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Biomedical subjects

J K Lodge

Publications and source records attributed to J K Lodge.

28 records · Page 2Linked to original sources
PubMed1994

Comparison of myristoyl-CoA:protein N-myristoyltransferases from three pathogenic fungi: Cryptococcus neoformans, Histoplasma capsulatum, and Candida albicans.

Myristoyl-CoA:protein N-myristoyltransferase (Nmt) transfers myristate from CoA to the N-terminal Gly residue of cellular proteins in an ordered reaction mechanism that first involves binding of myristoyl-CoA to the apoenzyme. The gene encoding Saccharomyces cerevisiae Nmt1p (NMT1) is essential for vegetative growth. Candida albicans, Cryptococcus neoformans var. neoformans, and Histoplasma capsulatum are the principal causes of systemic fungal infections in immunocompromised humans. Metabolic labeling studies indicate that they synthesize a small set of cellular N-myristoylproteins during exponential growth on rich media, the most prominent of which co-migrate with two essential functionally interchangeable S. cerevisiae N-myristoylproteins, ADP ribosylation factor-1 (Arf1p) and Arf2p. NMT and ARF genes have been recovered from C. neoformans and H. capsulatum using the polymerase chain reaction. They are single copy genes, interrupted by multiple introns. C. neoformans and H. capsulatum Nmts have approximately 50% amino acid sequence identity with the orthologous S. cerevisiae, C. albicans, and Homo sapiens N-myristoyltransferases, whereas C. neoformans and H. capsulatum Arfs are approximately 80% identical with C. albicans Arf and S. cerevisiae Arf1p and Arf2p. Functional studies of C. neoformans and C. albicans Nmts conducted in Escherichia coli reveal that (i) both efficiently acylate S. cerevisiae Arf2p; (ii) C. neoformans Arf is a substrate for C. neoformans Nmt; and (iii) substitution of an Asp for a Gly located 5 residues from the C terminus of these two enzymes causes marked temperature-dependent reductions in their catalytic efficiency, just as it does with S. cerevisiae and H. sapiens Nmts. Wild type C. neoformans, C. albicans, and H. sapiens NMTs can fully complement the lethal phenotype of a S. cerevisiae nmt1 null allele at 24 and 37 degrees C when the GAL1-10 promoter controlling their expression is induced by galactose. Only the C. albicans enzyme is able to do so when the promoter is repressed with glucose. This complementation profile likely arises, at least in part, from differences in the protein substrate specificities of the orthologous Nmts. A Gly-->Asp mutation in S. cerevisiae, C. neoformans, C. albicans, and H. sapiens Nmts produces temperature-sensitive growth arrest in isogenic S. cerevisiae strains with a nmt1 null allele. Growth of strains producing the mutant C. albicans or H. sapiens, but not the C. neoformans, enzyme can be rescued by myristate at the non-permissive temperature (37 degrees C) even in the presence of cerulenin, an inhibitor of fatty acid synthetase.(ABSTRACT TRUNCATED AT 400 WORDS)

Acyltransferases↗
PubMed1993

Broad-spectrum virus resistance in transgenic plants expressing pokeweed antiviral protein.

Exogenous application of pokeweed antiviral protein (PAP), a ribosome-inhibiting protein found in the cell walls of Phytolacca americana (pokeweed), protects heterologous plants from viral infection. A cDNA clone for PAP was isolated and introduced into tobacco and potato plants by transformation with Agrobacterium tumefaciens. Transgenic plants that expressed either PAP or a double mutant derivative of PAP showed resistance to infection by different viruses. Resistance was effective against both mechanical and aphid transmission. Analysis of the vacuum infiltrate of leaves expressing PAP showed that it is enriched in the intercellular fluid. Analysis of resistance in transgenic plants suggests that PAP confers viral resistance by inhibiting an early event in infection. Previous methods for creating virus-resistant plants have been specific for a particular virus or closely related viruses. To protect plants against more than one virus, multiple genes must be introduced and expressed in a single transgenic line. Expression of PAP in transgenic plants offers the possibility of developing resistance to a broad spectrum of plant viruses by expression of a single gene.

Base Sequence↗
PubMed1993

Aldehydes from metal ion- and lipoxygenase-induced lipid peroxidation: detection by 1H-n.m.r. spectroscopy.

The modification of lipoproteins by reactive aldehydes formed via lipid peroxidation is thought to be a key process in the pathogenesis of atherosclerosis. We show that 1H-n.m.r. spectroscopy can readily be used to detect a variety of different aldehydes resulting from peroxidation of liposomes induced by Fenton's reagent or lipoxygenase, and aldehydes arising from copper-induced reactions of low-density lipoprotein. There is a clear contrast between the major aldehydic products arising from metal-ion- and lipoxygenase-induced reactions.

Aldehydes↗
PubMed1992

4-oxatetradecanoic acid is fungicidal for Cryptococcus neoformans and inhibits replication of human immunodeficiency virus I.

Candida albicans and Cryptococcus neoformans are major causes of systemic fungal infections, particularly in patients with acquired immunodeficiency syndrome. Metabolic labeling studies revealed that these organisms synthesize a small number of N-myristoylproteins, the most prominent being 20-kDa ADP-ribosylation factors (Arfs). C. albicans Arf has approximately 80% identity with the essential Arf1 and Arf2 proteins of Saccharomyces cerevisiae. [3H]Myristic acid analogs with oxygen for -CH2- substitutions at C4, C6, C11, and C13 are incorporated into cellular N-myristoylproteins, phospholipids, and neutral lipids produced by these three yeasts during exponential growth at 30 degrees C in complex media. Analog- and organism-specific differences in the efficiency of labeling of proteins and lipid classes were observed. The effects of oxatetradecanoic acids with oxygen for -CH2- substitutions at C3-C13 on C. neoformans, C. albicans, and S. cerevisiae were assessed during mid-log phase growth at 30 degrees C. A single dose of 3-oxa-, 4-oxa-, 5-oxa- or 6-oxatetradecanoic acid (O3-O6, final concentration = 300 microM) was able to inhibit growth of C. neoformans in the order O4 greater than O5 greater than O3 approximately O6. The other compounds were inactive. 4-Oxatetradecanoic acid was fungicidal, producing a 10,000-fold reduction in viable cell number 1 h after administration and continued suppression of cell growth for 7 h. A clear dose response was observed over a concentration range of 100-300 microM. 4-Oxatridecanoic acid was 100-fold less potent in reducing cell viability than 4-oxatetradecanoic acid but more potent than 5-oxatridecanoic acid. O4 produced approximately 10-100-fold reductions in the viability of C. albicans and S. cerevisiae at 300-500 microM, respectively, whereas O5 and O6 were less active. Since N-myristoylation of the Pr55gag polyprotein precursor produced by human immunodeficiency virus I (HIV-I) is essential for its assembly, we also assessed the antiviral effects of 4-oxatetradecanoic acid. O4 is able to produce a 50% reduction in the replication of HIV-I in acutely infected human T-lymphocyte cell lines at a concentration of 18 microM. Together, these data suggest that (i) the position of the oxygen for methylene substitution is a critical determinant of the fungicidal activity of O4 and (ii) NMT may be an attractive therapeutic target for treating opportunistic fungal infections in patients infected with HIV-I.

ADP-Ribosylation Factor 1↗
PubMed1991

The second amino acid of alfalfa mosaic virus coat protein is critical for coat protein-mediated protection.

Transgenic plants expressing the coat protein (CP) of alfalfa mosaic virus (AIMV) are resistant to infection by AIMV. A mutation was introduced into the second amino acid of the cDNA for the CP of AIMV. Three different transgenic tobacco lines expressing the mutant CP and two different transgenic tobacco lines expressing the wild-type CP at similar levels were challenged with AIMV virions and viral RNA. Whereas the lines expressing the wild-type CP were highly resistant to infection by AIMV virions and viral RNA, the lines expressing the mutant CP were susceptible to infection by both. The binding affinity of the mutant and the wild-type CPs for the 3' terminal protein binding site on AIMV RNAs was similar, as determined by electrophoretic mobility shift assay. A mixture of AIMV genomic RNAs 1-3 was infectious on the plants expressing the mutant CP but not on vector control plants or plants expressing the wild-type CP, indicating that the mutant CP can activate the AIMV genomic RNAs for infection. These results demonstrate that the second amino acid of the AIMV CP is critical for protection from AIMV but not for the initial interaction between the AIMV RNA and CP, suggesting that this initial interaction does not play a major role in CP-mediated protection.

Journal Article↗
PubMed1991

Tn5 insertion specificity is not influenced by IS50 end sequences in target DNA.

The bacterial transposon Tn5 inserts into dozens of sites in a gene, some of which are used preferentially (hotspots). Features of certain sites and precedents provided by several other transposons had suggested that sequences in target DNA corresponding to the ends of Tn5 or of its component IS50 elements might facilitate transposition to these sites. We tested this possibility using derivatives of plasmid pBR322 carrying IS50 I or O end sequences. Tn5 inserted frequently into an IS50 I end at the major hotspot in pBR322, but not into either an I end or an O end 230 bp away from this hotspot. Adenine (dam) methylation at GATC sequences in the I end segment interferes with its use as the end of a transposon, but a dam- mutation did not affect Tn5 insertion relative to an I end sequence in target DNA. These results support models in which the ability of Tn5 to find its preferred sites depends on several features of DNA sequence and conformation, and in which target selection is distinct from recognition of the element ends during transposition.

Base Sequence↗
PubMed1990

Mutations that affect Tn5 insertion into pBR322: importance of local DNA supercoiling.

The major hot spot of transposon Tn5 insertion in plasmid pBR322 (hot spot I) is in the promoter for the tetracycline resistance gene (tet). We made a series of pBR322 derivatives with mutations in and around this promoter and assayed their effects on insertion of Tn5 into hot spot I. Those mutations which reduced transcription from the tet promoter also reduced the frequency of insertion into hot spot I. Transcription and translation of tet are thought to cause the formation of paired domains of negative and positive supercoiling in pBR322. An amber codon in tet, 345 base pairs from hot spot I, decreases the negative supercoiling of the DNA segment containing hot spot I because it terminates translation of tet prematurely. We report here that this amber mutation also reduces insertion into hot spot I. These results suggest that the ability of Tn5 to insert into its major hot spot in pBR322 depends directly on negative supercoiling of the target DNA.

Base Sequence↗
PubMed1989

Formation of supercoiling domains in plasmid pBR322.

Twin domains of positive and negative supercoiling are thought to form in DNA molecules whenever free rotation of a transcription complex around the DNA helix is impeded. Evidence for these domains has come from findings with Escherichia coli strains that are deficient in DNA topoisomerase I (top mutants) or that have been treated with DNA gyrase inhibitors. Plasmid pBR322 is highly supercoiled in these strains, whereas some of its deletion derivatives are not. The studies of pBR322 derivatives presented here show that high negative supercoiling in top strains requires translation as well as transcription of the first 98 codons of the tet gene and does not require the divergently transcribed amp gene. The N-terminal region of the TetA protein is thought to insert into the inner membrane. Our results favor models in which supercoiling domains are created when DNA segments are anchored to a large cellular structure via coupled transcription, translation, and membrane insertion of a nascent protein.

DNA Mutational Analysis↗
PubMed1988

Transposon Tn5 target specificity: preference for insertion at G/C pairs.

The procaryotic transposon Tn5 inserts into many different sites within a single gene, but some sites (hotspots) are targeted repeatedly. Hotspots are not closely related in sequence, but most have G/C pairs at the ends of the nine base pairs duplicated by Tn5 insertion. In pBR322, the major hotspot coincides with the "-10 region" of the tet promoter. We mutated the G/C pairs at this hotspot and assayed for insertion into hotspot I, resistance to tetracycline, and plasmid supercoiling. We found that changing the G/C pairs to A/T pairs reduced the frequency of insertion into the hotspot by at least fivefold. The reduction in hotspot use caused by these G/C to A/T changes was not attributable to changes in plasmid supercoiling or tet promoter strength.

Base Composition↗