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Biomedical subjects

C F Earhart

Publications and source records attributed to C F Earhart.

At least 37 records · Page 2Linked to original sources
PubMed1981

Specific inhibition of Escherichia coli ferrienterochelin uptake by a normal human serum immunoglobulin.

Normal human serum contains an enterochelin-specific antibody which presumably acts with transferrin to hinder iron assimilation by enterochelin-producing pathogens. This antibody can be isolated from serum by sodium sulfate fractionation or affinity chromatography by employing an enterochelin-derived ligand (2,3-dihydroxy-N-benzoyl-L-serine) attached to aminohexyl Sepharose 4B. In assays of iron uptake by whole cells, the antibody inhibited enterochelin-directed uptake but not that mediated by citrate or ferrichrome. Also, the growth stimulatory effect of enterochelin on an Ent- strain of Escherichia coli was blocked by the immunoglobulin. This antibody has a high affinity for enterochelin; various elution procedures employing high salt concentrations and low pH failed to remove it from affinity columns. Elution with 3 M sodium thiocyanate or 13 mM 2,3-dihydroxybenzoic acid proved successful. Two pieces of evidence indicate the enterochelin-specific antibody is primarily of the immunoglobulin A (IgA) isotype. It could be removed from serum with goat antihuman IgA and was present only in sodium sulfate fractions of serum known to contain IgA.

Antibodies, Bacterial↗
PubMed1981

Characterization of the predominant Azotobacter vinelandii envelope protein.

A protein with a molecular weight of 60,000 (60K) constitutes approximately 20% of the envelope protein of Azotobacter vinelandii. This protein was removed from cells and purified from other proteins by a simple washing procedure that had no effect on cell viability. Anti-60K antiserum blocked azotophage A-22 adsorption and agglutinated both vegetative cells and cysts; ferritin-conjugated antibodies used in indirect labeling studies bound uniformly to the periphery of vegetative cells. We conclude that 60K is present on the outer surface of vegetative cells and cysts. The protein is similar to the surface protein alpha of Acinetobacter ssp. in molecular weight, reassociation characteristics, and high ratio of acidic to basic amino acids. We propose that 60K forms a layer external to the outer membrane of A. vinelandii.

Agglutination Tests↗
PubMed1980

Alterations in the cell envelope of Escherichia coli late in bacteriophage T4 infection.

The cell envelope of Escherichia coli was examined for changes during late stages of bacteriophage T4 infection. Late events in T4 infection are shown to result in (i) a reduction in the effectiveness of membrane separation procedures employing either isopycnic sucrose gradient centrifugation or selective solubilization of inner membrane by detergent (Sarkosyl or Triton X-100), (ii) the appearance of a 54 000 dalton host protein in membrane preparations, (iii) the adventitious presence of detergent-resistant phage morphogenetic structures in membrane preparation, and (iv) a decrease in the activity of NADH oxidase and an apparent alteration in its association with inner membrane. These modifications occur regardless of the state of the e and t genes of T4.

Bacterial Proteins↗
PubMed1980

Bacteriostatic enterochelin-specific immunoglobulin from normal human serum.

Heat-inactivated normal human serum produces iron-reversible bacteriostasis of a number of microorganisms. This inhibitory effect was abolished by adsorption of serum with ultraviolet-killed cells of species that produce the siderophore enterochelin. Bacteriostasis also was alleviated by adsorption of serum with 2,3-dihydroxy-N-benzoyl-L-serine, a degradation product of enterochelin, bound to the insoluble matrix AH-Sepharose 4B. The adsorption process did not add iron or enterochelin to serum, nor did it remove transferrin. The immunoglobulin fraction from normal human serum was isolated; when added to a defined medium (M199) prepared so as to mimic normal human serum, the immunoglobulin rendered the medium inhibitory to an enterochelin-defective strain of Salmonella typhimurium. Adsorption of this medium with AH-Sepharose 4B-2,3-dihydroxy-N-benzoyl-L-serine removed the inhibition. Our results indicate that enterochelin-specific immunoglobulins exist in normal human serum. These immunoglobulins may act synergistically with transferrin to effect bacteriostasis of enterochelin-producing pathogens.

Adsorption↗
PubMed1977

Coordinate regulation by iron of the synthesis of phenolate compounds and three outer membrane proteins in Escherichia coli.

The biosynthesis of the low-molecular-weight iron carrier enterochelin and of three outer membrane polypeptides appears to be coordinately regulated by the amount of cell-associated iron in Escherichia coli K-12. Measurements of iron acquisition made throughout the growth cycle in iron-deficient media indicate that a very rapid accumulation of iron occurs in the first 2 h of growth; there is comparatively little iron uptake during exponential growth, which results in a gradual decrease in the cellular iron content with each generation. When this level falls below 400 ng of iron per mg (dry weight) of cells, there is a simultaneous onset of synthesis of the three outer membrane polypeptides and of enterochelin. This coordinate regulation was also observed in cells able to transport iron actively using only citrate as an iron-carrier.

Bacterial Proteins↗
PubMed1976

Phleomycin-induced solubilization of deoxyribonucleic acid in uninfected and T bacteriophage-infected Escherichia coli B.

Phleomycin (PM) induces rapid solubilization of deoxyribonucleic acid (DNA), inhibition of cellular mass increase, and loss of viability when added either to growing cultures of Escherichia coli B or an endonuclease I-defective derivative of B. Nonetheless, bacteriophage of the T-even series are produced, albeit to a reduced extent, when PM is added with phage or after infection to E. coli cells. Bacteriophage T4 infection inhibits the ability of PM to bring about the solubilization of DNA; this effect appears to account for most of the resistance of T-even phage replication to PM. The patterns of inhibition of phage yields obtained when PM is added at infection suggest that glucosylation of T-even phage DNA may also have a protective effect. However, PM induces the solubilization of glucosylated and nonglucosylated DNA at approximately the same rate and thermal denaturation studies indicate that the antibiotic binds both types of DNA equally well. The latter experiments also provided evidence that PM dissociates from DNA at temperatures greater than 80 C.

Anti-Bacterial Agents↗
PubMed1975

Effect of ribonuclease on the association of deoxyribonucleic acid with the membrane in Escherichia coli.

The Mg-2+-Sarkosyl crystals (M band) procedure was used to study the effect of ribonuclease (RNase) A on the association of Escherichia coli deoxyribonucleic acid (DNA) with membrane. Incubation of gently prepared cell extracts with RNase results in the release of DNA from membrane. This effect appears to result from the activation, by RNase, of endonuclease I and subsequent limited activity of this deoxyribonuclease. In support of this explanation, it is demonstrated (i) that the extent of the RNase-induced loss of DNA from membrane is directly correlated with the endogenous level of endonuclease I, and (ii) that endonucleolytic activity occurs when gently lysed cell preparations are incubated in the presence of RNase.

Cell Membrane↗
PubMed1974

Localization of membrane protein synthesized after infection with bacteriophage T4.

The synthesis of membrane protein after infection with bacteriophage T4 was examined. Protein constituents of both the cytoplasmic and outer membrane are made during the infective cycle. In addition, newly synthesized membrane protein is found in material which has a buoyant density greater than that of either of the two host membrane fractions. Polyacrylamide gel analyses and solubilization studies using the detergent Sarkosyl indicate that synthesis of most of the membrane proteins made during the first 5 min of infection is directed by bacterial genes. New membrane proteins synthesized at times greater than 6 min after infection appear to be distinct from those of the host, and new proteins of the outer membrane are different from those of the inner. Proteins in the new dense membrane fraction are similar to those of the outer membrane.

Bacterial Proteins↗
PubMed1974

Early synthesis of membrane protein after bacteriophage T4 infection.

Proteins that associate with cellular membrane during the first 5 min after infection with bacteriophage T4 were examined. Several procedures, including electrophoretic separations in three sodium dodecyl sulfate polyacrylamide gel systems and inhibition of host protein synthesis by UV irradiation, were employed to distinguish host-specified proteins from those induced by T4. Residual host protein synthesis was found to account for much of the new protein in preparations of the total membrane and for almost all of the newly synthesized protein in the outer membrane. Preliminary evidence indicates that the synthesis of some host membrane proteins is shut off less rapidly than is host synthesis of soluble protein. One host-directed polypeptide of the outer membrane was unique in that its synthesis or incorporation into the membrane was preferentially inhibited by infection. Also, it was found that the detergent Sarkosyl solubilizes all early T4 membrane proteins; this observation provides the basis for a simple procedure for distinguishing phage proteins from host outer membrane proteins.

Bacterial Proteins↗
PubMed1973

Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate.

The sensitivity of the outer and cytoplasmic membranes of Escherichia coli to detergent was examined by isopycnic sucrose density gradient centrifugation. Sodium lauryl sarcosinate (Sarkosyl) was found to disrupt the cytoplasmic membrane selectively under conditions in which Triton X-100 and dodecyl sodium sulfate solubilized all membrane protein. These results were verified by gel electrophoresis; membrane proteins solubilized by Sarkosyl were identical to those of the cytoplasmic membrane. The presence of Mg(2+) during treatment with Sarkosyl was found to afford partial protection of the cytoplasmic membrane from dissolution.

Bacterial Proteins↗
PubMed1973

Effect of inhibition of macromolecule synthesis on the association of bacteriophage T4 DNA with membrane.

The "Mg(2+)-Sarkosyl crystals" (M band) technique distinguishes between membrane-bound and free intracellular DNA. This procedure was employed to investigate the nature of the reactions necessary to convert input T4 DNA to a rapidly sedimenting form. Energy poisoning inhibits this attachment reaction. Neither protein nor DNA synthesis appears to be required, but experiments with rifampin and extensively irradiated T4 suggest that RNA synthesis is involved. These results were confirmed by a second procedure for the determination of rapidly sedimenting DNA.

Azides↗