Conformationally constrained opioid peptide analogs with novel activity profiles

Novel conformationally constrained opioid peptide analogs with antagonist, mixed agonist/ antagonist or agonist properties were developed. TIP(P)-related antagonists showed unprecedented antagonist potency and receptor selectivity, and may have potential for use in analgesia in combination with agonists. A definitive model of their receptor-bound conformation was developed. Three prototype mixed agonist/ antagonists were discovered. They represent the only known compounds with this pharmacological profile and, as expected, one of them was shown to be a potent analgesic and to produce no dependence and less tolerance than morphine. Novel dipeptide derivatives turned out to be potent and selective agonists. Because of their low molecular weight and lipophilic character, these compounds may cross the blood-brain barrier and, thus, may have potential as centrally acting analgesics.     

The Solution Structure of [d(CGC)r(amamam)d(TTTGCG)]2

The solution structure and hydration of a DNARNA hybrid chimeric duplex [d(CGC)r(a_ma_ma_m)d(TTTGCG)]₂ in which the RNA adenines were substituted by 2-O-methylated riboadenines was determined using two-dimensional NMR, simulated annealing, and restrained molecular dynamics. Only DNA residue 7T in the 2-OMe-RNA DNA junction adopted an O4-endo sugar conformation, while the other DNA residues including 3C in the DNA 2-OMe-RNA junction, adopted C1-exo or C2-endo conformations. The observed NOE intensity of 2-O-methyl group to H1 proton of 4a_m at the DNA 2-OMe-RNA junction is much weaker than those of 5a_m and 6a_m. The 2-O-methyl group of 4a_m was found to orient towards the minor groove in the trans domain while the 2-O- methyl groups of 5a_m and 6a_m were found to be in the gauche (+) domain. In contrast to the long-lived water molecules found close to the RNA adenine H2 and H1 protons and the methyl group of 7T in the RNA-DNA junction of [d(CGC)r(aaa)d(TTTGCG)]₂, there were no long-lived water molecules found in [d(CGC)r(a_ma_ma_m)d(TTTGCG)]₂. This is probably due to the hydrophobic enviroment created by the 2-O-methylated riboadenines in the minor groove or due to the wider minor groove width in the middle of the structure. In addition, the 2-O-methylation of riboadenines in pure chimeric duplex increses its melting temperature from 48.5°C to 51.9°C. The characteristic structural features and hydration patterns of this chimeric duplex provide a molecular basis for further therapeutic applications of DNARNA hybrid and chimeric duplexes with 2-modified RNA residues.     

Interactions between esterified whey proteins (alpha-lactalbumin and beta-lactoglobulin) and DNA studied by differential spectroscopy

Spectroscopic study of interactions between esterified whey proteins and nucleic acids, at neutral pH, showed positive differential spectra over a range of wavelength between 210 and 340 nm. In contrast, native forms of whey proteins added to DNA did not produce any differential spectra. The positive difference in UV absorption was observed after addition of amounts of proteins as low as 138 molar ratio (MR) of protein/DNA, indicating high sensitivity of the applied method to detect interactions between basic proteins and DNA. UV-absorption differences increased with MR of added whey protein up to saturation. The saturation points were reached at relatively lower MR in the case of methylated forms of the esterified protein as compared to its ethylated form. Saturation of nucleic acid (2996 bp long) was achieved using 850 and 1100 MR of methylated -lactoglobulin and of methylated -lactalbumin, respectively. Saturation with ethylated forms of the proteins was reached at MR of 3160 and 2750. Lysozyme, a native basic protein, showed a behavior similar to what was observed in the case of methylated forms of the dairy proteins studied. However, in the case of lysozyme, saturation was achieved at relatively lower MR (700). Methylated -casein failed to give positive spectra at pH 7 in the presence of DNA. It interacted with DNA only when the pH of the medium was lowered to 6.5, below its pI. Generally, amounts of proteins needed to saturate nucleic acid were much higher than those needed to neutralize it only electrostatically, demonstrating the presence on DNA of protein-binding sites other than the negative charges on the sugar-phosphate DNA backbones. Addition of 0.1% SDS to the medium suppressed totally all spectral differences between 210–340 nm. The presence of 5 M urea in the medium reduced only the spectral differences between 210–340 nm, pointing to the role played by hydrophobic interactions. Peptic hydrolysates of esterified and native proteins or their cationic fractions (pH > 7) produced negative differential spectra when mixed with DNA. The negative differences in UV absorption spectra were the most important in the case of peptic hydrolysates of methylated derivatives of whey proteins.     

Evidence for disaggregation of oligomeric G(o)alpha induced by guanosine-5;-3-O-(thio)triphosphate activation

Myristoylated G_o was expressed in and highly purified from Escherichia coli strain JM109 cotransformed with pQE60 (G_o) and pBB131 (N-myristoyltransferase, NMT). Non-denaturing gel electrophoresis and gel filtration analysis revealed that the G_o, in its GDP-bound form, could form oligomers involving dimer, trimer, tetramer, pentamer, or hexamer and guanosine 5"-3-O-(thio)triphosphate (GTPS) activation induced disaggregation of the G_o oligomers to monomers. The G_o was crosslinked by a cross-linker, N,N"-1,4-phenylenedimaleimide (p-PDM), yielding multiple crosslinked products. In contrast, no obvious cross-linking occurred when G_o was pretreated with GTPS. Immunoblot analysis also demonstrated oligomerization of the purified G_o proteins and its disaggregation triggered by GTPS. These results provided direct evidence for the disaggregation–coupling theory and the disaggregation action of GTPS may further elucidate the regulatory role of GDP/GTP exchange in G protein-coupled signal transduction pathways.     

Evidence for a non-antioxidant,dose-dependent role of α -lipoic acid in caspase-3 and ERK2 activation in endothelial cells

Endothelial cell apoptosis contributes to atherosclerosis and may be exacerbated by oxidative stress. Results from clinical trials using antioxidant supplementation are equivocal and could be enhanced by antioxidants with additional non-antioxidant properties such as -lipoic acid and -tocopherol. The aim of this study was to investigate the effects of these antioxidants on cytoprotective pathways and endothelial apoptosis. Endothelial cells were incubated with -lipoic acid and -tocopherol, alone or in combination, prior to incubation with H₂O₂ or staurosporine. -lipoic acid pre-treatment alone increased caspase-3 activity in a dose-dependent manner. Both H₂O₂ and staurosporine increased DNA fragmentation and caspase-3 activity and pre-treatment of cells with -lipoic acid and/or -tocopherol failed to prevent stress-induced apoptosis. Neither antioxidant treatments nor apoptotic inducers alone altered expressions of Bcl-2, Bax, HSP70 or pERK1/2 or pJNK. -lipoic decreased pERK2 in staurosporine-treated cells in a dose-dependent manner. These findings indicate that pre-incubation with -lipoic acid and -tocopherol, alone or in combination, does not protect against oxidative- or non-oxidative-induced apoptosis in endothelial cells. Moreover, we have demonstrated a non-antioxidant, dose-dependent role of -lipoic acid in caspase-3 and ERK2 activation. These data provide an insight and indicate caution in the use of high doses of -lipoic acid as an antioxidant.     

Suppression of cytotoxic T lymphocyte activity by γ/δ T cells in tumor-bearing mice

Spleen cells derived from tumor-bearing mice prove useful for the elucidation of the mechanism determining how tumor cells evade cytotoxic T lymphocytes (CTL) in tumor-bearing hosts. Our data indicate that inactive CTL or precursor CTL specific for tumor antigens are present among lymphocytes of tumor-bearing mice. However, their activity is inhibited by a soluble factor produced by other cells present in the same source. Inhibition of the cytolytic reaction was also detected in the culture supernatant of spleen cells obtained from normal mice, precultured in the presence of tumor cell culture supernatant and interleukin-2 (IL-2). Cell-depletion and cell-purification studies let us conclude that cells that produced the CTL-inhibitory factor (CTL-IF) were / T cells. The / T cells that were activated in vivo in tumor bearers were able to produce CTL-IF after isolation and in vitro culture. Maximum activation of / T cells was achieved by antigenic stimulation and by suppression of cells that interfered with the activation of / T cells. CTL-IF, which was assayed by use of CTL clones, did not show antigen specificity. Inhibition depended on a relatively heat- and acidstable, but alkali-labile molecule with a molecular mass of less than 10 kDa. The latter characteristics imply that CTL-IF does not resemble any of the known lymphokines produced by / T cells. These observations emphasize the crucial role of the / T cells in the escape of tumor cells from the attack of tumorspecific CTL.     

Evaluation of hydrophobicity versus chaperonelike activity of bovine alphaA- and alphaB-crystallin

Calf lens A-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than B-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to A-crystallin than to B-crystallin at room temperature. Bis-ANS binding to both A- and B-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of A-crystallin is lower than that of B-crystallin whereas at higher temperatures, A-crystallin shows significantly higher protection against aggregation of substrate proteins compared to B-crystallin. Therefore, calf lens A-crystallin is more hydrophobic than B-crystallin and chaperone-like activity of -crystallin subunits is not quantitatively related to their hydrophobicity.     

Properties and synthesis de novo of auxin-induced α-amylase in pea cotyledons

Analysis of starch-degrading enzymes in a crude extract of detached cotyledons of Pisum sativum L. by polyacrylamide gel electrophoresis (PAGE) demonstrated the presence of one band of -amylase (EC 3.2.1.1) activity. The activity of only this amylase was promoted in cotyledons incubated with 2,4-dichlorophenoxyacetic acid (2,4-D). The auxin-induced -amylase from pea cotyledons was purified to homogeneity, as judged by the criterion of a single band after PAGE. The relative molecular mass (M_r), estimated by gel filtration, was approx. 42 000 and the enzyme contained no carbohydrate moiety. Sodium dodecylsulfate-PAGE yielded a single band that corresponded to an M_r of 41 000. The isoelectric point was 5.85 and the aminoacid composition was similar to that of -amylase from other plants. When [³H]leucine was fed to detached dry cotyledons prior to incubation, the radioactivity in -amylase from cotyledons incubated in the presence of 2,4-D was found to be approx. 10-fold higher than that from cotyledons incubated in distilled water. When -amylase from cotyledons incubated with ²H₂O that contained 2,4-D and the tritiated amylase were centrifuged together in a CsCl density gradient, the peak of enzymatic activity of deuterated -amylase was shifted to a denser fraction than the peak of radioactivity of the tritiated enzyme. These results show that auxin-induced -amylase in pea cotyledons is synthesized de novo.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PAS periodic acid-Schiff - pI isoelectric point - SDS sodium dodecyl sulfate We are very grateful to Mr. Kazuo Itoh and Mrs. Matsumi Doe for carrying out the analysis of amino-acid composition.     

Differential maturation of μ and δ opioid receptors in the chick embryonic brain

The developmental profiles of the binding of and opiate receptors agonists was investigated using the chick embryo brain. Binding of opioids was performed at embryonic days 5, 6, 15, 18, and 20 in the developing chick embryo brain. [³H]dihyromorphine was used as a ligand and with 5×10^–7 M levorphanol for non-specific binding, and [³H](d-Ala²-d-Leu⁵)-enkephalin was used as a with 5×10^–7 M (d-Ser-Gly-Phe-Leu-Thr)-enkephalin for non-specific binding. Crude membranes were prepared from whole brain at days, 5, 6 and cerebral hemispheres at days 15, 18, and 20 of embryonic age. Both and opiate receptors were present during early embryogenesis and as early as day 5. Analysis of binding sites revealed high and low affinity sites during early embryogenesis but only one site. By 18 days of embryonic age, only one site remained. This developmental change is interpreted as a transitory state of the receptor to the adult pattern. The presence of only one site is constant throughout embryonic age; it is high during early embryogenesis reaching a lower level by 18 days. The presence of a dual binding site pattern for the receptor in early embryogenesis is implicated to have a functional significance in the pluripotential role of the endogenous opioids in early development.     

Unexpected Transformations of Natural Chlorins under Treatment with Dichlorodicyanobenzoquinone

The treatment of natural chlorins with 2,3-dichloro-5,6-dicyanobenzoquinone resulted not only in the intramolecular cyclization of the propionic acid residue in position 17 with the formation of an additional -lactone cycle at the pyrrole ring D, but also in the oxygen-assisted oxidation of 8-ethyl group in ring B to an -methoxyethyl substituent.     

δ-Toxin,unlike melittin,has only hemolytic activity and no antimicrobial activity: Rationalization of this specific biological activity

The antimicrobial activity of a synthetic peptide corresponding to -hemolysin had been examined. The peptide didnot exhibit antimicrobial activity against gram negative and gram positive micro-organisms unlike other hemolytic peptides like melittin. This lack of antibacterial activity arises due to the inability of -hemolysin to perturb the negatively charged bacterial cell surface and permeabilize the bacterial plasma membrane. However, the red blood cell surface has a structure considerably different from bacteria, and does not act as a barrier to molecules reaching the lipid membrane. Hence -toxin can lyse erythrocytes. Thus, the specificity in biological activity has been rationalized in terms of differences, in the interaction of the toxin with the bacterial and red blood cell surfaces.     

In vitro secretion of transforming growth factor alpha (TGF-α): A comparison of the A431 cell line with three human oesophageal squamous cell carcinoma lines

Transforming growth factor alpha (TGF-) is a single chain polypeptide which exists in a variety of forms differing in molecular weight. These forms are variously present in normal and neoplastic cells. Of particular interest are TGF-'s well-known mitogenic properties. The transition from a normal to a neoplastic cellular state results from signalling defects that may depend upon,iter alia, abonormal levels of expression and secretion of TGF-. It is known that the secretion of TGF- may be enhanced appreciably by agents such as phorbol 12-myristate 13-acetate (PMA), serum factors and epidermal growth factor (EGF). Here, we compare the efficacy of these three agents in the elevation of TGF- secretion in the well studied A431 cell line with their previously undocumented efficacy in certain interesting, but little known, human oesophageal squamous cell carcinoma (SCC) lines.     

Volume-sensitive taurine transport in fish erythrocytes

Summary Taurine plays an important role in cell volume regulation in both vertebrates and invertebrates. Erythrocytes from two euryhaline fish species, the eel (Anguilla japonica) and the starry flounder (Platichthys stellatus) were found to contain high intracellular concentrations of this amino acid ( 30 mmol per liter of cell water). Kinetic studies established that the cells possessed a saturable high-affinity Na⁺-dependent -amino-acid transport system which also required Cl^– for activity (apparentK _m (taurine) 75 and 80 m;V _max 0.85 and 0.29 mol/g Hb per hr for eel (20°C) and flounder cells (10°C), respectively. This -system operated with an apparent Na⁺/Cl^–/taurine coupling ratio of 211. A reduction in extracellular osmolarity, leading to an increase in cell volume, reversibly decreased the activity of the transporter. In contrast, low medium osmolarity stimulated the activity of a Na⁺-independent nonsaturable transport route selective for taurine, -amino-n-butyric acid and small neutral amino acids, producing a net efflux of taurine from the cells. Neither component of taurine transport was detected in human erythrocytes. It is suggested that these functionally distinct transport routes participate in the osmotic regulation of intracellular taurine levels and hence contribute to the homeostatic regulation of cell volume. Volume-induced increases in Na⁺-independent taurine transport activity were suppressed by noradrenaline and 8-bromoadenosine-3, 5-cyclic monophosphate, but unaffected by the anticalmodulin drug, pimozide.     

Emission of gaseous nitrogen oxides from an extensively managed grassland in NE Bavaria, Germany.

We analysed the stable isotope composition of emitted N₂O in a one-year field experiment (June 1998 to April 1999) in unfertilized controls, and after adding nitrogen by applying slurry or mineral N (calcium ammonium nitrate). Emitted N₂O was analysed every 2–4 weeks, with additional daily sampling for 10 days after each fertilizer application. In supplementary soil incubations, the isotopic composition of N₂O was measured under defined conditions, favouring either denitrification or nitrification. Soil incubated for 48 h under conditions favouring nitrification emitted very little N₂O (0.024 mol g_dw ^–1) and still produced N₂O from denitrification. Under denitrifying incubation conditions, much more N₂O was formed (0.91 mol g_dw ^–1 after 48 h). The isotope ratios of N₂O emitted from denitrification stabilized at ¹⁵N = –40.8 ± 5.7 and ¹⁸O = 2.7 ± 6.3. In the field experiment, the N₂O isotope data showed no clear seasonal trends or treatment effects. Annual means weighted by time and emission rate were ¹⁵N = –8.6 and ¹⁸O = 34.7 after slurry application, ¹⁵N = –4.6 and ¹⁸O = 24.0 after mineral fertilizer application and ¹⁵N = –6.4 and ¹⁸O = 35.6 in the control plots, respectively. So, in all treatments the emitted N₂O was ¹⁵N-depleted compared to ambient air N₂O (¹⁵N = 11.4 ± 11.6, ¹⁸O = 36.9 ± 10.7). Isotope analyses of the emitted N₂O under field conditions per se allowed no unequivocal identification of the main N₂O producing process. However, additional data on soil conditions and from laboratory experiments point to denitrification as the predominant N₂O source. We concluded (1) that the isotope ratios of N₂O emitted from the field soil were not only influenced by the source processes, but also by microbial reduction of N₂O to N₂ and (2) that N₂O emission rates had to exceed 3.4 mol N₂O m^–2 h^–1 to obtain reliable N₂O isotope data.     

Rapid and Efficient Synthesis of Peptides Containing α,α-dialkylamino acids employing KOBt

Several Fmoc-,-dialkylamino acids and their acid chlorides have been prepared, isolated and characterised. The synthesis of peptides containing sterically hindered dialkylamino acids has been accomplished using acid chloride/KOBt in dichloromethane. The yields as well as the purity of the peptides were satisfactory.     

Modulation of Potassium-Evoked [3H]Dopamine Release from Rat Striatal Slices by Voltage-Activated Calcium Channel Ligands: Effects of ω-Conotoxin-MVIIC

Neurochemical Research - We examined the involvement of voltage-activated Ca2+ channels (VACCs) on K+(50 mM)-evoked [3H]dopamine ([3H]DA) release from superfused rat striatal slices. Neither...