Seasonal and physiological aspects of the isolation of tobacco protoplasts

The viability of tobacco ( N. tabacum – L. cv. Xanthi-nc) protoplasts isolated during the winter months is affected by membrane calcium, total calcium, plant age and supplementary lighting. Feeding calcium nitrate and calcium chloride helps to increase protoplast stability. Feeding is important because it increases membrane calcium, but it also has other beneficial effects which may be connected with increased nitrate and chloride (anion) uptake and cation/anion interaction. Whatever optimal feeding regime is chosen, it must be used in conjunction with the correct harvesting practice, which depends on whether or not supplementary light is given. Plants grown without supplementary light are best harvested between 8–9 weeks. If supplementary light is given, older plants (with and without supplementary calcium feeding) of approx. ten weeks are better for protoplast isolation. Plants grown under supplementary light have low leaf shape index (LSI) values (i.e. wider leaves) compared to plants in low light.
Spring and summer calcium feeding treatments had no significant effect on increasing total or membrane calcium. Likewise, there was no significant correlation between total/membrane calcium and percentage protoplast viability from days 0–5; and protoplasts isolated from plants grown in shade and fed with calcium salts, were no more stable than protoplasts from control plants. Irrespective of the feed treatments given, spring and summer plants older than about fifty days and grown under shaded and normal light conditions, produced poor protoplasts despite the fact that there was more calcium present in the membranes compared to protoplasts of younger plants. This decrease in percentage protoplast viability appears to be associated with changes in leaf shape. As plants age from 49–63 days, the leaf shape index (LSI) values increase (i.e. leaves are becoming narrower) and % protoplast viability decreases.     

Some aspects of hepatic function in feral brown trout, Salmo trutta, living in metal contaminated water

Brown trout, Salmo trutta, exposed to heavy metals (mainly Cd and Zn) for at least 2 years in the Eagle River, Colorado, were examined for liver size and activity of the growth-promoting enzyme, ornithine decarboxylase (ODC) and compared to trout living in an uncontaminated site. Liver-somatic index (LSI) was greater for trout living in the uncontaminated site with the LSI of females being significantly greater than that of males. The LSI for females at the uncontaminated site was greater than that of females at the contaminated site, but males were not different statistically. ODC activity in the livers of both males and females was lower at the contaminated site. However, males and females did not differ with respect to ODC activity. These data suggest that chronic exposure to heavy metals may have important implications for growth and reproduction and possibly survival. The activity of ODC in liver might serve as a useful biomarker when assessing chronic toxicity of metals to naturally reproducing fish populations.     

Thyroid function and polyamines. III. Changes in ornithine decarboxylase activity and polyamine contents in the rat thyroid during hyperplasia and involution

Changes in ornithine decarboxylase (ODC) activity and in polyamine contents of the rat thyroid were studied under various experimental conditions. Methylthiouracil (MTU) treatment produced several-fold increases in the thyroid ODC activity and in the content of putrescine, spermidine and spermine within a week. While serum thyrotropin (TSH) levels increased gradually up to 3 weeks, the content of both putrescine and spermidine tended to reach a plateau after 2 weeks of the goitrogen treatment; spermine content continued to increase progressively for 3 weeks. Discontinuance of MTU at 7 days resulted in a rapid decline in the elevated thyroid ODC activity, followed by a diminution of putrescine, spermidine and RNA contents. Thyroidal putrescine, spermidine and RNA responded more sensitively to both introduction and withdrawal of TSH stimulation than thyroidal spermine and DNA. Excess iodide, having no effect on the basal level of thyroid ODC, suppressed the MTU-induced increase in this enzyme activity without affecting circulating TSH, thyroxine (T4) and triiodothyronine (T3) levels. There was a significant negative correlation between the ODC activity and intrathyroidal concentration of iodine in MTU-pretreated rats. Theophylline increased the thyroid weight and ODC activity when given to rats fed with a subeffective dose of MTU. Analyses of serum TSH, T4, T3 and of thyroidal iodine revealed that TSH-induced thyroid ODC activity was suppressed by increased circulating thyroid hormones and/or intrathyroidal iodine. Furthermore, it was suggested that thyroid hormones and excess iodide acted directly on the thyroid to alter polyamine biosynthesis, possibly by changing the responsiveness of the gland to TSH.     

Seasonal patterns of ornithine decarboxylase activity and levels of polyamines in relation to the cytology of germinal cells during spermatogenesis in the sea star, Asterias vulgaris

The activity of ornithine decarboxylase (ODC) and levels of polyamines were measured in the testes of Asterias vulgaris collected throughout an annual spermatogenic cycle. Samples of the testes were prepared for light and electron microscopy to observe the associated changes in the cytology of germinal cells. The specific activity of ODC increased at the onset of testicular growth, decreased during the coldest period of the winter when testicular growth was minimal, and increased again early in the spring when testes grew maximally. Increased activity of ODC resulted in increased levels of polyamines and was correlated with either mitotic or meiotic germinal cell divisions, or both. Spermine levels were always greater than putrescine, followed by spermidine. Highest levels of polyamine synthesis coincided with the onset of spermatogonial proliferation during the fall and with the period of meiotic differentiation and spermiogenesis in the spring. Mid-summer (July) testes were small (0.3-0.5 gonad index (GI)) and contained amitotic spermatogonia arrested in G(1) of the cell cycle. Mitotic and pre-meiotic testes (October/November) increased slightly in size (0.3-1.4 GI) and contained actively dividing spermatogonia, most of which differentiated into primary spermatocytes. Testes from February and March were large (1-6.75 GI), but the proliferative status of their spermatogonia and primary spermatocytes varied. Spermatogonia and primary spermatocytes from early February testes were not dividing. In testes obtained in March, both spermatogonial mitosis and meiosis of spermatocytes resumed, coincident with increased field water temperatures.     

Temporal Profiles of Proteins Responsive to Transient Ischemia

The responses of long and short half-lived proteins to ischemia were measured in rat brain during 6 days of recovery from 30 min of transient forebrain ischemia produced by four-vessel occlusion. At the end of the ischemic interval, the neocortical activities of four vulnerable enzymes [ornithine (ODC) and S-adenosylmethionine (SAMDC) decarboxylases, and RNA polymerases I and II] were unchanged, but within 30 min of reperfusion, their activities dropped by 25-50%. The loss of substance P in the striatum and substantia nigra was slower, reaching about 50% by 12 h. On the other hand, the activities of 5 long half-lived enzymes did not change in the neocortex at 5 and 15 h of reperfusion and regional protein concentrations were essentially unaffected over 6 days survival. The rate and extent of normalization of the amounts or activities of the vulnerable proteins varied. RNA polymerase II and ODC activities were restored within 4 h, and ODC showed a biphasic increase in activity, with peaks at 10 h and 2-3 days. RNA polymerase I and SAMDC activities were restored by 18 h and 5 days, respectively, whereas substance P concentrations did not completely recover, even at 6-15 days. The greater the regional reduction of blood flow during ischemia, the larger the net change (gain or loss) of SAMDC or ODC activity and the longer the time required to normalize the activities of these enzymes. The average rate of proteolysis, assessed by measuring the rate of clearance of 14C from protein prelabeled with [14C]bicarbonate, was abnormal during the first 2 days of reperfusion. Postischemic changes in both protein synthesis and degradation could affect the amounts of some of the proteins responsive to transient ischemia.     

Effects of feeding frequency and voluntary salt intake on fluid and electrolyte regulation in athletic horses.

The effect of feeding frequency and voluntary sodium intake (VSI) on fluid shifts and plasma aldosterone concentration (PAC) were studied at rest and after exercise in six athletic horses. The horses were fed twice a day (2TD) and six times a day (6TD) for 25 days for each protocol, according to a changeover design. VSI was measured by weighing each horse's salt block daily. Feeding 2TD or 6TD caused no major alterations in fluid shifts, but in the 2TD treatment there was a postprandial increase in plasma protein concentration and osmolality that lasted <1 h. PAC and VSI were not affected by feeding frequency. VSI ranged from 0 to 62 mg x kg body weight-1 x day-1 and caused significant alterations in PAC. At VSI <26 mg x kg body weight-1 x day-1, a diurnal rhythm for PAC was noted. Water intake, fecal concentrations of sodium and potassium, and packed cell volume during exercise were influenced by VSI. The response to exercise did not differ between treatments. In conclusion, VSI, but not feeding frequency, has significant effects on fluid and electrolyte regulation in athletic horses.     

Early events in the response of fast skeletal muscle to chronic low-frequency stimulation. Polyamine biosynthesis and protein phosphorylation.

The concentrations of putrescine, spermidine and spermine and the activities of ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (SAM-D) were investigated in fast muscle subjected to chronic low-frequency electrical stimulation. Both ODC and SAM-D activities increased markedly between 18 and 48 h of stimulation. Changes in enzyme activities were followed by phasic elevations in the concentrations of putrescine, spermidine and spermine. Peak levels were reached first by putrescine at 3-4 days, followed by spermidine at about 9 days and then by spermine at about 11 days. A possible relationship was sought between these events and changes produced in vitro in the phosphorylation pattern of cytoplasmic proteins and the total activity of cyclic AMP-dependent protein kinase. However, during the early stages of stimulation, no prominent changes were seen either in the phosphorylation pattern or in the activity of cyclic AMP-dependent protein kinase. These characteristics changed significantly at a later stage (by 12 days of stimulation) and became indistinguishable from those of slow muscle by 3 to 4 weeks of stimulation.     

Response of muscle-based biochemical condition indices to short-term variations in food availability in post-flexion reared sea bass Dicentrarchus labrax (L.) larvae

Six condition indices based on RNA, total soluble protein and two metabolic enzymes [lactate dehydrogenase (LDH) and citrate synthase (CS)] were analysed in muscle tissue of individual larvae, post-flexion reared sea bass Dicentrarchus labrax using DNA and total soluble protein as standards for size. In addition, the effect of 2 days of food deprivation on the cell proliferation rates was assessed. The RNA:DNA best reflected short-term changes in feeding conditions. If standardized by DNA content, LDH activity was a better indicator of condition than any other index but RNA:DNA. Further, the analysis of cell proliferation rates in muscle from 26 day-old larvae proved useful in distinguishing continuously fed larvae from individuals subjected to 2 days of fast.     

Effect of dexamethasone on the activity and expression of ornithine decarboxylase in rat liver and thymus

A single intraperitoneal injection of the synthetic glucocorticoid dexamethasone into rats resulted in a marked stimulation (more than 60-fold) of hepatic ornithine decarboxylase (ODC) at 4 h after the injection, whereas the enzyme activity in thymus was almost totally (about 95%) depressed at the same time. The stimulation of ODC activity in liver was in all likelihood attributable to a greatly enhanced accumulation of mRNA species for the enzyme as revealed by Northern blot and dot-blot hybridization analyses. ODC activity in thymus, in response to dexamethasone, was only 5% of that found in control animals, but this decrease was apparently not accompanied by similar reductions of the levels of ODC message, which was in fact decreased only by 50% at the maximum. In addition to two mRNA species (2.1 and 2.6 kilobases; kb), typical to mouse cells, rat tissues seemed to contain a third hybridizable message for ODC, smaller (1.6 kb) than the above-mentioned species and not seen in samples obtained from mouse or human cells. Interestingly, these smaller poly(A)+ RNA sequences, hybridizable with cDNA complementary to mouse ODC mRNA, were apparently constitutively expressed, as the treatment with glucocorticoid altered the amount of these sequences only slightly.     

The effect of a mealybug infestation on the activity of amino acid decarboxylases in orchid leaves

Changes in the activity of lysine decarboxylase (LDC), tyrosine decarboxylase (TyDC), and ornithine decarboxylase (ODC) within orchid (Phalaenopsis × hybridum ‘Innocence’) leaves, infested by two mealybug species: Pseudococcus longispinus (Targ. Tozz.) and Pseudococcus maritimus (Ehrh.) were quantified. The pattern of changes was dependent on the insect species and duration of infestation. P. longispinus feeding increased LDC and TyDC activity after one week during the total period of observations. This species inhibited ODC activity after one week but increased later. P. maritimus decreased LDC activity in orchid leaves at all studied terms. TyDC action also went up during the first week of the infestation and was reduced after two weeks, while ODC was decreased after one day and induced later. The mechanism for the participation of analysed amino acid decarboxylases in local and/or systemic steps of orchid responses to mealybug infestation is discussed.     

Fasting does not abolish the diurnal oscillation of ornithine decarboxylase activity in Morris hepatoma 5123-C.

The activities of ornithine decarboxylase (ODC) and tyrosine aminotransferase (TAT) were determined under conditions of feeding or fasting in the hepatomas and livers of rats bearing Morris hepatoma 5123-C. Prior to killing, the animals were entrained to a schedule of 12 hours of light followed by 12 hours of darkness with food (60% protein) available only during the first two hours of the dark period. With food available, ODC and TAT activities displayed diurnal oscillations in hepatomas and host livers, and in the livers of control (non-tumor bearing) animals, characterized by rapid increases in enzyme activity coincident with the onset of feeding followed by a decline to pre-feeding levels. When food was withheld the increase in ODC activity in host and control livers, and TAT activity in hepatoma, host and control livers was not evident. However, withholding food did not abolish the diurnal oscillation of ODC activity in hepatoma 5123-C.     

Expression of ornithine decarboxylase in pancreatic development

We examined the relationship between ornithine decarboxylase (ODC) and growth and differentiation in the developing rat exocrine pancreas. The ODC activity profile showed 2 distinct stages of increases with the first occurred at 14–16 days of age, and a second at 21–23 days of age. Growth parameters evaluated as gains in tissue mass, protein and DNA content in the pancreas indicated a low growth rate soon after birth with a transition to a much more rapid growth rate around the age of 20–21 days, a time corresponded to the second rise in ODC activity. Differentiation parameters evaluated as the accumulation of trypsinogen, amylase and lipase showed different temporal changes. While the rate of accumulation of all three enzymes was relatively low following birth, a rapid rate of accumulation of trypsinogen and amylase started around 15–16 days, a time corresponding to the first rise in ODC activity. Lipase, however, did not show an increase in its accumulation until around age 20 days. These results indicate that a rise in ODC activity is closely associated with growth and differentiation in the developing rat pancreas. To further examine this issue, the steady state levels of ODC mRNA in developing rats were evaluated by Northern blots probed with an ODC cDNA. The developmental profile of ODC mRNA showed a broad peak with a pronounced shoulder occurring at 10 days of age. A higher peak was reached around 20 days of age, then dropped precipitously to a very low level at the age of 24 days. This temporal changes in the level of ODC mRNA show good relationship to the changes in ODC activity suggesting that the control of ODC expression occurs at least in part at the pre-translational level.     

Uterine growth responses of the mature castrate rat to estradiol-17B

To examine estrogen-stimulated uterine growth we have monitored changes in uterine DNA synthesis, ornithine decarboxylase (ODC) activity and protein content as well as luminal epithelial (LE) cell mitotic index and ultrastructural changes. We have utilized this model to examine castrate mature rat uterine growth as a function of time between 18 and 40 hours following a single injection of 25.0 ug of estradiol-17B. LE cell mitotic index and protein content increases were maximally elevated as early as 18 hours postinjection while uterine ODC activity was maximal at 28 hours; uterine DNA synthesis increases continued throughout the experiment. In addition, the infusion of either 1 or 2 ug E2 plus progesterone over a 24 hour period, stimulated elevated ODC activity under both treatment regimens and LE cell mitotic index which was inversely related to E2 dose.     

Diurnal variations in activity of four pyridoxal enzymes in rat liver during metabolic transition from high carbohydrate to high protein diet.

The diurnal variations in enzyme activities including tyrosine aminotransferase (TAT), ornithine decarboxylase (ODC), ornithine aminotransferase (OAT) and serine dehydratase (SDH) have been studied in rats trained to a 2 hour meal feeding schedule (″2+22″) during metabolic transition from 12.5 to 60% protein diets over a period of 21 days. Although the maximal TAT activity on the first day was slightly lower compared with other days, both TAT and ODC activities adapted rapidly to the increased dietary protein from the first day. The responses of TAT and ODC to the food were so rapid that the maximal value was observed only 4 hrs after the onset of feeding. After each feeding ODC activity decreased rapidly after 4 hours, while TAT activity declined only after 6 hours had elapsed. No clear diurnal rhythm was observed in either OAT or SDH, though OAT activity tended to decrease from the beginning of the dark period and to resume a slow adaptation after about four hours. In contrast to ODC and TAT both OAT and SDH required about 7 days to fully adapt to the high protein diet. The activities of the four enzymes were also compared after 4 groups of rats had been adapted to the ″2+22″ feeding of 12.5, 30 and 60% protein diets and to 60% diet, $\text{ad}$$\text{libitum}$, respectively. The enzyme activities were not directly proportional to the protein content of the diets although higher activity was observed on the high protein diets. The diurnal variations in both TAT and ODC were observed in all ″2+22″ groups although the timing of the peak values were slightly different from each other. The maximal activities of TAT were found at earlier times in 12.5 and 30% protein groups than in the 60% protein group. The peak time for ODC activity was found at a later time in the 12.5% protein group than in rats fed 30% and 60% protein. $\text{Ad}$$\text{libitum}$ rats fed 60% protein maintained relatively high levels of TAT activity compared to the rats on the schedule. However, the maximal activity of ODC on the 60% ″2+22″ protein diet $\text{ad}$$\text{libitum}$ was so low that a diurnal rhythm was not clearly evident.     

Developmental regulation of ornithine decarboxylase (ODC) in rat testis: comparison of changes in ODC activity with changes in ODC mRNA levels during testicular maturation.

These studies were undertaken to analyze the changes in testicular ornithine decarboxylase (ODC) mRNA levels and ODC activity in rats from birth to maturity. Levels of ODC mRNA were initially low in animals aged 10-17 days. Beginning at 21 days, ODC mRNA levels began to rise, reaching maximal levels by 40 days (p less than 0.01). The size of the 2.2- and 2.6-kb ODC mRNAs did not appear to change with age, as determined by Northern blot analysis. The increase in ODC mRNA that began at 21 days paralleled the increase in testis weight. This increase in ODC mRNA preceded the appearance of rat protamine-1 mRNA, a germ cell-specific mRNA found in round spermatids, which was first detected on Day 40. In contrast, levels of sulfated glycoprotein-2 mRNA, which, in the testis, is found exclusively in Sertoli cells, were highest at Day 17 and thereafter declined gradually with age. Unlike the increase in ODC mRNA levels, ODC activity was highest in 10-day-old animals and thereafter declined steadily with age, reaching minimal levels by 40 days (p less than 0.01). Thus, the increase in testicular ODC mRNA levels was in marked contrast to the decrease in testicular ODC activity. Incubation of cytosolic extract from 40-day-old animals with that from 10- or 17-day-old animals inhibited ODC activity approximately 50%, when compared to cytosols from 10- or 17-day-old animals. Dialysis of cytosol from 40-day-old animals prior to incubation with cytosol from 10-day-old animals relieved this inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular effects of adrenosterone feeding to juvenile carp, Cyprinus carpio L., effect on liver, kidney, brain and muscle protein and nucleic acids

Different groups of juvenile common carp (3–4 g) were fed diets supplemented with adrenosterone at doses of 1.0, 2.5 and 10.0 mg kg^-1 for 60 days and the effect on food conversion efficiency and cellular growth responses of liver, kidney, brain and muscle were investigated. At the end of the hormone feeding phase an increase of 40–75% was observed in food conversion efficiency. No change in hepato-somatic and viscero-somatic index was observed, but brain and kidney weights in relation to body weight was decreased compared with the controls. Variable changes in protein, RNA/DNA, protein/RNA and protein/DNA were seen in liver, kidney, brain and muscle. Feeding of the steroid for 60 days increased the water content of the muscle but ash contents of the muscle were decreased. No change in the total muscle lipids and total nitrogen content were observed. After the removal of the drug from the feed, some of the changes reported were ameliorated.     

Digestive secretions of lake sturgeon, Acipenser fulvescens, during early development

Digestive secretions of lake sturgeon were analysed for 60 days post-hatch. Three developmental phases were observed. During the yolk-sac stage (days 1–16) the digestive system differentiated and digestive enzyme concentrations were low. Initiation of gastric secretion was concurrent with establishment of active feeding (days 14–18). Peptic and tryptic activities continued to increase thereafter. Amylolytic and lipolytic activities were highest during the larval feeding phase and then declined following metamorphosis of the larvae to juveniles (days 24–30). Changes in the digestive enzyme complement were age related, not dietarily induced, and appeared to correspond with the different feeding habits and, possibly, nutritional requirements of the distinct life history stages.     

Induction of ornithine decarboxylase, RNA, and protein synthesis in macrophage cell lines stimulated by immunoadjuvants

Early biochemical changes associated with adjuvant stimulation of macrophage protein synthesis were studied using two murine macrophage cell lines, PU5-1.8 and J774.1. An induction of ornithine decarboxylase (ODC) was detected 2 hours after exposure of PU5-1.8 and J774.1 cells to two crude immunoadjuvants, BCG cell walls (BCGcw) and lipopolysaccharides from Escherichia coli (LPS). The chemically defined immunoadjuvant glycopeptide, N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDPL) also promoted an increase in ODC activity at 2 hours that was maximal after 4 hours, while little or no effect was observed with the D-alanyl analog (MDPD) that is devoid of adjuvant activity. The increase in ODC activity promoted by BCGcw in PU5-1.8 and J774.1 cells returned toward control levels by 6 to 8 hours. BCGcw also stimulated RNA and protein synthesis which remained elevated for at least 24 hours and was associated with a decrease in DNA synthesis and cell proliferation. ODC induction by BCGcw and MDPL was enhanced by the addition of PGE2 in both cell lines. Indomethacin slightly depressed the magnitude of ODC stimulation by BCGcw in J774.1 cells but failed to alter the response of PU5-1.8 cells. Additional observations indicated that the induction of ODC by BCGcw in both cell lines was preceded by an activation of cyclic AMP-dependent protein kinase. These observations suggest that a cyclic AMP-mediated induction of ODC may be an early biochemical marker of adjuvant stimulation in macrophages.     

Cell-cycle-dependent regulation of ornithine decarboxylase activity in the unicellular green alga Chlamydomonas reinhardtii

Polyamines play an important role in the control of cell growth and cell division. In the unicellular green alga Chlamydomonas reinhardtii as in animal cells, biosynthesis of the 3 commonly occurring polyamines (putrescine, spermidine and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. Therefore, we have investigated the regulation of ODC activity during the cell cycle of Clamydomonas reinhardtii using synchronized cultures. A 2.5–3-fold increase in ODC activity was observed during the transition to the cell division phase. This up-regulation of ODC activity was not due to an increased level of ODC-mRNA as revealed by northern-blot analyses, but correlated with an increased half-life of this particular enzyme (from 1.1 to 3.2 h). Addition of the DNA topoisomerase II inhibitor nalidixic acid during the second half of the growth period caused a transient decrease of ODC activity and a considerable delay of cell divisions. After cell division, a down-regulation of ODC activity was observed which was faster in the dark than in the light and also correlated with changes of the ODC half-life.     

Liver dehydrogenase levels in rainbow trout, Salmo gairdneri, fed cyclopropenoid fatty acids and aflatoxin B1

Cyclopropenoid fatty acids in the diet of rainbow trout caused significant reductions in liver protein and activity of glucose-6-phosphate dehydrogenase, NADP-linked isocitrate dehydrogenase, lactate dehydrogenase, and malate dehydrogenase. Changes in total activity were usually accompanied by similar changes in specific activity. The activity of glucose-6-phosphate dehydrogenase appeared to be more sensitive to the ingestion of cyclopropenoid fatty acids than the other dehydrogenases studied. Feeding 20 ppb aflatoxin B(1) to rainbow trout did not significantly change the activity of the dehydrogenases except for a small increase in the activity of glucose-6-phosphate dehydrogenase after 21 days of feeding. Relationships of these changes to the cocarcinogenicity of cyclopropenoid fatty acids and the carcinogenicity of aflatoxin are discussed.