Improvement in the oxidative folding of endothelin-1 by a Lys-Arg extension at the amino terminus: Implication of a salt bridge between Arg-1 and Asp8

Summary An amino-terminal extension of endothelin-l by the lys-Arg dipeptide in the prosequence (KR-ET-1) greatly increased the ratio of native-type to non-native-type disulfide isomer (96/4 versus 71/29) during the oxidative folding reaction. This improvement was completely abolished by substituting Asn for Asp at position 8 (D8N-KR-ET-1), whereas most of it was maintained with similar carboxamide analogues replaced at Glu¹⁰ or Asp¹⁸. Structure analyses by circular dichroism spectroscopy revealed that (i) in the carboxylate state, the α-helical content of the native-type isomer of KR-ET-l is higher than that of the native-type isomer of ET-1, while such a variation is not observed in the corresponding non-native-type isomer of KR-ET-l; and (ii) the enhanced α-helicity resulting from the Lys-Arg extension is largely diminished in D8N-KR-ET-l. From these results and our previous findings that the helical structure in KR-ET-l is stabilized by a particular salt bridge between the extended Arg⁻¹ basic moiety and either the Asp⁸ or Glu¹⁰ acidic side chain in Et-1 [Aumelas, A. et al., Biochemistry, 34 (1995) 4546], we conclude that the formation of a specific salt bridge between the side chains of Arg⁻¹ and Asp⁸ in KR-ET-1 is critical for the predominant generation of the native-type disulfide isomer, probably because it stabilizes the helical structure of parental ET-1.     

Three-dimensional structure of α-conotoxin EI determined by 1H NMR spectroscopy H resonance assignments of α-conotoxin EI

-Conotoxin EI is an 18-residue peptide (RDOCCYHPTCNMSNPQIC; 4–10, 5–18) isolated from the venom of Conus ermineus, the only fish-hunting cone snail of the Atlantic Ocean. This peptide targets specifically the nicotinic acetylcholine receptor (nAChR) found in mammalian skeletal muscle and the electric organ Torpedo, showing a novel selectivity profile when compared to other -conotoxins. The 3D structure of EI has been determined by 2D-NMR methods in combination with dynamical simulated annealing protocols. A total of 133 NOE-derived distances were used to produce 13 structures with minimum energy that complied with the NOE restraints. The structure of EI is characterized by a helical loop between Thr⁹ and Met¹² that is stabilized by the Cys⁴-Cys¹⁰ disulfide bond and turns involving Cys⁴-Cys⁵ and Asn¹⁴-Pro¹⁵. Other regions of the peptide appear to be flexible. The overall fold of EI is similar to that of other 4/7-conotoxins (PnIA/B, MII, EpI). However, unlike these other 4/7-conotoxins, EI targets the muscular type nAChR. The differences in selectivity can be attributed to differences in the surface charge distribution among these 4/7-conotoxins. The implications for binding of EI to the muscular nAChR are discussed with respect to the current NMR structure of EI.     

Inhibition of Tryptase TL2 from Human T4+ Lymphocytes and Inhibition of HIV-1 Replication in H9 Cells by Recombinant Aprotinin and Bikunin Homologues

The serine esterase TL₂ from human T4+ lymphocytes is a binding component to HIV-1 glycoprotein gp120 and seems to play a role in the HIV-1 infection mechanism. Recombinant variants of the Kunitz-type serine proteinase inhibitor aprotinin were investigated for their ability to inhibit tryptase TL₂ and the binding of gp120 to this enzyme. Furthermore, the viral replication of HIV-1 was investigated in H9 cell cultures under the influence of recombinant aprotinin and bikunin variants. In contrast to native aprotinin, the recombinant variant [Arg¹⁵, Phe¹⁷, Glu⁵²]aprotinin with a reactive-site sequence homologous to the V3 loop of HIV-1 gp120 showed a specific inhibition of tryptase TL₂ (>80%). However, the [Leu¹⁵, Phe¹⁷, Glu⁵²]aprotinin variant with hydrophobic subsites was the most potent inhibitor of the binding of gp120 to tryptase TL₂ (68%). Our results show that the enzyme activity of purified tryptase TL₂ is inhibited not only by variants with basic amino acids, but also those with hydrophobic residues in the reactive-site region. Therefore, tryptase TL₂ is not a typical trypsin-like or chymotrypsin-like protease. Investigations on inhibition of HIV-1 replication in H9 cell cultures showed that tryptase TL₂ is involved in the mechanism of virus internalization into human lymphocytes. The [Leu¹⁵, Phe¹⁷, Glu⁵²]aprotinin showed a significant retardation of syncytium formation over a period of 5 days in a 1 M concentration. Similar investigations were performed with recombinant variants of bikunin, the light chain of human inter--trypsin inhibitor. Only the single-headed variant [Arg⁹⁴]⁸²bikunin inhibited slightly the syncytium formation over a period of 2 days in a 2.2 M concentration. Wild-type bikunin and all full-length variants showed no effect, possibly due to steric hindrance by the second domain of the double-headed inhibitor.     

Novel hemoglobin components and their amino acid sequences from the crab-eating macaque (Macaca fascicularis)

Summary We found two types of hemoglobin, T and R, from the crab-eating macaque and compared those to A and Q previously reported. The 22 animals studied showed six different phenotypes, A, R, QA, QT, QAT, and QAR. Analysis of the complete amino acid sequences for the chains of hemoglobins Q, A, T, and R revealed that amino acids at four positions, 8, 55, 71, and 78 from the N-terminal, are variable. In the ^A chain, Thr, Val, Gly, and Gln occupy these positions, and in the ^Q chain the analogous amino acids are Thr, Val, Asp, and Gln, respectively. In the newly found ^T chain they are Thr, Val, Gly, and His; and in the ^R chain, they are Ser, Ile, Gly, and His, respectively. Two amino acids (8 Thr and 79 Gln) in ^A of the crab-eating macaque were found to be different from those in the chain of the Japanese macaque.     

Induction of interleukin-2 receptor by tumor necrosis factor α on cultured ovarian tumor-associated lymphocytes

Summary We have recently reported that autologous tumor-specific cytotoxic T lymphocyte (CTL) lines and clones can be developed from lymphocytes infiltrating ovarian malignant ascites (TAL). In this study, we investigated the biological effects of tumor necrosis factor (TNF) in the induction, expansion, long-term proliferation and lytic function of CD8⁺ TAL. TNF up-regulated the IL-2 receptor (IL-2R) chain (Tac antigen) on the surface of CD3⁺ CD8⁺ CD4^– TAL, enhanced the proliferation of autologous tumor-specific CTL, and potentiated their lytic function in long-term cultures. Furthermore, in the induction and expansion phase of CD8⁺ TAL, the presence of TNF was associated with a selective increase in CD8⁺ IL-2R⁺ (Tac⁺) cells, and subsequent decrease in CD4⁺ IL-2R⁺ (Tac⁺) cells. These results suggest that the observed facilitation of the outgrowth of CD8⁺ cells in TAL cultures may be due, at least in part, to the up-regulation of IL-2R, and indicate the usefulness of TNF in the analysis of signalling in autologous tumor-reactive CTL.     

Molecular recognition between 4a<Emphasis Type="Italic">S/R</Emphasis>-galanthamine diastereoisomers and α-cyclodextrin

Molecular recognition between 4aS/R-galanthamine diastereoisomers (1: 4aS-galanthamine; 2: 4aR-galanthamine) and -cyclodextrin (-CD) were studied by use of docking and molecular dynamics (MD) simulation approaches. The binding energy of constructed 2···-CD complexes is ~17 kcal mol^–1 lower than that of 1···-CD, implying a stronger binding ability of 2 with -CD than that of 1. The theoretical modeling result is consistent with our previous CZE result, which demonstrated that -CD is an efficient chiral additive for separating 1 and 2. The modeling result also indicates that both hydrophobic interaction and H-bond force may work as major factors for molecular recognition between the galanthamine diastereoisomers and -CD. Figure Chemical structures of 4aS-galanthamine (left) and 4aR-galanthamine (right)Abbreviations Galanthamine 4aS,6R,8aS-4a,5,9,10,11,12-Hexahydroxy-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-e,f]benzazepin-6-ol     

Heteronuclear correlation experiments for the determination of one-bond coupling constants

Spin-state selective experiments, HSQC-/ and CT-HMQC-/, are proposed for the simple and rapid measurement of scalar one-bond coupling constants in two-dimensional,¹ H-detected ¹⁵N-¹H or¹³ C-¹H correlation experiments based on HSQC and HMQC schemes. Pairs of subspectra are obtained, containing either the high-field or the low-field component of the doublet representing the one-bond coupling constant. The subspectral editing procedure retains the full sensitivity of HSQC and HMQC spectra recorded without heteronuclear decoupling during data acquisition, with a spectral resolution similar to that of decoupled spectra.     

Study of amino acid formation during palmitate oxidation in rat brain mitochondria

The interrelation of palmitate oxidation with amino acid formation in rat brain mitochondria has been investigated in purified mitochondria of nonsynaptic origin by measuring the formation of aspartate, -ketoglutarate, and glutamate during palmitate oxidation, and also by assaying¹⁴C-products of [1-¹⁴C]palmitate oxidation. Oxidation of palmitate (or [1-¹⁴C]palmitate) resulted in the formation of aspartate (or¹⁴C-aspartate), and the oxidation was inhibited by aminooxyacetate (an inhibitor of transaminase), Palmitate oxidation also resulted in -ketoglutarate formation, which was sensitive to the effect of aminooxyacetate. Addition of NH₄Cl was found to increase¹⁴C-products and formation of -ketoglutarate, whereas glutamate formation was not increased unless the rate of palmitate oxidation was reduced by 50% by aminooxyacetate or -ketoglutarate was added exogenously. Exogenous -ketoglutarate was found to decrease¹⁴C-products, but not aspartate formation. These results indicated that palmitate oxidation was closely related to aspartate formation via aspartate aminotransferase. During palmitate oxidation without aminooxyacetate or added -ketoglutarate, however, -ketoglutarate was not available for glutamate formation via glutamate dehydrogenase. We discuss the possibility that this was because (a) oxidative decarboxylation of -ketoglutarate to form succinyl-CoA was favored over glutamate formation for the competition for -ketoglutarate in the same pool, and (b) the pool of -ketoglutarate produced in the aspartate aminotransferase reaction did not serve as substrate for glutamate formation.     

Interaction of Duodenase with Serpins from Human Blood Serum

The interaction of duodenase, a new serine protease from a small group of Janus-faced proteases, with serpins, ₁-protease inhibitor (₁-PI) and antichymotrypsin (ACT) from human blood serum, was studied. The stoichiometry of the inhibition process was found to be 1.2 and 1.3 mol/mol for ₁-PI and ACT, respectively. The presence of a stable enzyme–inhibitory complex duodenase–₁-PI was confirmed by SDS-PAGE. The formation of the duodenase–ACT complex was not demonstrated; instead, the band of the cleaved inhibitor indicated the ACT hydrolysis. The suicide mechanism of the duodenase interaction with the human blood serpins was proved. The association rate constants (k × 10⁵, ^–1 s^–1) were 2.4 ± 0.3 × 10⁵ for ₁-PI and 3.0 ± 0.4 × 10⁵ for ACT. These results indicate the possibility of the regulation of duodenase activity by endogenous serpins.     

The regions of alpha-neurotoxin binding on the extracellular part of the alpha-subunit of human acetylcholine receptor

A set of 18 synthetic uniform overlapping peptides spanning the entire extracellular part (residues 1–210) of the -subunit of human acetylcholine receptor were studied for their binding activity of¹²⁵I-labeled -bungarotoxin and cobratoxin. A major toxin-binding region was found to reside within peptide 122–138. In addition, low-binding activities were obtained with peptides 34–49 and 194–210. It is concluded that the region within residues 122–138 constitutes a universal major toxin-binding region for acetylcholine receptor of various species.     

Role of the N-terminal region of the A chain in beta 1-bungarotoxin from the venom of Bungarus multicinctus (Taiwan-banded krait)

The N-terminal -amino groups of ₁-bungarotoxin (₁-Bgt) fromBungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the -amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between ₁-Bgt and 8-antilinonaphthalene sulfonate (ANS) and revealed that ₁-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca²⁺ and ANS, indicating that the N-terminal region is not involved in Ca²⁺ and substrate binding. A fluorescence study revealed that the -amino group of the A chain was in the vicinity of substrate binding site and that the TNP -amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for ₁-Bgt.     

The effects of interleukin-1 therapy on peripheral blood granulocyte function in humans

During a phase I trial of interleukin-1 (IL-1) in patients with ovarian carcinomas, the effects of this treatment on blood granulocyte respiratory burst and locomotive responses were examined. Differences in baseline granulocyte function in patients as well as dose-related effects of IL-1 treatment were observed. Patients enrolled early in the trial (low-dose patients) had significantly lower locomotive responses before treatment than their paired controls; these low responses normalized after 5 days of continuous-infusion IL-1 treatment. Patients enrolled later (high-dose patients) had normal locomotive responses before treatment and IL-1 treatment was associated with suppression of responses to selected stimuli at the end of treatment. Pretreatment respiratory burst responses in both low-and high-dose patient groups were essentially normal, but the rates of granulocyte H₂O₂ production following phorbol myristate acetate stimulation became significantly less than control values at the end of treatment. In vitro exposure of either patient or control cells to 150 U/ml IL-1 did not alter their locomotive or respiratory burst responses, suggesting the observed in vivo effects were not mediated directly by IL-1. Treatment with IL-1 is associated with changes in ex vivo granulocyte function that are related to the IL-1 dose. Treatment with low doses of IL-1 may provide a means of normalizing abnormal polymorphonuclear leukocyte function in some patients with ovarian malignancies.     

Chemoattractant activity of Staphylococcus aureus serine proteinase modified human plasma α-1-proteinase inhibitor

S. aureus serine proteinase inactivates human -1-proteinase inhibitor (-1-PI) by attacking a single peptide bond between Glu³⁵⁴ and Ala³⁵⁵ giving a modified inhibitor which is a tight complex of Mr=4,000 and 48,000 fragments. In the present paper we show that this proteolytically inactivated -1-PI is a potent chemotactic factor for human neutrophiles at a nanomolar concentration, and we discuss its potential involvement in the inflammatory reaction due to S. aureus infections.     

Measurement of 15N-1H coupling constants in uniformly 15N-labeled proteins: Application to the photoactive yellow protein

A modified HNHB experiment is presented that allows thedetermination of J(NH) coupling constants directly from the ratio ofcross-peak to diagonal-peak intensities. The experiment was applied to thephotoactive yellow protein (PYP) and yielded the magnitude of 117³J(NH) coupling constants. In addition, 29³J(NH^(i–1)) coupling constantscould be measured, providing information about the backbone angle .These data, in conjunction with the magnitudes of the³J(H^NH) coupling constantsobtained from the HNHA spectrum, effectively discriminate the twopossibilities for the stereospecific assignment of theH resonances in glycine residues. For all eight glycineresidues in PYP that were not subject to conformational averaging and hadnon-degenerate H resonance frequencies, the J-couplingdata, together with limited NOE data, yielded the stereospecific assignmentof the H resonances for these residues. In addition,reliable and precise , dihedral constraints were also derived forthese residues from the J-coupling data.     

Conformational stability of α-lactalbumin missing a peptide bond between Asp66 and Pro67

The peptide bond between Asp⁶⁶-Pro67 of -lactalbumin was cleaved with formic acid (cleaved-lactalbumin). Secondary structural changes of the cleaved-lactalbumin, in which the two separated polypeptides were joined by disulfide bridges, were examined in solutions of sodium dodecyl sulfate (SDS), urea, and guanidine hydrochloride. The structural changes of the cleaved-lactalbumin were compared with those of the intact protein. The relative proportions of secondary structures were determined by curve fitting of the circular dichroism spectrum. The cleaved-lactalbumin contained 29%-helical structure as against 34% for the intact protein. Some helices of the cleaved-lactalbumin which had been disrupted by the cleavage appeared to be reformed upon the addition of SDS of very low concentration (0.5mM). In the SDS solution, the helicities of both the intact and cleaved proteins increased, attaining 44% at 4mM SDS. On the other hand, the helical structures of the cleaved-lactalbumin began to be disrupted at low concentrations of guanidine hydrochloride and urea compared with that of the intact protein. However, no diffrence was observed in the thermal denaturations of the intact and cleaved proteins, except for the difference in the original helicities. The helicities of both proteins decreased with an increase of temperature up to 65°C and recovered upon cooling.     

Synergistic effects of endothelin-1 (ET-1) and transforming growth factor alpha (TGF-α) or epidermal growth factor (EGF) on DNA replication and G1 to S phase transition

The cooperative cell kinetic actions of ET-1 with TGF- or EGF in normal rat kidney fibroblasts (NRK-49F) and KNRK cells (Kirsten MSV transformed) were analyzed by [³H]-thymidine incorporation assay and flow cytometry. A marked synergistic effect of TGF- and ET-1 (or EGF and ET-1) on DNA synthesis and G1 to S transition was observed in NRK cells; 15–20% S for TGF- and 12% S for ET-1 alone but 45–50% S in combination. There was no detectable effect on cell cycle kinetics by TGF- (1 ng/ml) or EGF (1 ng/ml) plus ET-1 (1 ng/ml) in KNRK cells treated for 22 hours. Insulin, insulin-like growth factor I (IGF-I), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), and transforming growth factor (TGF-) were also tested and found to have no significant synergistic effects on ET-1 actions. Our findings suggest that the combination of TGF- (EGF) and ET-1 is an important part of an intricate network which coordinates progression of G1 to S phase in normal cells.     

Induction of lymphokine-activated killer activity in mice by prothymosin α

We have recently demonstrated that prothymosin (ProT) when administered intraperitoneally (i.p.) protects DBA/2 mice against the growth of syngeneic leukemic L1210 cells through the induction of tumoricidal peritoneal cells producing high levels of tumor necrosis factor (TNF) [Papanastasiou et al. (1992) Cancer Immunol Immunother 35: 145]. In this report we tested further immunological alterations that may be caused by the administration of ProT in vivo. We demonstrate that i.p. injections of ProT enhance natural killer (NK) cell activity and induce lymphokine-activated (LAK) activity in vivo. Thus, splenocytes from ProT-treated DBA/2 animals exhibited significantly higher cytotoxic activity (up to threefold) against the NK-sensitive YAC cell line and the NK-resistant P815 and L1210 syngeneic tumor cells, as compared to splenocytes from syngeneic control mice. The enhancement of the cytotoxic profile of DBA/2 splenocytes was associated with increased percentages of CD8⁺ cells, NK cells and activated CD3⁺ cells. The ProT-induced effect persisted for 30 days after the end of the ProT treatment period and returned to normal levels 20 days later. SPlenocytes from non-treated DBA/2 animals generated high NK and LAK activities in response to ProT in vitro. The ProT-induced NK an LAK activities reached 84% and 75% respectively of what was obtained with interleukin-2 (IL-2). High concentrations of TNF and IL-2 were generated in response to ProT in LAK cultures. These findings suggest that ProT may provide an overall protective effect against tumor growth in vivo through induction of NK and LAK activities possibly indirectly via the production of IL-2 and TNF in the spleen, peritoneal cavity and probably other lymphoid organs.This work was supported by a CEC grant to M. Papamichail     

Circular Dichroism Studies of the Interaction of Tat Analogues Substituted in the Arg52 Position with TAR RNA HIV-1

The Tat wild-type fragment of sequence Arg⁴⁹-Lys-Lys-Arg⁵²-Arg-Gln-Arg-Arg-Arg⁵⁷-NH₂ (labelled as Tat1) and three analogues of this fragment with the substitution Arg⁵² D-Arg⁵² (labelled as Tat2) or L-citrulline (Cit) (labelled as Tat3) or L-ornithine (Orn) (labelled as Tat4) were synthesized to study Tat-TAR RNA HIV-1 (27-nucleotide fragment of sequence 5-AGAUCUGAGCCUGGAGCUCUCU-3) interactions by circular dichroism. -helical structure was the most readily adopted by the Tat3 analogue with Arg⁵² Cit substitution. All the peptides investigated caused conformational changes in the TAR structure. The most dramatic changes were observed for the Tat2-TAR complex.