Biochemical strategy of survival of the freshwater mollusc <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis> at near-zero temperatures

Effect of an autumn-winter decrease of the environmental temperature on changes of the content of free amino acids and reducing sugars was studied in tissues fluids of the freshwater mollusc L. stagnalis. At a decrease of the temperature to 4 and 0°C at the autumn period, concentration of free alanine rose several times; an increase of histidine, glutamate, glycine, and serine concentrations was less pronounced. Accumulation of methionine, leucine, isoleucine, tyrosine, and phenylalanine was revealed at 0°C. In the state of long hypobiosis (at 4°C), pools of all amino acids fell 4–8 times; essential amino acids, except for lysine, were not detected at all. Both at the summer and at the autumn periods, high concentrations of free fructose and glucose were revealed. In the process of hypobiosis the fructose pool decreased almost 35, while the glucose pool—12 times. Maltose that was absent in summer was found at 4 and 0°C as well as after 2.5 months of hypobiosis. The changes of the studied parameters in L. stagnalis in response to hypothermia are compared with the corresponding parameters of changes in cryoresistant animals in response to negative temperatures.     

Seasonal variation in the content of free reducing sugars in body fluids of freshwater mollusk Lymnaea stagnalis

Accumulation of certain mono-and disaccharides in body fluids of ectotherms in autumn and winter suggests their protective role in adaptation to near-zero temperatures. In this context, the effect of seasonal temperature variation on the content of free reducing sugars was studied in freshwater snail Lymnaea stagnalis. The summer and autumn carbohydrate spectrum essentially featured high concentrations of fructose and glucose. After 2.5-month hypobiosis (4°C), the concentration of fructose and glucose decreased 35 and 12 times, respectively. Maltose and other disaccharides (presumably cellobiose) undetectable in summer became detectable in autumn before hypobiosis started. The subsequent level of maltose increased to almost reach the sharply decreased glucose level and to exceed the fructose level after 2.5-month hypobiosis. The involvement of maltose in the mechanisms of L. stagnalis adaptation to near-zero temperatures as a membrane protectant is proposed.     

Variation in the content of free amino acids in body fluids of freshwater mollusk Lymnaea stagnalis during seasonal adaptation to low positive temperatures

The effect of autumn and winter decrease in environmental temperature on the content of free amino acids in body fluids has been studied in freshwater snail Lymnaea stagnalis. In autumn, when temperature drops to 4 and 0°C, the highest increase in alanine concentration was observed and its pool was almost three times that in summer. A less pronounced accumulation of glutamate, glycine, histidine, and serine was observed in the same temperature range. Cysteine was detected at 0°C. The accumulation of essential amino acids methionine, leucine, isoleucine, tyrosine, and phenylalanine took place at 0°C, while only traces of these amino acids were detectable at 4°C. At the same time, free lysine undetectable in summer has been revealed in autumn at 4°C and its concentration increased as temperature decreased to 0°C. In winter, when the mollusks were hypermetabolic for 2.5 months, the pools of all amino acids decreased 4–8 times, while essential amino acids (except lysine) were undetectable. The involvement of alanine and, possibly, lysine in L. stagnalis adaptation to near-zero temperature is proposed.     

Food reserves of scots pine (Pinus sylvestris L.)

Summary Starch, soluble sugars, triacylglycerols, diacylglycerols and free fatty acids were measured in 30-year-old Scots pine (Pinus sylvestris L.) trees during an annual cycle in the sapwood (youngest ten xylem rings). The radial distribution of carbohydrates and lipids was studied in the trunkwood of 90 -to 150-year-old Scots pine trees collected at the end of the growing season. Determination of the compounds was performed using specific enzymatic assays, capillary gas chromatography and thin layer chromatography. The amounts of glucose, fructose, sucrose, and galactose/arabinose in the sapwood were slightly higher in winter than in summer. Raffinose/stachyose increased up to 5-fold during the cold period. At the beginning of the growing season starch amounts rose, and then decreased in summer and autumn. No concentration changes were observed in the total amounts of diacylglycerols and fatty acids throughout the year. Triacylglycerol levels were slightly higher in February than in summer and autumn. Relative frequencies of individual fatty acids were similar in all lipid fractions. Glucose, fructose, sucrose, starch and triacylglycerols disappeared almost entirely at the transition zone from sapwood to heartwood. In contrast, free fatty acids and galactose/arabinose rose in centripetal direction, and diacylglycerols remained constant across trunk cross-sections. The relative amounts of individual fatty acids changed markedly in the free fatty acid fraction and in the triacylglycerols when crossing the sapwood-heartwood boundary. Concentration changes of reserve materials are discussed in relation to season, mobilization and translocation processes, dormancy, frost resistance, and heartwood formation. The results are compared to those found in needles.     

Reserve Mobilization During Germination of Tagetes minuta L.

Levels of albumins, globulins and starch were measured in dryTagetes minuta L. achenes (seeds) germinating at 25 °C andin those thermo-inhibited at 35 °C. In addition, the compositionof amino acids, fatty acids and free sugars was determined. In germinating seeds, albumins increased, while globulins decreased.At 35 °C, only a delayed decrease in globulins occurred.However, in both, the amino acids serine and glutamate increased.Lipid composition remained constant except for a slight declinein linoleic acid in the germinating seeds. At 25 °C, starchlevels increased with time while at 35 °C, there was onlya transient increase. Sucrose was the major free sugar foundin the seeds at all times, but its levels declined in the germinatingseeds. This was accompanied by an increase in the proportionsof glucose and fructose. No such changes in sugar compositionwere observed in the thermo-inhibited seeds. Tagetes minuta L., seed germination, reserve mobilization, thermo-inhibition     

Characteristics of myocardial metabolism during induction and prolongation of artificial hypobiosis

Glucose, free fatty acids and lipid fatty acid spectrum were studied in arterial and right atrial blood and myocardium of 122 rats during induction and prolongation of artificial hypobiosis (3 and 24 hours) at body temperature of 30 and 20 degrees C. Prolongation of hypobiosis was shown to be accompanied by enhanced participation of free fatty acids in the myocardial energy metabolism. Lipid fatty acid spectrum in the myocardium was characterized by the decrease in linoleic and palmitooleic acid content and the increase in oleic, palmitic, stearic and arachidonic acid levels.     

Change in content of sugars and free amino acids in potato tubers under short-term storage at low temperature and the effect on acrylamide level after frying

Changes in the sugar and amino acid contents of potato tubers during short-term storage and the effect on the acrylamide level in chips after frying were investigated. The acrylamide content in chips began to increase after 3 days of storage at 2 degrees C in response to the increase of glucose and fructose contents in the tubers. There was strong correlation between the reducing sugar content and acrylamide level, R(2)=0.873 for fructose and R(2)=0.836 for glucose. The sucrose content had less correlation with the acrylamide content because of its decrease after 4 weeks of storage at 2 degrees C, while the reducing sugar in potato tubers and the acrylamide in chips continued to increase. The contents of the four amino acids, i.e., asparatic acid, asparagine, glutamic acid and glutamine, showed no significant correlation with the acrylamide level. These results suggest that the content of reducing sugars in potato tubers determined the degree of acrylamide formation in chips. The chip color, as evaluated by L* (lightness), was correlated well with the acrylamide content.     

Seasonal Changes of the Metabolites in the Leaves, Bark and Xylem Tissues of Olive Tree (Olea europaea. L) I. Nitrogenous Compounds

The changes of total nitrogen and free amino compounds wereexamined in leaves, bark and xylem tissues of olive tree duringa complete annual cycle. In leaves total nitrogen decreasedin spring/early-summer but reached the highest level in autumn-winterperiods. In bark tissues total nitrogen fluctuations were mainlyrelated to the periodical cambial activities. In xylem tissuestotal nitrogen was detected in low levels with no fluctuationsover the year. The free amino compounds in leaves showed seasonalpeaks during the spring and autumn stem elongations, while aconsiderable restriction of their levels was detected in summer.Glutamate, aspartate, proline, alanine, serine and -aminobutyrateare the predominant amino acids detected in leaves. Glutamateand aspartate increased considerably in spring and autumn periodsbut disappeared in summer. Proline remained almost unchangedover the year. Alanine, in addition to the spring and autumnfluctuations, also fluctuated in summer, -aminobutyrate wasdetected at highest levels in winter. In bark tissues, the patternof fluctuations and the composition of the free amino compoundswere similar to those of leaves. In xylem tissues free aminocompounds were detected in high levels over the year exceptfor the drops in spring and summer cambial activity periods.Xylem was the most important reservoir for the readily transportablesoluble nitrogen being accumulated during the maturing of theproduced new xylem. Arginine, glutamine, alanine, glutamate,aspartate, serine, -aminobutyrate and proline are the most prevalentamino compounds in xylem. Arginine and glutamine showed extensiveinterchanges. Arginine increased in autumn while glutamine andalanine showed low levels in the same period. Olive tree (Olea europaea L), amino acids     

Bud-eating by bullfinches, Pyrrhulapyrrhula in relation to the chemical constituents of two pear cultivars

Damage to pear trees of the cultivars Conference and Doyenne du Cornice caused by Bullfinches feeding on the flower buds was measured during January-April 1981. Bud-eating was concentrated on trees near woodland, and occurred at a steady rate within each row, though progressively more rows were attacked. Levels of total soluble protein, free amino acids, starch, glyeeride-glycerol, fructose, glucose and phosphorus were low until mid March, when the amounts per bud (and concentrations of starch and total soluble protein) rose sharply, coinciding with an increase in the size of buds. Sucrose did not occur at detectable levels. Conference buds contained significantly higher concentrations of fructose and free amino acids than Cornice. The implications for understanding bullfinches' bud-feeding habits are discussed.     

Multiple component system of sugars and polyols in the overwintering spruce bark beetle, Ips typographus

Overwintering adults of the spruce bark beetle, Ips typographus (L.) showed an unusually complex sugar/polyol cryoprotectant system. The major components of the multiple system were: glucose (177.6 mmolL(-1), March); trehalose (175.0 mmolL(-1), December); sorbitol (147.9 mmolL(-1), January); mannitol (81.2 mmolL(-1), March); and erythritol (40.7mmolL(-1), March) (in the parentheses, the maximum concentrations are shown and the month when they were reached). Other minor components were glycerol, fructose, threitol, myo-inositol, arabinitol and ribitol. Distinct seasonal patterns of accumulation/depletion in various components were found. Glycerol, trehalose and glucose started to accumulate first, during early autumn, when the air temperatures fluctuated between 20 and 0 degrees C, and diapause beetles continued in feeding. Glycerol was depleted, glucose remained stable and trehalose continued in accumulation during late autumn when the temperatures oscillated around 0 degrees C. During early winter severe frosts reaching -20 degrees C came, the beetles terminated their diapause and trehalose was partially depleted, while mannitol, sorbitol, fructose, threitol and erythritol started to accumulate. Cold weather continued also during late winter when the beetles remained quiescent. During this period, trehalose was re-accumulated, threitol and erythritol continued to increase, mannitol remained stable and sorbitol, fructose decreased. All cryoprotectans were finally cleared in the beetles which were spontaneously leaving bark during early spring. The seasonal maximum of total concentration of all cryoprotectants (578.2 mOsmol L(-1)) was reached in March. Such a concentration results in colligative depression of melting point of body fluids down by 1.08 degrees C only. It suggests that the potential cryoprotective effect of accumulated sugars and polyols was related rather to their non-colligative functions.     

Food reserves of Scots pine (Pinus sylvestris L.)

Summary The amounts of starch, soluble sugars, triacylglycerols, diacylglycerols and free fatty acids were studied in Scots pine (Pinus sylvestris L.) during an annual cycle in current-year needles and in 1-, 2- and 3-year-old needles collected shortly after bud break. Determination of the compounds was performed using specific enzymatic assays, capillary gas chromatography and thin layer chromatography. Newly emerging needles contained relatively large amounts of starch, but only trace amounts of fat. During autumn and winter, fat content rose, while starch content decreased; amounts of both these reserve materials were very high the next spring shortly before bud break and decreased again during shoot elongation. Concentration of intermediates in triacylglycerol biosynthesis (diacylglycerols and free fatty acids), were low in summer and high in winter. The same pattern was observed for fructose and glucose (the predominant soluble sugars), galactose/arabinose and raffinose/melibiose. In contrast, sucrose concentrations were highest in spring and in autumn. Mature needles of different ages collected in May showed significant differences only in their triacylglycerol and starch content. Concentration changes of reserve materials are discussed in relation to season, mobilization and translocation processes, dormancy, frost resistance and the possibility of carbohydrate-fat interconversions.     

Gas chromatography-mass spectrometry analysis of 13C labeling in sugars for metabolic flux analysis

Metabolic flux analysis, using 13C labeled substrates, has become a powerful methodology for quantifying intracellular fluxes. Most often, analysis is restricted to nuclear magnetic resonance or mass spectrometry measurement of 13C label incorporation into protein amino acids. However, amino acid isotopomer distribution insufficiently covers the entire network of central metabolism, especially in plant cells with highly compartmented metabolism, and analysis of other metabolites is required. Analysis of label in saccharides provides complementary data to better define fluxes around hexose, pentose, and triose phosphate pools. Here, we propose a gas chromatography-mass spectrometry (GC-MS) method to analyze 13C labeling in glucose and fructose moieties of sucrose, free glucose, fructose, maltose, inositol, and starch. Our results show that saccharide labeling for isotopomer quantification is better analyzed by chemical ionization than by electron ionization. The structure of the generated fragments was simulated and validated using labeled standards. The method is illustrated by analysis of saccharides extracted from developing rapeseed (Brassica napus L.) embryos. It is shown that glucose 6-phosphate isomerase and plastidial glucose 6-phosphate transport reactions are not at equilibrium, and light is shed on the pathways leading to fructose, maltose, and inositol synthesis.     

The effect of prolonged exposure of pregnant rats to high ambient temperature on several constituents of the genital tract's fluids

1. Various constituents of the genital tract's (GT) fluids were measured in heat-exposed rats (kept for at least 30 days at 35 +/- 1 degree C) and control rats (maintained at 22 +/- 2 degrees C). 2. There were no differences between the groups in the GT fluid volume, protein, free amino acids and glucose contents. 3. Arterial and venous blood glucose levels, pO2 and pH values were similar in both groups. 4. GT fluid protein hydrolysate from heat-exposed rats showed significantly reduced contents of glycine and alanine and elevated contents of valine and lysine as compared with controls. 5. The GT fluid's free amino acid components showed reduced glycine and elevated valine and isoleucine in the heat-exposed group as compared with controls. 6. Progesterone levels in GT fluid of heat-exposed rats was 60% higher than that of controls. 7. It is suggested that the higher progesterone concentration and the altered relative contents of several free amino acids with a possible change in the proteins of the GT fluid may affect the development of the embryo in heat-exposed rats.     

Effects of storage temperature on the contents of sugars and free amino acids in tubers from different potato cultivars and acrylamide in chips

To clarify the effects of storage temperature on potato components and acrylamide in chips, tubers from five cultivars were stored at various temperatures (2, 6, 8, 10, and 18 degrees C) for 18 weeks, and the contents of sugars, free amino acids in tubers, and acrylamide in chips after frying were analyzed. At temperatures lower than 8 degrees C, the contents of reducing sugars increased markedly in all cultivars, with similar increases in the acrylamide level and dark brown chip color. Free amino acids showed little change at the storage temperatures tested and varied within certain ranges characteristic of each cultivar. The contents of reducing sugars correlated well with the acrylamide level when the fructose/asparagine molar ratio in the tubers was <2. When the fructose/asparagine ratio was >2 by low-temperature storage, the asparagine content, rather than the reducing sugar content, was found to be the limiting factor for acrylamide formation.     

Effect of heating in the presence or absence of glucose on digestive and metabolic utilization of amino acids of casein]

The effect of heating casein with and without glucose was studied with growing rats. Apparent digestibility of dry matter, organic matter, crude protein and amino acids was recorded. The utilization of amino acids was studied by estimating food consumption, growth and blood and muscle free amino acids. Heating casein with glucose at 90 degrees C during 24 hours resulted in significant loss of lysine and affected food intake, protein efficiency ratio and protein digestibility. Threonine and lysine availabilities were lowered.     

Effect of temperature on Pseudomonas fluorescens chemotaxis.

The effects of temperature and attractants on chemotaxis in psychrotrophic Pseudomonas fluorescens were examined using the Adler capillary assay technique. Several organic acids, amino acids, and uronic acids were shown to be attractants, whereas glucose and its oxidation products, gluconate and 2-ketogluconate, elicited no detectable response. Chemotaxis toward many attractants was dependent on prior growth of the microorganism with these compounds. However, the organic acids, malate and succinate, caused strong chemotactic responses regardless of the carbon source used for growth of the bacteria. The temperature at which the cells were grown (30 or 5 degrees C) had no significant detectable effect on chemotaxis to the above attractants. The temperature at which the cells were assayed appeared to affect the rate but the extent of the chemotactic response, nor the concentration response curves. The ratios of the rate of accumulation of cells to the attractant malate were approximately 2, 4, and 1 at 30, 17, and 5 degrees C, respectively. Strong chemotactic responses were observed with cells assayed at temperatures approaching 0 degree C and appeared to be functional over a broad temperature range of 3 to 35 degrees C.     

Temperature regulation of glucose metabolism in red blood cells of the freeze-tolerant wood frog.

The low-temperature metabolism of erythrocytes from the freeze-tolerant frog Rana sylvatica was investigated by (13)C and (31)P NMR spectroscopy. Erythrocytes readily took up high concentrations of the natural cryoprotectant, glucose, at both high (12 and 17 degrees C) and low (4 degrees C) temperatures but glucose was apparently not metabolized at 4 degrees C. Strong inhibition of glucose catabolism at low temperature would facilitate the maintenance of the very high concentrations of glucose (approximately 200 mM) that are accumulated to provide cryoprotection during freezing in wood frogs. Analysis of (13)C labeling of glycolytic intermediates at 4 degrees C showed mixing of label primarily in hexose (fructose) and hexose phosphate (glucose 6-phosphate, fructose 6-phosphate) pools but little label incorporation into triose phosphate intermediates. These data are consistent with a profound low-temperature-induced inhibition of phosphofructokinase (PFK). Investigations into potential PFK control mechanisms were undertaken. (31)P NMR analysis showed that the intracellular pH of erythrocytes increased from 7.0 to 7.3 as temperature decreased from 17 to 4 degrees C in a manner consistent with alphastat regulation. This change is exactly opposite to that expected if overall PFK activity was regulated by changes in cellular pH since PFK is less active at lower pH values in vitro. Other factors must, therefore, operate to regulate PFK at lower temperatures.     

Hexitols as major intermediates of glucose assimilation by mycelium of Puccinia graminis

Mycelium of Puccinia graminis was grown for 4 d on 200 mM D-[U-¹⁴C]glucose followed by a cold chase for 30 h. Analysis of cellular metabolites during the chase indicated significant turnover only in carbohydrates soluble in 80% (w/v) ethanol. A kinetic analysis of the depletion of [¹⁴C] in pools of free sugars and sugar alcohols indicated that the trehalose pools and a small proportion (12–16%) of the mannitol and glucitol pools did not turn over, whilst pools of glucose, fructose, and the remainder of the hexitols became totally,depleted of label during the chase. Because the [¹⁴C] was totally lost from the pools of glucose and fructose prior to the hexitols, it was deduced that both of these hexoses were precursors of the hexitols. Estimation of the carbon fluxes through pools indicated that 52, 36 and 16% of the carbon from glucose was assimilated via glucitol, fructose and mannitol respectively, demonstrating that glucitol could not have originated from fructose as sole precursor. After offering D-[U-¹⁴C]glucitol, [¹⁴C] was assimilated into trehalose phosphate, glucans and amino acids, but not into free glucose or fructose. These data indicate that hexitols are quantitatively important intermediates during the assimilation of glucose by Puccinia graminis.     

Seasonal dynamics of carbohydrate and nitrogenous components in the roots of perennial weeds

Abstract Chicory (Cichorium intybus L.) and dandelion (Taraxacum officinale L.) are persistent weeds, the aerial portions of which do not survive in winter. However, subterranean tissues remain viable and facilitate the rapid resumption of growth in early spring. The source of nutrients for growth prior to the establishment of foliage is the roots. Carbohydrate and N reserves are accrued during late summer and autumn, respectively. Hydrolysis of fructans during late autumn occurs coincidentally with increments in sucrose, the latter providing a readily accessible C pool. Nitrate, free amino acids and soluble protein all play substantial roles in nitrogen storage. Asparagine is the predominant amino acid in the free pool during winter, followed by glutamine, ornithine, serine, aspartic acid and glutamic acid. Storage reserves remain at peak levels throughout winter and deeline prior to the resumption of growth. The patterns observed here provide evidence that N is an important currency of storage metabolism and, thus, a framework has been provided for the examination of regulation of N storage in perennial weeds.     

桃果实不同发育时期细胞内糖酸分布对甜酸风味的影响

为了解桃果实发育过程中细胞内糖酸的分布、变化规律对果实甜酸风味的影响,采用区室分析方法研究了‘白凤’桃(Prunus persica‘Hakuho’)果实不同发育时期细胞内糖酸组分、含量及其分布对甜酸风味的影响。结果表明,成熟果实中(花后100 d)可溶性糖(蔗糖、葡萄糖、果糖和山梨醇)在液泡、细胞质和细胞间隙中的含量分别为27.3、11.6、9.0 mg/g,有机酸(苹果酸、柠檬酸、奎宁酸和莽草酸)含量为2.09、0.94、0.35 mg/g;未成熟果实中(花后60 d)可溶性糖在液泡、细胞质和细胞间隙中的含量分别为0.97、2.2、2.3 mg/g,有机酸含量为0.25、0.44、0.82 mg/g。‘白凤’桃果肉细胞内不同的糖酸分布对成熟果实的甜酸风味具有显著影响,而对未成熟果实影响较小。成熟果实中糖酸在液泡、细胞质和细胞间隙三者之间的分布差异可能是导致果实甜度变化的主要原因。