Differential centrifugation, calcium precipitation, and ultrasonic disruption of midgut cells of Erinnyis ello caterpillars. Purification of cell microvilli and inferences concerning secretory mechanisms

1986 ◽  
Vol 64 (2) ◽  
pp. 490-500 ◽  
Author(s):  
Custódio D. Santos ◽  
Alberto F. Ribeiro ◽  
Walter R. Terra
Keyword(s):  
Subcellular Fractions ◽  
Soluble Form ◽  
Differential Centrifugation ◽  
Posterior Midgut ◽  
Brush Borders ◽  
Columnar Cells ◽  
Differential Precipitation ◽  
Anterior Midgut ◽  
Erinnyis Ello ◽  
Calcium Precipitation

Subcellular fractions of the cells from the first and last third of midguts from Erinnyis ello caterpillars were obtained by conventional homogenization, followed by differential centrifugation or differential calcium precipitation, as well as by partial ultrasonic disruption. Aminopeptidase was enriched in the subcellular fractions, which in the electron microscope display mainly microvilli from the columnar cells (obtained by differential centrifugation and ultrasonic disruption), and also in the microvilli fraction obtained by differential precipitation. To account for the enzyme activities that sedimented with vesicles displaying brush borders, major amounts of the soluble glycosidases (cellobiase, N-acetylglucosaminidase, maltase, and trehalase) are assumed to be loosely bound to the cell glycocalyx, from where they are set free by homogenization and (or) freezing–thawing. Intracellular glycosidases seem to be bounded by membranes, which sediment together with vesicles that resemble secretory vesicles. The soluble form of amylase occurred mainly associated with the microvilli of anterior midgut cells and is supposed to be contained inside small vesicles, which are seen budding along columnar cell microvilli and fusing one with the other and with the tips of the microvilli from the anterior midgut cells. Secretory mechanisms are discussed in the light of the evidence that the posterior midgut secretes whereas the anterior midgut absorbs water.

Ultrastructural features of the gut of Sitophilus granarius (L.) (Coleoptera: Curculionidae) with notes on distribution of proteinases and amylases in crop and midgut

1984 ◽  
Vol 62 (7) ◽  
pp. 1251-1259 ◽  
Author(s):  
J. E. Baker ◽  
S. M. Woo ◽  
R. V. Byrd
Keyword(s):  
Plasma Membranes ◽  
Cell Types ◽  
Sitophilus Granarius ◽  
Posterior Midgut ◽  
Functional Differences ◽  
Basal Plasma ◽  
Dark Staining ◽  
Different Cell Types ◽  
Columnar Cells ◽  
Anterior Midgut

In addition to typical columnar cells, dark-staining cells characterized by deep invaginations of basal plasma membranes were found throughout the midgut of adult Sitophilus granarius (L.). These invaginations formed intracellular channels that extended to the perinuclear region and indicated an involvement of these cells in secretion and (or) absorption. Cells with large vacuolelike structures that occasionally filled the entire supranuclear region were found in the anterior midgut, while multicellular crypts and cells that formed apical extrusions into the lumen were common in the posterior midgut. Fine structure of gastric caeca indicated functional differences between those located in anterior and posterior midgut regions. Numerous dark-staining granules were found in apical regions of cells of the anterior caeca, whereas elongated mitochondria were found in microvilli that made up the brush border of posterior caecal cells. Thus, although the midgut is not obviously differentiated into zones, there are different cell types in different regions of the gut involved in digestive and nutrient absorption processes. Evidence indicated that amylase in S. granarius is secreted by salivary glands whereas trypsin and aminopeptidase are secreted by midgut. A slow, continuous secretion of amylase occurs whereas proteinases are secreted in response to ingested food.

Changes in microvilli and Golgi-associated membranes of lepidopteran cells induced by an insecticidally active bacterial δ-endotoxin

1989 ◽  
Vol 93 (2) ◽  
pp. 337-347
Author(s):  
NANCY J. LANE ◽  
J. B. HARRISON ◽  
W. M. LEE
Keyword(s):  
Bacillus Thuringiensis ◽  
Gap Junctions ◽  
Manduca Sexta ◽  
Golgi Complex ◽  
Intramembranous Particle ◽  
Structural Alterations ◽  
Particle Population ◽  
Consistent Increase ◽  
Columnar Cells ◽  
Anterior Midgut

The δ-endotoxin from Bacillus thuringiensis var. kurstaki was fed to late larvae of the tobacco hornworm, Manduca sexta, to determine its effect on the anterior midgut epithelium, which was examined ultrastructurally at intervals thereafter. The cells of the midgut are primarily of the columnar and goblet variety and the effects of the toxin were more pronounced on the former. Tissues examined after only 1–5 min of exposure to the toxin already revealed fine-structural alterations. These were most notably changes in the microvilli and membranes associated spatially with the Golgi complex: vacuoles associated with its maturing face became enlarged. This effect was intensified with more-extensive exposure to the toxin, resulting in an increase in both vacuoles and the number of lysosomal bodies, many containing myelin-like formations; some of these arose as autophagic vacuoles. There seemed to be no consistent increase in endocytotic activity at the apical border, however. The intramembranous particle population of the microvilli of the columnar cells showed some slight changes with toxin treatment; alterations in microvillar contours also occured. The intercellular septate and gap junctions on the lateral borders were sometimes disrupted and with time often became internalized. It seems, then, that the toxin initially modifies the microvillar membranes and subsequently the Golgi-associated saccules are affected, giving rise to vacuoles and lysosomes.

20 alpha-Hydroxysteroid dehydrogenase and 17 beta-estradiol dehydrogenase localize in cytosol of human term placenta

1982 ◽  
Vol 242 (3) ◽  
pp. E178-E183
Author(s):  
R. C. Strickler ◽  
B. Tobias
Keyword(s):  
Electron Microscopy ◽  
Dehydrogenase Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Subcellular Fractions ◽  
Enzyme Assays ◽  
Marker Enzyme ◽  
Differential Centrifugation ◽  
Experimental Conditions ◽  
Human Term Placenta ◽  
Term Placenta

The 20 alpha-hydroxysteroid dehydrogenase activity in human term placenta has been localized by different investigators to nuclear, mitochondrial, microsomal, and cytosolic subcellular fractions. Furthermore, in the cytosol, 20 alpha-hydroxysteroid dehydrogenase activity may be a second function of the enzyme that mediates 17 beta-estradiol dehydrogenase activity. To search for a unique 20 alpha-hydroxysteroid dehydrogenase, human placental villous tissue, homogenized in three different buffer systems, was fractionated by differential centrifugation, and the 17 beta- and 20 alpha-activities were measured by radioisotope conversion assay. The enrichment and purity of the subcellular fractions were shown by marker enzyme assays and electron microscopy studies. Under all experimental conditions, 20 alpha-hydroxysteroid dehydrogenase activity was identified only in the 105,000 g placental cytosol: intact, osmotically ruptured, and acetone-extracted mitochondria, nuclei, and microsomes did not convert progesterone to 20 alpha-dihydroprogesterone. Furthermore, because 17 beta-estradiol dehydrogenase activity was in large part soluble in the cytosol, these localization studies are consistent with the hypothesis that the 20 alpha- and 17 beta-oxidoreductase activities in human placenta reside on one soluble protein.

THE SUBCELLULAR DISTRIBUTION OF 17β-HYDROXYSTEROID DEHYDROGENASE IN THE HUMAN TERM PLACENTA

1976 ◽  
Vol 82 (1) ◽  
pp. 150-163 ◽  
Author(s):  
C. M. G. Thomas ◽  
J. H. Veerkamp
Keyword(s):  
Dehydrogenase Activity ◽  
Subcellular Distribution ◽  
Medium Composition ◽  
Subcellular Fractions ◽  
Differential Centrifugation ◽  
Marker Enzymes ◽  
Cytosol Fraction ◽  
Human Term Placenta ◽  
Term Placenta ◽  
Buffer Medium

ABSTRACT Human term placenta tissue homogenates were subjected to differential centrifugation procedures. The composition of the subcellular fractions was monitored with a number of marker enzymes and the effectiveness of these enzyme systems was evaluated. The subcellular fractions were tested for their 17β-hydroxy dehydrogenase activity on testosterone, oestradiol and the synthetic substrate retrotestosterone. Buffer medium composition showed a direct influence upon enzyme distribution patterns of all fractions during the same differential centrifugation procedure. All enzyme activities tested became less sedimentable when glycerol was present in the fractionation buffer. Glycerol stabilized soluble 17β-hydroxy dehydrogenase activity during fractionation. The activity of steroid-converting enzymes was inhibited by the presence of glycerol in the medium. Subcellular distribution of marker enzymes did not sustain the presence of mitochondrial 17β-hydroxy dehydrogenase but related it to microsomal contamination. In general the proportion of 17β-hydroxy dehydrogenase activity in the particulate fractions showed a decrease in the substrate order retrotestosterone > testosterone > oestradiol which was independent from the buffer medium used. Specific activities for both particle-bound and soluble 17β-hydroxy dehydrogenase increased in the substrate order retrotestosterone < testosterone < oestradiol. The particulate enzyme activity was maximal with NAD+ for the three substrates tested, but in the cytosol fraction NADP+ was the preferential co-enzyme only when oestradiol was used as the substrate.

Characteristics of rodent intestinal mucin Muc3 and alterations in a mouse model of human cystic fibrosis

2001 ◽  
Vol 280 (6) ◽  
pp. G1321-G1330 ◽  
Author(s):  
Ismat A. Khatri ◽  
Catherine Ho ◽  
Robert D. Specian ◽  
Janet F. Forstner
Keyword(s):  
Cystic Fibrosis ◽  
Mouse Model ◽  
Oligonucleotide Probe ◽  
Goblet Cells ◽  
Soluble Form ◽  
Density Fractions ◽  
Intestinal Mucin ◽  
Apical Membranes ◽  
Columnar Cells

Human mucin MUC3 and rodent Muc3 are widely assumed to represent secretory mucins expressed in columnar and goblet cells of the intestine. Using a 3′-oligonucleotide probe and in situ hybridization, we observed expression of rat Muc3 mostly in columnar cells. Two antibodies specific for COOH-terminal epitopes of Muc3 localized to apical membranes and cytoplasm of columnar cells. An antibody to the tandem repeat (TR) sequence (TTTPDV)3, however, localized to both columnar and goblet cells. On CsCl gradients, Muc3 appeared in both light- and heavy-density fractions. The lighter species was immunoreactive with all three antibodies, whereas the heavier species reacted only with anti-TR antibody. Thus Muc3 is expressed in two forms, a full-length membrane-associated form found in columnar cells (light density) and a carboxyl-truncated soluble form present in goblet cells (heavy density). In a mouse model of human cystic fibrosis, both soluble Muc3 and goblet cell Muc2 were increased in amount and hypersecreted. Thus Muc2 and Muc3 contribute to the excess intestinal luminal mucus of cystic fibrosis mice.

The gene serpent has homeotic properties and specifies endoderm versus ectoderm within the Drosophila gut

Development ◽  
1994 ◽  
Vol 120 (5) ◽  
pp. 1123-1135 ◽  
Author(s):  
R. Reuter
Keyword(s):  
Posterior Part ◽  
Posterior Region ◽  
Homeotic Gene ◽  
Gap Gene ◽  
Posterior Midgut ◽  
Endodermal Differentiation ◽  
Anterior Midgut

The gut of Drosophila consists of ectodermally derived foregut and hindgut and endodermally derived midgut. Here I show that the gene serpent plays a key role in the development of the endoderm. serpent embryos lack the entire midgut and do not show endodermal differentiation. They gastrulate normally and form proper amnioproctodeal and anterior midgut invaginations. However, the prospective anterior midgut cells acquire properties that are usually found in ectodermal foregut cells. In the posterior region of the embryo, the prospective posterior midgut forms an additional hindgut which is contiguous with the normal hindgut and which appears to be a serial duplication, not a mere enlargement of the hindgut. The fate shifts in both the anterior and the posterior part of the srp embryo can be described in terms of homeotic transformations of anterior midgut to foregut and of posterior midgut to hindgut. serpent appears to act as a homeotic gene downstream of the terminal gap gene huckebein and to promote morphogenesis and differentiation of anterior and posterior midgut.

CONVERSION OF ANDROST-4-ENE-3,17-DIONE-4-14C TO OESTROGENS BY SUBCELLULAR FRACTIONS OF THE HUMAN CORPUS LUTEUM OF THE NORMAL CYCLE

1967 ◽  
Vol 56 (2) ◽  
pp. 225-230 ◽  
Author(s):  
Rosalinda B. Arceo ◽  
Kenneth J. Ryan
Keyword(s):  
Subcellular Localization ◽  
Corpus Luteum ◽  
Paper Chromatography ◽  
Specific Activity ◽  
Subcellular Fractions ◽  
Corpora Lutea ◽  
Differential Centrifugation ◽  
Normal Cycle ◽  
Generating System ◽  
Human Corpus Luteum

ABSTRACT The subcellular localization of the aromatizing enzymes from four human corpora lutea was investigated. Subcellular fractions were prepared by differential centrifugation, and incubations of each fraction were carried out with androst-4-ene-3,17-dione-4-14C and a TPNH generating system. Oestrogen metabolites were characterized by phenolic separation, repeated paper chromatography, incubation with freshly prepared placental 17β-ol dehydrogenase, methylation and recrystallization to constant specific activity. The experimental results indicate that the aromatizing enzymes were localized mainly in the microsomal fractions of homogenates of human corpora lutea.

Diacylglycerol acyltransferase in maturing sunflower seeds

2000 ◽  
Vol 28 (6) ◽  
pp. 689-692 ◽  
Author(s):  
S. Triki ◽  
J. Ben Hamida ◽  
P. Mazliak
Keyword(s):  
Enzyme Inhibition ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Diacylglycerol Acyltransferase ◽  
Subcellular Fractions ◽  
Tween 20 ◽  
Differential Centrifugation ◽  
Sunflower Seeds ◽  
Triton X

Developing sunflower seeds exhibit a high diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) activity. The distribution of the enzyme has been studied in subcellular fractions prepared by differential centrifugation of seed homogenate. Its activity was characterized using [1-14C]oleoyl-CoA and diolein dispersed in Tween 20. Some properties of the microsomal fraction of DAGAT were investigated. Hyperbolic kinetics were observed, the apparent Km was 60 μM and the specific activity of the reaction 15 pmol/min/mg of protein. Addition of BSA (0.1%) stimulated oleate incorporation, which was not dependent on the presence of exogenous diacylglycerol. Detergents which might solubilize DAGAT, Triton X-100 and CHAPS, were tested for enzyme inhibition, and CHAPS was found to be the least denaturing.

Processing of the insulin-like growth factor-II–mannose 6-phosphate receptor in isolated liver subcellular fractions

2001 ◽  
Vol 79 (4) ◽  
pp. 469-477
Author(s):  
Khadija Tahiri ◽  
Laurence Cam ◽  
Bernard Desbuquois ◽  
Geneviève Chauvet
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Subcellular Fractions ◽  
Soluble Form ◽  
Dependent Manner ◽  
Insulin Like Growth Factor ◽  
Factor Ii ◽  
Mannose 6 Phosphate ◽  
Isolated Liver

A truncated, soluble form of the insulin-like growth factor-II–mannose 6-phosphate (IGF-II–M6P) receptor has been identified in serum and shown to be released from cultured tissues and cells, liver being the main contributor to serum receptor in adult rats. In the present study, the processing of the IGF-II–M6P receptor has been characterized in isolated liver subcellular fractions using ligand binding, affinity crosslinking, and Western immunoblotting techniques. The receptor in plasma membrane fractions differed from that in Golgi-endosomal fractions by: (i) a lower molecular size upon reducing polyacrylamide gel electrophoresis (245 vs. 255 kDa); (ii) a less tight membrane association as judged upon extractibility by NaCl; and (iii) the inability to recognize antibody anti-22C, directed against the cytoplasmic domain of the receptor. Incubation of cell fractions at 30°C led to a pH- and time-dependent release of the receptor into the medium. The pH optimum for release was 5.5 in the Golgi-endosomal fraction and 7.5 in plasma membrane fractions; at this pH, approximately 2% and 20%–30% of total receptors were released per hour, respectively. Receptor release was inhibited in a dose-dependent manner by aprotinin, benzamidine, and leupeptin in the Golgi-endosomal fraction, and by 1,10 phenanthroline in plasma membrane fractions, although high concentrations were required for inhibition. The receptor released from Golgi-endosomes showed a 5–10 kDa reduction in size and a loss of ability to recognize antibody anti-22C, but that released from plasma membranes showed little or no changes in size. We conclude that soluble, carboxy-terminally truncated forms of the IGF-II–M6P receptor are generated from the intact receptor in isolated Golgi-endosomal and plasma membrane fractions. However, receptor processing in these fractions exhibits different properties, suggesting the involvement of different proteases.Key words: insulin-like growth factor-II–mannose 6-phosphate (IGF-II–M6P) receptor, liver, plasma membrane, Golgi apparatus, endosomes.

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