[Immune anticancer response: recent advances in the treatment of renal cell carcinoma]. / Réponse immunitaire antitumorale: nouvelles perspectives dans le traitement du carcinome à cellules rénales.

Recent advances in the field of immunobiology have provided many opportunities for anticancer-immunotherapy. Because they express tu-mor antigen, tumor cells can be kill by T cells. Renal Cell Carcinoma (RCC) is an immunogenic tumor and metastatic RCC is presently treated by cytokines. Anticancer immunity may be achieved by different strategies: allogeneic hematopoietic cell transplantation, vaccination with peptides, vaccination with loaded dendritic cells or adoptive cellular therapy in which specific T cells are isolated and expanded in vitro and then infused to patients. In our group, we have chosen the adoptive transfer of in vitro activated T cells with autologous tumor antigen loaded dendritic cells. To determine the best strategy of anticancer-immunotherapy, we need rigorous control of the specificity and the phenotype of the cell therapy product linked with the immunological status of the patient (before and after infusion) and with the clinical response.

Biochemical characterization and nuclear magnetic resonance structure of novel alpha-conotoxins isolated from the venom of Conus consors.

Two novel alpha-conotoxins were purified and characterized from the venom of the fish-hunting cone snail Conus consors. These peptides were identified by screening HPLC fractions of the crude venom and by binding experiments with Torpedo nicotinic acetylcholine receptor. The toxins named alpha-CnIA and alpha-CnIB exhibited sequences of 14 and 12 amino acids, respectively. The alpha-CnIA represents the main alpha-conotoxin contained in the venom, whereas alpha-CnIB is present in a relatively small amount. Chemical synthesis of alpha-CnIA was carried out using the Fmoc methodology by selective disulfide bond formation. The biological activity of the toxin was assessed in fish and mice. The alpha-CnIA inhibited the fixation of iodinated alpha-bungarotoxin to Torpedo nicotinic acetylcholine receptors with an IC50 of 0.19 microM which can be compared to the IC50 of 0.31 microM found for the previously characterized alpha-MI isolated from the piscivorous Conus magus. The synthetic alpha-CnIA blocked spontaneous and evoked synaptic potentials in frog and mouse isolated neuromuscular preparations at sub-micromolar concentrations. Solution NMR of this toxin indicated a conformational heterogeneity with the existence of different conformers in solution, at slow and intermediate exchange rates relative to the NMR chemical shift time scale, similar to that reported for alpha-GI and alpha-MI. NMR structures were calculated for the major NMR signals representing more than 80% of the population at 5 degrees C.

Consequence of the removal of evolutionary conserved disulfide bridges on the structure and function of charybdotoxin and evidence that particular cysteine spacings govern specific disulfide bond formation.

Scorpion toxins are miniglobular proteins containing a common structural motif formed by an alpha-helix on one face, an antiparallel beta-sheet on the opposite face, and three disulfide bonds making up most of its internal volume. We have investigated the role of these evolutionary conserved bonds by replacing each couple of bridged cysteine residues of the scorpion charybdotoxin by a pair of nonbridging L-alpha-aminobutyric acid (Aba) residues. Three analogues were obtained by solid-phase synthesis, Chab I, Chab II, and Chab III, containing the Aba residues in positions 7 and 28, 13 and 33, 17 and 35, respectively. Circular dichroism analysis showed that the purified Chab II acquired a conformation similar to that of charybdotoxin, while the Chab I and Chab III possess decreased nativelike characteristics. All analogues block single high-conductance Ca(2+)-activated K+ channels from rat skeletal muscle inserted into planar lipid bilayers, but with different potencies. Chab II is the most active analogue (KD = 8.0 x 10(-8) M), with a 9-fold lower affinity as compared to native charybdotoxin. Chab I and Chab III have, respectively, 180- and 580-fold lower affinity. Therefore, the removal of evolutionary conserved disulfide bridges does not prevent the toxin to adopt a functional and presumably nativelike structure. However, removal of one disulfide bond affects the yields of formation of correct pairing between the remaining cysteine residues, and only Chab I preserves the ability to form the native disulfide pairings with high efficiency. This is the only analogue to preserve particular spacings of three and one residue between the cysteines, which have been described to thermodynamically disfavor disulfide bond formation between the cysteines [Zhang R., and Snyder, G. H. (1989) J. Biol. Chem. 264, 18472-18479]. Therefore, we conclude that the position of the cysteine residues in the sequence of charybdotoxin, by disfavoring specific pairings and favoring others, may govern selective formation of specific disulfide bonds, thus, explaining the efficient folding properties of Chab I and of native charybdotoxin. The structural properties of the Chab analogues and the discovered role of the cysteine spacings have interesting implications in protein design and engineering.

Cytotoxicity of copper complexes of 2-furaldehyde oxime derivatives in murine and human tissue cultured cell lines.

The copper complexes of furan oxime derivatives were found to be potent cytotoxic agents in both murine and human tissue cultured cell lines which were either suspended or solid tumors. The ED50 values were frequently improved over the clinically useful antineoplastic agents. These copper complexes of 2-furaldehyde oximes were effective inhibitors of L1210 lymphoid leukemia DNA synthesis followed by RNA synthesis. Purine synthesis regulatory enzyme activities were markedly reduced by the compounds with marginal inhibition of t-RNA polymerase, and nucleoside kinases activities. L1210 DNA topoisomerase II activity was markedly reduced with IC50 values better than the standard VP-16, etoposide. Yet, the copper complexes caused no further protein linked breaks than VP-16 did, but did block phosphorylation activation of the topoisomerase II enzyme.

A potassium-channel toxin from the sea anemone Bunodosoma granulifera, an inhibitor for Kv1 channels. Revision of the amino acid sequence, disulfide-bridge assignment, chemical synthesis, and biological activity.

The potassium channel toxin secreted by the sea anemone Bunodosoma granulifera (BgK) is a 37-amino-acid peptide containing three disulfide bridges. Because a synthetic peptide corresponding to the reported sequence of BgK was found not to fold properly, the sequence was determined again. The new sequence differed from the previous one in the C-terminal tetrapeptide, which contains two cysteines involved in disulfide bridging. The revised sequence is: V C R D W F K E T A C R H A K S L G N C R T S Q K Y R A N C A K T C E L C. The toxin BgK was synthesized according to the new sequence and folded successfully. Disulfide bridges were assigned by peptide mapping on both natural and synthetic forms to be between Cys2-Cys37, Cys11-Cys30 and Cys20-Cys34. The toxin contains a C-terminal free carboxylate as shown by comparing the native toxin with two synthetic peptides containing the C-terminus in either the carboxylate or carboxamido form. Synthetic BgK inhibits binding of 125I-alpha-dendrotoxin to rat brain synaptosomal membranes, similarly to natural BgK (nanomolar range). No activity was observed on maxi-K+ channels incorporated into planar lipid bilayers. The ability of BgK to block voltage-dependent K+ channels was determined from recordings of whole cell currents in Xenopus oocytes injected with cRNA encoding three cloned Kv1 channels (Kv1.1, Kv1.2, Kv1.3) and one Kv3 (Kv3.1) channel. The Shaker-related Kv1 channels are equally affected by BgK, while the Shaw-related channel Kv3.1 is insensitive up to 0.125 microM toxin. Indeed, half blockage of the current through the three Kv1 channels tested occurred in the same concentration range (Kd = 6 nM for Kv1.1, 15 nM for Kv1.2, 10 nM for Kv1.3). The specificity of BgK for the Shaker-related K+ channels indicates that BgK is able to discriminate a large group of neuronal Kv1 channels in situ. The sequence, the disulfide bridge pattern, the secondary structure and the biological activity of BgK demonstrated that the sea anemone toxins, i.e. BgK, ShK and Kaliseptine, constitute novel molecular probes useful for investigating K+ channel properties.

Erythrocyte spermine levels: a prognostic parameter in childhood common acute lymphoblastic leukemia.

Polyamines have been implicated to play a role in cell proliferation and in cancer development. Ninety percent of the circulating spermidine (Spd) and spermine (Spm) are transported by red blood cells (RBC). RBC Spd and Spm levels were prospectively determined in 63 unselected children with common acute lymphoblastic leukemia. The Spm and Spd levels were not correlated with white blood cell (WBC) count. On the basis of the polyamine levels it was possible to discriminate four groups with P< 10(-3). In C1, C2, C3 and C4 group the Spm level was respectively 90 (39-597), 3.75 (1-7.45), 9.95 (2.9-12.6) and 17(6.3-33.8). The probability of relapse-free survival (RFS) of the 58 children who entered complete remission was 55% +/- 9. For the groups C1 (n = 6), C2 (n = 16), C3 (n = 21) and C4 (n= 15) groups, the RFS was 25% +/- 20, 73% +/- 12, 73% +/- 13 and 32% +/- 13 respectively. For children with Spm levels <13/> or = 13nmol/8 x 10(9) RBC, event-free survival (EFS) was 54% +/- 11/33% +/- 10 and RFS was 64% +/- 12/38% +/- 11 respectively (P < 0.03, P < 0.005). Our clinical study shows clearly that an RBC spermine level could be used as parameter of prognosis at the time of diagnosis, particularly for patients with intermediary WBC count.

Determination of erythrocyte polyamines as a predictive method in tumour diagnosis. An animal study with chemically induced tumours.

There have been numerous attempts in the past to use polyamine determinations in body fluids for tumour diagnosis. Since spermidine (Spd) and spermine (Spm) are mainly transported in blood by erythrocytes, this study was concerned with the diagnostic possibilities of red blood cell (RBC) polyamine determinations. In tumour-grafted animals we observed that RBC polyamine levels correlated with the tumour mass progression and increased before the tumour was palpable. Discrepancies between the evolution of RBC polyamine levels in tumour-grafted animals and in cancer patients were probably due to the non-continuous growth of the tumours in patients. Therefore, an animal model was sought which mimicked the clinical situation. In the present experiments, ethylnitrosourea induced tumours were used which, in analogy to the clinical situation, had an undetermined time of the appearance in a non-predetermined proportion of the animals. RBC polyamines were determined over a period of 7 months in 154 rats. A total of 2,290 RBC polyamine determinations were performed during this study. The data clearly demonstrate the appearance of elevated Spd concentrations in advance of tumour diagnosis by conventional clinical methods. In 71% of the rats which later developed a tumour, abnormal Spd levels (> 40 nmol/8.109 RBC) preceded, by 35 +/- 31 days, the first clinical symptoms for the presence of a tumour. In 29% of the animals, abnormal RBC Spd concentrations were observed at the time of tumour diagnosis. Elevation of Spm concentrations (> 6 nmol/8.10(9) RBC) was less frequent. RBC polyamine levels did not allow discrimination between malignant and non malignant tumours. This confirms earlier findings that RBC polyamines are markers of the cell proliferation rate, but not for the presence of a malignant tumour. Elevated RBC polyamine concentrations are an index of the intensity of hyperplastic processes, which can be clinically used for the early detection of proliferative phases of tumours, thus allowing timely therapeutic measures.

The association of three subunits with yeast RNA polymerase is stabilized by A14.

RNA polymerase I of Saccharomyces cerevisiae is composed of 14 subunits. All of the corresponding genes have been cloned with the exception of the RPA14 gene encoding A14, a specific polypeptide of this enzyme. We report the cloning and the characterization of RPA14. The A14 polypeptide was separated from the other RNA polymerase I subunits by reverse-phase high pressure liquid chromatography and digested with proteinase K. Based on the amino acid sequence of one of the resulting peptides, a degenerate oligonucleotide was synthesized and used to isolate the RPA14 gene from a yeast subgenomic DNA library. RPA14 is a single copy gene that maps to chromosome IV and is flanked by CYP1 and HOM2. Disruption of RPA14 is not lethal, but growth of the rpa14::URA3 mutant strain is impaired at 37 and 38 degrees C. RNA polymerase I was purified from the rpa14::URA3 strain. After two purification steps, the enzyme did not contain the subunits A14, ABC23, and A43. This form of the enzyme was not active in a nonspecific in vitro transcription assay. These results demonstrate that A14 is a genuine subunit of RNA polymerase I and suggest that A14 plays a role in the stability of a subgroup of subunits.

The cloning of a cDNA encoding a protein (latrodectin) which co-purifies with the alpha-latrotoxin from the black widow spider Latrodectus tredecimguttatus (Theridiidae).

A cDNA encoding a polypeptide of 88 amino acids was cloned following the rapid amplification of cDNA ends (RACE) procedure using mRNA isolated from the venom glands of the Mediterranean black widow spider (Latrodectus tredecimguttatus) and oligonucleotides based on the sequence of a tryptic fragment putatively from alpha-latrotoxin. Apart from a potential signal peptide, the rest of this small protein, named latrodectin, was highly hydrophilic, having a calculated molecular mass of 7945 Da and a pI of 4.3. Northern-blot analysis showed that the mRNA was specifically expressed in the venom gland of L. tredecimguttatus and that it was well conserved between two geographically remote species (L. geometricus and L. indistinctus). A polyclonal serum raised in rabbits against the C-terminal sequence of latrodectin detected cross-reactive proteins in the venom fluid, venom gland extracts, and in purified alpha-latrotoxin, suggesting that latrodectin is intimately associated with alpha-latrotoxin. Finally, we produced a recombinant protein in a cell system infected with baculovirus and developed an immunoaffinity purification procedure for latrodectin to facilitate further structural and functional analyses of the molecule.

A diversified family of 12-kDa proteins with a high amino acid sequence similarity to macrophage migration-inhibitory factor (MIF).

Two isoforms of a bovine-brain-derived 12-kDa protein (designated p12a and p12b) whose N-termini have a high amino acid sequence similarity with the glycosylation-inhibiting factor (GIF) and macrophage migration-inhibitory factor (MIF) were purified to homogeneity. The complete amino acid sequence of bovine p12a (pI 9.5) was determined by Edman degradation of the intact molecule and overlapping fragments generated by proteolytic cleavage. The p12a isoform has nine and ten conservative substitutions versus human GIF (hGIF) and human MIF (hMIF), respectively. Molecular filtration revealed that both isoforms of p12 exist as monomers in aqueous solution. Circular dichroism spectra indicate that both isoforms of p12 consist of 39 +/- 3% alpha helix, 23 +/- 3% beta structure and 15 +/- 3% beta turns. Although the N-terminal parts of p12a and p12b have weak amino acid sequence similarity with that of glutathione S-transferase (GST) neither isoform of p12 was bound to a GST-affinity gel nor had GST activity. Despite a high amino acid sequence similarity with human MIF neither of the p12 isoforms inhibited migration of the mouse monocyte-macrophage cells P338D1.

Synthesis of charybdotoxin and of two N-terminal truncated analogues. Structural and functional characterisation.

Charybdotoxin and two N-terminal truncated peptides, corresponding to the 2-37 and 7-37 sequences, were obtained by stepwise solid-phase synthesis using N alpha-t-butyloxycarbonyl and benzyltype side-chain protection. While this strategy was generally useful, the S-acetamidomethyl protecting group used for the six cysteines was not completely stable under HF treatment and its subsequent removal by mercury(II) treatment was neither complete nor devoid of side reactions. The completely deprotected native and truncated sequences were folded efficiently in the presence of glutathione and were finally purified by high-pressure liquid chromatography with overall yields of 4.0-5.0%. Each protein was characterised chemically, structurally and functionally. 1H-NMR spectroscopy was used and a complete assignment of all the protons of the three synthetic proteins was achieved. NMR data show that synthetic charybdotoxin is indistinguishable from the natural protein. The two truncated proteins contain the same elements of secondary structure and a similar overall three-dimensional structure, in agreement with circular dichroic measurements. The shortest analogue, however, may have local structural perturbations and/or higher flexibility. Biological activity on dog epithelial Ca(2+)-activated K+ channels and on rat brain synaptosomal voltage-dependent K+ channels show that synthetic charybdotoxin was as potent as the natural toxin on both channels. For both channels, deletion of the first amino acid, 5-oxoproline (pyroglutamic acid) decreased only slightly the potency of the inhibitor, while deletion of the entire 1-6 segment reduced potency much more. We conclude that the N-terminal region of charybdotoxin plays a functional role in tuning the toxin's biological activity but is not essential for the folding and stability of the structure. The structure of the shortest analogue represents an interesting example of how a well organised and stable alpha/beta fold can be engineered with only 31 amino acid residues.

Two additional common subunits, ABC10 alpha and ABC10 beta, are shared by yeast RNA polymerases.

Yeast nuclear RNA polymerases are multisubunit enzymes that contain in common some small subunits. We show that the smallest, a 10-kDa component of three enzymes (A10, B10, and C10), is heterogeneous. In each case, it can be resolved into two distinct polypeptides (alpha and beta) by reverse-phase chromatography. A10 alpha, B10 alpha, and C10 alpha were indistinguishable on the basis of their electrophoretic and chromatographic behaviors, characteristic silver staining, and tryptic peptide analysis. All three polypeptides are blocked at their amino termini. By the same criteria, A10 beta, B10 beta, and C10 beta were also indistinguishable. The amino-terminal sequence of A10 beta and C10 beta corresponded to that of subunit B10 recently cloned by Woychik and Young (Woychik, N. A., and Young, R. A. (1990) J. Biol. Chem. 265, 17816-17819). Thus, the three forms of RNA polymerase share two additional and distinct polypeptides, ABC10 alpha and ABC10 beta, that therefore can be considered bona fide subunits of these enzymes. Interestingly, these two subunits bind zinc.

Interaction of modified neurotoxins from Naja nigricollis with the nicotinic acetylcholine receptor from Torpedo marmorata. A Raman spectroscopy study.

Two derivatives of alpha-toxin from Naja nigricollis venom were used in order to study, by resonance Raman spectroscopy, its interaction with the nicotinic acetylcholine (AcCho) receptor from membranes of Torpedo marmorata electrocytes. The two modified toxins carry either an NO2 group bound to Tyr25 or a nitrophenylthioether (NPS) bound to Trp29. The comparison of the spectra of the free and bound derivatized toxins indicates that the environment of Tyr25 is not perturbed upon binding to the AcCho receptor; but the surroundings of NPS bound to Trp29 are changed. This result indicates that Tyr25 is not involved in binding, while Trp29 of the alpha-toxin may be in contact with the AcCho receptor. Examination of the spectrum of the AcCho receptor membrane after binding of the NPS-Trp toxin discloses some modifications of the vibrations of the tryptophan and cysteine disulfide bridge of the receptor. These residues are possibly involved in toxin binding.

Structure and chemical modifications of neurotoxin from Naja nigricollis studied by Raman spectroscopy.

Raman spectroscopy was used to determine structural features of the native toxin alpha from Naja nigricollis, which contains only one Trp and one Tyr, and of chemically modified toxins having chromophores added to these two conserved aromatic amino acids. The percentages of secondary structure were determined by using amide I polypeptidic vibration analysis and are in agreement with X-ray structure [Low et al. (1976) Proc. Natl. Acad Sci. U.S.A. 73, 2991-2994] as well as with the geometry of the disulfide bridges estimated by using the v(S-S) vibrations. In the native toxin alpha, the single invariant tyrosine 25 appears to be buried in the structure and involved in a strong hydrogen bond. We have chemically modified these two invariant aromatic side chains by addition of chromophores. The presence of a (nitrophenyl)sulfenyl (NPS) chromophore bound to the Trp does not perturb the secondary structure of the toxin as shown by the analysis of the polypeptidic amide I vibrations; however, the environment of this Trp and the geometry of a disulfide bridge seem to be modified. The secondary structure is not affected by the presence of the NPS chromophore; therefore, the decrease in binding affinity observed after modification of Trp-29 by the reagent NPS-Cl [Faure et al. (1983) Biochemistry 22, 2068-2076] is due to an alteration of the environment of this aromatic amino acid and/or a steric hindrance and not to an overall modification of the toxin structure. The binding assays of [nitrotyrosyl]toxin show that after nitration the affinity toward the monoclonal antibody M alpha 1 is unchanged and that the affinity toward the cholinergic receptor (AcChR) from Torpedo marmorata remains high. We concluded that the structure of toxin alpha after adding the NO2 chromophore to Tyr-25 is the same as it is in native toxin.

Delineation of the functional site of a snake venom cardiotoxin: preparation, structure, and function of monoacetylated derivatives.

Toxin gamma, a cardiotoxin from the venom of the cobra Naja nigricollis, was modified with acetic anhydride, and the derivatives were separated by cation-exchange and reverse-phase chromatography. Nine monoacetylated derivatives were obtained, and those modified at positions 1, 2, 12, 23, and 35 were readily identified by automated sequencing. The overall structure of toxin gamma, composed of three adjacent loops (I, II, and III) rich in beta-sheet, was not affected by monoacetylation as revealed by circular dichroic analysis. Trp-11, Tyr-22, and Tyr-51 fluorescence intensities were not affected by modifications at Lys-12 and Lys-35, whereas Trp-11 fluorescence intensity slightly increased when Lys-1 and Lys-23 were modified. The cytotoxic activity of toxin gamma to FL cells in culture was unchanged after modification at positions 1 and 2, whereas it was 3-fold lower after modification at Lys-23 and Lys-35. The derivative modified at Lys-12 was 10-fold less active than native toxin. Using two isotoxins, we found that substitutions at positions 28, 30, 31, and 57 did not change the cytotoxic potency of toxin gamma. A good correlation between cytotoxicity, lethality, and, to some extent, depolarizing activity on cultured skeletal muscle cells was found. In particular, the derivative modified at Lys-12 always had the lowest potency. Our data show that the site responsible for cytotoxicity, lethality, and depolarizing activity is not diffuse but is well localized on loop I and perhaps at the base of loop II. This site is topographically different from the AcChoR binding site of the structurally similar snake neurotoxins.

Clinical importance of erythrocyte polyamine level determination during bone marrow transplantation in children.

Previous studies have shown that red blood cell (RBC) spermidine (Spd) and spermine (Spm) concentrations appear to be a reliable index of cell proliferation. Our aim was to study the RBC polyamine level evolution (Spd and Spm) in bone marrow (BM) transplanted children. Because of our interest in the finding of an early blood criteria of BM regeneration, our study was based upon the chemotherapy - induced post-transplant aplasia period. After BM transplantation, two main periods were observed: the first (A-period) corresponded to abnormally low Spd levels. This period ended with an increasing amount of Spd reaching normal values and with an inversion in the Spd/Spm ratio which became greater than 1. The second (B) period was usually linked to abnormally high RBC Spd concentrations and a Spd/Spm ratio greater than 1. The end of the B-period was characterized by an increase in the granulocyte count (reaching 0.5 X 10(9) cells/l). Since the A- and B-periods are considered as a post-transplant aplasia period (only according to leukocyte count) and since normal RBC Spd levels occurred 14 days (SD = 4) after BM transplantation and 16 days (SD = 12) before granulocyte rise, these data led us to consider erythrocyte polyamine levels to be an earlier biological criteria of bone marrow engraftment than the number of circulating granulocytes.

Separation of intermediates in the refolding of reduced erabutoxin b by analytical isoelectric focusing in layers of polyacrylamide gel.

Isoelectric focusing in a thin layer of polyacrylamide gel is shown to be a suitable method for the resolution of intermediates trapped during the refolding process of reduced cystine-containing proteins. The method has been applied to the well-characterized snake neurotoxin erabutoxin b. An explanation is offered for the relatively low rate of refolding of this polypeptide.

Refolding of reduced short neurotoxins: circular dichroism analysis.

The four disulfide bonds of nine homologous short curare-like polypeptides are cleaved by reduced dithiothreitol. Air oxidation renaturations of the reduced compounds are followed by far-ultraviolet circular dichroism analysis, and the kinetics of refolding thus determined are compared. They indicate that three toxins refold 4--10 times more slowly than the six others. It is shown that a significant difference between the refolding kinetics still subsists when renaturations are made in the presence of various concentrations of thiol-disulfide exchange reagents or at various pH values. From an examination of the toxin sequences, it is proposed that a single additional amino acid insertion is responsible for the difference in the observed kinetics. This proposal is supported by temperature studies of renaturation kinetics.

[Rate of structural renaturation of curare-like polypeptide from cobra venom]. / Etude des vitesses de renaturation structurale des polypeptides curarisants des venins des cobras.

After breakage of their four disulfide bonds, snake curare-like toxins recover spontaneously their native conformation at different rates depending on the number of residues in the sequence.