Density dependent expression of PDGF-A modulates the angiotensin II dependent proliferation of rat cardiac fibroblasts.

The proliferative effect of angiotensin II (Ang-II) on primary cardiac fibroblasts is not well understood and controversially discussed. Results described here show that fibroblasts from adult rat hearts exhibit a cell density dependent Ang-II induced cell proliferation. Whereas we could not detect a proliferative effect of Ang-II on confluent cells, which are still able to divide as shown by stimulation with platelet derived growth factor (PDGF), we observed an Ang-II induced cell division of approximately 20 % in non-confluent cells.

Isolation of full-size mRNA from cells sorted by flow cytometry.

Gene expression is one key mechanism to regulate cell growth and differentiation. It is usually determined by Northern blotting or RT-PCR. However, studies with primary cell cultures are frequently hampered due to contaminating cells such as fibroblasts.

Insulin stimulates the L-type Ca2+ current in rat cardiac myocytes.

Objective: The aim was to study the L-type calcium current (ICa,L) in cardiac myocytes as a possible target of insulin in the regulation of cardiac function.

Method: Using the whole-cell configuration of the patch-clamp technique, we investigated the stimulation of ICa,L by insulin in isolated rat ventricular myocytes.

Results: The stimulation of ICa,L by insulin was dose-dependent (EC50 = 33 nM) and reversible.

Early response kinase and PI 3-kinase activation in adult cardiomyocytes and their role in hypertrophy.

The present study investigated the role of early response kinase (ERK) and phosphatidylinositol 3 (PI 3)-kinase in ventricular cardiomyocytes from adult rat for the hypertrophic response to alpha-adrenoceptor stimulation. Parameters of the hypertrophic response were stimulation of protein synthesis and induction of creatine kinase BB. The alpha-adrenoceptor agonist phenylephrine (10 micromol/l) activated ERK2 and PI 3-kinase.

Differential expression of Listeria monocytogenes virulence genes in mammalian host cells.

We have used RT-PCR and GFP-mediated fluorescence to analyse the regulation of PrfA-dependent virulence genes of Listeria monocytogenes during proliferation in mammalian host cells. Our data show that most of the PrfA-regulated virulence genes are more efficiently expressed, as measured by transcript levels, when L. monocytogenes is grown in macrophages and macrophage-like cells rather than in epithelial cells, hepatocytes or endothelial cells.

Determination of copy number of c-Myc protein per cell by quantitative Western blotting.

The protooncogene c-Myc plays a key role in growth control, differentiation, and apoptosis. An abnormally high expression of c-myc has been found to be associated with many neoplasms. c-Myc gene expression is usually measured at the mRNA level.

Gene expression in rod shaped cardiac myocytes, sorted by flow cytometry.

Objective: Primary cardiac myocyte cultures are usually contaminated with variable parts of different cell types, such as fibroblasts, endothelial cells and smooth muscle cells. Thus, the objective of our study was to analyse the gene expression in a pure population.

Methods: To obtain an homogeneous population, cardiac myocytes from adult rats were fixed with ethanol and sorted by flow cytometry.

The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice.

In this work we identified and characterized a gene cluster containing three internalin genes of Listeria monocytogenes EGD. These genes, termed inlG, inlH and inlE, encode proteins of 490, 548 and 499 amino acids, respectively, which belong to the family of large, cell wall-bound internalins. The inlGHE gene cluster is flanked by two listerial house-keeping genes encoding proteins homologous to the 6-phospho-beta-glucosidase and the succinyl-diaminopimelate desuccinylase of E.

Advanced glycated albumin impairs protein degradation in the kidney proximal tubules cell line LLC-PK1.

Advanced glycation end-products (AGEs) are assumed to play a major role in the genesis of diabetic nephropathy and other diabetic complications. We studied the potential effect of AGEs on protein turnover and lysosomal proteinase activities in LLC-PK1 cells, a pig kidney proximal tubules cell line. Advanced glycated bovine serum albumin (AGE-BSA) was used as a model of AGEs and its action was compared to that of nonglycated BSA.

Late signals from the PDGF receptors leading to the activation of the p70S6-kinase are necessary for the transition from G1 to S phase in AKR-2B cells.

Platelet-derived growth factor AB (PDGF-AB) has to be permanently present in the culture medium to achieve full proliferation (>90%) of AKR-2B fibroblasts. Upon removal after 1 h incubation time, only a small number of cells (<20%) entered the cell cycle. Concomitantly there was no increase in RNA- and protein-synthesis.

Activation of p70(S6) kinase by beta-adrenoceptor agonists on adult cardiomyocytes.

Cardiac cellular hypertrophy plays an important role in cardiovascular diseases. Up until now, little has been known about the regulation of cellular growth on the level of intracellular signalling. Here, the implication of the p70(S6)-kinase (p70(S6K)) in the hypertrophic response after beta-adrenergic stimulation of cardiac myocytes from adult rats was investigated.

Mesangial cell hypertrophy induced by NH4Cl: role of depressed activities of cathepsins due to elevated lysosomal pH.

Enhanced ammoniagenesis is currently thought to play an important role in renal hypertrophy and subsequent tubulointerstitial fibrosis. Under certain conditions glomeruli also may be affected by ammonia toxicity. Exposure of glomeruli to augmented ammonia levels may occur: (i) in advanced liver diseases due to elevated blood ammonia concentrations; (ii) in conditions of enhanced tubular ammoniagenesis following cortical "trapping;" and (iii) due to increased ammonia formation in the glomeruli in the presence of impaired renal function.

Copy number, epigenetic state and expression of the rRNA genes in young and senescent rat embryo fibroblasts.

The recent cloning of the gene that causes the premature aging in Werner syndrome patients has evoked speculations that deficits in expression of the ribosomal RNA genes could be related to cellular aging in general. Here we compare the state of the rRNA genes and the rRNA metabolism in young and senescent (aged) rat embryo fibroblasts (REF). Southern blot analysis revealed that the copy number and the methylation state of the genes did not change significantly with increasing cumulative population doublings (CPD) of the culture.

Advanced glycation endproducts stimulate the MAP-kinase pathway in tubulus cell line LLC-PK1.

Advanced glycation endproducts (AGEs) are suggested to play an important role in diabetic nephropathy. They induce specific cellular responses such as the release of cytokines in different cell lines. The effect of AGEs on signal transduction pathways was investigated in the renal tubulus cell line LLC-PK1.

Cell death of AKR-2B fibroblasts after serum removal: a process between apoptosis and necrosis.

AKR-2B cells disintegrate after serum removal. After a delay of approximately 90 minutes, cell death began and reached after six hours a plateau of 40-50% remaining living cells. We used time-lapse video microscopy to monitor dynamic structural changes and to measure the time span of individual cells to die.

PDGF-AA, a potent mitogen for cardiac fibroblasts from adult rats.

The heart responds to increased haemodynamic load with growth of the ventricles. The rise in ventricle mass is due to increasing mass of the myocytes and proliferation of fibroblasts and smooth muscle cells. The accompanying adaptation and remodelling of the interstitium, e.

Okadaic acid induces cellular hypertrophy in AKR-2B fibroblasts: involvement of the p70S6 kinase in the onset of protein and rRNA synthesis.

At low concentrations (50 nM), okadaic acid (OA), an inhibitor of phosphatases 1 and 2A, inhibits platelet-derived growth factor-induced cell proliferation in late G1 (A. Simm et al., Exp.

Oncogene-associated transformation of rodent fibroblasts is accompanied by large morphologic and metabolic alterations.

Cellular growth, proliferative activity, cell volume and metabolism of four differently transformed cell lines were investigated. Studies were carried out with spontaneously immortalized and poorly tumorigenic Rat1 cells, c-mycl-transfected and non-tumorigenic M1 fibroblasts, as well as their T24Ha-ras-(co)-transfected counterparts Rat1-T1 and MR1. Ras-transfection of both Rat1 and M1 cells, which is associated with aggressive tumor growth in vivo, caused significant morphological alterations, namely a 30-50% decrease in cell volume.

Differential activation by platelet-derived growth factor-BB of mitogen activated protein kinases in starved or nonstarved AKR-2B fibroblasts.

More than 90% of serum-deprived (starved) AKR-2B mouse fibroblasts are stimulated to divided by the addition of platelet-derived growth factor (PDGF)-BB. In density-arrested (nonstarved) cells, PDGF-BB affords protection from cell death without stimulation of cell division. In both cultivation conditions the cells express similar amounts of PDGF beta-receptors and the receptor kinase activity was identical as judged by its autophosphorylation capacity.

Proliferative and metabolic capacity of rat embryo fibroblasts immortalized by c-myc depends on cellular age at oncogenic transfection.

It is well known that secondary rat embryo fibroblasts are immortalized and transformed with respect to requirements of growth factors by transfection with an overexpressed c-myc protooncogene. On the other hand, c-myc expression of nontransformed cells was shown to be independent of cellular age in vitro. In order to elucidate further the role of the c-myc protooncogene in the process of aging of rat embryo fibroblasts, we have transfected these cells at low (< or = 2) and at high (> or = 16) cumulative population doublings with SV40-promoter/enhancer-driven murine c-myc.

Platelet-derived growth factor isoforms prevent cell death during starvation of AKR-2B fibroblasts.

Confluent AKR-2B fibroblasts rapidly desintegrate upon removal of serum until a final density of approximately 50% of the initial value was reached after 12 h. This density remained unchanged for at least 48 h. Platelet-derived growth factor (PDGF)-BB stimulated more than 95% of these cells to divide.

Okadaic acid blocks PDGF-induced proliferation of AKR-2B fibroblasts at the transition from G1- to S-phase.

Okadaic acid (OA) at 100 ng/ml completely inhibited platelet-derived growth factor (PDGF)-BB-induced DNA synthesis but had no effect on early signals, i.e., PDGF receptor autophosphorylation or stimulation of inositolphosphate turnover.