, 20 or 25%) were investigated. The results of the physical analysis indicate that as the amount of sugar and fruit increases, there is an increase in the first dripping time period, viscosity values and the overrun. However, complete melting times decreased in parallel with the increase in sugar content and fruit concentration. It was observed that an increase in the amount of sugar in vanilla frozen yogurt softened the structure. In strawberry ice‐cream‐type frozen yogurt, the structure hardened in parallel with the increase in fruit concentration. In addition, the results show that frozen yogurts with high sugar (V3) and fruit (St3) concentrations are the type most preferred by the panellists. The results of the sensory analysis suggest that frozen yogurt can be an alternative to other dairy products such as yogurt and ice cream.
Technology options for new nutritional concepts
Authors: Korhonen Hannu
Published: March 2003

Recent advances in the food and nutrition sciences support the concept that the diet has a significant role in the modulation of various functions in the body. The diet and/or its components may contribute to an improved state of well‐being, a reduction of risks related to certain diseases and even an improvement in the quality of life. These new concepts have led to the introduction of a new category of health‐promoting foodstuffs, i.e. functional foods.

The concern about health embraces a number of driving issues, needs and opportunities which may be approached by designing specific diets from various food raw materials. These tailor‐made products provide physiological benefits that are targeted at particular consumer groups.

The functionality of functional foods is based on bioactive components, which may be contained naturally in the product but usually require formulation by appropriate technologies in order to optimise the desired beneficial properties. To this end, it is often necessary to develop and apply novel technologies, e.g. membrane separation, high hydrostatic pressure and supercritical fluid extraction techniques. Also the minimal processing concept could be employed in this context.

This review discusses the current technological options available and the future challenges faced in the area. Particular attention is paid to the exploitation of bovine colostrum and milk‐derived bioactive compounds for the development of functional foods.
Microbiological quality of white‐brined cheeses: a review
Authors: Bintsis T , Papademas P
Published: March 2003

White‐brined cheeses are widely produced in the North‐east Mediterranean area and the Balkans. Traditionally, they were manufactured as artisanal cheeses, and nowadays they are manufactured on an industrial scale, and rigorous control of the production and maturation processes is essential. Aspects of the microbiology of white‐brined cheeses and their significance with respect to the quality and safety of the final products are discussed in this review.
Health information: an evaluation of the use of touch screen kiosks in two hospitals
Authors: Nicholas David , Huntington Paul , Williams Peter , Vickery Pam
Published: March 2003

An exit questionnaire survey of 150 hospital users of a touch screen health information kiosk was undertaken. It was compiled by Royal Cornwall Hospitals NHS to evaluate kiosk use and was reanalysed by researchers at City University to determine whether the information obtained went anyway to meeting patients’ information needs. The study established two effective outcome variables on kiosk use: whether the information found answered the users’ questions and if users intended, after using the kiosk, to find more information elsewhere. Two‐thirds of respondents said that the information found had answered their question, whilst 16% said that after using the kiosk they still had further questions and intended to talk to a health professional to obtain this information. The variables found to have an impact on the information outcome included: gender, navigational ease, object of search, and ease of reading and understanding the information.
Developing evidence‐based librarianship: practical steps for implementation*
Authors: Crumley Ellen , Koufogiannakis Denise
Published: March 2003

Evidence‐based librarianship (EBL) is a relatively new concept for librarians. This paper lays out a practical framework for the implementation of EBL. A new way of thinking about research in librarianship is introduced using the well‐built question process and the assignment of librarian research questions to one of six domains specific to librarianship. As a profession, librarianship tends to reflect more qualitative, social sciences/humanities in its research methods and study types which tend to be less rigorous and more prone to bias. Randomised controlled trials (RCT) do not have to be placed at the top of an evidence ‘hierarchy’ for librarianship. Instead, a more encompassing model reflecting librarianship as a whole and the kind of research likely to be done by librarians is proposed. ‘Evidence’ from a number of disciplines including health sciences, business and education can be utilized by librarians and applied to their practice. However, access to and availability of librarianship literature needs to be further studied. While using other disciplines (e.g. EBHC) as a model for EBL has been explored in the literature, the authors develop models unique to librarianship. While research has always been a minor focus in the profession, moving research into practice is becoming more important and librarians need to consider the issues surrounding research in order to move EBL forward.
Evaluation of distance learning delivery of health information management and health informatics programmes: a UK perspective
Authors: Urquhart Christine , Chambers Mary , Connor Samantha , Lewis Leo , Murphy Jeannette , Roberts Ruth , Thomas Rhian
Published: March 2003

The aim of the article is to review evaluations of distance learning programmes in health information management, in order to identify the critical success factors for such programmes and discuss future directions. The emphasis is on the UK experience, based partly on reflections on the experience of one programme at University of Wales Aberystwyth (now over 10 years old), and partly on a policy review conducted for the NHS Information Authority and the NHS Information Policy Unit. The methods are, as far as possible, those of a systematic review of existing research, with, additionally, an overview of relevant policy developments for lifelong learning. The topics covered include the aims, objectives and educational philosophies of the programmes. The evidence, combined with the UWA experience, indicates the importance of face‐to‐face interaction, to complement distance or virtual learning. A student‐centred approach to curriculum design and delivery is essential.
Hydrogen Peroxide Activation of Multiple Mitogen‐Activated Protein Kinases in an Oligodendrocyte Cell Line
Authors: Bhat Narayan R. , Zhang Peisheng
Published: March 2003

Abstract : Oxidative stress is known to induce cell death in a wide
variety of cell types, apparently by modulating intracellular signaling
pathways. In this study, we have examined the activation of mitogen‐activated
protein kinase (MAPK) cascades in relation to oxidant‐induced cell death in an
oligodendrocyte cell line, central glia‐4 (CG4). Exposure of CG4 cells to
hydrogen peroxide (H2O2) resulted in an increased
tyrosine phosphorylation of several protein species, including the abundantly
expressed platelet‐derived growth factor (PDGF) receptor and the activation of
the three MAPK subgroups, i.e., extracellular signal‐regulated kinase (ERK),
p38 MAPK, and c‐Jun N‐terminal kinase (JNK). Dose‐response studies showed
differential sensitivities of PDGF receptor phosphorylation (>1
mM) and ERK/p38 MAPK (>0.5 mM) and JNK (>0.1
mM) activation by H2O2. The activation of ERK
was inhibited by PD98059, a specific inhibitor of the upstream kinase, MAPK or
ERK kinase (MEK). H2O2 also activated MAPK‐activated
protein kinase‐2, and this activation was blocked by SB203580, a specific
inhibitor of p38 MAPK. The oxidant‐induced cell death was indicated by
morphological changes, decreased
3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reduction, and
DNA fragmentation. These effects were suppressed dose‐dependently by the MEK
inhibitor PD98059. The results demonstrate that H2O2 induces the activation of multiple MAPKs in oligodendrocyte progenitors and that the activation of ERK is associated with oxidant‐mediated cytotoxicity.
Caspase Activation Accompanying Cytochrome c Release from Mitochondria Is Possibly Involved in Nitric Oxide‐Induced Neuronal Apoptosis in SH‐SY5Y Cells
Authors: Uehara Takashi , Kikuchi Yukio , Nomura Yasuyuki
Published: March 2003

Abstract : It is well known that caspases are produced as proforms, which are proteolytically cleaved and activated during apoptosis or programmed cell death. We report here that caspases are activated during apoptosis by treatment with NOC18, a nitric oxide (NO) donor. Our present experiments have examined the way in which NO induces neuronal cell death, using a new type of NO donor that spontaneously releases only NO without enzymatic metabolism. NOC18 induced apoptosis in human neuroblastoma SH‐SY5Y cells in a concentration‐and time‐dependent manner as estimated by DNA fragmentation assay, FACScan analysis, and nuclear morphology. Oxyhemoglobin, an NO trapper, suppressed NOC18‐triggered DNA fragmentation, indicating that NO from NOC18 is a real activator in this study. Upon the induction of apoptosis, an increase in caspase‐3‐like protease activity, but not caspase‐1, was observed. Procaspase‐2 protein, an inactive form of caspase‐2, decreased dramatically. In addition, NOC18 also resulted in poly (ADP‐ribose) polymerase (PARP) cleavage, yielding an 85‐kDa fragment typical of caspase activity. Oxyhemoglobin blocked the decrease of procaspase‐2 and the cleavage of PARP by NOC18 in a concentration‐dependent manner. Moreover, NO elicited the release of cytochrome c into the cytosol during apoptosis. These results suggest that both stimulation of caspase activity and cytochrome c release are partly involved in NO‐induced neuronal apoptosis.
Influence of Age and Strain on Striatal Dopamine Loss in a Genetic Mouse Model of Lesch‐Nyhan Disease
Authors: Jinnah H. A. , Jones M. D. , Wojcik B. E. , Rothstein J. D. , Hess E. J. , Friedmann T. , Breese G. R.
Published: March 2003

Abstract : Lesch‐Nyhan disease is a neurogenetic disorder caused by deficiency of the purine salvage enzyme hypoxanthine‐guanine phosphoribosyltransferase (HPRT). Affected individuals exhibit a characteristic pattern of neurological and behavioral features attributable in part to dysfunction of basal ganglia dopamine systems. In the current studies, striatal dopamine loss was investigated in five different HPRT‐deficient strains of mice carrying one of two different HPRT gene mutations. Caudoputamen dopamine concentrations were significantly reduced in all five of the strains, with deficits ranging from 50.7 to 61.1%. Mesolimbic dopamine was significantly reduced in only three of the five strains, with a range of 31.6‐38.6%. The reduction of caudoputamen dopamine was age dependent, emerging between 4 and 12 weeks of age. Tyrosine hydroxylase and aromatic amino acid decarboxylase, two enzymes responsible for the synthesis of dopamine, were reduced by 22.4‐37.3 and 22.2‐43.1%, respectively. These results demonstrate that HPRT deficiency is strongly associated with a loss of basal ganglia dopamine. The magnitude of dopamine loss measurable is dependent on the genetic background of the mouse strain used, the basal ganglia sub‐region examined, and the age of the animals at assessment.
Relationship of Glycosyltransferases and mRNA Levels to Ganglioside Expression in Neuroblastoma and Melanoma Cells
Authors: Ruan Shutian , Raj Mohan , Lloyd Kenneth
Published: March 2003

Abstract : Most human neuroblastoma tumors are characterized by the high expression of GD2 (or GD2 and/or GM2) gangliosides, whereas melanomas characteristically express GD3 ganglioside. The molecular basis for these patterns was investigated by examining the relationship between ganglioside levels, glycosyltransferase (GM2/GD2 synthase and GD3 synthase) activity, and corresponding mRNA levels in a panel of human neuroblastoma and melanoma cell lines. In general, the ganglioside patterns could be explained by the levels of the transferases and their mRNA, indicating control at the level of transcription. A key role was noted for GD3 synthase. Notably, it was found that neuroblastoma cell lines with high GD2 ganglioside levels had low levels of GD3, its synthase, and mRNA for the enzyme even though this step provides the substrate for GD2 synthesis. The key role for GD3 synthase was also examined by stably transfecting GD3 synthase cDNA into a neuroblastoma cell line (SH‐SY5Y) not expressing GD3 and GD2. The resulting cell line had high levels of GD2 ganglioside and altered morphology and growth characteristics.
Chronic Ethanol Increases the Cannabinoid Receptor Agonist Anandamide and Its Precursor N‐Arachidonoylphosphatidylethanolamine in SK ‐ N ‐ SH Cells
Authors: Basavarajappa Balapal S , Hungund Basalingappa L
Published: March 2003

Abstract : In an earlier study, we demonstrated that chronic ethanol
(EtOH) exposure down‐regulated the cannabinoid receptors (CB1) in mouse brain
synaptic plasma membrane. In the present study, we investigated the effect of
chronic EtOH on the formation of anandamide (AnNH), an endogenous
cannabimimetic compound, and its precursor
N‐arachidonoylphosphatidylethanolamine (N‐ArPE) in SK‐N‐SH cells that
were prelabeled with [3H]arachidonic acid. The results indicate
that exposure of SK‐N‐SH cells to EtOH (100 mM) for 72 h
significantly increased levels of [3H]AnNH and
[3H]N‐ArPE (p < 0.05) (1.43‐fold for
[3H]AnNH and 1.65‐fold for [3H]N‐ArPE). Exposure of
SK‐N‐SH cells to EtOH (100 mM, 24h) inhibited initially the formation
of [3H]AnNH at 24 h, followed by a progressive increase, reaching a
statistical significance level at 72 h (p < 0.05).
[3H]N‐ArPE increased gradually to a statistically significant level
after 48 and 72 h (p < 0.05). Incubation with exogenous
ethanolamine (7 mM) and EtOH (100 mM, 72 h) did not result
in an additive increase in the formation of [3H]AnNH. The formation
of [3H]AnNH and [3H]N‐ArPE by EtOH was enhanced by the
Ca2+ ionophore A23187 or by the depolarizing agent veratridine and
the K+ channel blocker 4‐aminopyridine. Further, the EtOH‐induced
formation of [3H]AnNH and [3H]N‐ArPE was inhibited by
exogenous AnNH, whereas only [3H]AnNH formation was inhibited by
the CB1 receptor antagonist SR141716A and pertussis toxin, suggesting that the
CB1 receptor and Gi/o protein mediated the regulation of AnNH levels. The observed increase in the levels of these lipids in SK‐N‐SH cells may be a mechanism for neuronal adaptation and may serve as a compensatory mechanism to counteract the continuous presence of EtOH. The present observation taken together with our previous results indicate the involvement of the endocannabinoid system in mediating some of the pharmacological actions of EtOH and may constitute part of a common brain pathway mediating reinforcement of drugs of abuse including EtOH.
Internalization of G Protein‐Coupled Receptors in Single Olfactory Receptor Neurons
Authors: Rankin Michele L. , Alvania Rebecca S. , Gleason Evanna L. , Bruch Richard C.
Published: March 2003

Abstract : Desensitization of many G protein‐coupled receptors after
ligand binding generally involves phosphorylation of the receptors and
internalization of the ligandbound, phosphorylated receptors by a
clathrin‐mediated endocytic pathway. Olfactory receptor neurons from the
channel catfish (Ictalurus punctatus) express the G protein‐coupled
odorant receptors and metabotropic glutamate receptors. To determine whether a
clathrin‐dependent receptor internalization pathway exists in olfactory
receptor neurons, western blotting and immunocytochemistry were used to
identify and localize clathrin and dynamin in isolated olfactory neurons.
Clathrin and dynamin immunoreactivity was found in the cell bodies, dendrites,
and dendritic knobs of the neurons. Using the activity‐dependent fluorescent
dye FM1‐43 to monitor receptor internalization, we show that single olfactory
neurons stimulated with the odorant amino acid l‐glumate internalized the dye. Odorant‐stimulated neurons showed a consistent pattern of internalized FM1‐43 fluorescence localized in the cell bodies and dendritic knobs. Odorant‐stimulated internalization was unaffected by the caveolae activator okadaic acid and was significantly decreased by a metabotropic glutamate receptor antagonist, suggesting that a functional, clathrindependent, receptor‐mediated internalization pathway exists in olfactory receptor neurons.
F4 ‐ Isoprostanes as Specific Marker of Docosahexaenoic Acid Peroxidation in Alzheimer's Disease
Authors: Nourooz‐Zadeh Jaffar , Liu Edwin H. C. , Yhlen B. , Änggåard Erik E. , Halliwell Barry
Published: March 2003

Abstract : F2‐isoprostanes are prostaglandin‐like
compounds derived from free radical‐catalysed peroxidation of arachidonic
acid. Peroxidation of eicosapentaenoic acid produces
F3‐isoprostanes, whereas peroxidation of docosahexaenoic acid would
give F4‐isoprostanes. This study demonstrates the presence of
esterified F4‐isoprostanes in human brain and shows that levels are
elevated in certain brain cortex regions in Alzheimer's disease. Our data with
Alzheimer's disease suggest that analysis of F4‐isoprostanes will provide new opportunities to study lipid peroxidation in the neurodegenerative diseases.
Nuclear Localization of the δ Subunit of Ca2+/Calmodulin‐Dependent Protein Kinase II in Rat Cerebellar Granule Cells
Authors: Takeuchi Yusuke , Yamamoto Hideyuki , Matsumoto Kazuya , Kimura Takemi , Katsuragi Shoichi , Miyakawa Taihei , Miyamoto Eishichi
Published: March 2003

Abstract : To examine the physiological roles of the δ subunit
of Ca2+/calmodulin‐dependent protein kinase ∥ (CaM kinase
∥δ) in brain, we examined the localization of CaM kinase
∥δ in the rat brain. A specific antibody to CaM kinase
∥δ1‐δ4 isoforms was prepared by immunizing rabbits with a
synthesized peptide corresponding to the unique carboxyl‐terminal end of these
isoforms. The prepared antibody did not recognize the α, β, and
γ subunits, which were each overexpressed in NG108‐15 cells. Immunoblot
analysis on various regions and the nuclear fractions from rat brains
suggested that some isoforms of CaM kinase ∥δ1‐δ4 were
abundant in the nucleus in the cerebellum. Total RNA from the cerebellum was
analyzed by RT‐PCR with a primer pair from variable domain 1 to variable
domain 2. We detected the three PCR products δ3.1, δ3.4, and
δ3 that contained the nuclear localization signal. These CaM kinase
∥δ3 isoforms were localized in the nuclei in transfected NG108‐15
cells. Immunohistochemical study suggested the existence of these isoforms in
the nuclei in cerebellar granule cells. These results suggest that CaM kinase
∥δ3 isoforms are involved in nuclear Ca2+ signaling in cerebellar granule cells.
Modeling of the Interaction of Na+ and K+ with theBinding of Dopamine and [3H]WIN 35,428 to the Human Dopamine Transporter
Authors: Li Li‐Bin , Reith Maarten E. A.
Published: March 2003

Abstract : Although much is known about the effects of
Na+, K+, and Cl‐ on the functional activity
of the neuronal dopamine transporter, little information is available on their
role in the initial event in dopamine uptake, i.e., the recognition step. This
was addressed here by studying the inhibition by dopamine of the binding of
[3H]WIN 35,428
{2β‐carbomethoxy‐3β‐(4‐fluorophenyl)[3H]tropane}, a
phenyltropane analogue of cocaine, to the cloned human dopamine transporter
expressed in HEK‐293 cells. The decrease in the affinity of dopamine (or WIN
35,428) binding affinity with increasing [K+] could be fitted to a
competitive model involving an inhibitory cation site (1) overlapping with the
dopamine (or WIN 35,428) domain. The K+ IC50 for
inhibiting dopamine or WIN 35,428 binding increased linearly with
[Na+], indicating a KD,Na+ of 30‐44
mM and a KD,K+ of 13‐16 mM
for this cation site. A second Na+ site (2), distal from the WIN
35,428 domain but linked by positive allosterism, was indicated by model
fitting of the WIN 35,428 binding affinities as a function of
[Na+]. No strong evidence for this second site was obtained for
dopamine binding in the absence or presence of low (20 mM)
Cl‐ and could not be acquired for high [Cl‐] because of
the lack of a suitable substitute ion for Na+. The
KD but not Bmax of [3H]WIN
35,428 binding increased as a function of the [K+]/[Na+]
ratio regardless of total [Cl‐] or ion tonicity. A similar plot was
obtained for the Ki of dopamine binding, with
Cl‐ at ≥ 140 mM decreasing the Ki.
At 290 mM Cl‐ and 300 mM Na+ the
potency of K+ in inhibiting dopamine binding was enhanced as
compared with the absence of Cl‐ in contrast to the lack of effect
of Cl‐ up to 140 mM (Na+ up to 150
mM). The results indicate that Cl‐ at its extracellular
level enhances dopamine binding through a mechanism not involving site 1. The
observed correspondence between the WIN 35,428 and dopamine domains in their
inclusion of the inhibitory cation site explains why many of the previously
reported interrelated effects of Na+ and K+ on the binding site of radiolabeled blockers to the dopamine transporter are applicable to dopamine uptake in which dopamine recognition is the first step.
p38 Kinase Is Activated in the Alzheimer's Disease Brain
Authors: Hensley Kenneth , Floyd Robert A. , Zheng Nai‐Ying , Nael Raha , Robinson Kent A. , Nguyen Xuan , Pye Quentin N. , Stewart Charles A. , Geddes James , Markesbery William R. , Patel Ela , Johnson Gail V. W. , Bing Guoying
Published: March 2003

Abstract: The p38 mitogen‐activated protein kinase is a stress‐activated enzyme responsible for transducing inflammatory signals and initiating apoptosis. In the Alzheimer's disease (AD) brain, increased levels of phosphorylated (active) p38 were detected relative to age‐matched normal brain. Intense phospho‐p38 immunoreactivity was associated with neuritic plaques, neuropil threads, and neurofibrillary tangle‐bearing neurons. The antibody against phosphorylated p38 recognized many of the same structures as an antibody against aberrantly phosphorylated, paired helical filament (PHF) tau, although PHF‐positive tau did not cross‐react with the phospho‐p38 antibody. These findings suggest a neuroinflammatory mechanism in the AD brain, in which aberrant protein phosphorylation affects signal transduction elements, including the p38 kinase cascade, as well as cytoskeletal components.
Adenosine Prevents the Death of Mesencephalic Dopaminergic Neurons by a Mechanism that Involves Astrocytes
Authors: Michel Patrick P. , Marien Marc , Ruberg Merle , Colpaert Francis , Agid Yves
Published: March 2003

Abstract: The purinergic nucleoside adenosine effectively prevented the death of dopaminergic neurons that occurs spontaneously and progressively in cultures of rat mesencephalon. Adenosine also significantly increased dopamine uptake, attesting to the state of differentiation and functional integrity of the neurons that were rescued. The effects of adenosine were (a) specific to the dopaminergic neurons in these cultures, (b) long‐lived, (c) still observed when the treatment was delayed after plating, (d) potentiated by inhibition of adenosine deaminase, and (e) abolished when this enzyme was added in excess to the culture medium. The action of adenosine was mimicked by 5′‐(N‐ethylcarboxamido)adenosine and dibutyryl‐cyclic AMP, but not by CGS‐21680, suggesting the possible involvement of A2B subtype purinergic receptors. ATP was also neuroprotective, but its action resulted principally from conversion to adenosine by ectonucleotidases. Several anticancer drugs, including cytosine arabinoside, have been shown previously to prevent apoptosis in cultured dopaminergic neurons by a mechanism that requires the inhibition of proliferating astrocytes. In the presence of adenosine, astrocytes were more differentiated, and their proliferation rate was significantly reduced, suggesting that the neurotrophic effect of the adenine nucleoside resulted also from the repression of the astroglial cells. We did not find evidence of a trophic intermediary in adenosine‐treated cultures, however, leading to the hypothesis that limitation of astrocyte replication in itself and/or ensuing changes in the glial phenotype were crucial. Our results suggest that molecules that modulate adenine nucleotide/nucleoside release may be useful for the treatment of chronic neurodegenerative conditions affecting dopaminergic neurons, such as Parkinson's disease.
Dopamine Inhibition of Human Tyrosine Hydroxylase Type 1 Is Controlled by the Specific Portion in the N‐Terminus of the Enzyme
Authors: Nakashima Akira , Mori Keiji , Suzuki Takahiro , Kurita Hideki , Otani Motohiko , Nagatsu Toshiharu , Ota Akira
Published: March 2003

Abstract: Tyrosine hydroxylase (TH), which converts L‐tyrosine to L‐DOPA, is a rate‐limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by feedback inhibition by catecholamine products including dopamine. To investigate the specific portion of the N‐terminus of TH that determines the efficiency of dopamine inhibition, wild‐type and N‐terminal 35‐, 38‐, and 44‐amino acid‐deleted mutants (del‐35, del‐38, and del‐44, respectively) of human TH type 1 were expressed as a maltose binding protein fusion in Escherichla coli and purified as a tetrameric form by affinity and size‐exclusion chromatography. The fused‐form wild‐type enzyme possessed almost the same specific enzymatic activity as the previously reported recombinant nonfused form. Although maximum velocities of all N‐terminus‐deleted forms were about one‐fourth of the wild‐type value, there was no difference in Michaeiis constants for L‐tyrosine or (6R)‐(L‐erythro‐1,1′,2′‐dihydroxypropyl)‐2‐amino‐4‐hydroxy‐5,6,7,8‐tetrahydropteridine (6RBPH4) among the four enzymes. The iron contents incorporated into the three N‐terminus‐deleted mutants were significantly lower than that of wild type. However, there was no substantial difference in incorporated iron contents among the three mutants. The deletion of up to no less than 38 amino acid residues in the N‐terminus made the enzyme more resistant to dopamine inhibition than the wild‐type or del‐35 TH form. Dopamine bound to the del‐38 more than to the del‐35 TH form. However, when incubation with dopamine was followed by further inhibition with the cofactor 6RBPH4 dopamine was expelled more readily from the del‐38 than from the del‐35 TH form. These observations suggest that the amino acid sequence Gly36‐Arg37‐Arg38 plays a key role in determining the competition between dopamine and 6RBPH4 and affects the efficiency of dopamine inhibition of the catalytic activity.
Stimulation of Adenylyl Cyclase and Induction of Brain‐Derived Neurotrophic Factor and TrkB mRNA by NKH477, a Novel and Potent Forskolin Derivative
Authors: Morinobu Shigeru , Fujimaki Koichiro , Okuyama Naoyuki , Takahashi Michihiro , Duman Ronald S.
Published: March 2003

Abstract: The present study was undertaken to examine whether NKH477, a novel and potent water‐soluble forskolin derivative, stimulates adenylyl cyclase and regulates brain‐derived neurotrophic factor (BDNF) and TrkB expression in the rat brain. Administration of NKH477 at a dose of 1.0 mg/kg, but not 0.1 mg/kg, increased levels of cyclic AMP (cAMP) in a time‐dependent manner in frontal cortex and hippocampus. Repeated administration of NKH477 (1.0 mg/kg) for 7 or 14 days also increased levels of cAMP in these two brain regions, indicating that the response does not desensitize with chronic treatment. In addition, administration of NKH477 at the 1 mg/kg dose increased the expression of BDNF and TrkB mRNA in frontal cortex and hippocampus. This effect was observed after single, as well as repeated (7 or 14 days), administration of NKH477. These results demonstrate that NKH477 administration rapidly increases cAMP levels in brain and provides evidence that stimulation of this second messenger system increases the expression of BDNF and TrkB mRNA.
Glutamate Neurotoxicity in Rat Cerebellar Granule Cells Involves Cytochrome c Release from Mitochondria and Mitochondrial Shuttle Impairment
Authors: Atlante Anna , Gagliardi Sabatina , Marra Ersilia , Calissano Pietro , Passarella Salvatore
Published: March 2003

Abstract: To gain some insight into the mechanism by which glutamate neurotoxicity takes place in cerebellar granule cells, two steps of glucose oxidation were investigated: the electron flow via respiratory chain from certain substrates to oxygen and the transfer of extramitochondrial reducing equivalents via the mitochondrial shuttles. However, cytochrome c release from intact mitochondria was found to occur in glutamate‐treated cells as detected photometrically in the supernatant of the cell homogenate suspension. As a result of cytochrome c release, an increase of the oxidation of externally added NADH was found, probably occurring via the NADH‐b5 oxidoreductase of the outer mitochondrial membrane. When the two mitochondrial shuttles glycerol 3‐phosphate/dihydroxyacetone phosphate and malate/oxaloacetate, devoted to oxidizing externally added NADH, were reconstructed, both were found to be impaired under glutamate neurotoxicity. Consistent early activation in two NADH oxidizing mechanisms, i.e., lactate production and plasma membrane NADH oxidoreductase activity, was found in glutamate‐treated cells. In spite of this, the increase in the cell NADH fluorescence was found to be time‐dependent, an index of the progressive damage of the cell.
GSH Transport in Immortalized Mouse Brain Endothelial Cells
Authors: Kannan Ram , Mittur Aravind , Bao Yuzhou , Tsuruo Takashi , Kaplowitz Neil
Published: March 2003

Abstract: We have previously shown GSH transport across the blood‐brain barrier in vivo and expression of transport in Xenopus laevis oocytes injected with bovine brain capillary mRNA. In the present study, we have used MBEC‐4, an immortalized mouse brain endothelial cell line, to establish the presence of Na+‐dependent and Na+‐independent GSH transport and have localized the Na+‐dependent transporter using domain‐enriched plasma membrane vesicles. In cells depleted of GSH with buthionine sulfoximine, a significant increase of intracellular GSH could be demonstrated only in the presence of Na+. Partial but significant Na+ dependency of [35S]GSH uptake was observed for two GSH concentrations in MBEC‐4 cells in which γ‐glutamyltranspeptidase and γ‐glutamylcysteine synthetase were inhibited to ensure absence of breakdown and resynthesis of GSH. Uniqueness of Na+‐dependent uptake in MBEC‐4 cells was confirmed with parallel uptake studies with Cos‐7 cells that did not show this activity. Molecular form of uptake was varified as predominantly GSH, and very little conversion of [35S]cysteine to GSH occurred under the same incubation conditions. Poly(A)+ RNA from MBEC expressed GSH uptake with significant (∼40‐70%) Na+ dependency, whereas uptake expressed by poly(A)+ RNA from HepG2 and Cos‐1 cells was Na+ independent. Plasma membrane vesicles from MBEC were separated into three fractions (30, 34, and 38% sucrose, by wt) by density gradient centrifugation. Na+‐dependent glucose transport, reported to be localized to the abluminal membrane, was found to be associated with the 38% fraction (abluminal). Na+‐dependent GSH transport was present in the 30% fraction, which was identified as the apical (luminal) membrane by localization of P‐glycoprotein 170 by western blot analysis. Localization of Na+‐dependent GSH transport to the luminal membrane and its ability to drive up intracellular GSH may find application in the delivery of supplemented GSH to the brain in vivo.
A Proline‐ and Glutamine‐Rich Protein Promotes Apoptosis in Neuronal Cells
Authors: Gomes Ignatius , Xiong Wen , Miki Toru , Rosner Marsha Rich
Published: March 2003

Abstract : During development, excess neurons are eliminated by

programmed cell death. Similarly, conditionally immortalized

(SV40‐Tts) rat hippocampal and septal cells undergo cell death following differentiation with several factors such as fibroblast growth factor, constitutively activated Raf‐1, or phorbol esters. The mechanism by which cell death occurs has not been identified. Using RNA differential display, we have identified and characterized a novel immediate early gene (denoted PQR for proline‐ and glutamine‐rich) induced during differentiation of both rat hippocampal and septal cell lines. The 44‐kDa PQR protein, rich in PQ, PH, and QQ repeats, is homologous to a murine protein (TDAG51) required for Fas‐mediated apoptosis in T cells. To determine whether PQR acts as a mediator of apoptosis in neuronal cells, the hippocampal H19‐7 cells were microinjected with either a plasmid expressing PQR cDNA or an antibody against PQR. Microinjection of differentiating H19‐7 cells with a neutralizing antibody against PQR increased the number of surviving cells by 50%. Transient expression of PQR in both differentiating and nondifferentiating H19‐7 cells decreased the number of surviving cells by 35‐50% ; this reduction was reversed by microinjection of PQR antibody. Finally, levels of Fas transcripts are not increased in the neuronal cells, indicating that the mechanism of action differs from that in T cells. These results demonstrate that PQR can be induced by growth factors and differentiating agents and can itself induce apoptosis in hippocampal H19‐7 cells. Furthermore, these data suggest that PQR can function more generally as a mediator of apoptosis and provide a possible mechanism for induction of programmed cell death during neuronal development.
Inhibition of Neutral Sphingomyelinase Activation and Ceramide Formation by Glutathione in Hypoxic PC12 Cell Death
Authors: Yoshimura Shin‐ichi , Banno Yoshiko , Nakashima Shigeru , Hayashi Katsuhiko , Yamakawa Haruki , Sawada Motoshi , Sakai Noboru , Nozawa Yoshinori
Published: March 2003

Abstract : Reduced glutathione (GSH) and N‐acetylcysteine

(NAC), but not other antioxidative or reducing agents, were found to inhibit

cell death, both apoptosis and necrosis, induced by hypoxia in naive and nerve

growth factor‐differentiated PC12 cells. The level of intracellular total GSH

decreased time‐dependently during hypoxia, but exogenously added GSH prevented

such a decrease in GSH. Pretreatment of cells with exogenous GSH or NAC

resulted in inhibition of both neutral sphingomyelinase (SMase) activation and

ceramide formation during hypoxia. In the in vitro assay system, neutral SMase

activity was inhibited dose‐dependently by GSH and NAC. Activation of

caspase‐3 induced by hypoxia was also inhibited by either GSH or NAC. NAC but

not GSH inhibited caspase‐3 activation induced by C2‐ceramide. These results suggest that GSH protects cells from hypoxic injury by direct inhibition of neutral SMase activity and ceramide formation, resulting in inhibition of caspase‐3 activation, and that NAC exerts an additional inhibitory effect(s) downstream of ceramide.