scholarly journals The site of cellulose synthesis. Hormone treatment alters the intracellular location of alkali-insoluble beta-1,4-glucan (cellulose) synthetase activities.

1975 ◽  
Vol 64 (3) ◽  
pp. 557-571 ◽  
Author(s):  
G Shore ◽  
G A Maclachlan
Keyword(s):  
Cell Structure ◽  
Hormone Treatment ◽  
Cellulose Synthesis ◽  
Marker Enzyme ◽  
Tissue Slices ◽  
Intact Cells ◽  
Intracellular Location ◽  
Sites Of Action

Membrane preparations from growing regions of 8-day old Pisum sativum epicotyls contain multiple beta-1,4-glucan (cellulose) synthetase activities (UDP- or GDP-glucose: beta-1,4-glucan-glucosyl transferase), and the levels of some of these are influenced by treatments with the growth hormone, indoleacetic acid (IAA). When membranes from control epicotyl segments (zero time) are fractionated by isopycnic sedimentation in sucrose density gradients, all of the synthetase activities are associated mainly with Golgi membrane (density 1.55 g/cm3). After decapitation and treatment of epicotyls with IAA, synthetases also appear in a smooth vesicle fraction (density 1.11 g/cm3) which is rich in endoplasmic reticulum (ER) marker enzyme. Major fractions of these synthetases are not recovered in association with plasma membrane or washed cell walls. When [14-C]sucrose is supplied in vivo to segments +/- IAA, radioactive cellulose is deposited only in the wall. Cellulose or cellodextrin precursors do not accumulate in those membranes in which synthetase activities are recovered in vitro. In experiments where tissue slices containing intact cells are supplied with [14C]sugar nucleotide in vitro, alkali-insoluble beta-1,4-glucan is synthesized (presumably outside the protoplast) at rates which greatly exceeded (20-30 times) those obtained using isolated membrane preparations. Progressive disruption of cell structure results in increasing losses of this high activity. These results are consistent with the interpretation that Golgi and ER-associated synthetases are not themselves loci for cellulose synthesis in vivo, but represent enzymes in transit to sites of action at the wall:protoplast omterface. There they operate only if integrity of cellular organization is maintained.

Studies on the mechanism of acute inhibition of thyroglobulin hydrolysis by iodine

1985 ◽  
Vol 108 (4) ◽  
pp. 511-517 ◽  
Author(s):  
Nandalal Bagchi ◽  
Birdie Shivers ◽  
Thomas R. Brown
Keyword(s):  
Time Course ◽  
Period 2 ◽  
Iodotyrosine Deiodinase ◽  
Rate Of Hydrolysis ◽  
Underlying Mechanisms ◽  
Excess Iodide ◽  
Sites Of Action ◽  
Hydrolysis Of

Abstract. Iodine in excess is known to acutely inhibit thyroidal secretion. In the present study we have characterized the time course of the iodine effect in vitro and investigated the underlying mechanisms. Labelled thyroid glands were cultured in vitro in medium containing mononitrotyrosine, an inhibitor of iodotyrosine deiodinase. The rate of hydrolysis of labelled thyroglobulin was measured as the proportion of labelled iodotyrosines and iodothyronines recovered at the end of culture and was used as an index of thyroidal secretion. Thyrotrophin (TSH) administered in vivo acutely stimulated the rate of thyroglobulin hydrolysis. Addition of Nal to the culture medium acutely inhibited both basal and TSH-stimulated thyroglobulin hydrolysis. The effect of iodide was demonstrable after 2 h, maximal after 6 h and was not reversible upon removal of iodide. Iodide abolished the dibutyryl cAMP induced stimulation of thyroglobulin hydrolysis. Iodide required organic binding of iodine for its effect but new protein or RNA synthesis was not necessary. The inhibitory effects of iodide and lysosomotrophic agents such as NH4C1 and chloroquin on thyroglobulin hydrolysis were additive suggesting different sites of action. Iodide added in vitro altered the distribution of label in prelabelled thyroglobulin in a way that suggested increased coupling in the thyroglobulin molecule. These data indicate that 1) the iodide effect occurs progressively over a 6 h period, 2) continued presence of iodide is not necessary once the inhibition is established, 3) iodide exerts its action primarily at a post cAMP, prelysosomal site and 4) the effect requires organic binding of iodine, but not new RNA or protein synthesis. Our data are consistent with the hypothesis that excess iodide acutely inhibits thyroglobulin hydrolysis by increasing the resistance of thyroglobulin to proteolytic degradation through increased iodination and coupling.

Investigation of the Thermal and Injury Behavior During Microwave Thermal Therapy of Porcine Kidney

2002 ◽  
Author(s):  
Xiaoming He ◽  
Shawn Mcgee ◽  
James E. Coad ◽  
Paul A. Iaizzo ◽  
David J. Swanlund ◽  
...  
Keyword(s):  
Thermal Model ◽  
Histological Study ◽  
Kidney Cortex ◽  
Cellular Injury ◽  
Bioheat Equation ◽  
Tissue Slices ◽  
Thermal Histories ◽  
Microwave Probe

In this paper, we report on the characterization of microwave therapy of normal porcine kidneys both in vitro and in vivo. This technology is being developed for eventual use in the treatment of small renal cell carcinoma (RCC) by minimally invasive procedures. During experiments, microwave energy was applied through an interstitial microwave probe (Urologix, Plymouth, MN) to the kidney cortex with occasional involvement of the kidney medulla. The thermal histories at several locations were recorded. After treatment, the kidneys were bisected and small tissue slices were cut out at approximately the same depth as the thermal probes. The tissue slices were further processed for histological study. Both cellular injury and the area of microvascular stasis were quantitatively evaluated by histology. Absolute rate kinetic models of cellular injury and vascular stasis were developed and fit to this data. A 3-D finite element thermal model based on the Pennes Bioheat equation was developed and solved using a commercial software package (ANSYS, V5.7). The Specific Absorption Rate (SAR) of the microwave probe was measured experimentally in tissue equivalent gel-like solution. The thermal model was first validated by the measured in vitro thermal histories. It was then used to determine the blood perfusion term in vivo.

Surfactant metabolism in surfactant protein A-deficient mice

1997 ◽  
Vol 272 (3) ◽  
pp. L479-L485 ◽  
Author(s):  
M. Ikegami ◽  
T. R. Korfhagen ◽  
M. D. Bruno ◽  
J. A. Whitsett ◽  
A. H. Jobe
Keyword(s):  
Lung Tissue ◽  
Protein A ◽  
Surfactant Protein ◽  
Normal Lung ◽  
Tissue Slices ◽  
Normal Lung Function ◽  
Deficient Mice ◽  
Surfactant Metabolism

In the present study we asked if surfactant metabolism was altered in surfactant protein (SP) A-deficient mice in vivo. Although previous studies in vitro demonstrated that SP-A modulates surfactant secretion and reuptake by type II cells, mice made SP-A deficient by homologous recombination grow and reproduce normally and have normal lung function. Alveolar and lung tissue saturated phophatidylcholine (Sat PC) pools were 50 and 26% larger, respectively, in SP-A(-/-) mice than in SP-A(+/+) mice. Radiolabeled choline and palmitate incorporation into lung Sat PC was similar both in vivo and for lung tissue slices in vitro from SP-A(+/+) and SP-A(-/-) mice. Percent secretion of radiolabeled Sat PC was unchanged from 3 to 15 h, although SP-A(-/-) mice retained more labeled Sat PC in the alveolar lavages at 48 h (consistent with the increased surfactant pool sizes). Clearance of radiolabeled dipalmitoylphosphatidylcholine and SP-B from the air spaces after intratracheal injection was similar in SP-A(-/-) and SP-A(+/+) mice. Lack of SP-A had minimal effects on the overall metabolism of Sat PC or SP-B in mice.

scholarly journals Biochemistry of postharvest spoilage of sweet potato (Ipomoea batatas L.). 2. Comparison of cellulolytic enzyme production in cultures and fungi-infected sweet potato tubers

2006 ◽  
pp. 48-57
Author(s):  
R. C. Ray
Keyword(s):  
Sweet Potato ◽  
Enzyme Production ◽  
Ipomoea Batatas ◽  
Rhizopus Oryzae ◽  
Cellulose Synthesis ◽  
Cellulolytic Enzyme ◽  
Optimum Temperature ◽  
Potato Tubers

The study was conducted to determine the production in vitro and in vivo of cellulases by Botrydiplodia theobromae and Rhizopus oryzae. Isolates of these organisms were obtained from the postharvest decay of sweetpotato tubers. Results revealed that B. theobrornae and R. oryzae which were isolated from postharvest spoilage of sweetpotato tubers produced endo-13-1,4-glucanase and exo-V-1 ,4-glucanase in culture and in fungi-infected tissues of sweetpotato tubers. The optimum temperature and pH for cellulose synthesis and activity were 30°C and pH 6.5, respectively.

scholarly journals In Vitro and In Vivo Neutralization of the Relapsing Fever Agent Borrelia hermsii with Serotype-Specific Immunoglobulin M Antibodies

2001 ◽  
Vol 69 (2) ◽  
pp. 1009-1015 ◽  
Author(s):  
Alan G. Barbour ◽  
Virgilio Bundoc
Keyword(s):  
Monoclonal Antibodies ◽  
Antibody Response ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Relapsing Fever ◽  
Borrelia Hermsii ◽  
Intact Cells ◽  
Whole Cells

ABSTRACT The antigenic variation of the relapsing fever agent Borrelia hermsii is associated with changes in the expression of the Vlp and Vsp outer membrane lipoproteins. To investigate whether these serotype-defining proteins are the target of a neutralizing and protective antibody response, monoclonal antibodies were produced from spleens of infected mice just after clearance of serotype 7 cells from the blood. Two immunoglobulin M monoclonal antibodies, H7-7 and H7-12, were studied in detail. Both antibodies specifically agglutinated serotype 7 cells and inhibited their growth in vitro. Administered to mice before or after infection, both antibodies provided protection against infection or substantially reduced the number of spirochetes in the blood of mice after infection. Whereas antibody H7-12 bound to Vlp7 in Western blotting, enzyme-linked immunosorbent assay, and immunoprecipitation assays, as well as to whole cells in other immunoassays, antibody H7-7 only bound to wet, intact cells of serotype 7. Antibody H7-7 selected against cells expressing Vlp7 in vitro and in vivo, an indication that Vlp7 was a conformation-sensitive antigen for the antibody. Vaccination of mice with recombinant Vlp7 with adjuvant elicited antibodies that bound to fixed whole cells of serotype 7 and to Vlp7 in Western blots, but these antibodies did not inhibit the growth of serotype 7 in vitro and did not provide protection against an infectious challenge with serotype 7. The study established that a Vlp protein was the target of a neutralizing antibody response, and it also indicated that the conformation and/or the native topology of Vlp were important for eliciting that immunity.

Intracellular regulation of 17β-hydroxysteroid dehydrogenase type 2 catalytic activity in A431 cells

1997 ◽  
Vol 153 (3) ◽  
pp. 453-464 ◽  
Author(s):  
C H Blomquist ◽  
B S Leung ◽  
C Beaudoin ◽  
D Poirier ◽  
Y Tremblay
Keyword(s):  
Reductase Activity ◽  
Maximum Velocity ◽  
Hydroxysteroid Dehydrogenase ◽  
A431 Cells ◽  
Intact Cells ◽  
Cell Monolayers ◽  
Intracellular Regulation

Abstract There is growing evidence that various isoforms of 17β-hydroxysteroid dehydrogenase (17-HSD) are regulated at the level of catalysis in intact cells. A number of investigators have proposed that the NAD(P)/NAD(P)H ratio may control the direction of reaction. In a previous study, we obtained evidence that A431 cells, derived from an epidermoid carcinoma of the vulva, are enriched in 17-HSD type 2, a membrane-bound isoform reactive with C18 and C19 17β-hydroxysteroids and 17-ketosteroids. The present investigation was undertaken to confirm the presence of 17-HSD type 2 in A431 cells and to assess intracellular regulation of 17-HSD at the level of catalysis by comparing the activity of homogenates and microsomes with that of cell monolayers. Northern blot analysis confirmed the presence of 17-HSD type 2 mRNA. Exposure of cells to epidermal growth factor resulted in an increase in type 2 mRNA and, for microsomes, increases in maximum velocity (Vmax) with no change in Michaelis constant (Km) for testosterone and androstenedione, resulting in equivalent increases in the Vmax/Km ratio consistent with the presence of a single enzyme. Initial velocity data and inhibition patterns were consistent with a highly ordered reaction sequence in vitro in which testosterone and androstenedione bind only to either an enzyme–NAD or an enzyme–NADH complex respectively. Microsomal dehydrogenase activity with testosterone was 2- to 3-fold higher than reductase activity with androstenedione. In contrast, although cell monolayers rapidly converted testosterone to androstenedione, reductase activity with androstenedione or dehydroepiandrosterone (DHEA) was barely detectable. Lactate but not glucose, pyruvate or isocitrate stimulated the conversion of androstenedione to testosterone by monolayers, suggesting that cytoplasmic NADH may be the cofactor for 17-HSD type 2 reductase activity with androstenedione. However, exposure to lactate did not result in a significant change in the NAD/NADH ratio of cell monolayers. It appears that within A431 cells 17-HSD type 2 is regulated at the level of catalysis to function almost exclusively as a dehydrogenase. These findings give further support to the concept that 17-HSD type 2 functions in vivo principally as a dehydrogenase and that its role as a reductase in testosterone formation by either the Δ4 or Δ5 pathway is limited. Journal of Endocrinology (1997) 153, 453–464

scholarly journals Degradation of Myogenic Transcription Factor MyoD by the Ubiquitin Pathway In Vivo and In Vitro: Regulation by Specific DNA Binding

1998 ◽  
Vol 18 (10) ◽  
pp. 5670-5677 ◽  
Author(s):  
Ossama Abu Hatoum ◽  
Shlomit Gross-Mesilaty ◽  
Kristin Breitschopf ◽  
Aviad Hoffman ◽  
Hedva Gonen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Specific Inhibitor ◽  
Inhibitory Effect ◽  
26S Proteasome ◽  
Intact Cells ◽  
Free System ◽  
Ubiquitin System

ABSTRACT MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

F-actin serves as a template for cytokeratin organization in cell free extracts

2002 ◽  
Vol 115 (7) ◽  
pp. 1373-1382 ◽  
Author(s):  
Kari L. Weber ◽  
William M. Bement
Keyword(s):  
Time Course ◽  
Early Time ◽  
Dynamic Imaging ◽  
Actin Network ◽  
Intact Cells ◽  
Time Points ◽  
Egg Extracts ◽  
Actin Cables

The microtubule, F-actin, and intermediate filament systems are often studied as isolated systems, yet the three display mutual interdependence in living cells. To overcome limitations inherent in analysis of polymer-polymer interactions in intact cells, associations between these systems were assessed in Xenopus egg extracts. In both fixed and unfixed extract preparations, cytokeratin associated with F-actin cables that spontaneously assembled in the extracts. Time-course experiments revealed that at early time points cytokeratin cables were invariably associated with F-actin cables,while at later time points they could be found without associated F-actin. In extract samples where F-actin assembly was prevented, cytokeratin formed unorganized aggregates rather than cables. Dynamic imaging revealed transport of cytokeratin by moving F-actin as well as examples of cytokeratin release from F-actin. Experimental alteration of F-actin network organization by addition of α-actinin resulted in a corresponding change in the organization of the cytokeratin network. Finally, pharmacological disruption of the F-actin network in intact, activated eggs disrupted the normal pattern of cytokeratin assembly. These results provide direct evidence for an association between F-actin and cytokeratin in vitro and in vivo, and indicate that this interaction is necessary for proper cytokeratin assembly after transition into the first mitotic interphase of Xenopus.

Recent findings on oogenesis of Drosophila melanogaster

Development ◽  
1977 ◽  
Vol 38 (1) ◽  
pp. 125-138
Author(s):  
F. Giorgi ◽  
J. Jacob
Keyword(s):  
Drosophila Melanogaster ◽  
Fat Body ◽  
Radioactive Tracer ◽  
Superficial Layer ◽  
Ruthenium Red ◽  
Time Interval ◽  
Intracellular Location ◽  
Short Time

Vitellogenic ovaries from Drosophila melanogaster flies have been exposed, either in in vivo or in vitro conditions, to various extracellular tracers in an attempt to determine the possible route of entry of the yolk precursors. Ruthenium red and lanthanum nitrate have been shown to gain access to the oocyte surface by initially passing through the intercellular spaces of the follicle layer. Both these tracers, however, never attain an intracellular location within any of the cells forming the ovarian chamber. Colloidal Thorotrast when injected into adult females has never been detected within any of the ovarian chambers examined, irrespective of their stage. Vitellogenic oocytes exposed to peroxidase in in vivo conditions exhibit the oolemma and all the structural elements present in the cortical ooplasm well labelled within a very short time after the injection. Moreover, with gradually increasing exposure times to peroxidase, the labelled yolk platelets increase progressively in number. At each time interval after the injection, the label over the yolk platelets remains restricted to the superficial layer and never gets into the associated body. The pattern of tritiated lysine incorporation into vitellogenic oocytes has been studied over a period of 20 h. A few hours after injection of the radioactive tracer, the silver grains located over the ooplasm appear distributed at random. A predominant labelling of the yolk platelets as compared to the rest of the ooplasm, becomes evident only with a 6 h delay since the time of injection. When analysed by electrophoresis and isolectrofocusing, the vitellogenic ovary is seen to exhibit a number of protein bands which are common to those of other tissues as, for instance, haemolymph and fat body. The evidence obtained in the present study is discussed in relation to the hypothesis of an extraovarian origin of the yolk precursors and their sequestration into forming yolk platelets.

Ligand-induced phosphorylation of the colony-stimulating factor 1 receptor can occur through an intermolecular reaction that triggers receptor down modulation

1990 ◽  
Vol 10 (4) ◽  
pp. 1664-1671
Author(s):  
M Ohtsuka ◽  
M F Roussel ◽  
C J Sherr ◽  
J R Downing
Keyword(s):  
Colony Stimulating Factor ◽  
Intact Cells ◽  
Atp Binding Site ◽  
Truncation Mutant ◽  
Intermolecular Mechanism ◽  
Carboxy Terminal ◽  
Lysine Substitution ◽  
Stimulating Factor

Ligand-induced tyrosine phosphorylation of the human colony-stimulating factor 1 receptor (CSF-1R) could involve either an intra- or intermolecular mechanism. We therefore examined the ability of a CSF-1R carboxy-terminal truncation mutant to phosphorylate a kinase-defective receptor, CSF-1R[met 616], that contains a methionine-for-lysine substitution at its ATP-binding site. By using an antipeptide serum that specifically reacts with epitopes deleted from the enzymatically competent truncation mutant, cross-phosphorylation of CSF-1R[met 616] on tyrosine was demonstrated, both in immune-complex kinase reactions and in intact cells stimulated with CSF-1. Both in vitro and in vivo, CSF-1R[met 616] was phosphorylated on tryptic peptides identical to those derived from wild-type CSF-1R, suggesting that receptor phosphorylation on tyrosine can proceed via an intermolecular interaction between receptor monomers. When expressed alone, CSF-1R[met 616] did not undergo ligand-induced down modulation, but its phosphorylation in cells coexpressing the kinase-active truncation mutant accelerated its degradation.

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