Catalytic activity of caspase-3 is required for its degradation: stabilization of the active complex by synthetic inhibitors.

The activation of caspase-3 represents a critical step in the pathways leading to the biochemical and morphological changes that underlie apoptosis. Upon induction of apoptosis, the large (p17) and small (p12) subunits, comprising active caspase-3, are generated via proteolytic processing of a latent proenzyme dimer. Two copies of each individual subunit are generated to form an active heterotetramer. The tetrameric form of caspase-3 cleaves specific protein substrates within the cell, thereby producing the apoptotic phenotype. In contrast to the proenzyme, once activated in HeLa cells, caspase-3 is difficult to detect due to its rapid degradation. Interestingly, however, enzyme stability and therefore detection of active caspase-3 by immunoblot analysis can be restored by treatment of cells with a peptide-based caspase-3 selective inhibitor, suggesting that the active form can be stabilized through protein-inhibitor interaction. The heteromeric active enzyme complex is necessary for its stabilization by inhibitors, as expression of the large subunit alone is not stabilized by the presence of inhibitors. Our results show for the first time, that synthetic caspase inhibitors not only block caspase activity, but may also increase the stability of otherwise rapidly degraded mature caspase complexes. Consistent with these findings, experiments with a catalytically inactive mutant of caspase-3 show that rapid turnover is dependent on the activity of the mature enzyme. Furthermore, turnover of otherwise stable active site mutants of capase-3 is rescued by the presence of the active enzyme suggesting that turnover can be mediated in trans.

Conformational difference between PDE4 apoenzyme and holoenzyme.

The type 4 cAMP-specific phosphodiesterases (PDE4s) are Mg(2+)-dependent hydrolases that catalyze the hydrolysis of 3', 5'-cAMP to AMP. Previous studies indicate that PDE4 exists in two conformations that bind the inhibitor rolipram with affinities differing by more than 100-fold. Here we report that these two conformations are the consequence of PDE4 binding to its metal cofactor such as Mg(2+). Using a fluorescence resonance energy transfer (FRET)-based equilibrium binding assay, we identified that L-791,760, a fluorescent inhibitor, binds to the apoenzyme (free enzyme) and the holoenzyme (enzyme bound to Mg(2+)) with comparable affinities (K(d) approximately 30 nM). By measuring the displacement of the bound L-791,760, we have also identified that other inhibitors bind differentially with the apoenzyme and the holoenzyme depending upon their structure. CDP-840, SB-207499, and RP-73401 bind preferentially to the holoenzyme. The conformational-sensitive inhibitor (R)-rolipram binds to the holoenzyme and apoenzyme with affinities (K(d)) of 5 and 300 nM, respectively. In contrast to its high affinity (K(d) approximately 2 microM) and active holoenzyme complex, cAMP binds to the apoenzyme nonproductively with a reduced affinity (K(d) approximately 170 microM). These results demonstrate that cofactor binding to PDE4 is responsible for eliciting its high-affinity interaction with cAMP and the activation of catalysis.

Evaluation of canine lumbosacral stenosis using intravenous contrast-enhanced computed tomography.

The objective of this study was to evaluate intravenous contrast-enhanced computed tomography as a technique for predicting the within-level location(s) of compressive soft tissues in the canine lumbosacral spine. Pre-operative intravenous contrast-enhanced computed tomography of the L5-S3 vertebral levels was performed in 12 consecutive large breed dogs with lumbosacral stenosis. The images were evaluated for enhancement of soft tissues by two radiologists who were unaware of the surgical findings. For each within-level location (dorsal canal, ventral canal, right lateral recess, left lateral recess) enhancement was classified as present, absent or equivocal. The results were compared with the results of surgical exploration and histopathology of excised tissues. The positive predictive values of intravenous contrast-enhanced computed tomography for compressive soft tissues involving the dorsal canal, ventral canal and lateral recesses were 83%, 100%, and 81% respectively. Negative predictive values for compressive soft tissues involving these locations were 29%, 50%, and 40% respectively.

Fetal plasma iron and restoration of red blood cell mass after hemorrhage of the ovine fetus.

OBJECTIVE: Our purpose was to determine whether the restoration of fetal red blood cell mass after acute hemorrhage of 40% of the fetal blood volume is related to fetal plasma iron concentration. STUDY DESIGN: Ten chronically catheterized ovine fetuses were monitored for 10 days beginning at 125 +/- 1 (SE) days of gestation. After a 3-day control period 40% of the fetal blood was removed over 2 hours at a rate of approximately 1 ml/min. Fetal plasma iron and erythropoietin concentrations, hematocrit, blood volume, and red blood cell mass were measured daily before and for 7 days after fetal hemorrhage. Statistical analysis was by analysis of variance, correlation, and regression. RESULTS: Although blood volume was restored within 3 days of the hemorrhage (101.0% +/- 1.4% of prehemorrhage volume), red blood cell mass was not (81.8% +/- 2.8%). Only 6 of 10 fetuses restored their red blood cell mass to prehemorrhage levels by the end of the 7-day posthemorrhage period. On day 10 red blood cell mass correlated positively with prehemorrhage (r = 0.74, p = 0.015) and posthemorrhage (r = 0.69, p = 0.045) plasma iron concentration and negatively with posthemorrhage erythropoietin concentration (r = -0.68, p = 0.047). CONCLUSION: Fetal plasma iron concentration is an important factor in restoration of fetal red blood cell mass after loss of blood. The negative correlation of erythropoietin concentration with posthemorrhagic red blood cell mass suggests that iron, not erythropoietin, may be the limiting factor in recovery from hemorrhage-induced anemia. Thus iron supplementation of the fetus may be of benefit in the treatment of some types of fetal anemia.

Intrapericardial teratoma in a twin fetus: diagnosis and management.

BACKGROUND: A rare cause of hydrops fetalis, intrapericardial teratoma is invariably associated with a pericardial effusion. In fetal life, the effusion or mass effect may cause cardiac tamponade, hydrops, and death. After delivery, ventilation and cardiac output may be compromised. CASE: One fetus of twins was diagnosed at 20 weeks' gestation with an intrapericardial teratoma. The affected twin underwent two intrauterine pericardiocentesis and had the tumor resected after delivery at 35 weeks' gestation. One year later, both twins are alive and well and have no evidence of tumor recurrence. CONCLUSION: Pericardiocentesis for tamponade secondary to a fetal intrapericardial teratoma, even when complicating a twin pregnancy, may prevent fetal death and allow delay of delivery until adequate fetal lung maturity has been achieved.

Enzymatic preparation of biosurfactants from sugars or sugar alcohols and fatty acids in organic media under reduced pressure.

Biosurfactants were prepared by enzymatic esterification of sugars and sugar alcohols in nonaqueous media. Sorbitol monooleate was produced in pure molten substrates, with reduced pressure to remove water. The results were compared to synthesis in organic solvent, with and without water removal. Synthesis in organic solvent with water removal, obtained by refluxing through a desiccant under reduced pressure, proved to be the most efficient method in terms of total yield and side-products formation. This process was applied to the production of different surfactants, by changing the nature of the hydrophilic and hydrophobic moieties. Yields above 90% of monoesters were obtained after 24 h when the reaction was carried out in 2-methyl-2-butanol with Novozym 435 (Type B lipase from Candida antarctica) with an excess of hydroxyl donor.

Magnesium status and excretion in age-matched subjects with normal and elevated blood pressures.

Plasma, erythrocyte, and leucocyte magnesium (Mg) concentration, as well as urinary Mg, calcium (Ca), and phosphorus (P) excretion were determined in male subjects with elevated diastolic blood pressure (DBP) > 90 mmHg. These parameters were compared to those in normotensive age-matched (DBP < 85 mmHg) and drug-treated hypertensive subjects. Mg and Ca were determined by atomic absorption spectroscopy and P was determined by a colorimetric method. Urinary excretion was expressed in terms of creatinine (Cr) excretion. There were no significant differences in plasma, erythrocyte, or leucocyte Mg concentrations. Both urinary Ca and Mg were significantly decreased in the group with elevated blood pressure (BP) and the drug-treated group compared to the normotensive group. Urinary P was elevated in the drug-treated hypertensive group. Of all the variables studied, urinary Mg was the only one that was significantly inversely correlated with both systolic (SBP) and DBP. These data suggest that decreased urinary Mg, which may be indicative of a decreased Mg intake, is associated with hypertension.

Effects of low copper and high zinc intakes and related changes in Cu,Zn-superoxide dismutase activity on DMBA-induced mammary tumorigenesis.

The effect of low copper and high zinc intakes on Cu,Zn-superoxide dismutase (Cu,Zn-SOD) activity and mammary tumorigenesis induced by 9,10-dimethyl-1,2-benzanthracene (DMBA) was investigated. Groups of 40 weanling female Sprague-Dawley rats were fed a modified AIN-76 diet containing the following (/kg diet): 1 mg Cu (0.016 mmol) and 30 mg Zn (0.459 mmol); 6 mg Cu (0.094 mmol) and 30 mg Zn (0.459 mmol) (control); or 6 mg Cu (0.094 mmol) and 150 mg Zn (2.295 mmol) for 21 wk. At 5 wk, 30 rats/group were given 4 mg (15.6 mumol) DMBA in corn oil intragastrically, and controls (10/group) received corn oil alone. Erythrocyte Cu,Zn-SOD activity was measured at 3, 5 (just before DMBA), 9, 13, 17, and 21 wk. The group fed the high-Zn diet had a slightly lower weight gain and food consumption. DMBA treatment had no effect on these parameters. Plasma and liver Cu concentration decreased in the low-Cu group. Femur zinc was significantly elevated in the high-Zn group. Erythrocyte Cu,Zn-SOD activity was decreased in the low-Cu group from 3 to 21 wk and was significantly elevated in the high-Zn group at 3 and 5 wk. In the low-Cu group, there were 5 nonmalignant adenomas and 3 malignant adenocarcinomas; in the control group, there were 4 adenomas and 3 adenocarcinomas; in the high-Zn group, there were 5 adenomas and 3 adenocarcinomas. No relationship between Cu,Zn-SOD activity and the presence of tumors could be found.

An evaluation of plasma and erythrocyte magnesium concentration and the activities of alkaline phosphatase and creatine kinase as indicators of magnesium status.

Rats were fed diets containing magnesium at concentrations ranging from 3.3 to 26.7 mmol/kg of diet (80 to 650 mg/kg). The magnesium concentration of their plasma and erythrocytes, and the activities of plasma alkaline phosphatase and creatine kinase were investigated to determine their usefulness as indices of magnesium status. All the indices increased with increasing dietary magnesium levels. The best correlations were observed between dietary intake and plasma concentration of magnesium (r = 0.846, p less than 0.001) and between intake and femur concentration (r = 0.811, p less than 0.001). There was an extremely high correlation between plasma concentration and femur concentration (r = 0.930, p less than 0.001). Although significant, the correlations between intake and the enzyme activities were not strong. It is concluded that plasma magnesium concentration is the most useful indicator of magnesium status and that the activities of the two magnesium-requiring enzymes can only be used for the purpose of diagnosing severely deficient magnesium status.

Effect of dietary iodine on autoimmune thyroiditis in the BB Wistar rats.

Diabetes-prone BB Wistar rats were fed a modified AIN-76 diet providing the following amounts of iodine for 10 wk: 0.2 mg/kg diet (recommended amount); 1.0 mg/kg; 2.0 mg/kg; or 3.0 mg/kg. The thyroids were examined for gross and microscopic changes and sera were assayed for antibodies to triiodothyronine (T3), thyroxine (T4), and thyroglobulin (Tg). The body weights and food consumption of the rats fed 0.2 mg of iodine/kg were significantly lower than those of the animals fed higher amounts. Urinary iodine excretion reflected dietary intakes. The thyroids from animals fed 2.0 and 3.0 mg/kg were significantly (P less than 0.01) larger than those from animals fed 0.2 mg/kg. One rat fed 0.2 mg/kg and 2 rats in each group fed 2.0 and 3.0 mg/kg had extensive lymphocytic thyroiditis. Three rats fed 1.0 mg/kg, 6 fed 2.0 mg/kg and 6 fed 3.0 mg/kg had enlarged thyroids. Two rats fed 0.2 mg/kg, 2 fed 2.0 mg/kg and 6 fed 3.0 mg/kg had detectable Tg antibodies. These data suggest that high iodine intakes increase Tg antibodies, which may be associated with an increase in autoimmune thyroiditis in these animals.

Effects of dietary magnesium on sodium-potassium pump action in the heart of rats.

Sprague-Dawley rats were fed a basal AIN-76 diet containing 80, 200, 350, 500 or 650 mg of magnesium per kilogram of diet for 6 wk. Ventricular slices, as well as microsomal fractions, were prepared from the hearts and were used to determine sodium-potassium pump activity. Sodium-potassium pump activity was assessed in the microsomal membranes by determining the ouabain-inhibitable Na+, K+-ATPase activity and [3H]ouabain binding, and in the ventricular slices, by determining ouabain-sensitive 86Rb uptake under K+-free conditions. The ATPase activity increased with increasing dietary magnesium, so that in the hearts of those animals that were fed 500 and 650 mg of magnesium/kg diet, it was significantly greater than the activity in the hearts of the animals fed 80 and 200 mg/kg diet. Similarly, 86Rb uptake by heart slices from rats fed 500 and 650 mg of magnesium/kg diet was significantly greater than the uptake by heart slices from animals fed 80 and 200 mg/kg diet. [3H]Ouabain binding did not change with increasing dietary magnesium. Thus, magnesium deficiency appears to have no effect on the number of sodium-potassium pump sites, but does decrease the activity of the pump. It is suggested that this leads to an increase in intracellular Na+, resulting in a change in the membrane potential, and may contribute to the arrhythmias associated with magnesium deficiency.

Colorimetric determination of total iodine in foods by iodide-catalyzed reduction of Ce+4.

An earlier acid digestion determination of iodine in foods was modified to provide an improved detection limit and to allow for the analysis of a greater variety and larger amounts of foods. The organic material in the sample was oxidized overnight by concentrated nitric acid, followed by digestion in a mixture of concentrated sulfuric and 70% perchloric acid. The iodine was determined by an automated colorimetric method based on the iodide-catalyzed reduction of Ce+4 by As+3. The method had an average relative standard deviation of 3.1% for the samples analyzed, and a detection limit of 0.1 ng/mL in the digested solution and 5 ng/g in a 2 g sample prior to digestion. The recovery of added iodine ranged from 90.3 to 101.3%, using external standards. Samples analyzed included NBS Standard Reference Material 1549, and composites of a variety of dairy products, meat, eggs and fish, cereals, and potatoes. The iodine detected in these samples ranged from 9 ng/g for the potato group to 3360 ng/g for the standard reference material.

Effect of zinc supplementation on copper status in adult man.

The effects of zinc supplementation on the copper status of healthy adult men, as assessed by the activities of the copper-metalloenzymes, plasma ferroxidase (ceruloplasmin), and erythrocyte Cu,Zn-superoxide dismutase, were determined. The subjects were given either two daily doses of 25 mg zinc or placebo for 6 wk. No significant differences in the plasma copper levels or the ferroxidase activities between the supplemented and control groups could be detected at 2, 4, or 6 wk. Plasma zinc increased and erythrocyte Cu,Zn-superoxide dismutase decreased in the supplemented group, the difference between the groups becoming significant at 6 wk (p less than 0.05). This suggested that the zinc supplements decreased the copper status of the experimental group.

Brainstem evoked response audiometry.

Brainstem evoked response audiometry is useful in the identification of threshold levels as well as in the diagnosis of eighth nerve tumors. The instrumentation is a modification of the electroencephalograph and an averaging computer; 3,000 clicks, 16/s, are presented and averaged for each printed response. Evoked responses can be obtained from a patient who is unable or unwilling to respond to conventional behavioral testing; best results are obtained from quiet or asleep patients.

Effects of zinc on mucosal copper binding and on the kinetics of copper absorption.

The kinetics of serosal copper transfer and the distribution of copper between metallothionein (MT) and a high-molecular-weight protein fraction (HMWPF) within the mucosal cells were examined, using isolated duodenal segments from rats fed different amounts of zinc. No difference in the Vmax or Km for serosal transfer could be detected between the different zinc groups, suggesting that zinc did not affect this aspect of copper absorption. When intestinal segments from rats fed the low zinc diet were incubated in high copper media, the majority of the copper was associated with the HMWPF, while those from rats fed the high zinc diet had the largest proportion bound to MT. All the intestines incubated in the low copper media, regardless of rat zinc status, had the majority of copper associated with MT. These results suggested that with the low zinc diet, the limited amount of MT present was saturated, and the excess copper was bound to the HMWPF. With larger intakes of zinc, MT synthesis was induced, and the larger amount of this protein was not as readily saturated, resulting in less copper binding to the HMWPF and more to MT. It is suggested that zinc interferes with copper absorption by inducing MT, which sequesters copper in the mucosal cells, making it unavailable for serosal transfer. The copper bound to the HMWPF is available for transfer.

The effect of dietary zinc on intestinal copper absorption.

Everted duodenal segments, tied into sacs, taken from animals fed different amounts of zinc were used to investigate the antagonistic effect of dietary zinc on copper absorption. The intestinal segments taken from animals fed low amounts of zinc transferred more copper from a nutrient medium across the mucosal cells than did intestines from rats fed high levels of zinc. The mucosal cells from animals fed low amounts of zinc retained less copper than the cells from animals fed high amounts of the element. This retained copper was bound to a protein fraction having a molecular weight similar to that of metallothionein. The data suggest that zinc exerts its antagonistic effect by inducing the synthesis of a copper-binding ligand, probably a thionein, in the mucosal cells which sequesters copper from the nutrient medium, making it unavailable for serosal transfer. This may be a possible mechanism by which dietary zinc decreases copper absorption and leads to a decreased copper status.

The effect of dietary copper and zinc on cholesterol metabolism.

The effects of different amounts of dietary zinc and copper on cholesterol metabolism in the rat were investigated. The levels of the minerals used were comparable to those likely to occur in a normal mixed North American diet. Although the different levels affected the serum, liver, and femur concentrations of the minerals, they had no effect on the levels of serum and liver, total, esterified or free cholesterol, or on high density lipoprotein cholesterol. It was concluded that dietary copper and zinc, at levels likely to occur in a normal mixed diet, are not significant factors in cholesterol metabolism.