Incorporation Of Labeled Thymidine Research Articles

Specific titin and myomesin domains stimulate myoblast proliferation

Myofibrillar proteins titin and myomesin stimulated myoblast proliferation as determined by MTT-test and labelled thymidine incorporation in the DNA. Specific Fn type III and Ig-like domains of these proteins were able to exert mitogenic effects as well. Proliferative effect of Fn type III domains was highly sensitive to inhibition of Ca2+/calmodulin dependent protein kinase, whereas the effect of Ig-like domains showed greater sensitivity to the inhibition of adenylyl cyclase – cAMP – PKA pathway. IGF-1 autocrine signalling inhibition partially suppressed mitogenic effects revealed by both domain types.

Sphingosine 1-phosphate stimulates proliferation and migration of satellite cells: Role of S1P receptors

Satellite cells are resident stem cells of skeletal muscle; they are normally quiescent but upon post-trauma activation start to proliferate and fuse with damaged fibers contributing to muscle regeneration. In this study the effect of the bioactive sphingolipid sphingosine 1-phosphate (S1P) on the proliferative and migratory response of murine satellite cells has been examined. S1P was found to stimulate labeled thymidine incorporation in a phosphatidylinositol 3-kinase-dependent manner. Moreover, by employing selective S1P receptor agonists and antagonists and silencing individual S1P receptors, the mitogenic action of S1P in satellite cells was shown to depend on S1P2 and S1P3. Notably, by using different experimental approaches S1P was found to positively influence satellite cell migration, necessary for their recruitment at the site of muscle damage. Interestingly, the specific silencing of individual S1P receptor subtypes demonstrated the pivotal role of S1P1 and S1P4 in mediating the S1P migratory effect. This latter result demonstrates for the first time that S1P4 receptor has a role in skeletal muscle cells, supporting the notion that this receptor subtype plays a biological action broader than that so far identified in lymphoid tissue. On the contrary, S1P2 was found to negatively regulate cell migration. Collectively, these results are in favour of an important function of S1P in satellite cell biology that could in principle be exploited as novel pharmacological target for improving skeletal muscle regeneration.

Cudrania tricupidata Bureau (CTB) Glycoprotein Inhibits Proliferation by Di(2-ethylhexyl) phthalate in Primary Splenocytes: Responses in Cell Proliferation Signaling

The aim of the present study was to evaluate inhibitory effect of CTB glycoprotein isolated from Cudrania tricuspidata Bureau on DEHP-induced cell proliferation in lymphocytes. Our results revealed that DEHP increased lymphocyte proliferation as confirmed by increasing [3H]thymidine incorporation, and proliferating cell nuclear antigen (PCNA), cyclin D1, and cyclin-dependent kinase (CDK)-4 expression. This was accompanied by induced intracellular Ca2+ level, protein kinase C (PKC) translocation from cytosol to membrane, ERK1/2 phosphorylation, and nuclear factor (NF)-κB transcriptional activation in DEHP-treated cells. However, CTB glycoprotein (100 μg/ml) reduced labeled thymidine incorporation and PCNA expression in DEHP-treated cells. Additionally CTB glycoprotein reduced Ca2+ level, PKC translocation, ERK1/2 phosphorylation, NF-κB transcriptional activation, cell cycle proteins (cyclin D1 and CDK4) expression in cells. The activation of NF-κB was collectively blocked by pretreatment with PKC inhibitor (staurosporine) and ERK1/2 inhibitor (PD98059), respectively. The results from these experiments indicate that CTB glycoprotein inhibits cell proliferation via down regulations of Ca2+/PKC, ERK1/2, and cell cycle proteins induced by DEHP. Therefore, we suggest that the CTB glycoprotein might be one component for prevention of cell proliferation-related immune diseases.

Sphingosine 1‐Phosphate Mediates Proliferation and Survival of Mesoangioblasts

Mesoangioblasts are stem cells capable of differentiating in various mesodermal tissues and are presently regarded as suitable candidates for cell therapy of muscle degenerative diseases, as well as myocardial infarction. The enhancement of their proliferation and survival after injection in vivo could greatly improve their ability to repopulate damaged tissues. In this study, we show that the bioactive sphingolipid sphingosine 1-phosphate (S1P) regulates critical functions of mesoangioblast cell biology. S1P evoked a full mitogenic response in mesoangioblasts, measured by labeled thymidine incorporation and cell counting. Moreover, S1P strongly counteracted the apoptotic process triggered by stimuli as diverse as serum deprivation, C2-ceramide treatment, or staurosporine treatment, as assessed by cell counting, as well as histone-associated fragments and caspase-3 activity determinations. S1P acts both as an intracellular messenger and through specific membrane receptors. Real-time polymerase chain reaction analysis revealed that mesoangioblasts express the S1P-specific receptor S1P3 and, to a minor extent, S1P1 and S1P2. By using S1P receptor subtype-specific agonists and antagonists, we found that the proliferative response to S1P was mediated mainly by S1P2. By contrast, the antiapoptotic effect did not implicate S1P receptors. These findings demonstrate an important role of S1P in mesoangioblast proliferation and survival and indicate that targeting modulation of S1P-dependent signaling pathways may be used to improve the efficiency of muscle repair by these cells. Disclosure of potential conflicts of interest is found at the end of this article.

Sphingosine 1‐phosphate regulates myogenic differentiation: a major role for S1P 2 receptor

In this study a novel biological activity of sphingosine 1-phosphate (S1P) in C2C12 myoblasts was identified. In these cells the bioactive lipid profoundly regulated myogenesis exerting an antimitogenic activity, by reducing serum-induced cell proliferation, and acting as powerful prodifferentiating agent by enhancing the expression of myogenic differentiation markers such as myogenin, myosin heavy chain, and caveolin-3. The S1P-dependent diminution of serum-induced labeled thymidine incorporation was abrogated by antisense oligodeoxyribonucleotides (ODN) to S1P2, but not to S1P1 or S1P3 receptor, also expressed in C2C12 cells, implicating S1P2 in the biological response. Using antisense ODN and short interfering RNA treatment, we highlighted the key role played by S1P2 in the S1P-dependent induction of muscle-specific gene products. Notably, S1P2 overexpression increased the content of myogenic markers and hastened the onset of differentiated muscle phenotype in comparison with control cells. Cell treatment with pertussis toxin did not affect the biological responses to S1P, ruling out the involvement of Gi-mediated events in the signaling promoted by the sphingolipid. Among the various signaling pathways activated by S1P, the activation of ERK1/ERK2 and p38 MAPK, both identified as downstream effectors of S1P2, was required for the inhibition of cell proliferation and the stimulation of myogenic differentiation, respectively.

On the determination of a conversion factor from labelled thymidine incorporation by bacteria to cell production in a sub-tropical estuary: preliminary results

Faculdade de Ciencias Ambientais - Oceanografia. Centro Universitario Monte Serrat - UNIMONTE. (Campus Ponta da Praia, Av. Saldanha da Gama, 89, 11030-220, Santos, SP, Brasil) Bacterial carbon production has become a key parameter in quantifying carbon flows through aquatic food webs (Smith & Azam, 1992). Measurement of

Pharmacodynamics of mycophenolic acid in heart allograft recipients: correlation of lymphocyte proliferation and activation with pharmacokinetics and graft histology.

Assays of drug blood levels are used for therapeutic immunosuppressive drug monitoring (pharmacokinetics, PK). We monitored lymphocyte functions (pharmacodynamics, PD) in allograft recipients treated with mycophenolic acid (MPA) to determine its mechanisms and the relationships among dose levels, PK, PD, and histological severity of graft rejection. Lewis rats transplanted with Brown Norway (BN) rat hearts were treated with different dose levels of MPA for 8, 15, or 29 days at which times grafts were removed and scored for rejection grade. Blood was analyzed (high-performance liquid chromatography) for MPA plasma concentrations (area under the concentration-time curve0-24 hr, C6 hr, trough) and for lymphocyte functions using concanavalin A-stimulated whole blood assays to measure lymphocyte proliferation (tritium labeled thymidine incorporation and flow cytometric bivariate proliferating nuclear cell antigen/DNA analysis) and activation (percent lymphocytes expressing CD25 or CD134). PD values were AUE0-24 hr (area under the PD effect-time curve), maximum inhibition and trough. MPA equipotently suppressed (by flow cytometry) both proliferation and activation and these effects correlated with MPA plasma levels (r2=0.80-0.91). Relationships among MPA dose levels, PK and PD were clear, direct, and reproducible. Correlation coefficients after 8 days of MPA treatment were: 0.90, 0.87, and 0.49 for MPA PK (AUC0-24 hr, C6 hr and trough) versus rejection scores; 0.80-0.89, 0.86-0.92, and 0.25-0.52 for PD flow cytometric assays (AUE0-24 hr, maximum inhibition, and trough) versus rejection scores. MPA inhibits both lymphocyte proliferation and activation. PD by flow cytometry (FCM) correlates highly with severity of graft rejection, showing that PD of MPA measured in peripheral blood predicts immune cell activity in graft tissue.

Direct Correlation Between Cell Loss and Depression of Labeled Thymidine Incorporation After Irradiation. I. Spleen

The object of this investigation was to determine the relationship between depression of DNA synthesis and cell loss in the spleen after single small radiation doses and to determine whether DNA synthesis after exposure to different radiation modalities can be used as a measure of radiation sensitivity and relative biological effectiveness (RBE). The results presented illustrate, for the first time, that depression of DNA synthesis and cell loss in the spleen after small single doses of irradiation are directly correlated. This correlation indicates that depression of labeled thymidine incorporation and cell loss can be used to determine tissue sensitivity to irradiation. Advance determination of depression of labeled thymidine incorporation or cell loss, using small radiation doses from different radiation modalities, can establish cell killing efficiency and RBE values for each proliferating tissue.

Uterine-associated serine protease inhibitors stimulate deoxyribonucleic acid synthesis in porcine endometrial glandular epithelial cells of pregnancy.

Protease inhibitors are major secretory components of the mammalian uterus that are thought to mediate pregnancy-associated events primarily by regulating the activity of proteolytic enzymes. In the present study, we examined the mitogenic potentials of two serine protease inhibitors, namely secretory leukocyte protease inhibitor (SLPI) and uterine plasmin/trypsin inhibitor (UPTI) in primary cultures of glandular epithelial (GE) cells isolated from early pregnant (Day 12) pig endometrium, using the [(3)H]thymidine incorporation assay. Purified porcine SLPI (pSPLI), porcine UPTI (pUPTI), or recombinant human SLPI (rhSLPI), all of which exhibited anti-trypsin activity, increased (p < 0.05) labeled thymidine incorporation into DNA of serum-deprived GE cells when tested at a range of 10-1000-ng/ml concentrations. Polyclonal antibodies directed against either hSLPI or pSLPI abrogated the effect of SLPI. Co-addition of pSLPI and pUPTI increased DNA synthesis in these cells to a level higher (p < 0.05) than that observed with either protease inhibitor. The glycosaminoglycan heparin, which has been previously shown to increase the anti-protease activity of SLPI, exhibited a tendency (p = 0.08) to enhance SLPI and UPTI induction of cellular DNA synthesis. Reverse transcription-polymerase chain reaction indicated that the messenger RNAs for both protease inhibitors were present in the endometrium throughout pregnancy and, within this tissue, in GE cells to a greater extent (p < 0.05) than in stromal fibroblastic cells. Results demonstrate that, in addition to their well-documented anti-protease activities, SLPI and UPTI may constitute autocrine growth promotants for the uterine epithelium. These data suggest a novel mechanism whereby locally produced protease inhibitors may modulate periimplantation events and embryo-maternal communication.

Release of a mitogenic factor by adult rat lung slices in culture.

In an effort to develop a suitable bioassay for testing lung growth factors that might be operative during compensatory lung growth following partial pneumonectomy, a simple and inexpensive lung organ culture system was characterized. The culture employs lung tissue slices obtained by means of a device allowing thicknesses of 500 microns to be cut reproducibly. To avoid the collapse of the organ, the alveolar spaces were filled prior to culture with Noble agar-containing Eagle's-Dulbecco's modified medium. Lung tissue sections could be maintained ultrastructurally intact for at least one week. The results showed that upon culture, a part of the type II pneumocytes undergo differentiation into type I pneumocytes, thus demonstrating that the culture system may be suited for differentiation studies. One surprising feature of this culture system was the mitogenic impulse associated with culture. Radioactively labeled thymidine incorporation was strongly stimulated in the culture, mainly affecting the epithelial cells, as could be established by "back-to-back" autoradiography. With a reconstruction experiment, it was possible to demonstrate the local release of a mitogenic factor following slicing, mincing, or dissection of the lung tissue, which could be assayed by its ability to induce serum-starved Balb/c 3T3 cells to synthesize DNA in culture.

Atherogenic potential of the blood serum in patients with type 1 diabetes mellitus

The task of this research was to study exogenous insulin effect on blood serum atherogenic potential in patients with type I diabetes mellitus. Under study were relationship between blood serum atherogenic potential in diabetics and daily insulin dose and duration of therapy, as well as exogenous insulin effect on the major atherosclerotic manifestations in tissue cultures, such as intracellular cholesterol content in cultured cells and labeled thymidine incorporation in cellular nuclei DNA. Blood serum atherogenicity did not correlate with insulin dose and duration of insulin therapy both in adult patients and children. Insulin prescription to newly detected patients with type I condition did not influence blood serum atherogenic potential. Exogenic insulin did not influence the major atherosclerotic manifestations at the cellular level, namely, on intracellular cholesterol content in cultured cells and these cells proliferative activity; it had no influence on blood serum atherogenic potential of the patients with type I condition.

THE USE OF INTERNALIZABLE DERIVATIVES OF CHLORIN E6 FOR INCREASING ITS PHOTOSENSITIZING ACTIVITY

Experiments with human hepatoma PLC/PRF/5 cells and human embryo skin fibroblasts involving the use of three different tests (colony formation, Trypan blue exclusion, labeled thymidine incorporation) have demonstrated a significantly higher photosensitizing activity of chlorin e6 conjugates with internalizable ligands as compared to that of chlorin e6 itself. Receptor-mediated internalization of chlorin e6 conjugates ensures a greater photosensitization of cells than binding of those conjugates to cell surface receptors. The suitability of such conjugates that permit the delivery of a photosensitizer to sensitive intracellular targets is discussed.

Protozoan Grazing and the Size–Activity Structure of Limnetic Bacterial Communities

We measured bacterial growth rates by labelled thymidine incorporation, grazing loss rates with fluorescent latex particles, and bacterial cell size and abundance within narrow size-fractions of freshwater bacterioplankton. Contrary to the predictions of standard allometric relationships, the smallest bacteria showed the lowest incorporation rate per cell and per unit DNA content. Cells trapped by 1- and 5-μm filters grew the fastest and were responsible for 86% of detected thymidine biosynthesis. Grazing studies in six lakes showed that grazing pressure from flagellated protozoans and mixotrophic algae on large bacterial cells was probably 2–40 times more intense than that on the smallest cells. We suggest that reduced grazing pressure on the smallest bacteria allows them to dominate numerically, despite their slower growth.

Biological Activity of Synthetic Juvenile Hormone Analogues (JHA) for Trypanosoma cruzi

Abstract Juvenile Hormone Analogues. Trypanosoma cruzi, Growth Inhibition Several juvenile hormone analogues, among them Fenoxycarb and authentic JH-III, were tested as growth inhibitors for Trypanosoma cruzi cultivated in vitro. The same compounds were also assayed for inhibition of tritium labelled-thymidine incorporation into T. cruzi cells. The results indicated that those analogues containing sulfur showed important activity against the development of the cells. Interesting inhibition values were also found for the remaining fenoxy-fenoxy derivatives assayed.

A Study on the lack of [methyl-3H] thymidine uptake and incorporation by chemolithotrophic bacteria

Five chemolithotrophic bacteria were tested for their ability to incorporate [methyl-(3)H] thymidine. None of the bacteria incorporated the label, even after incubation for 24 hours. The inability of these bacteria to incorporate thymidine appears to be due to an absence of transport mechanisms for exogenous nucleosides. As a result of these findings, it is concluded that activities deduced from labeled thymidine incorporation measurements probably do not include the activity of chemolithotrophic bacteria.

Inosine monophosphate dehydrogenase activity in acute leukaemia

Inosine monophosphate dehydrogenase (IMPD) is an important enzyme in de-novo purine synthesis. The level of IMPD activity has been suggested to determine whether acute leukaemia cells proliferate (if the activity is high) or differentiate (if IMPD activity is low). IMPD activity measured by the conversion of inosine monophosphate to xanthine monophosphate ranged from 12.5 to 87.0 (mean 49.4) pmol/h/10 6 cells in normal bone marrow. The levels were significantly raised in AML (range 14–374, mean 184 pmol/h/10 6 cells) and ALL (range 65–228, mean 172 pmol/h/10 6 cells). Normal tonsillar (B) lymphocytes showed higher levels (range 78–159, mean 110 pmol/h/10 6 cells) than resting peripheral blood T lymphocytes (range 8.8–51.2, mean 28.1 pmol/h/10 6 cells). In CLL, the results (range 19–173, mean 64.3 pmol/h/10 6 cells) were comparable to those of normal tonsillar B lymphocytes. IMPD levels could be related to cell cycle in PHA-stimulated lymphocytes, since IMPD activity increased in parallel with increase in DNA synthesis measured by labelled thymidine incorporation. On the other hand, IMPD activity did not correlate with the proportion of proliferating cells measured on a FACS sorter in either AML or ALL or in normal tonsillar B cells. We conclude that IMPD levels are higher in B than T lymphocytes and in acute leukaemia blasts compared to more differentiated mixed bone marrow cells. The results do not suggest, however, that IMPD assay will be of value in differentiation of the various subtypes of acute leukaemia or of malignant haemopoietic cells from the equivalent normal cell at the same level of differentiation.

Use of serum-free hormonally defined media to evaluate the effects of growth factors and inhibitors on proliferation of estrogen-responsive mammary and pituitary tumor cells in culture

Serum-free defined media have been developed for assay of the mitogenic effects of growth factors on human MCF-7, human T-47D, and mouse COMMA-D mammary cells as well as for identification of mitogens and inhibitors of GH4C1 rat pituitary tumor cell growth. These lines were shown to grow in vivo in response to a variety of hormones including estrogens and thyroid hormones. With mammary cells, complete hormonally and nutritionally defined media were established that supported continuous passage at 50 to 90% of the serum stimulated rate. The strategy used to measure mitogens for mammary cells was to identify nutritional conditions where the growth rate was reduced greatly without impairing the response to picomolar to nanomolar concentration of growth factors. The effects of polypeptide growth factors and tissue extracts were estimated by their addition to basal medium and measuring cell number increases or labeled thymidine incorporation into DNA. In a variation of this methodology, the MTW9/PL2 rat mammary cells were used to identify secreted autocrine growth factors; nutritionally defined conditions were sought for growth of these rat cells in the complete absence of exogenous growth factors. The factors secreted into the medium were detected by bioassays with COMMA-D or MCF-7 mammary cell lines. The effects of growth factors-inhibitors on pituitary cells were measured by a related method; the GH4C1 cells were grown at less than optimal rates in a defined medium designated TRM-1. Addition of mitogens to TRM-1 stimulated pituitary cell growth whereas addition of inhibitors caused reduced levels of growth. The methods described in this report offer new means of assaying growth factors-inhibitors for a range of mammary and pituitary tumor cells.

Multiple myeloma plasma cell kinetics: rapid and reliable evaluation using 5-bromo-2-deoxyuridine (BrdUrd) DNA incorporation detected by an anti-BrdUrd monoclonal antibody.

In 19 patients with monoclonal gammopathy, plasma cell proliferative activity was evaluated using 5-bromo-2-deoxyuridine (BrdUrd) incorporation, as revealed by an anti-BrdUrd monoclonal antibody. A simultaneous standard labelled thymidine incorporation was carried out in all patients as reference test. The BrdUrd method was confirmed as a reliable test and is recommended for routine clinical application in monoclonal gammopathies.

The effects of growth factors and serum on DNA synthesis and differentiation in thyroid cells in culture

The effects of three putative growth factors and serum on [ Me- 3H]thymidine and Na 125I incorporation into thyroid cell cultures have been examined. We found that serum and EGF could stimulate radioactively labelled thymidine incorporation into confluent cultures. However, both factors completely inhibited iodine uptake and organification at low concentrations. Insulin also stimulated [ Me- 3H]thymidine incorporation but had no adverse effect on thyroid differentiated function. TSH examined under the same conditions was not a growth factor but was essential to maintain differentiated functions. We conclude that thyroid growth and differentiation are not mutually exclusive processes. However, EGF and serum inhibit thyroid differentiated function at very low concentrations. Elucidation of the physiological role of these factors and their mechanism of action may lead to a greater understanding of thyroid hormone biosynthesis.

Pulsed electromagnetic fields increase the rate of rat liver regeneration after partial hepatectomy.

Pulsed extremely low-frequency electromagnetic fields interact with rat liver regeneration following partial hepatectomy when delivered to the rats immediately after the operation and every 12 hr thereafter. This interaction results first in an increased ornithine decarboxylase activity, an enzyme used as an early marker of cell growth. The rate of labeled thymidine incorporation into DNA is also increased by the treatments with magnetic fields during the early phases of liver regeneration. Glycogen depletion and lipid accumulation, two well-known early peculiar phenomena of liver regeneration following partial hepatectomy, are quantitatively decreased by the treatments with electromagnetic fields. The recovery to normal glycogen and lipid contents is completed within 5 days after surgery, instead of 7 days as found in control rats.