Intracellular pathways and degradation of endosomal contents in basal epithelial cells of freshwater sponges (Porifera, Spongillidae)

 The digestive system expressed by basal epithelial cells of the freshwater sponges Spongilla lacustris and Ephydatia muelleri is mainly represented by a population of 30–50 preexisting lysosomes located in the close vicinity of the central nucleus. The strongly acidic vacuoles (pH 4–4.5) vary in size between 1 and 3 μm, and contain a set of different lysosomal enzymes. Immunocytochemical studies succeeded in the detection of β-hexosaminidase, cathepsin D, acid phosphatase, and α-glucosidase. Endosomes resulting from fluid-phase macropinocytosis, receptor-mediated endocytosis, or phagocytotic activity deliver their exogenous contents to the preexisting lysosomes for enzymatic degradation. Macropinosomes and phagosomes follow a rather reduced intracellular pathway by immediate fusion with the lysosomal compartment, whereas substances conveyed by coated vesicles pass through two additional vacuolar stages, namely early and late endosomes. Early endosomes serve as sorting organelles and segregate various constituents of complex ligands (BSA-AU_6, BSA-AU₁₂) by size into individual late endosomes, which then coalesce with preexisting lysosomes. As a whole, the intracellular pathways and hydrolytic processing of endosomal and phagosomal contents in freshwater sponge cells share certain similarities with the respective mechanisms in cells of higher eukaryotes. Accepted: 7 October 1997     

Evidence for a clathrin-mediated recycling of albumin in human term placenta

During human pregnancy, the trophoblast layer is in direct contact with maternal albumin. In contrast to immunoglobulins, albumin does not cross the placental barrier. However, albumin affects the trophoblast placental lactogen and chorionic gonadotroph secretion. The present study investigated the interaction between albumin and syncytiotrophoblast using human term placental explants. Bovine serum albumin, labeled with either 125I or fluorescein isothio-cyanate, was taken up rapidly by placental explants. This process was temperature-sensitive. The internalized labeled BSA quickly outflowed from the tissue at the maternal side, largely without any major modification in molecular weight. Colchicine (1 mM), which disrupts the microtubule network, or cytochalasin B (40 microM), which disassembles filamentous actin, did not interfere with the placental transmembrane movements of labeled BSA. Megalin, clathrin, and caveolin 1 are three membrane proteins associated with albumin endocytosis in other tissues, but only megalin and clathrin were detected in the syncytiotrophoblast layer by immunohistochemistry. The uptake of labeled BSA into placental explants was not modified by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (1 mM) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM), two pharmacological tools known to disturb megalin-mediated albumin endocytosis. By contrast, methyl-beta-cyclodextrin (10 mM) and chlorpromazine (1.4 mM), both of which disrupt the clathrin-mediated endocytotic system, significantly reduced the uptake of labeled BSA. These data suggest, to our knowledge for the first time, that maternal albumin is actively internalized into the human trophoblast according to an apical recycling pathway. This temperature-sensitive process does not depend on an intact cytoskeleton, but it is associated with a clathrin-mediated endocytotic system.     

Study of the vesicular cycle in nerve structures in somatic muscle of earthworm (<Emphasis Type="Italic">Lumbricus terrestris</Emphasis>)

In the muscle wall of the earthworm Lumbricus terrestris, with the aid of fluorescent endocytotic dyes FM1-43, FM2-10, and FM4-64, there are revealed fluorescent spots 1–2 μm in diameter that represent clusters of “synaptic boutons.” Application takes place onto ganglia of the abdominal nerve chain of the Dil membrane probe capable of translocation by axoplasmic transport; the subsequent (next day) staining of nerve structures with the endocytotic marker FM4-64 showed the complete superposition of fluorescence of these dyes fluorescing in different specter areas. The fluorescent marker DiBAC₄(3) revealed an enhancement of fluorescence of nerve elements with increase of K⁺ concentration in the extracellular medium. Use of FM2-10 showed that, the higher the K⁺ content in solution and, accordingly, the nerve cell depolarization, the faster the release of the marker and, on the contrary, the slower the process in the absence of K⁺ in the medium. In the Ca²⁺-free solution and in the presence of the Ca²⁺ chelator BAPTA or BAPTA-AM, there uptake and release of FM2-10 are blocked, but only after preliminary 40-min incubation in such solution. In clusters of synaptic boutons, exo- and endocytosis processes take place that are also preserved under conditions of rest. This vesicular cycle depends on the membrane potential of nerve structures and on the content of K⁺ and Ca²⁺ in the medium, the calcium sensor working most likely by the “all or nothing” principle.     

Proton transport and membrane shuttling in turtle bladder epithelium

Summary Proton secretion in the urinary bladder of the freshwater turtle is mediated by proton pumps located in the apical membrane of carbonic-anhydrase (CA)-rich cells. It has been proposed that the rate of proton transport is regulated by endocytotic and exocytotic fusion processes which alter the apical membrane area, and hence number of exposed pumps. Three techniques were used to study this process. Analyses of transepithelial impedance provided estimates of transport-associated changes in net membrane area, as well as other electrical parameters. Electron microscopy allowed visualization of the endocytotic vesicles thought to be involved in the process. Finally, uptake of a florescent fluid-phase markerprovided measurements of the rates of endocytosis. We report the following: (i) endocytotic and exocytotic processes occur primarily in the CA-rich cells; (ii) inhibition of proton transport resulting from 0.5mm acetazolamide (AZ) results in a decrease in the apical membrane area of approximately 0.47 cm²/cm² tissue; (iii) the apical membrane specific conductance of the CA-rich cells is approximately 220 S/F, and possibly represents a Cl^– conductance that may function in counter-ion flow; (iv) the decline in transport following AZ is not directly proportional to the decline in apical membrane area, suggesting that changes in pump kinetics are also involved in the regulation of transport; (v) the CA-rich cells exhibit a high rate of constitutive pinocytosis, and hence membrane shuttling, which appears to be independent of the rate of transport; (vi) AZ induces a transient increase in the rates of endocytosis and shuttling; and (vii) the transport-associated changes in apical membrane area may reflect an effect of AZ on a regulated endocytotic pathway which is distinct from the pinocytotic process.     

Comparison of tetrahydrofuran,fetal calf serum,and Tween 40 for the delivery of astaxanthin and canthaxanthin to HepG2 cells

The present investigation aimed to compare fetal calf serum (FCS) and Tween 40 with the commonly employed tetrahydrofuran (THF) with respect to cytotoxicity, stability of the solubilized carotenoids, and uptake and accumulation of the xanthophylls astaxanthin (AX) and canthaxanthin (CX) in cultured human liver cells (HepG2). Incubation of HepG2 cells for 24 h with THF (≥1.25%) or FCS (≥11.25%) with or without AX (≥25 μmol/L) or CX (≥25 μmol/L) did not affect cell viability. Tween 40 (0.25–1.25% in medium) reduced cell viability by 75–99%. The stabilities of AX and CX in cell-free RPMI 1640 medium for ≤24 h were higher when delivered with THF instead of FCS. The dose- and time-dependent accumulations of AX and CX (1–10 μmol/L) in HepG2 cells were higher when carotenoids were delivered with FCS compared to THF. In conclusion, FCS and THF, but not Tween 40, were suitable solvent systems for the delivery of AX and CX to HepG2 cells. In our experiments FCS was superior with regard to the uptake and accumulation of both carotenoids.     

Influence of Environmental Salinity on Messenger RNA Levels of Growth Hormone, Prolactin, and Somatolactin in Pituitary of the Channel Catfish (Ictalurus punctatus)

Pituitary growth hormone (GH), prolactin (PRL), and somatolactin (SL) messenger RNA levels in channel catfish (Ictalurus punctatus) were examined under various environmental and physiological conditions. Catfish were sampled following salinity challenge, during the winter (December) and spring or summer (April or July), and at different sizes (15–18 g, 620–664 g, and 956–1134 g). When catfish (956–1134 g) were transferred from freshwater to saline water containing 8 ppt NaCl, their plasma [Na⁺] increased significantly above values in the freshwater control group until they were transferred back to freshwater. Pituitary GH mRNA levels were low for the first 24 hours following transfer to saline water, but thereafter were significantly elevated above control values until the fish were transferred back to freshwater. Pituitary GH mRNA levels were highest in July and lowest in December. Growth hormone mRNA levels were also elevated in the size groups 15–18 g and 956–1134 g in July when compared with December values. Pituitary PRL mRNA levels increased for the first 24 hours following transfer to saline water (956–1134 g), but thereafter were significantly lower than control values until the fish were transferred back to freshwater. Pituitary PRL mRNA levels were highest in April and July and lowest in December, and were also elevated in the size groups 620–664 g and 956–1134 g. Pituitary SL mRNA levels were unaffected in catfish transferred to saline water; however, levels were significantly elevated in catfish of the 956–1134-g size group sampled in April when compared with December. These results suggest the involvement of GH in adaptation to brackish water and of PRL in adaptation to freshwater in the catfish, and seasonal and size-related differences in pituitary GH, PRL, and SL mRNA levels. Received May 17, 2000; accepted October 30, 2000     

Elevated cadmium accumulation in marine organisms from Terra Nova Bay (Antarctica)

 As a contribution towards identification of the principal environmental factors involved in cadmium accumulation in Antarctic marine organisms and the establishment of a baseline near the Italian Antarctic Station “Baia Terra Nova”, surface sediments, plankton and benthic organisms were studied in coastal waters of Terra Nova Bay (Ross Sea). The cadmium content of sediments was similar to that regarded as background in most marine coastal areas, whereas in surface water, phyto- and zooplankton it was similar to values measured in areas of enhanced upwelling. Algal and animal taxa dominating benthic associations had a higher cadmium content than related species from other seas. Very high concentrations of the metal were found in sponges (10–80 μg/g dw) and in the digestive gland of molluscs (up to 345 μg/g in Neobuccinum eatoni). The rapid regeneration of cadmium and its natural occurrence and bioavailability in highly productive coastal waters seem to be responsible for cadmium accumulation in the tissues of marine organisms near the “Baia Terra Nova” station. Received: 6 June 1995/Accepted: 23 November 1995     

Epicatechin gallate increases glutamate uptake and S100B secretion in C6 cell lineage

There is a current interest in dietary compounds, such as green tea polyphenols, that can favor protection against a variety of brain disorders, including Alzheimer’s disease, ischemia, and stroke. The objective of the present study was to investigate the effects of (^_)-epicatechin-3-gallate (ECG), one of three three major green tea antioxidants, on C6 lineage cells. Here, we evaluated cell morphology and integrity and specific astrocyte activities; glutamate uptake and secretion of S100B in the presence of 0.1, 1 and 10 μM ECG. During 6 h of incubation, cell morphology was altered only at 10 μM ECG; however, after 24 h of treatment, cells become stellate in the presence of all concentrations of ECG. Loss of cell integrity was observed after 24 h with 10 μM ECG and represented only 6% of cells, in contrast with 2% observed at basal conditions. ECG (1–10 μM) induced a decrease (about 36%) in glutamate uptake after 1 h of incubation. After 6 h, an opposite effect occurred and ECG induced a sustained increase in glutamate uptake of about 70% from 0.1 μM. In addition, a significant increase in S100B was observed at 1 μM ECG (36%) and 10 μM ECG (69%) after 1 h, in contrast to 6 h of treatment, where all doses of ECG induced a significant increase (about 60%) in S100B secretion. These data demonstrate that ECG induces a significant improvement in glutamate uptake and S100B secretion in C6 cells, indicating that ECG could contribute to the neuroprotective role of astroglial cells.     

Stimulated endocytosis in penetratin uptake: effect of arginine and lysine

Cell-penetrating peptides can deliver macromolecular cargo into cells and show promise as vectors for intracellular drug delivery. Internalization occurs predominantly via endocytosis, but the exact uptake mechanisms are not fully understood. We show quantitatively how penetratin, a 16-residue cationic peptide, stimulates fluid-phase endocytosis and triggers its own uptake into Chinese hamster ovarian cells, using a 70 kDa dextran to indicate macropinocytosis. The total cellular endocytotic rate is significantly less affected and we therefore propose up-regulation of macropinocytosis to occur at the expense of other types of endocytosis. By comparing penetratin to its analogs PenArg and PenLys, enriched in arginines and lysines, respectively, we show how these side-chains contribute to uptake efficiency. The degree of peptide and dextran uptake follows similar patterns regarding peptide concentration and arginine/lysine content (PenArg > penetratin > PenLys), indicating that a high content of arginines is beneficial but not necessary for stimulating endocytosis.     

Long-Term Cultivation of Primmorphs from Freshwater Baikal Sponges <Emphasis Type="Italic">Lubomirskia baikalensis</Emphasis>

The work was aimed at performing long-term cultivation of primmorphs in vitro from freshwater sponge Lubomirskia baikalensis (Pallas 1776), collected from Lake Baikal, obtaining its long-term primmorph culture in both natural (NBW) and artificial (ABW) Baikal water and at identifying the impact of different environmental factors on formation and growth of primmorphs. The first fine aggregates of L. baikalensis are formed in vitro 10–15 min after dissociation of sponge cells. Epithelization of aggregates begins 4 h later after the dissociation. Young primmorphs are formed 1 or 2 days later. The surface of primmorphs is covered with a layer of exopinacocytes. The primmorphs remain viable for more than 10 months at 3–6°C. Over 50% of primmorphs in NBW and 25% in ABW are attached to the substrate and grow like adult sponges. Thus, the long-term primmorph cultivation in vitro allows the creation of a controlled live model system under experimental conditions. The results of this work will allow the creation of a cell culture collection of Baikal freshwater sponges for studying morphogenesis of primmorphs during cultivation at different stages and transdifferentiation of their cells, physiological functions of sponge cells, processes of spiculogenesis, identification of proteins involved in biomineralization process, decoding of their genes, as well as a spectrum of secondary metabolites.     

Endocytosis and exocytosis of phytohemagglutinin (PHA) cell surface receptors of human lymphocytes during blast transformation

The ultrastructural distribution of T lymphocyte surface membrane receptors for phytohemag-glutinin (PHA) during blast transformation is examined using PHA covalently coupled to ferritin (PHA-Fe). Human peripheral blood lymphocytes from normal donors were enriched for T cells by nylon wool elution and cultured in vitro with PHA-Fe at a concentration known to cause maximal stimulation of DNA synthesis as measured by [³H]thymidine incorporation. Over the course of a 72 h incubation period, cell samples were harvested at regular intervals and examined by transmission electron microscopy. Within several minutes of culture at 37 °C the majority of the ferritin (Fe)-labeled PHA surface receptors on almost all cells undergo rapid endocytosis; some Fe label remains at the cell surface. After several hours, endocytotic vesicles containing Fe-labeled receptors coalesce and undergo condensation. Within 36–48 h, most endocytotic vesicles transform into multivesicular bodies (MVBs). After 48–72 h, 70–80% of the cells had the ultrastructural appearance of blast transformation as characterized by increased size, euchromatic nuclei, nucleolonema and polyribosomes. In 40 % of the blast cells the Fe-labeled MVBs are exocytosed to the cell surface; cytoplasmic MVBs in the remaining portion of the blasts and non-blast lymphocytes do not appear to undergo exocytosis. Although endocytosis and exocytosis of lymphocyte surface receptors during mitogen-induced blast transformation are observed, the role and significance of receptor redistribution to cell activation remains unclear.     

Experimental silicon demand by the sponge Hymeniacidon perlevis reveals chronic limitation in field populations

Dissolved silicon (DSi) is a key marine nutrient. Sponges and diatoms are relevant DSi consumers, but sponges appear to have a less efficient uptake system that requires higher ambient DSI concentrations for maximum uptake. We experimentally tested whether a sponge adapted to live at the intertidal (Hymeniacidon perlevis) also shows such a need for high DSi. Under laboratory conditions, sponges were exposed to both the natural DSi concentration (10 μM) and much higher levels (25, 40, and 70 μM) for 36 h, being water samples taken at 6 h intervals to infer DSi uptake. Uptake rates shifted over time (particularly in high DSi treatments) and showed moderate inter-individual variability. Average DSi uptake rate at 70 μM was twice higher than those at 40 and 25 μM, which in turn were not significantly different from each other, but were twice higher than the uptake rate at 10 μM. Therefore, H. perlevis needs, for efficient uptake, ambient DSi concentrations two to four times higher than the maximum available in its natural habitat. From an eco-physiological point of view, it means that the skeletal growth in the populations of H. perlevis is chronically limited by DSi availability, a limitation that may favor sponge evolution toward skeletal slimming.     

In vitro culture of Phyllanthus caroliniensis (Euphorbiaceae)

An efficient micropropagation protocol was developed for the medicinal plant Phyllanthus caroliniensis (Euphorbiaceae) using nodal segments for axillary shoot proliferation. Maximum multiplication (21–23 shoots per explant) was achieved on MS or AR media supplemented with either 5.0 μM BA, 1.25–5.0 μM kinetin or 2.5–5.0 μM 2iP. Rooting was achieved with 80–100% of the microshoots on MS medium without growth regulators, although 1.25 μM NAA and 1.25–5.0 μM IAA promoted significant increases in the number of roots per explant. Regenerated plants were successfully acclimatized and about 88% of plantlets survived under ex vitro conditions. Flowering was observed on in vitro grown plantlets and after 3–4 weeks of acclimatization. High frequency callus initiation and growth was achieved when nodal segment explants were inoculated in the vertical position on MS medium supplemented with 5.0 μM 2,4-D. Root cultures were successfully established on MS medium containing 1.1 μM NAA. The optimized micropropagation, callus and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolism studies. This revised version was published online in June 2006 with corrections to the Cover Date.     

Are bioleaching rates determined by the available particle surface area concentration?

It is well known that pulp density and particle size determine the available surface area concentration and have an influence in the overall rate of bioleaching of minerals. As metal solubilization takes place through the surface area of the particles, it can be expected that different combinations of pulp densities and particle sizes giving the same surface area concentration would determine the same leaching rate. The objective of this work was to test this hypothesis on the effect of surface area concentration, pulp density and particle size of the biooxidation of a pyritic gold concentrate by the thermophilic Archaeon Sulfolobus metallicus in shake flasks. The gold concentrate was used at 2.5%, 5%, 10%, and 15% w/v pulp density and at four size fractions: 150–106, 106–75, 75–38 and –38 μm. Temperature was 68°C and the initial pH was 2.0. Results showed that the volumetric productivities of iron and sulfate depend not only on the surface area concentration but also on pulp density and particle size considered separately. These two variables not only determine surface area but also exert additional effects on the process, so the hypothesis was not confirmed. Maximum attained iron productivity was 1.042 g/l day with the 75–38 μm fraction at 5% pulp density. Maximum sulfate productivity was 4.279 g/l day with the 75–38 μm fraction at 10% pulp density.     

Differential Effects of selenium on the proliferation of human pulmonary adenocarcinoma cells and human embryonic lung diploid cells in vitro

The effect of different concentrations of Na₂SeO₃ on human pulmonary adenocarcinoma cells and human embryonic lung diploid cells in vitro was investigated. For human pulmonary adenocarcinoma cells, mitotic index and cell count decreased with increasing selenium concentrations. At 1 μg/mL sodium selenite, mitotic activity and growth of human lung cancer cells were partially inhibited, and the progression of human lung cancer cell cycle was partially arrested. When human embryonic lung diploid cells were treated with 1 μg/mL sodium selenite for five continuous days, cell counts of the treated group were closely parallel to those of the control group. After treating human embryonic lung diploid cells with 1–5μg/mL sodium selenite for 1–3 d, the mitotic index (MI), labeled index (LI), and average silver grain (SG) number per 20 labeled nuclei were the same as those of the control. In mixed cultures of human embryonic lung diploid cells and human pulmonary adenocarcinoma cells, treated with 3 and 5 μg/mL sodium selenite for 24 h, the lung diploid cells showed a normal fusiform morphology, whereas the lung cancer cells showed heavily vacuolated cytoplasms and distorted nuclei.     

Effect of temperature on endocytosis and degradation of sulphated proteoglycans by cultured skin fibroblasts

Temperature up to 16‡C reduced endocytosis of [³⁵S]-proteoglycans by human skin fibroblasts to less than 15% of that at 37‡C. At temperatures between 20–26‡C endocytosis was more than 50%. At temperatures below 26‡C, the relative rate of degradation of endocytosed [³⁵S]-proteoglycans was several fold less than the rate of endocytosis. Codistribution of endocytosed [³⁵S]-proteoglycans and the lysosomal marker enzyme Β-hexosaminidase upon subcellular fractionation indicated that endocytotic vesicles containing [³⁵S]-proteoglycans had fused with lysosomes at 37‡C and at 16‡C. The prolonged halflives of endocytosed [³⁵S]-proteoglycans at 16–26‡C could not be explained merely by a temperature dependent reduction of catalytic activity of lysosomal enzymes participating in the degradation of sulphated proteoglycans.     

Response of murine erythroleukemia cells to cis-and trans-diamminedichloro-platinum (II)

Summary Cis-diamminedichloroplatinum II (cis-DDP), an antitumor drug and the inactive trans-isomer were studied to evaluate their effects on cell multiplication, DNA synthesis, and surface morphology of the murine erythroleukemia cells (clone 6A11A). Short-term treatment of cells (1h) with 5 and 10μg/ml of cis-DDP resulted in a significant inhibition of cell multiplication. Continuous treatment with cis-DDP (up to 144 h) significantly arrested cell growth at 1,5, and 10μg/ml. The cells exposed to 10 μg/ml trans-DDP exhibited a slight decrease in cell multiplication; however, the 25-μg/ml treatments showed a modest inhibition of cell growth. Continuous treatment with cis-DDP resulted in a concentrationdependent decrease in DNA synthesis, although low-dose treatment (0.05 and 0.1 μg/ml), with a few exceptions, had no relative inhibitory effect. Likewise, trans-DDP treatments decreased tritiated thymidine incorporation; however, this inhibitory effect was not as drastic as with corresponding concentrations of cis-DDP. Scanning electron microscope studies revealed the formation of many giant cells and blebs at all short-term treatment concentrations of cis-DDP past the 48 h interval. Continuous treatment of cis-DDP at 1 μg/ml concentration produced giant cells with minute holes, whereas the 5 and 10 μg/ml exposure resulted in the formation of blebs and large holes and reduction of microvilli past the 48-h treatment period. At higher concentrations the continuous treatment of cis-DDP completely destroyed the cells. The surface morphology of trans-isomer treated cells, in most instances, resembled the corresponding untreated control cells.     

Degradation efficiency and molecular size alteration during the aerobic microbial treatment of lignite pyrolysis deposit water

 Investigations into aerobic biological degrada-tion were carried out as part of an extensive programme designed to facilitate the cheap remediation of a pyrolysis waste-water deposit. Attention was focused on the processes of carbon conversion by different populations. The susceptibility of a body of lignite-processing deposit water to microbiological degradation was examined in batch investigations in a Sapromat system and in continuous bench-scale fermenter cultivations, with respect to nutrient supply, inoculation culture and molecular size distribution. It was found that degradation best occurs with an adapted mixed culture. The autochthonous culture removes 30% less dissolved organic carbon (DOC) and has a 40% higher specific oxygen demand. A shortage of phosphorus, investigated with a view to avoiding additional eutrophication problems in the open water in the case of in situ remediation, causes reduced DOC degrada-tion and significantly higher specific oxygen demand. The biological process is overlapped by abiotic oxidation. During aerobic treatment, a concentration of colour-giving aromatic substances of between 0.5 kDa and 5 kDa was observed. This phenomenon is caused by the oxidation of low- and high-molecular-mass compounds. The removal of DOC is limited to 65% and mainly occurs in the range below 0.5 kDa (30%) and in the 0.5–1 kDa range (12%); the removal is negligible in the ranges 1–3 kDa (0.8%) and 3–5 kDa (2%) and a little higher in the ranges 5 kDa–0.3 μm (5%) and above 0.3 μm (6%). In the investigations it was discovered that DOC removal causes in the ranges below 0.5 kDa, 0.5–1 kDa and 5 kDa–0.3 μm mainly as a result of degradation, but the range above 0.3 μm is chiefly caused by bioadsorption. Aerobic microbiological treatment is able to remove most low-molecular-mass substances. In order to remove the macromolecular and colour-giving part of the deposit water, an additional treatment stage, e.g. flocculation, is required. Received: 10 October 1995/Received revision: 9 January 1996/Accepted: 15 January 1996     

Pericardial cell ultrastructure in the tobacco hornworm: Manduca sexta L. (Lepidoptera: Sphingidae)

The ultrastructure of the pericardial athrocytes of fifth instar tobacco hornworm Manduca sexta L. (Lepidoptera: Sphingidae) was examined by transmission electron microscopy. These cells are highly specialized for the maintenance of hemolymph homeostasis by synthesis and secretion of some proteins, and uptake and degradation of others. We observed a striking radial zonation with numerous labyrinthine channels extending into the cell providing a large surface area for enhanced efficiency of endocytosis. Granular material was imported into the endosomal compartment by fusion of endocytotic vesicles from the basal region. Anatomical evidence supports the hypothesis that proteins are transported from the endosome to the lysosome, by maturation of the early endosome to form the late endosome/lysosome, as opposed to vesicular transport. The presence of catalase in athrocyte lysosomes is reported for the first time. Endocytosis in the athrocytes of insects is compared with endocytosis in vertebrate systems.     

Tracheids in white spruce seedling's long lateral roots in response to nitrogen availability

This study examined how the availability of inorganic nitrogen (N) modified the anatomical characteristics of white spruce (Picea glauca (Moench) Voss) roots related to their hydraulic properties. Seedlings were grown for one growing season in 4 L capacity pots filled with sand under one of three N levels: low (10 ppm), medium (50 ppm) and high (125 ppm). First order lateral roots with intact tips were sampled from dormant seedlings in October. Root segments were collected from 4, 10, and 14 cm distances above the root tip for fixation and sectioning and for maceration. Additional specimens were collected from the 4 and 14 cm distances for maceration and scanning electron microscopy of xylem pits. Root diameter and surface area occupied by the xylem in root cross sections increased basipetally in all treatments but exceptions were found. Higher N-levels significantly increased root diameter and surface area occupied by the xylem. In the two higher N treatments secondary root development was more advanced near the root tip than in the low N treatment. There was a strong positive correlation between root diameter and cross-sectional root area occupied by the xylem (30–50% of the root cross section) but not in portions with little secondary development. Non-conducting space within the xylem occupied 10–13% of its cross-sectional surface. Tracheids of the primary xylem were larger, had larger lumens but thinner cell walls than those of the secondary xylem. Low N treatment seedling tracheids had smaller total cross-sectional area, less lumen, and less cell wall surface area than the two other N treatments. Tracheid diameter means were between 19–20 μm in the high and medium N treatments, and 15.2 μm in the low N treatment. The range was 4.5–51.3 μm. Tracheid length was not significantly affected by N. The average tracheid was about 1000 μm long, and the range was 110–3530 μm. Pit-border diameters ranged between 4.1–20.6 μm (average 10–11 μm) and were not affected by the N treatment. Pit aperture diameters were within 0.62–10.2 μm range (average between 3–4 μm) and were also not significantly affected by the N treatment, although tracheids from the medium N-treatment roots tended to have larger apertures. The pit border diameter equals that of the margo while the aperture size should be similar to that of the torus of the pit membrane. If the capacity for axial water transport in spruce roots is affected by N, it would be by its impact on conduit diameter and, possibly on the pit-membrane pore sizes but not by changes to conduit length and to the size of the pit membrane surface area. This revised version was published online in June 2006 with corrections to the Cover Date.