scholarly journals Cellular effects of deoxynojirimycin analogues: uptake, retention and inhibition of glycosphingolipid biosynthesis

2004 ◽  
Vol 381 (3) ◽  
pp. 861-866 ◽  
Author(s):  
Howard R. MELLOR ◽  
David C. A. NEVILLE ◽  
David J. HARVEY ◽  
Frances M. PLATT ◽  
Raymond A. DWEK ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Culture Medium ◽  
Cultured Cells ◽  
Complete Inhibition ◽  
Side Chain ◽  
Short Term ◽  
Cellular Effects ◽  
Novel Technique ◽  
Ceramide Glycanase ◽  
Glycosphingolipid Biosynthesis

Deoxynojirimycin (DNJ) analogues are inhibitors of ceramide glucosyltransferase (CGT), which catalyses the first step in the glucosphingolipid (GSL) biosynthetic pathway. We have synthesized a series of DNJ analogues to study the contribution of N-alk(en)yl side chains (C4, C9 or C18) to the behaviour of these analogues in cultured HL60 cells. When cells were treated for 16 h at non-cytotoxic concentrations of inhibitor, a 40–50% decrease in GSL levels was measured by HPLC analysis of GSL-derived oligosaccharides following ceramide glycanase digestion of GSL and 2-aminobenzamide labelling of the released oligosaccharides. Using a novel technique for short-term [14C]galactose labelling of cellular GSL, we used compound inhibition of GSL biosynthesis as a marker for compound uptake into cells. Surprisingly, the uptake of all three of the DNJ analogues was extremely rapid and was not dependent upon the length of the N-alk(en)yl moiety. Compound uptake occurred in less than 1 min, as shown by the complete inhibition of GSL labelling in cells treated with all the DNJ analogues. Greatly increased cellular retention of N-cis-13-octadecenyl-DNJ was observed relative to the shorter-chain compounds, N-butyl-DNJ and N-nonyl-DNJ, as indicated by complete inhibition of CGT 24 h after removal of inhibitor from the culture medium. The present study further characterizes the properties of N-alk(en)ylated DNJs, and demonstrates that increasing the length of the side chain is a simple way of improving imino sugar retention and therefore inhibitory efficacy for CGT in cultured cells.

Porcine granulosa cells in suspension culture. II. Luteinization and hCG responsiveness

1981 ◽  
Vol 240 (6) ◽  
pp. E622-E629 ◽  
Author(s):  
A. R. LaBarbera ◽  
R. J. Ryan
Keyword(s):  
Adenylate Cyclase ◽  
Granulosa Cells ◽  
Culture Medium ◽  
Cultured Cells ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Short Term ◽  
Progesterone Secretion ◽  
Porcine Granulosa Cells ◽  
Ovine Prolactin

Granulosa cells from small follicles were cultured as suspensions in spinner flasks for 10 days in the absence or presence of follicle-stimulating hormone (FSH). With or without FSH, the cultured cells ultrastructurally resembled luteinized cells to different degrees. FSH increased progesterone accumulation in the culture medium. Ovine prolactin potentiated the effect of FSH in terms of the quantity of progesterone produced and the duration of accumulation. FSH increased acute human chorionic gonadotropin (hCG)-responsive progesterone secretion in short-term incubations of cultured granulosa cells. Responsiveness of FSH-cultured cells was maximal at day 4; that of control cultured cells was maximal at day 6. Adenylate cyclase activity of homogenates of cells cultured for 4, 6, or 8 days was measured. FSH induced in cultured cells an hCG sensitivity of the adenylate cyclase enzyme. These results indicate that FSH induced hCG-responsive progesterone secretion and hCG-responsive adenylate cyclase activity that correlate with ultrastructural signs of luteinization and with the previously reported FSH induction of hCG receptors.

IN VITRO METHODS FOR THE STUDY OF THE ADENOHYPOPHYSIAL FUNCTIONS TO SECRETE GONADOTROPHIN

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S27-S40 ◽  
Author(s):  
T. Kobayashi ◽  
T. Kigawa ◽  
M. Mizuno ◽  
T. Watanabe
Keyword(s):  
Cell Culture ◽  
Organ Culture ◽  
Cultured Cells ◽  
Cell Sheet ◽  
Short Term ◽  
Hypothalamic Extract ◽  
Numerous Cell ◽  
Single Cell Culture ◽  
Almost All

ABSTRACT There are several in vitro methods to analyse the function of the adenohypophysis or the mechanisms of its regulation. The present paper deals with single cell culture, organ culture and short term incubation techniques by which the morphology and gonadotrophin-secreting function of the adenohypophysis were studied. In trypsin-dispersed cell culture, the adenohypophysial cells showed extensive propagation to form numerous cell colonies and finally develop into a confluent monolayer cell sheet covering completely the surface of culture vessels. Almost all of the cultured cells, however, became chromophobic, at least at the end of the first week of cultivation, when gonadotrophin was detectable neither in the culture medium nor in the cells themselves. After the addition of the hypothalamic extract, gonadotrophin became detectable again, and basophilic or PAS-positive granules also reappeared within the cells, suggesting that the gonadotrophs were stimulated by the extract to produce gonadotrophin. In organ culture and short term incubation, the incorporation of [3H] leucine into the adenohypophysial cells in relation to the addition of hypothalamic extract was examined. It was obvious that the ability to incorporate [3H] leucine into the gonadotrophs in vitro was highly dependent upon the presence of the hypothalamic extract.

scholarly journals A Novel Technique for Short-Term Load Forecasting using Sequential Models and Feature Engineering

IEEE Access ◽  
2021 ◽  
pp. 1-1
Author(s):  
Abdul Wahab ◽  
Muhammad Anas Tahir ◽  
Naveed Iqbal ◽  
Adnan Ul-Hasan ◽  
Faisal Shafiat ◽  
...  
Keyword(s):  
Load Forecasting ◽  
Feature Engineering ◽  
Short Term ◽  
Novel Technique ◽  
Short Term Load Forecasting ◽  
Sequential Models

Proliferation and agglutinability of primary and transformed human epithelial cells in culture

1976 ◽  
Vol 21 (3) ◽  
pp. 553-561
Author(s):  
M.A. Ricard ◽  
R.J. Hay
Keyword(s):  
Culture Medium ◽  
Monolayer Culture ◽  
Primary Cultures ◽  
Short Term ◽  
Chloromethyl Ketone ◽  
Normal Populations ◽  
Short Term Exposure ◽  
Cell Strains ◽  
Human Placentae

Primary epithelial populations (HAM) were obtained by dissociation of the amniotic membrane stripped from human placentae. Agglutinability of cells from such normal populations and of cells from the transformed epithelial line WISH was then compared using concavanalin A as mediator. Extensive similar studies have previously been reported with cell strains isolated from other species. Freshly dissociated HAM cells from primary cultures agglutinated much less readily than did cells from WISH populations. Furthermore, the former exhibited a drastic decline in agglutinability as a function of time in suspension culture after trypsinization. Short-term exposure (60 h) of HAM cells in monolayer culture to 5-bromodeoxyuridine (BrdU) elicited heightened agglutinability detectable through 22 days in vitro. Addition of the protease inhibitors n-tosyl-L-lysyl-chloromethyl ketone (TLCK) or p-tosyl-L-arginine-methyl ester (TAME) to the culture medium inhibited proliferation of the WISH line by 40–50% while effecting only a 10–15% inhibition of HAM cells. These results also confirm data with other cell species indicating that high proteolytic activity at the surface of transformed cells may be related to the rapid proliferation rate.

scholarly journals Glass beads load macromolecules into living cells

1987 ◽  
Vol 88 (5) ◽  
pp. 669-678
Author(s):  
P.L. McNeil ◽  
E. Warder
Keyword(s):  
Culture Medium ◽  
Small Volume ◽  
Cultured Cells ◽  
Cell Monolayer ◽  
Cell Loss ◽  
Living Cells ◽  
Glass Beads ◽  
Cell Type ◽  
Large Numbers ◽  
Micron Diameter

We describe and characterize an exceptionally rapid and simple new technique for loading large numbers of cultured cells with large macromolecules. The culture medium of the cell monolayer is replaced by a small volume of the macromolecule to be loaded. Glass beads (75–500 micron diameter) are then sprinkled onto the cells, the cells are washed free of beads and exogenous macromolecules, and ‘bead-loading’ is completed. The conditions for bead-loading can readily be modified to accommodate cell type and loading objectives: for example, the amount of loading per cell increases if bead size is increased or if beads are agitated after sprinkling onto the monolayer, but at the expense of increased cell loss. As many as 97% of a population of bovine aortic endothelial (BAE) cells were loaded with a 10,000 Mr dextran; and 79% with a 150,000 Mr dextran using bead-loading. Various cell lines have been loaded using glass beads. Moreover, bead-loading has the advantage of producing loaded cells that remain adherent and well-spread, thus minimizing recovery time and permitting immediate microscopic examination.

Responses of growth cones to changes in osmolality of the surrounding medium

1991 ◽  
Vol 98 (4) ◽  
pp. 507-515
Author(s):  
D. Bray ◽  
N.P. Money ◽  
F.M. Harold ◽  
J.R. Bamburg
Keyword(s):  
Hydrostatic Pressure ◽  
Growth Cone ◽  
Culture Medium ◽  
Vapor Pressure Deficit ◽  
Surrounding Medium ◽  
Culture Fluid ◽  
Short Term ◽  
Area Reduction ◽  
Osmotic Response ◽  
Upper Limits

The possible involvement of osmotically generated hydrostatic pressure in driving actin-rich extensions of the cell surface was examined using cultures of chick neurons. Estimation of the excess internal osmotic pressure of chick neural tissue by vapor pressure deficit osmometry, and of the excess internal hydrostatic pressure in cultured chick neurons using a calibrated pressure pipette, gave upper limits of 10 mosM and 0.1 atmosphere (1 atmosphere = 101325 Pa), respectively. Increases in the osmolality of the medium surrounding cultured neurons by addition of sucrose, mannitol or polyethylene glycol by amounts that should eliminate any internal pressure not only failed to arrest the growth of filopodia but caused them to increase in length up to twofold in 3–5 min. Lamellipodia remained unchanged following hyperosmotic shifts of 20 mosM, but higher levels caused a small decrease in area. Reduction of osmolality by the addition of water to the culture fluid down to 50% of its normal value failed to show any detectable change in either filopodial length or lamellipodia area. These observations argue against an osmotic mechanism for growth cone extension and show that the growth of filopodia, in particular, is unlikely to be driven by osmotically generated hydrostatic pressure. In contrast to the short-term effects on growth cone morphology, the slower elongation of the neuritic cylinder showed a consistent osmotic response. Growth rates were reduced following addition of osmolytes and increased in rate (as much as sixfold) following addition of water to the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Effects of mrpigG on Development and Secondary Metabolism of Monascus ruber M7

2020 ◽  
Vol 6 (3) ◽  
pp. 156
Author(s):  
Li Li ◽  
Fusheng Chen
Keyword(s):  
Biosynthetic Pathway ◽  
Gene Deletion ◽  
The Other ◽  
Side Chain ◽  
Asian Countries ◽  
Monascus Ruber ◽  
Monascus Pigments ◽  
The World ◽  
Carbon Side Chain ◽  
Yellow Pigments

Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries and are now used throughout the world via Asian catering. The MP biosynthetic pathway has been well-illustrated, but the functions of a few genes, including mrpigG, in the MP gene cluster are still unclear. In the current study, in order to investigate the function of mrpigG in M. ruber M7, gene deletion (ΔmrpigG), complementation (ΔmrpigG::mrpigG) and overexpression (M7::PtrpC-mrpigG) mutants were successfully obtained. The morphologies and biomasses, as well as the MP and citrinin production, of these mutants were analyzed. The results revealed that the disruption, complementation and overexpression of mrpigG showed no apparent defects in morphology, biomass or citrinin production (except MP production) in ΔmrpigG compared with M. ruber M7. Although the MP profiles of ΔmrpigG and M. ruber M7 were almost the same—with both having four yellow pigments, two orange pigments (OPs) and two red pigments (RPs)—their yields were decreased in ΔmrpigG to a certain extent. Particularly, the content of rubropunctatin (an OP) and its derivative rubropunctamine (an RP) in ΔmrpigG, both of which have a five-carbon side chain, accounted for 57.7%, and 22.3% of those in M. ruber M7. On the other hand, monascorubrin (an OP) and its derivative monascorubramine (an RP), both of which have a seven-carbon side chain, were increased by 1.15 and 2.55 times, respectively, in ΔmrpigG compared with M. ruber M7. These results suggest that the MrPigG protein may preferentially catalyze the biosynthesis of MPs with a five-carbon side chain.

Immunocytochemistrical Studies on Cytoskeletal Change in Cultured Cardiomyocyte of Tilapia

2000 ◽  
Vol 6 (S2) ◽  
pp. 888-889
Author(s):  
L. C. Tung
Keyword(s):  
Culture Medium ◽  
Cultured Cells ◽  
Calf Serum ◽  
Amino Acid Mixture ◽  
Penicillin G ◽  
Acid Mixture ◽  
Minimal Essential Medium ◽  
Cultured Fish ◽  
Cytoskeletal Change

In the present study, the myofibril regeneration in the long-term cultured fish cardiomyocytes was studied with immunocytochemistry.Adult Tilapia heart was dissociated into a single-cell suspension with collagenase and protease-minced tissue method. The culture medium was Eagle's minimal essential medium (MEM) with Earle's salts, supplemented with 10% fetal calf serum, 1 x nonessential amino acid mixture, 100 IU/ml penicillin G, and 100 μg/ml streptomycin. The cultured cells were grown in a humidified CO2 incubator at 28°Cand in a medium without glutamine for eliminating fibroblast contamination. In the initial 24 h culture, the elongated-shape cells gradually shortened from their both ends and rounded up. Over 5 to 6 days postcultivation, the cells attached to the bottom of the culture flask and began to protrude pseudopodia. The cells could not be subcultured and also proliferated indefinitely. The life span of cells in culture was 30 to 60 days.

An ultrasound-guided procedure to administer a label of DNA synthesis into fetal sheep

1999 ◽  
Vol 11 (5) ◽  
pp. 303 ◽  
Author(s):  
Paul L. Greenwood ◽  
Ramona M. Slepetis ◽  
Alan W. Bell ◽  
John W. Hermanson
Keyword(s):  
Bolus Dose ◽  
S Phase ◽  
Fetal Weight ◽  
Ultrasound Guided ◽  
Short Term ◽  
Fetal Sheep ◽  
Novel Technique ◽  
Cellular Events ◽  
High Degree

A novel technique was developed to deliver a bolus dose of a DNA label into the peritoneal cavity of fetal sheep at 85–130 days gestation. Use of markers to identify the site of injection in fetuses from litters up to quadruplets, and immunohistochemistry to detect the DNA label, 5-bromo-2¢-deoxyuridine (BrdU), confirmed the procedure was successful in 85% of cases. Duration of the procedure was (mean SD) 44 16 min, and recovery from anaesthesia was rapid and uneventful in all cases. Fetal weight was estimated with a high degree of accuracy (residual standard deviation (RSD) = 297 g and r 2 = 0.93, P<0.001) and the dose of label administered (110 33 mg BrdU/kg fetal weight) was adequate in all cases. BrdU detected in fetal nuclei following injection into amniotic fluid highlights the need for positive identification of the injection site in timed, short-term studies, and suggests potential to further develop the technique to investigate cellular events in fetal sheep younger than 85 days of gestation. The results demonstrate that the procedure can be used to determine in vivo whether or not nuclei have entered the S-phase of the cell cycle.

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