Ethylene-induced chitinase and β-1,3-glucanase accumulate specifically in the lower epidermis and along vascular strands of bean leaves

We have studied the spatial pattern of accumulation of chitinase (EC 3.2.1.14) and -1,3-glucanase (EC 3.2.1.39) in ethylene-treated leaves of bean (Phaseolus vulgaris L.). Electron-microscopical examination of chemically fixed tissue demonstrated the presence of large electron-dense aggregates in the vacuoles of ethylene-treated leaf cells. No such vacuolar structures were observed in untreated control cells. Immunogold labelling with antisera directed against the basic forms of chitinase and -1,3-glucanase indicated that the vacuolar aggregates were the major site of accumulation of chitinase and -1,3-glucanase. The chitinase- and -1,3-glucanase-containing vacuolar aggregates were not randomly distributed within the leaf tissue but were restricted to the lower epidermal cells and to parenchyma cells adjacent to vascular strands. In addition, heavy -1,3-glucanase labelling was observed over spongy plugs of expanded middle-lamella material that appear to occlude the transition regions between the airspaces underlying the stomata and those throughout the rest of the leaf. Some labelling was also seen to extend along the surface layer of the cell walls lining all of the airspaces. Protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting as well as enzyme-activity measurements showed that the peeled lower epidermis of the ethylene-treated leaves contained on a protein and on a per-weight basis several times more chitinase and -1,3-glucanase than the remainder of the leaf.Abbreviation in Text SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Abbreviations in Micrographs AS air space - C chloroplast - EP (epidermal) cell - G guard cell - P parenchyma cell - S stoma - V vacuole - VE] vein - VP vascular parenchyma cell - W cell wall - X xylem We thank Dr. L.A. Hadwiger, Pullman, Wash., and Dr. U. Vögeli, Lexington, Ky., for their kind gifts of antibodies. This work was supported by the National Science Foundation grant DCB-8615763 to L.A.S.     

Accumulation of alkaloids in plant vacuoles does not involve an ion-trap mechanism

Alkaloid uptake into vacuoles isolated from a Fumaria capreolata L. cell suspension culture was investigated. The uptake is carrier-mediated as shown by its substrate saturation, its sensitivity to metabolic inhibitors and especially by its exclusive preference for the (S)-forms of reticuline and scoulerine while the (R)-enantiomers which do not occur in this plant species were strictly discriminated. The carrier has a high affinity for (S)-reticuline with a K _m=0.3 M. The rate of alkaloid uptake was 6 pmol·h^-1·l^-1 vacuole, and 0.03 mg alkaloid·mg^-1 vacuolar protein were taken up. Transport was stimulated five-to seven-fold by ATP and was inhibited by the ATPase inhibitors N,N-dicyclohexylcarbodiimide and 4-4-diisothiocyanatostilbene-2,2 disulfonic acid, as well as by the protonophore carbonyl cyanide m-chlorophenylhydrazone. A number of alkaloids did not compete with labelled (S)-reticuline for uptake into vacuoles. The uptake system is absolutely specific for alkaloids indigenous to the plant from which the vacuoles were isolated. Slight modifications of the topography of an alkaloid molecule even with full retention of its electrical charge results in its exclusion. Alkaloid efflux was also shown to be mediated by a highly specific energy-dependent carrier. These results contradict the previously proposed ion-trap mechanism for alkaloid accumulation in vacuoles. A highly specific carrier-mediated and energy-dependent proton antiport system for alkaloid uptake and release is postulated.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DIDS 4-4-diisothiocyanatostilbene-2,2 disulfonic acid Dedicated to Professor Harry Beevers, Santa Cruz, on the occasion of his 60^th birthday     

Development of the resting sporangia ofSynchytrium endobioticum,the causal agent of potato wart disease

Summary An ultrastructural study of the development of the resting sporangium ofSynchytrium endobioticum (Schilb.) Perc. infecting potato cells is presented. The resting sporangium is found to have a single large, centrally placed nucleus with a prominent nucleolus through its entirein situ development. The cytoplasmic organization of the resting sporangium is further characterized by numerous membrane-bound lipid bodies and osmiophilic bodies. The latter have a characteristic sieve-like appearance, probably because certain storage components have been extracted during preparation for electron microscopy. Because of the similar location and appearance of these osmiophilic bodies it is suggested that they are identical to what has earlier (based on light microscopy) been described as chromatin granules; and the ultrastructural studies presented here show that nucleolar discharge which was described from light microscopic observations as leading to chromatin granules in the cytoplasm, and finally forming the nuclei of the zoospores (bally 1912,curtis 1921,percival 1910) simply does not occur.The appearance of dense fibrillar-like structures on the sporangial surface at an early stage of resting sporangium development ultrastructurally distinguishes the resting sporangium from the zoosporangium. The development of the layered portion of the thick sporangial wall is shown to be due to the fusion of vacuoles containing pre-made wall fibrils with the cell membrane. It is suggested that the inner compact wall layer which is essentially substructureless is formed by the membrane itself.The characteristic wings of the matureS. endobioticum resting sporangium originate from the potato host cell wall. Remnants of host cell organelles in the outermost layer of the resting sporangium wall show that degradation of the host cell cytoplasm contributes to wall formation of the parasite.     

Observations of the water expulsion vacoule ofPhytophthora palmivora

Summary Characteristic changes in the ultrastructure of the green algaScenedesmus armatus, grown in batch culture in the presence of aqueous fuel-oil extract (AFOE) have been observed. The changes affected mainly chloroplasts and mitochondria. The regular arrangement of the thylakoid stacks became distorted and the whole chloroplast lobed. Plastoglobules were more numerous in the treated cells than in the controls, especially after long-term exposure to AFOE.The mitochondrial matrix cells exposed to AFOE were more electron-translucent. An increase in the number of small mitochondrial profiles was observed after prolonged treatment with AFOE.The number and size of osmophilic bodies increased markedly in the cytoplasm of the treated cells. The cytochemical reaction of these bodies with Sudan black B indicated their lipid composition.Plasmalemma invagination into the cytoplasm and vacuoles, cytoplasmic layers, and an increase in size of the vacuolar compartment were observed in cells exposed to AFOE for a long time.The possibility that detoxification, involving microbody activity, may have occurred inScenedesmus is suggested.Abbreviation AFOE aqueous fuel-oil extract     

An ultrastructural study of phagocytosis and shrinkage in nutritive phagocytes of the sea urchin <Emphasis Type="Italic">Anthocidaris crassispina</Emphasis>

The ultrastructural mechanisms of waste-sperm phagocytosis and postspawning shrinkage were studied for accessory cells (nutritive phagocytes; NPs) of the sea urchin Anthocidaris crassispina. Sperm cells were phagocytosed by NPs; they penetrated into the cytoplasm of the NPs inside heterophagosomes formed by an invagination of the cell membrane. Single-sperm-containing heterophagosomes aggregated to form large multisperm heterophagosomes that were accompanied by cytoplasmic vesicles and lipids. Two types of vesicle, viz., Golgi-complex-derived electron-dense vesicles (zymogen granules) and smooth-endoplasmic-reticulum-derived electron-lucent vesicles, were incorporated within multisperm heterophagosomes. Completed multisperm heterophagosomes were transformed into electron-dense remnant bodies, the content of which underwent destruction, resulting in empty vacuoles inside the remnant body. The empty vacuoles were then compressed by the surrounding cytoplasm. Shrinkage of NPs occurred upon completion of sperm degeneration in gonad tubules. This process was undertaken by structures termed cell-size-reducing autolysosomes, which performed two types of autolysis, and resulted in the formation of cheese-hole-like vacuoles in the cytoplasm of NPs. Subsequent cytoplasmic compression of these vacuoles was required for the reduction in size of NPs, an essential event for remodeling the cell for the next gametogenetic cycle.We are grateful to the Research Committee, City University of Hong Kong, for the award of a Strategic Grant (no. 7000989). This work also was supported by a grant from Minpromnayka NS 1219.2003.4     

Movement and compartmentation of abscisic acid in guard cells of Valerianella locusta: Effects of osmotic stress,external H+-concentration and fusicoccin

Epidermal peels of Valerianella locusta were acid-treated for 1 h at pH 3.9 to kill all cells other than guard cells. These guard-cell preparations were used to explore the steady-state one-way fluxes and the cytoplasmic and vacuolar contents of abscisic acid (ABA). The method of compartmental analysis has been applied. The intracellular ABA concentrations were surprisingly high. At an external pH of 5.8 the cytoplasm contained 1.28 mmol·dm^-3 of ABA, twice of the amount which accumulated in the vacuoles (0.57 mmol·dm^-3). The fluxes of ABA at the plasmalemma (_oc=_oc=0.43 fmol · cell ^–1 · h ^–1) were higher than those at the tonoplast (_cv=_vc=0.12 fmol · cell ^–1 · h ^–1). Moderate stress (0.1 and 0.3 mol·dm^-3 sorbitol in the medium) caused a change in the kinetics of ABA movement. The rate constants of the fluxes from the cytoplasm into the vacuole (_cv) and into the apoplast (_co) were increased while the rate constant of the flux from the vacuoles into the cytoplasm (_vc) was decreased. As a consequence the amount of ABA sequestered in the vacuole remained unchanged; the cytoplasmic ABA content, however, was reduced to only 20% of that found in the control treatments (no sorbitol in the medium). Under moderate stress, one Valerianella guard cell released rapidly about 0.36 fmol·cell^-1 to its direct cell-wall space. This surprising result is discussed in regard to rapid stomatal closure under reduced water supply.Abbreviations ABA abscisic acid - FC fusicoccin     

Reactions of seven basic fluorochromes with unfixed cells obtained from the salivary glands of the dipteran fly Megaselia scalaris Loew (Phoridae)

Summary Seven basic fluorochromes with varying specificities were used to stain the large squamous epithelial cells isolated from the larval salivary glands of Megaselia scalaris (Phoridae). Although the EDTA-based method selected for isolating the cells produced permeabilization and a loss of viability of the cells, consistent results were obtained with the various fluorochromes. The classical pattern of green nuclear and red cytoplasmic fluorescence observed in cells stained with acridine orange could be changed to green cytoplasmic and red nuclear fluorescence by pretreatment with RNase. The predominantly cytoplasmic and nucleolar fluorescence obtained with pyronine Y could be changed to mainly nuclear fluorescence by RNase pretreatment. The other five fluorochromes tested were not affected appreciably by extraction with RNase. Quinacrine mustard, dicarbocyanine (DiOC₆(3)), and rhodamine 123 produced primarily cytoplasmic and nucleolar fluorescence, while nile red revealed mainly cytoplasmic lipid droplets. Phosphine 3R initially stained lipid droplets but very rapidly redistributed throughout the cytoplasm and nucleus. Because of their large size, flatness, and content of histochemically demonstrable components, the cells of Megaselia are especially appropriate for use as optical objects or controls in various studies. New methods of isolating the cells, however, will be needed to prevent permeabilization and loss of viability of the cells.     

Gamete Structure and Fertilization in the Barents Sea Sponge Leucosolenia complicata

The ultrastructure of male and female gametes of asconoid sponge Leucosolenia complicata(Calcispongiae, Calcaronea), a hermaphrodite species that reproduces in autumn, is described. The mature sponge's oocytes were up to 70 m in diameter, had no coatings, and contained a nucleus about 31 m in diameter with large nucleoli (up to 6.6 m). There were vacuoles with fibrillar contents typical of calcareous sponges in ooplasm. During vitellogenesis, a cluster of a great number of nurse cells developed above each oocyte from transformed choanocytes. Mature spermia of L. complicatalooked like orbicular cells about 2.5 m in diameter, with no acrosome or tail. The spermium nucleus (diameter about 2.2 m) was formed by incompletely condensed chromatin and was surrounded with a thin layer of cytoplasm of nonuniform thickness. In the thick layer of cytoplasm beyond the ribosomes, there were two or three mitochondria, dictyosomes, and electron-dense protein bodies lying freely under the nucleus. Fertilization occurred with the aid of a carrier cell. During spawning (mass release of spermia), any nurse cell complex can seize a spermium and transform into a carrier cell in situ. The transformation of a seized spermium into a spermiocyst was connected with the rapid isolation of the spermium nucleus from the protein body. Fertilization began with the penetration of the protein body into the oocyte cytoplasm. Only after this did the spermium's nucleus penetrate into the oocyte.     

Vacuolar pH in radish cotyledonal mesophyll cells

The vacuolar pH in cotyledonal mesophyll cells from radish (Raphanus sativus L. var. sativus) seedlings was determined from vacuoles, isolated from protoplasts through osmotic shock, by means of measurement of vacuole extracts with a pH meter and the methylamine method, and gave mean pH values of 6.28 and 6.26, respectively. Direct in situ measurements of the vacuolar pH from intact leaf tissue were recorded with pH-sensitive microelectrodes and gave a mean value of 6.0. The results are discussed with respect to possible erroneous pH measurements and the vacuolar location of specific anabolic reactions.     

Protein-accumulating cells and dilated cisternae of the endoplasmic reticulum in three glucosinolate-containing genera: Armoracia,Capparis, Drypetes

Three glucosinolate-containing species, Armoracia rusticana Gaertner, Meyer et Scherbius (Brassicaceae), Capparis cynophallophora L. (Capparaceae) and Drypetes roxburghii (Wall.) Hurusawa (Euphorbiaceae), are shown by both light and electron microscopy to contain protein-accumulating cells (PAC). The PAC of Armoracia and Copparis (former myrosin cells) occur as idioblasts. The PAC of Drypetes are usual members among axial phloem parenchyma cells rather than idioblasts. In Drypetes the vacuoles of the PAC are shown ultrastructurally to contain finely fibrillar material and to originate from local dilatations of the endoplasmic reticulum. The vacuoles in PAC of Armoracia and Capparis seem to originate in the same way; but ultrastructurally, their content is finely granular. In addition, Armoracia and Capparis are shown by both light and electron microscopy to contain dilated cisternae (DC) of the endoplasmic reticulum in normal parenchyma cells, in accord with previous findings for several species within Brassicaceae. The relationship of PAC and DC to glucosinolates and the enzyme myrosinase is discussed.Abbreviations ABB aniline blue black - DC dilated cisternae - EM electron microscopy - ER endoplasmic reticulum - GMA glycolmethacrylate - LM light microscopy - MBB mercuric bromphenol blue - PAC protein-accumulating cells - PAS periodic acid-Schiff Recipient of an Alexander von Humboldt Award and in residence at the University of Heidelberg during the period when this research was carried out. Permanent address: Department of Botany and Cell Research Institute, University of Texas, Austin, Texas 78712, USA     

Efficient developmental mis-targeting by the sporamin NTPP vacuolar signal to plastids in young leaves of sugarcane and <Emphasis Type="Italic">Arabidopsis</Emphasis>

Plant vacuoles are multi-functional, developmentally varied and can occupy up to 90% of plant cells. The N-terminal propeptide (NTPP) of sweet potato sporamin and the C-terminal propeptide (CTPP) of tobacco chitinase have been developed as models to target some heterologous proteins to vacuoles but so far tested on only a few plant species, vacuole types and payload proteins. Most studies have focused on lytic and protein-storage vacuoles, which may differ substantially from the sugar-storage vacuoles in crops like sugarcane. Our results extend the evidence that NTPP of sporamin can direct heterologous proteins to vacuoles in diverse plant species and indicate that sugarcane sucrose-storage vacuoles (like the lytic vacuoles in other plant species) are hostile to heterologous proteins. A low level of cytosolic NTPP-GFP (green fluorescent protein) was detectable in most cell types in sugarcane and Arabidopsis, but only Arabidopsis mature leaf mesophyll cells accumulated NTPP-GFP to detectable levels in vacuoles. Unexpectedly, efficient developmental mis-trafficking of NTPP-GFP to chloroplasts was found in young leaf mesophyll cells of both species. Vacuolar targeting by tobacco chitinase CTPP was inefficient in sugarcane, leaving substantial cytoplasmic activity of rat lysosomal -glucuronidase (GUS) [ER (endoplasmic reticulum)-RGUS-CTPP]. Sporamin NTPP is a promising targeting signal for studies of vacuolar function and for metabolic engineering. Such applications must take account of the efficient developmental mis-targeting by the signal and the instability of most introduced proteins, even in storage vacuoles.     

Histochemical demonstration of Δ5-3 ß-OHD activity in the granulosa cells of ovarian follicles of immature and mature mice correlated with some ultrastructural observations

Summary A systematic study of ⁵-3-hydroxysteroid dehydrogenase in the granulosa cells of immature and mature mice was made. The histochemical results were compared with the ultrastructural findings on the same cells in an attempt to determine whether the granulosa cells are capable of a steroidogenic role.In newborn and immature mice the granulosa cells of a great amount of follicles demonstrated a moderate or strong histochemical activity. In mature mice the granulosa cells demonstrated a weak or moderate activity normally only in preovulatory follicles and in some other atretic follicles. The granulosa cells of the normal developing follicles did not show such activity. In addition the histological control of numerous parallel sections demonstrated particularly in immature ovaries the presence of a great amount of atretic follicles.In the cytoplasm of the granulosa cells of the follicles in immature ovaries only clusters of lipid droplets with ribosomes were noted; while in the preovulatory follicles of mature animals there started to appear mitochondria with tubular cristae, smooth membranes of the endoplasmic reticulum and irregular lipid droplets. In the obviously atretic follicles several granulosa cells as well as theca interna cells showed numerous lipid droplets and ribosomes together with different degenerating organelles. The granulosa cells of the normal developing follicles showed a well developed Golgi complex, granular endoplasmic reticulum and free ribosomes.The histochemical and ultrastructural findings suggest a steroidogenic role of the granulosa cells only in the larger preovulatory follicles (probably related to an early luteinization of this layer) but this role was not demonstrated in the same cells in normal developing follicles.In addition, since an histochemical positivity was demonstrated also in the granulosa cells of some obviously atretic follicles, it is possible that many of the follicles having granulosa cells filled with lipid droplets and attached ribosomes and histochemically positive might be, in the immature ovaries, in a very precocious stage of atresia.It is to precise for these cells whether a cytoplasm with these two strictly correlated components (lipid droplets and attached ribosomes) and showing an histochemical positivity could carry-on all the biochemical steps involved in steroid biosynthesis or only has only a temporary capability to produce some precursors of steroids.The present results were partially presented to the 56^ème Congrès de l'Association des Anatomistes (Nantes, 4–8/4/1971) and to the 66° Verhandlungen der Anatomischen Gesellschaft (Zagreb, 2/4/1971).     

An electron microscopical study of absorbing cells in the posterior caput epididymidis of rabbits

Summary The columnar cells in regions 3 and 4 of the ductus epididymidis in rabbits display ultrastructural features characteristic of absorbing cells. The stereocilia show basal anastomoses and often a fibrillar core continuous with a fibrillar web in the apical cytoplasm. Numerous invaginations of the slightly downy apical cell membrane and many thick-walled apical vesicles and vacuoles contain an opaque substance similar to that seen in the lumen. The vacuoles often contain small vesicles or bodies, probably formed from the vacuolar wall by budding. Numerous bodies or vacuoles with moderately dense contents are seen in the Golgi area and in the supranuclear and intranuclear cytoplasm in region 3. In region 4 they are denser and mainly seen above the nucleus. A high acid phosphatase activity was demonstrated in most dense and some light bodies. India ink introduced by way of the rete testis was taken up from the lumen into apical invaginations, vesicles and vacuoles and slowly transferred to denser bodies below the Golgi apparatus.These observations are interpreted as evidence for a resorption of substances from the lumen by a pinocytotic process, and for their storage and perhaps digestion in the dense bodies, which appear to have a lysosomal character. The Golgi apparatus is large with many vesicles of two types and empty cisternae but few typical Golgi vacuoles. The partly granular endoplasmic reticulum is very well developed and has opaque contents. Microtubules run from the terminal bar region into the Golgi area. Thick-walled vesicles occur throughout the cytoplasm, sometimes in continuity with the cell membrane. The basal parts of the cell borders often interdigitate.Supported by a grant from the Swedish State Medical Research Council.     

On the histology and ultrastructure of the Harderian gland in rabbits

Summary The Harderian gland of rabbits has been studied with light and electron microscopes. The red part contains relatively wide alveoli with an irregular cuboidal epithelium. The stainability of the cytoplasm is poor. The white part has smaller alveoli with a low columnar epithelium. The cells show cytoplasmic basophilia removable with ribonuclease. The cytoplasm of both kinds of cells is very dense when examined in the electron microscope. The mitochondria show branched, closely packed cristae and a dense matrix. The Golgi apparatus displays few lamellae and rows of vacuoles. The endoplasmic reticulum is very finemeshed and partly associated with ribonucleoprotein particles. Both kinds of cells contain numerous lipid droplets, leaving vacuoles in the sections prepared for electron microscopy. They are fewer but distinctly larger in the red part. In both lobes pictures suggesting a secretion of lipid droplets have been observed. Cells showing signs of degeneration with subsequent discharge of the detritus have been observed in both lobes, but this process does not correspond to the holocrine secretion in sebaceous glands. Likewise, no apocrine secretion was observed.     

Demonstration of the existence of a single morphological type of gonadotrophic cell in Ellobius lutescens (Microtinae) by an ultrastructural analysis of their development under various physiological and experimental conditions

Summary Ultrastructural studies of pituitaries from Ellobius lutescens (immature males and females, adult hypogonadic males, and virgin and pregnant females) show that the gonadotrophic cells are characterized by a lamellar or vacuolar rough endoplasmic reticulum (RER), a spirally-arranged Golgi apparatus, elongated mitochondria and secretory granules of variable density and size (150–500 m). Ultrastructural differences between gonadotrophic cells previously determined by light microscopy correspond to changes in the development of the protein synthetic apparatus and in the intensity of hormonal discharge. Type 2 gonadotrophs always appear to be more active than type 1 gonadotrophs. After castration, all gonadotrophic cells develop into the same form of castration cell, although type 1 gonadotrophs change more slowly than type 2 cells. Treatment with testosterone induces an inverse development of the gonadotrophic cells which take on the appearance of resting cells similar to those found in immature animals, where the two cell types are also identical. Thus, only one morphological type of gonadotrophic cell can be identified in Ellobius lutescens. Moreover, the gonadotrophic cells of the hypogonadic adult male have the same appearance as those of the female two months after castration, which proves that the negative feedback mechanism which regulates gonadotrophic function is defective in adult male Ellobius lutescens.     

Surface properties of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> and their relevance to γ-decalactone formation from methyl ricinoleate

The surface of the lipid-degrading yeast, Yarrowia lipolytica, was characterized by contact angle and zeta potential () measurements. The cells were mainly hydrophilic with a negative charge that was only affected from pH 2 to 4. To study the effects of the surface charges on the biotransformation of methyl ricinoleate into the aroma compound, -decalactone, the values of the substrate droplets were modified by adding a cationic surfactant into the medium at concentrations that did not diminish cell viability: the adhesion of the lipid substrate to the cells was increased but not the overall performance of the process, therefore the adhesion is not the rate limiting here. Our methodology offers interesting perspectives for further applications.Revisions requested 6 January 2005; Revisions received 28 January 2005     

Immunocytochemical and ultrastructural study of the frog (Rana pipiens) pars distalis with special reference to folliculo-stellate cell function during in vitro superfusion

Summary The colloidal gold immunocytochemical technique was used to determine the ultrastructural features of the glandular cells in the pituitaries of male frogs, Rana pipiens, both in vivo and after superfusion in vitro. Specific reactions to antisera against bullfrog gonadotropins, human prolactin, and synthetic 1–39 corticotropin allowed identification of the 3 corresponding types of glandular cells. No immunoreaction was obtained with antisera against human or ovine-growth hormone, human -thyrotropin hormone, and bovine S-100 protein. General morphological features of these immunocytochemically identified glandular cells were similar to those of equivalent cells previously described in other amphibian species. Non-glandular folliculo-stellate cells were distinctive. In freshly removed pituitaries, these folliculo-stellate cells contained lysosome-like structures, but did not show phagocytic vacuoles in the cytoplasm; they contained many mitochondria, and the Golgi complex and endoplasmic reticulum were relatively undeveloped. After 4 or 18 h of superfusion, some immunoreactive gonadotropic, prolactin, and corticotropic cells showed degeneration and destruction. In the same gland, folliculo-stellate cells retained a viable appearance, but showed phagocytic vacuoles containing secretory granule-like structures which were immunoreactive to gonadotropic, prolactin, and corticotropic antibodies. Some folliculo-stellate cells showed phagocytic vacuoles containing complete glandular cells. These results suggest that superfusion causes a destruction of some of the glandular cells, and that folliculo-stellate cells act as phagocytes when cellular debris or moribund cells are present in the intercellular space in the pituitary parenchyma.Supported by grant DCB 8710462 from the National Science Foundation, grant 2148-83 from the CAICYT (Spain) and the Junta de Andalucia (Spain)     

Cortical cell fluxes and transport to the stele in excised root segments of Allium cepa L.

Summary From compartmental analysis of radioisotope elution measurements, concentrations and fluxes of Mg²⁺ were estimated for cortical cells in root segments of onion, Allium cepa L., relative to a complete nutrient solution containing 0.25 mM Mg²⁺. Five compartments for Mg²⁺ in the cortex were found and, in order of increasing rates of exchange, identified with the vacuoles and the cytoplasm of the cortical parenchyma, the Donnan free space, the water free space, and the superficial film of solution on the segments. With the Ussing-Teorell flux ratio equation as the criterion, it was concluded that Mg²⁺ entered the cytoplasm passively and was actively pumped back across the plasmalemma. Mg²⁺ concentration in the vacuole could be estimated only as lying between wide limits (1.3 to 14.3 eq ml^-1), but whatever the concentration within this range, it was concluded that Mg²⁺ was passively distributed across the tonoplast. Net flux was zero and the vacuolar concentration commensurate with this was found to be 6.6 eq ml^-1. The transported fraction of total efflux, appearing at the segment cut ends, was estimated separately. Magnesium was found to be transported almost exclusively in the basipetal direction.     

Exocytosis and uptake of bacteria by isolated haemocyte populations of two crustaceans: evidence for cellular co-operation in the defence reactions of arthropods

Summary The role of exocytosis in the cellular defence reactions of arthropods was investigated using in vitro cultures of isolated haemocytes (blood cells) from the freshwater crayfish Pacifastacus leniusculus, and the shore crab Carcinus maenas. In both species, activated lysates of those cell types that contain the prophenoloxidase activating system (granular cells of crab and crayfish and semigranular cells of crayfish) were found to induce degranulation (exocytosis) of semigranular and granular cells. A cell lysate, in which the prophenoloxidase system was kept inactive, did not have this effect. Limited degranulation of granular cells of crab was also induced by lipopolysaccharides as has earlier been shown for crayfish semigranular cells. The phagocytic capability of semigranular cells from crayfish was lost after exocytosis induced by the Ca²⁺ ionophore A23187, and under no conditions were the granular cells of crabs or crayfish seen to ingest bacteria in vitro. An opsonic function for the attaching proteins of a 1,3-glucan-activated haemocyte lysate was demonstrated using the phagocytic hyaline cells from crabs. Phenoloxidase appeared to lack opsonic properties.We suggest that, in crustaceans, opsonization takes place through hierarchically stimulated exocytotic release, and biochemical activation of the prophenoloxidase activating system: first from lipopolysaccharide-sensitive cells (semigranular cells of crayfish or granular cells of crabs) and then from granular cells, triggered by the initially released and activated prophenoloxidase system. Finally, sticky proteins of the activated prophenoloxidase system coat the invader, rendering it susceptible to the phagocytes (hyaline cells in both crab and crayfish and, to a lesser extent, semigranular cells of crayfish). These processes would, together, constitute a cellular communication pathway not previously demonstrated for invertebrates.Abbreviations DMSO dimethyl sulfoxide - L-DOPA L-dihydroxy-phenylalanine - GLS granular cell lysate supernatant - HLS haemocyte lysate supernatant - HyLS hyaline cell lysate supernatant - LPS lipopolysaccharide - proPO prophenoloxidase - SGLS semigranular cell lysate supernatant - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid disodium salt     

Evidence of steroidogenesis in postovulatory follicles of the tilapia,Oreochromis mossambicus

Summary Postovulatory follicles of the tilapia, Oreochromis mossambicus, were examined with electron microscopy and enzyme histochemistry for evidence of steroid-hormone production. Light microscopy was also used to examine changes in the ovary with time after spawning. Electron microscopy detected the presence of smooth endoplasmic reticulum, lipid droplets, and mitochondria with tubular cristae in certain cells of the theca interna. These structures are suggestive of cells that synthesize steroid hormones. Granulosa cells also contained some smooth endoplasmic reticulum, along with an augmentation of Golgi complexes, vesicles, microvilli, and microfilaments within 5–7 days after spawning. Enzyme histochemistry demonstrated an intense reaction of 5, 3-hydroxysteroid dehydrogenase (3-HSD) in variably placed thecal cells up to 7 days after spawning. At this time, the thecal cells of vitellogenic oocyte follicles also began to show strong 3-HSD activity. During the first 7 days after spawning, there was an increase in young primary oocytes and recruitment of some of these to vitellogenic oocytes. By 10 days after spawning, certain thecal cells in the follicles of these vitellogenic oocytes showed an intense 3-HSD reaction, while the postovulatory follicular tissue demonstrated a weak reaction. This arrangement continued for the lifespan of the postovulatory follicular tissue. Postovulatory follicles had a lifespan of up to 25 days after spawning in females that continued to hold the developing fry inside their mouths, i.e., mouthbrooders. At 25 days after spawning, the postovulatory follicular tissue showed signs of degeneration with the presence of vacuoles and lysosomes. In females that ate the zygotes, therefore exhibiting no parental behavior, the postovulatory follicular tissue showed signs of degeneration at l0days after spawning. In these females, the next clutch of eggs also developed at a higher rate than in mouthbrooders.