Changes in the pattern of protein synthesis during differentiation of the Ascomycete Sphaerostilbe repens

The vegetative mycelium of Sphaerostilbe repens Berkeley and Broome (strain CBS 275-60) gives rise, within 48 h, to aggregated organs composed of coremia and rhi-zomorphs. Developmental changes in polypeptide patterns were studied by one- and two-dimensional polyacrylamide gel electrophoresis after cells had been induced to undergo synchronized differentiation. One-dimensional gel electrophoresis revealed only minor changes during the morphogenesis. Of the 300 polypeptides resolved by two-dimensional gel electrophoresis, nearly 12% either increased or decreased during coremium and rhizomorph differentiation. Some polypeptides appeared to be unique to one or the other of the cell preparations and represented apparent qualitative differences. During the first 24 h of differentiation, about 20 polypeptide spots appeared, 6 were enhanced, 4 were reduced and 32 disappeared. Over the next 24 h changes in the population of proteins were less marked: 14 new proteins were revealed and 9 increased in intensity while 15 declined and 9 were no longer detectable. Five proteins which were present at a significant level only during the first stages of differentiation, may therefore, putatively be designated as aggregation-specific polypeptides.     

Biochemical pathways and mechanisms nitrogen, amino acid, and carbon metabolism

For both nitrogen and carbon metabolism there exist specific regulatory mechanisms to enable cells to assimilate a wide variety of nitrogen and carbon sources. Superimposed are regulatory circuits, the so called nitrogen and carbon catabolite regulation, to allow for selective use of “rich” sources first and “poor” sources later. Evidence points to the importance of specific regulatory mechanisms for short term adaptations, while generalized control circuits are used for long term modulation of nitrogen and carbon metabolism. Similarly a variety of regulatory mechanisms operate in amino acid metabolism. Modulation of enzyme activity and modulation of enzyme levels are the outstanding regulatory mechanisms. In prokaryotes, attenuation and repressor/operator control are predominant, besides a so called “metabolic control” which integrates amino acid metabolism into the overall nutritional status of the cells. In eukaryotic cells compartmentation of amino acid metabolites as well as of part of the pathways becomes an additional regulatory factor; pathway specific controls seem to be rare, but a complex regulatory network, the “general control of amino acid biosynthesis”, coordinates the synthesis of enzymes of a number of amino acid biosynthetic pathways.     

Influence of the nitrogen source and concentration on N fractions and free amino acid levels of grape vine explants

The influence of the source of inorganic nitrogen (KNO₃, (NH₄)₂SO₄ and NH₄NO₃) and its concentration (5, 10, 20 and 30 mM N) on total N incorporation, as well as on N distribution into different fractions (amminiacal, amino, amide and protein) and on free amino acid levels has been determined in grape vine explants cultured in vitro.Increasing concentrations of the nitrogen source resulted in increased total N content in tissues. This effect was small for KNO₃, higher for (NH₄)₂SO₄ and maximal for NH₄NO₃. In addition, nitrate promoted an increase in amino-N only, whereas ammonium increased both the ammoniacal-N and the amino-N fractions. Incorporation of N into amide-N and protein-N were not affected significantly by the N sources tested.The application of increasing quantities of N enhanced the accumulation of most free amino acids, especially arginine, alanine and proline, but to different extents, depending on both the N source and its concentration. The combination of ammonium and nitrate resulted in a higher accumulation of amino acids than that observed with either one of the two forms alone.     

The nitrogen status of Austrian forest ecosystems as influenced by atmospheric deposition,biomass harvesting and lateral organomass exchange

Measurements of the deposition rates of atmospheric trace constituents to forest ecosystems in Austria have shown that the deposition of plant utilizable nitrogen compounds is in the range from 12 kg N to more than 30 kg N ha^-1 a^-1. Locally, even higher deposition rates are encountered as a consequence of point sources or special deposition mechanisms such as fog interception, hoar frost formation, and accumulation in snow drifts. In order to place these values into perspective, they are compared with the nitrogen demand of past and present forest land use and with natural processes of nitrogen depletion and accumulation in forest ecosystems. During wind erosion of forest litter, woody material with a wide C/N-ratio remains on the windward side of ridges, while nutrient-rich material with a narrow C/N-ratio is deposited on the leeward side. As a result, total nitrogen storage in the forest soil as well as overall C/N-ratios change dramatically along a transect over a ridge, thus indicating a strong influence of litter C/N ratio on nitrogen retention in the forest soil. A study of nitrogen stores in the soil of beech ecosystems of the same yield class in the Vienna Woods showed a significant correlation of total N-content with base saturation. These results suggest that nitrogen storage capacity of forest soils may be managed by liming and tree species selection. As knowledge is still meagre, a special study on factors which determine nitrogen storage in forest soils is proposed within the FERN-programme.     

Effect of nitrogen nutrition on amino acid composition of xylem sap and stem wood in Alnus glutinosa

Citrulline was the major amino acid in root pressure sap, stem sap and stem wood from Alnus glutinosa L. Gaertn. plants relying on fixed nitrogen or, partly or wholly, on mineral nitrogen for growth. Glutamine increased in prominence in plants assimilating mineral nitrogen but asparagine remained a relatively insignificant component. Differences in the relative amounts of the free amino compounds of stem sap from nitrogen-fixing and mineral nitrogen-fed plants were usually small compared to differences between plants fed different sources of mineral nitrogen. In contrast, relatively high values for the ratios of citrulline/total free amino nitrogen compounds and particularly of citrulline/amides in root pressure sap distinguished nitrogen-fixing plants from those receiving mineral nitrogen. Although the amino acid ratios of stem wood extracts showed closer similarity to those for root pressure sap than stem sap, the seasonal accumulation of citrulline, possibly as a storage amino acid, in stem wood from field-grown plants negated the possibility of utilising stem wood analyses as an indicator of the form of nitrogen assimilation. Comparative data on the levels of citrulline or other free amino acids in Alnus glutinosa are unlikely to be useful as an index of nitrogen fixation, under most experimental conditions.     

The nitrogen mineralization rate of legume residues in soil as influenced by their polyphenol,lignin, and nitrogen contents

A 12-week greenhouse experiment was conducted to determine the effect of the polyphenol, lignin and N contents of six legumes on their N mineralization rate in soil and to compare estimates of legume-N release by the difference and ¹⁵N-recovery methods. Mature tops of alfalfa (Medicago sativa L.), round leaf cassia (Cassia rotundifolia Pers., var. Wynn), leucaena (Leucaena leucocephala Lam., deWit), Fitzroy stylo (Stylosanthes scabra Vog., var Fitzroy), snail medic (Medicago scutellata L.), and vigna (Vigna trilobata L., var verde) were incorporated in soil at the rate of 100 mg legume N kg^-1 soil. The medic and vigna were labeled with ¹⁵N. Sorghum-sudan hybrid (Sorghum bicolor, L. Moench) was used as the test crop. A non-amended treatment was used as a control. Net N mineralization after 12 weeks ranged from 11% of added N with cassia to 47% of added N for alfalfa. With the two legumes that contained less than 20 g kg^-1 of N, stylo and cassia, there was net N immobilization for the first 6 weeks of the experiment. The legume (lignin + polyphenol):N ratio was significantly correlated with N mineralization at all sampling dates at the 0.05 level and at the 0.01 level at 6 weeks (r²=0.866). Legume N, lignin, or polyphenol concentrations or the lignin:N ratio were not significantly correlated with N mineralization at any time. The polyphenol:N ratio was only significantly correlated with N mineralization after 9 weeks (r²=0.692). The (lignin + polyphenol):N ratio appears to be a good predictor of N mineralization rates of incorporated legumes, but the method for analyzing plant polyphenol needs to be standardized. Estimates of legume-N mineralization by the difference and ¹⁵N recovery methods were significantly different at all sampling dates for both ¹⁵N-labeled legumes. After 12 weeks, estimates of legume-N mineralization averaged 20% more with the difference method than with the ¹⁵N recovery method. This finding suggests that estimates of legume N available to subsequent crops should not be based solely on results from ¹⁵N recovery experiments.     

Uptake of free plasma amino acids by the lactating cow''s udder and amino acid composition of udder lymph

1. Total α-amino N and the amounts of 24 ninhydrin-positive substances were determined in several samples of plasma and lymph from the cow's udder. The arteriovenous differences of these substances across the mammary glands were measured in several experiments performed on lactating cows and in one experiment on a `dry' cow. Udder lymph obtained from live lactating cows by a lymph fistula and taken after killing lactating cows was analysed. 2. The concentrations of the individual free amino acids in udder lymph obtained from the live cow were similar to those found in cow's plasma. The concentrations of many amino acids in udder lymph taken immediately after death were two- to four-fold higher than those of the corresponding amino acids in udder lymph obtained from the live cow. 3. Most amino acids of the blood showed a considerable decrease in concentration by passage across the lactating mammary gland. Ornithine, a non-casein amino acid, showed arteriovenous differences of up to 60% of the arterial plasma concentration. No substantial amino acid uptake by the udder could be demonstrated in the experiment on the non-lactating cow. 4. The arteriovenous differences obtained for arginine, glutamine, isoleucine, leucine, lysine, valine, threonine and histidine were probably large enough to provide all the respective amino acid residues in milk protein. 5. The uptake of aspartic acid, asparagine, glutamic acid, serine and proline by the lactating cow's udder was not sufficient to account for all these respective amino acid residues found in milk protein.     

The fatty acid composition of milk fat as influenced by feeding oilseeds

The fatty acid composition of bovine milk fat can be substantially altered by feeding lipid sources which alter the fatty acid profile of lipid entering the intestine from the rumen. As long-chain fatty acids of dietary origin can be incorporated directly into milk fat the opportunity exists to alter the ratio of short and long-chain fatty acids as well as the degree of saturation of milk fat. In practice our ability to alter the fatty acid profile of milk fat is limited not by the synthetic capacity of the mammary gland, but rather by the challenge of achieving effective protection of unsaturated dietary fatty acids from biohydrogenation in the rumen, as well as keeping the level of polyunsaturated fatty acids within the range where the organoleptic quality and shelf-life of milk and dairy products are not compromised. The fatty acid composition of oilseeds such as canola are considered desirable from a human health perspective and thus their inclusion in the diet of dairy cattle as a means of achieving a more desirable fatty acid profile in milk fat may enhance the nutritive quality of milk.     

Dependence of amino acid composition upon nitrogen availability in birch (Betula pendula)

Small birch plants ( Betula pendula Roth .) were grown at different rates of exponentially increasing nitrogen supply. This resulted in plants with different relative growth rates and different internal nitrogen concentrations. Within a nitrogen treatment, both of these variables remained constant with time.
Free amino acids were measured in leaves and roots of the seedlings at two different harvests. At greater nitrogen supply, higher concentrations of total amino acid nitrogen were found in roots and leaves. The ratio of amino acid nitrogen to total nitrogen was low albeit greater at higher nitrogen supply. Higher concentrations of amino acid nitrogen were mainly due to high concentrations of citrulline, glutamine, γ-aminobuitric acid and arginine.
Greater leaf concentrations of amino acid nitrogen at higher nitrogen supply may be related lo increased concentrations in the xylem sap and/or may be indicative of small excesses of nitrogen with respect to current nitrogen usage in protein synthesis.     

Drug-induced changes in the composition of the cerebral free amino acid pool

The effects of insulin, hydroxybutyrate, deoxypyridoxine, chlorpromazine, codeine, morphine, puromycin, and cycloheximide on the composition of the free amino acids in mouse and rat brain were tested. Significant changes occurred in a number of amino acids with most compounds tested; the largest was of alanine (a 50% increase with glucose, a 50% decrease with drugs); histidine was often increased, and the nonessential amino acids were mostly decreased. The pattern of changes was somewhat different in the mouse brain from that in the rat brain. Changes of amino acid levels may participate in the pharmacological action of a number of compounds.     

Effect of parenteral nitrogen feeding on the free amino acid composition of the blood serum and liver tissue in irradiated rats

A considerable change in the free amino acid composition of blood serum and hepatic tissue was noted on the 7th and 14th days following total-body X-irradiation of rats with a dose of 2.9 Gy. The total free amino acid content of blood serum increased and that of hepatic tissue decreased by 85% (on an average) as compared to the intact controls. Quantitative changes in the content of individual amino acids were analysed. Polyamine injected enterally for 7 days and parenterally for 3 days after irradiation aids the elimination of the postirradiation changes in the amino acid balance.     

Changes in the free and total amino acid composition of ripening chestnut seeds

The distribution of free and protein amino acids during seed development is described for three chestnut varieties. Asparagine accumulation was shown to correlate with protein biosynthesis. Accumulation of free amino acids takes place before that of proteins in ripening seeds. A temporary decrease was generally accompanied by protein biosynthesis and followed by final accumulation of both protein and free amino acids.     

Submerged culture conidial germination and conidiation of the bioherbicideColletotrichum truncatum are influenced by the amino acid composition of the medium

Summary Submerged culture experiments were conducted to determine the optimal nitrogen source for rapidly producing conidia of the bioherbicide,Colletotrichum truncatum. Germination ofC. truncatum conidial inocula in submerged culture occurred most rapidly (>95% in 6 h) in media provided with a complete complement of amino acids. When (NH₄)₂SO₄, urea, or individual amino acids were provided as the sole nitrogen source, conidial germination was less than 20% after 6 h incubation. Conidia production was delayed inC. truncatum cultures grown in media with urea or individual amino acids as nitrogen sources compared to cultures supplied with Casamino acids or complete synthetic amino acid nitrogen sources. The use of methionine, lysine, tryptophan, isoleucine, leucine or cysteine as a sole nitrogen source severely inhibitedC. truncatum conidia production. Media with synthetic amino acid mixtures less these inhibitory amino acids produced significantly higher conidia yields compared to media with amino acid mixtures containing these amino acids. When various amounts of each individual inhibitory amino acid were added to media which contained amino acid mixtures, cysteine and methionine were shown to be most effective in reducing conidiation. An optimal nitrogen source forC. truncatum conidiation in submerged culture should contain a complete mixture of amino acids with low levels of cysteine, methionine, leucine, isoleucine, lysine and tryptophan for rapid conidiation and optimal conidia yield.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Utilization of adenine and guanine as nitrogen sources by Chlamydomonas reinhardtii cells

Chlamydomonas reinhardtü Dangeard, adenine or guanine can be used as the sole nitrogen source for growth by means of an inducible system which is repressed by ammonia. Cells grown on either adenine or guanine were able to take up both purines, although the adenine uptake rate was always about 40% of the guanine uptake rate. Both adenine and guanine were taken up by an inducible system(s) exhibiting hyperbolic kinetics with identical apparent A, values of 3-2 mmol m^?3 for adenine and 3-2mmol m^?3 for guanine. Adenine and guanine utilization depended on pH, with similar optimal pH values of 7·3 and 7·4, respectively. Adenine and guanine each acted as a competitive inhibitor of the other's uptake, and their utilization was also inhibited by hypoxanthine, xanthine and urate. Inhibition of adenine uptake by guanine and hypoxanthine was competitive, with A′, values of 5·5 and 1. 6 mmol m^?3 respectively. Guanine uptake was also inhibited competitively by adenine (K₁= 1·3mmol m^?3) and hypoxanthine (K₁= 3. 3 mmol m^?3). Utilization of both adenine and guanine was inhibited by cyanide, azide, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, 2,4-dinitrophenol and carbonylcyanide m-chlorophenylhydrazone, and was also sensitive to p-hydroxymercuribenzoate and N-ethyl-maleimide. On the basis of these results, taken together, the possibility that adenine and guanine are translocated into Chlamydomonas by a common system is discussed.     

Photoautotrophic growth of soybean cells in suspension culture IV. Free amino acid pools and the effect of nitrogen on chlorophyll levels

A photoautotrophic soybean suspension culture was used to study free amino acid pools during a subculture cycle. Free amino acid analysis showed that the intracellular concentrations of asparagine, serine, glutamine, and alanine reached peaks of 200, 10, 9 and 7 mM, respectively, at specific times in the 14-day subculture cycle. Asparagine and serine levels peaked at day 14 but glutamine level rose quickly after subculture, peaking at day three and then declined gradually. Roughly similar patterns were found in the conditioned culture medium although the levels were 1000-fold lower than those found in cells. Photoautotrophic (SB-P) and photomixotrophic (SB-M) cultures were quantitatively similar with regard to free asparagine and serine but not glutamine or free ammonia. Heterotrophic (SB-H) cells had 81–85% less free asparagine on day seven than did SB-M or SB-P cells. Hence, similar to the phloem sap of a soybean plant, asparagine, glutamine, alanine and serine were the predominant amino acids in photoautotrophic soybean cell cultures. Varying the amount of total nitrogen in culture medium for two subcultures at 10, 25, 50, and 100% Of normal levels showed that growth was inhibited only at the 10 and 25% levels but that growth on medium containing 50% of the normal nitrogen was as good as that on 100% nitrogen. Moreover, cellular chlorophyll content correlated exceptionally well with initial nitrogen content of the medium. Thus, the photosynthesis of SB-P cells was not limited by chlorophyll content. SB-P cells grown for two subcultures on 10% nitrogen contained very low free amino acid levels and only 1% of the free ammonia levels found in cells growing on a full nitrogen complement.Abbreviations SB-P photoautotrophic soybean cells (no sucrose, high CO₂, high light) - SB-M photomixotrophic soybean cells (1% w/v sucrose, high light) - SB-H heterotrophic soybean cells (3% sucrose, dark)     

Utilization of aromatic amino acids as nitrogen sources in Brevibacterium linens: an inducible aromatic amino acid aminotransferase

The first step of the utilization of the aromatic amino acids as sole nitrogen sources by Brevibacterium linens strain 47 was found to be a transamination. The deaminated metabolites of the amino acids were detected in culture supernatants, and the enzyme activity was identified in cell free extracts. The cells contained increased aromatic amino acid aminotransferase activities on growth on the aromatic amino acids as sole nitrogen sources. Two aromatic aminotransferases (AT-I and AT-II) were separated upon diethylaminoethyl-Trisacryl M column chromatography of cell free extracts. Only AT-I was responsible for the increased level of aromatic amino acid aminotransferase activity of induced cells. The results suggested a catabolic role of AT-I in vivo.Abbreviations DNP dinitrophenyl - HPLC high performance liquid chromatography - PLP pyridoxal-5-phosphate     

Concomitant accumulation of intracellular free calcium and arachidonic acid in the ischemic-reperfused rat heart

This study was designed to elucidate the relationship between enhanced cytoplasmic calcium levels (Ca²⁺ _i) and membrane phospholipid degradation, a key step in the loss of cellular integrity during cardiac ischemia/reperfusion-induced damage. Isolated rat hearts were subjected to 15 min ischemia followed by 30 min reperfusion. Ca²⁺ _i was estimated by the Indo-1 fluorescence ratio technique. Degradation of membrane phospholipids as indicated by the increase of tissue arachidonic acid content was assessed in tissue samples taken from the myocardium at various points of the ischemia/reperfusion period. The hemodynamic parameters showed almost complete recovery during reperfusion. Fluorescence ratio increased significantly during ischemia, but showed a considerable heart-to-heart variation during reperfusion. Based upon the type of change of fluorescence ratio during reperfusion, the hearts were allotted to two separate subgroups. Normalization of fluorescence ratio was associated with low post-ischemic arachidonic acid levels. In contrast, elevated fluorescence ratio coincided with enhanced arachidonic acid levels. This observation is suggestive for a relationship between the Ca²⁺-related fluorescence ratio and arachidonic acid accumulation probably due to a calcium-mediated stimulation of phospholipase A₂.     

Changes in nitrogen levels and free amino acids in rooting cuttings of mulberry (Moms alba)

Total and protein nitrogen in bark and wood of parent stems of mulberry ( Morns alba L. cv. Ichinose) decreased readily and to the same extent during leafing-out of the buds, but the decrease in wood was less marked than in bark. Simultaneously, soluble nitrogen in both bark and wood also declined but the depletion was less marked than that of total and protein nitrogen. During the same period total nitrogen in the new shoots and adventitious roots increased drastically; however, the increase in total nitrogen in the growing parts during rooting was almost the same as the decrease in total nitrogen in the parent stems. Proline, the prevalent amino acid in wood and bark of the parent stems, decreased drastically during rooting, whereas during the same period asparagine in the developing buds, callus and adventitious roots increased markedly and became the predominant amino acid. The amount of arginine was relatively high in bark of the parent stems but Low in wood and the buds. The level of arginine in bark decreased considerably during the experiments (as did that of proline). The results suggest that the nitrogen required by the growing parts (sinks) in the rooting cuttings comes mainly from protein breakdown in bark of the parent stems (source), although stored protein in wood (source) and soluble nitrogen in bark and wood (sources) also play a part in storage of nitrogen. Asparagine is suggested to be the main nitrogen transport compound in the new growth of the tree and the initiating roots of cuttings.