ALBERT

Notes: Abstract The impact of the grape leafhopper,Empoasca vitis, on leaf gas exchange, plant growth, yield, fruit quality and carbohydrate reserves of the grapevines,Vitis vinifera L., was studied. Gas exchange was measured on the discolored (red) and the green parts of infested main leaves and on leaves from uninfested vines. Photosynthesis and mesophyll conductance were severely reduced on main leaves showing leafhopper feeding symptoms. The stomatal conductance of the red leaf section of infested main leaves was lower than on undamaged control leaves. Additionally, the red leaf section of infested main leaves showed lower transpiration rates when compared to the green parts of the same leaves and to undamaged control leaves. Gas exchange processes of lateral leaves were not affected by leafhopper feeding. Leafhopperload on main leaves was correlated to visual damage symptoms. At 71.8 leafhopper-days per leaf up to 40% of the main leaf area of the infested plants was discolored from the borders towards the center. Lateral leaves showed no feeding symptoms. Shoot diameter, pruning weight and carbohydrate reserves in the wood were not affected by leafhoppers. Lateral leaf area growth was significantly stimulated on plants infested by leafhoppers. No decrease in yield and fruit quality with leafhopper-loads up to 71.8 leafhopper-days per leaf were observed.

Notes: Abstract This study concerns the inhibitory effects of acid pH and nickel on growth, nutrient (NO3 - and NH4 +) uptake, carbon fixation, O2 evolution, electron transport chain and enzyme (nitrate reductase and ATPase) activities of acid tolerant and wild-type strains of Chlorella vulgaris. Though a general reduction in all these variables was noticed with decreasing pH, the tolerant strain was found to be metabolically more active than the wild-type. A reduced cation (NH4 +, Na+, K+ and Ca2+) uptake, coupled with a facilitated influx of anions (NH4 +, PO4 3- and HCO3 -), suggested the development of a positive membrane potential in acid tolerant Chlorella. Nevertheless, a tremendous increase in ATPase activity at decreasing pH revealed the involvement of superactive ATPase in exporting H+ ions and keeping the internal pH neutral. A difference in Na+ and K+ efflux of the two strains at decreasing pH suggests there is a difference in membrane permeability. The low toxicity of Ni in the acid tolerant strain may be due to the low Ni uptake brought about by a change in membrane potential as well as in permeability. Hence, the development of superactive ATPase and a change in both membrane potential and permeability not only offers protection against acidity, but also co-tolerance to metals.

Notes: Abstract This tutorial was designed for nonbiologists requiring an introduction to the nature and general timescales of phytoplankton responses to physical forcing in aquatic environments. As such, an effort was made to highlight biological markers which might assist in identifying, measuring and/or validating physical processes controlling the variability in the distribution, abundance, composition and activity of phytoplankton communities. Given the recent advances in environmental optics and remote sensing capabilities, a special emphasis was placed on the nature and utility of phytoplankton optical properties in current bio-optical modelling efforts to predict temporal and spatial variability in phytoplankton productivity and growth.

Notes: Abstract Toxicological responses of the filamentous N2-fixing cyanobacteriumNostoc calcicola Bréb. towards Hg2+ were studied to enumerate the decisive lethal events. In low-dose, long-term experiments (0.05–0.25 μm Hg2+, 10 days), photoautotrophic growth was severely inhibited with concurrent loss of photosynthetic pigments (phycocyanin〉chlorophyll α〉carotenoids) and nucleic acids. The termination of growth after a day 4 exposure to 0.25 μm Hg2+ has been attributed to the complete inhibition ofin vivo photosynthetic activity in the cyanobacterium (O2 evolution〉14CO2 incorporation). The elevated Hg2+ concentrations irreversibly damaged the cell membrance as observed under light microscopy, and as indicated by the leakage of intracellular electrolytes and phycocyanin. In high-dose, short-term experiments (0.5–20.0 μm Hg2+, up to 6 h), thein vivo activities of selected enzymes (glutamine synthetase 〉 nitrate reductase 〉 nitrogenase) were less inhibited by Hg2+ than the uptake of nutrient ions (NH 4 + 〉NO 3 − 〉PO 4 3− ).

Notes: Abstract Since the beginning of this century evidence has accumulated which demonstrates that non-mammalian vertebrates possess photoreceptors situated deep within the brain. While many attempts have been made to localize these sensory cells, studies have either failed or been inconclusive. In this report we have used several experimental approaches to localize the deep brain photoreceptors of the lizard Anolis carolinensis. Using 3 antibodies that bind vertebrate cone opsins, we have immunolabelled cerebrospinal fluid (CSF)-contacting neurons located at the ventricular border within the nucleus ventromedialis of the septum. Western blot analysis indicates that these antibodies recognized a single 40 kD protein in ocular, anterior brain, and pineal extracts. Immunoblots of rodent brain did not show a similar protein band. We have also identified specific retinoids associated with phototransduction (11-cis and all-trans-3,4-didehydroretinaldehyde) within anterior brain extracts. This combined data provides the most detailed analysis of deep brain photoreceptors in any vertebrate. Consequently, we feel Anolis provides an excellent model to study this unexplored sensory system of the vertebrates.

Notes: Abstract A protein solubilized from a membrane preparation of the gonad of Aplysia californica has been isolated by affinity chromatography, using bag cell egg-laying hormone (ELH) as the bound ligand, and partially purified and characterized by gel electrophoresis. The protein has an apparent molecular weight of 52 kDa and consists of two disulfide-linked subunits of about 30 kDa each. The protein is glycosylated and has an acidic pI. Approximately 10–15 μg of this protein can be isolated from a single ovotestis, representing less than 1% of the total protein in the gonad; but the protein could not be detected in buccal mass or body wall, tissues which do not have apparent response to ELH. Antibodies generated against this ELH-binding protein (ELHBP) were used to localize sites in the ovotestis which might contain this molecule and thus represent targets for egg-laying hormone. Immunocytochemical results indicate that the oocytes are a rich source of this protein, since their cytoplasm was the only detectable site of immunoreactivity. Whether this binding protein represents an egg-laying hormone receptor is uncertain, but its prevalence in oocytes suggests that ELH plays a signaling role on these gametes.

Notes: Abstract Vascular disintegration mainly of medulla rays of spruce roots is of major significance in root rot disease of spruce caused byH. annosum. Using seedling roots as an experimental model, the possible routes and initial host reactions preceding invasion of vascular tissues was investigated. Transmission electron microscopy showed that penetration through the endodermis was an obvious route but not without host resistance. Using antibodies againstH. annosum hyphal materials, some labelling of vascular tissues remote from sites of fungal colonization suggest the release of fungal secretory products partly active in tissue disintegration. Similarly, intense labelling was also observed in severely colonized host tissues at late stages of infection. Strong labelling recorded at 3 d p.i. mainly on fungal hyphae and scant gold particles on invaded host tissues could imply that induction of host antifungal metabolites may have been a late event. A correlation was found between total antigenic material in root homogenates measured by ELISA, density of tissue labelling by immunocytochemistry and severity of disease symptoms. The importance of this in relation to diagnosis of biotic root rot diseases in the field is discussed.

Notes: Abstract 1. The world consumption of energy is roughly 250 EJ. It increases with the level of technology and gross national product of a country. More than 83% of world energy consumption is used by the industrialized countries with one third of the world population; not quite 17% is used by the developing countries with two thirds of the world population. The world's resources of fossil fuels are estimated at 364,560 EJ; about 5–13% of this, and 31% in the case of natural gas, are considered reserves that are economically recoverable and utilizable with current technologies. 2. Agriculture's share of the economy's energy consumption in the Federal Republic of Germany is about 3.4%. It was five times higher per hectare of agricultural land in 1975 than in 1880, but the productivity of the energy was only half as high because of the enormous increase in productivity per unit of labor and area. In absolute terms, however, energy production per unit area increased tremendously, with gross agricultural production two and a half times its earlier size. 3. As a producer of plant material, agriculture qualifies as an energy producer, while as a producer of livestock it also is an energy consumer. In fact, through plant production agriculture becomes the only branch of our economic system that produces more energy than it consumes as fossil energy. Agriculture uses about 40% of its energy requirement for fuel, about 20% for machinery repair and replacement, 30% for mineral fertilizers, about 10% for electricity, and 1–2% for chemical crop protection. Forestry can be evaluated as particularly favorable from the energy viewpoint, while hothouse crops are very unfavorable. Agricultural chemicals support the energy output of green plants; agriculture as a whole is on balance energetically. 4. Solar energy and photosynthesis are the primary sources of energy to our plant. About 3 million EJ solar energy are radiated to the earth annually; 3000 EJ are fixed photosynthetically (2 ⋅ 1011 tons vegetable matter); the food requirement of 4 billion people is 15EJ. Another item of interest on the periphery of the energy balance is the enrichment of our atmosphere with oxygen, which has been accomplished for millions of years solely by the photosynthesis of green plants. 5. Through their additional yield effect, mineral fertilizers increase the energy output of plants more strongly than just the equivalent of the energy input. They cause the plant to produce more foliage and thereby promote more intensive assimilation, which means that mineral fertilizers enable the plant to utilize free solar energy better. A calculation of the energy involved in long-term field trials in cereals disclosed energy input: energy output ratios of 1:5.8 and 1:6.1. 6. Chemical crop protection has a similar effect since it protects against loss of plantproduced energy. Based on an average energy expenditure of 263 MJ ha−1 per kg ha−1 ‘typical’ active ingredient for a crop protection product, additional yields of only 4–4.5% — or considerably less in the case of high-energy crops such as cereals or sugar beets — would be sufficient to cover the energy expenditure; as a rule, however, the productivity of the chemical crop protectants is higher. The biological potential of our crops to utilize solar energy also has been improved considerably compared to earlier times — with cereals, for example, from 0.25% per unit area during the Middle Ages to 1.5% today; theoretically 4% is possible. The thesis that agrochemical aids in agriculture and horticulture are a waste of energy is unjustified. 7. Biomass also creates energy. Experts estimate the utilizable annual production of biomass in the Federal Republic of Germany to be 30 million tons mineral coal units (1 coal unit = 29.3 MJ), whereby undersized and refuse wood, straw and biogas are of special significance. Especially “fuel forests' of, for example, willows, poplars and alders could produce the equivalent of 486,000 MJ by way of 30 tha−1 biomass, contributing sizably to the fuel supply of the nation; at the moment, the conventional form of forestry produces only 30,240 MJ. It is considered feasible in Sweden to supply the entire energy requirement of the country from 93,000 km2 of ‘fuel forest’, and it must be remembered that mineral fertilization could be used to increase the productivity of land used for this purpose relatively quickly if the need were to become acute. The extraction of alcohol from crops offers other interesting aspects; the currently highest yield fuel crops (sugar beet, sugarcane and cassava) produce between 4,900 and 10,700 l ha−1 alcohol. 8. The energy problem of modern economies will not find its complete answer in the green plant. Prudent and well contemplated use of the green plant, however, may eventually do much to take the edge off today's energy dilemma.

Notes: Abstract The effects of aflatoxin B1, (0.5–25 µg ml−1) on in vitro root and shoot development in young tobacco explants were investigated. Despite an initial apparent stimulatory effect on most measured parameters at 0.5 µg ml−1 AFB1, the number of leaves, root and leaf mass per plantlet were progressively inhibited with increasing AFB1 concentration. The number of explants developing roots was reduced to 34% at the highest (25 µg ml−1) AFB1 concentration, following 3 weeks exposure to the toxin. Leaf chlorophyll content at this toxin concentration was significantly lower than that measured for control plantlets. Thin layer chromatography confirmed the absorption of AFB1 by the plantlets. Using immunocytochemical techniques, AFB1 was immunolocated predominantly in the vacuoles, the nucleus and the cytoplasm (possibly intravesicularly). The results are discussed in terms of this immunolocation within the cell.

Notes: Abstract Diurnal gas exchange characteristics were measured simultaneously in two mangrove species, Avicennia marina and Bruguiera gymnorrhiza, over 7 d in summer (February–March), to compare their productivity. The study was undertaken in the Beachwood Mangroves Nature Reserve, Durban, South Africa, using fully expanded leaves of young and mature trees at the top of the canopy. Gas exchange was strongly influenced by photosynthetic photon flux density (PPFD), leaf temperature and the accompanying leaf to air vapour pressure deficit (Δ w). Carbon dioxide exchange was saturated at a PPFD of about 600 μmol m-2s-1 in B. gymnorrhiza compared to 800 μmol m-2s-1 in A. marina. Maximal CO2 exchange occurred between 12h00 and 14h00 and was consistently greater in A. marina (8.8 μmol m-2s-1) than in B. gymnorrhiza (5.3 mu;mol m-2s-1). Mean internal CO2 concentrations ( ci) were 260 μl l-1 in A. marina and 252 μl l-1 in B. gymnorrhiza. Photorespiratory activity was 32% in A. marina and 30% in B. gymnorrhiza. Mean water use efficiency (WUE) was 8.0 μmol mmol-1 in A. marina and 10.6 μmol mmol-1 in B. gymnorrhiza. Diurnal leaf water potentials ranged from –0.8 to –3.5 MPa and were generally lower in A. marina.

Notes: Abstract Data on stand structure and rates of photosynthesis were used to estimate net canopy carbon fixation and carbon accumulation as living biomass in mangrove forests in Hinchinbrook Channel, Australia. Total annual canopy net carbon fixation was estimated to be about 29 t C ha−1 yr−1. This equates to about 204,000 t C yr−1 for all mangrove forests in Hinchinbrook Channel. Of this, only about 12% was stored as living plant biomass. Although it is not yet possible to present a robust carbon balance for mangrove trees, the remainder is presumably lost through plant respiration, litter fall, root turnover and exudation of organic compounds from roots.

Notes: Abstract Photosynthetic characteristics were investigated in the geographically isolated and restricted mangrove species, P.rhizophoreae. Gas exchange measurements were made on two to seven years old hydroponically grown plants maintained in 10%, 50% and 100% seawater. CO2 exchange in the 50% and 100% seawater treatments was reduced by 10% and 26%, respectively, compared to the 10% seawater treatment. CO2 response curves indicated that carboxylation efficiency was greater in 10% than in 50% seawater, while stomatal limitation increased from 11% to 16% as salinity increased from 10% to 50% seawater. Carbon losses via photorespiration (31% and 41%) and CO2 compensation point (67 and 81 μ11−1) were greater in 50% than in the 10% seawater treatment. Maximal CO2 exchange occurred at 30 °C with no differences among the salinity treatments. The results indicate that P. rhizophoreae exhibits many gas exchange characteristics previously reported for other mangroves.

Notes: Summary Vitellin was purified from eggs of the silkworm,Bombyx mori, by a new method in which vitellin was extracted from isolated yolk granules. The purified vitellin had a molecular weight of 540,000. An antibody against purified vitellin was prepared in rabbits. It reacted with the hemolymph vitellogenin as well as with purified vitellin, but not with other proteins in the hemolymph or in the extract from yolk granules. The anti-vitellin IgG was used to immunocytochemically locate vitellin in theBombyx non-diapause egg during early developmental stages. In the egg, just after oviposition, vitellin was located in internal yolk granules and in small yolk granules of the periplasm. During the early developmental stages studied, vitellin was not metabolized uniformly throughout the egg. The vitellin of the internal yolk granules located at the posterior-dorsal part and of the small peripheral yolk granules was utilized in 16 h and 2 days, respectively, after oviposition. A thin, very vitellin-poor layer was located between the periplasm and the vitellin-rich interior in the newly laid egg. it was always in close contact with the periphery where blastoderm and germ-band cells developed.

Notes: Abstract A one-year study of phytoplankton, primary production and related physical and chemical factors was made in a Swiss basin of Lake Lugano (Lago di Lugano). The chlorophylls and 12 carotenoids were analyzed with a TLC technique. The carotenoid monitoring was considered to be particularly interesting, because the role of these pigments in freshwater algae is still very poorly documented by field studies. The dependence of photosynthesis on several factors was statistically evaluated. Evidence was found of light-adaptation phenomena. The variations of photosynthetic activity and efficiency largely depended on the light regime in the few days before the field observations and on the cellular content of chlorophylls and single carotenoids, whose concentrations in their turn were closely linked with light, temperature, average cell size, and with the actual species assemblage.

Notes: Summary The developmental profile of the major haemolymph proteins (ceratitins) inCeratitis capitata was studied. Ceratitin concentration in the haemolymph decreases dramatically during the last days of pupal life, while the amounts of ceratitins in whole organism extracts remain unchanged. By electrophoretic, immunological and immunofluorescence techniques it was revealed that ceratitins are reabsorbed by the fat body and a fraction of them is deposited in the cuticle. The possible role of ceratitins is discussed.

Notes: Summary The immunocytochemical localization of the egg-jelly macromolecule, a fucose sulphate glycoconjugate (FSG) that induces the acrosome reaction in spermatozoa, was investigated in ovaries of the sea urchin Hemicentrotus pulcherrimus by use of a polyclonal antibody. The polyclonal antibody reacted with the accessory cells and oocytes in the ovarian lumen. In the accessory cells, evidence of an intense immunohistochemical reaction was observed in many globules of variable density. Products of the specific immunohistochemical reaction were frequently observed in the surface region of oocytes, at a distance from the ovarian wall. At the ultrastructural level, the polyclonal antibody was found to react with the material present in the vacuole-like structures of the globules in the accessory cells. Many gold particles, demonstrating specific immunolabelling, were associated with well-developed microvilli on the vitellogenic oocytes. In the mature oocytes, intense labelling was observed in the jelly coat but not in the vitelline coat. By contrast, oogonia and early oocytes were barely labelled. Quantitative data indicated that the extent of immunolabellings in the surface region of oocytes was very high in the vitellogenic and mature oocytes. In all cases, neither the oocyte cytoplasm nor the subcellular organelles were labelled. These results suggest that FSG is produced by the accessory cells and is deposited initially on the surface of vitellogenic oocytes for the formation of jelly. These findings may provide a new insight into the role of the accessory cells in the reproductive process of the sea urchin.

Notes: Abstract In the sea urchin Hemicentrotus pulcherrimus, the egg-jelly macromolecule, a fucose sulphate glycoconjugate (FSG) that induces the acrosome reaction in spermatozoa, originates from the accessory cells in the ovary. In the present study we examined the seasonal variations in the distribution of FSG in the ovary by immunocytochemistry with a polyclonal antibody. An enzyme-linked immunosorbent assay indicated that FSG was present in supernatants of extracts of ovaries throughout the development of the ovary. However, the immunohistochemical study showed that there are marked seasonal changes in the distribution of FSG in ovaries. The polyclonal antibody reacted strongly with globules of accessory cells before the beginning of the breeding season (August to December). During the breeding season (February to April), the immunohistochemical reaction was found on the surface of oocytes but was weak in the accessory cells. At the ultrastructural level, the antibody reacted with globules of variable density in accessory cells. Intense immunolabelling was observed in the vacuole-like structures of the globules. Sometimes, products of the specific immunocytochemical reaction were found in the Golgi apparatus in these globules. Quantitative examination indicated that FSG was actively produced by the accessory cells from the late non-breeding season to the pre-breeding season. These results suggest that there are marked seasonal variations in the production of FSG by the accessory cells in the sea urchin ovary. These findings also provide new evidence that accessory cells exhibit dynamic changes during the reproductive process in the sea urchin.

Notes: Abstract  Trees represent a, probably the, major component of the biosphere and have a unique place in the history of Mankind. One of their most fascinating features is the process of secondary growth which is effected principally by the secondary vascular system, the developmental continuum of secondary phloem, vascular cambium, and secondary xylem. However, for too long assumptions about the developmental biology of trees have had to be based upon studies of primary growth systems within annual, herbaceous species because study of the secondary vascular system had been largely ignored. Even when attempts are made to understand some of the most fundamental features of the secondary vascular system, such as xylogenesis, the current model system, isolated Zinnia mesophyll cells, is not entirely appropriate to the situation in the intact tree. Some deficiencies of the Zinnia system are discussed, and the advantages of the genus Populus as a model for study of the hardwood secondary vascular system are considered. Some of the new approaches which are poised to lead to significant advances in our knowledge of the cell bio-logy of the secondary vascular system of trees – spe-cifically of the cell wall, the plasmalemma, and the cytoskeleton – are discussed. The value of one of these new techniques – immunocytochemistry – is demonstrated by a consideration of recent work on the role of the cytoskeleton in the hardwood secondary vascular system.

Notes: Summary Expression of androgen receptor (AR) in mouse osteoclast-like multi-nucleated cells (OCs) was examined with immunocytochemical techniques. Murine OCs were obtained by co-culturing mouse osteoblastic cells and bone marrow cells. Three preparations of polyclonal anti-AR antibody which were raised in rabbit against different parts of the human AR were employed for the experiments. Specific staining for AR was demonstrated in the nuclei and the perinuclear area of mouse OCs. This is the first report demonstrating the presence of AR in osteoclast-like cells.

Notes: Summary Growth hormone (GH) may regulate tooth formation and bone remodeling associated with tooth eruption. This study reports the distribution of growth hormone receptor/binding protein in developing rat molars and adjacent alveolar bone by immunocytochemistry using well-characterized anti-growth hormone receptor monoclonal antibodies. These tissues represent an excellent model for studying the ontogenic changes that occur in odontogenic and osteogenic cells, as these cells are found in linear arrays displaying the various stages of morphological and functional differention, and differentiated function. Immunoreactivity was first seen in precementoblasts in contact with the epithelial root sheath, and preodontoblasts. However, growth hormone receptor immunoreactivity was associated primarily with the cytoplasm of odontogenic and osteogenic cells forming their respective matrices. Thus, cementoblasts and odontoblasts at sites of new matrix formation showed intense immunoreactivity whereas cementocytes and mature odontoblasts at later stages of tooth development were nonreactive. Osteoblasts engaged in intramembranous ossification in the alveolar bone were positive, although osteocytes and endosteal cells were immunonegative. Osteoclasts at sites of alveolar bone remodeling resorption were also immunopositive. These patterns of receptor expression parallel the ontogenic sequences of odontogenic and osteogenic cells and suggest that GH promotes the functional state of these cells. Our results also imply that GH may influence differentiation or differentiated functions associated with odontogenesis, osteogenesis, and bone remodeling independent of systemic insulin-like GF-I.

Notes: Abstract The immunogold silver staining method (IGSS) is widely used as a sensitive and specific immunohistochemical visualisation technique. IGSS involves the specific deposition of metallic silver at the site of immunogold labelling and provides a means of visualisation at low magnification by light or electron microscopy. Silver developers for IGSS rapidly deposit metallic silver only at the site of heavy metals, including gold and silver, because of their catalytic activity. The developing solution contains the silver ions and reducing agent necessary for this reaction. Using different silver salts as ion donors and by selecting an appropriate temperature and pH, visible amounts of silver can be deposited in a few minutes at the site of colloidal gold labelling while little non-specific background deposition occurs. Inclusion of protective colloids in the solution can also be used to control the reaction. Although studies of the chemical basis of silver deposition around unlabelled colloidal gold date back to 1939, immunogold enhancement by silver was established in 1983. The IGSS method evolved from the combination of disparate photographic, histochemical and immunogold techniques which have been effectively combined and optimised over the last 10 years to provide a visualisation system which is well suited to many immunohistochemical studies.

Notes: Abstract  The immunogold silver staining method (IGSS) is widely used as a sensitive and specific immunohistochemical visualisation technique. IGSS involves the specific deposition of metallic silver at the site of immunogold labelling and provides a means of visualisation at low magnification by light or electron microscopy. Silver developers for IGSS rapidly deposit metallic silver only at the site of heavy metals, including gold and silver, because of their catalytic activity. The developing solution contains the silver ions and reducing agent necessary for this reaction. Using different silver salts as ion donors and by selecting an appropriate temperature and pH, visible amounts of silver can be deposited in a few minutes at the site of colloidal gold labelling while little non-specific background deposition occurs. Inclusion of protective colloids in the solution can also be used to control the reaction. Although studies of the chemical basis of silver deposition around unlabelled colloidal gold date back to 1939, immunogold enhancement by silver was established in 1983. The IGSS method evolved from the combination of disparate photographic, histochemical and immunogold techniques which have been effectively combined and optimised over the last 10 years to provide a visualisation system which is well suited to many immunohistochemical studies.

Notes: Abstract  The aim of this study was to characterise growth and photosynthetic capacity in plants adapted to long-term contrasting atmospheric CO2 concentrations (C a). Seeds of Agrostis canina L. ssp. monteluccii were collected from a natural CO2 transect in central-western Italy and plants grown in controlled environment chambers at both ambient and elevated CO2 (350 and 700 μmol mol−1) in nutrient-rich soil. Seasonal mean C a at the source of the plant material ranged from 610 to 451 μmol CO2 mol−1, derived from C4 leaf stable carbon isotope discrimination (δ13C). Under chamber conditions, CO2 enrichment stimulated the growth of all populations. However, plants originating from elevated C a exhibited higher initial relative growth rates (RGRs) irrespective of chamber CO2 concentrations and a positive relationship was found between RGR and C a at the seed source. Seed weight was positively correlated with C a, but differences in seed weight were found to explain no more than 34% of the variation in RGRs at elevated CO2. Longer-term experiments (over 98 days) on two populations originating from the extremes of the transect (451 and 610 μmol CO2 mol−1) indicated that differences in growth between populations were maintained when plants were grown at both 350 and 700 μmol CO2 mol−1. Analysis of leaf material revealed an increase in the cell wall fraction (CWF) in plants grown at elevated CO2, with plants originating from high C a exhibiting constitutively lower levels but a variable response in terms of the degree of lignification. In vivo gas exchange measurements revealed no significant differences in light and CO2 saturated rates of photosynthesis and carboxylation efficiency between populations or with CO2 treatment. Moreover, SDS-PAGE/ LISA quantification of leaf ribulose bisphosphate carboxylase/oxygenase (Rubisco) showed no difference in Rubisco content between populations or CO2 treatments. These findings suggest that long-term adaptation to growth at elevated CO2 may be associated with a potential for increased growth, but this does not appear to be linked with differences in the intrinsic capacity for photosynthesis.

Notes: Abstract We have examined the properties and subcellular localization of phytohemagglutinin (PHA), the major lectin of the common bean (Phaseolus vulgaris.), in the axis cells of nearly mature and imbibed mature seeds. On a protein basis the axis contained about 15% as much PHA as the cotyledons. Localization of PHA was done with an indirect immunolabeling method (rabbit antibodies against PHA, followed by colloidal gold particles coated with goat antibodies against rabbit immunoglobulins) on ultra-thin cryosections which were embedded in plastic on the grids after the immunolabeling procedure. The embedding greatly improved the visualization of the subcellular structures. The small (4 nm) collodial gold particles, localized with the electron microscope, were found exclusively over small vacuoles or protein bodies in all the cell types examined (cortical parenchyma cells, vascular-bundle cells, epidermal cells). The matrix of these vacuoles-protein bodies appears considerably less dense than that of the protein bodies in the cotyledons, but the results confirm that in all parts of the embryo PHA is localized in similar structures.

Notes: Abstract The ultrastructure of the storage parenchyma cells of the cotyledons of developing bean (Phaseolus vulgaris L.) seeds was examined in ultrathin frozen sections of specimens fixed in a mixture of glutaraldehyde, formaldehyde and acrolein, infused with 1 M sucrose, and sectioned at-80° C. Ultrastructural preservation was excellent and the various subcellular organelles could readily be identified in sections which had been stained with uranyl acetate and embedded in Carbowax and methylcellulose. The cells contained large protein bodies, numerous long endoplasmic reticulum cisternae, mitochondria, dictyosomes, and electron-dense vesicles ranging in size from 0.2 to 1.0 μm. Indirect immunolabelling using rabbit immunoglobulin G against purified phaseolin (7S reserve protein), and ferritin-conjugated goat immunoglobulin G against rabbit immunoglobulin G was used to localize phaseolin. With a concentration of 0.1 mg/ml of anti-phaseolin immunoglobin G, heavy labeling with ferritin particles was observed ober the protein bodies, the cisternae of the endoplasmic reticulum, and the vesicles. The same structures were lightly labeled when the concentration of the primary antigen was 0.02 mg/ml. Ferritin particles were also found over the Golgi bodies. The absence of ferritin particles from other organelles such as mitochondria and from areas of cytoplasm devoid of organelles indicated the specificity of the staining, especially at the lower concentration of anti-phaseolin immunoglobulin G.

Notes: Abstract The localization of phosphoenol pyruvate carboxylase (EC 4.1.1.3.1.) in the leaf cells of Sorghum vulgare was investigated by using three techniques: the conventional aqueous and non aqueous methods gave conflicting results; the immunocytochemical techniques clearly showed that the enzyme is predominantly located in the cytoplasm of mesophyll cells.

Notes: Summary The growth and photosynethetic responses to atmospheric CO2 enrichment of 4 species of C4 grasses grown at two levels of irradiance were studied. We sought to determine whether CO2 enrichment would yield proportionally greater growth enhancement in the C4 grasses when they were grown at low irradiance than when grown at high irradiance. The species studied were Echinochloa crusgalli, Digitaria sanguinalis, Eleusine indica, and Setaria faberi. Plants were grown in controlled environment chambers at 350, 675 and 1,000 μl 1-1 CO2 and 1,000 or 150 μmol m-2 s-1 photosynthetic photon flux density (PPFD). An increase in CO2 concentration and PPFD significantly affected net photosynthesis and total biomass production of all plants. Plants grown at low PPFD had significantly lower rates of photosynthesis, produced less biomass, and had reduced responses to increases in CO2. Plants grown in CO2-enriched atmosphere had lower photosynthetic capacity relative to the low CO2 grown plants when exposed to lower CO2 concentration at the time of measurement, but had greater rate of photosynthesis when exposed to increasing PPFD. The light level under which the plants were growing did not influence the CO2 compensation point for photosynthesis.

Notes: Summary Disagreement has arisen over the presence of actin-containing microfilaments (Mfs) in angiosperm generative cells and sperm (GSP). In order to address this issue, we subjected GSP of Tradescantia virginiana, Nicotiana tabacum and Rhododendron laetum to a series of localizations using different antiactins, rhodamine phalloidin and antimyosin. Coordinate staining with antitubulin and Hoechst 33258 defined the status of the microtubule (Mt) cytoskeleton and stages of generative cell division. Additional experiments utilized cytochalasin D (CD). In no instance could Mfs be detected in GSP of the three species. Instead, Mfs seen at the periphery of GSP appear to be continuous with vegetative Mfs and thus are in the vegetative cytoplasm. Mfs are not seen in the constriction zone of dividing T. virginiana generative cells, nor are they indicated in the phragmoplast of N. tabacum and R. laetum. Myosin localizations reveal punctate staining in the vegetative cytoplasm and a thin line of fluorescence around the the outside of the generative cell. While CD seems to delay generative cell division, cytokinesis still takes place. CD-induced Mf fragments are evident in the vegetative cytoplasm but not in GSP. The weight of evidence therefore indicates that GSP do not contain Mfs. The implications of this conclusion for the behavior of GSP and the mechanism of cytokinesis in dividing generative cells are considerable.

Notes: Summary We have examined the effects of light on circadian locomotor rhythms in retinally degenerate mice (C57BL/6J mice homozygous for the rd allele: rd/rd). The sensitivity of circadian photoreception in these mice was determined by varying the irradiance of a 15 min light pulse (515 nm) given at circadian time 16 and meauring the magnitude of the phase shift of the locomotor rhythm. Experiments were performed on animals 80 days of age. Despite the loss of visual photoreceptors in the rd/rd retina, animals showed circadian responses to light that were indistinguishable from mice with normal retinas (rd/+ and +/+). While no photoreceptor outersegments were identified in the retina of rd/rd animals (80–100 days of age), we did identify a small number of perikarya that were immunoreactive for cone opsins, and even fewer cells that contained rod opsin. Using HPLC, we demonstrated the presence and photoisomerization of the rhodopsin chromophore 11-cis retinaldehyde. The rd/rd retinas contained about 2% of 11-cis retinaldehyde found in +/+ retinas. We have yet to determine whether the opsin immunoreactive perikarya or some other unidentified cell type mediate circadian light detection in the rd/rd retina.

Notes: Abstract Immunogold labelling techniques on ultrathin sections of low temperature embedded cells yielded evidence for the periplasmic location of the respiratory enzymes N2O reductase and nitrite reductase (cytochrome cd 1) in Pseudomonas stutzeri strain ZoBell. Cell fractionation by spheroplast preparation and two-dimensional electrophoresis showed the absence of a membrane association of these enzymes. Immunocytochemical localization of N2O reductase in a mutant strain deficient in the chromophore of N2O reductase showed the gold label at the cell periphery, indicating that the copper chromophore processing takes place after export of this protein's apoform.

Notes: Abstract The effect of SO2 on the photosynthesis ofClethra barbinervis collected from a smoke-polluted area near the Ashio copper smelter in Tochigi Prefecture was compared withC. barbinervis collected from a nonpolluted district in Tsukuba, Ibaraki Prefecture andQuercus mongolica var.grosseserrata grown in a nonpolluted field in Nagano Prefecture. The plants were exposed to 0.5–1.5 p.p.m. SO2 for 90 min (short-term) and to 0.3 p.p.m. SO2 for 31–39 days (long-term). TheClethra plants from both sites had a lower intrinsic stomatal conductance and photosynthetic rate thanQuercus plants. Short-and long-term fumigation caused stomatal closure inQuercus plants, but had little effect on the stomatal conductance ofClethra plants. Under short-term fumigation, nonstomatal photosynthetic inhibition per unit of absorbed SO2 was smallest inClethra plants from Ashio. Long-term fumigation caused photosynthetic decline and visible foliar injury toQuercus plants, but had no effect onClethra plants from Ashio. Consequently,Clethra plants from Ashio had a higher photosynthetic rate thanQuercus plants after long-term fumigation. These results suggest thatC. barbinervis populations in the smoke-polluted area of Ashio had evolved high SO2 resistance connected with SO2 detoxification ability in mesophyll cells.

Notes: Abstract Light saturated net photosynthesis was measured in bracts and leaves ofCarpinus laxiflora, the major species in secondary forests in cool and intermediate temperate zones in Japan. The maximum net photosynthesis of leaves and bracts was essentially constant from May to early August and decreased gradually thereafter. For bracts, it was 3.2 μmol m−2s−1, approximately half that for the leaves. The photosynthesis of bracts would thus appear to contribute significantly to seed maturity. The estimated production of bract based on the photosynthesis would make seeds (3 mg dry weight) mature for 37 days, assuming all photosynthate of the bracts to have been distributed in the seeds only. This was quite consistent with the growth curve for the seeds. A mast year phenomenon is discussed in relation to bract photosynthesis and leaf number.

Notes: Abstract Aucuba japonica varieties are common evergreen understory shrubs in Japan.Aucuba japonica var.borealis is distributed on the Sea of Japan side of Honshu and Hokkaido where heavy snow cover lasts for more than 3 months in winter.Aucuba japonica var.japonica is distributed in areas with shallow or no snow on the Pacific Ocean side of Honshu and Shikoku. The ecophysiological characteristics of var.borealis were compared with those of var.japonica to examine the effects of heavy and long-term snow cover on the life cycle of var.borealis. Shoots of both varieties were shaded in crushed ice for 110 days, but their photosynthetic activities, chlorophyll contents and the chlorophylla/b ratio was not affected. The leaves of var.borealis were no less frost tolerant than those of var.japonica. In spite of the difference in environmental factors, both varieties had similar characteristics in seasonal changes of photosynthesis, respiration and chlorophylla/b ratio. These results suggest that var.japonica could survive in areas with heavy snow where it does not normally occur. Leaf net production (LNP) was estimated based on the microclimatic data and seasonal photosynthetic and respiration rates. The difference in the annual LNP between the two varieties was equivalent to the difference in the LNP during the snow season. One of the major effects of snow cover is to interrupt and reduce the production period of var.borealis.

Notes: Abstract The impacts of juglone on plant growth and several other physiological functions were evaluated in this study. Juglone inhibitedLemna minor growth, chlorophyll content, and net photosynthesis at treatments between 10 and 40μM. Soybean leaf disks vacuum infiltrated with as little as 10μM juglone had reduced photosynthesis. Oxygen evolution by chloroplasts isolated fromPisum sativum was inhibited by juglone with an I50 of 2μM. Micromolar treatments of juglone stimulated oxygen uptake in mitochondria isolated fromGlycine max. These data suggest perturbations of chloroplast and mitochondrial functions may contribute to plant growth reductions observed in juglone-mediated allelopathy.

Notes: Abstract In closed-chamber fumigation experiments dry matter partitioning and chlorophyll fluorescence of wheat were studied, analysing the effects of ozone during different stages of plant development. Ozone causes enhanced leaf senescence, leading to a loss of green leaf area and, consequently to a decreased supply of assimilates, affecting (in increasing order of severeness) stem, ear and grain productivity because of reduced storage pools for translocation. Leaves of plants before shooting stage were most sensitive but the lack of green leaf area after ear emergence had the most pronounced effects on grain yield. Measurements of photochemical capacity showed that evidence for negative ozone effects could be found in changes of chlorophyll fluorescence parameters in leaf sections not yet showing visible ozone injury. Negative effects on photosynthesis were more distinct with increasing accumulated ozone dose, with increasing age of leaf tissue and with increasing ozone sensitivity of the cultivar. The changes in chlorophyll fluorescence are most likely to be explained by a decreased pool size of plastoquinones caused by ozone.

Notes: Abstract A second locus (Lhb1B) encoding Photosystem II Type I chlorophyll a/b-binding (CAB) polypeptides was identified in Arabidopsis thaliana. This locus carries two genes in an inverted orientation. The predicted sequences of the polypeptides encoded by these two genes show substantial divergence in their amino termini relative to each other and to the proteins encoded by the three Lhb1 CAB genes previously characterized [10], but little divergence within the predicted primary structure of the mature protein. DNA probes derived from seven additional types of tomato CAB genes, encoding chlorophyll a/b-binding polypeptides of several antenna systems of the photosynthetic apparatus, were tested against A. thaliana. Each of these hybridized in Southern blots to unique DNA fragment(s), demonstrating the existence of each of these different types of CAB genes in the genome of A. thaliana. The number of genes encoding each CAB type in A. thaliana was estimated to be similar to that of tomato.

Notes: Abstract The activity and location of carbonic anhydrase has been modified by transformation of tobacco with antisense and over-expression constructs. Antisense expression resulted in the inhibition of up to 99% of carbonic anhydrase activity but had no significant impact on net CO2 assimilation. Stomatal conductance and susceptibility to water stress appeared to increase in response to the decline in carbonic anhydrase activity. An over-expression construct designed to increase cytosolic carbonic anhydrase abundance resulted in a significant increase in net activity, a small increase in stomatal conductance but little impact on CO2 assimilation. Chloroplastic carbonic anhydrase activity was enhanced by the expression of an additional construct which targeted the polypeptide to the organelle. The increase in chloroplastic carbonic anhydrase appeared to be accompanied by a concomitant increase in Rubisco activity.

Notes: Abstract The genes encoding the photosynthetic cytochrome b 6 (petB) and subunit 4 (petD) have been cloned and sequenced from the unicellular, photoheterotrophic, transformable cyanobacterium Synechococcus sp. PCC 7002, formerly designated Agmenellum quadruplicatum. The gene arrangement was found to be similar to that reported in the cyanobacterium Nostoc PCC 7906. The DNA and derived protein sequences were compared to chloroplast and the other cyanobacterial sequences. By pulsed-field electrophoresis, the petBD operon and the petCA operon, encoding the Rieske iron-sulfur protein and cytochrome f, were found to be located on separate, unlinked,Not I-digested DNA fragments. ThepetBD operon was found on the third largest Not I fragment (NC-325) while the petCA operon was found on the second largest Not I fragment (NB-370). These results suggest the two operons are not in proximity. The 1.35 kb transcript was shown to be light-regulated. Transcripts from cells grown under constant illumination showed a decrease in petB transcript levels to undetectable levels within 2 h after the cells were placed in the dark. Upon reillumination, transcript levels rose to three-fold over that seen initially under constant illumination.

Notes: Abstract Detailed molecular mechanisms of electron transfer-driven translocation of ions and of the generation of electric fields across biological membranes are beginning to emerge. The ideas inherent in the early formulations of the chemiosmotic hypothesis have provided the framework for this understanding and have also been seminal in promoting many of the experimental approaches which have been successfully used. This article is an attempt to review present understanding of the structures and mechanisms of several osmoenzymes of central importance and to identify and define the underlying features which might be of general relevance to the study of chemiosmotic devices.

Notes: Abstract A mutant strain of the cyanobacterium Synechocystis sp. PCC (Pasteur Culture Collection) 6803 has been developed in which psbB, the gene coding for the chlorophyl a-binding protein CP47 in Photosystem II (PSII), has been deleted. This deletion mutant can be used for the reintroduction of modified psbB into the cyanobacterium. To study the role of a large hydrophilic region in CP47, presumably located on the lumenal side of the thylakoid membrane between the fifth and sixth membrane-spanning regions, specific deletions have been introduced in psbB coding for regions within this domain. One psbB mutation leads to deletion of Gly-351 to Thr-365 in CP47, another psbB mutation was targeted towards deletion of Arg-384 to Val-392 in this protein. The deletion from Gly-351 to Thr-365 results in a loss of PSII activity and of photoautotrophic growth of the mutant, but the deletion between Arg-384 and Val-392 retains PSII activity and the ability to grow photoautotrophically. The mutant strain with the deletion from Gly-351 to Thr-365 does not assemble a stable PSII reaction center complex in its thylakoid membranes, and exhibits diminished levels of CP47 and of the reaction center proteins D1 and D2. In contrast to the Arg-384 to Val-392 portion of this domain, the region between Gly-351 and Thr-365 appears essential for the normal structure and function of photosystem II.

Notes: Abstract We studied assembly of the PsaE subunit of photosystem I into photosynthetic membranes of cyanobacterial mutant strains that lack specific photosystem I subunits. Radiolabeled PsaE was incubated with photosynthetic membranes, and their binding and assembly were assayed by resistance to removal by chaotropic agents and proteolytic digestion. PsaE incorporated into the wild-type membranes was resistant to these treatments. In the absence of PsaD, it was resistant to proteolytic digestion, but was removed by NaBr. When the membranes were isolated from a mutant strain in which the psaF and psaJ genes have been inactivated, PsaE assembled in vitro could not be removed. PsaE could associate with the membranes of the strain DF in which the psaD, psaJ and psaF genes have been mutated. However, the radiolabeled PsaE associated with these membranes was removed both by the proteolytic as well as by the chaotropic agents. Characterization of PsaE present in vivo revealed similar results. These observations suggest that PsaD and PsaF/J may interact with PsaE and stabilize it in the photosystem I complex.

Notes: Abstract The unicellular cyanobacterium Synechococcus sp. PCC 7942 has three psbA genes encoding two different forms of the photosystem II reaction centre protein D1 (D1:1 and D1:2). The level of expression of these psbA genes and the synthesis of D1:1 and D1:2 are strongly regulated under varying light conditions. In order to better understand the regulatory mechanisms underlying these processes, we have constructed a strain of Synechococcus sp. PCC 7942 capable of over-producing psbA mRNA and D1 protein. In this study, we describe the over-expression of D1:1 using a tac-hybrid promoter in front of the psbAI gene in combination with lacI Q repressor system. Over-production of D1:1 was induced by growing cells for 12 h at 50 μmol photons m-2 s-1 in the presence of 40 or 80 μg/ml IPTG. The amount of psbAI mRNA and that of D1:1 protein in cells grown with IPTG was three times and two times higher, respectively. A higher concentration of IPTG (i.e., 150 μg/ml) did not further increase the production of the psbAI message or D1:1. The over-production of D1:1 caused a decrease in the level of D1:2 synthesised, resulting in most PSII reaction centres containing D1:1. However, the over-production of D1:1 had no effect on the pigment composition (chlorophyll a or phycocyanin/number of cells) or the light-saturated rate of photosynthesis. This and the fact that the total amounts of D1 and D2 proteins were not affected by IPTG suggest that the number of PSII centres within the membranes remained unchanged. From these results, we conclude that expression of psbAI can be regulated by using the tac promoter and lacI Q system. However, the accumulation of D1:1 protein into the membrane is regulated by the number of PSII centres.

Notes: Abstract ADP-ribosylation factor (ARF) is a highly conserved, low molecular mass (ca. 21 kDa) GTP-binding protein that has been implicated in vesicle trafficking and signal transduction in yeast and mammalian cells. However, little is known of ARF in plant systems. A putative ARF polypeptide was identifed in subcellular fractions of the green alga Chlamydomonas reinhardtii, based on [32P]GTP binding and immunoblot assays. A cDNA clone was isolated from Chlamydomonas (Arf1), which encodes a 20.7 kDa protein with 90% identity to human ARF1. Northern blot analyses showed that levels of Arf1 mRNA are highly regulated during 12 h/12 h light/dark (LD) cycles. A biphasic pattern of expression was observed: a transient peak of Arf1 mRNA occurred at the onset of the light period, which was followed ca. 12 h later by a more prominent peak in the early to mid-dark period. When LD-synchronized cells were shifted to continuous darkness, the dark-specific peak of Arf1 mRNA persisted, indicative of a circadian rhythm. The increase in Arf1 mRNA at the beginning of the light period, however, was shown to be light-dependent, and, moreover, dependent on photosynthesis, since it was prevented by DCMU. We conclude that the biphasic pattern of Arf1 mRNA accumulation during LD cycles is due to regulation by two different factors, light (which requires photosynthesis) and the circadian clock. Thus, these studies identify a novel pattern of expression for a GTP-binding protein gene.

Notes: Abstract Over-expression of the psbAIII gene encoding for the D1 protein (form II; D1:2) of the photosystem II reaction centre in the Synechococcus sp. PCC 7942 was studied using a tac promoter and the lacI Q system. Over-expression was induced with 40 μg/ml IPTG in the growth medium for either 6 or 12 h at growth irradiance (50 μmol photons m-2 s-1). This treatment doubled the amount of psbAII/III mRNA and the D1:2 protein in membranes but decreased the amount of psbAI messages and the D1:1 protein. The total amount of both heterodimeric reaction centre proteins, D1 and D2, remained constant under growth light conditions, indicating that the number of PSII centres in the membranes was not affected, only the form of the D1 protein was changed from D1:1 to D1:2 in most centres. When the cells were photoinhibited either at 500 or 1000 μmol photons m-2 s-1, in the presence or absence of the protein synthesis inhibitor lincomycin, the D1:2 protein remained at a higher level in cells in which over-expression had been induced by IPTG. These cells were also less prone to photoinhibition of PSII. It is suggested that the tolerance of cells to photoinhibition increases when most PSII reaction centres contain the D1:2 protein at the beginning of high irradiance. This tolerance is further strengthened by maintaining psbAIII gene over-expression during the photoinhibitory treatment.

Notes: Abstract Phosphoenolpyruvate carboxylase (PEPC) genes from Corynebacterium glutamicum (cppc), Escherichia coli (eppc) or Flaveria trinervia (fppc) were transferred to Solanum tuberosum. Plant regenerants producing foreign PEPC were identified by Western blot analysis. Maximum PEPC activities measured in eppc and fppc plants grown in the greenhouse were doubled compared to control plants. For cppc a transgenic plant line could be selected which exhibited a fourfold increase in PEPC activity. In the presence of acetyl-CoA, a known activator of the procaryotic PEPC, a sixfold higher activity level was observed. In cppc plants grown in axenic culture PEPC activities were even higher. There was a 6-fold or 12-fold increase in the PEPC activities compared to the controls measured in the absence or presence of acetyl-CoA, respectively. Comparable results were obtained by transient expression in Nicotiana tabacum protoplasts. PEPC of C. glutamicum (PEPC C.g.) in S. tuberosum leaf extracts displays its characteristic K m(PEP) value. Plant growth was examined with plants showing high expression of PEPC and, moreover, with a plant cell line expressing and antisense S. tuberosum (anti-sppc) gene. In axenic culture the growth rate of a cppc plant cell line was appreciably diminished, whereas growth rates of an anti-sppc line were similar or slightly higher than in controls. Malate levels were increased in cppc plants and decreased in antisense plants. There were no significant differences in photosynthetic electron transport or steady state CO2 assimilation between control plants and transformants overexpressing PEPC C.g. or anti-sppc plants. However, a prolonged dark treatment resulted in a delayed induction of photosynthetic electron transport in plants with less PEPC. Rates of CO2 release in the dark determined after a 45 min illumination period at a high proton flux density were considerably enhanced in cppc plants and slightly diminished in anti-sppc plants. When CO2 assimilation rates were corrected for estimated rates of mitochondrial respiration in the light, the electron requirement for CO2 assimilation determined in low CO2 was slightly lower in transformants with higher PEPC, whereas transformants with decreased PEPC exhibited an appreciably elevated electron requirement. The CO2 compensation point remained unchanged in plants (cppc) with high PEPC activity, but might be increased in an antisense plant cell line. Stomatal opening was delayed in antisense plants, but was accelerated in plants overexpressing PEPC C.g. compared to the controls.

Notes: Abstract CP 47, a component of photosystem II (PSII) in higher plants, algae and cyanobacteria, is encoded by the psbB gene. Site-specific mutagenesis has been used to alter a portion of the psbB gene encoding the large extrinsic loop E of CP 47 in the cyanobacterium Synechocystis 6803. Alteration of a lysine residue occurring at position 321 to glycine produced a strain with altered PSII activity. This strain grew at wild-type rates in complete BG-11 media (480 µM chloride). However, oxygen evolution rates for this mutant in complete media were only 60% of the observed wild-type rates. Quantum yield measurements at low light intensities indicated that the mutant had 66% of the fully functional PSII centers contained in the control strain. The mutant proved to be extremely sensitive to photoinactivation at high light intensities, exhibiting a 3-fold increase in the rate of photoinactivation. When this mutant was grown in media depleted of chloride (30 µM chloride), it lost the ability to grow photoautotrophically while the control strain exhibited a normal rate of growth. The effect of chloride depletion on the growth rate of the mutant was reversed by the addition of 480 µM bromide to the chloride-depleted BG-11 media. In the presence of glucose, the mutant and control strains grew at comparable rates in either chloride-containing or chloride-depleted media. Oxygen evolution rates for the mutant were further depressed (28% of control rates) under chloride-limiting conditions. Addition of bromide restored these rates to those observed under chloride-sufficient conditions. Measurements of the variable fluorescence yield indicated that the mutant assembled fewer functional centers in the absence of chloride. These results indicate that the mutation K321G in CP 47 affects PSII stability and/or assembly under conditions where chloride is limiting.

Notes: Abstract The two operons atp1 and atp2, encoding the subunits of the FOF1 ATP-synthase, have been cloned and sequenced from the cyanobacterium Synechocystis sp. PCC 6803. The organization of the different genes in the operons have been found to resemble that of the cyanobacteria Synechococcus sp. PCC 6301 and Anabaena sp. PCC 7120. The Synechocystis FOF1 ATP-synthase has nine subunits. A tenth open reading frame with unknown function was detected at the 5′ end of atp1, coding for a putative gene product similar to uncI in Escherichia coli. A promoter structure was inferred for the Synechocystis atp operons and compared to other known promoters of cyanobacteria. Even though the operon structure of atp1 and atp2 in Synechocystis resembles the corresponding operons of Synechococcus, the amino acid sequences of individual gene products show marked differences. Genetic distances between cyanobacterial genes and genes for ATP-synthase subunits from other species have been calculated and compiled into evolutionary trees.

Notes: Abstract A shift in the ratio of chlorophyll (Chl) a and Chl b is an early indicator of heat bleaching in Euglena gracilis. This observation prompted us to consider whether or not changes in steady-state levels of chloroplast transcripts and in transcriptional activity could limit the synthesis of Chl a-binding proteins in bleaching plastids. We found that the mature transcripts for CP47 and CP43, the Chl a-binding apoproteins of the proximal antenna of photosystem II, decline sharply very early during bleaching. Our study also shows that transcription of psbB and psbC, the chloroplast genes encoding CP47 and CP43, remains essentially unchanged during the same interval. We conclude that posttranscriptional events, such as mRNA stability, could play a major role in initiating an irreversible loss of chloroplast function in Euglena at a moderately elevated temperature. Lack of these transcripts would eventually impair the assembly of photosystem II in thylakoids.

Notes: Abstract The levels of chlorophyll a/b-binding protein (Cab) gene polysomal poly(A)+ mRNA were quantitated throughout the development of Glycine max L. Cab mRNAs were abundant in young expanding leaves, representing 6.1% of the leaf mRNA population. Lower Cab mRNA levels were present in embryos, stems, and cotyledons of developing seedlings; the lowest levels were found in roots where they accounted for 0.04% of the polysomal poly(A)+ mRNA of this organ. To determine the contribution of different members of the Cab gene family to the Cab mRNA populations, a quantitative S1 nuclease reconstruction assay was developed. Cab3, Cab4, and Cab5 mRNAs were detected in all stages examined during soybean development but their levels underwent differential changes. Cab3 encodes the most abundant Cab mRNA in young leaves, developing embryos, and in Stage VII cotyledons from the developing soybean seedling. The levels of Cab mRNAs were compared to the levels of ribulose-1,5-bisphosphate carboxylase small subunit gene mRNA and differences in their patterns of accumulation were noted. Collectively these data indicate that during soybean embryogenesis developmental control mechanisms supersede light-regulatory signals.

Notes: Abstract DNA sequence, copy number, expression and phylogenetic relevance of the psbA gene from the abundant marine prokaryote P. marinus CCMP 1375 was analyzed. The 7 amino acids near the C-terminus missing in higher plant and in Prochlorothrix hollandica D1 proteins are present in the derived amino acid sequence. P. marinus contains only a single psbA gene. Thus, this organism lacks the ability to adapt its photosystem II by replacement of one type of D1 by another, as several cyanobacteria do. Phylogenetic trees suggested the D1-1 iso-form from Synechococcus PCC 7942 as the next related D1 protein and place P. Marinus separately from Prochlorothrix hollandica among the cyanobacteria.

Notes: Abstract Part of the chlL gene encoding a component involved in light-independent protochlorophyllide reduction was deleted in wild type and in a photosystem I-less strain of Synechocystis sp. PCC 6803. In resulting mutants, chlorophyll biosynthesis was fully light-dependent. When these mutants were propagated under light-activated heterotrophic growth conditions (in darkness except for 15 min of weak light a day) for several weeks, essentially no chlorophyll was detectable but protochlorophyllide accumulated. Upon return of the chlL - mutant cultures to continuous light, within the first 6 h chlorophyll was synthesized at the expense of protochlorophyllide at a rate independent of the presence of photosystem I. Chlorophyll biosynthesized during this time gave rise to a 685 nm fluorescence emission peak at 77 K in intact cells. This peak most likely originates from a component different from those known to be directly associated with photosystems II and I. Development of 695 and 725 nm peaks (indicative of intact photosystem II and photosystem I, respectively) required longer exposures to light. After 6 h of greening, the rate of chlorophyll synthesis slowed as protochlorophyllide was depleted. In the chlL - strain, greening occurred at the same rate at two different light intensities (5 and 50 μE m-2s-1), indicating that also at low light intensity the amount of light is not rate-limiting for protochlorophyllide reduction. Thus, in this system the rate of chlorophyll biosynthesis is limited neither by biosynthesis of photosystems nor by the light-dependent protochlorophyllide reduction. We suggest the presence of a chlorophyll-binding ‘chelator’ protein (with 77 K fluorescence emission at 685 nm) that binds newly synthesized chlorophyll and that provides chlorophyll for newly synthesized photosynthetic reaction centers and antennae.

Notes: Summary The region of the chloroplast genome of Chlamydomonas reinhardii containing the gene of the thylakoid polypeptide D2 (psbD) has been sequenced. A unique open reading frame of 350 codons exists in this region. Because the first ATG is followed 11 codons downstream by a second one, the D2 polypeptide consists of either 339 or 350 amino acids. Comparison of the sequences of D2 and the 32K dalton polypeptides, both of which are associated with photosystem II, reveals partial homology. Although, the overall homology of these two polypeptides is only 27%, they contain several related regions and their hydropathic profiles are strikingly similar. These data suggest that the two polypeptides may have related functions and/or that their genes may have originated from a common ancestor. Alternatively, convergent evolution of these polypeptides may be due to structural constraints in the thylakoid membrane. Limited sequence homology is also observed between the D2 polypeptide and some of the subunits of the reaction centers of photosynthetic bacteria.

Notes: Abstract To identify amino acid residues of the D2 protein that are critical for functional photosystem II (PS II), sodium bisulfite was utilized for in vitro random mutagenesis of the psbDI gene from Synechocystis sp. PCC 6803. Sodium bisulfite reacts specifically with cytosine in single-stranded regions of DNA and does not attack double-stranded DNA. Using a hybrid plasmid that was single-stranded in the region to be mutagenized and that was double-stranded elsewhere, mutations were targeted to a specific psbDI region coding for the lumenal A-B loop of the D2 protein. Several mutants were isolated with a total of 15 different amino acid changes in the loop. The majority of these mutations did not result in a loss of photoautotrophic growth or in significantly altered PS II function. However, mutation of Glu-69 to Lys, Ser-79 to Phe, and Ser-88 to Phe were found to influence photosystem II activity; the importance of the latter two residues for proper PS II function was unexpected. Cells carrying the double mutation S79F/S88F in D2 did not grow photoautotrophically and had no functionally active PS II centers. The single mutant S79F was also incapable of photoautrophic growth, but displayed reasonably stable oxygen evolution, while PS II function in the single mutant S88F appeared to be close to normal. Because of the more pronounced phenotype of the S79F/S88F strain as compared to the single mutants, both Ser residues appear to affect stable assembly and function of the PS II complex. The mechanism by which the S79F mutant loses photoautotrophic growth remains to be established. However, these results show the potential of targeted random mutagenesis to identify functionally important residues in selected regions of proteins.

Notes: Abstract. An antiserum raised against the purified 33-kDa β-1,3-glucanase of wheat (Triticum aestivum L.) was employed to investigate the ultrastructural localization of the enzyme in wheat leaves infected with Puccinia recondita Rob. ex Desm. f.sp. tritici Eriks. and Henn. using a post-embedding immunogold labelling technique. In both compatible and incompatible interactions, β-1,3-glucanase was detected in the host plasmalemma and in the domain of the host cell wall near the plasmalemma of the mesophyll cells, but higher concentrations of the enzyme were detected in infected resistant wheat leaves than in infected susceptible ones. β-1,3-Glucanase was also found in the secondary thickening of xylem vessels and in the walls of guard cells, epidermal cells and phloem elements, while no labelling was observed in host organelles, viz. vacuoles, mitochondria, endoplasmic reticulum, Golgi bodies, nuclei and chloroplasts. A low concentration of the enzyme was detected on the intercellular hyphal wall and in the hyphal cytoplasm. In the compatible interaction, β-1,3-glucanase was demonstrated to accumulate predominantly in the haustorial wall and extrahaustorial matrix. In the incompatible interaction, strong labelling for β-1,3-glucanase was found in host cell wall appositions, in the extracellular matrix in the intercellular space, and in electron-dense structures of host origin which occurred in the incompatible interaction only.

Notes: Summary The effect of the pore-forming antibiotic gramicidin on pure lipid membranes is well characterized. We studied its action in protein-rich thylakoid membranes that contain less than 25% (wt/wt) acyl lipids. A transmembrane voltage was induced by flashing light, and its decay was measured and interpreted to yield the distribution of gramicidin over thylakoids, its dimerization constant and its single-channel conductance in this membrane. The distribution of gramicidin over the ensemble of thylakoids was immediately homogeneous when the antibiotic was added under stirring, while it became homogeneous only after 20 min in a stirred suspension that was initially heterogeneous. The dimerization constant, 5×1014 cm2/mol, was about 10 times larger than in pure lipid membranes. This was attributed to the upconcentration of gramicidin in the small fractional area of protein free lipid bilayer and further by a preference of gramicidin for stacked portions of the membrane. The latter bears important consequences with regard to bioenergetic studies with this ionophore. As gramicidin was largely dimerized from a concentration of 1 nm (in the suspension) on, the membrane's conductance then increased linearly as a function of added gramicidin. When the negative surface potential at the thylakoid membrane was screened, the conductance of a single gramicidin dimer agreed well with figures reported for bilayers from neutral lipid (about 0.5 pS at 10 mm NaCl). The modulation of the conductance by the surface potential in spinach versus pea thylakoids and between different preparations is discussed in detail.

Notes: Summary The rate of inorganic carbon uptake and its steadystate accumulation ratio (intracellular/extracellular concentration) was determined in the cyanobacteriumAnabaena variabilis as a function of extracellular pH. The free energy of protons ( $$\Delta \overline \mu _{H^ - }$$ ) across the plasmalemma was calculated from determinations of membrane potential, and intracellular pH, as a function of the extracellular pH. While inward proton motive force decreased with increasing extracellular pH from 6.5 to 9.5, rate of HCO 3 − influx and its accumulation ration increased. The latter is several times larger than would be expected should HCO 3 − influx be driven by $$\Delta \overline \mu _{H^ + }$$ . It is concluded that HCO 3 − transport in cyanobacteria is not driven by the proton motive force.

Notes: Abstract Aquaporin 2 is a collecting duct water channel that is located in apical vesicles and in the apical plasma membrane of collecting duct principal cells. It shares 42% identity with the proximal tubule/thin descending limb water channel, CHIP28. The present study was aimed at addressing three questions concerning the location and behavior of the AQP2 protein under different conditions. First, does the AQP2 channel relocate to the apical membrane after vasopressin treatment? Our results show that AQP2 is diffusely distributed in cytoplasmic vesicles in collecting duct principal cells of homozygous Brattleboro rats that lack vasopressin. In rats injected with exogenous vasopressin, however, AQP2 became concentrated in the apical plasma membrane of principal cells, as determined by immunofluorescence and immunogold electron microscopy. This behavior is consistent with the idea that AQP2 is the vasopressin-sensitive water channel. Second, is the cellular location of AQP2 modified by microtubule disruption? In normal rats, AQP2 has a mainly apical and subapical location in principal cells, but in colchicine-treated rats, it is distributed on vesicles that are scattered throughout the entire cytoplasm. This is consistent with the dependence on microtubules of apical protein targeting in many cell types, and explains the inhibitory effect of microtubule disruption on the hydroosmotic response to vasopressin in sensitive epithelia, including the collecting duct. Third, is AQP2 present in neonatal rat kidneys? We show that AQP2 is abundant in principal cells from neonatal rats at all days after birth. The detection of AQP2 in early neonatal kidneys indicates that a lack of this protein is not responsible for the relatively weak urinary concentrating response to vasopressin seen in neonatal rats.

Notes: Summary We used physiological recordings, intracellular dye injections and immunocytochemistry to further identify and characterize neurons in the buccal ganglia of Aplysia calif ornica expressing Small Cardioactive Peptide-like immunoreactivity (SCP-LI). Neurons were identified based upon soma size and position, input from premotor cells B4 and B5, axonal projections, muscle innervation patterns, and neuromuscular synaptic properties. SCP-LI was observed in several large ventral neurons including B6, B7, B9, B10, and B11, groups of s1 and s2 cluster cells, at least one cell located at a branch point of buccal nerve n2, and the previously characterized neurons B1, B2 and B15. B6, B7, B9, B10 and B11 are motoneurons to intrinsic muscles of the buccal mass, each displaying a unique innervation pattern and neuromuscular plasticity. Combined, these motoneurons innervate all major intrinsic buccal muscles (I1/I3, I2, I4, I5, I6). Correspondingly, SCP-LI processes were observed on all of these muscles. Innervation of multiple nonhomologous buccal muscles by individual motoneurons having extremely plastic neuromuscular synapses, represents a unique form of neuromuscular organization which is prevalent in this system. Our results show numerous SCPergic buccal motoneurons with widespread ganglionic processes and buccal muscle innervation, and support extensive use of SCPs in the control of feeding musculature.

Notes: Summary The activity of acid phosphatase produced in pure culture by the endomycorrhizal fungus Hymenoscyphus ericae (Read) Korf & Kernan (H. ericae LPA 2) was inhibited by high phosphorus levels, alkaline pH, fluoride, molybdate and mannosidase, and activated by concanavalin A. Over 80% of the enzyme activity was due to two wall-bound acid phosphatase isozymes with the characteristics of mannose-rich glycoproteins. Antiserum was raised against the major, low-molecular-weight wall isozyme and its activity tested by immunoblotting and ELISA. The antiserum cross reacted 100% with exocellular (excreted) and 28% with cytoplasmic cellular fractions of H. ericae (LPA 2) cultures, and showed high reactivity with other strains of H. ericae but not with fungal isolates from Erica hispidula L. or E. mauritanica L. Ultrastructural localization of acid phosphatase by cytoenzymology and indirect immunogold labelling confirmed its association with the fungal wall in pure culture and showed that the influence of a high phosphorus level, fluoride and molybdate is through inactivation of the enzyme. Intense acid phosphatase activity, sensitive to the latter inhibitors, was also present on external hyphae growing over a host or non-host root but it was weak or absent from intracellular hyphae where these developed within a host root. Indirect immunolabelling confirmed that this acid phosphatase was of fungal origin and that the specific inhibitory effect of host cells is due to inactivation of the enzyme rather than repression of its synthesis. Possible implications of fungal acid phosphatase in ericoid endomycorrhizal infection processes are discussed together with mechanisms that may be regulating the enzyme activity.

Notes: Abstract We produced three monoclonal antibodies, SG1, SG2 and SG3, specific for human seminal γ-glutamyltransferase when characterized by enzyme-linked immunosorbent assay and immunoblotting. Seminal γ-glutamyltransferase was localized, by immunostaining, to the epithelial cells of the ductus epididymidis, seminal vesicle and prostate gland with SG1, those of the prostate gland with SG2, and those of the seminal vesicle with SG3. Rabbit polyclonal anti-seminal γ-glutamyltransferase serum reacted with the proximal convolution of the kidney and the bile capillaries of the liver, and with the epithelial cells of the reproductive organs. However, immunoreactivity was not observed in the kidney or liver with the monoclonal antibodies. Thus, these monoclonal antibodies are probably all specific to seminal γ-glutamyltransferase but recognize different epitopes.

Notes: Abstract Mammary-derived growth inhibitor (MDGI) has previously been localized in the mammary parencyma, dependent on the stage of differentiation of the mammary gland. Here, we have elucidated the distribution of MDGI in the mammary stroma by a combined immunohisto-and cytochemical analysis with antibodies raised against MDGI. Distinct staining of capillary endothelial cells has been revealed. Although its subcellular distribution resembles former observations in secretory epithelial cells, the expression of MDGI in capillary endothelial cells clearly precedes that in secretory epithelial cells. On the other hand, no endothelial MDGI staining has been detected in bovine heart, which contains a fatty acid-binding protein almost identical to MDGI. The localization of MDGI in the mammary capillary endothelium is discussed in terms of its possible involvement in the intracellular transport of hydrophobic ligands or in the regulation of endothelial cell proliferation.

Notes: Abstract. The present study provides light- and electron-microscopic immunocytochemical data on the presence of neurons that are immunoreactive to the C-terminal flanking peptide of neuropeptide Y, C-PON, in the neostriatum of the hedgehog (Erinaceus europaeus). Positive neurons have mostly fusiform or round perikarya from which two to four poorly branched processes arise. Immunostained fibers and puncta are also evenly distributed throughout the neostriatum. Ultrastructurally, each neuron exhibits a deeply invaginated nucleus surrounded by abundant cytoplasm with a well-developed rough endoplasmic reticulum and Golgi apparatus. Positive neurons receive symmetric and asymmetric synapses from unlabeled terminals. The results of this study can be correlated with previous findings, as the C-PON-positive neurons of the hedgehog resemble medium-sized neostriatal neurons that are known to be local circuit neurons exhibiting C-PON in the rat. Thus, a high degree of C-PON neuronal system phylogenetic conservation and function can be postulated for the neostriatum of mammals.

Notes: Abstract Serotonin- and ten peptide-immunoreactive (IR) cell types were identified in the digestive tract of sea bass (Dicentrarchus labrax L.) larvae of four morphofunctional phases ranging in age from hatching to 61 days. The sequence of appearance and location of endocrine cells during ontogenetic development of the larvae was determined. The differentiation of endocrine cells followed a distal-proximal gradient in the gut which paralleled the morphofunctional differentiation. Serotonin-IR cells were identified in the last portion of the digestive tract from phase I onwards and in the gastric region from phase III, before these regions were morphofunctionally differentiated; met-enkephalin-IR cells were identified from phase II onwards in both the differentiated rectum and the undifferentiated intestine; cholecystokinin (CCK)- and synthetic human gastrin-34-IR cells were located only in the intestine and first found in the undifferentiated intestine of phase II; human gastrin-17-, peptide YY (PYY)- and neuropeptide Y (NPY)-IR cells appeared in the intestine from phase II and in stomach in phase IV, when it showed gastric glands; pancreatic polypeptide (PP)- and glucagon-IR cells were observed in both intestine and stomach, but insulin- and somatostatin-IR cells only in stomach, from phase III, during which the intestine but not the stomach was differentiated. PP- and PYY-, PP- and glucagon-, and PYY- and glucagon-like immunoreactivities coexisted from their first appearance in some cells of the gut.

Notes: Abstract. Serotonin- and ten peptide-immunoreactive (IR) cell types were identified in the digestive tract of sea bass (Dicentrarchus labrax L.) larvae of four morphofunctional phases ranging in age from hatching to 61 days. The sequence of appearance and location of endocrine cells during ontogenetic development of the larvae was determined. The differentiation of endocrine cells followed a distal-proximal gradient in the gut which paralleled the morphofunctional differentiation. Serotonin-IR cells were identified in the last portion of the digestive tract from phase I onwards and in the gastric region from phase III, before these regions were morphofunctionally differentiated; met-enkephalin-IR cells were identified from phase II onwards in both the differentiated rectum and the undifferentiated intestine; cholecystokinin (CCK)- and synthetic human gastrin-34-IR cells were located only in the intestine and first found in the undifferentiated intestine of phase II; human gastrin-17-, peptide YY (PYY)- and neuropeptide Y (NPY)-IR cells appeared in the intestine from phase II and in stomach in phase IV, when it showed gastric glands; pancreatic polypeptide (PP)- and glucagon-IR cells were observed in both intestine and stomach, but insulin- and somatostatin-IR cells only in stomach, from phase III, during which the intestine but not the stomach was differentiated. PP- and PYY-, PP- and glucagon-, and PYY- and glucagon-like immunoreactivities coexisted from their first appearance in some cells of the gut.

Notes: Abstract. Triple fluorescence labelling was employed to reveal the distribution of chemically identified neurons within the pontine laterodorsal tegmental nucleus and dorsal raphe nucleus which supply branching collateral input to the central nucleus of the amygdala and hypothalamic paraventricular nucleus. The chemical identity of neurons in the laterodorsal tegmental nucleus was revealed by immunocytochemical detection of choline- acetyltransferase or substance P; in the dorsal raphe nucleus, the chemical content of the neurons was revealed with antibody recognizing serotonin. The projections were defined by injections of two retrograde tracers, rhodamine- and fluorescein-labelled latex microspheres, in the central nucleus of the amygdala and paraventricular nucleus, respectively. Neurons projecting to both the central nucleus of the amygdala and the paraventricular nucleus were distributed primarily within the caudal extensions of the laterodorsal tegmental nucleus and dorsal raphe nucleus. Approximately 11% and 7% of the labelled cells in the laterodorsal tegmental nucleus and dorsal raphe nucleus projected via branching collaterals to the paraventricular nucleus and central nucleus of the amygdala. About half of these neurons in the laterodorsal tegmental nucleus were cholinergic, and one-third were substance-P-ergic; in the dorsal raphe nucleus, approximately half of the neurons containing both retrograde tracers were serotonergic. These results indicate that pontine neurons may simultaneously transmit signals to the central nucleus of the amygdala and paraventricular nucleus and that several different neuroactive substances are found in the neurons participating in these pathways. This coordinated signalling may lead to synchronized responses of the central nucleus of the amygdala and paraventricular nucleus for the maintenance of homeostasis. Interactions between different neuroactive substances at the target site may serve to modulate the responses of individual neurons.

Notes: Abstract. Haemocytes of the hard tick Ixodes ricinus were characterized on the basis of their ultrastructure, their ability to ingest foreign material, and to produce or store molecules of the immune defence. Distinction was made between types of haemocytes according to the absence or presence of granular inclusions, shape and size of the lysosomal compartment or the rough endoplasmic reticulum, and ultrastructural and functional similarity to the corresponding haemocytes of insects. Three types of haemocytes were found in adult ticks: plasmatocytes and type-I and type-II granular haemocytes, respectively. The precipitated reaction product of acid phosphatase activity revealed the shape of the lysosomal compartment. The additional injection of particulate materials into the haemocoel further revealed the endocytic activity of the haemocytes. The lysozyme-like immunoreactivity of the haemocytes suggests bactericidal potential. Detection of immunoreactivity in haemocytes to a 25 kDa antigenic protein involved in cuticle formation further suggests their involvement in wound healing and encapsulation.

Notes: Abstract. In bird skin, nerve fibres develop in the dermis but do not enter the epidermis. In co-cultures of 7-day-old chick embryo dorsal root ganglia and epidermis, the neurites also avoid the epidermis. Previous studies have shown that chondroitin sulphate proteoglycans may be involved. Chondroitin sulphate has therefore been visualized by immunocytochemistry, using the monoclonal antibody CS-56, both in vivo and in vitro using light and electron microscopy. Its distribution was compared to those of 2 other chondroitin sulphate epitopes and to that of the growing nerve fibres. In cultures of epidermis from 7-day-old embryonic chicks, immunoreactivity is found uniformly around the epidermal cells while at 7.5 days the distribution in dermis is heterogeneous, and particularly marked in feather buds. In vivo, chondroitin sulphate immunoreactivity is detected in the epidermis, on the basal lamina, on the surfaces of fibroblasts and along collagen fibrils. This localization is complementary to the distribution of cutaneous nerves. Chondroitin sulphate in the basal lamina could prevent innervation of the epidermis and the dermal heterogeneities could partly explain the nerve fibres surrounding the base of the feathers. Chondroitin sulphate could therefore be important for neural guidance in developing chick skin.

Notes: Abstract. Stanniocalcin-immunoreactive cells were localized in the corpuscles of Stannius of a holostean fish, the garpike (Lepisosteus osseus), using antisera against salmon and trout stanniocalcins and the peroxidase-antiperoxidase and protein A-gold immunohistochemical methods. The stanniocalcin-immunoreactive cells were periodic acid-Schiff-positive, and antibody staining was abolished if the antiserum was preabsorbed with corpuscle homogenate. Immunocytochemistry revealed two reactive cell types in the glandular parenchyma, and immunoreactivity was confined to the secretory granules. Staining of the granules was also abolished when the antisera were blocked with crude corpuscle homogenate. When corpuscle extracts from garpike were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis, a single dense band was evident with a molecular weight of ∼68 kDa under non-reducing conditions, whereas three bands were observed (∼29, ∼31, and ∼34 kDa) under reducing conditions. Staining of all bands disappeared following preabsorption of the antiserum with salmon stanniocalcin, trout stanniocalcin, or garpike corpuscle extract. The results are compared with stanniocalcins from another extant holostean, the bowfin (Amia calva), and from more modern bony fishes, the teleosts.

Notes: Abstract Stanniocalcin-immunoreactive cells were localized in the corpuscles of Stannius of a holostean fish, the garpike (Lepisosteus osseus), using antisera against salmon and trout stanniocalcins and the peroxidase-antiperoxidase and protein A-gold immunohistochemical methods. The stanniocalcin-immunoreactive cells were periodic acid-Schiff-positive, and antibody staining was abolished if the antiserum was preabsorbed with corpuscle homogenate. Immunocytochemistry revealed two reactive cell types in the glandular parenchyma, and immunoreactivity was confined to the secretory granules. Staining of the granules was also abolished when the antisera were blocked with crude corpuscle homogenate. When corpuscle extracts from garpike were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis, a single dense band was evident with a molecular weight of ∼68 kDa under non-reducing conditions, whereas three bands were observed (∼29, ∼31, and ∼34 kDa) under reducing conditions. Staining of all bands disappeared following preabsorption of the antiserum with salmon stanniocalcin, trout stanniocalcin, or garpike corpuscle extract. The results are compared with stanniocalcins from another extant holostean, the bowfin (Amia calva), and from more modern bony fishes, the teleosts.

Notes: Abstract Expression of certain cytokeratins can be indicative of the state of differentiation of epithelial cells. The basal cells in the normal adult human prostatic epithelium are characterized by the expression of cytokeratins 5 and 14, whereas the secretory luminal cells contain cytokeratins 8 and 18. Cells cultured from the prostatic epithelium expressed cytokeratins 5, 8, and 18, and thus had features of both basal and luminal cells. Certain growth-inhibitory conditions altered keratin expression in conjunction with growth modulation. Deletion of peptide factors and hormones from the culture medium induced the expression of cytokeratins 1 and 10, associated with a squamous phenotype. These same squamous keratins were found in very dense, stratified cultures that were maintained at confluency in standard, complete medium for extended periods. Retinoic acid enhanced the expression of secretory luminal cell-associated cytokeratins 8 and 18 in semi-confluent cultures. Other growth inhibitory factors such as suramin, transforming growth factor-β, and interferon-γ had no effect on keratin expression. These observations indicate that the differentiation of prostatic epithelial cells can be directed toward alternate pathways, either squamous or secretory, by different growth-inhibitory conditions. However, not all growth inhibitory factors altered differentiation, demonstrating that growth inhibition in itself is not a sufficient inducer of differentiation.

Notes: Abstract The localization of an acrosomal protein was studied using a monoclonal antibody MN7 raised against mouse spermatozoa. MN7 specifically recognized the anterior acrosome of several mammalian (mouse, rat, hamster) spermatozoa fixed with paraformaldehyde. An immunoblot study with periodate treatment showed that MN7 recognized a carbohydrate region of a 90 kDa protein in an extract of mouse and rat cauda epididymal spermatozoa. The change in distribution of the MN7 antigen during acrosome development was investigated in the rat testis using the pre-embedding immunoperoxidase technique. The antigen first appeared in the proacrosomic granules of spermatids in steps 1–2. Small vesicles adjacent to the outer acrosomal membrane and the developing acrosomic system were immunoreactive during steps 4–7. The majority of the antigen was then redistributed to the head-cap portion during steps 8–18, and finally restricted to the anterior acrosome in the step 19-spermatid. These results suggest that the antigen is transported to the acrosome by way of the vesicles that originate from the Golgi apparatus during early spermiogenesis, and are then delivered to the final destination within the acrosome by the intra-acrosomal migration during late spermiogenesis.

Notes: Abstract. The light yellow neuropeptidergic cell system of the basommatophoran snail Lymnaea stagnalis is homologous to the R3-R14 system of the opisthobranch Aplysia californica, and produces three different neuropeptides. Systems homologous to the light yellow cells of Lymnaea stagnalis have been investigated morphologically in two Basommatophora (Lymnaea ovata, Bulinus truncatus) and three Stylommatophora (Helix aspersa, Cepaea nemoralis, Deroceras reticulatum). To this end, an antibody to synthetic light-yellow-cell peptide-II and oligonucleotides to mRNAs encoding parts of peptide-I and peptide-III, were used. The in situ hybridization probes gave negative results. On the other hand, neuronal cell clusters were observed in the central nervous system of all species studied by immunocytochemistry. These clusters were located in the ganglia of the visceral complex. The neurons project axons into all nerves of these ganglia, especially into the pallial nerves, into the connective tissue of the central nervous system, and into the neuropile of various ganglia. The morphology of the systems is similar to that of the light-yellow-cell system of Lymnaea stagnalis. In all species, the wall of the aorta was innervated by immunoreactive axons. Peripheral innervation by the light-yellow-cell system was investigated in Helix aspersa and Deroceras reticulatum. Serial and alternate sections of whole snails were studied. Reconstructions were made of the heart-kidney-lung complex of these animals. In both species, the muscular vessels of the pulmonary system at the right side of the body were strongly innervated by immunoreactive axons. Furthermore, immunopositive innervation was observed to muscles in the secondary ureter-pneumostome area. The light-yellow-cell system of pulmonates is thus probably involved in the regulation of blood pressure and urine release.

Notes: Abstract Reissner's fibers are secretions produced by different ependymal areas of the chordate brain, viz., in adult vertebrates, by the dorsal subcommissural organ, and in all stages of cephalochordates (Branchiostoma lancelets), by the ventral infundibular organ. Fibers produced by these different organs are seemingly identical and the two fiber sources also share some immunocytochemical and lectin-binding properties. The secretions in these two glands are, however, not identical; the infundibular organ cells are strongly reactive with antibodies against vertebrate Reissner's fibers, but they do not react with antibodies raised against the source of the vertebrate fibers, viz., the subcommissural organ. The results support the possibility that, in adult vertebrates, the Reissner's fibers are composed of material not only from the subcommissural organ, but also from another, not yet identified, source that is identical or equivalent to the infundibular organ of the lancelet. There are indications that the infundibular organ is immunocytochemically closely akin to some secretory cells in the vertebrate embryonic brain and also to those that produce the juvenile vertebrate Reissner's fibers, viz., secretory cells in the flexural organ.

Notes: Abstract Triple fluorescence labelling was employed to reveal the distribution of chemically identified neurons within the pontine laterodorsal tegmental nucleus and dorsal raphe nucleus which supply branching collateral input to the central nucleus of the amygdala and hypothalamic paraventricular nucleus. The chemical identity of neurons in the laterodorsal tegmental nucleus was revealed by immunocytochemical detection of choline-acetyltransferase or substance P; in the dorsal raphe nucleus, the chemical content of the neurons was revealed with antibody recognizing serotonin. The projections were defined by injections of two retrograde tracers, rhodamine-and fluorescein-labelled latex microspheres, in the central nucleus of the amygdala and paraventricular nucleus, respectively. Neurons projecting to both the central nucleus of the amygdala and the paraventricular nucleus were distributed primarily within the caudal extensions of the laterodorsal tegmental nucleus and dorsal raphe nucleus. Approximately 11% and 7% of the labelled cells in the laterodorsal tegmental nucleus and dorsal raphe nucleus projected via branching collaterals to the paraventricular nucleus and central nucleus of the amygdala. About half of these neurons in the laterodorsal tegmental nucleus were cholinergic, and one-third were substance-P-ergic; in the dorsal raphe nucleus, approximately half of the neurons containing both retrograde tracers were serotonergic. These results indicate that pontine neurons may simultaneously transmit signals to the central nucleus of the amygdala and paraventricular nucleus and that several different neuroactive substances are found in the neurons participating in these pathways. This coordinated signalling may lead to synchronized responses of the central nucleus of the amygdala and paraventricular nucleus for the maintenance of homeostasis. Interactions between different neuroactive substances at the target site may serve to modulate the responses of individual neurons.

Notes: Abstract. Gamma-aminobutyric acid (GABA)-like immunoreactive neurons were studied in the central and peripheral nervous system of Helix pomatia by applying immunocytochemistry on whole-mount preparations and serial paraffin sections. GABA-immunoreactive cell bodies were found in the buccal, cerebral and pedal ganglia, but only GABA-immunoreactive fibers were found in the viscero-parietal-pleural ganglion complex. The majority of GABA-immunoreactive cell bodies were located in the pedal ganglia but a few could be found in the buccal ganglia. Varicose GABA-ir fibers could be seen in the neuropil areas and in distinct areas of the cell body layer of the ganglia. The majority of GABA-ir axonal processes run into the connectives and commissures of the ganglia, indicating an important central integrative role of GABA-immunoreactive neurons. GABA may also have a peripheral role, since GABA-immunoreactive fibers could be demonstrated in peripheral nerves and the lips. Glutamate injection did not change the number or distribution of GABA-immunoreactive neurons, but induced GABA immunoreactivity in elements of the connective tissue ensheathing the muscle cells and fibers of the buccal musculature. This shows that GABA may be present in different non-neural tissues as a product of general metabolic pathways.

Notes: Abstract The first component of complement $$C\bar 1s$$ has been shown to degrade type I and type II collagens (Yamaguchi et al. 1990), the latter of which is a major constituent of the cartilage matrix. In order to understand the physiological roles of $$C\bar 1s$$ in cartilage resorption, the expression of C1s was examined by immunohistochemistry in the primary ossification center where the matrix is removed and replaced by bone marrow. Hypertrophic chondrocytes, endothelium and hematogenous elements in the capillary buds were intensely stained by a monoclonal antibody against C1s. Matrix metalloproteinase 9 (MMP-9, 92kDa gelatinase/type IV collagenase) was also immunolocalized in hypertrophic chondrocytes, mesenchymal cells in the primitive bone marrow and the cartilage matrix adjacent to the marrow. In addition, $$C\bar 1s$$ was found to activate the zymogen of MMP-9. These observations suggest that $$C\bar 1s$$ and MMP-9 coordinately participate in matrix degradation in cartilage.

Notes: Abstract Collagen IV and laminin are important constituents of the basement membrane (BM). By use of immunocytochemistry we examined the occurrence and distribution of these two components in the BM beneath normal, mucoid and metaplastic epithelium of large bronchi in 22 adults suffering from chronic nonspecific lung diseases. Both collagen IV and laminin were expressed as a thin and continuous layer beneath the epithelium in most tissue specimens with normal epithelium. In a few specimens the layer showed interruptions with a patchy distribution of the immunoreactivity. Three patterns of distribution of BM components were found under the metaplastic epithelium. Total absence of immunoreactive collagen IV and laminin was the most common variant. Weak and scarce staining for both proteins in the BM characterized the second pattern. The third variant showed strong collagen IV immunoreactivity but lack of laminin. The BM beneath the mucoid epithelium was characterized by irregular distribution of collagen IV and laminin. We suggest that the occurrence and distributional pattern of the BM components are related to the type of overlying epithelium and connected with an altered synthesis of these components.

Notes: Abstract Gamma-aminobutyric acid (GABA)-like immunoreactive neurons were studied in the central and peripheral nervous system of Helix pomatia by applying immunocytochemistry on whole-mount preparations and serial paraffin sections. GABA-immunoreactive cell bodies were found in the buccal, cerebral and pedal ganglia, but only GABA-immunoreactive fibers were found in the viscero-parietal-pleural ganglion complex. The majority of GABA-immunoreactive cell bodies were located in the pedal ganglia but a few could be found in the buccal ganglia. Varicose GABA-ir fibers could be seen in the neuropil areas and in distinct areas of the cell body layer of the ganglia. The majority of GABA-ir axonal processes run into the connectives and commissures of the ganglia, indicating an important central integrative role of GABA-immunoreactive neurons. GABA may also have a peripheral role, since GABA-immunoreactive fibers could be demonstrated in peripheral nerves and the lips. Glutamate injection did not change the number or distribution of GABA-immunoreactive neurons, but induced GABA immunoreactivity in elements of the connective tissue ensheathing the muscle cells and fibers of the buccal musculature. This shows that GABA may be present in different non-neural tissues as a product of general metabolic pathways.

Notes: Abstract The present study provides light- and electronmicroscopic immunocytochemical data on the presence of neurons that are immunoreactive to the C-terminal flanking peptide of neuropeptide Y, C-PON, in the neostriatum of the hedgehog (Erinaceus europaeus). Positive neurons have mostly fusiform or round perikarya from which two to four poorly branched processes arise. Immunostained fibers and puncta are also evenly distributed throughout the neostriatum. Ultrastructurally, each neuron exhibits a deeply invaginated nucleus surrounded by abundant cytoplasm with a well-developed rought endoplasmic reticulum and Golgi apparatus. Positive neurons receive symmetric and asymmetric synapses from unlabeled terminals. The results of this study can be correlated with previous findings, as the C-PON-positive neurons of the hedgehog resemble medium-sized neostriatal neurons that are known to be local circuit neurons exhibiting C-PON in the rat. Thus, a high degree of C-PON neuronal system phylogenetic conservation and function can be postulated for the neostriatum of mammals.

Notes: Abstract Haemocytes of the hard tick Ixodes ricinus were characterized on the basis of their ultrastructure, their ability to ingest foreign material, and to produce or store molecules of the immune defence. Distinction was made between types of haemocytes according to the absence or presence of granular inclusions, shape and size of the lysosomal compartment or the rough endoplasmic reticulum, and ultrastructural and functional similarity to the corresponding haemocytes of insects. Three types of haemocytes were found in adult ticks: plasmatocytes and type-I and type-II granular haemocytes, respectively. The precipitated reaction product of acid phosphatase activity revealed the shape of the lysosomal compartment. The additional injection of particulate materials into the haemocoel further revealed the endocytic activity of the haemocytes. The lysozyme-like immunoreactivity of the haemocytes suggests bactericidal potential. Detection of immunoreactivity in haemocytes to a 25 kDa antigenic protein involved in cuticle formation further suggests their involvement in wound healing and encapsulation.

Notes: Abstract The light yellow neuropeptidergic cell system of the basommatophoran snail Lymnaea stagnalis is homologous to the R3-R14 system of the opisthobranch Aplysia californica, and produces three different neuropeptides. Systems homologous to the light yellow cells of Lymnaea stagnalis have been investigated morphologically in two Basommatophora (Lymnaea ovata, Bulinus truncatus) and three Stylommatophora (Helix aspersa, Cepaea nemoralis, Deroceras reticulatum). To this end, an antibody to synthetic light-yellow-cell peptide-II and oligonucleotides to mRNAs encoding parts of peptide-I and peptide-III, were used. The in situ hybridization probes gave negative results. On the other hand, neuronal cell clusters were observed in the central nervous system of all specias studied by immunocytochemistry. These clusters were located in the ganglia of the visceral complex. The neurons project axons into all nerves of these ganglia, especially into the pallial nerves, into the connective tissue of the central nervous system, and into the neuropile of various ganglia. The morphology of the systems is similar to that of the light-yellow-cell system of Lymnaea stagnalis. In all species, the wall of the aorta was innervated by immunoreactive axons. Peripheral innervation by the light-yellow-cell system was investigated in Helix aspersa and Deroceras reticulatum. Serial and alternate sections of whole snails were studied. Reconstructions were made of the heart-kidney-lung complex of these animals. In both species, the muscular vessels of the pulmonary system at the right side of the body were strongly innervated by immunoreactive axons. Furthermore, immunopositive innervation was observed to muscles in the secondary ureter-pneumostome area. The light-yellow-cell system of pulmonates is thus probably involved in the regulation of blood pressure and urine release.

Notes: Abstract The distribution of Merkel cells in fetal and adult terminal hair follicles of human scalp was studied immunohistochemically using cytokeratin (CK) 20 as a specific Merkel cell marker. In hair follicles of adult scalp, abundant Merkel cells were found enriched in two belt-like clusters, one in the deep infundibulum and one in the isthmus region. No Merkel cells were found in the deep follicular portions including the bulb, or in the dermis. In early fetal hair follicles (bulbous peg stage), Merkel cells were only detected in the basal layer of the developing infundibulum but not in deeper follicular areas. In later stages, Merkel cells were also present in the isthmus and bulge. No Merkel cells were seen in the dermis around developing hair follicles. Nerve growth factor receptor was not only present in nerves but was found to be widely distributed within fetal skin. In adult skin, this receptor was localized to the basal cell layers of the outer root sheath of the bulb and the suprabulbar area, but was not detectable in the areas containing Merkel cells. The present study localizing Merkel cells within the permanent hair follicle structures close to their possible stem cells suggests that they have paracrine functions.

Notes: Abstract In bird skin, nerve fibres develop in the dermis but do not enter the epidermis. In co-cultures of 7-day-old chick embryo dorsal root ganglia and epidermis, the neurites also avoid the epidermis. Previous studies have shown that chondroitin sulphate proteoglycans may be involved. Chondroitin sulphate has therefore been visualized by immunocytochemistry, using themonoclonal antibody CS-56, both in vivo and in vitro using light and electron microscopy. Its distribution was compared to those of 2 other chondroitin sulphate epitopes and to that of the growing nerve fibres. In cultures of epidermis from 7-day-old embryonic chicks, immunoreactivity is found uniformly around the epidermal cells while at 7.5 days the distribution in dermis is heterogeneous, and particularly marked in feather buds. In vivo, chondroitin sulphate immunoreactivity is detected in the epidermis, on the basal lamina, on the surfaces of fibroblasts and along collagen fibrils. This localization is complementary to the distribution of cutaneous nerves. Chondroitin sulphate in the basal lamina could prevent innervation of the epidermis and the dermal heterogeneities could partly explain the nerve fibres surrounding the base of the feathers. Chondroitin sulphate could therefore be important for neural guidance in developing chick skin.

Notes: Abstract. Mammary-derived growth inhibitor (MDGI) has previously been localized in the mammary parenchyma, dependent on the stage of differentiation of the mammary gland. Here, we have elucidated the distribution of MDGI in the mammary stroma by a combined immunohisto- and cytochemical analysis with antibodies raised against MDGI. Distinct staining of capillary endothelial cells has been revealed. Although its subcellular distribution resembles former observations in secretory epithelial cells, the expression of MDGI in capillary endothelial cells clearly precedes that in secretory epithelial cells. On the other hand, no endothelial MDGI staining has been detected in bovine heart, which contains a fatty acid-binding protein almost identical to MDGI. The localization of MDGI in the mammary capillary endothelium is discussed in terms of its possible involvement in the intracellular transport of hydrophobic ligands or in the regulation of endothelial cell proliferation.

Notes: Summary This study was conducted to determine the functional and/or developmental relationships among three heterogeneous types of prolactin cells (I, II and III) in rats. Rats were injected subcutaneously daily with estradiol or testosterone propionate on days 10–20 after birth. Estradiol increased the proportion of cell types II and III, increased serum PRL levels 12-fold in males and 15-fold in females, and increased pituitary levels of prolactin 12-fold in males and 5-fold in females. Testosterone mainly increased the proportion of the Type-II cells, decreased serum levels of prolactin in males only, and did not change pituitary levels of prolactin. In a second experiment, treatment of rats with nafoxidine for five days after E2 treatment (days 10–20 after birth) increased the proportion of Type-I cells and decreased the proportion of Type-III cells and decreased serum and pituitary levels of prolactin by 50% in females and by 15 and 45% in males. In a third experiment utilizing adult male rats, estradiol and testosterone were found to modulate the relative ratios of the different types of PRL cells as they did in immature animals. The data taken as a whole suggest the possibility of an estrogen-stimulated conversion of one cell type to another, which may be a reflection of prolactin secretory activity.

Notes: Summary Antigenic markers characteristic of astrocytes and their differentiative states (i.e., glial fibrillary acidic protein (GFAP), vimentin, and M1 and C1 antigens) were investigated in the pineal gland of mouse and rat using double immunolabeling techniques. In both species the socalled interstitial cells as characterized by TEM were shown to be astrocytes, since they expressed vimentin, but neither fibronectin (a marker for fibroblasts and endothelial cells) nor the neuron-specific L1 antigen or tetanus toxin receptors. Subpopulations of vimentin-positive pineal astrocytes were also GFAP- and C1- antigen-positive. M1- antigenpositive cells were not detected. It is concluded that a considerable proportion of interstitial cells in the pineal gland of rat and mouse are immature astrocytes which, in contrast to other parts of the central nervous system, persist into adulthood.

Notes: Summary The subcommissural organs (SCO) of 76 specimens belonging to 25 vertebrate species (amphibians, reptiles, birds, mammals) were studied by use of the immunoperoxidase procedure. The primary antiserum was obtained by immunizing rabbits with bovine Reissner's fiber (RF) extracted in a medium containing EDTA, DTT and urea. Antiserum against an aqueous extract of RF was also produced. The presence of immunoreactive material in cell processes and endings was regarded as an indication of a possible route of passage. Special attention was paid to the relative development of the ventricular, leptomeningeal and vascular pathways established by immunoreactive structures. The SCO of submammalian species is characterized by (i) a conspicuous leptomeningeal connection established by ependymal cells, (ii) scarce or missing hypendymal cells, and (iii) a population of ependymal cells establishing close spatial contacts with blood vessels. The SCO of most mammalian species displays the following features: (i) ependymal cells lacking immunoreactive long basal processes, (ii) hypendymal secretory cells occurring either in a scattered arrangement or forming clusters, (iii) an occasional leptomeningeal connection provided by hypendymal cells, and (iv) in certain species numerous contacts of secretory cells with blood vessels. In the hedgehog immunoreactive material was missing in the ependymal formation of the SCO, but present in hypendymal cells and in the choroid plexuses. The SCO of several species of New-and Old-World monkeys displayed immunoreactive material, whereas that of anthropoid apes (chimpanzee, orangutan) and man was completely negative with the antisera used.

Notes: Summary In 76 specimens (amphibians, reptilians, mammals) belonging to 25 different vertebrate species, the region of the subcommissural organ (SCO) was investigated with the use of a primary antiserum raised against an extract of bovine Reissner's fiber+the immunoperoxidase procedure according to Sternberger et al. (1970). In the SCO of a toad (Bufo arenarum) and several species of reptiles (lacertilians, ophidians, crocodilians), the ependymal cells were the only type of secretory cell displaying vascular contacts, whereas in mammals ependymal and hypendymal cells established intimate spatial contacts with blood vessels. In Bufo arenarum, but especially in the reptilian species examined, the ependymo-vascular relationship was exerted by a population of ependymal cells having a rather constant location within the SCO and projecting to capillaries that showed a remarkably constant pattern of anatomical distribution. In the SCO of mammals the modality and degree of the structural relationships between secretory cells and blood vessels varied greatly from species to species. In the SCO of the armadillo and dog the secretory tissue was organized as a thick, highly vascularized layer with most of the cells oriented toward the capillaries. A rather opposite situation was found in the SCO of New-and Old-World monkeys, where vascular contacts were restricted to a few ependymal cells.

Notes: Summary The distribution of α-melanocyte-stimulating hormone (α-MSH) was studied in the brain of the lizard Lacerta muralis by means of immunocytochemical staining methods. α-MSH-like containing cells were found in the ventro-lateral preoptic area and the paraventricular and supraoptic nuclei. Some scattered cells staining for α-MSH were also detected in the mesencephalo-diencephalic boundary region, while numerous α-MSH-like nerve fibres were localized in the medial eminence. No reaction was observed after the use of antiserum preabsorbed with synthetic antigen. These findings suggest that an α-MSH-like peptidergic system could possibly be involved in the hypothalamo-hypophysial regulation and/or play a role as neurotransmitter in this animal.

Notes: Summary In order to study the distribution of neuropeptide Y-like immunoreactivity in the human hypothalamus, an immunocytochemical localization of this peptide was performed. Using antibodies developed against synthetic porcine neuropeptide Y (NPY), we have been able to localize immunoreactivity in neuronal cell bodies located exclusively in the infundibular nucleus. Immunostained fibers were found in several regions in the hypothalamus with a high concentration in the periventricular areas. Fibers were also found in the neurovascular zone of the median eminence, the pituitary stalk and the posterior pituitary. These results suggest that immunoreactive material related to porcine NPY is present in the human hypothalamus, with a distribution similar to that observed in the rat.

Notes: Summary Three neuronal systems of the pond snail Lymnaea stagnalis were immunocytochemically investigated at the ultrastructural level with the unlabeled peroxidase-antiperoxidase technique. Preliminary electrophysiological and cell-filling investigations have shown that a cluster of neurons which reacts positively with an antiserum against the molluscan cardio-active peptide FMRFamide, sends axons to the penis retractor muscle. In this muscle anti-FMRF-amide (aFM) positive axons form neuro-muscular synapses with (smooth) muscle fibers. The morphological observations suggest the aFM immunoreactive system to be involved in peptidergic neurotransmission. In the right parietal ganglion a large neuron (LYAC) is penetrated by aFM positive axons which form synapse-like structures (SLS) with the LYAC. The assumption that the SLS represent the morphological basis for peptidergic transmission is sustained by the observation that iontophoretical application of synthetic FMRFamide depolarizes the LYAC. The axons of a group of pedal anti-vasopressin (aVP) positive cells run in close vicinity to the cerebral ovulation (neuro-)-hormone producing cell system (CDC system) Synapses or SLS between the two systems were not observed. The fact that (bath) application of arg-vasopressin induces bursting in the CDC, may indicate that the vasopressin-like substance of the aVP cells is released non-synaptically.

Notes: Summary The ultrastructural localization of the glycoprotein D2 in rat adrenal gland was investigated using immunohistochemical methods, and D2 localization in cultures of adult bovine chromaffin cells was studied by immunofluorescence. D2 was found to be situated on nerve fibers passing through the adrenal cortex and in the medulla zone, and also on the surface of all chromaffin cells. In addition, it was strongly expressed on the surface of glial (Schwann) cells. Cortical cells were unreactive to the antiserum. In cultures, all adrenalin and noradrenalin [dopamine-β-hydroxylase (DBH)-positive] cells were surface labelled for D2. A less frequent second cell type was recognized in vitro which was DBH negative but D2 positive. Such cells were presumed to be Schwann cells. These data are discussed in terms of the developmental origin of the cells and with regard to the putative functional rôle of D2 in cell adhesion phenomena.

Notes: Summary The pars intermedia of S. mossambicus contains two different endocrine-cell types. The predominant cell type is lead-haematoxyline-positive and assumed to synthesize MSH and related peptides. The second cell type is PAS positive and its function and product(s) are unknown. Staining of light-microscopic and ultrathin sections with antisera against α-MSH, ACTH 1–24 and human β-endorphin revealed that only the lead-haematoxyline-positive cells of the pars intermedia react with these antisera, and that the secretory granules of these cells contain compounds that were immunoreactive to all three antisera. These findings are in line with the hypothesis that α-MSH, ACTH and endorphins are derived from the same precursor molecule. No specific reaction with one of the antisera could be detected in the PAS positive cells.

Notes: Summary Gastrin, pancreatic polypeptide and somatostatin immunoreactive cells in the gut of two fish with stomachs (perch and catfish) and a stomachless fish (carp) were studied by immunocytochemistry. In the gastric mucosa of perch and catfish, cells showing gastrin and somatostatin-like immunoreactivity are found, scattered among the surface mucous cells and mucous neck cells. No pancreatic polypeptide (P.P.) immunoreactive cells are detected in the gastric mucosa. Cells showing gastrin and P.P.-like immunoreactivity are observed in the intestinal mucosa of perch, catfish and carp. In this location no somatostatin immunoreactive cells are found.

Notes: Summary The brain of the frog Rana temporaria was studied at the light microscopic level with the use of a double immunocytochemical staining method. The telencephalon, diencephalon and rhombencephalon contain somatostatin perikarya and fibers. In the telencephalon, the location of the somatostatin neurons largely corresponds to that of mammals. In the hypothalamus, the somatostatin perikarya are located in and near the magnocellular preoptic nucleus and also in the pars ventralis of the tuber cinereum. Like the somatostatin neurons of the rat hypothalamus, they form a separate subpopulation, different from the neurons producing neurohypophysial hormones. In Rana, somatostatin neurons are also present in (as well as in the vicinity of) the subfornical organ, in the thalamus, the tectum opticum, the interpeduncular nucleus and the caudal end of the roof of the calamus scriptorius. A precise localization of the perikarya of most somatostatin fibers, including those found in the median eminence and the neural lobe, was not attained.

Notes: Summary In the brain of Rana temporaria, two distinct systems reactive with α- and β-endorphin antisera, respectively, and with a met-enkephalin antiserum, have been detected immunohistochemically. Neurons reacting with α- and β-endorphin antisera are located (1) in the preoptic nucleus, and (2) in the pars ventralis of the tuber cinereum. Immunoreactive nerve fibres of both groups of perikarya end in the infundibular floor near the capillaries and the preoptico-hypophysial tract. Control reactions have shown that the immunoreactivity is suppressed by the corresponding antigens, but also by β-LPH. In view of these results the immunoreactive systems examined correspond to an α/β-endorphin system or a lipotropinergic system. Neurons reacting with the met-enkephalin antiserum are located in the paraventricular organ. Intense immunofluorescence was observed in the infundibular floor. Controls show that the labelling by met-enkephalin antiserum is exclusively suppressed by met-enkephalin. In the pituitary gland, on the other hand, α- and β-endorphin antisera reveal: 1) the MSH/ACTH-like cells of the pars intermedia and 2) the ACTH-like cells of the pars distalis.

Notes: Summary Neuronal pathways in the retrocerebral complex and thoracico-abdominal ganglionic mass of the blowflyCalliphora vomitoria have been identified immunocytochemically with antisera against the extended-enkephalins, Met-enkephalin-Arg6-Phe7 (Met-7) and Met-enkephalin-Arg6-Gly7-Leu8 (Met-8). Neurons of the hypocerebral ganglion, immunoreactive to Met-8, have axons in the crop duct nerve and terminals in muscles of the crop and its duct. Certain neurons of the hypocerebral ganglion are also immunoreactive to Met-7, and axons from these cells innervate the heart. Met-8 immunoreactive nerve terminals invest the cells of the corpus allatum. The source of this material is believed to ve a single pair of lateral neurosecretory cells in the brain. There is no Met-7 immunoreactive material in the corpus allatum. In the corpus cardiacum neither Met-7 nor Met-8 immunoreactivity is present in the cells. However, in the neuropil of the gland certain fibres, with their origins elsewhere, do contain Met-8 immunoreactivity. The most prominent neurons in the thoracic ganglion are the Met-7 immunoreactive ventral thoracic neurosecretory cells, axons from which project to neurohaemal areas in the dorsal neural sheath and also, via the ventral connective, to the brain. Co-localisation studies show that the perikarya of these cells are immunoreactive to antisera raised against several vertebrate-type peptides, such as Met-7, gastrin/cholecystokinin and pancreatic polypeptide. However, their axons and terminals show varying amounts of the peptides, suggesting differential transport and utilisation. Only a few cells in the thoracic ganglion are immunoreactive to Met-8 antisera. These lie close to the nerve bundles suppling the legs. In the abdominal ganglion, Met-8 immunoreactive neurons project to the muscles of the hindgut. This study suggests that the extended enkephalin-like peptides ofCalliphora may have a variety of different roles: as neurotransmitter or neuromodulator substances; in the direct innervation of effector organs; and as neurohormones.

Notes: Summary Immunocytochemical tests with eight monoclonal antibodies against either bovine or human insulin and seven polyclonal antibodies against bovine insulin were carried out to determine the presence of insulin-like neuropeptides in the brain and affiliated neuroendocrine structures of the insect Leucophaea maderae. Reaction products identified in the brain, subesophageal ganglion, and corpus cardiacum-corpus allatum complex indicate the presence of materials resembling mammalian insulins in its antigenic properties. The immunostaining observed with monoclonal antibodies appears to indicate the occurrence of an insulin-related peptide that shows sequential similarities with parts of both the A- and B-chains of mammalian insulin molecules. These suppositions are supported by the results of dot-blot and two-site time-resolved immunofluorometric assay (TR-IFMA) screenings of fractions of Leucophaea tissue extracts obtained by chromatography. The polyclonal antibodies yielded reaction products in some of the same areas and in additional parts of the neuroendocrine system not visualized by the monoclonal antibodies. Immunoreaction was observed in the following areas: the pars intercerebralis of the protocerebrum, the nervi corporis cardiaci I transporting insulin-like material to the corpus cardiacum, the dorsolateral protocerebral area and the optic lobes, the deutocerebrum, the tritocerebrum, and the subesophageal ganglion. In addition, smaller cell bodies with immunoreactive deposits occur at the border between proto- and deutocerebrum, and in the central area of the protocerebrum. The distribution of reactive material in the corpus cardiacum-corpus allatum complex after use of both groups of antibodies was the same. The fact that polyclonal and monoclonal antibodies yielded reaction products in different cells of the brain suggests either that the two groups of antibodies recognize different epitopes of the same molecule, or that they reveal two different types of immunoreactive molecules related to mammalian insulins. Together with the biochemical data reported by Nagasawa and coworkers (PNAS 83, 1986) the present immunocytochemical analysis has established a closer relationship between mammalian and insect “insulins” than was previously known.