Glutaric acidemia type II. Heterogeneity in beta-oxidation flux, polypeptide synthesis, and complementary DNA mutations in the alpha subunit of electron transfer flavoprotein in eight patients.

We studied metabolic, polypeptide and genetic variation in eight glutaric acidemia type II (GA II) patients with electron transfer flavoprotein (ETF) deficiency. As measured by 3H-fatty acid oxidations in fibroblasts, beta-oxidation pathway flux correlated well with clinical phenotypes. In six patients with severe neonatal onset GA II, oxidation of [9,10(n)-3H]-palmitate ranged from 2% to 22% of control and of [9,10(n)-3H]myristate, from 2% to 26% of control. Of two patients with late onset GA II, one had intermediate residual activities with these substrates and the other normal activities. Radiolabeling and immunoprecipitation studies revealed that three of the six neonatal onset GA II patients had greatly diminished or absent alpha- and beta-ETF subunits, consistent with a failure to assemble a stable heterodimer. Another neonatal onset patient showed normal synthesis of beta-ETF but decreased synthesis of alpha-ETF. Two neonatal onset and two late onset GA II patients showed normal synthesis of both subunits. Analysis of the pre-alpha-ETF coding sequence revealed seven different mutations in the six patients with neonatal onset GA II. The most common mutation was a methionine for threonine substitution at codon 266 found in four unrelated patients, while all the other mutations were seen in single patients. No mutations were detected in the two patients with late onset GA II.

An atypical case of cytochrome c oxidase deficiency with biochemical heterogeneity in fibroblasts.

A patient presenting in the first year of life with feeding difficulties and failure to grow had variable but persistent lactic acidemia noted at age 20 months. Nonspecific nutritional and biochemical therapy was accompanied by improvement in general clinical status, growth, gait, and development. However, she died in a catastrophic illness at the end of the third year of life. Studies in intact fibroblast mitochondria were consistent with an isolated but partial defect in cytochrome c oxidase. On direct assay of this enzyme complex in fibroblast homogenates and mitochondria, activity was much more severely depressed (less than or equal to 8% of control). Her fibroblasts normally synthesized the three cytochrome c oxidase subunits encoded on the mitochondrial genome. These data confirm that this patient had cytochrome c oxidase deficiency and demonstrate significant biochemical heterogeneity, since the results of the intact mitochondrial studies correlate better with her clinical course than do those of the direct enzymatic assays.

Effect of kernel density on the apparent and true metabolizable energy value of corn for chickens.

Five samples of corn selected to vary widely in kernel density (test weight per unit volume) were assayed for AMEn with male broiler chickens at 4 wk of age by regression analysis of a multilevel assay and for TMEn with adult White Leghorn roosters. The kernel densities (kilograms per hectoliter), AMEn, and TMEn (kilocalories per gram of DM) values of the corn were, respectively, 72, 3.68, and 3.96; 71, 3.72, and 3.95; 68, 3.66, and 3.90; 62, 3.64, and 3.88; 60, 3.54, and 3.68. Positive relationships were established between AMEn and TMEn (r = .974, P less than .01) or kernel density (r = .875, P less than .05). The assay for TMEn provided a simple, rapid and sensitive method for estimating the AMEn content of corn for chickens. The small (4%) variation in AMEn relative to a large (20%) variation in kernel density precludes the use of kernel density for estimating the AMEn content of corn.

Effect of the previous diet, body weight, and duration of starvation of the assay bird on the true metabolizable energy value of corn.

Two experiments were conducted with 36 adult White Leghorn roosters to determine the effect of previous intake of protein (Experiment 1) or purified cellulose (Experiment 2), body weight, and duration of starvation of the assay bird on the true metabolizable energy (TME) value of ground yellow corn. The mean TME value (+/- SE) of the corn was 4.06 +/- .03 and 4.06 +/- .04 kcal/g of dry matter in Experiment 1 and 2, respectively. Dietary levels of 10, 20, and 30% protein or 0, 7.5, and 15.0% supplementary cellulose, and body weight distribution had no significant (P greater than .05) effect on the TME value of the corn. Starvation for 12 hr before force feeding resulted in a significant (P less than .05) decrease of 4% in the TME value of the corn, whereas fasting periods of 24 or 48 hr had no significant effect. It is suggested that the assay birds be paired on the basis of body weight and starved for a minimum period of 24 hr prior to force feeding to reduce the variation associated with the estimate of TME.

Rate of passage of corn-canola meal and corn-soybean meal diets through the gastrointestinal tract of broiler and White Leghorn chickens.

The effect of corn-canola meal and corn-soybean meal diets on the form and function of the gastrointestinal tract of broiler (meat-type) and White Leghorn (egg-type) cockerels was measured from 14 to 44 and 14 to 86 days of age or 203 to 1,844 and 115 to 1,777 g of body weight, respectively. Dry weights of the empty crop (P less than .01), gizzard (P less than .001), and ceca (P less than .001) relative to live body weight (g/kg) were lighter in broilers than in Leghorns. Canola meal at 370 g/kg diet was associated with increased (P less than .001) dry weight of the gizzard and jejunum relative to body weight. Soybean meal at 370 g/kg diet was associated with increased (P less than .001) dry weight of the ceca relative to body weight. The lengths, relative to a power of body weight of the duodenum (cm/g.187) and jejunum plus ileum (cm/g.240), were longer (P less than .001) in broilers than in Leghorns. The canola meal diet was associated with an increase (P less than .001) in length of the jejunum plus ileum (cm/g.240) relative to a power of body weight. Mean retention time (MRT) of a particle marker, 103ruthenium phenanthroline, increased with body weight in the entire gastrointestinal tract (P less than .001) and in each of its segments except in the proventriculus, where it was not affected by body weight (P greater than .05), and in the gizzard, where it decreased (P less than .05) with body weight. The MRT, adjusted for body weight in the entire gastrointestinal tract of broilers (338.0 +/- 10.8 min) and Leghorns (359.9 +/- 10.8 min), was similar (P greater than .05) but varied significantly in segments of the gut for both type of chicken and diet. Adjusted MRT was shorter in the crop (P less than .001) and gizzard (P less than .001) and longer in the duodenum (P less than .001) and ileum (P less than .01) of broilers than Leghorns. The soybean meal diet was retained for 2.3 min longer in the duodenum (P less than .001) and 84.2 min longer in the ceca (P less than .001) than the canola meal diet, which accounted for the longer (P less than .001) retention of the soybean meal diet in the entire gastrointestinal tract (388.0 +/- 10.6 vs. 309.8 +/- 10.8 min). Segments of the gastrointestinal tract vary in length, weight, and MRT of digesta with dietary composition and type and body weight of chicken.

The isolation of HTC variant cells which can replicate in butyrate. Changes in histone acetylation and tyrosine aminotransferase induction.

We have obtained a number of variant HTC cells which are capable of vigorous replication in the presence of 6 mM sodium butyrate. These cells show characteristic changes in histone acetylation. H2A/H2B are no longer modified and the turnover of histones H3/H4 acetate is about 4-fold greater than in control HTC cells at the same butyrate concentration. Histone deposition continues successfully even though histones H3/H4 become hyperacetylated upon association with the chromatin. Prompt deacetylation of new histones does not appear to be a prerequisite for successful deposition processes. Initial enzymatic studies indicate that not only do the butyrate-resistant cells show an increased deacetylase activity (on a per cell basis), but also the enzyme is less sensitive to sodium butyrate under in vitro assay conditions. In contrast to control HTC cells in 6 mM butyrate in which dexamethasone induction of tyrosine aminotransferase is inhibited, the butyrate-resistant variant cells are capable of tyrosine aminotransferase induction even in the presence of butyrate. The implications of these observations are discussed.

Nonuniform behavior of multiple isoactins in the same cell is a cell-dependent phenomenon.

The functional significance of multiple isoactins in the same cell is still not understood. To address this question, we examined the response of smooth muscle and cardiac muscle alpha-isoactins to a serial extraction procedure applied to both muscle and nonmuscle cell types. We compared these extraction results with results obtained with the beta- and gamma-nonmuscle actin isoforms from the same cells. In differentiated BC3H1 nonfusing muscle cells (smooth muscle alpha-isoactin), in human rhabdomyosarcoma cells (cardiac alpha-isoactin), and in chick skeletal muscle cells (cardiac alpha-isoactin), different fractions were found selectively enriched in either the nonmuscle or the muscle-specific actin isoforms compared with their relative abundance in whole cell extracts. Conversely, when these same isoactins were examined either in undifferentiated BC3H1 cells or in mouse nonmuscle cells stably transfected with a cardiac alpha-isoactin gene, no enrichment of these isoforms above their relative abundance in whole cell extracts was observed. These results indicate that within the muscle or muscle-like cells examined, the different actin isoforms were either selectively utilized or localized. These results further show that isoactin-specific responses observed were apparently related to the cell type in which they were found and not to differences in inherent physical properties such as solubility of the different isoactins examined.

Studies on highly metabolically active acetylation and phosphorylation of histones.

The capacity to effectively label tumor cell hostones using very short pulses of [3-H]acetate and [32-P]phosphate (1 to 10 min) has been developed. Four histone fractions F3, F2a1, F2a2, and F2b are extensively acetylated in short time periods. About 70% of the acetate accumulated on the histone during a short pulse is removed with a half-life of similar to 3 min. The rest of the metabolically active acetate is removed with a half-life of 30 to 40 min. Histones F2a1, F2a2, and F1 are acetylated at the NH2 terminus and this modification is metabolically stable. In short pulses, histones are labeled with 32-P in the order F2a2 greater than F1 greater than F3 greater than F2a1 greater than F2b. All fractions have a fairly rapid turnover time (t1/2 similar 20 to 40 min) except F1 phosphate which turns over some 5 times more slowly.

New histones found in mature mammalian testes.

Two new histones have been found in sexually mature testes of several mammals. The new histones have been identified as a new F1 (TF1) and F2b (TF2b) on the basis of their behavior upon chemical fractionation procedures and electrophoresis at several urea concentrations. The new testis-specific histones are absent in very immature animals and in somatic tissues. However, by day 20, in the rat, the histone pattern is that of the adult, displaying nearly a full complement of TF1 and TF2b. The new histone complement evidently is characteristic of prespermatid germ cells. The degree of evolutionary variations in these histones and in other basic sperm proteins, as detected by change in electrophoretic mobility, appears to be much greater than that seen in somatic histones.

Only a small fraction of avian erythrocyte histone is involved in ongoing acetylation.

We have studied histone acetylation in chicken erythrocytes. We find that about 30% of the histone in these cells is acetylated, however the majority of these histones are not in a dynamic steady state typical of other chicken cells and of mammalian cells, but rather are frozen in this state of modification. A very small fraction of erythrocyte histones are being modified normally but cannot be detected as shifting to higher levels of acetylation upon treatment with butyrate because the amount of histone so modified is small. Nonetheless, chicken erythrocytes incorporate 3H-acetate into histones about 40% as well as seen in the dynamically active HTC cells. This is most likely due to the formation of very high specific activity Acetyl CoA pools in erythrocytes which have very low levels of coenzyme A. We conclude that these genetically inactive cells are involved in only a minor way with histone acetylation.

Histone hypoacetylation following release of HTC cells from butyrate.

Exposure of HTC cells to 6 mM sodium butyrate for 20 h results in significantly reduced amounts of nuclear histone acetyltransferase. While the direct butyrate inhibition of histone deacetylases is rapidly reversed upon removal of cells from butyrate, histone acetyltransferase activity increases only gradually following release of cells from butyrate, requiring several hours to regain control cell levels. As a result, HTC cells released from a 20-h exposure to butyrate display normal kinetics of histone acetate turnover but markedly reduced rates of histone acetylation, the imbalance in these two processes produces histone hypoacetylation.