Filament-carrying tubules demonstrated by negative staining in various mammalian cell types

Summary Among other tubular elements obviously representing the endoplasmic reticulum, tubules carrying filaments with a diameter of about 4 m were found in negatively stained specimens of a variety of mammalian cell strains. They have been found in strains of epithelial and fibroblastic, normal and malignant, human and animal origin. So far, it is not possible to identify the filament-carrying tubules with equivalent structures in thin sections.     

The Sertoli cell of the water buffalo (Bubalus bubalis) during the spermatogenic cycle

Summary Ultrastructural features and morphometric evaluations of buffalo Sertoli cells are reported for the six phases of the spermatogenic cycle. The phases of the tubular seminiferous epithelium are identified according to characteristic cellular associations with completed spermiation as demarcation between two cycles. Average tubular diameter (245 m) and epithelial height (61 m) do not vary significantly during the cycle. The relative Sertoli cell volume in the seminiferous epithelium varies between 30% (phase 4) and 39% (phase 8). The calculated volume of a single Sertoli cell increases from a nadir of 7118 m³ in phase 3 abruptly to a maximum of 8968 m³ in phase 4 and is then gradually reduced during the following phases. The Sertoli cell surface area shows a similar trend: it amounts to 11105 m² in phase 3 and to 14260 m² in phase 4. The contact area of the Sertoli cell with adjacent cells and structures is subject to characteristic changes; from the expansion of basal Sertoli-Sertoli contacts it is concluded that the blood-testis barrier in the buffalo is particularly tight during phases 8, 1 and 2. The irregularly contoured nucleus contains a vesicular nucleolus, has a calculated volume from 465 m³ to 543 m³ and occupies 5 to 7% of the cell. Volume percentages of mitochondria (4%), Golgi apparatus and lysosomal bodies are rather constant during the cycle. Whorls and orderly arranged aggregates of the smooth endoplasmic reticulum occur in basal location as well as in close association with elongating spermatids. Smooth ER is the organelle that exhibits the most prominent changes during the Sertoli cell cycle: it occupies 5.79% in phase 3 and 20.9% in phase 4 of the total cellular volume. Phagocytosis of residual bodies is insignificant in this species and a lipid cycle is absent in buffalo Sertoli cells.     

Fine structure of abscission zones

Summary Electron micrographs of the zone of separation in flower pedicels of Lycopersicon esculentum and Nicotiana tabacum are presented with particular reference to the indentation of epidermal tissue in the abscission zone, subcellular organelles, and the cell wall. The indentation or groove which delineates the abscission zone extends some distance into the pedicel with branchings off the main groove. These branches are approximately 20 m in width while the main groove averages approximately 200 m in width. Invaginations of the plasmalemma are observed with considerable frequency. within these invaginations one observes a material of about the same density as the cell wall except that it is more fibrillar. Plasmodesmata are also observed, with considerable branching into middle lamellae of cells comprising the abscission zones. Microbodies with crystalloid cores appear with considerable frequency in cells of the abscission zone. The crystalloids appear to be cubical in shape and are composed of parallel sheets of osmiophilic material. The sheets average about 6 m in thickness and are spaced at 4 m intervals. The microbodies with crystalloid cores are observed to be characteristically of two size groupings. In tobacco the microbodies average 900 m and 1,500 m in profile. In tomato they average 300 m and 500 m. Chloroplasts contain a granular component which is membrane-enclosed. The component is large in comparison with the plastid in which it occurs, averaging 1.2–1.4 in diameter in chloroplasts ranging from 1.6 to 2.2 in diameter. The inner membrane of the chloroplast is highly invaginated, and DNA- and phytoferritin-like materials are observed within the plastids. Microtubules with an average diameter of 20 m are observed adjacent and parallel to the plasmalemma, primarily in the corners of the cells. Micrographs of other normally occurring cytoplasmic inclusions are also presented.     

Ontogeny of the antennal glands in the crayfish Astacus leptodactylus (Crustacea, Decapoda): anatomical and cell differentiation

The ontogeny of the antennal glands was studied during the embryonic and post-embryonic development of Astacus leptodactylus. The future glands arising from undifferentiated columnar cells were detectable at the metanauplius stage EI 150 m (EI: eye index; approximately 440 m at hatching). The tubule and labyrinth differentiated in embryos at EI 190 m, and the bladder and coelomosac at EI 250 m. At EI 350 m, the tubule lengthened and divided into proximal and distal sub-regions. In later stages, the gland retained the same morpho-anatomy but the differentiation and size of each part increased. The cells of the coelomosac displayed the cytological features of podocytes in late embryonic development at EI 440 m. Only small apical microvilli and a few mitochondria were observable in the labyrinth cells at EI 250 m; by EI 440 m, these cells presented well-shaped apical microvilli, formed bodies, basal infoldings and mitochondria. In the cells of the tubules and bladder, mitochondria and basal infoldings occurred at EI 440 m and EI 250 m, respectively. The differentiation of the tubules and bladder cells suggested that they were involved in active transport at EI 440 m. Following hatching, the differentiation of the cells and the size of the glands increased. The ontogeny of the antennal glands thus starts in early embryos, the specific cellular functional features being differentiated in the various parts of the glands by EI 440 m. The antennal glands are probably functional just before hatching, i.e., before the juveniles are confronted with the low osmolality of freshwater.Thanks are due to the University of Tarbiat Modarres and Ministry of Science, Research and Technology, Islamic Republic of Iran, for financial aid and support. Special thanks are also extended to the Société Française dExportation des Ressources Educatives (SFERE) for a scholarship to S.K.     

Ultrastructural analysis ofSpinacia oleracea sperm cells isolated from mature pollen grains

Summary In isolated condition, the sperm cells ofSpinacia oleracea are no longer arranged in pairs as in the pollen grain. The vegetative membrane, which surrounds a sperm cell pair in a mature pollen grain, is lost during the isolation procedure. The sperm cells become spherical in shape.The isolated sperm cell is surrounded by an intact plasma membrane. The heterochromatic or euchromatic sperm cell nucleus is located in the cell center. Mitochondria are round to oval and have distinct cristae. Often they are clustered in groups of 5 to 10 mitochondria. Dictyosomes are present in the cytoplasm and consist of 4 to 5 cisterns. Endoplasmatic reticulum is mostly situated at the sperm cell periphery, as single cisterns very near the plasma membrane.From diameters of sectioned sperm cells in electron micrographs, it is possible to calculate the average diameter of the whole sperm cell. This average diameter is 3.66 m with a variation of 3.0 m to 4.2 m, resulting in an average volume of 25.6 m³. The nuclear volume is 12.8 m³ (50.0% of the whole cell) and the mitochondrial volume is 0.7 m³ (2.5% of the whole cell). The frequency distribution of the isolated sperm cells diameters shows only one peak with a normal distribution, indicating that there is no dimorphism in volume.     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

Fine structure of the spermatozoon of the sea cucumber,Leptosynapta clarki (Echinodermata: Holothuroidea)

Summary The spermatozoon of the holothurian Leptosynapta clarki has a small circular head measuring about 3.0 at the greatest diameter, a midpiece containing a single mitochondrion and a tail flagellum measuring between 35 and 45 in length. The acrosomal region contains a granule measuring 0.7 in diameter which consists of electron dense material arranged in concentric lamellae. Five concentric very electron dense lamellae alternate with areas of much less electron dense material in the central region of the granule. This granule rests in an anterior nuclear depression.The nucleus is circular in shape and contains one or two unbound vacuoles which frequently contain a fine granular material. Posteriorly the nucleus is bounded by a large mitochondrion and an occasional Golgi complex. The proximal centriole which contains a lateral arm of dense material lies in a deep fossa projecting into the nucleus. The distal centriole lies posteriorly in the mitochondrial mass and gives rise to nine satellite projections and their Y-shaped connective extensions.The tail contains the 9 + 2 tubule arrangement and tapers at its distal end.This investigation was supported by a National Research Council grant to F. S. Chia.     

The influence of temperature on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the regulation of these enzymes in a mesophilic and a thermophilic cyanobacterium

The activities and kinetics of the enzymes G6PDH (glucose-6-phosphate dehydrogenase) and 6PGDH (6-phosphogluconate dehydrogenase) from the mesophilic cyanobacterium Synechococcus 6307 and the thermophilic cyanobacterium Synechococcus 6716 are studied in relation to temperature. In Synechococcus 6307 the apparent K _m's are for G6PDH: 80M (substrate) and 20M (NADP⁺); for 6PGDH: 90M (substrate) and 25M (NADP⁺). In Synechococcus 6716 the apparent K _m's are for G6PDH: 550M (substrate) and 30M (NADP⁺); for 6PGDH: 40M (substrate) and 10M (NADP⁺). None of the K _m's is influenced by the growth temperature and only the K _m's of G6PDH for G6P are influenced by the assay temperature in both organisms. The idea that, in general, thermophilic enzymes possess a lower affinity for their substrates and co-enzymes than mesophilic enzymes is challenged.Although ATP, ribulose-1,5-bisphosphate, NADPH and pH can all influence the activities of G6PDH and 6PGDH to a certain extent (without any difference between the mesophilic and the thermophilic strain), they cannot be responsible for the total deactivation of the enzyme activities observed in the light, thus blocking the pentose phosphate pathway.Abbreviations G6PDH glucose-6-phosphate, dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - G6P glucose-6-phosphate - 6PG 6-phosphogluconate - RUDP ribulose-1,5-bisphosphate - Tricine N-Tris (hydroxymethyl)-methylglycine     

Axillary shoot induction and plant regeneration in Plantago ovata Forssk

Axillary shoot induction and plant regeneration were obtained in Plantago ovata. The optimum medium for inducing axillary shoots was Murashige & Skoog (MS) medium [5] supplemented with 4.6 M kinetin and 0.05 M NAA. Rooting of shoots was best on half-strength MS medium containing 5.0 M IBA and 0.05 M kinetin. The regenerated plants were similar to the control plants in karyotypic and phenotypic details.     

Ultrastructural analysis of the sperm cells of mature pollen of maize,Zea mays

Summary Ultrastructural analysis of the mature viable unhydrated pollen of maize,Zea mays from dehiscent anthers shows that the sperm cells are physically distant, each bounded by an envelope comprising their own plasma membrane and the inner plasma membrane of the vegetative cell. The chondriome is unusual in containing one or more filamentous complexes, up to 12m in length appressed to the side of the sperm nucleus. The extensions at each end of the elongate sperm cells contain longitudinally-oriented arrays of endoplasmic lamellae. In a three-dimensional reconstruction of serial thin sections, there is a long J-shaped sperm, c. 35 × 5m and up to 1m in thickness, sited within pointed evaginations of the vegetative nucleus and a second shorter sperm c. 20 × 5m and up to 3.5m in thickness.Abbreviations PA-TCH-SP periodic acid-thiocarbohydrazide-silver proteinate - DAPI 4,6-diamino-2-phenylindole - SC sperm cell - Sn sperm nucleus - Ua-Pb Uranyl acetate-lead citrate staining - ER endoplasmic reticulum     

Ultrastructural analysis of cell component distribution in the apical cell ofCeratodon protonemata

Summary A distinctive feature of tip-growing plant cells is that cell components are distributed differentially along the length of the cell, although most ultrastructural analyses have been qualitative. The longitudinal distribution of cell components was studied both qualitatively and quantitatively in the apical cell of dark-grown protonemata of the mossCeratodon. The first 35 m of the apical cell was analyzed stereologically using transmission electron microscopy. There were four types of distributions along the cell's axis, three of them differential: (1) tubular endoplasmic reticulum was evenly distributed, (2) cisternal endoplasmic reticulum and Golgi vesicles were distributed in a tip-to-base gradient, (3) plastids, vacuoles, and Golgi stacks were enriched in specific areas, although the locations of the enrichments varied, and (4) mitochondria were excluded in the tipmost 5 m and evenly distributed throughout the remaining 30 m. This study provides one of the most comprehensive quantitative, ultrastructural analyses of the distribution of cell components in the apex of any tip-growing plant cell. The finding that almost every component had its own spatial arrangement demonstrates the complexity of the organization and regulation of the distribution of components in tip-growing cells.Abbreviations CER cisternal endoplasmic reticulum - ER endoplasmic reticulum - N_d numerical density - SE standard error - S_v surface density - TEM transmission electron microscopy - TER tubular endoplasmic reticulum - V_v volume fraction     

The endomembrane system of differentiating carposporangia in the red algaChondria tenuissima: Occurrence and participation in secretion of polysaccharidic and proteinaceous substances

Summary The endomembrane system during carposporogenesis inChondria tenuissima was studied using electron microscopy and histochemistry. Profiles of the nucleus are convoluted, resulting in a highly increased surface area. Stacked cisternae are found within the peripheral part of the nucleus. Vesicles, tubules and membrane bound fibrillar bodies occur within the nucleoplasm. The endoplasmic reticulum surrounds the nuclear envelope.The endoplasmic reticulum and the Golgi apparatus, together with small transition vesicles, represent a functional unit. They form two different secretory substances during carposporogenesis. In young stages, carbohydrates are produced by normal dictyosomes within large, normal exocytotic Golgi vesicles. They do not react positively with PAS or Thiéry method and are believed to represent cell wall material. In later stages, the central area of the Golgi cisternae becomes filled with electron dense material. The individual cisternae are transformed into cored vesicles at the trans-face of the dictyosomes. The dense core of the vesicles is proteinaceous and stains with coomassie brilliant blue R. The peripheral fibrillar material is polysaccharidic and reacts positively using the Thiéry method. The contents of the cored vesicles are believed to participate in carpospore attachment. The ER gives rise to cytolysosomes in which starch grains are sequestrated and digested. Mucilaginous sacs seem to be similarly formed.     

Three-dimensional architecture of the tubular endocytic apparatus and paramembranous networks of the endoplasmic reticulum in the rat visceral yolk-sac endoderm

The three-dimensional architecture of the tubular endocytic apparatus and the endoplasmic reticulum in the rat yolk-sac endoderm was investigated after loading with horseradish peroxidase-conjugated concanavalin A by intrauterine administration. After 30 min, small vesicles (50–150 nm in diameter), small tubules (80–100 nm in diameter) and large vacuoles (0.2–1.0 m in diameter) in the apical cytoplasm were labeled with the tracer, but lysosomes (1.0–3.5 m in diameter) in the supranuclear cytoplasm were not labeled until 60 min after loading. Stereo-viewing of the labeled small tubules in thick sections revealed that they were not isolated structures but formed three-dimensional anastomosing networks, which were also confirmed by scanning electron microscopy after maceration with diluted osmium tetroxide. Their earlier labeling with the endocytic tracer, localization in the apical cytoplasm and three-dimensional network formation indicated that the labeled small tubules represented tubular endosomes (tubular endocytic apparatus). These well-developed membranous networks provided by the tubular endosomes are suggested to facilitate the receptor-mediated endocytosis and transcytosis of the maternal immunoglobulin in the rat yolk-sac endoderm. Scanning electron microscopy further revealed lace-like networks of the smooth endoplasmic reticulum near the lateral plasma membrane. Their possible involvement in transport of small molecules or electrolytes is discussed.     

The Effect of Phenylalanine on DOPA Synthesis in PC12 Cells

DOPA synthesis from phenylalanine was studied in PC12 cells incubated with m-hydroxybenzylhydrazine, to inhibit aromatic L-amino acid decarboxylase. DOPA synthesis rose with increasing concentrations of either phenylalanine or tyrosine; maximal rates (~55 pmol/min/mg protein for tyrosine; ~40 pmol/min/mg protein for phenylalanine) occurred at a medium concentration of ~10 M for either amino acid. The K_m for either amino acid was about 1 M (medium concentration). At tyrosine concentrations above 30 M, DOPA synthesis declined; inhibition was observed at higher concentrations for phenylalanine (300 M). These effects were most notable in the presence of 56 mM potassium. Measurements of intracellular phenylalanine and tyrosine suggested the K_m for either amino acid is 20–30 M; maximal synthesis occurred at 120–140 M. In the presence of both phenylalanine and tyrosine, DOPA synthesis was inhibited by phenylalanine only at a high medium concentration (1000 M), regardless of medium tyrosine concentration. The inhibition of DOPA synthesis by high medium tyrosine concentrations was antagonized by high medium phenylalanine concentrations (100, 1000 M). Together, the findings indicate that for PC12 cells, phenylalanine can be a significant substrate for tyrosine hydroxylase, is a relatively weak inhibitor of the enzyme, and at high concentrations can antagonize substrate inhibition by tyrosine.     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Plant regeneration from somatic embryos ofTaxus brevifolia

Summary Taxusbrevifolia is the source of paclitaxel (Taxol®), an anticancer drug. A method for regeneration ofTaxus brevifolia from immature zygotic embryos via somatic embryogenesis is described. Embryogenic callus tissues were obtained by culturing immature zygotic embryos on Lloyd and McCown medium (MCM) supplemented with 160 M 2,4-dichlorophenoxyacetic acid (2,4-D) + 5 M benzylaminopurine (BA) + 5 M naphthaleneacetic acid (NAA) for 4 weeks. Putative embryoids were obtained following transfer of cultures to MCM medium supplemented with 4 M BA + 5 M kinetin + 1 M NAA for 6 to 8 weeks. Conversion of embryos was obtained on MCM medium supplemented with 40 M abscisic acid (ABA) + 1% activated charcoal. Development of bipolar structures with recognizable shoot and root apices was observed in somatic embryos. Five percent of somatic embryos were regenerated into plantlets on half-strength growth regulator-free MCM medium.     

Two sites for adenine-nucleotide regulation of ATP-sensitive potassium channels in mouse pancreatic β-cells and HIT cells

Summary ATP-inhibited potassium channels (K(ATP)) were studied in excised, inside-out patches from cultured adult mouse pancreatic -cells and HIT cells. In the absence of ATP, ADP opened K(ATP) channels at concentrations as low as 10 m and as high as 500 m, with maximal activation between 10 and 100 m ADP in mouse -cell membrane patches. At concentrations greater than 500 m, ADP inhibited K(ATP) channels while 10 mm virtually abolished channel activity. HIT cell channels had a similar biphasic response to ADP except that more than 1 mm ADP was required for inhibition. The channel opening effect of ADP required magnesium while channel inhibition did not. Using creatine/creatine phosphate solutions with creatine phosphokinase to fix ATP and ADP concentrations, we found substantially different K(ATP)-channel activity with solutions having the same ATP/ADP ratio but different absolute total nucleotide levels. To account for ATP-ADP competition, we propose a new model of channel-nucleotide interactions with two kinds of ADP binding sites regulating the channel. One site specifically binds MgADP and increases channel opening. The other, the previously described ATP site, binds either ATP or ADP and decreases channel opening. This model very closely fits the ADP concentration-response curve and, when incorporated into a model of -cell membrane potential, increasing ADP in the 10 and 100 m range is predicted to compete very effectively with millimolar levels of ATP to hyperpolarize -cells.The results suggest that (i) K(ATP)-channel activity is not well predicted by the ATP/ADP ratio, and (ii) ADP is a plausible regulator of K(ATP) channels even if its free cytoplasmic concentration is in the 10–100 m range as suggested by biochemical studies.We would like to thank Mr. Louis Stamps for expert technical assistance and Dr. Wil Fujimoto and Ms. Jeanette Teague for generously providing HIT cells obtained from Dr. Robert Santerre at Eli Lilly. We would also like to thank Dr. Michel Vivaudou for providing the program ALEX. Support was provided by the NIH and the Department of Veterans Affairs.     

Callus proliferation from the protoplasts of embryogenic cells of Quercus serrata

Embryogenic culture was induced from the immature embryos of Quercus serrata using Marashige and Skoog's medium (MS) containing 0.1 M each of 2,4-d and BAP, and subcultured for seven months before isolation of protoplasts by using 1% Cellulase RS in 0.6 M mannitol solution. Efficient colony formation was obtained when protoplasts were cultured in a liquid MS medium containing 0.6 M mannitol, 3% sucrose and combination of 0.1 M or 1 M each of 2,4-d and BAP. Excluding ammonium nitrate from the MS medium resulted in the decrease of the percentage of colony formation. From colonies, both agar culture and liquid culture were sustained in the MS media without mannitol containing no plant growth regulator, or containing 0.1 M of BAP in combination with 0.1 M or 1 M of 2,4-d.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog (1962).     

Shoot,root and flower morphogenesis on tomato inflorescence explants

Regeneration of de novo shoots, roots and flowers has been obtained on inflorescence explants of tomato (Lycopersicon esculentum Mill.). Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and -naphthaleneacetic acid (NAA) were added in a 3×3×3 factorial combination with kinetin, each at 0.001, 0.1 and 10 M concentrations. Direct shoot formation occurred on media with 10 M kinetin and 0.001 M IAA or NAA. Root formation was observed on media with 0.1–10 M IAA, IBA or NAA. Flower formation occurred on elongated shoots with several leaves on media with 10 M IAA and 0.1 M kinetin. Shoot organogenesis was increased by substituting 10 M zeatin or N⁶-benzyladenine (BA) for kinetin. Eleven tomato cultivars were tested for their ability to undergo de novo shoot regeneration on the improved medium. All tomato cultivars were capable of shoot morphogenesis with a mean number of shoots per explant that ranged from 1.3 (Red Alert) to 5.3 (Large Red Cherry). Histological studies revealed that active cell divisions occurred in subepidermal and cambial tisue during the first week of culture. Meristematic centers of dividing cells were evident by day 14, and well-developed shoot apices and leaf structures were observed on 50% of the explants 28 days after culture initiation.Abbreviations BA N⁶-benzyladenine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - 2iP N⁶-[²-isopentyl]adenine - NAA -naphthaleneacetic acid - PGR plant growth regulator