bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolutionary aspects of positioning and identification of vertebrate limbs
Authors: TAMURA KOJI , KURAISHI RITSU , SAITO DAISUKE , MASAKI HIDEKI , IDE HIROYUKI , YONEI‐TAMURA SAYURI
Published: March 2003

abstract

Emerging developmental studies contribute to our understanding of vertebrate evolution because changes in the developmental process and the genes responsible for such changes provide a unique way for evaluating the evolution of morphology. Endoskeletal limbs, the locomotor organs that are unique to vertebrates, are a popular model system in the fields of palaeontology and phylogeny because their structure is highly visible and their bony pattern is easily preserved in the fossil records. Similarly, limb development has long served as an excellent model system for studying vertebrate pattern formation. In this review, the evolution of vertebrate limb development is examined in the light of the latest knowledge, viewpoints and hypotheses.
Development of the arterial pattern in the upper limb of staged human embryos: normal development and anatomic variations
Authors: RODRÍGUEZ‐NIEDENFÜHR M. , BURTON G. J. , DEU J. , SAÑUDO J. R.
Published: March 2003

A total of 112 human embryos (224 upper limbs) between stages 12 and 23 of development were examined. It was observed that formation of the arterial system in the upper limb takes place as a dual process. An initial capillary plexus appears from the dorsal aorta during stage 12 and develops at the same rate as the limb. At stage 13, the capillary plexus begins a maturation process involving the enlargement and differentiation of selected parts. This remodelling process starts in the aorta and continues in a proximal to distal sequence. By stage 15 the differentiation has reached the subclavian and axillary arteries, by stage 17 it has reached the brachial artery as far as the elbow, by stage 18 it has reached the forearm arteries except for the distal part of the radial, and finally by stage 21 the whole arterial pattern is present in its definitive morphology. This differentiation process parallels the development of the skeletal system chronologically. A number of arterial variations were observed, and classified as follows: superficial brachial (7.7%), accessory brachial (0.6%), brachioradial (14%), superficial brachioulnar (4.7%), superficial brachioulnoradial (0.7%), palmar pattern of the median (18.7%) and superficial brachiomedian (0.7%) arteries. They were observed in embryos belonging to stages 17–23 and were not related to a specific stage of development. Statistical comparison with the rates of variations reported in adults did not show significant differences. It is suggested that the variations arise through the persistence, enlargement and differentiation of parts of the initial network which would normally remain as capillaries or even regress.
Comparing human skeletal muscle architectural parameters of cadavers with in vivo ultrasonographic measurements
Authors: MARTIN D. C. , MEDRI M. K. , CHOW R. S. , OXORN V. , LEEKAM R. N. , AGUR A. M. , McKEE N. H.
Published: March 2003

The purpose of this study was to document and compare the architectural parameters (fibre bundle length, angle of pennation) of human skeletal muscle in cadaveric specimens and live subjects. The medial (MG) and lateral (LG) gastrocnemius, and posterior (PS) and anterior (AS) soleus were examined bilaterally in 5 cadavers (mean age 72.6, range 65–83 y) and 9 live subjects (mean age 76.3, range 70–92 y). Data were obtained from direct measurement of cadaveric specimens and from ultrasonographic scans of the live subjects. In cadaveric muscle, fibre bundles were isolated; their length was measured in millimetres and pennation angles were recorded in degrees. In live muscle, similar measurements were taken from ultrasonographic scans of relaxed and contracted muscle. For the scans of relaxed muscle, subjects were positioned prone with the foot at a 90° angle to the leg, and for scans of contracted muscle, subjects were asked to sustain full plantarflexion during the scanning process. Fibre bundle length and angle of pennation were compared at matched locations in both groups. It was found that the relationship between cadaveric and in vivo values for fibre length and angle of pennation varied between muscle parts. The cadaveric architectural parameters did not tend to lie consistently towards either extreme of relaxation or contraction. Rather, within MG, PS and AS, cadaveric fibre bundle lengths lay between those for relaxed and contracted in vivo muscle. Similarly both the anterior and posterior cadaveric fibre angles of pennation lay between the in vivo values within LG and PS. In summary, architectural characteristics of cadaveric muscle differ from both relaxed and contracted in vivo muscle. Therefore, when developing models of skeletal muscle based on cadaveric studies, the architectural differences between live and cadaveric tissue should be taken into consideration.
The cellular networks of normal ovine medial collateral and anterior cruciate ligaments are not accurately recapitulated in scar tissue
Authors: Lo Ian K. Y. , Ou Yong , Rattner John‐Paul , Hart David A. , Marchuk Linda L. , Frank Cyril B. , Rattner Jerome B.
Published: March 2003

The purpose of this study was to characterize the cellular organization of the ovine medial collateral ligament (MCL) and anterior cruciate ligament (ACL) and compare this organization with that found in ligaments undergoing healing. Indirect immunofluorescence microscopy, used in combination with antibodies to cytoskeletal proteins, was employed to visualize individual ligament cells. Normal ligaments contained fusiform cells arranged in rows, which were stacked at regular intervals across the body of the ligament forming a three‐dimensional cellular lattice. Each cell exhibited prominent cytoplasmic processes that extended for long distances through the extracellular matrix to adjacent cells, and these processes contained gap junctions. Thus the cells in rows and between rows were interconnected. The cells of the MCL and ACL scars were also arranged in rows, but these rows were shorter, irregularly arranged and closely packed into bundles resulting in tissue with a higher cellular density. In addition, cells transiting the cell cycle were detected in the scar but not in normal ligament. While the rows of cells in the normal ligament extended along the long axis of the ligament, the bundles of rows of ligament scar cells had a random orientation with respect to one another and to the region outside the scar. Over time both the ACL and the MCL scars displayed discontinuities in their cellular rows. In contrast to the scars of the MCL, which contained discontinuities filled with cellular projections and gap junctions, ACL scars contained discontinuities that were devoid of cells and gap junctions. These discontinuities as well as the differences between normal and scar cytoarchitecture may represent features of an inadequate healing response and/or may provide the structural basis for the altered biomechanics of healing ligaments.
Understanding Schwann cell–neurone interactions: the key to Charcot–Marie–Tooth disease?*
Authors: Maier Marcel , Berger Philipp , Suter Ueli
Published: March 2003

Charcot–Marie–Tooth disease (CMT) comprises a heterogeneous group of disorders. The most frequent subtype is caused by increased PMP22 gene dosage or missense point mutations affecting the PMP22 gene (CMT type 1A; CMT1A). Animal models in rat and mouse with the corresponding PMP22 alterations are available and mimic many aspects of the human diseases. Detailed examinations of the animal mutants, together with complementary data from patients, point towards altered Schwann cell–neurone interactions as a major underlying mechanism of CMT1A and related hereditary neuropathies. This is evident from the finding that mutated proteins affecting either Schwann cells or neurones have a profound influence on their partner cells. Recently, a number of novel genes causing various forms of CMT have been identified which are expressed either mainly by Schwann cells and/or by the accompanying neurones. These genes can be viewed, in analogy to classic experiments routinely performed in lower vertebrates, as the result of a ‘functional screen’ revealing crucial players in the interactions between Schwann cells and neurones. Studying how Schwann cell and axon‐encoded proteins are functionally interconnected will be an exciting task for the future. It will not only yield insights into the molecular and cellular basis of neuropathies but also provide crucial information about the interplay between Schwann cells and neurones in general.
Change in endothelial cell shape in response to ischaemia in the cardiac and retinal capillary beds
Authors: Glyn M. C. , Lawrenson J. G. , Ward B. J.
Published: March 2003

The change in shape of cardiac capillary endothelial cells in response to ischaemia and ischaemia followed by reperfusion is well established. We investigated the possibility that this process is an active rather than a passive process and that the change might be prevented or attenuated in order to minimise reperfusion injury and ‘no‐reflow’. We have also compared the change in shape of cardiac capillaries with those of the retina. We used an isolated heart preparation in which global ischaemia was induced by cessation of flow. Retinal capillaries were made ischaemic by one of 2 methods, using firstly superfusion with an oxygen depleted medium, and secondly by occlusion of upstream vessels with starch spheres. Ultrastructural and morphometric analysis of cardiac and retinal capillaries demonstrated that the change in shape seen in cardiac capillaries in response to ischaemia does not occur in retinal capillaries reflecting the different metabolic activities of the 2 tissues. Immunocytochemical localisation of _‐actin showed no apparent difference in the retinal capillaries between control and ischaemic capillaries. Cardiac capillaries, however, showed an irregular redistribution of _‐actin in ischaemic vessels which could be attenuated by exposure to phalloidin. Resin casts of ischaemic capillaries showed focal narrowings which were not present in controls. From these results we conclude that the change in endothelial cell shape in response to ischaemia is not a passive process and can be modulated by agents which target the microfilament system.
Functional and morphological consequences of removing astrocytes from the in vivo blood–brain barrier
Authors: Ray D. E. , Mavroudis G. , Prior M. J. W. , Guérin C. J. , Nolan C. C.
Published: March 2003

Our purpose was to attempt to determine the role of astrocytes in the maintenance and repair of the blood–brain barrier in adult F344 rats. Systemic administration of the glyceraldehyde‐3‐phosphate dehydrogenase inhibitor S‐alpha‐chlorohydrin produces selective destruction of astrocytes within specific brain stem nuclei (Cavanagh et al. Neuropath. Appl. Neurobiol. 19, 1993). At appropriate dose levels (140–160 mg/kg i.p.) other brain areas are undamaged, which allows the full time course of the response of sensitive areas to be studied in rats showing relatively little functional impairment. All experiments were carried out in accordance with UK Home Office requirements. Lesion development was followed by in vivo Magnetic Resonance Imaging, by post mortem immunohistochemistry and confocal microscopy, or electron microscopy. Barrier function was studied by the permeation of fluorescent conjugated dextrans of 3–500 KDa molecular weight given intravenously 1 h before perfusion with fixative, and visualised by confocal microscopy (Guérin et al. Neurosci. 103, 2001).

Astrocytic death was seen throughout areas such as the inferior colliculi and vestibular nuclei over 6–24 h and was followed by secondary neuronal death and a microglial reaction from 48 h. After a delay of about 12 h following the loss of astrocytes, endothelial cells become permeable to dextran for up to 4 d and then recovered. This recovery was seen in the absence of direct contact with astrocytes, as determined by immunohistochemistry and electron microscopy.

Since chlorohydrin does not increase endothelial monolayer permeability in vitro (Romero et al. Neurotoxicol. 18, 1997), we hypothesise that this transient loss of barrier function in vivo represents a partial dedifferentiation in response to loss of astrocytic contact. The subsequent restoration of endothelial barrier function in areas devoid of astrocytes represents the first in vivo demonstration that direct astrocytic contact is not needed for establishment of the blood–brain barrier.
Complementary actions of VEGF and angiopoietins on blood vessel permeability and growth in mice
Authors: Thurston G.
Published: March 2003

The families of vascular endothelial growth factors (VEGFs) and angiopoietins regulate blood vessel growth, maturation and function via distinct endothelial‐cell specific tyrosine kinase receptors. We have gained some insight into the actions of these growth factors by comparing 2 systems in which VEGF or angiopoietin‐1 (Ang1) was overexpressed in mice. In one set of studies we compared the blood vessels in the skin of transgenic mice in which VEGF, angiopoietin‐1, or both factors, were expressed locally under the keratin‐14 (K14) promoter. Mice overexpressing VEGF (K14‐VEGF) had numerous tortuous capillary‐sized skin vessels which were leaky to the plasma tracer Evans blue under baseline conditions and became even leakier in response to inflammatory stimuli such as topical mustard oil. The dermis of K14‐VEGF mice was increased in thickness and contained numerous infiltrating leukocytes. In contrast mice overexpressing angiopoietin‐1 had increased size of vessels in the skin without a significant increase in vessel number. Furthermore the skin vessels in K14‐Ang1 mice were less leaky than those of wild‐type mice in response to inflammatory stimuli and did not leak under baseline conditions. The dermal thickness was normal and there were normal dermal leukocytes. In double transgenic mice overexpressing both factors, both the size and number of skin vessels were increased, however, the vessels were not abnormally leaky under baseline or inflammatory conditions and the dermis was relatively normal. In a second set of studies we compared the effects of systemically delivered VEGF or angiopoietin‐1 using an adenoviral‐mediated system to produce the factors in vivo. Intravenous injection of adenovirus encoding VEGF (Ad‐VEGF) into mice resulted in widespread tissue oedema within 1–2 d after administration. In contrast injection i.v. of Ad‐Ang1 resulted in the skin vessels becoming less leaky in response to topical inflammatory stimuli or local injection of VEGF. The decreased leakage in the skin vessels was found at one day after Ad‐Ang1 injection, was not accompanied by morphologic changes, and was reproduced by systemic administration of recombinant Ang1 proteins. Thus overexpression of VEGF appears to promote growth of new vessels accompanied by plasma leakage and inflammation, whereas overexpression of Ang1 promotes the enlargement of existing vessels and a decrease in inflammation induced leakage. Further understanding of the interrelationship of these factors during normal development could lead to their application in the treatment of ischaemic diseases.

Studies were supported in part by NIH HL59157.
Aquaporin‐4 water channel expression in human vasogenic brain oedema
Authors: Geeson F. , Saadoun S. , Papadopoulos M. C. , Bell B. A. , Davies D. C.
Published: March 2003

A number of brain lesions result in brain oedema formation. This oedema is considered to be vasogenic in nature, resulting from breakdown of the blood–brain barrier. Brain microvessels are surrounded by astrocyte foot processes that express the water channel protein aquaporin 4 (aqp4). Aqp4 has been implicated in the formation of vasogenic oedema in animal models, but little is known about its involvement in vasogenic oedema in man. Therefore formalin‐fixed samples of a variety of brain lesions that give rise to brain oedema were obtained from 17 patients who underwent craniotomy at atkinson morley’s hospital during 1993–9. Immunocytochemistry was then performed on the samples using a polyclonal aqp4 antibody (ab3068, chemicon) visualised with immunoperoxidase and diaminobenzidine hydrochloride. In histologically normal cerebral cortex (n = 1) aqp4 immunoreactivity was restricted to astrocyte foot processes lining the pial membrane and surrounding some microvessels. In contused cortex (n = 3) astrocyte aqp4 expression was up‐regulated in accordance with the degree of tissue damage and the severity of oedema, assessed by contrast enhancement on ct. Aqp4 immunoreactivity was not present in nonastrocytic tumour cells including adenocarcinoma (n = 4), meningioma (n = 4), oligodendroglioma (n = 1) and medulloblastoma (n = 1). However diffuse aqp4 immunoreactivity was present in cells with the morphology of reactive astrocytes at the periphery of these tumours and occasionally ‘trapped’ within them. In addition up‐regulation of aqp4 immunoreactivity was observed in astrocyte end feet surrounding the microvessels in the perimeningioma cortex. Diffuse aqp4 immunoreactivity was observed in a subependyal astrocytoma (n = 1). Strong aqp4 immunolabelling of astrocytes was observed in cerebral cortex affected by bacterial meningitis (n = 1). Up‐regulation of aqp4 immunoreactivity was also observed in the foot processes of astrocytes lining the pial membrane and surrounding microvessels in this tissue. Up‐regulation of aqp4 immunoreactivity was not observed in brain tissue affected by subarachnoid haemorrhage (n = 1), from a patient who did not show any evidence of oedema on ct. Therefore brain lesions with different aetiologies that result in vasogenic brain oedema appear to cause a similar up‐regulation of aqp4 expression in astrocytes associated with the lesions. These results suggest that aqp4 up‐regulation in astrocytes may be part of a ‘final common pathway’ leading to oedema formation.
Patterning of the axial musculature in the developing chick embryo (Gallus)
Authors: Adds P. J. , Robson L. G.
Published: March 2003

While the development and patterning events of the skeletal, myogenic and connective tissues of the developing limb buds of the chick have been relatively well studied, there is little known about the formation of the epaxial muscles and tendons. The epaxial muscles form the postvertebral muscle groups and develop from the myotome of the somite. The myotome develops from myogenic precursors migrating from the dorsomedial lip of the dermomyotome. These myogenic cells differentiate in a cranial to caudal sequence with the muscle fibres also orientated in a cranial to caudal direction.

Here we use immunohistological staining both in whole mount and in transverse sections of chick embryos from various stages of development. Antibodies to the myosin heavy chain or tenascin were used to visualise the development of the epaxial muscles and tendons.

The first myotomal muscle fibres are differentiated by Hamburger and Hamilton (H & H) stage 10 in the cranial somites, and differentiation proceeds caudally until at least H & H stage 22–23. Further development of the epaxial muscles does not take place until H & H stage 26–27 with the splitting of the myotome into the individual muscles. We demonstrate how the myotome splits into the individual muscles and how some muscle fibres become reorientated into a more oblique orientation. This delayed development and reorientation of muscle fibres is unique to the epaxial muscles.
The F‐actin cytoskeleton of neurons following microinjection with the small GTPase V14 RhoA
Authors: Bird M. M.
Published: March 2003

A recently refined technique for microinjecting proteins into small cells (Bird et al. J. Anat.198, 2001) has been used to examine changes in the morphology and F‐actin cytoskeleton of cultured spinal cord neurons following microinjection of IgG alone or in conjunction with V14 Rhoa, a small gtpase known to cause changes in the actin cytoskeleton.

Dissociated cell cultures from embryonic chick spinal cord (E7) were plated out onto glass coverslips in Ham’s F12 culture medium, incubated at 37 °C and re‐fed at intervals of 72 h. After 10 d in culture iindividual neurons were microinjected with V14RhoA (1 mg/mL), and/or IgG (to identify injected cells). The cultures were incubated at 37 °C for a further 20 min before fixation in 3.7% formaldehyde. The cells were washed permeabilised with 0.2% Triton X‐100 in PBS and then washed prior to immersion in buffer containing TRITC‐labelled phalloidin and FITC‐labelled anti rat IgG. The cultures were mounted in Mowiol containing a grain of antiquench and viewed on a confocal laser microscope.

Microinjected cells showed spread of injectate throughout the cell body and into the proximal parts of the major dendrites. On the rare occasions when the nucleus was injected IgG did not escape into the cytoplasm. In control cells the F‐actin was concentrated close to the cell membrane in cell bodies and in specific parts of the growth cone. When cells were injected with V14RhoA there was an increase in the number of actin containing microspikes on the cell soma and major dendrites close to the origin of the presumptive axon. Near the substrate increased numbers of podosomes were also observed. More varicosities were found in both axons and dendrites after injection with V14RhoA.

The changes in the F‐actin cytoskeleton after V14RhoA injections were observed within 30 min of the injections. Although some of the changes were similar to those observed in fibroblasts others, such as the presence of varicosities, were unique to neurons.
Astrocyte–endothelial interactions and blood–brain barrier permeability*
Authors: Abbott N. Joan
Published: March 2003

The blood–brain barrier (BBB) is formed by brain endothelial cells lining the cerebral microvasculature, and is an important mechanism for protecting the brain from fluctuations in plasma composition, and from circulating agents such as neurotransmitters and xenobiotics capable of disturbing neural function. The barrier also plays an important role in the homeostatic regulation of the brain microenvironment necessary for the stable and co‐ordinated activity of neurones. The BBB phenotype develops under the influence of associated brain cells, especially astrocytic glia, and consists of more complex tight junctions than in other capillary endothelia, and a number of specific transport and enzyme systems which regulate molecular traffic across the endothelial cells. Transporters characteristic of the BBB phenotype include both uptake mechanisms (e.g. GLUT‐1 glucose carrier, L1 amino acid transporter) and efflux transporters (e.g. P‐glycoprotein). In addition to a role in long‐term barrier induction and maintenance, astrocytes and other cells can release chemical factors that modulate endothelial permeability over a time‐scale of seconds to minutes. Cell culture models, both primary and cell lines, have been used to investigate aspects of barrier induction and modulation. Conditioned medium taken from growing glial cells can reproduce some of the inductive effects, evidence for involvement of diffusible factors. However, for some features of endothelial differentiation and induction, the extracellular matrix plays an important role. Several candidate molecules have been identified, capable of mimicking aspects of glial‐mediated barrier induction of brain endothelium; these include TGFβ, GDNF, bFGF, IL‐6 and steroids. In addition, factors secreted by brain endothelial cells including leukaemia inhibitory factor (LIF) have been shown to induce astrocytic differentiation. Thus endothelium and astrocytes are involved in two‐way induction. Short‐term modulation of brain endothelial permeability has been shown for a number of small chemical mediators produced by astrocytes and other nearby cell types. It is clear that endothelial cells are involved in both long‐ and short‐term chemical communication with neighbouring cells, with the perivascular end feet of astrocytes being of particular importance. The role of barrier induction and modulation in normal physiology and in pathology is discussed.
Quantifying temporal bone morphology of great apes and humans: an approach using geometric morphometrics
Authors: Lockwood Charles A. , Lynch John M. , Kimbel William H.
Published: March 2003

The hominid temporal bone offers a complex array of morphology that is linked to several different functional systems. Its frequent preservation in the fossil record gives the temporal bone added significance in the study of human evolution, but its morphology has proven difficult to quantify. In this study we use techniques of 3D geometric morphometrics to quantify differences among humans and great apes and discuss the results in a phylogenetic context. Twenty‐three landmarks on the ectocranial surface of the temporal bone provide a high level of anatomical detail. Generalized Procrustes analysis (GPA) is used to register (adjust for position, orientation and scale) landmark data from 405 adults representing Homo, Pan, Gorilla and Pongo. Principal components analysis of residuals from the GPA shows that the major source of variation is between humans and apes. Human characteristics such as a coronally orientated petrous axis, a deep mandibular fossa, a projecting mastoid process, and reduced lateral extension of the tympanic element strongly impact the analysis. In phenetic cluster analyses, gorillas and orangutans group together with respect to chimpanzees, and all apes group together with respect to humans. Thus, the analysis contradicts depictions of African apes as a single morphotype. Gorillas and orangutans lack the extensive preglenoid surface of chimpanzees, and their mastoid processes are less medially inflected. These and other characters shared by gorillas and orangutans are probably primitive for the African hominid clade.
Sonic hedgehog: restricted expression and limb dysmorphologies
Authors: Hill Robert E. , Heaney Simon J. H. , Lettice Laura A.
Published: March 2003

Sonic hedgehog, SHH, is required for patterning the limb. The array of skeletal elements that compose the hands and feet, and the ordered arrangement of these bones to form the pattern of fingers and toes are dependent on SHH. The mechanism of action of SHH in the limb is not fully understood; however, an aspect that appears to be important is the localized, asymmetric expression of Shh. Shh is expressed in the posterior margin of the limb bud in a region defined as the zone of polarizing activity (ZPA). Analysis of mouse mutants which have polydactyly (extra toes) shows that asymmetric expression of Shh is lost due to the appearance of an ectopic domain of expression in the anterior limb margin. One such polydactylous mouse mutant, sasquatch (Ssq), maps to the corresponding chromosomal region of the human condition pre‐axial polydactyly (PPD) and thus represents a model for this condition. The mutation responsible for Ssq is located 1 Mb away from the Shh gene; however, the mutation disrupts a long‐range cis‐acting regulator of Shh expression. By inference, human pre‐axial polydactyly results from a similar disruption of Shh expression. Other human congenital abnormalities also map near the pre‐axial polydactyly locus, suggesting a major chromosomal region for limb dysmorphologies. The distinct phenotypes range from loss of all bones of the hands and feet to syndactyly of the soft tissue and fusion of the digits. We discuss the role played by Shh expression in mouse mutant phenotypes and the human limb dysmorphologies.
Copper‐binding proteins in ectomycorrhizal fungi
Authors: HOWE REBECCA , EVANS R. L. , KETTERIDGE S. W.
Published: March 2003

The presence of copper‐binding proteins produced in response to added copper was examined in isolates of Laccaria laccata (Scop, ex Fr.) Cooke and Paxillus involutus (Batsch ex Fr.) Fr. taken from copper‐contaminated and uncontaminated sites, and in a single isolate of Scleroderma citrinum Pers. from a contaminated site. Two isolates of Laccaria (GLac4 and ELacl) grew better in 1–5 miu and 2–5 niM copper than a third (Lac3G) and were considered to be more tolerant. Amongst five isolates of P. involutus, three (WJPax2R, GPaxRSp2 and Pax4) were capable of growth in media containing 4–0 mM copper and were regarded as tolerant. All isolates of both Laccaria and Paxillus were capable of some growth in 2–5 mM copper, but S. citrinum was much more copper‐sensitive and the concentration had to be reduced at least 10‐fold before any growth occurred. Tolerance of isolates was not related to whether they were taken from copper‐contaminated or uncontaminated sites. Copper‐binding proteins were detected in response to copper in the culture media in the two tolerant isolates of Laccaria (GLac4 and ELacl) but not in the least tolerant isolate. In Paxillus, similar proteins were found in two tolerant isolates (GPaxRSp2 and Pax4) but not in WJPax2R, which was also regarded as tolerant, nor in any of the less tolerant isolates. Copper‐binding proteins were not detected in S. citrinum. The copper‐binding protein purified from the Laccaria isolate ELacl appeared as a single band in modified SDS‐PAGE electrophoresis. Its molecular mass and spectral characteristics were consistent with it being a metallothionein.
Effects of ambient ozone on injury and yield of Phaseolus vulgaris at four rural sites in the Netherlands as assessed by using ethylenediurea (EDU)
Authors: TONNEIJCK A. E. G. , VAN DIJK C. J.
Published: March 2003

To assess adverse effects of ambient ozone on injury and yield, EDU (ethylenediurea) and non‐EDU‐treated plants of bean (Phaseolus vulgaris L. cv. Lit) were exposed to ambient air at four rural sites in the Netherlands in 1994. After 6 wk of exposure, the proportion of leaves injured in non‐EDU‐treated plants was higher than that in plants treated with the antioxidant. A maximal degree of 35 % injury was observed. The highest ozone levels accumulated at each site for three consecutive days before injury clearly exceeded the proposed short‐term critical level for injury development. Both injury in untreated plants and the protective effect of EDU differed between sites and were not related to the measured ozone levels. EDU increased the d. wt of dry (mature) pods after 11 wk of exposure but not of green (marketable) pods after 6 wk. The effect of EDU in enhancing dry pod yield did not differ between sites and was 20% on average. The proportional yield increases in EDU‐treated plants compared with those of non‐EDU‐treated plants at the sites were not correlated with the measured ozone levels known to adversely affect bean yield. The available evidence, however, suggests that ambient ozone was involved in the observed yield increase by EDU. When the dry pods were harvested, EDU‐treated plants had more leaves than non‐EDU‐treated ones. The effect of EDU in delaying senescence appears to be an important factor to consider when attempting to evaluate the effect of ambient ozone on pod weight in beans using the EDU approach.
Effects of soil compaction on plant growth phosphorus uptake and morphological characteristics of vesicular—arbuscular mycorrhizal colonization of Trifolium subterraneum
Authors: NADIAN H. , SMITH S. E. , ALSTON A. M. , MURRAY R. S.
Published: March 2003

We investigated the effect of soil compaction and phosphorus (P) application on morphological characteristics of mycorrhizal colonization and growth responses, to determine the reasons for reduced responses observed in our previous work with compacted soil. Growth, phosphorus (P) uptake and intensity of vesicular–arbuscular (VA) mycorrhizal colonization were studied in clover plants (Trifolium subterraneum L.) with and without VA mycorrhizal colonization at two P applications and three levels of soil compaction. Phosphorus was supplied either at constant mass concentration (mg P kg−1 soil) or at constant volume concentration (mg P dm−3 soil). Increasing bulk density of the soil from 1·1 to 1.6 Mg m−3 significantly decreased root length and shoot d. wt, but increased the diameter of both main axes and first order lateral roots regardless of P application. Total P uptake and shoot d. wt of clover plants colonized by Glomus intraradices (Schenck & Smith) were significantly greater than those of non‐mycorrhizal plants at all levels of soil compaction and both P applications. However, soil compaction to a bulk density of 1.6 Mg m−3 (penetrometer resistance = 3.5 MPa at a matric potential of − 33 kPa) significantly decreased mycorrhizal growth response. There was no evidence that the increased volume concentration of P at high bulk densities was responsible for the reduced responses. Soil compaction had no significant effect on the fraction of root length containing arbuscules and vesicles, but total root length colonized by arbuscules, vesicles or by any combination of arbuscules, vesicles and intra‐radical hyphae significantly decreased as soil compaction was increased. The air‐filled porosity of highly compacted soil, which varied from 0.07 to 0.11 over the range of matric potentials encountered (− 33 and − 100 kPa), had no significant effect on the intensity of internal colonization.
Topological scaling and plant root system architecture: developmental and functional hierarchies
Authors: BERNTSON GLENN M.
Published: March 2003

Topology is an important component of the architecture of whole root systems. Unfortunately, most commonly applied indices used for characterizing topology are poorly correlated with one another and thus reflect different aspects of topology. In order to understand better how different methods of characterizing topology vary, this paper presents an exploration of several different methods for assigning order within branched root systems on the basis of (a) developmental (centrifugal) vs. functional (centripetal) ordering sequences and (b) whether orders are assigned to individual links or groups of adjacent links (segments). For each ordering system, patterns of scaling in relation to various aspects of link and segment size are explored using regression analyses. Segment‐based ordering systems resulted in better fits for simple scaling relationships with size, but these patterns varied between developmental vs. functional ordering as well as the different size metrics examined. The functional (centripetal), link‐based ordering system showed complex, non‐linear scaling in relation to numbers of links per order. Using a simple simulation model of root growth, it is demonstrated that this method of characterizing root topology in relation to root size might be a more powerful tool for characterizing root system architecture than in the use of simple, single‐index characterizations of topology.
Effects of ozone and drought stress on the physiology and growth of two clones of Norway spruce (Picea abies)
Authors: KARLSSON P. E. , MEDIN E. L. , WALLIN G. , SELLDÉN G. , SKÄRBY L.
Published: March 2003

Cuttings of two clones of Norway spruce,Picea abies(L.) Karst., were exposed to three different ozone concentrations in open‐top chambers during four months in the summer of 1990. The treatments were charcoal‐filtered air, non‐filtered air and non‐filtered air with extra ozone(c.30 ppb) added daily between 1100–1800 local time. During the last 4 wk of the exposure period, half the seedlings were drought‐stressed, while the remainder were well watered. Biomass, gas exchange and water potential were measured during the drought stress period. The ozone treatments affected the two clones very differently. High ozone reduced the rate of biomass increase in the faster‐growing clone (clone M) whereas ozone generally stimulated the rate of biomass increase in the slower‐growing clone (clone L). At the end of the measuring period, the high‐ozone treatments reduced the rate of root biomass increase of the well watered seedlings of the M‐clone, but it had no effect on the drought‐stressed seedlings of the same clone, probably because the root growth was already to a large extent inhibited by the drought stress.

The treatment with the highest concentration of ozone partly protected the seedlings of the M clone against the drought stress, presumably by delaying shoot growth and thus delaying the increase in the total transpiring needle area. As a result, stomata tended to close less during the drought period in this treatment.

The results are discussed in relation to the suggested critical level for ozone effects on trees.
Inter‐ and intraspecific variation in the ITS region of rDNA of ectomycorrhizal fungi in Fennoscandia as detected by endonuclease analysis
Authors: KÅRÉN OLA , HÖGBERG NILS , DAHLBERG ANDERS , JONSSON LENA , NYLUND JAN‐ERIK
Published: March 2003

Interspecific and intraspecific variation in the internal transcribed spacer (ITS) region of rDNA of ectomycorrhizal fungi of 44 species in 17 genera were examined using the restriction fragment length polymorphism (RFLP) method. For each species, two to five herbarium vouchers (mainly basidiocarps), collected throughout Fennoscandia, were examined. The ITS region was amplified using the polymerase chain reaction (PCR) and the universal primers ITS1 and ITS4, and subjected to RFLP analysis with three endonucleases. Intraspecific polymorphisms in the ITS region were found in seven species (in nine of the 132 herbarium vouchers). Polymorphisms were due to length mutations, ranging from 5 to 15 bp in four of the seven polymorphic species and mutations in endonuclease restriction sites in six species, mostly affecting only one endonuclease, but in two species two endonucleases. Using a single endonuclease, a unique RFLP pattern could be obtained for more than half the investigated species. By combining different endonucleases, 34 (77%) of the species could be distinguished from another. The remaining RFLP types occurred in one genus. On the basis of the low intra‐ but high interspecific variation in the ITS region, it is concluded that most ectomycorrhizas formed by the 44 investigated species should be recognized by comparison with this dataset, if the mycorrhizas are sampled from a site located in Fennoscandia. However, in datasets from even larger geographical areas encompassing a higher degree of intraspecific variation in the ITS region, or when mycorrhizas from several sites distant from each other are compared, it might be necessary to include local reference species.
Changes in shikimate dehydrogenase and the end products of the shikimate pathway, chlorogenic acid and lignins, during the early development of seedlings of Capsicum annuum
Authors: DÍAZ JOSÉ , BARCELÓ A. ROS , DE CÁCERES F. MERINO
Published: March 2003

The developmental regulation and organ‐specific expression of shikimate dehydrogenase was studied in seedlings of Capsicum annuum L. during their early development. The results obtained suggest that there is both a developmental regulation and an organ‐specific expression of shikimate dehydrogenase in seedlings of C. annuum, seen mainly in the cotyledons. For example, shikimate dehydrogenase isoenzyme 3 was differentially expressed in cotyledons, but not at all in roots or hypocotyls. When the amounts of shikimate dehydrogenase were compared with the end‐products of the shikimate pathway, chlorogenic acid and lignins, it was observed that the fall in shikimate dehydrogenase activity in cotyledons coincided with a decrease in the concentrations of phenolics during primary leaf development. However, this did not seem to be the case with the hypocotyls, where the fall in phenolics was not related to the amount of shikimate dehydrogenase but rather to an increase in lignin deposition, supporting the view that chlorogenic acid might act as a precursor of the aromatic moiety of cinnamoyl alcohols during cell wall lignification.
Variations of the natural abundances of nitrogen and carbon isotopes in Triticum aestivum, with special reference to phloem and xylem exudates
Authors: YONEYAMA T. , HANDLEY L. L. , SCRIMGEOUR C.M. , FISHER D. B. , RAVEN J. A.
Published: March 2003

summary

This work explored whether the natural abundances of carbon and nitrogen isotopes could be used to describe the movement of C and X within wheat plants; we also considered whether isotopic analyses of aphids or their honeydew would substitute for direct analysis of phloem exudate.

The δ13C of ears and roots (sinks) most closely matched those of the sugars + organic acids fraction (sources) in both growth stages; phloem δ13C matched that of leaf blade sugars. Xylem exudate δ13C matched no other putative (and measured) source in the ear‐forming stage and matched that of whole roots and ears in the grain‐filling stage. The δ15N of grain and roots (sinks) resembled that of leaf amino acids (sources) in the ear‐forming stage. In the gram‐filling stage, ear δ15N continued to resemble that of leaf amino acids, and δ15N of roots most closely resembled that of whole leaves. In the grain‐filling stage, phloem δ15N fell between that of leaf blade amino acids and that of whole leaves and was 15X‐depleted relative to internal and external NO, ‐N. In both growth stages, xylem exudate δ15N was less than that of soil NO3−‐N and more than that of residual soil N after mineral N extraction. The isotopic values are generally in agreement with data from other approaches, such as isotope labelling; they show NO3−‐N reduction in both shoots and roots of wheat and significant N recycling (root‐shoot‐phloem‐root) and C movement.

Aphids might serve as a substitute for isotopic analysis of phloem δ15N. having the same value as their food source. Their excreta was 15N‐enriched relative to phloem.
Correlating molecular markers with physiological expression in Hordeum, a developing approach using stable isotopes
Authors: HANDLEY L. L. , ROBINSON D. , SCRIMGEOUR C. M. , GORDON D. , FORSTER B. P. , ELLIS R. P. , NEVO E.
Published: March 2003

summary

This paper describes a developing approach for correlating molecular markers with phenotypic expression in barley, and research examples are given. This approach is intended to elucidate the connection between denned parts of the barley genome and their physiological expressions. To illustrate our approach (up to the point of marker correlation), the materials, methods and results of an experiment are presented. Briefly, the natural abundance levels of carbon and nitrogen isotopes (δ13C δ15and N) serve as the primary screening tools for assessing barley genotypes for their responses to imposed abiotic stresses. The isotopic responses are then correlated with molecular markers as the first step in identifying the mechanisms relating genetic variation with phenotypic expression.

A well‐described mechanistic model exists for interpreting plant δ13C, and this enables partitioning of the physiological processes contributing to the whole plant δ13C. Although plant δ15N appears to be equally well correlated with genotype and environment, there is no mechanistic model explaining this isotopic signature. Hence, δ15N is used empirically and in parallel with attempts to understand its underlying mechanism(s).
Dehydrins: genes, proteins, and associations with phenotypic traits
Authors: CAMPBELL SCOTT A. , CLOSE TIMOTHY J.
Published: March 2003

summary

Dehydrin proteins (late embryogenesis abundant (LEA) D11 family) are produced in a wide variety of plant species in response to environmental stimuli with a dehydrative component, including drought, low temperature, salinity, and developmental stages such as seed and pollen maturation. Despite their widespread occurrence and abundance in cells under dehydrative conditions, the biochemical role of dehydrins remains elusive. The subcellular location of dehydrins is consistent with a biochemical role as an intracellular stabilizer, possibly with surfactant characteristics, acting upon targets in both the nucleus and cytoplasm. In some species, dehydrin loci are located within quantitative trait loci (QTL) intervals for important phenotypic traits including winter hardiness in barley (Hordeum vulgare L.) and anthesis–silking interval in maize (Zea mays L.). Dehydrin loci tend to be multigenic and occur in clusters on more than one chromosome. Investigations are currently under way in our laboratory and others' to move beyond protein accumulation studies and correlations with QTL to uncover direct cause–and–effect relationships between dehydrin (dhn) genes and phenotypes associated with physiological responses to stress.
Potential NH4+ and NO3− uptake in seven Sphagnum species
Authors: JAUHIAINEN J. , WALLÉN B. , MALMER N.
Published: March 2003

The rate of nitrogen uptake by seven Sphagnum species, which from a gradient from hummock to hollow and from ombrotrophic to minerotrophic conditions, was measured as the decrease in the concentrations of NH4+ and NO3− from solutions in which capitula were grown under laboratory conditions.

The highest uptake rate was by individuals of each species with large capitula and a high number of ion exchange sites, i.e. lawn species (S. pulchrum, S. fallax, S. papillosum and S. magellanicum). On a dry‐mass basis, the most effective species were the hummock species (S. fuscum and S. rubellum), even though these species have a low dry mass. Hummock species, which occur in high densities and have high potential N‐uptake rates on a dry‐mass basis, were the most effective species in retaining available nitrogen.
Developmental and morphological responses of mycelial systems of Stropharia caerulea and Phanerochaete velutina to soil nutrient enrichment
Authors: DONNELLY DAMIAN P. , BODDY LYNNE
Published: March 2003

Patterns produced by foraging mycelial systems of decomposer fungi might be the visual evidence of strategies for obtaining differently distributed carbon sources. Mycelial systems might also be responsive to unevenly distributed mineral nutrients in the soil. The nature of the mycelial response might differ between species inhabiting eutrophicated disturbed sites, such as Stropharia caerulea (Kriesel), and inhabitants of more typical woodland soils, such as Phanerochaete velutina DC.: (Pers.) Parmasto. We examined the effect of enrichment of soil by addition of different concentrations of N, P and potassium on inoculum decay rate and on extension rate, biomass production and fractal dimension (determined by image analysis) of extra‐resource mycelia of S. caerulea and P. velutina extending from wood blocks across compacted non‐sterile soil in trays.

S. caerulea was more responsive than P. velutina to both type and quantity of soil nutrient enrichment. P. velutina grew more rapidly, produced more biomass and decayed inocula more rapidly than S. caerulea under most conditions, rates of growth and resource decay of the former remaining stable over a range of soil nutrient enrichments. In contrast, morphology of S. caerulea altered on enriched soils compared to controls with greater and more pronounced fanning (divergence of separate hyphae) of the foraging mycelial front. These changes were reflected in fractal values. Extension rate and biomass production of S. caerulea was less than controls on N‐amended soil, but greater on P‐amended soils, with the greatest biomass produced on 0·13–0·27 mg P g−1 air‐dried soil. Decay rate of inocula was often positively related with mycelial production rates. These responses indicate extra‐resource mycelium of S. caerulea is able to respond rapidly to a range of soil nutrient enrichments, and this is discussed in terms of the different foraging strategies exhibited by the two fungi.