A Newer Method for Isolation of the 7S Globulin in Soybean Seeds

The effects of freezing on the proteolysis of beef during storage at 4°C after being thawed was investigated.

A sarcoplasmic 32-kDa protein in frozen as well as unfrozen beef decreased rapidly during storage at 4°C, and a more than 100-kDa protein appeared in both beef samples. And the increment of peptides in the frozen beef during the storage at 4°C was larger than that in the unfrozen beef, suggesting that the proteolysis was faster during the storage of the former than the latter. However, its increment in the frozen beef for 10 weeks during the storage at 4°C became smaller than that of the one frozen for less than 5 weeks.

To discover an indicator for evaluation of the conditioning of frozen and unfrozen beef, peptides produced during the storage of beef at 4°C were surveyed. A peptide, APPPPAEVPEVHEEV, was detected and seemed to be available as an indicator in the conditioning of beef.     

STUDIES ON THE MECHANISM OF DEMYELINATION: MYELIN AUTOLYSIS IN NORMAL AND EDEMATOUS CNS TISSUE

The effects on myelin of autolysis in situ after death and after purification were studied in normal brains and spinal cords and in those made edematous as a result of chronic triethyl tin (TET) feeding. Myelin prepared from normal and edematous brains and spinal cords autolyzed for 12 h at 4°C contained only slightly less basic protein than that prepared from freshly killed animals. The amounts of a light lipid-protein fraction (dissociated myelin) usually obtained during purification of myelin from edematous CNS were about the same in tissue from freshly killed rats and those autolyzed for 12 h at 4°C. Autolysis for 12 h at room temperature resulted in formation of large amounts of dissociated myelin and loss of basic protein, but more dissociation and basic protein loss occurred in CNS from edematous brains and spinal cords than from the normal. Purified myelin prepared from freshly-killed normal and TET-fed rats was incubated at 37°C in media of several ionic strengths. In Krebs-Ringer bicarbonate (physiological extracellular fluid) extensive dissociation of myelin occurred with much less in 0.04 M-Tris buffer, pH 7.2, and only small amounts were formed in 0.01 M-Tris. In all cases myelin from edematous CNS formed more dissociated fraction than did the normal myelin. Basic protein loss was also proportional to the ionic strength of the media, but there was no difference in loss between normal and TET-myelin. Two different factors, proteolysis and physical extraction of basic protein by salt solutions, may be contributing to myelin dissociation and loss of basic protein.     

Glial fibrillary acidic protein from bovin and rat brain Degradation in tissues and homogenates

Compared with human material glial fibrillary acidic protein isolated from bovine, rat and mouse brain was remarkably homogeneous and migrated as a single band at 54 000 mol. wt. on sodium dodecyl sulfate gel electrophoresis. The protein was extremely susceptible to proteolysis and lower molecular weight components were invariably isolated together with the major species when the brain was not rapidly frozen. Further degradation of the 54 000 mol. wt. polypeptide in bovine tissues incubated at 24 °C resulted in preparations essentially identical to those previously isolated from human autopsy material and separating into a series of immunologically active polypeptides ranging in molecular weight from 54 000 to approximately 40 500. The gel band pattern obtained after progressively longer periods of autolysis suggested that small fragments were cleaved from the original polypeptide in successive steps of degradation. As in human brain, the lower molecular weight products in the 45 000–40 500 range were more resistant to proteolysis and still present after prolonged periods of tissue autolysis. The effect of the pH and of proteinase inhibitors on degradation was studied in homogenates of bovine brain stem incubated at 37 °C. At pH 8.0 proteolysis of the glial fibrillary acidic protein followed essentially the same pattern as in tissue. Cleavage of the major species was not prevented by the addition of proteinase inhibitors. At pH 6.0 and 6.5 a different type of degradation was observed, with rapid breakdown of the protein and loss of immunological activity. Increased solubility in buffer solutions was another effect of autolysis. Compared with cerebral cortex and brain stem, where most of the protein was water soluble, only a small fraction was extracted with buffer from bovine white matter. However, the solubility markedly increased following incubation and comparable amounts were extracted in buffer and in 6 M urea.     

Cryoprotectant removal temperature as a factor in the survival of frozen rice and sugarcane cells

Removal of cryoprotective additives through use of a room temperature (22 °C) washing step, instead of 0 °C, was found to improve the recovery of sugarcane suspension culture and rice callus tissues. Cultured cells were cryoprotected by gradual addition of a mixture of polyethylene glycol, glucose, and DMSO (PGD) to a final concentration of 10%-8%-10%, w/v, respectively, added at either 0 or 22 °C. After a programmed slow freezing of the cells, they were thawed rapidly and the cryoprotectants were gradually diluted and washed out using a 22 or 0 °C washing medium. Viability of suspension cultured sugarcane cells protected with PGD was greatly diminished when a cold washing solution was used, whether the cells had been frozen (?23 °C) or not. Two mutant lines of rice callus when frozen to ?196 °C in PGD and thawed showed less growth than unfrozen cells, but their growth was improved by washing the thawed cells with a 22 °C solution. With all cultures tested, the addition of PGD at 0 °C and post-thaw washing out at 22 °C gave improved survival. Particularly with the rice lines, optimizing the addition and washing procedures allowed culture survival of liquid nitrogen freezing not otherwise attained.     

Distribution of Acid Phosphatase During Autolysis in Bovine Liver

The percentage distribution of acid phosphatase between lysosomes and cytoplasm in bovine liver during autolysis at 37°C was investigated. The share of the cytoplasmic acid phosphatase activity of the total acid phosphatase activity in liver tissue increased during autolysis being before incubation 28–42 % and after 24 hrs.' incubation at 37°C 63–94 %. Microbiological contamination increased the proportion of cytoplasmic acid phosphatase. When bovine liver was incubated at 37°C for 24 hrs., the activity of the total acid phosphatase decreased to about 50 % of the initial activity. During a 16 days' incubation at 4°C the total acid phosphatase activity of bovine liver, however, remained unchanged.     

Susceptibility of the Myelin-Associated Glycoprotein and Basic Protein to a Neutral Protease in Highly Purified Myelin from Human and Rat Brain

Abstract: Incubation of highly purified human myelin at 25° and pH 8 in ammonium bicarbonate buffer resulted in the conversion of the myelin-associated glycoprotein (MAG) to a smaller derivative (dMAG) with an apparent molecular weight about 10,000 less. dMAG was stable and was not degraded to lower-molecular-weight breakdown products. Incubation of myelin under these conditions also resulted in the degradation of basic protein, but at a much slower rate. Half of the MAG was converted to dMAG in about 30 min, whereas degradation of half of the basic protein required 18 h of incubation. There was no significant loss of proteolipid, the Wolfgram doublet, or other myelin proteins during incubation for up to 18 h under these conditions. The formation of dMAG and the degradation of basic protein appear to be mediated by similar enzymatic activities; both processes exhibited broad pH optima in the neutral range, were prevented by briefly heating the myelin to 70° before incubation, and were stimulated by ammonium bicarbonate and other salts. Incubation of purified rat myelin also resulted in the formation of dMAG and the degradation of basic protein, but the conversion to dMAG occurred much more slowly than in human myelin preparations. In the rat, the percentage decreases in intact MAG and in basic protein were similar to each other and proceeded at rates comparable to the loss of basic protein in human myelin. These studies confirm and extend earlier demonstrations of neutral protease activity in purified myelin, and show that cleavage of MAG is one of the effects of this activity. The proteolytic activity affecting MAG and basic protein was not significantly reduced by further purification of the myelin on sucrose or CsCl gradients, suggesting that the neutral protease may be a myelin-related enzyme. The very high susceptibility of human MAG to this enzyme indicates that the effect of neutral protease on this glycoprotein should be considered in connection with demyelinating diseases.     

Fertilizability of cryopreserved zona-free hamster ova

Mature unfertilized ova from superovulated hamsters were freed from all investments and frozen at ?50°C. They were cooled at about 1°C/min to 0°C then at 0.8° to 0.6°C/min to ?50°C. At 0°C, dimethyl sulfoxide was added to a final concentration of 1.25 M. The ova were stored at ?50°C for up to four months. Thawing was performed at 2–4°C/min and followed by several washes with insemination medium. Approximately 90% of the ova were normal in appearance after thawing. The frozen and thawed ova with normal appearance could be penetrated by hamster or human spermatozoa at a rate comparable to unfrozen controls. The ability of hamster ova to tolerate storage at a relatively convenient temperature (?50°C) for long periods (tested for up to four months) makes possible their shipment at low cost to institutions lacking this resource. There they can be used for basic biological studies of sperm–egg interaction or in the clinical assessment of human sperm quality.     

New method of FACS analyzing and sorting of intact whole ovarian fragments (COPAS) after long time (24 h) cooling to 5 °C before cryopreservation

As recently announced by the American Society for Reproductive Medicine (ASRM), human ovarian tissue cryopreservation is an established option for fertility preservation in prepubertal girls and young women undergoing gonadotoxic treatments for cancer as well as some autoimmune diseases. Proper ovarian tissue assessment before and after cryopreservation is essential to increase success rates. Ovarian fragments from 16 patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following two groups. Pieces of Group 1 (n?=?16) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n?=?16) after operation were cooled to 5 °C for 24 h, then frozen after 24 h pre-cooling to 5 °C, thawed and just after thawing their quality was analyzed. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development and viability of follicles (Calcein-AM and Propidium Iodide) using complex object parametric analyzer and sorter machine (COPAS). Positive effect of cooling of cells to low supra-zero temperatures on their future development after re-warming has been observed. New flow cytometry- technique is suitable for the evaluation and sorting of cryopreserved whole human whole intact ovarian fragments. Long time (24 h) cooling of ovarian tissue to 5 °C before cryopreservation has a trend of a cell viability increasing.

     

Characterization of γ-glutamyltranspeptidase in the liver of the frog: 3. Response to freezing and thawing in the freeze-tolerant wood frog Rana sylvatica

The freeze tolerant wood frog Rana sylvatica was studied to determine the impact of the freezing and thawing of this frog on the activity of γ-glutamyltranspeptidase in the liver. On exposure to ?2·5°C, for 1, 12 and 24 h, frogs were found to be cool, covered with ice crystals and frozen, respectively. Thawing for 24 h at 4°C recovered the frogs completely. A 45 per cent decrease in the liver weight: body weight ratio was notable after 1 h at ?2·5°C, suggestive of an early hepatic capacitance response. A glycemic response to freezing was observed: blood glucose levels exhibited a 55 per cent decrease after 1 h at ?2·5°C on cooling; a 10·5-fold increase after 12 h at ?2·5°C on the initiation of freezing; and a 22-fold increase after 24 h at ?2·5°C in the fully frozen state. Blood glucose levels remained elevated four-fold in the thawed state. Plasma insulin levels were increased twofold in the frozen state and 1·8-fold in the thawed state, while plasma ketone levels were increased 1·8-fold in the frozen state and 1·5-fold in the thawed state. Plasma total T₃ levels were decreased by 22 per cent in the frozen state and normalized on thawing. In homogenates and plasma membranes isolated from the livers of Rana sylvatica, the activity of γ-glutamyltranspeptidase was found to be elevated at all stages of the freeze–thaw process. After 1, 12 and 24 h at ?2·5°C, activities were increased 2·5-, 2·3-, 2·4-fold respectively in the homogenates and 2·5-, 2·2-, 2·4-fold respectively in the plasma membranes. After thawing, activities were still increased 1·9-fold in both homogenates and plasma membranes. In homogenates prepared from the kidneys of Rana sylvatica, the activity of γ-glutamyltranspeptidase was increased 1·4-fold after 1 h at ?2·5°C after which it returned to normal. The role of thyroid hormone in producing the increase in γ-glutamyltranspeptidase in the liver of Rana sylvatica in response to freezing is discussed as is the significance of the enzyme increase in terms of hepatic cytoprotection and freeze tolerance.     

STUDIES OF THE MECHANISM OF DEMYELINATION REGIONAL DIFFERENCES IN MYELIN STABILITY IN VITRO1

—Incubation of slices of rat central nervous system in Krebs-Ringer bicarbonate buffer produced a lipoprotein fraction which floated on 10·5% sucrose after homogenization of the slices and centrifugation. This fraction was not found after homogenization and centrifugation of fresh tissue and appeared to depend upon incubation. The amount of the light fraction increased in the following order per 100-mg slice: cerebrum < thalamic area < cerebellum < brain stem < spinal cord. The lipid composition of this fraction was similar to that of myelin, but contained a lower protein content compared to myelin of the corresponding area. This fraction was termed ‘dissociated myelin’. Upon incubation of slices a portion of the basic protein was lost from myelin subsequently isolated, and the dissociated fraction was slightly enriched in basic protein. The distribution of myelin protein among the characteristic three groups (basic, proteolipid and high mol. wt.) was quite different in myelin from spinal cord compared to that from other CNS area. Spinal cord myelin contained about 17% protein compared to about 23% in cerebrum, with brain stem myelin intermediate (19%), and the difference appeared to be due to lesser amounts of proteolipid in the caudal areas. The amount of dissociation after incubation was about 3–5 per cent of the total myelin in the cerebral cortex, 10 per cent in the thalamic area, 20 per cent in cerebellum, 35 per cent in the brain stem, and around 45 per cent in spinal cord. The smaller amount of proteolipid protein in spinal cord myelin may result in a deficiency of cohesive forces holding lipids and proteins together, thus causing greater instability and dissociation. Myelin dissociation increased with time of incubation up to 3 h, was augmented by Ca²⁺, and was substantial at pH 11, reaching a peak at pH 7, then decreased in the acid range. A similar fraction has been isolated previously from fresh CNS tissue made edematous by chronic treatment of rats with triethyl tin. The possible relationship of swelling in the disease process and myelin dissociation are discussed.     

The effect of glutathione on the motility and fertility of frozen bull sperm

Adding glutathione (GSH, 5 mM) to frozen bull sperm increased the motility of the thawed sperm and decreased the release of aspartate aminotransferase from the sperm. After 3 h of incubation at 37°C, the addition of GSH increased the percentage of normal acrosomes and the motility of the spermatozoa. GSH also increased the fertility of the thawed bull sperm.     

Effect of thaw rates on motility,survival and acrosomal integrity of buffalo spermatozoa frozen in medium French straws

The objective was to determine the effect of different thaw rates on motility, survival and acrosomal integrity of buffalo spermatozoa frozen in medium French straws. Sixteen ejaculates from four mature buffalo bulls of Murrah breed were tested in a 4 × 4 × 4 factorial combination. Semen was extended in Tris-egg yolk-glycerol extender, frozen in 0.5 ml polyvinyl chloride straws in liquid nitrogen vapour and stored in liquid nitrogen for 24 h. Straws were thawed at water bath temperatures of 30°, 37° or 75°C for 30 s, 15 or 30 s, and 9 s respectively. Semen was incubated at 37°C for 6 h and evaluated at hourly intervals for percentage of motile spermatozoa (% MOT), percentage of total spermatozoa with intact acrosomes (PIA) and percentage of spermatozoa with intact, healthy acrosomes (PIHA) after 0 and 3 h of incubation. The initial post-thaw motility (0 h) averaged 66.9, 66.6, 72.1 and 64.6% for the four thaw rates respectively. Differences were significant between thaw rates for % MOT at 0 h (P < 0.05) and 1 h (P < 0.01) evaluation, post-thaw sperm survival at 37°C and absolute index of sperm survival. Bulls also differed (P < 0.01) for % MOT at 1, 2, 3 and 4 h evaluation, post-thaw sperm survival at 37°C and absolute index of sperm survival. Significant (P < 0.01) interaction of thaw rate × bull for % MOT at 1 h evaluation was observed. Neither treatments nor bulls had any significant effect on PIA and PIHA after 0 and 3 h incubation. Thaw rate of 37°C for 30 s was comparatively superior to other rates studied.     

Analysis and Prediction of the Effective Thermal Conductivities of Meats

Published data of the effective thermal conductivities of meats were analyzed in relation to approximate compositions of the meats. On the basis of series heat conduction model, the “intrinsic” thermal conductivity value of meat protein was estimated to be 0.342 [W/m · °C] when unfrozen, and 0.581 [W/m · °C] when frozen. Using these “intrinsic” values and the series heat conduction model, the effective thermal conductivities of meats were reversely predicted from the contents of water and fat. Standard deviations of the published data from the predictions were ± 7.0% for unfrozen meats and ± 15.4% for frozen meats. If heat flow is parallel to the meat grain, published data for frozen meats are higher than the predictions by about 20% as a mean.     

The in vitro biosynthesis and secretion of glycerol by larval fat bodies of chilled Ostrinia nubilalis

Fat bodies from diapausing fifth-instar larvae of Ostrinia nubilalis were incubated in vitro at 5 or 23°C in Grace's medium and the glycerol contents of the organ and incubation medium determined. Fat bodies from diapausing larvae chilled 3 weeks at 5°C secreted glycerol into the medium at 5°C at a net rate of approx. 0.75 nmol/mg fat body dry wt/h for at least 96 h while the tissue levels remained essentially constant. Depending upon the experiment, from 6 to 15 times more glycerol was produced in 24 h at 5°C by these fat bodies than by those taken from diapausing unchilled larvae and incubated at either 5 or 23°C. A minimal chilling period of 10–12 days was recognized as necessary for chilled larval fat bodies to demonstrate rates of glycerol synthesis greater than those of unchilled larvae and the lag showed a temporal correlation with changes in haemolymph glycerol concentrations. These results suggest that this response to chilling by O. nubilalis is relatively slow. While incubation, at 23°C, of fat bodies from previously chilled larvae did not result in cessation of glycerol secretion, the rate of its appearance in the culture medium decreased during the 24-h incubation period. Although the ability of chilled fifth-instar larvae to accumulate glycerol is not dependent upon the diapause state results show that clearance of glycerol from the haemolymph by rewarmed O. nubilalis is related to diapause intensity.     

Glial fibrillary acidic protein from bovine and rat brain. Degradation in tissues and homogenates.

Compared with human material glial fibrillary acidic protein isolated from bovine, rat and mouse brain was remarkably homogeneous and migrated as a single band at 54 000 mol. wt. on sodium dodecyl sulfate gel electrophoresis. The protein was extremely susceptible to proteolysis and lower molecular weight components were invariably isolated together with the major species when the brain was not rapidly frozen. Further degradation of the 54 000 mol wt. polypeptide in bovine tissues incubated at 24 degrees C resulted in preparations essentially identical to those previously isolated from human autopsy material and separating into a series of immunologically active polypeptides ranging in molecular weight from 54 000 to approximately 40 500. The gel band pattern obtained after progressively longer periods of autolysis suggested that small fragments were cleaved from the original polypeptide in successive steps of degradation. As in human brain, the lower molecular weight products in the 45 000-40 500 range were more resistant to proteolysis and still present after prolonged periods of tissue autolysis. The effect of the pH and of proteinase inhibitors on degradation was studied in homogenates of bovine brain stem incubated at 37 degrees C. At pH 8.0 PROTEOLYSIS OF The glial fibrillary acidic protein followed essentially the same pattern as in tissue. Cleavage of the major species was not prevented by the addition of proteinase inhibitors. At pH 6.0 and 6.5 a different type of degradation was observed, with rapid breakdown of the protein and loss of immunological activity. Increased solubility in buffer solutions was another effect of autolysis. Compared with cerebral cortex and brain stem, where most of the protein was water soluble, only a small fraction was extracted with buffer from bovine white matter. However, the solubility markedly increased following incubation and comparable amounts were extracted in buffer and in 6 M urea.     

A RADIOIMMUNOASSAY FOR MYELIN BASIC PROTEIN AND ITS USE FOR QUANTITATIVE MEASUREMENTS

—A specific radioimmunoassay (RIA) for myelin basic protein is described which is sensitive to 10⁻⁹ g of basic protein. The amount of basic protein detected in isolated myelin by the RIA and by SDS-gel electrophoresis and spectrophotometric quantitation agree to within experimental error. In contrast to isolated myelin, the major portion of the basic protein in fresh tissue is not accessible to its antibody. It is shielded from its antibody in a complex which is disrupted by heat, organic solvents, and various detergents. Maximum antibody binding was obtained with tissue heated to 100°C for 10 min. It is possible to calculate that the RIA quantitatively detects basic protein in boiled tissue. Boiled adult rat brain contains approximately 2·5 μg of basic protein/mg wet wt of cerebral cortex. The antibody to basic protein has no capacity to bind non-neural tissues.     

The “Intrinsic” Thermal Conductivity of Some Wet Proteins in Relation to Their Hydrophobicity: Analysis on Gelatin Gel

Since the intrinsic thermal conductivity of protein cannot be measured directly, the apparent “intrinsic” thermal conductivity of gelatin was estimated on the basis of the most suitable heat conduction model. Among four models of heat conduction through heterogeneous material, the series layers model best described the experimental results for frozen gelatin gels of different concentrations. Then, the “intrinsic” thermal conductivity of frozen wet gelatin was estimated to be 0.61 [W/m·°C]. The “intrinsic” thermal conductivity of unfrozen wet gelatin was estimated on the basis of Filippov’s equation to be 0.38 [W/m·°C], since the unfrozen gelating gel seemed to be as homogeneous as the solution.     

Effect of osmolality of skim-milk diluents and thawing rate on cryosurvival of ram spermatozoa

The effect of osmolality of skim-milk diluents (200, 320, 450, 600, and 750 mOsm/kg water) on the survival of ram spermatozoa frozen in straws were investigated after thawing in 39 °C water or in 20 °C air.Spermatozoa motility improved with increasing osmolality of the freezing diluent, irrespective of thawing rate. Diluents of 600 and 750 mOsm resulted in highest motility immediately after thawing and after 60 min incubation at 39 °C. A significant decrease in spermatozoa motility was observed when straws were thawed at 20 °C air with the magnitude of decrease inversely related to osmolality of the freezing diluent. Fertility of progestagen synchronized ewes inseminated with semen frozen in the 600 mOsm hypertonic skim-milk diluent was comparable to that obtained with fresh semen.     

Administration of heparin causes in vitro release of non-esterified fatty acids in human plasma

The objective of this study was to investigate the extent to which $\text{in vitro}$ hydrolysis of endogenous triglycerides contributes to the elevated concentrations of non-esterified fatty acids (NEFAs) which have been reported after heparin administration. Heparin is known to induce the release of lipases which hydrolyze endogenous substrate both $\text{in vivo}$ and $\text{in vitro}$. Four patients undergoing diagnostic cardiac catheterization, who routinely receive heparin, were studied. Blood samples were obtained before and at 5 and 30 minutes after an intravenous bolus of heparin (46 U/kg) was administered. Determinations of NEFAs in plasma were carried out immediately and again various times after the samples had incubated at 24°C and at 0°C. In addition, an aliquot of each sample was frozen quickly, stored for 5–7 days, thawed, and incubated at 24°C for 180 minutes. As expected, there were no significant increases after incubation in the concentrations of NEFAs in the samples obtained before heparin administration. In contrast, in the samples obtained after heparin administration, incubation at 24°C produced significant increases in the concentrations of NEFAs. For example, in the plasma samples obtained 5 minutes after administration of heparin, concentrations of NEFAs increased 50, 160 and 300% after 5, 60, and 180 minutes of incubation compared to pre-heparin concentrations. When assayed immediately, the concentrations of NEFAs increased only 15% over pre-heparin concentrations. Incubating the samples at 0°C slowed lipase activity. Freezing the samples stopped the lipase activity; however, when the thawed samples were incubated at 24°C, concentrations of NEFAs continued to rise. This study suggests that much of the reported increases in the $\text{in vivo}$ concentrations of NEFAs after administration of heparin may be due to $\text{in vitro}$ formation from continued lipase activity on endogenous substrate. Moreover, studies relating increases in the concentrations of NEFAs after administration of heparin to changes in drug binding to plasma proteins should be re-examined for possible $\text{in vitro}$ artifacts.     

Accumulation and turnover of abscisic acid in osmotically stressed cotton leaf tissue in relation to temperature

Leaf discs of cotton (Gossypium hirsutum L. cv. Deltapine 70) were osmotically stressed by floating them on solutions of polyethylene glycol 8000. The tissue produced copious amounts of abscisic acid (ABA) when stressed. Accumulation of ABA depended strongly upon temperature during the incubation, displaying a maximum at 20°C. At 35°C, the amount of ABA accumulated after 24 h was 45–80% less than at 20°C. Temperature did not affect leakage of ABA into the medium. Turnover rate of [¹⁴C]ABA was more than 3 times greater at 35°C than at 20°C. This rapd turnover at 35°C could account for the decreased ABA accumulation. Three ¹⁴C-containing metabolites of ABA were extracted from the tissue. At 20°C, two of these accumulated and retained substantial ¹⁴C over 16 h. At 35°C, though, the ¹⁴C in one of these compounds was almost completely lost during the last 8 h of the incubation. Although the metabolites are not identified, the results show some specific effects of temperature on ABA metabolism. The strong effect of temperature on ABA accumulation may contribute to patterns of ABA-dependent processes (such as stomatal closure) during water stress.