Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene,and expression of inactive mutant enzyme protein in E. coli

Summary A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G⁹⁸⁵. The same assay consistently revealed A⁹⁸⁵ in genomic DNA from 26 control individuals. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD. All the experiments are consistent with the contention that the G⁹⁸⁵ mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.     

Dynamics of a hydrophobic peptide in membrane bilayers by solid-state nuclear magnetic resonance

Solid-state NMR studies of the dynamics of a synthetic hydrophobic peptide, tert-butyloxycarbonylleucylphenylalanine methyl ester (Boc-Leu-Phe-OMe), in phospholipid bilayers are described. Motionally averaged powder pattern line shapes from 2H- and 15N-labeled backbone and side-chain sites of the peptide in phospholipid bilayers demonstrate the presence of both overall and internal reorientations of substantial amplitude. The peptide motions are shown to be strongly influenced by the motions of the lipids.     

The role of erythropoietin in the production of principal erythrocyte proteins other than hemoglobin during terminal erythroid differentiation

Erythropoietin (EP) controls the terminal phase of differentiation in which proerythroblasts and their precursors, the colony forming units-erythroid (CFU-e), develop into erythrocytes. Biochemical studies of this hormone-directed terminal differentiation have been hindered by the lack of a homogeneous population of erythroid cells at the developmental stages of CFU-e and proerythroblasts that will synchronously differentiate in response to EP. Such a population of cells can be prepared from the spleens of mice with the acute erythroblastosis resulting from infection with anemia-inducing Friend virus (FVA). Using these FVA-infected erythroid cells, which were induced to differentiate with EP, four proteins other than hemoglobin that have key functions in mature erythrocytes were monitored during the 48-hour period of terminal differentiation. Synthesis of spectrin and membrane band 3 proteins were determined by immunoprecipitation and SDS-polyacrylamide gel electrophoresis; accumulation of the cytoskeletal protein band 4.1 was monitored by immunoblotting; carbonic anhydrase activity was measured electrometrically. Band 3 synthesis and band 4.1 accumulation could be detected only after exposure of the cells to EP. Spectrin synthesis was ongoing prior to culture with EP, but it did increase after exposure to the hormone. Carbonic anhydrase-specific activity changed very little throughout the terminal differentiation process. These results reveal at least three patterns of production of principal erythrocyte proteins during EP-mediated terminal differentiation of FVA-infected erythroid cells. Depending on the specific protein examined, de novo synthesis can be induced by EP, an ongoing production can be enhanced by EP, or the production of a protein can be completed at a developmental stage prior to EP-mediated differentiation in these cells.     

Phenylacetic acid in an anaerobic swine manure digester

Summary The presence of phenylacetic acid (PAA) in an anaerobic swine manure digester was determined by gas chromatography of the butyl ester and confirmed by mass spectroscopy. PAA concentration increased during start-up of a digester and with low carbon, high nitrogen loading. Unlike acetate, propionate and butyrate, the concentration of PAA varied little through the day in a stable digester loaded once per day. The laboratory scale digester was loaded at 4 g of swine manure solids/liter digester volume per day. The retention time and temperature were 15 days and 37°C. PAA is a microbial intermediate which is produced by one group of anaerobic bacteria and converted to methane by other members of the bacterial community in the digester. As such, it may be a useful indicator of the relative metabolic activity of the bacterial groups and thus of the overall stability of the anaerobic process.     

Control of sugar transport in human fibroblasts independent of glucose metabolism or carrier-substrate interaction

Transport regulation by different metabolizable and nonmetabolizable sugars was studied in human fibroblasts. Sugars were classed as glucose-like (D-mannose, 3-0-methyl-D-glucose, thio-D-glucose, and D-allose) and starvation-like (D-galactose, D-fructose, L-glucose, D-xylose, 6-deoxy-D-glucose and 2-deoxy-D-glucose) based on their competence in curbing glucose starvation enhanced transport. No significant correlation existed between the ability of a sugar to curb hexose transport and the KI of that sugar in inhibiting hexose transport. Independence of the transport curb from glucose metabolism was observed since nonmetabolizable analogs of D-glucose when substituted for D-glucose in the culture medium effected glucose [i.e. 3-0-methyl-D-glucose (3-OMG)] and starvation-like (i.e. 6- and 2-deoxy-D-glucose) effects. The KI of inhibition pf 2-deoxy-D-glucose transport for 3-OMG was 8.5 mM, similar to those obtained for 6-deoxyglucose and 2-deoxyglucose on 2-deoxyglycose transport (7.5 and 3.5 mM, respectively) and on 3-0-methylglucose transport (3.5 and 2.5 mM, respectively). An equimolar mixture of D-glucose and 3-OMG (5.55 mM each) was more effective than 11.1 mM D-glucose or 3-OMG alone in curbing hexose transport or reversing hexose starvation induced increases in transport. The effect of 3-OMG may be independent of glucose metabolism but it is possible that 3-OMG structurally mimics a metabolite of glucose that may interact with intracellular regulators of carrier degradation and or expression.     

Phosphorylation of histone H1 through the cell cycle of Physarum polycephalum. 24 sites of phosphorylation at metaphase

H1 phosphorylation has been studied through the precise nuclear division cycle of Physarum polycephalum. The number of sites of phosphorylation of Physarum H1 is very much larger than the number of sites reported for mammalian H1 molecules which is consistent with the larger molecular weight of Physarum H1. At metaphase all of the Physarum H1 molecules contain 20-24 phosphates. Immediately following metaphase, these metaphase-phosphorylated H1 molecules undergo rapid dephosphorylation to give an intermediate S phase set of phosphorylated H1 molecules containing 9-16 phosphates. Progressing into S phase newly synthesized H1 is phosphorylated and eventually merges with the old dephosphorylated H1 to give a ladder of bands 1-20. By the end of S phase or early G2 phase, there is a ladder of bands 1-16 all of which undergo phosphate turnover. Further into G2 phase the bands move to higher states of phosphorylation, and by prophase all of the H1 molecules contain 15-24 phosphates which increases to 20-24 phosphates at metaphase. These results support the proposals that H1 phosphorylation is an important factor in the process of chromosome condensation through G2 phase, prophase to metaphase.     

Natural Selection VS. Random Drift: Evidence from Temporal Variation in Allele Frequencies in Nature

We have obtained monthly samples of two species, Drosophila pseudoobscura and Drosophila persimilis, in a natural population from Napa County, California. In each species, about 300 genes have been assayed by electrophoresis for each of seven enzyme loci in each monthly sample from March 1972 to June 1975. Using statistical methods developed for the purpose, we have examined whether the allele frequencies at different loci vary in a correlated fashion. The methods used do not detect natural selection when it is deterministic (e.g., overdominance or directional selection), but only when alleles at different loci vary simultaneously in response to the same environmental variations. Moreover, only relatively large fitness differences (of the order of 15%) are detectable. We have found strong evidence of correlated allele frequency variation in 13-20% of the cases examined. We interpret this as evidence that natural selection plays a major role in the evolution of protein polymorphisms in nature.     

Glucocorticoid inhibition of immunoreactive beta-endorphin release from the anterior lobe of the rat pituitary: in vitro and in vivo studies

Glucocorticoid control of pituitary beta-endorphin (beta-END) release was investigated in vitro and in vivo. Cultured cells of both rat anterior (AL) and neurointermediate (NIL) lobe released beta-END-like immunoreactivity (beta-END-LI) in response to epinephrine (10(-7) M); however, only the response of AL cells was prevented by corticosterone (10(-8)-10(-6) M) or dexamethasone (10(-9)-10(-7) M). Gel chromatographic analysis (Sephadex G-50) revealed that the major forms of beta-END-LI released by AL cells corresponded to beta-END and beta-lipotropin (beta-LPH) in molecular size, whereas virtually all of the immunoreactivity released by NIL cells resembled beta-END. In vivo administration of dexamethasone attenuated the stress-induced release of beta-END-LI in a dose- and time-related fashion, having a more pronounced effect on plasma levels of beta-END-LI corresponding to beta-LPH in molecular size. Metyrapone (100 mg/kg), an inhibitor of glucocorticoid synthesis, evoked a rapid (20-40 min) four- to sixfold increase in total plasma beta-END-LI and 75% of this rise was due to immunoreactivity resembling beta-LPH in size. This response was diminished by coadministration of either dexamethasone (0.05-1.25 mg/kg) or corticosterone (0.05-1.25 mg/kg) and completely prevented by 4-hr pretreatment with dexamethasone (50 micrograms/kg). The briskness of the plasma beta-END-LI response to acute changes in glucocorticoid status suggests that a "rapid" feedback mechanism operates in the physiologic control of pituitary beta-END-LI secretion. Moreover, the ability of glucocorticoids to selectively inhibit AL release of beta-END-LI in vitro and their pronounced effect on plasma levels of beta-END-LI resembling beta-LPH, a marker of AL secretion, together indicate that glucocorticoids exert a selective influence over the secretion of AL corticotrophs in vivo. This demonstration of differential regulation of the AL versus IL secretion of beta-END-LI in vivo most likely reflects a phenomena having biologic importance related to the different physiologic actions of the several molecular forms of beta-END-LI secreted by the two tissues.