Heavy metals in the terrestrial isopod Porcellio scaber Latreille. II. Subcellular fractionation of metal-accumulating lysosomes from hepatopancreas

Two populations of metal contaminated Porcellio scaber Latreille were studied: one consisting of animals that had been fed heavy metals in the laboratory for several months, and one from a metal-polluted site in the field (Braubach, FRG). Density gradient centrifugation was performed on hepatopancreas homogenates in order to identify cellular fractions and their association with lead, copper and cadmium. Marker enzymes were used for localisation of cellular fractions in the density gradient. Two lysosomal fractions, called the light and heavy fraction, were separated. They contained mainly lead, but also copper and some cadmium.Abbreviations AP acid phosphatase - ER endoplasmic reticulum - LDH lactic dehydrogenase - MDH malic dehydrogenase - NADH nicotinamide adenine dinucleotide - P. scaber Porcellio scaber     

Vergleichende fluoreszenzmikroskopische und elektronenmikroskopische Untersuchungen am zentralen Nervensystem von Planorbarius corneus L. (Basommatophora)

Zusammenfassung Durch Vergleich fluoreszenz- und elektronenmikroskopischer Untersuchungen am zentralen Nervensystem von Planorbarius corneus L. wird nachgewiesen, daß in den Schlundringganglien Neurosekretzellen vorkommen (Nachweis mit Pseudoisocyaninchlorid), die mit Nervenzellen nicht identisch sind, die durch ihren hohen Gehalt an biogenen Aminen auffallen (Nachweis durch die Methode von Falck und Owman, 1965). Es können daher im Schlundring von Planorbarius corneus peptiderge und aminerge Neurosekretzellen unterschieden werden. Die PSC-positiven Neurosekretzellen enthalten elektronendichte Elementargrana und die aminergen Neurone dense-core Vesikel. Der Nachweis biogener Amine in einigen Nervenzellen von Planorbarius corneus spicht für deren chemische Identität mit Transmittersubstanzen, ihre hohe Konzentration aber für eine Abgabe in die Körperflüssigkeit.

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Comparative fluorescence- and electron microscopic studies of the central nervous system of Planorbarius corneus L. (Basommatophora)

Summary The neurosecretory system of the snail Planorbarius corneus has been investigated by fluorescence and electron microscopy. With the fluorochrome Pseudoisocyanin the established neurosecretory system in the cerebral ganglia and single neurosecretory cells in the other ganglia show an intensive yellow fluorescence. Electron micrographs reveal the presence of electron dense granules (elementary granules) in the pericarya and the axons of neurones which have the same localisation in the ganglia as the pseudoisocyanin-positive cells. The fluorescence technique for biogenic amines produces yellow and green fluorescence within neurons and in the neuropil and nerves. The fluorescence obtained in determinable areas and neurones correlates well with the electron microscopic location of dense-core vesicles within the pericarya and axons of cells in even the same areas. It is discussed, that in this animal both types of cells are so-called neurosecretory cells, because the high content of elementary granules in the peptidergic neurosecretory cells and of dense-core vesicles in the aminergic neurosecretory cells is an indication for secretion of these products in neurohaemal areas (circulatory channels).

     

Ultrastructural observations on the marine fouling diatomAmphora

Ecological and Scanning electron microscope (S. E. M.) studies indicated that the diatomAmphora was an important constituent in the initial colonization of test panels coated with a copper antifouling composition.Amphora was also found as the dominant fouling diatom species on paint samples from in-service supertankers and yachts. Associated with the diatom was copious amounts of mucilaginous material, which often encapsulated the cells. Histochemical analysis of the mucilage indicates that it is predominantly polysaccharide in nature. Using the Transmission electron microscope (T. E. M.) and electron microscope cytochemistry the intracellular origin of the adhesive was investigated. T. E. M. and S. E. M. observations of acid-cleaned-cells indicate that the mucilage may be secreted through specialized regions of the frustule. Material isolated from antifouling panels was compared with laboratory culturedAmphora spp. for copper resistance and internal accumulation using TEMSCAN — X ray analytical equipment.     

The ultrastructure of the male and female prostate of Praomys (Mastomys) natalensis

Summary Male ventral and female prostates of Praomys (Mastomys) natalensis were examined with the electron microscope. The findings support and add to information obtained with the light microscope on tissues from normal, castrated and ovariectomised animals.Our results indicate that although the female prostate may be considered a homologue of the male ventral prostate anatomically and histologically, there are differences in sub-cellular morphology and hormone dependence.Cells of the intact ventral prostate of the male are characterised by prominent dilated Golgi vesicles and electron-dense mature secretory granules seen in the apical region of the cell. In the cells of the female prostate these features are absent. These morphological differences reflect the influence of hormones upon the cells, as shown by the reduction of the dilated Golgi vesicles in the castrated male and conversely, their occasional presence in the cells of the oestrous female.Comparison of castrated and ovariectomised animals shows that the male ventral prostate is much more dependent on androgens than the female is on ovarian hormones.There are several modes of secretion in the male ventral and the female prostate. These are by acellular and cellular blebbing, by a variety of secretory vesicles into the acinar lumina, and by a system of double walled vesicles not previously described.We are grateful to Dr. R.C.B. Pugh of the Department of Pathology, St. Peter's Hospitals for helpful discussion, to Mr. P. Chaloner and Miss P. Gunter for technical assistance and also the Department of Medical Art of the Institute of Urology     

Cytochemical localization of β-galactosidase in resident and inflammatory peritoneal macrophages from C57BL mice

Summary A cytochemical method for the detection of -galactosidase (-Gase) in mouse peritoneal macrophages was used to study the ultrastructural localization of this enzyme in these cells. It was found that the reaction product for -Gase was localized in the perinuclear cisternae, the endoplasmic reticulum, the Golgi complex, lysosomes, vesicles and on the cell surface of peritoneal macrophages from untreated C57BL mice. When examined by X-ray microanalysis the crystalline reaction product was found to contain bromine, an element present in the indolyl substrate which was used to identify -Gase. Injection of Proprionibacterium acnes (P. acnes) intraperitoneally or BCG intravenously caused a visible loss in -Gase from all the organelles and from the cell surface of the macrophages.Abbreviations used -Gase -galactosidase - RP reaction product - PNC perinuclear cisternae - RER rough endoplasmic reticulum     

Dense-core vesicles in motor nerve terminals

Summary Motor nerve terminals on white and intermediate muscle fibers of the Atlantic hagfish (Myxine glutinosa, L.) contain translucent synpatic vesicles and about 1–2% dense-core vesicles. Terminals on red muscle fibers contain up to 40% dense-core vesicles with diameter 800–1100 Å. Examinations for formaldehyde-induced fluorescence indicate yellow fluorescence (5-HT ?) apparently corresponding with terminal axons on red muscle fibers in craniovelar muscles. Possibly red muscle fibers of Myxine receive monoaminergic innervation.The author is indebted to Dr. Finn Walvig, Biological station, University of Oslo, Drøbak, for supply of hagfishes.     

The development of synapses in vitro between previously dissociated chick spinal cord neurons

Summary The formation and development of synaptic contacts between dissociated chick spinal cord neurons has been investigated. By the 6th day in vitro immature profiles with few vesicles were observed. By 14–18 days mature types with numerous vesicles were found, indistinguishable from those of newly hatched chick spinal cord. After this period degeneration occurred, and was especially marked in the post-synaptic element. Such degeneration could be postponed by the addition of small numbers of somatic muscle cells. The Kanaseki and Kadota (1969) technique was applied to the study of coated vesicles at various stages of synaptic development.     

Organ-specific crystalline structures of ferritin cores inβ-thalassemia/hemoglobin E

Summary The cores of ferritins isolated from different organs of human subjects with-thalassemia/hemoglobin E (-thal/HbE) disease have different size distributions and crystallinities depending on the source organ. These patients have not been treated by hypertransfusion regimen or iron chelation therapy.-Thal/HbE spleens and livers yield ferritin cores which are less crystalline than those isolated from normal spleens and livers, reflecting the more rapid deposition of iron in the diseased state. Ferritins isolated from the hearts and pancreases of-thal/HbE subjects were found to have larger, more crystalline cores than those from the-thal/HbE livers and spleens, possibly as a consequence of the role of the heart and pancreas as long-term iron deposition sites in this iron overload pathology.     

Electron microscopical microprobe analysis of freeze dried and unstained mineralized epiphyseal cartilage

Summary Electron micrographs have been taken of unfixed, freeze dried, unstained epiphyseal cartilage. In the mineralized long septa round to elliptic clusters (up to 0.6 m in diameter) consisting mainly of dots and needles could be observed. The clusters were surrounded by microareas with a low contrast consisting mainly of ribbon plate-like crystallites. With the aid of scanning mode (STEM) of a transmission electron microscope, equipped with a Si(Li)detector system, both regions were analyzed for calcium and phosphorus by electronprobe X-ray microanalysis. In ten series of 106 measurements in each region, it could be determined by registration of the CaK and PKX-ray counts that the mineral content in the clusters was in the range of 30–100 % higher than in the light regions. The question of the sequence of the epiphyseal plate mineralization is discussed and whether the dense clusters represent the mineralized matrix vesicles.The authors thank the Deutsche Forschungsgemeinschaft for financial supportDedicated to Professor Dr. G. Pfefferkorn on the occasion of his 65th birthday     

Localization of sulphated polysaccharides by X-ray microanalysis in Laminaria saccharina

Summary Analysis of thick sections of Laminaria thallus by X-ray electron microscope microanalysis using EMMA 4 shows that sulphated polysaccharide is located in secretory canals, the middle lamella of cell walls and on the thallus surface. Sulphation occurs in the Golgi bodies of specialised secretory cells. These results confirm previous reports using histochemical and autoradiographic techniques.     

Localization of calcium on the stigma surface of Ruscus aculeatus L.

By use of chlorotetracycline and X-ray microanalysis it is demonstrated that the receptive surface of the stigma of Ruscus aculeatus is rich in calcium. The high level of calcium is found in the epidermal cells and in the exudate covering the stigma. These results indicate that in vivo, as in vitro, calcium takes part in the regulation of pollen grain germination.Abbreviations CTC chlorotetracycline - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid     

Ultrastructure of motor nerve terminals on different types of muscle fibers in the atlantic hagfish (Myxine glutinosa,L.)

Summary White and intermediate parietal muscle fibers of Myxine are innervated focally at one end. Most synaptic vesicles are empty. These terminals also contain 1–2% large 800–1.100 Å dense-core vesicles. Red fibers of parietal and craniovelar muscle are innervated in a distributed fashion, and the presynaptic profiles contain a higher number of large dense-core vesicles (averaging 9% and 15%, respectively; up to 37%). For all terminals the synaptic gap is 450–600 Å wide, and postsynaptic folds are absent.Empty synaptic vesicles exist as round or elongated profiles. The proportion of elongated profiles increases by formation from round ones when increasing the molarity of the buffer in the aldehyde fixative. Furthermore, the proportion of elongated vesicle profiles in terminals on Myxine white fibers at different buffer molarities, is identical with that in mammalian motor terminals at similar molarities. On this basis the significance and mode of formation of elongated vesicle profiles is discussed. The conclusion is made that the susceptibility of flattening depends on the osmotic pressure of the vesicle contents once the aldehyde has influenced the vesicle membrane.The different vesicle populations in terminals on different types of muscle fibers are significant. Terminals on red fibers probably contain serotonin (5-HT) either as sole transmitter or in addition to acetylcholine.The author is indebted to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supply of hagfishes, and to Mrs. Jorunn Line Vaaland for expert technical assistance.     

Radicle ofEchinocactus platyacanthus (Cactaceae)

Radicle of matureEchinocactus platyacanthus embryo is approximately 320 m long and represents less then 1/7 of the embryonal axis length. The radicle-hypocotyl boundary can be distinguished according to the striking difference in the size and shape of cells in protoderm and procambium, as well as discontinuity and different number of the cell files in the ground meristem. The root cap is small, consists of 4 layers of cells covering the apex of the radicle. The upper limit of the root cap is approximately 100 m closer towards the radicle tip than the radicle-hypocotyl boundary. Ultrastructure of radicle cells showed numerous lipid bodies as is typical for other oily seeds. Protein bodies of variable structure were also present together with other cell structures. Striking differences in protein body structure were found when protoderm and ground meristem were compared. Several small globoid crystals were present in each protein body of the protoderm, while protein bodies in the radicle ground meristem mostly contained one large globoid crystal. X-ray microanalysis revealed presence of P, K and Mg in all analyzed globoid crystals. Fe, Ca and Zn were detected in some of them.Abbreviations EDX microanalysis energy-dispersive X-ray microanalysis - GC(s) globoid crystals - ICP spectroscopy ion-coupled plasma spectroscopy - LM light microscopy - PB(s) protein bodies - SEM scanning electron microscopy - TEM transmission electron microscopy     

Ultrastructural localization of cellular compartments involved in secretion of the low molecular weight,alkaline xylanase by Trichoderma reesei

The intracellular location of the low-molecular weight, alkaline xylanase (XYN II) of Trichoderma reesei RUT C-30 was investigated during growth on xylan, using immunoelectron microscopy. A monoclonal antibody, produced against XYN II, was used for this purpose. The enzyme was found at the endoplasmic reticulum and in electron dense 0.2 to 0.8 m vesicles, as well as in the vacuole, at the plasma membrane and in the fungal cell-wall. No staining occured in the cytoplasm, the mitochondria and the nucleus. No Golgi-like structures could be seen. Addition of the carboxylic ionophore monensin blocked xylanase as well as total protein secretion. The results are discussed with respect to XYN II being secreted by T. reesei via a pathway involving the endoplasmic reticulum and secretory vesicles and/or the vacuole.     

In vitro uptake of cadmium by basidiomycetesPleurotus ostreatus

Summary The mycelium of BasidiomycetesPleurotus ostreatus was grown in liquid cultures of malt broth enriched with increasing amounts of cadmium also in the presence of copper and glutathione. Cadmium, up to 150 g/ml gradually inhibited mycelial development but never blocked it completely. Cadmium accumulated to a higher degree (20 mg/g dry wt) when administered alone and was mostly bound (80%) to hyphal cell walls. Interactions with copper may play an important role in cadmium tolerance.     

Electrostatic surface properties of plasmalemma vesicles from oat and wheat roots. Ion binding and screening investigated by 9-aminoacridine fluorescence

Right-side-out and sealed plasmalemma vesicles were isolated from roots of spring wheat (Triticum aestivum L. cv. Drabant) and oat (Avena sativa L. cv. Brighton) by two-phase partition in a medium containing sucrose (0.25 mol l^-1). Oat root plasmalemma vesicles were discovered to contain a strongly fluorescent compound with an emission maximum at 418 nm. The surface potential of the membranes was monitored by 9-aminoacridine fluorescence and the effect of protein concentration, mannitol versus sucrose, absence of osmoticum, concentrations of salt, and titrations with chelators investigated. It is concluded that i) protein concentrations of less than 50 g ml^-1 for oat and 100 g ml^-1 for wheat plasmalemma vesicles should be used to avoid serious problems with non-linearity of response of 9-aminoacridine fluorescence, ii) mannitol can be used instead of sucrose as the osmoticum, iii) the vesicles were ruptured in the absence of osmoticum allowing us to monitor both sides of the membranes, iv) plasmalemma vesicles from oat roots are more negative than vesicles from wheat roots, and v) oat and wheat root plasmalemma vesicles are isolated with about the same amounts of bound Ca²⁺ and Mg²⁺. These bound divalent cations may not, however, reflect the in-vivo conditions since the tissues were homogenised in the presence of ethylenediaminetetraacetic acid.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - c1/2 value concentration at which half of the maximum effect is observed - Mops 3-(N-morpholino)propanesulfonic acid     

Possible sites of release of neurosecretory granules in the sinus gland of the crayfish,Orconectes nais

Summary The sinus gland is a neurohemal organ located in the crayfish eyestalk and represents a storage site for neurohormones prior to their release into the circulation. The sinus gland contains three classes of electron dense, membrane-limited granules. Class 3 granules are the largest and most electron dense of the granules found in the sinus gland. Granules of class 1 are the smallest, while those of class 2 are the most abundant. Generally, all granules undergo similar changes during their release.Release of neurosecretory material may be initiated by a preliminary fragmentation of the parent granules into smaller granules. Following the formation of numerous smaller granules, these move to the plasma membrane and their limiting membrane apparently fuses with it thus releasing its contents into the external lamina which is applied to the sinusoidal surface of the axon terminals. Granule release does not appear to occur along the entire plasma membrane adjacent to the blood sinus but, instead, probably occurs only at specific active sites on the membrane. The active sites are characterized in part by an accumulation of small granules and clear vesicles against the cytoplasmic side of the plasma membrane. At the site of release of the neurohormone, there is often an accumulation of dense homogeneous material beneath the axolemma.Occasionally, axon endings filled with large, electron lucent vesicles are seen. These clear granules vary from 1150–1750 Å in diameter and often exhibit broken limiting membranes. Few small vesicles are seen near the plasma membrane of these endings; however, instances of invaginations of the plasma membrane occur. The significance of endings filled with clear granules is discussed.Supported by a grant from the National Research Council of Canada (No. A-4675).     

Cytokinesis in the green alga Eremosphaera viridis: Plasmalemma formation from “open membranes”

Summary Cytokinesis in the unicellular chlorococcalean alga Eremosphaera viridis de Bary has been investigated by electron microscopy of thin sections. The new plasmalemmata of the daughter cells in this organism form centrifugally within a phycoplast. Unlike other cell division systems each new plasmalemma is formed, not by the fusion of vesicles, but rather by the fusion of open membranes which are characteristically heavily stained. Measurements of these open membranes reveal that they are 11 nm thick with a central 4,5 nm unstained portion. The possible origin of these open membranes as burst-open vesicles has been suggested from the presence of intensely straining vesicles in the vicinity of the cell equator. Calculations of vesicle and open membrane surface areas support this contention.     

Freeze-fracture studies on the cockroach hemocyte membrane complex: Symmetric and asymmetric membrane particle distributions

Summary Freeze-fracture studies were conducted on the membranes of normal cockroach hemocytes. The plasmalemma is asymmetric with the A fracture face containing 80–100 Å membrane intercalated particles at a concentration of 2500/². The B fracture face contains 120–150 Å particles with a relatively low density (800/²). The nuclear envelope displays an asymmetry with the A fracture face containing 1500 particles/² and the B face containing 300/ ². No significant particle size differences were observed in nuclear envelope fracture faces. Two types of symmetric membranes were also found in these cells. Both A and B fracture faces of the membrane surrounding the numerous cytoplasmic inclusion bodies contain particle sizes and concentrations similar to the B face of the plasmalemma. A second type of symmetry was observed in cells apparently engaged in exocytosis. Vesicles (0.1 D) from this process were completely particle free on both fracture faces. Such particle free vesicles could be found in the cytoplasm, attached to the plasmalemma, or completely separated from the cell.Supported by a Pharmaceutical Manufacturers Association Foundation Fellowship.The author wishes to thank Ms. Annalena K. Charla for assistance in plate preparation, Dr. Julius Schultz and the Papanicolaou Cancer Research Institute for use of the freeze-etch device, and Dr. David Smith for the electron microscope facilities.     

Heterogeneity of calcium compartmentation: Electron probe analysis of renal tubules

Summary The objective of this study has been to determine the intracellular localization of calcium in cryofixed, cryosectioned suspensions of kidney proximal tubules using quantitative electron probe X-ray microanalysis. Two populations of cells have been identified: 1) Viable cells, representing the majority of cells probed, are defined by their relatively normal K/Na concentration ratio of 41. Their measured Ca content is 4.1±1.4 (sem) mmol/kg dry wt in the cytoplasm and 3.1 ± 1.1 mmol/kg dry wt in the mitochondria, or an average cell calcium content of 3.8 mmol/kg dry wt. 2) Nonviable cells, defined by the presence of dense inclusions in their mitochondria and a K/Na concentration ratio of 1. The Ca content is 15±2 mmol/kg dry wt in the cytoplasm and 685±139 mmol/kg dry wt in the mitochondria of such cells. Assuming 25 to 30% of the cell volume is mitochondrial, the overall calcium content of such nonviable cells is 210 mmol/kg dry wt. The presence of these inclusions in 4 to 5% of the cells would account for the average total Ca content measured in perchloric acid extracts of isolated proximal tubule suspensions ( 18 nmol/mg protein or 12.6 mmol/kg dry wt). Whole kidney tissues display a large variability in toal Ca content (4.5 to 18 nmol/mg protein, or 3.4 to 13.5 mmol/kg dry wt), which could be accounted for by inclusion in 0 to 4% of the cells. The electron probe X-ray microanalysis (EPXMA) data conclusively demonstrate that thein situ mitochondrial Ca content of viable cells from the kidney, proximal tubule is low and support the idea that mitochondrial Ca may regulate dehydrogenase activity but probably does not normally control cytosolic free Ca.