Effect of hexadecane on piglet epidermal ornithine decarboxylase activity

Topical application of hexadecane has been shown to induce hyperproliferation and hyperkeratosis in rodent skin. The application of hexadecane to epidermis from the backs of piglets less than 1 week old resulted in a rapid biphasic-rise in the level of ornithine decarboxylase (ODC) activity. The second phase of the elevation of activity was suppressed by cycloheximide indicating that it resulted from de novo protein synthesis. The first, cycloheximide-insensitive phase presumably represents activation of existing enzyme. The activation of this latent ODC by hexadecane was independent of extracellular calcium. A similar degree of activation was observed using the bivalent-cation ionophore A23187 which augmented the hexadecane effect implicating a rise in intracellular calcium concentration as a possible cause for the activation possibly via the receptor-mediated phospholipid hydrolysis. The time-course of the ODC activation also corresponded with a rapid fall in cAMP levels indicating a possible role for cAMP in ODC regulation.     

Flow cytometric identification of Paclitaxel-accumulating subpopulations

An immunofluorescence procedure was developed for paclitaxel quantification at the single cell level via flow cytometry in Taxus cuspidata suspension cultures. Intracellular staining was validated via fluorescence microscopy. Paclitaxel content of isolated cells and protoplasts was compared to total paclitaxel levels measured via HPLC. Paclitaxel accumulation was significantly increased by elicitation with methyl jasmonate (100 microM) on day 7 post-transfer as compared to unelicited cultures. Maximum accumulation was observed by day 12 post-transfer in both total paclitaxel (approximately 0.25 mg/L) and the percentage of paclitaxel-accumulating cells (approximately 95%). A similar trend was observed with isolated protoplasts, although protoplasts accumulated only ca. 40-75% of the paclitaxel present in single cells. In unelicited cell cultures, a small subpopulation (ca. 3-5%) of single cells was shown to accumulate paclitaxel. Although nearly all cells were observed to accumulate paclitaxel in methyl jasmonate-elicited cell cultures, a high degree of cell-to-cell variation was observed in paclitaxel content. The identified subpopulations represent targets for cell sorting, which may be applied to develop higher-accumulating cell lines. The quantification of single cell paclitaxel content is useful for characterizing production variability in cell cultures and can be utilized to develop rational strategies to increase paclitaxel production.     

Immunocytochemical detection of ornithine decarboxylase.

Ornithine decarboxylase (ODC), a regulatory enzyme of polyamine biosynthesis, is involved in cell growth and differentiation. Lack of information about the exact cellular and subcellular localization of ODC is one of the main obstacles to precise interpretation of the biological roles of the ODC/polyamine system. Here we describe the development and optimization of an immunocytochemical method to detect ODC in cells and tissues. For this purpose a monoclonal antibody (MP16-2) against a defined epitope of ODC protein was developed. Specificity of the antibody for ODC was substantiated by Western blotting and ELISA analysis using cell and tissue homogenates. In cultured cells, optimal staining results were obtained after fixation with crosslinking fixatives followed by permeabilization with methanol. In rat tissues, ODC immunoreactivity was best preserved in paraffin sections fixed with Bouin's fixative. Antigen retrieval using SDS and citrate buffer substantially increased ODC immunostaining and decreased background staining. Localization studies of ODC in different cell lines showed that strongest staining for ODC was found in the nucleoplasm of mitotic cells, whereas confluent cells showed moderate perinuclear staining. Immunocytochemical studies of various rat tissues showed high cytoplasmic immunostaining of ODC in epithelial cells of kidney, prostate, and adrenal medulla of testosterone-treated rats, in glandular epithelium of small intestine, and in pancreas of neonatal and adult rats. (J Histochem Cytochem 47:1395-1404, 1999)     

Early changes in ornithine decarboxylase activity during friend leukemia cell differentiation

Agents such as dimethylsulfoxide, N,N′-dimethylformamide and bisacetyldiaminopentane that induce erythroid differentiation of Friend leukemia cells, cause a rapid increase in ornithine decarboxylase (EC 4.1.1.17) activity in intact cells during the ‘latent’ period preceding the accumulation of hemoglobin-containing cells. Blockage of erythroid differentiation with 5-bromo-2′-deoxyuridine did not prevent these alterations in enzyme activity. Addition of each chemical inducer in the extracts of these cells stimulate the basal levels of ornithine decarboxylase activity. These data indicate that the chemical inducers of differentiation modify the normal pattern of ornithine decarboxylase activity in this system.     

Induction of ornithine decarboxylase activity by 1-oleoyl-2-acetyl-glycerol in isolated mouse epidermal cells

1-Oleoyl-2-acetyl-glycerol induced a rise in ornithine decarboxylase activity in isolated epidermal cells in a concentration-dependent manner. The time course of the induction of ornithine decarboxylase by 1-oleoyl-2-acetyl-glycerol was similar to that by 12-O-tetradecanoylphorbol-13-acetate. A23187 did not enhance the enzyme induction caused by 1-oleoyl-2-acetyl-glycerol. Palmitoyl-DL-carnitine prevented the induction of the enzyme either by 1-oleoyl-2-acetyl-glycerol or 12-O-tetradecanoyl-phorbol-13-acetate. These results suggest that the activation of protein kinase C is an initial and essential event in the process of ornithine decarboxylase induction caused by 12-O-tetradecanoyl-phorbol-13-acetate.     

Posttranscriptional regulation of ornithine decarboxylase activity

We have used a Chinese hamster ovary cell line (DF3) that overproduces ornithine decarboxylase (ODC) to examine various parameters in the cell cycle-dependent regulation of this enzyme. Under a variety of conditions, alterations in the activity of ODC were accompanied by parallel changes in the levels of the protein, as measured by immunologically cross-reactive material (CRM). While putrescine has been known to suppress the induction of ODC, we have found that in DF3 cells 10(-4)M ornithine completely suppresses ODC activity. We also show that the levels of ODC mRNA are not modulated when the levels of ODC activity and CRM change drastically. The data can be interpreted in terms of models involving either an effect of putrescine on the translation of ODC mRNA, or on the activity of a relatively specific protease with ODC as its target.     

Location of renal ornithine decarboxylase activity

Renal ornithine decarboxylase (ODC) activity was found to be more prevalent in the medulla of the normal rat kidney than in the cortex. When renal ODC activity was stimulated by ethanol, growth hormone, ACTH, or corticosterone, proportional increases were observed in both medulla and cortex. After hypophysectomy, ODC activities fell equally in both areas of the kidney. The administration of cycloheximide, which is known to cause a rebound increase after six hours in overall renal ODC activity, was followed by an increase of medullary ODC activity while cortical activity remained suppressed.     

Flow cytometric sorting and analysis of human epidermal stem cell candidates

ESC (epidermal stem cells) play a central role in the regeneration of human epidermis. These cells are also responsible for wound healing and neoplasm formation. Efficient isolation of ESC allows their use in medicine and pharmacy as well as in basic science. Cultured keratinocytes and ESC may be used as biological dressing in burn injuries, chronic wounds and hereditary disorders. Therefore, the isolation and characterization of ESC have been goals in biomedical science. Here, we present a flow cytometric method for the isolation and analysis of human ESC candidates. The strategy presented for the isolation of ESC combines previously proposed enzymatic digestion and FACS‐sorting of the obtained cell suspension that utilizes morphological features, integrin‐β1 expression and Rh123 (Rhodamine 123) accumulation of the cells. We also performed a flow cytometric analysis of sorted cells using a cell tracer.     

Early changes in ornithine decarboxylase activity during Friend leukemia cell differentiation.

Agents such as dimethylsulfoxide, N,N'-dimethylformamide and bisacetyldiaminopentane that induce erythroid differentiation of Friend leukemia cells, cause a rapid increase in ornithine decarboxylase (EC 4.1.1.17) activity in intact cells during the 'latent' period preceding the accumulation of hemoglobin-containing cells. Blockage of erythroid differentiation with 5-bromo-2'-deoxyuridine did not prevent these alterations in enzyme activity. Addition of each chemical inducer in the extracts of these cells stimulate the basal levels of ornithine decarboxylase activity. These data indicate that the chemical inducers of differentiation modify the normal pattern of ornithine decarboxylase activity in this system.     

Peroxynitrite inhibits the activity of ornithine decarboxylase

Polyamines are required during cell proliferation, whereas NO has anti-proliferative properties. Ornithine decarboxylase (ODC) is a critical enzyme for the synthesis of polyamines. We tested the hypothesis that the modification of ODC by peroxynitrite (OONO-), a short-lived free radical formed from NO and superoxide produces a fall in ODC activity, and therefore polyamine synthesis and cell proliferation. The treatment of a rat recombinant ODC (rODC) with OONO- resulted in a dose-dependent inhibition of rODC activity with an IC50 of approximately 100 microM. A Western blot employing a specific antibody to nitrotyrosine revealed a dose-dependent nitration of rODC tyrosine residues. When intact IEC-6 cells were treated with ONOO-, ODC activity decreased by 49%. These data suggest a correlation between ODC activity and nitration, and a possible mechanism by which NO synthesis may modulate polyamine synthesis.     

Effect of epidermal growth factor on ornithine decarboxylase activity and urea synthesis in isolated rat hepatocytes.

Ornithine decarboxylase (ODC) activity is induced by protein-synthesis independent mechanisms in freshly isolated rat hepatocytes, incubated either without or with a mixture of amino acids in the incubation medium. Urea synthesis rates were two- to three-fold higher in those hepatocytes incubated in the presence of amino acids that in those lacking amino acids in the medium. Epidermal growth factor (EGF) delayed ODC induction, but only in the presence of amino acids. EGF significantly decreased ureagenesis when hepatocytes were incubated in the presence of amino acids and only endogenous substrates were available. No evidence of any link between ODC induction and urea synthesis was found.     

Central dopaminergic regulation of adrenomedullary ornithine decarboxylase activity

The administration of the two dopamine receptor agonists apomorphine (APM) and piribedil (PBD) to rats leads to an increase in ornithine decarboxylase (ODC) activity in the adrenal medulla. In this work, we have tried to elucidate the neural pathways involved in the regulation of this enzyme. The treatments used are: unilateral splanchnicotomy, spinal cord section, intraventricular injection of the neurotoxin 6-hydroxydopamine and section of the brain at various levels. Unilateral splanchnicotomy reduces very significantly the induction of ODC produced by either APM or PBD. Spinal cord section at either of two different levels (T₅ or T₂) also lowers the response to APM. Intracerebroventricular injection of 6-hydroxydopamine, on the other hand, elevates the mean response to APM, although not to a statistically significant extent. Section of the mesencephalon well below the periaqueductal gray does not alter the response of adrenomedullary ODC to APM. Transection of the diencephalon almost prevents it whereas hypothalamic deafferentation and incomplete diencephalic transection potentiate the effect of this drug. These observations strongly suggest that adrenomedullary ODC activity is predominantly regulated by a central system, originating mainly in the diencephalon-telencephalon and including a facilitatory dopaminergic component.     

Analysis of cell cycle subpopulations from cytometric data

As a result of experimental error, measurements of the DNA content of cells from proliferating populations give rise to histograms in which the G1-phase, S-phase and G2-phase distributions overlap. A new method for distinguishing these subpopulations is proposed, based on a model in which the S-phase subpopulation is made up of a number of uniformly overlapping log-normal curves, whose composite has a rectangular central part and sloping ends. The ratio between certain parameters of the slopes and the height of the rectangle is shown to be closely related to the degree of overlap of the constituents by a defined cubic polynomial. It is then possible to calculate the numbers of cells in each of the three phases of the cell cycle, even when only a few hundred cells have been measured.