scholarly journals Changes in glycosaminoglycan biosynthesis during differentiation in vitro of human monocytes

1983 ◽  
Vol 210 (3) ◽  
pp. 661-667 ◽  
Author(s):  
S O Kolset ◽  
L Kjellén ◽  
R Seljelid ◽  
U Lindahl
Keyword(s):  
Culture Medium ◽  
Negative Charge ◽  
Morphological Changes ◽  
Paper Electrophoresis ◽  
Exchange Chromatography ◽  
Adherent Cells ◽  
The Novel ◽  
Dermatan Sulphate ◽  
Negative Charge Density

Monocytes isolated from human blood were maintained in vitro on plastic culture dishes. After 3-4 days, adherent cells displayed morphological changes previously attributed to differentiation of the cells into histiocytes. 35S-labelled glycosaminoglycans were isolated after incubation of the cells with inorganic [35S]sulphate. Polysaccharide recovered from the culture medium after labelling from day 0 to day 2 or from day 5 to day 7 in vitro was approximately 90% galactosaminoglycan (resistant to deamination by HNO2), irrespective of labelling period. Whereas day-0-2 material was extensively degraded to disaccharide on incubation with the bacterial eliminase chondroitinase AC, a significant portion, about 30%, of the day-5-7 material resisted degradation under the same conditions. The resistant portion was readily depolymerized by treatment with chondroitinase ABC and may be dermatan sulphate. Paper electrophoresis and paper chromatography of the disaccharides obtained by eliminase digestion identified the day-0-2 labelled galactosaminoglycan as chondroitin 4-sulphate. In contrast, the corresponding day-5-7 material yielded approximately 20% disulphated disaccharide, both on digestion with chondroitinase AC and on subsequent enzymic degradation of the chondroitinase AC-resistant fraction. Further treatment of the disulphated disaccharide with chondro-4-sulphatase and chondro-6-sulphatase indicated that both sulphate groups were located on the N-acetylgalactosamine residue. In accordance with these findings, the day-5-7 polysaccharide showed a higher negative charge density than the day-0-2 material on ion-exchange chromatography. It is concluded that the novel properties acquired by the monocyte during prolonged culturing on plastic include the ability to synthesize glycosaminoglycan(s) containing 4,6-disulphated N-acetylgalactosamine units.

scholarly journals Effect of Lignocaine Concentration on Human Fibroblasts Growth in Eyes Undergoing Trabeculectomy: An in vitro Study

2018 ◽  
Vol 3 (3) ◽  
pp. 1-10 ◽  
Author(s):  
Madhuravasal Krishnan Janani ◽  
Venkatakrishnan Jaichandran ◽  
Hajib Narahari Rao Madhavan ◽  
Lingam Vijaya ◽  
Ronnie Jacob George ◽  
...  
Keyword(s):  
Culture Medium ◽  
In Vitro Model ◽  
Morphological Changes ◽  
Human Fibroblasts ◽  
Annexin V ◽  
In Vitro Study ◽  
Tenon’S Capsule ◽  
Tenon's Capsule ◽  
Vitro Model

Purpose: To evaluate the effect of lignocaine on growth and apoptosis indication of primary human Tenon’s capsule fibroblast (HTFs) in an in vitro model. Patients and Methods: Tenon’s capsule tissue obtained from patients undergoing trabeculectomy were grown in cell culture medium. The effect of different concentrations of lignocaine (0.5, 1.0, 1.5, and 2%) on the morphology and growth of the fibroblasts was studied using microscopy, cell viability, and proliferation assay, and apoptosis was detected using the FITC Annexin V Apoptosis Kit. Results: Morphological changes similar to those of apoptotic cells, including cytoplasmic vacuolation, shrinkage, and rounding were visualized in the cells treated with concentrations greater than 1.0% (i.e., 1.5, 2.0%). Though proliferation inhibition was found with all four concentrations (0.5–2.0%), the viability of cells decreased from 1.0% lignocaine. Conclusion: 0.5% lignocaine prevents proliferation of fibroblasts without causing apoptosis in vitro.

scholarly journals Experimental conversion of virulent RH Toxoplasma gondii tachyzoites in vitro

2001 ◽  
Vol 7 (1-2) ◽  
pp. 181-188
Author(s):  
N. A. Hammouda ◽  
I. R. Ibrahim ◽  
E. D. Elkerdany ◽  
A. Y. Negm ◽  
S. R. Allam
Keyword(s):  
Toxoplasma Gondii ◽  
Culture Medium ◽  
Dna Analysis ◽  
Morphological Changes ◽  
Control Group ◽  
Image Analyser

We aimed to induce conversion of RH-stain tachyzoites to bradyzoites by changing the pH of the culture medium. Alkalization of the medium to pH 8 induced morphological changes in the cultured tachyzoites. The majority of the organism increased in size and changed from a regular crescent shape to a rounded or ovoid shape. Cyst-like structures were formed. Using a computerized image analyser, significant differences in the size of the whole organisms and in their nuclei were observed compared to the control group. The converted organisms also showed significant differences from the control group by quantitative DNA analysis, and did not infect mice.

Paracrystalline bundles of large tubules, induced in vitro by Mebendazole in Cysticercus cellulosae

Parasitology ◽  
1981 ◽  
Vol 83 (3) ◽  
pp. 513-518 ◽  
Author(s):  
J. P. Laclette ◽  
Marie Therese Merchant ◽  
Kaethe Willms ◽  
L. Cañedo
Keyword(s):  
Electron Microscopy ◽  
Culture Medium ◽  
Morphological Changes ◽  
Bladder Wall ◽  
Secretory Cells ◽  
External Diameter ◽  
Nematode Larvae ◽  
Transmission Electron ◽  
Cysticercus Cellulosae

SUMMARYThe effect of the anthelmintic Mebendazole on Cysticercus cellulosae maintained in culture medium was studied by transmission electron microscopy. In addition to the well-known morphological changes induced by Mebendazole in other cestode and nematode larvae, it also induced the cytoplasmic appearance of paracrystalline bundles in the secretory cells of the bladder wall. These bundles were formed by groups of large parallel tubules arranged in a hexagonal-like pattern. The tubules, which had an external diameter of about 50 nm and a length that might exceed 5 μm, were surrounded by a matrix and a distance between neighbouring tubules of 80–120 nm centre to centre was estimated. The tubules were stable to colchicine and low temperature. The temporary appearance of bundles is described and some alternative explanations on their origin are advanced.

scholarly journals Confirmation of d-aspartic acid in the novel dipeptide β-aspartylglycine isolated from tissue extract of Aplysia kurodai

1989 ◽  
Vol 263 (2) ◽  
pp. 617-620 ◽  
Author(s):  
M Sato ◽  
T Yamaguchi ◽  
N Kanno ◽  
Y Sato
Keyword(s):  
Aspartic Acid ◽  
Body Wall ◽  
Optical Resolution ◽  
Paper Electrophoresis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Optical Rotatory Dispersion ◽  
The Novel ◽  
Reactive Compound ◽  
High Concentrations

A novel o-phthalaldehyde-reactive compound was found in the h.p.l.c. chromatogram of Aplysia kurodai extract. This compound was isolated by ion-exchange chromatography and preparative high-voltage paper electrophoresis. It was shown by optical-rotatory-dispersion spectrum and optical-resolution h.p.l.c. analysis that this compound consisted of equimolar amounts of D-aspartic acid and glycine. This compound resisted cleavage in the Edman reaction. This peptide was inferred to be beta-D-aspartylglycine, and this was confirmed by synthesis. beta-D-Aspartylglycine was detected in all tissues of Aplysia kurodai, with especially high concentrations in body wall (skin and muscle) and gill.

102. CHARACTERISATION OF THE GTPase DYNAMIN THROUGHOUT MURINE SPERM MATURATION

2010 ◽  
Vol 22 (9) ◽  
pp. 20
Author(s):  
A. T. Reid ◽  
S. D. Roman ◽  
R. Aitken ◽  
B. Nixon
Keyword(s):  
Protein Trafficking ◽  
Molecular Mechanisms ◽  
Protein Translocation ◽  
Morphological Changes ◽  
Sperm Maturation ◽  
The Novel ◽  
Epididymal Maturation ◽  
Distinct Cell ◽  
Molecular Machinery

Throughout sperm maturation distinct remodelling events occur that imbue the cells with both the ability to bind the zona pellucida and undergo the acrosome reaction. Of long standing interest to our laboratory is the elucidation of the molecular mechanisms that underpin the attainment of these key functional attributes. This process begins with a complex range of morphological changes that accompany spermatogenesis, and is continued through post-testicular phases of maturation in both the male (epididymal maturation) and female (capacitation) reproductive tracts. However, among these changes only those occurring during the initial stages of spermatogenesis are intrinsically driven. The fact that the majority of sperm remodelling is extrinsically stimulated, and occurs in the absence of new protein synthesis, highlights the potential importance of processes such as intracellular protein trafficking. This has directed our focus towards the dynamin family of protein traffickers. The GTPase dynamin exists in three isoforms, namely Dnm1, Dnm2 and Dnm3 and is an integral part of the molecular machinery required for vesicle mediated protein translocation. Recent research from our laboratory has demonstrated the presence of these three isoforms in distinct, cell-specific locations during murine spermatogenesis. Immunofluorescence on mouse testis revealed that both Dnm1 and 2 are present within a region corresponding to the developing acrosome in maturing sperm, whilst Dnm3 appears to reside solely within pre-meiotic germ cells. Interestingly, Dnm1 and Dnm2 are both retained within the peri-acrosomal region of the sperm head in mature spermatozoa. Additionally, upon the induction of capacitation in vitro, staining for both Dnm1 and 2 becomes significantly reduced. Collectively these data support the novel hypothesis that dynamin not only participates in sperm remodelling events during spermatogenesis but may also have a previously unappreciated role in capacitation-associated priming of the sperm surface for interaction with the oocyte.

Serum deprivation induces apoptotic cell death in a subset of Balb/c 3T3 fibroblasts

1994 ◽  
Vol 107 (5) ◽  
pp. 1169-1179 ◽  
Author(s):  
G.V. Kulkarni ◽  
C.A. McCulloch
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Time Dependent ◽  
Cell Diameter ◽  
Adherent Cells ◽  
Serum Withdrawal ◽  
3T3 Fibroblasts ◽  
Mean Diameter

Little is known about the regulation of apoptosis in fibroblasts although several model systems including serum deprivation and treatment with staurosporine or topoisomerase inhibitors have been used to induce apoptosis in vitro. To validate a reproducible in vitro model for the study of apoptosis in fibroblasts, we cultured density-inhibited monolayer cultures of Balb/c 3T3 fibroblasts in Dulbecco's modified essential medium plus 15% fetal calf serum and then withdrew serum. Time-lapse video microscopy demonstrated that within minutes of serum withdrawal, cells lost substrate attachment and floated to the top of the liquid growth medium. There was a time-dependent increase in the number of non-adherent cells. Some of these cells regained attachment and spread momentarily, but they eventually rounded up and lost attachment permanently. In contrast to serum-containing cultures in which similar morphological changes were followed by mitosis, in serum-free cultures repeated attempts at mitosis were followed by permanent attachment loss and presumably cell death. To assess whether all the non-adherent cells were in fact dead, the percentages of cells that continued to proliferate upon return to serum-supplemented conditions was computed. After various periods of serum starvation a decreasing proportion (approx. 75% at 30 minutes; < 2% at 24 hours) of the non-adherent cells could be rescued by addition of serum. Transmission electron microscopy of cells 3 hours after serum withdrawal showed that the majority (approximately 60%) of non-adherent cells exhibited marked intranuclear chromatin condensation but maintained integrity of cell and nuclear membranes and cell organelles, morphological changes consistent with those of apoptotic cell death. Scanning electron microscopy of cultures 3 hours following serum withdrawal showed rounded cells with marked surface blebbing. Fluorescence and confocal microscopy revealed increased intensity of nuclear staining with DAPI while actin filaments became indistinct or collapsed around the nucleus. After cycloheximide treatment to inhibit protein synthesis, there was no reduction of apoptosis. Gel electrophoresis of DNA from both control and 3 hour-serum-deprived cells showed intact DNA with no oligonucleosomal length fragmentation. After serum withdrawal, intracellular calcium was reduced by about 32% over 5 minutes as measured by fura2 ratio fluorimetry in single cells. Serum-starved cells showed a time-dependent shrinkage in mean cell diameter compared to trypsinized, adherent control cells (at 0 hours, mean diameter = 18.0 microns--viable; at 4 hours, mean diameter = 15.5 microns--apoptotic). Flow cytometric analysis showed increased propidium iodide staining and reduced fluorescein diacetate uptake over 3 hours, changes that were contemporaneous with the reduction of cell diameter.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of serum on the mitochondrial active area on developmental days 1 to 4 in in vitro-produced bovine embryos

Zygote ◽  
2011 ◽  
Vol 19 (4) ◽  
pp. 297-306 ◽  
Author(s):  
M. Crocco ◽  
R.H. Alberio ◽  
L. Lauria ◽  
M.I. Mariano
Keyword(s):  
Electron Microscopy ◽  
Culture Medium ◽  
Morphological Changes ◽  
Mitochondrial Dynamics ◽  
Developmental Changes ◽  
Bovine Embryos ◽  
Functional Changes ◽  
Transmission Electron ◽  
Subcellular Level

SummaryCertain morphological changes at the subcellular level caused by the current techniques for in vitro embryo production seem to affect mitochondria. Many of these, including dysfunctional changes, have been associated with the presence of serum in the culture medium. Thus, the aim of the present work was to assess the mitochondrial dynamics occurring in embryos during the first 4 days of development, in order to analyze the most appropriate time for adding the serum. We used transmission electron microscopy (TEM) micrographs to calculate the embryo area occupied by the different morphological types of mitochondria, and analyzed them with Image Pro Plus analyzer. The results showed hooded mitochondria as the most representative type in 1- to 4-day-old embryos. Swollen, on-fusion, orthodox and vacuolated types were also present. When analyzed in embryos cultured without serum, the dynamics of the different mitochondrial types appeared to be similar, a fact that may provide evidence that the developmental changes control the mitochondrial dynamics, and that swollen mitochondria may not be completely inactive. In contrast, in culture medium supplemented with serum from estrous cows, we observed an increased area of hooded mitochondria by developmental day 4, a fact that may indicate an increased production of energy compared with previous days. According to these results, the bovine serum added to the culture medium seems not to be responsible for the functional changes in mitochondria.

An endogenous peptide that stimulates lanthanum-resistant calcium uptake in vascular tissue

1987 ◽  
Vol 65 (9) ◽  
pp. 1991-1995 ◽  
Author(s):  
William D. McCumbee ◽  
Peter Johnson ◽  
Peter J. Kasvinsky ◽  
Gary L. Wright
Keyword(s):  
High Performance ◽  
Calcium Uptake ◽  
Vascular Tissue ◽  
Paper Electrophoresis ◽  
Exchange Chromatography ◽  
Calcium Dependent ◽  
Significant Stimulation ◽  
Cation Exchange Column ◽  
Residue Concentration

A recent report has described the preparation of an extract from hemolyzed erythrocytes that has a stimulatory effect on lanthanum-resistant calcium uptake by vascular tissue in vitro and a hypertensive effect when injected into normotensive rats. The compound having a stimulatory effect on calcium uptake was further fractionated by molecular sieve and ion exchange chromatography, precipitation with CaCl2, high voltage paper electrophoresis, and high performance liquid chromatography (HPLC). HPLC yielded only a single fraction containing biological activity. This fraction was ninhydrin positive and acid labile. The amino acid composition was as follows: Asp/Asn (1.41), Ser (1.02), Glu/Gln (1.00), and Gly (2.00). Based on the assumption that the compound contains a single glutamic acid or glutamine residue, concentration–response data indicated that only nanomolar amounts of material were necessary to achieve significant stimulation. There was a marked increase in stimulatory activity of the resolubilized compound following calcium precipitation. The compound became inactive or showed a reduction in activity after being applied to a cation exchange column to remove calcium. Subsequent reprecipitation with CaCl2 and resolubilization restored the lost activity. Thus, we conclude that the compound is a small, acidic, calcium-dependent peptide that is extremely potent in stimulating lanthanum-resistant calcium uptake in vascular tissue.

scholarly journals Effect of benzyl γ-d-xyloside on the biosynthesis of chondroitin sulphate proteoglycan in cultured human monocytes

1986 ◽  
Vol 238 (1) ◽  
pp. 209-216 ◽  
Author(s):  
S O Kolset ◽  
J Ehlorsson ◽  
L Kjellén ◽  
U Lindahl
Keyword(s):  
Culture Medium ◽  
Regulatory Mechanism ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Morphological Changes ◽  
Control Material ◽  
Chondroitin Sulphate Proteoglycan ◽  
Dose Dependent Fashion ◽  
Dose Dependent

Monocytes isolated from human blood differentiate into macrophage-like cells when maintained in vitro for 3-5 days on plastic or glass culture dishes. In the process the cells display characteristic morphological changes, and in addition, a transition in glycosaminoglycan biosynthesis, from the production of chondroitin 4-sulphate to the formation of a polysaccharide containing 20% 4,6-disulphated disaccharide units [Kolset, Kjellén, Seljelid & Lindahl (1983) Biochem. J. 210, 661-667]. Cells were incubated with inorganic [35S]sulphate on day 1 or day 6 in culture, in the presence or absence of benzyl beta-D-xyloside, and labelled polysaccharide was isolated from the culture medium. In the presence of xyloside, the secretion of proteoglycans (90% galactosaminoglycan) was inhibited in a dose-dependent fashion and replaced by release of single polysaccharide chains, the size of which decreased with increasing dose of xyloside. The single polysaccharide chains produced on day 6 in the presence of 0.5 mM-xyloside showed the same proportion of disulphated disaccharide units as did the corresponding control material. Day-1 polysaccharide contained negligible amounts of this component, irrespective of the presence or absence of xyloside. It is concluded that the regulatory mechanism that induces ‘oversulphation’ during the differentiation process operates independently of any association between the polysaccharide chains and the core protein. Moreover, cells maintained in the presence of 0.5 mM-xyloside throughout a 6-day culture period showed the same morphological change, indicative of differentiation into macrophage-like cells, as did untreated control cells. The xyloside did not significantly affect the cytotoxicity of the monocytes, or of the differentiated macrophage-like cells, toward tumour cells.

scholarly journals Rabbit Normal Regenerating Pancreas Extract induces rat BM-MSCs into Pancreatic Islet-like Structure Cells in vitro

2019 ◽  
Vol 3 (1) ◽  
pp. 1-12
Author(s):  
Huifeng Wang ◽  
Yichen Xu ◽  
Liqing Wu ◽  
Yali Kou ◽  
Zhuo Wu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Culture Medium ◽  
The Novel ◽  
Insulin Producing Cells ◽  
Specific Morphology ◽  
Islet Tissue ◽  
New Strategy ◽  
Marrow Mesenchymal Stem Cells ◽  
Three Stages

Bone marrow mesenchymal stem cells (BM-MSCs) could differentiate into Insulin Producing Cells(IPCs) with notable advantages. The present study tried to develop a method may be able to use a normal regenerating pancreas extract(N-RPE) medium to induce BM-MSCs into islet phenotype, in tests to assess how efficient method and simple duplicate the novel condition medium protocol is. Isolate and purify MSCs from rat bone marrow. BM-MSCs were differentiated into Adipogenic, Osteogenic and Myocardium and the lineages were assessed its multi-lineage potential. Islet differentiation medium, blending rabbit conditioned medium N-RPE, was administered to rat BM-MSCs. After 15 days, differentiation was evaluated by lineage-specific morphology and three stages could be observed: induced cells, islet like cells(ILCs) and islet like structures(ILSs). The morphology, SEM, DTZ staining, Mallory staining, HE staining and glucose stimulation demonstrated that the N-RPE could stimulate suitable development microenvironment to supply the islet differentiate from BM-MSCs. In addition, islet-related genes (ins/glu) expression and proteins(insulin/glucagon) expression suggested that culture medium rabbit N-RPE enhanced the rat BM-MSCs transdifferentiation efficiency. N-RPE derived from normal pancreas tissue could promote pancreas development of microenvironment and significantly enhance the transdifferentiation of BM-MSCs into ILSs. Results from this work will contribute to optimize the conditions of BM-MSCs and supply a new strategy for the development of islet tissue engineering.

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