A 27,000-D core of the Dictyostelium 34,000-D protein retains Ca(2+)-regulated actin cross-linking but lacks bundling activity.

Actin cross-linking proteins are important for formation of isotropic F-actin networks and anisotropic bundles of filaments in the cytoplasm of eucaryotic cells. A 34,000-D protein from the cellular slime mold Dictyostelium discoideum mediates formation of actin bundles in vitro, and is specifically incorporated into filopodia. The actin cross-linking activity of this protein is inhibited by the presence of micromolar calcium. A 27,000-D fragment obtained by digestion with alpha-chymotrypsin lacks the amino-terminal six amino acids and the carboxyl-terminal 7,000 D of the intact polypeptide. The 27,000-D fragment retains F-actin binding activity assessed by cosedimentation assays and by 125I-[F-actin] blot overlay technique, F-actin cross-linking activity as assessed by viscometry, and calcium binding activity. Ultrastructural analyses indicate that the 27,000-D fragment is deficient in the bundling activity characteristic of the intact 34,000-D protein. Actin filaments are aggregated into microdomains but not bundle in the presence of the 27,000-D fragment. A polarized light scattering assay was used to demonstrate that the 34,000-D protein increases the orientational correlation among F-actin filaments. The 27,000-D fragment does not increase the orientation of the actin filaments as assessed by this technique. A terminal segment(s) of the 34,000-D protein, lacking in the 27,000-D fragment, contributes significantly to the ability to cross-link actin filaments into bundles.

Measurement of the cytoplasmic pH of Dictyostelium discoideum using a low light level microspectrofluorometer.

Pyranine was employed as a sensitive pH indicator in a low light level microspectrofluorometer. The in vivo and in vitro standard curves of the 460/410-nm fluorescence excitation ratio of pyranine as a function of pH are identical. Therefore, pyranine is specifically sensitive to cytoplasmic pH in Dictyostelium. The cytoplasmic pH of single cells in a population of Dictyostelium discoideum amoebae was obtained for the first time. The median cytoplasmic pH of vegetative amoebae was 7.19. Carbonyl cyanide m-chlorophenylhydrazone, a mitochondrial uncoupler and a protonophore, lowered the median cytoplasmic pH to 6.12 when the extracellular pH was 6.1. This result is in accord with the protonophore activity of carbonyl cyanide m-chlorophenylhydrazone. Interest in the cytoplasmic pH of Dictyostelium has been greatly stimulated by the theory that cytoplasmic acidification promotes development of pre-stalk cells, while cytoplasmic alkalinization favors the pre-spore pathway (Gross, J. D., J. Bradbury, R. R. Kay, M. J. Peacey. 1983. Nature (Lond.). 303:244-245). The theory postulates that diethylstilbestrol (DES), an inducer of stalk cell differentiation and a plasma membrane proton translocating ATPase inhibitor, should cause acidification of the cytosol. Previous measurements of the effects of stalk cell inducers including DES on intracellular pH using 31P nuclear magnetic resonance measurements have failed to confirm the predictions of the theory, and have suggested that significant modification of the model may be required. Using pyranine as the pH indicator, we find that the median cytoplasmic pH in cells treated with 10 microM DES dropped from 7.19 to pH 6.02. This effect is consistent with the pharmacological action of DES and with the proposal that DES, a stalk cell inducer, should acidify the cytosol. These results provide direct support for the theory that cytoplasmic pH is an essential regulator of the developmental pathway in Dictyostelium.

Cytoplasmic pH of Dictyostelium discoideum amebae during early development: identification of two cell subpopulations before the aggregation stage.

Development of the cellular slime mold Dictyostelium discoideum is initiated by the removal of nutrients, and results in formation of a mature fruiting body composed of two cell types, the stalk and spore cells. A considerable body of evidence supports the hypothesis that cytoplasmic pH may be an essential regulator of the choice to differentiate in either the prestalk or prespore pathway. We have devised methods for measurement and analysis of intracellular pH in developing Dictyostelium amebae in order to assess directly the potential role of cytoplasmic pH in regulating the pathway of differentiation. The intracellular pH of single D. discoideum amebae during development and in intact slugs has been measured using the pH-sensitive indicator pyranine in a low light level microspectrofluorometer. We have used the ATP-mediated loading method to introduce pyranine into these cells. Cells loaded by the ATP method appear healthy, have no detectable defects in development, and exhibit a similar population distribution of intracellular pH to those loaded by sonication. The intracellular pH of populations comprised of single amebae was found to undergo a transient acidification during development resulting in a bimodal distribution of intracellular pH. The subpopulations were characterized by fitting two gaussian distributions to the data. The number of cells in the acidic intracellular pH subpopulation reached a maximum 4 h after initiation of development, and had returned to a low level by 7 h of development. In addition, a random sample of single amebae within a slug had a median intracellular pH of 7.2, nearly identical to the median pH (7.19) of similarly treated vegetative cells. No gradient of intracellular pH along the anterior to posterior axis of the slug was detected. Our data demonstrate the existence of two distinct subpopulations of cells before the aggregation stage of development in Dictyostelium, and offers support for the hypothesis that changes in intracellular pH contribute to development in D. discoideum.

Inhibition of actin filament depolymerization by the Dictyostelium 30,000-D actin-bundling protein.

We have studied the effect of the Dictyostelium discoideum 30,000-D actin-bundling protein on the assembly and disassembly of pyrenyl-labeled actin in vitro. The results indicate that the protein is a potent inhibitor of the rate of actin depolymerization. The inhibition is rapid, dose dependent, and is observed at both ends of the filament. There is little effect of 30-kD protein on the initial rate of elongation from F-actin seeds or on the spontaneous nucleation of actin polymerization. We could detect little or no effect on the critical concentration. The novel feature of these results is that the filament ends are free for assembly but are significantly impaired in disassembly with little change in the critical concentration at steady state. The effects appear to be largely independent of the cross-linking of actin filaments by the 30-kD protein. Actin cross-linking proteins may not only cross-link actin filaments, but may also differentially protect filaments in cells from disassembly and promote the formation of localized filament arrays with enhanced stability.

Dictyostelium discoideum cells lacking the 34,000-dalton actin-binding protein can grow, locomote, and develop, but exhibit defects in regulation of cell structure and movement: a case of partial redundancy.

Cells lacking the Dictyostelium 34,000-D actin-bundling protein, a calcium-regulated actin cross-linking protein, were created to probe the function of this polypeptide in living cells. Gene replacement vectors were constructed by inserting either the UMP synthase or hygromycin resistance cassette into cloned 4-kb genomic DNA containing sequences encoding the 34-kD protein. After transformation and growth under appropriate selection, cells lacking the protein were analyzed by PCR analyses on genomic DNA, Northern blotting, and Western blotting. Cells lacking the 34-kD protein were obtained in strains derived from AX2 and AX3. Growth, pinocytosis, morphogenesis, and expression of developmentally regulated genes is normal in cells lacking the 34-kD protein. In chemotaxis studies, 34-kD- cells were able to locomote and orient normally, but showed an increased persistence of motility. The 34-kD- cells also lost bits of cytoplasm during locomotion. The 34-kD- cells exhibited either an excessive number of long and branched filopodia, or a decrease in filopodial length and an increase in the total number of filopodia per cell depending on the strain. Reexpression of the 34-kD protein in the AX2-derived strain led to a "rescue" of the defect in the persistence of motility and of the excess numbers of long and branched filopodia, demonstrating that these defects result from the absence of the 34-kD protein. We explain the results through a model of partial functional redundancy. Numerous other actin cross-linking proteins in Dictyostelium may be able to substitute for some functions of the 34-kD protein in the 34-kD cells. The observed phenotype is presumed to result from functions that cannot be adequately supplanted by a substitution of another actin cross-linking protein. We conclude that the 34-kD actin-bundling protein is not essential for growth, but plays an important role in dynamic control of cell shape and cytoplasmic structure.

Homogeneity of wheat flour in 5ml containers for certified reference materials.

We aimed to develop certified reference materials that could be used for well-type HPGe detector. We chose wheat flour as a sample and evaluated the homogeneity of the sample in well-type container (5ml). Results showed that inhomogeneity was sufficiently small for validation checks of well-type HPGe detector (uhom = 0.44%).

Proficiency testing with uncertainty evaluation for measuring activities per unit mass of 134Cs and 137Cs in brown rice in Japan.

In Japan, we conducted proficiency testing of activity measurement by using high-purity germanium detectors for 134Cs and 137Cs in brown rice grains. Among 176 reported results, 86 % (for 134Cs) and 93 % (for 137Cs) of the results satisfied |En| â‰¦ 1. However, 58 reports for 134Cs and 51 reports for 137Cs had some failures in their evaluations of uncertainties. The proficiency testing was effective to improve the ability of uncertainty evaluation.

Arthroscopic decompression with indigo carmine for treating paralabral cysts in the shoulder.

Paralabral cysts in the shoulder are a relatively rare pathology. It is sometimes difficult to detect the location of a paralabral cyst in the shoulder using arthroscopy, and it can be difficult to confirm sufficient decompression by arthroscopy. We describe the case of a 64-year-old woman who underwent arthroscopic decompression for a paralabral cyst in the shoulder. Indigo carmine was injected into the cyst under ultrasonography guidance just before the operation. The leakage point of indigo carmine was detected using arthroscopy. Arthroscopic decompression was performed until the indigo carmine was completely discharged. Her shoulder pain, limited range of motion, and muscle weakness during abduction and external rotation improved postoperatively. Magnetic resonance imaging confirmed the disappearance of the cyst. Arthroscopic decompression using an ultrasonography-guided injection of indigo carmine is a useful treatment for a paralabral cyst in the shoulder.

Elongation factor 1beta is an actin-binding protein.

A 17 kDa polypeptide found in association with actin in cellular extracts of Dictyostelium discoideum was identified as a proteolytic fragment of eEF1beta. Antibody elicited against the 17 kDa protein reacted with a single 29 kDa polypeptide in Dictyostelium, indicating that the 17 kDa peptide arises from degradation of a larger precursor. The cDNA isolated from a Dictyostelium library using this antibody as a probe encodes Dictyostelium elongation factor 1beta. Amino acid degradation of the 17 kDa protein fragment confirmed the identity of the protein as eEF1beta. Direct interaction of eEF1beta with actin in vitro was further demonstrated in mixtures of actin with the 17 kDa protein fragment of Dictyostelium eEF1beta, recombinant preparations of Dictyostelium eEF1beta expressed in Escherichia coli, and the intact eEF1betagamma complex purified from wheat germ. Localization of eEF1beta in Dictyostelium by immunofluorescence microscopy reveals both diffuse cytoplasmic staining, and some concentration in the cortical and hyaline cytoplasm. The results support the existence of physical and functional interactions of the translation apparatus with the cytoskeleton, and suggest that eEF1beta may function in a dual role both to promote the elongation phase of protein synthesis, and to interact with cytoplasmic actin.

The structure, function, and assembly of actin filament bundles.

The cellular organization, function, and molecular composition of selected biological systems with prominent actin filament bundles are reviewed. An overall picture of the great variety of functions served by actin bundles emerges from this overview. A unifying theme is that the actin cross-linking proteins are conserved throughout the eukaryotic kingdom and yet assembled in a variety of combinations to produce actin bundles of differing functions. Mechanisms of actin bundle formation in vitro are considered illustrating the variety of physical and chemical driving forces in this exceedingly complex process. Our limited knowledge regarding the formation of actin filament bundles in vivo is contrasted with the elegant biophysical studies performed in vitro but nonetheless reveals that interactions with membranes, nucleation sites, and other organizational components must contribute to formation of actin bundles in vivo.

Clinical application of radial magnetic resonance imaging for evaluation of rotator cuff tear.

BACKGROUND: Magnetic resonance imaging is useful for evaluating the rotator cuff, but some tendinous insertions cannot be assessed using oblique sagittal, oblique coronal, and axial magnetic resonance (MR) images because of the presence of the partial volume effect. HYPOTHESIS: The purpose of this study was to determine whether radial-slice MR images could reveal normal rotator cuff insertions and rotator cuff tears more clearly than conventional MR images. PATIENTS AND METHODS: The study included 18 subjects with normal rotator cuffs and 30 with rotator cuff tears. MR images of rotator cuff insertions sliced into radial, oblique coronal, and axial sections were obtained. The extent to which normal rotator cuff insertions and rotator cuff tears were visualized in each of the three MR images was evaluated. RESULTS: The top to posterior portions of the rotator cuff insertions from 0° to 120° could be visualized in the radial MR images. In comparison, the posterior portions of the rotator cuff insertions could not be visualized around 45° in both the oblique coronal and axial MR images. DISCUSSION: These findings demonstrate that radial MR images are superior to the oblique coronal and axial MR images regarding their ability to accurately visualize rotator cuff insertions. Radial MR images also revealed greater detail around 45° in the posterior area of the rotator cuff tears than the oblique coronal and axial MR images. Radial MR images are particularly useful for visualizing clinically important posterosuperior rotator cuff tears. LEVEL OF EVIDENCE: Level III - Diagnostic study.

Age-related changes in contact photosensitivity differ among mouse strains.

There are differences among mouse strains in the age-related changes in reactivity to the contact photosensitizer tetrachlorosalicylanilide (TCSA). We found a tendency to lower reactions in older mice, with some strains showing declines from an early age (BALB/cJ, MRL/MpJ +/+, MRL/MpJ lpr/lpr and SJL/J). Others had increasing reactions until about 30-50 weeks of age before declining (DBA/1J, C3H/HeJ, and A/J) and one strain (C57BL/6J) had increased reactivity with age. There are also differences in the role of cyclophosphamide-sensitive T-suppressor cells in these age-related changes. In some mouse strains, BALB/cJ, C57BL/6J, A/J, DBA/1J and C3H/HeJ, age-related changes in reactivity to TCSA are independent of changes in cyclophosphamide-sensitive suppressor cells. In other strains, MRL/MpJ +/+, MRL/MpJ lpr/lpr and SJL/J, the development of cyclophosphamide-sensitive suppressor cells is responsible for the initial, though not later, stages of the age-related decline in reactivity.

Adenosine pretreatment for prolonged cardiac storage. An evaluation with St. Thomas' Hospital and University of Wisconsin solutions.

Adenosine pretreatment has been shown to be beneficial in several models of ischemia-reperfusion. We wished to evaluate whether adenosine pretreatment is cardioprotective for prolonged cardiac storage and whether the presence of adenosine in the storage media affects the results. Isolated rodent hearts were obtained from Sprague-Dawley rats, mounted on a Langendorff apparatus, instrumented with an intraventricular balloon, and ventricularly paced at 300 beats/min. Four groups of hearts were studied in a 2 x 2 factorial experiment (n = 8 to 12 per group). Hearts were subjected to normal perfusion or to solution supplemented with adenosine 50 mumol/L for 10 minutes followed by adenosine-free perfusion for 10 minutes. Hearts then were stored for 8 hours at 0 degrees C in either University of Wisconsin solution (adenosine 5 mmol/L) or St. Thomas' Hospital II solution (adenosine free). Adenosine pretreatment increased tissue levels of adenosine triphosphate before storage (p = 0.04). Nonfunction was less common after storage (1/19 versus 6/20 hearts, p < 0.05), and diastolic function was better preserved in the adenosine groups in the reperfusion phase (p = 0.01). The beneficial effects of adenosine pretreatment were independent of which storage solution was used. Developed pressure was increased (p < 0.05) and release of creatine kinase and lactate dehydrogenase was reduced (p < 0.0001) in hearts treated with University of Wisconsin solution compared with those treated with St. Thomas' Hospital solution. These studies suggest that adenosine pretreatment improves recovery after prolonged hypothermic storage and that the presence of adenosine in the preservation solution does not alter the results. The experiments provide further evidence that extended myocardial protection is better enhanced with University of Wisconsin solution than with St. Thomas' Hospital II solution.

Cardiac storage with University of Wisconsin solution, calcium, and magnesium.

BACKGROUND: Previous investigations from this institution and others support the role of University of Wisconsin solution for prolonged hypothermic cardiac storage. Modification of the divalent cation concentrations has been beneficial for cardioplegic investigations and may enhance cardiac recovery after extended preservation. METHODS: To investigate this hypothesis, isolated rodent hearts were obtained from Sprague-Dawley rats and mounted on a Langendorff apparatus. Rat hearts were flushed (15 ml/kg) and stored (30 ml) for 8 hours at 0 degree C in unmodified University of Wisconsin solution (n = 16/group) or University of Wisconsin solution with calcium concentrations of 0.025 to 10 mmol/L or magnesium concentrations of 10 to 20 mmol/L (six to eight hearts/group). Finally, combinations of calcium and magnesium were examined. Rat hearts were studied before storage and after 45 minutes of reperfusion with an intraventricular balloon. RESULTS: Developed pressure (mean +/- standard deviation) was increased with calcium 0.1 mmol/L (University of Wisconsin solution: 69.2% +/- 7.0%; Ca++ 0.1 mmol/L: 78.9% +/- 6.1%, p < 0.05), whereas only the addition of the highest calcium concentration (10 mmol/L) was significantly harmful (developed pressure: 58.3% +/- 8.4%, p < 0.05; creatine kinase release: 408 +/- 200 versus 170 +/- 104 IU/gm, p < 0.05; lactate dehydrogenase release: 103 +/- 43 versus 37 +/- 26 IU/gm, p < 0.05). Coronary flow recovered to control values with magnesium 15 mmol/L, which was significantly greater than that achieved with unmodified University of Wisconsin solution (97.1% +/- 14.6% versus 72.1% +/- 8.4%, p < 0.05). Of the calcium-magnesium combinations tested, developed pressure was increased compared with unmodified University of Wisconsin solution with calcium 0.1 and magnesium 20 mmol/L (76.8% +/- 6.4%, p < 0.05). Diastolic function was reduced in all groups (p < 0.0001) and not significantly different between groups. CONCLUSIONS: The experiments indicated that recovery after storage with University of Wisconsin solution is enhanced with the addition of calcium and magnesium. The addition of high concentrations of calcium (> or = 2.5 mmol/L) appears harmful.

Radioimmunodetection of cancer using antibodies to alpha-fetoprotein and carcinoembryonic antigen.

This study reports the use of radiolabeled antibody to AFP and CEA for the detection and localization of AFP- or CEA-producing tumors. Thirty-one patients received 131I-labeled anti-AFP or anti-CEA antibodies. Photoscans were taken at 24 and 48 hours after injection of radioantibodies. In three of six patients with CEA-producing tumors, radioimmunodetection with anti-CEA antibody showed positive scans. In AFP-producing tumors, 7 of 15 patients had positive findings on immunoscintigraphy using polyclonal anti-AFP antibody, and two of nine patients had positive findings when monoclonal antibodies were used. Analysis of radioantibody in the blood after injection showed both complex and free antibody with immunoreactivity in the circulation, and smaller complexes were seen to form after administration of monoclonal antibodies.

Effects of butanedione monoxime and temperature on prolonged cardiac storage.

BACKGROUND: The optimal temperature for cardiac allograft storage remains controversial. We conjectured that supplementation of the potent cardioprotective agent 2,3-butanedione monoxime with calcium may improve allograft storage and make the precise storage temperature less critical. METHODS: Hearts were harvested from Sprague-Dawley rats (250 to 350 g), mounted on a Langendorff apparatus, and instrumented with an intraventricular balloon. Hearts were flushed and stored with either unmodified University of Wisconsin solution (UWS) or UWS supplemented with 10 mmol/L of 2,3-butanedione monoxime and calcium 0.1 mmol/L (BDM). Hearts were then subjected to 12 hours of storage at one of five temperatures (0 degree, 4 degrees, 8 degrees, 12 degrees, or 16 degrees C) in a complete 2 x 5 factorial design (n = 6/group). Data are reported either as a percentage of the prestorage results or as an absolute value (mean +/- standard deviation). RESULTS: Recovery of developed pressure (p < 0.0001), coronary flow (p < 0.0001), and diastolic volume (p < 0.001) were significantly enhanced, whereas creatine kinase (p < 0.0001) and lactate dehydrogenase release (p < 0.0001) were reduced in the BDM versus the UWS groups. In both the BDM and UWS storage groups, recovery was better at temperatures of 8 degrees C or less than at 12 degrees C or more. The single preferred temperature was 4 degrees C, significantly better than 0 degree C with unmodified UWS, while similar to 0 degree and 8 degrees C with BDM. Adenine nucleotide values were decreased equally in the BDM and UWS hearts, but preservation was enhanced at 0 degree C compared with all warmer temperatures. CONCLUSIONS: We conclude that 4 degrees C is the preferred temperature for prolonged cardiac storage with UWS and that the inclusion of 2,3-butanedione monoxime with calcium 0.1 mmol/L markedly enhances recovery for storage temperatures of 8 degrees C or less.

Cardiac storage with UW solution and glucose.

Previous investigations from our institution using an isolated human cardiomyocyte model concluded that glucose supplementation of University of Wisconsin solution (UWS) was beneficial with respect to adenine nucleotide and protein recovery. We wished to confirm these results using an isolated heart model. Rodent hearts were frozen in liquid nitrogen (control) or flushed and stored in UWS for 8 hours at 0 degrees C or UWS supplemented with 10, 20, or 30 mmol/L glucose. Experimental hearts were assessed at end-storage or after 45 minutes of reperfusion on a Langendorff apparatus. Adenine nucleotides were assessed by high performance liquid chromatography. In parallel experiments, ventricular function was assessed before and after storage in Langendorff-perfused hearts instrumented with a left ventricular balloon. Glucose supplementation was associated with greater poststorage (20 and 30 mmol/L glucose) and postreperfusion (10, 20, and 30 mmol/L glucose) adenosine triphosphate levels than unmodified UWS. Developed pressure (expressed as a percentage of control values) was increased with 10 mmol/L glucose (75.2% +/- 7.9%, mean +/- standard deviation) compared with unmodified UWS (64.6% +/- 6.6%; p < 0.05). Coronary flow was greater with 10 (72.6% +/- 10.7%) or 20 mmol/L (71.2% +/- 12.5%) versus 0 mmol/L glucose (58.6% +/- 12.1%, p < 0.05). The data support previous in vitro findings and suggest that the addition of 10 mmol/L glucose to UWS is associated with enhanced recovery after prolonged hypothermic storage.

Comparison of two experimental models for assessment of cardiac preservation.

Previous studies from this institution using human cell cultures have suggested that University of Wisconsin solution is preferred for prolonged hypothermic storage for cardiac transplantation. The primary objective of this study was to evaluate the effectiveness of extended cardiac preservation with University of Wisconsin solution by assessing the time-related changes of purine metabolites using two different models of cold storage. Isolated rat hearts (n = 6/group) or human ventricular myocyte cultures (n = 7 dishes/group) were assessed after 0, 6, 12, and 24 hours in University of Wisconsin solution at 0 degrees C using high-performance liquid chromatography. Adenosine triphosphate content decreased from 18.1 +/- 5.4 to 9.6 +/- 2.7 mumol/g dried weight by 12 hours and to 1.0 +/- 0.6 mumol/g by 24 hours (p < 0.0001 by analysis of variance) in the rat model. Adenosine triphosphate content decreased from 0.64 +/- 0.42 to 0.14 +/- 0.11 nmol/micrograms DNA at 6 hours and to 0.04 +/- 0.03 nmol/micrograms DNA by 24 hours (p < 0.00001) in the cardiomyocytes. Inosine monophosphate content increased from 0.1 +/- 0.2 to 10.8 +/- 1.0 by 24 hours (p < 0.0001) in the rat studies. Inosine monophosphate values tended to increase up to 12 hours (p = 0.06) in the cell cultures and then declined. Adenosine concentration increased from 0.3 +/- 0.3 to 2.3 +/- 0.9 mumol/g at 6 hours and declined thereafter (p < 0.0005) in the rodent hearts. Adenosine concentration increased from 0.03 +/- 0.02 to 1.53 +/- 0.72 nmol/micrograms DNA at 6 hours (p < 0.0001) in the cardiomyocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

The limits of cardiac preservation with University of Wisconsin solution.

Previous studies from this institution have suggested that University of Wisconsin solution is preferred for prolonged cardiac storage and preserves high-energy phosphates better than other storage fluids. University of Wisconsin solution contains adenosine (5 mmol/L), which may maintain the concentration of myocardial adenine nucleotides. Cultures of human adult myocytes were grown from left ventricular biopsy specimens obtained from patients undergoing coronary bypass procedures. Cells (seven to nine dishes per group) were rinsed of culture medium and stored at 0 degrees C in University of Wisconsin solution. Cells were analyzed for adenine nucleotide content after 1, 6, 12, and 24 hours of storage by high-performance liquid chromatography (units = nmol/microgram DNA) and compared with control samples (0 hour). Adenosine concentration increased from 0.03 +/- 0.02 (mean +/- standard deviation) to 1.77 +/- 1.03 by 1 hour (p less than 0.0001, analysis of variance) and remained increased thereafter. Adenosine was largely degraded to inosine (0 hours, 0.03 +/- 0.03; 6 hours, 0.88 +/- 0.56; p less than 0.001) and hypoxanthine (0 hours, 0.01 +/- 0.01; 6 hours, 0.15 +/- 0.09; p = 0.004). Measured levels of xanthine and uric acid were extremely low at all time intervals. Adenosine triphosphate levels were maintained at 1 hour (0 hours, 0.64 +/- 0.38; 1 hour, 0.67 +/- 0.45) but declined thereafter (6 hours, 0.21 +/- 0.21; 12 hours, 0.11 +/- 0.09; 24 hours, 0.04 +/- 0.03; p less than 0.0001). Levels of adenosine diphosphate (p = 0.007) and adenosine monophosphate (p less than 0.05) decreased to approximately 25% of original values by 24 hours.(ABSTRACT TRUNCATED AT 250 WORDS)