Electron microscopic and cytochemical study on the role of Golgi elements and plasma membrane of enterocytes in the intestinal lipid transport

Summary Intestinal lipid absorption and transport were investigated in albino rats. The observations point towards the existence of a continuity between plasma membrane invaginations and elements of the Golgi complex on its mature face. They also suggest a segregation of lipid droplets by paired Golgi membranes and plasma membrane invaginations. The following way for lipid transport is deduced: lipid droplets moving inside the smooth endoplasmic reticulum accumulate progressively and are condensed in Golgi cisternae of the forming face. Their limiting membrane ruptures and liberated lipid droplets are segregated by paired Golgi membranes of the mature face or by plasma membrane invaginations. Subsequently the inner of the two segregating membranes disappears while the lipid droplet is moved towards the intercellular space inside a canal communicating with this space. The suggestion is made that the Golgi apparatus is of double origin: one component representing a terminal plication of the endoplasmic reticulum; the second one—a terminal plication of the plasma membrane invagination. This concept explains the ultrastructural and histochemical differences between Golgi membranes of the forming and mature faces of the complex.     

Multiple rough endoplasmic cisternae in “C” cells

Summary Multiple rough endoplasmic cisternae were found in C cells of the adult rat, at interphase. They are considered to be normal constituents of C cells. Their morphological relation to rough endoplasmic reticulum and their close proximity to mitochondria, Golgi dictyosomes and secretory granules suggest that they may have a role in the secretory activity of this endocrine cell.     

LH-producing cells in the ovine pituitary

Summary An immunocytochemical technique using the peroxidase-antiperoxidase complex (PAP) was applied to identify and characterize the LH-secreting cells in the ovine pituitary at the ultrastructural level. These cells, round or oval in shape, possessing flattened cisternae of the rough endoplasmic reticulum, contain one class of secretory granules (mean diameter 250 nm) and large dense bodies (600 to 800 nm in diameter). LH molecules and the two subunits LH and LH were localized on the secretory granules and on the small granules near the Golgi complex. The large dense bodies, the cisternae of the endoplasmic reticulum and the saccules of the Golgi complex showed no reaction product.Abbreviations used in this Article O-LH ovine luteinizing hormone - b-LH bovine luteinizing hormone - p-LH porcine luteinizing hormone - p-LH porcine LH subunit - p-LH porcine LH subunit - O-FSH ovine follicle stimulating hormone - b-TSH bovine thyrotropic hormone - A-b LH antiserum to bovine LH - A-pLH antiserum to porcine LH subunit - A-pLH antiserum to porcine LH subunit     

Steady state changes in mitochondrial electrical potential and proton gradient in perfused liver from rats fed a high fat diet

In this work the protonmotive force (p), as well as the subcellular distribution of malate, ATP, and ADP were determined in perfused liver from rats fed a low fat or high fat diet, using density gradient fractionation in non acqueous solvents.Rats fed a high fat diet, despite an enhanced hepatic oxygen consumption, exhibit similar p to that found in rats fed a low fat diet, but when we consider the two components of p, we find a significant decrease in mitochondrial/cytosolic pH difference (pH_m) and a significant increase in mitochondrial membrane potential (_m) in rats fed a high fat diet compared to rats fed a low fat diet, which tend to compensate each other. In rats fed a high fat diet the concentration ratio of malate and ATP/ADP does not reflect the changes in pH_m and _m, which represent the respective driving force for their transport.The findings are in line with an increase in substrate supply to the respiratory chain which is, however, accompanied by a higher energy turnover in livers from HFD rats. By this way the liver could contribute to the lack of weight gain from the high caloric intake in HFD rats.     

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.     

Cloned β1,4N-acetylgalactosaminyltransferase: subcellular localization and formation of disulfide bonded species

Cloned human 1,4N-acetylgalactosaminyltransferase (GalNAcT) catalyses the synthesis of the glycosphingolipids GM2, GD2, and gangliotriosylceramide. To determine the subcellular location of this enzyme and whether it exists in intermolecular disulfide bonded species, we stably transfected Chinese hamster ovary (CHO) cells with three myc epitope-tagged forms of the GalNAcT gene: the native enzyme; the lumenal domain of GalNAcT fused to the cytoplasmic and transmembrane domains ofN-acetylglucosaminyltransferase I (GNT); and the transmembrane and lumenal domains of GalNAcT fused to the cytoplasmic domain of the Iip33 form of human invariant chain in order to retain the enzyme in the endoplasmic reticulum (ER). Immunoelectron microscopic analysis with anti-myc revealed that GalNAcT/myc was present throughout the Golgi stack, the GNT/GalNAcT/myc form was restricted primarily to the medial Golgi cisternae, and the Iip33/GalNAcT/myc form was restricted to the ER. Cells transfected with each of the three constructs contained high levels of GM2 synthase activityin vitro, but only the GalNAcT/myc form and the GNT/GalNAcT/myc forms were able to synthesize the GM2 productin vivo. The enzyme produced by all three constructs was present in the transfected cells in a disulfide bonded form having a molecular size consistent with that of a homodimer or higher aggregate.Abbreviations GSL glycosphingolipid(s) - CHO Chinese hamster ovary - GSL structures: GM2 GalNAc1,4(NeuAc2,3)Gal1,4GlcCer - GD2 GalNac1,4(NeuAc2,8NeuAc2,3)Gal1,4GlcCer - GM3 NeuAc2,3Gal1,4GlcCer - Gg₃ GalNAc1,4Gal1,4GlcCer - LacCer Gal1,4GlcCer - GlcCer glucosylceramide - PBS-BSA phosphate buffered saline pH 7.4 containing 1% bovine serum albumin - GalNAcT N-acetylgalactosaminyltransferase - GNT N-acetylglucosaminyltransferase I - Iip33 p33 form of human invariant chain - HPTLC high performance thin layer chromatography - PCR polymerase chain reaction - BFA Brefeldin A This paper is dedicated to Professor Sen-itiroh Hakomori on the occasion of his 65th birthday.     

Fine structural localization of acid and alkaline phosphatase activities in the absorbing cells of the duodenum of rodents

Summary The morphology of the absorbing cells of the duodenal villi in the mouse, the rat, the hamster and the guinea-pig is described. The polymorphism of the dense bodies is pointed out. The fine localization of acid and alkaline phosphatase is investigated and compared. In all the species, acid phosphatase activity is observed in the dense bodies, Golgi vesicles and rare smooth endoplasmic profiles. Alkaline phosphatase is localized on the microvilli, Golgi apparatus, some smooth endoplasmic cisternae and numerous dense bodies. The presence of an alkaline phosphatase reaction in the dense bodies, probably lysosomes, of the absorbing cells is discussed. It is assumed that this enzyme follows a catabolic pathway and is finally degraded in the lysosomes.Abbreviations used AlPase alkaline phosphatase - AcPase acid phosphatase This work was done thanks to the contract C.E.N./A.I.E.A. N 347/RB and thanks to grants from the Fonds de la Recherche scientifique fondamentale collective.     

Subcompartment sugar residues of gastric surface mucous cells studied with labeled lectins

Summary We examined the intracellular localization of sugar residues of the rat gastric surface mucous cells in relation to the functional polarity of the cell organellae using preembedding method with several lectins.In the surface mucous cells, the nuclear envelope and rough endoplasmic reticulum (rER) and cis cisternae of the Golgi stacks were intensely stained with Maclura pomifera (MPA), which is specific to -Gal and GalNAc residues. In the Golgi apparatus, one or two cis side cisternae were stained with MPA and Dolichos biflorus (DBA) which is specific to terminal -N-acetylgalactosamine residues, while the intermediate lamellae were intensely labeled with Arachis hypogaea (PNA) which is specific to Gal 1,3 GalNAc. Cisternae of the trans Golgi region were also stained with MPA, Ricinus communis I (RCA I) which is specific to -Gal and Limax flavus (LFA) which is specific to -NeuAc. Immature mucous granules which are contiguous with the trans Golgi lamellae were weakly stained with RCA I, while LFA stained both immature and mature granules.The differences between each lectin's reactivity in the rough endoplasmic reticulum, in each compartment of the Golgi lamellae and in the secretory granules suggest that there are compositional and structural differences between the glycoconjugates in the respective cell organellae, reflecting the various processes of glycosylation in the gastric surface mucous cells.     

Interactive effects of salinity and irradiance on photoprotection in acclimated seedlings of two sympatric mangroves

The synergistic effects of irradiance and salinity on leaf angle, the photochemical efficiency of photosystem II and photosynthetic pigment composition of mangroves were studied in a factorial experiment. Seedlings of Aegiceras corniculatum (L.) Blanco (Myrsinaceae) and Avicennia marina (Forstk.) Vierh var. australasica (Walp.) Moldenke (Avicenniaceae) were grown under salinity treatments (0, 5, 25, 50, 75, and 100% artificial seawater), in full sunlight or under shade cloth (transmitting 30 or 70% sunlight), during summer and autumn. Significant species differences and effects of salinity and growth irradiance were found for key measures. Depressions in F_v/F_m due to salinity and growth irradiance were chronic, they were least in 25% seawater and in 30% sunlight, and greater in low and high salinity, and higher irradiance. A diurnal depression of F_v/F_m was superimposed on the chronic depression, and was greater for Ae. corniculatum than Av. marina. Increases in leaf angle; and increases in the size, and de-epoxidation state of the xanthophyll cycle pigment pool afforded protection from adverse effects of excess excitation energy. Adverse effects of the highest salinities on ,-carotene and ,-carotene biosynthetic pathways were suggested, particularly in Ae. corniculatum. The ecological significance of differences in species extent and temporal patterns of response are discussed.     

Quantitative relationship between active sodium transport,expansion of endoplasmic reticulum and specialized vacuoles (“scalloped sacs”) in the outermost living cell layer of the frog skin epithelium (Rana temporaria)

Summary When an isolated frog skin (Rana temporaria) is exposed to a hydrostatic pressure difference between inside and outside bathing solutions (inside pressure higher than outside) of 20–50 cm of H₂O and if under these conditions the skin is short-circuited electrically, small vacuoles appear light-microscopically in the outermost living cell layer in the epithelium. The number of such vacuoles shows a linear dependency on the rate of active sodium transport as measured by the short-circuit current. Electron-microscopically, the vacuoles are interpreted as previously undescribed organelles, the scalloped sacs which are about 0.5 in diameter, with a wrinkled surface and bounded by a unit membrane. This organelle is in intimate contact with sacs and tubules of smooth endoplasmic reticulum. The observed increase in the number of scalloped sacs usually is accompanied by a significant expansion of the whole system of endoplasmic reticulum. Some of the vacuoles seen light-microscopically must indeed be expanded cisternae of endoplasmic reticulum. The findings are discussed in light of the possibility that the scalloped sacs and the endoplasmic reticulum may be involved in active transport of sodium ions.     

Electron microscope observations on the early development of the rat

Summary The early development of the rat, from the mature oocyte through fertilization until the 8-cell stage, has been studied with the electron microscope. The fine structure is described and discussed, with particular reference to the following topics. The middle piece of the spermatozoon is found in every stage studied, within the ovum cytoplasm; it is not significantly altered by this situation. The nucleoli are numerous during the 1-cell stage and often present in positions that suggest their extrusion into the cytoplasm; in subsequent stages a branching structure develops around them. The dividing cell presents the mitotic apparatus with its centrioles, hollow looking fibers, chromosomes, but without centromeres; in the cytoplasm the small granules align in rows. Mitochondria are evenly distributed during the 1-cell stage and can be found in the 8-cell stage constricted as if dividing. The multivesicular bodies constitute an abundant population of cytoplasmic elements that may be related to the endoplasmic reticulum or the Golgi complex, neither of which is clearly recognizable. In the 8-cell stage the cytoplasm segregates into two zones, one of which contains all the multivesicular bodies, while the mitochondria are found in both of them; this distinction provides some basis to adscribe to the multivesicular bodies the properties of the so called metachromatic particles.The support of the Gildemeister Foundation is gratefully acknowledged.     

Adoptions expérimentales de larves entre des fourmis de genres différents (II):Myrmica laevinodis Nylander etAnergates atratulus Schenck

Résumé Nous avons fait élever des larves d'Anergates atratulus par des ouvrières deMyrmica laevinodis à 22°C. Pour y parvenir, il n'est pas utile de faire hivernerensemble les larves d'Anergates et les ouvrières deMyrmica. La présence de larves autochtones n'empêche pas lesMyrmica d'élever des larves d'Anergates. Dans toutes les expériences lesMyrmica ont été soumises au fridavant de recevoir des larves d'Anergates. Aucune reine deMyrmica n'a été utilisée dans ces expériences.Sur les 64 larves d'Anergates que nous avons utilisées, 38 se sont transformées en imagos. C'est au début de l'adoption et au moment des métamorphoses que périrent la plupart des 26Anergates perdus. Les femelles vécurent en général 2 ou 3 jours et cherchèrent très tôt à quitter le nid natal. Les mâles vécurent 2 à 3 semaines.

---

Summary Larvae ofAnergates atratulus were experimentally reared by workers ofMyrmica laevinodis, at 22°C. An overwintering of both larvae ofAnergates and workers ofMyrmica is not necessary for the success of that experiment. The presence of larvae ofMyrmica does not keep theMyrmica from rearing larvae ofAnergates. The workers ofMyrmica have been cooled, in all the experiments, before receiving larvae ofAnergates. No queen ofMyrmica have been used in that experiments.38 of the 64 larvae ofAnergates used became imagos. Most of the 26 lostAnergates died at the beginning of the adoption and during the metamorphosis. The females lived generally 2 or 3 days and tried, very early, to leave their native nest. The males lived 2 or 3 weeks.

---

Anergates atratulus Myrmica laevinodis, 22 . bmecme Anergates Myrmica. Myrmica Anergates. Myrmica Anergates. Myrmica . 64 Anergates , 38 . 26 Anergates 2 3 . 2 3 .

     

Expression of a chimeric stilbene synthase gene in transgenic wheat lines

A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno T_Oplant were obtained and, except one Combi T_Oplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_{_o}plants. Two of the respective T₁progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation.     

Meiotic studies ofElymus nutans andE. jacquemontii (Poaceae,Triticeae) and their hybrids withPseudoroegneria spicata and seventeenElymus species

Hybridizations ofElymus nutans andE. jacquemontii were carried out with one species ofPseudoroegneria (S genome), and 20Elymus species, each containing either of the SH, SY, SYH, or SYW genomes. Chromosome configurations were analysed at metaphase I of the two target taxa and their interspecific hybrids. It is concluded that (i)E. nutans is an allohexaploid containing the SYH genomes, andE. jacquemontii is an allotetraploid having the SY genomes; (ii) the genomic affinity is associated with the geographic distance between the species studied; (iii) minor genomic structural rearrangements have occurred within the hexaploid taxon ofE. nutans.     

Synthesis of Gal-β-(1→4)-GlcNAc-β-(1→6)-[Gal-β- (1→3)]-GalNAc-α-OBn oligosaccharides bearing O-methyl or O-sulfo groups at C-3 of the Gal residue: specific acceptors for Gal: 3-O-sulfotransferases

Our recent studies have revealed the existence of two distinct Gal: 3-O-sulfotransferases capable of acting on the C-3 position of galactose in a Core 2 branched structure, e.g., Gal14GlcNAc16(Gal13)GalNac1OBenzyl as acceptor to give 3-O-sulfoGal14GlcNAc13(Gal13)GalNAc1OB 20 and Gal14GlcNAc16(3-O-sulfoGal13)GalNAc1OB 23. We herein report the synthesis of these two compounds and also that of other modified analogs that are highly specific acceptors for the two sulfotransferases. Appropriately protected 1-thio-glycosides 7, 8, and 10 were employed as glycosyl donors for the synthesis of our target compounds.     

Ultrastructure of differentiating hemocytes in the embryo of Oncopeltus fasciatus Dallas (Insecta,Heteroptera)

Summary The hemocytes of Oncopeltus differentiate rather early during embryogenesis. They are segregated by the mesoderm soon after its formation (about 50h after egg deposition). Newly segregated hemocytes show the typical features of embryonic cells: many free ribosomes, a few strands of rough ER, the cisternae of which are considerably distended, electron lucent vacuoles around the periphery, and glycogen deposits. A few hours thereafter the hemocytes undergo striking subcellular changes. First, glycogen, electron lucent vacuoles and rough ER disappear and phagocytotic activity can be observed. Golgi complexes become well expressed and give rise to electron dense vesicles which fuse to larger bodies. Then, rough ER develops again and occupies large areas of the cytoplasm. Its cisternae are often considerably distended by proteinaceous secretions. All hemocytes undergo the same steps of differentiation.Embryonic hemocytes obviously play a decisive role in the elimination of waste products, in particular of tissue debris that results from programmed cellular death. The significance of the conspicuous protein secretions is not fully understood. They may participate in the deposition of the acellular connective tissue, or may have some of the other functions ascribed to insect blood cells.Larvae and imagines of Oncopeltus have four types of hemocytes, which agree rather well with those found in Rhodnius (Lai-Fook, 1970). All embryonic hemocytes, aside from the newly segregated ones, represent plasmatocytes but, unlike plasmatocytes of postembryonic stages, they contain no large inclusion bodies. Newly segregated embryonic hemocytes, in addition to their typical embryonic features, have some similarities with larval and adult prohemocytes. Oenocytoids and granulocytophagous cells are absent in the embryo. Some aspects concerning the differentiation and classification of hemocytes are discussed.Supported by research grant Do 163 from the Deutsche ForschungsgemeinschaftThe author is grateful to Ms. K. Schmidtke and Ms. M. Ullmann for technical assistance     

In-vivo observations of a spherical aggregate of endoplasmic reticulum and of Golgi vesicles in the tip of fast-growing Chara rhizoids

In-vivo differential interference contrast microscopy was used to detect individual Golgi vesicles and a new structure in the tip of fast-growing rhizoids of Chara fragilis Desvaux. This structure is a spherical clear zone which is free of Golgi vesicles, has a diameter of 5 m and is positioned in the center of the apical Golgi-vesicle accumulation (Spitzenkörper). After glutaraldehyde fixation and osmium tetroxide-potassium ferricyanide staining of the rhizoid, followed by serial sectioning and three-dimensional reconstruction, the spherical zone shows a tight accumulation of anastomosing endoplasmic reticulum (ER) membranes. The ER membranes radiate from this aggregate towards the apical plasmalemma and to the membranes of the statolith compartments. Upon gravistimulation the ER aggregate changes its position according to the new growth direction, indicating its participation in growth determination. After treatment of the rhizoid with cytochalasin B or phalloidin the ER aggregate disappears and the statoliths sediment. It is concluded that the integrity of the ER aggregate is actin-dependent and that it is related to the polar organisation of the gravitropically growing cell tip.Abbreviations CB cytochalasin B - DIC differential interference contrast microscopy - DMSO dimethyl sulfoxide - ER endoplasmic reticulum     

Fine structural localization of 3beta-hydroxysteroid dehydrogenase in rat corpus luteum

Synopsis A procedure for the ultracytochemical demonstration of 3-hydroxysteroid dehydrogenase (3-HSD) is described and the results for the localization of this enzyme in the lutein cells of the rat corpus luteum are presented.The procedure involved pre-fixation with aldehydes and incubation of small blocks. Short fixation (maximum 30 min) in 0.1% glutaraldehyde, 2% depolymerized paraformaldehyde or in 6.25% hydroxyadipaldehyde was found to be the best compromise for the preservation of both 3-HSD activity and fine structure. The method utilizes 3-hydroxy-5-androstan-17-one as substrate, tetranitro blue tetrazolium as a final electron acceptor, and phenazine methosulphate or menadione as an intermediate electron carrier to bypass the NADH₂-diaphorase. Control and inhibitor (cyano-ketone) experiments provide evidence for the specificity of the cytochemical reaction.Results showed that 3-HSD activity is localized on or in membranes of smooth endoplasmic reticulum, in outer compartments of mitochondria, and possibly within smooth endoplasmic reticulum cisternae and intracristal spaces of mitochondria.Localization of NADH₂-diaphorase activity in the same tissue was also examined. With the ferricyanide techniques, the reaction product was found to be associated mainly with the mitochondria.