PubMed Health⌕ Search

Biomedical subjects

W D Stuart

Publications and source records attributed to W D Stuart.

34 records · Page 2Linked to original sources
PubMed1990

Apolipoprotein J: structure and tissue distribution.

The primary structure of apolipoprotein J (apoJ) was deduced by the combined strategies of protein sequencing and cDNA cloning and sequencing. ApoJ, an apolipoprotein associated with discrete subclasses of high-density lipoproteins, is encoded by a single gene in both the human and mouse genomes. ApoJ is synthesized as a 427 amino acid polypeptide that is posttranslationally cleaved at an internal bond between Arg-205 and Ser-206. The subunits of apoJ are designated apoJ alpha, corresponding to residues 1-205, and apoJ beta, corresponding to resides 206-427. The subunits are associated through disulfide bonds. Analysis of the primary structure of apoJ predicts the existence of amphiphilic helices, which may account for the association of apoJ with lipoproteins, and heparin-binding motifs in both subunits. ApoJ appears to be the human analogue of a rat protein present in high concentrations in the testis, sulfated glycoprotein 2. ApoJ mRNA (1.9 kb) is expressed in all but one tissue examined. The mRNA is present in relatively high levels in brain, ovary, testis, and liver, is less abundant in heart, spleen, lung, and breast, and is absent in T-lymphocytes. ApoJ is unique among previously characterized human apolipoproteins in its structure and tissue distribution.

Amino Acid Sequence↗
PubMed1988

Cloning of mtr, an amino acid transport gene of Neurospora crassa.

Translocation of neutral aliphatic and aromatic amino acids across the plasma membrane of the ascomycete Neurospora crassa requires a functional gene product of the mtr locus. Mutations at this locus are defective in transport of those amino acids. We have cloned the mtr+ gene of Neurospora crassa from an ordered cosmid library of genomic DNA and produced a preliminary restriction map of 2.9 kilobases of genomic DNA that encompasses the mtr coding region. We have confirmed that the cloned DNA regions contain the mtr gene sequence by restriction fragment length polymorphism mapping and have determined that the cloned sequence codes for a messenger RNA transcript of approximately 1200 nucleotides in length.

Amino Acids↗
PubMed1987

Plasma cholesteryl ester-triglyceride transfer protein. The catalytic domain is a low molecular weight proteolipid.

The lipid transfer protein complex (LTC) isolated from human plasma by immunoaffinity chromatography transfers cholesteryl esters (CE), triglycerides, and phosphatidylcholine (PC) between lipoproteins in vitro. The molecular weight of this lipid transfer catalyst in sodium dodecyl sulfate-polyacrylamide gels was 65,000. When resolved on a gel filtration column by high performance liquid chromatography (HPLC), LTC was composed of fractions of high (greater than 150,000) to low (18,000) molecular weight, although sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of each fraction revealed bands at Mr 65,000 (major) and 52,000 (minor). The CE and triglyceride transfer activity of the low Mr HPLC fraction (1049 nmol of triglyceride/mg/h and 244 nmol of CE/mg/h) was significantly greater than that of the high Mr HPLC fraction (15-27 nmol of triglyceride/mg/h and 20-30 nmol of CE/mg/h). The PC transfer activity of the HPLC fractions was not determined. LTC proteins were separated by dialysis in acidified chloroform:methanol solution into dialysand and dialysate proteins. The dialysate contained a low Mr proteolipid, designated the catalytic domain Cd, which catalyzed CE and triglyceride transfer at equivalent rates (11.0 versus 9.5 mumol/mg/h, respectively). PC transfer activity was approximately 10% of these levels (1.5 mumol/mg/h). The dialysand consisted of a protein, designated the transfer protein TP, which facilitated CE (3.4 mumol/mg/h) preferentially over triglyceride and PC (1.0 mumol/mg/h) transfer, and a catalytically inactive protein, designated the heparin-binding domain Hd. We propose a model of the LTC protein (based on catalytic activities, monoclonal antibody reactivities, and heparin-binding capacities of the isolated proteins) in which both Hd (approximately 13 kDa) and Cd (approximately 3 kDa) originate from a single lipid transfer protein, TP.

Amino Acids↗
PubMed1983

Isolation of parafluorophenylalanine-resistant mutants from HeLa cell cultures.

This report describes a method to isolate temperature-conditional phenylalanine transport mutants from the transformed human cell line HeLa. Using ultraviolet light as a mutagenic agent and DL-parafluorophenylalanine (PFPA), a poisonous analogue of L-phenylalanine, as a selective agent, mutagenized cells were selected for survival in the presence of PFPA at a temperature of 39 degrees C. Survivors of the mutagenesis and selection procedures were removed from the culture dishes by trypsin and cloned at a temperature of 35 degrees C. Seven of these lines isolated demonstrated continued resistance to PFPA at 39 degrees C. These lines were tested for uptake of L-phenylalanine at an external concentration of 100 microM and for continued resistance to PFPA at two concentrations. Cells were tested at 35 and at 39 degrees C. The data were compared to those obtained for the parental HeLa cell line under identical conditions. The seven mutant cell lines demonstrated varying resistances to PFPA and varying levels of accumulation of L-phenylalanine when tested at 35 and 39 degrees C. Three mutant lines were additionally tested for L-phenylalanine tRNA charging levels and for transport of L-arginine. The lines had parental cell levels of tRNA charging and L-arginine transport which suggest that the induced genetic defect affects a specific L-phenylalanine transport system.

Drug Resistance↗
PubMed1981

Location of the 18/28S ribosomal RNA genes in two Hawaiian Drosophila species by monoclonal immunological identification of RNA.DNA hybrids in situ.

Using both heterologous rabbit antisera and mouse monoclonal antibody to RNA.DNA hybrids, we have mapped the in situ hybridization locus of the 18/28S ribosomal RNA fraction to a single large band on polytene autosome 3 in Drosophila heteroneura and Drosophila silvestris. This portion of the chromosome is not physically connected with the nucleolus at the end of larval salivary gland development. In mature larvae, little or no hybridization with the material in the nucleolus can be detected. In younger larvae, hybridization of the ribosomal RNA probe to the nucleolus itself can be observed. The chromosome 3 locus is the only band in the polytene genome that shows variation in size and intensity of staining between populations and species. The interband chromosome regions that are immediately distal or proximal to the 18/28S rRNA locus have been involved in a disproportionately large number of natural inversion breaks observed in the euchromatic portion of the polytene chromosome. In 104 species of Hawaiian Drosophila in which chromosome 3 polytene sequences have been determined, 15 breaks occur in these two regions. On a random basis, only one such break is expected. We propose that this locus may be flanked by substantial heterochromatic blocks which are not represented in the salivary gland chromosome.

Animals↗
PubMed1977

New class of ribonucleic acid in Neurospora associated with the outer cell envelope.

Extrinsic ribonucleic acid (RNA) can be isolated from a KCl extract of Neurospora crassa conidial cell surface products. It is heterogeneous in size. The bulk of this RNA travels as a broad band, trailing the 5.8S ribosomal marker RNA on electrophoretic gels. The extrinsic RNA, when denatured, exhibites several discrete lengths between 50,000 and 130,000 daltons. Melting profiles confirm the heterogeneity of the RNA and indicate that 58% of the bases are involved in hydrogen bonding. Analyses of alkaline hydrolysis products reveal no extensive methylation and few or none of the unusual bases present in transfer RNA. The bases are present in approximately equivalent amounts. Extrinsic RNA represents 2 to 3% of the total cellular RNA. Since this membrane-associated class of RNA does not resemble ribosomal RNA, messenger RNA, or transfer RNA and since it is extracted from the cell exterior by methods used to remove extrinsic membrane molecules, we have designated it extrinsic RNA.

Adenine↗
PubMed1975

Effects of ribonuclease A on amino acid transport in Neurospora crassa.

Incubation of Neurospora crassa conidia with ribonuclease (RNase) A reduces transport of L-phenylalanine by those cells. Under similar conditions, oxidized RNase A, RNase T1, and RNase T2 do not have this effect. Incubation of conidia with active RNase covalently attached to polyacrylamide beads reduces L-phenylalanine transport. This indicates that the site of enzymatic action is at the cell surface. At the lower concentration of enzyme used in this study, incubation with RNase A reduces transport of L-phenylalanine by the general (G) amino acid permease. Increasing the enzyme concentration results in reduction of transport by the neutral aromatic (N)-specific permease. The increased transport activity that accompanies onset of conidial germination is also sensitive to incubation with RNase A. Application of the enzyme to actively transporting cells does not release amino acid transported prior to enzyme addition. Cells cultured on media supplemented with [2-14C] uridine release isotopic activity after RNase A incubation. Analogous treatments with Pronase, RNase T1, RNase T2, or deoxyribonuclease I do not release isotope activity. Pronase treatment does reduce L-phenylalanine transport. Incubation of conidia with RNase A also inhibits germination of those conidia.

Amino Acids↗
PubMed1972

Disruption of an amino acid transport mutant of Neurospora crassa by KCl.

A double amino acid transport-deficient mutant (Pm (-)NB) of Neurospora crassa is shown to be altered in the molecular structure of its cell wall or membrane. This alteration was revealed by a high degree of cellular disruption and cell-cell interaction following extraction by a high molar concentration of KCl.

Amino Acids↗
PubMedDate not supplied

A proposal that malignancies represent genetic changes in cell surface RNA.

A great number of experimental studies have now been performed with malignant cells. The mass of data generated presents a confusing and often apparently contradictory picture of the fundamental molecular biological defect which most scientists sense must be the cause of transformation of a normal cell into a malignant one. This paper proposes the hypothesis that RNA on the exterior of the cell membrane organizes the various functional molecular aggregates such as transport complexes (permeases), lectin receptor sites, transmembrane microfilament attachment sites, hormone receptor complexes, and the elusive contact inhibition components. Furthermore it is proposed that a defect in the production of or competition for the assembly of exterior-organizer RNA (exoRNA) complexes is the primary molecular defect inherent in all malignancies. The defect could be caused by deletion of a chromosome region producing or controlling exoRNA, mutation of the genes involved in exoRNA production or by insertion of viral genetic material into the genome thereby producing an incomplete viral RNA (vRNA) which competes for exoRNA binding sites in cell surface complexes. Such changes would be genetically transmitted and could represent a range of genetic change between chromosomal deletion and point mutation. Several experiments are suggested to directly test the hypothesis.

Biological Transport↗