bluuebunny/crossref_metadata_2025 huggingface dataset:
Ordering of Compartments in the Yeast Endocytic Pathway
Authors: Prescianotto‐Baschong Cristina , Riezman Howard
Published: March 2003

We have characterized the morphology of the yeast endocytic pathway leading from the plasma membrane to the vacuole by following the trafficking of positively charged nanogold in combination with compartment identification using immunolocalization of t‐SNARE proteins. The first endocytic compartment, termed the early/recycling endosome, contains the t‐SNARE, Tlg1p. The next compartment, the prevacuolar compartment, contains Pep12p. After transport to the prevacuolar compartment, where vacuolar enzymes are seen on their way to the vacuole, endocytic content is delivered to the late endosome and on to the vacuole, both of which are devoid of Pep12p immunolabel. Traffic to the prevacuolar compartment is reduced in strains mutant for the Rab5 homologs, Vps21p, Ypt52p, and Ypt53p and in vps27 mutant cells. On the other hand, traffic to the early recycling endosome is less dependent on Rab5 homologs and does not require Vps27p.
Endocytosis Via Caveolae
Authors: Pelkmans Lucas , Helenius Ari
Published: March 2003

Caveolae are flask‐shaped invaginations present in the plasma membrane of many cell types. They have long been implicated in endocytosis, transcytosis, and cell signaling. Recent work has confirmed that caveolae are directly involved in the internalization of membrane components (glycosphingolipids and glycosylphosphatidylinositol‐anchored proteins), extracellular ligands (folic acid, albumin, autocrine motility factor), bacterial toxins (cholera toxin, tetanus toxin), and several nonenveloped viruses (Simian virus 40, Polyoma virus). Unlike clathrin‐mediated endocytosis, internalization through caveolae is a triggered event that involves complex signaling. The mechanism of internalization and the subsequent intracellular pathways that the internalized substances take are starting to emerge.
Cycling of Early Golgi Proteins Via the Cell Surface and Endosomes Upon Lumenal pH Disruption
Authors: Puri Sapna , Bachert Collin , Fimmel Claus J. , Linstedt Adam D.
Published: March 2003

The cis‐Golgi protein GPP130 reversibly redistributes to endosomes upon pH disruption, but the identity of the endosomes and the involved cycling route are unknown. It is also unknown whether any other early Golgi proteins participate in this pathway. Here, we analyze GPP130 and the structurally related Golgi protein GP73. Unlike the TGN marker TGN38/46, GPP130 and GP73 colocalized in the early Golgi and redistributed to the ER after brefeldin A treatment. Nevertheless, after pH disruption by monensin, GPP130 and GP73 redistributed to endosomes containing redistributed TGN38/46, but not other endosomal markers. In common with TGN38/46, the redistribution involved transient appearance on the plasma membrane, and upon monensin washout, the proteins moved back to the Golgi along a microtubule‐ and PI3 kinase‐independent route. Although GP73 did not associate with GPP130, its steady‐state Golgi targeting was also mediated by a lumenal predicted coiled‐coil stem domain. These findings indicate that at least two early Golgi proteins, each containing stem domain Golgi targeting determinants, cycle to the cell surface and back along the late endosome independent TGN38/46 pathway.
Drosophila Sec10 is Required for Hormone Secretion but not General Exocytosis or Neurotransmission
Authors: Andrews Hillary K. , Zhang Yong Q. , Trotta Nick , Broadie Kendal
Published: March 2003

The sec6/8, or exocyst, complex is implicated in trafficking of secretory vesicles to fusion sites in the plasma membrane. Genetic analyses have been done primarily in yeast, where mutation of the eight protein subunits similarly disrupts polarized vesicle fusion. The goal of this study was to assay the sec6/8 complex in Drosophila, and specifically to test its widely hypothesized functions in synaptogenesis and neurotransmission. We used a transgenic RNAi approach to remove the most highly conserved complex component, Drosophila sec10 (dSec10). Ubiquitous dSec10 RNAi resulted in early postembryonic lethality, demonstrating that dSec10 is essential. Surprisingly, tissue‐specific dSec10 RNAi revealed no essential requirement in nervous system, musculature, gut or epidermis. Assays of polarized secretion in all these tissues failed to reveal any role for dSec10. In particular, the neuromuscular synapse showed no defects in morphogenesis or vesicle trafficking/fusion underlying neurotransmission. The essential requirement for dSec10 was restricted to the ring gland, the Drosophila organ specialized for endocrine function. The developmental arrest of dSec10 RNAi animals was partially rescued by feeding ecdysone, suggesting dSec10 mediates steroid hormone secretion. We conclude that dSec10 has no detectable role in most forms of polarized trafficking/exocytosis, including neurotransmission, but rather is essential for endocrine secretion.
Apoptosis of periodontal ligament cells induced by mechanical stress during tooth movement
Authors: Hatai T , Yokozeki M , Funato N , Baba Y , Moriyama K , Ichijo H , Kuroda T
Published: March 2003

The mechanical force generated during tooth movement creates compressed and cell‐free areas in the periodontal membrane. The way in which periodontal ligament cells disappear at the compressed area during tooth movement remains unclear. In the present study we examined whether periodontal ligament cells undergo apoptosis by mechanical stress during tooth movement using the terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP‐biotin nick‐end‐labelling method (TUNEL). TUNEL‐positive stainings of periodontal ligament cells began to appear at the compressed areas 12 h after tooth movement, and the number of those cells reached maximum at 24 h after tooth movement. Thereafter TUNEL‐positive cells disappeared at 48 h, and direct and undermining bone resorption began at the same area 72 h after tooth movement. These results showed that compressed periodontal ligament cells were eliminated by apoptosis in the early phase of tooth movement.
Identification of the AgNORs, PCNA and ck16 proteins in oral lichen planus lesions
Authors: Fonseca LM da Silva , Do Carmo MAV
Published: March 2003

The expression of proliferating cell nuclear antigen (PCNA), cytokeratin 16 (ck16) and Ag nucleolar organizer regions (AgNORs) were assessed in 20 cases of lichen planus, 20 cases of keratosis and 20 cases of normal oral mucosa in order to evaluate the rate of keratinocyte proliferation in these tissues. Three hundred cells were counted in each sample: 100 basal cells, 100 suprabasal cells and 100 squamous cells. The mean number of AgNORs and the percentage of PCNA positive cells were calculated. Except from similar staining of suprabasal cells of lichen planus and keratosis, PCNA and AgNORs values were higher in all layers of lichen planus than in both keratosis and normal oral mucosa. The three groups showed similar ck16 immunostaining: all of the cells were positive, except those of the basal layer. The results suggest that the keratinocyte proliferation index is higher in lichen planus than in keratosis and normal mucosa. Besides, ck16 should not be used to differentiate the entities studied.
Viral infections in the mouth
Authors: Teo CG
Published: March 2003

Oral hairy leukoplakia (OHL) and Kaposi's sarcoma (KS) are commonly encountered in the HIV‐infected patient. A unique feature of OHL is non‐cytolytic high level of replication of Epstein–Barr virus (EBV) in the glossal epithelium. The expression of viral‐encoded anti‐apoptotic proteins concomitant to replicative proteins probably underlies this phenomenon. The question of whether OHL arises from activation of EBV latent in the tongue, or from superinfection by endogenous EBV shed via non‐glossal sites or by exogenous EBV remains unresolved. Human herpesvirus 8 (HHV8) is now seen as necessary but not sufficient cause of KS. Expression of HHV8‐encoded oncogenic proteins in endothelial cells probably explains the aberrant proliferation of these cells in KS lesions. Studies into why KS is so commonly observed at the palate in HIV‐infected patients may provide important clues to its pathogenesis.
Congenital sinus of the upper lip with idiopathic precocious puberty
Authors: Sumitomo S , Ogawa S , Hirai K , Takai Y
Published: March 2003

An extremely rare case of congenital sinus of the upper lip is presented in a 22‐year‐old woman with idiopathic precocious puberty. The sinus has a pin‐like orifice located in the midline of the upper lip philtum just below the base of the columella. Sexual precocity was found out when she was 7 years old. A series of exact examinations had been performed at that time. Neither neurogenic lesions of the central nervous system nor other systemic abnormalities were observed.
Origins and benefits of dental hygiene practice in Europe
Authors: Luciak‐Donsberger C.
Published: March 2003

Abstract:  Origins and benefits of the practice of dental hygiene were investigated in order to provide guidelines to countries where initiatives are being taken to introduce the profession. In Europe, so far the profession has been introduced in the Czech Republic, Denmark, Great Britain, Finland, Italy, Latvia, Lithuania, the Netherlands, Norway, Portugal, Sweden, Switzerland and Spain. Programmes in Ireland, Poland, and Romania are not presented in this article. Information for this study was obtained using questionnaires and followed up by e‐mail correspondence with additional experts, supporting studies and reference literature. All experts consulted are involved in the professional and educational organisation of dental hygiene in their countries. Results show that dentists and dental hygienists who had been inspired by the delivery of preventive care in the US, initiated the European dental hygiene movement. In some countries, opposition of organised dentistry had to be overcome. In countries where the population has limited access to qualified dental hygiene care, such as in Austria, Belgium, Germany and France, a high prevalence of untreated periodontal disease has been reported. There, the lucrative practice of delegating dental hygiene tasks to dental assistants without qualifying education has slowed efforts to implement the profession and resulted in negative health and vocational outcomes. This leads to the conclusion that an implementation of legislation governing the practice and the educational process of dental hygiene in the EU and beyond would contribute to an equitable standard of health care as well as to equal opportunities in education and employment.
Evidence for a direct interaction between the Thr11 residue of vasoactive intestinal polypeptide and Tyr184 located in the first extracellular loop of the VPAC2 receptor
Authors: NACHTERGAEL Ingrid , VERTONGEN Pascale , LANGER Ingrid , PERRET Jason , ROBBERECHT Patrick , WAELBROECK Magali
Published: March 2003

We developed previously VPAC1 [vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (PACAP) receptor]>VPAC2 receptor selective ligands. Replacement of the VIP-Thr11 by an Arg11 in these ligands contributed to their selectivity: Arg11-VIP had a 200-fold lower affinity when compared with VIP at VPAC2 receptors as opposed to 3- to 5-fold higher affinity at VPAC1 receptors. Comparison of the binding and functional properties of related VIP analogues suggested that the VPAC1 selectivity of Arg11-VIP was due to the loss of a hydrogen bond between the hydroxy group of Thr residue and the VPAC2 receptor, steric hindrance between the Arg side chain and the VPAC2 receptor and charge attraction by the VPAC1 receptor. Comparison of the ability of VIP analogues to activate adenylate cyclase through chimaeric VPAC1/VPAC2 and VPAC2/VPAC1 receptors indicated that the first extracellular receptor loop carried most of the VPAC2 receptors’ ability to discriminate VIP from Arg11-VIP. Based on results obtained for a truncated VPAC2 receptor and the closely related PACAP-preferring receptor (PAC1) and secretin receptors, we hypothesized that Thr11 interacted with the VPAC2 receptor Tyr184 (similar to the VPAC1 receptor Phe200 residue). The Y184F (Tyr184→Phe) VPAC2 mutant lost the ability to discriminate VIP from Val11-VIP, and the F200Y VPAC1 mutant acquired the ability to discriminate the natural peptide from Val11-VIP. These results support the hypothesis that the hydroxy group of the native VIP-Thr11 side chain can indeed form a hydrogen bond with the Tyr side chain in the VPAC2 receptor.
Degradation of mutant initiator protein DnaA204 by proteases ClpP, ClpQ and Lon is prevented when DNA is SeqA-free
Authors: SLOMINSKA Monika , WAHL Anne , WEGRZYN Grzegorz , SKARSTAD Kirsten
Published: March 2003

A mutant form of the Escherichia coli replication initiator protein, DnaA204, is unstable. At low growth rates, the dnaA204 mutant cells experience a limitation of initiator protein and grow with reduced initiation frequency and DNA concentration. The mutant DnaA protein is stabilized by the lack of SeqA protein. This stabilization was also observed in a dam mutant where the chromosome remains unmethylated. Since unmethylated DNA is not bound by SeqA, this indicates that DnaA204 is not stabilized by the lack of SeqA protein by itself, but rather by lack of SeqA complexed with DNA. Thus the destabilization of DnaA204 may be due either to interaction with SeqA—DNA complexes or changes in nucleoid organization and superhelicity caused by SeqA. The DnaA204 protein was processed through several chaperone/protease pathways. The protein was stabilized by the presence of the chaperones ClpA and ClpX and degraded by their cognate protease ClpP. The dnaA204 mutant was not viable in the absence of ClpY, indicating that this chaperone is essential for DnaA204 stability or function. Its cognate protease ClpQ, as well as Lon protease, degraded DnaA204 to the same degree as ClpP. The chaperones GroES, GroEL and DnaK contributed to stabilization of DnaA204 protein.
cDNA Array Analysis of Pineal Gene Expression Reveals Circadian Rhythmicity of the Dominant Negative Helix‐Loop‐Helix Protein‐Encoding Gene, Id‐1
Authors: Humphries A. , Klein D. , Baler R. , Carter D. A.
Published: March 2003

The pineal gland is a major output of the endogenous vertebrate circadian clock, with melatonin serving as the output signal. In many species, elevated nocturnal melatonin production is associated with changes in pineal gene expression. In the current study, cDNA array analysis was used in an attempt to identify additional genes that exhibit day/night differential expression in the rat pineal gland. This revealed 38 candidate genes, including Id‐1 (inhibitor of DNA binding and differentiation). Id‐1 encodes a helix‐loop‐helix (HLH) protein that lacks a basic DNA binding domain and could affect pineal physiology via a dominant negative trans‐acting regulatory activity. For this reason Id‐1 was selected for further analysis. Id‐1 was expressed in a major population of pineal cells and the Id‐1 protein was associated with a nuclear complex. The levels of Id‐1 mRNA and protein exhibit approximately six‐fold day/night rhythms. In contrast, the related genes Id‐2 and Id‐3 do not exhibit marked day/night differences in pineal expression. Rhythmic Id‐1 expression is primarily limited to a C‐terminally extended splice variant of Id‐1, which would restrict the functional output of the rhythm to protein binding partners of this isoform of Id‐1. Our findings add to the body of evidence indicating that transcriptional regulators play a role in neuroendocrine rhythms, and extend this by introducing the concept of a dominant negative HLH involvement. The rhythm in Id‐1 in the pineal gland provides an experimental opportunity to identify Id‐1‐binding partners which may also be involved in Id‐1 activity in other functional contexts.
Mitogen‐Activated Protein Kinase Mediates Epidermal Growth Factor‐Induced Morphogenesis in Pituitary GH3 Cells
Authors: Lewis M. D. , Ham J. , Rees D. A. , Lewis B. M. , Scanlon M. F.
Published: March 2003

Epidermal growth factor (EGF) causes pituitary GH3 cells to change from their normal predominantly rounded morphology to much more elongated cells with extensive filopodia, and this effect is accompanied by a parallel increase in cell volume. In view of this, and because EGF receptor expression is increased in some pituitary tumours, we examined the mechanism of this EGF‐induced morphological effect as it may play a role in tumour invasiveness. The effect of treatment of the cells with EGF (1 nm, 4 days) was determined visually (expressed as percent non round cells) and by measuring the cell volume by Coulter Counter analysis. EGF treatment caused the cells to change their morphology with percent non round cells increasing from 37% in control cells to 74% in EGF‐treated cultures; this was accompanied by a parallel increase in cell volume. Treatment of the cells with EGF in the presence of the MEK1 inhibitor (PD98059) completely blocked the EGF‐induced morphological changes, showing that activation of the mitogen‐activated protein kinase (MAPK) pathway is necessary to mediate this effect. Transfection of the cells with a constitutively activated mutant of MEK1 produced a similar morphological change to that produced by EGF treatment, with the proportion of non round cells increasing to 62% with a parallel increase in cell volume compared to cells transfected with the empty vector, demonstrating that direct activation of MAPK pathway is sufficient to mediate the observed morphological effects. The effects produced by activated MEK1 transfection could be blocked by PD98059. EGF had opposing effects on prolactin and growth hormone (GH) secretion by the cells, increasing prolactin release and inhibiting GH release. Transfection of the cells with activated MEK1 produced similar effects on hormone release as EGF treatment. In conclusion, the morphological effects of EGF on GH3 cells are mediated by activation of the MAPK pathway as blockade of this pathway abolished the observed effect, and direct activation of this pathway by transfection with an activated mutant of MEK1 was able to duplicate these effects. This mechanism may contribute to the growth and possibly local invasiveness of some pituitary tumours that express the EGF receptor.
The role of glycine betaine in the protection of plants from stress: clues from transgenic plants
Authors: Sakamoto A. , Murata N.
Published: March 2003

The acclimation of a plant to a constantly changing environment involves the accumulation of certain organic compounds of low molecular mass, known collectively as compatible solutes, in the cytoplasm. The evidence from numerous investigations of the physiology, genetics, biophysics and biochemistry of plants strongly suggests that glycine betaine (GB), an amphoteric quaternary amine, plays an important role as a compatible solute in plants under various types of environmental stress, such as high levels of salts and low temperature. Plant species vary in their capacity to synthesize GB and some plants, such as spinach and barley, accumulate relatively high levels of GB in their chloroplasts while others, such as Arabidopsis and tobacco, do not synthesize this compound. Genetic engineering has allowed the introduction into GB‐deficient species of biosynthetic pathways to GB from both micro‐organisms and higher plants; this approach has facilitated investigations of the importance of GB in stress protection. In this review, we summarize recent progress in the genetic manipulation of the synthesis of GB, with special emphasis on the relationship between the protective effects of GB in vivo and those documented in vitro.
Effects of long‐term exposure to elevated CO2 and N fertilization on the development of photosynthetic capacity and biomass accumulation in Quercus suber L.
Authors: Maroco J. P. , Breia E. , Faria T. , Pereira J. S. , Chaves M. M.
Published: March 2003

The effects of long‐term (4 year) CO2 enrichment (70 Pa versus 35 Pa) and nitrogen nutrition (8 mm versus 1 mm NO3–) on biomass accumulation and the development of photosynthetic capacity in leaves of cork oak (Quercus suber L., a Mediterranean evergreen tree) were studied. The evolution of photosynthetic parameters with leaf development was estimated by fitting the biochemical model of Farquhar et al. (Planta 149, 78–90, 1980) with modifications by Sharkey (Botanical Review 78, 71–75, 1985) to A–Ci response curves. CO2 enrichment had a small reduction effect on the development of the maximum CO2 fixation capacity by Rubisco (VCmax), and no effect over maximum electron transport capacity (Jmax), day‐time respiration (Rd) and Triose‐P utilization (TPU). However, there was a statistically significant effect of N fertilization and the interaction CO2 × N over the evolution of VCmax, Jmax and TPU. Relative stomatal limitation (estimated from A–Ci curves) was higher (+20%) for plants grown under ambient CO2 than for plants grown under elevated CO2. There was a significant effect of CO2 and N fertilization over total biomass accumulation as well as leaf area. Plants grown at elevated CO2 had 27% more biomass than plants grown at ambient CO2 when given high N. However, for plants grown under low N there was no significant effect of CO2 enrichment on biomass accumulation. Plants grown under low N also had significantly higher root : shoot ratios whereas there were no differences between CO2 treatments. The larger biomass accumulation of Q. suber under elevated CO2 is attributable to a higher availability of CO2 coupled to a larger leaf area, with no significant decrease in photosynthetic capacity under CO2 enrichment and elevated N fertilization. For low N fertilization, the effects of CO2 enrichment over leaf area and biomass accumulation are lost, suggesting that in native ecosystems with low N availability, the effects of CO2 enrichment may be insignificant.
IgG4 breaking the rules
Authors: Aalberse Rob C. , Schuurman Janine
Published: March 2003

Immunoglobulin G4 (IgG4) antibodies have been known for some time to be functionally monovalent. Recently, the structural basis for this monovalency has been elucidated: the in vivo exchange of IgG half‐molecules (one H‐plus one L‐chain) among IgG4. This process results in bispecific antibodies that in most situations will behave as functionally monovalent antibodies. The structural basis for the abnormal behaviour of IgG4 seems to be largely the result of a single amino acid change relative to human IgG1: the change of a proline in core hinge of IgG1 to serine. This results in a marked shift in the equilibrium between interchain disulphide bridges and intrachain disulphide bridges, which for IgG4 results in 25–75% absence of a covalent interaction between the H‐chains. Because of strong non‐covalent interactions between the CH3 domains (and possibly also between the CH1 domain and the trans‐CH2 domain) IgG4 is a stable four‐chain molecule and does not easily exchange half‐molecules under standard physiological conditions in vitro. We postulate that the exchange is catalysed in vivo by protein disulphide isomerase (PDI) and/or FcRn (the major histocompatibility complex (MHC)‐related Fc receptor) during transit of IgG4 in the endosomal pathway in endothelial cells. Because IgG4 is predominantly expressed under conditions of chronic antigen exposure, the biological relevance of this exchange of half‐molecules is that it generates antibodies that are unable to form large immune complexes and therefore have a low potential for inducing immune inflammation. In contrast to monovalent immunoglobulin fragments, these scrambled immunoglobulins have a normal half‐life. The significance of the ensuing bispecificity needs further evaluation, because this will be relevant only in situations where high IgG4 responses are found to two unrelated antigens that happen to be present in the body at the same time and place. In this context the significance of IgG4 autoreactivity might have to be re‐evaluated. The main function of IgG4, however, is presumably to interfere with immune inflammation induced by complement‐fixing antibodies, or, in the case of helminth infection or allergy, by IgE antibodies.
Intralobular ducts of human major salivary glands contain leptin and its receptor
Authors: De Matteis R. , Puxeddu R. , Riva A. , Cinti S.
Published: March 2003

Leptin, a 16‐kDa hormone, plays an important role in the control of food intake and in energy homeostasis both in rodents and in man. Leptin is mainly produced and secreted by adipocytes, but other tissues and gastric glands have also recently been shown to produce it in a dual (endocrine and exocrine) mode. In addition, a leptin receptor has been detected in taste cells of mouse circumvallate papillae and in rat intestinal epithelium. These data prompted us to carry out a detailed study of human salivary glands as potential leptin‐producing organs. Biopsies of salivary glands (submandibular and parotid) obtained from male and female patients during surgery for different clinical indications were subjected to immunohistochemical study for the presence of leptin, its functional receptor, insulin and glucagon. The presence and cellular distribution of glucocorticoid receptor in leptin‐secreting cells were also investigated. Double immunohistochemical staining (silver–gold intensification and avidin–biotin–peroxidase) was used for the visualization of glucocorticoid receptor and leptin labelling, respectively. The results show that intralobular duct cells of submandibular and parotid glands are immunoreactive for leptin, leptin receptor and glucagon but not for insulin. Leptin was also detected in some microglobules in whole saliva obtained from four healthy volunteers. Co‐localization for leptin, leptin receptor and glucocorticoid receptor in the same cell type suggested a functional relationship between glucocorticoid hormone and leptin secretion also at the level of the salivary glands.
Shading by shrubs in a desert system reduces the physiological and demographic performance of an associated herbaceous perennial
Authors: Forseth I. N. , Wait D. A. , Casper B. B.
Published: March 2003

A 2‐year field study examined the demographic consequences of association with shrubs in an herbaceous perennial, Cryptantha flava. Physiological data were collected to evaluate whether shrub effects were mediated primarily through water, nutrient or light availability.

 Microclimatic conditions under the north side of shrubs differed from open microhabitats, primarily in light availability. Due to little photosynthetic acclimation to light, daily photosynthesis for plants under shrubs was reduced proportionally to the light regime.

 Shading did not reduce stomatal conductance proportionally to photosynthesis, which led to decreased water use efficiency for plants under shrubs. Few differences were found in leaf water potential between microhabitats, indicating that little competition for water was occurring.

 There was little evidence for shrub‐induced nutrient island effects. Soil nitrogen, phosphorus and organic content did not differ between open and shrub microhabitats. Leaf nitrogen content also differed little between plants in the two microhabitats.

 Growth and flowering responses of individuals under shrubs were reduced relative to those in the open, even for plants located on the south side of shrubs.

 Over this 2‐year period of average to above‐average rainfall, association of C. flava with shrubs was dominated by competition for light, rather than for water or nutrients. Future investigations will address whether this asymmetric competitive interaction changes during years with below‐average rainfall to a facilitative interaction, or one of increased competition for water.
Importance of physiological integration of dwarf bamboo to persistence in forest understorey: a field experiment
Authors: Saitoh Tomoyuki , Seiwa Kenji , Nishiwaki Aya
Published: March 2003

Sasa spp., dwarf bamboo which dominate the undergrowth of temperate forests in Japan occur as clonal fragments in which ramets in light gaps to are connected to those in shaded understoreys by long rhizomes. We test whether persistence under shaded conditions is supported by translocation of assimilates from illuminated ramets. A dense population of Sasa palmata growing at an open site, was exposed to two light conditions (homogeneous: open–open and heterogeneous: open–shaded) and two rhizome connection treatments (intact and severed) in a full factorial design.

Ramet mass, and the mass of many parts of the clonal fragments, was much lower in the shade than in the open, but this effect was less marked when the rhizome connection was intact than when it was severed. Clone parts in shade may therefore be supported by translocation from connected clone parts in the open, with such physiological integration enhancing persistence where light supply is heterogeneous as in the gap–understorey continuum.

Above‐ground biomass was reduced sooner than that below ground. Clonal fragments of S. palmata recover via dormant buds on rhizomes, whose longer persistence would therefore enhance performance of the clonal fragment.

Specific leaf area (SLA) was greater in shade than in the open, irrespective of rhizome connection, suggesting that individual leaves show morphological plasticity independently of only physiological integration.
Relative labelling index: a novel stereological approach to test for non‐random immunogold labelling of organelles and membranes on transmission electron microscopy thin sections
Authors: Mayhew T. M. , Lucocq J. M. , Griffiths G.
Published: March 2003

Simple and efficient protocols for quantifying immunogold labelling of antigens localized in different cellular compartments (organelles or membranes) and statistically evaluating resulting labelling distributions are presented. Two key questions are addressed: (a) is compartmental labelling within an experimental group (e.g. control or treated) consistent with a random distribution? and (b) do labelling patterns vary between groups (e.g. control vs. treated)? Protocols rely on random sampling of cells and compartments. Numbers of gold particles lying on specified organelle compartments provide an observed frequency distribution. By superimposing test‐point lattices on cell profiles, design‐based stereology is used to determine numbers of points lying on those same compartments. Random points hit compartments with probabilities determined by their relative sizes and so provide a convenient internal standard, namely, the expected distribution if labelling is purely random. By applying test‐line lattices, and counting sites at which these intersect membrane traces, analogous procedures provide observed and expected labelling distributions for different classes of membranes. Dividing observed golds by expected golds provides a relative labelling index (RLI) for each compartment and, for random labelling, the predicted RLI = 1. In contrast to labelling densities of organelles (golds µm−2) or membranes (golds µm−1), RLI values are estimated without needing to know lattice constants (area per point or length per intersection) or specimen magnification. Gold distributions within a group are compared by chi‐squared analysis to test if the observed distribution differs significantly from random and, if it is non‐random, to identify compartments which are preferentially labelled (RLI > 1). Contingency table analysis allows labelling distributions in different groups of cells to be compared. Protocols are described and illustrated using worked specimen examples and real data.
The prevention of the staurosporine‐induced apoptosis by Bcl‐XL, but not by Bcl‐2 or caspase inhibitors, allows the extensive differentiation of human neuroblastoma cells
Authors: Yuste Víctor J. , Sánchez‐López Isabel , Solé Carme , Encinas Mario , Bayascas Jose R. , Boix Jacint , Comella Joan X.
Published: March 2003

Staurosporine is one of the best apoptotic inducers in different cell types including neuroblastomas. In this study we have compared the efficiency and final outcome of three different anti‐apoptotic strategies in staurosporine‐treated SH‐SY5Y human neuroblastoma cells. At staurosporine concentrations up to 500 nm, z‐VAD.fmk a broad‐spectrum, noncompetitive inhibitor of caspases, reduced apoptosis in SH‐SY5Y cells. At higher concentrations, z‐VAD.fmk continued to inhibit caspases and the apoptotic phenotype but not cell death which seems to result from oxidative damage. Stable over‐expression of Bcl‐2 in SH‐SY5Y protected cells from death at doses of staurosporine up to 1 µm. At higher doses, cytochrome c release from mitochondria occurred, caspases were activated and cells died by apoptosis. Therefore, we conclude that Bcl‐2 increased the threshold for apoptotic cell death commitment. Over‐expression of Bcl‐XL was far more effective than Bcl‐2. Bcl‐XL transfected cells showed a remarkable resistance staurosporine‐induced cytochrome c release and associated apoptotic changes and survived for up to 15 days in 1 µm staurosporine. In these conditions, SH‐SY5Y displayed a remarkable phenotype of neuronal differentiation as assessed by neurite outgrowth and expression of neurofilament, Tau and MAP‐2 neuronal specific proteins.
Effect of endogenous serotonin on the binding of the 5‐HT1A PET ligand 18F‐MPPF in the rat hippocampus: kinetic β measurements combined with microdialysis
Authors: Zimmer Luc , Mauger Gweltas , Le Bars Didier , Bonmarchand Gregory , Luxen André , Pujol Jean‐François
Published: March 2003

By using a combination of an original β+‐sensitive␣intracerebral probe and microdialysis, the effect of increased endogenous serotonin on specific binding of 18F‐MPPF [4‐(2′‐methoxyphenyl)‐1‐[2′‐[N‐(2′′‐pyridinyl)‐p‐fluorobenzamido]ethyl]piperazine] to the serotonin‐1A (5‐HT1A) receptors was investigated in the hippocampus of the anaesthetized rat. Our β‐sensitive probe prototype was sensitive enough to obtain specific 18F‐MPPF time–activity curves in the rodent (hippocampus/cerebellum ratio ≈ 2). The serotonin neuronal release was pharmacologically enhanced using fenfluramine at three different doses (1, 2 and 10 mg/kg intravenous) multiplying by 2–15 the extracellular serotonin in the hippocampus. These extracellular variations of extracellular serotonin resulted in dose‐ranging decreases in 18F‐MPPF‐specific binding in the same rat. Our results showed for the first time that 18F‐MPPF binding could be modulated by modifications of extracellular serotonin in the rat hippocampus. These results were confirmed by the enhancement of extracellular radioactivity collected in dialysates after the displacement of 18F‐MPPF by fenfluramine. After modelization, 18F‐MPPF binding could constitute an interesting radiotracer for positron emission tomography in evaluating the serotonin endogenous levels in limbic areas of the human brain.
Relevance of different striatal markers in assessment of the MPP+‐induced dopaminergic nigrostriatal injury in rat
Authors: Barc Stéphanie , Page Guylène , Barrier Laurence , Garreau Lucette , Guilloteau Denis , Fauconneau Bernard , Chalon Sylvie
Published: March 2003

Many striatal dopaminergic markers are available for estimating the degree of the nigrostriatal lesion by MPTP/MPP+, but the changes of these markers are not perfectly matched. In this study we investigated different striatal markers and determined which ones closely reflected the nigrostriatal alteration. The in vivo binding of (E)‐N‐(3‐iodoprop‐2‐enyl)‐ 2‐β‐carbomethoxy‐3β‐(4′‐methylphenyl)nortropane (PE2I), a selective and potent inhibitor of the neuronal dopamine transporter (DAT) was considered as the reference index of injury of striatal dopaminergic nerve‐endings. Rats received a 10‐µg MPP+ injection in the right substantia nigra and were killed at 7 days after lesion. The results were as follows: (i) a decrease (66%) of the biodistribution of [125I]PE2I; (ii) a great reduction of the DAT expression measured by the binding of [125I]PE2I in striatal membranes (Bmax decreased by 54%) and in cerebral slices (88%); (iii) an 80% inhibition of the vesicular monoamine transporter expression revealed by the binding of [3H]dihydrotetrabenazine in cerebral slices; (iv) a robust decrease in the quantity of DA and its metabolites (about 50–60%); (v) a slight modification of the DAT activity with a decreased number of functional sites (Vmax decreased by 12%, p < 0.05) without change of the affinity in striatal synaptosomes. Among these markers the binding of [125I]PE2I in membrane homogenates and the content of DA, and its metabolites, in striatum could be the most relevant in vitro indexes of the degenerative state of the nigrostriatal pathway after MPP+ lesion.
Local and systemic increase in lipid peroxidation after moderate experimental traumatic brain injury
Authors: Praticò Domenico , Reiss Peter , Tang Lina X. , Sung Syan , Rokach Joshua , McIntosh Tracy K.
Published: March 2003

Traumatic brain injury is a common event associated with neurological dysfunction. Oxidative damage, may contribute to some of these pathologic changes. We used a specific and sensitive marker of lipid peroxidation, the isoprostane 8,12‐iso‐iPF2α‐VI, to investigate whether local and also systemic lipid peroxidation were induced following lateral fluid percussion (FP) brain injury in the rat. Animals were anesthetized and subjected to lateral FP brain injury of moderate severity, or to sham injury as controls. Urine was collected before anesthesia (baseline), 6 and 24 h after injury. Blood was collected at baseline, 1, 6 and 24 h after injury. Animals were killed 24 h after surgery and their brains removed for biochemical analysis. No significant difference was observed at baseline (preinjury) for urine and plasma 8,12‐iso‐iPF2α‐VI levels between injured and sham‐operated animals. By contrast, plasma and urinary levels increased significantly already at 1 and further increased 24 h following brain injury, when compared to sham‐operated animals. Finally, compared with sham, injured animals had a significant increase in brain 8,12‐iso‐iPF2α‐VI levels. These results demonstrate that moderate brain injury induces widespread brain lipid peroxidation, which is accompanied by a similar increase in urine and plasma. Peripheral measurement of 8,12‐iso‐iPF2α‐VI levels after brain injury may be a reliable marker of brain oxidative damage.
High rates of extracellular superoxide production in bryophytes and lichens, and an oxidative burst in response to rehydration following desiccation
Authors: Minibayeva Farida , Beckett Richard Peter
Published: March 2003

The mechanism of extracellular superoxide radical (O2·−) formation and the role of the oxidative burst in response to desiccation stress is reported here in bryophytes and lichens from habitats of contrasting water availability.

Rates of extracellular production of O2·− radicals were measured, before and after desiccation stress, by the oxidation of epinephrine to adrenochrome, determined spectrophotometrically.

Several desiccation‐sensitive lichens and bryophytes that grow in very wet microhabitats produced O2·− extracellularly at high rates, even when they were not stressed. In addition, some species showed a powerful burst of O2·− production during rehydration following desiccation. Production of high levels of O2·− and the existence of an inducible oxidative burst was best developed in cyanobacterial lichens, a hornwort and the two thalloid liverworts tested. Extracellular production of O2·− was almost absent from all mosses tested, a leafy liverwort, a filmy fern and from desiccation‐tolerant lichens.

Patterns of O2·− production are discussed in terms of their possible role as a defence against pathogenic fungi and bacteria.
A model coupling foliar monoterpene emissions to leaf photosynthetic characteristics in Mediterranean evergreen Quercus species
Authors: Niinemets Ülo , Seufert Günther , Steinbrecher Rainer , Tenhunen John D.
Published: March 2003

A model was developed and parameterized to simulate monoterpene emission rates per unit leaf area (E) dependent on foliar photosynthetic potentials and activity of monoterpene synthases in the evergreen sclerophylls Quercus coccifera L. and Q. ilex L.

 Assuming that total activity of monoterpene synthases controls the pathway flux, the product of leaf dry mass per unit area E was culculated as the fraction of total electron flow used for monoterpene synthesis (ɛ), the rate of photosynthetic electron transport (J) per unit leaf dry mass and the reciprocal of the electron cost of monoterpene synthesis. In the model, light effects on E result from light responses of J, and the temperature relationship of E combines temperature dependencies of J and the specific activity of monoterpene synthase (SS).

 Having determined J from leaf photosynthesis data and deriving an estimate of SS from in vitro laboratory measurements, good fits to diurnal time‐courses of monoterpenoid emission rates were obtained using a single leaf‐dependent coefficient, the total monoterpene synthase activity in the leaves.

 Our analysis demonstrates that using J as a surrogate of E leads to a realistic description of E, especially under stress conditions where the previous models fail. However, analysis of daily time courses of E indicated that storage of monoterpenoids in nonspecific leaf compartments might alter the correspondence between monoterpenoid synthesis and emission rates, especially after rapid environmental change.
Calcium signalling in stomatal responses to pollutants
Authors: McAinsh Martin R. , Evans Nicky H. , Montgomery Lucy T. , North Kathryn A.
Published: March 2003

Stomatal responses to air pollutants are complex, varying among species and with concentration, environmental conditions and age. In general, short‐term exposure to sulphur dioxide (SO2) promotes stomatal opening, whereas longer‐term exposure can cause partial stomatal closure. By contrast, the effects of oxides of nitrogen (NOx) are often small or insignificant. The effects of ozone, and oxidative stress, are equally complex. Short‐term exposure to ozone stimulates a rapid reduction in stomatal aperture, whilst longer‐term exposure causes stomatal responses to become sluggish. The response of stomata to abscisic acid (ABA) has been shown to be slower in plants exposed to a combination of SO2 and NO2 suggesting an adverse effect on guard cell ABA signal transduction. In addition, ozone causes a reduction in stomatal closure under drought conditions. There is an increasing body of evidence to suggest that air pollutants and oxidative stresses can have a marked effect on the Ca2+ homeostasis of guard cells and the intracellular machinery responsible for stomatal movements. Here we discuss the effects of air pollutants on stomatal responses and their possible effects on Ca2+ based signalling in guard cells focusing on the effects of ozone and oxidative stress.
The recent history of Puccinia striiformis f.sp. tritici in Denmark as revealed by disease incidence and AFLP markers
Authors: Justesen A. F. , Ridout C. J. , Hovmøller M. S.
Published: March 2003

Disease observations and amplified fragment length polymorphism (AFLP) markers were used to study recent developments in the Puccinia striiformis f.sp. tritici population in Denmark. The fungus appeared spontaneously at 10 locations in Denmark in 1997 after it was not observed under natural conditions in 1996. The pattern of disease development and prevailing winds suggested that the fungus reappeared by airborne spores from the south or west. In 1998, disease incidence was more evenly distributed throughout the country. Forty‐eight single lesion isolates were collected from most crops where the disease was observed in these years; all except one from 1997 belonged to two pathotypes that were not previously detected in the country, and both possessed the newly discovered Yr17 virulence. The isolates were characterized with AFLP markers together with 28 isolates representing eight of 13 pathotypes observed prior to 1996. Initial screening of 240 PstI/MseI AFLP primer combinations on four isolates showed that a primer combination, on average, revealed 0·4 polymorphisms between any isolate pair. A selection of 21 primer combinations resulted in 28 AFLP markers, which revealed 16 AFLP phenotypes among all 76 isolates. The two Yr17‐virulent pathotypes consisted of three AFLP phenotypes, which were observed in both 1997 and 1998; the two most frequent AFLP phenotypes occurred at most sampling locations and often within the same crop. AFLP diversity was larger among samples collected prior to 1996, and also in this period most AFLP phenotypes were observed at different sampling locations. These results are consistent with the features of an entirely asexually reproducing pathogen dispersed by aerial spores across large areas.
Mechanisms of immunity to Haemonchus contortus infection in sheep
Authors: Balic A. , Bowles V. M. , Meeusen E. N. T.
Published: March 2003

In two separate experiments, sheep were immunized by nine to 12 weekly immunizing infections with 4000 Haemonchus contortus third stage larva (L3), drenched with anthelminthics and maintained free of H. contortus infection for a further 12 weeks. The anamnestic cellular immune responses in both the abomasal lymph node (ALN) and mucosa of the immunized sheep were examined 3 and 5 days post challenge with 50 000 H. contortus L3. Sheep in the two experiments clearly segregated out in two distinct groups, one in which challenge larvae were obviously present in the tissues of all 12 sheep at 3 and 5 days post challenge while no challenge larvae were detected in tissues of seven of the eight sheep in the other group. In sheep in which no tissue larvae were detected, very few changes were noted in either the ALN or mucosa. In contrast, dramatic changes were observed in the cellular profiles of the ALN and mucosa after challenge infection in sheep in which larvae were observed in the abomasal tissues. In the ALN, these changes were characterized by an increase in the relative percentage of γδ‐TCR+ T cells and B cells and an increase in the proportion of CD4+ T cells coexpressing the activation markers MHC class II and CD25. In the abomasal mucosa, an increase in the number of infiltrating CD4+ and γδ‐TCR+ T cells and B cells was observed by 3 days postinfection and these levels were further increased at 5 days postinfection. This infiltration of the abomasal mucosa by lymphocytes was accompanied by a dramatic increase in the number of infiltrating eosinophils, which were often in intimate association with the surface of H. contortus larvae. None of these changes occurred in the mucosa of the sheep that showed no sign of challenge larvae in the tissues; however, a transient increase in γδ T cells in the ALN and a drop in intraepithelial globule leucocytes were uniquely observed in these sheep at 5 days post challenge. These results suggest that two different types of immune responses can be generated after challenge infection of immunized sheep, one where tissue larvae are excluded from their tissue niche as observed previously and which is associated with changes in globular leucocyte population but no mobilization of the local immune system. In contrast, when challenge larvae reach their tissue niche, dramatic changes in the local immune system occur, including a pronounced infiltration of eosinophils. These two immune mechanisms may be associated with the rapid and delayed rejection of parasite infections in immune sheep.