Primary structure of diphtheria toxin fragment B: structural similarities with lipid-binding domains.

Two different lipid-associating domains have been identified in the B fragment of diphtheria toxin using automated Edman degradation of its cyanogen bromide peptides, secondary structure prediction analysis, and comparisons with known phospholipid-interacting proteins. The first domain is located in the highly hydrophilic (polarity index [PI] = 61.0%) 9.00-dalton N-terminal region of fragment B. This region shows primary and predicted secondary structures dramatically similar to those found for the phospholipid headgroup-binding domains of human apolipoprotein A1 (surface lipid-associating domain). The second domain is located in the highly hydrophobic (PI = 32.4%) middle region of fragment B. Its structure resembles that found for the membranous domain of intrinsic membrane proteins (transverse lipid-associating domain). In contrast, the hydrophilic C-terminal 8,000-dalton region of fragment B (PI = 53.8%) does not show structural similarity with lipid-associating domains.

Isolation and characterization of the cyanogen bromide peptides from the B fragment of diphtheria toxin.

Homogeneous fragment B, obtained through nicking of diphtheria toxin with insoluble trypsin, was cleaved with cyanogen bromide in 70% formic acid. After citraconylation, the cleavage products were separated by gel filtration on Sephadex G--75 and purified by gel filtration, ion-exchange and thin-layer or paper chromatography. Six CNBr peptides were characterized, the composition of which account for the total amino acid content of fragment B. Their apparent molecular weights are: CB 1, 12 000; CB 2, 14 000; CB 3, 8000; CB 4a, 2400; CB 4b, 2200; CB 5, 2200. CB 4a is the NH2--terminal peptide; it contains the cysteine residue of the disulfide bridge linking fragment B to fragment A. CB 3 is the COOH--terminal peptide; it bears the disulfide bridge of fragment B. Characterization of two CNBr--derived overlapping peptides provided the positioning of CB 4b and CB 2 and allowed an alignment of the CNBr peptides of fragment B to be proposed.

Diphtheria toxin induces fusion of small unilamellar vesicles at low pH.

Model membrane systems such as phospholipid vesicles have been extensively used to study the mechanism of membrane fusion at the molecular level. We report here on the capacity of diphtheria toxin to induce fusion of small unilamellar vesicles of dipalmitoylphosphatidylcholine at low pH. Fluorescence polarization and differential scanning calorimetry make it possible to demonstrate the mixing of the lipid phase. Mixing of the internal aqueous compartments of liposome was established using the terbium fluorescence technique. The analogy of structure and properties between melittin and a diphtheria toxin fragment is discussed.

Renal epidermal growth factor precursor: proteolytic processing in an in vitro cell-free system.

The enzymatic processing of the membrane-bound renal epidermal growth factor precursor (proEGF) could be an important step in the control of nephrogenic repair consecutive to kidney insult. The enzyme machinery responsible for that processing was examined in a cell-free system consisting of renal membranes isolated from kidney homogenates by differential centrifugation, and incubated in vitro. After a 24-h incubation at 37 degrees C, 6-14% of membrane-bound proEGF was processed and soluble products with EGF immunoreactivity were released. As revealed by HPLC and Western blotting analysis, the products of proEGF proteolysis consisted of 6 kDa EGF (the molecular weight of mature EGF) and two polypeptides with molecular weights around 45 kDa. Interestingly the 45 kDa EGF forms, like the 6 kDa EGF, exhibited mitogenic activity toward growth-arrested NRK-52E renal cell line. The kinetic study of proEGF degradation gave data consistent with the 45 kDa product(s) being processing intermediate(s) between proEGF and 6 kDa EGF. The enzymatic activity responsible for proEGF nicking was inhibited by divalent heavy metal ions (Cu2+ or Zn2+) and several protease inhibitors (aprotinin, PMSF, leupeptin, soybean trypsin inhibitor), suggesting that proEGF is processed by kallikrein-like serine proteases present in the membrane preparations. Along with previous studies, the current observations suggest that renal kallikreins might play a role in renal tubular regeneration by promoting the release of soluble EGF in renal tissue.

The complete amino acid sequence of diphtheria toxin fragment B. Correlation with its lipid-binding properties.

The complete amino acid sequence of fragment B from diphtheria toxin has been determined. The polypeptide chain was split with cyanogen bromide, o-iodosobenzoic acid, clostripain and trypsin; all amino acid sequence analyses were made by automated Edman degradation. Fragment B, which corresponds to the carboxy terminus of the toxin molecule, contains 342 amino acids and has an Mr of 37240. The proposed amino acid sequence fully confirms the structure recently deduced from the nucleotide sequence of the structural gene. The complete sequence is analyzed in relationship with the role of fragment B in the transfer of diphtheria toxin fragment A from the extracellular medium into the cell cytoplasm.

Proteomics of bronchoalveolar lavage fluid.

Lung diseases are essentially multi-factorial diseases that require a global analysis, and thus, cannot be understood through the sole analysis of individual or small numbers of genes. Proteome analysis has rapidly developed in the post-genome era and is now widely accepted as the obligated complementary technology for genetic profiling. It has been shown to be a powerful tool for the study of human diseases and for identifying novel prognostic, diagnostic and therapeutic markers. During last years, proteomic approaches applied to lung diseases are centred on the analysis of proteins in samples, such as cell cultures, biopsies and physiological fluids like serum and, especially, bronchoalveolar lavage fluid (BALF). BALF is presently the most common way of sampling the components of the epithelial lining fluid (ELF) and the most faithful reflect of the protein composition of the pulmonary airways. This review focuses on the state of the investigations of BALF proteome and its powerful contribution both to a better knowledge of the lung structure at the molecular level and to the study of lung disorders at the clinical level.

Sequences of the genes encoding argininosuccinate synthetase in Escherichia coli and Saccharomyces cerevisiae: comparison with methanogenic archaebacteria and mammals.

The nucleotide (nt) sequences of the genes encoding argininosuccinate synthetase from Escherichia coli K-12 (argG) and Saccharomyces cerevisiae (ARG1) were determined. The deduced amino-acid sequences were compared to each other and to their counterparts in two methanogens and in mammals. Three regions are highly conserved. Two of them appear to contain possible Walker-type nt-binding sites [Walker et al., EMBO J. 1 (1982) 945-951] and are therefore candidates for ATP-binding sites. The third region shows some similarity to a short portion of the N-proximal part of the PurA enzyme which catalyses an analogous reaction.

Localization in diphtheria toxin fragment B of a region that induces pore formation in planar lipid bilayers at low pH.

Like diphtheria toxin and the N-terminal (Mr 23 000) region of fragment B, CB1 (Mr 13 000), the cyanogen bromide peptide located in the middle region of fragment B is able to induce pore formation in lipid bilayer membrane at low pH. These two peptides (Mr 23 000 and 13 000) share a common segment (Mr 6300) containing the predicted amphipathic, alpha-helical, transverse lipid-associating domain (Mr 2750) of fragment B [J. Cell Biol. (1980) 87, 837-840]. Therefore, we postulated this domain to be responsible for the pore formation ability of diphtheria toxin [Proc. Natl. Acad. Sci. USA (1981) 78, 172-176]. A relationship between the pH dependency of pore formation and the presence of a cluster of prolines in the C-terminal region of CB1 is proposed.

Secondary structure changes of diphtheria toxin interacting with asolectin liposomes: an infrared spectroscopy study.

Until now, the study of the secondary structure of diphtheria toxin (DT) in the presence of phospholipid vesicles as a function of the pH has been prevented by the optical turbidity of the solution. In the present paper, this problem has been overcome by the use of IR attenuated total reflection (IR-ATR) spectroscopy. Incubation of DT with asolectin liposomes at pH 7.3 results in the binding of DT on to the liposomes and in a 10% increase in its alpha-helix content. At pH 4, in the presence of asolectin liposomes, the secondary structure of DT is characterized by the appearance of a beta-sheet structure with strengthened hydrogen bonds, which did not exist before lowering of the pH. This new type of beta-sheet (low frequency beta-sheet) involves 25% of the amino acid residues of the protein.

Proteomics as the tool to search for lung disease markers in bronchoalveolar lavage.

Most lung disorders are known to be associated to considerable modifications of surfactant composition. Numerous of these abnormalities have been exploited in the past to diagnose lung diseases, allowing proper treatment and follow-up. Diagnosis was then based on phospholipid content, surface tension and cytological features of the epithelial lining fluid (ELF), sampled by bronchoalveolar lavage (BAL) during fiberoscopic bronchoscopy. Today, it appears that the protein content of ELF displays a remarkably high complexity, not only due to the wide variety of the proteins it contains but also because of the great diversity of their cellular origins. The significance of the use of proteome analysis of BAL fluid for the search for new lung disease marker proteins and for their simultaneous display and analysis in patients suffering from lung disorders has been examined.

Changes in protein expression in the vestibular nuclei during vestibular compensation.

In the guinea pig, labyrinthectomy induces an immediate depression of the resting discharges in the neurons of the ipsilateral vestibular nuclei. Later on, in spite of the persistent deprivation of their ipsilateral labyrinthine input, a spontaneous restoration of activity, which is complete within 1 week, occurs in these neurons. Here, by using computer-assisted quantitative two-dimensional gel analysis, we have detected three proteins whose expressions were increased in the ipsilateral vestibular nuclei 1 week after unilateral labyrinthectomy. The spatio-temporal pattern of this phenomenon was compatible with a role for it in the restoration of activity in the vestibular neurons deprived of their ipsilateral labyrinthine input. Furthermore, the N-terminal amino acid sequences of two of these expressed proteins were obtained.

Structure-activity relationships of the B fragment of diphtheria toxin: the lipid-binding domains.

Two different lipid-associating domains have previously been identified in diphtheria toxin fragment B: one of the surface type in the N-terminus of B and one of the transverse type in its middle region. We have now determined about 85% of the primary structure of fragment B and show, here, that the middle part of fragment B contains a highly hydrophobic region of 72 amino acid residues (polarity index: 295) which includes the transverse lipid-associating domain. That this domain is actually involved in a process of membrane penetration is suggested by lipid bilayer conductance measurements of the CNBr peptides of fragment B and trypsin treatment of fragment B-multilamellar liposome complexes.

Aggregation and fusion of lipid vesicles induced by diphtheria toxin at low pH: possible involvement of the P site and the NAD+ binding site.

Model membranes have been used to study the interaction between diphtheria toxin and lipids. We report here on the ability of this toxin to induce, at low pH, fusion and aggregation of asolectin lipid vesicles. Resonance energy transfer experiments using lipid fluorescent probes make it possible to discriminate between these two processes.

Characterization of a cell line established from diethylstilbestrol-induced renal tumors in Syrian hamsters.

This article describes HKT-1097, a new cell line established from renal tumors induced by the protracted administration of diethylstilbestrol (DES) to male Syrian golden hamsters. Cell culture was initiated from tumor samples obtained from two 14-mo.-old animals which had undergone exposure to DES for a period of 11 mo. The HKT-1097 cell line was characterized between Passages 16 and 22 with respect to cell morphology, growth properties, karyology, and the presence of estrogen receptors. Moreover, immunostaining with a panel of antisera was performed to identify the cytological profile of the cell line and establish a parallel with tumor tissue in vivo. HKT-1097 cells are fibroblastoid; their most distinctive feature is that they exhibit strikingly long processes. The HKT-1097 cell line grows as a monolayer with a tendency toward a less stringent density-dependent inhibition of growth. The modal chromosome number is 44, but more than 50% of the cells are aneuploid, suggesting a substantial degree of karyotype instability. HKT-1097 cells express estrogen receptors. They contain immunoreactive vimentin and desmin, but appear negative upon cytokeratin immunostaining. In addition, these cells express glial fibrillary acidic protein and other markers of the neuroectodermal lineage, but lack neurofilament protein. Insofar as the same lineage markers have been demonstrated in DES-induced Syrian hamster kidney tumors (SHKT), we conclude that HKT-1097 cells retain some of the original tumor cell phenotype. The current observations suggest that estrogen-induced SHKT derive from the renal interstitium and point to an involvement of neuroectodermal cells in the development of these neoplasms.

Demonstration of estrogen receptors and of estrogen responsiveness in the HKT-1097 cell line derived from diethylstilbestrol-induced kidney tumors.

This study was undertaken in order to examine the estrogen sensitivity of HKT-1097, an established cell line recently derived from diethylstilbestrol (DES)-induced kidney tumors in Syrian hamsters. Estrogen receptor (ER) level in HKT-1097, determined by enzyme-linked immunoassay, was 67 fmol/mg protein, i.e., a value approx. 30% lower than that found in Syrian hamster kidney tumors. ER immunostaining in cells fixed with Carnoy's mixture, as well as ER demonstration by Western blotting, suggested DES-induced nuclear translocation or stabilization of the receptor within the nucleus. Kinetic parameters of estrogen binding to ER in HKT-1097 cells were 8.4 x 10(-11) M and 60.8 fmol/mg protein for Kd and Bmax, respectively. The Kd of estrogen binding to ER in HKT-1097 was close to that evaluated for the receptor in breast cancer-derived MCF-7 cell line, whereas the Bmax value was approx. seven times lower in HKT-1097 as compared to MCF-7. In HKT-1097 cells, antiestrogens ICI 182,780 and RU 58,668 induced ER downregulation and competed with estrogen binding to the receptor. As demonstrated by Western blot analysis, DES exposure led to an increased expression of progesterone receptor (PgR) in HKT-1097 cells. Addition of DES to estrogen-free medium produced a stimulation of growth in both HKT-1097 and MCF-7 cells, but the mitogenic effect was less marked for HKT-1097. Despite the fact that ICI 182,780 and RU 58,668 clearly interact with HKT-1097 cell ER, they appeared unable to suppress DES-induced stimulation of growth and increase of PgR expression.

Transforming growth factor-alpha and epidermal growth factor in hamster tissues: biochemical and immunohistochemical studies.

In Syrian golden hamster kidneys and submaxillary glands, the levels of EGF, determined by radioimmunoassay, were much lower than in the same organs of two other rodent species, mouse and rat. In submaxillary glands, the EGF/TGF-alpha receptor-binding activities were also much lower in hamster than in mouse and rat. In contrast, the TGF-alpha content of hamster kidneys, determined by radioimmunoassay, was higher than in the kidneys of the other animals, as was the EGF/TGF-alpha receptor-binding activity. Using immunohistochemistry, the TGF-alpha immunoreactivity in hamster kidneys was localized both in proximal and distal tubules with the exception of the macula densa area. The levels of TGF-alpha in the submaxillary glands were very low in all the animals tested. Hamster kidney extracts contained a specific immunoreactive protein with the M(r) and the N-terminal amino acid sequence (VVSHFNECPD) expected for mature hamster TGF-alpha. Western blot analysis of hamster renal solubilized membrane proteins using anti-EGF receptor antibodies revealed three immunoreactive protein bands of which one had the M(r) expected for the EGF/TGF-alpha receptor. The immunohistochemical pattern of this receptor in hamster kidneys proximal tubular cells was very similar to the other tested rodent species.

Sublethal alterations and sustained cell proliferation associated with the diethylstilbestrol-induced renal carcinogenesis in male Syrian golden hamsters.

The current study was initiated to explore the sublethal alterations and the tissue damage occurring in the hamster kidney during diethylstilbestrol-induced renal carcinogenesis. A total of 49 male Syrian golden hamsters (35 treated and 13 control animals) was utilized in the experimental procedure. Chronic exposure to diethylstilbestrol was achieved by s.c. insertion of implants containing 25 mg diethylstilbestrol. For long-term observation, adequate blood level of diethylstilbestrol was insured by renewing the implant every 2 months. Experimental groups (n = 4 to 9) were terminated 1, 2, 4, 6, 9 and 11 months after initial implantation for morphological examination of the kidney. Diethylstilbestrol carcinogenicity in this experimental model was confirmed by the observation that most animals undergoing drug exposure for 6 months or more exhibited renal neoplasms. The most striking nonneoplastic morphological abnormality disclosed by histological and cytological examination consisted in the accumulation of granular inclusions in proximal tubule cells. In renal tissue, the extent of cell proliferation determined by PCNA labeling progressively increased along with the duration of diethylstilbestrol exposure and suggested a sustained proliferative response in altered proximal tubules. The present data suggest that an impairment of functional tubular regeneration could promote, as well as the estrogen genotoxic effect, the tumorigenicity of diethylstilbestrol in the kidney of male hamsters.

Immunological and structural relatedness of catabolic ornithine carbamoyltransferases and the anabolic enzymes of enterobacteria.

Purified catabolic ornithine carbamoyltransferase of Pseudomonas putida and anabolic ornithine carbamoyltransferase (argF product) of Escherichia coli K-12 were used to prepare antisera. The two specific antisera gave heterologous cross-reactions of various intensities with bacterial catabolic ornithine carbamoyltransferases formed by Pseudomonas and representative organisms of other bacterial genera. The immunological cross-reactivity observed only between the catabolic ornithine carbamoyltransferases and the anabolic enzymes of enterobacteria suggests that these proteins share some structural similarities. Indeed, the amino acid composition of the anabolic ornithine carbamoyltransferase of E. coli K-12 (argF and argI products) closely resembles the amino acid compositions of the catabolic enzymes of Pseudomonas putida, Aeromonas formicans, Streptococcus faecalis, and Bacillus licheniformis. Comparison of the N-terminal amino acid sequence of the E. coli anabolic ornithine carbamoyltransferase with that of the A. formicans and Pseudomonas putida catabolic enzymes shows, respectively, 45 and 28% identity between the compared positions; the A. formicans sequence reveals 53% identity with the Pseudomonas putida sequence. These results favor the conclusion that anabolic ornithine carbamoyltransferases of enterobacteria and catabolic ornithine carbamoyltransferases derive from a common ancestral gene.

pH dependent insertion of a diphtheria toxin B fragment peptide into the lipid membrane: a conformational analysis.

A peptide of diphtheria toxin B fragment (residues 147-266) has been shown to induce pore formation in lipid bilayer membranes at low pH. Such an effect was obtained at a much lower extent or not at all at pH = 7. The region localized between residues 225 and 246 is highly hydrophobic (27.3% polarity) and characterized by a high concentration of proline residues. Since proline cis-trans isomerization is highly sensitive to the pH of the medium, we investigated the capability of the cis and trans isomers to penetrate into the lipid matrix. Obviously, the cis-trans isomerization of proline 242 and 245, assumed to be imposed by a low pH, uncovers the hydrophobic region and induces its insertion into a lipid layer of dipalmitoylphosphatidylcholine. The lipid matrix destabilization resulting from this process could promote the penetration into the lipid bilayer of an amphipatic structure (153-178) similar to the transverse lipid associating domains of membrane proteins.

Purification and preliminary characterization of a frog-derived proteinaceous chemoattractant eliciting prey attack by checkered garter snakes (Thamnophis marcianus).

A potent proteinaceous chemoattractant, eliciting prey attack by checkered garter snakes (Thamnophis marcianus) was isolated from aqueous washes of the common frogRana temporaria and purified by preparative continuous-elution electrophoresis. The biological activity of the frog crude extract or of the purified chemoattractive protein, measured by a snake bioassay, was unaffected by freezing, lyophilization, or dialysis but was lost after proteolytic digestion. The purified chemoattractant is glycosylated, has an apparent molecular mass of 24 kDa, estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and a pI of 4.8. It gave one spot in two-dimensional electrophoresis. The bioassay showed that this protein is highly attractive to snakes. The lowest concentration yielding positive responses in the snake bioassay was approximately 25 µg/ml. These results suggest that a water-soluble Mr 24 kDa glycoprotein molecule produced by the common frog may be a vomeronasal stimulus used by checkered garter snakes for prey recognition.