bluuebunny/crossref_metadata_2025 huggingface dataset:
Reproductive development of blackberry (Rubus cuneifolius), as influenced by ozone
Authors: Chappelka A. H.
Published: March 2003

The effects of ozone on reproductive development of blackberry ( Rubus cuneifolius
)
in situ are reported here and, in particular, changes over time of flower and fruit production, and the size and time to maturity of fruits produced.

Blackberry, a common associate in early successional communities in the southern USA, was exposed to subambient, ambient (both chambered and nonchambered plots) and elevated (2× ambient) ozone concentrations for two growing seasons (1994–95). Competition from other plant species was not excluded, and reproductive development was examined in April–May of the second year on the floricanes.

An initial acceleration in flowering resulted from exposure to elevated ozone with more flowers initiated and peak production occurring sooner than all other treatments. Total numbers of fruits were not significantly different among treatments. Numbers of large and ripe fruits were greatest in carbon‐filtered and unfiltered chambers and least in the 2× and ambient air treatments.

Possible implications related to changes in community structure and function are discussed.
Signals from the cuticle affect epidermal cell differentiation
Authors: Bird Susannah M. , Gray Julie E.
Published: March 2003

Studies of Arabidopsis wax biosynthesis mutants indicate that the control of cell fate in the aerial epidermis is dependant upon the synthesis of the waxy cuticle that overlies the epidermal layer. Several cer mutants, originally isolated as wax deficient, not only affect cuticular wax composition but also exhibit large increases in stomatal numbers. Stomatal numbers are also affected in hic mutant plants, but despite HIC encoding a putative wax biosynthetic enzyme the hic phenotype of increased stomatal numbers is more subtle, and only seen at elevated CO2 concentrations. This suggests that environmental effects on stomatal number may be mediated through cuticular wax composition. Other putative wax biosynthetic genes, FDH and LCR, have effects on the number of trichomes that develop in the epidermis, indicating that trichome development may also be affected by cuticle composition. Thus signals from the cuticle may influence how trichome and stomatal numbers in the epidermis are determined. Wax components could be the developmental signalling molecules, or could be the mediating medium for such signals, stimulated by environmental cues, which affect epidermal cell fate.

Contents

Summary
9

I.
Introduction
10

II.
Cuticle structure
10

III.
Cuticular waxes
10

IV.
Cell patterning in the epidermis
11

V.
Stomatal development
12

VI.
Stomatal development in dicotyledonous plants
12

VII.
Mutants in stomatal development
14

VIII.
Control of Stomatal Development
14

IX.
Cuticle composition affects stomatal development
14

X.
The HIC – HI gh Carbon dioxide gene
15

XI.
Fatty acid elongases
17

XII.
The cuticle: an alternative signalling medium?
17

XIII.
Trichome development
18

XIV.
Cuticle composition affects trichome development
19

XV.
Cuticle composition affects pollen germination
20

XVI.
Conclusions
20

Acknowledgements
21

References
21
Consumers, cars and communities: the challenge of sustainability
Authors: Auld John W.
Published: March 2003

The huge subsidization of sprawling development and the unbridled use of the private automobile needs to be brought to the forefront of public policy debate, not left to the excesses of the market‐place. To do this we need citizens‐in‐the‐community, not consumers‐in‐the‐market. The present costly and unsustainable situation of our human settlements is a result of a profound failure in public policy; a failure that resulted when we left the fate of our human settlements to the vagaries of the market‐place. We can no longer rely on the present subsidized, distorted, market‐place system approach to designing, building and operating our human settlements. The consequences have been disastrous for human welfare, health and safety, and the natural environment upon which all life depends.
The food pyramid challenge
Authors: Hertzler Ann A. , Mitchell Bridgit , Poole Kathleen
Published: March 2003

Pyramid Power is a 1‐hour programme designed to teach food choices, serving sizes and nutrition. Participants (19 women and 8 men) were challenged to follow the recommendation in a step‐by‐step manner. Evaluation, two months after the programme, resulted in two major areas of change. Participants improved their scoring of three food groups (bread‐cereal‐rice‐pasta, vegetable and fruit) and decreased the number of fat–sweet mentions. The programme and challenge provides a way for nutritionists to identify what participants are eating, to track progress and to identify future programme needs.
Antecedents and consequences of consumer satisfaction/dissatisfaction with the performance of apparel products at purchase and after consumption: a comparison of male and female South Korean consumers
Authors: Chen‐Yu Jessie , Hong Keum‐Hee
Published: March 2003

The purpose of the study is to investigate antecedents and consequences of consumer satisfaction/dissatisfaction (CS/D) with the performance of apparel products at purchase, and after consumption, for male and female South Korean consumers. An experimental research technique with a 2 × 2 × 2 between‐subjects factorial design with gender (female, male), expectation for future performance, (high, low) and perception of product consumption performance (good, poor) was used. Both similarities and differences between genders were found. Implications and applications of results are discussed.
Does price reflect emotional, structural or performance quality?
Authors: Chowdhary Usha
Published: March 2003

Three denim jeans products from the same manufacturer with differentiated pricing and labels (antiqued, sandblasted, and stonewashed) were used to determine the relationship between price and quality. Both qualitative and quantitative procedures were used to analyse the garments. All three jeans were made of twill weave with 3 × 1 repeat. Both structural and performance characteristics were compared using standardized tests. Analysis of variance was used to compare the selected structural and performance characteristics among the three jeans products. Tukey’s HSD tests were used for post hoc multiple comparisons for three jeans. The findings revealed that despite the fact that all three jeans products were from the same store, and that the fabrics used the same weave, degree and direction of twist, the jeans differed significantly for several structural and performance characteristics. Several possibilities for future research were explored.
Young people's perceptions of complaints procedures in local government
Authors: Brennan Carol , Galloway Alison , Thompson Susan
Published: March 2003

This study examined 46 young people aged 16–24 years and evaluated their knowledge and awareness of the formal complaints procedures used by local government. Two areas in Scotland, one city (Edinburgh) and one town (Stirling), were chosen to participate in the study. Six focus groups, three in each area, were carried out to identify the level of awareness among the respondents and to permit a cross‐section of educational backgrounds to be obtained. A questionnaire was used to assemble a profile on each participant. After analysis of the focus groups, a focused interview with the Corporate Complaints Officers from two councils was undertaken. Each interview incorporated an in‐depth discussion regarding the formal complaints procedure while focusing on the young people within their constituency. The research revealed that young people's knowledge and awareness of local government complaints procedures is low, regardless of educational background and area of residence. For a minority, social factors such as confidence and competence do play a role although it is mainly organizational barriers, such as lack of information and access, which are the main causes of the problem. A number of young people indicated that they would complain if they knew how to execute a complaint successfully. The service providers were knowledgeable that awareness is low among this age group.
Proteolysis levels of white cheeses salted and ripened in brines prepared from various salts
Authors: Güven M , Karaca O B
Published: March 2003

This study was carried out to investigate the effects of salting and ripening periods in brines, prepared from various salts, on the properties of white cheeses. The brines were made from a mixture of NaCl with CaCl2, KCl and MgCl2 at 1 : 1 (molar basis) ratio. The NaCl solution alone was used as a control. The effects of different brines on total nitrogen, water‐soluble nitrogen, nonprotein nitrogen, casein nitrogen and proteose‐peptone nitrogen contents of cheeses were found to be insignificant, whereas the effect of ripening period on these properties was found to be significant (P < 0.05).
Current trends in whey processing and utilization in Greece
Authors: Philippopoulos Charalabos D , Papadakis Marios T
Published: March 2003

Whey treatment began in Greece in 1998; in previous years, whey was considered only as effluent from cheese production, a part of which yielded whey cheeses. In 1998, the first whey powder plant started to operate successfully. The main physicochemical characteristics of whey from feta cheese production are illustrated, together with those of the ‘cooked’ whey, ie the serum remaining after whey cheese production. Quality standards for both types of whey are proposed. The drive for efficient whey processing is the unrelenting demand for whey products in the local market and the current status is described.
Some properties of urfa cheese (a traditional white‐brined Turkish cheese) produced from bovine and ovine milks
Authors: Ozer Barbaros , Atasoy Ferit , Akin Serdar
Published: March 2003

In this study, the basic composition and ripening profile of traditional urfa cheese made from ovine and bovine milks were investigated. While cheese made from ovine milk had higher total solids, fat‐in‐dry matter and total nitrogen, the titratable acidity, salt‐in‐dry matter, pH, total mesophilic colony count and total yeasts and moulds counts were found to be close to each other. During storage, whilst the total solids content of cheese produced from ovine milk gradually decreased, the variation in the total solids content of cheese made from bovine milk was found to be insignificant. The salt penetration into the cheeses was rapid during the first two weeks of ripening, and it continued to diffuse into the samples throughout storage. Proteolysis developed faster in the cheese made from ovine milk than in cheese of bovine milk. The former sample had higher water soluble nitrogen, nonprotein nitrogen, phosphotungustic acid soluble nitrogen, Proteose‐peptone nitrogen and tyrosine levels throughout storage, and the ripening index was higher as well.
Effect of addition of amino acids, treatment with β‐galactosidase and use of heat‐shocked cultures on the acetaldehyde level in yoghurt
Authors: Özer Barbaros , Atasoy Ferit
Published: March 2003

In this study, the biochemical activities of nonviscous and viscous yoghurt starter cultures were investigated. Yoghurt samples produced with nonviscous and viscous cultures, and viscous cultures + methionine (10 and 30 mg/100 mL milk), + threonine (5 and 10 mg/100 mL milk), + β‐galactosidase (1 mg/100 mL milk), and with a heat‐shocked culture were analysed. In the experimental yoghurts, the pH, titratable acidity, lactic acid, tyrosine and acetaldehyde contents and the number of total starter organisms were determined. According to the results obtained, the samples produced with the viscous culture had the lowest acetaldehyde levels, whereas the highest acetaldehyde level was found in the samples manufactured with the nonviscous culture. Compared with the samples inoculated with the viscous culture alone, the amino acid supplementation, lactose hydrolysis and heat‐shock treatments caused a significant increase in the level of acetaldehyde.
Review of systems for monitoring curd setting during cheesemaking
Authors: O’Callaghan Donal J , O’Donnell Colm P , Payne F A
Published: March 2003

This paper reviews the current state of development of various techniques for monitoring coagulum formation in cheesemaking, and the implications of recent research findings. The techniques, which have become available for on‐line use on modern cheese vats include hot wire probes, vibrating probes and several types of optical probe. Recent research has focused on comparing the various techniques against a background of cheese manufacture from milk with seasonal variation. The findings indicate that on‐line techniques can improve the consistency of coagulum at cutting in a modern cheese factory.
Some properties of yoghurt produced by adding mulberry pekmez (concentrated juice)
Authors: Celik Serafettin , Bakirci Ihsan
Published: March 2003

Fruit‐flavoured yoghurt was made by adding 2.5, 5.0, 7.5 and 10.0% mulberry pekmez (MP) into milk. The effects of the MP on the quality and fermentation process of the yoghurt were determined. The titratable acidity, pH, viscosity, whey separation and lactic acid bacteria (LAB) counts were determined at weekly intervals for 28 days. The pH range of the MP yoghurts was 4.65–5.57 and the pH of the plain yoghurt was 4.47 (P < 0.05). The addition of MP led to an increase in the fermentation time and a decrease in the viscosity of the yoghurts. Statistically significant differences were found between the plain and MP yoghurts in terms of pH (4.01 and 4.35), viscosity (5429 and 3175 cP) and number of LAB (7.07 and 6.48 log cfu). During storage, the titratable acidity, viscosity and LAB counts of MP yoghurts were lower and the whey separations higher than those of controls.
Diversification of Drosophila Chloride Channel Gene by Multiple Posttranscriptional mRNA Modifications
Authors: Semenov Eugene P. , Pak William L.
Published: March 2003

Abstract : We have identified and analyzed a Drosophila
melanogaster gene that encodes a chloride channel subunit
(DrosGluCl‐α) previously shown to function as a glutamategated chloride
channel in an in vitro expression system. Sequence analysis of several cDNAs
corresponding to the gene revealed sequence diversity in their open reading
frames at seven specific sites. Site‐specific A‐to‐G variations between cDNA
and genomic sequences, consistent with RNA editing, were detected at five
nucleotide positions. In addition, sequence variations among cDNA clones
consistent with alternative splicing of mRNA were found at two different
sites. In the 5′ region, two small adjacent exons, containing similar
but distinct modular sequences, are alternatively incorporated into the mature
mRNA. In the 3′ region, alternative splicing generates a variant
encoding a protein with four additional amino acids just upstream of the
fourth transmembrane domain. Combinations of RNA editing and alternative
splicing can lead to extensive diversification of transcripts. These results
give the first example of RNA editing in neurotransmitter‐gated chloride
channel genes or of alternative splicing in a glutamate‐gated chloride channel
gene of Drosophila.
Proteolysis of SNAP‐25 Isoforms by Botulinum Neurotoxin Types A, C, and E
Authors: Vaidyanathan Vadakkanchery V , Yoshino Ken‐ichi , Jahnz Michael , Dörries Christos , Bade Steffen , Nauenburg Sonja , Niemann Heiner , Binz Thomas
Published: March 2003

Abstract : Tetanus toxin and the seven serologically distinct
botulinal neurotoxins (BoNT/A to BoNT/G) abrogate synaptic transmission at
nerve endings through the action of their light chains (L chains), which
proteolytically cleave VAMP (vesicle‐associated membrane
protein)/synaptobrevin, SNAP‐25 (synaptosome‐associated protein of 25 kDa), or
syntaxin. BoNT/C was reported to proteolyze both syntaxin and SNAP‐25. Here,
we demonstrate that cleavage of SNAP‐25 occurs between Arg198 and
Ala199, depends on the presence of regions Asn93 to
Glu145 and Ile156 to Met202, and requires
about 1,000‐fold higher L chain concentrations in comparison with BoNT/A and
BoNT/E. Analyses of the BoNT/A and BoNT/E cleavage sites revealed that changes
in the carboxyl‐terminal residues, in contrast with changes in the
amino‐terminal residues, drastically impair proteolysis. A proteolytically
inactive BoNT/A L chain mutant failed to bind to VAMP/synaptobrevin and
syntaxin, but formed a stable complex (KD = 1.9 ×
10‐7M) with SNAP‐25. The minimal essential domain of
SNAP‐25 required for cleavage by BoNT/A involves the segment
Met146‐Gln197, and binding was optimal only with
full‐length SNAP‐25. Proteolysis by BoNT/E required the presence of the domain
Ile156‐Asp186. Murine SNAP‐23 was cleaved by BoNT/E and,
to a reduced extent, by BoNT/A, whereas human SNAP‐23 was resistant to all
clostridial L chains. Lys185Asp or Pro182Arg mutations of human SNAP‐23 induced susceptibility toward BoNT/E or toward both BoNT/A and BoNT/E, respectively.
Localization and Subcellular Distribution of N‐Copine in Mouse Brain
Authors: Nakayama Takashi , Yaoi Takeshi , Kuwajima Goro
Published: March 2003

Abstract : N‐Copine is a novel protein with two C2 domains.
Its expression is brain specific and up‐regulated by neuronal activity such as
kainate stimulation and tetanus stimulation evoking hippocampal CA1 long‐term
potentiation. We examined the localization and subcellular distribution of
N‐copine in mouse brain. In situ hybridization analysis showed that
N‐copine mRNA was expressed exclusively in neurons of the hippocampus
and in the main and accessory olfactory bulb, where various forms of synaptic
plasticity and memory formation are known to occur. In immunohistochemical
analyses, N‐copine was detected mainly in the cell bodies and
dendrites in the neurons, whereas presynaptic proteins such as synaptotagmin I
and rab3A were detected in the regions where axons pass through. In
fractionation experiments of brain homogenate, N‐copine was
associated with the membrane fraction in the presence of Ca2+ but
not in its absence. As a GST‐fusion protein with the second C2 domain of
N‐copine showed Ca2+ ‐dependent binding to
phosphatidylserine, this domain was considered to be responsible for the
Ca2+ ‐dependent association of N‐copine with the membrane.
Thus, N‐copine may have a role as a Ca2+ sensor in postsynaptic events, in contrast to the known roles of “double C2 domain‐containing proteins,” including synaptotagmin I, in presynaptic events.
Dynamic Interaction Between Soluble Tubulin and C‐Terminal Domains of N‐Methyl‐D‐Aspartate Receptor Subu
Authors: Van Rossum Denise , Kuhse Jochen , Betz Heinrich
Published: March 2003

Abstract : The cytoplasmic C‐terminal domains (CTs) of the NR1 and
NR2 subunits of the NMDA receptor have been implicated in its anchoring to the
subsynaptic cytoskeleton. Here, we used affinity chromatography with
glutathione S‐transferase‐NR1‐CT and ‐NR2B‐CT fusion proteins to
identify novel binding partner(s) of these NMDA receptor subunits. Upon
incubation with rat brain cytosolic protein fraction, both NR1‐CT and NR2B‐CT,
but not glutathione S‐transferase, specifically bound tubulin. The respective fusion proteins also bound tubulin purified from brain, suggesting a direct interaction between the two binding partners. In tubulin polymerization assays, NR1‐CT and NR2B‐CT significantly decreased the rate of microtubule formation without destabilizing preformed microtubules. Moreover, only minor fractions of either fusion protein coprecipitated with the newly formed microtubules. Consistent with these findings, ultrastructural analysis of the newly formed microtubules revealed a limited association only with the CTs of the NR1 and NR2B. These data suggest a direct interaction of the NMDA receptor channel subunit CTs and tubulin dimers or soluble forms of tubulin. The efficient modulation of microtubule dynamics by the NR1 and NR2 cytoplasmic domains suggests a functional interaction of the receptor and the subsynaptic cytoskeletal network that may play a role during morphological adaptations, as observed during synaptogenesis and in adult CNS plasticity.
Antioxidative Effects of Docosahexaenoic Acid in the Cerebrum Versus Cerebellum and Brainstem of Aged Hypercholesterolemic Rats
Authors: Hossain Md. Shahdat , Hashimoto Michio , Gamoh Shuji , Masumura Sumio
Published: March 2003

Abstract : Female Wistar rats (100 weeks old) were divided into two
groups ; one group was fed a high‐cholesterol diet (HC) and the other a
high‐cholesterol diet plus docosahexaenoic acid (HC‐fed DHA rats). Fatty acid
concentrations in brain tissues were analyzed by gas chromatography. In the
HC‐fed DHA rats, brain catalase (CAT), GSH, and glutathione peroxidase (GPx)
increased in the cerebrum but not in the brainstem or cerebellum. The rate of
increase was 23.0% for CAT, 24.5% for GSH, and 26.3% for GPx compared with
that in the HC animals (p < 0.05). In the cerebrum of the HC‐fed DHA rats, CAT and GPx increased, with an increase in the ratio of DHA to arachidonic acid. The cerebrum, unlike the other areas of the brain, seems to be more sensitive to DHA in stimulating CAT and GPx. We suggest that DHA plays an important role in inducing an antioxidative defense against active oxygen by enhancing the cerebral activities of CAT, GPx, and GSH.
Abnormal Purine and Pyrimidine Nucleotide Content in Primary Astroglia Cultures from Hypoxanthine—Guanine Phosphoribosyltransferase‐Deficient Transgenic Mice
Authors: Pelled Dori , Sperling Oded , Zoref‐Shani Esther
Published: March 2003

Abstract : Lesch‐Nyhan syndrome is a pediatric metabolic‐neurological syndrome caused by the X‐linked deficiency of the purine salvage enzyme hypoxanthine‐guanine phosphoribosyltransferase (HGPRT). The cause of the metabolic consequences of HGPRT deficiency has been clarified, but the connection between the enzyme deficiency and the neurological manifestations is still unknown. In search for this connection, in the present study, we characterized purine nucleotide metabolism in primary astroglia cultures from HGPRT‐deficient transgenic mice. The HGPRT‐deficient astroglia exhibited the basic abnormalities in purine metabolism reported before in neurons and various other HGPRT‐deficient cells. The following abnormalities were found : absence of detectable uptake of guanine and of hypoxanthine into intact cell nucleotides ; 27.8% increase in the availability of 5‐phosphoribosyl‐1‐pyrophosphate ; 9.4‐fold acceleration of the rate of de novo nucleotide synthesis ; manyfold increase in the excretion into the culture media of hypoxanthine (but normal excretion of xanthine) ; enhanced loss of label from prelabeled adenine nucleotides (loss of 71% in 24 h, in comparison with 52.7% in the normal cells), due to 4.2‐fold greater excretion into the media of labeled hypoxanthine. In addition, the HGPRT‐deficient astroglia were shown to contain lower cellular levels of ADP, ATP, and GTP, indicating that the accelerated de novo purine synthesis does not compensate adequately for the deficiency of salvage nucleotide synthesis, and higher level of UTP, probably due to enhanced de novo synthesis of pyrimidine nucleotides. Altered nucleotide content in the brain may have a role in the pathogenesis of the neurological deficit in Lesch‐Nyhan syndrome.
Mitochondrial Iron Sequestration in Dopamine‐Challenged Astroglia: Role of Heme Oxygenase‐1 and the Permeability Transition Pore
Authors: Schipper H. M. , Bernier L. , Mehindate K. , Frankel D.
Published: March 2003

Abstract: Little is currently known concerning the mechanisms responsible for the excessive deposition of redox‐active iron in the substantia nigra of subjects with Parkinson's disease (PD). In the present study, we demonstrate that dopamine promotes the selective sequestration of non‐transferrin‐derived iron by the mitochondrial compartment of cultured rat astroglia and that the mechanism underlying this novel dopamine effect is oxidative in nature. We also provide evidence that up‐regulation of the stress protein heme oxygenase‐1 (HO‐1) is both necessary and sufficient for mitochondrial iron trapping in dopamine‐challenged astroglia. Finally, we show that opening of the mitochondrial transition pore (MTP) mediates the influx of non‐transferrin‐derived iron into mitochondria of dopamine‐stimulated and HO‐1‐transfected astroglia. Our findings provide an explanation for the pathological iron sequestration, mitochondrial insufficiency, and amplification of oxidative injury reported in the brains of PD subjects. Pharmacological blockade of transition metal trapping by “stressed” astroglial mitochondria (e.g., using HO‐1 inhibitors or modulators of the MTP) may afford effective neuroprotection in patients with PD and other neurological afflictions.
Analysis of the Neuroprotective Effects of Various Nitric Oxide Donor Compounds in Murine Mixed Cortical Cell Culture
Authors: Vidwans Aniruddha S. , Kim Sungmee , Coffin Deborah O. , Wink David A. , Hewett Sandra J.
Published: March 2003

Abstract: Nitric oxide (NO) has been implicated in both the pathogenesis of and protection from NMDA receptor‐mediated neuronal injury. This apparent paradox has been attributed to alternate redox states of nitrogen monoxide, whereby, depending on the redox milieu, nitrogen monoxide can be neuroprotective via nitrosation chemistry or react with superoxide to form secondary toxic species. In our murine mixed cortical cell culture system, the NONOate‐type NO donors diethylamine/NO complex sodium (Dea/NO), (Z)‐[N‐(3‐ammoniopropyl)‐N‐(n‐propyl)amino]diazen‐1‐ium‐1,2‐diolate (Papa/NO), and spermine/NO complex sodium (Sper/NO), as well as the S‐nitrosothiols S‐nitroso‐L‐glutathione (GSNO) and S‐nitroso‐N‐acetyl‐D,L‐penicillamine (SNAP) (NO+ equivalents), decreased NMDA‐induced neuronal injury in a concentration‐dependent manner. 8‐Bromo‐cyclic GMP did not mimic the inhibitory effects of the donors, suggesting that the neuroprotection was not the result of NO‐stimulated neuronal cyclic GMP production. Furthermore, neuronal injury induced by exposure of cultures to H2O2 was not altered by the presence of Dea/NO, indicating the absence of a direct antioxidant effect. NONO‐ates did, however, reduce NMDA‐stimulated uptake of 45Ca2+, whereas high potassium‐induced 45Ca2+ accumulation, a measurement of entry via voltage‐gated calcium channels, was unaffected. The parallel reduction of 45Ca2+ accumulation and NMDA neurotoxicity by NONOates mimicked that seen with an NMDA receptor antagonist. Electrochemical measurements of NO via an NO‐sensitive electrode demonstrated that neuroprotective concentrations of all donors produced appreciable amounts of NO over the 5‐min time frame. Determination of the formation of NO+ equivalents, as assessed by N‐nitrosation of 2,3‐diaminonaphthylene, revealed little or no observable N‐nitrosation by Sper/NO, GSNO, and SNAP with significant N‐nitrosation observed by Papa/NO and Dea/NO. However, addition of ascorbate (400 μM) effectively prevented the nitrosation of 2,3‐diaminonaphthylene produced by Dea/NO and Papa/NO without altering their neuroprotective properties or their effects on 45Ca2+ accumulation. Present results indicate that the intrinsic NO/NO+ characteristics of NO donor compounds may not be a good predictor of their ability to inhibit NMDA receptor‐mediated neurotoxicity at the cellular level.
Lack of Causal Relationship Between Clusterin Expression and Photoreceptor Apoptosis in Light‐Induced Retinal Degeneration
Authors: Jomary Catherine , Darrow Ruth M. , Wong Paul , Organisciak Daniel T. , Neal Michael J. , Jones Stephen E.
Published: March 2003

Abstract: Induction of apoptosis in the retina leads to cellular death by molecular mechanisms that are not well understood. Clusterin expression is increased in tissues undergoing apoptosis, including retinal neurodegenerative states, but the causal relationships remain to be clarified. To gain insight into clusterin's role in photoreceptor apoptosis, the cellular distribution of clusterin mRNA was compared with the pattern of apoptotic nuclear labelling in a rat model of light‐induced retinal degeneration. In control retinal sections, clusterin mRNA was localized to the retinal pigment epithelium cells, photoreceptor inner segments, inner nuclear layer, and ganglion cell layer. Clusterin expression decreased in photoreceptors and retinal pigment epithelium cells, which progressively degenerated, and increased in preserved inner nuclear layer, in proportion to the duration of light exposure in both cyclic light‐ and dark‐reared animals. These results suggest that clusterin is not causally involved in apoptotic mechanisms of photoreceptor death, but may relate to cytoprotective functions.
A Nitric Oxide/Cyclic GMP‐Dependent Protein Kinase Pathway Alters Transmitter Release and Inhibition by Somatostatin at a Site Downstream of Calcium Entry
Authors: Gray D. Bruce , Polo‐Parada Luis , Pilar Guillermo R. , Eang Peau , Metzger Ryan R. , Klann Eric , Meriney Stephen D.
Published: March 2003

Abstract: We have examined the somatostatin‐mediated modulation of acetylcholine release from intact chick embryo choroid tissue and compared these data with those obtained using acutely dissociated neuronal cell bodies from the chick ciliary ganglion. Acetylcholine release, evoked in a calcium‐dependent manner by a high potassium (55 mM KCl) stimulation in both preparations, was inhibited almost completely by 100 nM somatostatin. Measurement of intracellular calcium in these neurons revealed that somatostatin blocked the large calcium transient that was observed in control neurons following KCl exposure. The modulatory effect of somatostatin on transmitter release was significantly attenuated by pretreatment with pharmacologic agents that selectively block cyclic GMP (cGMP)‐dependent protein kinase (PKG) or nitric oxide (NO) synthase. It is interesting that this prevention of somatostatin‐mediated acetylcholine release inhibition occurred without reversal of the somatostatin‐mediated block of the KCl‐evoked calcium transient. Furthermore, a NO donor or cGMP analogue could block KCl‐evoked acetylcholine release, but only cGMP could reduce the KCl‐evoked calcium transient. Although cGMP could reduce the KCl‐evoked calcium transient, a cGMP analogue was shown to reduce calcium ionophore‐evoked transmitter release. Thus, somatostatin reduces acetylcholine release by modulating calcium influx, but the NO‐PKG pathway can inhibit acetylcholine release, and alter somatostatin‐mediated inhibition, by affecting transmitter release at some point after calcium entry.
Regional Distribution and Cellular Expression of Tryptophan Hydroxylase Messenger RNA in Postmortem Human Brainstem and Pineal Gland
Authors: Austin Mark C. , O'Donnell Sandra M.
Published: March 2003

Abstract: The characterization and cellular localization of tryptophan hydroxylase mRNA in the human brainstem and pineal gland were investigated by using northern blot analysis and in situ hybridization histochemistry. Northern analysis of human pineal gland revealed the presence of two mRNA species that were absent in RNA isolated from human raphe. In situ hybridization experiments revealed very dense hybridization signal corresponding to tryptophan hydroxylase mRNA in cells throughout the pineal gland. In contrast, tryptophan hydroxylase mRNA was heterogeneously distributed in neurons in the dorsal and median raphe nuclei. Within the dorsal raphe, the ventrolateral and interfascicular subnuclei contained the greatest number of tryptophan hydroxylase mRNA‐positive neurons. Also, the cellular concentration of tryptophan hydroxylase mRNA varied widely within the dorsal and median raphe. Comparison of the cellular concentration of tryptophan hydroxylase mRNA between the pineal gland and the raphe nuclei revealed an 11‐ and 46‐fold greater average grain density of tryptophan hydroxylase mRNA positive cells in the pineal gland compared with the dorsal and median raphe, respectively. These findings are the first to demonstrate the cellular localization of tryptophan hydroxylase mRNA in the human brain and pineal gland as well as heterogeneity in the cellular concentration within and between these tissues.
Conserved Extracellular Cysteine Pair in the M3 Muscarinic Acetylcholine Receptor Is Essential for Proper Receptor Cell Surface Localization but Not for G Protein Coupling
Authors: Zeng Fu‐Yue , Soldner Andrea , Schöneberg Torsten , Wess Jürgen
Published: March 2003

Abstract: Most G protein‐coupled receptors contain a conserved pair
of extracellular cysteine residues that are predicted to form a disulfide bond
linking the first and second extracellular loops. Previous studies have shown
that this disulfide bond may be critical for ligand binding, receptor
activation, and/or proper receptor folding. However, the potential importance
of the two conserved cysteine residues for proper receptor cell surface
localization has not been investigated systematically. To address this issue,
we used the rat M3 muscarinic receptor as a model system. Most
studies were carried out with a modified version of this receptor subtype
(lacking potential N‐glycosylation sites and the central portion of
the third intracellular loop) that could be readily detected via western blot
analysis. Cys→Ala mutant receptors were generated, transiently expressed
in COS‐7 cells, and then examined for their subcellular distribution and
functional properties. ELISA and immunofluorescence studies showed that the
presence of both conserved cysteine residues (corresponding to C140 and C220
in the rat M3 muscarinic receptor sequence) is required for
efficient expression of the M3 muscarinic receptor on the cell surface. On the other hand, these residues were found not to be essential for protein stability (determined via immunoblotting) and receptor‐mediated G protein activation (studied in second messenger assays). These results shed new light on the functional role of the two extracellular cysteine residues present in most G protein‐coupled receptors.
Sp1/Egr1 Motif
Authors: Papanikolaou Nikolaos A. , Sabban Esther L.
Published: March 2003

Abstract: The rat tyrosine hydroxylase (TH) promoter contains a GC box (Sp1 motif) whose function is currently unclear. In this study, we examined the effect of immobilization (IMO) stress on binding of adrenomedullary transcription factors to that site. DNase I footprinting analysis reveals the binding of proteins to the Sp1 motif. Extracts from the adrenal medulla of IMO‐treated rats generate a footprint on the Sp1 motif that is smaller than that generated by control extracts. Electrophoretic mobility shift assays using an oligonucleotide containing the Sp1 region show that two Sp1 protein‐containing complexes (I and III) form with control extracts. In contrast, extracts from the adrenal medulla of IMO‐treated rats form a novel complex (II) that contains the Egr1 protein. This is the first report to reveal changes in the binding pattern on the TH Sp1 motif in response to stress and to identify an overlapping Egr1 site.
Induction of an Immediate Early Gene egr‐1 by Zinc Through Extracellular Signal‐Regulated Kinase Activation in Cortical Culture
Authors: Park Jeong Ae , Koh Jae‐Young
Published: March 2003

Abstract : Egr‐1 is one of the immediate early transcription factors that are induced after brain insults. However, the mechanism and the role of Egr‐1 induction are not yet determined. In the present study, using mouse cortical cultures, we examined the ionic mechanism of Egr‐1 induction and its role in neuronal death. Although zinc, NMDA, or ionomycin induced comparable neuronal death in cortical culture, only zinc increased Egr‐1 expression, which was attenuated by blocking zinc influx. It is intriguing that brief exposure to zinc induced sustained extracellular signal‐regulated kinase (Erk) activation. PD098059, an inhibitor of the Erk 1/2 upstream kinase mitogen‐activated protein kinase kinase 1 (MEK1), blocked Erk 1/2 activation, Egr‐1 induction, and neuronal death by zinc. The present study has demonstrated that zinc, rather than calcium, induces lasting Egr‐1 expression in cortical culture by activating Erk 1/2, which is part of a cascade that may play an active role in zinc neurotoxicity. We propose that translocation of endogenous zinc may be the key mechanism of Egr‐1 induction and neuronal death in brain ischemia.
HIV‐1 Tat‐Mediated Activation of Glycogen Synthase Kinase‐3β Contributes to Tat‐Mediated Neurotoxicity
Authors: Maggirwar Sanjay B , Tong Ning , Ramirez Servio , Gelbard Harris A , Dewhurst Stephen
Published: March 2003

Abstract : Human immunodeficiency virus type 1 (HIV‐1) Tat induces neuronal apoptosis. To examine the mechanism(s) that contribute to this process, we studied Tat's effects on glycogen synthase kinase‐3β (GSK‐3β), an enzyme that has been implicated in the regulation of apoptosis. Addition of Tat to rat cerebellar granule neurons resulted in an increase in GSK‐3β activity, which was not associated with a change in protein expression and could be abolished by the addition of an inhibitor of GSK‐3β (lithium). Lithium also enhanced neuronal survival following exposure to Tat. Coprecipitation experiments revealed that Tat can associate with GSK‐3β, but direct addition of Tat to purified GSK‐3β had no effect on enzyme activity, suggesting that Tat's effects might be mediated indirectly. As the activation of platelet activating factor (PAF) receptors is critical for the induction of neuronal death by several candidate HIV‐1 neurotoxins, we determined whether PAF can also activate GSK‐3β. Application of PAF to neuronal cultures activated GSK‐3β, and coincubation with lithium ameliorated PAF‐induced neuronal apoptosis. These findings are consistent with the existence of one or more pathways that can lead to GSK‐3β activation in neurons, and they suggest that the dysregulation of this enzyme could contribute to HIV‐induced neuronal apoptosis.
Regional Distribution, Ontogeny, Purification, and Characterization of the Ca2+‐Independent Phospholipase A2 from Rat Brain
Authors: Yang Hsiu‐Chiung , Mosior Marian , Ni Binhui , Dennis Edward A.
Published: March 2003

Abstract : We purified an 80‐kDa Ca2+‐independent phospholipase A2 (iPLA2) from rat brain using octyl‐Sepharose, ATP‐agarose, and calmodulin‐agarose column chromatography steps. This procedure gave a 30,000‐fold purification and yielded 4 μg of a near‐homogeneous iPLA2 with a specific activity of 4.3 μmol/min/mg. Peptide sequences of the rat brain iPLA2 display considerable homology to sequences of the iPLA2 from P388D1 macrophages, Chinese hamster ovary cells, and human B lymphocytes. Under optimal conditions, the iPLA2 revealed the following substrate preference toward the fatty acid chain in the sn‐2 position of phosphatidylcholine : linoleoyl > palmitoyl > oleoyl > arachidonoyl. The rat brain iPLA2 also showed a head group preference for choline ≥ ethanolamine ≫ inositol. The iPLA2 is inactivated when exposed to pure phospholipid vesicles. The only exception is vesicles composed of phosphatidylcholine and phosphatidylinositol 4,5‐bisphosphate. Studies on the regional distribution and ontogeny of various phospholipase A2 (PLA2) types in rat brain indicate that the iPLA2 is the dominant PLA2 activity in the cytosolic fraction, whereas the group IIA secreted PLA2 is the dominant activity in the particulate fraction. The activities of these two enzymes change during postnatal development.
Effect of Social Experience on Dopamine‐Stimulated Adenylyl Cyclase Activity and G Protein Composition in Chick Forebrain
Authors: Reiser Michael , Poeggel Gerd , Schnabel Reinhild , Schröder Helmut , Braun Katharina
Published: March 2003

Abstract: The stimulation of adenylyl cyclase (AC) by dopamine was investigated in membrane fractions of the forebrain areas mediorostral neostriatum/hyperstriatum ventrale (MNH) and lobus parolfactorius (LPO) of 8‐day‐old domestic chicks that had been raised under different social conditions : group A, socially isolated ; group B, imprinted on an acoustic stimulus ; group C, trained but nonimprinted ; and group D, reared in small groups. Only in the brain of the socially experienced groups could cyclic AMP (cAMP) synthesis be stimulated by dopamine, but not in the socially isolated animals (group A). Ligand binding studies of dopamine D1‐ and D2‐type receptors in membrane fractions did not reveal differences between socially experienced and isolated animals. Forskolin stimulation of total AC in MNH and LPO membrane fractions revealed a significantly enhanced AC stimulation in the socially reared but not in the imprinted group compared with isolated controls. Stimulation of AC by the G protein activator guanylylimidodiphosphate was significantly increased in the MNH and the LPO of socially reared chicks compared with isolated control animals. These results suggest that early postnatal social experience modulates the rate of cAMP synthesis and that these lasting changes are not due to changes of dopamine receptors but are related to increased AC activities and to increased sensitivity of Gsprotein.
Noninvasive Measurements of [1‐13C] Glycogen Concentrations and Metabolism in Rat Brain In Vivo
Authors: Choi In‐Young , Tkáčc Ivan , Ugurbil Kâmil , Gruetter Rolf
Published: March 2003

Abstract : Using a specific 13C NMR localization method, 13C label incorporation into the glycogen C1 resonance was measured while infusing [1‐13C]glucose in intact rats. The maximal concentration of [1‐13C]glycogen was 5.1 ± 0.6 μmol g‐1 (mean ± SE, n = 8). During the first 60 min of acute hyperglycemia, the rate of 13C label incorporation (synthase flux) was 2.3 ± 0.7 μmol g‐1 h‐1 (mean ± SE, n = 9 rats), which was higher (p <0.01) than the rate of 0.49 ± 0.14 μmol g‐1 h‐1 measured ≥2 h later. To assess whether the incorporation of 13C label was due to turnover or net synthesis, the infusion was continued in seven rats with unlabeled glucose. The rate of 13C label decline (phosphorylase flux) was lower (0.33 ± 0.10 μmol g‐1 h‐1) than the initial rate of label incorporation (p < 0.01) and appeared to be independent of the duration of the preceding infusion of [1‐13C]glucose (p > 0.05 for correlation). The results implied that net glycogen synthesis of ~3 μmol g‐1 had occurred, similar to previous reports. When infusing unlabeled glucose before [1‐13C]glucose in three studies, the rate of glycogen C1 accumulation was 0.46 ± 0.08 μmol g‐1 h‐1. The results suggest that steady‐state glycogen turnover rates during hyperglycemia are ~1% of glucose consumption.
Characterization of Transcripts from the Synapsin III Gene Locus
Authors: Porton Barbara , Teh Kao Hung , Greengard Paul
Published: March 2003

Abstract : Synapsin III, the most recently described member of the synapsin gene family, displays a gene structure and protein domain structure similar to those of synapsins I and II. In this report, however, we describe major differences in the temporal‐ and tissue‐specific expressions of synapsin III. Whereas synapsins I and II each give rise to two isoforms that are expressed predominantly in adult brain, there are at least six synapsin III transcripts (synapsin IIIa‐IIIf) that differ with respect to tissue‐ and developmental stage‐specific expression. Three of the neuronal transcripts are detected in fetal and to a lesser extent in adult brain (IIIa‐IIIc), whereas one (IIId) is detected only in fetal brain. Two additional transcripts (IIIe and IIIf) are detected only in nonneuronal tissues. A putative second promoter, which is contained within an intron in the synapsin III gene locus, appears to generate the nonneuronal synapsin IIIe and IIIf transcripts. This level of genome complexity is far greater than that described previously for the synapsin I and II genes and suggests that synapsin III may have functions distinct from those described for synapsins I and II.
Activation and Degradation of the Transcription Factor C/EBP During Long‐Term Facilitation in Aplysia
Authors: Yamamoto Naoki , Hegde Ashok N. , Chain Daniel G. , Schwartz James H.
Published: March 2003

Abstract : Long‐term facilitation (LTF) of the sensory‐to‐motor

synapses that mediate defensive reflexes in Aplysia requires

induction of the transcription factor Aplysia CCAAT/enhancer binding protein (ApC/EBP) as an early response gene. We examined the time course of ApC/EBP DNA binding during the induction of LTF : Binding activity was detected within 1 h of the sensitization treatment with serotonin, reached a maximum at 2 h, and decreased after 6 h. How are DNA binding and the turnover of ApC/EBP regulated ? We find that phosphorylation of ApC/EBP by mitogen‐activated protein (MAP) kinase is essential for binding. MAP kinase appears to be activated through protein kinase C. We also showed that ApC/EBP is degraded through the ubiquitin‐proteasome pathway but that phosphorylation by MAP kinase renders it resistant to proteolysis. Thus, phosphorylation by MAP kinase is required for ApC/EBP to act as a transcription activator as well as to assure its stability early in the consolidation phase, when genes essential for the development of LTF begin to be expressed.
Peroxynitrite‐ and Nitrite‐Induced Oxidation of Dopamine : Implications for Nitric Oxide in Dopaminergic Cell Loss
Authors: LaVoie Matthew J. , Hastings Teresa G.
Published: March 2003

Abstract : Increased nitric oxide (NO) production has been

implicated in many examples of neuronal injury such as the selective

neurotoxicity of methamphetamine and

1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine to dopaminergic cells, presumably

through the generation of the potent oxidant peroxynitrite (ONOO). Dopamine

(DA) is a reactive molecule that, when oxidized to DA quinone, can bind to and

inactivate proteins through the sulfhydryl group of the amino acid cysteine.

In this study, we sought to determine if ONOO could oxidize DA and participate

in this process of protein modification. We measured the oxidation of the

catecholamine by following the binding of [3H]DA to the sulfhydryl‐rich protein alcohol dehydrogenase. Results showed that ONOO oxidized DA in a concentration‐ and pH‐dependent manner. We confirmed that the resulting DA‐protein conjugates were predominantly 5‐cysteinyl‐DA residues. In addition, it was observed that ONOO decomposition products such as nitrite were also effective at oxidizing DA. These data suggest that the generation of NO and subsequent formation of ONOO or nitrite may contribute to the selective vulnerability of dopaminergic neurons through the oxidation of DA and modification of protein.
Human Apolipoprotein E Isoform‐Specific Differences in Neuronal Sprouting in Organotypic Hippocampal Culture
Authors: Teter B. , Xu P.‐T. , Gilbert J. R. , Roses A. D. , Galasko D. , Cole G. M.
Published: March 2003

Abstract : The apolipoprotein E (ApoE) ε4 allele is a major risk factor for neurodegenerative conditions, including Alzheimer's disease. A role for ApoE is implicated in regeneration of synaptic circuitry after neural injury. In the in vitro mouse organotypic hippocampal slice culture system, we previously showed that cultures derived from ApoE‐knockout mice are defective in mossy fiber sprouting into the dentate gyrus molecular layer. This sprouting defect was rescued in cultures from transgenic mice expressing ApoE3 under the control of the human promoter and in ApoE‐knockout cultures treated with ApoE3‐conditioned media. Although the ApoE3 transgene fully restored sprouting, ApoE4 restored sprouting to only 58% of ApoE3 levels. These data indicate that ApoE isoform‐specific effects on neuroregeneration may contribute to its genetic risk for Alzheimer's disease.