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Biomedical subjects

G M Ihler

Publications and source records attributed to G M Ihler.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1986

Entry of Bartonella bacilliformis into erythrocytes.

Bartonella bacilliformis, which causes the human diseases Oroya fever and verruga peruana, binds to human erythrocytes in vitro and produces substantial and long-lasting deformations in erythrocyte membranes, including cone-shaped depressions, trenches, and deep invaginations. The deforming force is probably provided by the polar flagella of these highly motile bacteria. Deep invaginations containing bacteria are commonly seen, and membrane fusion at the necks of the invaginations leads to the formation of intracellular vacuoles containing bacteria. Fluorescent compounds present externally render the vacuoles fluorescent and, occasionally, lightly fluorescent cells are seen, suggesting that the vacuoles sometimes rupture to admit the bacteria to the cytoplasm. Vacuoles present in fluorescent erythrocytes prepared by preloading the erythrocytes with fluorescent compounds are seen as dark areas from which the fluorescent marker is excluded. Entry of the bacteria appears to be the result of a process of forced endocytosis.

Bacterial Adhesion↗
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

Detection of Plasmodium falciparum using a synthetic DNA probe.

A labeled synthetic polynucleotide representing a repetitive sequence from Plasmodium falciparum was hybridized with genomic DNA spotted on nitrocellulose. After an overnight exposure, 0.1 ng of P. falciparum DNA was specifically detected and 0.01 ng was detected after an exposure of 1 week. The synthetic probe showed no cross-hybridization with host DNA or with DNA isolated from other species in the phylum Apicomplexa, P. vivax and Babesia species. Since synthetic DNA is easily prepared, the observed sensitivity and specificity suggests that synthetic DNA probes would be generally useful in diagnosis.

Animals↗
PubMed1985

Erythrocyte carriers.

The properties of erythrocytes used as carriers for drugs, enzymes, and DNA will be reviewed. One potential application is delivery of these substances to cells responsible for or capable of erythrophagocytosis and are located primarily in the liver and the spleen. A second potential application depends on the ability of loaded cells to survive for substantial periods of time in the circulation after reinfusion. Circulating cells used as drug carriers may be able to modify the pharmacokinetics of administered drugs and if used as enzyme carriers, they may be able to alter the level of various substances in the plasma. Erythrocytes in vitro may fuse with recipient cells, introducing their contents in a functional form into recipient cells. Nucleic acids, either RNA or DNA, as well as enzymes or other entrapped substances, may be transferred in this manner.

Amphotericin B↗
PubMed1985

Visualization of fluorescent erythrocytes in the microcirculation.

Erythrocytes loaded internally with FITC-BSA can be readily visualized in the microcirculation by using a television camera and a fluorescence microscope. Flow properties of the erythrocytes and their adherence to the vascular endothelium or erythrophagocytic cells can be observed. This procedure should also be useful to delineate the microcirculation under circumstances where infused free FITC-BSA can escape into the interstitial tissue.

Animals↗
PubMed1984

Long range base-pairing in the leftward transcription unit of bacteriophage lambda. Characterization by electron microscopy and computer-aided sequence analysis.

Restriction fragments of bacteriophage lambda DNA corresponding to the major leftward transcription unit were purified, denatured to form single-stranded DNA, self-annealed, and examined by electron microscopy. Three intrastrand stem and loop secondary structures were observed reproducibly and the locations of the paired regions were determined. A method for computer-aided sequence analysis of these regions is presented and used to identify sets of base-pairings likely to account for the observed structures. One loop observed within gene Ea47 is postulated to involve pairing of sequences which include the polypeptide initiation and termination codons. Another loop is postulated to involve pairing of sequences in gene int with sequences located in the gam-cIII region. A third loop appears to involve sequences in and to the right of gene Ea22 paired with sequences located in the bet-gam region. A general discussion of base-pairing which gives rise to long range interactions is presented along with possible effects of the postulated models on gene expression.

Bacteriophage lambda↗
PubMed1984

Proteinase sensitivity of bacteriophage lambda tail proteins gpJ and pH in complexes with the lambda receptor.

Previous studies have shown that bacteriophage lambda initially binds to liposomes bearing its receptor protein by the tip of the tail fiber (type 1 complex). It then associates more directly so that the hollow tail tube is in direct contact with the membrane (type 2 complex). DNA can be injected across the lipid bilayer into the liposome from type 2 complexes. We show here that gpJ, the tail fiber protein, becomes more sensitive to proteolytic degradation in type 2 complexes, indicating that the tail fiber does not pass into the liposome and that the tail fiber may undergo a conformational change in type 2 complexes. Another bacteriophage protein, pH, is sensitive to proteolytic degradation in free bacteriophage, type 1 complexes, or type 2 complexes formed with free receptor, but is resistant to proteinases in type 2 complexes formed with liposomes. This finding suggests that pH associates with the membrane. We suggest that this association is part of the mechanism by which a transmembrane hole for DNA entry is formed.

Bacterial Outer Membrane Proteins↗
PubMed1983

Comparison of three methods for measuring locations of long range base pairing in single-stranded DNA by electron microscopy.

A comparison was made of three procedures used for measuring the locations of long range base pairing in single-stranded DNA. All three procedures gave equivalent results for the location of a transposon, Tn903, and for a loop created by pairing between the nut and operator segments on an EcoRI restriction fragment of lambda DNA. DNA molecules prepared by a T4 gene 32 protein procedure had the best contrast. The highest frequency of measurable loops resulted from a procedure which utilized ammonium acetate/formamide spreading conditions. This procedure also appeared to be the simplest and most satisfactory procedure. Only one procedure revealed the fact that the nutR and oR regions are separated by several hundred bases, but this procedure, which involves crosslinking with trioxsalen, gave generally unsatisfactory DNA morphology. None of the procedures revealed the existence of a short "bulge" loop of single-stranded DNA located between nutL and oL and a longer "bulge" loop located between nutR and oR.

Acetates↗
PubMed1983

Injection of DNA into liposomes by bacteriophage lambda.

Small unilamellar vesicles (75-100 nm diameter) and large liposomes (greater than 1 micron in diameter) were prepared containing the lamB protein, an outer membrane protein of Escherichia coli and Shigella which serves as the receptor for bacteriophage lambda. Bacteriophage were observed to bind to these liposomes and vesicles by their tails and in most cases the heads of the bound bacteriophage appeared empty or partially empty of DNA. The lambda DNA was usually only partially ejected from the bacteriophage head when small unilamellar liposomes were used, presumably because the vesicles are too small to contain all the DNA. The partially ejected DNA was not susceptible to DNase unless the vesicle bilayer was first disrupted suggesting that DNA injection of phage DNA into the vesicle had occurred. After disruption of these vesicles on electron microscope grids, the bacteriophage are seen to have partially empty heads and a small mass of DNA associated with their tails. Using larger liposomes prepared by the fusion of lamB bearing vesicles with polyethylene glycol and n-hexyl bromide, the heads of most of the bound bacteriophage appeared to be completely empty of DNA. Disruption of these preparations on electron microscope grids revealed circular arrays of empty-headed bacteriophage surrounding DNA which had apparently been contained within the intact liposomes. These results indicate that high molecular weight DNA can be entrapped within liposomes with high efficiency by ejection from bacteriophage lambda. The possible use of these DNA-containing liposomes to facilitate gene transfer in eukaryotic cells is discussed.

Bacteriophage lambda↗
PubMed1983

Morphology of complexes formed between bacteriophage lambda and structures containing the lambda receptor.

Two types of complexes can be formed between bacteriophage lambda and structures bearing the lambda receptor, either liposomes or rod-shaped particles. Type 1 complexes involve binding between the tip of the lambda tail fiber and the receptor, so that the hollow tail is positioned an average of 17 nm from the surface of the receptor-bearing structures. In type 2 complexes, the hollow tail is in direct contact with the membrane of the liposome or surface of the rod-shaped particle. Type 1 complexes are the precursors for type 2 complexes whose formation is necessary for normal DNA ejection.

Bacterial Outer Membrane Proteins↗