Ted T. Sakai

NMR study of in vivo RIF-1 tumors. Analysis of perchloric acid extracts and identification of 1H, 31P and 13C resonances

Perchloric acid extracts of radiation-induced fibrosarcoma (RIF-1) tumors grown in mice have been analyzed by multinuclear NMR spectroscopy and by various chromatographic methods. This analysis has permitted the unambiguous assignment of the 31P resonances observed in vivo to specific phosphorus-containing metabolites. The region of the in vivo spectra generally assigned to sugar phosphates has been found in RIF-1 tumors to contain primarily phosphorylethanolamine and phosphorylcholine rather than glycolytic intermediates. Phosphocreatine was observed in extracts of these tumor cells grown in culture as well as in the in vivo spectra, indicating that at least some of the phosphocreatine observed in vivo arises from the tumor itself and not from normal tissues. In the 31P-NMR spectra of the perchloric acid extract, resonances originating from purine and pyrimidine nucleoside di- and triphosphate were resolved. HPLC analyses of the nucleotide pool indicate that adenine derivatives were the most abundant components, but other nucleotides were present in significant amounts. The 1H and 13C resonance assignments of the majority of metabolites present in RIF-1 extracts have also been made. Of particular importance is the ability to observe lactate, the levels of which may provide a noninvasive measure of glycolysis in these cells in both the in vitro states. In addition, the aminosulfonic acid, taurine, was found in high levels in the tumor extracts.

De novo design of peptides targeted to the EF hands of calmodulin.

This report describes the use of the concept of inversion of hydropathy patterns to the de novo design of peptides targeted to a predetermined site on a protein. Eight- and 12-residue peptides were constructed with the EF hands or Ca2+-coordinating sites of calmodulin as their anticipated points of interaction. These peptides, but not unrelated peptides nor those with the same amino acid composition but a scrambled sequence, interacted with the two carboxyl-terminal Ca2+-binding sites of calmodulin as well as the EF hands of troponin C. The interactions resulted in a conformational change whereby the 8-mer peptide-calmodulin complex could activate phosphodiesterase in the absence of Ca2+. In contrast, the 12-mer peptide-calmodulin complex did not activate phosphodiesterase but rather inhibited activation by Ca2+. This inhibition could be overcome by high levels of Ca2+. Thus, it would appear that the aforementioned concept can be used to make peptide agonists and antagonists that are targeted to predetermined sites on proteins such as calmodulin.

Monitoring the bioenergetics of cardiac allograft rejection using in vivo P-31 nuclear magnetic resonance spectroscopy

Monitoring human cardiac allograft rejection is currently accomplished by endomyocardial biopsy. Available noninvasive methods for identifying rejection have lacked the necessary sensitivity or specificity, or both, for routine clinical application. In vivo phosphorus-31 (P-31) nuclear magnetic resonance (NMR) spectroscopy has been used for monitoring phosphorus metabolism in both animal models and humans. In the present study this technique was employed as a noninvasive means to assess the bioenergetic processes that occur during cardiac allograft rejection in a rat model. Brown Norway rat hearts were transplanted subcutaneously into the anterior region of the neck of Lewis rat recipients (allografts). Control isografts employed Lewis donors and recipients. Phosphocreatine to inorganic phosphate (PCr/Pi), phosphocreatine to beta-adenosine triphosphate (PCr/ATP beta), beta-adenosine triphosphate to inorganic phosphate (ATP beta/Pi) ratios and pH of the transplanted hearts were monitored using surface coil P-31 NMR spectroscopy (at 4.7 tesla) daily for 7 days. To allow recovery from the compromise induced by the surgical procedure, the measurements obtained on day 2 were taken as a baseline. PCr/Pi was unchanged or increased in the isografts but decreased continually in allografts, with the difference becoming significant by day 4 when compared with levels in day 2 allografts (p less than 0.005) and by day 3 when compared with levels in the isograft group (p less than 0.05). PCr/ATP beta in isografts did not change throughout the study; however, allografts demonstrated a significant decrease as early as day 3 (p less than 0.01), although a significant difference between isografts and allografts did not become manifest until day 4 (p less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Bleomycin-A2 complexes with poly(dA-dT): A proton nuclear magnetic resonance study of the nonexchangeable hydrogens

Abstract Binding of bleomycin-A2 (Bleo-A2) to poly(dA-dT) in 100 mM sodium phosphate (pH 6.8) in D2O has been investigated by 1H NMR spectroscopy at 270 and 360 MHz. Significant spectral perturbations were observed only when the nucleic acid was in the duplex state. Of the Bleo-A2 resonances, the two bithiazole peaks exhibited the largest spectral shiffs and line broadening effects. The high field shift of these resonances was very small near room temperature and reached a maximum of about 0.27 ppm just below the thermal denaturation temperature of the nucleic acid (Tm = 60 ± 1°C). The temperature dependence of spectral perturbations may be accounted for by the formation of at least two types of Bleo-A2 complexes with the polynucleotide. Other perturbed resonances of bleomycin are the S-C H 3 and S-C H 2 of the terminal amine, the C H 2-N resonance of the bithiazole residue, and the C H (CH3)CO of the methylvaleric acid residue. The significantly perturbed resonances of the nucleic acid originate from the A(H-2), A(H-8), T(H-6) and one of the H-2′ hydrogens. Binding of the C-terminal tripeptide fragment of Bleo-A2 to poly(dA-dT) is accompanied by selective broadening of the bithiazole group. These experiments have identified potential loci of interaction on the Bleo-A2 and poly(dA-dT) molecules.

Proton NMR study of iron (II)-bleomycin: Assignment of resonances by saturation transfer experiments

Abstract The assignment of the paramagnetically shifted resonances of the Fe(II)-bleomycin complex in D 2 O has been accomplished using the transfer of saturation method. A number of additional resonances arising from labile N H protons which are shifted by the metal ion are observed in the 1 H spectrum of the complex in H 2 O. The temperature dependence of the chemical shifts is consistent with the formation of an isolated 1:1 complex, but does not obey either the Curie Law or the Curie-Weiss Law. The magnitude of the shifts suggests that the valeric acid hydroxyl (or carbonyl) group, the α-amino group, the imidazole N π , the carbamoyl oxygen, the pyrimidine N 1 and/or the secondary amino group may be coordinated to the iron(II).

Bleomycin interactions with DNA studies on the role of the C-terminal cationic group of bleomycin A2 in association with and degradation of DNA

The role of the cationic dimethylsulfonium group of bleomycin A2 in the binding of the drug to poly(dA-dT) has been investigated by proton NMR studies on the S-demethylated derivative. In contrast to the parent drug, the demethyl congener shows no intercalation of the aromatic bithiazole group which is adjacent to the former cationic group. However, chemical studies show that the demethyl derivative retains the capability to degrade DNA in the presence of iron(II), albeit at a reduced rate and to a lesser extent than the intact bleomycin A2. Thus, the cationic group is necessary for the intercalation of the bithiazole portion of the drug molecule; however, intercalation is not essential for the degradation of DNA.

Expression of functional recombinant scorpion β-neurotoxin Css II in E. coli

Abstract The gene for a β-neurotoxin [ Centruroides suffusus suffusus toxin II (Css II)] from the scorpion C. suffusus suffusus was synthesized by recursive PCR and cloned into the expression vector, pET15b. This recombinant vector was transformed into a thioredoxin mutant host bacterial cell, AD 494(DE3)pLysS, and expression was induced with isopropyl thiogalactoside (IPTG). Although the level of expression was low, the recombinant toxin was found only in the soluble fraction with no evidence for the formation of inclusion bodies as had been observed previously with other scorpion toxins. The recombinant Css II was purified by successive ion-exchange and hydrophobic interaction chromatography. Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectral measurements indicate that the protein has a native structure with no indication of denatured species. The recombinant neurotoxin inhibits the uptake of [ 3 H]GABA [γ-aminobutyric acid (GABA)] in neuronal cells as effectively as natural β-toxins.

Studies on Bleomycin-DNA and Bleomycin-Iron Interactions

The bleomycins, a group of antitumor antibiotics (Figure 1), cause the degradation of DNA by a process requiring iron(II) and dioxygen (1,2). DNA degradation appears to involve two steps: association of the drug with the nucleic acid and degradation of the DNA. As part of studies directed toward achieving an understanding of how the bleomycins degrade DNA, we have examined various properties of the drug using a variety of chemical and physico-chemical techniques, including NMR and Mössbauer spectroscopy. We have studied both the interaction of the antibiotic with its target (DNA) as well as its association with its metal ion cofactor. This work has been performed on the intact drug and its derivatives as well as on synthetic models of the parent drug. This paper reviews and updates the recent work from this laboratory on the bleomycins.

Bleomycin-induced life prolongation of mice infected with Trypanosoma brucei brucei EATRO 110

The antitumour antibiotic bleomycin, supplied as commercial BlenoxaneR (a mixture of bleomycinic acids), at 7 or 14 mg/kg prolonged life greater than 30 days beyond death of controls without relapse or sign of trypanosomes in the peripheral blood of mice infected with Trypanosoma b. brucei EATRO 110. Control mice died in three to four days. The purified A2 and B2 bleomycin congeners were also active at this dose. Spermidine, spermine and hirudonine (1,8-diamidinospermidine) but not putrescine, co-administered with drug, annulled the curative effects of these compounds, as signalled by appearance of trypanosomes in the blood and death of the animals.

Synthesis and properties of some novel anti-calmodulin drugs.

The preparation and properties of some novel inhibitors of calmodulin function are described. The compounds are cationic derivatives of phenyl-substituted thiazoles which inhibit the calmodulin stimulation of cyclic-AMP phosphodiesterase and are active against animal tumor cells in culture. These derivatives form the basis for the preparation of new, more potent inhibitors of calmodulin function which could take advantage of the reported elevated levels of calcium-bound calmodulin in tumor cells and show preferential anti-tumor activity.