Plasmodesmatal frequency in relation to short-distance transport and phloem loading in leaves of barley (Hordeum vulgare). Phloem is not loaded directly from the symplast

We investigated the phloem loading pathway in barley, by determining plasmodesmatal frequencies at the electron microscope level for both intermediate and small blade bundles of mature barley leaves. Lucifer yellow was injected intercellularly into bundle sheath, vascular parenchyma, and thin-walled sieve tubes. Passage of this symplastically transported dye was monitored with an epifluorescence microscope under blue light. Low plasmodesmatal frequencies endarch to the bundle sheath cells are relatively low for most interfaces terminating at the thin- and thick-walled sieve tubes within this C₃ species. Lack of connections between vascular parenchyma and sieve tubes, and low frequencies (0.5% plasmodesmata per μm cell wall interface) of connections between vascular parenchyma and companion cells, as well as the very low frequency of pore-plasmodesmatal connections between companion cells and sieve tubes in small bundles (0.2% plasmodesmata per μm cell wall interface), suggest that the companion cell-sieve tube complex is symplastically isolated from other vascular parenchyma cells in small bundles. The degree of cellular connectivity and the potential isolation of the companion cell-sieve tube complex was determined electrophysiologically, using an electrometer coupled to microcapillary electrodes. The less negative cell potential (average –52 mV) from mesophyll to the vascular parenchyma cells contrasted sharply with the more negative potential (–122.5 mV) recorded for the companion cell-thin-walled sieve tube complex. Although intercellular injection of lucifer yellow clearly demonstrated rapid (0.75 μm s^-1) longitudinal and radial transport in the bundle sheath-vascular parenchyma complex, as well as from the bundle sheath through transverse veins to adjacent longitudinal veins, we were neither able to detect nor present unequivocal evidence in support of the symplastic connectivity of the sieve tubes to the vascular parenchyma. Injection of the companion cell-sieve tube complex, did not demonstrate backward connectivity to the bundle sheath. We conclude that the low plasmodesmatal frequencies, coupled with a two-domain electropotential zonation configuration, and the negative transport experiments using lucifer yellow, precludes symplastic phloem loading in barley leaves.     

Effect of Water Deficit and Membrane Destruction on Water Diffusion in the Tissues of Maize Seedlings

We investigated diffusion of water in maize seedlings (Zea mays L. cv. Dnepropetrovskaya) following addition of polyethylene glycol (PEG) 6000 (osmotic potential –0.1 and –0.3 MPa) to the root medium by NMR method with pulsed gradient of magnetic field. Diffusion coefficients of different water phases in plant tissues (water of apoplast and vacuoles, water transported through the membranes) have been estimated from multicomponent decays of echo amplitude. Different signs of changes of water diffusion coefficients of fast and slow components of diffusional echo decay in roots and leaves under the influence of PEG-induced water deficits were shown. It has been supposed that under water deficit a sharing of water flows takes places through the different pathways (apoplastic, symplastic and transmembrane). In roots, 1-h water deficit increased the rate of fast diffusing water (water of apoplasm, vacuoles and, perhaps, water contained in intercellular endoplasm system), and decreased the rate of slowly diffusing water (water passing across the membranes). A long-term water deficit increased to a small extent the rate of water transmembrane transfer in root tissue. Leaf response to water stress was in the intensification of rate of transmembrane water transport that could be connected with the expression of water channels, and in the decrease of apoplastic water flow and flow along endoplasm. The possibility of estimation of plant tissue (membrane) integrity on the basis of diffusional data has been demonstrated.     

Lead accumulation and its translocation barriers in roots of Allium cepa L.—autoradiographic and ultrastructural studies

Abstract Lead migrating through the tissues of Allium cepa L. was found, by electron microscopy, autoradiography and other methods, to encounter at least three barriers to penetration. The layers of protoderm and hypodermic meristematic cells in the root meristematic zone and the layer of endodermis in the mature root zone were barriers to apoplastic transport. The central zone was a barrier to apoplastic and symplastic transport. It comprises the quiescent centre in the root meristem and the central part of the root cap. The cells of the deepest ground meristematic tissue layers seemed to act as a barrier, which keeps lead away from the procambium. Lead accumulated in roots but it was not uniformly distributed between their various tissues. The largest amount of lead accumulated both in ground meristematic and cortex tissues.     

Comparative Microscopical Studies on the Patterns of Calcium Oxalate Distribution in the Needles of Various Conifer Species

The distribution of calcium oxalate crystals in various conifer needles is visualized by light and electron microscopy. Such crystals occur (1) in the vascular bundle, either intracellularly in the xylem or phloem parenchyma, or extracellularly within the radial phloem walls; (2) extracellularly on the outside of the walls of mesophyll cells which face the intercellular spaces; (3) and finally as numerous small crystals within the cell walls of the epidermal cells, especially in the cuticular layer. The development and distribution of these apoplastic crystals is described in detail. Some hypotheses are finally presented for interpretations of these unusual patterns of the crystallization of Ca-oxalate outside the vacuole. Possible evolutionary aspects of this feature among the different conifer families are also discussed.     

Structure and function of leaf minor veins in trees and herbs

Summary The structure of leaf minor veins in 700 species from 140 families of dicotyledons, monocotyledons and conifers has been studied by light and electron microscopy. The presence of several structural types of minor veins has been shown. The main types are open and closed veins characteristic of trees and herbs, respectively. These vein types differ by the structure of intermediate cells, and by the mechanisms of phloem loading and sugar transport. Most woody plants have intermediate cells with numerous plasmodesmal fields, symplastic transport as the main phloem loading mechanism, as well as oligosaccharides and other complex sugars as the main phloem transport substances. By contrast, the majority of herbs have intermediate cells without plasmodesmal connections, and apoplastic loading of sucrose occurs only by membrane proton cotransport. The closed type is divided into three subtypes, differing in the degree of development of the structures used for sugar uptake from the apoplast. A list of the plants investigated with their vein types is given. The evolution of the minor vein structure and phloem loading mechanism are discussed in relation to the evolution of life forms of higher plants.     

Electromagnetic Field of Microtubules: Effects on Transfer of Mass Particles and Electrons

Biological polar molecules and polymer structures with energy supply (such as microtubules in the cytoskeleton) can get excited and generate an endogenous electromagnetic field with strong electrical component in their vicinity. The endogenous electrical fields through action on charges, on dipoles and multipoles, and through polarization (causing dielectrophoretic effect) exert forces and can drive charges and particles in the cell. The transport of mass particles and electrons is analyzed as a Wiener-Lévy process with inclusion of deterministic force (validity of the Bloch theorem is assumed for transport of electrons in molecular chains too). We compare transport driven by deterministic forces (together with an inseparable thermal component) with that driven thermally and evaluate the probability to reach the target. Deterministic forces can transport particles and electrons with higher probability than forces of thermal origin only. The effect of deterministic forces on directed transport is dominant.     

Anatomical and Chemical Alterations but not Photosynthetic Dynamics and Apoplastic Transport Changes are Involved in the Brittleness Culm Mutation of Rice

Brittleness culm is an important agronomic trait that has a potential usefulness in agricultural activity as animal forage although the developmental mechanism is not clear yet. In the present study, the anatomical and chemical characteristics as well as some ecophysiological features in the brittleness culm mutation of rice (Oryza sativa L.) were investigated. Compared with the wild type (WT), the brittleness culm mutant (bcm) exhibited higher culm vascular bundle distance and lower culm wall thickness, leaf interveinal distance and leaf thickness. Ratio of bundle sheath cell/whole bundle and areas of whole vascular bundles and bundle sheath of leaves were reduced while ratios of xylem and phloem to whole bundles were elevated in bcm. The Fourier transform infrared (FTIR) microspectroscopy analysis and further histochemical and physiological measurements revealed that the different contents and depositions of cell wall components such as pectins, lignin, suberin and cellulose all participated in the mutation of brittleness. However, the mutant presented no significant changes in leaf photosynthetic dynamics and apoplastic transport ability. These results strongly indicate that the alterations in anatomical and chemical characteristics, rather than changes in major ecophysiological features such as photosynthesis and apoplastic transport were involved in the brittleness mutation of rice.     

Potassium ion influx measurements on cultured Chinese hamster cells exposed to 60-Hertz electromagnetic fields

Potassium ion influx was measured by monitoring ⁴²KCI uptake by Chinese hamster ovary (CHO) cells grown in suspension culture and exposed in the culture medium to 60–Hz electromagnetic fields up to 2.85 V/m. In the presence of the field CHO cells exhibited two components of uptake, the same as previously observed for those grown under normal conditions; both these components of influx were decreased when compared to sham-exposed cells. Although decreases were consistently observed in exposed cells when plotted as log_c of uptake, the differences between the means of the calculated fluxes of exposed and sham-exposed cells were quite small (on the order of 4–7%). When standard deviations were calculated, there was no significant difference between these means; however, when time-paired uptake data were analyzed, the differences were found to be statistically significant. Cells exposed only to the magnetic field exhibited similar small decreases in influx rates when compared to sham-exposed cells, suggesting that the reduction in K⁺ uptake could be attributed to the magnetic field. Additionally, intracellular K⁺ levels were measured over a prolonged exposure period (96 h), and no apparent differences in intracellular K⁺ levels were observed between field-exposed and shamexposed cultures. These results indicate that high-strength electric fields have a small effect on the rate of transport of potassium ions but no effect on long-term maintenance of intracellular K⁺.     

Sugar transport across the plasma membranes of higher plants

The fluxes of carbohydrates across the plasma membranes of higher-plant cells are catalysed mainly by monosaccharide and disaccharide-H⁺ symporters. cDNAs encoding these different transporters have been cloned recently and the functions and properties of the encoded proteins have been studied extensively in heterologous expression systems. Several of the proteins have been identified biochemically in these expression systems and their location in plants has been shown immunohistochemically or with transgenic plants which were transformed with reporter genes, expressed under the control of the promoters of individual transporter genes. In this paper we summarize the current knowledge on the molecular biology and biochemistry of higher-plant sugar transport proteins.     

Identifying the pathways for foliar water uptake in beech (Fagus sylvatica L.): a major role for trichomes

Foliar water uptake (FWU), the direct uptake of water into leaves, is a global phenomenon, having been observed in an increasing number of plant species. Despite the growing recognition of its functional relevance, our understanding of how FWU occurs and which foliar surface structures are implicated, is limited. In the present study, fluorescent and ionic tracers, as well as microcomputed tomography, were used to assess potential pathways for water entry in leaves of beech, a widely distributed tree species from European temperate regions. Although none of the tracers entered the leaf through the stomatal pores, small amounts of silver precipitation were observed in some epidermal cells, indicating moderate cuticular uptake. Trichomes, however, were shown to absorb and redistribute considerable amounts of ionic and fluorescent tracers. Moreover, microcomputed tomography indicated that 72% of empty trichomes refilled during leaf surface wetting and microscopic investigations revealed that trichomes do not have a cuticle but are covered with a pectin‐rich cell wall layer. Taken together, our findings demonstrate that foliar trichomes, which exhibit strong hygroscopic properties as a result of their structural and chemical design, constitute a major FWU pathway in beech.     

Causes and effects of changes in xylem functionality in apple fruit

BACKGROUND AND AIMS: The xylem in fruit of a number of species becomes dysfunctional as the fruit develops, resulting in a reduction of xylem inflow to the fruit. Such a reduction may have consequential effects on the mineral balance of the fruit. The aim of this study was to elucidate the dynamics and nature of xylem failure in developing apples (Malus domestica) showing differing susceptibilities to bitter pit, a calcium-related disorder. METHODS: Developmental changes in xylem functionality of the fruit were investigated in "Braeburn" and "Granny Smith" apples by using a dye infusion technique, to stain the vasculature along the path of dye movement. The vascular bundles were clearly visible in transverse section when fruit were sectioned equatorially. The intensity of staining of the vascular bundles in the fruit was recorded at regular intervals throughout the season. Tissue containing dysfunctional bundles was fixed and embedded in wax for subsequent sectioning and examination. KEY RESULTS: As the season progressed, an increasing proportion of vascular bundles failed to show any staining, with the most marked change occurring in the primary bundles, and in nearly all bundles with increasing distance from the stalk end of the fruit. Decreased conductance in the primary bundles of "Braeburn" occurred earlier than in "Granny Smith". Microscopy revealed that the xylem in vascular bundles of the fruit suffered substantial damage, indicating that the mode of dysfunction was via the physical disruption of the xylem caused by expansion of the flesh. CONCLUSIONS: Results support the view that the relative calcium deficiency of apple fruit is due to a progressive breakdown of xylem conductance caused by growth-induced damage to the xylem strand in the bundle. The earlier onset of xylem dysfunction in the cultivar more susceptible to bitter pit suggests that the relative growth dynamics of the fruit may control the occurrence of calcium-related disorders.     

Sugar Transport into Storage Tubers of Stachys sieboldii Miq.: Evidence for Symplastic Unloading and Stachyose Uptake into Storage Vacuoles by an H+-Antiport Mechanism*

Following assimilation of ¹⁴CO₂ by leaves of Stachys sieboldii, ¹⁴C-stachyose is translocated into the tubers. Stachyose is accumulated and stored in the vacuoles of the pith parenchyma. Protoplasts and vacuoles were isolated and the uptake of sugars was examined. Uptake of sucrose and sucrosyl oligosaccharides of the raffinose family by protoplasts was very low compared to glucose. Transport parameters for glucose indicated a carrier mediated transport in the lower concentration range which was superimposed by diffusion at higher concentrations (> 10 mM). The very low sugar uptake by protoplasts and the sparse enzyme activities of stachyose synthase in the storage parenchyma as well as acid invertase and α-galactosidase in the cell walls indicated symplastic unloading of stachyose in the tubers. Experiments on ¹⁴C-stachyose uptake by isolated vacuoles confirmed previous observations by Keller (1992). Isolated vacuoles exhibited ATP and PP hydrolysis and were capable of generating a proton gradient across the tonoplast by a V-type H⁺-ATPase and H⁺-PPase. This was demonstrated by fluorescence quenching of quinacrine. Fluorescence could be restored by the addition of gramicidin and partly recovered by the addition of stachyose; mannitol, sorbitol and glucose had no effect. Fluorescence recovery depended on the concentration of stachyose and revealed saturation kinetics (K_m = 28 mM). Comparable results have been obtained with tonoplast vesicles by Greutert and Keller (1993). Experimental data presented here provide circumstantial evidence for symplastic unloading of stachyose in the tubers of Stachys sieboldii and demonstrate that the stachyose concentration in the cytoplasm of storage parenchyma cells is kept low by active stachyose transport into the vacuoles. The results suggest a stachyose/H⁺-antiport system.     

Tetracycline accumulates in Iberis sempervirens L. through apoplastic transport inducing oxidative stress and growth inhibition

Environmental antibiotic contamination is due mainly to improper and illegal disposal of these molecules that, yet pharmacologically active, are excreted by humans and animals. These compounds contaminate soil, water and plants. Many studies have reported the bioaccumulation of antibiotics in plants and their negative effects on photosynthesis, cell growth and oxidative balance. Therefore, the principal objective of this paper was the study of antibiotic accumulation sites in plants and its uptake modality. Iberis sempervirens L., grown in soil and in agar in the presence or absence of tetracycline, were used as a model system. Using confocal and transmission electron microscopy, we demonstrated that tetracycline was absorbed and propagated in plants through apoplastic transport and also accumulated in intercellular spaces. Tetracycline was rarely detected inside cells (in cytoplasm and mitochondria where, coherent to its pharmacological activity, it probably affected ribosomes), except in stomata. Moreover, we verified and clarified further the phytotoxic effects of tetracycline on plants. We observed that the antibiotic induced a large reduction in plant growth and development and inhibition of photosynthetic activity. As tetracycline may lead to oxidative stress in plants, plant cells tried to balance this disequilibrium by increasing the amount and activity of some endogenous enzyme antioxidant agents (superoxide dismutase 1 and catalase) and levels of antiradical secondary metabolites.     

磁场对肿瘤细胞的抑制作用

目的 :用磁场进行小鼠荷瘤和人体的离体癌细胞抑制试验。方法 :用装备有NdFeB永磁材料的仪器产生非均匀磁场 (磁通密度为 0 .2 0T ,梯度为 0 .0 7T/cm)对荷瘤小鼠和人体的离体癌细胞每天作用0 .5小时 ,1 2天后解剖检查试验结果。结果 :磁场对荷瘤小鼠的癌细胞抑止率达 61 % (p <0 .0 0 2 ) ,胸腺平均比化疗组重 8mg(p <0 .0 1 ) ,癌组织的纤维包膜是对照组的 2～ 3倍。谷丙转氨酶 (SGPT)低于化疗组的平均值 (p <0 .0 0 1 ) ,肝、心肌细胞无损伤 ;白细胞 (WBC)高于化疗组的平均值 (p <0 .0 2 ) ,免疫功能提高 ;血红蛋白 (Hb)低于化疗组的平均值 (p <0 .0 5 ) ,局部缺氧 ,癌细胞呈片状坏死 ,部分癌组织呈孤岛状。磁疗组无明显毒副作用反应。重复性实验结果相近。小鼠和人体离体癌细胞实验显著性检验无显著性意义。结论 :磁场在一定的梯度范围是可以有效的抑制小鼠肿瘤的生长。     

Characterization of the multiple transport routes for methotrexate in L1210 cells using phthalate as a model anion substrate

Summary o-Phthalate is actively transported into L1210 cells and the primary route for cell entry is the same transport system which mediates the influx of methotrexate and other folate compounds. The identity of the influx route has been established by the following observations: (A) Phthalate influx is competitively inhibited by methotrexate and the inhibition constant (K _i ) is comparable to theK _i for half-maximal influx of methotrexate; (B) Various anions inhibit the influx of phthalate and methotrexate with comparableK _i values; (C) The influx of phthalate and methotrexate both fluctuate in parallel with changes in the anionic composition of the external medium; and (D) A specific covalent inhibitor of the methotrexate transport system (NHS-methotrexate) also blocks the transport of phthalate. In contrast, the efflux of phthalate does not occur via the methotrexate influx carrier, but rather by two separate processes which can be distinguished by their sensitivities to bromosulfophthalein. Efflux via the bromosulfophthalein-sensitive route constitutes 75% of total efflux and is enhanced by glucose and inhibited by oligomycin. The inability of phthalate to exit via the methotrexate influx carrier is due to competing intracellular anions which prevent phthalate from interacting with the methotrexate binding site at the inner membrane surface.     

Pulsed electromagnetic fields accelerate proliferation and osteogenic gene expression in human bone marrow mesenchymal stem cells during osteogenic differentiation

Osteogenesis is a complex series of events involving the differentiation of mesenchymal stem cells to generate new bone. In this study, we examined the effect of pulsed electromagnetic fields (PEMFs) on cell proliferation, alkaline phosphatase (ALP) activity, mineralization of the extracellular matrix, and gene expression in bone marrow mesenchymal stem cells (BMMSCs) during osteogenic differentiation. Exposure of BMMSCs to PEMFs increased cell proliferation by 29.6% compared to untreated cells at day 1 of differentiation. Semi‐quantitative RT‐PCR indicated that PEMFs significantly altered temporal expression of osteogenesis‐related genes, including a 2.7‐fold increase in expression of the key osteogenesis regulatory gene cbfa1, compared to untreated controls. In addition, exposure to PEMFs significantly increased ALP expression during the early stages of osteogenesis and substantially enhanced mineralization near the midpoint of osteogenesis. These results suggest that PEMFs enhance early cell proliferation in BMMSC‐mediated osteogenesis, and accelerate the osteogenesis. Bioelectromagnetics 31:209–219, 2010. © 2009 Wiley‐Liss, Inc.     

Acute exposure to a 60 Hz magnetic field increases DNA strand breaks in rat brain cells

Acute (2 h) exposure of rats to a 60 Hz magnetic field (flux densities 0.1, 0.25, and 0.5 mT) caused a dose-dependent increase in DNA strand breaks in brain cells of the animals (assayed by a microgel electrophoresis method at 4 h postexposure). An increase in single-strand DNA breaks was observed after exposure to magnetic fields of 0.1, 0.25, and 0.5 mT, whereas an increase in double-strand DNA breaks was observed at 0.25 and 0.5 mT. Because DNA strand breaks may affect cellular functions, lead to carcinogenesis and cell death, and be related to onset of neurodegenerative diseases, our data may have important implications for the possible health effects of exposure to 60 Hz magnetic fields. Bioelectromagnetics 18:156–165, 1997. © 1997 Wiley-Liss, Inc.     

Cell Transplantation for Parkinson's Disease: Present Status

1. Parkinson's disease (PD) is a neurodegenerative disorder caused by the loss of neurons in the substantia nigra pars compacta and a striatal deficiency of dopamine. PD typically affects people in late middle age and progresses slowly. In the early stages of the disease, treatment targeting the dopaminergic network is effective. However, with disease progression, transplantation is an option for repairing and replacing missing dopaminergic neurons. 2. In this review, we evaluate the tissue grafts and cellular therapies that have and are being considered. Clinical trials were originally derived from transplants of adrenal medullary chromaffin cells and embryonic nigral dopaminergic neurons in patients with PD. 3. Recently, novel molecular and cellular treatments are being utilized in animals and these include embryonic stem cells, fetal cells from pigs, or transfected cells. In spite of new molecular techniques and some 20 years of experience, the transplantation therapy for PD has today the same problems and results as the first reports which used neural fetal tissue or adrenal grafts.     

Distinct mechanisms of hypoxanthine and inosine transport in membrane vesicles isolated from Chinese hamster ovary and Balb 3T3 cells

Both enzyme-mediated group translocation and facilitated diffusion have been proposed as mechanisms by which mammalian cells take up purine bases and nucleosides. We have investigated the mechanisms for hypoxanthine and inosine transport by using membrane vesicles from Chinese hamster ovary cells (CHO), Balb/c 3T3 and SV3T3 cells prepared by identical procedures. Uptake mechanisms were characterized by analyzing intravesicular contents, determining which substrates could exchange with the transport products, assaying for hypoxanthine phosphoribosyltransferase activity, and measuring the stimulation of uptake of hypoxanthine by phosphoribosyl pyrophosphate ($\text{P}\text{Rib}\text{-PP}$).We found that the uptake of hypoxanthine in Balb 3T3 vesicles was stimulated 3–4-fold by $\text{P}\text{Rib}\text{-PP}$. The intravesicular product was predominantly IMP. The hypoxanthine phosphoribosyltransferase activity copurified with the vesicle preparation. These results suggest the possible involvement of this enzyme in hypoxanthine uptake in 3T3 vesicles. In contrast to the 3T3 vesicles, CHO vesicles prepared under identical procedures did not retain hypoxanthine phosphoribosyltransferase activity and did not demonstrate $\text{P}\text{Rib}\text{-PP}$-stimulated hypoxanthine uptake. The intravesicular product of hypoxanthine uptake in CHO vesicles was hypoxanthine. These results and data from our kinetic and exchange studies indicated that CHO vesicles transport hypoxanthine via facilitated diffusion. An analogous situation was observed for inosine uptake; CHO vesicles accumulated inosine via a facilitated diffusion mechanism, while in the same experiments SV3T3 vesicles exhibited a purine nucleoside phosphorylase-dependent translocation of the ribose moiety of inosine.