Ultrastructural evaluation of the effects induced by a macrolide antibiotic (LY281389) on L6 muscle cells in vitro

1991 ◽  
Vol 49 ◽  
pp. 80-81
Author(s):  
J. W. Horn ◽  
S. L. White ◽  
D. A. Laska ◽  
M. K. Buening ◽  
M. N. Novilla ◽  
...  
Keyword(s):  
Macrolide Antibiotic ◽  
Uniform Density ◽  
Solution Ph ◽  
L6 Cells ◽  
Vacuolar Change ◽  
Interference Range ◽  
Cacodylate Buffer ◽  
Ultrathin Sections ◽  
L6 Muscle Cells

Toxic doses of LY281389 (9-N(n-propyl)erythromycylamine), a cationic amphophilic macrolide antibiotic, induce a generalized cytoplasmic vacuolar change in tissues of rats and dogs. The present in vitro study using L6 cells (a line derived from neoplastic skeletal muscle) was done to evaluate sequentially early cytologic changes.The L6 cells seeded on tissue culture plates were allowed to grow to a uniform density. The culture medium was then replaced with control medium or medium containing 0.25 mg/ml LY281389 for exposures of 0.5, 2, 6, 12, 24, or 48 hours. The L6 cells were then fixed for 1-hour in modified Karnovsky's solution (pH 7.2), rinsed with 0.1 M sodium cacodylate buffer (pH 7.2) and postfixed for an additional hour in 2% osmium tetroxide (pH 7.2). Following a second buffer rinse, the L6 cells were dehydrated in graded ethanol solutions. The cells on each plate were embedded with epoxy resin (EPON 812), selected areas were scribed out and re-embedded for orientation. Ultrathin sections (silver to gold color interference range) were mounted on copper 200 mesh grids, counterstained, and examined using a Philips EM410LS electron microscope at 60kV.

Ultrastructural autoradiography of L6 skeletal-muscle cells exposed to a tritiated macrolide antibiotic (LY237216) in vitro

1992 ◽  
Vol 50 (1) ◽  
pp. 612-613
Author(s):  
S.L. White ◽  
C.B. Jensen ◽  
D.D. Giera ◽  
D.A. Laska ◽  
M.N. Novilla ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Quantitative Analysis ◽  
Macrolide Antibiotic ◽  
Circle Method ◽  
Apical Surface ◽  
Polycarbonate Membrane ◽  
L6 Cells ◽  
Low Viscosity ◽  
Erythromycin A

In vitro exposure to LY237216 (9-Deoxo-11-deoxy-9,11-{imino[2-(2-methoxyethoxy)ethylidene]-oxy}-(9S)-erythromycin), a macrolide antibiotic, was found to induce cytoplasmic vacuolation in L6 skeletal muscle myoblast cultures (White, S.L., unpubl). The present study was done to determine, by autoradiographic quantitative analysis, the subcellular distribution of 3H-LY237216 in L6 cells.L6 cells (ATCC, CRL 1458) were cultured to confluency on polycarbonate membrane filters (Millipore Corp., Bedford, MA) in M-199 medium (GIBCO® Labs) with 10% fetal bovine serum. The cells were exposed from the apical surface for 1-hour to unlabelled-compound (0 μCi/ml) or 50 (μCi/ml of 3H-LY237216 at a compound concentration of 0.25 mg/ml. Following a rapid rinse in compound-free growth medium, the cells were slam-frozen against a liquid nitrogen cooled, polished copper block in a CF-100 cryofixation unit (LifeCell Corp., The Woodlands, TX). Specimens were dried in the MDD-C Molecular Distillation Drier (LifeCell Corp.), vapor osmicated and embedded in Spurrs low viscosity resin. Ultrathin sections collected on formvar coated stainless steel grids were counter-stained, then individually mounted on corks. A monolayer of Ilford L4 nuclear emulsion (Polysciences, Inc., Warrington, PA) was placed on the sections, utilizing a modified “loop method”. The emulsions were exposed for 7-weeks in a light-tight box at 4°C. Autoradiographs were developed in Microdol-X developer and examined on a Philips EM410LS transmission electron microscope. Quantitative analysis of compound localization employed the point and circle approach of Williams; incorporating the probability circle method of Salpeter and McHenry.

Fine structure of human sperm entry into zona-free hamster oocyte

1978 ◽  
Vol 36 (2) ◽  
pp. 540-541
Author(s):  
C. Barros ◽  
J. González ◽  
E. Herrera ◽  
E. Bustos-Obregón
Keyword(s):  
Osmium Tetroxide ◽  
Human Sperm ◽  
Ultrastructural Level ◽  
Uranyl Acetate ◽  
Human Spermatozoa ◽  
Low Viscosity ◽  
Cacodylate Buffer ◽  
Ultrathin Sections ◽  
Hamster Oocyte

Zona-free mammalian oocytes have been observed to fuse, under in vitro conditions, With non-homologous spermatozoa. Taking advantage of this heterologous gamete fusion, we designed a bioassay to test -by means of zona-free hamster oocytes- the fertile ability of human spermatozoa, from semen samples of patients attending an Infertility Clinic. To further validate our bioassay, which Was reported elsewhere, we studied the behavior of gamete membranes during fusion at the ultrastructural level.Zona-free hamster oocytes were mixed in vitro with human spermatozoa. At different times after the start of incubation, oocytes were fixed in 1% glutaraldehyde in 0. 25M cacodylate buffer pH 7. A and post-fixed in 196 osmium tetroxide. After dehydration in acetone, they were embedded in a low viscosity epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Phillips 300 electron microscope.

Opening images of synaptic vesicles and calcium localization by means of microwave fixation method

1990 ◽  
Vol 48 (2) ◽  
pp. 182-183
Author(s):  
Vinci Mizuhira ◽  
Hiroshi Hasegawa
Keyword(s):  
Synaptic Vesicles ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Sampling Procedure ◽  
Fixation Method ◽  
Potassium Oxalate ◽  
X Ray ◽  
Cacodylate Buffer ◽  
Ultrathin Sections ◽  
Microwave Fixation

Microwave irradiation (MWI) was applied to 0.3 to 1 cm3 blocks of rat central nervous system at 2.45 GHz/500W for about 20 sec in a fixative, at room temperature. Fixative composed of 2% paraformaldehyde, 0.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4, also contained 2 mM of CaCl2 , 1 mM of MgCl2, and 0.1% of tannic acid for conventional observation; and fuether 30-90 mM of potassium oxalate containing fixative was applied for the detection of calcium ion localization in cells. Tissue blocks were left in the same fixative for 30 to 180 min after MWI at room temperature, then proceeded to the sampling procedure, after postfixed with osmium tetroxide, embedded in Epon. Ultrathin sections were double stained with an useal manner. Oxalate treated sections were devided in two, stained and unstained one. The later oxalate treated unstained sections were analyzed with electron probe X-ray microanalyzer, the EDAX-PU-9800, at 40 KV accelerating voltage for 100 to 200 sec with point or selected area analyzing methods.

Structural integrity after cold storage of human epidermal model in hypothermosol: A correlative ultrastructural and fluorescent assay

1994 ◽  
Vol 52 ◽  
pp. 270-271
Author(s):  
Henry H. Eichelberger ◽  
John G. Baust ◽  
Robert G. Van Buskirk
Keyword(s):  
Cold Storage ◽  
Structural Integrity ◽  
Culture Media ◽  
Cell Structure ◽  
Natural State ◽  
Sodium Cacodylate ◽  
Ruthenium Tetroxide ◽  
Cacodylate Buffer ◽  
Before And After

For research in cell differentiation and in vitro toxicology it is essential to provide a natural state of cell structure as a benchmark for interpreting results. Hypothermosol (Cryomedical Sciences, Rockville, MD) has proven useful in insuring the viability of synthetic human epidermis during cold-storage and in maintaining the epidermis’ ability to continue to differentiate following warming.Human epidermal equivalent, EpiDerm (MatTek Corporation, Ashland, MA) consisting of fully differentiated stratified human epidermal cells were grown on a microporous membrane. EpiDerm samples were fixed before and after cold-storage (4°C) for 5 days in Hypothermosol or skin culture media (MatTek Corporation) and allowed to recover for 7 days at 37°C. EpiDerm samples were fixed 1 hour in 2.5% glutaraldehyde in sodium cacodylate buffer (pH 7.2). A secondary fixation with 0.2% ruthenium tetroxide (Polysciences, Inc., Warrington, PA) in sodium cacodylate was carried out for 3 hours at 4°C. Other samples were similarly fixed, but with 1% Osmium tetroxide in place of ruthenium tetroxide. Samples were dehydrated through a graded acetone series, infiltrated with Spurrs resin (Polysciences Inc.) and polymerized at 70°C.

scholarly journals Unusual derivatives from Hypericum scabrum

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sara Soroury ◽  
Mostafa Alilou ◽  
Thomas Gelbrich ◽  
Marzieh Tabefam ◽  
Ombeline Danton ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
2D Nmr ◽  
Moderate Activity ◽  
Trypanosoma Brucei Rhodesiense ◽  
L6 Cells ◽  
Aerial Parts ◽  
Hypericum Scabrum ◽  
New Compounds ◽  
Absolute Structure

AbstractThree new compounds (1–3) with unusual skeletons were isolated from the n-hexane extract of the air-dried aerial parts of Hypericum scabrum. Compound 1 represents the first example of an esterified polycyclic polyprenylated acylphloroglucinol that features a unique tricyclo-[4.3.1.11,4]-undecane skeleton. Compound 2 is a fairly simple MPAP, but with an unexpected cycloheptane ring decorated with prenyl substituents, and compound 3 has an unusual 5,5-spiroketal lactone core. Their structures were determined by extensive spectroscopic and spectrometric techniques (1D and 2D NMR, HRESI-TOFMS). Absolute configurations were established by ECD calculations, and the absolute structure of 2 was confirmed by a single crystal determination. Plausible biogenetic pathways of compounds 1–3 were also proposed. The in vitro antiprotozoal activity of the compounds against Trypanosoma brucei rhodesiense and Plasmodium falciparum and cytotoxicity against rat myoblast (L6) cells were determined. Compound 1 showed a moderate activity against T. brucei and P. falciparum, with IC50 values of 3.07 and 2.25 μM, respectively.

Decreased Platelet Vitamin C in Diabetes Mellitus; Possible Role in Peraggregatoon

1979 ◽  
Author(s):  
K.E. Sarji ◽  
J. Gonzalez ◽  
H. Hempling ◽  
J.A. Colwell
Keyword(s):  
Ascorbic Acid ◽  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Vitamin C ◽  
Platelet Rich Plasma ◽  
Solution Ph ◽  
Dose Dependent Fashion ◽  
Insulin Dependent Diabetic ◽  
Induced Aggregation

To determine whether Vitamin C might relate to the increased platelet sensitivity in the diabetic, we have measured levels of platelet Vitamin C and studied the effects of Vitamin C on platelet aggregation. Ascorbic acid levels in washed platelets from diabetics were significantly lower than from normals (4s.2±3 μg/1010 platelets vs. 2s.s±2 μg/1010 platelets, p<.001). The effects of ascorbic acid on platelet aggregation in vitro were studied by adding ascorbic acid in buffered solution (pH 7.35) prior to-aggregating agents. Ascorbic acid in platelet-rich plasma consistently inhibited platelet aggregation with threshold concentrations of ADP, epinephrine, and collagen. With washed platelets, ascorbic acid inhibited arachidonic, acid-induced aggregation. When platelets were incubated at 37°C for 10 minutes with varying concentrations of ascorbic acid, rewashed, and aggregation with arachidonic acid tested, aggregation was inhibited in a linear dose-dependent fashion. Oral ingestion of ascorbic acid (2 gm/day) for seven days by normal non-smoking males produced a marked inhibition of aggregation. In a similar study, platelets from an insulin-dependent diabetic showed no change in aggregation. These results suggest that platelet levels of ascorbic acid may relate to the hyperaggregat ion of platelets from diabetics.

pH-induced hysteretic properties of phosphofructokinase purified from rat myocardium

1986 ◽  
Vol 250 (3) ◽  
pp. R505-R511 ◽  
Author(s):  
S. C. Hand ◽  
J. F. Carpenter
Keyword(s):  
Catalytic Activity ◽  
Metabolic Regulation ◽  
Inverse Correlation ◽  
Rat Myocardium ◽  
Solution Ph ◽  
Reversible Inactivation ◽  
Ph Range ◽  
Hysteretic Loss ◽  
Fructose 1,6 Bisphosphate

Phosphofructokinase (PFK) purified from the rat myocardium is reversibly inactivated under a pH regime approximating that reported for ischemic hearts. At pH 6.5 and 37 degrees C, the enzyme displays a hysteretic loss of activity during 60-min incubations, declining to 48% of control (pH 7.1, 37 degrees C) values. Citric acid increases the degree of inactivation (28% of control), whereas fructose 1,6-bisphosphate reduces the decline in activity. Simultaneous measurements of 90 decreases light scattering and catalytic activity suggest the inactivation is temporally linked to dissociation of active tetrameric enzyme into an inactive form of lower molecular weight. Fluorescence enhancement of the extrinsic probe sodium mansate, which binds preferentially to dimeric PFK, indicates that the equilibrium dimer concentration (cp1 infinity) increases as pH is lowered. This increase in cp1 infinity exhibits a strong inverse correlation (r = 0.984) with catalytic activity across the pH range of 8.0 to 6.5. Returning solution pH to 7.0 or above promotes a time-dependent reactivation and repolymerization of PFK. The rate of reactivation is increased at higher enzyme concentrations and in the presence of trimethylamine-N-oxide, a nitrogenous osmolyte noted for its ability to promote protein aggregation reactions. Thus these results demonstrate the capacity of rat heart PFK to undergo reversible inactivation and dissociation in vitro and represent the first phase of a two-part study testing the hypothesis that these pH-induced hysteretic processes are operative in the ischemic myocardium. The data are evaluated in terms of the potential roles of hysteretic enzymes in metabolic regulation.

The Study of Albumin Release from Silica/Albumin as a Potential Drug Delivery Carrier

2014 ◽  
Vol 69 (5) ◽  
Author(s):  
Shafiyah Pondi ◽  
Jon Efendi ◽  
Ho Chin Siong ◽  
Lai Sin Yuan ◽  
Sheela Chandren ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Fumed Silica ◽  
In Vitro Release ◽  
Phosphate Buffer Solution ◽  
Buffer Solution ◽  
Solution Ph ◽  
Drug Delivery Carrier ◽  
High Interaction ◽  
Uv Visible

The drug-delivery field has been an attractive as well as challenging area for research. With the emerging of new formulated drugs and pharmaceutical compounds, development of good drug-delivery system (DDS) is crucially required. This study aims to utilize albumin as the drug template in silica/albumin/drug (S/A/D) system. Prior to designing this system, the interaction between silica and albumin was investigated. It is hypothesized that high interaction between silica and albumin may result in slower drug release over time, which is preferred for a good DDS. Silica and albumin (S/A) materials were prepared by using fumed silica and tetraethyl orthosilicate (TEOS) as the silica precursors. Three different S/A samples were prepared; fumed silica with albumin (FS/A), fumed silica with pre-treated albumin by sodium borohydrate (FS/A-N), and silica sol (TEOS) with albumin (SS/A). In-vitro release of albumin in phosphate buffer solution (pH 7) was carried out to examine the interaction between albumin and silica. The concentration of albumin was detected at 280 nm by UV-visible spectrophotometer. All samples were characterized by diffuse reflectance-UV-visible spectrophotometer (DR-UV), Fourier transform infrared spectrophotometer (FTIR) dan thermogravimetric-differential thermal analysis (TG-DTA). DR-UV results show that SS/A exhibited the lowest absorption intensity at 280 nm, which indicates better interaction between silica and albumin. This result was supported by the presence of Si-O stretching band of silanol at 952 cm-1 from the FTIR spectrum. Release study of albumin demonstrated that the release of albumin from SS/A was slowest compared to those of FS/A and FS/A-N. 

scholarly journals Sodium cacodylate as antimitotic agent

2014 ◽  
Vol 57 (3) ◽  
pp. 329-340
Author(s):  
Jadwiga A. Tarkowska
Keyword(s):  
Allium Cepa ◽  
Mitotic Spindle ◽  
Sodium Cacodylate ◽  
Antimitotic Agent ◽  
Ultrastructural Studies ◽  
Meristematic Cells ◽  
Cacodylate Buffer ◽  
Root Meristematic Cells ◽  
Dividing Cells

The effect of pure sodium cacodylate on dividing cells was studied. The root meristematic cells of <em>Allium cepa</em> L. (the roots were squashed in acetoorcein) and endosperm cells of <em>Haemanthus katherinae</em> Bak. (<em>in vitro</em> observations) were used. Serious disturbances in karyokinesis and cytokinesis were found that led most often to the formation of polyploid or multinucleate (<em>A. cepa</em>) cells. These results point to damage of the mitotic spindle and phragmoplast. Careful use of cacodylate buffer in ultrastructural studies of microtubules is advised.

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