Comparison of adenosine metabolism in leaves of several mangrove plants and a poplar species

A possible increased demand for ATP in salt- tolerant mangrove plants was studied by the comparison of metabolic fates of [8-¹⁴C] adenosine in leaf disks of several mangrove plants and of poplar. In mangrove trees, Rhizophora stylosa, Bruguiera gymnorrhiza, Kandelia candel and Sonneratia alba, 56–92% of [8-¹⁴C]adenosine taken up by leaf disks was converted during 3 h incubation to salvage products, i.e., nucleotides and RNA. Synthesis of nucleotides including ATP was stimulated by salt stress induced by 250 mM NaCl. In leaf disks of Avicennia marina, a mangrove shrub that produces glycinebetaine as compatible solutes, 46% of radioactivity entered salvage products when [8-¹⁴C] adenosine was continuously supplied to the leaf disks. Hydrolysis of adenosine to adenine was extremely active in this mangrove shrub. This is probably due to the high activity of adenosine nucleosidase (EC 3.2.2.7). In leaf disks of another mangrove shrub, Lumnitzera racemosa, only limited amounts of [8-¹⁴C]adenosine were metabolised (< ca. 30% taken up by leaf disks), but synthesis of ATP and ADP was stimulated by salt stress. In Pemphis acidula leaf disks, adenosine salvage activity was low and more than 30% of adenosine was hydrolysed to adenine. In leaf disks of poplar, a non-salt-resistant plant, ca. 40% of [8-¹⁴C] adenosine was converted to salvage products during 3 h of incubation, but the rate was slightly reduced by treatment with 250 mM NaCl. The present results suggest that large mangrove trees generally have efficient adenosine salvage ability, which is stimulated by salt. Lesser salvage activity is found in small size mangrove shrubs, although salt generally still enhances salvage activity.     

Effect of surplus glucose on physiological and biochemical characteristics of sugar beet leaves in relation to the age of the leaf and the whole plant

Effect of surplus glucose on physiological and biochemical parameters of leaves of different age was investigated in sugar beet (Beta vulgaris L., subsp. saccharifera) plants in the stages of vegetative growth (SVG). Early and late SVG were differentiated by the ratio between the weights of roots and aboveground organs (0.10 and 0.35, respectively). The excess of Glu was produced by incubation of the disks excised from detached leaves in water or 0.1 M Glu at radiant flux density of 250 μmol/(m² s) with the light regime pattern described as night/day/night/light (8/16/8/3 h). In all the leaf disks incubated in water and glucose solution, the content of Glu and other soluble carbohydrates considerably increased as compared with their content in the leaves they were taken from. After disk incubation in water and glucose solution, the content of chlorophyll (a + b) rose as compared with its level in respective leaves in early SVG; in late SVG, it declined. In early SVG, the rate of the O₂ photosynthetic evolution (Ph) in the ageing leaves under saturating concentration of NaHCO₃ after incubation in water and Glu solution declined more considerably than in young leaves. In late SVG, incubation of leaf disks in water and Glu solution weakly affected P _n. The rate of O₂ dark consumption in the leaf disks of all the types of treatment increased after incubation in water and especially in Glu solution. Activity of soluble carbonic anhydrase (sCA) in the extracts from young leaves in early SVG after their incubation in water and Glu solution was essentially the same, but after the incubation of ageing leaves in Glu solution, it reliably decreased. In late SVG, sCA activity sharply decreased after incubation in water and Glu solution irrespective of the leaf age. In late SVG, activity of Rubisco in the young leaves did not change after their incubation in water but decreased after incubation of the leaves of the three ages in Glu solution. In early SVG, nonphotochemical fluorescence quenching (NPQ) in the young intact leaf was lower than in the ageing leaf, and after leaf incubation in water and Glu solution, it rose. In late SVG, the value of NPQ was greater than in early SVG and, in contrast to the leaves of early SVG, it declined after leaf incubation; in water, this decline was more pronounced than in the Glu solution. In early SVG, efficient quantum yield of photosystem II (PSII) was much greater than in late SVG and it declined in the leaves incubated with Glu. It was concluded that surplus Glu can maintain biosynthetic processes in the young leaves of young sugar beet plants (trophic function). A decline in the level of chlorophyll and the activities of sCA and Rubisco in the course of leaf development and senescence is considered as a symptom of the suppression of biosynthesis of proteins of chlorophyll-protein complexes and the enzymes (Rubisco and sCA).     

Effects of lactic acid fermentation and gamma irradiation of barley on antinutrient contents and nutrient digestibility in mink (Mustela vison) with and without dietary enzyme supplement

The experiment was conducted to study the effects of fermentation of barley, using two different strains of lactic acid bacteria, a Lactobacillus plantarum/pentosus strain isolated from spontaneously fermented rye sourdough (AD2) and a starch-degrading Lactobacillus plantarum (AM4), on contents of mixed-linked (1 --> 3) (1 --> 4)-beta-glucans, alpha-amylase inhibitor activity, inositol phosphates, and apparent digestibility of macronutrients in mink. Effects of fermentation were compared with effects of gamma irradiation (gamma-irradiation: 60Co gamma-rays at 25 kGy). The diets were fed to mink with and without a supplementary enzyme preparation. Both lactic acid fermentation and gamma-irradiation followed by soaking and incubation, reduced concentrations of soluble beta-glucans, phytate and alpha-amylase inhibitor activity. Dietary enzyme supplementation increased significantly digestibility of crude protein, fat, starch and crude carbohydrate (CHO). Fermentation of the barley increased digestibility of starch and CHO. Fermentation with lactic acid bacteria AD2 resulted in higher starch and CHO digestibility than strain AM4, and had greater effect than gamma-irradiation, soaking and incubation. The highest digestibility of starch and CHO was obtained after AD2 fermentation followed by enzyme supplementation. It is concluded that both lactic acid fermentation of barley and enzyme supplementation have positive nutritional implications in the mink by limiting the effects of antinutrients and improving digestibility and energy utilization.     

Effects of lactic acid fermentation and gamma irradiation of barley on antinutrient contents and nutrient digestibility in mink (Mustela vison) with and without dietary enzyme supplement

Abstract

The experiment was conducted to study the effects of fermentation of barley, using two different strains of lactic acid bacteria, a Lactobacillus plantarum/pentosus strain isolated from spontaneously fermented rye sourdough (AD2) and a starch-degrading Lactobacillus plantarum (AM4), on contents of mixed-linked (1 → 3) (1 → 4)-β-glucans, α-amylase inhibitor activity, inositol phosphates, and apparent digestibility of macronutrients in mink. Effects of fermentation were compared with effects of gamma irradiation (γ-irradiation: ⁶⁰Co γ-rays at 25 kGy). The diets were fed to mink with and without a supplementary enzyme preparation. Both lactic acid fermentation and γ-irradiation followed by soaking and incubation, reduced concentrations of soluble β-glucans, phytate and α-amylase inhibitor activity. Dietary enzyme supplementation increased significantly digestibility of crude protein, fat, starch and crude carbohydrate (CHO). Fermentation of the barley increased digestibility of starch and CHO. Fermentation with lactic acid bacteria AD2 resulted in higher starch and CHO digestibility than strain AM4, and had greater effect than γ-irradiation, soaking and incubation. The highest digestibility of starch and CHO was obtained after AD2 fermentation followed by enzyme supplementation. It is concluded that both lactic acid fermentation of barley and enzyme supplementation have positive nutritional implications in the mink by limiting the effects of antinutrients and improving digestibility and energy utilization.     

The effect of cell turgor on sugar uptake in strawberry fruit cortex tissue

A reduction in cell turgor has been shown to stimulate sugar uptake in several plant sink tissues and it may regulate the import of assimilate into the sink apoplast, as well as maintain cell turgor. To determine whether cell turgor influences sugar uptake by strawberry (Fragaria x ananassa Duch. cv. Brighton) fruit cortex tissue, disks were cut from greenhouse-grown primary fruit at the green-white stage of development and placed in buffered incubation solutions containing either mannitol or ethylene glycol as an osmoticum. Cell turgor of fruit disks was calculated from the difference between the water potential of bathing solution and tissue solute potential after incubation at various osmolarities. Cell turgor increased when tissue disks were placed into mannitol incubation solutions more dilute than the water potential of fresh tissue (about 415 mOsmol kg^?1). The rate of uptake of [¹⁴C]-sucrose or [¹⁴C]-glucose decreased as osmolarity of the incubation solution increased, i.e. as cell turgor declined. Cell turgor and the rate of [¹⁴C]-sucrose uptake were unaffected when rapidly permeating ethylene glycol was used as an osmoticum. A decrease in cell turgor reduced both the V_max of the saturable (carrier mediated) kinetic component of sucrose uptake, and the slope of the linear (diffusional) component. The sulfhydryl binding reagent p-chloromercuibenzenesulfonic acid, an inhibitor of the plasma membrane sucrose carrier, strongly inhibited only the saturable component of sucrose uptake. Increased uptake of the nonmetabolizable sugar, O-methyl-glucose, at high turgor was similar to that of glucose, indicating that carrier activity was influenced by cell turgor, not cell metabolism. Turgor did not influence efflux of [¹⁴C]-sucrose from disks and had no effect on cell viability. Strawberry fruit cells do not possess a sugar uptake system that is stimulated by a reduction in turgor.     

Changes in ribosomal activity during incubation of Daucus carota root disks

Cytoplasmic monoribosomes from freshly cut and ‘aged’ carrot root disks were characterized relative to the Mg²⁺ optima for poly U (polyuridylic acid)-directed phenylalanine incorporation, the ease of dissociation by KCl in the presence of Mg²⁺, the ability to bind ³H-poly U, and acrylamide gel fractionation of the ribosomal proteins. The differences in in vitro amino acid incorporation by ribosomes and supernatant from fresh and ‘aged’ disks were confined to the ribosome fraction. The Mg²⁺ optima for poly U-directed ¹⁴C-phenylalanine incorporation was 16 mM for ribosomes from ‘aged’ disks compared to 20 mM for ribosomes from fresh disks. Monoribosomes from the fresh disks were easily dissociated into subunits (0·2 M KCl in 5 mM Mg²⁺) while the ribosomes from ‘aged’ disks were not completely dissociated even in 0·5 M KCl. Ribosomes from ‘aged’ disks were more effective in binding ³H-poly U than ribosomes from fresh disks. When the disks were subjected to an anaerobic environment prior to ribosome extraction (to strip monoribosomes of peptidyl-t RNA) the above effects of ‘aging’ were reversed. These results suggest that increased monoribosome activity associated with ‘aging’ may be related in part to an increase in the level of peptidyl-tRNA associated with the ribosomes. Acrylamide gel electrophoresis profiles of ribosomal proteins extracted from ribosomes of fresh and ‘aged’ tissue suggest that a change in the protein complement may also be important to the observed changes in ribosomal activity. The ribosomes from ‘aged’ disks contained at least two components not associated with ribosomes from fresh disks.     

In vitro evaluation of starch degradation from feeds with or without various heat treatments

An in vitro method was used to evaluate starch degradation from various feeds with or without heat treatments in four studies. The method was based on incubation of feed samples with a buffered rumen fluid solution and subsequent enzymatic analysis of the remaining starch. In all studies, heat treatment of the feed samples increased rate or extent of starch degradation to glucose. In Study 1, measurements of remaining starch, after 5 h in vitro incubations, demonstrated substantial effects of cooking on starch degradation in potatoes, and a trend to faster degradation from autoclaving peas. Up to 0.60 of the starch remaining after a 5 h of incubation was not recovered by centrifugation at 3000 × g for 10 min. In Study 2, cooking increased in vitro starch degradation rate from isolated potato starch (from 0.038 to 0.197/h). Intact starch in barley and wheat grain had similar rates of degradation (0.117 and 0.109/h, respectively). In Study 3, both autoclaving time (15, 30, 60 min) and temperature (115, 130 and 145°C) affected in vitro starch degradation rates in peas, and, in no case did autoclaving for only 15 min increase degradation rates. For the 30 min autoclaving time, only the highest temperature (145°C) increased the degradation rate of the pea starch compared to the untreated peas (0.175 versus 0.110/h). When autoclaving for 60 min, both 130 and 145°C resulted in a considerable increase in starch degradation rate (0.211 and 0.193/h, compared to 0.110/h for the untreated peas). In Study 4, the proportion of starch degraded at 8 h of in vitro incubation was increased by heat treatment of pure potato starch (0.155 versus 0.870), peas (0.491 versus 0.815), barley (0.686 versus 0.913) and maize (0.351 versus 0.498). Measurements of volatile fatty acid production in the fermentation tubes showed a lower acetate:propionate ratio for the faster fermenting heat-treated feeds. Heat treatment generally increased starch degradation in vitro.     

Wound-induced phenylalanine ammonia-lyase in potato tuber tissue. Development of enzyme activity and effects of antibiotics.

Phenylalanine ammonia-lyase [EC 4.3.1.5.] activity increased rapidly after a 3-hr lag period in potato tuber (Solanum tuberosum L. cv. May Queen) disks incubated in a suitable medium in the dark at 25 degrees. The activity reached a maxinum after incubation for about 40 hr. The effects of actinomycin D, 6-methylpurine, cycloheximide, chloramphenicol, and mitomycin C on the induction of phenylalanine ammonia-lyase were investigated during incubation of the disks. Actinomycin D, 6-methylpurine, and cycloheximide all inhibited the formation of phenylalanine ammonia-lyase, though cycloheximide was the most effective at low concentrations. Application of actinomycin D for the initial lag period (3 hr) caused strong inhibition; however, if it was supplied later it did not inhibit but actually increased phenylalanine ammonialyase formation. In contrast, cycloheximide was effective over most of the incubation period. Chloramphenicol and mitomycin C did not inhibit phenylalanine phenylalanine ammonialyase induction, but markedly stimulated it. Light was not an essential factor for phenylalanine ammonia-lyase induction in the wounded tissue.     

Effects of nitrate adaptation by rumen inocula donors and substrate fiber proportion on in vitro nitrate disappearance,methanogenesis, and rumen fermentation acid

A study was conducted to evaluate the main effects of dietary nitrate adaptation by cattle and alfalfa cell wall to starch ratio in in vitro substrates on nitrate disappearance and nitrite and volatile fatty acid (VFA) concentrations, as well as hydrogen (H₂) and methane (CH₄) accumulations. Rumen fluid from steers fed diets containing urea or nitrate was added into in vitro incubations containing sodium nitrate as the sole nitrogen source and 20 cell wall : 80 starch or 80 cell wall : 20 starch as the carbohydrate source. The results showed that during 24 h incubation, rumen fluid inoculums from steers adapted to dietary nitrate resulted in more rapid nitrate disappearance by 6 h of incubation (P < 0.01), no significant effect on nitrite concentration and diminished CH₄ accumulation (P < 0.05). Cell wall to starch ratio did not affect nitrate disappearance, CH₄ accumulation and total VFA concentration. The higher cell wall ratio had the lower total gas production and H₂ concentration (P < 0.05). Ammonia-N (NH₃-N) concentration increased because of adaptation of donors to nitrate feeding (P < 0.05). Nitrate adaptation did not alter total VFA concentration, but increased acetate, and decreased propionate and butyrate molar proportions (P < 0.01).     

The influence of growth regulating substances on the development of enhanced metabolic rates in thin slices of beetroot storage tissue

Freshly cut disks of beetroot tissue develop high rates of respiration, uptake of phosphate and activity of the enzyme invertase after having been washed for 18 hours in 0.01 m potassium maleate.Incubation of the disks in solutions of indole-3-acetic acid or kinetin completely prevented the development of the higher activities in all 3 systems assayed, while incubation in gibberellic acid had no inhibitory effect. Using a series of synthetic plant growth regulating compounds it was possible to establish that there was no correlation between the activity of the compound as an auxin and the ability of the compound to prevent the development of the enhanced rates of metabolism.     

Action of Rice α-Glucosidase on Maltose and Starch

Rice seeds possess α-glucosidase I and II, and the action of the α-glucosidases on maltose and starch was studied. The activity on starch was increased 2.3~2.6 times in both enzymes at the concentration of 50 mM of potassium chloride. Such activation was also caused by mono and di-valent cations. The activity on maltose was not influenced by the cations. In mixed substrate experiments, liberation of ¹⁴C-glucose from ¹⁴C-maltose was not inhibited in the presence of starch, and this was also the case with that from ¹⁴C-starch in the existence of maltose. From these results, it was suggested that the α-glucosidases possess maltose-hydrolyzing site and starch-hydrolyzing site separately, and also probably regulatory. The α-glucosidases liberated only glucose from starch, and were presumed to complete hydrolysis of starch after longer incubation.     

Response of lactic acid bacteria to amino acid derivatives. II. Glycine

The response of five lactobacilli (for which glycine is an essential nutrient) to two tripeptides, five dipeptides, and seventeen other glycine derivatives has been tested over a range of concentrations and at four or five incubation times from 18 to 229 hours. In general the activities decreased with increasing concentrations and incubation times although in some cases they remained nearly constant, increased, or increased and then decreased. Hippuric acid, all of the dipeptides, and the tripeptide, l-leucylglycylglycine, exhibited greater activity than glycine for one or more of the organisms. These results may be interpreted to signify that (a) the apparent decrease in apparent glycine in hydrolyzed urines may be accounted for in part by the higher activity of hippuric acid in unhydrolyzed urines, and (b) some peptides may be utilized directly by lactic acid bacteria under some conditions.     

The depletion of starch from the sapwood of the ash (Fraxinus excelsior) and its relation to attack by powder-post beetles (Lyctus spp.)

The depletion of starch in the living sapwood of ash was examined as a possible means of rendering converted timber immune from attack by Lyctus (powder-post beetle).
Observations on disks of timber kept under controlled conditions showed that depletion is conditioned by access of oxygen; thus although in the standing tree depletion proceeds from without inwards, it can be induced in any part of the sapwood, and in any direction, by permitting access of oxygen, i.e. there is no polarity in depletion. The optimum temperature range for depletion in ash is from 31 to 36 C.: above 45 C. death of the cells may interrupt depletion.
The presence of β-indoly acetic acid does not influence rate of depletion. Reformation of the starch in the depleted wood in the presence of cane sugar could not be induced. The enzyme concerned in mobilization of the starch appears to be a labile one with an optimum in the neighbourhood of 40 C. and to be produced during the active respiration of the cells, starch depletion ceasing when oxygen is withdrawn.
In transversely cut disks the rate of respiration at 33 C. ceases to be proportional to the volume of tissue after a thickness of about 6 mm. has been attained. At 20 C. disks 10 mm. thick may be evenly depleted. Infestation experiments upon timber undergoing depletion showed that the attack by Lyctus is circumscribed by starch-level and not by total nitrogen or soluble sugars.
Under correct conditions of kilning, 1 in. sapwood plank can be rendered starch-free in about 20 days: with larger sizes depletion is uncertain and probably uneconomic.
The methods of starch and sugar analysis used in the work are appended.     

ATP-Dependent Proteolytic Activity from Spinach Leaves

Spinach (Spinacia oleracea CV Bloomsdale Long Standing) leaf cytoplasmic starch phosphorylase and rabbit muscle phosphorylase a were inactivated by incubation with partially purified leaf extract in the presence of ATP and Mg²⁺. The inactivating factor(s) were heat stable and susceptible to protease attack. Phosphorylase inactivation was prevented by incubation in the presence of p-aminobenzamidine and phenylboronic acid, or prolonged treatment with phenylmethylsulfonyl fluoride or leupeptin for the ATP-stimulated inhibitory activity. Mg²⁺ -dependent inactivation was prevented by incubation with leupeptin, phenylmethylsulfonyl fluoride, p-aminobenzamidine, or 5′-adenylate. ATP-mediated inactivation of phosphorylase was stimulated by Mg²⁺ with a reduction in the apparent K_m for ATP. Casein-degrading activities with the same properties of ATP and/or Mg²⁺ stimulation, heat stability, and susceptibility to proteinase inhibitors were detected suggesting that phorphorylase inactivation was due to proteolysis. The activity was greatest at about the time of flowering and also appeared to depend on the light regime.     

Growth of Saccharomycopsis fibuliger and Candida utilis in mixed culture on pectic materials

Hydrolysis of pectin by Saccharomycopsis fibuliger and cell growth on the products of hydrolysis by Candida utilis require different incubation conditions. A three-stage sequential culture is described in which S. fibuliger was first grown under aerobic conditions to generate cell mass. The concentration of dissolved oxygen was then reduced to promote pectolytic activity and reduce the number of viable cells in the culture. Finally culture conditions were adjusted to promote the growth of C. utilis in mixed culture with S. fibuliger. The presence of C. utilis increased the rate of pectolytic activity by S. fibuliger. A yeast product, containing 98% C. utilis cells, was obtained from the mixed culture grown on 10 g l⁻¹ pectin. Cell yields using starch or an equal mixture of starch and pectin were similar to those reported in the Symba process, although lower cell yields were recorded using pectin alone.     

Crosslinking of glucoamylases via carbohydrates hardly affects catalysis but impairs stability

Mild oxidation of glucoamylases from Aspergillus niger (E.C.3.2.1.3. ) with periodate, followed by incubation with adipic acid dihydrazide, covalently linked enzyme molecules via their glycosyl groups. Size exclusion chromatography demonstrated and electron microscopy confirmed the formation of tetramers and octamers. Heat inactivation studies in the range of 60 degrees to 80 degrees C indicated that, in contrast to a priori expectations, crosslinking via the carbohydrates decreased rather than increased thermostability. The covalently linked species, even the octamers, displayed similar activity as the native forms toward maltose and soluble starch, but activity toward raw starch was completely lost.     

Juvenile hormone and DNA synthesis in imaginal disks of Calliphora erythrocephala: results of a new incubation technique.

An in vitro incubation technique in which imaginal disks are exposed to juvenile hormone and some of its analogues is presented. These substances were shown to have an inhibitory effect on the incorporation of tritiated thymidine (³HTdR) during the post-feeding period of the last larval instar of Calliphora. The technique makes it possible to investigate the nature of the effects of ecdysterone and juvenile hormones on the DNA synthesis in imaginal disks of exo- and endopterygote insects.