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L J Evans

Publications and source records attributed to L J Evans.

18 recordsLinked to original sources
PubMed2000

Colicin pore-forming domains bind to Escherichia coli trimeric porins.

Colicin N kills sensitive Escherichia coli cells by first binding to its trimeric receptor (OmpF) via its receptor binding domain. It then uses OmpF to translocate across the outer membrane and in the process it also needs domains II and III of the protein TolA. Recent studies have demonstrated sodium dodecyl sulfate- (SDS) dependent complex formation between trimeric porins and TolA-II. Here we demonstrate that colicin N forms similar complexes with the same trimeric porins and that this association is unexpectedly solely dependent upon the pore-forming domain (P-domain). No binding was seen with the monomeric porin OmpA. In mixtures of P-domain and TolA with OmpF porin, only binary and no ternary complexes were observed, suggesting that binding of these proteins to the porin is mutually exclusive. Pull-down assays in solution show that porin-P-domain complexes also form in the presence of outer membrane lipopolysaccharide. This indicates that an additional colicin-porin interaction may occur within the outer membrane, one that involves the colicin pore domain rather than the receptor-binding domain. This may help to explain the role of porins and TolA-II in the later stages of colicin translocation.

Amino Acid Sequence↗
PubMed1998

Discovery of critical Tol A-binding residues in the bactericidal toxin colicin N: a biophysical approach.

Colicins translocate across the Escherichia coli outer membrane and periplasm by interacting with several receptors. After first binding to outer membrane surface receptors via their central region, they interact with TolA or TonB proteins via their N-terminal regions. Finally, the toxic C-terminal region is inserted into or across the cytoplasmic membrane. We have measured the binding of colicin N to TolA by isothermal titration microcalorimetry (ITC) and tryptophan fluorescence. The isolated N-terminal domain exhibits a higher affinity for TolA (Kd = 1 microM) than does the whole colicin (18 microM), and similar behaviour has been observed when the N-terminal domain of the g3p protein of the bacteriophage fd, which also binds TolA, is examined in isolation and in situ. This may indicate a similar mechanism in which a cryptic TolA binding site is revealed after primary receptor binding. The isolated colicin N N-terminal domain appears to be unstructured in circular dichroism and fluorescence studies. We have used mutagenesis and ITC to characterize the TolA binding site and have shown it to be of a different sequence and much further from the N-terminus than previously thought.

Amino Acid Sequence↗
PubMed1996

The central domain of colicin N possesses the receptor recognition site but not the binding affinity of the whole toxin.

Colicin N is a three-domain pore-forming colicin which kills enterobacterial cells following an initial binding to its receptor, the outer membrane porin OmpF. The receptor-binding domain of colicin N alone, and attached to the translocation domain, was overexpressed and purified using a hexahistidine tag. The receptor domain attached to the pore-forming domain was obtained by enzymatic digestion. Circular dichroism spectroscopy showed that the domains have structure in keeping with the known structure of colicin N. The receptor domain was stable, retaining both secondary and tertiary structure in 2 M guanidine hydrochloride and at low pH. It bound to both OmpF and PhoE porin-producing Escherichia coli with no toxicity and protected sensitive E. coli against intact colicin N toxicity at high domain/ colicin N ratios. Its in vitro affinity for OmpF, as determined by isothermal titration microcalorimetry, was found to be approximately 50-fold weaker than that of native colicin N. The receptor domain was readily out-competed by native colicin N in in vivo fluorescence assays which, coupled with its structural stability, suggests that its interaction with OmpF is one of weak, reversible binding. Since neither of the double domain constructs shows wild-type binding affinity either, it appears that the molecular recognition is a property of the receptor domain but that affinity is influenced by the entire molecule.

Bacterial Outer Membrane Proteins↗
PubMed1996

Different sensitivities to acid denaturation within a family of proteins: implications for acid unfolding and membrane translocation.

Colicins A, B, and N form a family of membrane pore-forming toxins with > 50% sequence identity in their toxic C-terminal domains. The colicin A C-terminal domain has been shown to insert into model membranes via an acidic molten-globule insertion intermediate, and thus this family provides a means to compare acid unfolding of related proteins. Unlike the domains of colicins A and B which are acidic, that of colicin N is very basic with fewer Asp and Glu residues. If surface positive charge density is the crucial factor in acidic molten globule formation, colicin N should begin to unfold at higher pH values than colicins A or B. However, comparison of their CD spectra reveals that colicins A and B both form acidic molten globules but colicin N does not. None of the proteins forms a denaturant-induced molten globule at neutral pH where the proteins exhibit very similar stabilities. The acidic unfolding cannot therefore be due to excess positive surface charge and may be caused by a subset of acidic residues as has been predicted for myoglobin. The difference between the colicins is confirmed by their in vivo membrane insertion, with colicins A and B inserting much faster than colicin N. Stopped-flow circular dichroism measurements of colicin A insertion into vesicles confirmed that a molten globule insertion intermediate occurs at the membrane surface.

Amino Acid Sequence↗
PubMed1996

Direct measurement of the association of a protein with a family of membrane receptors.

A specific receptor is a requirement for most protein toxins and OmpF, a trimeric porin, was previously considered to be the unique membrane-receptor for colicin N. We show by qualitative in vivo analysis that the related porins OmpC or PhoE act as much less effective receptors. To elucidate receptor function, the in vitro binding of the 42 kDa toxin to each of the 120 kDa porin trimers was determined quantitatively using isothermal titration calorimetry. Colicin N binds to OmpF with Ka approximately 5 x 10(5) M-1 and a stoichiometry consistent with about three per trimer but it also binds to PhoE and OmpC with surprisingly similar affinities and stoichiometry. However, thermodynamic analysis of these hitherto unmeasured interactions suggests an unexpected entropic difference between these protein import receptors.

Bacterial Outer Membrane Proteins↗
PubMed1993

Experiences with a science hotline.

Scientific literacy of the population may be enhanced by outreach programs conducted by universities. One such program, the University of Kentucky Science Hotline, has been successful in attracting and answering many inquiries from students, teachers, and others. The authors describe the organization and management of this hotline. The operation is inexpensive and efficient, with 35 faculty and staff members in 21 departments responding to 515 calls during the first year of operation. Fifty-four outreach visits were arranged through the hotline. Other benefits have included a more positive public image of scientists and the creation of links between academic scientists and precollege teachers.

Hotlines↗
PubMed1984

High-performance liquid chromatographic determination of nafazatrom in human plasma using fluorescence detection.

A rapid, sensitive, and selective high-performance liquid chromatographic assay was developed for determination of the pyrazole derivative nafazatrom (Bay g 6575, NFZ) in human plasma. Separation was obtained using a normal-phase Si-60 column and a mobile phase of methylene chloride--methanol (90:10, v/v) containing 0.25% water. The fluorescence of NFZ was monitored at excitation and emission wavelengths of 232 and 362 nm, respectively. The recovery of NFZ extracted from plasma with methylene chloride was 109 +/- 5% (mean +/- S.D.) in the concentration range from 5.0 to 500 ng/ml. The assay was applied to the determination of plasma concentrations of NFZ following administration of the compound to patients in a Phase I clinical trial.

Chromatography, High Pressure Liquid↗
PubMed1984

Phase I and pharmacologic evaluation of nafazatrom in patients with cancer.

Nafazatrom was evaluated in escalating daily oral doses ranging from 0.25 to 8.0 g/m2 without producing significant toxicities. Malabsorption proved dose limiting at 8.0 g/m2 as a single daily dose, but splitting the same total dose into two or four doses circumvented this problem. Doses of 2.0 g/m2 at 6-h intervals or 4.0 g/m2 every 12 h are reasonable for Phase II and adjuvant trials. Pharmacologic evaluation of nafazatrom confirmed malabsorption at the highest single daily dose level tested and suggests that absorption was impaired in patients with extensive liver metastases.

Adult↗
PubMed1981

Measurement of the fetal biparietal diameter by ultrasound is not an accurate method of detecting fetal growth retardation.

Seventy-one patients had serial ultrasound examinations performed during the third trimester of pregnancy. Diameter, circumference and area measurements were made of the fetal head, thorax and abdomen. In three of five infants with birthweights below the 10th percentile no biparietal diameter measurements were below the 10th percentile whereas all five infants had at least the last two abdominal circumference measurements below the 10th percentile. The correlation coefficient between the various fetal parameters, measured within two weeks of delivery and birthweight averaged 0.83 for the thorax measurements and 0.85 for the abdomen measurements but was only 0.60 for the biparietal diameter. It is recommended that fetal abdominal measurements should be used for diagnosing growth retardation and not biparietal diameter measurements.

Abdomen↗
PubMed1981

Measurement of the uterus and gestation sac by ultrasound in early normal and abnormal pregnancy.

Uterine volumes measured by two different ultrasonic methods, and gestation sac volumes in early normal pregnancy are reported. The results obtained for uterine volume measurements are compared. Methods using measurements obtained from only a longitudinal scan were simpler but slightly less accurate. Uterine volumes were also calculated in a series of patients with pregnancy complicated by threatened abortion. The accuracy of the prediction of the outcome of the pregnancy, based solely on uterine volume was 71 percent. Uterine volume measurement is most useful in identifying cases of missed abortion where the period of gestation is known.

Abortion, Threatened↗
PubMed1981

Prediction of the extremes of birth weight from a single ultrasound examination at 34 weeks of gestation.

One hundred and forty patients were subjected to ultrasound examination at about 34 weeks of gestation. Using the product of the biparietal diameter measurement and either the fetal abdominal circumference or abdominal area measurement, it was possible to predict three-quarters of both large for dates infants (false positive rate, 21.3%) and small for dates infants (false positive rate, 15.7%). Using the presence of a head and/or abdomen measurement above the 90th percentile for gestation, it was possible to predict 62.5% of large for dates infants (false positive rate, 15.7%). Using the presence of any measurement below the 10th percentile, it was possible to predict 81.3% of small for dates infants (false positive rates, 21.3%).

Anthropometry↗
PubMed1977

Measurement of intragastric oxygen concentration for the diagnosis of H-type tracheoesophageal fistula.

The demonstration of an H-type tracheoesophageal fistula before surgical correction is frequently difficult. Currently utilized procedures are so irregularly successful that surgical exploration is sometimes necessary without prior demonstration of the fistula. A case is reported in which the diagnosis was made by measurement of intragastric oxygen concentration, which increased and declined in response to the endotracheal insuffation of 100% oxygen and room air, respectively. The fistula was subsequently demonstrated by barium esophagram and was successfully treated surgically. In the presence of the classic clinical triad (choking during feedings, abdominal distention, and pneumonitis), a positive intragastric oxygen test may be sufficient indication for surgical exploration.

Female↗