bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Streptococcus pneumoniae enoyl-(acyl-carrier protein) reductase (FabK)
Authors: MARRAKCHI Hedia , DeWOLF Walter E. , QUINN Chad , WEST Joshua , POLIZZI Brian J. , SO Chi Y. , HOLMES David J. , REED Shannon L. , HEATH Richard J. , PAYNE David J. , ROCK Charles O. , WALLIS Nicola G.
Published: March 2003

The enoyl-(acyl-carrier protein) (ACP) reductase catalyses the last step in each cycle of fatty acid elongation in the type II fatty acid synthase systems. An extensively characterized NADH-dependent reductase, FabI, is widely distributed in bacteria and plants, whereas the enoyl-ACP reductase, FabK, is a distinctly different member of this enzyme group discovered in Streptococcus pneumoniae. We were unable to delete the fabK gene from Strep. pneumoniae, suggesting that this is the only enoyl-ACP reductase in this organism. The FabK enzyme was purified and the biochemical properties of the reductase were examined. The visible absorption spectrum of the purified protein indicated the presence of a flavin cofactor that was identified as FMN by MS, and was present in a 1:1 molar ratio with protein. FabK specifically required NADH and the protein activity was stimulated by ammonium ions. FabK also exhibited NADH oxidase activity in the absence of substrate. Strep. pneumoniae belongs to the Bacillus/Lactobacillus/Streptococcus group that includes Staphylococcus aureus and Bacillus subtilis. These two organisms also contain FabK-related genes, suggesting that they may also express a FabK-like enoyl-ACP reductase. However, the genes did not complement a fabI(Ts) mutant and the purified flavoproteins were unable to reduce enoyl-ACP in vitro and did not exhibit NAD(P)H oxidase activity, indicating they were not enoyl-ACP reductases. The restricted occurrence of the FabK enoyl-ACP reductase may be related to the role of substrate-independent NADH oxidation in oxygen-dependent anaerobic energy metabolism.
Docking sites on mitogen-activated protein kinase (MAPK) kinases, MAPK phosphatases and the Elk-1 transcription factor compete for MAPK binding and are crucial for enzymic activity
Authors: BARDWELL A. Jane , ABDOLLAHI Mahsa , BARDWELL Lee
Published: March 2003

Mitogen-activated protein kinase (MAPK) cascades control gene expression patterns in response to extracellular stimuli. MAPK/ERK (extracellular-signal-regulated kinase) kinases (MEKs) activate MAPKs by phosphorylating them; activated MAPKs, in turn, phosphorylate target transcription factors, and are deactivated by phosphatases. One mechanism for maintaining signal specificity and efficiency is the interaction of MAPKs with their substrates and regulators through high-affinity docking sites. In the present study, we show that peptides corresponding to the MAPK-docking sites of MEK1, MEK2, Ste7, Elk-1 and MAPK phosphatase (MKP)-2 potently inhibit MEK2 phosphorylation of ERK2, ERK2 phosphorylation of Elk-1, and MKP-1 dephosphorylation of ERK2. Each peptide inhibited multiple reactions; for example, the MEK2 peptide inhibited not only MEK2, but also ERK2 and MKP-1. In addition, these docking-site peptides inhibited MEK2—ERK2 binding. The MAPK-docking site of MEK1 also potently stimulated ERK2-mediated phosphorylation of a target site on the same peptide. Control peptides with mutations of conserved basic and hydrophobic residues of the MAPK-docking site consensus lacked biological activity. We conclude that MEKs, MKPs and the Elk-1 transcription factor compete for binding to the same region of ERK2 via protein—protein interactions that are crucial for kinase/phosphatase activity.
Heat-shock protein 90 and Cdc37 interact with LKB1 and regulate its stability
Authors: BOUDEAU Jérôme , DEAK Maria , LAWLOR Margaret A. , MORRICE Nick A. , ALESSI Dario R.
Published: March 2003

LKB1 is a widely expressed serine/threonine protein kinase that is mutated in the inherited Peutz—Jeghers cancer syndrome. Recent findings indicate that LKB1 functions as a tumour suppressor, but little is known regarding the detailed mechanism by which LKB1 regulates cell growth. In this study we have purified LKB1 from cells and establish that it is associated with the heat-shock protein 90 (Hsp90) chaperone and the Cdc37 kinase-specific targetting subunit for Hsp90. We demonstrate that Cdc37 and Hsp90 bind specifically to the kinase domain of LKB1. We also perform experiments using Hsp90 inhibitors, which indicate that the association of Hsp90 and Cdc37 with LKB1 regulates LKB1 stability and prevents its degradation by the proteasome. Hsp90 inhibitors are being considered as potential anti-cancer agents. However, our observations indicate that prolonged usage of these drugs could possibly lead to tumour development by decreasing cellular levels of LKB1.
Hippocampal Serotonin Responses in Short and Long Attack Latency Mice
Authors: Van Riel E. , Meijer O. C. , Veenema A. H. , Joëls M.
Published: March 2003

Short and long attack latency mice, which are selected based on their offensive behaviour in a resident–intruder model, differ in their neuroendocrine regulation as well as in aspects of their brain serotonin system. Previous studies showed that the binding capacity and expression of serotonin‐1A receptors in the hippocampal CA1 field of long attack latency mice are significantly lower than that found in short attack latency mice. We tested whether the functional responses of CA1 hippocampal cells to serotonin are also reduced in long attack latency mice. To this end, serotonin‐induced changes in the membrane potential and input resistance were recorded in vitro with microelectrodes in CA1 pyramidal neurones of long and short attack latency mice. The data show that in long attack latency mice, along with a reduction of the serotonin‐1A receptor mRNA expression, the serotonin‐induced membrane hyperpolarization and decrease in resistance are attenuated. Basal membrane properties of CA1 neurones in the two mice lines were comparable. Plasma corticosterone levels in response to a novelty stress were elevated in long compared to short attack latency mice and inversely related to the serotonin‐induced responses. We tentatively conclude that long attack latency mice show attenuated functional responses to serotonin in the hippocampus, possibly linked to a chronic perturbation of hormonal levels.
Critical role of divalent cations and Na+ efflux in Arabidopsis thaliana salt tolerance
Authors: Elphick C. H. , Sanders D. , Maathuis F. J. M.
Published: March 2003

Detrimental effects of salinity on plants are known to be partially alleviated by external Ca2+. Previous work demonstrated that the Arabidopsis SOS3 locus encodes a Ca2+‐binding protein with similarities to CnB, the regulatory subunit of protein phosphatase 2B (calcineurin). In this study, we further characterized the role of SOS3 in salt tolerance. We found that reduced root elongation of sos3 mutants in the presence of high concentrations of either NaCl or LiCl is specifically rescued by Ca2+ and not Mg2+, whereas root growth is rescued by both Ca2+ and Mg2+ in the presence of high concentrations of KCl. Phenocopies of sos3 mutants were obtained in wild‐type plants by the application of calmodulin and calcineurin inhibitors. These data provide further evidence that SOS3 is a calcineurin‐like protein and that calmodulin plays an important role in the signalling pathways involved in plant salt tolerance. The origin of the elevated Na : K ratio in sos3 mutants was investigated by comparing Na+ efflux and influx in both mutant and wild type. No difference in Na+ influx was recorded between wild type and sos3; however, sos3 plants showed a markedly lower Na+ efflux, a property that would contribute to the salt‐oversensitive phenotype of sos3 plants.
Responses to different external light rhythms by the circadian rhythm of Crassulacean acid metabolism in Kalanchoe daigremontiana
Authors: Bohn A. , Geist A. , Rascher U. , Lüttge U.
Published: March 2003

Time series of net CO2 exchange (JCO2) and leaf conductivity for water vapour (gH2O) were measured and subsequently analysed mathematically in the Crassulacean acid metabolism (CAM) plant Kalanchoe daigremontiana (Hamet et Perrier de la Bâthie) under constant environmental conditions and under imposed external rhythms of lower and higher light intensity. The time series were analysed by Fourier methods and a correlation analysis considering the first time derivatives of JCO2, gH2Oand photosynthetically active photon flux density (PPFD). The ratio of internal to external CO2 (ci/ca) was also considered in the analysis, leading to a discussion of the interaction of stomata and carbon assimilation under periodic stimulation. It is suggested that for stimulation with frequencies close to the endogeneous circadian period, stomatal conductance and carbon assimilation oscillate synchronously, guard‐cell movements trailing behind changes in internal CO2 with a delay of 10–15 min. For stimulation frequencies far shorter than the endogeneous period, this synchrony can be disturbed due to independent responses of stomata and assimilation to light pulses, leading to an arrhythmic gas exchange pattern. These results are discussed in the context of understanding circadian oscillations as the output of a multioscillator, multisignalling pathway system on the organismic and metabolic level.
Effects of age and ontogeny on photosynthetic responses of a determinate annual plant to elevated CO2 concentrations
Authors: Lewis J. D. , Wang X. Z. , Griffin K. L. , Tissue D.T.
Published: March 2003

Plant responses to elevated CO2 concentrations ([CO2]) may be regulated by both accelerated ontogeny and allocational changes as plants grow. However, isolating ontogeny‐related effects from age‐related effects are difficult because these factors are often confounded. In this study, the roles of age and ontogeny in photosynthetic responses to elevated [CO2] were examined on Xanthium strumarium L. grown at ambient (365 µmol mol−1) and elevated (730 µmol mol−1) [CO2]. To examine age‐related effects, six cohorts were planted at 5‐day intervals. To examine ontogeny‐related effects, all plants were induced to flower at the same time; ontogeny in Xanthium is relatively unaffected by growth in elevated [CO2]. Growth in elevated [CO2] increased net photosynthetic rates by approximately 30% throughout vegetative growth (i.e. active carbohydrate sinks), approximately 10% during flowering (i.e. minimal sink activity), and approximately 20% during fruit production (i.e. active sinks). At the harvest, the ratio of source to sink tissue significantly decreased with increasing plant age and was correlated with leaf soluble sugar concentration. Leaf soluble sugar concentration was negatively correlated with the relative photosynthetic response to elevated [CO2]. These results suggest that age and ontogeny independently affect photosynthetic responses to elevated [CO2] and the effects are mediated by reversible changes in source : sink balance.
Hunting disturbance on a large shallow lake: the effectiveness of waterfowl refuges
Authors: Evans Darren M. , Day Keith R.
Published: March 2003

Disturbance is most likely to have an impact during those periods of the annual cycle when food is scarce and birds have difficulty in meeting their energy requirements. The provision of disturbance‐free refuges has been shown to enhance the abundance and diversity of waterfowl wintering on such sites. Lough Neagh, a large shallow lake in Northern Ireland, hosts internationally important numbers of wintering waterfowl, the most numerous being Common Pochard Aythya ferina, Tufted Duck A. fuligula, Greater Scaup A. marila and Common Goldeneye Bucephala clangula. Hunting is the predominant winter recreational activity. A small number of refuges have been provided to limit the effects of shooting disturbance on the wintering populations. The use of one of these refuges (Doss Bay) was studied during the winter of 1997/8 and compared with a non‐refuge site (Brockish Bay) during the winter of 1998/9. Shooting intensity was greater at weekends than midweek, and significantly more birds used the refuge at weekends than midweek during the shooting season. In contrast, significantly fewer birds were observed in Brockish Bay at weekends. This trend ceased when the shooting season closed, suggesting that the effect was due to shooting disturbance. Furthermore, significantly fewer birds used Doss Bay after the shooting season had closed, whereas significantly more birds were observed at Brockish Bay. Shooting disturbance had the greatest impact on dabbling duck species and rails at both sites. Diving ducks moved away from shoreline disturbance to shallow areas where they could still feed. The value of shoreline refuges for waterfowl populations on lakes is discussed.
Leapfrogging revisited in Andean birds: geographical variation in the Tody‐tyrant superspecies Poecilotriccus ruficeps and P. luluae
Authors: Johnson Ned K.
Published: March 2003

I analysed geographical variation in size, colour and pattern quantitatively in the Rufous‐crowned Tody‐Tyrant Poecilotriccus ruficeps and Lulu Von Hagen’s Tody‐Tyrant P. luluae, two component species of a superspecies inhabiting the northern Andes of South America. The conspicuously bridled P. r. melanomystax, here reported for the first time from the Andes of Venezuela and the Eastern Andes of Colombia, leapfrogs southward over populations of the plain‐headed P. r. ruficeps in the Eastern Andes of Colombia, re‐occurs in the Central Andes of Colombia, leapfrogs over populations of P. r. ruficeps in south‐western Colombia and Ecuador, then ‘re‐appears’ as the similarly bridled P. r. peruvianus in southern Ecuador and northern Peru. In a second example of leapfrogging, the recently described P. luluae from the Eastern Andes of northern Peru is separated from superficially similar populations of P. r. ruficeps by the distribution of P. r. peruvianus. Because of their striking plumage differences and lack of interbreeding, the two bridled forms, P. r. melanomystax and P. r. peruvianus, may together constitute a species separable from the plain‐headed forms, P. r. ruficeps and P. r. rufigenis. Vocally, however, all forms of P. ruficeps are similar. I recognize Poecilotriccus r. rufigenis provisionally pending the accumulation of new material. Geographical variation in size among populations of P. ruficeps is weakly clinal or chaotic. Crown and back colour are geographically uniform, however, and are identical among all populations of both P. ruficeps and P. luluae. In colour of belly and face, P. luluae is exceptionally stable and unique. This contrasts with striking geographical variation in facial colour and pattern in P. ruficeps. Although differences in song syllables are apparent, samples of tape recordings are too small to justify conclusions about the potential role of voice in reproductive isolation.
Activation of the immune system by bacterial CpG‐DNA
Authors: Häcker Georg , Redecke Vanessa , Häcker Hans
Published: March 2003

The past decade has seen a remarkable process of refocusing in immunology. Cells of the innate immune system, especially macrophages and dendritic cells, have been at the centre of this process. These cells had been regarded by some scientists as non‐specific, sometimes perhaps even confined to the menial job of serving T cells by scavenging antigen and presenting it to the sophisticated adaptive immune system. Only over the last few years has it become unequivocally clear that cells of the innate immunity hold, by variation of context and mode of antigen presentation, the power of shaping an adaptive immune response. The innate immune response, in turn, is to a significant degree the result of stimulation by so‐called pathogen‐associated molecular patterns (PAMPs). One compound with high stimulatory potential for the innate immune system is bacterial DNA. Here we will review recent evidence that bacterial DNA should be ranked with other PAMPs such as lipopolysaccharide (LPS) and lipoteichoic acid. We will further review our present knowledge of DNA recognition and DNA‐dependent signal transduction in cells of the immune system.
Mycobacterial antigens induce apoptosis in human purified protein derivative‐specific αβ T lymphocytes in a concentration‐dependent manner
Authors: Soruri Afsaneh , Schweyer Stefan , Radzun Heinz‐Joachim , Fayyazi Afshin
Published: March 2003

The morbidity and lethality of tuberculosis is partially the result of an ineffective delayed‐type hypersensitivity reaction which causes caseating granulomas in the lung and other organs. Recently we showed that during caseation besides macrophages numerous Fas+ FasL+ lymphocytes undergo apoptosis and postulated that this phenomenon may be due to activation‐induced cell death (AICD) as a consequence of T‐lymphocyte reactivation via bacillary antigens. As purified protein derivative of Mycobacterium tuberculosis (Mtb‐PPD) provokes caseation in tuberculosis patients, the question arose as to whether bacillary antigens are responsible for AICD within caseous areas. In the present study Mtb‐PPD‐specific T helper 1 (Th1)‐differentiated T lymphocytes were generated in vitro. Reactivation of these cells with Mtb‐PPD resulted in a concentration‐dependent hyporesponsiveness, which was due to an increase in apoptosis of γδ+, αβ+ CD4+ as well as αβ+ CD8+ T lymphocytes as assessed by the demonstration of the apoptosis‐associated mitochondrial membrane protein 7A6 and DNA fragmentation. Blocking experiments demonstrated that Mtb‐PPD antigens exploited the Fas/FasL system to induce apoptosis in Mtb‐PPD‐specific T lymphocytes. These results may support the hypothesis that in tubercle granulomas with caseation T lymphocytes undergo AICD following reactivation by bacillary antigens, thus contributing to the persistence of tuberculosis.
Evans Blue Dye as an in vivo marker of myofibre damage: optimising parameters for detecting initial myofibre membrane permeability
Authors: Hamer P. W. , McGeachie J. M. , Davies M. J. , Grounds M. D.
Published: March 2003

Evans Blue Dye (EBD) is widely used to study cellular membrane permeability and has recently been utilised in mdx mice to identify permeable skeletal myofibres that have become damaged as a result of muscular dystrophy. EBD has the potential to be a useful vital stain of myofibre permeability in other models of skeletal muscle injury and membrane‐associated fragility. The parameters for its use for such purposes were optimised in the present study. Of particular interest is the use of EBD to identify the onset of muscle damage. This study compared intravenous vs. intraperitoneal injection; tissue fixation; volume of EBD; time of availability in tissue; and persistence after injection in mdx mice (with endogenous muscle damage) and control mice. Satisfactory labelling of permeable myofibres was seen in frozen sections viewed with fluorescence microscopy when intraperitoneal injection of a 1% EBD solution injected at 1% volume relative to body mass was administered between 16 and 24 h prior to tissue sampling. EBD labelling was then assessed in three mouse models of experimental injury and repair – cut injury, whole muscle grafts, and exercise‐induced muscle damage. These experiments demonstrated that (i) following a cut injury across myofibres, EBD penetrated up to 150 µm from the injury site over a 20‐h period; (ii) EBD was present throughout myofibres of avascular whole muscle graft by one day after transplantation; and (iii) damaged myofibres were detected within 20 min after controlled lengthening–contraction exercise. This simple and inexpensive technique has sensitivity for the detection of increased myofibre permeability and/or sublethal damage that has advantages over other traditional histological techniques at the light microscopy level.
Ultrastructural analysis of pancreatic acinar cells from mice fed on genetically modified soybean
Authors: Malatesta Manuela , Caporaloni Chiara , Rossi Luigia , Battistelli Serafina , Rocchi Marco B. L. , Tonucci Francesco , Gazzanelli Giancarlo
Published: March 2003

No direct evidence that genetically modified (GM) food may represent a possible danger for health has been reported so far; however, the scientific literature in this field is quite poor. Therefore, we investigated the possible effects of a diet containing GM soybean on mouse exocrine pancreas by means of ultrastructural, morphometrical and immunocytochemical analyses. Our observations demonstrate that, although no structural modification occurs in pancreatic acinar cells of mice fed on GM soybean, quantitative changes of some cellular constituents take place in comparison to control animals. In particular, a diet containing significant amount of GM food seems to influence the zymogen synthesis and processing.
Weak cross‐species relationships between body size and trophic level belie powerful size‐based trophic structuring in fish communities
Authors: Jennings Simon , Pinnegar John K. , Polunin Nicholas V. C. , Boon Trevor W.
Published: March 2003

Body size determines rates of respiration and production, energy requirements, mortality rates, patterns of predation and vulnerability to mortality. Body size distributions are often used to describe structure and energy flux in communities and ecosystems.

If clear relationships can be established between body size and trophic level in fishes, they may provide a basis for integrating community and ecosystem analyses based on size spectra, food webs and life histories.

We investigated relationships between the body sizes (weight and length) of north‐east Atlantic fishes and their trophic level. The abundance of15N, as determined by stable isotope analysis, was used as an index of trophic level.

Cross‐species and comparative analyses demonstrated that body size was unrelated or weakly related to trophic level. Thus allometric relationships between body size and trophic level could not be used to predict the trophic structure of fish communities.

The results of the cross‐species analyses contrasted with patterns in the size and trophic structure of entire fish communities. When fish communities were divided into size classes, there were strong positive relationships between size class and trophic level. The slope suggested a mean predator : prey body mass ratio of 80 : 1.

Our results suggest that body size does not provide a useful surrogate of trophic level for individual species, but that body size is an excellent predictor of trophic level within the community, providing an empirical basis for integrating community analyses based on models of trophic structure and body size distributions.
Interactive effects of parental age and environmental variation on the breeding performance of Tengmalm’s owls
Authors: Laaksonen Toni , Korpimäki Erkki , Hakkarainen Harri
Published: March 2003

Two main mechanisms have been proposed to account for the changes in breeding performance of long‐lived animals with age: progressive appearance or disappearance of different quality individuals within a cohort and within‐individual improvements with age.

We studied age‐related breeding performance of vole‐eating Tengmalm’s owls (Aegolius funereus, L.), whose prey populations fluctuate in a cyclic manner in western Finland. In years of low vole abundance, most yearling owls were not able to breed. In years of initially intermediate but increasing vole abundance, clutch size increased with female age and partners of old males initiated egg‐laying earlier than those of young males. In years of initially high but decreasing vole abundance, the differences in laying dates were still detectable, whereas age‐related differences in clutch size of females tended to disappear.

Within‐cohort analyses did not indicate quality differences between surviving and dying males of the same age in years of increasing vole abundance, whereas in years of decreasing vole abundance early breeding males were more likely to survive than late‐breeding males. This novel finding indicates that quality differences between individuals may induce observed age‐related differences in only some years, and especially that differential mortality may occur in a different year when the difference in breeding performance is detectable.

Within‐individual analyses showed that individual males advanced their nest initiation with age, and there was a trend that females laid larger clutches with increasing age. In nests of long‐lived males, a significant decline in the clutch size from mid‐age (2–4 years) to older age (6–10 years) was found.

We conclude that environmental variation may mask age‐related differences in breeding performance at the population level, since the differences appeared to emerge more clearly in poor and intermediate food conditions. Some evidence for the differential mortality‐hypothesis was found for males, which are mainly responsible for subsisting their families during the entire breeding season. Substantial improvements in competence or increased reproductive effort from 1 to 3 years of age, and deteriorated skills due to senescence at older ages may account for within‐individual changes in breeding performance of males.
Aapa mires as a carbon sink and source during the Holocene
Authors: Mäkilä Markku , Saarnisto Matti , Kankainen Tuovi
Published: March 2003

The vertical and horizontal dynamics of peat carbon accumulation in Ruosuo aapa mire, north‐central Finland, were investigated in an attempt to understand variations in carbon accumulation over time.

Paludification began around 8700 BP (9600 cal BP). The long‐term net carbon accumulation rate, based on vertical and lateral growth, was 8.0 g C m−2 year−1. Variations in carbon accumulation rates can be explained to a great extent by natural succession and variations in local conditions, but the role of climate cannot be ignored.

The marked decline in carbon accumulation rates after 6100 BP (7000 cal BP) may indicate a relatively dry and warm climate. The levelling out and subsequent increase in carbon accumulation rates after 4000 BP (4500 cal BP) indicates the development of a cooler and moister climate.

The low long‐term net carbon accumulation rate and the high carbon content per unit volume in the layer underlying the surficial peat is typical for a wet aapa mire. The continuous penetration of roots, mostly sedge, results in carbon input into the underlying peat layer and contributes to the intensive decay of the surficial peat due to high biological activity.

Although one‐third of the apparent carbon has accumulated during the last 2000 years, most of Ruosuo is approaching the stage where primary production does not adequately compensate for overall carbon loss. The declining net carbon accumulation rates from the base up to the depth of 80 cm may indicate a real delayed trend in net carbon accumulation rates.

We suggest that under the present climatic conditions the northern circumpolar wet mires are significant sources of greenhouse gases. In addition to high methane production, northern aapa mires also have lower carbon accumulation rates than more southern raised bogs.

Our results, which are based on horizontal and vertical carbon accumulation rates, suggest that the earlier published carbon accumulation rates in Finnish aapa mires are overestimated.
Effects of streptozotocin‐diabetes on the hippocampal NMDA receptor complex in rats
Authors: Gardoni F. , Kamal A. , Bellone C. , Biessels G. J. , Ramakers G. M. J. , Cattabeni F. , Gispen W. H. , Di Luca M.
Published: March 2003

In animal models of diabetes mellitus, such as the streptozotocin‐diabetic rat (STZ‐rat), spatial learning impairments develop in parallel with a reduced expression of long‐term potentiation (LTP) and enhanced expression of long‐term depression (LTD) in the hippocampus. This study examined the time course of the effects of STZ‐diabetes and insulin treatment on the hippocampal post‐synaptic glutamate N‐methyl‐d‐aspartate (NMDA) receptor complex and other key proteins regulating hippocampal synaptic transmission in the post‐synaptic density (PSD) fraction. In addition, the functional properties of the NMDA‐receptor complex were examined. One month of STZ‐diabetes did not affect the NMDA receptor complex. In contrast, 4 months after induction of diabetes NR2B subunit immunoreactivity, CaMKII and Tyr‐dependent phosphorylation of the NR2A/B subunits of the NMDA receptor were reduced and αCaMKII autophosphorylation and its association to the NMDA receptor complex were impaired in STZ‐rats compared with age‐matched controls. Likewise, NMDA currents in hippocampal pyramidal neurones measured by intracellular recording were reduced in STZ‐rats. Insulin treatment prevented the reduction in kinase activities, NR2B expression levels, CaMKII–NMDA receptor association and NMDA currents. These findings strengthen the hypothesis that altered post‐synaptic glutamatergic transmission is␣related to deficits in learning and plasticity in this animal model.
Elevated hepatic and depressed renal cytochrome P450 activity in the Tg2576 transgenic mouse model of Alzheimer's disease
Authors: Van Ess P. J. , Pedersen W. A. , Culmsee C. , Mattson M. P. , Blouin R. A.
Published: March 2003

Recent studies indicate that the Tg2576 transgenic mouse model of Alzheimer's disease [tg(hAPP)] demonstrates disturbances in plasma glucose and neuroendocrine function reminiscent of Alzheimer's disease (AD). Alterations in any one of these systems can have a profound effect on hepatic cytochrome P450 (CYP) expression. Additionally, the recent discovery that amyloid beta 1–42 can induce the expression of CYP reductase in neuronal cultures further suggests that hepatic CYP‐related metabolism may be affected by the expression of mutant human amyloid precursor protein in these tg(hAPP) mice. Therefore, the current study was conducted to investigate the activity and protein content of several CYP isoforms in the livers and kidneys of aged (20‐month‐old) tg(hAPP) mice. tg(hAPP) mice exhibit significant elevations in hepatic CYP2B, CYP2E1‐, CYP3A‐ and CYP4A‐associated activities and CYP4A immunoreactive protein compared with wild‐type. In contrast to the liver, a significant depression in renal CYP2E1‐ and CYP4A‐associated activities were demonstrated in tg(hAPP) mice. The presence of the mutant hAPP protein was detected in the brain, kidney and livers of tg(hAPP) mice.
Lithium protection against glutamate excitotoxicity in rat cerebral cortical neurons: involvement of NMDA receptor inhibition possibly by decreasing NR2B tyrosine phosphorylation
Authors: Hashimoto Ryota , Hough Christopher , Nakazawa Takanobu , Yamamoto Tadashi , Chuang De‐Maw
Published: March 2003

The therapeutic mechanisms of lithium for treating bipolar mood disorder remain poorly understood. Recent studies demonstrate that lithium has neuroprotective actions against a variety of insults. Here, we studied neuroprotective effects of lithium against excitotoxicity in cultured cerebral cortical neurons. Glutamate‐induced excitotoxicity in cortical neurons was exclusively mediated by NMDA receptors. Pre‐treatment of cortical neurons with LiCl time‐dependently suppressed excitotoxicity with maximal protection after 6 days of pre‐treatment. Significant protection was observed at the therapeutic and subtherapeutic concentration of 0.2–1.6 mm LiCl with almost complete protection at 1 mm. Neuroprotection was␣also elicited by valproate, another major mood‐stabilizer. The neuroprotective effects of lithium coincided with inhibition␣of␣NMDA receptor‐mediated calcium influx. Lithium pre‐treatment did not alter total protein levels of NR1, NR2A and NR2B subunits of NMDA receptors. However, it did markedly reduce the level of NR2B phosphorylation at Tyr1472 and this was temporally associated with its neuroprotective effect. Because NR2B tyrosine phosphorylation has been positively correlated with NMDA receptor‐mediated synaptic activity and excitotoxicity, the suppression of NR2B phosphorylation by lithium is likely to result in the inactivation of NMDA receptors and contributes to neuroprotection against excitotoxicity. This action could also be relevant to its clinical efficacy for bipolar patients.
The human histamine H2‐receptor couples more efficiently to Sf9 insect cell Gs‐proteins than to insect cell Gq‐proteins: limitations	of Sf9 cells for the analysis of receptor/Gq‐protein coupling
Authors: Houston Christine , Wenzel‐Seifert Katharina , Bürckstümmer Tilmann , Seifert Roland
Published: March 2003

The human histamine H2‐receptor (hH2R) couples to Gs‐proteins to activate adenylyl cyclase and to Gq‐proteins to activate phospholipase C, but phospholipase C activation has not consistently been observed. The aim of this study was to compare coupling of hH2R to insect and mammalian Gs‐ and Gq‐proteins in Spodoptera frugiperda (Sf9) cells. Interaction of hH2R with mammalian G proteins was assessed with coexpressed proteins or receptor‐Gα fusion proteins that enhance coupling efficiency. hH2R efficiently coupled to insect Gs‐proteins to activate adenylyl cyclase. However, hH2R poorly coupled to insect Gq‐proteins as assessed by the lack of enhancement of histamine‐stimulated steady‐state GTP hydrolysis by regulators of G protein signaling (RGS proteins). In contrast, RGS‐proteins efficiently enhanced GTP hydrolysis stimulated by the human platelet‐activating factor receptor (PAFR) and the histamine H1‐receptor (H1R) from man and guinea pig. The measurement of intracellular free Ca2+ concentration was not useful for studying receptor/Gq‐protein coupling. hH2R also efficiently interacted with mammalian Gs‐proteins, specifically with fused Gsα as assessed by guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS)‐sensitive high‐affinity agonist binding, agonist‐stimulated [35S]GTPγS binding and adenylyl cyclase activation. In contrast, coupling of hH2R to coexpressed and fused mammalian Gqα was poor. However, our inability to reconstitute efficient coupling of PAFR and H1R to mammalian Gqα indicated that a large portion of the expressed G protein was functionally inactive. Taken together, our data show that hH2R couples more efficiently to insect cell Gs‐proteins than to insect cell Gq‐proteins. Unfortunately, there are significant limitations in the usefulness of Sf9 cells for comparing the coupling of receptors to mammalian Gs‐ and Gq‐proteins and assessing Gq‐mediated activation of effector systems.
A homeostatic mechanism counteracting K+‐evoked choline release in adult brain
Authors: Klein Jochen , Weichel Oksana , Ruhr Judith , Dvorak Claudia , Löffelholz Konrad
Published: March 2003

Choline (Ch) is an essential nutrient as the biosynthetic precursor of acetylcholine (ACh) and phospholipids. Under resting conditions, the intracellular accumulation of Ch (above 10‐fold), which is positively charged, is governed by the membrane potential and follows the Nernst equation. Accordingly, in synaptosomes from adult rats during depolarization, we observed a linear relationship between release of free cytoplasmic Ch and KCl concentration (2.7–120 mm). The K+‐evoked Ch release was Ca2+‐independent and did not originate from ACh or phospholipid hydrolysis. In superfused brain slices of adult rats, however, a K+‐induced Ch efflux was absent. Also, under in vivo conditions, 30–60 mm KCl failed to increase the extracellular Ch level as shown by microdialysis in adult rat hippocampus. On the contrary, in brain slices from 1‐week‐old rats, high K+ as well as 4‐aminopyridine evoked a marked Ch efflux in a concentration‐dependent fashion. This phenomenon faded within 1 week. Hemicholinium‐3 (HC‐3, 1␣and 10 µm), a blocker of cellular choline uptake, caused a marked efflux of choline from adult rat slices but no or significantly less release from immature slices. We conclude that depolarization of synaptic endings causes a Ca2+‐independent release of free cytoplasmic Ch into the extracellular space. In adult rat brain, this elevation of Ch is counteracted by a homeostatic mechanism such as uptake into brain cells.
Effect of depleting vesicular and cytoplasmic dopamine on methylenedioxymethamphetamine neurotoxicity
Authors: Yuan Jie , Cord Branden J. , McCann Una D. , Callahan Brian T. , Ricaurte George A.
Published: March 2003

The mechanism by which 3,4‐methylenedioxymethamphetamine (MDMA) produces serotonin (5‐HT) neurotoxicity is unknown but considerable evidence suggests that endogenous brain dopamine (DA) is involved. However, it has recently become apparent that some of the data implicating brain DA in MDMA neurotoxicity may be confounded by drug effects on thermoregulation. The purpose of the present studies was to examine the role of DA in MDMA neurotoxicity, while controlling for possible confounding effects of drug‐ induced changes in core temperature. Rats were treated with reserpine, alone and in combination with α‐methyl‐p‐tyrosine (AMPT), to deplete vesicular and cytoplasmic stores of DA. When drug‐induced hypothermia was averted (by raising ambient temperature), the 5‐HT neuroprotective effects of reserpine and AMPT were no longer apparent. The lack of neuroprotection by AMPT and reserpine, alone and in combination, in studies that control for the effects of these drugs on core temperature, suggests that DA per se is not essential for the expression of MDMA‐induced 5‐HT neurotoxicity.
PK 11195 attenuates kainic acid‐induced seizures and alterations in peripheral‐type benzodiazepine receptor (PBR) protein components in the rat brain
Authors: Veenman L. , Leschiner S. , Spanier I. , Weisinger G. , Weizman A. , Gavish M.
Published: March 2003

Peripheral‐type benzodiazepine receptors (PBR) are located in glial cells in the brain and in peripheral tissues. Mitochondria form the primary location for PBR. Functional PBR appear to require at least three components: an isoquinoline binding protein, a voltage‐dependent anion channel, and an adenine nucleotide carrier. In the present study, rats received intraperitoneal kainic acid injections, which are known to cause seizures, neurodegeneration, hyperactivity, gliosis, and a fivefold increase in PBR ligand binding density in the hippocampus. In the forebrain of control rats, hippocampal voltage‐dependent anion channel and adenine nucleotide carrier abundance was relatively low, while isoquinoline binding protein abundance did not differ between hippocampus and the rest of the forebrain. One week after kainic acid injection, isoquinoline binding protein abundance was increased more than 20‐fold in the hippocampal mitochondrial fraction. No significant changes were detected regarding hippocampal voltage‐dependent anion channel and adenine nucleotide carrier abundance. Pre‐treatment with the isoquinoline PK11195, a specific PBR ligand, attenuated the occurrence of seizures, hyperactivity, and increases in isoquinoline binding protein levels in the hippocampus, which usually follow kainic acid application. These data suggest that isoquinoline binding protein may be involved in these effects of kainic acid injections.
Direct evidence that release‐stimulating α7* nicotinic cholinergic receptors are localized on human and rat brain glutamatergic axon terminals
Authors: Marchi Mario , Risso Francesca , Viola Concetta , Cavazzani Paolo , Raiteri Maurizio
Published: March 2003

The existence on glutamatergic nerve endings of nicotinic acetylcholine receptors (nAChRs) mediating enhancement of glutamate release has often been suggested but not demonstrated directly. Here, we study the effects of nAChR agonists on [3H]‐d‐aspartate ([3H]‐d‐ASP) release from synaptosomes superfused in conditions known to prevent indirect effects. Nicotinic receptor agonists, while unable to modify the basal [3H]‐d‐ASP release from human neocortex or rat striatal synaptosomes, enhanced the Ca2+‐dependent exocytotic release evoked by K+ (12 mm) depolarization. Their rank order of potency were anatoxin‐a > epibatidine > nicotine > ACh (+ atropine). The anatoxin‐a effect, both in human and rat synaptosomes, was antagonized by mecamylamine, α‐bungarotoxin or methyllycaconitine. The basal release of [3H]ACh from human cortical synaptosomes was increased by (–)‐nicotine (EC50 = 1.16 ± 0.33 µm) or by ACh plus atropine (EC50 = 2.0 ± 0.04 µm). The effect of ACh plus atropine was␣insensitive to α‐bungarotoxin, methyllycaconitine or α‐conotoxin MII, whereas it was totally antagonized by mecamylamine or dihydro‐β‐erythroidine. To conclude, glutamatergic axon terminals in human neocortex and in rat striatum possess α7* nicotinic heteroreceptors mediating enhancement of glutamate release. Release‐enhancing cholinergic autoreceptors in human neocortex are nAChRs with a pharmacological profile compatible with the α4β2 subunit combination.
Intrinsic water‐use efficiency and heterotrophic investment in tropical leaf growth of two Neotropical pioneer tree species as estimated from δ13C values
Authors: Terwilliger V. J. , Kitajima K. , Le Roux‐Swarthout D. J. , Mulkey S. , Wright S. J.
Published: March 2003

13C enrichment in emerging leaves and its effect on carbon isotopic composition (δ13C) of mature leaves were investigated in the neotropical pioneer tree species, Cecropia longipes and Urera caracasana, in Panama.

Leaves of all ages were analysed for δ13C, gas exchange, nitrogen, and leaf mass per area. Low intercellular to atmospheric carbon dioxide (CO2) partial pressure (pi : pa); and high δ13C of CO2 in air, intrinsic water‐use efficiency, internal resistance, and carboxylation capacity were discounted as causes of 13C enrichment.

13C enrichment might occur when leaf growth is from imported organic carbon but might not reflect δ13C values at the leaf producing the carbon. Results support hypotheses that: de novo sucrose synthesis causes 13C enrichment of mobile sugars after export from source leaves; and high ratios of PEP carboxylase (PEPc) : PEPc + Rubisco cause emerging leaves to be 13C enriched relative to their growth substrate. Carbon contributions of 13C‐enriched early growth could yield inaccurate pi : pa estimates from δ13C in mature leaves.

A model estimated investment of imported organic carbon to leaf growth and improved estimates of pi : pa from mature leaves. With such adjustment, δ13C analyses provide valuable information about age‐related source–sink relations in leaves.
Remobilized old‐leaf nitrogen predominates for spring growth in two temperate grasses
Authors: Bausenwein Ursula , Millard Peter , Raven John A.
Published: March 2003

The importance of N derived from internal remobilization and root uptake to growth in spring is reported here for the perennial grasses Festuca rubra and Agrostis capillaris derived from seminatural grasslands in Scotland, UK.

Plants grown in sand culture, received 15N‐enriched nutrient solution during the first year of growth and nutrient solution with N at natural abundance during the subsequent spring and summer when destructive harvests were taken.

Labelled N was recovered in new growth of overwintering tillers and new tillers. Remobilized N contributed 70% and 82% for F. rubra and A. capillaris, respectively, to the total N in new above‐ground growth in early spring, declining to 34% and 45%, respectively, by mid June. Species showed similar patterns of remobilization on a new growth biomass basis. The root system did not remobilize N to support new above‐ground growth. Labelled N was derived from senescing leaves present on overwintering tillers. Net balances of labelled N suggest that N was translocated between tillers; reproductive tillers acted as sinks, vegetative tillers as the source of N.

Initial growth in spring is largely independent of N uptake from the soil, provided that overwintering leaves are present on the plants.
Functional genomics: tools of the trade
Authors: Colebatch G. , Trevaskis B. , Udvardi M.
Published: March 2003

Functional genomics is transforming the way biological research is done in the 21st century. Functional genomics brings together high‐throughput genetics with multiparallel analyses of gene transcripts, proteins, and metabolites to answer the ultimate question posed by all genome‐sequencing projects: what is the biological function of each and every gene? Functional genomics is driving a shift in the research paradigm away from vertical analysis of single genes, proteins, or metabolites, towards horizontal analysis of full suites of genes, proteins, and metabolites. By identifying and measuring many, if not all of the molecular players that participate in a given biological process, functional genomics offers the prospect of obtaining a truly holistic picture of life. This review describes the tools that are currently being used for functional genomics work and considers the impact that this new discipline is likely to have in the future.
Specification of stomatal fate in Arabidopsis: evidences for cellular interactions
Authors: Serna Laura , Torres‐Contreras Javier , Fenoll Carmen
Published: March 2003

Stomata are bi‐celled epidermal structures distributed in predictable patterns providing plants with pathways for gas exchange with the atmosphere. In Arabidopsis thaliana, stomatal formation is emerging as an elegant and powerful model system to study the genetic and molecular control of cell fate specification and pattern formation in multicellular organisms. In this review, we describe the mechanisms that regulate stomatal distribution in this model plant. The emerging view indicates that cellular interactions play a relevant role during stomatal pattern formation. These cellular interactions are not restricted to a cell layer and signalling within the epidermis, between the epidermis and the underlying tissues and between organs seem to play a relevant role during stomatal formation. Whatever the nature of the different signals, the stomatal pattern must arise as the result of the integration by the epidermal cells of multiple inputs. Uncovering the molecular nature of such signals and understanding the specific role during stomatal development provides a formidable task for the future.
Fasciola hepatica cathepsin L suppresses sheep lymphocyte proliferation in vitro and modulates surface CD4 expression on human and ovine T cells
Authors: Prowse Rhoda k. , Chaplin Paul , Robinson H. Clem , Spithill Terry W.
Published: March 2003

Fasciola hepatica infection has been shown to suppress sheep lymphocyte proliferation in vitro and this is at least partially attributable to excretory/secretory products (ES) released by F. hepatica parasites. We identified a suppressive component in ES by analysing the effect of ES fractions, separated by gel filtration, on the proliferation of sheep T cells in vitro. A major proportion of the suppressive activity in ES was shown to coelute with the cathepsin L proteases: E64, a cysteine protease inhibitor, blocked the suppressive activity of cathepsin L. In order to identify possible mechanisms by which cathepsin L could suppress T cell proliferation, the effect of ES and F. hepatica recombinant cathepsin L (rFhCatL) on the expression of 22 different sheep T cell surface markers was analysed by flow cytometry. Incubation of sheep T cells with ES or two rFhCatL signicantly reduced surface CD4 expression and this effect was prevented in the presence of E64. In similar experiments with human T lymphocytes, ES and rFhCatL were shown to down regulate surface CD4 expression. These results show that F. hepatica cathepsin L both suppresses sheep T cell proliferation and reduces surface CD4 expression on both human and ovine T cells.