bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitrogen assimilation in Lolium perenne colonized by the arbuscular mycorrhizal fungus Glomus fasciculatum
Authors: FAURE SANDRINE , CLIQUET JEAN‐BERNARD , THEPHANY GAELLE , BOUCAUD JEAN
Published: March 2003

To investigate nitrogen assimilation in Lolium perenne L. colonized by the arbuscular mycorrhizal (AM) fungus Glomus fasciculatum (Thax. sensu Gerd.), nitrate uptake, key enzyme activities, and 15N incorporation into free amino acids were measured. After a 4‐h labelling period with [15N]nitrate, 15N content was higher in roots and shoots of AM‐plants than in those of control plants. Glutamine synthetase (GS) and nitrate reductase (NR) activities were increased in shoots of AM‐plants, but not in roots. More label was incorporated into amino acids in shoots of AM plants. Glutamine, glutamate, alanine and γ‐aminobutyric acid were the major sinks for 15N in roots and shoots of control and AM plants. Interactions between mycorrhizal colonization, phosphate and nitrate nutrition and NR activity were investigated in plants which received different amounts of phosphate or nitrate. In shoots of control plants, NR activity was not stimulated by high levels of phosphate nutrition but was stimulated by high levels of nitrate. At 4 mM nitrate in the nutrient solution, NR activity was similar in control and AM plants. We concluded that mycorrhizal effects on nitrate assimilation are not mediated via improved phosphate nutrition, but could be due to improved nitrogen uptake and translocation.
How does the C4 grass Eragrostis pilosa respond to elevated carbon dioxide and infection with the parasitic angiosperm Striga hermonthica?
Authors: WATLING J. R. , PRESS M. C.
Published: March 2003

Eragrostis pilosa (Linn.) P Beauv., a C4 grass native to east Africa, was grown at both ambient (350 μmol mol−1 and elevated (700 μmol mol−1) CO2 in either the presence or absence of the obligate, root hemi‐parasite Striga hermonthica (Del.) Benth. Biomass of infected grasses was only 50% that of uninfected grasses at both CO2 concentrations, with stems and reproductive tissues of infected plants being most severely affected. By contrast, CO2 concentration had no effect on growth of E. pilosa, although rates of photosynthesis were enhanced by 30–40% at elevated CO2. Infection with S. hermonthica did not affect either rates of photosynthesis or leaf areas of E. pilosa, but did bring about an increase in root∶shoot ratio, leaf nitrogen and phosphorus concentration and a decline in leaf starch concentration at both ambient and elevated CO2. Striga hermonthica had higher rates of photosynthesis and shoot concentrations of soluble sugars at elevated CO2, but there was no difference in biomass relative to ambient grown plants. Both infection and growth at elevated CO2 resulted in an increase in the Δ13C value of leaf tissue of E. pilosa, with the CO2 effect being greater. The proportion of host‐derived carbon in parasite tissue, as determined from δ13C values, was 27% and 39% in ambient and elevated CO2 grown plants, respectively. In conclusion, infection with S. hermonthica limited growth of E. pilosa, and this limitation was not removed or alleviated by growing the association at elevated CO2.
Nitrogen and phosphorus acquisition by the mycelium of the ectomycorrhizal fungus Paxillus involutus and its effect on host nutrition
Authors: BRANDES BETTINA , GODBOLD DOUGLAS L. , KUHN ARND J. , JENTSCHKE GEORG
Published: March 2003

The contribution of the extramatrical mycelium to N and P nutrition of mycorrhizal Norway spruce (Picea abies (L.) Karst.) was investigated. Seedlings either inoculated with Paxillus involutus (Batsch) Fr. or non‐mycorrhizal were grown in a two compartment sand culture system where hyphae were separated from roots by a 45 μm nylon net. Nutrient solution of the hyphal compartment contained either 1.8 mm NH4+ and 0.18 mm H2PO4− or no N and P. Aluminium added to the hyphal compartment as a tracer of mass flow was not detected in the plant compartment, indicating that measurements of N and P transfer by the mycelium were not biased by solute movement across the nylon net.

The addition of N and P to the hyphal compartment markedly increased dry weight, N and P concentration and N and P content of mycorrhizal plants. Calculating uptake from the difference in input and output of nutrient in solution confirmed a hyphal contribution of 73% and 76% to total N and P uptake, respectively. Hyphal growth was increased at the site of nutrient solution input.
The effect of light on the growth and reproduction of Floerkea proserpinacoides
Authors: MCKENNA MARGARET F. , HOULE GILLES
Published: March 2003

Floerkea proserpinacoides (Limnanthaceae) is a spring ephemeral annual species that grows in deciduous forests throughout eastern North America. Seeds germinate from late November to December, although the first leaf emerges only from late March to early April. Growth begins in early April at the onset of favourable temperatures, following snowmelt, and continues through mid‐June. Senescence coincides with increasing air temperature and decreasing light level as a result of canopy closure. In this paper, we present the results of a growth chamber study designed to determine the effect of light level on growth, biomass allocation and reproduction of F. proserpinacoides. The study consists of two parts: in a first experiment, plants were grown at five constant photosynthetic photon fluence rates (PPFR: 90, 180, 360, 540 or 900 μmol m−2 s−1), and in a second experiment, PPFR was reduced from 900 μmol m−2 s−1 to 180 μmol m−2 s−1 after 0, 14, 21, 28 or 35 d of growth. Relative humidity, temperature, nutrient and water supply were kept constant in a hydroponic sand culture experiment. Total biomass, leaf mass and leaf area increased with increasing PPFR up to 540 μmol m−2 s−1. Plants grown at the highest (900 μmol m−2 s−1) and the lowest (90 μmol m−2 s−1) PPFR had a substantially lower biomass by the end of the 35‐d growth period than plants grown at intermediate PPFRs (360 or 540 μmol m−2 s−1). Despite differences in total biomass, there were no significant differences in seed production among treatments. The mean relative growth rate (RGR) increased with increasing light levels between 90–540 μmol m−2 s−1, and it was reduced at 900 μmol m−2 s−1. However, differences in RGR were not significant among treatments. Specific leaf area did not vary consistently as a function of light level, whereas leaf area ratio and leaf mass ratio tended to increase with increasing PPFR, reaching maximum values at 360–540 μmol m−2 s−1. However, none of these growth variables differed significantly across the range of PPFR levels. The transfer of plants to lower PPFR had no significant effect on any of the growth components. Biomass production for the species appeared to be optimized at PPFR of 360–540 μmol m−2 s−1. Growth might be restricted by an insufficient supply of photosynthates at low PPFR and by photoinhibitory processes at higher PPFRs.
The effect of abscisic acid on chlorophyll fluorescence in lichens under extreme water regimes
Authors: DIETZ S. , HARTUNG W.
Published: March 2003

Chlorophyll fluorescence measurements were used to test whether externally applied abscisic acid (ABA) has an effect on the desiccation tolerance of lichens and their sensitivity to constant water saturation. A surplus of ABA did not decrease the time required to regain full photosynthetic capacity after prolonged dry periods. However, an effect of ABA could be observed when lichens were permanently hydrated at moderately high temperatures. Lichens suffered less from constant saturation if ABA was added to the incubation medium. These results suggest a positive effect of ABA on membrane function.
Seasonal variation in heteroside concentrations of field‐collected Porphyra species (Rhodophyta) from different biogeographic regions
Authors: KARSTEN ULF
Published: March 2003

Eight different species of the red algal genus Porphyra from different biogeographic regions in Europe, Africa, North America, Asia and Australia were collected over the course of the respective growth season and surveyed for the qualitative and quantitative occurrence of the low molecular weight carbohydrates, floridoside, D‐isofloridoside and L‐isofloridoside. Except for Porphyra saldanhae from South Africa, all species tested exhibited clear seasonal variations in their heteroside composition. Whereas Porphyra dioica and Porphyra umbilicalis from the North Sea showed highest carbohydrate concentrations in spring and summer, those of Porphyra leucosticta from Spain were highest in winter. The red algae studied exhibited highest concentrations of heteroside at different seasons, some in winter, others in spring or summer. The composition of the three compounds varied among the species studied. In P. columbina from Australia, L‐isofloridoside was always quantitatively dominant, while floridoside was the major component in P. dioica. Usually D‐isofloridoside was present in small concentrations, except in P. perforata from the Pacific coast of the USA where it occurred in equal concentrations with floridoside and L‐isofloridoside. The results point to species‐specific different enzymic activities of the underlying anabolic pathways. Correlations between the heteroside concentrations and various prevailing environmental parameters are detectable but do not reveal a general response of Porphyra. Data from the literature prove that numerous physical factors determine the growth patterns of various Porphyra species in different biogeographic regions. Of these, photoperiod, temperature and nutrients are the most important. Highest heteroside concentrations in the Porphyra species studied always coincided with enhanced growth, and this relationship determined that growth, though related to abiotic conditions, was also promoted by maximum carbohydrate content. The multiple physiological function of floridoside, D‐isofloridoside and L‐isofloridoside as osmolytes, compatible solutes and carbon reserves is discussed in relation to the environmental stresses that Porphyra species usually experience in their upper intertidal habitats.
Glomales SSUrRNA gene diversity
Authors: Schüßler Arthur
Published: March 2003

The arbuscular mycorrhiza (AM) is one of the most important symbioses on earth, formed between about 80% of vascular plants and AM fungi (Smith & Read, 1997), which are placed in the order Glomales (Morton & Benny, 1990). A discussion has recently arisen about the rDNA‐gene and general genome organization of this fungal group, which, as far as is known, does not possess sexuality (Sanders, 1999). This discussion mainly arose as a result of the assumption that one spore of an AM fungus (Scutellospora castanea) contains small subunit (SSU) rRNA genes that belong, phylogenetically, to different families within the Glomales, namely the Gigasporaceae and Glomaceae (Hosny et al., 1999). Questions about genetic drift, infrequent recombination, concerted evolution and the validity of phylogenetic techniques have arisen (Hosny et al., 1999; Sanders, 1999). However, this assumption might well be based on the sequence of an Ascomycete contaminant.
Geographical and seasonal influences on the distribution of fungal endophytes in Quercus ilex
Authors: COLLADO J. , PLATAS G. , GONZÁLEZ I. , PELÁEZ F.
Published: March 2003

A systematic survey of the endophytic assemblages of Quercus ilex in central Spain has been performed, with the goal of evaluating the importance of geographical and seasonal factors on these fungal communities. Four sampling sites were selected; one of them was sampled twice, in the spring and the autumn. The collected plant material consisted of bark, twigs and leaves from eight trees per site. Fungal strains were isolated with the use of a surface‐sterilization method with sodium hypochlorite. A total of 2921 fungal strains grouped into 149 ‘species’ or morphological types were recovered. The 10 dominant species, with isolation frequencies >1.5%, were Pyrenochaeta sp., Periconiella anamorph of Biscogniauxia mediterranea (De Not.) Kuntze, Pseudonectria sp., Cryptosporiopsis quercina Petrak, Alternaria alternata (Fr:) Keissl., two undetermined coelomycetes, Penicillium funiculosum Thom, Diplodia mutila Fr. apud Mont. and Ascochyta sp. Medians of fungal species per tree were significantly different among the sampled sites. The isolation frequencies of the dominant species, as well as other less frequent species, were significantly dependent on the sampling site. The degree of endophytic infection and the diversity of fungal species were significantly higher in the spring. The frequencies of all dominant species at one of the sites depended significantly on the season, except for C. quercina, Acremonium sclerotigenum (F & V Moreau ex Valenta) Gams. and D. mutila. Cluster analysis of the whole endophytic mycoflora of the sampled trees suggested that the geographical factor affects the endophytic distribution patterns more significantly than the seasonal factor.
Utilization of dissolved inorganic carbon (DIC) and the response of the marine flagellate Isochrysis galbana to carbon or nitrogen stress
Authors: CLARK D. R. , MERRETT M. J. , FLYNN K. J.
Published: March 2003

The growth of the marine flagellate Isochrysis galbana was followed in batch cultures at four concentrations of dissolved inorganic carbon (DIC), from C‐ and N‐replete lag phase into C‐ and/or N‐deplete stationary phase. Organic buffers were omitted from the growth medium, and culture pH was maintained at 8.30±0.05 by the addition of acid or alkali. The responses of the flagellate to N stress included an increase in the C∶N ratio, and decreases in the ratios of glutamine (Gln)∶glutamate (Glu) and Chl a∶C, and the cell Chl a quota. Conversely, the responses to C stress included a decrease in the C∶N ratio, and increases in the ratios of Gln∶Glu and Chl a∶C, and the cell Chl a quota. The relationship between carbon‐specific growth rate (C‐μ), and the concentration of extracellular DIC, [DIC]ext, exhibited Michaelis–Menten type kinetics with a half saturation constant, KG(DIC), of 81 μM. Comparative studies of the diatom Phaeodactylum tricornutum showed similar results, although the value of KG(DIC) was lower at 30 μM.
Raman spectroscopy of pigments and oxalates in situ within epilithic lichens: Acarospora from the Antarctic and Mediterranean
Authors: HOLDER J. M. , WYNN‐WILLIAMS D. D. , RULL PEREZ F. , EDWARDS H. G. M.
Published: March 2003

Fourier Transform laser Raman spectroscopy was used to generate diagnostic spectra for pigments and biodegradative calcium oxalate in situ in two yellow‐pigmented species of the lichen genus Acarospora from contrasting sites in the Antarctic and the Mediterranean. This non‐intrusive technique was used to identify the photoprotective pigments rhizocarpic acid and β‐carotene by their unique Raman spectral fingerprints. The use of low energy near‐IR excitation at 1064 nm eliminated interference from autofluorescence of photosynthetic pigments. The insensitivity of the technique to water permitted the use of field‐fresh material. The dominant yellow pigment, rhizocarpic acid, gave a diagnostic pattern of corroborative bands at wavenumbers (ν) 1596, 1665, 1620 and 1000 cm−1. It was possible to discriminate between hydration states of calcium oxalate; the monohydrate (whewellite) featured a ν(CO) stretching band at 1493 cm−1 whereas the dihydrate (weddellite) had a contrasting ν(CO) stretching band at 1476 cm−1. Fourier Transform deconvolution and intensity measurements were used to obtain relative quantitative data for rhizocarpic acid by using its ν(CO) and ν(CONH) amide modes, for carotenoid pigment by its ν(C = C) band at 1520 cm−1 and for calcium oxalates by their ν(CO) bands. ν(CO), ν(CONH) and ν(C = C) are the vibrational stretching modes of the carbonyl C = O, protein amide 1 and alkenyl C = C moieties, respectively, in the pigments and metabolic products of the Acarospora lichens. The ability to determine the precise (20 μm spot diameter) spatial distribution of these key functional molecules in field‐fresh thallus profiles and variegations has great potential for understanding the survival strategies of lichens, which receive high insolation, including elevated levels of UV‐B, under extremes of desiccation and temperature in hot and cold desert habitats.
Severe photobiont injuries of lichens are strongly associated with air pollution
Authors: TARHANEN SARI , POIKOLAINEN JARMO , HOLOPAINEN TOINI , OKSANEN JARI
Published: March 2003

The photobiont ultrastructure of the epiphytic lichens Bryoria fuscescens and Bryoria fremontii was studied along
the pollution gradient from two Cu‐Ni smelters in Nikel and Monchegorsk in northern Finland and north‐western
Russia. The relationship between ultrastructural characteristics of B. fuscescens and environmental factors (i.e.
climate, atmospheric SO2 and bark element concentrations) was studied by using a principal component analysis
(PCA) aiming to assess the air quality in a northern environment. Based on PCA, increased plasmolysis and
mitochondrial changes in the Trebouxia photobiont were significantly correlated with elevated pollutant concentrations. Degenerated cells, showing altered chloroplasts and electron‐translucent pyrenoglobuli, occurred in lichens growing 35–50 km from the Monchegorsk smelter. Cell wall and cytoplasmic lipid volumes, and size of pyrenoglobuli, positively correlated with the distance from the Monchegorsk smelter. Vacuoles and electron‐opaque vacuolar deposits were significantly increased at the Finnish site in the vicinity of a pulp mill. Swelling of mitochondrial cristae and thylakoids showed little correlation with environmental factors, but indicated of initial stage of injuries and were observed at several slightly polluted sites in northern Finland and north‐western Russia. The results suggest that the severe photobiont injuries of lichens are strongly associated with poor air quality.
Root structure and function in an ecological context
Authors: Eissenstat D.
Published: March 2003

Ecological theories concerning the relationship of suites of traits with resource supply and plant potential growth rate have concentrated primarily on leaf structure and physiology – much less is known about interspecific patterns of variation in root structure and physiology. In this issue, Wahl & Ryser reduce this knowledge gap by demonstrating important correlations of root structure with potential growth rate and height among a range of perennial temperate grass species adapted to sites of a wide range of fertility (see pp. 459–471).
Ecotypic variation in selenium accumulation among populations of Stanleya pinnata
Authors: Feist Laura J. , Parker David R.
Published: March 2003

• Ecotypic variation in selenium (Se) hyperaccumulation in plants is reported here among populations of Stanleya pinnata (Brassicaceae), which has a broad biogeographical range in the western USA.

• In a glasshouse study, Se and sulfur accumulation were examined in 16 populations of S. pinnata. Plants grown from seed (collected from sites representing the species range) were subjected to five treatments differing in selenate (SeO42−) and sulfate (SO42−) concentrations.

• The populations differed in shoot Se concentration by 1.4‐ to 3.6‐fold, depending on the treatment, and these concentrations were positively correlated with the indigenous soil Se levels at the collection sites. Shoot S concentrations varied by less than two‐fold, and did not correlate with the shoot Se levels. All populations accumulated SeO42− preferentially over SO42−. By contrast, Brassica juncea seedlings grown in a similar solution series consistently accumulated SO42− preferentially over SeO42−. Biomass production differed up to three‐fold between populations.

•  S. pinnata is a primary Se accumulator, but populations exhibit significant ecotypic differences in Se accumulation. Environmental concerns about Se are common, and the broad adaptation of S. pinnata makes it an attractive candidate for phytoremediation.
Nutritive value and the nitrogen dynamics of Trifolium subterraneum and Phalaris aquatica under warmer, high CO2 conditions
Authors: Lilley J. M. , Bolger T. P. , Peoples M. B. , Gifford R. M.
Published: March 2003

Will changes in nutritive values and N‐relations offset initial gains in forage productivity under potential climate change observed for grass–legume pastures of south‐eastern Australia?

 Herbage nutritive value and symbiotic nitrogen fixation were investigated for pure and mixed swards of subterranean clover (Trifolium subterraneum) and phalaris (Phalaris aquatica) in field tunnels at ambient and 690 µmol mol−1 CO2 concentrations and at ambient and warmed (+3.4°C) air temperatures.

 Elevated CO2 increased the nonstructural carbohydrate content of herbage whereas warming tended to decrease it. These effects were mainly on soluble carbohydrates in phalaris and starch in clover herbage. The N concentration of both species was decreased by elevated CO2 but unaffected by warming. The proportion of clover‐N derived from N2 fixation was increased by 12% under elevated CO2 but decreased by 6% under warming.

 Concurrent warming and high‐CO2 conditions are expected to lead to improved herbage nutritive value for ruminants due to increased nonstructural soluble carbohydrate content. Longer term effects on nutritive value and N‐dynamics via species persistence and competition require further study.
Basal hymenomycetes belonging to the Sebacinaceae are ectomycorrhizal on temperate deciduous trees
Authors: Selosse M.‐A. , Bauer R. , Moyersoen B.
Published: March 2003

• Heterobasidiomycetous species of the Sebacinaceae family, previously considered as saprophytes or parasites, are shown here to form ectomycorrhizas on temperate forest trees.

• Ectomycorrhizas were collected under sebacinoid sporophores and near root systems of Neottia nidus‐avis, an orchid symbiotic with sebacinoids. To identify the partners each ectomycorrhiza was submitted to amplification and sequencing of the plant and fungal internal transcribed spacer (ITS), and further investigated by light and electron microscopy whenever a sebacinoid ITS was found.

• Molecular and microscopic analyses correlated well. Two sebacinoids of divergent rDNA sequences were demonstrated to form similar ectomycorrhizas, with a well‐developed Hartig net and a hyphal mantle having thick‐walled outer mantle hyphae. The ultrastructure of the septal pore (dolipore with imperforate caps) was typical for sebacinoids. In one case, intracellular colonization was seen. The ectomycorrhizal host range of these sebacinoids was not specific and included Betulaceae, Fagaceae and Tiliaceae.

• Sebacinoids probably represent an overlooked ectomycorrhizal group and ectomycorrhizal symbiosis may be common among basal lineages of hymenomycetes.
Possible Involvement of Interleukin‐1 in Cyclooxygenase‐2Induction After Spinal Cord Injury in Rats
Authors: Tonai Takeharu , Taketani Yutaka , Ueda Natsuo , Nishisho Takehiko , Ohmoto Yasukazu , Sakata Yasuyo , Muraguchi Masahiro , Wada Kaori , Yamamoto Shozo
Published: March 2003

Abstract : A standardized compression injury of rat spinal cord
brought about a time‐dependent biphasic production of thromboxane
A2 (detected as thromboxane B2) and prostaglandin
I2 (detected as 6‐ketoprostaglandin F1α.
Thromboxane B2 was predominant during the first 1 h, whereas the
6‐ketoprostaglandin F1α level exceeded that of thromboxane
B2 at 8 h postinjury. As examined by inhibitor experiments and
northern blotting, cyclooxygenase‐1 was responsible for the first phase, and
cyclooxygenase‐2 was involved in the second phase. On compression injury the
levels of interleukin‐1α and ‐1β detected as mRNA and protein
increased and peaked at 2‐4 h. Injection of exogenous interleukin‐1 α
into the spinal cord resulted in an increase of cyclooxygenase‐2 mRNA content
and a predominant production of 6‐ketoprostaglandin F1α resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin‐1α injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti‐cyclooxygenase‐2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase‐1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase‐2 mediated by interleukin‐1 α.
Molecular Cloning of a Putative Cyclic Nucleotide‐Gated Ion Channel cDNA from Limulus polyphemus
Authors: Chen Frank H , Ukhanova Maria , Thomas David , Afshar Ghazaleh , Tanda Soichi , Battelle Barbara‐Anne , Payne Richard
Published: March 2003

Abstract : Cyclic nucleotide‐gated channels have been proposed to
mediate the electrical response to light in the ventral photoreceptor cells of
the horseshoe crab, Limulus polyphemus. However, a cyclic
nucleotide‐gated channel has not been identified from Limulus. We
have cloned a putative full‐length cyclic nucleotide‐gated channel cDNA by
screening cDNA libraries constructed from Limulus brain using a probe
developed from Limulus ventral eye nerves. The putative full‐length
cDNA was derived from two overlapping partial cDNA clones. The open reading
frame encodes 905 amino acids ; the sequence shows 44% identity to that of the
α subunit of the bovine rod cyclic GMP‐gated channel over the region
containing the transmembrane domains and the cyclic nucleotide binding domain.
This Limulus channel has a novel C‐terminal region of ~200 amino
acids, containing three putative Src homology domain 3 binding motifs
and a putative coiled‐coil domain. The possibility that this cloned channel is
the same as that detected previously in excised patches from the
photoreceptive membrane of Limulus ventral photoreceptors is discussed in terms of its sequence and its expression in the ventral eye nerves.
Antioxidative and Proapoptotic Effects of Riluzole on Cultured Cortical Neurons
Authors: Koh Jae‐Young , Kim Dae‐Kyong , Hwang Jee Yeon , Kim Yang Hee , Seo Ji Heui
Published: March 2003

Abstract : Riluzole is used clinically in patients with amyotrophic
lateral sclerosis. As oxidative stress, in addition to excitotoxicity, may be
a major mechanism of motoneuron degeneration in patients with amyotrophic
lateral sclerosis, we examined whether riluzole protects against
nonexcitotoxic oxidative injury. Probably reflecting its weak antiexcitotoxic
effects, riluzole (1‐30 μM) attenuated submaximal neuronal death
induced by 24‐h exposure to 30 μM kainate or NMDA, but not that by
100 μM NMDA, in cortical cultures. Riluzole also attenuated
nonexcitotoxic oxidative injury induced by exposure to FeCl3 in the
presence of MK‐801 and CNQX. Consistent with its antioxidative effects,
riluzole reduced Fe3+‐induced lipid peroxidation, and inhibited
cytosolic phospholipase A2. By contrast, riluzole did not attenuate
neuronal apoptosis induced by staurosporine. Rather unexpectedly, 24‐48‐h
exposure to 100‐300 μM riluzole induced neuronal death accompanied
by nuclear and DNA fragmentations, which was attenuated by caspase inhibitor
carbobenzyloxy‐Val‐Ala‐Asp‐fluoromethyl ketone but not by protein synthesis
inhibitor cycloheximide. The present study demonstrates that riluzole has
direct antioxidative actions, perhaps in part by inhibiting phospholipase
A2. However, in the same neurons, riluzole paradoxically induces neuronal apoptosis in a caspase‐sensitive manner. Considering current clinical use of riluzole, further studies are warranted to investigate its potential cytolethal effects.
Oxidized Low‐Density Lipoprotein Induces Neuronal Death
Authors: Keller Jeffrey N. , Hanni Keith B. , Markesbery William R.
Published: March 2003

Abstract:Low‐density lipoprotein (LDL) exists within the brain and is highly vulnerable to oxidative modifications. Once formed, oxidized LDL (oxLDL) is capable of eliciting cytotoxicity, differentiation, and inflammation in nonneuronal cells. Although oxLDL has been studied primarily for its role in the development of atherosclerosis, recent studies have identified a possible role for it in neurological disorders associated with oxidative stress. In the present study application of oxLDL, but not LDL, resulted in a dose‐ and time‐dependent death of cultured rat embryonic neurons. Studies using pharmacological inhibitors implicate the involvement of calcium, reactive oxygen species, and caspases in oxLDL‐induced neuronal death. Coapplication of oxLDL with either amyloid β‐peptide or glutamate, agents that enhance oxidative stress, resulted in increased neuronal death. Taken together, these data demonstrate that oxLDL induces neuronal death and implicate a possible role for oxLDL in conditions associated with increased levels of reactive oxygen species, including Alzheimer's disease.
Effect of ATP Depletion on the Palmitoylation of Myelin Proteolipid Protein in Young and Adult Rats
Authors: Bizzozero Oscar A. , Sanchez Paul , Tetzloff Sabine U.
Published: March 2003

Abstract: The present study was designed to determine whether the
palmitoylation of the hydrophobic myelin proteolipid protein (PLP) is
dependent on cellular energy. To this end, brain slices from 20‐ and
60‐day‐old rats were incubated with [3H]palmitate for 1 h in the
presence or absence of various metabolic poisons. In adult rats, the
inhibition of mitochondrial ATP production with KCN (5 mM),
oligomycin (10 μM), or rotenone (10 μM) reduced the
incorporation of [3H]palmitate into fatty acyl‐CoA and
glycerolipids by 50‐60%, whereas the labeling of PLP was unaltered. Incubation
in the presence of rotenone (10 μM) plus NaF (5 mM)
abolished the synthesis of acyl‐CoA and lipid palmitoylation, but the
incorporation of [3H]palmitate into PLP was still not different from that in controls. In rapidly myelinating animals, the inhibition of both mitochondrial electron transport and glycolysis obliterated the palmitoylation of lipids but reduced that of PLP by only 40%. PLP acylation was reduced to a similar extent when slices were incubated for up to 3 h, indicating that exogenously added palmitate is incorporated into PLP by ATP‐dependent and ATP‐independent mechanisms. Determination of the number of PLP molecules modified by each of these reactions during development suggests that the ATP‐dependent process is important during the formation and/or compaction of the myelin sheath, whereas the ATP‐independent mechanism is likely to play a role in myelin maintenance, perhaps by participating in the periodic repair of thioester linkages between the fatty acids and the protein.
Treatment of C6 Glioma Cells and Rats with Antidepressant Drugs Increases the Detergent Extraction of Gsα from Plasma Membrane
Authors: Toki Sadamu , Donati Robert J. , Rasenick Mark M.
Published: March 2003

Abstract : Results from previous studies suggested that chronic treatment of rats or C6 glioma cells with antidepressants augments the coupling between Gs and adenylyl cyclase. As these effects on C6 glioma cells are seen in the absence of presynaptic input, several antidepressant drugs may have a direct “postsynaptic” effect on their target cells. It was hypothesized that the target of antidepressant action was some membrane protein that may regulate coupling between G proteins and adenylyl cyclase. To test this, C6 glioma cells were treated with amitriptyline, desipramine, iprindole, or fluoxetine for 3 days. Chlorpromazine served as a control for these treatments. Membrane proteins were extracted sequentially with Triton X‐100 and Triton X‐114 from C6 glioma cells. Triton X‐100 extracted more Gsα in membranes prepared from antidepressant‐treated C6 glioma cells than from control groups. In addition, cell fractionation studies revealed that the amount of Gsα in caveolin‐enriched domains was reduced after antidepressant treatment and that adenylyl cyclase comigrated with Gsα in the gradients. These data suggest that some postsynaptic component that increases availability of Gs to activate effector molecules, such as adenylyl cyclase, might be a target of antidepressant treatment.
Inhibitors of Mitochondrial Respiration, Iron (II), and Hydroxyl Radical Evoke Release and Extracellular Hydrolysis of Glutathione in Rat Striatum and Substantia Nigra
Authors: Han Jilin , Cheng Fu‐Chou , Yang Zhaoliang , Dryhurst Glenn
Published: March 2003

Abstract : In this investigation, microdialysis has been used to

study the effects of 1‐methyl‐4‐phenylpyridinium (MPP+), an

inhibitor of mitochondrial complex I and α‐ketoglutarate dehydrogenase

and the active metabolite of the dopaminergic neurotoxin

1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), on extracellular

concentrations of glutathione (GSH) and cysteine (CySH) in the rat striatum

and substantia nigra (SN). During perfusion of a neurotoxic concentration of

MPP+ (2.5 mM) into the rat striatum or SN, extracellular

concentrations of GSH and CySH remain at basal levels (both ~2

μM). However, when the perfusion is discontinued, a massive but

transient release of GSH occurs, peaking at 5,000% of basal levels in the

striatum and 2,000% of basal levels in the SN. The release of GSH is followed

by a slightly delayed and smaller elevation of extracellular concentrations of

CySH that can be blocked by the γ‐glutamyl transpeptidase (γ‐GT)

inhibitor acivicin. Low‐molecular‐weight iron and extracellular hydroxyl

radical (OH·) have been implicated as participants in the

mechanism underlying the dopaminergic neurotoxicity of MPTP/MPP+.

During perfusion of Fe2+ (OH·) into the rat striatum and SN,

extracellular levels of GSH also remain at basal levels. When perfusions of

Fe2+ are discontinued, a massive transient release of GSH occurs

followed by a delayed, small, but progressive elevation of extracellular CySH

level that again can be blocked by acivicin. Previous investigators have noted

that extracellular concentrations of the excitatory/excitotoxic amino acid

glutamate increase dramatically when perfusions of neurotoxic concentrations

of MPP+ are discontinued. This observation and the fact that

MPTP/MPP+ causes the loss of nigrostriatal GSH without

corresponding increases of glutathione disulfide (GSSG) and the results of the

present investigation suggest that the release and

γ‐GT/dipeptidase‐mediated hydrolysis of GSH to glutamate, glycine, and

CySH may be important factors involved with the degeneration of dopamine

neurons. It is interesting that a very early event in the pathogenesis of

Parkinson's disease is a massive loss of GSH in the SN pars compacta that is

not accompanied by corresponding increases of GSSG levels. Based on the

results of this and prior investigations, a new hypothesis is proposed that

might contribute to an understanding of the mechanisms that underlie the

degeneration of dopamine neurons evoked by MPTP/MPP+, other agents that impair neuronal energy metabolism, and Parkinson's disease.
Palmitoyl Protein Thioesterase 1 Protects Against Apoptosis Mediated by Ras—Akt—Caspase Pathway in Neuroblastoma Cells
Authors: Cho Seongeun , Dawson Glyn
Published: March 2003

Abstract: Palmitoyl protein thioesterase (PPT) 1 is an enzyme
involved in deacylation of palmitoylated proteins. A deficiency in PPT1
results in a genetic disease, infantile neuronal ceroid lipofuscinosis,
associated with massive death of cortical neurons. The role of PPT1 in
neuronal survival and apoptosis was studied in human neuroblastoma (LA‐N‐5)
cells overexpressing PPT1. Overexpression of PPT1 was shown both by the
200‐350% increase in depalmitoylating activity over basal level (as determined
by an in vitro PPT assay) and by western blot analysis of transiently
expressed epitope‐tagged PPT1. Overexpressed PPT1 showed the same acidic pH
optimum (pH 4.0) as the endogenous enzyme, when assayed with a
Po‐derived octapeptide substrate, and reduced the growth rate by
30%. LA‐N‐5 cells underwent apoptosis, as evidenced by increased caspase
3‐like activity and increased DNA fragmentation, when challenged with either
C2‐ceramide or a phosphatidylinositol 3‐kinase inhibitor
(LY294002). Overexpression of PPT1 inhibited this C2‐ceramide‐ or
LY294002‐mediated activation of caspase‐3 by 50%. There was also a concomitant
decrease in DNA fragmentation and cell death. Consistent with increased
resistance to apoptosis, we found increased phosphorylation of the
antiapoptotic protein Akt (protein kinase B) in PPT1‐overexpressing cells.
p21Ras is known to be dynamically palmitoylated and depalmitoylated
and is involved in both growth and cell death. The
C2‐ceramide‐induced membrane association of p21Ras was reduced by 30‐50% in PPT1‐overexpressing cells compared with control. PPT overexpression also led to reduced membrane association of another palmitoylated protein, GAP‐43, a neuron‐specific protein. Our studies suggest that protein palmitoylation could be a physiological regulator of apoptosis.
Expression of a Dominant‐Negative Mutant of p21ras Inhibits Induction of Nitric Oxide Synthase and Activation of Nuclear Factor‐κB in Primary Astrocytes
Authors: Pahan Kalipada , Liu Xiaojuan , McKinney Michael J. , Wood Charles , Sheikh Faruk G. , Raymond John R.
Published: March 2003

Abstract: The present study underlines the importance of p21ras in regulating the inducible nitric oxide synthase (iNOS) in primary astrocytes. Bacterial lipopolysaccharides induced the GTP loading of p21ras, and the expression of a dominant‐negative mutant of p21ras (Δp21ras) inhibited lipopolysaccharide‐induced GTP loading in rat primary astrocytes. To delineate the role of p21ras in the induction of iNOS, we examined the effect of Δp21ras on the expression of iNOS and the production of nitric oxide. It is interesting that expression of Δp21ras markedly inhibited the production of nitric oxide and the expression of iNOS in lipopolysaccharide‐ and proinflammatory cytokine (tumor necrosis factor‐α, interleukin‐1β; interferon‐γ)‐stimulated rat and human primary astrocytes. Inhibition of iNOS promoter‐derived chloramphenicol acetyltransferase activity by Δp21ras suggests that p21ras is involved in the transcription of iNOS. As activation of nuclear factor‐κB (NF‐κB) is necessary for the transcription of iNOS, we examined the effect of Δp21ras on the activation of NF‐κB. Expression of Δp21ras inhibited the DNA binding as well as the transcriptional activity of NF‐κB in activated astrocytes, suggesting that Δp21ras inhibits the expression of iNOS by inhibiting the activation of NF‐κB. These studies also suggest that inhibitors of p21ras may be used as therapeutics in nitric oxide‐ and cytokine‐mediated neuroinflammatory diseases.
High‐Affinity Anti‐Ganglioside IgG Antibodies Raised in Complex Ganglioside Knockout Mice
Authors: Lunn Michael P. T. , Johnson L'Aurelle A. , Fromholt Susan E. , Itonori Saki , Huang Jian , Vyas Alka A. , Hildreth James E. K. , Griffin John W. , Schnaar Ronald L. , Sheikh Kazim A.
Published: March 2003

Abstract : Gangliosides, sialic acid‐bearing glycosphingolipids, are

highly enriched in the vertebrate nervous system. Anti‐ganglioside antibodies

are associated with various human neuropathies, although the pathogenicity of

these antibodies remains unproven. Testing the pathogenic role of

anti‐ganglioside antibodies will be facilitated by developing high‐affinity

IgG‐class complement‐fixing monoclonal anti‐bodies against major brain

gangliosides, a goal that has been difficult to achieve. In this study, mice

lacking complex gangliosides were used as immune‐naive hosts to raise

anti‐ganglioside antibodies. Wild‐type mice and knockout mice with a disrupted

gene for GM2/GD2 synthase (UDP‐N‐acetyl‐D‐galactosamine :

GM3/GD3 N‐acetyl‐D‐glactosaminyltransferase) were immunized with GD1a conjugated to keyhole limpet hemocyanin. The knockout mice produced a vigorous anti‐GD1a IgG response, whereas wildtype littermates failed to do so. Fusion of spleen cells from an immunized knockout mouse with myeloma cells yielded numerous IgG anti‐GD1a antibody‐producing colonies. Ganglioside binding studies revealed two specificity classes ; one colony representing each class was cloned and characterized. High‐affinity monoclonal antibody was produced by each hybridoma : an IgG1 that bound nearly exclusively to GD1a and an IgG2b that bound GD1a, GT1b, and GT1aα. Both antibodies readily readily detected gangliosides via ELISA, TLC immune overlay, immunohistochemistry, and immunocytochemistry. In contrast to prior reports using anti‐GD1a and anti‐GT1b IgM class monoclonal antibodies, the new antibodies bound avidly to granule neurons in brain tissue sections and cell cultures. Mice lacking complex gangliosides are improved hosts for raising high‐affinity, high‐titer anti‐ganglioside IgG antibodies for probing for the distribution and physiology of gangliosides and the pathophysiology of anti‐ganglioside antibodies.
2‐Deoxy‐D‐Glucose Prevents and Nicotinamide Potentiates 3,4‐Methylenedioxymethamphetamine‐Induced Serotonin Neurotoxicity
Authors: Hervías Isabel , Lasheras Berta , Aguirre Norberto
Published: March 2003

Abstract: Neurotoxicity induced by different substituted amphetamines has been associated with the exhaustion of intracellular energy stores. Accordingly, we examined the influence of 2‐deoxy‐D‐glucose (2‐DG), a competitive inhibitor of glucose uptake and metabolism, and nicotinamide, an agent that improves energy metabolism, on 3,4‐methylenedioxymethamphetamine (MDMA)‐induced 5‐hydroxytryptamine (5‐HT; serotonin) deficits. Administration of MDMA (15 mg/kg i.p.) produced a significant hyperthermia, whereas 2‐DG caused a profound hypothermia that lasted throughout the experiment. When MDMA was given to 2‐DG‐treated rats, an immediate but transient hyperthermia occurred and was followed by a return to hypothermia. 2‐DG had no effect on 5‐HT concentrations in the frontal cortex, hippocampus, and striatum but prevented the neurotoxicity induced by MDMA. When rats were injected with 2‐DG/MDMA and were warmed to prevent hypothermia, the protection afforded by 2‐DG was abolished. Nicotinamide had no effect on body temperature of the rats, and the hyperthermia induced by the nicotinamide/MDMA treatment was similar to that of the saline/MDMA‐treated rats. However, the long‐term 5‐HT deficits induced by MDMA were potentiated by nicotinamide in all the brain regions examined. Finally, no change on ATP concentrations in the frontal cortex, hippocampus, and striatum was observed up to 3 h after a single dose of MDMA. These results suggest that an altered energy metabolism is not the main cause of the neurotoxic effects induced by MDMA.
Phagocytic Clearance of Apoptotic Neurons by Microglia/Brain Macrophages In Vitro
Authors: Witting Anke , Müller Peter , Herrmann Andreas , Kettenmann Helmut , Nolte Christiane
Published: March 2003

Abstract: Microglia, the tissue macrophages of the brain, play a

crucial role in recognition and phagocytic removal of apoptotic neurons. The

microglial receptors for recognition of apoptotic neurons are not yet

characterized. Here we established a co‐culture model of primary microglia and

cerebellar granule neurons to examine the receptor systems involved in

recognition/uptake of apoptotic neurons. Treatment with 100 μM

S‐nitrosocysteine induced apoptosis of cerebellar neurons as indicated by

nuclear condensation and phosphatidylserine exposure to the exoplasmic leaflet

of the plasma membrane. Microglial cells were added to neurons 2 h after

apoptosis induction and co‐cultured for 6 h in the presence of ligands that

inhibit recognition by binding to respective receptors. Binding/phagocytosis

was determined after combined 4′,6‐diamidino‐2‐phenylindole/propidium

iodide (for apoptotic/necrotic neurons) and lectin staining (for microglia).

Uptake of apoptotic neurons was reduced by N‐acetylglucosamine or

galactose, suggesting that recognition involves asialoglycoprotein‐like

lectins. Furthermore, the inhibition of microglial binding/uptake of apoptotic

neurons by RGDS peptide suggests a role of microglial vitronectin receptor. As

microglia selectively bind lipid vesicles enriched in phosphatidylserine and

O‐phospho‐L‐serine interfered with the uptake of apoptotic neurons, an involvement of phosphatidylserine receptor is rather likely. Apoptotic neurons do not release soluble signals that serve to attract or activate microglia. Collectively, these results suggest that apoptotic neurons generate a complex surface signal recognized by different receptor systems on microglia.
In Alzheimer's Disease, Heme Oxygenase Is Coincident with Alz50, an Epitope of τ Induced by 4‐Hydroxy‐2‐Nonenal Modification
Authors: Takeda Atsushi , Smith Mark A. , Avilá Jesus , Nunomura Akihiko , Siedlak Sandra L. , Zhu Xiongwei , Perry George , Sayre Lawrence M.
Published: March 2003

Abstract: In this study, we compared the neuronal induction of the antioxidant heme oxygenase‐1 (HO‐1) in Alzheimer's disease with abnormalities in τ marked by antibodies recognizing either phosphorylation (AT8) or conformational change (Alz50). The epitope recognized by Alz50 shows a complete overlap with HO‐1‐containing neurons, but AT8 recognized these neurons as well as neurons not displaying HO‐1. These findings suggest that τ phosphorylation precedes the HO‐1 response and that HO‐1 is coincident with the Alz50 epitope. This led us to consider whether oxidative damage plays a role in forming the Alz50 epitope. We found that 4‐hydroxy‐2‐nonenal (HNE), a highly reactive product of lipid peroxidation, reacts with normal τ and induces the Alz50 epitope in τ. It is important that the ability of HNE to create the Alz50 epitope not only is dependent on lysine residues of τ but also requires τ phosphorylation because neither methylated, recombinant, nor dephosphorylated τ reacts with HNE to create the Alz50 epitope. Supporting the in vivo relevance of this observation, endogenous paired helical filament‐τ isolated from subjects with Alzheimer's disease was immunoreactive with an antibody to a stable HNE‐lysine adduct, as were all vulnerable neurons in subjects with Alzheimer's disease but not in control individuals. Together, these findings support the involvement of oxidative damage early in neurofibrillary tangle formation in Alzheimer's disease and also suggest that HNE modification contributes to the generation of the τ conformation defining the Alz50 epitope. These findings provide evidence that an interplay between phosphorylation of τ and neuronal oxidative stress‐induced pathology is important in the formation of neurofibrillary tangles.
Altered Intracellular Calcium Homeostasis in Cerebellar Granule Cells of Prion Protein‐Deficient Mice
Authors: Herms Jochen W. , Korte Stefan , Gall Stefan , Schneider Ilka , Dunker Stefanie , Kretzschmar Hans A.
Published: March 2003

Abstract: Previous studies have indicated that recombinant cellular
prion protein (PrPC), as well as a synthetic peptide of
PrPC, affects intracellular calcium homeostasis. To analyze whether
calcium homeostasis in neurons is also affected by a loss of PrPC,
we performed microfluorometric calcium measurements on cultured cerebellar
granule cells derived from prion protein‐deficient (Prnp0/0) mice.
The resting concentration of intracellular free calcium
([Ca2+]i) was found to be slightly, but significantly,
reduced in Prnp0/0 mouse granule cell neurites. Moreover, we
observed a highly significant reduction in the [Ca2+]i
increase after high potassium depolarization. Pharmacological studies further
revealed that the L‐type specific blocker nifedipine, which reduces the
depolarization‐induced [Ca2+]i increase by 66% in
wild‐type granule cell somas, has no effect on [Ca2+]i
in Prnp0/0 mouse granule cells. Patch‐clamp measurements, however,
did not reveal a reduced calcium influx through voltage‐gated calcium channels
in Prnp0/0 mice. These data clearly indicate that loss of
PrPC alters the intracellular calcium homeostasis of cultured cerebellar granule cells. There is no evidence, though, that this change is due to a direct alteration of voltage‐gated calcium channels.