Biomedical subjects

W C Johnson

Publications and source records attributed to W C Johnson.

At least 55 records · Page 3Linked to original sources

PubMed1995

Design and synthesis of amphipathic antimicrobial peptides.

A large proportion of antimicrobial peptides share a common structural feature that is critical to their antimicrobial activity, i.e. amphipathic alpha-helices. The amphipathy of a polypeptide chain can be quantitated through the value of the hydrophobic moment. Generally, antimicrobial peptides are characterized by high hydrophobic moment and low hydrophobicity values. Using these criteria we have identified two short segments that possess hydrophobic moment properties associated with known antimicrobial peptides. Using in vitro assays the segment derived from the protein perforin displays no antifungal or antibacterial activity and, while showing no alpha-helicity in buffer or liposomes, exhibits a modest degree of alpha-helical structure in the presence of the alpha-helical inducer, 2,2,2-trifluoroethanol. However, rational modifications result in a derivative which assumes an alpha-helical conformation in the presence of liposomes, exhibits potent antifungal activity against plant fungal pathogens, has significant antibacterial activity, effects leakage of a fluorescent dye from acidic liposomes and is devoid of hemolytic activity. Results are also presented for a segment derived from the human immunodeficiency virus envelope protein. We suggest that the identification of putative amphipathic structures in proteins may provide a useful starting strategy in the design and synthesis of antimicrobial peptides.

Amino Acid Sequence↗

PubMed1994

Infrared linear dichroism reveals that A-, B-, and C-DNAs in films have bases highly inclined from perpendicular to the helix axis.

Infrared linear dichroism has been employed to investigate the inclination of the bases in films of poly[d(AC)].poly[d(GT)], poly[d(AG)].poly[d(CT)], and natural DNAs (from Escherichia coli and calf thymus). All DNAs investigated assume the B-form at high (> 94%) relative humidity. Poly[d(AC)].poly[d(GT)], E. coli DNA, and calf thymus DNA assume the A-form at low (75%) relative humidity, whereas poly[d(AG)].poly[d(CT)] assumes the C-form at low (66%) relative humidity. Infrared linear dichroism demonstrates that the bases for DNA in films are highly inclined from perpendicular to the helix axis, even for B-DNA. C-DNA has almost same inclinations as in B-DNA, and the inclinations are slightly increased in A-DNA. These inclination angles confirm our earlier UV linear dichroism results for the orientation of the bases for DNA in solution. Infrared linear dichroism has also been used to obtain conformational angles for the phosphodiester backbone geometry of the A-, B-, and C-forms of DNA.

Animals↗

PubMed1994

Importance of environment in determining secondary structure in proteins.

We report here the effect of bulk solvent environment on the secondary structure of several peptides. In previous work, equivocal peptide sequences that are predicted to be alpha-helical from amino acid preference but are found to be beta-strand in their proteins were shown to be alpha-helical in alcohol solvents and beta-strand in nonmicellar sodium dodecyl sulfate (SDS) by circular dichroism (CD) spectroscopy [Zhong, L., & Johnson, W. C., Jr. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4462-4465]. Here we show that equivocal sequences that are predicted to be beta-strand but are found to be alpha-helical follow the same pattern; they are alpha-helical in alcohol solvents and beta-strand in nonmicellar SDS. Furthermore, we investigated a control sequence with only a strong alpha-helical propensity and a control sequence with only a strong beta-strand propensity. Both of these well-behaved sequences followed the same pattern as the equivocal sequences. The exceptionally stable Y(EAAAK)3A is an alpha-helix in all solvents, but analyses of the CD spectra indicate the loss of helix with an increase in beta-strand and other structures on changing solvent from trifluoroethanol (TFE) to SDS, similar to the other peptides. We find that solvent is a very important factor in determining the secondary structure of an amino acid sequence in vitro and can override the propensity for a secondary structure due to sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

A CD study of the alpha-helix nucleation hypothesis.

One of the dilemmas in predicting the secondary structure of proteins from their amino acid propensity for a given conformation is the presence of all amino acids in all types of secondary structure, regardless of their propensity for that specific structure. One explanation is the nucleation hypothesis that only a few residues with a strong propensity for the secondary structure, such as the alpha-helix structure, initiates its formation and propagates the structure through indifferent sequences until strong breakers terminate the growth on both ends. Eight 15-mer peptides were studied to examine the alpha-helix nucleation hypothesis. The nucleation sequence of VAEAK, with high helix propensity, was mixed with an indifferent sequence of TSDSR in all possible permutations. From the percent alpha-helix structure derived from the CD at 222 nm, it appears that helicity does not propagate through the indifferent sequence.

Amino Acid Sequence↗

PubMed1994

Flow linear dichroism and Fourier transform IR spectra reveal geometry for X-form DNA.

Flow linear dichroism measurements extended into the vacuum uv region yield inclinations for the base normal from the helix axis of 21 degrees for dA and 40 degrees for dT in the X-form of poly(dAdT).poly(dAdT). These inclination angles are similar to the B form of the synthetic polymer, but the axes around which the bases incline are different. Hydrogen-bonded base pairs are consistent with the geometry for the standard B, C, D, and Z forms of natural DNA, but will not fit into the A form. Fourier transform ir spectra indicate that the X form has sugar pucker and phosphate geometry similar to B-form DNA, and supports the dinucleotide repeat with two kinds of phosphates seen in earlier work, in analogy to Z-form DNA. Clearly, X-form DNA has a unique geometry.

Biopolymers↗

PubMed1994

The lateral approach to the proximal popliteal artery for popliteal to anterior tibial artery bypass.

The exposure of the proximal popliteal artery through a lateral incision for bypasses to the anterior tibial artery is simple and logical. It avoids the need to direct the graft from the medial to the lateral side of the leg and, therefore, is advantageous when limited autogenous vein is available. The lesser saphenous vein is usually long enough to reach the most distal anterior tibial artery.

Humans↗

PubMed1994

Pentoxifylline therapy for chronic claudication: are patients dependent on therapy?

BACKGROUND: The purpose of this study was to evaluate the efficacy of long-term pentoxifylline therapy. METHODS: Fifty consecutive patients on long-term pentoxifylline therapy entered a weaning program. The standard dose of 400 mg three times a day was reduced to 400 mg twice a day for 1 month, 400 mg daily for an additional month, and then withdrawn completely for 3 months. Subjective evaluation of each patient's symptoms and objective measurements (treadmill testing for initial claudication distance, maximum walking distance, and ankle/brachial indexes) were evaluated during treatment withdrawal. RESULTS: Twenty-seven patients tolerated withdrawal of therapy without symptomatic deterioration. Seven of nine patients who did not tolerate weaning had deterioration of treadmill ICD/MWD parameters; there was a significant absolute decrease (p = 0.016) in MWD from 430 +/- 78 to 221 +/- 23 feet. Patients with an initial low MWD were less likely to tolerate weaning. Sixty percent of the patients with successful weaning had clinically asymptomatic deterioration of treadmill ICD/MWD parameters. CONCLUSIONS: A substantial number of patients who have been on long-term successful pentoxifylline therapy for claudication can be weaned from the drug. Asymptomatic deterioration of treadmill test parameters is not an indication for resuming therapy.

Aged↗

PubMed1993

Biologically active Arg-Gly-Asp oligopeptides assume a type II beta-turn in solution.

The sequence Arg-Gly-Asp (RGD) has been found to be the consensus sequence of matrix proteins for binding cell surface receptors (integrins). Studies with synthetic peptides containing the RGD sequence show that the biological activity of these oligopeptides is removed upon a conservative substitution of Glu for Asp in the RGD sequence. Two-dimensional 1H NMR methods were used to investigate the secondary structures in aqueous solution for two such oligopeptides of differing biological activity. The sequence Tyr-Gly-Arg-Gly-Asp-Ser-Pro, which binds to selected integrins, is found to assume a type II beta-turn at both pH 4 and 7. In contrast, the sequence Tyr-Gly-Arg-Gly-Glu-Ser-Pro, which does not interfere with integrin-mediated cell attachment, is found to assume a type I or III beta-turn at both pH 4 and 7. This comparison confirms not only that oligopeptides can assume a secondary structure in aqueous solution, but also that these structures may be important to biological functions.

Amino Acid Sequence↗

PubMed1993

Linear dichroism demonstrates that the bases in poly[d(AC)].poly[d (GT)] and poly[d(AG)].poly[d(CT)] are inclined from perpendicular to the helix axis.

Flow linear dichroism is used to measure specific inclinations for each of the four bases in poly[d(AC)].poly[d(GT)] and poly[d(AG)].poly[d(CT)] in both the B and A forms. For the B form in solution the bases are found to have a sizable inclination. Inclination is increased in the A form, as expected. In all cases the pyrimidines are more inclined than the purines.

Adenine↗

PubMed1993

Effects of relative band intensity on prediction of protein secondary structure from CD.

Increasing the magnitude of a protein CD spectrum obviously increases the magnitude of each predicted secondary structure by the same amount. However, increasing the magnitude of the negative, long-wave-length portion of a protein CD spectrum usually has the opposite effect from increasing the positive, short-wave-length portion. Thus small distortions in the CD spectra of proteins at short wavelength can have a significant effect on the analysis for secondary structure. This measurement error and its effect on the analysis are systematically investigated for 16 proteins of known structure. The results demonstrate that a two-point calibration of a CD instrument is mandatory to avoid serious errors when estimating secondary structure from protein CD spectra.

Calibration↗

PubMed1992

Circular dichroism and molecular modeling yield a structure for the complex of human immunodeficiency virus type 1 trans-activation response RNA and the binding region of Tat, the trans-acting transcriptional activator.

Transcription in the human immunodeficiency virus type 1 (HIV-1) retrovirus is regulated by binding the viral Tat protein (trans-acting transcriptional activator) to the trans-activation response (TAR) RNA sequence. Here, vacuum UV circular dichroism (VUV-CD) is used to study the structure of TAR and its complex with two peptide fragments that are important for Tat binding to TAR. The VUV-CD spectrum of TAR is typical of A-form RNA and is minimally perturbed when bound to either the short or the long Tat peptide. The CD spectra of the complexes indicate an extended structure in the arginine-rich region of Tat from amino acid residue 47 through residue 58 and a short alpha-helix within the adjacent 59-72 region. Models of TAR and its peptide complexes are constructed to integrate these spectroscopic results with current biochemical data. The model suggests that (i) the arginine-rich 49-58 region is primarily responsible for electrostatic interactions with the phosphates of the RNA, (ii) the arginine side chains can additionally interact with substituent groups of the nucleotide bases to confer base recognition in the complex, (iii) the recognition of uracil-23 in TAR is facilitated by the peptide backbone, and (iv) the glutamine-rich face of an alpha-helix within the 59-72 region pairs to bases UGG at nucleotide positions 31-33 in the TAR loop and thus provides an additional motif in the Tat trans-activating protein to recognize TAR RNA.

Amino Acid Sequence↗

PubMed1992

Intrinsic pigment cell stimulating activity in the skin of the leopard frog, Rana pipiens.

Consistent with the concept that specific pigment patterns of amphibians might result from the highly localized distribution of stimulators and inhibitors of pigment cell expression in the skin, the spot pattern of the leopard frog, Rana pipiens, was examined through the use of the Xenopus neural tube explant assay system (Fukuzawa and Ide, 1988). Media conditioned with pieces of skin from dorsal black spotted areas promoted melanization of neural crest cells at a significantly higher level than did media conditioned with dorsal interspot skin in the absence of extra tyrosine. All conditioned media contained exceedingly low concentrations of tyrosine. With the addition of supplemental tyrosine, the melanization capacity of conditioned media from the interspot areas was elevated to that of the spotted skin. Control media conditioned with ventral frog skin inhibited melanization, as usual, because of the presumed presence of melanization inhibiting factor (MIF). It is considered that dorsal skin contains a melanization stimulating factor (MSF) which is present in significantly higher levels in spotted skin than in interspot areas and that expression of the particular pigmentary pattern of this leopard frog is regulated by the relative distribution of MIF, MSF, and possibly other intrinsic substances present in the skin.

Animals↗

PubMed1992

Environment affects amino acid preference for secondary structure.

Three equivocal amino acid sequences were synthesized that are predicted to be alpha-helical from amino acid preference but are found to be primarily beta-strand from x-ray diffraction of their respective proteins. In some solvent systems we recover the alpha-helical structure predicted by amino acid preference, whereas in other systems we mimic the interior of the protein and produce a beta-strand. These results are experimental proof that the environment is important in determining the secondary structure formed by an amino acid sequence; therefore schemes that predict secondary structure from amino acid sequence alone can never be totally successful.

Amino Acid Sequence↗

PubMed1992

Structural properties of arrestin studied by chemical modification and circular dichroism.

A unique conformation of arrestin is crucial for its interaction with phosphorylated photolyzed rhodopsin. Conformational changes in arrestin were investigated using chemical modification and circular dichroism. We studied the kinetics of sulfhydryl modification of bovine arrestin in order to determine whether its conformation is altered by the presence of ligands or salts at different ionic strengths. We found that all three cysteines (stoichiometry was 2.7 +/- 0.06 3-carboxy-4-nitrophenyl sulfide (NbS)/arrestin) are accessible for modification by NbS2. Under pseudo-first-order conditions (30-100-fold excess of NbS2 over arrestin), the modifications of the 3 cysteines are indistinguishable. At higher concentrations of NbS2 (150-300-fold excess), the pseudo-first-order plot is not linear, and the reaction can be resolved into two processes that involve two classes of sulfhydryl groups. Addition of CaCl2, MgCl2, inorganic phosphate, MgATP, or MgGTP had little effect on the rate of modification of the cysteine residues; however, heparin and inositol hexakisphosphate, which have been shown to induce conformational changes in arrestin, block modification of one sulfhydryl group of arrestin and accelerate the modification of the remaining two. Analysis of CD spectra revealed that arrestin has virtually no alpha-helical structure, about 40% beta-structure, about 18% beta-turns, and about 40% other structure. The CD spectrum for arrestin did not change in the presence of heparin. These studies suggest that arrestin exists in equilibrium between two or more conformational states. However, it is proposed that conversion between these conformations occur without altering significantly the secondary structure of arrestin.

Adenosine Triphosphate↗

PubMed1992

Extending CD spectra of proteins to 168 nm improves the analysis for secondary structures.

The CD spectra for 10 proteins with known secondary structure have been extended from 178 to 168 nm. Combined with the data for 6 other proteins investigated previously, this produces a basis set of 16 proteins, which can be used to analyze CD spectra for secondary structure. Extending the spectra adds another CD band to the data and increases the information content from the equivalent of five to six. Analyzing the CD for each of the 16 proteins in the basis set with the 15 other proteins shows a modest improvement in the prediction of secondary structure with the extended CD spectra.

Circular Dichroism↗

PubMed1992

Multivalent ions are necessary for poly[d(AC).d(GT)] to assume the Z form: a CD study.

In previous work, it was shown that poly[d(AC).d(GT)] could be forced into the Z form by strong dehydrating conditions, provided EDTA was not present. Presumably multivalent impurities were also necessary for the transition. In order to gain control over the B to Z transition for this DNA, we carefully removed all divalent contaminants from the sample and asked the obvious question: What ions are necessary for the transition under dehydrating conditions? We systemically investigated the effect of various multivalent ions. The common contaminants Ca2+, Mg2+, and Fe3+ will not cause the transition, but Co2+ and Ni2+ facilitate the transition, undoubtedly because of their well-known propensity to bind to purine N7. Since the transition also depends on the synergistic dehydrating action of sodium perchlorate and ethanol, we include CD spectra for the independent variations of these two factors. In addition, vacuum-uv CD spectra for the A form and various B forms of poly[d(AC).d(GT)] are presented for the first time.

Circular Dichroism↗

PubMed1992

Stacking interactions of ApA analogues with modified backbones.

CD spectra have been measured as a function of temperature for a number of ApA analogues with modified backbones. Oligonucleotides with these modified backbones are being used as antisense agents having potential as viral therapeutics. Results of these studies show that when a carbonyl is substituted for the phosphate to produce an uncharged backbone, the analogues that have either sugar or morpholino substitution do not stack. In contrast, when a morpholino group is substituted for the sugar and the phosphate is modified so as to be uncharged, there is strong base stacking. Stacking interactions in the phosphorus-linked morpholino analogues are at least as strong as those found in d(ApA). The stacking interactions in ApA are weak by comparison. Singular value decomposition demonstrates that the stacking is two state, and Taylor series decomposition yields a coefficient that measures base stacking interactions. The van't Hoff equation is applied to the base stacking coefficient from the Taylor series fitting to give thermodynamic parameters.

Circular Dichroism↗

PubMed1992

Preliminary biological characterization of a melanization stimulating factor (MSF) from the dorsal skin of the channel catfish, Ictalurus punctatus.

A two step fractionation of conditioned media made from the darkly pigmented dorsal skin of the channel catfish, Ictalurus punctatus, has produced fractions that contain a melanization stimulating factor (MSF). Isolated neural tubes of Xenopus laevis embryos exposed to conditioned media and to specific fractions exhibit greater melanization (increased numbers of melanized cells and elevated percentages of melanized cells), a greater number of dendrites per melanized cell, and a greater number of emigrated neural crest cells than control neural tubes. The presence of MSF activity in the darkly pigmented dorsal integument suggests a role for a molecule or molecules in the development and maintenance of the dorsal/ventral pigment pattern of this piscine species and possibly of other vertebrates.

Animals↗