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T D Edlind

Publications and source records attributed to T D Edlind.

At least 37 records · Page 2Linked to original sources
PubMed1992

The beta-tubulin gene from rat and human isolates of Pneumocystis carinii.

The development of new drugs for treating Pneumocystis carinii infections in AIDS patients is hampered by the lack of long-term culture systems, and by our generally limited knowledge of this organism. Recently, however, we observed significant activity of various benzimidazoles against growth of this organism in short-term cultures. Benzimidazoles inhibit microtubule polymerization; there is strong evidence that the primary target is the beta-tubulin subunit. To understand the basis for benzimidazole activity against P. carinii, and to examine the apparent relatedness of this organism to fungi, we have cloned and sequenced the single beta-tubulin gene from a rat P. carinii isolate. There was 89-91% identity at the amino acid level to beta-tubulins from filamentous fungi, but only 79-82% identity to yeast and protozoal beta-tubulins. Also, eight introns were distributed throughout the P. carinii beta-tubulin gene in a pattern characteristic of filamentous fungi. Specific residues previously implicated in benzimidazole sensitivity were conserved in P. carinii beta-tubulin. The polymerase chain reaction was used to amplify a segment of P. carinii beta-tubulin DNA from bronchoalveolar lavages obtained from two patients with AIDS. There was considerable divergence at the DNA level between the human and rat sequences, but 100% identity at the amino-acid level.

Amino Acid Sequence↗
PubMed1992

Antimicrotubule benzimidazoles inhibit in vitro growth of Pneumocystis carinii.

Nine antimicrotubule benzimidazole derivatives tested in a Pneumocystis carinii culture system with human embryonic lung fibroblast monolayers inhibited organism proliferation. The concentrations of drugs inhibitory in culture ranged from 10 to 0.1 micrograms/ml, with thiabendazole being the least effective (10 micrograms/ml) and parbendazole being the most effective (0.1 microgram/ml). The parent compound, benzimidazole, was inactive at 10 micrograms/ml. Demonstration that this group of compounds has activity against P. carinii provides a new potential target that can be exploited, the microtubules. Also, the variability in the effectiveness of the compounds provides the basis for studies of structure-activity relationships, which were initiated in this study.

Antiprotozoal Agents↗
PubMed1992

Molecular epidemiology of Pseudomonas cepacia determined by polymerase chain reaction ribotyping.

Traditional ribotyping detects genomic restriction fragment length polymorphisms by probing chromosomal DNA with rRNA. Although it is a powerful method for determining the molecular epidemiology of bacterial pathogens, technical difficulties limit its application. As an alternative, polymorphisms were sought in the 16S-23S spacer regions of bacterial rRNA genes by use of the polymerase chain reaction (PCR). Chromosomal DNA from isolates of Pseudomonas cepacia was used as a template in the PCR with oligonucleotide primers complementary to highly conserved sequences flanking the spacer regions of the rRNA genes. Length polymorphisms in the amplified DNA distinguished unrelated isolates of P. cepacia. Isolates of P. cepacia previously implicated in person-to-person transmission were shown to have identical amplification patterns. These data demonstrate the utility of this new PCR ribotyping method for determining the molecular epidemiology of bacterial species.

Bacterial Typing Techniques↗
PubMed1991

Enhanced antiparasitic activity of lipophilic tetracyclines: role of uptake.

It was previously noted that the inhibitory activities of lipophilic tetracyclines against the growth of Giardia lamblia in vitro were up to 40-fold greater than those of nonlipophilic tetracyclines (50% inhibitory concentration [IC50] = 1.8 to 71 micrograms/ml) (T. D. Edlind, Antimicrob. Agents Chemother. 33:2144-2145, 1989). We have now extended this observation to Trichomonas vaginalis (IC50 = 2.9 to 200 micrograms/ml), Entaoeba histolytica (IC50 = 3.8 to 36 micrograms/ml), and Leishmania major promastigotes (IC50 = 21 to 250 micrograms/ml; one strain only). The basis for these differential tetracycline activities was investigated with G. lamblia. In a cell-free protein synthesis system, lipophilic and nonlipophilic tetracyclines had similar, relatively low activities (IC50 = 170 to 500 micrograms/ml). On the other hand, tetracycline uptake into intact cells after a 1-h incubation varied dramatically: the ratios of intracellular to extracellular drug concentrations were 1.7 to 7.2 for nonlipophilic tetracyclines and 47 to 112 for lipophilic derivatives. Thus, the variable effects of tetracyclines on the growth of G. lamblia can be fully accounted for by differences in uptake. Passive diffusion probably plays a more important role than active transport in uptake of lipophilic tetracyclines, since similar results were obtained with cells rendered nonviable by metronidazole treatment.

Animals↗
PubMed1991

A novel approach to insertional mutagenesis of Haemophilus influenzae.

Insertional mutagenesis of the Haemophilus influenzae chromosome was accomplished by a novel method employing a 2.2-kbp element, TSTE. This element, consisting of the neo gene of Tn5 flanked by Haemophilus-specific uptake sequences, was ligated to circularized chromosomal fragments before transformation into the homologous strain. Eight mutants defective in the production of haemocin were detected. This strategy provides an efficient mechanism for the insertional mutagenesis of H. influenzae.

DNA Transposable Elements↗
PubMed1990

Ribosomal RNA of the primitive eukaryote Giardia lamblia: large subunit domain I and potential processing signals.

The cytoplasmic ribosomal RNA (rRNA) from the intestinal protozoan, Giardia lamblia, is unusually short; the large subunit (LS) and small subunit RNA and the 5.8S RNA are only 70-80% of the length found in typical protozoa, and are even smaller than most of their prokaryotic counterparts. Flanking regulatory DNA and processed rRNA sequences are similarly compact in size. To shed light on the origins and implications of this 'minimal' rRNA, the nucleotide sequence encoding the 5.8S RNA and domain I of LS RNA was determined. Secondary structure analysis revealed that an evolutionarily variable internal hairpin is partially 'deleted' in G. lamblia 5.8S RNA; the 3'-terminal pairing with LS RNA is conserved. Previously characterized eukaryotic 'expansion' regions are extensively shortened within the LS RNA; in one case, a hairpin is precisely 'deleted'. The short sequences flanking the mature 5.8S RNA that are removed by RNA processing (ITS1 and ITS2) are C-rich; our analysis suggests that the sequence GCGCCCC, in a hairpin configuration, may function as the processing signal.

Animals↗
PubMed1990

Activity of the anthelmintic benzimidazoles against Giardia lamblia in vitro.

In vitro growth of the protozoan parasite Giardia lamblia was highly sensitive to certain anthelmintic benzimidazoles. Albendazole and mebendazole were 30- to 50-fold more active than metronidazole and 4- to 40-fold more active than quinacrine. Thiabendazole, a noncarbamate benzimidazole, was less active. Since lack of intestinal absorption makes mebendazole an attractive new antigiardial agent, its in vitro activity was further characterized. At low concentrations (0.05 micrograms/ml) mebendazole had a static effect on G. lamblia growth; however, lethal activity was observed at a concentration fivefold lower (0.3 micrograms/ml) than necessary for the cidal agent metronidazole. Two observations are consistent with a microtubule target for mebendazole. First, attachment of cells to the culture tube, mediated by the ventral disk and flagella, was rapidly disrupted by mebendazole treatment. Second, the characteristic cell structure was grossly distorted by treatment. No mebendazole-resistant G. lamblia were detected in a population of 10(8) cells.

Albendazole↗
PubMed1989

Tetracyclines as antiparasitic agents: lipophilic derivatives are highly active against Giardia lamblia in vitro.

Comparisons of the inhibitory activities of different tetracyclines have been reported for Plasmodium falciparum but no other parasites. The in vitro response of the intestinal parasite Giardia lamblia to six tetracyclines in current use was determined. In addition, the experimental drug thiacycline (EMD 33,330) was evaluated. Three groups were discerned, with representative 50 and 90% inhibitory concentrations of, respectively, 36 and 130 (tetracycline), 6.4 and 22 (doxycycline), and 1.8 and 3.4 (thiacycline) micrograms/ml. These dramatic differences in activity correlate with increased lipophilicity.

Animals↗
PubMed1989

Susceptibility of Giardia lamblia to aminoglycoside protein synthesis inhibitors: correlation with rRNA structure.

The very limited development of antiparasitic agents targeting protein synthesis stems in part from the belief that parasite and host ribosomes are sufficiently similar to preclude selective toxicity. However, recent studies have revealed that Giardia lamblia rRNA has an unusual size and sequence; consequently, this organism and its homogeneous rRNA provide a useful model for the development of protein synthesis inhibitors with antiparasitic activity. In this study, I determined the sequence and secondary structure of the 3' end of the small-subunit RNA, the target for aminoglycoside inhibitory activity. The primary structure of these 140 nucleotides includes two blocks of sequence highly conserved among other organisms; the remaining sequence, although not conserved, can be folded into a secondary structure common to all rRNAs. The presence of U-1495 within one of the conserved blocks predicts hygromycin susceptibility. Also, a specific base pair (C-1409.G-1491) implicated in paromomycin susceptibility is present; whereas all procaryotes have this base pair, it is absent in many eucaryotes (including mammals). Conversely, kanamycin and apramycin resistance can be predicted from substitution of A-1408 with G. A growth inhibition assay was used to test the susceptibility of G. lamblia to a variety of aminoglycosides. After 48 h, 8 of 11 aminoglycosides tested failed to inhibit growth at a concentration of 200 micrograms/ml. Paromomycin and hygromycin, however, inhibited growth of three strains tested by 50% at 50 to 60 micrograms/ml and by close to 90% at 120 micrograms/ml. These results correlate well with the sequence and secondary-structure analyses. Paromomycin may be clinically useful when the toxicity of standard antigiardial drugs is of concern.

Aminoglycosides↗
PubMed1988

Heat shock and stress response in Giardia lamblia.

The heat shock response was studied as a model for control of gene expression and protein synthesis in Giardia lamblia. Cultured trophozoites were metabolically labelled with [35S]methionine, and proteins were analysed by SDS-polyacrylamide gel electrophoresis and fluorography. A temperature shift from 37 degrees C to 43 degrees C resulted in the depression of normal protein synthesis, and the enhanced synthesis of four major heat shock proteins of 100, 83, 70 and 30 kDa. This response resembles that seen in other organisms of wide phylogenetic diversity. An examination of the kinetics of induction and recovery from heat shock suggests that the individual heat shock proteins are independently regulated. In vitro translation of messenger RNA isolated from heat shocked cells further indicates that regulation occurs at both transcriptional and translational levels. The response of G. lamblia to other stresses including cysteine deprivation, exposure to oxygen, ethanol, hydrogen peroxide, and the chemotherapeutic drugs metronidazole and quinacrine was also investigated. The induction of two or more of the heat shock proteins was generally observed; however, certain treatments inhibited synthesis of all proteins including heat shock proteins.

Animals↗
PubMed1988

A broad-spectrum probe for molecular epidemiology of bacteria: ribosomal RNA.

We investigated the use of ribosomal RNA (rRNA) as a probe for molecular epidemiology of bacterial pathogens. The chromosomal DNA of Escherichia coli, Pseudomonas cepacia, and nontypable Haemophilus influenzae was digested with EcoRI. Agarose gel electrophoresis, Southern blotting, and hybridization by 32P-labeled rRNA revealed eight to 13 bands. The P. cepacia and H. influenzae banding patterns, observed by using an E. coli rRNA probe, were identical to those produced with homologous rRNA probes. Polymorphism of several hybridization bands distinguished all E. coli isolates, nine of 10 H. influenzae isolates, and seven of eight P. cepacia isolates. Two to four bands were common to all P. cepacia and E. coli isolates. The banding patterns of H. influenzae isolates cultured from the trachea and blood of an infant and from the mother's cervix were identical. These data demonstrate that this method is a widely applicable system for determining the molecular epidemiology of genetically diverse gram-negative organisms.

DNA Restriction Enzymes↗
PubMed1987

Unusual ribosomal RNA of the intestinal parasite Giardia lamblia.

The anaerobic protozoan Giardia lamblia is a common intestinal parasite in humans, but is poorly defined at molecular and phylogenetic levels. We report here a structural characterization of the ribosomal RNA (rRNA) and rRNA genes of G. lamblia. Gel electrophoresis under native or non-denaturing conditions identified two high molecular weight rRNA species corresponding to the 16-18S and 23-28S rRNAs. Surprisingly, both species (1300 and 2300 nucleotides long, respectively) were considerably shorter than their counterparts from other protozoa (typically 1800 and 3400 nucleotides), and from bacteria as well (typically 1540 and 2900 nucleotides long). Denaturing polyacrylamide gel electrophoresis identified a major low molecular RNA of 127 nucleotides and several minor species, but no molecules with the typical lengths of 5.8S (160 nucleotides) and 5S (120 nucleotides) rRNA. The G. lamblia 1300, 2300, and 127 nucleotide RNAs are encoded within a 5.6 kilobase pair tandemly repeated DNA, as shown by Southern blot analysis and DNA cloning. Thus, the rRNA operon of this eukaryotic organism can be no longer than a typical bacterial operon. Sequence analysis identified the 127 nucleotide RNA as homologous to 5.8S RNA, but comparisons to archaebacterial rRNA suggest that Giardia derived from an early branch in eukaryotic evolution.

Animals↗
PubMed1986

Detection of Babesia bovis using DNA hybridization.

Plasmids containing inserts of Babesia bovis DNA were prepared and clones suitable for use in the diagnosis of B. bovis infections were isolated. Dot blot hybridization with DNA from these plasmids, which probably contain repetitive sequences, can detect after an overnight exposure 100 pg of B. bovis DNA, which corresponds to the amount of DNA present in 50 microliters of 0.01% parasitemic erythrocytes. No detectable cross-hybridization was observed with Babesia microti, Plasmodium falciparum, Plasmodium vivax, Boophilus, or cow DNA. A small amount of cross-hybridization was observed with 10 ng Babesia bigemina DNA. Use of these probes in a hybridization assay may be helpful in the diagnosis of babesiosis in cattle and ticks, in the confirmation of strain identities, and in correlating virulence with particular strains of Babesia.

Animals↗
PubMed1985

The DNA between Rz and cosR in bacteriophage lambda is nonessential.

Near the right end of phage lambda DNA, between gene Rz and the cos site, are 2050 bp of apparently non-coding DNA. We have cloned a lambda DNA fragment containing this DNA into a plasmid and constructed a deletion, omega l, extending from a site within the Rz gene to a site about 560 bp from cos. This deletion could be recombined into viable lambda phage at a frequency equal to that observed for the undeleted sequence. Recombinant phage lambda carrying the omega l deletion were demonstrated to have the same burst size and kinetics of phage production as undeleted lambda. The omega l deletion can be used to extend the capacity of lambda cloning vectors and to provide a region for the insertion of heterologous DNA which should exhibit controllable high level expression from the lambda late promoter, p'R.

Bacteriophage lambda↗
PubMed1985

Cloning of Physarum actin sequences in an exonuclease-deficient bacterial host.

A genomic library of Physarum was constructed in the replacement vector EMBL3. Efficient propagation of the recombinant phages occurred only on the recBC-sbcB- host Escherichia coli CES200, which is deficient in the exonucleases I and V. Thirteen different recombinants with actin-related sequences were detected and 10 were purified from 90,000 plaques (the equivalent of 6 Physarum genomes) on strain CES200. Comparison of the plating efficiencies of the library and the actin-related isolates suggests that palindromic DNA sequences are responsible for the instability of Physarum DNA in E. coli. In one of these isolates, lambda PpA10, and in a 2.81-kilobase subclone of that isolate in plasmid pBR322, a deletion of 360 base pairs was detected that led to stable propagation of the recombinant DNA molecules in Rec+ E. coli. Electron microscopic analysis of the 2.81-kilobase fragment, after denaturation and self-hybridization, revealed secondary structures consistent with "foldback" structures. Restriction and DNA blot analysis of lambda PpA10 suggest that the unstable DNA segment is in close proximity to, if not part of, the previously defined actin-gene locus ardA.

Actins↗