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Biomedical subjects

L J Wilson

Publications and source records attributed to L J Wilson.

At least 37 records · Page 2Linked to original sources
PubMed1993

Use of Aspergillus overproducing mutants, cured for integrated plasmid, to overproduce heterologous proteins.

Aspergillus niger var. awamori was previously transformed with a vector designed to express a fused glucoamylase-prochymosin gene and bearing the Neurospora crassa pyr4 gene as a selectable marker. Mutant strains that overproduced the glucoamylase-prochymosin fusion protein were derived from one of the transformants. Despite the fact that the expression vector was integrated into the genome of these strains it was possible to obtain strains from which the vector sequences had been removed. This was performed by selection against the pyr4 gene present on the expression vector using 5-fluoroorotic acid. The cured strains were retransformed in order to investigate production of heterologous proteins using other expression vectors. In addition to the glucoamylase-prochymosin fusion protein, the mutant Aspergillus strains also overproduced Rhizomucor miehei aspartic proteinase but not preprochymosin produced as a non-fusion protein. The ability to select for loss of integrated plasmid from Aspergillus transformants may prove to be important for a variety of applications.

Aspartic Acid Endopeptidases↗
PubMed1992

The anti-hepatitis B virus activities, cytotoxicities, and anabolic profiles of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine.

The anti-hepatitis B (anti-HBV) activities of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (2'-deoxy-3'-thia-5-fluorocytosine [FTC]) were studied by using an HBV-transfected cell line (HepG2 derivative 2.2.15, subclone P5A). The (-) isomer was found to be a potent inhibitor of viral replication, with an apparent 50% inhibitory concentration of 10 nM, while the (+) isomer was found to be considerably less active. Both isomers showed minimal toxicity to HepG2 cells (50% inhibitory concentration, > 200 microM) and showed minimal toxicity in the human bone marrow progenitor cell assay. In accord with the cellular antiviral activity data, the 5'-triphosphate of (-)-FTC inhibited viral DNA synthesis in an endogenous HBV DNA polymerase assay, while the 5'-triphosphate of the (+) isomer was inactive. Unphosphorylated (-)-FTC did not inhibit product formation in the endogenous assay, suggesting that the antiviral activity of the compound is dependent on anabolism to the 5'-triphosphate. Both (-)- and (+)-FTC were anabolized to the corresponding 5'-triphosphates in chronically HBV-infected HepG2 cells. The rate of accumulation and the steady-state concentration of the 5'-triphosphate of (-)-FTC were greater. Also, (-)-FTC was not a substrate for cytidine deaminase and, therefore, is not subject to deamination and conversion to an inactive uridine analog. The (+) isomer is, however, a good substrate for cytidine deaminase.

Animals↗
PubMed1992

Identification of donor melanoma in a renal transplant recipient.

A patient with chronic renal failure received a closely matched cadaveric kidney. Approximately 3 months after transplantation, the patient developed a metastatic malignant melanoma. A large retroperitoneal mass consisting of large pleomorphic polygonal neoplastic cells was found close to the donated kidney. This tumor was diagnosed as a malignant melanoma. DNA analysis of this tumor, the donated kidney, and the recipient indicated that the melanoma originated from the donor. Although this is not the first report of a donated melanoma, it is the first report of definitive DNA analysis of the origin of the malignant cells.

DNA, Neoplasm↗
PubMed1991

Treatment acceptability of alternative sex therapies: a comparative analysis.

Eighty female subjects rated therapy descriptions of a case involving the treatment of inhibited female orgasm in group and individual formats. Half of the treatment descriptions described the patient's husband participating in some of the homework exercises. After each treatment description, subjects completed a series of treatment acceptability ratings (Treatment Evaluation Inventory, Semantic Differential, Credibility Rating Scale) designed to evaluate the alternative treatment dimensions. A case history of primary anorgasmia, representative of actual clients and problems typically seen in sex therapy clinics, was presented to subjects via written material and audiotape players. A clear preference for individual format over group format was consistently found in credibility and acceptability ratings. Inclusion of the identified patient's partner in therapy was not a salient factor in acceptability ratings for the female subjects. Implications of this research paradigm are discussed in light of the current state of the art in sex therapy, and future research needs are identified.

Adolescent↗
PubMed1990

Functional morphology of the telson-uropod stretch receptor in the sand crab Emerita analoga.

The telson-uropod stretch receptor in Emerita analoga belongs to a segmental array of axial-coxal receptors having sensory neurons with central somata. It consists of an elastic strand innervated by four giant, nonspiking, mechanoreceptive neurons. We examined the elastic (sensory) strand by light and electron microscopy to discover whether 1) functional differences between the four sensory neurons could be surmised from their peripheral terminations and 2) the process of mechano-electric transduction in this type of receptor might be revealed by the ultrastructure of the dendritic terminals. The elastic strand is divided into nonoverlapping domains by the mechanoreceptive neurons. We discerned no differences other than their serial order along the strand. Each neuron has three morphological zones in the periphery. In the zone of dendrite entry, the dendrite bifurcates into 40-60 microns primary branches that enter the strand at acute to right angles and turn rostrally and caudally along the strand's axis. The zone of branching consists of 4-35 microns diameter dendritic branches within the strand. Approximately 21,000 dendritic tips (diameters, 0.1-0.7 micron; lengths, 3-20 microns) per sensory neuron arise via short stubs from all levels of the zone of branching and constitute the zone of dendrite termination. The repeated bifurcations of dendrites within the zone of branching take place by radial intrusion of longitudinally oriented, partitions of extracellular matrix each of which extends the length of the pair of dendritic branches it divides. Tips project into these partitions. Since they change shape when the elastic strand is stretched, which compresses the extracellular matrix of the partitions, they are probably the site of mechanosensory transduction. Cross-sectional profiles of dendritic tips are significantly larger but less numerous in stretched than in relaxed receptors. We propose a model in which stretch compresses distal portions of tips until they are too small to be recognized. The large profiles are the proximal portions of tips that have been expanded by hydrostatic pressure. The very large number of dendritic tips/nonspiking mechanoreceptor may be what endows each neuron with 1) high sensitivity to stretch of the elastic strand and 2) ability to respond to a broader range of tensions than is usual for single sensory cells (the entire range experienced by the stretch receptor).

Animals↗
PubMed1990

Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awamori.

We have cloned genomic pepA sequences encoding the aspartic proteinase aspergillopepsin A (PEPA) from Aspergillus awamori using a synthetic oligodeoxyribonucleotide probe. Nucleotide sequence data from the pepA gene revealed that it is composed of four exons of 320, 278, 249, and 338 bp. Three introns which interrupt the coding sequence are 51, 52, and 59 bp in length. Directly downstream from the putative start codon lies a sequence encoding 69 amino acids (aa) which are not present in mature PEPA. Based on similarities to other aspartic proteinases, this region may represent a 20-aa signal peptide followed by a 49-aa propeptide that is rich in basic aa residues. Northern blots of total cellular RNA extracted from A. awamori cells indicate that pepA is transcribed as a single 1.4-kb mRNA. Mutants of A. awamori lacking the pepA structural gene were derived by the following gene replacement strategy. First, we constructed a plasmid in which a 2.4-kb SalI fragment containing the entire pepA coding region was deleted from a 9-kb Eco RI genomic DNA clone and replaced by a synthetic DNA polylinker. Second, a selectable argB gene was inserted into the polylinker. Third, the EcoRI fragment which contained the argB marker flanked by pepA sequences was excised from the plasmid and used to transform an argB auxotroph of A. awamori. From 16-40% of the resulting prototrophic transformants were found to have a PEPA-deficient phenotype when screened with an immunoassay using antibodies specific for PEPA. Southern hybridization experiments confirmed that these mutants resulted from a gene replacement event at the pepA locus.

Amino Acid Sequence↗
PubMed1990

Cloning, characterization, and expression of two alpha-amylase genes from Aspergillus niger var. awamori.

Using synthetic oligonucleotide probes, we cloned genomic DNA sequences encoding an alpha-amylase gene from Aspergillus niger var. awamori (A. awamori) on a 5.8 kb EcoRI fragment. Hybridization experiments, using a portion of this cloned fragment to probe DNA from A. awamori, suggested the presence of two alpha-amylase gene copies which were subsequently cloned as 7 kb (designated as amyA) and 4 kb (amyB) HindIII fragments. DNA sequence analysis of the amyA and amyB genes revealed the following: (1) Both genes are arranged as nine exons and eight introns; (2) The nucleotide sequences of amyA and amyB are identical throughout all but the last few nucleotides of their respective coding regions; (3) The amyA and amyB genes from A. awamori share extensive homology (greater than or equal to 98% identity) with the genes encoding Taka-amylase from A. oryzae. In order to test whether both amyA and amyB were functional in the genome, we constructed vectors containing gene fusions of either amyA and amyB to bovine prochymosin cDNA and used these vectors to transform A. awamori. Transformants which contained either the amyA- or amyB-prochymosin gene fusions produced extracellular chymosin, suggesting that both genes are functional.

Amino Acid Sequence↗
PubMed1990

Improved production of chymosin in Aspergillus by expression as a glucoamylase-chymosin fusion.

We have extended the work on chymosin production in Aspergillus by constructing an expression vector in which the cDNA encoding bovine prochymosin B was fused in frame immediately following the codon for the last amino acid of the A. awamori glucoamylase (glaA) gene. Transformation of A. awamori with this plasmid led to the secretion of considerably higher amounts of chymosin than obtained with previous chymosin expression vectors. We present evidence that mature chymosin is autocatalytically released from the glucoamylase-chymosin fusion protein after secretion.

Amino Acid Sequence↗
PubMed1988

The oliC3 gene of Aspergillus niger: isolation, sequence and use as a selectable marker for transformation.

The oliC3 gene of Aspergillus niger has been isolated and sequenced. This gene encodes an oligomycin-resistant variant of the mitochondrial ATP synthase subunit 9. In transformation experiments the gene can serve as a semi-dominant selectable marker for A. niger. It was possible to recognize transformants in which oliC3 had integrated at the homologous oliC locus, as opposed to elsewhere in the genome, by observation of phenotypes on medium containing oligomycin. DNA sequencing has allowed comparison of the deduced amino acid sequence with subunit 9 proteins from other species and comparison of 5' untranslated sequences with those from other fungi.

Amino Acid Sequence↗
PubMed1987

Sequence and centromere proximal location of a transformation enhancing fragment ans1 from Aspergillus nidulans.

The Aspergillus nidulans sequence ans1, previously known to enhance transformation frequencies of pyr4-based vectors, was shown to enhance the efficiency of argB and trpC-based vectors. Increased efficiencies could be obtained by constructing vectors containing argB and ans1 or by cotransforming selectable plasmids (containing argB, trpC, or pyr4) with the non-selectable ans1 sequence. The preponderance of evidence suggests that the mechanism of ans1 activity does not involve homologous recombination events, in spite of the presence of multiple regions of homology in the A. nidulans genome. Genetic mapping localized ans1 to the vicinity of the centromere of linkage group I. The nucleotide sequence of a 1.8 Kb functional subclone of ans1 was determined and found to be highly A + T rich (81%).

Aspergillus nidulans↗
PubMed1987

Transformation of Aspergillus nidulans with the hygromycin-resistance gene, hph.

Aspergillus nidulans strain G191 was transformed to hygromycin resistance using plasmid pDH25, which contains the bacterial hygromycin B phosphotransferase gene (hph) fused to promoter elements of the A. nidulans trpC gene. Southern hybridizations of transformants revealed multiple, integrated copies of the vector. A pleiotropic effect conferring increased hygromycin B sensitivity was found to be associated with the A. nidulans pyrG89 allele. Plasmid pDH25 features a ClaI site immediately preceding the hph start codon thus permitting convenient replacement of the trpC sequences with other eukaryotic promoters.

Anti-Bacterial Agents↗
PubMed1986

Primary structure of Mucor miehei aspartyl protease: evidence for a zymogen intermediate.

The gene encoding the aspartyl protease of the filamentous fungus Mucor miehei has been cloned in Escherichia coli and the DNA sequenced. The deduced primary translation product contains an N-terminal region of 69 amino acid (aa) residues not present in the mature protein. By analogy to the evolutionarily related mammalian gastric aspartyl proteases it is inferred that the primary secreted product is a zymogen containing a 47-aa propeptide. This propeptide is presumably removed in the later steps of the secretion process or upon secretion into the medium. To study the effects of modifications of the protease structure on its maturation by enzyme-engineering methods, an efficient expression system was sought. In E. coli, transcription of the preproenzyme coding sequence from a bacterial promoter results primarily in the accumulation of unsecreted, enzymatically inactive polypeptides, immunologically related to the authentic protease. In Aspergillus nidulans expression of the cloned gene, probably from its own promoter, results in the secretion into the culture medium of polypeptides which, compared to the authentic protease, are similar in specific activity, but differ in the character of their asparagine-linked oligosaccharides.

Amino Acid Sequence↗
PubMed1986

Acrylamide and iodide fluorescence quenching as a structural probe of tryptophan microenvironment in bovine lens crystallins.

Fluorescence quenching using acrylamide and iodide quenchers has been used to investigate the microenvironments of tryptophan residues in bovine alpha-, beta-, and gamma-crystallin fractions. Acrylamide quenching is very sensitive to the degree of tryptophan accessibility to the solvent containing the acrylamide. Since acrylamide is able to diffuse into the interior of the protein, accessibility to acrylamide may result from Trp residues lying at the surface of the protein or from the existence of channels leading to the interior of the protein. Iodide ion is hydrated and is limited by its large size and charge to quenching of tryptophan residues lying at or near the surface of proteins. Tryptophan residues in the lens crystallin fractions were found to be highly accessible to acrylamide, yet the rate of quenching by acrylamide was very low, indicating that the tryptophan residues of the lens crystallin fractions occupy predominately hydrophobic environments. The high accessibility to acrylamide likely results from diffusion of acrylamide into the interior of the protein. Accessibility to iodide was much lower, as was the rate of quenching by iodide, adding support to the conclusions from acrylamide quenching.

Acrylamide↗