Structural characteristics of an abnormal protein influencing its proteolytic susceptibility.

Structural properties of two similar beta-galactosidase fragments were investigated to determine how they influence the fragments' degradation rate in Escherichia coli. Both fragments resulting from a C-terminal nonsense mutation in lacZ, the CSH11 polypeptide and its 90 kDa degradative intermediate, exist predominantly as monomer subunits instead of in the tetrameric form characteristic of the native enzyme. However, both fragments appear to produce trace amounts of dimers and tetramers. The tetramer and higher molecular weight aggregates formed by the wild-type subunit confer greater protection for the enzyme's N-terminal auto-alpha polypeptide than does the monomer state of the beta-galactosidase fragments. The thermally induced aggregation of both beta-galactosidase fragments correlates with their sensitivity to alpha-chymotrypsin. The relatively low thermal stability of the 90 kDa degradative intermediate appears to be the cause of the significant increase in its proteolytic susceptibility at moderately high temperatures.     

Investigation of the mechanism of protein turnover in HeLa S-3 cells by incubation at elevated temperatures

An apparent paradox relating to the degradation of endogenous proteins in HeLa S-3 cells occurs at 45 degrees C, at which their proteolysis is considerably enhanced in vitro but completely inhibited in vivo. No significant differences in rates of degradation of short-lived (nascent) and long-lived ('existing') proteins synthesised at 37 degrees C were found when chased at temperatures up to 43 degrees C, but at 45 degrees C degradation of both categories was reduced to zero in vivo. Synthesis of protein was suppressed at temperatures above 41 degrees C, being reduced by up to 60% at 43 degrees C. Proteolysis in vitro proceeded 1.6-1.7 times faster at 45 degrees C than at 37 degrees C and neutral pH. Evidence is presented for the involvement of the basal system; the findings both in vivo and in vitro do not seem to implicate the lysosomal system, no firm indication being obtained of its 'induction' at elevated temperatures. The results are discussed in terms of the arrest of intracellular circulation at elevated temperatures, thereby reducing the delivery rate of proteins as substrates of the intracellular basal proteolytic enzyme system to negligible levels (i.e., to the frequency of encounters due solely to the diffusion of protein molecules with the cytoplasm).     

嗜盐古菌启动子DNA片段的功能检测

将来源于嗜盐古菌染色体DNA的启动子片段RM07或RM13插入到启动子探针载体pYLZ_2的报告基因lacZ之前,通过β_半乳糖苷酶酶活性的检测,进一步确证RM07和RM13片段在大肠杆菌(Escherichia coli)中的启动功能。同时用微量热技术检测了大肠杆菌DH5α及其重组菌株在LB培养基中37℃生长过程的热输出功率。T2(pYLZ_2)、TE07(pYL726)、TE07_2(pYL702)、TE131(pYL131)和TE132(pYL132)菌株的生长速率分别比大肠杆菌DH5α降低了6.5%、11%4、1.1%4、7.5%和42.7%。当启动子启动了基因表达时,菌株的生长速率显著降低,热力学参数与酶活性检测结果有较好的一致性。微量热结果表明基因的表达比质粒DNA的复制过程需要消耗更多的能量,对细菌的生理代谢有较大改变。微量热技术为检测基因的表达和转录调控提供了新的方法和思路。     

Effects of heat and other inducers of the stress response on protein degradation in Chinese hamster and Drosophila cells

Many recent studies have suggested that heat and other inducers of the heat shock (stress) response in eukaryotic cells might result in the generation of abnormal proteins which would result in the overloading of protein degradation systems and the stabilization of proteins involved in positively regulating heat shock (hs) gene expression. In this study we have examined the effects different heat treatments and other hs inducers have on protein degradation in Chinese hamster ovary (CHO) and Drosophila Kc and Schneider cells. We have found that intermediate temperatures which induced the hs response (42 degrees C in CHO and 34 degrees C in Kc cells) did increase protein degradation rates whereas, higher temperatures which also induced the hs response (45 degrees C in CHO and 37 degrees C in Kc cells) initially increased but then decreased protein degradation rates. While these results are consistent with a model in which the protein degradation system is being overloaded and/or components of it are being depleted, we have found several conditions which induce hs proteins which rule out this mechanism. Exposure of either cell type to amino acid analogs (5 mM canavanine or 5 mM S-aminoethyl cysteine) resulted in the rapid degradation of those proteins which had incorporated the analogs in both CHO and Drosophila cells. However, the addition of analogs had little or no effect on the degradation of preexisting proteins, indicating that the introduction of abnormal proteins probably didn't overload the protein degradation system(s). The addition of 100 microM cadmium sulfate or 100 microM sodium arsenite had little or no effect on protein degradation rates in CHO cells even though both were good inducers of the hs proteins. Thus, exposure to inducers of the hs response does not universally increase protein degradation rates nor does it stabilize preexisting proteins. Therefore, the degradation of abnormal proteins is probably not involved in inducing the hs genes.     

Decreased intracellular proteolysis correlates with the maintenance of a specific isoenzyme of cytochrome P-450

The rates of intracellular protein degradation, of identically labelled populations of proteins, were compared in hepatocytes cultured at 37 degrees (on an adsorbed collagen layer) and in cells preserved on gelatin gels at 10 degrees C. The half-lives of the long-lived proteins were 35.4+/-8.6 h (N=4) and 692.9+/-216.9 h (N=4) respectively. Proteolysis was substantially decreased at 10 degrees C but the rate of decrease remained constant. Hepatocytes rapidly removed resorufin from the culture medium. The resorufin was not being conjugated or accumulated within the cells. Dicumarol, a potent inhibitor of quinone oxidoreductase, at high concentration (500 microm ) caused only a 72% decrease in the utilization of resorufin. The microsomal detoxifying enzyme, cytochrome P-450 1A1 remained at a constant level in the preserved hepatocyte monolayers. The results of this study strongly favour storing hepatocytes at 10 degrees C rather than at 4 degrees or 37 degrees C.     

Thermal instability in the endoplasmic reticulum of the rat hepatocyte

The rates of heat denaturation of a protein component of endoplasmic reticulum, NADPH cytochrome c reductase, measured in the temperature range of 37-47 degrees C, show that this protein is highly unstable in the range of temperatures used in clinical hyperthermia. The rate of enzyme inactivation increases some 20-fold as the temperature increases from 37 degrees C to 45 degrees C. The enzyme has an in vitro half-life of only 7 h due to thermal inactivation at 37 degrees C. This suggests a general scheme for the physiological degradation pathway of intracellular proteins in which the first step is rapid thermal denaturation of the molecule followed by a second and slower step of proteolytic degradation. The rapid physiological turnover of intracellular proteins may be an unavoidable cost of maintaining metabolic precision in the presence of thermal noise at normal body temperature.     

Mechanism-based fragmentation of coenzyme A transferase. Comparison of alpha 2-macroglobulin and coenzyme A transferase thiol ester reactions

The plasma proteins, alpha 2-macroglobulin and complement components 3 and 4, contain an internal thiol ester involving a glutamyl and cysteinyl residue. The thiol ester is susceptible to cyclization at greater than 37 degrees C and forms an unstable 5-oxyproline intermediate. The latter can be hydrolyzed to produce two peptide fragments. We propose that enzymes having activated glutamyl residues as part of their catalytic mechanisms may undergo an analogous cyclization and peptidyl cleavage. As a model, we have investigated pig heart succinyl-CoA:3-keto acid transferase. When the CoA-enzyme thiolester intermediate is heated at pH 7.4 and 70 degrees C for 1 h, approximately 60% of the Mr = 60,000 subunits are cleaved to give Mr = 40,000 and 20,000 fragments. We have shown that formation of the enzyme thiolester is an obligate precursor for the protein fragmentation. However, the reaction was incomplete with a maximum of approximately 65% cleavage at times greater than 60 min. These results suggest that there is a competing, deactivation reaction; namely, the thiol ester and oxyproline intermediates are hydrolyzed to regenerate the active site glutamic acid. Although the maximum rate of cleavage is at 70 degrees C, approximately 15% autolysis also occurs at 37 degrees C. The Mr = 40,000 fragment had the same amino terminal sequence as the Mr = 60,000 subunit, (Trp-Lys-Phe-Tyr-Thr-Asp-Ala-Val-Glu-Ala-). No amino terminal could be detected for the Mr = 20,000 fragment, even after digesting the fragment with pyroglutaminase. Peptide maps of the fragments and the uncleaved subunit indicate that the fragments are generated in parallel. The size of the fragments puts the active site about two-thirds of the way from the amino terminal of the protein.     

Limited proteolysis. Early steps in the processing of large premature termination fragments of beta-galactosidase in Escherichia coli

The mechanism by which large premature termination fragments of beta-galactosidase were degraded in Escherichia coli was studied using quantitative immunoprecipitation techniques. Two different lacZ nonsense mutants which produced apparent primary translation products of 96,000 and 109,000 daltons, respectively, were both shown to produce a second beta-galactosidase-related polypeptide of Mr = 90,000. These 90,000-dalton polypeptides appeared to be the same in both strains since they co-migrated when analyzed as a mixture on sodium dodecyl sulfate-polyacrylamide gels and were indistinguishable when analyzed by one-dimensional peptide mapping. Pulse-chase experiments established a stoichiometric precursor-product relationship between the primary mutant gene products (called the A polypeptides) and the common 90,000-dalton polypeptide (called the B polypeptide). No intermediates were detected between the A and B polypeptides. We propose that there is a common pathway for the degradation of these different large fragments of beta-galactosidase. According to this model, the first step would be a specific endoproteolytic cleavage of the primary translation product which produces the 90,000-dalton polypeptide as a common intermediate. The kinetic analysis demonstrated a first order decay of both A and B polypeptides but, surprisingly, the first order rate constant for the decay of A appeared dependent upon the induction regimen. This result suggested that degradation may possibly be autoregulated either by the intracellular level of A or by other intermediates in the degradation pathway.     

Relationship between thermal tolerance and protein degradation in temperature-sensitive mouse cells.

The induction of thermotolerance was studied in a temperature sensitive mouse cell line, ts85, and results were compared with those for the wild-type FM3A cells. At the nonpermissive temperature of 39 degrees C, ts85 cells are defective in the degradation of short-lived abnormal proteins, apparently because of loss of activity of a ubiquitin-activating enzyme. The failure of the ts85 cells to develop thermotolerance to 41-43 degrees C after incubation at the nonpermissive temperature of 39 degrees C correlated with the failure of the cells to degrade short-lived abnormal proteins at 39 degrees C. However, the failure of the ts85 cells to develop thermotolerance to 43 degrees C during incubation at 33 degrees C after either arsenite treatment or heating at 45.5 degrees C for 6 or 10 min did not correlate with protein degradation rates. Although the rate of degrading abnormal protein was reduced after heating at 45.5 degrees C for 10 min, the rates were normal after arsenite treatment or heating at 45.5 degrees C for 6 min. In addition, when protein synthesis was inhibited with cycloheximide both during incubation at 33 degrees C or 39 degrees C and during heating at 41-43 degrees C, resistance to heating was observed, but protein degradation rates at 39 degrees C or 43 degrees C were not altered by the cycloheximide treatment. Therefore, there is apparently no consistent relationship between rates of degrading abnormal proteins and the ability of cells to develop thermotolerance and resistance to heating in the presence of cycloheximide.     

Diacytosis of 125I-asialoorosomucoid by rat hepatocytes. A non-lysosomal pathway insensitive to inhibition by inhibitors of ligand degradation

Diacytosis of 125I-asialoorosomucoid by rat hepatocytes was studied by preincubating the cells with the labelled ligand at 37 degrees C for 30 min or 18 degrees C for 2 h, washing free of cell surface receptor-bound tracer at 4 degrees C and then reincubating at 37 degrees C. The cells preloaded at 37 degrees C released a maximum of 18% of the total intracellular ligand as undegraded molecules after 1 h of incubation with an apparent first-order rate constant of 0.018 min-1 (t1/2 = 39 min). When the preloaded cells were incubated in the presence of 100 micrograms/ml unlabelled asialoorosomucoid or 5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, the amount of the released ligand increased to 32 and 37%, respectively, without apparent change in kinetics, indicating that these agents prevented rebinding of the released ligand. In the presence of 5 microM colchicine, 20 microM cytochalasin B, 20 microM chloroquine, 10 mM NH4Cl, 10 microM monensin or 20 microM leupeptin, degradation of the preloaded ligand was inhibited, whereas the release of the ligand was either slightly increased or unchanged. Similar effects of leupeptin, colchicine and asialoorosomucoid were observed with cells preloaded at 18 degrees C. These results indicate that diacytosis of 125I-asialoorosomucoid occurs from a prelysosomal compartment via a route insensitive to inhibition by the inhibitors of ligand degradation.     

Degradation of Escherichia coli beta-galactosidase fragments in protease-deficient mutants of Salmonella typhimurium.

The degradation rates of several mutationally generated fragments of Escherichia coli beta-galactosidase were determined in wild-type strains of Salmonella typhimurium and in mutant Salmonella strains lacking several proteases and peptidases. Three termination fragments (produced by lacZ545, lacZ521, and lacZX90) and one internal reinitiation (restart) fragment [lacZpi(1)] are degraded in wild-type Salmonella strains at the same rates observed in wild-type Escherichia coli strains. Mutations that lead to loss of peptidases N, A, B, P, and Q or to loss of protease I or II do not affect the decay rates of any of these fragments. In addition, all of these peptidases and proteases are present in E coli mutants carrying deg mutations (deg mutations are known to stabilize beta-galactosidase fragments). Apparently, none of the proteases and peptidases that are currently accessible to direct genetic analysis plays a role in the early steps of the degradation of protein fragments.     

Increasing yeast secretion of heterologous proteins by regulating expression rates and post-secretory loss

Heterologous protein secretion involves the coupled processes of protein synthesis, protein folding, and secretory trafficking. A more complete understanding of how these processes interrelate could help direct optimization of secretion systems. Here we provide a detailed study regarding the dynamics of heterologous protein secretion from yeast in terms of intracellular protein levels, secreted protein levels, and unfolded protein response (UPR). Three different protein expression induction temperatures (20, 30, and 37 degrees C) were investigated as a means to modulate expression rates and thus cellular responses. Inducing at 20 degrees C yielded the slowest initial secretion rate, but the highest absolute level of product. Correspondingly, the level and the rate of both intracellular protein accumulation and unfolded protein response (UPR) activation were also the lowest at 20 degrees C. In addition, secretion ceased after approximately 22 h at 30 and 37 degrees C, respectively, while it was continuous until nutrient depletion at 20 degrees C. Maxima in secretion levels were observed that were a result of the additive effects of secretion cessation and post-secretory protein loss. The post-secretory loss of protein did not appear to result from solution phase proteolysis or aggregation, but required the presence of yeast cells. Refeeding of both yeast nitrogen base and casamino acids successfully prevented the post-secretory loss of protein at both high (37 degrees C) and low (20 degrees C) temperatures, and further increased secretion levels 1.5-fold at 20 degrees C where the secretory pathway was still functioning. Taken together, these findings suggest that there exists an appropriate balance between protein synthesis, processing and secretion rates required for secretion optimization.     

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells

Receptor sites for insulin on GH3 cells were characterized. Uptake of 125I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37 degrees C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with 125I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for 125I-labeled insulin binding sites. 125I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. 125I-labeled insulin was degraded at both 4 and 37 degrees C by GH3 cells, but not by medium conditioned by these cells. After a 5 min incubation at 37 degrees C, products of 125I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of 125I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of 125I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of 125I-labeled insulin, as well as intact hormone, were recovered from GH3 cells. After 30 min incubation at 37 degrees C, 80% of the cell-bound radioactivity was not extractable from GH3, cells with acetic acid.     

PCR fragmentation of DNA

A method has been developed to prepare random DNA fragments using PCR. First, two cycles are carried out at 16 degrees C with the Klenow's fragment and oligonucleotides (random primers) with random 3'-sequences and the 5'-constant part containing the site for cloning with the site-specific endonuclease. The random primers can link to any DNA site, and random DNA fragments are formed during DNA synthesis. During the second cycle, after denaturation of the DNA and addition of the Klenow's fragment, the random primers can link to newly synthesized DNA strands, and after DNA synthesis single-stranded DNA fragments are produced which have a constant primer sequence at the 5'-end and a complementary to it sequence at the 3'-end. During the third cycle, the constant primer is added and double-stranded fragments with the constant primer sequences at both ends are formed during DNA synthesis. Incubation for 1 h at 37 degrees C degrades the oligonucleotides used at the first stage due to endonuclease activity of the Klenow's fragment. Then routine PCR amplification is carried out using the constant primer. This method is more advantageous than hydrodynamic methods of DNA fragmentation widely used for "shotgun" cloning.     

Inter-molecular degradation of signal peptidase I in vitro.

Highly purified preparations of signal peptidase I (36 kDa) were found to undergo an apparent inter-autocatalytic degradation at 4 degrees C and 37 degrees C. The disappearance of the 36 kDa protein coincided with the stable appearance of a 31 kDa and a 5 kDa species. Amino-terminal sequencing of the 31 kDa product indicated a site specific cleavage following Ala38-Gln-Ala of signal peptidase I. The 31 kDa fragment was purified and shown to have 100-fold less activity than the native enzyme, with pre-maltose binding protein as a substrate.     

Proteolytic response to the expression of an abnormal \-galactosidase in Escherichia coli

Summary Because induction of proteolytic activity and stress-response proteins can significantly affect expression levels in recombinant Escherichia coli, the influence of low-level expression of a mutant \-galactosidase was investigated. A single copy of the well-characterized CSH11 mutant of the lacZ gene was integrated into the chromosome. Induction of expression of the mutant \-galactosidase caused a measurable increase in ATP-dependent intracellular proteolytic activity but resulted in no significant change in ATP-independent proteolytic activity. Growth at temperatures above 40°C resulted in a significant decrease in the level of ATP-independent proteolytic activity compared to growth at 37°C, and the ATP-dependent activity increased 2.5-fold from 30 to 42°C. Synthesis of stress-response proteins was evident in two-dimensional gel electrophoresis analysis of proteins in the strain expressing the abnormal \-galactosidase at 37°C, but no such response was evident when mutant \-galactosidase expression was induced at 30°C. In separate experiments, stress proteins were overexpressed by inducing expression of the htpR gene on a plasmid. Resulting increases in stress-protein levels correlated with an increase in ATP-dependent proteolytic activity with no significant change in the intracellular ATP-independent proteolytic activity. These data suggest that even very low levels of abnormal protein can substantially influence protease levels and stress response in E. coli. These responses were reduced by induction' at lower temperatures. Correspondence to: J. E. Bailey     

Analysis of pBR322 replication kinetics and its dependency on growth rate

Accurate estimates of plasmid copy number in a cell are a prerequisite for predicting plasmid stability and protein production. A refined version of a structured model for the pBR322 plasmid replication mechanism is described. The model is capable of accurately predicting pBR322 plasmid copy number in Escherichia coli B/r for a wide range of growth rates. The refinements include better estimates of promoter strength, the degradation rate of RNA species, binding constant of RNAI-RNAII reaction, and dependency of promoter strength on growth rate. The predictions of the model are verified by recent experimental observations but differ from some previous reports. This model can also be used to predict the binding constant of the RNAI-RNAII reaction of ColE1 type plasmids. At 37 degrees C, the binding constant is estimated to be 77 +/- 11 x 10(-13) mL/molecule-h for pBR322.     

Anaerobic biodegradation of 2,4-dichlorophenol in freshwater lake sediments at different temperatures

Anaerobic degradation of 2,4-dichlorophenol (2,4-DCP) between 5 and 72 degrees C was investigated. Anaerobic sediment slurries prepared from local freshwater pond sediments were partitioned into anaerobic tubes or serum vials, which then were incubated separately at the various temperatures. Reductive 2,4-DCP dechlorination occurred only in the temperature range between 5 and 50 degrees C, although methane was formed up to 60 degrees C. In sediment samples from two sites and at all tested temperatures from 5 to 50 degrees C, 2,4-DCP was transformed to 4-chlorophenol (4-CP). The 4-CP intermediate was subsequently degraded after an extended lag period in the temperature range from 15 to 40 degrees C. Adaptation periods for 2,4-DCP transformation decreased between 5 and 25 degrees C, were essentially constant between 25 and 35 degrees C, and increased in the tubes incubated at temperatures between 35 and 40 degrees C. The degradation rates increased exponentially between 15 and 30 degrees C, had a second peak at 35 degrees C, and decreased to about 5% of the peak activity by 40 degrees C. In tubes from one sediment sample, incubated at temperatures above 40 degrees C, an increase in the degradation rate was observed following the minimum at 40 degrees C. This suggests that at least two different organisms were involved in the transformation of 2,4-DCP to 4-CP. Storage of the original sediment slurries for 2 months at 12 degrees C resulted in increased adaptation times, but did not affect the degradation rates.     

Anaerobic biodegradation of 2,4-dichlorophenol in freshwater lake sediments at different temperatures.

Anaerobic degradation of 2,4-dichlorophenol (2,4-DCP) between 5 and 72 degrees C was investigated. Anaerobic sediment slurries prepared from local freshwater pond sediments were partitioned into anaerobic tubes or serum vials, which then were incubated separately at the various temperatures. Reductive 2,4-DCP dechlorination occurred only in the temperature range between 5 and 50 degrees C, although methane was formed up to 60 degrees C. In sediment samples from two sites and at all tested temperatures from 5 to 50 degrees C, 2,4-DCP was transformed to 4-chlorophenol (4-CP). The 4-CP intermediate was subsequently degraded after an extended lag period in the temperature range from 15 to 40 degrees C. Adaptation periods for 2,4-DCP transformation decreased between 5 and 25 degrees C, were essentially constant between 25 and 35 degrees C, and increased in the tubes incubated at temperatures between 35 and 40 degrees C. The degradation rates increased exponentially between 15 and 30 degrees C, had a second peak at 35 degrees C, and decreased to about 5% of the peak activity by 40 degrees C. In tubes from one sediment sample, incubated at temperatures above 40 degrees C, an increase in the degradation rate was observed following the minimum at 40 degrees C. This suggests that at least two different organisms were involved in the transformation of 2,4-DCP to 4-CP. Storage of the original sediment slurries for 2 months at 12 degrees C resulted in increased adaptation times, but did not affect the degradation rates.     

The effect of straw size, freezing rate and thawing rate upon post-thaw quality of dog semen

Optimal freeze-thaw processes for dog semen will yield a maximal number of insemination doses from an ejaculate. The objectives of this study were to compare the effects of two straw sizes (0.25- and 0.5-mL French), two freezing rates (straws suspended 3.5 and 8 cm above liquid nitrogen) and two thawing rates (in water at 37 and 70 degrees C) upon post-thaw quality of dog semen, and to determine the best treatment combination. Quality was expressed in terms of the percentage progressively motile sperm 5 and 60 min after thawing and the percentage of abnormal acrosomes 5 min after thawing. One ejaculate from each of eight dogs was frozen. Two straws from each ejaculate were exposed to each of the eight treatment combinations. Data were analyzed by means of a repeated measures factorial analysis of variance and means compared using Bonferroni's test. Dog affected each response variable (P < 0.01). Neither straw size, nor freezing rate, nor thawing rate affected motility 5 min after thawing (P > 0.05). Half-milliliter straws resulted in 5.7% more progressively motile sperm 60 min after thawing and 6.5% fewer abnormal acrosomes than 0.25-mL straws (P < 0.05, n = 64). The percentage progressively motile sperm 60 min after thawing tended to be higher for semen thawed at 70 degrees C compared to 37 degrees C (P < 0.06, n = 64). Semen thawed in water at 70 degrees C had 6.6% fewer abnormal acrosomes than semen thawed in water at 37 degrees C (P < 0.05, n = 64). Freezing rate interacted with thawing rate (P < 0.05) in their effects upon acrosomal morphology and freezing 8 cm above liquid nitrogen and thawing in water at 70 degrees C was best. Dog semen should be frozen in 0.5-mL straws, 8 cm above liquid nitrogen and thawed in water at 70 degrees C.