Controlled growth of colloidal gold particles and implications for labelling efficiency

Summary A new method is reported for the preparation of colloidal gold particles with diameters ranging between 5 and 12 nm. The initial gold particle population, with an average diameter of 5.6±0.9 nm, is prepared by reduction of chloroauric acid with white phosphorous. An increase in particle diameter by growth is obtained by reduction of chloroauric acid with white phosphorous in the presence of colloidal gold particles. The labelling efficiency of these gold particles, conjugated with protein A, in indirect immunolabelling experiments is investigated by labelling of -galactosidase on ultrathin cryosections of Escherichia coli cells. We demonstrate that the labelling efficiency is at least dependent on particle diameter, probe concentration and preparation method. In addition it is shown, that with this new method, gold particle populations can be prepared with minor overlap in diameter spreading. Therefore these gold probes are suitable for qualitative double labelling experiments. The quantitative aspect of immunolabelling is discussed.     

A post-embedding immunoelectron-microscopic demonstration of apolipoprotein-B-containing lipoprotein particles in hepatocytes of fetal rats

Summary We used the protein-A gold technique to demonstrate the presence of apolipoprotein-B in ultrathin sections of fetal rat liver tissue. It was possible to show for the first time that the electron-dense, osmiophilic particles with diameters of 20–20 nm located within the RER cisternae and Golgi complexes of fetal rat hepatocytes contain apolipoprotein-B components and therefore are lipoproteins. After specific labelling an accumulation of gold label was observed on the RER cisternae, Golgi cisternae and the Golgi-associated secretory vesicles of hepatocytes. The specifity of this labelling pattern was assessed by comparison with cytochemical controls. Our qualitative findings were confirmed by a quantitative analysis of the mean labelling intensity (mean number of gold particles per square micron of the surface area of a particular cellular compartment) on the RER, Golgi complexes, mitochondria, nuclei and the remaining cytoplasm of hepatocytes. It is concluded that the hepatocytes of fetal rats are capable of forming apolipoprotein-B-containing lipoprotein particles. With respect to the size-distribution pattern of the observed intrahepatic lipoprotein particles, we suggest that the hepatocytes of fetal rats produce lipoproteins of the low- and very low-density-lipoprotein type.Abbreviations GA Golgi complex - RER rough endoplasmic reticulum - M mitochondria - N nuclei - LP lipoprotein partieles - L lipid droplet - SV secretory vesicle - BCP blood cell precursor - dm dense intracisternal and intravesicular material - LDL low density lipoproteins - VLDL very low density lipoproteins     

The subcellular distribution of transferrin in rat choroid plexus studied with immunogold labelling of ultracryosections

Summary Choroid plexus epithelium from third ventricle choroid plexus of 2–3-week-old rats was examined for transferrin-like immunoreactivity. In 5 µm paraffin sections most epithelial cells exhibited a pronounced immunoperoxidase staining for transferrin. The ultrastructure of the epithelium in question was examined by conventional electron microscopy. Immunolabelling of ultracryosections with IgG-gold, protein-A gold or protein-A gold—antiprotein-A protein-A gold showed an intense labelling of the basal extracellular space. The lateral intercellular space and the luminal surface showed a more variable labelling; no labelling of the tight junction zone was seen. Intracellularly a distinct labelling of the uptake and disposal pathway (the endosomal—lysosomal system) was observed, but also the synthetic machinery (rough endoplasmic reticulum, stacked Golgi membranes) showed a characteristic labelling. Thus it seems likely that both uptake and synthesis of transferrin occur in choroid plexus epithelial cells.     

Electronmicroscopical observations on yolk and yolk formation in Ophryotrocha labronica LaGreca and Bacci

Summary In Ophryotrocha labronica LaGreca & Bacci mature yolk granules are found only in the ovocyte. Other typical yolk elements are lipid droplets, small vesicular bodies, multivesicular bodies and dense bodies. The two last-mentioned also appear in the accompanying nurse cell and from there obviously pass over unchanged into the ovocyte through a specific intercellular bridge, the fusome.The mature yolk granules are considered as aggregates of mitochondrial, endoplasmic and Golgi material, to which also is added pinocytotically incorporated external material. Mitochondria apparently play a fundamental role in the process, as the multivesicular bodies, most likely the direct precursors to the yolk granules, in all probability represent transformed mitochondria.Labelling with ³H-thymidine during vitellogenesis reveals presence of DNA in the yolk granules. From the labelling pattern, which shows DNA-synthesis both in the ovocyte and the nurse cell nucleus, it is concluded that the labelled material present in the cytoplasm of both cells — most of it in yolk granules and dense bodies — is of nuclear origin. The possible mitochondrial nature of yolk granule DNA is discussed.The author is indebted to Dr. Bertil Åkesson, Zoological Institute, Lund, for kindly supplying the initital material for the Ophryotrocha cultures. The excellent technical assistance of Mrs. Mariann Carleson is gratefully acknowledged. My thanks are also due to Mrs. Siv Nilsson for skilful assistance with the photography. This work has been supported by the Swedish Natural Science Research Council and Kungliga Fysiografiska Sällskapet, Lund.     

Elimination of the non-specific binding of avidin to tissue sections

Summary A simple procedure is described for eliminating non-specific staining with avidin—peroxidase conjugates. Murine ovaries were embedded in either paraffin wax or epoxy resin and, after blocking endogenous peroxidase activity, were treated with 10 µg/ml biotinylatedPisum sativum agglutinin. Avidin—peroxidase conjugates (5 µg/ml), diluted in standard 0.05m tris-buffered saline, pH 7.6, containing 0.139m NaCl, produced considerable background coloration and intense mast cell staining in controls without the lectin. This background diminished as the ionic strength of the buffer was raised. At 0.125m Tris-buffered saline (containing 0.347m NaCl) the background was completely unstained, with elimination of all binding to mast cells and only minimal loss of specific lectin binding.     

The application of a biotin-anti-biotin gold technique providing a significant signal intensification in electron microscopic immunocytochemistry: a comparison with the ultrasmall immunogold silver staining procedure

 A three-step biotin-anti-biotin gold-detection system (method A) has been applied for ultraimmunocytochemistry using ultrasmall colloidal gold (0.8 nm) linked to anti-biotin antibodies which were visualized and enhanced by silver reduction. The reactivity for glucagon in human pancreatic islets and for cytochrome-c oxidase in heart mitochondria has been compared to a two-step ultrasmall immunogold technique (method B). For both antigens, method A provided significantly higher labelling indices (P<0.001): the labelling density for cytochrome-c oxidase was 223/μm² using method A and 78/μm² using method B. For glucagon, the labelling density was 1455/μm² with method A and 322/μm² with method B. The results demonstrate that the silver-intensified biotin-anti-biotin gold-detection system is a valuable immunocytochemical method for signal enhancement. The method utilizes biotinylated antibodies from different species, allowing its broad application at the electron microscopic level. Accepted: 24 June 1997     

Animal and worker exposure to dust and biological particles in animal care houses

An aerobiological study was performed to evaluate the potential exposure of animals and workers to dust constituents generated during routine animal house work. Different rooms of air conditioned (A, control) and passively ventilated (B, non-air conditioned) animal facilities were sampled, in order to evaluate total airborne culturable fungi and bacteria, fungal spore concentrations and particle levels. Airborne room particles were analyzed gravimetrically and for endotoxin content. All parameters, except for culturable fungi, were higher in facility B and statistically significant, with respect to those from the control facility A. Median values for airborne particle concentration, endotoxin and fungal spores in facility B were: 115 µg m^–3, 25 EU m^–3, and 2173 spores m^–3, respectively. Median values for facility A were: 66 µg m^–3, 9 EU m^–3, and 248 fungal spores m^–3. Broncheoalveolar lavage from rats kept in the rat room of B, presented median concentrations of total cells and lactate dehydrogenase, higher than those found in the control facility (4.4 × 10⁵ vs. 1.1 × 10⁵ and 2.7 UmL^-1 vs. 0.39 UmL^–1, respectively). Values of total and biological particles of both facilities, as well as the time spent in different rooms, showed that worker exposure was higher during cage washing. It was especially high in the passively ventilated facility (airborne particles 686 µg m^–3 3.5 h^–1 vs. 976 µg m^–3 3.5 h^–1, endotoxin 70 EU m^–3 3.5 h^–1 vs. 108 EU m^–3 3.5 h^–1). The type of basidiospores and ascospores found, as well as the significant correlation between particle levels and endotoxin contents suggests that wood chip bedding disturbance during cage washing is an important source for airborne biological particles. The changes in broncheoalveolar lavage components found in rats from these facilities and previously reported changes in pro-inflammatory cellular responses found in workers, indicate that these relatively low levels of exposure are enough to induce a biological response. Studies considering the composition of mixed organic dusts, would be needed to reevaluate current occupational standards.     

An evaluation of the conditions necessary for optimal protein A-gold labelling of capsular antigen in ultrathin methacrylate sections of the bacteriumPasteurella haemolytica

Summary The protein A-gold (PAG) probe is a particulate immunocytochemical probe that is eminently suitable for quantification. In order to obtain critical results from the technique, a specific and reproducible probe is needed. To this end, the concentration of probe, the variation of labelling different sections within a single grid, the effect of washing procedures, the variation of labelling with time and temperature and the effect of different storage conditions on the probe have been investigated using PAG labelling of capsular antigen on ultrathin methacrylate sections of the bacteriumPasteurella haemolytica.The results indicate that in this antigen-antibody system, and using a 20nm probe, optimal results are achieved with 2×10¹² particles/ml, a labelling time of 60min at room temperature and the PAG probe, which will have been stored at 4°C, should be between 1-and 5-weeks-old. The efficiency of the probe is tested by evaluating different primary antibody concentrations, by evaluating cross reactions of the primary antibody and by evaluating the relative amounts of antibody against internal components of the bacterium present in different antisera.     

Impact of in vitro Cultivation Conditions on Stress Responses and on Changes in Thylakoid Membrane Proteins and Pigments of Tobacco during ex vitro Acclimation

Four physiologically and phenotypically diversified tobacco (Nicotiana tabacum L. cv. Samsun) plantlet variants had been generated by cultivation on media either lacking or containing sucrose (0 and 3 %, m/v) under two different photon flux densities (PFD), 50 µmol m^–2 s^–1 (LL) and 200 µmol m^–2 s^–1 (HL). Plantlets were transferred into soil without any pre-acclimation and grown either under PFD of 200 µmol m^–2 s^–1 or 700 µmol m^–2 s^–1. Sucrose feeding in vitro resulted in reduced degree and duration of wilting after transfer. The highest readiness for ex vitro acclimation was found in 3 % HL plants, in which changes of photosynthetic apparatus and stress responses were the smallest. On the contrary, the steepest decline of Fv/Fm ratio on the first day after transplantation, doubled chlorophyll content and almost tripled D1/LHC 2 ratio after 7 d of ex vitro growth under 700 µmol m^–2 s^–1 characterized 0 % HL plants, which had suffered chronic photoinhibition in vitro. Remarkably high abscisic acid content at the end of in vitro cultivation and during acclimation as well as increased synthesis of both D1 and LHC 2 proteins even at the end of analyzed acclimation period were found only in 0 % LL plants. Increase of D1/LHC 2 ratio and chlorophyll contents demonstrate that in vitro developed leaves of all plant variants are able to acclimate to new environment. The most surprising result in the whole study is the drop of D1 protein synthesis in all plants on the 3^rd day. Five times decline of photoprotection level of xanthophylls in plants after ex vitro transfer into the same PFD showed stress character of in vitro cultures.     

Trophic structure and productivity of the lagoonal communities of Tikehau atoll (Tuamotu Archipelago,French Polynesia)

Carbon standing stocks and fluxes were studied in the lagoon of Tikehau atoll (Tuamotu archipelago, French Polynesia), from 1983 to 1988.The average POC concentration (0.7–2000 µm) was 203 mg C m^–3. The suspended living carbon (31.6 mg C m^–3) was made up of bacteria (53%), phytoplankton < 5 µm (14.2%), phytoplankton > 5 µm (14.2%), nanozooplankton 5–35 µm (5.7%), microzooplankton 35–200 µm (4.7%) and mesozooplankton 200–2000 µm (7.9%). The microphytobenthos biomass was 480 mg C m^–2.Suspended detritus (84.4% of the total POC) did not originate from the reef flat but from lagoonal primary productions. Their sedimentation exceeded phytobenthos production.It was estimated that 50% of bacterial biomass was adsorbed on particles. the bacterial biomass dominance was explained by the utilisation of 1) DOC excreted by phytoplankton (44–175 mg C m^–2 day ^–1) and zooplankton (50 mg Cm^–2 day^–1)2) organic compounds produced by solar-induced photochemical reactions 3) coral mucus.50% of the phytoplankton biomass belongs to the < 5 µm fraction. This production (440 mg C m^–2 day^–1) exceeded phytobenthos production (250 mg C m^–2 day^–1) when the whole lagoon was considered.The zooplankton > 35 µm ingested 315 mg C m^–2 day^–1, made up of phytoplankton, nanozooplankton and detritus. Its production was 132 mg C m^–2 day^–1.     

Hämatologische Beobachtungen an Regenbogenforellen (Salmo gairdneri RICH.). V. Die Erythozytenverteilungskurve

Zusammenfassung Für die Fischpathologie wird als differentialdiagnostisches Verfahren die Erythozytenmessung vorgeschlagen. Zur Darstellung der Erythrozytenverteilungskurve wird wegen der elliptischen Form der Fisch-Erythrozyten die in der Humanmedizin übliche Technik modifiziert.An 102 Regenbogenforellen (Salmo gairdneri) beiderlei Geschlechts im Alter von 1 bis 3 Jahren wurde versucht, die Normalkurve für gesunde Tiere dieser Population zu ermitteln. Die Größenverteilung der Erythrozyten ergibt eine Gauss-Glockenkurve, deren Gipfel in die Größenklasse 30–35 µm²/ fällt, und deren Basis von 15–20 µm²/ bis 55–60 µm²/ reicht.Zwischen den Price-Jones-Kurven der Milchner (52 Individuen) und Rogner (50 Individuen) bestehen keine signifikanten Unterschiede. Ebenso konnte keine Beeinflussung dieser Normalkurve durch unterschiedliches Alter oder jahreszeitliche Einflüsse — untersucht wurden in dieser Hinsicht die F₂ — beobachtet werden.

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Summary As differential-diagnostic feature in the fish pathology it is suggested to measure the red blood cells. The normal technique used in human medicine for plotting the distribution curve of the red blood cells is modified, because the red blood cells of the fish are of elliptical shape.102 rainbow trouts (Salmo gairdneri), both, male and female, and 1–3 years of age, were examined to find the normal curve for healthy animals of this population. The size distribution of the red blood cells show a bell-shaped Gauss-curve, the peak of which is 30–35 µm²/, and the basis of which reaches from 15–20 µm²/ to 55–60 µm²/.The Price-Jones-curves show no significant differences between the milters (52 individuals) and the spawners (50 individuals). Besides, changes caused by different age or seasonal influences could not be observed on this normal curve. Analyzed were the F₂ individuals.

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Institut für Siedlungswasserbau und Wassergütewirtschaft der Universität Stuttgart Biologische Abteilung - Arbeitsgruppe Haider     

The effects of bioturbation on particle redistribution in Mediterranean coastal sediment. Preliminary results

In order to quantify bioturbation processes in a coastal Mediterranean ecosystem, experiments were performed to determine sediment mixing rates resulting from macrobenthos activity. Particle flux was measured in situ for 22 days using luminophores, which are colored sediment particles with sizes ranging from 10 to 200 µm.In sediment depths from 0–5 cm, particle mixing was intensive due to high macrobenthos abundance. A small quantity of luminophores was transported down to a depth of 14 ± 2 cm, where the macrofauna was represented principally by Polychetes. In a control experimental structure — without benthic fauna — no transfer of luminophores into the sediment was recorded.Sediment particle mixing measured in the ecosystem studied is intensive, and is the result of high macrobenthos activity. Different mixing modes occur with scales and rates depending on the organisms present. The luminophore profile resulting from bioturbation processes is explained by an intensive bioadvection sediment mixing added to a biodiffusive mixing with an order of magnitude of 10^–6 cm² s^–1. Tracer accumulations between 1 and 2 ± 1 cm and between 4 and 5 ± 1 cm are attributed to bioadvection activity of two or more distinct populations. Studies over a larger time scale have been undertaken to monitor developments in the observed subsurface maxima.     

Blood parameters of two hill stream cobitids in relation to their habitat

The blood of two fresh water cobitids — Botia lohachata, an exclusive water breathing form and Lepidocephalus guntea a dual breather, — showed a comparatively higher range of Hb (16.0–19.0 g%), Hct (50.0–61.1%) and number of RBC (2.71–6.7 millions/mm³) than many other water and air breathing fishes. Significant sexual difference exists in these characteristics (P > 0.05).The impact of life in oxygen depleted water, also inhabited by L. guntea as a result of its air breathing habit, is well reflected in its greater RBC size (11.86 × 8.66 µm) and their larger surface area (83.96 µm²) compared to that of Botia (53.16 µm²). The smaller size (9.92 × 6.45 µm) and consequently greater number of erythrocytes (4.67 millions/mm³) in Botia, are related to its active mode of life in the swiftly flowing water of hilly rivers. Though the higher nucleo-cytoplasmic ratio of 1 : 5.2 in Botia and 1 : 6.9 in Lepidocephalus suggest a slower red cell metabolism, the greater number of erythrocytes seems to have compensated for their active mode of life.     

An histochemical approach to characterization of anionic constituents in mast cell secretory granules

We used cationized colloidal gold in order to investigate the distribution of anionic sites in different secretory granules of rat and mouse mast cells. The localization of the anionic sites was performed by post-embedding labeling of thin sections of rat peritoneal cells or mouse skin tissue, fixed in Karnovsky's fixative and OsO₄ and embedded in Araldite or LR white, respectively. In all cases anionic sites were demonstrated with a high density variation depending on cell type. In all mast cell secretory granules we have observed the highest density (ca. 500–900 gold particles/m²), while in other peritoneal cell granules it was about 10 times less (ca. 40–80 gold particles/m²). Pretreatment of the LR white sections with heparinase I and III resulted in a reduction of 97% and 72%, respectively, in the binding of the gold particles to the granules, indicating that the majority of the gold binding reactivity is due to heparin. Correlation of section profile area with labeling density revealed that the smaller granules were significantly more labeled when compared to the larger profiles. On the basis of these observations it seems that a post-translational change (mainly sulfation of heparin) of secretory content influences the granule anionic charge and thus may affect the intragranule buffer capacity.     

Near-coronal fluid flow patterns and food cell manipulation in the rotifer Brachionus calyciflorus

Brachionus calyciflorus is able to differentially capture or deflect potential food cells depending on the overall density (numbers µl^–1) and size of suspended particles the rotifer encounters while feeding. This has been shown to correlate with behavioral regulation of pseudotrochal structures, and primarily by large medial tufts of compound cilia (cirri). When pseudotrochal cirri are extended laterally, inferred fluid flow pathlines show that only cells in the central 25 µm of the coronal disk are subject to ingestion. When pseudotrochal cirri form medially-oriented screens, fluid flow is directed away from the pseudotrochas and central corona, an effect apparent up to 70 µm in front of a restrained animal. Fluid flow in all areas of the B. calyciflorus corona is at very low Reynolds Number (Re), with calculated values always less than 10^–1. Accordingly, removal of cells from feeding currents probably is not due to sieving but is more likely due to direct interception of particles by individual ciliary elements. Certain large cells — such as Euglena gracilis — are retained in the inner pseudotrochal space for up to 500 msec, ample time for B. calyciflorus to assess the physical and chemical characteristics of this or other potential foods.     

Production of planktonic Rotatoria in Ormajärvi,an eutrophicated lake in southern Finland

The production of planktonic rotifers was studied in eutrophic Lake Ormajärvi. Of the total annual production of rotifers (2.9 g org. C m^–2 or 231 mg dry weight m^–3) 49% was achieved during one month (July) and 88% during 3 months of summer. The most important producers were Keratella cochlearis (1.2 g C m^–2), Asplanchna priodonta (0.8 g C m^–2) and Conochilus unicornis (0.6 g C m^–2). The P/B ratio for the total rotifer community during the growing season (7 months) was 25.0; monthly P/ B values varied between 0.3 and 5.2. The daily P/ B values were highest among species of Collotheca. The relationships of rotifers to some biotic and abiotic factors (invertebrate predators — Mesocyclops, Cladocera, planktonic Protozoa and temperature) are briefly discussed.     

Development of eye enlargement of domestic fowl subjected to low intensity light

Week-old White Leghorn chicks were randomly divided into two light control chambers. One chamber was equipped with clear incandescent light with a maximum intensity of 0.66µW cm^–2 mµ ^–1 and the remaining chamber was equipped with blue light with a maximum intensity of 0.015µW cm^–2 mµ ^–1. Both groups received a daily photoperiod of 14 hours' light and 10 hours' dark. Low intensity blue light caused a definite eye enlargement after 7 weeks of exposure.The eye enlargement was not related to an increase release rate of thyroidal¹³¹I or oxygen consumption. Intraocular pressure was no greater in the enlarged eyes than in control eyes.Length of exposure to light and dark cycles caused a daily rhythm in intraocular pressure which was not different in the two light treatments. Associated with the eye enlargement was an exophthalmic condition during the developmental stages, a slight flattening of the cornea, and definite alterations in visual parameters resulting in an axial length myopia. Some lenticular contribution to the myopia was inferred by reason of an increase in size of the lens.

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Zusammenfassung Eine Woche alte Leghorn-Küken wurden nach einer Zufallsverteilung in zwei Lichtkammern gegeben.Die eine Kammer war ausgerüstet mit klaren Glühlampenlicht mit einer Maximalintensität von 0,66µW cm^–2mµ ^–1, und die andere Kammer mit blauem Licht mit einer Maximalintensität von 0,015µW cm^–2mµ ^–1. Beide Gruppen erhielten täglich 14 Stunden Licht bei 10 Stunden Dunkelheit. Blaues Licht geringer Intensität verursachte nach 7 Wochen eine eindeutige Augenvergrösserung.Sie war nicht mit einer Zunahme der¹³¹I Ausscheidungrate aus der Schilddrüse oder des Sauerstoffverbrauchs verbunden. Der Augeninnendruck war bei den Versuchstieren nicht grösser als bei den Kontrolltieren. Die Länge der Lichteinwirkung sowie der Dunkelperioden verursachte einen Tagesrhythmus des Augeninnendruckes,der bei den Tieren in den beiden Lichtkammern gleich war.Mit der Augenvergrössung verbunden war während des Entwicklungsstadiums ein Exophthalmus, eine leichte Abflachung der Cornea und eine sichere Veränderung des Visus durch Myopie. Eine Beteiligung von Linsenveränderungen an der Myopie wird wegen des Anstiegs der Linsengrösse angenommen.

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Resume On a placé des poussins Leghorn choisis au hasard dans deux chambres lumineuses. La première de ces chambres était équipée de lampes à incandescence claires ayant une intensité maximum de 0,66µW cm^–2 mµ ^–1, la seconde de lampes bleues d'intensité maximum de 0,015µW cm^–2 mµ ^–1. Chacun des deux groupes reçut de la lumière durant 14 heures avec une période d'obscurité de 10 heures.La lumière bleue de faible intensité a provoqué après 7 semaines un agrandissement très net des yeux des poussins. Cet agrandissement ne fut cependant pas lié à une augmentation de la sécrétion thyroïdale de¹³¹I ou de la consommation en oxygène. La pression interne de l'oeil ne fut en outre pas supérieure à celle des bêtes de contrôle. La durée d'action de la lumière et de l'obscurité ont provoqué un rythme journalier de la pression interne de l'oeil identique pour les bêtes des deux chambres lumineuses. Pendant le stade de croissance, on a constaté — parallèlement à l'agrandissement des yeux — de l'exophtalmie, un léger aplatissement de la cornée et une modification certaine de la vue par myopie. On admet que la myopie est due à une modification de la lentille suite de l'agrandissement de celle-ci.

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This investigation was supported in part by funds from Medical and Biological Research by State of Washington Initiative No. 171, and by Research Grant No. NB03 770-05 from the Division of General Medical Sciences, U.S. Public Health Office. Scientific Paper No. 3183, College of Agriculture, Washington State University, Pullman. Project Nos. 1677 and 1895.     

Nitrite uptake by Chlamydomonas reinhardtii cells immobilized in calcium alginate

When an initial cell loading of about 30–40 µg chlorophyll (Chl)·g^–1 gel and alginate suspension of 3% (w/v) were used for immobilization of Chlamydomonas reinhardtii, the resulting cell beads showed optimum nitrite uptake rate, at 30° C and pH 7.5, of 14 µmol NO _inf2 ^sup– ·mg^–1 Chl·h^–1, the photosynthetic and respiratory activities being about 120 µmol O₂ produced·mg^–1 Chl·h^–1, and 40 µmol O₂ consumed ·mg^–1 Chl·h^–1, respectively. The nitrite uptake activity required CO₂ in the culture and persisted after 8 days of cells immobilization, or in the presence of 0.2 mm ammonium in the medium. Our data indicate that alginate-entrapped C, reinhardtii cells may provide a stable and functional system for removing nitrogenous contaminants from waste-waters.Correspondence to: C. Vílchez     

Immuno-gold localisation of arabinogalactan protein in the developing style of Nicotiana alata

Arabinogalactan-protein was localised ultrastructurally in the transmitting tissue of the Nicotinana alata Link & Otto style from the bud stage to anthesis using monoclonal antibody directed to terminal α-L-arabinosyl residues followed by goat anti-mouse IgG linked to 20 nm gold particles. Gold particles were localised on the Golgi apparatus, multivesicular bodies, cell wall and intercellular matrix with the highest concentration over multivesicular bodies. During flower development there was an increase in the total number of gold particles with a relative decrease on the multivesicular bodies and an increase on the intercellular matrix. There was also an increase during development in the proportion of intercellular matrix relative to all other components of the transmitting tissue. Vesiculation of the endoplasmic reticulum and the production of cup-shaped vesicles by the Golgi bodies were observed during development. It is concluded that secretion of arabinogalactan-protein from the intracellular to the extracellular location occurs via multivesicular bodies produced by the Golgi apparatus and possibly the endoplasmic reticulum.     

Analysis of Qualitative Contribution of Assimilatory and Non-Assimilatory De-Excitation Processes to Adaptation of Photosynthetic Apparatus of Barley Plants to High Irradiance

The adaptation of barley (Hordeum vulgare L. cv. Akcent) plants to low (LI, 50 µmol m^–2 s^–1) and high (HI, 1000 µmol m^–2 s^–1) growth irradiances was studied using the simultaneous measurements of the photosynthetic oxygen evolution and chlorophyll a (Chl a) fluorescence at room temperature. If measured under ambient CO₂ concentration, neither increase of the oxygen evolution rate (P) nor enhancement of non-radiative dissipation of the absorbed excitation energy within photosystem 2 (PS2) (determined as non-photochemical quenching of Chl a fluorescence, NPQ) were observed for HI plants compared with LI plants. Nevertheless, the HI plants exhibited a significantly higher proportion of Q_A in oxidised state (estimated from photochemical quenching of Chl a fluorescence, q_P), by 49–102 % at irradiances above 200 µmol m^–2 s^–1 and an about 1.5 fold increase of irradiance-saturated PS2 electron transport rate (ETR) as compared to LI plants. At high CO₂ concentration the degree of P stimulation was approximately three times higher for HI than for LI plants, and the irradiance-saturated P values at irradiances of 2 440 and 2 900 µmol m^–2 s^–1 were by 130 and 150 % higher for HI plants than for LI plants. We suggest that non-assimilatory electron transport dominates in the adaptation of the photosynthetic apparatus of barley grown at high irradiances under ambient CO₂ rather than an increased NPQ or an enhancement of irradiance-saturated photosynthesis.