Effects of arachidonic acid and its cyclo-oxygenase and lipoxygenase products on lymphatic vessel contracility in vitro

Lymphatic vessels exhibit rhythmical contractility $\text{in vivo}$ and $\text{in vitro}$ and this activity appears ti regulate lymph flow. A technique for measuring the cicurlar muscle contractions of isolated bovine mesenteric lymphatic vessel segments has been devised and utilized to study the pharmacological properties of these vessels. Non-contracting lympahtic vessels can be induced to contract rhythmically with a variety of mediators, the most potent being a stable PGH₂ analogue (compound U46619), and the leukotrienes B₄, C₄ and D₄ (threshold concentrations in the nanomolar range). Prostagladin F_2α, noradrenaline, serotonin and histamine also elicited rhythmical activity but much higher concentrations were required. PGE₂ and PGE₁ were potent inhibitors of spontaneous contractions or those induced with U46619. In keeping with the diverse pharmacological effects of the metabolites of arachidonic acid, the addition of arachidonate to an isolated lymphatic vessel generated both stimulatory and inhibitory activities.It is concluded that arachidonic acid products (produced in the lymphatic vessel or entering the vessel in lymph draining the tissues) regulate lymph flow through their effects on lymphatic smooth muscle.     

Sphingosine 1-Phosphate Receptor Signaling Regulates Proper Embryonic Vascular Patterning

Sphingosine 1-phosphate (S1P) binds G-protein-coupled receptors (S1P_1–5) to regulate a multitude of physiological effects, especially those in the vascular and immune systems. S1P receptors in the vascular system have been characterized primarily in mammals. Here, we report that the S1P receptors and metabolic enzymes are conserved in the genome of zebrafish Danio rerio. Bioinformatic analysis identified seven S1P receptor-like sequences in the zebrafish genome, including duplicated orthologs of receptors 3 and 5. Sphingolipidomic analysis detected erythrocyte and plasma S1P as well as high plasma ceramides and sphingosine. Morpholino-mediated knockdown of s1pr1 causes global and pericardial edema, loss of blood circulation, and vascular defects characterized by both reduced vascularization in intersegmental vessels, decreased proliferation of intersegmental and axial vessels, and hypersprouting in the caudal vein plexus. The s1pr2 gene was previously characterized as a regulator of cell migration and heart development, but its role in angiogenesis is not known. However, when expression of both s1pr1 and s1pr2 is suppressed, severely reduced vascular development of the intersegmental vessels was observed with doses of the s1pr1 morpholino that alone did not cause any discernible vascular defects, suggesting that s1pr1 and s1pr2 function cooperatively to regulate vascular development in zebrafish. Similarly, the S1P transporter, spns2, also cooperated with s1pr1. We propose that extracellular S1P acts through vascular S1P receptors to regulate vascular development.     

Distinct signalling pathways regulate sprouting angiogenesis from the dorsal aorta and the axial vein

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is critical to most physiological processes and many pathological conditions. During zebrafish development, angiogenesis expands the axial vessels into a complex vascular network that is necessary for efficient oxygen delivery. Although the dorsal aorta and the axial vein are spatially juxtaposed, the initial angiogenic sprouts from these vessels extend in opposite directions, indicating that distinct cues may regulate angiogenesis of the axial vessels. We found that angiogenic sprouts from the dorsal aorta are dependent on vascular endothelial growth factor A (Vegf-A) signalling, and do not respond to bone morphogenetic protein (Bmp) signals. In contrast, sprouts from the axial vein are regulated by Bmp signalling independently of Vegf-A signals, indicating that Bmp is a vein-specific angiogenic cue during early vascular development. Our results support a paradigm whereby different signals regulate distinct programmes of sprouting angiogenesis from the axial vein and dorsal aorta, and indicate that signalling heterogeneity contributes to the complexity of vascular networks.     

Characterization and ontogeny of PGE2 and PGF2α receptors on the retinal vasculature of the pig

The vasoconstrictor effects of PGE₂ and PGF_2α are less pronounced on retinal vessels of the newborn than of the adult pig. We tested the hypothesis that the decreased vasomotor response to these prostaglandins might be due to relatively fewer receptors and/or different receptor subtypes (in the case of PGE₂) on retinal vessels of the newborn animal. Binding studies using [³H]PGE₂ and [³H]PGF_2α revealed that PGE₂ (EP) and PGF_2α (FP) receptor densities in retinal microvessel membrane preparations from newborn animals were approximately 25% of those found in vessels from the adult. The K_d for PGF_2α did not differ; however, the K_d for PGE₂ was less in newborn than in adult vessels. Competition binding studies using AH 6809 (EP₁ antagonist), butaprost (EP₂ agonist), M&B 28,767 (EP₃ agonist), and AH 23848B (EP₄ antagonist) suggested that the retinal vessels of the newborn contained approximately equal number of EP₁ and EP₂ receptor subtypes whereas the main receptor subtype in the adult vessels was EP₁. In addition, PGE₂ and butaprost produced comparable increases in adenosine 3′,5′-cyclic monophosphate synthesis in newborn and adult vessels. PGE₂, 17-phenyl trinor PGE₂ (EP₁agonist) and PGF_2α caused a 2.5 to 3-fold greater increase in inositol1,4,5-triphosphate (IP₃) formation in adult than in newborn preparations. It is concluded that fewer PGF_2α receptors and an associated decrease in receptor-coupled IP₃ formation in the retinal vessels of the newborn could lead to weaker vasoconstrictor effects of PGF_2α on retinal vessels of the newborn than of adult pigs; fewer EP₁ receptors (associated with vasoconstriction) and a relatively greater proportion of EP₂ receptors (associated with vasodilation) might be responsible for the reduced retinal vasoconstrictor effects of PGE₂ in the newborn.     

Immunolocalization of hemicelluloses in Arabidopsis thaliana stem. Part II: Mannan deposition is regulated by phase of development and its patterns of temporal and spatial distribution differ between cell types

Microdistribution of mannans in Arabidopsis stem was examined using immunolocalization with mannan-specific monoclonal antibodies (LM21 and LM22). Mannan labeling in secondary xylem cells (except for protoxylem vessels) was initially detected in the cell wall during S₂ formation and increased gradually during development. Labeling in metaxylem vessels (vessels) was detected earlier than that in xylary fibers (fibers), but was much weaker than fibers. The S₁ layer of vessels and fibers showed much less labeling than the S₂ layer. Some strong labeling was also detected in pit membranes of vessel pits. Interfascicular fibers (If-fibers) showed more heterogeneous labeling patterns than fibers by LM21. Unlike fibers, If-fibers also revealed some strong labeling in the cell corner of the S₁ layer, indicating different mannan labeling patterns between If-fibers and fibers. Interestingly, protoxylem vessels (proto-vessels) showed strong labeling at the early stage of secondary xylem formation with more intense labeling in the outer- than inner cell wall even though fibers and vessels showed no or very low labeling at this stage. Labeling intensity of proto-vessels was also much stronger than vessels and stronger or slightly weaker than fibers by LM21 and LM22, respectively. Using pectinase and mild alkali treatment, the presence of mannans in parenchymatous cells was also confirmed. Together our observations indicate that there are temporal and spatial variations in mannan labeling between cell types in the secondary xylem of Arabidopsis stems. Some similar features of mannan labeling between Arabidopsis and poplar are also discussed.     

The use of multiple-vessel,open flow systems to investigate carbon flow in anaerobic microbial communities

Five vessels, connected in series, were used for a continuous flow system to model carbon flow in anaerobic microbial communities. Two such 5-vessel systems were constructed, the inflows containing 10 mM sulfate and either 10 mM glucose or benzoate. Dilution was slow (D=0.0018 h^?1 for the whole system). Analyses of dissolved organic and inorganic carbon, and of CO₂ and CH₄, showed that the systems attained steady states in which biomass was constant, although there was net biosynthesis in the early vessels and net mineralization in succeeding vessels. Examination of the distributions of sulfate reduction, methanogenesis, and of H₂+CO₂-utilizing fatty acid-forming bacteria revealed spatial separation of these functional groups of bacteria in different vessels of the array, resembling the vertical spatial separation found in many natural sediments. Such model systems should, therefore, prove valuable in investigating the many microbial activities that contribute to the flow of carbon in anaerobic microbial communities.     

Antigen stimulation of human pulmonary smooth muscle: An in vitro model of inflammation

Human airways in vitro contract when stimulated by anti-IgE, whereas human pulmonary vessels relax. Leukotriene D₄ (LTD₄) induced a contractile response in the airways, while in pulmonary vessels both contractions and relaxations were observed. The LTD₄ contractions in airways were blocked by cysLT₁ receptor antagonists (MK 571, ICI 198615, and BAY x7195). In contrast none of the compounds affected the LTD₄ contractions of pulmonary veins. These results suggest that the leukotrienes which are released during antigen challenge of airways and pulmonary vessels may be acting at distinct receptors in the human lung.Abbreviations NE noradrenaline - LT leukotriene     

Influence of carbon dioxide on the impregnation of the nervous tissue by silver

It has been shown that 4% carbon dioxide (CO₂) in the air above reaction mixture inhibits the initiation of the formation of silver nanoparticles from complexes with biogenic amines (noradrenaline and serotonin). At the same concentration of CO₂ in the air above solution of AgNO₃, which is used for staining nerve tissues by the method of Golgi, neurons are preferentially stained, whereas at a concentration of 0.06%, vessels are stained. It is suggested that the entry of free silver ions to neurons is due to the inhibition of sites of initiation of silver nanoparticles in vessels at high CO₂ concentrations, while the lack of inhibition leads to silver precipitation in vessels at low CO₂ concentrations. It can be assumed that, for stable silver impregnation, the concentration of CO₂ must be controlled.     

Signal transduction and protein phosphorylation in smooth muscle contraction

Smooth muscles are important constituents of vertebrate organisms that provide for contractile activity of internal organs and blood vessels. Basic molecular mechanism of both smooth and striated muscle contractility is the force-producing ATP-dependent interaction of the major contractile proteins, actin and myosin II molecular motor, activated upon elevation of the free intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, whereas striated muscles display a proportionality of generated force to the [Ca²⁺]_i level, smooth muscles feature molecular mechanisms that modulate sensitivity of contractile machinery to [Ca²⁺]_i. Phosphorylation of proteins that regulate functional activity of actomyosin plays an essential role in these modulatory mechanisms. This provides an ability for smooth muscle to contract and maintain tension within a broad range of [Ca²⁺]_i and with a low energy cost, unavailable to a striated muscle. Detailed exploration of these mechanisms is required to understand the molecular organization and functioning of vertebrate contractile systems and for development of novel advances for treating cardiovascular and many other disorders. This review summarizes the currently known and hypothetical mechanisms involved in regulation of smooth muscle Ca²⁺-sensitivity with a special reference to phosphorylation of regulatory proteins of the contractile machinery as a means to modulate their activity.     

Xylem parenchyma cells deliver the H<Subscript>2</Subscript>O<Subscript>2</Subscript> necessary for lignification in differentiating xylem vessels

Lignification in Zinnia elegans L. stems is characterized by a burst in the production of H₂O₂, the apparent fate of which is to be used by xylem peroxidases for the polymerization of p-hydroxycinnamyl alcohols into lignins. A search for the sites of H₂O₂ production in the differentiating xylem of Z. elegans stems by the simultaneous use of optical (bright field, polarized light and epi-polarization) and electron-microscope tools revealed that H₂O₂ is produced on the outer-face of the plasma membrane of both differentiating (living) thin-walled xylem cells and particular (non-lignifying) xylem parenchyma cells. From the production sites it diffuses to the differentiating (secondary cell wall-forming) and differentiated lignifying xylem vessels. H₂O₂ diffusion occurs mainly through the continuous cell wall space. Both the experimental data and the theoretical calculations suggest that H₂O₂ diffusion from the sites of production might not limit the rate of xylem cell wall lignification. It can be concluded that H₂O₂ is produced at the plasma membrane in differentiating (living) thin-walled xylem cells and xylem parenchyma cells associated to xylem vessels, and that it diffuses to adjacent secondary lignifying xylem vessels. The results strongly indicate that non-lignifying xylem parenchyma cells are the source of the H₂O₂ necessary for the polymerization of cinnamyl alcohols in the secondary cell wall of lignifying xylem vessels.     

Detection of 15-hydroxyprostaglandin dehydrogenase in bovine mesenteric blood vessels

Two types of 15-hydroxyprostaglandin dehydrogenase (NAD⁺ and NADP⁺ dependent) were demonstrated in bovine mesentric arteries and veins. The 15-hydroxyprostaglandin dehydrogenase activity was found in the high-speed supernatant, suggesting that these enzymes are associated with the cytoplasmic fraction of the blood vessels. The levels of activities of both NAD⁺- and NADP⁺-dependent dehydrogenases were similar in mesentric blood vessels. Prostaglandin F_2α was preferred to the prostaglandin E₂ as subtrate by both NAD⁺ and NADP⁺ dependent enzymes. The presence of 15-hydroxyprostaglandin dehydrogenase in blood vessels may play a siginificant role in the regulation of intracellular levels of prostaglandins of the E and F series in blood vessels.     

Roles of Rho GTPases in leucocyte and leukaemia cell transendothelial migration

Leucocytes migrate into and out of blood vessels at multiple points during their development and maturation, and during immune surveillance. In response to tissue damage and infection, they are rapidly recruited through the endothelium lining blood vessels into the tissues. Leukaemia cells also move in and out of the bloodstream during leukaemia progression. Rho GTPases are intracellular signalling proteins that regulate cytoskeletal dynamics and are key coordinators of cell migration. Here, we describe how different members of the Rho GTPase family act in leucocytes and leukaemia cells to regulate steps of transendothelial migration. We discuss how inhibitors of Rho signalling could be used to reduce leucocyte or leukaemia cell entry into tissues.     

Water relations of Robinia pseudoacacia L.: do vessels cavitate and refill diurnally or are R‐shaped curves invalid in Robinia?

Since 2005, an unresolved debate has questioned whether R‐shaped vulnerability curves (VCs) might be an artefact of the centrifuge method of measuring VCs. VCs with R‐shape show loss of stem conductivity from approximately zero tension, and if true, this suggests that some plants either refill embolized vessels every night or function well with a high percentage of vessels permanently embolized. The R‐shaped curves occur more in species with vessels greater than half the length of the segments spun in a centrifuge. Many have hypothesized that the embolism is seeded by agents (bubbles or particles) entering the stem end and travelling towards the axis of rotation in long vessels, causing premature cavitation. VCs were measured on Robinia pseudoacacia L. by three different techniques to yield three different VCs; R‐shaped: Cavitron P₅₀ = 0.30 MPa and S‐shaped: air injection P₅₀ = 1.48 MPa and bench top dehydration P₅₀ = 3.57 MPa. Stem conductivity measured in the Cavitron was unstable and is a function of vessel length when measured repeatedly with constant tension, and this observation is discussed in terms of stability of air bubbles drawn into cut‐open vessels during repeated Cavitron measurement of conductivity; hence, R‐shaped curves measured in a Cavitron are probably invalid.     

Differences in nitric oxide production in porcine resistance arteries and epicardial conduit coronary arteries

The purpose of this study was to test the hypothesis that endothelial cells from resistance arteries and epicardial conduit coronary arteries differ in their expression of nitric oxide synthase (NOS) and calcium metabolism, and that these differences contribute to the mechanism underlying disparate physiological vasodilator responses observed between the two populations of vessels. The functional vasodilator responses of isolated resistance arteries and epicardial conduit coronary arteries were compared in vitro using both the receptor-independent agonist A23187 ionophore to increase intracellular calcium and the receptor-dependent agonist bradykinin. Constitutive NOS (cNOS) activity in monocultures of endothelial cells derived from resistance arteries and conduit arteries was assayed using a fibroblast-reporter cell method. Intracellular calcium concentration was assessed using fura-2 microfluorometry. Nitric oxide production was determined using a chemiluminescence technique, while cNOS protein was quantitated by Western blot analysis. A23187 was a less potent vasodilator of resistance arteries studied in vitro, compared to epicardial conduit arteries (EC₅₀ = 1.6 μM, resistance artery vs. EC₅₀ = 0.03 μM, conduit artery); however, bradykinin was more potent in resistance arteries (EC₅₀ = 0.3 nM, resistance artery vs. EC₅₀ = 2 nM, conduit artery). In pure monocultures of endothelium, nitric oxide production measured by chemiluminescence both basally and in response to A23187 was significantly less in resistance arteries (6.1 ± 0.5, basal vs. 10.80 ± 0.55, stimulated nmol/μg protein), compared to conduit arteries (7.7 ± 0.5, basal vs. 17.00 ± 1.52, stimulated nmol/μg protein; P < 0.05 resistance artery endothelium vs. conduit artery endothelium). cNOS enzyme activity assessed by cGMP production in reporter cell fibroblasts was also lower in resistance arteries compared to conduit arteries (0.17 ± 0.03 vs. 0.33 ± 0.05 fmol cGMP/μg protein, respectively; P < 0.05 resistance artery endothelium vs. conduit artery endothelium). Conduit arteries expressed 2.1 × more cNOS protein than resistance arteries, as assessed by Western blotting of cellular homogenates. No significant differences were found with microfluorimetry in either basal or ionophore-stimulated intracellular calcium concentrations. The results signified that porcine resistance arteries expressed less NOS and produced less nitric oxide than epicardial conduit arteries both basally and in response to an increase in intracellular calcium. This difference was reflected functionally as a decreased vasodilatory response to increased intracellular calcium in resistance arteries that could not be explained on the basis of differences in the metabolism of intracellular calcium. In contrast, the functional vasodilator response of intact vessels to a receptor-mediated agonist was enhanced in resistance arteries compared to conduit arteries, suggesting an important role of signal transduction mechanisms in specific physiological responses. Thus, the ability of the endothelium to regulate on a regional basis the expression of NOS and integrate receptor-mediated responses with these differences may provide a mechanism for diverse vasomotor responses in different populations of vessels. © 1996 Wiley-Liss, Inc.     

The Blood Platelet as a Model for Regulating Blood Coagulation on Cell Surfaces and Its Consequences

Platelets actively participate in regulating thrombin production following physical or chemical injury to blood vessels. Injury to blood vessels initiates activation of the large numbers of platelets that appear in the subendothelium where they become exposed to tissue factor and to molecules adhesive for platelets and normally found in the extracellular matrix. The complex of plasma factor VIIa with extravascular tissue factor both initiates and localizes thrombin production on platelets and on extravascular cells. Thrombin production at these sites in turn enhances platelet activation and the subsequent hemostatic plug formation to minimize bleeding. Thrombin production and platelet activation also initiate the process of wound healing requiring thrombin-dependent cell activation and platelet-dependent formation of new blood vessels (angiogenesis). Activated platelets release from their storage granules several proteins and other factors that regulate local thrombin formation and the responses of blood vessel cells to injury to assure hemostasis and effective wound healing. Failure to localize and adequately regulate thrombin production and/or platelet activation can have pathological consequences, including the development and propagation of atherosclerosis and enhancement of tumor development. The primary basis for the pathological consequences of the failure to adequately regulate thrombin production is that the multi-functional thrombin activates several types of cells to initiate their mitogenesis. Mitogenesis precedes many of the undesirable consequences of poorly regulated thrombin production and platelet activation. In addition, activated platelets release a variety of products which influence the functions of several cell types to the extent that inadequate regulation of platelet activation (by excessive thrombin production) could contribute to the pathogenesis of acute and chronic arterial thrombosis and to tumor development. Activated platelets participate in tumor development by releasing several factors that positively (and negatively) regulate blood vessel formation.     

Interaction between pericytes and endothelial cells leads to formation of tight junction in hyaloid vessels

The hyaloid vessel is a transient vascular network that nourishes the lens and the primary vitreous in the early developmental periods. In hyaloid vessels devoid of the support of astrocytes, we demonstrate that tight junction proteins, zonula occludens-1 and occludin, are regularly expressed at the junction of endothelial cells. To figure out the factor influencing the formation of tight junctions in hyaloid vessels, we further progress to investigate the interactions between endothelial cells and pericytes, two representative constituent cells in hyaloid vessels. Interestingly, endothelial cells interact with pericytes in the early postnatal periods and the interaction between two cell types provokes the up-regulation of transforming growth factor β1. Further in vitro experiments demonstrate that transforming growth factor β1 induces the activation of Smad2 and Smad3 and the formation of tight junction proteins. Taken together, in hyaloid vessels, pericytes seem to regulate the formation of tight junctions by the interaction with endothelial cells even without the support of astrocytes. Additionally, we suggest that the hyaloid vessel is a valuable system that can be utilized for the investigation of cell-cell interaction in the formation of tight junctions in developing vasculatures.     

The effects of prostaglandins, prostaglandin inhibitors, and oxygen on the closure of the ductus arteriosus, pulmonary arteries and umbilical vessels in vitro

Three prostaglandins (PGF₂α and PGE₁, PGE₂) have been found in maternal and fetal circulation during labour. Two of these prostaglandins (PGF₂α and PGE₂) are present in elevated levels in maternal circulation during labour and their presence in fetal vessels has been shown.These three prostaglandins have been tested for their effects on fetal vessels in vitro (umbilical artery and vein, ductus arteriosus, and smaller pulmonary artery). These vessels were selected as being crucial in the conversion from fetal to extra-uterine circulation in mammalian species. Responses of these vessels to the prostaglandins under varying oxygen regimes have been examined as well as their responses to prostaglandin inhibitors. Activity of vessels of varying gestational ages exposed to PGF₂α was also examined. The following results were obtained:

1. All vessels, with the exception of pulmonary arteries, contracted in the presence of oxygen over the range 20–100mmHg pO₂. At a pO₂ of < 20mmHg the ductus arteriosus remained inactive or dilated. Pulmonary arteries dilated at high pO₂.

2. All vessels contracted in response to exogenous PGF₂α with the exception of the pulmonary arteries which dilated. In the presence of PGF₂α, the umbilical veins dilated under low (< 20mmHg) pO₂ and contracted at higher levels. Contraction also occurred at lower levels after a period of time.

3. Although PGF₂α was capable of causing contraction in the ductus arteriosus at near zero pO₂, oxygen, (or possibly the products of oxygenation), appear to be required for continued contraction in the presence of PGF₂α. A synergistic relationship between oxygen and PGF₂α responses was found as oxygen tensions increased. A synergistic response between PGF₂α and oxygen with umbilical arteries which did not increase with increased pO₂ was also found. Oxygen tension appeared to have little effect on the response of other vessels to PGF₂α.

4. PGE₁ caused dilations in all vessels examined. Such dilations appearing to be independent of the oxygen regime prevailing. However, an increase in oxygen during experiments reversed any dilation caused by the prostaglandins.

5. PGE₂ caused contractions in umbilical vessels which were independent of oxygen. PGE₂ caused contraction of pulmonary arteries. However, in the ductus arteriosus, PGE₂ caused an initial contraction followed by a strong dilation. This dilation became weaker as pO₂ increased.

6. Additions of prostaglandin inhibitors (Naproxen and Indomethacin) to the bathing solution in which the ductus arteriosus and umbilical arteries were contracting (in response to PGF₂α, or oxygen alone) caused a decrease in contractions, and sometimes a slight decrease when the vessel had been pretreated with PGF₂α suggesting a possible need for endogenously synthesised prostaglandins for the maintenance of oxygen mediated contractions (in vivo).

7. Vessels responsed to PGF₂α at an early gestational age. A role for prostaglandins and oxygen in the closure of fetal vessels is discussed.

     

Evaluation of a closed system,diffusive and humidity-induced convective throughflow ventilation on the growth and physiology of cauliflower in vitro

The effects of ethylene inhibitors (silver nitrate – AgNO₃ and silver thiosulphate – Ag₂S₂O₃ as inhibitors of ethylene activity, cobalt chloride – CoCl₂ as inhibitor of ethylene biosynthesis) and ethylene stimulator (aminocyclopropane-1-carboxylic acid – ACC) were studied on the growth of cauliflower (Brassica oleracea L.) seedlings cultured in closed vessels (60 cm³). The addition of ethylene inhibitors have significant stimulatory effects on the growth and development of seedlings and the effects were greatest with 10 μM AgNO₃, the fresh weight of leaves was 2.6×, and the leaf area 2.8× those of the control (no additives). The effects of various methods of ventilation (humidity-induced convective through-flow ventilation, diffusive ventilation and sealed condition) on the growth and physiology of in vitrocauliflower seedlings were also investigated. The seedlings were cultured either in the presence or absence of AgNO₃ (inhibitors of ethylene activity) and ACC (a precursor). Ethylene and CO₂ levels in the head-space of the culture vessels were monitored. The humidity-induced through-flow ventilation system has shown to be effective for improving growth, leaf chlorophyll content and the rate of net photosynthesis and preventing symptoms of hyperhydricity, such as leaf epinasty, and franginess, reduction of leaf area etc. In contrast, the results also indicated that the sealing of culture vessels could have serious inhibitory effects on growth and development, induce hyperhydricity and reduce leaf chlorophyll content. In the light period, CO₂ depletion occurred in the head-space of the sealed vessels (ca. 40 μl l^-1), the CO₂ concentration increased with increasing efficiency of the ventilation. No ethylene accumulation was noticed in the head-space of the culture vessels when humidity-induced throughflow ventilation was applied; however, high ethylene accumulation occurred in sealed vessels. This revised version was published online in June 2006 with corrections to the Cover Date.     

Risso's dolphins alter daily resting pattern in response to whale watching at the Azores

Behavioral responses of Risso's dolphins (Grampus griseus) to whale watching vessels were studied off Pico Island, Azores. Dolphin behavior was studied from a land‐based lookout, enabling observations of groups in the absence and presence of vessels. The number of whale watching vessels showed a clear seasonal pattern, dividing the whale watching period into a low season and a high season. During the low season, Risso's dolphins rested mainly in the morning and afternoon. During the high season, Risso's dolphins rested less and did so mainly at noon, when the number of active vessels was lowest. Data analysis using a generalized additive mixed model indicated that this change in resting behavior was associated with vessel abundance. When more than five vessels were present, Risso's dolphins spent significantly less time resting and socializing. During the high season, this vessel abundance was exceeded during 20% of observation days. While we cannot be sure that the observed changes in behavior have fitness consequences for Risso's dolphins, reduced resting and socializing rates can have negative impacts on the build‐up of energy reserves and on reproductive success. We suggest the adoption of precautionary management measures to regulate the timing and intensity of whale watching activities.     

Effect of heavy metals and temperature on the oxalate oxidase activity and lignification of metaxylem vessels in barley roots

The effect of Cd on oxalate oxidase (OxO) activity and its localisation were analysed in barley root. In Cd-treated roots OxO activity was strongly induced in the region 2–4 mm behind the root tip and in the area toward the root base. In situ analyses showed that Cd-induced OxO activity was localised to the cell wall (CW) of early metaxylem vascular bundles and surrounding parenchyma cells and was accompanied by lignification of metaxylem vessels. OxO activation was also observed during treatment with other heavy metals (HMs), salt treatment and at elevated non-optimal temperature. In contrast to HM activation of OxO and lignification, high temperature and NaCl indeed activated OxO but did not induce lignification of metaxylem vessels. These results suggest that oxalate oxidase as an H₂O₂-generating enzyme is activated in response to several stresses, however the ectopic lignification of metaxylem vessels is activated specifically by HMs. This HM-induced premature root xylogenesis due to ectopic lignification of metaxylem vessels probably causes shortening of the root elongation zone and therefore a reduction in root growth.