ALBERT

Notes: Summary Cytodifferentiation of inner enamel epithelium and the adjacent connective tissue from the tip of the cervical loop to the initiation of enamel elaboration in twoMacaca species was examined. Ten- to twelve-month-old specimens were fixed by perfusion and the permanent tooth buds were prepared for transmission electron microscopy. At the cervical loop proper, inner enamel epithelium cells have lobed nuclei, a paucity of cytoplasm, and wide extracellular spaces; the basal lamina facing the dental papilla is straight. With increasing distance from the tip of the cervical loop, the following changes occur gradually: (a) preameloblasts elongate from 15 to 45 µm, and their organelles, particularly mitochondria and profiles of rough endoplasmic reticulum, become more numerous; (b) extracellular spaces decrease between preameloblasts starting at the basal (infranuclear) end; (c) the basement membrane becomes convoluted and associated with aperiodic fibers; (d) preodontoblast projections penetrate the aperiodic fibers; (e) collagen fibers subjacent to the basement membrane increase in density, with particularly thick fibers paralleling the aperiodic fibers. These modifications occur within three-fourths of the distance from the tip of the cervical loop to the mineralization front. The condensation of preodontoblasts is followed immediately by predentin synthesis. Concomitantly, the basement membrane breaks down and the aperiodic fibers are engulfed by preameloblasts. Preameloblast projections penetrate junctional predentin, contact mineralized dentin, and enamel synthesis ensues. At this stage the ameloblast is 45 µm long, the nucleus is central or basal, the Golgi apparatus has migrated apically, but the Tomes' process has not yet formed. The results indicate that odontogenesis inMacaca monkeys more closely resembles human odontogenesis than does that in the murine rodents.

Notes: Abstract Effects of SO2, aqueous fluoride (NaF) and a solution of nitrogen compounds (NH4NO3) on the visible symptoms, pollutant accumulation and ultrastructure of Scots pine (Pinus sylvestris L.) and Norway spruce [Picea abies (L.) Karst.] seedlings were studied in an open-air experiment lasting for 3 consecutive years. Visible injury symptoms were most pronounced in combination exposures and whenever F was applied. Visible symptoms correlated well with needle pollutant concentrations. Exposure to NaF increased needle F contents particularly when F was applied with SO2 or NH4NO3. This suggests that a reduction in N or SO2 emissions, in F polluted areas, could improve the condition of conifers via decreased accumulation of phytotoxic F in the needles. Norway spruce needles accumulated 2–10 times as much S and F as those of Scots pine. Microscopic observations showed various changes in the needle mesophyll cell ultrastructure. In both species, exposure to SO2 increased significantly the amount of cytoplasmic vacuoles, suggesting detoxification of excess sulphate or low pH. F treatments resulted in a significant enlargement of plastoglobuli in Scots pine and a darkening of plastoglobuli in Norway spruce. All exposures enhanced the accumulation of lipid bodies. An increased portion of translucent plastoglobuli was most pronounced in N treatments. Many of the ultrastructural changes and visible symptoms appeared only as number of years exposed increased, indicating that long-term experiments are needed. Both visible symptoms and ultrastructural changes pointed to the more pronounced sensitivity of Norway spruce compared to Scots pine. Ultrastructural results mostly supported earlier qualitative observations of F, N and SO2 effects on needle mesophyll cell ultrastructure. However, no reduction of thylakoids in SO2 containing exposure or curling of thylakoids in F exposure could be detected in the present study.

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 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: Abstract  Effects of SO2, aqueous fluoride (NaF) and a solution of nitrogen compounds (NH4NO3) on the visible symptoms, pollutant accumulation and ultrastructure of Scots pine (Pinus sylvestris L.) and Norway spruce [Picea abies (L.) Karst.] seedlings were studied in an open-air experiment lasting for 3 consecutive years. Visible injury symptoms were most pronounced in combination exposures and whenever F was applied. Visible symptoms correlated well with needle pollutant concentrations. Exposure to NaF increased needle F contents particularly when F was applied with SO2 or NH4NO3. This suggests that a reduction in N or SO2 emissions, in F polluted areas, could improve the condition of conifers via decreased accumulation of phytotoxic F in the needles. Norway spruce needles accumulated 2 – 10 times as much S and F as those of Scots pine. Microscopic observations showed various changes in the needle mesophyll cell ultrastructure. In both species, exposure to SO2 increased significantly the amount of cytoplasmic vacuoles, suggesting detoxification of excess sulphate or low pH. F treatments resulted in a significant enlargement of plastoglobuli in Scots pine and a darkening of plastoglobuli in Norway spruce. All exposures enhanced the accumulation of lipid bodies. An increased portion of translucent plastoglobuli was most pronounced in N treatments. Many of the ultrastructural changes and visible symptoms appeared only as number of years exposed increased, indicating that long-term experiments are needed. Both visible symptoms and ultrastructural changes pointed to the more pronounced sensitivity of Norway spruce compared to Scots pine. Ultrastructural results mostly supported earlier qualitative observations of F, N and SO2 effects on needle mesophyll cell ultrastructure. However, no reduction of thylakoids in SO2 containing exposure or curling of thylakoids in F exposure could be detected in the present study.

Notes: Summary The ultrastructural localization of adenylate cyclase (E.C. 4.6.1.1.) and cAMP phosphodiesterase (PDE) (E.C. 3.1.4.17.) in the ectoderm of the developmental stage 4 chick embryo was studied. Adenylate cyclase was localized in the lateral surfaces of the ectodermal cells. In the primitive streak cells the enzymatic activity was observed on all the lateral surfaces, whereas in the periphery of the blastoderm the reaction product was localized in the apical parts of the lateral plasma membranes only. cAMP PDE localized in the apical cytoplasm of the ectodermal cells, with highest activity in the globular projections.

Notes: Summary The self-differentiation potency of the endoderm of the chick embryo was investigated mainly by transmission electron microscopy. Endodermal fragments isolated from 4- to 6-day stomach or small intestine were cultured in the absence of mesenchyme and were able to differentiate in vitro into organ-specific epithelia. Endodermal fragments isolated from the stomach region differentiated into a pseudo-stratified epithelium with periodic acid Schiff-positive mucous granules in the apical cytoplasm, while those from the small intestinal region differentiated into a simple columnar epithelium with a striated border which was positive in alkaline phosphatase activity. These features are comparable with those of the mucous secretory epithelium of the normal embryonic stomach and the absorptive epithelium of normal embryonic small intestine, respectively. Next, the self-differentiation potencies were investigated of the upper and lower layers of the blastoderms, at stages 1–5 of Hamburger and Hamilton (H. and H.). Both stomach-type and small-intestine-type epithelia developed only when fragments of the lower layer isolated from the blastoderms older than stage 3 of H. and H. were cultured, suggesting that cells possessing the potency to differentiate into the stomach- and small-intestine-type epithelia exist in the definitive endoderm at the beginning of its formation.

Notes: Summary In the eggs ofWachtliella persicariae the cleavage nuclei move relative to the surrounding ooplasm. This ‘active’ migration is caused by an organelle whose ultrastructure was studied throughout the mitotic cycle. It consists of a greatly enlarged polar cytaster derived from the mitotic apparatus, linked to the nucleus by 100 Å filaments. The microtubules of the cytaster were found only during periods of active nuclear migration, i.e., from the onset of anaphase to the early prophase of the next mitotic cycle. They are always solitary and follow the course of the astral rays, which are known to temporarily adhere to peripheral structures of the egg cell and to exert tractive forces. In contrast to the cytaster microtubules, the microtubules in the spindle are bundled and persist from early metaphase through late telophase. During ontogenesis the first migration cytaster is built up between 3 and 12 min after oviposition near the anterior egg pole, in the vicinity of the sperm nucleus. In non-inseminated eggs time lapse films show a migration cytaster to develop autonomously in a region free from nuclei, but it does not follow the normal path of the male pronucleus. In several cases the female pronucleus, which remains without a cytaster of its own, was observed to move to the cytaster generated in the absence of the male pronucleus. Whether or not it is adhering to a nucleus, the cytaster divides into two at the correct time, i.e, corresponding to the first cleavage division in fertilized eggs. In some non-inseminated eggs this type of ‘pseudocleavage’ has been observed to occur repeatedly, giving rise to an increasing number of anucleate cytasters.

Notes: Abstract  In one of his classical studies on insect metamorphosis, Weismann compared the imaginal anlagen of the ancestral phantom midge, Chaoborus, with those of advanced brachycerans. We have expanded his findings on the relationships between larval and imaginal organs using electron microscopy and cobalt backfilling of the antenna and leg anlagen and the axonal trajectories of corresponding larval sensilla. We show that both primordia are confluent with the larval antennae and ”leg” sensilla (an ancestral Keilin organ), respectively. These fully developed larval organs represent the distal tips of the imaginal anlagen rather than separate cell clusters. The axons of the larval antenna and leg sensilla project across the corresponding anlagen to their target neuromeres within the central nervous system (CNS). Within the discs, nerves composed of these larval axons, developing afferent fibres and efferences ascending from the CNS are found. Both the structure of the primordia and the axonal trajectories thus relate the situation found in advanced brachycerans with that seen in more ancestral insects. In addition, the larval antennae, legs, wings and even the eyes possess very similar afferent pioneer trajectories supporting the idea that the described pattern is generally used in the ontogeny of sensory systems.

Notes: Abstract Oocytes of hymenopterans are equipped with peculiar organelles termed accessory nuclei. These organelles originate from the germinal vesicle (oocyte nucleus) and gather preferentially at the anterior pole. To gain insight into the mechanism of uneven (asymmetrical) distribution of accessory nuclei, the organization of the microtubule cytoskeleton in the oocytes of two hymenopterans Chrysis ignita and Cosmoconus meridionator has been studied. It is shown that during late previtellogenesis two networks of microtubules are present along the contact zone between the oocyte and enveloping follicular epithelium. The external one is associated with belt desmosomes connecting neighbouring follicular cells. The internal network is composed of randomly orientated microtubules and separates transparent, organelle-free periplasm from the endoplasm. All cellular organelles and the germinal vesicle are localized in the endoplasm. Accessory nuclei are accumulated in the anterior endoplasm; they always lie in direct contact with the subcortical network. Treatment with colchicine results in the disappearance of the periplasm as well as in the redistribution of cellular organelles including accessory nuclei. Presented findings suggest that subcortical microtubules play an important role in the positioning of accessory nuclei throughout the ooplasm.

Notes: Summary Changes at the ultrastructural level during germ band extension in the embryo ofDrosophila melanogaster are described. Cytoplasmic connections between cells and the yolk sac are present during initial cellular movements. At this time, a continuous system of microfilaments is present adjacent to the membranes in the connections and at the periphery of the yolk sac. As germ band extension progresses, this system becomes discontinuous, and microfilaments are apparent only in the immediate vicinity of the connections. Cytoplasmic connections are disassembled at approximately the midpoint of extension; at the same time, extensive membrane associations develop between germ band cells and between these cells and adjacent yolk sac membranes. Positioning and orientation of cytoplasmic connections suggest that the yolk sac, via these connections, is actively involved in the cellular movements of early germ band extension.

Notes: Summary The ultrastructural localization and gradient of activity of alkaline phosphatase were studied with respect to cell differentiation, matrix synthesis, and matrix mineralization in the incisor and molar teeth of 4-day-old Sprague-Dawley rats. The animals were perfused intracardially at room temperature with 2.5% glutaraldehyde in 0.1M sodium cacodylate (pH 7.4) with 3–4% sucrose. The jaws were dissected, immersion-fixed for 24 h, and the incisor and molar tooth germs removed. These were demineralized in 10% EDTA in NaOH (pH 7.4) with 7% sucrose. After reactivation of the enzyme with 0.1M MgCl in Tris-maleate buffer (pH 7.4) at 4°C, the teeth were incubated for alkaline phosphatase in a medium consisting of 6 ml 3% sodiumβ-glycerophosphate, 4 ml 0.2M Tris-HCl buffer (pH 9.2), 3 ml 1.6% MgSO4, 12 ml 0.5% lead citrate (pH⋍12), and 2.1 g sucrose. The pH was adjusted to 9.2 with 0.2M HCl, the volume made up to 30 ml, and the solution centrifuged for 10 min at 5000 rpm. Control teeth were incubated in medium minus the substrate. Finally, the specimens were routinely post-fixed and embedded for sectioning and examination with a Philips 300 electron microscope. A gradient of alkaline phosphatase activity was mapped along the developing teeth in the cells of the stratum intermedium, the proximal borders of the ameloblasts, the early dentine matrix, the predentine-dentine border, matrix vesicles, and the plasma membranes of odontoblasts and subodontoblast cells. The gradient of alkaline phosphatase activity was evident in the forming tooth from the cervical loop to the crown apex and was related to the cellular events, matrix synthesis, and matrix mineralization occurring during odontogenesis.

Notes: Summary Using transmission and scanning electron microscopy, we have studied the ultrastructure of a number of urinary calculi, mainly composed of calcium phosphate. Three fundamental kinds of calcium phosphates were detected: nonstoichiometric carbonate apatite, nonhexagonal octacalcium phosphate, and calcium-magnesium whitlockite. The influence that the organic matter, substitutions in the phosphate lattice of CO3 and Mg, and apatitic stoichiometry have on the ultrastructure of the calcium phosphate calculi has been detailed. An originating apatitic unity named U2 is assumed to be the responsible for all the different structures of calcium apatites appearing in renal calculi. On the basis of our observations, a mechanism whereby apatites grow is postulated; magnesium functions as an inhibitor for the growing mechanism.

Notes: Abstract A technique to correlate the ultrastructural distribution of mineral with its organic material in identical sections of mineralized turkey leg tendon (MTLT) and human bone was developed. Osmium or ethanol fixed tissues were processed for transmission electron microscopy (TEM). The mineralized tissues were photographed at high, intermediate, and low magnifications, making note of section features such as fibril geometry, colloidal gold distribution, or section artifacts for subsequent specimen realignment after demineralization. The specimen holder was removed from the microscope, the tissue section demineralized in situ with a drop of 1 N HCl, then stained with 2% aqueous vanadyl sulfate. The specimen holder was reinserted into the microscope, realigned with the aid of the section features previously noted, and rephotographed at identical magnification used for the mineralized sections. A one to one correspondence was apparent between the mineral and its demineralized crystal “ghost” in both MTLT and bone. The fine structural periodic banding seen in unmineralized collagen was not observed in areas that were fully mineralized before demineralization, indicating that the axial arrangement of the collagen molecules is altered significantly during mineralization. Regions that had contained extrafibrillar crystallites stained more intensely than the intrafibrillar regions, indicating that the noncollagenous material surrounded the collagen fibrils. The methodology described here may have utility in determining the spatial distribution of the noncollagenous proteins in bone.

Notes: Summary The potassium pyroantimonate technique was utilized for the selective subcellular localization of calcium in the mandibular condylar cartilage of 1-day-old rats. Electron dense calcium pyroantimonate precipitates were localized principally in mitochondria and at the cell membrane of the chondrocytes. In addition, small intracellular vesicles 0.1–0.2µm in diameter were observed in proximity to the cell membrane of chondrocytes of the mid-hypertrophic zone. The results suggest that these vesicles were being extruded from the cell into the extracellular matrix. Energy-dispersive analysis by X-rays confirmed that calcium is the principal cation of the electron-dense precipitates.

Notes: Summary An original method for fractionating and preparing isolated crystals of homogeneous size was developed. It was demonstrated that enamel apatite crystals are at least 100 µm long. The flexibility of the very long crystallites was demonstrated. Crystal curvatures, accounting for the irregular course of the prisms through the enamel thickness, were visualized and measured. It was shown that in the deep forming enamel layer, lateral branches may grow out of the crystals and crystal fusing often occurs, inducing the crystallites to assume pyramidal shapes with their wide bases pointing toward the dentino-enamel junction and one or two tops toward Tomes' processes. During the maturation process, the two tops of the still immature crystals also fuse so that the mature crystals acquire a rodlike aspect, with parallel faces and steplike graduations along thec axis, allowing a close contact between the crystals. These results support the hypothesis that the crystallites would be continuous from the dentino-enamel junction to the surface.

Notes: Summary Diaphyseal tibial bone of 12.5 – 13-day and 19-day-old embryos and 20-day-old hatched chicks infected with retrovirus MAV.2-O were examined by transmission electron microscopy. The viruses were associated with lining osteoblasts and osteocytes. Whereas the infection of the osteoblast layer seemed to be a transient stage, virus association with osteocytes was a constant and main ultrastructural feature. The viruses were found either in the osteoid or in the periosteocytic space of the bone lacunae. They arose from dense cytoplasmic areas located near the cell plasmalemma via a budding process. The newly budded virus particles often had a large tail or a fine stalk-like process lost in the extracellular space. The viruses underwent calcification by deposition of inorganic material and were incorporated in the bone trabeculae. No production of virus was observed in typical osteoclasts with well-differentiated ruffled borders. The viral-induced avian osteopetrosis seemed to result from increased bone deposition through stimulation of osteoblast and osteocyte activities, whereas osteoclastic bone resorption seemed to be undisturbed.

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: 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: Abstract  The ultrastructure of egg cells in Abies alba was examined to elucidate the lack of maternal inheritance of plastids. Before fertilization, maternal plastids are absent in the perinuclar zone containing mainly mitochondria and smooth endoplasmic reticulum. During egg cell development the maternal plastids are transformed into large inclusions which are situated mostly towards the periphery of the egg cell, and finally disintegrate. As a consequence, they do not participate in zygote formation. RFLP analysis of cpDNA of parental trees and their F1 interspecific hybrids (A. alba×A. numidica, A. alba×A. nordmanniana, A. nordmanniana×A. Alba) using HindIII and BamHI showed a paternal mode of cpDNA inheritance. Paternal inheritance has also been found with PCR/RFLP analysis of cpDNA from parental trees and their hybrids (A. alba×A. pinsapo, A. pinsapo×A. alba, A. pinsapo×A. numidica) using ApaI and HaeIII digests, as well as in the crosses of A. cephalonica×A. nordmanniana, A. nordmanniana×A. cephalonica, A. cephalonica×A. numidica using TagI digests.

Notes: Abstract  In this study, megasporogenesis of the plant model Arabidopsis thaliana was investigated by electron microscopy for the first time. The data described here could constitute a reference for future investigations of Arabidopsis mutants. During the beginning of meiosis the megaspore mother cell shows a polarity created by unequal distribution of organelles in the cytoplasm. Plastids accumulate in the chalazal region and long parallel saccules of endoplasmic reticulum, small vacuoles and some dictyosomes are found in the micropylar region. Plasmodesmata are abundant in the chalazal cell wall. The nucleus is almost centrally localized and contains a prominent excentric nucleolus and numerous typical synaptonemal complexes. After the second division of meiosis the four megaspores are separated by thin cell walls crossed by numerous plasmodesmata and do not show significant cellular organization. The young functional megaspore is characterized by a large nucleus and a large granular nucleolus. The cytoplasm is very electron dense due to the abundance of free ribosomes and contains the following randomly distributed organelles: mitochondria, a few short saccules of endoplasmic reticulum, dictyosomes and undifferentiated plastids. However, there is no apparent polarity, except for the distribution of some small vacuoles which are more abundant in the micropylar region of the cell. The degenerating megaspores are extremely electron dense and do not show any substructure.

Notes: Abstract Ultrastructural changes during zygotic and somatic embryogenesis in pearl millet (Pennisetum glaucum [L.] R. Br.) were quantified using morphometric techniques. The total area per cell profile and the cell volume percentage of the whole cell, endoplasmic reticulum (ER), Golgi bodies, mitochondria, nuclei, lipids, plastids, starch grains and vacuoles were measured and comparisons made between three zygotic and three somatic embryo developmental stages. All measurements were taken from scutellar or scutellar-derived cells. Zygotic embryogenesis was characterized by increases in cell size, lipids, plastids, starch, Golgi bodies, mitochondria and ER. Somatic embryogenesis was characterized by two phases of cell development: (1) the dedifferentiation of scutellar cells involving a reduction in cell and vacuole size and an increase in cell activity during somatic proembryoid formation and (2) the development of somatic embryos in which most cell organelle quantities returned to values found in late coleoptile or mature predesiccation zygotic stages. In summary, although their developmental pathways differed, the scutella of somatic embryos displayed cellular variations which were within the ranges observed for later stages of zygotic embryogenesis.

Notes: Abstract  Ultrastructural changes during zygotic and somatic embryogenesis in pearl millet (Pennisetum glaucum [L.] R. Br.) were quantified using morphometric techniques. The total area per cell profile and the cell volume percentage of the whole cell, endoplasmic reticulum (ER), Golgi bodies, mitochondria, nuclei, lipids, plastids, starch grains and vacuoles were measured and comparisons made between three zygotic and three somatic embryo developmental stages. All measurements were taken from scutellar or scutellar-derived cells. Zygotic embryogenesis was characterized by increases in cell size, lipids, plastids, starch, Golgi bodies, mitochondria and ER. Somatic embryogenesis was characterized by two phases of cell development: (1) the dedifferentiation of scutellar cells involving a reduction in cell and vacuole size and an increase in cell activity during somatic proembryoid formation and (2) the development of somatic embryos in which most cell organelle quantities returned to values found in late coleoptile or mature predesiccation zygotic stages. In summary, although their developmental pathways differed, the scutella of somatic embryos displayed cellular variations which were within the ranges observed for later stages of zygotic embryogenesis.

Notes: Abstract  Asplenium trichomanes L. subsp. trichomanes spermatozoids are spirals of about five turns. Keels link the elements of the microtubular ribbon with the plates of the lamellar layer (LL) which are uninterrupted, parallel and curved with an inner angle of about 150°. Electron-opaque filaments connect the microtubules of the multilayered structure (MLS) and the osmiophilic crest, the LL and the MLS-associated mitochondrion and the latter and the plasmalemma. The nucleus occupies the 2.5–3 posterior turns and has an inner honeycomb-shaped chromatin mass and an outer highly condensed chromatin mass with randomly scattered electron-transparent areas. The basal bodies of the ca. 50 flagella are bounded by a reticulum of granular material which forms a plug inside their proximal region; the proximal region of the flagellum has a 9 + 0 pattern. The axoneme has a 9 + 2 pattern.

Notes: Abstract  A protocol for isolating viable eggs in Plumbago zeylanica by mechanical dissection is reported. The optimum solution for isolation was 0.8 M mannitol + 10 mM MOPS + 10 mM CaCl2, (pH 4.5–5.0) with an osmolality of 860–940 mmol/kg. Eggs retain their viability for at least 24 h. Isolated eggs were true protoplasts without cell walls and could tolerate osmolality of 437 mmol/kg to 965 mmol/kg. Observation of the isolated eggs using transmission electron microscopy indicated that they were well preserved and reflected the ultrastructure of physiologically active cells, displaying features similar to those of in vivo egg cells. Notable differences include the absence of a filiform apparatus and the accumulation of dense particles in the plastids, which was most conspicuous in egg cells that were damaged during isolation.

Notes: Abstract  The ultrastructure of the egg apparatus of the sexual (aestivum)-Salmon line (aS) and the isogenic but alloplasmic (kotschyi)-Salmon line (kS) of the Salmon system of wheat was studied by transmission electron microscopy 3 days before and during anthesis. Additionally, the zygotic stage of aS, 17 h after pollination, was included. Metabolic activity of egg cells from the sexual line aS was low 3 days before anthesis and increased dramatically after pollination and fertilization. This timing of increased activity was evident because of changes occurring in the egg cell nucleus and nucleolus, polysomes, endoplasmic reticulum and Golgi apparatus, and the completion of the cell wall around the zygote. In contrast to the sexual line, the egg cell of the parthenogenetic line showed high activity 3 days before anthesis. The metabolic and ultrastructural characters observed in the nucleus and cytoplasm of the kS line 3 days before and during anthesis corresponded with those of the isogenic sexual line aS during anthesis and 17 h after pollination, respectively. High metabolic activity observed in the persistent synergid of kS may be connected with the occurrence of additional embryos in seeds (twins) of this line.

Notes: Abstract The nucellar ultrastructure of apomictic Panicum maximum was analyzed during the meiocytic stage and during aposporous embryo sac formation. At pachytene the megameiocyte shows a random cell organelle distribution and sometimes only an incomplete micropylar callose wall. The chalazal nucellar cells are meristematic until the tetrad stage. They can turn into initial cells of aposporous embryo sacs. The aposporous initials can be recognized by their increased cell size, large nucleus, and the presence of many vesicles. The cell wall is thin with few plasmodesmata. If only a sexual embryo sac is formed, the nucellar cells retain their meristematic character. The aposporous initial cell is somewhat comparable to a vacuolated functional megaspore. It shows large vacuoles around the central nucleus and is surrounded by a thick cell wall without plasmodesmata. In the mature aposporous embryo sac the structure of the cells of the egg apparatus is similar to each other. In the chalazal part of the egg apparatus the cell walls are thin and do not hamper the transfer of sperm cells. Structural and functional aspects of nucellar cell differentiation and aposporous and sexual embryo sac development are discussed.

Notes: Abstract The organization ofPinus sylvestris pollen tubes during growth was studied by video microscopy of living cells and by electron microscopy after freeze-fixation and freeze-substitution (FF-FS). Pollen germinated and the tubes grew slowly for a total period of about 7 days. Some of the grains formed two tubes, while 10–50% of the tubes ramified. These features are in accordance with development in vivo. The cytoplasmic hyaline cap at the tip disappeared during the 2nd or 3rd day of culture. Aggregates of starch grains progressively migrated from the grain into the tube and later into the branches. Vacuoles first appeared at day 2 and eventually filled large parts of the tube. The tube nucleus was located at variable distances from the tip. Some of the organelles showed linear movements in a mostly circulatory pattern, but the majority of the organelles showed brownian-like movements. Rhodamine-phalloidin-stained actin filaments had a gross axial orientation and were found throughout the tube including at the tip. The ultrastructure of pollen tubes was well preserved after FF-FS, but signs of shrinkage were visible. The secretory vesicles in growing tips were not organized in a vesicle cone, and coated pits had a low density with only local accumulations, which is in accordance with slow growth. The mitochondria contained small cristae and a darkly stained matrix and were located more towards the periphery of the tube, indicating low respiratory activity and low oxygen levels. The dictyosomes carried typical trans-Golgi networks, but some contained less than the normal number of cisternae. Other elements of the cytoplasm were irregularly spaced rough endoplasmic reticulum, many multivesicular bodies, lipid droplets and two types of vacuoles. The typical organization associated with tip growth in angiosperm pollen tubes, e.g.Nicotiana tabacum, was not present inP. sylvestris pollen tubes. The different morphology may relate to the growth rate and not to the type of growth.

Notes: Abstract  The organization of Pinus sylvestris pollen tubes during growth was studied by video microscopy of living cells and by electron microscopy after freeze-fixation and freeze-substitution (FF-FS). Pollen germinated and the tubes grew slowly for a total period of about 7 days. Some of the grains formed two tubes, while 10–50% of the tubes ramified. These features are in accordance with development in vivo. The cytoplasmic hyaline cap at the tip disappeared during the 2nd or 3rd day of culture. Aggregates of starch grains progressively migrated from the grain into the tube and later into the branches. Vacuoles first appeared at day 2 and eventually filled large parts of the tube. The tube nucleus was located at variable distances from the tip. Some of the organelles showed linear movements in a mostly circulatory pattern, but the majority of the organelles showed brownian-like movements. Rhodamine-phalloidin-stained actin filaments had a gross axial orientation and were found throughout the tube including at the tip. The ultrastructure of pollen tubes was well preserved after FF-FS, but signs of shrinkage were visible. The secretory vesicles in growing tips were not organized in a vesicle cone, and coated pits had a low density with only local accumulations, which is in accordance with slow growth. The mitochondria contained small cristae and a darkly stained matrix and were located more towards the periphery of the tube, indicating low respiratory activity and low oxygen levels. The dictyosomes carried typical trans-Golgi networks, but some contained less than the normal number of cisternae. Other elements of the cytoplasm were irregularly spaced rough endoplasmic reticulum, many multivesicular bodies, lipid droplets and two types of vacuoles. The typical organization associated with tip growth in angiosperm pollen tubes, e.g. Nicotiana tabacum, was not present in P. sylvestris pollen tubes. The different morphology may relate to the growth rate and not to the type of growth.

Notes: Abstract Ultrastructure of the mating tube formed in yeast haplont of the heterobasidiomycete Tremella mesenterica was studied by electron microscopy. Cell wall of the mating tube emerged as evagination of the inner layers, rupturing outer layers of the mother cell wall. Comparison with budding cells suggested that the tube emergence place at bud scar and the process of tube emergence was the same as that of bud emergence. Electron transparent vesicles of 0.1 μm diameter were scattered in the cytoplasm of the mating tube. Nucleus-associated organelle was located at one side of the nuclear envelope which extended towards the mating tube. A few microtubules were detected in the mating tube, but their association with a nucleus was not clear. The cytoplasmic structure of the mating tube was discussed in comparison with that of hyphae of the filamentous fungi.

Notes: Abstract Microcysts of the myxomycete Didymium iridis were induced to excyst by transfer to 5mM potassium phosphate buffer. After 1 h in suspension, 90% of the microcysts had germinated into myxamoebae distinguishable by phase contrast microscopy and staining with Lugol's iodine. Both pH and osmolarity affected the kinetics of excystment. The rate and extent of excystment were decreased by cycloheximide but remained unaffected by actinomycin D, suggesting a requirement for protein synthesis but not RNA synthesis. Initially, the outer wall layers separated from the inner layer, which gradually expanded and loosened. The protoplast rehydrated and reverted to a vegetative morphology. Excysting cells were characterized by nucleolar inclusions, changes in the nuclear envelope and plasma membrane, appearance of ringed cisternal elements and microbodies in the cytoplasm, and formation of a densely fibrous zone adjacent to the site of emergence. Excysting populations have been classified into characteristic stages: mature, initiated, swollen, and pre-emergent microcysts.

Notes: Abstract The morphology and ultrastructure of the thermophilic cyanobacteriumMastigocladus laminosus were examined by scanning and transmission electron microscopy. Mature cultures consisted of relatively old, wide filaments that branched frequently to form younger, thinner filaments. The cells of the younger filaments had a consistently cylindrical morphology, while those of older filaments were rounded and pleomorphic. The internal ultrastructure of the cells depended somewhat on their age. As young cells became larger and wider, their thylakoids underwent slight rearrangement and spread out toward the center of the cytoplasm. Polyphosphate bodies, carboxysomes (polyhedral bodies), and lipid-body-like structures increased in number as the cells aged, but ribosomes and cyanophycin granules were depleted. Cell division involved septum formation followed by ingrowth of the outer membrane and sheath. Cells in older filaments were separated from each other by a complete layer of sheath material. Septum formation in older cells was also seen to occur parallel to the long axis of the filament, thereby confirming that true branching took place.

Notes: Abstract A new coccoid methanogen, Methanogenium tatii, was isolated and characterized. The mesophilic isolate can grow on and produce methane from H2:CO2 and formate. For growth acetate is strictly required. The cell shape, the G+C content of 54 mol% and DNA-DNA homology data suggest it to be a Methanogenium species.

Notes: Abstract The type strain of Streptomyces torulosus Lyons and Pridham (1971) was studied by scanning- and transmission electron microscope. Spore chains were formed in spirals by aerial mycelium. The spores were connected by nozzles in which small channels could be observed. The knobby ornamentations of the spores arised on a thin fibrous sheath, enveloping the spore chains. These irregular blunt projections, called knobs, had varying diameters of 100 to 250 nm. The base of the knob, consisting of globose to flattened electron dense material, was sitting directly on the sheath. It was covered by several small vesicles of the same material. Each hollow vesicle beared a thin bowlshaped shell of electron transparent material. In general, the cupular bowls and their supporting vesicles became easily depressed on their base, but not detached from the surface of the spores. This type of knobby spore ornamentation was suggested to be designated as a verrucate spore type.

Notes: Abstract The effects of nitrogen limitation on the ultrastructure of the unicellular cyanobacterium, Agmenellum quadruplicatum, were studied by thin sectioning transmission electron microscopy. Nitrogen became limiting for growth 14–15 h after transfer to nitrogen-limiting medium, but cultures retained full viability for at least 45 h. The c-phycocyanin: chlorophyll a ratio and cellular nitrogen content of the culture dropped rapidly after 14–15 h, as a progressive deterioration of major cell structures took place. Phycobilisomes were degraded first, followed by ribosomes and, then, thylakoid membranes. These structures were virtually depleted from the cells within 26 h. Intracellular polysaccharide accumulated in place of the normal cell structures throughout this period. Nitrogen limitation did not affect polyphosphate bodies, carboxysomes, lipid granules, the cell envelope, or the extra-cellular glycocalyx. All of the ultrastructural changes resulting from nitrogen limitation were reversed upon addition of nitrate to a starved culture. Most cell structures were restored within 3 h, and restoration was complete within 9 h.

Notes: Abstract A facultative methylotrophic bacterium was isolated from enrichment cultures containing methylamine as the sole carbon source. It was tentatively identified as an Arthrobacter species. Extracts of cells grown on methylamine or ethylamine contained high levels of amine oxidase (E.C. 1.4.3.) activity. Glucose- or choline-grown cells lacked this enzyme. Oxidation of primary amines by the enzyme resulted in the formation of H2O2; as a consequence high levels of catalase were present in methylamine-and ethylamine-grown cells. The significance of catalase in vivo was demonstrated by addition of 20 mM aminotriazole (a catalase inhibitor) to exponentially growing cells. This completely blocked growth on methylamine whereas growth on glucose was hardly affected. Cytochemical studies showed that methylamine-dependent H2O2 production mainly occurred on invaginations of the cytoplasmic membrane. Assimilation of formaldehyde which is generated during methylamine oxidation was by the FBP variant of the RuMP cycle of formaldehyde fixation. The absence of NAD-dependent formaldehyde and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formal-dehyde via hexulose phosphate synthase. Enzyme profiles of the organism grown on various substrates suggested that the synthesis of amine oxidase, catalase and the enzymes of the RuMP cycle is not under coordinate control.

Notes: Abstract The development of sclerotia of Claviceps purpurea was investigated by light and electron microscopy. During the first days after infection sterigma and conidiospores are formed. The spores show a moderately developed vacuolar system, they are thick walled and contain about 20% lipid (related to the cell volume) embedded in glycogen. The sterigma are cylindrical unicellular hyphae with electron dense cytoplasm and isolated strongly contrasted lipid droplets. In maturing sclerotia the hyphae become septated with increasingly thick cell walls and a large lipid content. The lipid forms small droplets in young cells, while in the mature sclerotium it occurs in the form of very large drops, occupying the major part of the cell. Simultaneously the composition of the lipid is changed. The mature cells have several nuclei. They are partially connected by osmiophilic substances, forming a network of intercellular spaces.

Notes: Abstract Nitrite oxidoreductase, the essential enzyme complex of nitrite oxidizing membranes, was isolated from cells of the nitrifying bacterium Nitrobacter hamburgensis. The enzyme system was solubilized and purified in the presence of 0.25% sodium deoxycholate. Nitrite oxidoreductase oxidized nitrite to nitrate in the presence of ferricyanide. The pH optimum was 8.0, and the apparent K m value for nitrite amounted to 3.6 mM. With reduced methyl-and benzylviologen nitrite oxidoreductase exhibited nitrate reductase activity with an apparent K m value of 0.9 mM for nitrate. NADH was also a suitable electron donor for nitrate reduction. The pH optimum was 7.0. Treatment with SDS resulted in the dissociation into 3 subunits of 116,000, 65,000 and 32,000. The enzyme complex contained iron, molydbenum, sulfur and copper. A c-type cytochrome was present. Isolated nitrite oxidoreductase is a particle of 95±30 Å in diameter.

Notes: Abstract Natural enrichments of magnetic bacteria from the Itaipu lagoon near Rio de Janeiro were dominated by coccoid-to-ovoid morphotypes that produced unusually large magnetosomes. To determine the phylogenetic position of these unusual microorganisms, 16S rRNA genes were retrieved from bacteria magnetically separated from sediment of the Itaipu lagoon by in vitro amplification and cloning of PCR products into a plasmid vector. Partial sequencing of the obtained clones revealed two clusters of closely related sequences affiliated to a distinct lineage consisting exclusively of magnetic bacteria within the α-subclass of Proteobacteria. For a detailed phylogenetic analysis, several almost complete sequences of the 16S rRNA genes were determined. One representative clone of each cluster provided a PCR template for the in vitro transcription of group-specific polynucleotide probes complementary to a variable region of the 16S rRNA molecule. At least three different morphotypes of magnetic bacteria were reliably identified by post-embedding hybridization of ultra-thin sections. Electron microscopic analyses of hybridized cells enabled for the first time a detailed description of the morphological variety and ultrastructure of phylogenetically identified, uncultured magnetic bacteria. Two distinct coccoid bacteria were identified by the transcript probe complementary to the 16S rRNA sequence mabrj12, whereas the probe complementary to the sequence mabrj58 allowed the identification of an ovoid morphotype that displayed magnetosomes with the largest volumes observed to date.

Notes: Abstract The sporogenesis of aerial spores in Streptomyces thermoviolaceus corresponded to a common sporulation type in the genus. The sporulation septum was composed of an outer ring-shaped constriction wall and an inner interspace septum arising by the inwards growth of a double annulus. In mature spores the wall was composed of two layers, the outer one was part of the parent hyphal wall and septum material, the inner one was formed de novo. The spore chains were enclosed by the thin breakable sheath containing small rod-like elements. The ornamentation in the form of knobs, which were a characteristic feature of the species originated from the sheath. The knobs were hemispherical particles with an inner electron dense core and an outer electron transparent shell. The term “cupular knobs” was suggested for this type of tuberculate ornamentation. Frequently, the knobs became detached from the surface in which case the inner core separated easily from the shell.

Notes: Abstract Two strains of desiccation-tolerant coccoid cyanobacteria, Chroococcus S24, a marine form, and Chroococcus N41, a cryptoendolith isolated from a hot-desert rock, have been characterized. The mol % DNA base compositions of the strains are 47.1 and 48.9% respectively. Plasmid DNA was not detected in either strain. The pigment contents and nutritional characteristics of the strains are identical. Both lack phycoerythrinoid pigments and, in culture, behave as slow-growing halotolerant marine forms with elevated requirements for Na+, Cl−, Mg2+ and Ca2+. Sucrose was the only carbon source of those tested that supported photoheterotrophic growth. Each strain synthesizes nitrogenase under anaerobic conditions but not in air. Morphologically the two strains are indistinguishable. They are considered to be independent isolates of the same cyanobacterial species. Chroococcus N41 was studied in detail with the electron microscope. When brought to equilibrium at matric water potentials of-168 MPa and lower (to-673 MPa=c0.12a w) the protoplast shrinks, but the cells maintain the same size and diameter as those at-2,156 kPa (MN medium; control); the sheath expands and remains attached to the cell wall outer membrane by fibrils. The cell wall, cell membrane, thylakoid membranes, cyanophycin granules and carboxysomes appeared intact in desiccated cells.

Notes: Abstract Microorganisms that hydrolyse the ester linkages between phenolic acids and polysaccharides in plant cell walls are potential sources of enzymes for the degradation of lignocellulosic waste. An anaerobic, mesophilic, spore-forming, xylanolytic bacterium with high hydroxy cinnamic acid esterase activity was isolated from the gut of the grass-eating termite Tumilitermes pastinator. The bacterium was motile and rod-shaped, stained gram-positive, had an eight-layered cell envelope, and formed endospores. Phylogenetic analysis based on 16S rRNA indicated that the bacterium is closely related to Clostridium xylanolyticum and is grouped with polysaccharolytic strains of clostridia. A wide range of carbohydrates were fermented, and growth was stimulated by either xylan or cellobiose as substrates. The bacterium hydrolysed and then hydrogenated the hydroxy cinnamic acids (ferulic and p-coumaric acids), which are esterified to arabinoxylan in plant cell walls. Three cytoplasmic enzymes with hydroxy cinnamic acid esterase activity were identified using non-denaturing gel electrophoresis. This bacterium possesses an unusual multilayered cell envelope in which both leaflets of the cytoplasmic membrane, the peptidoglycan layer and the S layer are clearly discernible. The fate of all these components was easily followed throughout the endospore formation process. The peptidoglycan component persisted during the entire morphogenesis. It was seen to enter the septum and to pass with the engulfing membranes to surround the prespore. It eventually expanded to form the cortex, verification for the peptidoglycan origin of the cortex. Sporogenic vesicles, which are derived from the cell wall peptidoglycan, were associated with the engulfment process. Spore coat fragments appeared early, in stage II, though spore coat formation was not complete until after cortex formation.

Notes: Abstract The effects of aluminium chloride (AICI3) treatments (50 and 150 mg/l) on 3-year-old Scots pine (Pinus sylvestris L.) seedlings were studied in a sand culture during 2 growing periods in an open field experiment. Even by the end of the first growing period, a decline was observed in the concentrations of Ca, Mg and P within the needles, and of Ca and Mg in the roots. After the second growing period, increased N and K concentrations were observed in the needles of Al-treated seedlings. Both the needles and roots of Al-treated seedlings showed, after the second growing period, a decline in growth and increased concentrations of AI as the amount of AICI3 in the nutrient solution increased. Al-induced changes in needle structure were found to be symptomatic of a nutrient imbalance, particularly of Mg and P. Al-stress did not result in any observable changes in root anatomy or in the number of mycorrhizas. Scots pine proved to be rather resistant to Al-stress, indicating that direct Al-injuries are not likely in the field, though Al-stress may be a contributing factor in the formation of nutrient imbalances.

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 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 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 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: 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: Summary The ultrastructure of gill epidermal cells of Diopatra neapolitana and their relationship with blood spaces are described. The existence of a basal infolding complex, related to the blood spaces, is also reported. A possible involvement of these cells in osmoregulation and ion interchange, apart from their well-known role in respiration, is suggested.

Notes: Summary This paper describes the unequal distribution of plastids in the developing microspores of Impatiens walleriana and Impatiens glandulifera which leads to the exclusion of plastids from the generative cell. During the development from young microspore to the onset of mitosis a change in the organization of the cytoplasm and distribution of organelles is gradually established. This includes the formation of vacuoles at the poles of the elongate-shaped microspores, the movement of the nucleus to a position near the microspore wall in the central part of the cell, and the accumulation of the plastids to a position near the wall at the opposite side of the cell. In Impatiens walleriana, the accumulated plastids are separated from each other by ER cisterns, and some mitochondria are also accumulated. In both Impatiens species, the portion of the microspore in which the generative cell will be formed is completely devoid of plastids at the time mitosis starts.

Notes: Abstract  Objective: A new co-culture system of rat articular chondrocytes and synoviocytes (HIG-82; cell line) was incubated with phorbol myristate acetate (PMA), H2O2 or a combination of Fe2+ and ascorbic acid to simulate inflammation-like radical attacks in articular joints. Methods: Chondrocytes were characterized by immunocytochemistry against collagen type II, transmission electron (TEM) and light microscopy. Lipid peroxidation was investigated by measuring thiobarbituric-acid-reactive material in the supernatants, cytotoxicity by determining release of lactate dehydrogenase and proliferation by measuring [3H]thymidine incorporation, culture protein and DNA. Results: PMA or Fe2+ and ascorbic acid induced lipid peroxidation in chondrocytes and synoviocytes that was decreased significantly in co-cultures. PMA and H2O2 dose dependently induced release of lactate dehydrogenase in chondrocytes, which was lowered in co-cultures or in previously co-cultured chondrocytes to a nearly basal level. In contrast, conditioned media of synoviocyte cultures showed no lowering effect on the radical-induced toxicity. Protection against H2O2-induced damage of cellular membranes by co-culturing was also shown by TEM. Synoviocytes released chondrocyte-stimulating growth factors spontaneously without previous interaction. Conclusion: Chondrocytes establish protective mechanisms against reactive oxygen species via an interaction with synoviocytes. Our co-culture model presents a possible way to study mechanisms of inflammation in articular joints under defined conditions.

Notes: Summary Degenerative changes in the digestive tract of zooids of Botryllus schlosseri were studied by light and electron microscopy. Three main processes occurred in the tissues: contraction, involution and phagocytosis. The contraction of epidermis and peribranchial epithelium in which cytoplasmic microfilaments probably participate, seemed to have a special role in compressing the underlying organs. During contraction most of the body cavities collapsed, the branchial walls disintegrated and the fragments were rapidly taken up by large phagocytes. The gut epithelium retained its apparent continuity longer, though isolated phagocytes infiltrated it to eliminate single cells. Cell degeneration came about chiefly either through swelling and lysis of cells or through loss of water and condensation of cytoplasm and nucleus. The fate of all regressed tissues was to be engulfed and digested by wandering phagocytes. However, it was also observed that numerous cells of different epithelia could act as fixed phagocytes by engulfing cell debris and entire cells into heterophagic vacuoles.

Notes: Summary Between days 8 and 11 of pregnancy spherical blastocysts from 0.3 to 10 mm in diameter were flushed from the uterine horns of Dutch Landrace pigs. A description of their ultrastructure is given, and the uptake of horseradish peroxidase and ferritin is demonstrated. The ultrastructure of the trophoblast was similar at all ages studied. The trophoblast which has many apical microvilli is able to take up and digest the macromolecules which were offered in the in vitro incubation medium. The hypoblast consists of flattened cells. In blastocysts 2 mm and larger, compact cells bearing microvilli are found below the embryoblast. Cell organelles indicating protein synthesis are found within hypoblast cells of such blastocysts. In the embryoblast, local concentrations of cell organelles are visible, indicating that differentiation has started. After the disappearance of Rauber's layer, which takes place when the blastocyst reaches a diameter of about 2 mm, superficial embryoblast cells develop short microvilli. The cells do not absorb ferritin or peroxidase but are dependent on the trophoblast for their food requirements. All cell layers in the blastocyst contain mitochondria that have characteristics of those found in steroidproducing cells. The significance of the uptake and digestion of macromolecules by trophoblast cells, the synthesis of protein by hypoblast cells and the possible synthesis of steroids is discussed with respect to the relationship between the cell layers of the blastocyst and in the context of conceptomaternal relationships.

Notes: Summary The fine structure of muscle fibres from m. vastus lateralis of nine healthy males (mean age 26 years) was investigated. Four individuals constituted non-exercised controls while five subjects participated in a two-months eccentric muscular training program. Specimens from the controls showed a well-preserved, regular myofibrillar band pattern while changes in the myofibrillar architecture were constantly found in specimens taken after the training program. These changes consisted of Z-band alterations, Z-bands being out of register, extra sarcomeres, Z-band extensions and bisected Z-bands. Between the separated Z-band halves, thin and thick myofilaments as well as abundant glycogen particles and/or ribosomes, were observed. Type-2 (fast-twitch) fibres were predominantly affected. Contrary to the controls the trained individuals constantly showed a greater variation in sarcomere lengths in Type-2 fibres than in Type-1 fibres. It is concluded that muscular work of high tension can induce fine-structural alterations. When repeated over a long period of time, extreme tension demands seem to initiate reorganization in the muscle fibres, predominantly in the, ultrastructurally defined, Type-2 fibres. This adaptation probably results in a better stretchability of the muscle fibres, reduces the risk for mechanical damage and brings about an optimal overlap between actin and myosin filaments.

Notes: Summary An ultrastructural and stereological examination was performed on stomach smooth muscle of the salamander Amphiuma. This tissue has very large cells, ranging up to 12×1500 μm when relaxed. The extracellular space is 31% of the tissue volume, and the tissue contains 84.6% water. These values are similar to those of other amphibian and mammalian gastrointestinal smooth muscle. The cells possess the usual smooth muscle organelles. Thick, thin and intermediate filaments are present, along with membrane-associated and cytoplasmic dense regions. There is a well-developed sarcoplasmic reticulum and many microtubules. Caveolae are found in rows along the cellular surface; the caveolae increase the cellular surface area by about 70%. The ratio mean volume: surface area of the cells is 1.26 μm. This tissue appears to be typical of gastrointestinal smooth muscle, with the exception of the very large size of the cells.

Notes: Summary Spermatogenesis and spermiogenesis in Lepidodermella squammata are confined to the postparthenogenic phase of the life cycle and coincide with developmental changes in the bilateral female gonads. Male stages are bilateral but asynchronous, in the lateral abdomen anterior to the female gonads. Maximum observed sperm production is two packets per side, or 64 sperm. Sperm formation occurs more rapidly at 27° C than at 20° C (p〈0.001), requiring as little as 1 day. Two spermatogonial mitotic divisions produce a clone of four primary spermatocytes connected by bridges (stage 1). Centrioles are absent. Development occurs within a cyst. Meiotic divisions produce 16 spermatids (stage 2), each containing a dense, elongate, tapered nucleus. Cytoplasmic membranes enclose one end of the nuclear rod, excluding all other organelles. Completion of this process results in stage 3, a packet of 16 sperm associated with one dense sphere, a modified ‘residual body’ containing cytoplasmic debris. The residual body then disappears, leaving the sperm packet of stage 4. Each mature sperm is a dense nuclear rod with surrounding membranes, lacking acrosome, mitochondrion, centrioles, and flagellum. Function of sperm has not been demonstrated. The spermatozoa are of a reduced type not previously described.

Notes: Summary Six small cells are present in each of the bilateral gonads of parthenogenically reproductive Lepidodermella squammata. Early in the extended postparthenogenic phase of the life history, these cells undergo limited proliferation followed by differentiation. Primary oocytes of three types are present 0.3 days after deposition of the final parthenogenic egg: small oocytes with presynaptic nuclei; intermediate oocytes with nuclei containing synaptonemal complexes; and larger oocytes with a germinal vesicle. Oocytes persist without further development at least until day four of the postparthenogenic phase. Older isolated animals may contain and even deposit an enlarged egg, but successful progeny does not result. Oocytes are located at the anterior pole of each of the bilateral gonads, adjacent to developing male tissues producing sperm. More posterior cells in the gonad are initially undifferentated in the postparthenogenic phase. Dorsal and central cells first show specialization for secretory activity, and by day four contain peripheral layers of RER and central accumulations of polymorphic secretion droplets. The posterior and ventral cells produce secretion droplets that aggregate into an enlarging bilobed structure called the X-body. Two or three cells in each gonad contribute secretions to the X-body, which is intracellular in a secondary syncytium formed by the contributing cells. Functions for the postparthenogenic gametes and for the X-body are not yet demonstrated.

Notes: Summary In the bullfrog, the meninges surrounding the central nervous system include an arachnoid mater that contains layers of cells with abundant intermediate filaments (IFs) having unique organizational characteristics. This membrane contains an inner lamina of cells that resemble fibroblasts and an outer lamina of flattened cells that are almost filled with IFs. The IFs of the outer arachnoid are arranged in compact, arching bundles that lie parallel to the outer surface of the central nervous system. Thus, sections cut tangentially to the membrane reveal bending of filament bundles, whereas transverse sections do not. In some cells bordering the subdural space, bundles of filaments are organized into highly-ordered spiral arrays. Attachments to the numerous desmosomes and, apparently, to the nuclear envelope suggest anchoring of cytoplasmic structures by the IF system. Microtubules occur primarily near the plasma membrane and the nucleus. Numerous caveolae also are associated with the plasma membrane. The unusual abundance, organization, and cytoplasmic relations of IFs in the bullfrog arachnoid suggest that this membrane may serve as an important model for study of fundamental cytoskeletal relations and function.

Notes: Summary The general structure, ultrastructure and innervation of the swimbladder of the smooth toadfish, Tetractenos glaber, were examined with light-microscopic, fluorescence-histochemical, and transmission electron-microscopic techniques. The structure of the swimbladder is similar to that of other euphysoclists. Fluorescence histochemistry showed adrenergic fibres in both the secretory and resorptive areas of the swimbladder. Transmission electron microscopy revealed two morphologically distinct axon profiles type-I profiles containing many small, flattened vesicles; type-II profiles containing both large, granular vesicles and rounded, small clear vesicles in varying proportions. The gas-gland cells and surrounding muscularis mucosae are innervated by both type-I and type-II fibres. Type-I fibres also innervate pre-rete arteries. The rete- and gas-gland capillaries do not appear to be innervated. Arteries running to the resorptive area are innervated by type-I fibres. Both type-I and type-II profiles make contact with the muscularis mucosae in the resorptive area. Only type-I fibres innervate the radial dilator muscle in the oval sphincter region, whereas only type II fibres innervate the circular muscle of the oval sphincter. Type-I fibres took up α-methyl-noradrenaline, and could not be found after pre-treatment with 6-hydroxydopamine. They are, therefore, assumed to be adrenergic. Type-II fibres were tentatively identified, by exclusion, as cholinergic.

Notes: Summary Scale regeneration has been studied in Hemichromis bimaculatus. The removed scale, which serves as a control, is covered by its surrounding scleroblasts as can be seen with scanning electron microscopy. Subsequently, during regeneration, a population of scleroblasts arises in the empty dermal pocket as shown with transmission electron microscopy. At first, an elongated papilla of regeneration forms, probably from the differentiation of dermal fibroblasts. A scale anlage composed of the osseous layer appears in the middle of the papilla, which becomes a regenerating bag. All the surrounding large scleroblasts are involved in scale formation, although later three populations of scleroblasts specialize according to their location around the scale. Superficial scleroblasts flatten when the final thickness of the osseous layer of the scale is attained; the deep scleroblasts are responsible for the formation of the basal plate whereas marginal scleroblasts increase the diameter of the osseous layer of the scale. During scale regeneration, scleroblasts are more numerous and larger than during scale ontogenesis. In particular, deep scleroblasts form a columnar epithelium when the basal plate is laid down, a feature which is not found during scale ontogenesis. Moreover, the regenerated basal plate exhibits an orthogonal “plywood” arrangement that is never seen in the embryonic scale where the “plywood” is of the intermediate type.

Notes: Summary The ductus epididymidis of the tammar is lined by an epithelium composed of principal, mitochondria-rich, apical and basal cells, and intraepithelial leucocytes. The epithelium is structurally differentiated into 6 zones referred to as the initial segment, middle segment (3 subdivisions) and terminal segment (2 subdivisions). The occurrence of the initial, middle and terminal segments corresponds quite closely to the anatomical differentiation of the epididymis into a head, body and tail. The initial segment epithelium in the tammar is lower and has shorter and more slender stereocilia than in other mammals which have been described. Otherwise, the structure of the epithelium has similar characteristics in the tammar to that described in other mammals. Spermatozoa begin to develop the capacity for motility within the initial segment, but only show structural signs of maturation in the middle segment. The sperm head rotates through 90 degrees in the proximal subdivision of the middle segment. The cytoplasmic droplet is detached and spermatozoa develop the capacity for motility in the middle subdivision of the middle segment. The cytoplasmic droplets are phagocytosed by the epididymal epithelium of the middle segment. Sperm storage appears to be the main function of the terminal segment.

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 Conspicuous cytological differences are found between specific secretory cells of the hypophysial pars tuberalis of Djungarian hamsters exposed to long and short photoperiods. The cells differ with respect to the shapes of perikarya and nuclei and show diverse amounts of secretory granules, lysosome-like bodies and glycogen.

Notes: Summary Pituitaries of the African catfish (Clarias lazera) were studied with immunocytochemical methods, at the light-microscopic and ultrastructural levels, for the characterization and localization of gonadotropic and thyrotropic cells. Two immunostaining procedures with the use of different markers were carried out: (i) with peroxidase-antiperoxidase, (ii) with protein A-gold. In routinely stained sections for light microscopy two types of basophils were identified in the proximal pars distalis: (1) large, round, purple cells, and (2) small, angular, light-blue cells. Both types were immunolabeled with antibodies against Clarias α,β-gonadotropin (GTH) and salmon G100-GTH. Only the large basophils were immunolabeled with anti-carp β-GTH, whereas the small basophils were the only cells immunolabeled with anti-human thyrotropin beta subunit (anti-h TSH-β). It was concluded that the large basophils represent the gonadotrops and the small basophils the thyrotrops. At the ultrastructural level the immunostaining of the GTH-cells was confined to three types of inclusions: (i) secretory vesicles, (ii) globules, and (iii) electron-dense, membrane-bound irregular masses. Especially the protein A- gold method, in combination with the use of a highly diluted homologous antiserum, resulted in a distinct localization of GTH. The presence of two types of nerve fibres, synaptically contacting the gonadotrops, is discussed with regard to the presence of a peptidergic (stimulatory) and an aminergic (inhibitory) control of GTH-secretion.

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 In the pineal gland of the pipistrelle bat two different populations of pinealocytes and glial cells were observed electron microscopically. The pinealocytes of populations I and II differ in their content of metabolically active cell organelles. In the pinealocytes of population I, granular vesicles originating from the Golgi apparatus were found in the perikaryon and especially in the endings of the pinealocyte processes. Granular vesicles appeared to be more numerous in hibernating nulliparous females. The pinealocytes of population II are characterized by the presence of small cytoplasmic vacuoles, probably originating from cisternae of the granular endoplasmic reticulum and containing flocculent material of moderate electron density. The classification of the pinealocytes belonging to population II is discussed.

Notes: Summary The morphology of the compound eye of the noctuid moth Spodoptera exempta was investigated by electron microscopy. This eucone superposition eye is composed of about 8000 ommatidia. Each ommatidium is surrounded by six secondary pigment cells showing pigment movement according to the state of adaptation. It contains four crystalline cone cells forming together a crystalline cone and tract, two primary pigment cells, which encompass the crystalline cone, and usually eight retinula cells. On the basis of their rhabdomeric structure, three types of retinula cells can be distinguished. According to the structure of the rhabdom, two types of ommatidia are found in different regions of the eye. The rhabdom of the lobed type, providing more than 80% of ommatidia, is composed of V-shaped rhabdomeres with fanwise arranged microvilli. The rhabdom of the square type, found in a small area in the dorsal region of the eye, consists of triangular rhabdomeres with parallel microvilli. The functional significance of this difference is discussed.

Notes: Summary The dioptric apparatus of the lateral eyes of the scorpion, Androctonus austrails, consists of a cuticular lens, but lacks a vitreous body. The retina is formed by (1) retinula cells displaying a contiguous network of rhabdoms; (2) arhabdomeric cells bearing a distal dendrite that contacts retinula cells via numerous projections and ends before the rhabdomere of the retinula cells; (3) pigment cells that ensheath retinula and arhabdomeric cells with the exception of the contact regions; and (4) neurosecretory fibres possibly originating in the supraesophageal ganglion. The ratio of the number of retinula to arhabdomeric cells is determined to be close to 2 ∶ 1 in the three larger anterolateral eyes, in contrast to the median eyes where the ratio is 5 ∶ 1. The construction of the dioptric apparatus as well as the anatomy of the retina imply that in the lateral eyes of Androctonus australis visual acuity is reduced. A certain degree of spatial discrimination, however, may be retained by the presence of a relatively high number of arhabdomeric cells. It is suggested that the lateral eyes of A. australis mainly function as light detectors, e.g., for Zeitgeber stimuli.

Notes: Summary The structure of the compound eyes of adult Cicindela tranquebarica Herbst was examined by use of light, scanning, and transmission electron microscopy. Each ommatidium of these photopic eyes is eucone and has a “subcorneal layer” situated between the corneal lens and crystalline cone. A distal rhabdomere consisting only of microvilli from retinula cell seven, a more proximal, rectangular, fused rhabdom formed from six retinula cells, and a basal, eighth retinula cell with a spherical rhabdomere comprise the light sensitive portions of the ommatidium. The “subcorneal layer” consists of lamellae of endocuticular microfibrils and, in surface view, shows 11 concave polygons. Proximal extensions of the crystalline thread form inter-retinular fibres containing microtubules between retinula cells 1/2, 3/4, 5/6, and 7/1. The primary pigment cells are devoid of pigment granules, but are rich in rough endoplasmic reticulum. Proximal to each retinula cell nucleus are two basal bodies, one perpendicular to the other. The more proximal basal body extends two fibrillar feet proximally which fuse to form a horizontally-banded ciliary rootlet extending the length of the retinula peripheral to the rhabdom. Each ommatidium is surrounded by 16 secondary pigment cells. Interfacetal mechanoreceptors between some adjacent lenses each have a single bipolar neuron, with a dendritic sheath, tubular body, cilium, outer and inner sheath cells, and an axon surrounded by a neurilemma sheath cell.

Notes: Summary Calcitonin cells are relatively numerous in the thyroid gland of the rat. In contrast, somatostatin cells are very scarce except at the time of birth and a few days thereafter, when they are conspicuously numerous. Somatostatin cells of the thyroid gland, which are ultrastructurally similar to somatostatin cells in gut and pancreas, also contain immunoreactive calcitonin. It is not clear whether somatostatin cells in the rat thyroid gland produce calcitonin or accumulate calcitonin from the environment.

Notes: Summary The preputial glands of male nude (nu/nu) mice were analyzed by a combination of electron microscopy and gas chromatography to determine whether or not they are affected, like developing hairs and nails, by the nu/nu genotype. Results of the analyses revealed no differences between the glands of nude and normal male mice in either their ultrastructural characteristics or lipid secretory products.

Notes: Summary To identify and describe ovarian follicles committed to undergo follicular degeneration (atresia), immature rats were primed with pregnant mare serum gonadotropin (PMSG). After PMSG treatment, preovulatory follicles develop but subsequently degenerate. Prior to the appearance of pyknotic nuclei (Stage I of atresia), degenerative changes were observed in focal areas of the granulosa cell layer. These changes include “blebbing” of the cytoplasm and alterations in the shape of the granulosa cells. The appearance of these degenerative changes coincides with a decrease in ovarian concentrations of estradiol and testosterone. Since estrogens and androgens maintain the follicle, the decline in estradiol and testosterone could be responsible for the further degenerative alterations that lead to complete deterioration of the preovulatory follicle. In Stage I atretic follicles, lysosome-derived autophagic vacuoles develop and macrophages invade both the thecal and granulosa cell layers. The combined actions of the autophagic vacuoles and macrophages could destroy both the granulosa-cell and thecal layers and thereby transform the preovulatory follicle into an ovarian cyst.

Notes: Summary Kidneys of 2 to 10 day-old rats of Wistar and Sprague-Dawley strains were fixed with glutaraldehyde by retrograde vascular perfusion and then prepared for observation in TEM and SEM. In addition methacrylate casts of differentiating glomerular capillaries were examined by SEM. Although the glomerular vascular pattern differs from one glomerulus to another, its differentiation proceeds according to the following general plan. First the glomerular capillary splits longitudinally, finally to form 3 to 5 lobules consisting of a capillary network, sustained centrally by the mesangium. In the present study the differentiation of glomerular capillaries is described in five successive arbitrarily selected stages. At Stage I a capillary loop penetrates between the lower limb and the middle segment of the S-shaped body, the rudimentary nephron. At Stage II the capillary undergoes a first subdivision, establishing the primitive lobulation of the glomerulus. At Stage III the vascular and urinary poles differentiate. At Stage IV the glomerulus assumes the aspect of a spherical body, and the capillaries in each lobule undergo subdivision. In Stage V the glomerular vascular pattern approaches its adult appearance, although the maturation processes continue for an extended period of time. Hence in the 10 day-old rat the best-differentiated glomeruli are half the size of adult glomeruli, and their capillary loops are proportionally less well-developed. The capillaries of adjacent lobules may communicate with each other, but a direct vascular shunt between the afferent and efferent vessels cannot be demonstrated.

Notes: Summary The Stannius bodies of killifish and goldfish were ultrastructurally investigated after exposure of the fish to media of different osmolality and calcium content. In both species two structurally different secretory cell types are found. Type-1 cells predominate. They contain an extensive granular endoplasmic reticulum, some large Golgi areas, and secretory granules with a mean diameter of about 0.4 μm. These cells are activated by transfer of freshwater fish to seawater, but not by transfer to low-calcium seawater. Type-2 cells often contain cytoplasmic processes that penetrate between the type-1 cells and are ending on the basal lamina. In this contact area granule release takes place. Type-2 cells contain some strands of granular endoplasmic reticulum, several small Golgi areas, and secretory granules with a mean diameter between 0.15 and 0.20 μm. Type-2 cells are not activated after transfer of fish to seawater. In killifish seawater exposure leads to a reduction of type-2 cells. The differences between the reactions of both cell types to different media point to functional differences between their secretory products. Type-1 cells may produce a hypocalcemic factor. It is concluded that type-2 cells are typical for freshwater fish or euryhaline fish spending part of their life cycle in freshwater. The proper function of these cells is unclear.

Notes: Summary The thymus of wild young and adult bank voles (Clethrionomys glareolus) was examined by histological methods for the presence of developing erythroid cells. Nucleated erythroid cells were observed in 26% of the glands examined by light microscopy and in 69% of the glands examined by electron microscopy. The largest number of developing erythroid cells was observed in the thymus of pregnant females, also showing raised reticulocyte counts (3.1–10.2%). However, erythropoiesis could also be found in breeding and non-breeding, first year and older animals. Erythroid cells were mainly located in the cortex, sometimes in small groups interspersed between lymphoid cells, and also randomly scattered in the cortex. Occasionally, macrocytic erythroid cells were also present. Pyknotic cells were commonly present, and granulopoiesis was frequently observed.

Notes: Abstract. A pair of antagonistic motoneurons, one excitatory and one inhibitory, innervates the distal accessory flexor muscle in the walking limb of the crayfish Pro- cambarus clarkii. The number and size of synapses formed by these two axons on the muscle fibers (neuromuscular synapses) and on each other (axo-axonal synapses) were estimated using thin-section electron microscopy. Although profiles of nerve terminals of the two axons occur in roughly equal proportions, the frequency of occurrence of neuromuscular synapses differed markedly: 73% were excitatory and 27% were inhibitory. However, inhibitory synapses were 4–5 times larger than excitatory ones, and consequently, the total contact areas devoted to neuromuscular synapses were similar for both axons. Axo-axonal synapses were predominantly from the inhibitory axon to the excitatory axon (86%), and a few were from the excitatory axon to the inhibitory axon (14%). The role of the inhibitory axo-axonal synapse is presynaptic inhibition, but that of the excitatory axo-axonal synapse is not known. The differences in size of neuromuscular synapses between the two axons may reflect intrinsic determinants of the neuron, while the similarity in total synaptic area may reflect retrograde influences from the muscle for regulating synapse number.

Notes: Abstract. The spermatogonial stem cell line in prepubertal and adult bovine testis was studied by electron microscopy and protein gene product 9.5 immunohistochemistry. Three successive spermatogonia precursor cell configurations were observed. Small basal stem cells were found to possess a spherical shape and nuclei with two to three nucleoli. They were observed in prepubertal testes (25 and 30 weeks) and in low numbers during all the stages of the seminiferous epithelial cycle in the adult. Aggregated spermatogonia precursor cells are the dominating germ cell type in the 25-week-old and 30-week-old calf. In the adult seminiferous epithelium, they cause expansion of the basal tubular compartment as they form dense groups containing up to 15 cells. These groups are observed concomitantly with cycling A-spermatogonia and preleptotenes at the beginning of spermatocytogenesis. At the end of A-spermatogonia propagation, the aggregated spermatogonia precursor cells separate and intermingle with cycling A-spermatogonia. The spermatogonia precursor cells can later be found together with I-spermatogonia as members of an interconnected cellular network of medium-sized cells. When the I-spermatogonia divide to form the smaller B-spermatogonia, the precursor cells, which stay connected with the cycling spermatogonial population, pass through a growth phase. They can now be considered as committed spermatogonia precursor cells and are continuously being transformed into A1-spermatogonia to start a new round of spermatocytogenesis. Ultrastructurally, all members of the precursor cell line are similar. However, a number of features have been found to show a quantitative increase (endoplasmic reticulum, mitochondria) or to exhibit a rising degree of complexity (nucleolus) during the progression from basal stem cells to committed spermatogonia precursor cells.

Notes: Abstract The spermatogonial stem cell line in prepubertal and adult bovine testis was studied by electron microscopy and protein gene product 9.5 immunohistochemistry. Three successive spermatogonia precursor cell configurations were observed. Small basal stem cells were found to possess a spherical shape and nuclei with two to three nucleoli. They were observed in prepubertal testes (25 and 30 weeks) and in low numbers during all the stages of the seminiferous epithelial cycle in the adult. Aggregated spermatogonia precursor cells are the dominating germ cell type in the 25-week-old and 30-week-old calf. In the adult seminiferous epithelium, they cause expansion of the basal tubular compartment as they form dense groups containing up to 15 cells. These groups are observed concomitantly with cycling A-spermatogonia and preleptotenes at the beginning of spermatocytogenesis. At the end of A-spermatogonia propagation, the aggregated spermatogonia precursor cells separate and intermingle with cycling A-spermatogonia. The spermatogonia precursor cells can later be found together with I-spermatogonia as members of an interconnected cellular network of medium-sized cells. When the I-spermatogonia divide to form the smaller B-spermatogonia, the precursor cells, which stay connected with the cycling spermatogonial population, pass through a growth phase. They can now be considered as committed spermatogonia precursor cells and are continuously being transformed into A1-spermatogonia to start a new round of spermatocytogenesis. Ultrastructurally, all members of the precursor cell line are similar. However, a number of features have been found to show a quantitative increase (endoplasmic reticulum, mitochondria) or to exhibit a rising degree of complexity (nucleolus) during the progression from basal stem cells to committed spermatogonia precursor cells.

Notes: Abstract. A specific monoclonal antiserum (Mab 6.17) inducing a strong immunostaining of the neuromuscular junction has been used to detect the possible occurrence of the corresponding antigen throughout the intact or lesioned central nervous system of adult rats. In intact animals, 6.17-immunolabeling was essentially detected in astrocyte-like structures located in white matter fasciculi of the brain, such as the optic tract, corpus callosum, fornix, and in the white matter of the spinal cord. The astroglial nature of such 6.17-immunolabeled profiles was verified by performing double or triple immunofluorescent labeling with Mab 6.17 and with specific antisera against astrocytic markers, such as S100 protein, glial fibrillary acidic protein and vimentin. In the white matter, all the structures reactive to Mab 6.17 were also reactive to antibodies against S100 protein, glial fibrillary acidic protein and vimentin. On the other hand, astrocytes of the grey matter that were immunoreactive to S100 and glial fibrillary acidic protein but negative to vimentin, were devoid of 6.17-immunoreactivity. After lesions including stab wound through the diencephalon or transection of the spinal cord, a marked increase of 6.17-immunostaining was noted in the regions surrounding the lesions. In these regions, 6.17-immunolabeling was associated with S100-, GFAP- and vimentin-positive astrocytes constituting the glial scar. The ultrastructural localization of 6.17-immunoreactivity indicated that, similar to glial fibrillary acidic protein and vimentin, the recognized antigen was mainly associated with gliofilaments. These observations indicate that, in the central nervous system of adult rats, Mab 6.17 recognizes a molecule associated with gliofilaments, which is essentially associated to reactive astrocytes expressing high levels of vimentin.

Notes: Abstract. The oxyntic mucosa of the mammalian stomach is rich in endocrine cells, such as ECL cells, A-like cells, somatostatin cells, D1/P cells and, in some species, enterochromaffin cells. The various endocrine cell types can be distinguished on the basis of their characteristic cytoplasmic granules and vesicles. The ECL cells contain numerous large secretory vesicles and relatively few, small electron-dense granules and small clear microvesicles. We have suggested that in the rat the ECL cells contain most of the gastric histamine with the secretory vesicles as the major histamine storage site in these cells. α-Fluoromethylhistidine is an irreversible inhibitor of histidine decarboxylase, the histamine-forming enzyme. We have previously shown that this enzyme inhibitor depletes histamine from the ECL cells in the rat and reduces the number of secretory vesicles in the cytoplasm. In the present study, we have examined whether α-fluoromethylhistidine affects the ECL cells in other species and whether it affects other types of endocrine cells in the oxyntic mucosa of the rat. Mice, rats and hamsters were treated with the inhibitor (3 mg/kg per h) via minipumps subcutaneously for 24 h. This treatment lowered the oxyntic mucosal histamine concentration by 65–90% and the number and volume density of the secretory vesicles by 85–95% in the ECL cells of the three species examined. In contrast, the number and volume density of granules and microvesicles were not greatly affected. No evidence was found for an effect of α-fluoromethylhistidine on A-like cells, somatostatin cells or D1/P cells of the rat stomach, suggesting that, unlike the ECL cells, they do not contain histamine.

Notes: Abstract  The ECL cells are histamine- and peptide hormone-producing endocrine cells in the rat oxyntic mucosa. They are rich in secretory vesicles and also contain microvesicles and electron-dense granules. They operate under the control of circulating gastrin. In the present study, we examined the ECL-cell ultrastructure after long term treatment with omeprazole, which is known to induce hypergastrinemia, and after withdrawal of the drug. Rats received omeprazole (400 µmol/kg per day, orally) for 16 days and were killed 1, 5, 20, or 40 days after the last dose of the drug. Oxyntic mucosal specimens were processed for electron microscopy. Electron micrographs of ECL-cell profiles were analyzed planimetrically. The ECL-cell profile area increased promptly in response to omeprazole, the secretory vesicles and granules were reduced in number and volume density, the microvesicles were unchanged in number but reduced in volume density, and vacuoles appeared. Within a week after stopping the omeprazole treatment, the numbers and volume densities of secretory vesicles and microvesicles returned to pre-stimulation values. Also, the vacuoles disappeared promptly. The ECL-cell profile area decreased below the pre-stimulation level within five days after stopping treatment, while, in contrast, the granules increased in number and volume density. Somewhat surprisingly, the cell size and the granule compartment did not return to normal until 40 days after stopping treatment.

Notes: Abstract. In order to capture the exocytotic figures of gastrin cells in the pyloric antrum of the stomach, we examined antral cells of the mouse, rat, and guinea pig by electron microscopy following stimulation with the cholinergic secretagogue carbamylcholine. Increased numbers of omega profiles indicative of exocytosis were seen in the basal or lateral cell membrane after stimulation with carbamylcholine. The number of exocytotic figures in stimulated gastrin cells was higher in the guinea pig than in the mouse and rat. Coated and non-coated omega profiles and coated pits in the plasma membrane were smaller than the secretory granules. Omega profiles with or without electron-dense contents were seen. Coated and non-coated vesicles were often visible near the plasma membrane of stimulated gastrin cells in all three species, large cytoplasmic vacuoles also being found in the guinea pig. In the mouse pretreated with horseradish peroxidase, reaction deposits were observed in the omega profiles and in microvesicles near the plasma membrane. These results suggest that, after exocytosis, membrane retrieval and endocytosis occur in the gastrin cells.

Notes: Abstract  Integumental and branchial chloride cells of tilapia larvae (Oreochromis mossambicus) were studied at the light-microscopical and ultrastructural level. Total numbers and distribution of chloride cells were quantified after immunostaining of cross sections of the entire larvae with an antibody against the α-subunit of Na+/K+-ATPase. The majority (66%) of Na+/K+-ATPase-immunoreactive (ir) cells, i.e. chloride cells, of freshwater tilapia larvae were located extrabranchially up to 48 h after hatching. Five days after hatching, the majority (80%) of chloride cells were found in the buccal cavity. Transfer of 24-h-old larvae to 20% sea water speeded up this process; 24 h after transfer (i.e. 48 h after hatching), the majority (59%) of chloride cells were located in the buccal cavity. The branchial chloride cell population of 24-h- and 120-h-old larvae consisted of immature, mature, apoptotic and necrotic chloride cells. However, relatively more immature chloride cells were observed in freshwater larvae (42–63%) than in (previously studied) freshwater adults (21%), illustrating the developmental state of the gills. After transfer to sea water, the incidence of degenerative chloride cells did not change. Furthermore, the incidence of immature cells had decreased and a new subtype of chloride cells, the ”mitochondria-poor” cells, appeared more frequently. These mitochondria-poor chloride cells were characterised by an abundant tubular system and relatively few mitochondria, which were aligned at the border or concentrated in one part of the cytoplasm. Most of these cells did not contact the water. The function of their enhanced appearance after seawater transfer is unknown.

Notes: Abstract. Striated rootlets in ciliated cells are conical banded structures composed of longitudinally aligned filaments. The formation of striated rootlets during ciliognesis in the human oviduct epithelium was studied by electron microscopy. Primitive rootlets appeared at the proximal side of basal bodies before or at the same time as ciliary budding. After the formation of several striations, the tip of the rootlets extended deeply toward the interior of the cell and became differentiated into two distinct parts, viz., the proximal conical part connected to the basal body and the distal fibrillar part. The periodicity of the striations in the fibrillar part was 68.5±2.95 nm, about 5 nm longer than that of the conical part (63.9±2.25 nm). The dark band in the striation was thicker in the fibrillar part than in the conical part. Since the fibrillar part was not observed in the mature cilium, this part was considered as being either degraded or changed into the conical part during ciliogenesis.

Notes: Abstract  Interstitial cells of Cajal (ICCs) are believed to be a major element in generating the spontaneous rhythm of the gastrointestinal tract. A prominent problem in the study of these cells has been the difficulty in observing them in intact tissues. We used the lipophilic dye DiI to stain ICCs in the submucosal-circular muscle border of freshly dissected mouse colon. The placement of small DiI crystals in this area resulted in the labeling of ICC-like cells. Two main morphological cell types, viz., bipolar and multipolar, were noted. Bipolar cells had two primary processes emerging from the poles of an elongated soma. The mean length of these processes was 78.7 μm. These cells constituted 42.3% of the sample (n=105). Multipolar cells (54.3% of total) had a less elongated soma and extended 3–6 main processes whose mean length was 56.3 μm. These processes showed no preferred direction. The length of the primary processes of bipolar cells was 40% greater than that of multipolar cells (P〈0.02). Three cells (2.9%) had only one primary process. The DiI stain could be converted into a stable electron-opaque product. Electron-microscopic observations showed that these cells had the typical appearance of ICCs reported in previous studies. This staining method should be useful for physiological investigations of ICCs in gastrointestinal tissues.

Notes: Abstract  The cutaneous glands of the forehead and the metatarsus were studied by histological and histochemical methods and electron microscopy in adult male and female impalas in various seasons of the year. All glandular areas consist of apocrine and holocrine glands, which, however, occur in different proportions. Our findings in the apocrine gland cells suggest (1) the synthesis and exocytosis of a glycoproteinaceous secretory product stored in secretory granules, (2) typical apocrine secretion of the transformed apical cytoplasm, and (3) transepithelial fluid transport. The Golgi apparatus and apical membrane have binding sites for several lectins (PNA, HPA, RCA I, WGA). Cytokeratins 7, 14 and 19 are expressed at various intracellular localizations, suggesting an active role in the secretory mechanisms. The glands of the male forehead show marked seasonal changes in activity that are correlated with the main phases of the reproductive cycle, with the highest cellular activity occurring during the rut in April/May. The female forehead glands are only moderately developed and do not undergo seasonal changes. The metatarsal glands are of equal size in males and females and show no seasonal changes in activity. This study supports the hypothesis that (1) forehead glands in the male have a signaling role in the rut and (2) the metatarsal glands have a more general, probably social role maintaining and restoring contact between herd members.

Notes: Abstract Indirect immunocytochemical tests were used at the light- and electron-microscopic levels to investigate peripheral chemical synapses in identified sensory neurons of two types of cuticular mechanosensors in the spider Cupiennius salei Keys.: (1) in the lyriform slit-sense organ VS-3 (comprising 7–8 cuticular slits, each innervated by 2 bipolar sensory neurons) and (2) in tactile hair sensilla (each supplied with 3 bipolar sensory cells). All these neurons are mechanosensitive. Application of a monoclonal antibody against Drosophila synapsin revealed clear punctate immunofluorescence in whole-mount preparations of both mechanoreceptor types. The size and overall distribution of immunoreactive puncta suggested that these were labeled presynaptic sites. Immunofluorescent puncta were 0.5–6.8 μm long and located 0.5–6.6 μm apart from each other. They were concentrated at the initial axon segments of the sensory neurons, while the somata and the dendritic regions showed fewer puncta. Western blot analysis with the same synapsin antibody against samples of spider sensory hypodermis and against samples from the central nervous system revealed a characteristic doublet band at 72 kDa and 75 kDa, corresponding to the apparent molecular mass of synapsin in Drosophila and in mammals. Conventional transmissionelectron-microscopic staining demonstrated that numerous chemical synapses (with at least 2 vesicle types) were present at these mechanosensory neurons and their surrounding glial sheath. The distribution of these synapses corresponded to our immunofluorescence results.Ultrastructural examination of anti-synapsin-stained neurons confirmed that reaction product was associated with synaptic vesicles. We assume that the peripheral synaptic contacts originate from efferents that could exert a complex modulatory influence on mechanosensory activity.

Notes: Summary Axons in the duodenum, ileum and rectum of the domestic fowl were identified as catecholamine-containing (CA) on the basis of positive reactivity following chromaffin fixation for electron microscopy. CA-axons in association with blood vessels in all regions of the intestine and in non-vascular sites in the small intestine had a ‘ typical’ adrenergic appearance, in that they contained many small granular vesicles (SGV) and variable numbers of large granular vesicles (LGV). In the rectum the non-vascular CA-axon profiles were atypical, in that there were many elongated LGV and few SGV, and the chromaffin reactivity was weak. The nerve profiles in the rectum were dramatically reduced following 6-hydroxydopamine and reserpine treatment and were absent in rectum cultured in the absence of extrinsic ganglia. It was concluded that the profiles, in spite of their low chromaffin reactivity, truely represent CA-axons. The possibility was raised that the atypical morphology and reduced chromaffin reactivity is due to the presence of adrenaline.

Notes: Summary Corticotropes of rat fetuses aged 16, 18 and 21 days were localized by the indirect antibody-enzyme method on semithin sections of the pituitary. The development of the ultrastructure of these cells was observed on consecutive ultrathin sections. In comparison with previous data our present results show that identification of a fetal cell type cannot be based entirely on morphological criteria. The structural peculiarities of corticotropes obtained from studies in vivo are compared with those observed in cells maintained in vitro.

Notes: Summary Cyclophosphamide-induced changes in rodent odontogenesis were investigated by light and electron microscopy in four-day-old Sprague Dawley rats given one injection of 40 mg/kg of body weight of cyclophosphamide and killed at intervals of one hour, one day, one week and two weeks. Incisor and molar teeth were dissected from the animals, fixed in 2.0% glutaraldehyde in 0.1 M sodium cacodylate with 3.4% sucrose, and subsequently some were incubated for alkaline phosphatase reaction, and embedded in Spurr's medium for sectioning at light- and electron-microscopic levels. From three days a cell-sparse zone was created in the pulp in the growing end of the tooth and progressive cellular changes were observed which became more severe in the one-week and two-week specimens. Subodontoblast and adjacent pulpal cells were the most affected showing nuclear changes, damage to, or loss of, organelles, and inclusion bodies. Odontogenic epithelium was less affected and odontoblasts appeared to be unaffected by the drug. A new irregular matrix was laid down in the defect area and seemed to be the product of depolarized odontoblasts. This new matrix showed alkaline phosphatase activity, as did the cells embedded in it, and later it became mineralized. It is speculated that the polarity of odontoblasts might be maintained by an intact subodontoblastic layer; when this is lost the odontoblasts become depolarized and capable of secreting matrix from both ends.

Notes: Summary The ultrastructural changes in the spatial organization of nucleolar DNA in lymphocytes during phytohemagglutinin (PHA) stimulation was studied in guinea pigs by means of oxidized diaminobenzidine (DAB) at low pH as a differentially contrasting stain for nucleic acids and by the use of reconstruction of serial sections. The extended DNA filaments situated inside the fibrillar area originate from a large aggregation of heterochromatin, which is closely associated with the nucleolus, and from the perinucleolar shell of condensed chromatin. It is suggested that these two distinct regions of chromatin might be associated with different functions.

Notes: Summary Myeloid bodies (MBs) occur in the newt (Notophthalmus viridescens) retinal pigment epithelium (RPE) and are similar to areas of specialized endoplasmic reticulum found in a variety of other cell types. The function of these structures is unknown, although a role in lipid metabolism has been strongly suggested. Random samples from conventionally-fixed and sectioned newt RPE, obtained over a 24-hr cycle (LD 12∶12), were examined by electron microscopy. Myeloid bodies appear as stacks of flattened endoplasmic reticulum-associated saccules which increase in length and number as the RPE accumulates shed outer segment material, prior to increase in the amount of stored lipid. Associations of MBs with the nuclear envelope can be related to this increased length. Myeloid bodies decrease numerically in the cell as phagosomes are removed from the cytoplasm, but a decrease in mean sectional MB area, seen in the light phase, is counteracted in darkness where individual MBs are larger than those found in the light. The total sectional area of MBs within a cell and their mean length varied depending on the lighting condition; differences were also found between eyes after extended periods of continuous light and dark. Ribosomes were found in association with the surfaces of both flattened and circular MBs, but they were consistently more densely associated with the shorter concave surfaces of curved regions. A new hypothesis for MB function is presented, which is concerned with their role in isolating toxic lipids such as retinoids, which are accumulated during phagocytosis of shed outer segment tips, and which are capable of disrupting membrane-bound systems necessary for their eventual metabolism and safe storage.