Anti-inflammatory and cartilage-protecting effects of an intra-articularly injected anti-TNF{alpha} single-chain Fv antibody (ESBA105) designed for local therapeutic use.

OBJECTIVES: (1) To show that a single-chain Fv antibody (scFv) against tumour necrosis factor alpha (TNFalpha) (ESBA105) has efficacy comparable to a full length anti-TNFalpha IgG (infliximab); (2) to evaluate whether ESBA105 has all the properties required for the local treatment of arthritis; and (3) to investigate its discriminative tissue penetration properties. METHODS: In vivo efficacy was measured in arthritis of the knee joint induced by the intra-articular injection of recombinant human TNFalpha (rhTNFalpha) in Lewis rats. Cartilage penetration of scFv (ESBA105) and full length IgG (infliximab) were studied in bovine cartilage specimens ex vivo. Tissue penetration, biodistribution and pharmacokinetics of ESBA105 were followed and compared after intra-articular and intravenous administration. RESULTS: In cell culture, ESBA105 showed similar TNFalpha inhibitory potency to infliximab. In vivo, ESBA105 inhibited rhTNFalpha-induced synovial inflammation in rats with efficacy again comparable to infliximab. An 11-fold molar excess of ESBA105 over rhTNFalpha resulted in 90% inhibition of knee joint swelling, inflammatory infiltrates and proteoglycan loss from cartilage. In ex vivo studies of bovine cartilage, ESBA105 penetrated well into the cartilage whereas infliximab remained on the surface. In vivo, rapid penetration into the synovial tissue, cartilage and surrounding tissues was observed following intra-articular injection of [(125)I]-ESBA105 into the knee joint of rabbits. CONCLUSIONS: ESBA105 potently inhibits inflammation and prevents cartilage damage triggered by TNFalpha. In contrast to a full length IgG, ESBA105 also penetrates into cartilage and can be expected to reverse the TNFalpha-induced catabolic state of articular cartilage in arthritides.

Validation of leaf ozone symptoms in natural vegetation using microscopical methods.

Ozone injury to natural vegetation is being increasingly surveyed throughout the northern hemisphere. There exists a growing list of species showing visible 'ozone-like' symptoms which needs to be validated. This study presents the results from a test survey of ozone injury to forest vegetation in the light exposed sites of five Swiss level II plots, for the new ICP-Forests protocol. With AOT40 from 14 to 28 ppm-h in 2000, ten out of 49 woody plant species displayed typical symptoms, and four showed untypical symptoms. Symptom origin was investigated in nine and validated in seven species, using morphological, histological and cellular markers of oxidative stress and ozone-induced plant response. Independent of taxonomic position, ozone effects were characterized by the induction of oxidative stress in the mesophyll resulting in discrete and light-dependent hypersensitive-like responses and in accelerated cell senescence. The presented combination of cellular and morphological markers allows differential diagnosis of visible ozone injury.

Binding sites of Drosophila melanogaster sex peptide pheromones.

Drosophila melanogaster sex peptide (SP) and Ductus ejaculatorius peptide (DUP99B) are male pheromones transferred in the seminal fluid to the female during copulation. Both reduce sexual receptivity and stimulate oviposition in females. The presence of high-affinity SP and DUP99B binding sites in the female were investigated by incubation of cryostat tissue sections with (125)I-iodinated peptides and subsequent autoradiography. We found that in adult females radiolabeled SP and DUP99B bind to peripheral nerves, the subesophageal ganglion, the cervical connective, to discrete parts of the thoracic ganglion, and to the genital tract. Weak and uniform labeling was detected in the neuropil of the brain and the thoracic ganglion. The labeling pattern in the nervous system suggests binding of the peptides to sensory afferents or glial cells. Scatchard analysis of the binding of (125)I-DUP99B to antennal nerves yielded a dissociation constant K(d) of 6.4 nM. Competition experiments with peptide fragments show that the peptides bind with their homologous C-terminal regions. Binding sites in the nervous system of females are established throughout sexual maturation. Prominent binding of the peptides to afferent nerves suggests modification of sensory input.

Bicelle-based liquid crystals for NMR-measurement of dipolar couplings at acidic and basic pH values.

It is demonstrated that mixtures of ditetradecyl-phosphatidylcholine or didodecyl-phoshatidylcholine and dihexyl-phosphatidylcholine in water from lyotropic liquid crystalline phases under similar conditions as previously reported for bicelles consisting of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl-phosphatidylcholine (DHPC). The carboxy-ester bonds present in DMPC and DHPC are replaced by ether linkages in their alkyl analogs, which prevents acid- or base-catalyzed hydrolysis of these compounds. 15N-1H dipolar couplings measured for ubiquitin over the 2.3-10.4 pH range indicate that this protein retains a backbone conformation which is very similar to its structure at pH 6.5 over this entire range.

Characterization of magnetically oriented phospholipid micelles for measurement of dipolar couplings in macromolecules.

Weak alignment of solute molecules with the magnetic field can be achieved in a dilute liquid crystalline medium, consisting of an aqueous mixture of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl-phosphatidylcholine (DHPC). For a certain range of molar ratios, DMPC and DHPC can form large, disc-shaped particles, commonly referred to as bicelles (Sanders and Schwonek, 1992), which cooperatively align in the magnetic field and induce a small degree of alignment on asymmetrically shaped solute molecules. As a result, dipolar couplings between pairs of 1H, 13C or 15N nuclei are no longer averaged to zero by rotational diffusion and they can be readily measured, providing valuable structural information. The stability of these liquid crystals and the degree of alignment of the solute molecules depend strongly on experimental variables such as the DMPC:DHPC ratio and concentration, the preparation protocol of the DMPC/DHPC mixtures, as well as salt, temperature, and pH. The lower temperature limit for which the liquid crystalline phase is stable can be reduced to 20 degrees C by using a ternary mixture of DHPC, DMPC, and 1-myristoyl-2-myristoleoyl-sn-glycero-3-phosphocholine, or a binary mixture of DHPC and ditridecanoyl-phosphatidylcholine. These issues are discussed, with an emphasis on the use of the medium for obtaining weak alignment of biological macromolecules.

Measurement of dipolar couplings for methylene and methyl sites in weakly oriented macromolecules and their use in structure determination.

A simple and effective method is described for simultaneously measuring dipolar couplings for methine, methylene, and methyl groups in weakly oriented macromolecules. The method is a J-modulated 3D version of the well-known [1H-13C] CT-HSQC experiment, from which the J and dipolar information are most accurately extracted by using time-domain fitting in the third, constant-time dimension. For CH2-sites, the method generally yields only the sum of the two individual 13C-1H couplings. Structure calculations are carried out by minimizing the deviation between the measured sum, and the sum predicted for each methylene on the basis of the structure. For rapidly spinning methyl groups the dipolar contribution to the splitting of the outer 13C quartet components can be used directly to constrain the orientation of the C-CH3 bond. Measured sidechain dipolar couplings are in good agreement with an ensemble of NMR structures calculated without use of these couplings.

Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra.

Simple procedures are described for recording complementary in-phase and antiphase J-coupled NMR spectra. The sum and difference of these spectra contain only the upfield and the downfield components of a doublet, making it possible to measure the J splitting directly from these combinations without an increase in resonance overlap relative to the decoupled spectrum. The approach is demonstrated for measurement of 1JNH splittings and 2JHNC splittings in oriented and isotropic ubiquitin. Dipolar couplings obtained from differences in the splittings measured in the oriented and isotropic phases are in excellent agreement with dipolar couplings obtained from direct measurement of the splitting or from a conventional E. COSY-type measurement.

Multiple cycles of global unfolding of GroEL-bound cyclophilin A evidenced by NMR.

GroE, the chaperonin system of Escherichia coli, prevents the aggregation of partially folded or misfolded proteins by complexing them in a form competent for subsequent folding to the native state. We examined the exchange of amide protons of cyclophilin A (CypA) interacting with GroEL, using NMR spectroscopy. We have applied labeling pulses in H2O to the deuterated GroEL-CypA-complex. When ATP and GroES were added after the labeling pulse, refolding of CypA could be accelerated to rates comparable to the amide proton exchange. This allowed the calculation of protection factors (PF) for the backbone amide protons in the GroEL-bound substrate protein. A set of highly protected protons in the native state (PF 10(5) to 10(7)) was observed to be much less protected (PF 10(2) to 10(4)) in complex with GroEL and, in contrast to the native structure, the protection factors were found to be quite uniform along the sequence suggesting that CypA with native-like structure undergoes multiple cycles of unfolding while bound to GroEL, which are faster than unfolding in free solution. Because of the small sequence dependence of the protection factors, unfolding must be global, and in this way the chaperone appears to resolve off-pathway intermediates and to support protein folding by annealing. Although in the complex with GroEL native-like states still predominate over globally unfolded states, this equilibrium is shifted 10(2) to 10(4)-fold toward the unfolded state when compared to CypA in free solution. Repeated global unfolding may be a key step in achieving a high yield of correctly folded proteins.

The NMR solution conformation of unligated human cyclophilin A.

The nuclear magnetic resonance (NMR) solution structure of free, unligated cyclophilin A (CypA), which is an 18 kDa protein from human T-lymphocytes that was expressed in Escherichia coli for the present study, was determined using multidimensional heteronuclear NMR techniques. Sequence-specific resonance assignments for 99.5% of all backbone amide protons and non-labile hydrogen atoms provided the basis for collection of an input of 4101 nuclear Overhauser enhancement (NOE) upper distance constraints and 371 dihedral angle constraints for distance geometry calculations and energy minimization with the programs DIANA and OPAL. The average RMSD values of the 20 best energy-refined NMR conformers relative to the mean coordinates are 0.49 A for the backbone atoms and 0.88 A for all heavy atoms of residues 2 to 165. The molecular architecture includes an eight-stranded antiparallel beta-barrel that is closed by two amphipathic alpha-helices. Detailed comparisons with the crystal structure of free CypA revealed subtle but significant conformational differences that can in most cases be related to lattice contacts in the crystal structure. 15N spin relaxation times and NMR lineshape analyses for CypA in the free form and complexed with cyclosporin A (CsA) revealed transitions of polypeptide loops surrounding the ligand-binding site from locally flexible conformations in the free protein, some of which include well-defined conformational equilibria, to well-defined spatial arrangements in the CypA-CsA complex. Compared to the crystal structure of free CypA, where the ligand-binding area is extensively involved in lattice contacts, the NMR structure presents a highly relevant reference for studies of changes in structure and internal mobility of the binding pocket upon ligand binding, and possible consequences of this conformational variability for calcineurin recognition by the CypA-CsA complex.

Three-dimensional (1)H-TOCSY-relayed ct-[(13)C, (1)H]-HMQC for aromatic spin system identification in uniformly (13)C-labeled proteins.

Three-dimensional (1)H-TOCSY-relayed ct-[(13)C,(1)H]-HMQC is a novel experiment for aromatic spin system identification in uniformly (13)C-labeled proteins, which is implemented so that it correlates the chemical shift of a given aromatic proton with those of the directly attached carbon and all vicinal protons. The ct-HMQC scheme is used both for overlay of the indirect (1)H and (13)C chemical shift evolution periods and for the generation of (1)H-(1)H antiphase magnetization to accelerate the (1)H-TOCSY magnetization transfer at short mixing times. As an illustration, data recorded for the 18 kDa protein cyclophilin A are presented. Since transverse relaxation of (13)C-(1)H zero-quantum and double-quantum coherences is to first order insensitive to (13)C-(1)H heteronuclear dipolar relaxation, the new experiment should work also for proteins with molecular weights above 20 kDa.

Structure of the human CALM1 calmodulin gene and identification of two CALM1-related pseudogenes CALM1P1 and CALM1P2.

The human CALM1 calmodulin gene has been isolated and characterized. The gene contains six exons spread over about 10 kb of genomic DNA. The exon-intron structure is identical to that of the human CALM3 and of the rat CALM1 and CALM3 genes. A cluster of transcription-start sites was identified 200 bp upstream of the ATG translation-start codon, and several putative regulatory elements were found in the 5' flanking region as well as in intron 1. Sequence comparison with the rat CALM1 gene revealed significant similarities in the promoter regions of the two genes and an even more striking degree of identity (70%) in the available intron 1 sequences. A short CAG trinucleotide repeat region was identified in the 5' untranslated region of the human CALM1 gene; this sequence is not conserved in the rat counterpart. Expression of the CALM1 gene was detected in all human tissues tested, although at varying levels. A 1.7-kb mRNA was uniformly present at comparable levels, whereas a 4.2-kb mRNA species was particularly abundant in brain and skeletal muscle. Clones for two different CALM1-related pseudogenes CALM1P1 and CALM1P2 were also isolated and characterized. Both pseudogenes are intronless and non-functional as judged from the presence of mutations abolishing the open reading frame. Genomic Southern analysis indicates that the human CALM1 gene/pseudogene subfamily comprises at least three but probably no more than four members. The entire family consists of three bona fide CALM genes, at least one expressed calmodulin-like CALML gene as well as at least five pseudogenes.

The NMR solution structure of the pheromone Er-2 from the ciliated protozoan Euplotes raikovi.

The NMR structure of the pheromone Er-2 from the ciliated protozoan Euplotes raikovi has been determined in aqueous solution. The structure of this 40-residue protein was calculated with the distance geometry program DIANA from 621 distance constraints and 89 dihedral angle constraints; the program OPAL was employed for the energy minimization. For a group of 20 conformers used to characterize the solution structure, the average pairwise RMS deviation from the mean structure calculated for the backbone heavy atoms N, C alpha, and C' of residues 3-37 was 0.31 A. The molecular architecture is dominated by an up-down-up bundle of 3 short helices of residues 5-11, 14-20, and 23-33, which is similar to the structures of the homologous pheromones Er-1 and Er-10. Novel structural features include a well-defined N-cap on the first helix, a 1-residue deletion in the second helix resulting in the formation of a 3(10)-helix rather than an alpha-helix as found in Er-1 and Er-10, and the simultaneous presence of 2 different conformations for the C-terminal tetrapeptide segment, i.e., a major conformation with the Leu 39-Pro 40 peptide bond in the trans form and a minor conformation with this peptide bond in the cis form.

Structure comparison of the pheromones Er-1, Er-10, and Er-2 from Euplotes raikovi.

The NMR structures of the homologous pheromones Er-1, Er-10, and Er-2 from the ciliated protozoan Euplotes raikovi are compared. For all 3 proteins the molecular architecture is made up of an antiparallel 3-helix bundle. The preservation of the core part of the structure is directly manifested by similar patterns of slowed backbone amide proton exchange rates, hydrogen bond formation, and relative solvent accessibility. To align the 6 half-cystine residues in the individual sequences within the preserved 3-dimensional core structure, several deletions and insertions had to be introduced that differ from those previously proposed on the basis of the primary structures. Of special interest is a deletion in the second helix of Er-2, which is accommodated by a transition from an alpha-helix in Er-1 and Er-10 to a 3(10)-helix in Er-2. The most significant structural differences are located in the C-terminal part of the proteins, which may have an important role in specific receptor recognition.

Destabilization of the complete protein secondary structure on binding to the chaperone GroEL.

Protein folding in vivo is mediated by helper proteins, the molecular chaperones, of which Hsp60 and its Escherichia coli variant GroEL are some of the best characterized. GroEL is an oligomeric protein with 14 subunits each of M(r) 60K, which possesses weak, co-operative ATPase activity and high plasticity. GroEL seems to interact with non-native proteins, binding one or two molecules per 14-mer in a 'central cavity', but little is known about the conformational state of the bound polypeptides. Here we use nuclear magnetic resonance techniques to show that the interaction of the small protein cyclophilin with GroEL is reversible by temperature changes, and all amide protons in GroEL-bound cyclophilin are exchanged with the solvent, although this exchange does not occur in free cyclophilin. The complete secondary structure of cyclophilin must be disrupted when bound to GroEL.