bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessing leaf pigment content and activity with a reflectometer
Authors: GAMON J. A. , SURFUS J. S.
Published: March 2003

This study explored reflectance indices sampled with a ‘leaf reflectometer’ as measures of pigment content for leaves of contrasting light history, developmental stage and functional type (herbaceous annual versus sclerophyllous evergreen). We employed three reflectance indices: a modified normalized difference vegetation index (NDVI), an index of chlorophyll content; the red/green reflectance ratio (RRED∶RGREEN), an index of anthocyanin content; and the change in photochemical reflectance index upon dark–light conversions (ΔPRI), an index of xanthophyll cycle pigment activity. In Helianthus annuus (sunflower), xanthophyll cycle pigment amounts were linearly related to growth light environment; leaves in full sun contained approximately twice the amount of xanthophyll cycle pigments as leaves in deep shade, and at midday a larger proportion of these pigments were in the photoprotective, de‐epoxidized forms relative to shade leaves. Reflectance indices also revealed contrasting patterns of pigment development in leaves of contrasting structural types (annual versus evergreen). In H. annuus sun leaves, there was a remarkably rapid increase in amounts of both chlorophyll and xanthophyll cycle pigments along a leaf developmental sequence. This pattern contrasted with that of Quercus agrifolia (coast live oak, a sclerophyllous evergreen), which exhibited a gradual development of both chlorophyll and xanthophyll cycle pigments along with a pronounced peak of anthocyanin pigment content in newly expanding leaves. These temporal patterns of pigment development in Q. agrifolia leaves suggest that anthocyanins and xanthophyll cycle pigments serve complementary photoprotective roles during early leaf development. The results illustrate the use of reflectance indices for distinguishing divergent patterns of pigment activity in leaves of contrasting light history and functional type.
Relationships between photosynthesis, nitrogen and leaf structure in 14 grass species and their dependence on the basis of expression
Authors: GARNIER E. , SALAGER J.‐L. , LAURENT G. , SONIÉ L.
Published: March 2003

The relationships between leaf structure, nitrogen concentration and CO2 assimilation rate (A) were studied for 14 grass species grown in the laboratory under non‐limiting nutrient conditions. Structural features included leaf thickness and density, and the proportion of leaf volume occupied by different types of tissue (mesophyll, epidermis, vessels and sclerenchyma). Relationships were assessed for data expressed per unit leaf area and fresh mass. The latter was found to be closely related to leaf volume, which allowed us to use A per unit leaf fresh mass (Afm) as a surrogate of A per unit leaf volume. Assimilation rate per unit leaf area (Aa) was positively correlated with leaf thickness and with the amount of mesophyll per unit leaf area; the relationship with leaf nitrogen content per unit area was only marginally significant. Afm was negatively correlated with leaf thickness and positively with fresh mass‐based leaf organic nitrogen concentration. A multiple regression involving these two variables explained 81% of the variance in Afm. The value of Afm was also significantly related to the proportion of mesophyll in the leaf volume, but surprisingly the correlation was negative. This was because thin leaves with high Afm and nitrogen concentration had proportionally more mechanically supportive tissues than thick ones; as a consequence, they also had a lower proportion of mesophyll. These data suggest that, in addition to leaf nitrogen, leaf thickness has a strong impact on CO2 assimilation rate for the grass species studied.
Extraction of apoplastic sap from plant roots by centrifugation
Authors: YU Q. , TANG C. , CHEN Z. , KUO J.
Published: March 2003

A centrifugal method for extracting apoplastic sap from roots of lupin (Lupinus angustifolius) and pea (Pisum sativum) plants, and a method to analyse malic dehydrogenase in the sap using capillary electrophoresis, are described. Osmolality of apoplastic sap was relatively constant at relative centrifugal forces (RCFs) between 600 and 3000 g for lupin, and between 600 and 4000 g for pea. Electropherograms of a marker enzyme (malic dehydrogenase) and other components in apoplastic and symplastic saps revealed that contamination occurred at 7000 g. It is suggested that apoplastic sap expelled from plant roots at RCF between 600 and 3000 g is free from symplastic contamination, and is regarded as being of apoplastic origin. The proposed method was used to measure apoplastic pH changes in the plant roots in response to external pH, ammonium, nitrate and vanadate.
A model of the effects of water stress on seed advancement and germination
Authors: ROWSE H. R. , McKEE J. M. T. , HIGGS E. C.
Published: March 2003

A model of seed germination is proposed which uses a variable with the units of an osmotic potential (virtual osmotic potential) to integrate the effect of a constant or a varying water potential. This differs from existing models that describe the effects of fixed water potentials on germination, or the effects of fixed priming water potentials on the subsequent germination at a fixed water potential. When a seed is sown, the virtual osmotic potential is assumed to fall at a rate that depends on the ambient water potential, and on the difference between its current and a minimum value. Radicle growth is assumed to initiate when the difference between the ambient water potential and the virtual osmotic potential exceeds a threshold. The germination of carrot and onion seeds at various fixed potentials below 0 MPa was well described by the virtual osmotic potential model. The model was also used to simulate the results of experiments in which seeds were given a single step change in water potential.
The New Phytologist Forum
Authors: Ingram Jonathan
Published: March 2003

New Phytologist has, throughout its history, been a forum for open debate – indeed, in his opening editorial (1902), Tansley referred to ‘easy communication and discussion’ as being the main ideas for the journal launch. The Internet now offers exciting new ways for sharing information, and this has challenged all information providers to reassess what they offer. It is our intention that the new Forum section in the journal should formalize our standing commitment, and encourage serious dialogue – through scientific commentary, correspondence and opinion.

As the journal approaches its hundredth year, so the need for dialogue is stronger than ever. More and more science is published, making it harder, year on year, to keep abreast of important developments. Science is also global, making discussion confined to those within one country or economic bloc to be too restrictive. And as the pressure to publish is as strong as it has ever been, so the free flow of original and unorthodox ideas can too easily become stifled.

New Phytologist, as a high‐impact, high‐quality journal, with its international scope and fierce independence – the Trust is not answerable to any society or commercial publisher – is a respected vehicle for such material. We invite you to participate, and to join us in helping the Forum to develop.
Identification and characterization of an extracellular acid trehalase from the ectomycorrhizal fungus Amanita muscaria
Authors: WISSER G. , GUTTENBERGER M. , HAMPP R. , NEHLS U.
Published: March 2003

The non‐reducing disaccharide trehalose is a major storage compound in most fungi. For a better understanding of carbon metabolism in the ectomycorrhizal‐forming basidiomycete Amanita muscaria, trehalase activity was analysed from mycelium growing in liquid culture or on agar plates, and from mycorrhizas. Trehalase activity was found in both the culture medium and in mycelial extracts. The excreted trehalase was purified to apparent homogeneity by anion‐exchange chromatography, gel filtration and preparative gel electrophoresis. The identified enzyme belongs to the group of acid trehalases. The apparent molecular mass of the native enzyme was estimated to be 165 kDa by gel filtration and 135 kDa by SDS–PAGE. Isoelectrofocusing indicated an isoelectric point (pI) of approx. 3.7, which is typical for acid trehalases. The enzyme is highly specific for its substrate trehalose, with an apparent Km of 0.38 mM. Validamycin and sucrose act as competitive inhibitors with Ki values of 45 nM and 15 mM, respectively. The activity of acid trehalase excreted into the growth medium is independent of the carbon source (glucose or trehalose), revealing that the enzyme is not regulated by its substrate trehalose. Nevertheless, fungal hyphae grown in the absence of an external carbon source showed enhanced enzyme excretion. The biochemical characteristics of the trehalase activity measured in the mycelium are in the same range as those determined for the excreted enzyme. The enzyme is localized in the cell wall and its activity is strongly decreased in ectomycorrhizas.
Evolution of metal hyperaccumulation and phytoremediation hype
Authors: Ernst Wilfried H. O.
Published: March 2003

Cadmium accumulation and tolerance are discussed in a New Phytologist article by Krämer (2000), which comments
on a paper by Lombi et al. (2000). In this context, a number of additional points should be made, putting the role of humans in the evolution of metal resistance into context and emphasizing what is the ‘hype’ of phytoremediation.

It is important that sites created by humans should not be overemphasized in considering the evolution of metal resistance. Plants resistant to heavy metals have their primary sites not on these, but on soils where ores are outcropping, the so‐called metalliferous or orogenic soils (Ernst, 1974). Over thousands of years, natural exposure to a surplus of various metals, depending on the mineralization process, has driven the evolution of metal resistance in many plant species under the local environmental conditions. Many publications have shown that Thlaspi caerulescens can hyperaccumulate Zn (e.g.
Vázquez et al., 1992), and accumulate other heavy metals
such as Cu and Pb depending on soil chemistry (e.g. Baker
et al., 1994). It has been known for more than 30 years that
T. caerulescens gives a good response to experimentally
supplied high Zn levels (Ernst, 1968). One of the ecological
effects of hyperaccumulation of heavy metals is a defence
against herbivorous insects (Boyd & Martens, 1994). This
effect is enhanced by a preferential accumulation of heavy
metals in the epidermal leaf layer (Heath et al., 1997).
Traffic exposure increases natural 15N and heavy metal concentrations in mosses
Authors: PEARSON J. , WELLS D. M. , SELLER K. J. , BENNETT A. , SOARES A. , WOODALL J. , INGROUILLE M. J.
Published: March 2003

Mosses have been used as biomonitors of atmospheric pollution for some years, but few studies have been carried out on the effect of NOx emissions from traffic on moss tissue N. Eight species of moss (102 samples) growing on
walls or roofs next to roads exposed to different traffic densities were collected from urban and rural sites in the
UK. The shoots were sampled for total N, their stable isotope 15N/14N content (δ15N) and heavy metal content
(Pb, Zn). There was a lack of correlation between tissue total N and traffic exposure, but a very good correlation
between traffic exposure and tissue δ15N. Plants collected near motorways or busy urban roads had δ15N values
ranging between +6 and −1‰, while in rural areas with hardly any traffic these ranged from −2 to −12‰. In
a separate survey of mosses, the average δ15N of shoots from busy roadsides in London was +3.66‰, whereas from
samples collected from farm buildings near poultry or cattle pens it was −7.8‰. This indicates that the two main
atmospheric N sources, NOx and NHx, have different δ15N signatures, the former tending to be positive and the
latter negative. Tissue concentrations of both Pb and Zn show a strong positive correlation with traffic exposure,
with Zn in particular being greater than Pb. The results are discussed with regard to the use of moss tissue Zn
as a means for monitoring or mapping pollution from vehicles, and of δ15N as an aid to distinguish between urban
(NOx) and rural (NHx) forms of N pollution.
Seed weight and seed number affect subsequent fitness in outcrossing and selfing Primula species
Authors: TREMAYNE MICHELLE A. , RICHARDS A. J.
Published: March 2003

Using the outcrossing Primula farinosa and its autogamous selfing relatives
P. scotica, P. scandinavica and P.
stricta, we compared the fitness of light and heavy seeds. Heavy seeds germinated in greater numbers and more
quickly. In competition with seedlings grown from lighter seeds, heavy seeds produced larger rosettes. In P.
farinosa such seedlings went on to produce more seeds, and in two populations heavier seeds, than plants from
lighter seeds. After transplantation to natural populations, seedlings of P. farinosa derived from heavy seeds
produced larger rosettes, more flowers and seeds than those from lighter seeds in certain populations so that
seedlings born of heavy seeds were much fitter than seedlings from lighter seeds. Average seed weight varied in
inverse proportion to seed number per capsule. The autogamous species produced on average about twice as many
seeds per capsule as P. farinosa. In P. scotica and P. stricta this difference appears to be due in part to assured
fertilization, but this high fecundity did not cause disadvantageously light seeds. As these species produced fewer
capsules per scape, their overall seed production was on average no greater than for P. farinosa. P. farinosa traded‐off fitness between capsules with large seed numbers, which donated more offspring to the next generation, and those with small seed numbers, whose heavy seeds would be more likely to reproduce themselves in the next generation. We conclude that low fecundity in outcrossing species might at times be advantageous.
Glomus intraradices causes differential changes in amino acid and starch concentrations of in vitro strawberry subjected to water stress
Authors: HERNÁNDEZ‐SEBASTIÀ CINTA , SAMSON GUY , BERNIER PIERRE‐YVES , PICHÉ YVES , DESJARDINS YVES
Published: March 2003

The effect of colonization of tissue‐cultured strawberry (Fragaria×ananassa
Duch. cv. Kent) plantlets in vitro by
the arbuscular mycorrhizal fungus (AMF) Glomus intraradices on plantlet response to poly(ethylene glycol)
(PEG)‐8000‐induced water stress was investigated. The plantlets were inoculated axenically and co‐cultured with
the AMF for 4 wk, then transferred to 15% PEG‐8000 solutions for 4, 8 and 12 h. Relative water content, water
potential, osmotic potential, leaf conductance for water vapour diffusion and photosynthetic efficiency as estimated
by chlorophyll a fluorescence were all affected by the PEG treatment and its duration but not by the presence of
the intraradical phase of the AMF. However, distinct differences in PEG‐induced changes in amino acid content
were observed between nonmycorrhizal and mycorrhizal plantlets. In the latter, the treatment with PEG caused
a substantial decrease in asparagine levels in leaves that was accompanied by a marked increase in asparagine
concentration in roots. The opposite was observed in nonmycorrhizal plantlets. Furthermore, concentrations of
aspartic acid, serine, threonine, amino‐N‐butyric acid, alanine and starch increased in roots of mycorrhizal and decreased in nonmycorrhizal plantlets. Our results suggest the presence of a mobile pool of asparagine that can be translocated from leaves to roots or vice versa in response to PEG‐induced water stress, depending on the mycorrhizal status of the plantlets. These opposite patterns suggest different strategies of mycorrhizal and nonmycorrhizal plantlets to water stress, which seem to involve different adjustments in nitrogen and carbon metabolism.
Metabolism‐induced free radical activity does not contribute significantly to loss of viability in moist‐stored recalcitrant seeds of contrasting species
Authors: GREGGAINS VALERIE , FINCH‐SAVAGE WILLIAM E. , QUICK W. PAUL , ATHERTON NEIL M.
Published: March 2003

Because recalcitrant seeds are not desiccation‐tolerant they must be stored moist. Their limited storage potential presents significant practical problems, but the cause of viability loss is not known. It has been suggested that a stress‐induced metabolic imbalance can develop during storage that results in free‐radical generation and consequent damage. To investigate this hypothesis, the presence of a stable free radical, lipid peroxidation and representative enzymatic and nonenzymatic protection mechanisms against oxidative attack were monitored in nondormant recalcitrant seeds during moist storage. A comparison was made between seeds of a short‐lived sub‐tropical species (Avicennia marina) and two longer‐lived temperate species
(Quercus robur and Castanea sativa).
As a test of the hypothesis, seeds of both temperate species were held under conditions of elevated temperature
and oxygen concentration to develop different rates of respiration during storage. The number of normal seedlings
produced from seeds of the two temperate species declined during storage, but viability remained high, so effects
of ageing were not confounded with an increasing proportion of dead seeds in the population. Under these
conditions, lipid peroxidation changed little over the storage period, although there was evidence of accumulation
of a stable free radical in Q. robur axes. However, this response was not affected by storage conditions that elevated
respiration rates. In the shorter‐lived A. marina seeds viability declined soon after the start of storage, but the
significant increase in free radicals shown by EPR measurement was only evident when an increasing percentage
of the seed population was no longer viable. Changes in the activity of scavenging enzymes and the concentration
of antioxidants were time‐dependent and not related to respiration rates. Therefore, in the present work, no
consistent evidence was found to show that metabolism‐induced free‐radical activity was a significant contributing
factor to pre‐mortem deterioration in moist‐stored recalcitrant seeds.
Elevated CO2 and nitrogen supply alter leaf longevity of grassland species
Authors: Craine Joseph M. , Reich Peter B.
Published: March 2003

The longevity of green leaf area for 10 grassland species was measured to establish whether elevated CO2 and N fertilization alter leaf longevity, an important determinant of ecosystem leaf area and ecosystem carbon gain.

 Plants were selected from monocultures in their second year of growth in a field experiment that directly manipulated atmospheric CO2 (550 ppm and ambient) and nitrogen fertilization (4 g N m−2 and ambient). Leaves were censused biweekly over a 4‐month period.

 Leaf longevity increased under elevated CO2 (+3.4 d, P = 0.03) and decreased under elevated N (−4.2 d, P = 0.03). Leaf longevity increased under elevated CO2 for C3 species only; there was no change in leaf longevity of C4 species under elevated CO2. For both CO2 and N, changes in leaf longevity were congruent with expectations based on observed changes in N cycling.

 In addition to supplies of resources such as CO2 and N, site fertility and the development of ecoystem feedbacks appear to be important in determining leaf longevity.
Characterization of an Aux/IAA cDNA upregulated in Pinus pinaster roots in response to colonization by the ectomycorrhizal fungus Hebeloma cylindrosporum
Authors: Charvet‐Candela V. , Hitchin S. , Ernst D. , Sandermann H. , Marmeisse R. , Gay G.
Published: March 2003

• In an attempt to determine whether fungal auxin affects host plant gene expression during mycorrhizal formation, an auxin upregulated cDNA, Pp‐iaa88, was isolated by differential screening of a cDNA library made from auxin‐treated Pinus pinaster roots.

• Pp‐iaa88 codes for a polypeptide that shares extensive homology to auxin‐inducible Aux/IAA proteins, which are supposed to act as transcription factors. Cycloheximide did not inhibit auxin‐induced mRNA accumulation, indicating that Pp‐iaa88 upregulation is a primary (direct) auxin response.

• The level of Pp‐iaa88 transcripts in roots increased following inoculation with either an indoleacetic acid‐overproducing mutant or a wild‐type strain of the ectomycorrhizal fungus Hebeloma cylindrosporum. With both strains, mRNA accumulation was detectable as soon as fungal hyphae reached the root and it increased during differentiation of symbiotic structures. The kinetics of Pp‐iaa88 transcript accumulation was closely connected with the dynamics of symbiosis establishment and was more rapid with the mutant than with the wild‐type strain.

• As a putative transcription factor expressed at the very early stages of symbiosis establishment, Pp‐iaa88 could play a key role in mycorrhizal formation.
Lichens respond to heavy metals by phytochelatin synthesis
Authors: Pawlik‐Skowrońska Barbara , Di Toppi Luigi Sanità , Favali Maria Augusta , Fossati Fabrizia , Pirszel Jacek , Skowroński Tadeusz
Published: March 2003

The main aims of this paper are to investigate: if widespread lichen species ( Xanthoria parietina
,
Physconia grisea
and
Physcia adscendens , collected in Italy and Poland) exposed to cadmium (Cd), lead (Pb) and zinc (Zn) synthesize phytochelatins; whether phytochelatins are synthesized by the heterotrophic fungus (mycobiont) or by the photosynthetic alga (photobiont), or by both of the partners; and if there are significant differences in phytochelatin production in the same lichen species growing at different latitudes.

In the lichens investigated, Cd, Pb and Zn induced the biosynthesis of phytochelatins (PC 2
, PC
3
and PC
4
) and some DES‐GLYCIL derivatives. However, only the photobiont partners (the green microalgae of the genus
Trebouxia ) were capable of phytochelatin production and, by contrast, aposymbiotically grown mycobionts only produced glutathione.

The Cd‐exposed lichens grown in Italy produced significantly larger amounts of phytochelatins than the same species grown in Poland. An opposite trend was observed in Pb‐exposed lichens.

The trebouxioid lichens may gain an ecological advantage from their capability to counter heavy metal stress with prompt phytochelatin synthesis.
The effects of skirt lining properties on comfort and movement
Authors: Kawabata Hiroko , Obara Kazuyuki , Komiyama Hitomi , Narumi Taeko
Published: March 2003

This study was conducted to clarify the influences of the lining properties of skirts upon comfort and movement. The comfort of lining fabrics and real skirts with and without lining was rated by sensory tests on the basis of sensorial comfort on the skin and body movement. Actual body movements were investigated by motion analysis while the skirts were being worn. The results are described: (1) Comfort on the skin, movement and overall preference for the lining fabrics were evaluated by sensory testing, comparing skirts made with five kinds of lining fabrics. Conventional non‐stretch cupro lining was ranked first on the basis of smoothness and good feel to the skin. Two‐way stretch lining was also ranked highly in terms of ease of movement. We discuss how sensorial comfort on the skin and ease of movement related to overall preference for lining fabrics. (2) Sensory test of the actual skirts with and without lining clearly confirmed that lining played a role in improving comfort. (3) For a non‐stretch fabric skirt, conventional lining was ranked highly for good feel to the skin, and two‐way stretch lining was considered useful for ease of movement. There was no significant difference between the two lining fabrics in overall preference. (4) For a stretch fabric skirt, stretch lining is the most useful because it maintains easy extension of the outer fabric, thus allowing ease of body movement. (5) Motion analysis showed that a combination of knitted stretch skirts with stretch lining was the best for ease of movement. Comparing the sidelines and the hemline of the stretch fabric skirts demonstrated a better shape using lining.
Effect of growth phase on the subsequent growth kinetics of psychrotrophic bacteria of raw milk origin
Authors: Rowe Michael T , Dunstall George , Kilpatrick David , Wisdom G Brian
Published: March 2003

Psychrotrophs isolated at days 1, 3 and 5 from refrigerated raw milk were monitored for growth kinetics using conductance when they had been inoculated during their lag, log and stationary growth phases. The results indicated that there was a more rapid change in the microflora during the initial stages of storage when the lag phases of the 1, 3 and 5 day isolates were analysed. There was also evidence of the ‘suicide response’. In addition, the fact that the stationary phase cells showed the greatest exponential growth rate may be a contributory factor to their selective advantage.
The Swiss Federal Dairy Research Station
Authors: Rüegg Max
Published: March 2003

The Federal Dairy Research Station (FAM) was established at the beginning of the last century. It is one of six agricultural research stations of the Swiss Department of Agriculture and the leading national research institute in dairy technology and consulting. It is part of the Swiss Centre of Competence for Animal Production and Foods of Animal Origin. Its mission is to improve the competitive position of the Swiss milk producers and dairy industry. The institute is organized into three functional areas corresponding to its well‐established core competencies: (1) production of raw milk cheese and microorganisms for fermentation, (2) knowledge transfer and consultation for the dairy industry and beekeeping, and (3) testing of, and providing expertise on dairy and bee products. Research work is carried out using a multidisciplinary approach in a matrix organization. Two product‐ and customer‐orientated technology units and one unit for consulting, contractual work and other services for the dairy industry are supported in their projects by three scientific and technical units (microbiology; chemistry, physics and sensory analysis; engineering and production) as well as by a unit offering central services. The 4‐year research programmes are based on the strategic goals of the agricultural policy as well as on the current needs of the dairy industry. An advisory board oversees the preparation and execution of the projects. FAM is connected to an international network for both scientific work and supervisory tasks. Work is carried out within the background of the political strategy of a sustainable development including economic, ecological and social aspects. A primary research focus is therefore the avoidance of excessive processing of milk and dairy products, and keeping products natural and free of residues by applying only minimal, unavoidable treatments and additives.
Identification and Transgenic Analysis of a Murine Promoter that Targets Cholinergic Neuron Expression
Authors: Naciff Jorge M. , Behbehani Michael M. , Misawa Hidemi , Dedman John R.
Published: March 2003

Abstract : Choline acetyltransferase (ChAT) is a specific phenotypic
marker of cholinergic neurons. Previous reports showed that different upstream
regions of the ChAT gene are necessary for cell type‐specific expression of
reporter genes in cholinergic cell lines. The identity of the mouse ChAT
promoter region controlling the establishment, maintenance, and plasticity of
the cholinergic phenotype in vivo is not known. We characterized a promoter
region of the mouse ChAT gene in transgenic mice, using β‐galactosidase
(LacZ) as a reporter gene. A 3,402‐bp segment from the
5′‐untranslated region of the mouse ChAT gene (from ‐3,356 to +46, +1
being the translation initiation site) was sufficient to direct the expression
of LacZ to selected neurons of the nervous system ; however, it did
not provide complete cholinergic specificity. A larger fragment (6,417 bp,
from ‐6,371 to +46) of this region contains the requisite regulatory elements
that restrict expression of the LacZ reporter gene only in cholinergic neurons of transgenic mice. This 6.4‐kb DNA fragment encompasses 633 bp of the 5′‐flanking region of the mouse vesicular acetylcholine transporter (VAChT), the entire open reading frame of the VAChT gene, contained within the first intron of the ChAT gene, and sequences upstream of the start coding sequences of the ChAT gene. This promoter will allow targeting of specific gene products to cholinergic neurons to evaluate the mechanisms of diseases characterized by dysfunction of cholinergic neurons and will be valuable in design strategies to correct those disorders.
Neuroprotective Role of Na+/myo‐Inositol Cotransporter Against Veratridine Cytotoxicity
Authors: Yamashita Toshihide , Yamauchi Atsushi , Miyai Akiko , Taniguchi Masaaki , Yoshimine Toshiki , Tohyama Masaya
Published: March 2003

Abstract: Na+/myo‐inositol cotransporter has been shown to protect cells from the perturbing effects of hypertonic stress by the accumulation of myo‐inositol. Here we report a regulatory mechanism for the cotransporter. Induction of myo‐inositol cotransporter mRNA was observed after exposure to veratridine, a voltagegated sodium channel opener. The veratridine‐elicited induction was inhibited when Na+ was eliminated from the bath, although calcium chelation failed to modify the gene expression. Veratridine evoked an accumulation of Na+ in the cells, which paralleled the abundance of the mRNA. These results strongly suggested that an increase in Na+ influx due to sodium channel opening affected transcription of the cotransporter gene. Activity of the myo‐inositol cotransporter was also up‐regulated after veratridine exposure. To clarify the possible roles of myo‐inositol accumulation under veratridine exposure, we next examined the neurotoxic effects of veratridine when myo‐inositol uptake was blocked. Neither 30 μM veratridine nor 500 μM 2‐O,C‐methylene myo‐inositol, a competitive inhibitor of myo‐inositol, elicited apparent cytotoxicity. However, a combination of these agents markedly increased cytotoxicity in culture, suggesting that an adequate amount of myo‐inositol was necessary when the cells were stimulated with veratridine.
Specificity of the Dynorphin‐Processing Endoprotease: Comparison with Prohormone Convertases
Authors: Berman Yemiliya , Juliano Luiz , Devi Lakshmi A.
Published: March 2003

Abstract: The cleavage specificity of a monobasic processing dynorphin converting endoprotease is examined with a series of quench fluorescent peptide substrates and compared with the cleavage specificity of prohormone convertases. A dynorphin B‐29‐derived peptide, Abz‐Arg‐Arg‐Gln‐Phe‐Lys‐Val‐Val‐Thr‐Arg‐Ser‐Glneddnp (where Abz is o‐aminobenzoyl and eddnp is ethylenediamine 2,4‐dinitrophenyl), that contains both dibasic and monobasic cleavage sites is efficiently cleaved by the dynorphin converting enzyme and not cleaved by two propeptide processing enzymes, furin and prohormone convertase 1. A shorter prorenin‐related peptide, Dnp‐Arg‐Met‐Ala‐Arg‐Leu‐Thr‐Leu‐eddnp, that contains a monobasic cleavage site is cleaved by the dynorphin converting enzyme and prohormone convertase 1 and not by furin. Substitution of the P1’ position by Ala moderately affects cleavage by the dynorphin‐processing enzyme and prohormone convertase 1. It is interesting that this substitution results in efficient cleavage by furin. The site of cleavage, as determined by matrix‐assisted laser desorption/ionization time of flight mass spectrometry, is N‐terminal to the Arg at the P1 position for the dynorphin converting enzyme and C‐terminal to the Arg at the P1 position for furin and prohormone convertase 1. Peptides with additional basic residues at the P2 and at P4 positions also serve as substrates for the dynorphin converting enzyme. This enzyme cleaves shorter peptide substrates with significantly lower efficiency as compared with the longer peptide substrates, suggesting that the dynorphin converting enzyme prefers longer peptides that contain monobasic processing sites as substrates. Taken together, these results suggest that the cleavage specificity of the dynorphin converting enzyme is distinct but related to the cleavage specificity of the prohormone convertases and that multiple enzymes could be involved in the processing of peptide hormones and neuropeptides at monobasic and dibasic sites.
Endothelins Stimulate Expression of Cyclooxygenase 2 in Rat Cultured Astrocytes
Authors: Koyama Yutaka , Mizobata Tsutomu , Yamamoto Naoyoshi , Hashimoto Hitoshi , Matsuda Toshio , Baba Akemichi
Published: March 2003

Abstract : Endothelin (ET) is one of the active endogenous substances regulating the functions of astrocytes. In the present study, we examined effects of ET on cyclooxygenase (COX) expression in cultured astrocytes. ET‐3 (100 nM) caused transient increases in the expression of both COX2 mRNA and protein, but not those of COX1, in cultured astrocytes. ET‐induced COX2 mRNA expression was suppressed by 5 μg/ml actinomycin D, 30 μM BAPTA/AM, inhibitors of protein kinase C (1‐100 nM staurosporin and 100 μM H‐7), 2 μM dexamethasone, and prolonged treatment with 100 nM phorbol 12‐myristate 13‐acetate. ET‐3 stimulated production of prostaglandin (PG) E2 in cultured astrocytes. The effect of ET‐3 on the PGE2 production was diminished by actinomycin D. Indomethacin and NS398, a selective COX2 inhibitor, comparably decreased both the basal and the ET‐stimulated PGE2 production. Proliferation of cultured astrocytes was stimulated by 100 nM ET‐3, and the increased proliferation was reduced by co‐addition of 1 μM PGE2. Treatment with 1 μM PGE2 caused astrocytic morphological changes accompanied by disappearance of stress fibers, a prominent structure of organized cytoskeletal actin in cultured astrocytes. In the presence of 10 nM ET‐3, PGE2 did not show an effect on astrocytic actin organization. The present study shows that ET is an inducer of astrocytic COX2 and suggests that ET‐induced PGE2 production through COX2 may be involved in the regulation of astrocytic functions.
Increased Butyrylcholinesterase Levels in Microsomal Membranes of Dystrophic Lama2dy Mouse Muscle
Authors: Moral‐Naranjo María Teresa , Campoy Francisco Javier , Cabezas‐Herrera Juan , Vidal Cecilio J.
Published: March 2003

Abstract : The proportions and the glycosylation of butyrylcholinesterase (BuChE) forms in vesicles rich in sarcoplasmic reticulum from normal (NMV) and dystrophic (DMV) muscle were analyzed, using merosin‐deficient dystrophic mice. BuChE activity in DMV was two‐ to threefold that in NMV. Globular amphiphilic GA1, GA2, and GA4 and hydrophilic GH4 BuChE forms were identified in NMV and DMV. The amount of GA2 forms increased sevenfold in DMV, and the other forms increased about twofold. The higher BuChE level in DMV might reflect a maturational defect, with dystrophy preventing the down‐regulation of BuChE with muscle development. About half of GA1, GA2, and GH4 BuChE forms in NMV or DMV bound to Lens culinaris agglutinin (LCA), a higher fraction to wheat germ agglutinin (WGA), and little to Ricinus communis agglutinin (RCA). Most of the GA4 forms in NMV or DMV bound to LCA or WGA ; those from NMV failed to bind to RCA, whereas most of the variants in DMV bound to it, suggesting that the excess of tetramers in DMV is mainly RCA‐reactive. The differential interaction of lectins with BuChE components from muscle microsomes, serum, and nerves confirmed that the microsomal BuChE was muscle‐intrinsic. The results provide clues regarding the alterations that dystrophy produces in the biosynthesis of BuChE forms in muscle.
Extracellular Glucose Concentrations in the Rat Hippocampus Measured byZero‐Net‐Flux
Authors: McNay Ewan C. , Gold Paul E.
Published: March 2003

Abstract : The concentration of glucose in the brain's extracellularfluid remains controversial, with recent estimates and measurements rangingfrom 0.35 to 3.3 mM. In the present experiments, we used the methodof zero‐net‐flux microdialysis to determine glucose concentration in thehippocampal extracellular fluid of awake, freely moving rats. In addition, thepoint of zero‐net‐flux was measured across variations in flow rate to confirmthat the results for glucose measurement were robust to such variations. In3‐month‐old male Sprague‐Dawley rats, the concentration of glucose in thehippocampal extracellular fluid was found to be 1.00 ± 0.05mM, which did not vary with changes in flow rate. Three‐month‐old and24‐month‐old Fischer‐344 rats both showed a significantly higher hippocampalextracellular fluid glucose concentration, at 1.24 ± 0.07 and 1.21± 0.04 mM, respectively ; there was no significant differencebetween the two age groups. The present data demonstrate variation inextracellular brain glucose concentration between rat strains. When takentogether with previous data showing a striatal extracellular glucoseconcentration on the order of 0.5 mM, the data also demonstratevariation in extracellular glucose between brain regions. Traditional modelsof brain glucose transport and distribution, in which extracellularconcentration is assumed to be constant, may require revision.
Delineation of the Structural Determinants of the N‐Methyl‐D‐Aspartate Receptor Glycine Binding Site
Authors: Sandhu Satpal , Grimwood Sarah , Mortishire‐Smith Russell J. , Whiting Paul J. , Le Bourdellès Béatrice
Published: March 2003

Abstract: In this study, we have further delineated the domains of the N‐methyl‐D‐aspartate receptor NR1 subunit that contribute to the glycine co‐agonist binding site. Taking an iterative approach, we have constructed truncation mutants of the NR1 subunit, transiently expressed them in HEK‐293 cells, and determined the binding of the glycine site antagonist [3H]L‐689,560. Amino acids 380‐811 were sufficient to form a glycine binding site with affinities for [3H]L‐689,560 and glycine that were not significantly different from wild‐type NR1. More extensive deletions, from either the amino‐ or the carboxy‐terminal end, resulted in loss of ligand binding. Additional constructs were made starting from amino acids 380‐843 of NR1, replacing the transmembrane (TMI‐TMIII) domain with intervening linker sequences while retaining the TMIV domain so as to anchor the polypeptide to the membrane. Although robust amounts of polypeptides were synthesised by transfected cells, only low levels of [3H]L‐689,560 binding sites could be detected. This suggests that only a small proportion of the synthesised polypeptide folds in the appropriate manner so as to form a ligand binding site. These data indicate that although it is possible to reduce the glycine binding site to minimal so‐called S1 and S2 domains, efficient folding of the polypeptide so as to form a ligand binding site may require sequences within the TMI‐TMIII domain.
Intracellular Polyamine Levels Are Involved in NMDA‐Evoked Nitric Oxide Production in Chick Retina Cells
Authors: Ientile Riccardo , Pedale Santa , Ginoprelli Tiziana , Cannavò Letterio , Macaione Salvatore
Published: March 2003

Abstract: The NMDA‐sensitive glutamate receptor complex can be modulated by numerous drugs and endogenous substances such as polyamines. We studied the pathway of arginine/nitric oxide/cyclic GMP in cultured chick retina cells through NMDA receptor activation, seen as a function of both differentiation stages of culture and intracellular polyamine levels. In our experimental conditions, the nitric oxide synthase activity was stimulated by NMDA from three to four times between embryonic day (E) 8 plus 5 days in vitro (C) and E8C7. The NMDA response was blocked by MK‐801 (10 μM) by >60% at stage E8C5. During culture differentiation, the NMDA‐induced increase in nitric oxide synthase activity at the E8C5 stage was blocked by preliminary incubation (24 h) of the cells with α‐difluoromethylornithine, the inhibitor of polyamine biosynthesis. This effect was assessed by a reduction of NMDA‐evoked cyclic GMP formation in polyamine‐depleted retina cells. Thus, intracellular polyamine levels are involved in NMDA‐evoked nitric oxide production. Our results indicate that (a) the developmental pattern of polyamine levels can be associated with the modulation of NMDA‐evoked events and (b) the NMDA‐mediated effects have been reduced in α‐difluoromethylornithine‐treated cell cultures. These observations provide evidence for a physiological interaction between polyamines and NMDA‐sensitive glutamate receptors during differentiation stages of cultured chick retina cells.
The Monoamine Neurons of the Rat Brain Preferentially Express a Splice Variant of α1B Subunit of the N‐Type Calcium Channel
Authors: Ghasemzadeh M. Behnam , Pierce R. Christopher , Kalivas Peter W.
Published: March 2003

Abstract : The N‐type voltage‐dependent calcium channels play a

significant role in neurotransmitter release. The α1B subunit

of the N‐type calcium channel functions as the primary subunit that forms the

pore and contains the structural motifs that mediate the pharmacological and

gating properties of the channel. We report on an isoform of the

α1B subunit that is preferentially expressed by the

monoaminergic neurons of the rat brain. This isoform contains a 21‐amino acid

cassette in the synprint site present in the cytoplasmic loop between domains

IIS6 and IIIS1. RT‐PCR of micropunched tissue was used to show preferential

expression of this isoform in regions of the brain containing monoaminergic

neurons and to a lesser extent in the cerebellum. Double‐label in situ

hybridization was used to show expression of this isoform mRNA in dopaminergic

neurons of the ventral mesencephalon. The expression of two distinct N‐type

calcium channels containing these α1B subunit isoforms by the monoaminergic neurons may provide for synapse‐specific regulation of neurotransmitter release.
Opioids Transiently Prevent Activation of Apoptotic Mechanisms Following Short Periods of Serum Withdrawal
Authors: Dermitzaki E. , Chatzaki E. , Gravanis A. , Margioris A. N.
Published: March 2003

Abstract: Opioids exert a proapoptotic effect on several normal and
tumoral cells. The aim of the present article was to examine the effect of
opioids on the PC12 rat pheochromocytoma cell line, a model for the study of
chromaffin cell apoptosis. These cells produce δ‐ and κ‐opioid
agonists and their receptors. Our results were as follows: The κ‐ and
δ2‐opioid receptor agonists had a rapid but transient effect
on apoptosis at 3 h, whereas μ opioids did not. The effect of opioids was
reversible by the opioid antagonists naloxone and
nor‐binaltorphimine. The effect of opioids was protective, suppressing serum deprivation‐induced apoptosis to ∼50% of controls. The protective effect of opioids on PC12 apoptosis was measurable only under serum deprivation. The effect of opioids was remarkably reproducible and highly constant in timing, which did not appear to depend on the duration of the preceding serum deprivation. Finally, opioids prevented the elevation of the Bcl‐2 and Bak proteins following serum deprivation to the levels attained by serum supplementation. Our combined data suggest that opioids protect PC12 cells from entering a state of induced apoptosis following serum deprivation.
Novel Cathepsin D Inhibitors Block the Formation of Hyperphosphorylated Tau Fragments in Hippocampus
Authors: Bi Xiaoning , Haque Tasir S. , Zhou Jun , Skillman A. Geoffrey , Lin Bin , Lee Christine E. , Kuntz Irwin D. , Ellman Jonathan A. , Lynch Gary
Published: March 2003

Abstract: Lysosomal disturbances may be a contributing factor to Alzheimer's disease. We used novel compounds to test if suppression of the lysosomal protease cathepsin D blocks production of known precursors to neurofibrillary tangles. Partial lysosomal dysfunction was induced in cultured hippocampal slices with a selective inhibitor of cathepsins B and L. This led within 48 h to hyperphosphorylated tau protein fragments recognized by antibodies against human tangles. Potent nonpeptidic cathepsin D inhibitors developed using combinatorial chemistry and structure‐based design blocked production of the fragments in a dose‐dependent fashion. Threshold was in the submicromolar range, with higher concentrations producing complete suppression. The effects were selective and not accompanied by pathophysiology. Comparable results were obtained with three structurally distinct inhibitors. These results support the hypothesis that cathepsin D links lysosomal dysfunction to the etiology of Alzheimer's disease and suggest a new approach to treating the disease.
Adrenomedullin Receptor Is Found Exclusively in Noradrenaline‐Secreting Cells of the Rat Adrenal Medulla
Authors: Renshaw D. , Thomson L. M. , Michael G. J. , Carroll M. , Kapas S. , Hinson J. P.
Published: March 2003

Abstract: Adrenomedullin, originally identified in the adrenal
medulla, has binding sites in the adrenal gland; however, its role in the
adrenal medulla is unclear. This study was designed to characterise
adrenomedullin binding sites in the rat adrenal medulla, using ligand binding
studies, immunocytochemistry, and mRNA analysis. A single population of
specific adrenomedullin receptors was identified in adrenal medullary
homogenates. 125I‐Adrenomedullin was displaced only by
adrenomedullin1‐50 and not by calcitonin gene‐related peptide or
amylin at concentrations up to 100 nmol/L. The receptor KD
was 3.64 nmol/L with a receptor density of 570 fmol/mg of protein. Analysis of
mRNA revealed that the genes encoding both the putative adrenomedullin
receptors, termed calcitonin receptor‐like receptor (CRLR) and L1, were
expressed in the rat adrenal medulla. Dual‐colour indirect‐labelled
immunofluorescence was used to localise phenylethanolamine
N‐methyltransferase (PNMT) and the adrenomedullin receptor in the same section. PNMT is the enzyme that converts noradrenaline to adrenaline and is not expressed in noradrenaline‐secreting cells. These studies revealed that both CRLR and L1 were expressed only in cells that did not express PNMT, suggesting that adrenomedullin receptors are only found in noradrenaline‐secreting cells. Further evidence to support this conclusion was provided by the demonstration of colocalisation of adrenomedullin receptors with dopamine β‐hydroxylase, confirming the presence of the receptors in medullary chromaffin cells. Taken together, these data suggest that adrenomedullin acts through a specific adrenomedullin receptor in the rat adrenal medulla. RT‐PCR and northern blot analysis revealed greater abundance of mRNA for L1 than for CRLR, possibly suggesting that L1 may be the major adrenomedullin receptor expressed in this tissue. As it has been reported that adrenomedullin is synthesised predominantly by adrenaline‐secreting cells, it appears likely that adrenomedullin is a paracrine regulator in the adrenal medulla.
Effect of p‐Chloroamphetamine on 5‐HT1A and 5‐HT7 Serotonin Receptor Expression in Rat Brain
Authors: García‐Osta Ana , Frechilla Diana , Del Río Joaquín
Published: March 2003

Abstract: The aim of this study was to investigate if p‐chloroamphetamine (PCA), which is neurotoxic to serotonin (5‐HT) nerve terminals, was able to induce, like 3,4‐methylenedioxymethamphetamine, a region‐specific regulation of 5‐HT1A receptor mRNA expression. The effect of PCA on the expression of 5‐HT7 receptors, which share some pharmacological properties with 5‐HT1A receptors, was comparatively studied. PCA (2 × 5 mg/kg) produced a lasting depletion of 5‐HT content in the rat frontal cortex and hippocampus. In the hippocampus, the maximal 5‐HT depletion was found on day 21 (‐70%), whereas in the cortex, the highest 5‐HT depletion was found on day 14 (‐73%), with a partial but significant recovery on day 21. At the latter time point, 5‐HT1A receptor mRNA expression was increased by 80% in the cortex and decreased by 50% in the hippocampus. The 5‐HT1A receptor mRNA expression was also enhanced after exposure to PCA of rat cortical but not of hippocampal primary cultures. In regard to 5‐HT7 receptor mRNA expression, the most remarkable change after PCA was the great increase (+200%) in the brainstem. Binding studies to 5‐HT1A receptors matched the changes in receptor mRNA expression. Gel shift assays revealed enhanced nuclear protein binding to the κB sequence with use of cortical but not hippocampal extracts of PCA‐treated rats. Overall, the data show region‐specific changes in 5‐HT receptor‐type expression that may not be entirely dependent on the neurotoxic effect of PCA on 5‐HT terminals.