Tumor-targeting peptide-PNA-peptide chimeras for imaging overexpressed oncogene mRNAs.

We have optimized a method involving continuous solid phase synthesis of chelator-peptide-PNA-peptide probes in order to noninvasively image oncogene mRNAs overexpressed in tumors. The PNA (peptide nucleic acid) probes carry cyclized peptide ligand analogs specific for receptors overexpressed on malignant breast or colorectal cancer cells, and chelators to bind radioactive metal ions, or a fluorophore. In vivo scintigraphic imaging of MCF7 xenografts in immunocompromised mice indicated that CCND1 and MYC [99sTc] chelator-PNA-D (CSKC) probes concentrated in MCF7 cells up to 7 times more than the corresponding mismatch controls.

Effects of peptide and nonpeptide antagonists of bradykinin B2 receptors on the venoconstrictor action of bradykinin.

The isolated rabbit jugular and human umbilical veins respond to bradykinin (BK) by contractions that are mediated by the BK B2 type receptors. In this report, the pharmacology of recently developed BK B2 receptor antagonists is assessed by using these preparations. The nonpeptide kinin antagonist WIN 64338 (phosphonium, [[4-[[2-[[bis(cyclohexylamino)methylene]amino]- 3-(2-naphthalenyl)-1-oxopropyl]amino]phenyl]methyl]tributyl chloride monohydrochloride) demonstrates competitive and surmountable antagonism of BK in both the jugular and the umbilical veins (pA2 values of 6.14 and 5.99, respectively). WIN 64338 shows selectivity in its antagonist action as it does not inhibit the effect of various other contractile agents in either of the preparations. HOE-140 (D-Arg[hydroxyproline3,beta-thienylalanine5, D-Tic7, octahydroindol-2-yl-carbonyl residue8]-BK), a "second generation" peptide antagonist of BK, behaves as an insurmountable and irreversible antagonist in the rabbit jugular vein, but appears to be competitive in the umbilical vein (pA2 = 8.2). In the jugular vein, [L-Tic7]HOE-140 is an insurmountable antagonist about 2000-fold less potent than HOE-140; the L-Tic7 isomer demonstrates no significant antagonist activity on the umbilical vein at 30 microM. This study confirms that WIN 64338 behaves as a competitive and selective kinin antagonist of the BK B2 type receptors. The pharmacological profile of the L-Tic7 analog of HOE-140 may provide useful information in discerning the molecular interaction of noncompetitive BK antagonists with their receptors.

Synthesis, characterization, and conformational analysis of the D/L-Tic7 stereoisomers of the bradykinin receptor antagonist D-Arg0[Hyp3,Thi5,D-Tic7,Oic8]bradykinin.

D-Arg0[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (HOE-140) is a potent (Ki = 0.11 nM) inhibitor of [3H]bradykinin binding to bradykinin B2 receptors found on human IMR-90 fetal lung fibroblasts. During the synthesis of this compound, we isolated and unambiguously identified the L-Tic7 stereoisomer (WIN 65365), which exhibits a 2000-fold lower binding affinity (Ki = 130 nM) than HOE-140 to the bradykinin receptor. A similar decrease in potency is observed for WIN 65365 inhibition of bradykinin-stimulated 45Ca2+ efflux from IMR-90 cells. Both HOE-140 and WIN 65365 appear to be competitive antagonists at the IMR-90 bradykinin receptor. This is the first documentation of bradykinin binding and functional antagonist activity by a bradykinin peptide analogue with an L amino acid replacing Pro7. In an attempt to rationalize the differences in binding affinities of HOE-140 and WIN 65365, a conformational analysis of the peptides was undertaken using annealed molecular dynamics (AMD). Conformational analysis of HOE-140 reveals a strong preference for the formation of a type II' beta-turn in the carboxy-terminal region. Analogous modeling of WIN 65365 reveals that its conformation is strikingly different from HOE-140 in that the four carboxy-terminal residues of WIN 65365 do not form a beta-turn. These differences in low-energy conformations between the two peptides may lead to a better understanding of the molecular interaction of antagonists with the bradykinin receptor.

Fluorescence properties of calmodulin-binding peptides reflect alpha-helical periodicity.

A basic amphiphilic alpha-helix is a structural feature common to many calmodulin-binding peptides and proteins. A set of fluorescent analogues of a very tight binding inhibitor (dissociation constant of 200 picomolar) of calmodulin has been synthesized. The fluorescent amino acid tryptophan has been systematically moved throughout the sequence of this peptide. The fluorescence properties for the peptides repeat every three to four residues and are consistent with the periodicity observed for an alpha-helix.

Predicted calmodulin-binding sequence in the gamma subunit of phosphorylase b kinase.

A basic, amphiphilic alpha helix is a structural feature common to a variety of inhibitors of calmodulin and to the calmodulin-binding domains of myosin light chain kinases. To aid in recognizing this structural feature in sequences of peptides and proteins we have developed a computer algorithm which searches for sequences of appropriate length, hydrophobicity, helical hydrophobic moment, and charge to be considered as potential calmodulin-binding sequences. Such sequences occurred infrequently in proteins of known crystal structure. This algorithm was used to find the most likely site in the catalytic (gamma) subunit of phosphorylase b kinase for interaction with calmodulin (the delta subunit). A peptide corresponding to this site (residues 341-361 of the gamma subunit) was synthesized and found to bind calmodulin with approximately an 11 nM dissociation constant. A variant of this peptide in which an aspartic acid at position 7 in its sequence (347 of the gamma subunit) was replaced with an asparagine was found to bind calmodulin with approximately a 3 nM dissociation constant.

p-Benzoyl-L-phenylalanine, a new photoreactive amino acid. Photolabeling of calmodulin with a synthetic calmodulin-binding peptide.

A new photoreactive amino acid analog, p-benzoyl-L-phenylalanine, is described. Convenient methods for the preparation of this amino acid and its subsequent incorporation into synthetic peptides by the solid-phase technique are outlined. To illustrate its utility, p-benzoyl-L-phenylalanine was substituted in place of tryptophan in a 17-residue calmodulin-binding peptide. The substitution did not measurably affect the affinity of this peptide for calmodulin. When this peptide was photolyzed at 350 nm in a 1:1 molar ratio with calmodulin in the presence of 500 microM CaCl2, 70% of the calmodulin was derivatized. The specificity of the reaction was investigated by photolysis in the absence of CaCl2 where little binding occurs; under these conditions little or no photolabeling occurred.

The design, synthesis, and characterization of tight-binding inhibitors of calmodulin.

Based on a consideration of the probable structure of calmodulin and some natural peptides known to interact with it, two calmodulin-binding peptides were designed. These peptides bind to calmodulin in helical conformations and are capable of forming electrostatic and hydrophobic interactions with calmodulin. Their dissociation constants for binding (less than or equal to 210 and 400 pM) place them as the tightest-binding inhibitors of calmodulin thus far reported. The study of the interactions of these and similar peptides with calmodulin will provide valuable insights into the mechanisms whereby calmodulin binds to target enzymes, and it also serves as an excellent model system for exploring the physical chemistry of protein-protein interaction.

Exposure to pesticides in agriculture: a survey of spraymen using dimethoate in the Sudan.

There is a need for objective data on the exposure of spraymen to pesticides in agriculture in order to assess minimum requirements for practicable protection in tropical areas. To provide data, a survey was carried out in the Sudan on spraymen using dimethoate. No cholinesterase depressions were found and the calculated dose received by each man per day was within safe limits in the circumstances of this survey. Respiratory exposure was only a minor part of total exposure. The methodology of such surveys is discussed and the need for the use of a standard protocol is emphasized.

Exposure of apple thinners to parathion residues.

In studies of potential exposure of a volunteer working under controlled conditions during apple hand-thinning operations at 1, 24, 48, 72, 96, 168, and 240 hr after application of conventional 0.03% parathion spray, both dermal and respiratory exposure values were greater where water-wettable powder formulations were used than where emulsifiables were used. Residue levels of parathion on leaves from the two types of applications were about the same. Only trace amounts of paraoxon could be detected at one and seven days after application. Highest exposure values (14.2 mg/hr dermally and 0.15 mg/hr respiratorily) were obtained within 24 hr of application. Exposure was considerably less after residues were 72 hr old. Greatest exposure was on the forearms and hands. Urinary p-nitrophenol excretion indicated slightly more absorption following exposure in water-wettable powder experimental plots. Potential exposure values indicate that absorption could reach hazardous levels after one or two hr of work, even at the 96-hr residue period, if all the pesticide were absorbed. Considering that only a small fraction of the total amount would be absorbed, it is calculated that at 75-hr residue period poisoning should not occur. There was no significant change in blood cholinesterase activity of the volunteer worker. Variation in spray deposit within an orchard due to poor tank mixing did not appear to be great enough to be considered an important factor affecting exposure.