Lo Persson

Molecular genetics of polyamine synthesis in eukaryotic cells

The polyamines putrescine, spermidine and spermine are important cellular constituents involved in the regulation of cell growth and differentiation. Their intracellular levels are regulated by a multitude of mechanisms affecting their synthesis, degradation, uptake and excretion. As a result of the application of molecular biology techniques, some of these mechanisms are presently being unravelled, and are providing a basis for the rational development of novel agents effective against proliferative disorders and various parasitic diseases.

Smooth Muscle Cell Hypertrophy and Hyperplasia in the Rat Detrusor after Short-Time Infravesical Outflow Obstruction

Infravesical outflow obstruction of a duration of 3 days, 10 days and 6 weeks was induced in female rats by a standardized degree of urethral obstruction. A striking ability of the detrusor to respond to an acute obstruction with both smooth muscle cell hypertrophy and hyperplasia leading to an approximately 10-fold increase of the total muscle mass of the bladder wall after 6 weeks of obstruction was found. The maximum relative growth rate was greatest in the bladders subjected to obstruction for only 3 days, and this was also reflected by the concentrations of ornithine decarboxylase and the polyamines spermidine and spermine. The total amount of DNA in the detrusor was already significantly increased after 3 days, while a 9-fold increase was observed in the group subjected to obstruction for 6 weeks. At this time the smooth muscle cell nucleus volume also showed a considerable increase, and a comparison of the nucleus density and the DNA concentration suggested an increased mean DNA content per muscle cell nucleus. The concentration of RNA in the detrusor had already increased significantly after 3 days and also remained so after 10 days and 6 weeks of obstruction, a finding that coincided with the abundant appearance of nucleoli seen at electron microscopic investigation. The previously reported decreased ability to pressure production at small volumes in the rat urinary bladder subjected to an acute infravesical outflow obstruction might thus, at least in part, be due to changed contractile properties of the hypertrophic cells, and/or to an inefficient incorporation of the newly formed smooth muscle cells.

Translational regulation of ornithine decarboxylase by polyamines

The activity of ornithine decarboxylase (ODC), the first and rate‐limiting enzyme in the polyamine biosynthetic pathway, is dramatically increased in proliferating cells. In addition to transcriptional regulation of ODC, the present study shows that the enzyme is regulated at the translational level by putrescine and spermidine. ODC synthesis is inhibited by an increase and stimulated by a decrease in their cellular content. Spermidine is a more potent negative regulator than is putrescine. The effects of polyamines on ODC synthesis were not attributable to changes in the cellular content of ODC mRNA, thus demonstrating regulation at the translational level.

Arginine Consumption by the Intestinal Parasite Giardia intestinalis Reduces Proliferation of Intestinal Epithelial Cells

In the field of infectious diseases the multifaceted amino acid arginine has reached special attention as substrate for the host´s production of the antimicrobial agent nitric oxide (NO). A variety of infectious organisms interfere with this part of the host immune response by reducing the availability of arginine. This prompted us to further investigate additional roles of arginine during pathogen infections. As a model we used the intestinal parasite Giardia intestinalis that actively consumes arginine as main energy source and secretes an arginine-consuming enzyme, arginine deiminase (ADI). Reduced intestinal epithelial cell (IEC) proliferation is a common theme during bacterial and viral intestinal infections, but it has never been connected to arginine-consumption. Our specific question was thereby, whether the arginine-consumption by Giardia leads to reduced IEC proliferation, in addition to NO reduction. In vitro cultivation of human IEC lines in arginine-free or arginine/citrulline-complemented medium, as well as in interaction with different G. intestinalis isolates, were used to study effects on host cell replication by MTT assay. IEC proliferation was further analyzed by DNA content analysis, polyamine measurements and expressional analysis of cell cycle regulatory genes. IEC proliferation was reduced upon arginine-withdrawal and also in an arginine-dependent manner upon interaction with G. intestinalis or addition of Giardia ADI. We show that arginine-withdrawal by intestinal pathogens leads to a halt in the cell cycle in IECs through reduced polyamine levels and upregulated cell cycle inhibitory genes. This is of importance with regards to intestinal tissue homeostasis that is affected through reduced cell proliferation. Thus, the slower epithelial cell turnover helps the pathogen to maintain a more stable niche for colonization. This study also shows why supplementation therapy of diarrhea patients with arginine/citrulline is helpful and that citrulline especially should gain further attention in future treatment strategies.

Ornithine decarboxylase inhibitors increase the cellular content of the enzyme: implications for translational regulation.

Ehrlich ascites tumor cells grown in the presence of inhibitors of ornithine decarboxylase (EC 4.1.1.17) exhibited an elevated content of this enzyme. The increase could not solely be explained by a decrease in the degradation rate of the enzyme. Instead a stimulation of enzyme synthesis, probably mediated via the polyamine-depleting properties of the inhibitors, is suggested. The enhancement of cellular ornithine decarboxylase content was not accompanied by any significant changes in the amount of ornithine decarboxylase mRNA, indicating a regulation at the level of translation.

Multiple forms of rat stomach histidine decarboxylase may reflect posttranslational activation of the enzyme

Histidine decarboxylase (HDC) catalyzes the formation of histamine, which takes part in a variety of physiological processes including gastric acid secretion, neurotransmission and inflammation. While purified rat HDC is a homodimer of approximately 54 kDa subunits, molecular cloning of mammalian HDC has revealed that HDC mRNA encodes a 74 kDa protein. This discrepancy in molecular mass may be due to a posttranslational processing of the primary translated product of rat HDC mRNA. In the present study we demonstrate that full-length rat HDC expressed in Escherichia coli or in an in vitro transcription/translation system is enzymatically inactive, while expression of a C-terminus truncated HDC (reducing the molecular mass to 54 kDa) gave rise to a protein with high enzyme activity in the same expression systems. COS-7 cells expressing truncated HDC displayed high HDC activity, whereas COS-7 cells expressing full-length HDC displayed low activity. Western blot analysis of fetal rat liver and oxyntic mucosa of gastrin-stimulated rats revealed the presence of both full-length HDC (approximately 73 kDa) and a approximately 53 kDa subunit form in addition to an intermediate form of about 63 kDa. The results are in line with the view that rat HDC may be produced as an enzymatically inactive proenzyme which is processed to give rise to the active enzyme.

Reversibility of detrusor hypertrophy and hyperplasia after removal of infravesical outflow obstruction in the rat

The present investigation was performed in order to study the degree of reversibility of changes of the rat detrusor secondary to infravesical outflow obstruction for various periods of time. In a previous study it was shown that outflow obstruction induced both hypertrophy and hyperplasia of the smooth muscle cells. This was determined by means of morphometry and analyses of DNA and RNA. The results of the present investigation confirm the findings of previous studies that these changes to a considerable extent are reversible after removal of the outflow obstruction. This seems to be true both in animals obstructed for a short period of time (10 days) and in animals where the hypertrophy and hyperplasia processes have diminished (six weeks).

Localization of ornithine decarboxylase by immunocytochemistry

Abstract The present work describes for the first time the cellular localization of ornithine decarboxylase (EC 4.1.1.17) (ODC ★ ) by immunocytochemistry. The kidney of gonadectomized male mice contained no demonstrable immunoreactive ODC. Testosterone administration resulted in the appearance of numerous ODC-containing cells in the tubules of the renal cortex. The glomeruli and the medullary part of the kidney were devoid of immunoreactivity. Exposure of the antiserum to purified mouse kidney ODC effectively blocked the immunostaining of the cortical cells. Additional facts supporting the specificity of the ODC-immunoreaction are presented.

Histidine decarboxylase in rat stomach ECL cells: relationship between enzyme activity and different molecular forms.

Mammalian HDC mRNA encodes a protein with a molecular mass of 74 kDa. The reported molecular mass for the purified HDC subunit is 53-55 kDa. Western blot analysis of extracts of rat gastric mucosa and fetal rat liver has revealed the presence of at least three different forms of HDC immunoreactivity, having molecular masses of about 74, 63 and 53 kDa. There is evidence from previous studies that full length rat HDC is enzymatically inactive and that activation requires C-terminal truncation. In the present study we examined the various immunoreactive HDC forms in rat oxyntic mucosa and their response to treatments known to affect the HDC activity. Freely fed rats and hypergastrinemic rats (treated with gastrin or the proton pump inhibitor omeprazole) had higher oxyntic mucosal HDC activity and HDC mRNA level than fasted or untreated rats. The difference in HDC activity was greater than the difference in HDC mRNA level. Western blot analysis confirmed the existence of the 74, 63 and 53 kDa HDC forms in the oxyntic mucosa. All three forms were more abundant in the oxyntic mucosa of freely fed and hypergastrinemic rats than in the mucosa of fasted or untreated rats. Of the three HDC forms, the 63 kDa form was the predominant one, the 73 kDa form was quantitatively insignificant by comparison and the 53 kDa form was at or below the limit of detection in fasted rats. The activity of HDC was well correlated to the amount of the 63 kDa HDC form. Administration of cycloheximide to hypergastrinemic rats (undergoing omeprazole treatment) resulted in a rapid decline of the HDC activity (estimated half-life 1 h and 50 min). The 63 kDa HDC form disappeared with a rate that corresponded to the decline in HDC activity. The two other HDC forms seemed to have a slower turnover. Our findings suggest that the 63 kDa form is enzymatically active. The results do not allow any conclusion as to the functional activity of the 74 and 53 kDa forms.

Antileishmanial Effect of 3-Aminooxy-1-Aminopropane Is Due to Polyamine Depletion

ABSTRACT The polyamines putrescine, spermidine, and spermine are organic cations that are required for cell growth and differentiation. Ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthetic pathway, catalyzes the conversion of ornithine to putrescine. As the polyamine biosynthetic pathway is essential for the growth and survival of Leishmania donovani, the causative agent of visceral leishmaniasis, inhibition of the pathway is an important leishmaniacidal strategy. In the present study, we examined for the first time the effects of 3-aminooxy-1-aminopropane (APA), an ODC inhibitor, on the growth of L. donovani. APA inhibited the growth of both promastigotes in vitro and amastigotes in the macrophage model, with the 50% inhibitory concentrations being 42 and 5 μM, respectively. However, concentrations of APA up to 200 μM did not affect the viability of macrophages. The effects of APA were completely abolished by the addition of putrescine or spermidine. APA induced a significant decrease in ODC activity and putrescine, spermidine, and trypanothione levels in L. donovani promastigotes. Parasites were transfected with an episomal ODC construct, and these ODC overexpressers exhibited significant resistance to APA and were concomitantly resistant to sodium antimony gluconate (Pentostam), indicating a role for ODC overexpression in antimonial drug resistance. Clinical isolates with sodium antimony gluconate resistance were also found to overexpress ODC and to have significant increases in putrescine and spermidine levels. However, no increase in trypanothione levels was observed. The ODC overexpression in these clinical isolates alleviated the antiproliferative effects of APA. Collectively, our results demonstrate that APA is a potent inhibitor of L. donovani growth and that its leishmaniacidal effect is due to inhibition of ODC.