bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurement of a growth‐induced water potential gradient in tall fescue leaves
Authors: MARTRE PIERRE , BOGEAT‐TRIBOULOT MARIE‐BEATRICE , DURAND JEAN‐LOUIS
Published: March 2003

Spatial distribution of cell turgor pressure, cell osmotic pressure and relative elemental growth rate were measured in growing tall fescue leaves (Festuca arundinacea). Cell turgor pressure (measured with a pressure probe) was c. 0.55 MPa in expanding cells but increased steeply (+0.3 MPa) in cells where elongation had stopped. However, cell osmotic pressure (measured with a picolitre osmometer) was almost constant at 0.85 MPa throughout the leaf. The water potential difference between the growth zone and the mature zone (0.3 MPa) was interpreted as a growth‐induced water potential gradient. This and further implications for the mechanism of growth control are discussed.
Androdioecy in the entomophilous tree Fraxinus ornus (Oleaceae)
Authors: DOMMÉE BERTRAND , GESLOT ALAIN , THOMPSON JOHN D. , REILLE MAURICE , DENELLE NICOLE
Published: March 2003

Androdioecy (the coexistence of two genders, cosexuals and males, in a single population) is a rare breeding system. In terms of functional gamete production, androdioecy has been reported in a small number of wind‐pollinated and insect‐pollinated species. In this study we examine the floral biology, stability of gender, and fruit and seed production after self‐pollination and outcrossing pollination in a potentially androdioecious tree, Fraxinus ornus, in southern France. Individual trees have either hermaphrodite flowers or male flowers, the latter lacking a well‐developed gynoecium. The two genders produce morphologically similar pollen and were present in 1∶1 or slightly male‐biased ratios. Only hermaphrodites set fruit during 2–4 yr of observation at four different sites in southern France. Experimental pollinations of eight trees in two different sites showed that hermaphrodites produce viable pollen in dehiscent anthers and viable seeds. Thus, in terms of functional gamete production, F. ornus is a new case of functional androdioecy. Seven hermaphrodites were self‐compatible, and such self‐compatibility of hermaphrodites might improve the colonizing ability of F. ornus in a region where this species is actively expanding its range.
Natural 13C and 15N abundance of field‐collected fungi and their ecological implications
Authors: KOHZU A. , YOSHIOKA T. , ANDO T. , TAKAHASHI M. , KOBA K. , WADA E.
Published: March 2003

The natural abundance of 13C and 15N was measured in basidiocarps of at least 115 species in 88 genera of ectomycorrhizal, wood‐decomposing and litter‐decomposing fungi from Japan and Malaysia. The natural abundance of 13C and 15N was also measured in leaves, litter, soil and wood from three different sites. 15N and 13C were enriched in ectomycorrhizal and wood‐decomposing fungi, respectively, relative to their substrates. Ectomycorrhizal and wood‐decomposing fungi could be distinguished on the basis of their δ13C and δ15N signatures. Although there was high variability in the isotopic composition of fungi, the following isotope‐ enrichment factors (ε, mean±SD) of the fungi relative to substrates were observed:

ε ectomycorrhizal fungi/litter = 6.1±0.4‰15N

ε ectomycorrhizal fungi/wood = 1.4±0.8‰13C

ε wood‐decomposing fungi/wood = −0.6±0.7‰15N

ε wood‐decomposing fungi/wood = 3.5±0.9‰13C

The basis of isotope fractionation in C metabolism from wood to wood‐decomposing fungus is discussed.
Responses to ozone in 12 provenances of European beech (Fagus sylvatica): genotypic variation and chamber effects on photosynthesis and dry‐matter partitioning
Authors: PALUDAN‐MÜLLER G. , SAXE H. , LEVERENZ J. W.
Published: March 2003

Seedlings of 12 provenances of European beech (Fagus sylvatica) were exposed to ambient, non‐filtered air (NF) or NF+50 nl l−1 ozone (NF50) for 8 h d−1 in open‐top chambers (OTCs), from 1 June to 4 October 1995. In 1996 exposure was continued from 31 May to 1 October at four levels: charcoal‐filtered air (CF), NF, NF50 and NF+100 nl l−1 ozone (NF100). Provenances were grown for both seasons in outside reference plots. All treatments were replicated. Ozone did not affect gas exchange in the provenances until late in the second season. NF100 reduced photosynthesis by 18% in August 1996 compared to CF. In September, photosynthesis was reduced by 22% in NF50 and by 29% in NF100. After two seasons, ozone reduced the root:shoot ratio by 24% when comparing CF and NF100; this was caused by reductions in the root biomass. Ozone did not affect height growth or stem diameter, and there were no ozone×provenance interactions for any growth parameter. There was, however, a significant ozone×provenance interaction for photosynthesis, showing northwest European provenances to be more sensitive to ozone than southeast European provenances when comparing dose–response estimates. This is interpreted in terms of genetic adaptation of the photosynthetic apparatus to regional growing conditions. Seedlings in the chambers grew 45% taller, and had 28% more shoot biomass and 29% smaller root biomass, resulting in a 44% reduction of root:shoot ratios compared to seedlings outside. Increased temperature and decreased PAR inside the chambers relative to the outside were probably the main causes for the differences. The magnitude of the chamber effects in OTCs raises doubts about conclusions drawn from ozone exposures in such chambers. This and previous ozone experiments with OTCs may have reached inaccurate conclusions concerning the size of ozone responses due to chamber effects.
Effect of elevated CO2 on the stomatal distribution and leaf physiology of Alnus glutinosa
Authors: POOLE I. , LAWSON T. , WEYERS J. D. B. , RAVEN J. A.
Published: March 2003

Variation in stomatal development and physiology of mature leaves from Alnus glutinosa plants grown under
reference (current ambient, 360 μmol mol−1 CO2) and
double ambient (720 μmol mol−1 CO2) carbon dioxide (CO2)
mole fractions is assessed in terms of relative plant growth, stomatal characters (i.e. stomatal index and density)
and leaf photosynthetic characters. This is the first study to consider the effects of elevated CO2 concentration on
the distribution of stomata and epidermal cells across the whole leaf and to try to ascertain the cause of intraleaf
variation. In general, a doubling of the atmospheric CO2 concentration enhanced plant growth and significantly
increased stomatal index. However, there was no significant change in relative stomatal density. Under elevated
CO2 concentration there was a significant decrease in stomatal conductance and an increase in assimilation rate.
However, no significant differences were found for the maximum rate of carboxylation (Vcmax) and the light
saturated rate of electron transport (Jmax) between the control and elevated CO2 treatment.
Determining uptake of ‘non‐labile’ soil cadmium by Thlaspi caerulescens using isotopic dilution techniques
Authors: HUTCHINSON JULIAN J. , YOUNG SCOTT D. , MCGRATH STEVE P. , WEST HELEN M. , BLACK COLIN R. , BAKER ALAN J. M.
Published: March 2003

We assessed the ability of several populations of the metal‐hyperaccumulator species, Thlaspi caerulescens, to
mobilize non‐labile cadmium in soils historically contaminated by Pb/Zn mine spoil or sewage sludge. Radio‐
labile Cd was determined chemically as an ‘E‐value’, [CdE],
and biologically as an ‘L‐value’, [CdL]. For
comparison, chloride‐extractable Cd, [Cdchlor], was also determined using 1 M CaCl2 as a single‐step soil
extractant. Values of [CdL] were measured for six populations of T. caerulescens that varied substantially in their
ability to assimilate soil Cd, and a non‐accumulator species with a similar growth habit, Lepidium heterophyllum.
Seeds were sown in soil spiked with 109Cd and grown for 9–12 wk in a controlled environment room. Values of
[CdL] were determined from the specific activity of 109Cd and concentration of Cd in the plant leaves. For the six
soils studied, [CdE] ranged from 4.9 to 49% of total soil Cd [CdT]. Values of [CdL] were, in general, in close
agreement with both [CdE] and [Cdchlor] and substantially less than [CdT]. However, [CdL] showed no correlation
with the concentration of Cd in plant tissue, [Cdshoot]. This suggests that, in the soils studied, T. caerulescens did not mobilize non‐labile soil Cd by producing root exudates or altering rhizosphere pH. The results imply that there may be significant restrictions to metal bioavailability, even to hyperaccumulator species, in heavily contaminated soils in which a large proportion of the metal may be present in ‘non‐labile’ forms.
Effects of ectomycorrhizas and vesicular–arbuscular mycorrhizas, alone or in competition, on root colonization and growth of Eucalyptus globulus and E. urophylla
Authors: CHEN Y. L. , BRUNDRETT M. C. , DELL B.
Published: March 2003

Eucalyptus species are considered to have ectomycorrhizas (ECM), but many also have vesicular–arbuscular mycorrhizas (VAM) and their relative importance is unclear. Interactions between ECM and VAM fungi colonizing roots of Eucalyptus species were examined in a glasshouse experiment. This experiment investigated
competition between these two types of fungi and compared benefits provided to Eucalyptus globulus and E.
urophylla. Eucalyptus seedlings were inoculated with spores of the ECM fungus Laccaria lateritia and/or pot‐
culture soil for a VAM fungus (species of Glomus, Acaulospora or Scutellospora). Initial inoculum levels were
important, as VAM fungi became established much more rapidly than Laccaria. In plants with both types of
mycorrhizas, Laccaria mycorrhizas substantially increased after 2 or 3 months and the proportion of roots with
VAM declined. However, the proportion of roots with VAM also decreased significantly in plants without ECM
after 2 months. Thus, the relative susceptibility of eucalypt roots to these mycorrhizal associations changed.
Substantial growth responses to mycorrhizal inoculation occurred when a low concentration of phosphorus
fertilization was used (5 mg kg−1), but not at a higher concentration (10 mg kg−1). Treatments where Laccaria was
applied, alone or in combination with a VAM fungus, resulted in the largest growth increases relative to
nonmycorrhizal plants. An Acaulospora isolate was the most effective VAM fungus for E. urophylla although other
VAM fungi also increased growth relative to the control. VAM fungi alone had little effect on E. globulus growth,
but plants inoculated with both ECM and VAM fungi were larger than plants only inoculated with Laccaria. ECM fungi had a major impact on root system form by reducing the proportion of fine roots (specific root length) relative to nonmycorrhizal plants or those with VAM. Changes in root colonization patterns over time must be considered in studies of ECM/VAM interactions, as there can be substantial changes in the relative importance of the two associations.
Effects of altered water regimes on forest root systems
Authors: JOSLIN J. D. , WOLFE M. H. , HANSON P. J.
Published: March 2003

How ecosystems adapt to climate changes depends in part on how individual trees allocate resources to their components. A review of research using tree seedlings provides some support for the hypothesis that some tree species respond to exposure to drought with increases in root∶shoot ratios but little change in total root biomass. Limited research on mature trees over moderately long time periods (2–10 yr), has given mixed results with some studies also providing evidence for increases in root: shoot ratios. The Throughfall Displacement Experiment (TDE) was designed to simulate both an increase and a decrease of 33% in water inputs to a mature deciduous forest over a number of years. Belowground research on TDE was designed to examine four hypothesized responses to long‐term decreases in water availability; (1) increases in fine‐root biomass, (2) increases in fine root∶foliage ratio, (3) altered rates of fine‐root turnover (FRT), and (4) depth of rooting. Minirhizotron root elongation data from 1994 to 1998 were examined to evaluate the first three hypotheses. Differences across treatments in net fine‐root production (using minirhizotron root elongation observations as indices of biomass production) were small and not significant. Periods of lower root production in the dry treatment were compensated for by higher growth during favorable periods. Although not statistically significant, both the highest production (20 to 60% higher) and mortality (18 to 34% higher) rates were found in the wet treatment, resulting in the highest index of FRT. After 5 yr, a clear picture of stand fine‐root‐system response to drought exposure has yet to emerge in this forest ecosystem. Our results provide little support for either an increase in net fine‐root production or a shift towards an increasing root∶shoot ratio with long‐term drought exposure. One possible explanation for higher FRT rates in the wet treatment could be a positive relationship between FRT and nitrogen and other nutrient availability, as treatments have apparently resulted in increased immobilization of nutrients in the forest floor litter under drier conditions. Such hypotheses point to the continued need to study the interactions of water stress, nutrient availability and carbon‐fixation efficiency in future long‐term studies.
Flavonoids accumulate in leaves and glandular trichomes of Phillyrea latifolia exposed to excess solar radiation
Authors: TATTINI M. , GRAVANO E. , PINELLI P. , MULINACCI N. , ROMANI A.
Published: March 2003

Experiments were conducted on Phillyrea latifolia plants grown under a dense overstorey of Pinus pinea (shade
plants) or on seashore dunes (sun plants) in a coastal area of Tuscany (42° 46′ N, 10° 53′ E). Total integrated
photon flux densities averaged 1.67 and 61.4 m mol m−2 d−1 for shade and sun sites, respectively. A leaf
morphological–structural analysis, a qualitative and quantitative analysis of phenylpropanoids of leaf tissue and
leaf surface, and a histochemical localization of flavonoids were conducted. The area of sun leaves reached 57%
of that of shade leaves, whereas leaf angle (β), sclerophylly index (ratio of leaf d. wt:leaf area), and trichome
frequency (trichome number mm−2
) were markedly greater in leaves exposed to full solar radiation than in leaves
acclimated to shade. The total thickness of sun leaves was 78% higher than that of shade leaves, mostly owing to
a greater development of both palisade parenchyma and spongy mesophyll. The concentration, but not the
composition, of leaf tissue phenylpropanoids varied significantly between sun and shade leaves, with a marked
increase in flavonoid glycosides in sun leaves. Flavonoids occurred almost exclusively in the upper epidermal cells
of shade leaves. By contrast, flavonoids largely accumulated in the upper and lower epidermis, as well as in the
mesophyll tissue of leaves that were acclimated to full sunlight. Flavonoid glycosides were found exclusively in
the secretory products of glandular trichomes of P. latifolia leaves exposed to high levels of light; luteolin 7‐O‐
glucoside and quercetin 3‐O‐rutinoside were the major constituents. By contrast, verbascoside and an unidentified
caffeic acid derivative constituted 72% of total phenylpropanoids secreted by glandular trichomes of shade leaves,
whereas they were not detected in glandular trichomes of sun leaves. These findings suggest that the light‐induced
synthesis of flavonoids in glandular trichomes of P. latifolia probably occurs in situ and concomitantly inactivates
other branch pathways of the general phenylpropanoid metabolism. This is the first report of the key role of
glandular trichomes and of flavonoid glycosides in the integrated mechanisms of acclimation of P. latifolia to excess light.
Modelling a spring wheat crop under elevated CO2 and drought
Authors: Grossman‐Clarke S. , Pinter P. J. , Kartschall T. , Kimball B. A. , Hunsaker D. J. , Wall G. W. , Garcia R. L. , LaMorte R. L.
Published: March 2003

The simulation model DEMETER was used here to investigate which mechanisms led to a larger CO2 effect on biomass production and yield of a spring wheat crop under drought compared with unlimited water supply.

 Field data of the free‐air CO2 enrichment (FACE) wheat experiments in Arizona (1993–94) were used to test the model. The influence of a particular mechanism leading to a higher CO2 effect under drought was investigated by eliminating the influence of the other causes on the simulation results on selected days during the growing seasons.

 A larger CO2 effect under drought was caused in the model by the lower potential transpiration rate, higher root biomass and the nonlinear functional dependence of net assimilation rate on leaf internal CO2 concentration. The contribution of the different mechanisms changed in significance during the growing season depending on the degree of soil water limitation. The model successfully described the qualitative and quantitative behaviour of the crop under elevated CO2.

 A well‐tested simulation model can be a useful tool in understanding the complex interactions underlying observed ecosystem responses to stress under elevated CO2.
Immunocytochemical location of the (1→3) (1→4)‐β‐glucan lichenin in the lichen‐forming ascomycete Cetraria islandica (Icelandic moss)1
Authors: Honegger Rosmarie , Haisch Annette
Published: March 2003

Thalli of Cetraria islandica (Icelandic moss) and Evernia prunastri (oak moss) contain considerable amounts of the linear (1→3), (1→4)‐β‐glucan lichenin (lichenan), which different proportions of linkage groups in either species.

 The immunocytochemical location of lichenan within the thallus is reported using a monoclonal antibody (barley anti‐(1→3), (1→4)‐β‐glucan antibody) and low‐temperature scanning electron microscopy (LTSEM) techniques.

 The antibody labelled ultrathin sections of C. islandica, but not of E. prunastri. In C. islandica, lichenin was located in the extracellular matrix of the peripheral cortex and in a thick outer wall layer of medullary hyphae. LTSEM of fully hydrated and desiccated thalli of C. islandica showed that both the cortical extracellular matrix and the outer wall layer of medullary hyphae shrink dramatically during drought stress, indicating that these are major sites of water storage. A mycobiont‐derived, hydrophilic wall surface layer prevents the thallus interior from becoming water‐logged at full hydration.

 The LTSEM observations and immunocytochemical data strongly suggest that lichenin is a structural compound, rather than a storage product, with important functions in thallus water relations.
Are ectomycorrhizas more abundant than arbuscular mycorrhizas in tropical heath forests?
Authors: Moyersoen B. , Becker P. , Alexander I. J.
Published: March 2003

Tropical heath forests have accumulations of slowly decomposing organic matter at the soil surface. To test the hypothesis that ectomycorrhizas are more abundant than arbuscular mycorrhizas under such conditions, we compared the abundance of ectomycorrhizal (EcM) tree species, and the relative proportions of EcM and arbuscular mycorrhizal (AM) roots, in heath forest and lowland rain forest.

 The mycorrhizal status of trees in two heath forest and two lowland (mixed dipterocarp) forest plots in Brunei Darussalam was determined by literature and field survey. Fine‐root density, proportion of EcM roots, and fractional colonization of EcM and AM roots were measured in monoliths from organic and mineral soil.

 There was no difference in the relative basal area abundance (10–41%) of EcM trees, the proportion of EcM roots in monoliths (8–46%), or fractional colonization (90%) of EcM roots, between the two forest types. However, fractional colonization of AM roots was higher (54%) in heath forest than in mixed dipterocarp forest (27%).

 Our data do not support the hypotheses that ectomycorrhizas are more abundant in, or determine the floristic composition of, tropical heath forests.
Estimating age‐dependent costs and benefits of roots with contrasting life span: comparing apples and oranges
Authors: Bouma Tjeerd J. , Yanai Ruth D. , Elkin Adrienne D. , Hartmond Ulrich , Flores‐Alva Dora E. , Eissenstat David M.
Published: March 2003

The relation between root age and root function is poorly understood, despite its importance to root longevity.

 The effect of root age on respiration rates and 32P‐uptake kinetics was determined for roots excavated from mature apple and citrus trees (median root life spans of 30 vs 300 d). To evaluate whether root longevity maximizes the efficiency of nutrient capture, daily and lifetime efficiencies were calculated by dividing simulated P‐uptake benefits (solute transport model) by age‐specific respiration costs.

 We found that: respiration rates and P uptake capacity change with root age in a species‐specific way; and soil characteristics that determine the rate of nutrient depletion in the rhizosphere are as important as changes in root physiology in determining the age at which a root reaches its maximum efficiency.

 Further testing of the efficiency of nutrient capture as a predictor of root life span requires measurement of both soil properties and age‐specific physiology of roots including their mycorrhizal fungi.
Dual requirement of the LjSym4 gene for mycorrhizal development in epidermal and cortical cells of Lotus japonicus roots
Authors: Novero Mara , Faccio Antonella , Genre Andrea , Stougaard Jens , Webb K. Judith , Mulder Lonneke , Parniske Martin , Bonfante Paola
Published: March 2003

• The LjSym4 mutation leads to Lotus japonicus plants that are defective in arbuscular mycorrhiza (AM) development.

• Two alleles of LjSym4 with different phenotypic strength are compared here. The development of AM was assessed by considering five parameters related to fungal structures present in root segments from wild‐type and mutant plants. The distribution of intercellular hyphae was determined using semithin sections from resin‐embedded roots. Cellular interactions were investigated ultrastructurally, whereas cell wall components from the host plant were identified using immunogold labeling.

• In roots of Ljsym4‐1 mutant, fungal hyphae were mostly restricted to the intercellular spaces of the cortex, indicating a block to infection by mutant cortical cells, which resulted in a very low number of arbuscules.

• This observation suggests the presence of an additional, genetically defined ‘checkpoint’ for mycorrhizal development, located at the wall of cortical cells. The LjSym4 gene is therefore required for infection of both epidermal and cortical cells by AM fungi.
Developmentally regulated conversion of surface‐exposed chitin to chitosan in cell walls of plant pathogenic fungi
Authors: El Gueddari Nour Eddine , Rauchhaus Una , Moerschbacher Bruno M. , Deising Holger B.
Published: March 2003

Conversion of surface‐exposed chitin to chitosan in cell walls of in vitro
‐ and
in vivo
‐differentiated infection structures of two rust fungi, the wheat stem rust fungus
Puccinia graminis
f. sp.
tritici
and the broad bean rust fungus
Uromyces fabae
, and of the causal agent of maize anthracnose,
Colletotrichum graminicola , were studied.

Epi‐fluorescence microscopy with the fluorescence‐labeled lectin wheat germ agglutinin (WGA) revealed that surfaces of infection structures formed on the plant cuticle expose chitin, whereas surfaces of structures formed after invading the host do not.

To identify chitin modification by de‐ N
‐acetylation, we raised polyclonal antibodies specifically recognizing de‐
N ‐acetylated chitosan. These antibodies labeled only those infection structures that differentiate inside the plant, indicating that chitosan is exposed on cell wall surfaces post penetration.

Surface modification of the fungal cell walls by chitin de‐ N ‐acetylation is discussed as a fungal strategy to protect cell walls of pathogenic hyphae from enzymatic hydrolysis by host chitinases, and to avoid generation of an auto‐catalytic defense response system in the invaded host tissue.
Investigating indices to explain the impacts of ozone on the biomass of white clover (Trifolium repens L. cv. Regal) at inland and coastal sites in Spain
Authors: Bermejo V. , Gimeno B. S. , Granados I. , Santamaría J. , Irigoyen J. J. , Bermejo R. , Porcuna J. L. , Mills G.
Published: March 2003

Ozone (O 3
) phytotoxicity is driven by the concentrations the plants are exposed to, and also by meteorological and physiological variables. Thus, O
3
uptake might be a better indicator of the phytotoxicity of this pollutant than O
3 exposure.

An ozone biomonitoring system based on the biomass ratio between an ozone‐resistant (NC‐R) and an ozone‐sensitive (NC‐S) biotype of white clover ( Trifolium repens
cv. Regal) has been tested for 4 yr at four Spanish sites experiencing wide differences in their climatic and ozone exposure profiles. A complementary experiment was also carried out in open‐top chambers exposing plants of both biotypes to three O
3
treatments. Ozone exposure, air temperature, relative humidity and leaf stomatal conductance to water vapour were measured during the life span of both clover clones. These measurements enabled the total O
3
accumulated dose (CUMFO
3 ) for each biotype to be calculated.

The biomass ratio of both clones was best correlated with indices based on O 3
uptake, rather than with indices related to O
3
exposure. In fact, the best performance of the system was achieved when O
3
uptake > 40 nl l
−l
(CUMFO
3 40) was considered.

The results support the need to relate O 3
phytotoxicity to actual plant O
3 uptake.
Combined effect of whey protein and aS1‐casein genotype on the heat stability of goat milk
Authors: Morgan F , Micault S , Fauquant J
Published: March 2003

Goat milk is characterized by a very low heat stability, which could be related to compositional factors. In this paper, the role of the heat‐induced interaction between whey proteins and casein micelles is considered. The effect of the casein micelle structure is evaluated using milk samples with different αS1‐casein genotypes, and the protein interactions are studied at various pH values in order to take into account the strong pH‐dependence of the heat stability. The results suggest that the heat‐induced interaction of β‐lactoglobulin with κ‐casein is of minor importance at natural pH, but is promoted at more elevated pH.
Physicochemical, microbiological and coagulating properties of ewe's milk produced on the Calabrian Mount Poro plateau
Authors: Micari Pietro , Caridi Andrea , Colacino Teresa , Caparra Pasquale , Cufari Antonio
Published: March 2003

Three hundred individual ewe's milks and six bulk milks were analysed over two seasons. The results show that in spring but not in winter the milks examined conform substantially to the standards laid down by Italian legislation. Nevertheless, the data acquired constitute a good starting point from which the quality of ewe's milk produced on the Calabrian Mount Poro plateau should be improved, by performing selection of sheep for mastitis resistance based on somatic cell count, improving the hygiene standards at the dairy, using selected autochthonous lactic starter cultures and, only if strictly necessary, thermal treatment of the milk.
Site‐Specific Dephosphorylation of Tau of Apolipoprotein E‐Deficient and Control Mice by M1 Muscarinic Agonist Treatment
Authors: Genis I. , Fisher A. , Michaelson D. M.
Published: March 2003

Abstract : Apolipoprotein E (apoE)‐deficient mice have memory deficits that are associated with synaptic loss of basal forebrain cholinergic projections and with hyperphosphorylation of distinct epitopes of the microtubuleassociated protein tau. Furthermore, treatment of apoEdeficient mice with the M1 selective agonist 1‐methylpiperidine‐4‐spiro‐(2'‐methylthiazoline) [AF 150(S)] abolishes their memory deficits and results in recovery of their brain cholinergic markers. In the present study, we used a panel of anti‐tau monoclonal antibodies to further map the tau epitopes that are hyperphosphorylated in apoE‐deficient mice and examined the effects of prolonged treatment with AF 150(S). This revealed that tau of apoE‐deficient mice contains a distinct, hyperphosphorylated “hot spot” domain which is localized N‐terminally to the microtubule binding domain of tau, and that AF150(S) has an epitope‐specific tau dephosphorylating effect whose magnitude is affected by apoE deficiency. Accordingly, epitopes which reside in the hyperphosphorylated “hot spot” are dephosphorylated by AF 150(S) in apoE‐deficient mice but are almost unaffected in the controls, whereas epitopes which flank this tau domain are dephosphorylated by AF150(S) in both mice groups. In contrast, epitopes located at the N and C terminals of tau are unaffected by AF150(S) in both groups of mice. These findings suggest that apoE deficiency results in hyperphosphorylation of a distinct tau domain whose excess phosphorylation can be reduced by muscarinic treatment.
Identification of Amino Acids in the N‐Terminal Domain of Corticotropin‐Releasing Factor Receptor 1 that Are Important Determinants of High‐Affinity Ligand Binding
Authors: Wille Sandra , Sydow Sabine , Palchaudhuri Monika R , Spiess Joachim , Dautzenberg Frank M.
Published: March 2003

Abstract : The aim of the present study was to identify the
N‐terminal regions of human corticotropin‐releasing factor (CRF) receptor type
1 (hCRF‐R1) that are crucial for ligand binding. Mutant receptors were
constructed by replacing specific residues in hCRF‐R1 with amino acids from
the corresponding position in the N‐terminal region of the human vasoactive
intestinal peptide receptor type 2 (hVIP‐R2). In cyclic AMP stimulation and
CRF binding assays, it was established that two regions within the N‐terminal
domain were crucial for the binding of CRF receptor agonists and antagonists :
one region mapping to amino acids 43‐50 and a second amino acid sequence
extending from position 76 to 84 of hCRF‐R1. Recently, it was found that the
latter sequence plays a very important role in determining the high ligand
selectivity of the Xenopus CRF‐R1 (xCRF‐R1). Replacement of amino
acids 76‐84 of hCRF‐R1 with residues from the same segment of the hVIP‐R2 N
terminus markedly reduced the binding affinity of CRF ligands. Mutation of
Arg76 or Asn81 but not Gly83 of hCRF‐R1 to the corresponding amino acids of xCRF‐R1 or hVIP‐R2 resulted in 100‐1,000‐fold lower affinities for human/rat CRF, rat urocortin, and astressin. These data underline the importance of the N‐terminal domain of CRF‐R1 in high‐affinity ligand binding.
GTP Cyclohydrolase I Feedback Regulatory Protein Is Expressed in Serotonin Neurons and Regulates Tetrahydrobiopterin Biosynthesis
Authors: Kapatos Gregory , Hirayama Kei , Shimoji Mika , Milstien Sheldon
Published: March 2003

Abstract : Tetrahydrobiopterin, the coenzyme required for hydroxylation of phenylalanine, tyrosine, and tryptophan, regulates its own synthesis through feedback inhibition of GTP cyclohydrolase I (GTPCH) mediated by a regulatory subunit, the GTP cyclohydrolase feedback regulatory protein (GFRP). In the liver, L‐phenylalanine specifically stimulates tetrahydrobiopterin synthesis by displacing tetrahydrobiopterin from the GTPCH‐GFRP complex. To explore the role of this regulatory system in rat brain, we examined the localization of GFRP mRNA using double‐label in situ hybridization. GFRP mRNA expression was abundant in serotonin neurons of the dorsal raphe nucleus but was undetectable in dopamine neurons of the midbrain or norepinephrine neurons of the locus coeruleus. Simultaneous nuclease protection assays for GFRP and GTPCH mRNAs showed that GFRP mRNA is most abundant within the brainstem and that the ratio of GFRP to GTPCH mRNA is much higher than in the ventral midbrain. Two species of GFRP mRNA differing by ~20 nucleotides in length were detected in brainstem but not in other tissues, with the longer, more abundant form being common to other brain regions. It is interesting that the pineal and adrenal glands did not contain detectable levels of GFRP mRNA, although GTPCH mRNA was abundant in both. Primary neuronal cultures were used to examine the role of GFRP‐mediated regulation of GTPCH on tetrahydrobiopterin synthesis within brainstem serotonin neurons and midbrain dopamine neurons. L‐Phenylalanine increased tetrahydrobiopterin levels in serotonin neurons to a maximum of twofold in a concentration‐dependent manner, whereas D‐phenylalanine and L‐tryptophan were without effect. In contrast, tetrahydrobiopterin levels within cultured dopamine neurons were not altered by L‐phenylalanine. The time course of this effect was very rapid, with a maximal response observed within 60 min. Inhibitors of tetrahydrobiopterin biosynthesis prevented the L‐phenylalanine‐induced increase in tetrahydrobiopterin levels. 7,8‐Dihydroneopterin, a reduced pteridine capable of inhibiting GTPCH in a GFRP‐dependent manner, decreased tetrahydrobiopterin levels in cultures of both serotonin and dopamine neurons. This inhibition was reversed by L‐phenylalanine in serotonin but not in dopamine neurons. Our data suggest that GTPCH activity within serotonin neurons is under a tonic inhibitory tone mediated by GFRP and that tetrahydrobiopterin levels are maintained by the balance of intracellular concentrations of tetrahydrobiopterin and L‐phenylalanine. In contrast, although tetrahydrobiopterin biosynthesis within dopamine neurons is also feedback‐regulated, L‐phenylalanine plays no role, and therefore tetrahydrobiopterin may have a direct effect on GTPCH activity.
Down‐Regulation of the Expression of O‐Acetyl‐GD3 by theO‐Acetylesterase cDNA in Hamster Melanoma Cells
Authors: Birklé Stéphane , Ren Shulin , Slominski Andrzej , Zeng Guichao , Gao Luoyi , Yu Robert K.
Published: March 2003

Abstract : The composition of the gangliosides of hamster melanoma
cells is closely related to their cellular growth and degree of
differentiation, with slow‐growing, highly differentiated melanotic melanoma
MI cells expressing GM3 and fast‐growing, undifferentiated amelanotic Ab
melanoma cells having a preponderance of GD3 and O‐acetyl‐GD3. To
study the putative function of O‐acetyl‐GD3, we established stably
transfected AbC‐1 amelanotic hamster melanoma cells with
O‐acetylesterase gene from influenza C virus to hydrolyze the
O‐acetyl group from O‐acetyl‐GD3. The content of
O‐acetyl‐GD3 in the transfected cells expressing
O‐acetylesterase gene was reduced by >90%. These
O‐acetyl‐GD3‐depleted cells differed from the parental ones in their
cellular morphology, growth behavior, and melanogenesis activity. The absence
of O‐acetyl‐GD3 in the transfected cells was accompanied by increased
thick dendrite formation with an enlarged cell body, which is in striking
contrast to the control cells, which were rounded and flattened, with few
processes. Their growth was significantly slower than that of the control
cells. They also demonstrated significantly lower tyrosinase activity and
melanogenic potential. We suggest that the enhanced expression of
melanoma‐associated O‐acetyl‐GD3 ganglioside may stimulate cellular growth and suppress certain differentiated phenotypes such as dendrite formation but not melanogenesis.
Characterization of LY231617 Protection Against Hydrogen Peroxide Toxicity
Authors: Fuson Kimberly S. , Mark Robert J. , Panetta Jill A. , May Patrick C.
Published: March 2003

Abstract : The compound LY231617
{2,6‐bis(1,1‐dimethylethyl)‐4‐[[(1‐ethyl)amino]methyl]phenol hydrochloride}
has been reported to afford significant neuroprotection against hydrogen
peroxide (H2O2)‐induced toxicity in vitro and global
ischemia in vivo. We now report on further mechanistic studies of
H2O2 toxicity and protection by LY231617. Brief exposure
to H2O2 (15 min) elicited an oxidative insult comparable
with that generated by overnight treatment.
H2O2‐mediated cellular degeneration was characterized
using lactate dehydrogenase (LDH) release, changes in total glutathione, and a
new marker of oxidative stress, 8‐epiprostaglandin F2α
(8‐isoprostane). LY231617 attenuated H2O2‐mediated
degeneration under a variety of exposure conditions, including a more
clinically relevant posttreatment paradigm. Levels of 8‐isoprostane paralleled
LDH release under various treatment paradigms of 100 μM
H2O2± 5 μM drug. In contrast, despite affording significant protection, LY231617 had modest to no effects on cellular levels of glutathione. Taken together, these results are consistent with a membrane site of action for LY231617 and suggest that the compound affords cytoprotection via its antioxidant properties.
Amyloid β Protein (25‐35) Phosphorylates MARCKS Through Tyrosine Kinase‐Activated Protein Kinase C Signaling Pathway in Microglia
Authors: Nakai Masamichi , Hojo Kaori , Yagi Keiko , Saito Naoaki , Taniguchi Taizo , Terashima Akira , Kawamata Toshio , Hashimoto Takeshi , Maeda Kiyoshi , Gschwendt Michael , Yamamoto Hideyuki , Miyamoto Eishichi , Tanaka Chikako
Published: March 2003

Abstract : Myristoylated alanine‐rich C kinase substrate (MARCKS) is
a widely distributed specific protein kinase C (PKC) substrate and has been
implicated in membrane trafficking, cell motility, secretion, cell cycle, and
transformation. We found that amyloid β protein (Aβ) (25‐35) and
Aβ (1‐40) phosphorylate MARCKS in primary cultured rat microglia.
Treatment of microglia with Aβ (25‐35) at 10 nM or
12‐O‐tetradecanoylphorbol 13‐acetate (1.6 nM) led to
phosphorylation of MARCKS, an event inhibited by PKC inhibitors,
staurosporine, calphostin C, and chelerythrine. The Aβ (25‐35)‐induced
phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine
kinase inhibitors genistein and herbimycin A, but not with pertussis toxin.
PKC isoforms α, δ, and £ were identified in microglia by
immunocytochemistry and western blots using isoform‐specific antibodies.
PKC‐δ was tyrosine‐phosphorylated by the treatment of microglia for 10
min with Aβ (25‐35) at 10 nM. Other PKC isoforms α and £ were tyrosine‐phosphorylated by Aβ (25‐35), but only to a small extent. We propose that a tyrosine kinase‐activated PKC pathway is involved in the Aβ (25‐35)‐induced phosphorylation of MARCKS in rat microglia.
cDNA Cloning and Molecular Characterization of Human Brain Metalloprotease MP100
Authors: Huber Gerda , Thompson Annick , Grüninger Fiona , Mechler Hans , Hochstrasser Remo , Hauri Hans‐Peter , Malherbe Pari
Published: March 2003

Abstract : Metalloprotease MP100 was originally isolated as a
β‐secretase candidate from human brain using a β‐amyloid precursor
protein (β‐APP)‐derived p‐nitroanilide (pNA) peptide substrate.
Peptide sequences from purified MP100 were now found to resemble sequences
reported for a puromycin‐sensitive aminopeptidase (PSA) highly enriched in
brain, and cDNA cloning revealed nearly complete homology of MP100 to PSA,
with only a single bp difference resulting in an amino acid change at position
184. Another MP100 cDNA encoded a protein with a 36‐amino acid deletion
(positions 180‐217) and a two‐amino acid insertion after Val533.
Purified recombinant human MP100 cleaved the original pNA substrate as well as
a free β‐site‐spanning amyloid β (Aβ) peptide
(Aβ‐10/+10), generating Aβ1‐10. The latter
substrate, however, remained uncleaved, if N‐ and C‐terminally blocked, and
also purified β‐APP was not cleaved. Double immunoimaging revealed
partial, patchy, colocalization of β‐APP and MP100 in doubly transfected
human embryonic kidney cells (HEK cells) and in normal neuroblastoma cells,
and both proteins could be coimmunoprecipitated from rat brain extracts,
suggesting their close vicinity in vivo. Coexpression of MP100 and
β‐APP695, however, did not boost Aβ levels in HEK cells, although active enzyme was produced. Thus, MP100 does not exert true β‐secretase‐like function in cells, although it may well act as a secondary exoprotease in a complex β‐APP/Aβ metabolism.
Role of Calcineurin and Protein Phosphatase‐2A in the Regulation of DARPP‐32 Dephosphorylation in Neostriatal Neurons
Authors: Nishi Akinori , Snyder Gretchen L. , Nairn Angus C. , Greengard Paul
Published: March 2003

Abstract: DARPP‐32, a dopamine‐ and cyclic AMP‐regulated phosphoprotein of Mr 32 kDa, is phosphorylated on Thr34 by cyclic AMP‐dependent protein kinase, resulting in its conversion to a potent inhibitor of protein phosphatase‐1 (PP‐1). Conversely, Thr34‐phosphorylated DARPP‐32 is dephosphorylated and inactivated in vitro by calcineurin and protein phosphatase‐2A (PP‐2A). We have investigated the relative contributions of these protein phosphatases to the regulation of DARPP‐32 dephosphorylation in mouse neostriatal slices. Cyclosporin A (5 μM), a calcineurin inhibitor, maximally increased the level of phosphorylated DARPP‐32 by 17 ± 2‐fold. Okadaic acid (1 μM), an inhibitor of PP‐1 and PP‐2A, had a smaller effect, increasing phospho‐DARPP‐32 by 5.1 ± 1.3‐fold. The effect of okadaic acid on DARPP‐32 phosphorylation was shown to be due to inhibition of PP‐2A activity. Incubation of slices in the presence of cyclosporin A plus either okadaic acid or calyculin A, another PP‐1/PP‐2A inhibitor, caused a synergistic increase in the level of phosphorylated DARPP‐32. The use of Ca2+‐free/EGTA medium mimicked the effects of cyclosporin A on DARPP‐32 phosphorylation, supporting the conclusion that the action of cyclosporin on DARPP‐32 phosphorylation was attributable to blockade of the Ca2+‐dependent activation of calcineurin. The results indicate that calcineurin and PP‐2A, but not PP‐1, act synergistically to maintain a low level of phosphorylated DARPP‐32 in neostriatal slices.
Decreased GABA Enhancement of Benzodiazepine Binding After a Single Dose of Diazepam
Authors: Holt R. A. , Bateson A. N. , Martin I. L.
Published: March 2003

Abstract: The GABA and benzodiazepine binding sites on GABAA receptors are allosterically coupled. The in vitro binding of 2 nM [3H]flunitrazepam to cortical and cerebellar membranes prepared from drug‐naive rats was potentiated ∼1.6‐fold by 100 γM GABA. Potentiation in both regions was significantly reduced 4 or 12 but not 24 h after a single dose of 15 mg/kg diazepam. At 24 h after the last of 14 daily doses of diazepam, no differences in GABA poten‐tiation were observed. Diazepam‐induced changes in GABAA receptor δ2‐subunit gene transcription and α1‐, β2‐, and δ2‐subunit steady‐state mRNA levels did not appear to be temporally related to allosteric uncoupling.
TATA‐Driven Transcriptional Initiation and Regulation of the Rat Serotonin 5‐HT1A Receptor Gene
Authors: Storring John M. , Charest Alain , Cheng Peihua , Albert Paul R.
Published: March 2003

Abstract: The transcriptional initiation and regulation of the rat
serotonin 5‐HT1A receptor gene were characterized. By three types
of analyses, a single brain‐specific site of transcriptional initiation was
localized to ‐967 bp upstream of the translation initiation condon that is
utilized both in hippocampus and in the rat raphe RN46A cell line. This major
site of transcriptional initiation was located 58 bp downstream from a
consensus TATA element, suggesting TATA‐driven transcription of the rat
5‐HT1A receptor. To identify the promoter activity of the receptor
gene, progressive 5′ deletions of the ‐2,719/‐117‐bp fragment of the
5‐HT1A promoter linked to luciferase gene were transfected into
5‐HT1A‐negative (pituitary GH4C1, L6 myoblast, and C6 glioma) and
5‐HT1A‐positive (septal SN‐48 and raphe RN46A) cell lines. Enhancer
regions were identified within a fragment between nucleotides ‐426 and ‐117
that selectively enhanced transcription in 5‐HT1A‐positive cells. A
nonselective enhancer/promoter that mediated expression in all cell lines was
located upstream between ‐1,519 and ‐426 bp in a DNA segment containing
consensus TATA, CCAAT, SP‐1, and AP‐1 elements as well as a
poly‐GT26 dinucleotide repeat. Strong repression of transcription
in all cell lines was conferred by the region upstream of ‐1,519 bp that
contains a 152‐bp DNA segment with >80% identity to RANTES, tumor necrosis
factor‐β, and other immune system genes. Our results indicate that
TATA‐driven expression of the 5‐HT1A receptor is regulated by a novel proximal tissue‐specific enhancer region, a nonselective promoter, and an upstream repressor region that is distinct from previously identified neuron‐specific repressors.
Radicicol Potentiates Neurotrophin‐Mediated Neurite Outgrowth and Survival of Cultured Sensory Neurons from Chick Embryo
Authors: Sano Mamoru , Yoshida Minoru , Fukui Shigeyuki , Kitajima Satoko
Published: March 2003

Abstract: Radicicol, an antifungal antibiotic with markedly low
toxicity, is a potent inhibitor of the Src family of protein tyrosine kinases
and causes morphological reversion of v‐src‐transformed fibroblasts. Recently, this antibiotic was also found to inhibit Raf kinase. In the present study, we found that nanomolar concentrations of radicicol (10 ng/ml) enhanced the survival and neurite outgrowth of neurons from embryonic chick dorsal root ganglia (DRGs) and sympathetic ganglia. It potentiated the trophic effects of nerve growth factor, brain‐derived neurotrophic factor, and neurotrophin‐3 on the cultured DRG neurons. This concentration of radicicol did not alter the tyrosine phosphorylation of Trk receptors or the activity of mitogen‐activated protein (MAP) kinases. Wortmannin, an inhibitor of phosphatidylinositol 3‐kinase (P13‐kinase), did not inhibit radicicol, excluding the involvement of P13‐kinase in the radicicol‐dependent trophic actions. These results suggest that radicicol mediates neuronal growth presumably via a mechanism not involving the activation of Trk receptors, MAP kinase, or P13‐kinase.
L‐Glutamate and Insulin Enhance Glycogen Synthesis in Cultured Astrocytes from the Rat Brain Through Different Intracellular Mechanisms
Authors: Hamai Meiko , Minokoshi Yasuhiko , Shimazu Takashi
Published: March 2003

Abstract: The effects of L‐glutamate and insulin on glycogen synthesis in astrocytes were examined. L‐Glutamate and insulin both stimulated glycogen synthesis in primary cultures of rat astrocytes in a dose‐dependent manner, as measured by the incorporation of 14C from [14C]glucose into glycogen. D‐Aspartate also increased the incorporation of 14C into glycogen. When insulin and L‐glutamate were added together, the glycogen synthesis as well as glycogen content of the cells was additively increased. Wortmannin, an inhibitor of phosphatidylinositol 3‐kinase, had little effect on glycogen synthesis induced by L‐glutamate, whereas it suppressed the insulin‐induced glycogen synthesis. These results suggest that the insulin‐ and L‐glutamate‐induced glycogen syntheses are mediated by different intracellular mechanisms. In fact, insulin stimulated the conversion of glycogen synthase b to glycogen synthase a, which was suppressed by wortmannin. L‐Glutamate and D‐aspartate, however, did not increase the level of glycogen synthase a activity. By contrast, L‐glutamate increased 2‐deoxy‐D‐[3H]glucose uptake by the astrocytes, whereas insulin did not affect the uptake. These results suggest that insulin stimulates glycogen synthesis in astrocytes by activating glycogen synthase, which is dependent on a wortmannin‐sensitive signaling pathway. L‐Glutamate, however, enhances the glucose uptake, which contributes to the increase in glycogen synthesis in the cells.