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.     

Polymorphism in a ferritin H gene from chromosome 6p

Summary This paper addresses the question of whether abnormalities in ferritin expression in the iron storage disease hemochromatosis (HC) involve major deletions or alterations in regions containing the two ferritin H genes that lie near the disease locus on chromosome 6p. We present evidence from analyses of Southern blots that neither gene is deleted in hemochromatosis. We also describe a polymorphism in one of the genes that we have previously shown to be a processed pseudogene. This polymorphism does not correlate with the presence of HC. The PIC value for this polymorphism was calculated as 0.49.     

Simultaneous degradation of the herbicides 2,4-dichlorophenoxyacetic acid and 2-(2-methyl-4-chlorophenoxy)propionic acid by mixed bacterial cultures

The simultaneous degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-(2-methyl-4-chlorophenoxy)propionic acid (mecoprop) was achieved by two mixed cultures in the absence of any additional carbon or energy substrates. Mecoprop was not completely degraded by either of the two cultures, nor did addition of 2,4-D affect the degradation of mecoprop. The cultures completely degraded 2,4-D, and the degradation was uninfluenced by the addition of mecoprop. Nearly complete dechlorination of the mixture of two herbicides was achieved by both cultures, on the basis of the total amount of the two herbicides degraded. During the course of the reaction, however, the expected values of chloride were not met. Cell growth continued after the degradation of the parent substrates ceased. Although the mecoprop degradation did not continue to completion, spectral and growth data indicated that the metabolites which had accumulated during the reaction were degraded upon further incubation.     

Structure and function of disk aggregates of the coat protein of tobacco mosaic virus

Experiments have been carried out on the coat protein of tobacco mosaic virus (TMVP) to test for the occurrence of the previously postulated RNA-induced direct switching, during in vitro assembly of tobacco mosaic virus (TMV), of the subunit packing from the cylindrical bilayer disk to the virus helical arrangement. No evidence was found for such RNA-induced switching and no evidence for the direct participation of the bilayer disk in either the nucleation or elongation phases of the in vitro virus assembly. Instead, virus assembly proceeds by an initiation step involving the binding of the RNA to the previously characterized two-plus turn helical aggregate that is formed from small oligomers of subunits. However, a bilayer disk, which has been characterized in high ionic strength crystals, has been observed in low ionic strength virus assembly solutions only as a transient species upon depolymerization of dimers of bilayer disks formed in solution at high ionic strength, and not as an equilibrium species of TMVP.     

The effects of axotomy on electrophysiological properties of B cells of bullfrog sympathetic ganglia conditioned by a previous lesion

In bullfrog sympathetic B cells, axotomy decreases the amplitude and decay time of membrane afterhyperpolarization (AHP) and increases action potential (AP) duration. A second (test) axotomy, 7 days after an initial (conditioning) axotomy, did not amplify these changes. No recovery of AHP amplitude or AP duration occurred by 56 days post-axotomy, but AHP decay time recovered 21 days earlier than after test axotomy alone. Conditioning, previously shown to accelerate regeneration, speeds the return to normal of those membrane properties previously shown to recover after axotomy.     

Suppressors of the ras2 Mutation of SACCHAROMYCES CEREVISIAE

Saccharomyces cerevisiae contains two members of the ras gene family. Strains with disruptions of the RAS2 gene fail to grow efficiently on nonfermentable carbon sources. This growth defect can be suppressed by extragenic mutations called sra. We have isolated 79 independent suppressor mutations, 68 of which have been assigned to one of five loci. Eleven additional dominant mutations have not been assigned to a specific locus. Some sra1 and SRA4 and all SRA3 mutations were RAS independent, allowing growth of yeast cells that lack a functional RAS gene. Mutations in sra1, SRA3, SRA4 and sra6 are linked to his6, ino1, met3 and ade6, respectively. Some sra mutants have pleiotropic phenotypes that affect glycogen accumulation, sporulation, viability, respiratory capacity and suppression of two cell-division-cycle mutations, cdc25 and cdc35. The proposed functions of many of the suppressor genes are consistent with the model in which RAS activates adenylate cyclase.     

alpha-Difluoromethylornithine induces differentiation of a human embryonal carcinoma cell line in vitro

Human embryonal carcinoma cells could serve as a useful model system for analysis of early human development. A limited number of human embryonal carcinoma cell lines have been generated from in vivo tumors. We report here that alpha-difluoromethylornithine, a specific enzyme-activated inhibitor of ornithine decarboxylase activity, can induce differentiation in human embryonal carcinoma cells. The differentiated phenotype could be distinguished from undifferentiated cells by altered cellular morphology, biochemical and cell surface antigenic properties. These results suggest that alterations in the intracellular levels of polyamines may play a role in human embryonal carcinoma cell differentiation, and possibly human embryogenesis.     

Production of steroids and release of prostaglandins by spherical pig blastocysts in vitro

Levels of pregnenolone and progesterone in spherical pig blastocysts (near 4 and 15 microM respectively) exceeded respective levels in histotroph by about 400-fold. When blastocysts were cultured for 5 days in a synthetic medium containing pregnenolone sulfate (1 microM), daily rates of release of pregnenolone, progesterone, androstenedione, testosterone, oestrone and oestradiol were determined to be near 320, 45, 26, 27, 0.8 and 9.2 fmol per blastocyst respectively. Daily outputs of progesterone and testosterone (fmol per blastocyst) diminished (P less than 0.05) to 1.3 and undetectable levels (less than 2) respectively in the presence of Trilostane (94 microM). Increasing the content of pregnenolone sulfate in the culture medium (to 4.5 microM) resulted in higher daily rates of release of pregnenolone and progesterone (to near 1740 and 380 fmol per blastocyst respectively), verifying activity of 3 beta-hydroxy-delta 5-steroid dehydrogenase, and of arylsulfatase, in tissues of intact spherical pig blastocysts. Prostaglandin E2 was the predominant prostaglandin (PG) released by cultured blastocysts (about 1 fmol per blastocyst per hour), hourly rates of release of PGH2 (derived) and PGF2 alpha being near 0.1 and less than 0.06 fmol per blastocyst respectively. The data establish a capacity for spherical pig blastocysts to release a range of steroids and PGs of possible significance to embryonic growth and development in vivo.     

Spontaneous interstitial nephritis in kdkd mice. II. Characterization of a tubular antigen-specific, H-2K-restricted Lyt-2+ effector T cell that mediates destructive tubulointerstitial injury

In this report, we have examined the effector T cell repertoire in the spontaneous interstitial nephritis of kdkd mice. Lymph node cells from nephritic kdkd mice are capable of transferring this disease into thymectomized, irradiated, and bone marrow-reconstituted CBA/Ca recipients. CBA/Ca mice do not spontaneously develop interstitial nephritis and are normally resistant to the adoptive transfer of nephritic cells, a resistance that in the short term can be attenuated with low-dose cyclophosphamide. We therefore used delayed-type hypersensitivity responses and direct transfer of immune cells under the renal capsule to characterize nephritogenic effector cells from kdkd donor mice. Lyt-2+, L3T4- T cells from the peripheral lymphoid organs of nephritic kdkd mice, after adoptive transfer into cyclophosphamide-pretreated CBA/Ca recipients, mediate an antigen-specific delayed-type hypersensitivity response to renal tubular basement membrane antigens. These cells are restricted by gene products in H-2Kk; they are also present in nephritic, but not in control kidneys. We have also observed this same phenotypic subpopulation of kdkd lymphocytes mediate a destructive interstitial renal lesion within 7 days of being placed under the kidney capsule of CBA/Ca mice. These findings suggest that T lymphocytes reactive to a parenchymal tubular antigen are of substantial importance in the development of spontaneous interstitial nephritis in kdkd mice.