Peroxidases in Tobacco Abscission Zone Tissue: II. Time Course Studies of Peroxidase Activity during Ethylene-induced Abscission

Ethylene-induced abscission in flower pedicels of Nicotiana tabacum L. cv. Little Turkish causes a progressive increase in peroxidase activity during the first 4 hours of a 5-hour time course ethylene treatment period, with decrease in peroxidase activity occurring between 4 hours and 5 hours, when the supernatant extracts of abscission zone segments are tested spectrophotometrically for peroxidase activity, using guaiacol and hydrogen peroxide. Nonethylene-treated tissue has a much lower level of peroxidase activity over the same time course period. In ethylene-treated tissue the decline in break-strength correlates with the beginning of increase in peroxidase activity (3 hours). When the abscission zone area of the pedicel is further divided into proximal, abscission zone, and distal portions, respectively, the ethylene-treated tissue has the highest peroxidase activity in the abscission zone portion, with the maximum peak occurring at 4 hours and decreasing between 4 hours and 5 hours. Acrylamide gel electrophoresis of enzyme breis from ethylene-treated aand nonethylene-treated plants reveals that no new peroxidase isozymes are formed in response to ethylene, indicating an increase in the amount of one or in both of the two already existing isozyme banding patterns. The measurement of protein in the proximal, abscission zone, and distal segments, over a 5-hour ethylene treatment period, indicates that it is being translocated in a distal to proximal direction in the abscission zone pedicel. The possible participatory role for peroxidase in ethylene-induced tobacco flower pedicel abscission are discussed.     

Promotion of growth and invertase activity by gibberellic Acid in developing Avena internodes

Gibberellic acid (GA₃) induces invertase activity within 6 hours in Avena stem segments that are incubated in the dark at 23°. The maximum amount of promotion is about 5 times that of invertase activity in untreated segments. GA₃ causes significant promotion of invertase activity at concentrations as low as 3 × 10⁻⁵ μm GA₃. The increase in invertase activity elicited by GA₃ between 3 × 10⁻⁵ μm and 300 μm closely parallels the growth promotion that is caused by GA₃ over this concentration range. In control segments, invertase activity rises steeply during the first 6 hours of incubation, then decays slowly between 12 and 48 hours. In GA₃-treated segments, the invertase activity also rises during the first 6 hours, parallel to that in control segments and continues to rise during the next 42 hours. These changes in invertase activity during 48-hour incubation periods do not parallel the changes in growth that occur in control and GA₃-treated segments. Cycloheximide at 10 μg/ml abolishes all GA₃-promoted growth and invertase activity in these segments. Actinomycin D at 40 and 80 μg/ml decreases GA₃-promoted growth by 20% and invertase activity by 38 and 44%, respectively. The data clearly support the idea that protein synthesis is necessary for GA₃-promoted growth and invertase activity in Avena stem segments.     

Diurnal Changes in Maize Leaf Photosynthesis : III. Leaf Elongation Rate in Relation to Carbohydrates and Activities of Sucrose Metabolizing Enzymes in Elongating Leaf Tissue

Maize (Zea mays L. cv. Pioneer 3184) leaf elongation rate was measured diurnally and was related to diurnal changes in the activities of sucrose metabolizing enzymes and carbohydrate content in the elongating portion of the leaf. The rate of leaf elongation was greatest at midday (1300 hours) and was coincident with the maximum assimilate export rate from the distal portion of the leaf. Leaf elongation during the light period accounted for 70% of the total observed increase in leaf length per 24 hour period. Pronounced diurnal fluctuations were observed in the activities of acid and neutral invertase and sucrose phosphate synthase. Maximum activities of sucrose phosphate synthase and acid invertase were observed at 0900 hours, after which activity declined rapidly. The activity of sucrose phosphate synthase was substantially lower than that observed in maize leaf source tissue. Neutral invertase activity was greatest at midday (1200 hours) and was correlated positively with diurnal changes in leaf elongation rate. There was no significant change in the activity of sucrose synthase over the light/dark cycle. Sucrose accumulation rate increased during a period when leaf elongation rate was maximal and beginning to decline. Maximum sucrose concentration was observed at 1500 hours, when the activities of sucrose metabolizing enzymes were low. At no time was there a significant accumulation of hexose sugars. The rate of starch accumulation increased after the maximum sucrose concentration was observed, continuing until the end of the light period. There was no delay in the onset of starch mobilization at the beginning of the dark period, and essentially all of the starch was depleted by the end of the night. Mobilization of starch in the elongating tissue at night could account for a significant proportion of the calculated increase in the tissue dry weight due to growth. Collectively, the results suggested that leaf growth may be controlled by the activities of certain sucrose metabolizing enzymes and may be coordinated with assimilate export from the distal, source portion of the leaf. Results are discussed with reference to diurnal photoassimilation and export in the distal, source portion of the leaf.     

Change in invertase activity of sweet potato in response to wounding and purification and properties of its invertases

When root tissue of sweet potato (Ipomoea batatas Lam.) was sliced, acid invertase activity, initially absent in freshly sliced tissue, appeared after a 3- to 6-hour lag phase, rapidly reached a maximum in 18 hours, and thereafter decreased. The increase in invertase activity was accompanied by a decrease in sucrose content of the root tissue. Alkaline invertase activity was present in fresh root tissue, but changed little after wounding. Acid invertase in wounded tissue and alkaline invertase in fresh tissue were purified and their properties were investigated. The acid invertase was a ß-fructofuranosidase and was unaffected by substrate or by any of the cations and several metabolites. The alkaline invertase was more specific for sucrose, was inhibited by glucose and glucose 6-phosphate, and displayed non-Michaelis-Menten kinetics.     

Effect of heat and cold on the amylolytic and invertase activity of the small intestine of growing rats]

Acute experiments were conducted on ratlings; it was shown that the action of heat (40-41 degrees C), cold (5-6 degrees C), and ACTH injections (4 units per 100 g of body weight) during the first week after birth led to a sharp reduction of the total (alpha-gamma-conditioned) amylolytic and invertase activity of the homogenates and of the portions of the small intestine (turned inside out) of the growing rats. This depressed condition of the intestinal function was revealed directly after the arrest of the action of the unfavourable factors (7-day rats) and persisted for the subsequent two weeks of the animal life.     

Mechanisms of catecholamine synthesis disorders in the adrenals of rats subjected to physical fatigue]

There was a depression of transformation of noradrenaline, DOPA and thyrosine added in vitro, into catecholamines in the adrenal glands of rats after swimming for a period of 8 hours. This permitted to suppose a depression of the activity of phenylethanolamine-N-methyl-transpherase, DOPA-decarboxylase, and, possibly, of tyrosine hydroxylase under these conditions. After the end of swimming, in the presence of 1-tyrosine, there is at first an activation of noradrenaline synthesis, and then there occurs a gradual normalization (on the 7th day) of adrenaline formation. In rats trained for a period of 2 months the extent of reduction of the catecholamine synthesis in the adrenal glands in response to the 8-hour swimming was much less in comparison with the untrained aniamals.     

Further characterization of the distribution and metabolism of nitrofen in the pregnant rat

Nitrofen (2,4-dichloro-4'-nitrodiphenyl ether) is an herbicide with potent teratogenic activity in rodent species. The present study was an extension of previous efforts to characterize the distribution and metabolism of nitrofen in pregnant rats. Following a single p.o. exposure to radiolabeled compound on day 10 of pregnancy, maternal and embryonic tissues were collected at intervals from 1.5 to 72 hours. Radioactivity was accumulated and retained in maternal fat for over 72 hours. Peak levels were reached in other maternal organs at 3-12 hours. The half-life in maternal plasma was estimated to be 42 hours. Radioactivity was first detected in the embryonic compartment at 3 hours and continued to increase through the 72-hour time point. HPLC analysis indicated that the parent compound is initially deposited in maternal fat and after 48 hours redistributes to other maternal organs and to the embryo. The 5-hydroxy derivative was the major nitrofen metabolite found in maternal tissues, while the 4'-amino and 4'-acetylamine derivatives were found at lower levels and all exhibited single-phase kinetics. The parent compound alone was found in the embryo, and levels increased gradually as nitrofen redistributed from the fat at 48 hours after exposure. The results of this and other studies of nitrofen metabolism in pregnant rats suggest that its teratogenicity is not mediated via generation of mutagenic intermediates through nitroreduction of the parent compound. Rather, the embryo is exposed to the parent compound alone and appears to be a deep compartment for accumulation of nitrofen.     

Localization of cloned invertase in Saccharomyces cerevisiae directed by the SUC2 and MFalpha1 signal sequences

Protein localization in Saccharomyces cerevisiae was studied with two plasmid systems used as a model: one containing the SUC2 structural gene fused with the MFalpha1 (alpha-factor) promoter and signal-sequence, the other containing the entire SUC2 gene. Special emphasis was placed on the effect of promoter/signal-sequence (SUC2 vs. MFalpha1) on the efficiency of invertase transport. The MFalpha 1 and SUC2 signal sequences were capable of transporting, respectively, 83% and 77% of cloned invertase out of the cytoplasm. However, the SUC2 promoter was easier to control since a six-fold enhancement of the transported invertase activity associated with derepression was achieved in response to a glucose concentration change from 10 to 2 g/L Cloning on a multicopy plasmid resulted in a four-fold increase in total specific invertase activity over the wild type yeast strain (which harbors a single copy of the SUC2 gene on the chromosome), whereas the chromosomal site was more efficient for invertase localization yielding over 90% of the invertase transported out of the cytoplasm. Transient experiments done with the SUC2 signal-sequence-containing plasmid showed that the specific invertase activity in the periplasmic space reached a maximum three hours after derepression, then decreased very slowly with an accompanying gradual increase in invertase activity in the growth medium.     

The need of protein synthesis for the effect of LH--RH on the cAMP level in the anterior pituitary in vitro

The effect of LH--RH on cAMP accumulation in the anterior pituitary of male rats was investigated. The effect consisted of two phases. In the first phase of incubation (about 2 hours), there was no change in the cAMP level, while in the second phase a significant increase was observed. Cycloheximide (10(-5)M) given simultaneously with the LH--RH totally blocked the observed effect of LH--RH. If cycloheximide was given in the second phase of incubation, when the cAMP level was already elevated, its further rise was prevented and the elevated cAMP level remained unchanged for 2 or more hours. Actinomycin D (10(-5)M) added together with LH--RH totally abolished the action of the latter, but if actinomycin D was given 1 hour after LH--RH a significant increase of the cAMP level was found at the end of the 4-hour incubation.     

Calcium stimulates ornithine decarboxylase activity in cultured mammalian epithelial cells

Ornithine decarboxylase (ODC) activity usually rises to a peak a few hours after a trophic stimulus. The stimulation of ODC has been shown to depend on extracellular calcium in several in vitro eukaryotic systems. We have investigated the effect of calcium concentration on ODC activity and have found that ODC is stimulated when CaCl2 alone is added to calcium-deprived cells. Epithelial cells from calf esophagus were cultured and grown until stratified. Replacement of medium with fresh serum-free medium resulted in stimulation of ODC activity, which peaked at 4 hours and declined to basal level by 10 hours. Subsequent depletion of Ca2+ either by addition of ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) or by replacement of medium with Ca2+-free medium, resulted in obliteration of ODC activity 4 hours later. Conversely, cultures in which medium was replaced with Ca2+-free medium and at 10 hours were repleted with Ca2+ (either by addition of CaCl2 or by replacement of medium with Ca2+-containing medium) exhibited a pronounced elevation of ODC activity 4 hours later. ODC activity peaked at 6 hours after the addition of CaCl2 and declined by 8 hours. The effect was elicited by a wide range of concentrations of added Ca2+ from 0.1 mM to 4.0 mM, but was maximal at 1.0 mM. ODC activity was totally abolished if either cycloheximide (10 micrograms/ml) or putrescine (10 mM) was added to cultures immediately prior to Ca2+ addition. Actinomycin D (2, 5, or 10 micrograms/ml) added 30 minutes before Ca2+ did not prevent the stimulation of ODC by added Ca2+. Stimulation by Ca2+ is dependent on (1) absence of Ca2+ during the initial 10-hour incubation and (2) duration of incubation in Ca2+-free medium prior to Ca2+ replenishment. The results indicate that Ca2+ can increase ODC in epithelial cells exposed to Ca2+-depleted medium and that the increase in ODC depends on protein synthesis but is not inhibited by actinomycin D.     

Thoracic duct cannulation of the rhesus monkey (Macaca mulatta) for lymphocyte collection: thoracic approach.

The thoracic duct of the rhesus monkey (Macaca mulatta) was cannulated with medical grade tubing through a thoracatomy at right interspace 9-10. In 42 monkeys successfully cannulated, a mean of 1.194 x 10(9) lymphocytes was collected in the 70-hour period after surgery. A second collection, 1-3 weeks later, was possible for a shorter period (up to 60 hours) in a portion of these monkeys (15) with a smaller mean yield of lymphocytes (1.076 x 10(9). A third, but still shorter, collection period of up to 48 hours was possible in only five monkeys from which a mean yield of 0.698 x 10(9) lymphocytes was collected. A temporary postoperative weight loss in the monkeys was followed by a return to normal weight several months later.     

Fructan metabolism in wheat in alternating warm and cold temperatures

The objective of this research was to develop a system in which the direction of fructan metabolism could be controlled. Three-week-old wheat seedlings (Triticum aestivum L. cv Caldwell) grown at 25°C were transferred to cold temperature (10°C) to induce fructan synthesis and then were transferred to continuous darkness at 25°C after defoliation and fructan degradation monitored. The total fructan content increased significantly 1 day after transferring from 25°C to 10°C in both leaf blades and the remainder of the shoot tissue, 90% of which was leaf sheath tissue. Leaf sheaths contained higher concentrations of fructan and greater portions of high molecular weight fructan than did leaf blades. Fructan content in leaf sheaths declined rapidly and was gone completely within 48 hours following transfer to 25°C in darkness. In leaf blades the invertase activity fluctuated during cold treatment. The activity of sucrose:sucrose fructosyl transferase increased markedly during cold treatment, while fructan hydrolase activity decreased slightly. In leaf sheaths, however, the activity of invertase decreased rapidly upon transfer to cold temperature and remained low. Trends in sucrose:sucrose fructosyl transferase and hydrolase activity in sheaths were the same as those of leaf blades. Sheath invertase and hydrolase activity increased when plants were transferred back to darkness at 25°C, while sucrose:sucrose fructosyl transferase activity decreased. These results indicate that changing leaf sheath temperature can be utilized to control the direction of fructan metabolism and thus provide a system in which the synthesis or degradation of fructan can be examined.     

Effect of stress on parietal hydrolysis of saccharose in rats]

Experiments were conducted in albino rats of both sexes and different age; a study was made of the activity of the intestinal invertase directly after 4-hour immobilization. There was revealed a depressive effect of stress on the activity of membrane digestion and enzyme production in the intestinal epithelal cells, and a definite dependence of the observed effect on the animals' sex age. The latter was expressed in a greater fall of the invertase activity in young females and in its marked variations in old female rats.     

CYCLOHEXIMIDE ALTERS AXONAL TRANSPORT AND SUBCELLULAR DISTRIBUTION OF DOPAMINE-β-HYDROXYLASE ACTIVITY

—Administration of cycloheximide, 10 mg/kg s.c. led within 4 h to an approx 30% reduction of dopamine-β-hydroxylase (DBH) activity in the abdominal portion of rat sciatic nerves. At least two more hours elapsed before DBH activity in the distal part of these nerves began to fall. This pattern suggests reduced synthesis or delivery of DBH into axons but continued transport of previously delivered enzyme. Coinciding with the time at which DBH activity began to fall in distal segments of sciatic nerve, there was a marked reduction in the accumulation of DBH activity above a ligature in this region. Between 4 and 8 h after administration of cylcoheximide, 10 mg/kg, accumulation above a ligature was 70% less than in untreated nerves (P < 0.001), a reduction significantly greater (P < 0.05) than the accompanying 28% loss of baseline DBH activity. At the same time, the clearance of DBH activity from nerve regions distal to a ligature was greatly reduced. This pattern is consistent with the depletion of a minor but rapidly transported compartment of DBH. Six hours after administration of cylcoheximide, 10 mg/kg, the apparent subcellular distribution of DBH in distal regions of sciatic nerve was altered by a significant 36% loss in sedimentable DBH activity, with non-significant changes in othcr fractions. This suggests that rapidly transported DBH, depleted from the nerve by cycloheximide-induced inhibition of protein synthesis, is more highly associated with intraneuronal particles than is slowly transported or stationary DBH.     

The effect of high temperature and hydrocortisone on amylolytic and invertase activities of the mucous membrane of the small intestine in early ontogenesis of rats]

In experiments on 16-19 day rats, studies have been made of the effect of 2-hour heat exposure (40-41 degrees ) and hydrocortizone injection (4 mg per 100 g of body weight) on gamma-amylolytic and invertase activities of homogenized ad inside-turned pieces of proximal, middle and distal parts of the small intestine. It was shown that thermal stress results in a sharp increase of the enzymic activities of homogenates and total pieces of proximal and middle parts of the intestine. In distal parts, the enzymic induction is less significant or even completely absent. Hydrocortizone injections evoke similar changes, although the latter are more evident. On the basis of the data obtained, it is suggested that the effect of thermal factor on the synthesis of intestinal enzymes is realized via the hormones of suprarenal cortex.     

Increased density of fluorescent adrenergic fibers around the middle cerebral arteries of stroke-prone spontaneously hypertensive rats.

The distribution of fluorescent adrenergic nerve fibers in the proximal portion (horizontal segment, Hs) and the three distal portions (major branches) of the middle cerebral arteries (MCA) was examined in stroke-prone spontaneously hypertensive rats (SHRSP) aged 10, 30, 60, 90, and 180 days, by the glyoxylic acid method. The results were compared with those in age-matched normotensive Wistar Kyoto (WKY) rats. While the distribution pattern of fluorescent nerve fibers in the proximal portion of WKY rats changed from a straight linear arrangement at 10 and 30 days of age to a network-like arrangement after 60 days, those from SHRSP showed a constant meshwork pattern throughout the entire examination period. In the distal portions of the MCA of both SHRSP and WKY rats at all ages examined, fluorescent nerve fibers formed a coarse network. The distribution densities of adrenergic nerve fibers in the proximal and distal portions of the MCA of SHRSP were significantly higher (P less than 0.01 and 0.05) than those of WKY rats at all ages examined, except in the proximal portion at 90 and 180 days of age. The difference in nerve fiber density between SHRSP and WKY rats reached a peak at 30 days of age in both proximal and distal portions, and then gradually decreased with age. The present study suggests that sympathetic hyperinnervation is an important factor in the development of hypertension, and is involved in its maintenance in SHRSP.     

Increased density of fluorescent adrenergic fibers around the middle cerebral arteries of stroke-prone spontaneously hypertensive rats

The distribution of fluorescent adrenergic nerve fibers in the proximal portion (horizontal segment, Hs) and the three distal portions (major branches) of the middle cerebral arteries (MCA) was examined in stroke-prone spontaneously hypertensive rats (SHRSP) aged 10, 30, 60, 90, and 180 days, by the glyoxylic acid method. The results were compared with those in agematched normotensive Wistar Kyoto (WKY) rats. While the distribution pattern of fluorescent nerve fibers in the proximal portion of WKY rats changed from a straight linear arrangement at 10 and 30 days of age to a network-like arrangement after 60 days, those from SHRSP showed a constant meshwork pattern throughout the entire examination period. In the distal portions of the MCA of both SHRSP and WKY rats at all ages examined, fluorescent nerve fibers formed a coarse network. The distribution densities of adrenergic nerve fibers in the proximal and distal portions of the MCA of SHRSP were significantly higher (P<0.01 and 0.05) than those of WKY rats at all ages examined, except in the proximal portion at 90 and 180 days of age. The difference in nerve fiber density between SHRSP and WKY rats reached a peak at 30 days of age in both proximal and distal portions, and then gradually decreased with age. The present study suggests that sympathetic hyperinnervation is an important factor in the development of hypertension, and is involved in its maintenance in SHRSP.     

Inflection points of cardiovascular responses and oxygenation are correlated in the distal but not the proximal portions of muscle during incremental exercise.

To test whether there is a regional difference in the exercise pressor reflex within a given muscle, we investigated the relationship between the inflection points of cardiovascular responses and muscle oxygenation during exercise. Seven subjects performed incremental exercise, which consisted of incremental 30-s static knee extensions, each separated by 30 s of recovery. The workload started at 5% maximal voluntary contraction (MVC) and increased by 5% MVC for each increment until exhaustion. Changes (Delta) in the concentrations (denoted by brackets) of oxygenated Hb (O2Hb) and deoxygenated Hb (HHb) were monitored in proximal and distal portions of the vastus lateralis by near-infrared spectroscopy. The inflection points of mean arterial pressure (MAP), calf vascular resistance (CVR), and muscle deoxygenation index (Delta[O2Hb-HHb]) were calculated as the intersection point of two regression equations obtained at lower and higher workloads. The inflection point of Delta[O2Hb-HHb] differed significantly between proximal and distal portions (28.5 +/- 3.0 vs. 39.5 +/- 3.0%MVC, P < 0.05). Linear regression analysis showed significant correlations between the inflection point of Delta[O2Hb-HHb] in the distal portion and MAP (r = 0.89; P < 0.01) and CVR (r = 0.89; P < 0.05), but no significant relationship between the inflection point in the proximal portion and MAP or CVR. These data show that the inflection point of muscle deoxygenation differs between proximal and distal portions within the vastus lateralis during incremental exercise and suggest that the distal portion of the vastus lateralis contributes more to the pressor response than does the proximal portion.