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Biomedical subjects

D A Mitchell

Publications and source records attributed to D A Mitchell.

At least 55 records · Page 3Linked to original sources
PubMed1995

Discrimination between metachromatic leukodystrophy and pseudo-deficiency of arylsulfatase A by restriction digest of amplified gene fragments.

Mutations causing metachromatic leukodystrophy and pseudo-deficiency were detected in the arylsulfatase A gene by methods based on different wild-type and mutant restriction sites. After polymerase chain reaction amplification of fragments of the arylsulfatase A gene and digestion by the appropriate endonuclease, the mixtures were separated by polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. The common splice mutation in intron 2 (459 + 1G-->A) causing, in homozygosity, late-infantile metachromatic leukodystrophy and the common missense mutation in exon 8 (P426L) causing, in homozygosity, adult or juvenile metachromatic leukodystrophy were found to abolish Bst NI and Aci I sites, respectively. The polyadenylation pseudo-deficiency mutation (1619A-->G) was found to create a Mae III restriction site. The N-glycosylation pseudo-deficiency mutation (N350S) does not produce or destroy any known restriction site, and in this case, introduction of a single nucleotide mismatch in one of the primers enabled the authors to create a Bfa I site in the mutant allele.

Alleles↗
PubMed1995

Identification of a ras-activated enhancer in the mouse osteopontin promoter and its interaction with a putative ETS-related transcription factor whose activity correlates with the metastatic potential of the cell.

The role of RAS in transducing signals from an activated receptor into altered gene expression is becoming clear, though some links in the chain are still missing. Cells possessing activated RAS express higher levels of osteopontin (OPN), an alpha v beta 3 integrin-binding secreted phosphoprotein implicated in a number of developmental, physiological, and pathological processes. We report that in T24 H-ras-transformed NIH 3T3 cells enhanced transcription contributes to the increased expression of OPN. Transient transfection studies, DNA-protein binding assays, and methylation protection experiments have identified a novel ras-activated enhancer, distinct from known ras response elements, that appears responsible for part of the increase in OPN transcription in cells with an activated RAS. In electrophoretic mobility shift assays, the protein-binding motif GGAGGCAGG was found to be essential for the formation of several complexes, one of which (complex A) was generated at elevated levels by cell lines that are metastatic. Southwestern blotting and UV light cross-linking studies indicated the presence of several proteins able to interact with this sequence. The proteins that form these complexes have molecular masses estimated at approximately 16, 28, 32, 45, 80, and 100 kDa. Because the approximately 16-kDa protein was responsible for complex A formation, we have designated it MATF for metastasis-associated transcription factor. The GGANNNAGG motif is also found in some other promoters, suggesting that they may be similarly controlled by MATF.

3T3 Cells↗
PubMed1994

A polybasic domain allows nonprenylated Ras proteins to function in Saccharomyces cerevisiae.

Ras proteins undergo a series of posttranslational modifications prior to association with the cytoplasmic surface of the plasma membrane. The modification steps include farnesylation, proteolysis, methylesterification, and palmitoylation. A 4-amino acid residue motif known as the CaaX box (C is cysteine, a is generally aliphatic, and X is the carboxyl-terminal residue) is the sequence recognized by the prenyl transferase that initiates the modification pathway. As part of our studies to define the requirements for Ras membrane association, we directed mutagenesis to the yeast Ras2 protein CaaX box to assess the relative importance of prenylation, palmitoylation, and stretches of basic amino acids on the function of the protein. The wild type yeast Ras2 protein terminates in the sequence Cys-Cys-Ile-Ile-Ser. We have identified mutations that do not contain a CaaX box but still encode functional Ras proteins. These mutations replace the terminal serine of the CaaX box with the sequence -Lys-Leu-Ile-Lys-Arg-Lys. Three mutants have been analyzed in detail. Ras2(CCIIKLIKRK) functions at a level similar to wild type Ras2, whereas cells expressing only Ras2(SCIIKLIKRK) and Ras2(SSIIKLIKRK) forms of Ras2 protein grow more slowly at 30 degrees C. In addition, strains expressing only Ras2(SSIIKLIKRK) protein fail to grow at 37 degrees C. Replacement of the basic residues with neutral amino acids (Ras2(CCIISIIS)) completely abolishes their ability to support Ras-dependent growth. The extension mutants are not prenylated, but Ras2(CCIIKLIKRK) and Ras2(SCIIKLIKRK) are palmitoylated. These results demonstrate that a diverse set of carboxyl-terminal sequence motifs and posttranslational modifications lead to functional Ras proteins in yeast.

Amino Acid Sequence↗
PubMed1994

A bizarre facial sinus.

A 63-year-old Caucasian man presented to his general dental practitioner with a spontaneously exfoliated lower central incisor and hair growing from the socket. Examination revealed a hair-filled sinus from the skin of the median aspect of his chin to the socket of the exfoliated tooth. This was confirmed histopathologically following excision biopsy.

Chin↗
PubMed1993

Vectors for the inducible overexpression of glutathione S-transferase fusion proteins in yeast.

A rapid and convenient method of protein purification involves creating a fusion protein with glutathione S-transferase (GST) (Smith and Johnson, Gene 67, 31-40, 1988). In this report, we describe two vectors for the conditional expression of GST fusions in Saccharomyces cerevisiae. The parent plasmid is based on a high-copy, galactose-inducible shuttle vector previously described (Baldari et al., EMBO J. 6, 229-243, 1987). We have demonstrated the use of this system by creating fusions between GST and the yeast RAS2 gene. GST-Ras2 fusion proteins undergo the post-translational modifications required for Ras2p to become membrane localized. These vectors provide a useful system for the expression and purification of eukaryotic proteins requiring post-translational modification.

Base Sequence↗
PubMed1993

Intraosseous leiomyoma of the mandible: report of a case.

A rare case of intraosseous leiomyoma of the mandible is reported. This is the fourth recorded case involving the mandible. The literature is reviewed and the amenability of the lesion to simple local excision is highlighted.

Actins↗
PubMed1993

Operational parameters for packed beds in solid-state cultivation.

Packed bed cultivation systems have potential for widespread application in solid-state cultivation (SSC), but they are poorly characterized. The effects of particle size and substrate loading on the growth of Rhizopus oligosporus on sago-beads in packed bed bioreactors were investigated. Pressure drop and protein were monitored as indicators of fungal growth in cultivations performed in a large column (4.9 cm internal diameter and 60 cm height) and a system of small columns (4.2 cm internal diameter and 5.2 cm height). The differential pressure drop increased to a maximum between 34 and 44 h and then decreased again. The maximum differential pressure drop attained was greatest for the smallest particle size and for the lower substrate loadings. However, since the protein content continued to increase throughout the cultivation, pressure drop could not be used to monitor growth directly.

Journal Article↗
PubMed1993

An audit of 50 bitemporal flaps in primary facial trauma.

The bitemporal (bicoronal) flap is a well recognised technique for exposing the skull and upper face. Prospectively collected data over a 5-year period in which this technique was used for access in primary craniomaxillofacial trauma was analysed. Emphasis was placed on indications, complications and outcome. A total of 50 cases were audited. 84% were male. 74% had sustained midface or frontal bone trauma, 24% had lateral upper face injuries. Only 1 patient had a penetrating missile injury. Consistent complications of the procedure were altered sensation at the vertex in the early postoperative period and a small area of wound breakdown over the suture knot in the galea. In all cases the altered sensation greatly improved or resolved, 12% developed a wound infection, however, 83% of these were trivial requiring no active treatment. There was an isolated case of pressure necrosis over a drain site. 12% had a demonstrable transient deficit of the temporal branch of the facial nerve, all of which improved.

Adolescent↗
PubMed1992

Gram negative aerobic bacillus pneumonia in oral and maxillofacial surgery--a case for comment.

A case illustrating the potentially fatal complication of endogenous Gram Negative Aerobic Bacillus (GNAB) septicaemia secondary to nosocomial pneumonia is presented along with current theories as to its aetiology. The technique of selective decontamination of the digestive tract is designed and advocated to prevent such occurrences; oral and maxillofacial surgeons should be aware of this approach. It may be, however, that by using much simpler manoeuvres such as changes in policy regarding gastric stress ulcer prophylaxis, the already small risk of such an occurrence will be further reduced. Awareness of this condition will allow a higher index of suspicion when presented with catastrophic septic complications on the ITU and aid in more rational planning of antimicrobial therapy.

Carcinoma, Squamous Cell↗
PubMed1992

Mucolipidoses II and III variants with normal N-acetylglucosamine 1-phosphotransferase activity toward alpha-methylmannoside are due to nonallelic mutations.

Normal N-acetylglucosamine 1-phosphotransferase activity toward mono- and oligosaccharide acceptor substrates was detected in cultured skin fibroblasts from mucolipidoses II and III patients who were designated as variants (one of four mucolipidosis II and three out of six mucolipidosis III patients examined). The activity toward natural lysosomal protein acceptors was absent or deficient in cell preparations from all patients with classical as well as variant forms of mucolipidoses II and III. Complementation analysis, using fused and cocultivated mutant fibroblast combinations, revealed that, while cell lines with variant mucolipidosis III constituted a complementation group distinct from that of classical forms of mucolipidoses II and III, the variant mucolipidosis II cell line belonged to the same complementation group as did the classical forms. In contrast to the mutant enzyme from variant mucolipidosis III patients that failed to recognize lysosomal proteins as the specific acceptor substrates, the activity toward alpha-methylmannoside in the variant mucolipidosis II patient could be inhibited by exogenous lysosomal enzyme preparations (bovine beta-glucuronidase and human hexosaminidase A). These findings suggest that N-acetylglucosamine 1-phosphotransferase is composed of at least two distinct polypeptides: (1) a recognition subunit that is defective in the mucolipidosis III variants and (2) a catalytic subunit that is deficient or altered in the classical forms of mucolipidoses II and III as well as in the mucolipidosis II variant.

Alleles↗
PubMed1991

Triiodothyronine-induced accumulations of malic enzyme, fatty acid synthase, acetyl-coenzyme A carboxylase, and their mRNAs are blocked by protein kinase inhibitors. Transcription is the affected step.

Addition of triiodothyronine (T3) to chick-embryo hepatocytes in culture causes increased accumulations of malic enzyme, fatty acid synthase, acetyl-CoA carboxylase and their mRNAs. H-8 and other protein kinase inhibitors inhibited the T3-induced accumulations of these lipogenic enzymes and their mRNAs but had no effect on the activities of 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase, enzymes not induced by T3 in chick-embryo hepatocytes. H-8 also had no effect on the activities of malic enzyme, fatty acid synthase, and acetyl-CoA carboxylase in hepatocytes not treated with T3. Synthesis of soluble protein, levels of mRNAs for beta-actin and glyceraldehyde-3-phosphate dehydrogenase, and induction of metallothionein mRNA by Zn2+ were unaffected by H-8 at concentrations that inhibited the T3-induced accumulation of lipogenic enzymes and their mRNAs. H-8 inhibited T3-induced transcription of the genes for both malic enzyme and fatty acid synthase but had little effect on transcription of the beta-actin or glyceraldehyde-3-phosphate dehydrogenase genes or on total RNA synthesis in isolated nuclei. H-8 also had no effect on binding of T3 to its nuclear receptor. In isolated nuclei, H-8 inhibited phosphorylation of total protein by 15-20%. Phosphorylation of only one major protein was consistently and substantially inhibited, indicating that the effect of H-8 was selective. These results suggest that on-going protein phosphorylation is required specifically for stimulation of transcription of the lipogenic genes by T3.

Acetyl-CoA Carboxylase↗
PubMed1991

Stimulation of GM3 ganglioside sialidase activity by an activator protein in patients with mucolipidosis IV and controls.

An activator protein that stimulates the enzymic hydrolysis of sialic acid from gangliosides by ganglioside sialidase was fractionated from human liver. This fraction was distinct from those stimulating the hydrolysis of galactose from GM1 ganglioside by beta-galactosidase and the hydrolysis of N-acetylgalactosamine from GM2 ganglioside by hexosaminidase A. This fraction was highly specific for the hydrolysis of sialic acid from GM3 ganglioside, and was equally effective in fibroblasts from patients with mucolipidosis IV and in fibroblasts from controls.

Chromatography, Thin Layer↗
PubMed1990

Triiodothyronine stimulates transcription of the fatty acid synthase gene in chick embryo hepatocytes in culture. Insulin and insulin-like growth factor amplify that effect.

Hepatic fatty acid synthase is regulated by nutritional state. Starvation decreases and refeeding increases the activity of avian fatty acid synthase, principally by regulating transcription of the gene (Back, B. W., Goldman, M. J., Fisch, J.E., Ochs, R.A., and Goodridge, A.G. (1986) J. Biol. Chem. 261, 4190-4197). In chick embryo hepatocytes in culture, the stimulatory effect of feeding on fatty acid synthase activity is mimicked by adding triiodothyronine and insulin; the inhibitory effect of starvation is mimicked by adding glucagon or cyclic AMP. We now show that triiodothyronine alone stimulates transcription of fatty acid synthase by 4- to 6-fold, about the same as the increase in fatty acid synthase mRNA. When added alone, insulin has little or no effect on transcription, mRNA level, or enzyme activity. In combination with triiodothyronine, however, insulin amplifies the response to triiodothyronine by about 2-fold, leading to an overall increase of about 10-fold. Insulin-like growth factor 1 (IGF-1) has the same effect as insulin, no effect by itself, and amplification of the stimulation by triiodothyronine. A maximally effective dose of insulin has no effect in the presence of a maximally effective dose of IGF-1, suggesting regulation by a common pathway. It takes much less IGF-1 than insulin to achieve a given effect, suggesting that both insulin and IGF-1 may act through IGF-1 receptors. Plasma levels of IGF-1 are decreased by starvation and increased by feeding (reviewed by Froesch, E.R., and Zapf, J. (1985) Diabetologia 28, 485-493). Thus, IGF-1 may play a physiological role in the regulation of hepatic fatty acid synthase during transitions between the starved and fed states, roles previously assigned primarily to insulin and glucagon. IGF-1 regulates transcription of the fatty acid synthase gene. Insulin and IGF-1 also have similar effects on activity, mRNA abundance, and transcription of the malic enzyme gene. Glucagon or dibutyryl cyclic AMP inhibit fatty acid synthase activity and mRNA level in hepatocytes in culture by 70-80% and 60%, respectively, but have no effect on transcription of the fatty acid synthase gene, suggesting a post-transcriptional mode of regulation for cyclic AMP.

Animals↗
PubMed1990

Nutritional regulation and tissue-specific expression of the malic enzyme gene in the chicken. Transcriptional control and chromatin structure.

Refeeding starved chicks causes a 25- to 50-fold increase in the level of malic enzyme mRNA in liver. To define the regulated steps, we measured transcriptional activity of the malic enzyme gene using the nuclear run-on assay and a variety of DNA probes specific to the malic enzyme gene. Refeeding starved chicks stimulated transcription of the malic enzyme gene in liver by 40- to 50-fold. An increased transcription rate was detectable at 1.5 h, was maximal at 3 h, and remained high at 24 h of refeeding. The level of nuclear precursor RNA for malic enzyme assessed by hybridization with intron-specific probes was high in liver of refed birds, and barely detectable in that of starved birds. These results indicate that nutritional regulation of the level of malic enzyme mRNA is transcriptional. Low levels of malic enzyme mRNA in brain, kidney, and heart correlated well with low rates of transcription of the malic enzyme gene in these tissues. In contrast to liver, neither the rate of transcription nor the steady-state level of malic enzyme mRNA was affected by refeeding starved birds. A series of DNase I-hypersensitive sites were located within 4000 base pairs upstream of the transcription start site of the malic enzyme gene in liver. The DNase I-hypersensitive region extending from the start of transcription to 400 base pairs upstream was much more pronounced in the refed state than in the starved state. This change in DNase I hypersensitivity followed the same time course as increased transcription of the malic enzyme gene. This DNase I-hypersensitive region also was present at low intensity in kidney and heart independently of nutritional state. The three constitutive DNase I-hypersensitive sites further upstream were present in liver but not in kidney or heart.

Animal Nutritional Physiological Phenomena↗