Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in early Xenopus embryos induces cell dissociation and inhibits transition from the blastula to gastrula stage

Xenopus early embryos contain relatively low levels of S-adenosyl-methionine decarboxylase (SAMDC) and its mRNA. When SAMDC mRNA was injected into Xenopus embryos, it was preserved until the blastula stage and induced a large increase in SAMDC activity. The SAMDC-overexpressed embryos developed normally until the blastula stage but at the early gastrula stage cells which received the mRNA, dissociated autonomously and stopped synthesizing protein. In a hypotonic medium, the dissociated cells, and hence whole embryos, autolyzed. However, in isotonic media dissociated cells did not autolyze, although they did not divide and their DNA and RNA synthesis activity was greatly inhibited. The effects of SAMDC overexpression were abolished by coinjection of ethylglyoxal-bis(guanylhydrazone) (EGBG), a specific inhibitor of SAMDC. In SAMDC-overexpressed embryos the level of putrescine decreased and that of spermidine increased, though to limited extents, resulting in a considerable decrease in the putrescine/spermidine ratio. However, direct injection of spermidine did not mimic the effect of SAMDC overexpression, and putrescine coinjected with SAMDC mRNA to maintain the normal putrescine/spermidine ratio did not rescue the embryos. Conversely, the level of S-adenosylmethionine (SAM) greatly decreased and coinjection of SAM, which restored the level of SAM, rescued the embryos. We concluded that in SAMDC-overexpressed embryos a SAM-deficient state was induced and this caused cell dissociation and inhibition of transition from the blastula to gastrula stage. We suggest that the SAM-deficient embryos obtained in the present study provide a unique system for studying the cellular control mechanism underlying the blastula-gastrula transition.     

S-adenosylmethionine decarboxylase activity and utilization of exogenous putrescine are enhanced in colon cancer cells stimulated to grow by EGF

Polyamines spermidine and spermine and their precursor putrescine are necessary for cell growth. Polyamine content is high in rapidly growing malignant cells, due to enhanced putrescine synthesis by ornithine decarboxylase (ODC), and increased uptake. In contrast to other cells of the body, colon cancer cells are exposed to high putrescine concentrations from the lumen. AIMS: To investigate the utilization of luminal putrescine in colon cancer, we studied the effect of a potent mitogen, epidermal growth factor (EGF), on the activity of the enzyme responsible for putrescine conversion, S-adenosylmethionine decarboxylase (SAMDC), in Caco-2 cells. METHODS: Cell counts, ODC and SAMDC activities and intracellular polyamines were evaluated in the presence and absence of exogenous putrescine in concentrations resembling those normally present in the colonic lumen. RESULTS: ODC and SAMDC activity and putrescine uptake were strongly stimulated by EGF. Both synthesized and absorbed putrescine was rapidly converted to spermidine and spermine after EGF. Conversion pattern was identical in the cells stimulated with EGF only and EGF plus exogenous putrescine, indicating that, if stimulated to proliferate, colon cancer cells utilize the entire available putrescine pool. SAMDC inhibitor, methylglyoxal-bis-guanylhydrazone, induced growth arrest which was not reversed by exogenous putrescine, but only by high concentrations of spermidine. CONCLUSION: Enhanced proliferation in colon cancer cells is associated with increased SAMDC activity and rapid conversion of putrescine to spermidine and spermine. SAMDC might be a preferable target for therapeutic attempts to impair growth by reducing intracellular polyamine pools in colon cancer.     

Differential induction by methyl jasmonate of genes encoding ornithine decarboxylase and other enzymes involved in nicotine biosynthesis in tobacco cell cultures

A cDNA of tobacco BY-2 cells corresponding to an mRNA species which was rapidly induced by methyl jasmonate (MeJA) in the presence of cycloheximide (CHX) was found to encode ornithine decarboxylase (ODC). Another cDNA from a MeJA-inducible mRNA encoded S-adenosylmethionine synthase (SAMS). Although these enzymes could be involved in the biosynthesis of polyamines, the level of putrescine, a reaction product of ODC, increased slowly and while the levels of spermidine and spermine did not change following treatment of cells with MeJA. However, N-methylputrescine, which is a precursor of pyrrolidine ring of nicotine, started to increase shortly after MeJA-treatment of cells and the production of nicotine occured thereafter. The levels of mRNA for arginine decarboxylase (ADC), an alternative enzyme for putrescine synthesis, and that for S-adenosylmethionine decarboxylase (SAMDC), required for polyamine synthesis, were not affected by MeJA. In addition to mRNAs for ODC and SAMS, mRNA for putrescine N-methyltransferase (PMT) was also induced by MeJA. Unlike the MeJA-induction of ODC mRNA, MeJA-induction of SAMS and PMT mRNAs were blocked by CHX. The level of ODC mRNA declined after 1 to 4 h following MeJA treatment, while the levels of mRNAs for SAMS and PMT continued to increase. Auxin significantly reduced the MeJA-inducible accumulation of mRNAs for ODC, SAMS and PMT. These results indicate that MeJA sequentially induces expression of a series of genes involved in nicotine biosynthesis by multiple regulatory mechanisms.     

Sudden death in implantable cardioverter-defibrillator recipients: clinical context, arrhythmic events and device responses

Reports regarding the effect of all-trans-retinoic acid (RA) on the cell growth of retinal pigment epithelial cells (RPE) have been contradictory. The aims of this study are to clarify the in vitro effect of RA on RPE cells and to examine polyamine metabolism after RA stimulation. A 4-day incubation of fetal-calf-serum (FCS)-stimulated RPE cells with 10 or 25 microM RA significantly increased both cell number and [3H]thymidine incorporation. RPE cells grown over an extended period for 8 days also increased in number and reached full confluency. However, if the incubation was further extended to 12 days, no further increase in cell number was detected. RA treatment of FCS-stimulated RPE cells shifted the peak of ornithine decarboxylase (ODC) activity from 16 to 4 h. S-adenosylmethionine decarboxylase (SAMDC) activity and spermidine/spermine N1-acetyltransferase (SAT) activity of RA-treated RPE cells were significantly greater until 8 and 16 h after incubation, respectively. The putrescine content was significantly increased in RA-treated RPE cells up until 24 h, while spermidine, spermine and N1-acetylspermidine contents were significantly increased until 16 h. Our findings suggest that RA treatment increases the intracellular polyamine concentration of RPE cells via activation of ODC, SAMDC and SAT and that this results in the promotion of RPE cell growth until the cells reach full confluency.     

Effects of continuous light and darkness on the eyes of the troglobitic salamander Typhlotriton spelaeus

Larval Typhlotriton spelaeus collected from five caves in Pulaski Co., Missouri, were kept as larvae or induced to transform in darkness or continuous fluorescent illumination. Larvae maintained in darkness for 215 and 279 days had smaller eyes, smaller rod inner and outer segments, and fewer metaphase figures in the germinative zone of the neural retina than comparable larvae maintained in light (258 lux). Except for visual cell size, differences were small and for each characteristic exceptions were observed. One larva kept in light showed early retinal degeneration comparable to that in transformed adults to T. spelaeus. All larvae exhibited optomotor behavior both before and after the experiment. Among animals induced to transform by L-thyroxin and maintained in darkness 111 to 366 days, visual cell and pigment epithelium degeneration was more extensive and more frequent than in animals kept for the same length of time in light (237-298 lux). In darkness the frequency of animals with retinal degeneration increased between 111 and 366 days. In light some animals exhibited pigment epithelium reduction with normal visual cells, and others had free, pigmented cells in the subretinal space. These effects were not comparable to degeneration in darkness. Eyelids covered the eyes of only a few animals in both light and dark treatments. The extent of eyelid encroachment over the eye greater in darkness than in light. Most animals exhibited optomotor responses after experiments, but responses of animals kept in darkness were impaired in comparison to those of animals kept in light.     

Current concepts of economic interrelationships in the public health system

Effect of Hirudo therapy on the level of lipid peroxidation (LPO) products and catalase activity in the retina and pigmented epithelium (PE) after exposure to intensive illumination is assessed. Light injury to the eye (100,000 lux in 90 min) leads to accumulation of LPO products and suppression of catalase activity. Hirudo therapy after potent light exposure of the eye decreased the level of hydroperoxide and malonic dialdehyde and normalized retinal and PE catalase activity, thus exerting a therapeutic antioxidant effect. Hirudo therapy before illumination did not notably protect the eye as regards the content of LPO products and catalase activity.     

Polyamines regulate human medullary thyroid carcinoma TT-cell proliferation and secretion of calcitonin and calcitonin gene-related peptide

The significance of polyamines for the neoplastic proliferation and secretion of calcitonin (CT) and calcitonin-gene-related peptide (CGRP) by the human medullary thyroid carcinoma TT cell line was investigated. TT cells were cultured in vitro for 6 days with or without additions of pathway inhibitors of polyamine biosynthetic enzymes. Treatment of the cells with 1 mM of the specific L-ornithine decarboxylase (ODC) inhibitor DL-alpha-difluoromethylornithine (DFMO) resulted in a 97% decrease in ODC activity, lowered contents of putrescine (96%) and spermidine (85%) and cell proliferation rates (90%) along with a compensatory 15-fold increase in S-adenosyl-L-methionine decarboxylase (SAMDC) activity. DFMO treatment also led to a decrease in cellular content of CT (33%) and CGRP (26%), while the drug enhanced secretion of CT (31%) but depressed that of CGRP (26%), and elevated the ratio of CT to CGRP secreted into the medium by 74%. Ethylglyoxal bis(guanylhydrazone) (EGBG), a SAMDC inhibitor, at 100 microM evoked a similar reduction of cell proliferation and lowered the content of spermine by 81%. Furthermore, EGBG treatment caused a 34-fold increase in ODC activity and a subsequent 35-fold build-up of putrescine, but also seemed to stabilize SAMDC as evidenced by a highly enhanced SAMDC activity (approximately 200-fold) during enzyme assays in the absence of the inhibitor. EGBG exposure resulted in an increase in cellular CT content (110%) and secretion of the hormone (82%), while not affecting CGRP content or release.2+ EGBG effects were partially counteracted by DFMO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light-induced cGMP-phosphodiesterase activity in intact rat retinal rod cells as revealed by rapid-freezing enzyme cytochemistry

Cyclic guanosine monophosphate (cGMP)-phosphodiesterase (PDE) in the outer segment of vertebrate retinal rod cells is one of key enzymes mediating phototransduction. We report here on light-induced PDE activity in intact rat retinal rod cells processed by rapid-freezing enzyme cytochemistry, a new morphological technique that is a combination of rapid-freezing, freeze-substitution fixation, and subsequent enzyme cytochemistry. This technique quickly immobilizes and preserves both enzyme activity and the cell ultrastructure in a state approximating living conditions; consequently, it has proved useful for cytochemical detection of light-induced PDE activity. Using this technique we observed that the catalytic site of PDE molecules in rapid-frozen outer segments was predominantly located in the extradiscal spaces; PDE activity was significantly greater in light than in darkness; and illumination elicited marked increases in PDE activity in dark-adapted cells. Light-induced PDE activity was first cytochemically detected after 3 s of illumination and reached a peak within 5 s, at which time it was in virtually the same as that seen in fully light adapted cells. In addition, cytochemical guanosine triphosphatase (GTPase) activity in dark-adapted cells, as well as corresponding PDE activity, increased in a time-dependent manner with illumination duration; this acceleration in GTPase activity closely paralleled the PDE activity. Thus, our results suggest, in part, the existence of reciprocal regulation of PDE and activated transducin alpha subunit, thereby modulating light adaptation in rod cells.     

The effect of LH, FSH and pregnant mares' serum gonadotrophin on ornithine decarboxylase activity in thecal and granulosa tissue during follicular growth and atresia in laying hens (Gallus domesticus)

The effect of ovine LH, porcine FSH and pregnant mares' serum gonadotrophin (PMSG) on the activity of ornithine decarboxylase activity in theca and granulosa tissue during folliculogenesis in laying hens is described. The changes in the activity of ornithine decarboxylase induced by hormonal challenge was used to measure the sensitivity of the tissue to the hormone. Thecal tissue from small (< 6 mm) follicles showed a large increase in the activity of ornithine decarboxylase 3 h after treatment with LH, FSH and PMSG, in vivo, whereas ornithine decarboxylase activity in thecal tissue from large (> 8 mm) preovulatory follicles and atretic follicles did not respond to any of the hormonal treatments. Ornithine decarboxylase activity in granulosa tissue from the largest preovulatory follicle increased significantly 3 h after treatment with LH and PMSG in vivo; no effect was observed with FSH. Granulosa tissue from the third largest and fifth largest preovulatory follicles were refractory to the hormonal treatments. Basal activity of ornithine decarboxylase in granulosa tissue from preovulatory follicles increased as the follicles approached ovulation, whereas the activity in thecal tissue from the same follicles decreased. The difference in sensitivity of thecal tissue from small and large preovulatory follicles towards gonadotrophin treatment in vivo is correlated with the difference in the observed rate of atresia occurring within the two groups of follicles. Atresia is the common fate for small follicles, whereas it is a rare event for large preovulatory follicles under normal physiological conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro and in vivo antibacterial activities of TOC-50, a new parenteral cephalosporin, against Enterococcus faecalis

The object of the study was to compare resting pupil diameter in darkness and light, and the pupillary darkness and light reflexes between a group of young and elderly healthy subjects. Twelve young (eight men, four women; median age 19.5 years) and 14 elderly subjects (six men, eight women; median age 69 years) participated. Pupil diameter was monitored with an infra-red television pupillometer. Resting pupil size was measured in light (16 and 32 Cd m-2) and in darkness. The darkness reflex was elicited by switching off the ambient illumination (16 Cd m-2) for 1 s. The light reflex was elicited in darkness by short (200 ms) pulses of green (peak wavelength 565 nm) light at four ascending stimulus intensities (8.5 x 10(-3), 7.0 x 10(-2), 0.43 and 1.84 mW cm-2). The amplitude (mm) and maximum velocity (mm s-1) of the darkness reflex and the latency (ms), amplitude (mm), maximum constriction velocity (mm s-1) and 75% recovery time (s) of the light reflex were measured. The resting pupil diameter was found to be smaller in the elderly group at all three illumination levels (p = 0.001). The amplitude and maximum dilatation velocity of the darkness reflex were smaller for the elderly group (p = 0.001). The amplitude of the light reflex at the three highest light intensities and maximum constriction velocity at all light intensities were smaller in the elderly group (p = 0.002). Seventy-five per cent recovery time was longer in the elderly group (p = 0.02). There was no difference in the latency of the light reflex response between the two groups. The reduced pupil size, diminished darkness reflex amplitude and velocity, and prolonged recovery time of light reflex are consistent with sympathetic deficit in old age. Although the reductions in light reflex amplitude and constriction velocity in the elderly group at first sight would indicate a parasympathetic deficit in old age, they are more likely to be secondary to the grossly diminished pupil size.     

Reversible inactivation of the median raphe nucleus enhances consolidation and retrieval but not acquisition of passive avoidance learning in rats

Involvement of median raphe nucleus (MRN) in acquisition, consolidation and retrieval of passive avoidance (PA) was investigated with functional suppression of this area by lidocaine. Rats carrying a chronically implanted cannula aimed at the MRN were trained on a step-through passive avoidance task and received intra-MRN injection of lidocaine or saline 5 min before training or 5, 90 and 360 min after acquisition trial or 5 min before the retrieval test. Lidocaine MRN inactivation had no effect on PA learning. Lidocaine injected 5 and 90 min after the acquisition trial significantly enhanced avoidance of the dark compartment in comparison with the control group injected with saline. But PA retention was not affected by lidocaine injected 360 min after acquisition or 5 min before training. Retention latency significantly increased, when lidocaine injected 5 min before retrieval test. Step-through latency of naive rats was not affected by MRN blockade. Furthermore, reversible inactivation of MRN did not have a significant effect on locomotor activity. Our results indicate that the MRN contributes to PA consolidation at least until 90 min after acquisition and involves in PA retrieval. It is concluded that functional ablation of the MRN may disrupt the inhibitory actions of MRN projections to sub-cortical circuits participating in PA memorization and retrieval.     

Structure-activity relations of S-adenosylmethionine decarboxylase inhibitors on the growth of MCF-7 breast cancer cells

SAMDC is a key enzyme in the biosynthesis of spermidine and spermine, 2 polyamines that are essential for cell proliferation. Inhibition of polyamine biosynthesis is often targeted as a therapeutic strategy to suppress cancer cell growth as these cells contain elevated levels of polyamines. We examined the effect of a new group of SAMDC inhibitors, CGP33829, CGP35753, CGP36958, CGP39937, and CGP48664, (obtained from Ciba-Geigy, Basel, Switzerland), and their parent compound, MGBG, on the proliferation of MCF-7 breast cancer cells. MGBG had minimal effects on the proliferation of MCF-7 cells up to 6 microM concentration. In contrast, CGP48664 and CGP39937, containing 2 aromatic rings that delocalize the pi electron system of the backbone of MGBG, were potent inhibitors with 50% growth inhibition at 0.5 microM concentration. Other CGP compounds were less effective in inhibiting cell growth. The ability of CGP48664 to inhibit MCF-7 cell proliferation was related to its ability to inhibit SAMDC and to consequently deplete spermidine and spermine levels in the cell. Exogenous spermidine and spermine could reverse the growth inhibitory effects of this compound. CGP compounds also increased the activity of ODC, another enzyme involved in polyamine biosynthesis. Northern blot analysis of mRNA from MCF-7 cells progressing in cell cycle after G1 synchronization did not show an increase in ODC mRNA level by CGP48664. These data demonstrate structure-activity relationships of a series of MGBG derivatives on cell growth, enzyme activities, and polyamine biosynthesis in a hormone-responsive breast cancer cell line and suggest potential application of SAMDC inhibitors as therapeutic agents.     

Effect of matrine on mouse hepatitis and tumor necrosis factor production induced by Propionibacterium acnes/lipopolysaccharides

The effect of matrine (Mat) on lipopolysaccharides (LPS)-induced fatal hepatitis and tumor necrosis factor (TNF) production in Propionibacterium acnes (PA)-primed mice were studied. Mice were injected i.p. LPS (10 micrograms/mouse) 7 d after i.p. PA (0.5 ml/mouse) to induce fatal hepatitis. After i.p. LPS, serum TNF activity rose to 1657 +/- 406 kU.L-1 at 1.5 h and ALT activity increased up to 1,496 +/- 890 U.L-1 at 5 h. Six of 8 mice died within 5 h and the massive hemorrhagic necrosis of the liver was observed in all mice. Administration of Mat (10, 50 mg.kg-1, i.p., bid x 3 d) before the LPS injection markedly reduced the elevation of serum TNF and ALT activity in a dose-dependent manner, and diminished the mortality induced by LPS. Liver congestion and necrosis induced by LPS in PA-primed mice were ameliorated markedly by Mat pretreatment. Mat (62.5-250 mg.L-1) inhibited LPS-induced TNF release from PA-primed mouse peritoneal macrophage in vitro in a concentration-dependent manner. These results seggest that Mat protected PA-primed mice from the development of fatal hepatitis induced by LPS due to inhibition of TNF production.     

Differential sensitivity of membrane-associated pyrophosphatases to inhibition by diphosphonates and fluoride delineates two classes of enzyme

The participation of GABAergic mechanisms in the regulation of circadian rhythmicity by the suprachiasmatic nuclei (SCN) has been suggested from different lines of evidence. Little is known, however, whether GABA synthesis, release, uptake or content within the SCN may show a circadian pattern. The present results show that the activity of the GABAergic system within the SCN region of the rat exhibits circadian rhythmicity, which is manifested by correlative changes of the GABA content and the glutamic acid decarboxylase activity under the light/dark cycle, and by changes in the GABA content in animals kept under constant darkness.     

Tensile strength of lens capsules in eye-bank eyes

In this study, we investigated some factors contributing to renal regeneration after acute renal failure (ARF) induced by vitamin E (VE) deficiency and glutathione (GSH) depletion. Acute renal failure was induced by feeding rats a vitamin E-deficient diet for 6 weeks and then injecting buthionine sulfoximine (BSO), a glutathione-depleting agent. The level of hepatocyte growth factor (HGF), a renotropic factor for regeneration in the kidney, showed a transient increase at 5 hr after the BSO treatment. Subsequently, renal ornithine decarboxylase (ODC) activity, a marker of G1 phase, and labeling index (LI) of proliferating cell nuclear antigen (PCNA), a marker of DNA synthesis (S phase), reached peaks at 10 and 53 hr after the injection, respectively. Thus, it appears that the increase in ornithine decarboxylase activity and subsequent elevation in proliferating cell nuclear antigen labeling index following the increase in the hepatocyte growth factor level in the kidneys are closely related to the renal regenerative response after acute renal failure.     

High insulin-like growth factor binding protein 1 levels in cirrhosis: link with insulin resistance

The plasma membrane from Arabidopsis thaliana leaves (wild-type and F mutant) has been purified by two-phase partitioning and the H(+)-transport activity was tested in vitro in the presence of different concentrations of IAA. While 1 microM of IAA had no effect, higher concentrations (10 microM and 100 microM) were inhibitory in 25 short days grown wild-type plants. However, the activity was increased by about 16% after 10 supplementary short days of growth (from 25 to 35 SD) in the represence of 10 microM of IAA. No significant sensitivity to the tested concentrations of auxin was observed during the development in short days (30, 38, 42 SD) or even the plants were induced by a 24 h of continuous light. The variability was not observed for a given developmental stage, from one experiment to another, but it was also observed after light induction of F mutant plants.     

The utilization of anti-infective agents in primary care in La Rioja

The relationship between the rhythm in tissue nonprotein sulphydryl groups (NPSH) and that in 1,2-diamine (trans-I)-cyclohexane oxalatoplatinum (1-OHP) toxicity was investigated in a total of 266 male B6D2F1 mice, using buthionine sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase. Mice were synchronized with an alternation of 12 h light (L) and 12 h darkness (D; LD 12:12), and circadian time was expressed in hours after light onset (HALO). NPSH was measured in liver, jejunum and bone marrow at 0, 8 and 16 HALO. Dosing 1-OHP at these times achieved intermediate. high or low toxicity respectively. The physiological circadian rhythm in NPSH content was statistically significant in all tissues studied, with a maximum at the transition from D to L (0 HALO). BSO administration (450 mg/kg i.p., 4 h before sampling) induced a large depletion in liver and jejunum NPSH at their physiological peak (0 HALO), but exerted no significant effect at their trough (8 HALO). As a result, 24 h rhythm was suppressed in liver and jejunum, but remained similar to the physiological one in bone marrow. BSO enhanced 1-OHP-induced mortality and jejunal toxicity, but exerted no significant effect upon bone marrow toxicity. Despite these differences, 1-OHP remained least toxic at 16 HALO, near the middle of the dark span, which corresponds to maximum activity in the circadian rest/activity cycle. Our results show that mean NPSH levels in liver seem to account for the mean level of 1-OHP toxicity, while jejunal NPSH rhythm plays an important role in the intestinal toxicity rhythm of this drug.     

Intrauterine transfusions influence fetal leukocyte counts and subsets

Histamine is an important mediator in allergic reactions, gastric acid secretions, and neurotransmission in the central nervous system. Basophils and mast cells are the main sources of histamine, which is formed from L-histidine by histidine decarboxylase (HDC). However, the regulatory mechanism of HDC in these cells remains unclear. We examined the regulation of HDC activity and gene expression using a unique human mast cell line, HMC-1, after stimulation with phorbol 12-myristate 13-acetate (PMA) or ionomycin. HDC activity was increased from 52.1+/-0.4 (mean+/-standard deviation) to 154+/-6.9, or 105.6+/-6.2 pmol/min/mg protein (n = 3), 4 hours after stimulation with PMA (10 ng/mL) or ionomycin (10[-6] M). Although actinomycin D had no effect on this increase, cycloheximide completely inhibited the increase caused by these stimuli. The population of HMC-1 cells containing HDC protein was increased after stimulation with either PMA or ionomycin as evaluated by immunocytochemical analysis with anti-HDC antibody as a marker. HMC-1 constitutively expressed HDC mRNA, and its level was not increased with these stimuli. These results suggest that the increase of HDC activity in HMC-1 induced by PMA or ionomycin is regulated at the translational level.