scholarly journals Osteocalcin Production in Primary Osteoblast Cultures Derived from Normal and Hyp Mice

Endocrinology ◽  
1998 ◽  
Vol 139 (1) ◽  
pp. 35-43 ◽  
Author(s):  
Thomas O. Carpenter ◽  
Kathleen C. Moltz ◽  
Bruce Ellis ◽  
Monica Andreoli ◽  
Thomas L. McCarthy ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Specific Effect ◽  
Continuous Exposure ◽  
Primary Osteoblast ◽  
Characteristic Features ◽  
Species Specific ◽  
Osteocalcin Production

Abstract Rickets and osteomalacia are characteristic features of the Hyp mouse model of human X-linked hypophosphatemia. Hyp mice demonstrate elevated circulating osteocalcin levels, as well as altered regulation of osteocalcin by 1,25(OH)2D3. Whether this osteocalcin abnormality is intrinsic to the osteoblast, or mediated by the in vivo milieu, has not been established. We therefore characterized osteocalcin production and its regulation by 1,25(OH)2D3 in primary cultures of murine osteoblasts and examined osteocalcin and its messenger RNA in response to 1,25(OH)2D3 in cultures of Hyp mouse-derived osteoblasts. Cell viability and osteocalcin production are optimal when murine cells are harvested within 36 h of age. Murine primary osteoblast cultures mineralize and produce osteocalcin in a maturation-dependent fashion (as demonstrated in other species), and continuous exposure to 1,25(OH)2D3, beginning at day 9 of culture, inhibits osteoblast differentiation and osteocalcin production and prevents mineralization of the culture. However, in contrast to other species, exposure to 1,25(OH)2D3, added later (days 17–25) in culture, does not stimulate osteocalcin but arrests osteocalcin production at current levels. Ambient media levels of osteocalcin were no different in cultures from Hyp mice and their normal litter mates, and the down-regulatory response to 1,25(OH)2D3 was comparable in cultures from normal and Hyp mice. Furthermore, expression of osteocalcin messenger RNA in murine cultures is reduced with exposure to 1,25(OH)2D3, and there is no difference between normal and Hyp cultures in this response. Thus, primary murine osteoblasts manifest a species-specific effect of 1,25(OH)2D3 on osteocalcin production. Furthermore, the increased serum osteocalcin production seen in intact Hyp mice, and the altered response to 1,25(OH)2D3 in Hyp mice, are not observed in osteoblast cultures derived from the mutant strain. These data indicate that abnormalities of osteocalcin described in intact Hyp mice require factors other than those present in cultured cells.

scholarly journals In vitro and in vivo induction of brown adipocyte uncoupling protein (thermogenin) by retinoic acid

1996 ◽  
Vol 317 (3) ◽  
pp. 827-833 ◽  
Author(s):  
Pere PUIGSERVER ◽  
Francisca VÁZQUEZ ◽  
María L. BONET ◽  
Catalina PICÓ ◽  
Andreu PALOU
Keyword(s):  
Retinoic Acid ◽  
Cultured Cells ◽  
Uncoupling Protein ◽  
Primary Cultures ◽  
Brown Adipocyte ◽  
Brown Adipose ◽  
Adipocyte Cell ◽  
Differentiation Stage

The effects of retinoic acid (RA) isomers (all-trans-RA and 9-cis-RA) on the appearance of uncoupling protein (UCP; thermogenin), the only unequivocal molecular marker of the brown adipocyte differentiated phenotype, have been investigated in primary cultures of brown adipocytes, in the brown adipocyte cell line HIB 1B and directly in intact mice. The results obtained with cultured cells indicate that retinoids function as inducers of the appearance of UCP and, at the same time, partially inhibit brown adipocyte cell proliferation. The two RA isomers displayed similar effectiveness as UCP inducers, their effect being comparable with that triggered by noradrenaline, so far considered to be the main modulator of UCP gene expression. The effectiveness of retinoids as UCP inducers was dependent on the stage of brown adipocyte differentiation, being maximal in confluent primary cells and in the medium–late differentiation stage of HIB 1B cells. Corroborating the results obtained in vitro, we show that administration of all-trans-RA or 9-cis-RA to mice leads to an increase in their brown adipose tissue specific UCP content. 9-cis-RA treatment also prevented the loss of UCP on cold deacclimation. To our knowledge, this is the first report of a stimulatory effect of retinoid compounds on UCP induction in vivo.

scholarly journals Antenatal betamethasone attenuates the angiotensin-(1–7)-Mas receptor-nitric oxide axis in isolated proximal tubule cells

2017 ◽  
Vol 312 (6) ◽  
pp. F1056-F1062 ◽  
Author(s):  
Yixin Su ◽  
Jianli Bi ◽  
Victor M. Pulgar ◽  
Mark C. Chappell ◽  
James C. Rose
Keyword(s):  
Nitric Oxide ◽  
Proximal Tubule ◽  
Cell Model ◽  
Primary Cultures ◽  
Specific Effect ◽  
Renal Tubules ◽  
Proximal Tubule Cells ◽  
Mas Receptor ◽  
Tubule Cells

We previously reported a sex-specific effect of antenatal treatment with betamethasone (Beta) on sodium (Na+) excretion in adult sheep whereby treated males but not females had an attenuated natriuretic response to angiotensin-(1–7) [Ang-(1–7)]. The present study determined the Na+ uptake and nitric oxide (NO) response to low-dose Ang-(1–7) (1 pM) in renal proximal tubule cells (RPTC) from adult male and female sheep antenatally exposed to Beta or vehicle. Data were expressed as percentage of basal uptake or area under the curve for Na+ or percentage of control for NO. Male Beta RPTC exhibited greater Na+ uptake than male vehicle cells (433 ± 28 vs. 330 ± 26%; P < 0.05); however, Beta exposure had no effect on Na+ uptake in the female cells (255 ± 16 vs. 255 ± 14%; P > 0.05). Ang-(1–7) significantly inhibited Na+ uptake in RPTC from vehicle male (214 ± 11%) and from both vehicle (190 ± 14%) and Beta (209 ± 11%) females but failed to attenuate Na+ uptake in Beta male cells. Beta exposure also abolished stimulation of NO by Ang-(1–7) in male but not female RPTC. Both the Na+ and NO responses to Ang-(1–7) were blocked by Mas receptor antagonist d-Ala7-Ang-(1–7). We conclude that the tubular Ang-(1–7)-Mas-NO pathway is attenuated in males and not females by antenatal Beta exposure. Moreover, since primary cultures of RPTC retain both the sex and Beta-induced phenotype of the adult kidney in vivo they appear to be an appropriate cell model to examine the effects of fetal programming on Na+ handling by the renal tubules.

scholarly journals A Comparative Study on Delivery of Externally Attached DNA by Papillomavirus VLPs and Pseudoviruses

Vaccines ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 1501
Author(s):  
Sarah Brendle ◽  
Nancy Cladel ◽  
Karla Balogh ◽  
Samina Alam ◽  
Neil Christensen ◽  
...  
Keyword(s):  
Critical Role ◽  
Specific Effect ◽  
Dna Delivery ◽  
Dna Uptake ◽  
In Vivo Models ◽  
Cottontail Rabbit ◽  
Species Specific ◽  
Dna Delivery Vehicle

Human papillomavirus (HPV) 16 capsids have been chosen as a DNA delivery vehicle in many studies. Our preliminary studies suggest that HPV58 capsids could be better vehicles than HPV16 capsids to deliver encapsidated DNA in vitro and in vivo. In the current study, we compared HPV16, HPV58, and the cottontail rabbit papillomavirus (CRPV) capsids either as L1/L2 VLPs or pseudoviruses (PSVs) to deliver externally attached GFP-expressing DNA. Both rabbit and human cells were used to test whether there was a species-specific effect. DNA delivery efficiency was determined by quantifying either GFP-expressing cell populations or mean fluorescent intensities (MFI) by flow cytometry. Interestingly, CRPV and 58-VLPs and PSVs were significantly more efficient at delivering attached DNA when compared to 16-VLPs and PSVs. A capsid/DNA ratio of 2:1 showed the highest efficiency for delivering external DNA. The PSVs with papillomavirus DNA genomes also showed higher efficiency than those with irrelevant plasmid DNA. HPV16L1/58L2 hybrid VLPs displayed increased efficiency compared to HPV58L1/16L2 VLPs, suggesting that L2 may play a critical role in the delivery of attached DNA. Additionally, we demonstrated that VLPs increased in vivo infectivity of CRPV DNA in rabbits. We conclude that choosing CRPV or 58 capsids to deliver external DNA could improve DNA uptake in in vitro and in vivo models.

Chromium(VI)-induced damage to the cytoskeleton and cell death in isolated hepatocytes

2002 ◽  
Vol 30 (4) ◽  
pp. 748-751 ◽  
Author(s):  
M. Gunaratnam ◽  
M. H. Grant
Keyword(s):  
Cell Death ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Annexin V ◽  
Isolated Hepatocytes ◽  
Chromium Vi ◽  
Cell Staining ◽  
Cell Cytoskeleton ◽  
Low Concentrations

Cr(VI) is a known human carcinogen. Although it has been investigated widely, the mechanism(s) of its action is/are not fully understood. The aim of this study was to evaluate Cr(VI)-induced damage to the cell cytoskeleton and the mode of cell death in primary cultures of hepatocytes. Exposure of the cultured cells (105/cm2) to 1 and 5 μM Cr(VI) for 24 h resulted in loss of the cell cytoskeleton, and this was accompanied by membrane blebbing and shrinking of the cell. Staining of the cells with annexin V and propidium iodide showed that Cr(VI) induces apoptosis at low concentrations (5 μM), whereas at higher concentrations (25 μ) it induces necrosis. This study shows that Cr(VI) causes damage to the cell cytoskeleton, and induces apoptosis at low concentrations. However, the importance of necrosis and apoptosis in vivo, and the effects of longer exposure times, which simulate environmental and occupational exposure to Cr(VI), remain to be investigated.

scholarly journals Regulation of collagen production and collagen mRNA amounts in fibroblasts in response to culture conditions

1986 ◽  
Vol 239 (1) ◽  
pp. 179-183 ◽  
Author(s):  
S R Quinones ◽  
D S Neblock ◽  
R A Berg
Keyword(s):  
Type I Collagen ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Culture Conditions ◽  
Type I ◽  
Collagen Production ◽  
Tendon Cells ◽  
Chain Synthesis ◽  
Polypeptide Chains

Collagen synthesis and mRNA amounts for the alpha 1 and alpha 2 polypeptide chains of Type I collagen were measured in embryonic-chick tendons and in tendon cells both in suspension and in primary cultures. The percentage of protein production represented by collagen in suspension-cultured cells was initially the same as in the intact tendon; however, on an hourly basis, there was actually a steady decline in collagen production by suspended cells. Collagen production in primary cultures of chick tendon fibroblasts was decreased when compared with intact tendon, even though ascorbate-supplemented primary cultures were able to maintain higher rates of collagen production than were non-supplemented cultures. The amounts of mRNA for alpha 1(I) and alpha 2(I) polypeptide chains of collagen responded in similar fashions to different culture conditions and were compared with the amounts of mRNA for beta-actin. In primary cultures the available alpha 1 and alpha 2 collagen mRNAs support proportionately higher collagen production than in the intact tendon. However, the ratio of alpha 1/alpha 2 mRNA and polypeptide-chain synthesis did not remain 2:1, but increased with the concomitant production of Type I trimers composed of three alpha 1 chains. Removal of fibroblasts from their environment in vivo appears to alter the amounts of mRNA for alpha 1 and alpha 2 chains and to alter the utilization of those mRNAs for polypeptide synthesis.

scholarly journals Gene expression in Acetabularia. III. Comparison of stained cytosolic proteins and in vivo and in vitro translation products

1983 ◽  
Vol 60 (1) ◽  
pp. 1-12
Author(s):  
R.L. Shoeman ◽  
G. Neuhaus ◽  
H.G. Schweiger
Keyword(s):  
Messenger Rna ◽  
In Vitro Translation ◽  
Cytosolic Proteins ◽  
Molecular Weights ◽  
Translational Activity ◽  
Stage Of Development ◽  
Extended Analysis ◽  
Species Specific

A comparison of stained cytosolic proteins, in vivo 80 S ribosome translation products and in vitro translation products of poly(A)+ RNA from three species of Acetabularia was performed after characterization of their molecular weights and isoelectric points via two-dimensional electrophoresis. A total of 803 stained proteins, and 121 in vivo and 77 in vitro translation products, representing the most abundant proteins in each category, were analysed. In interspecies comparisons, approximately 10% of the stained proteins were common to all three species and more than 50% were found to be species-specific. Approximately 25% of the in vivo translation products were common to all three species and more than 30% were found to be species-specific. The majority of the in vivo and in vitro translation products were detected by one or both of the other methods employed. Even though the analysis was limited to the most abundant proteins detected by each of the three methods and to one stage of development, the results suggest that the translation of some proteins is not regulated, that the in vivo translation of others, whose mRNA is present and translated in vitro, is turned off while the translation in vivo of others is enhanced relative to the total. This feature makes them candidates for stage-specific proteins. The results provide a firm basis for the extended analysis of the biological activity of heterologous messenger RNA in Acetabularia cytoplasm and for a more complete cataloguing of the mRNA population and translational activity at different stages in the development of Acetabularia.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

The Infection of Cells by Bullet-Shaped Viruses

1969 ◽  
Vol 27 ◽  
pp. 372-373
Author(s):  
Frederick A. Murphy ◽  
Alyne K. Harrison ◽  
Sylvia G. Whitfield
Keyword(s):  
Electron Microscopy ◽  
Physical Properties ◽  
Host Cell ◽  
Thin Section ◽  
Cultured Cells ◽  
Cell Infection ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Section Electron Microscopy

The bullet-shaped viruses are currently classified together on the basis of similarities in virion morphology and physical properties. Biologically and ecologically the member viruses are extremely diverse. In searching for further bases for making comparisons of these agents, the nature of host cell infection, both in vivo and in cultured cells, has been explored by thin-section electron microscopy.

Ultrastructural modification of cellular interactions in cancer tumor

1978 ◽  
Vol 36 (2) ◽  
pp. 318-319
Author(s):  
N. P. Dmitrieva
Keyword(s):  
Cancer Cells ◽  
Human Thyroid ◽  
Adjacent Cell ◽  
Main Role ◽  
Cellular Interactions ◽  
Electron Microscopic ◽  
Cancer Tumor ◽  
Normal Human ◽  
Characteristic Features

One of the most characteristic features of cancer cells is their ability to metastasia. It is suggested that the modifications of the structure and properties of cancer cells surfaces play the main role in this process. The present work was aimed at finding out what ultrastructural features apear in tumor in vivo which removal of individual cancer cells from the cell population can provide. For this purpose the cellular interactions in the normal human thyroid and cancer tumor of this gland electron microscopic were studied. The tissues were fixed in osmium tetroxide and were embedded in Araldite-Epon.In normal human thyroid the most common type of intercellular contacts was represented by simple junction formed by the parallelalignment of adjacent cell membranees leaving in between an intermembranes space 15-20 nm filled with electronlucid material (Fig. 1a). Sometimes in the basal part of cells dilatations of the intercellular space 40-50 nm wide were found (Fig. 1a). Here the cell surfaces may form single short microvilli.

Preparation of cultured astrocytes for x-ray microanalysis

1989 ◽  
Vol 47 ◽  
pp. 988-989
Author(s):  
N.K.R. Smith ◽  
K.E. Hunter ◽  
P. Mobley ◽  
L.P. Felpel
Keyword(s):  
Cultured Cells ◽  
Elemental Content ◽  
Elemental Distribution ◽  
Sprague Dawley ◽  
X Ray ◽  
Cultured Astrocytes ◽  
Freeze Dried ◽  
Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

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