bluuebunny/crossref_metadata_2025 huggingface dataset:
Salivary gland delivery of pDNA‐cationic lipoplexes elicits systemic immune responses
Authors: Sankar V , Baccaglini L , Sawdey M , Wheeler CJ , Pillemer SR , Baum BJ , Atkinson JC
Published: March 2003

OBJECTIVE: To test the ability of two cationic lipoplexes, Vaxfectin and GAP‐DLRIE/DOPE, to facilitate transfection and elicit immune responses from plasmid DNAs (pDNAs) after retrograde instillation into salivary glands.

METHODS: Two pDNA expression vectors encoding either the influenza NP protein or human growth hormone (hGH) were complexed with the cationic lipid transfection reagents, GAP‐DLRIE/DOPE or Vaxfectin, and delivered to the submandibular glands of rats. Samples from rats receiving the influenza NP protein pDNA and cationic lipoplexes were analyzed for anti‐influenza NP‐specific IgG1, IgG2a, and IgG2b in serum, and IgA in saliva, by an enzyme‐ linked immunosorbent assay (ELISA). Cytotoxic T‐cell lymphocyte (CTL) assays were also performed. Transgene protein expression (hGH) was determined from extracts of submandibular glands of rats receiving hGH lipoplexes.

RESULTS: Serum antibodies (IgG) against the NP protein developed and were highest in all rats vaccinated with GAP‐DLRIE/DOPE or Vaxfectin. The major serum IgG subclass stimulated by this route of immunization was IgG2b, followed by IgG2a. CTL assay results showed statistically significantly higher percentage killing in the Vaxfectin group than controls (P < 0.05). No rats developed IgA antibodies to NP protein in saliva. Animals receiving plasmid encoding hGH and either lipoplex expressed significantly higher amounts of hGH compared with those receiving the hGH plasmid and water. Although hGH expression was higher in the animals receiving pDNA/Vaxfectin (∼ 30‐fold > pDNA/water), the difference with those receiving pDNA/GAP‐DLRIE/DOPE (∼10‐fold > pDNA/water) was not significant.

CONCLUSIONS: Retrograde instillation of pDNA complexed with Vaxfectin into the salivary glands can stimulate cytotoxic and humoral responses to the influenza NP protein antigen. Optimization of the conditions required to stimulate humoral and secretory antibody formation may facilitate use of this tissue for specific clinical applications of pDNA immunization.
Dento‐osseous changes as diagnostic markers in familial adenomatous polyposis families
Authors: Aggarwal VR , Sloan P , Horner K , Macfarlane TV , Clancy T , Evans G , Thakker N
Published: March 2003

BACKGROUND:  We have previously described a weighted index for determining the diagnostic significance of dento‐osseous changes observed on dental panoramic radiographs (DPRs) in individuals at 50% risk of inheriting Familial Adenomatous Polyposis (FAP). A diagnostic test based on this index (Dental Panoramic Radiograph Score, DPRS) was shown to have a sensitivity of 69% and specificity of 100%.

OBJECTIVES: To evaluate the validity of the diagnostic test in an independent sample of individuals at 50% risk of inheriting FAP.

DESIGN: A retrospective assessment of DPRs in individuals at 50% risk of inheriting FAP.

SUBJECTS AND METHODS:  A final year dental student assessed blindly and independently, DPRs from an independent sample (n = 119) of affected (n = 26), unaffected (n = 78) and clinically low risk individuals (n = 15). This revealed a sensitivity and specificity of 62 and 97% respectively which is in close agreement with results of the previous study. The dental student's training in assessing DPRs was previously tested using radiographs from 81 individuals from our original study. Weighted Kappa statistics were used to test for agreement. A kappa score of 0.82 (95% confidence interval 0.70–0.93) indicated almost perfect agreement.

MAIN OUTCOME: The DPRS is a reproducible and valid index for assessing the diagnostic significance of dento‐osseous changes, in individuals at 50% risk of FAP, even in relatively inexperienced hands.
The 37 kDa/67 kDa laminin receptor is required for PrPSc propagation in scrapie‐infected neuronal cells
Authors: Leucht Christoph , Simoneau Steve , Rey Clémence , Vana Karen , Rieger Roman , Lasmézas Corinne Ida , Weiss Stefan
Published: March 2003

The accumulation of PrPSc in scrapie‐infected neuronal cells has been prevented by three approaches: (i) transfection of ScMNB cells with an antisense laminin receptor precursor (LRP) RNA‐expression plasmid, (ii) transfection of ScN2a cells and ScGT1 cells with small interfering RNAs (siRNAs) specific for the LRP mRNA, and (iii) incubation of ScN2a cells with an anti‐LRP/LR antibody. LRP antisense RNA and LRP siRNAs reduced LRP/LR expression and inhibited the accumulation of PrPSc in these cells. The treatments also reduced PrPc levels. The anti‐LRP/LR antibody, W3, abolished PrPSc accumulation and reduced PrPc levels after seven days of incubation. Cells remained free of PrPSc after being cultured for 14 additional days without the antibody, whereas the PrPc level was restored. Our results demonstrate the necessity of the laminin receptor (LRP/LR) for PrPSc propagation in cultured cells and suggest that LRP/LR‐specific antibodies could be used as powerful therapeutic tools in the treatment of transmissible spongiform encephalopathies.
Hypoxia-inducible factor induction by tumour necrosis factor in normoxic cells requires receptor-interacting protein-dependent nuclear factor kappaB activation
Authors: JUNG YunJin , ISAACS Jennifer S. , LEE Sunmin , TREPEL Jane , LIU Zheng-gang , NECKERS Len
Published: March 2003

Tumour necrosis factor α (TNF-α) binds to its receptor (TNFR1) and activates both death- and inflammation/survival-related signalling pathways. The inflammation and survival-related signalling cascade results in the activation of the transcription factor, nuclear factor κB (NF-κB) and requires recruitment of receptor-interacting protein (RIP) to TNFR1. The indispensable role of RIP in TNF-induced NF-κB activation has been demonstrated in RIP-/- mice and in cell lines derived from such mice. In the present study, we show that the TNF-α-induced accumulation of hypoxia-inducible factor 1α (HIF-1α) protein in normoxic cells is RIP-dependent. Exposing fibroblasts derived from RIP-/- mice to either cobalt or PMA resulted in an equivalent HIF-1α induction to that seen in RIP+/+ fibroblasts. In contrast, RIP-/- cells were unable to induce HIF-1α in response to TNF-α. Further, transient transfection of NIH 3T3 cells with an NF-κB super-repressor plasmid (an inhibitor of NF-κB activation) also prevented HIF-1α induction by TNF-α. Surprisingly, although HIF-1α mRNA levels remained unchanged after induction by TNF, induction of HIF-1α protein by the cytokine was completely blocked by pretreatment with the transcription inhibitors actinomycin D and 5,6-dichlorobenzimidazole riboside. Finally, TNF failed to induce both HIF-1α, made resistant to von Hippel—Lindau (VHL), and wild-type HIF-1α transfected into VHL-/- cells. These results indicate that HIF-1α induction by TNF-α in normoxic cells is mediated by protein stabilization but is nonetheless uniquely dependent on NF-κB-driven transcription. Thus the results describe a novel mechanism of HIF-1α up-regulation and they identify HIF-1α as a unique component of the NF-κB-mediated inflammatory/survival response.
Failure to increase glucose consumption through the pentose-phosphate pathway results in the death of glucose-6-phosphate dehydrogenase gene-deleted mouse embryonic stem cells subjected to oxidative stress
Authors: FILOSA Stefania , FICO Annalisa , PAGLIALUNGA Francesca , BALESTRIERI Marco , CROOKE Almudena , VERDE Pasquale , ABRESCIA Paolo , BAUTISTA José M. , MARTINI Giuseppe
Published: March 2003

Mouse embryonic stem (ES) glucose-6-phosphate (G6P) dehydrogenase-deleted cells (G6pdΔ), obtained by transient Cre recombinase expression in a G6pd-loxed cell line, are unable to produce G6P dehydrogenase (G6PD) protein (EC 1.1.1.42). These G6pdΔ cells proliferate in vitro without special requirements but are extremely sensitive to oxidative stress. Under normal growth conditions, ES G6pdΔ cells show a high ratio of NADPH to NADP+ and a normal intracellular level of GSH. In the presence of the thiol scavenger oxidant, azodicarboxylic acid bis[dimethylamide], at concentrations lethal for G6pdΔ but not for wild-type ES cells, NADPH and GSH in G6pdΔ cells dramatically shift to their oxidized forms. In contrast, wild-type ES cells are able to increase rapidly and intensely the activity of the pentose-phosphate pathway in response to the oxidant. This process, mediated by the [NADPH]/[NADP+] ratio, does not occur in G6pdΔ cells. G6PD has been generally considered essential for providing NADPH-reducing power. We now find that other reactions provide the cell with a large fraction of NADPH under non-stress conditions, whereas G6PD is the only NADPH-producing enzyme activated in response to oxidative stress, which can act as a guardian of the cell redox potential. Moreover, bacterial G6PD can substitute for the human enzyme, strongly suggesting that a relatively simple mechanism of enzyme kinetics underlies this phenomenon.
Copper‐ and arsenate‐induced oxidative stress in Holcus lanatus L. clones with differential sensitivity
Authors: Hartley‐Whitaker J. , Ainsworth G. , Meharg A. A.
Published: March 2003

The biochemical responses of Holcus lanatus L. to copper and  arsenate  exposure were investigated in arsenate‐tolerant and ‐non‐tolerant plants from uncontaminated and arsenic/copper‐contaminated sites. Increases in lipid peroxidation, superoxide dismutase (SOD) activity and phytochelatin (PC) production were correlated with increasing copper and arsenate exposure. In addition, significant differences in biochemical responses were observed between arsenate‐tolerant and ‐non‐tolerant plants. Copper and arsenate exposure led to the production of reactive oxygen species, resulting in significant lipid peroxidation in non‐tolerant plants. However, SOD activity was suppressed upon metal exposure, possibly due to interference with metallo‐enzymes. It was concluded that in non‐tolerant plants, rapid arsenate influx resulted in PC production, glutathione depletion and lipid peroxidation. This process would also occur in tolerant plants, but by decreasing the rate of influx, they were able to maintain their constitutive functions, detoxify the metals though PC production and quench reactive oxygen species by SOD activity.
The temperature dependence of shoot hydraulic resistance: implications for stomatal behaviour and hydraulic limitation
Authors: Matzner S. , Comstock J.
Published: March 2003

Recent soil pressurization experiments have shown that stomatal closure in response to high leaf–air humidity gradients can be explained by direct feedback from leaf water potential. The more complex temperature‐by‐humidity interactive effects on stomatal conductance have not yet been explained fully. Measurements of the change in shoot conductance with temperature were made on Phaseolus vulgaris (common bean) to test whether temperature‐induced changes in the liquid‐phase transport capacity could explain these temperature‐ by‐humidity effects. In addition, shoot hydraulic resistances were partitioned within the stem and leaves to determine whether or not leaves exhibit a greater resistance. Changes in hydraulic conductance were calculated based on an Ohm’s law analogy. Whole‐plant gas exchange was used to determine steady‐ state transpiration rates. A combination of in situ psychrometer measurements, Scholander pressure chamber measurements and psychrometric measurements of leaf punches was used to determine water potential differences within the shoot. Hydraulic conductance for each portion of the pathway was estimated as the total flow divided by the water potential difference. Temperature‐induced changes in stomatal conductance were correlated linearly with temperature‐induced changes in hydraulic conductance. The magnitude of the temperature‐induced changes in whole‐plant hydraulic conductance was sufficient to account for the interactive effects of temperature and humidity on stomatal conductance.
The cooling of convolvulaceous flowers in a tropical environment
Authors: Patiño S. , Grace J.
Published: March 2003

The temperatures of flowers of Ipomoea pes‐caprae ssp. brasilensis, Ipomoea aquatica and Merremia borneensis in bright sunshine, were studied to determine the role of corollas and sepals in cooling the gynoecium. The corollas and sepals were prevented from transpiring by greasing, to investigate the extent of evaporative cooling. In the exposed natural habitats of these flowers the maximum temperatures of air and soil were high (32 and 42 °C, respectively) and corolla, sepal and gynoecium temperatures were often intermediate. Despite being almost astomatous, significant evaporative cooling was observed in the corolla. Between 20 and 80% of the energy absorbed by the corollas was dissipated as evaporation. The sepals were stomatous and their evaporative cooling was very important in reducing the temperature of the gynoecium. The temperatures of the non‐transpiring gynoecia and corollas were significantly higher than the temperatures of the normally transpiring corollas and gynoecia. Furthermore, the gynoecia temperatures were significantly higher with non‐transpiring corollas than with normally transpiring corollas, suggesting that the corollas alone play a role in maintaining the gynoecium within optimal temperatures levels. It was shown in an incubation experiment that temperatures exceeding 32 °C may damage the carpels, and temperatures exceeding 42 °C may damage sepals. Pollen grains were killed after 200 min of exposure to temperatures in the range 32 to 47 °C. It is concluded that the cooling mechanisms (evaporation and self‐shading) are critical for the reproductive success of these flowers in their natural environment.
‘Soil conditions and plant growth’
Authors: Passioura J. B.
Published: March 2003

Plants can respond to soil conditions in ways that can not readily be explained in terms of the ability of the roots to take up water and nutrients. Roots may sense difficult conditions in the soil and thence send inhibitory signals to the shoots which harden the plants against the consequences of a deteriorating or restrictive environment, especially if the plants’ water supply is at risk. Generally, this behaviour can be interpreted as feedforward responses to the soil becoming too dry or too hard, or to the available soil volume being very small as with bonsai plants, or to roots’ becoming infected with pathogens. However, soil that is too soft or in which the roots are forced to grow in very large pores can also induce large conservative responses, the significance of which is unclear. The inhibitory signals may affect stomatal conductance, cell expansion, cell division and the rate of leaf appearance. Their nature is still under debate, and the debate is becoming increasingly complex, which probably signifies that a network of hormonal and other responses is involved in attuning the growth and development of a plant to its environment.
Flower developmental stage and organ sensitivity of bell pepper (Capsicum annuum L.) to elevated temperature
Authors: Erickson A. N. , Markhart A. H.
Published: March 2003

High temperatures adversely affect crop productivity of several plant species including bell pepper (Capsicum annuum L. var. annuum). The objectives of this study were: (1) to determine whether flower ontogeny is adversely affected by high temperature during different phases of development, including pre‐ and post‐pollination events; (2) to determine the duration of high temperature exposure necessary to cause reduction in fruit set; and (3) to determine whether injury to the pistil or stamen during development is responsible for reduced fruit set during high temperature. We determined that flower buds at <2·5 mm in length, corresponding to microspore mother cell meiosis to tetrad dissolution, and flowers that reached anthesis during the high temperature exposure had reduced fruit set when exposed to 33 °C for 48 or 120 h. Flower buds at <2·5 mm in length also had reduced pollen viability when exposed to 33 °C for 120 h. Morphological examination demonstrated that meiocytes initiated tetrad formation, but after dissolution the microspores remained small and clumped without a thick exine. High temperature exposure at a late‐development, pre‐anthesis stage did not affect pistil or stamen viability, but high post‐pollination temperatures inhibited fruit set, suggesting that fertilization is sensitive to high temperature stress.
Environmental and stomatal control of photosynthetic enhancement in the canopy of a sweetgum (Liquidambar styraciflua L.) plantation during 3 years of CO2 enrichment
Authors: Gunderson C. A. , Sholtis J. D. , Wullschleger S. D. , Tissue D. T. , Hanson P. J. , Norby R. J.
Published: March 2003

Light‐saturated photosynthetic and stomatal responses to elevated CO2 were measured in upper and mid‐canopy foliage of a sweetgum (Liquidambar styraciflua L) plantation exposed to free‐air CO2 enrichment (FACE) for 3 years, to characterize environmental interactions with the sustained CO2 effects in an intact deciduous forest stand. Responses were evaluated in relation to one another, and to seasonal patterns and natural environmental stresses, including high  temperatures, vapour pressure deficits (VPD), and drought. Photosynthetic CO2 assimilation (A) averaged 46% higher in the +200 µmol mol−1 CO2 treatment, in mid‐ and upper canopy foliage. Stomatal conductance (gs) averaged 14% (mid‐canopy) and 24% (upper canopy) lower under CO2 enrichment. Variations in the relative responses of A and gs were linked, such that greater relative stimulation of A was observed on dates when relative reductions in gs were slight. Dry soils and high VPD reduced gs and A in both treatments, and tended to diminish treatment differences. The absolute effects of CO2 on A and gs were minimized whenever gs was low (<0·15 mol m−2 s−1), but relative effects, as the ratio of elevated to ambient rates, varied greatly under those conditions. Both stomatal and non‐stomatal limitations of A were involved during late season droughts. Leaf temperature had a limited influence on A and gs, and there was no detectable relationship between prevailing temperature and CO2 effects on A or gs. The responsiveness of A and gs to elevated CO2, both absolute and relative, was maintained through time and within the canopy of this forest stand, subject to seasonal constraints and variability associated with limiting air and soil moisture.
Seasonal variation in δ13C and δ18O of cellulose from growth rings of Pinus radiata
Authors: Barbour M. M. , Walcroft A. S. , Farquhar G. D.
Published: March 2003

Seasonal variation in δ13C and δ18O of cellulose (δ13Cc and δ18Oc) was measured within two annual rings of Pinus radiata growing at three sites in New Zealand. In general, both δ13Cc and δ18Oc increased to a peak over summer. The three sites differed markedly in annual water balance, and these differences were reflected in δ13Cc and δ18Oc. Average δ13Cc and δ18Oc from each site were positively related, so that the driest site had the most enriched cellulose. δ13Cc and δ18Oc were also related within each site, although both the slope and the closeness of fit of the relationship varied between sites. Supporting the theory, the site with the lowest average relative humidity also had the greatest change in δ18Oc‰ change in δ13Cc. Specific climatic events, such as drought or high rainfall, were recorded as a peak or a trough in enrichment, respectively. These results suggest that seasonal and between‐site variation in δ13Cc and δ18Oc are driven by the interaction between variation in climatic conditions and soil water availability, and plant response to this variation.
Different wintering strategies of two Palearctic migrants in West Africa – a consequence of foraging strategies?
Authors: Salewski Volker , Bairlein Franz , Leisler Bernd
Published: March 2003

The wintering strategies of Pied Flycatchers Ficedula hypoleuca and Willow Warblers Phylloscopus trochilus in their West African winter quarters were compared. Pied Flycatchers arrived early in the season (September) and stayed in the study area throughout the winter. They were territorial and showed a high return rate. Intraspecific relationships were mostly expressed by territorial behaviour. Interspecific relations seemed to be unimportant. Willow Warblers arrived relatively late (November) and were absent from the area for some weeks in January and February, a behaviour which was interpreted as itinerancy. Willow Warblers were non‐territorial and never returned to a site. Willow Warblers usually moved through the area in monospecific or mixed‐species flocks. Habitat and microhabitat choice of these species were similar but in feeding ecology they differed by the higher diversity of feeding substrates and feeding techniques of Pied Flycatchers. The differences in the winter strategies are explained by the ability of Pied Flycatchers to defend a territory because of their diversity in foraging behaviour, whereas Willow Warblers are more specialized and are therefore forced to be more mobile to find their patchily distributed food.
The effects of parasitism and body length on positioning within wild fish shoals
Authors: Ward A. J. W. , Hoare D. J. , Couzin I. D. , Broom M. , Krause J.
Published: March 2003

The influence of body length and parasitism on the positioning behaviour of individuals in wild fish shoals was investigated by a novel means of capturing entire shoals of the banded killifish (Fundulus diaphanus, Lesueur) using a grid‐net that maintained the two‐dimensional positions of individuals within shoals.

Fish in the front section of a shoal were larger than those in the rear.

Individuals parasitized by the digenean trematode (Crassiphiala bulboglossa, Haitsma) showed a tendency to occupy the front of shoals. Parasitized fish were also found more in peripheral positions than central ones in a significant number of shoals.

Shoal geometry was affected by the overall parasite prevalence of shoal members; shoals with high parasite prevalence displayed increasingly phallanx‐like shoal formations, whereas shoals with low prevalence were more elliptical.

There was no relationship between body length and parasite abundance or prevalence in the fish population which suggests body length and parasite status are independent predictors of positioning behaviour.

Solitary individuals found outside shoals were both more likely to be parasitized and had higher parasite abundance than individuals engaged in shoaling.

Differences in the shoaling behaviour of parasitized and unparasitized fish are discussed in the context of the adaptive manipulation hypothesis.
Is there really a drift paradox?
Authors: Humphries Stuart , Ruxton Graeme D.
Published: March 2003

Recent explanations for the persistence of freshwater invertebrates in the face of downstream drift (the so‐called ‘drift paradox’) have centred largely on recolonization by upstream flight by preovipositing adults.

Such explanations do not cover the many important species often found in the drift that lack an aerial phase. Here we demonstrate conclusively that long‐range aerial movements are not required to explain the drift paradox.

We show that small‐scale movements along the streambed of the order of centimetres, combined with density dependence at some stage of the lifecycle, are sufficient to maintain populations over geological timescales.

Further, we demonstrate that the principal previous modelling study of the drift paradox was parameterized in a way that is more compatible with such small‐scale movements than the long‐range flight for which it was intended.
A metadata classification schema for semantic content analysis of videos
Authors: Shotton D. M. , Rodríguez A. , Guil N. , Trelles O.
Published: March 2003

Simple ancillary metadata, such as those encompassed by the 15 elements of the Dublin Core, may be sufficient and entirely appropriate for basic coarse‐granularity cross‐domain resource discovery. However, they are insufficient and inappropriate for content description of complex data types such as videos, which require more detailed relational models. We propose a metadata classification schema for the characterization of items and events in videos that permits subsequent query by content. Following MPEG‐7 nomenclature, metadata intrinsic to the information content of the video are defined as either structural or semantic, where structural metadata are numerical feature primitives produced by analysing the colour, shape, texture, structure and motion within the video frames, whereas semantic metadata describe the locations and timings of individual items and particular actions or events in the video, and are thus of higher information value. In this paper, the semantic metadata required to describe the visual information content of videos are defined and classified into four distinct classes: Media Entities; Content Items; Events; and Supplementary Items, and three types of property tables are defined: Identity Tables; Spatio‐Temporal Position Tables; and Event Tables, in which these metadata may be stored in a relational database.
Neuroprotection by adenosine A2A receptor blockade in experimental models of Parkinson's disease
Authors: Ikeda Ken , Kurokawa Masako , Aoyama Shiro , Kuwana Yoshihisa
Published: March 2003

Adenosine A2A receptors are abundant in the caudate‐putamen and involved in the motor control in several species. In MPTP‐treated monkeys, A2A receptor‐blockade with an antagonist alleviates parkinsonian symptoms without provoking dyskinesia, suggesting this receptor may offer a new target for the antisymptomatic therapy of Parkinson's disease. In the present study, a significant neuroprotective effect of A2A receptor antagonists is shown in experimental models of Parkinson's disease. Oral administration of A2A receptor antagonists protected against the loss of nigral dopaminergic neuronal cells induced by 6‐hydroxydopamine in rats. A2A antagonists also prevented the functional loss of dopaminergic nerve terminals in the striatum and the ensuing gliosis caused by MPTP in mice. The neuroprotective property of A2A receptor antagonists may be exerted by altering the packaging of these neurotoxins into vesicles, thus reducing their effective intracellular concentration. We therefore conclude that the adenosine A2A receptor may provide a novel target for the long‐term medication of Parkinson's disease, because blockade of this receptor exerts both acutely antisymptomatic and chronically neuroprotective activities.
Mitochondrial permeability transition and calcium dynamics in striatal neurons upon intense NMDA receptor activation
Authors: Alano Conrad C. , Beutner Gisela , Dirksen Robert T. , Gross Robert A. , Sheu Shey‐Shing
Published: March 2003

Deregulation of the intracellular Ca2+ homeostasis by NMDA receptor activation leads to neuronal cell death. Induction of the mitochondrial permeability transition pore (MPT) by Ca2+ is a critical event in mediating cell death. In this study, we used fluorescent Ca2+ indicators to investigate the effect of high concentrations of NMDA on cytosolic and mitochondrial Ca2+ concentrations ([Ca2+]c and [Ca2+]m, respectively) in cultured striatal neurons. Exposure to NMDA resulted in an immediate, sustained increase in [Ca2+]c followed by a secondary increase in [Ca2+]c. This second increase of [Ca2+]c was prevented by pretreatment with N‐methyl‐valine‐4‐cyclosporin (NMV‐Cys). Exposure of neurons to NMDA also resulted in an increase in [Ca2+]m that was followed by a precipitous decrease in the rhod‐2 signal. This decrease followed the time frame of the secondary increase in [Ca2+]c. Preincubation of the neurons with NMV‐Cys prevented the decrease in rhod‐2 fluorescence. These dynamic changes in the rhod‐2 signal and [Ca2+]m in response to NMDA were confirmed by using confocal microscopy. The presented results indicate that MPT can be detected in living neurons using fluorescent Ca2+ indicators, which would allow the study of the physiological role of MPT in cell death.
Multiple mechanisms of transmitter release evoked by ‘pathologically’ elevated extracellular [K+]: involvement of transporter reversal and mitochondrial calcium
Authors: Raiteri Luca , Stigliani Sara , Zedda Luca , Raiteri Maurizio , Bonanno Giambattista
Published: March 2003

The release of [3H]GABA evoked by depolarization with various concentrations of KCl was studied using superfused rat cerebrocortex synaptosomes. Elevating [K+] produced release of [3H]GABA over basal which was increasingly less dependent on external Ca2+ but more sensitive to the GABA transporter blocker SKF 100330 A. Accordingly, the sensitivity to clostridial toxins of the depolarization‐evoked amino acid release was inversely correlated to the concentration of KCl used. However, at 50 mm K+, one‐third of the stimulated release remained which was external Ca2+‐independent but insensitive to SKF 100330 A. This release was prevented by BAPTA, thapsigargin or dantrolene; it also was inhibited by blocking in mitochondria the ATP production with oligomycin, the H+‐dependent Ca2+ uniporter with RU 360, the Na+/Ca2+ exchanger with CGP 37157 or by lowering extraterminal [Na+]. In fluorescence experiments with fura‐2/AM, 50 mm K+ (in␣Ca2+‐free medium) caused elevation of cytosolic [Ca2+] that was sensitive to thapsigargin or CGP 37157; these compounds produced partially additive effects. When exocytosis was monitored with the fluorescent dye acridine orange, the fluorescence elicited by 50 mm K+ was sensitive to thapsigargin or CGP 37157, which produced additive effects, and to low‐Na+ media. To conclude, extracellular K+ concentrations occurring in the CNS in certain pathological conditions provoke GABA release by mechanisms different from classical exocytosis. These include carrier‐mediated release and internal Ca2+‐dependent exocytosis; in the latter, mitochondrial Ca2+ seems to play a primary role.
Protease‐activated receptor‐2 (PAR‐2) in brain microvascular endothelium and its regulation by plasmin and elastase
Authors: Dömötör Eszter , Bartha Katalin , Machovich Raymund , Adam‐Vizi Vera
Published: March 2003

Protease‐activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. We have reported earlier that primary cultures of rat brain capillary endothelial (RBCE) cells express at least two receptors for thrombin: PAR‐1 and PAR‐3. In the present study we show that PAR‐2 activation by trypsin or by the PAR‐2 agonist peptide (SLIGRL) evokes [Ca2+]i signal in RBCE cells. Taking advantage of RBCE cells expressing PAR‐1 and PAR‐2, we show that trypsin activates both receptors. The relative agonist activity of trypsin and thrombin on PARs of RBCE cells compared with that of SLIGRL were 112% and 48%, respectively, whereas the potency of trypsin was 105‐fold higher than that of SLIGRL. Because under pathological conditions other proteases such as plasmin or leukocyte elastase may reach the cells of the blood–brain barrier, we investigated the effect of these proteases on RBCE cells. Elastase evoked a small increase in [Ca2+]i but preincubation of cells with elastase dose‐dependently reduced the trypsin‐induced [Ca2+]i signal. Plasmin had a 30% inhibitory effect on the trypsin‐induced response, and reduced the SLIGRL signal by 20%. It is concluded that PAR‐2 is functional in brain capillary endothelium, and that the main␣fibrinolytic proteases, plasmin and elastase, may regulate PAR‐2 signalling under pathological conditions.
Hypoxia induces mitochondrial DNA damage and stimulates expression of a DNA repair enzyme, the Escherichia coli MutY DNA glycosylase homolog (MYH), in vivo, in the rat brain
Authors: Lee Heung‐Man , Wang Cheng , Hu Zhaoyong , Greeley George H. , Makalowski Wojciech , Hellmich Helen L. , Englander Ella W.
Published: March 2003

Hypoxia‐associated, acutely reduced blood oxygenation can compromise energy metabolism, alter oxidant/antioxidant balance and damage cellular components, including DNA. We show in vivo, in the rat brain that respiratory hypoxia leads to formation of the oxidative DNA lesion, 8‐hydroxy‐2′‐deoxyguanosine (oh8dG), a biomarker for oxidative DNA damage and to increased expression of a DNA repair enzyme involved in protection of the genome from the mutagenic consequences of oh8dG. The enzyme is a homolog of the Escherichia coli MutY DNA glycosylase (MYH), which excises adenine residues misincorporated opposite the oxidized base, oh8dG. We have cloned a full‐length rat MYH (rMYH) cDNA, which encodes 516 amino acids, and by in situ hybridization analysis obtained expression patterns of rMYH mRNA in hippocampal, cortical and cerebellar regions. Ensuing hypoxia, mitochondrial DNA damage was induced and rMYH expression strongly elevated. This is the first evidence for a regulated expression of a DNA repair enzyme in the context of respiratory hypoxia. Our findings support the premise that oxidative DNA damage is repaired in neurons and the possibility that the hypoxia‐induced expression of a DNA repair enzyme in the brain represents an adaptive mechanism for protection of neuronal DNA from injurious consequences of disrupted energy metabolism and oxidant/antioxidant homeostasis.
Characterization of the cocaine‐ and amphetamine‐regulated transcript (CART) peptide gene promoter and its activation by a cyclic AMP‐dependent signaling pathway in GH3 cells
Authors: Dominguez Geraldina , Lakatos Anita , Kuhar Michael J.
Published: March 2003

Cocaine‐ and amphetamine‐regulated transcript (CART) peptides are regulated neuropeptides that play a role in a variety of physiological processes. CART mRNA is also highly regulated as its levels change in response to psychostimulant drugs and leptin. To understand the mechanisms involved in regulating CART mRNA levels, the mouse CART 5′‐flanking regulatory region was studied. The sequence of 3.4 kb of the mouse CART 5′‐flanking region revealed a proximal promoter that contains a cluster of transcription factor binding sites, including an overlapping STAT/CRE/AP1 site. In addition, the 5′‐most 320 bp of the CART promoter shares 83% nucleotide identity between mouse and human. Three luciferase expressing constructs containing varying amounts of CART 5′ upstream sequence were generated and tested for promoter activity. Transient transfection of GH3 cells with constructs containing 641 and 3451 bp of upstream sequence displayed strong promoter activity, producing 29‐fold and 51‐fold stimulation, respectively, while, a construct containing 102 bp of upstream sequence displayed a 5.4‐fold increase in activity. A␣construct containing the composite STAT/CRE/AP1 site was responsive to cyclic AMP induction by forskolin in GH3 cells. Forskolin treatment also resulted in a 4.5‐fold increase in CART mRNA levels after 6 h and the addition of H89, an inhibitor of protein kinase A, reduced the levels by 50%. These studies indicate that the CART proximal promoter lies within the 5′‐most 641 bp and that in GH3 cells the CART gene is regulated via a cyclic AMP‐dependent pathway.
Dβ3, an atypical nicotinic acetylcholine receptor subunit fromDrosophila: molecular cloning, heterologous expression and coassembly
Authors: Lansdell Stuart J. , Millar Neil S.
Published: March 2003

Insect nicotinic acetylcholine receptors (nAChRs) play a central role in mediating neuronal synaptic transmission and are the target sites for the increasingly important group of neonicotinoid insecticides. Six nicotinic acetylcholine receptor (nAChR) subunits (four α‐type and two β‐type) have been cloned previously from the model insect speciesDrosophila melanogaster. Despite extensive efforts, it has not been possible to generate functional recombinant nAChRs by heterologous expression of any combination of these six subunits. It has, however, been possible to express functional hybrid receptors whenDrosophilaα subunits are co‐expressed with vertebrate β subunits. This has led to the assumption that successful heterologous expression might require an, as yet, uncloned β‐type insect subunit. Examination of the recently completedDrosophilagenomic sequence data has identified a novel putative nAChR β‐type subunit. Here we report the molecular cloning, heterologous expression and characterization of this putativeDrosophilanAChR subunit (Dβ3). Phylogenetic comparisons with other ligand‐gated ion channel subunit sequences support its classification as a nAChR subunit but show it to be a distantly related member of this neurotransmitter receptor subunit family. Evidence that the Dβ3 subunit is able to coassemble with otherDrosophilanAChR subunits and contribute to recombinant nAChRs has been obtained by both radioligand binding and coimmunoprecipitation studies in transfectedDrosophilaS2 cells.
Signals in abscission
Authors: Taylor Jane E. , Whitelaw Catherine A.
Published: March 2003

Abscission is the term used to describe the process of natural separation of organs from the parent plant. This may be part of the highly programmed development of a plant, or in response to environmental stress. It enables temperate plants to overwinter and hence survive, but in agricultural or horticultural environments premature abscission can lead to significant crop losses. Abscission is the culmination of changes in gene expression, which result in the loosening of adjacent cell walls within the zone and subsequent cell separation. For many years it has been recognized that the balance between the plant hormones ethylene and auxin determine where, and when, separation takes place. As we begin to understand the mechanisms by which plant growth regulator signals are perceived and transduced, we can begin to understand how the process of abscission itself may be induced and regulated. This review details what we know of the signals that lead to the differentiation of zone cells; the environmental signals that promote cell separation, and the possible intracellular signalling events that culminate in organ shedding.

Contents

Summary
323

I.
Introduction
323

II.
Differentiation of the abscission zone
324

III.
Environmental and developmental signals that induce abscission
327

1. Senescence
327

2. Photoperiod
327

3. Water stress
328

4. Wounding and pathogen attack
328

5. Ozone
329

IV.
Inter‐ and intracellular signals
329

1. Role of ethylene and auxin
330

2. Mechanisms of plant hormone signal perception
330

3. Roles of other hormones and components of signal transduction
333

V.
Conclusions
335

Acknowledgements
336

References
336
Pollen competition between cultivated and wild rice species (Oryza sativaandO. rufipogon)
Authors: Song Zhiping , Lu Baorong , Zhu Yingguo , Chen Jiakuan
Published: March 2003

Post‐pollination competition is reported here in cultivated rice (Oryza sativa) and a perennial wild rice (O. rufipogon) to investigate the occurrence of crop‐to‐wild gene flow.

 Wild and cultivated rice (variety Minghui‐63) were grown in a common garden in Hunan province, China, and crop‐specific genetic markers were used to detect hybridization following hand‐pollinations. Using 11 sequential pollination treatments, the effects of the relative timing of pollination on the success of foreign pollen was investigated.

 Foreign pollen from the crop resulted in lower pollen germination, fewer pollen tubes per style, and a significant reduction of seed set, demonstrating a disadvantage of foreign pollen even in the absence of pollen competition. When 1 : 1 pollen mixtures were applied, only 2% of the resulting seeds were hybrids, revealing a much stronger disadvantage of foreign pollen when competing with conspecific pollen. Testing the effects of the relative timing of pollination on the success of foreign pollen suggested that conspecific pollen is often more successful than foreign pollen. Nonetheless, hybridization is possible following the deposition of pollen mixtures, especially when foreign pollen arrives earlier than conspecific pollen.

 Pollen competition between wild and cultivated rice could slow the rate of crop‐to‐wild gene flow, but even if pollen competition was ubiquitous it would not prevent gene flow from the crop.
Growth, nitrogen uptake, and resource allocation in the two tripartite lichens Nephroma arcticum and Peltigera aphthosa during nitrogen stress
Authors: Dahlman Lena , Näsholm Torgny , Palmqvist Kristin
Published: March 2003

Lichen responses towards nitrogen stress, both increased exposure and deprivation of N, were investigated by measuring N uptake, growth, ergosterol, chitin and Chla in two tripartite nitrogen‐fixing species, Nephroma arctium and Peltigera aphthosa.

 The lichens were irrigated with different N forms, enriched in 15N to assess N uptake, during 3 months in the field, with a total N dosage of 500 mg m−2. Nitrogen deprivation was induced by removing the nitrogen‐fixing cephalodia.

 The lichens took up 11–134 mg N m−2 of the added N, corresponding to 1–4% of their total thallus N. Uptake was 4 times higher for NH4+ than for NO3−, and the highest 15N concentrations were found in newly synthesized tissue. Both forms of N stress affected thallus expansion rates in both species.

 It is concluded that the two lichens were able to maintain a balanced tissue N concentration despite large variations in N supply, and that assimilated N might be transported to growing apices. Alternatively, N assimilation from external sources might be greater in the margins than in the mature thallus. Thallus expansion was sensitive to N stress, apparently being tightly coupled to N assimilation.
Reading a CO2 signal from fossil stomata
Authors: Beerling D. J. , Royer D. L.
Published: March 2003

The inverse relationship between atmospheric CO2 and the stomatal index (proportion of epidermal cells that are stomata) of vascular land plant leaves has led to the use of fossil plant cuticles for determining ancient levels of CO2. In contemporary plants the stomatal index repeatedly shows a lower sensitivity atmospheric CO2 levels above 340 ppm in the short term. These observations demonstrate that the phenotypic response is nonlinear and may place constraints on estimating higher‐than‐present palaeo‐CO2 levels in this way. We review a range of evidence to investigate the nature of this nonlinearity. Our new data, from fossil Ginkgo cuticles, suggest that the genotypic response of fossil Ginkgo closely tracks the phenotypic response seen in CO2 enrichment experiments. Reconstructed atmospheric CO2 values from fossil Ginkgo cuticles compare well with the stomatal ratio method of obtaining a quantitative CO2 signal from extinct fossil plants, and independent geochemical modelling studies of the long‐term carbon cycle. Although there is self‐consistency between palaeobiological and geochemical CO2 estimates, it should be recognized that the nonlinear response is a limitation of the stomatal approach to estimating high palaeo‐CO2 levels.
Recent experience with human immunodeficiency virus transmission by cellular blood products in Germany: antibody screening is not sufficient to prevent transmission
Authors: Dreier J. , Götting C. , Wolff C. , Petersen N. , Kleesiek K.
Published: March 2003

Background and ObjectivesA case of transfusion‐related human immunodeficiency virus‐1 (HIV‐1) transmission was not detected by standard HIV antibody screening.

Materials and MethodsIn a look‐back procedure, the preceding donations were extensively analyzed by nucleic acid amplification technology (NAT) screening and alternative serological tests.

ResultsThe chain of infection could be demonstrated by the analysis of HIV‐specific amplicon sequences from the donor and the recipient.

ConclusionThis case report clearly indicates that the remaining risk of transfusion‐related transmission of HIV could be severely reduced, not only by the use of NAT screening but even by HIV antigen screening or more sensitive HIV antibody assays.
Serum immunoglobulin E response in calves infected with the lungworm Dictyocaulus viviparus and its correlation with protection
Authors: Kooyman F. N. J. , Yatsuda A. P. , Ploeger H. W. , Eysker M.
Published: March 2003

Protection of a primary Dictyocaulus viviparus infection was measured against a homologueous challenge infection in two independent experiments and this was correlated with serum immunoglobulin (Ig)E responses. A primary infection of 30 third stage larvae (L3) of D. viviparus on day 0 protects calves for 70% against a challenge infection of 2000 L3 on day 35 compared to calves with no primary infection. The variation in post mortem worm counts within this group (n = 6) was very large with mean worm counts of 145 (range 3–446) lungworms. Parasite specific IgA, IgE, IgG1 and IgG2 and total IgE levels in serum were measured by ELISA. Parasite specific IgA, IgG1 and IgG2 were elevated after infection, but correlation with protection was only found with IgG1 levels on day 42 and with IgG2 levels on day 70. IgE was measured in a sandwich ELISA using antisheep IgE that cross‐reacts with cattle IgE. No parasite specific IgE could be detected. However, total serum IgE was elevated after infection and total serum IgE levels before and on the day of challenge correlated with protection (P < 0·05). Total serum IgE also correlates with peripheral eosinophil counts between days 14 and 28 after primary infection. Western blots with three different parasite antigen preparations, L1, excretory/secretory products and crude worm adult antigens, were used to detect parasite specific IgE in sera depleted of IgG and IgM. These depleted sera from protected calves contained parasite specific IgE, while sera from nonprotected calves were negative. A band of approximately 100 kDa was recognized in all three antigens. In a second experiment, primary doses of 30, 60, 120, 240, 480 and 960 L3 of D. viviparus were used and necropsy was 11 days after challenge. This experiment confirmed the correlation between protection and total IgE levels before and on the day of challenge. The rapid and strong IgE responses in protected animals after such a low infection might be caused by the specific characteristics of the lungworm antigens or by the somatic migration of the worm and might be involved in the rapid development of protection against lungworm reinfections in cattle.