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Biomedical subjects

D M Hunt

Publications and source records attributed to D M Hunt.

At least 145 records · Page 8Linked to original sources
PubMed1984

Aberrant polyadenylation by a vesicular stomatitis virus mutant is due to an altered L protein.

TsG16(I) is a temperature-sensitive mutant of vesicular stomatitis virus, Indiana serotype. Our stocks of this mutant overproduce polyadenylic acid in an in vitro transcription system. The overproduction of polyadenylic acid occurs at all temperatures tested (27, 31, 35, and 39 degrees C) and is apparently not due to an alternation in the N protein-RNA template. To characterize the altered moiety in tsG16(I) responsible for this phenotype, virions were fractionated and the polyadenylation phenotype in homologous and heterologous reconstitution assays was determined. The aberrant polyadenylation phenotype correlated with the presence of ts L protein but not ts NS or ts M protein fractions. Results of experiments in which solubilized tsG16(I) and wild-type virion components were mixed indicated that the altered moiety behaved as if present in stoichiometric amounts relative to active L protein. The effects of raising the temperature from 31 to 39 degrees C in such mixes were as would be predicted upon the assumption that the polyadenylation phenotype was associated with a thermosensitive transcriptase component [the L protein of tsG16(I) is known to be thermosensitive]. We conclude that the data strongly support the hypothesis that L is the altered protein responsible for the aberrant polyadenylation phenotype of tsG16(I).

Animals↗
PubMed1983

A study of intestinal copper-binding proteins in mottled mice.

The substantial retention of Cu2+ and to a lesser extent Zn2+, in the gut mucosa of neonatal MO mutant mice is largely associated with a low molecular weight protein tentatively identified as metallothionein. [35S]Cysteine incorporation into this protein in mutant mice is elevated, indicating that Cu2+ retention in the gut is associated with an increase in the synthesis of metallothionein. The high Cu2+ levels of mutant gut tissue decline rapidly with age to reach an approximately normal level by 24 days of age; this decline cannot be prevented by dietary supplementation and it is suggested that gut 'closure' and consequent reduced uptake by pinocytosis are important factors in this decline.

Aging↗
PubMed1983

Genetic studies on metallothionein synthesis in the mouse: the induction of metallothionein by cadmium in inbred strains.

Differences in the Cd-induced levels of liver metallothionein in inbred mouse strains are described. All strains examined show substantial increases in metallothionein production after the administration of 25 mumol CdCl2/kg, but the maximal levels achieved after 48 hr differed such that CBA/Ca greater than DBA/2 greater than C57BL/10 = C3H/He greater than BALB/c. These differences were generally not paralleled by differences in the amount of Cd2+ bound to metallothionein or in the total tissue content of Cd2+. Incorporation of [35S]cysteine into metallothionein suggest that differential synthesis affecting both isoproteins (MT1 and MT2) equally is largely responsible for the strain difference between C57BL/10 and DBA/2, and interstrain crosses between these two strains reveal codominant inheritance of Cd2+-induced metallothionein level and indicate that a single gene, either linked or identical to the cdm gene on chromosome 3, is involved.

Animals↗
PubMed1983

Metallothionein and the development of the mottled disorder in the mouse.

Copper accumulates in kidney tissue of mottled (Mo) mice largely in association with a low MW cytosol protein, and the reduced copper levels in neonatal mutant liver are largely the result of a reduction in the amount of copper associated with this same protein. On the basis of ion-exchange chromatographic profile, heat stability, absence of a 280nm absorption peak, and the binding of Cd109 and Zn65 the protein mutants in the kidney is identified as metallothionein (MT). Amino acid analysis, however, failed to confirm this, and it is suggested that the high copper content of the mutant protein results in its oxidative degradation during purification, even when normal anaerobic precautions are taken. Estimates of thionein protein content of tissues from mutant and normal mice demonstrated that the levels are significantly elevated in both young and adult mutant kidney and depressed in young mutant liver, in parallel therefore with the changes in tissue copper levels. In adult mutant liver tissue, however, thionein levels are significantly raised, even though tissue copper content is normal. The synthesis and degradation of MT was examined in some detail. Incorporation of S35-cysteine in kidney MT was significantly raised in both young and adult mutant mice, while in adult tissue the rate of degradation of MT was significantly depressed. The elevated kidney MT levels arise therefore in young mutant mice from an increased rate of synthesis and in adult mice from the combined effects of increased synthesis and reduced degradation.

Animals↗
PubMed1983

Vesicular stomatitis virus mutant with altered polyadenylic acid polymerase activity in vitro.

In vitro RNA synthesis by purified virions of a stock of tsG16(I) was aberrant compared with that of wild-type (wt) vesicular stomatitis virus. RNA made in vitro by tsG16(I) contained a larger proportion of A residues in polyadenylic acid [poly(A)] tracts than did RNA synthesized by wt virus, tsG13(I), tsG21(II) or tsG41(IV). Experiments to determine whether the aberrant polyadenylation was correlated with the known thermolability of the tsG16(I) L protein were inconclusive. Total product RNA made by tsG16(I) was methylated to almost the same extent as wt RNA, contained the same major methylated 5' cap structure as wt RNA, and was translated as well in a reticulocyte cell-free system, yielding the same molecular weight proteins in similar ratios. Most polyadenylated [poly(A)+] RNA made by tsG16(I) was considerably larger than wt poly(A)+ RNA and richer in AMP:UMP residues; however, the protein-coding capacities of mutant and wt poly(A)+ RNAs were similar. This suggested that most mRNAs made in vitro by tsG16(I) might possess very long poly(A)+ tracts, and digestion of RNA by T1 RNase supported this. It appeared, therefore, that a virally coded component of vesicular stomatitis virus could affect polyadenylation. This could be the poly(A) polymerase itself, a protein involved in control of polyadenylation, or a protein which affects an event spatially and temporally connected with polyadenylation (such as initiation of the subsequent mRNA).

Methylation↗
PubMed1983

The effect of freezing on peripheral nerve repair.

It has been suggested that where a double lesion to a peripheral nerve exists, the results of regeneration of the nerve is actually improved. This has been studied by comparing the results of nerve section and repair with and without an accompanying second lesion in the form of freezing of the nerve proximal to the anastomosis. While the effects of freezing does not adversely affect the results of nerve repair it has not been shown to produce any significant improvement.

Animals↗
PubMed1982

The distribution of copper in neonatal mottled mutant mice after exposure to copper and penicillamine.

Tissue copper levels of brindled (Mobr) mice and normal litter-mates after single and repeated dosing with CuCl2 and/or D-penicillamine are examined, together with a study of the cytosol distribution of copper after CuCl2 treatment. The results confirm that the mutant mouse kidney is capable of extensive copper accumulation in association with the low MW copper-binding protein. Deficient tissues such as brain, heart and spleen are able to sequester sufficient of the exogenous copper to raise their levels to the normal control level, whereas mutant liver levels, even after copper treatment, remain below normal, indicating that the Mo gene affects the ability of the liver to retain copper.

Animals↗
PubMed1982

Haemoglobin Constant Spring has an unstable alpha chain messenger RNA.

Haemoglobin Constant Spring (Hb CS) is a variant with an elongated alpha-chain associated with an alpha + thalassaemia phenotype. The amount of alpha mRNA relative to beta mRNA in reticulocytes was reduced in carriers of Hb CS by an amount equivalent to the reduction observed in carriers of alpha + thalassaemia. In a patient with Hb CS-H disease there was greater alpha/beta mRNA ratio in bone marrow nuclear RNA than in the peripheral blood. Furthermore, all the alpha mRNA in the patient's peripheral blood was derived from the alpha 1 (alpha A) gene. The data suggest that alpha CS mRNA is unstable and degraded in the cytoplasm. This instability may be due to destabilization of a specific sequence in the 3' non-coding region during translation.

Female↗
PubMed1981

Hemoglobin H disease and mental retardation: a new syndrome or a remarkable coincidence?

Each of three families of northern European origin contains a mentally retarded son with hemoglobin H (Hb H) disease. One parent is a carrier of mild alpha-thalassemia and the other is normal, suggesting that this form of Hb H disease results from the interaction between an inherited defect of alpha-chain production and one member of the pair in chromosome 16 and a new mutation on the other. Restriction-enzyme analysis indicated that the new mutation was not the same in the other three patients, and demonstrated at least two hitherto undescribed lesions involving the alpha-globin gene cluster. Unless the association between the Hb H disease and mental retardation is fortuitous, the new mutations may also be related to the development changes in these children. Since the mutations only came to light because there was concurrent inheritance of an additional alpha-thalassemia determinant, this type of mutation of chromosome 16 may have been overlooked in other mentally retarded patients.

Adult↗
PubMed1980

Copper and neurological function.

The role of copper in maintaining normal neurological function has been examined in animals copper-deficient by dietary means, and in the genetic disorders of copper homeostasis -- Menkes' kinky-hair disease in humans and the mottled (Mo) mutants in the mouse. With the exception of the disorder in Mo mice, reduced myelination is a constant feature of these copper diseases but there is otherwise a lack of conformity in the structural defects produced in different species. Dietary copper-deficient animals show a reduction in noradrenaline and dopamine concentrations, together with a depressed tyrosine 3-monooxygenase activity (EC 1.14.16.2). Noradrenaline concentrations are also reduced in brain tissue of Mo mice and this reduction is associated with a decrease in the vivo activity of the copper metalloenzyme, dopamine beta-monooxygenase (EC 1.14.17.1). Many tissues contain potent inhibitors of dopamine beta-monooxygenase activity, and assays of this enzyme have utilized cupric ions to inactivate these inhibitors. The elevated in vitro activities of dopamine beta-monooxygenase obtained for both Mo brain and adrenal tissue may therefore reflect either a reduced inactivation of these endogenous inhibitors in the intact animal or the activation in vitro of apoenzyme. Concentrations of dopamine and tyrosine 3-monooxygenase are unchanged in Mo mice. The reduction in dopamine and tyrosine 3-monooxygenase activity in dietary copper-deficient animals may therefore reflect neuronal loss rather than reduced catalytic activity of the catecholamine biosynthetic pathway. The possible effects of depressed activities of cytochrome c oxidase (EC 1.9.3.1) and superoxide dismutase (EC 1.15.1.1) in the development of neurological dysfunction are also discussed, and attention is drawn to the possible significance of the elevated uptake of neutral amino acids, especially tyrosine and tryptophan, by Mo brain tissue.

Aging↗
PubMed1980

Pyrimidine biosynthesis in the dumpy mutants of Drosophila melanogaster.

The status of de novo pyrimidine synthesis in the dp mutant of Drosophila melanogaster was examined by measuring the activity of the rate-limiting orotate phosphribosyl transferase (OPRT) enzyme. Activity is significantly elevated in late third instar larvae of 5 different dp mutant strains. A more detailed analysis of a dpovc allele has shown that this elevation arises at about mid-larval life and persists until pupation. A low nucleotide diet causes a depression in OPRT activity in dpovc larvae which can be reversed by dietary supplementation of uracil. However, neither the low nucleotide diet nor uracil supplementation results in a change in the expressivity of the dp mutant phenotypes. Changes in expressivity are produced by 6-azauracil and by elevated temperature although, in those cases, the effect on OPRT activity is minimal. The significance of the observations is discussed in relation to the role of pyrimidine biosynthesis in dp expressivity and chitin synthesis.

Diet↗
PubMed1980

Determination of alpha thalassaemia phenotypes by messenger RNA analysis.

The possibility of using alpha/beta globin messenger RNA (mRNA) ratios to distinguish between the carrier states for different forms of alpha thalassaemia has been explored. Alpha/beta globin mRNA ratios were determined in the red cells of a series of normal individuals and in members of four Cypriot families, one Thai and one Chinese family in which at least one person has haemoglobin H disease. It was found that there was a clear distinction in the ratios between normals, alpha thalassaemia 1 carriers, alpha thalassaemia 2 carriers, and those with haemoglobin H disease. This method should be a valuable addition to haematological analysis, haemoglobin synthesis and restriction mapping of DNA for the further elucidation of the genetics of alpha thalassaemia.

DNA↗
PubMed1980

The genetic basis of Hb Q-H disease.

A Chinese family has been studied in which two siblings have haemoglobin Q-H disease. Using a combination of haematological and haemoglobin analysis, globin chain synthesis, analysis of alpha/beta globin messenger RNA ratios and restriction endonuclease mapping, it has been shown that each of these siblings has received one chromosome on which both alpha chain genes have been deleted and another on which there is only a single alpha chain locus which carries the alpha Q mutation. Their genotype is thus --/-alpha Q. Despite the fact that the haemoglobin Q mutation in this family is carried on a chromosome with a single alpha chain locus, heterozygous carriers for the variant have only 25% or less haemoglobin Q. Our observations indicate that the molecular basis for haemoglobin Q-alpha thalassaemia is similar to that for the common form of haemoglobin H disease in Orientals. Furthermore, they provide clear evidence that the level of an alpha chain variant in heterozygous carriers is not a reliable reflection of the number of alpha globin genes.

Aged↗
PubMed1979

A study of the copper-binding proteins in liver and kidney tissue of neonatal normal and mottled mutant mice.

The Cu2+-binding proteins from liver and kidney tissue of 7--8-day-old brindled (Mobr) mice and their normal littermates were compared. (1) Separation over Bio-Gel P-10 showed that the differences in the Cu2+ content of mutant tissues were largely associated with a low-molecular-weight protein fraction (mol.wt. 14 500). (2) Further purification of this low-molecular-weight fraction by anion-exchange chromatography revealed four subfractions. The Cu2+ content of each subfraction reflected the Cu2+ status of the tissue of origin; the Cu2+ contents of the mutant kidney subfractions were elevated and those of the mutant liver were depressed compared with normal. In contrast, the protein contents of the subfractions were less variable and did not reflect the differing Cu2+ contents. (3) Amino acid analysis of the four subfractions from CuCl2-treated mutant and normal animals revealed clos similarities. The proteins showed high glycine, glutamic acid, serine, alanine and lysine contents and a rather variable cysteine content. Differences were apparent in the normal liver subfractions, which showed a higher cysteine content and lower glutamic acid content than did either the mutant liver or normal and mutant kidney subfractions. These observations, together with the recorded presence of aromatic amino acids, indicated that these proteins are not thioneins.

Amino Acids↗
PubMed1979

Negro alpha-thalassaemia is caused by deletion of a single alpha-globin gene.

Studies in two Jamaican Negro families, including haematological and haemoglobin analysis, haemoglobin synthesis, and globin messenger-RNA assay, have defined two alpha-thalassaemia phenotypes which resemble the severe (alpha-thalassaemia 1) and mild (alpha-thalassaemia 2) forms of the disorder described in Orientals. Genetic analysis suggests that subjects with the alpha-thalassaemia-1 phenotype are homozygous for the alpha-thalassaemia-2 determinant. Restriction-endonuclease mapping shows that alpha-thalassaemia-2 results from the deletion of one of the linked pair of alpha-chain genes. Hence the genotypes of the alpha-thalassaemia heterozygotes and homozygotes in these families are -alpha/alpha alpha and -alpha/-alpha respectively. If these are the usual alpha-thalassaemia genotypes in Negroes, these findings explain the difference in clinical expression of the disorder between Orientals and Negroes--in particular, the absence of haemoglobin Bart's hydrops and the rarity of haemoglobin-H disease in Negroes.

Black People↗