Streptococcal cell wall-induced arthritis and adjuvant arthritis in F344----Lewis and in Lewis----F344 bone marrow chimeras

Streptococcal cell wall (SCW)-induced arthritis and adjuvant arthritis (AA) are rat models for chronic, erosive polyarthritis. Both models can be induced in susceptible Lewis rats, whereas F344 rats are resistant. In AA as well as in SCW arthritis, antigen-specific T lymphocytes have been demonstrated to be crucial for chronic disease. In this communication we describe our studies to probe the cellular mechanism responsible for the difference in susceptibility of Lewis and F344, using bone marrow chimeras. By transplanting bone marrow cells from F344 into lethally irradiated Lewis recipients, Lewis rats were rendered resistant to SCW arthritis induction. F344 rats reconstituted with Lewis bone marrow, i.e., Lewis----F344 chimeras, develop an arthritis upon SCW injection. For AA comparable results were obtained. These data suggest that both resistance and susceptibility to bacterium-induced chronic arthritis are mediated by hemopoietic/immune cells and that the recipiental environment does not influence the susceptibility to chronic joint inflammation.     

NMR studies of lantibiotics. The structure of nisin in aqueous solution.

Nisin is a posttranslationally modified protein of 34 amino acids, and is a member of the class of bacteriocidal polypeptides known as lantibiotics, that contain the unusual amino acid lanthionine. Its structure in aqueous solution has been determined on the basis of NMR data, i.e. interproton distance constraints derived from nuclear Overhauser enhancement spectroscopy and torsion angle constraints derived from double-quantum-filtered correlated spectroscopy. Translation of the NMR constraints into a three-dimensional structure was carried out with the distance-geometry program DISMAN, followed by restrained energy minimization using CHARMm. The internal mobility of the peptide chain prohibited the determination of a precise overall folding of the molecule, but parts of the structure could be obtained, albeit sometimes with low resolution. The structure of nisin can best be defined as follows. The outermost N-terminal and C-terminal regions of nisin appear quite flexible, the remainder of the molecule consists of an amphiphilic N-terminal fragment (residues 3-19), joined by a flexible 'hinge' region to a rigid double-ring fragment formed by residues 23-28. The latter fragment has the appearance of a somewhat overwound alpha-helix. It is suggested, by assuming the presence of a (transient) alpha-helical structure in this part of prenisin, that the coupling between residues 23 and 26, as well as between 25 and 28, by thioether bridges, and the inversion of the C alpha chiralities at positions 23 and 25, can be rationalized.     

Isoforms of human cytochrome-c oxidase. Subunit composition and steady-state kinetic properties.

The subunit pattern and the steady-state kinetics of cytochrome-c oxidase from human heart, muscle, kidney and liver were investigated. Polyacrylamide gel electrophoresis of immunopurified cytochrome-c oxidase preparations suggest that isoforms of subunit VIa exist, which show differences in staining intensity and electrophoretic mobility. No differences in subunit pattern were observed between the other nucleus-encoded subunits of the various cytochrome-c oxidase preparations. Tissue homogenates, in which cytochrome-c oxidase was solubilised with laurylmaltoside, were directly used in the assays to study the cytochrome-c oxidase steady-state kinetics. Cytochrome-c oxidase concentrations were determined by immunopurification followed by separation and densitometric analysis of subunit IV. When studied in a medium of low ionic strength, the biphasic kinetics of the steady-state reaction between human ferrocytochrome c and the four human cytochrome-c oxidase preparations revealed large differences for the low-affinity TNmax (maximal turnover number) value, ranging from 77 s-1 for kidney to 273 s-1 for liver cytochrome-c oxidase at pH 7.4, I = 18 mM. It is proposed that the low-affinity kinetic phase reflects an internal electron-transfer step. For the steady-state reaction of human heart cytochrome-c oxidase with human cytochrome c, Km and TNmax values of 9 microM and 114 s-1 were found, respectively, at high ionic strength (I = 200 mM, pH 7.4). Only minor differences were observed in the steady-state activity of the various human cytochrome-c oxidases. The interaction between human cytochrome-c oxidase and human cytochrome-c proved to be highly specific. At high ionic strength, a large decrease in steady-state activity was observed when reduced horse, rat or bovine cytochrome c was used as substrate. Both the steady-state TNmax and Km parameters were strongly affected by the type of cytochrome c used. Our findings emphasize the importance of using human cytochrome c in kinetic assays performed with tissues from patients with a suspected cytochrome-c oxidase deficiency.     

Reduction of the amount of periplasmic hydrogenase in Desulfovibrio vulgaris (Hildenborough) with antisense RNA: direct evidence for an important role of this hydrogenase in lactate metabolism.

To establish the function of the periplasmic Fe-only hydrogenase in the anaerobic sulfate reducer Desulfovibrio vulgaris (Hildenborough), derivatives with a reduced content of this enzyme were constructed by introduction of a plasmid that directs the synthesis of antisense RNA complementary to hydrogenase mRNA. It was demonstrated that the antisense RNA technique allowed specific suppression of the synthesis of this hydrogenase in D. vulgaris by decreasing the amount of hydrogenase mRNA but did not result in the complete elimination of the enzyme, as is usual with most conventional mutagenesis techniques. The hydrogenase content in these antisense RNA-producing D. vulgaris clones was two- to threefold lower than in the parental strain when the strains were grown in batch cultures with lactate as a substrate and sulfate as a terminal electron acceptor. Under these conditions, several differences in growth parameters were measured between the hydrogenase-suppressed clones and wild-type D. vulgaris: growth rates of the clones decreased two- to threefold, and at excess lactate, growth yields were reduced by 20%. Furthermore, the amount of hydrogen measured in the culture headspaces was reduced three- to fivefold for the clones. These observations indicate that this hydrogenase has an important function during growth on lactate and is involved in hydrogen production from protons and electrons originating from at least one of the two oxidation reactions in the conversion of lactate to acetate. The implications for the energy metabolism of D. vulgaris are discussed.     

Hydrolysis and reduction of factor 390 by cell extracts of Methanobacterium thermoautotrophicum (strain delta H).

Cell extracts of Methanobacterium thermoautotrophicum (strain delta H) were found to perform a hydrogen-dependent reduction of factor 390 (F390), the 8-adenylyl derivative of coenzyme F420. Upon resolution of cell extracts, F390-reducing activity copurified with the coenzyme F420-dependent hydrogenase. This indicates that F390 serves as a substrate of that enzyme. Activity towards F390 was approximately 40-fold lower than that towards coenzyme F420 (0.12 and 5.2 mumol.min-1.mg of protein-1, respectively). In addition, cell extracts catalyzed the hydrolysis of F390 to AMP and coenzyme F420. This hydrolysis required the presence of thiols (6 mM) and much ionic strength (1 M KCl) and was reversibly inhibited by oxygen. The reaction proceeded optimally at pH 8.2 and was Mn dependent. Conditions for F390 hydrolysis in cell extracts are in many respects opposite to those previously described for F390 synthesis.     

Pathogen-induced proteins with inhibitory activity toward Phytophthora infestans.

A bioassay using Phytophthora infestans was developed to determine whether inhibitory proteins are induced in pathogen-inoculated plants. Using this bioassay, AP24, a 24-kilodalton protein causing lysis of sporangia and growth inhibition of P. infestans, was purified from tobacco plants inoculated with tobacco mosaic virus. Analysis of the N-terminal amino acid sequence identified AP24 as the thaumatin-like protein osmotin II. The sequence was also similar to NP24, the salt-induced protein from tomato. Subsequently, we purified a protein from tomato plants inoculated with P. infestans that had inhibitory activities identical to those of the tobacco AP24. The N-terminal amino acid sequence of this protein was also similar to those of osmotin and NP24. In general, both the tobacco and tomato AP24 caused lysis of sporangia at concentrations greater than 40 nanomolar and severely inhibited hyphal growth at concentrations greater than 400 nanomolar. Because both proteins were induced by pathogen inoculation, we discussed the possible involvement of these proteins as a plant defense mechanism.     

The effect of driving force on intramolecular electron transfer in proteins. Studies on single-site mutated azurins.

An intramolecular electron-transfer process has previously been shown to take place between the Cys3--Cys26 radical-ion (RSSR-) produced pulse radiolytically and the Cu(II) ion in the blue single-copper protein, azurin [Farver, O. & Pecht, I. (1989) Proc. Natl Acad. Sci. USA 86, 6868-6972]. To further investigate the nature of this long-range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single-site mutation of the wild-type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine residue (Met44) that is proximal to the copper coordination sphere has been replaced by a positively charged lysyl residue ([M44K]azurin), while in the second mutant, another residue neighbouring the Cu-coordination site (His35) has been replaced by a glutamine ([H35Q]azurin). Though both these substitutions are not in the microenvironment separating the electron donor and acceptor, they were expected to affect the LRET rate because of their effect on the redox potential of the copper site and thus on the driving force of the reaction, as well as on the reorganization energies of the copper site. The rate of intramolecular electron transfer from RSSR- to Cu(II) in the wild-type P. aeruginosa azurin (delta G degrees = -68.9 kJ/mol) has previously been determined to be 44 +/- 7 s-1 at 298 K, pH 7.0. The [M44K]azurin mutant (delta G degrees = -75.3 kJ/mol) was now found to react considerably faster (k = 134 +/- 12 s-1 at 298 K, pH 7.0) while the [H35Q]azurin mutant (delta G degrees = -65.4 kJ/mol) exhibits, within experimental error, the same specific rate (k = 52 +/- 11 s-1, 298 K, pH 7.0) as that of the wild-type azurin. From the temperature dependence of these LRET rates the following activation parameters were calculated: delta H++ = 37.9 +/- 1.3 kJ/mol and 47.2 +/- 0.7 kJ/mol and delta S++ = -86.5 +/- 5.8 J/mol.K and -46.4 +/- 4.4 J/mol.K for [H35Q]azurin and [M44K]azurin, respectively. Using the Marcus relation for intramolecular electron transfer and the above parameters we have determined the reorganization energy, lambda and electronic coupling factor, beta. The calculated values fit very well with a through-bond LRET mechanism.     

The EGF receptor is an actin-binding protein

In a number of recent studies it has been shown that in vivo part of the EGF receptor (EGFR) population is associated to the actin filament system. In this paper we demonstrate that the purified EGFR can be cosedimented with purified filamentous actin (F-actin) indicating a direct association between EGFR and actin. A truncated EGFR, previously shown not to be associated to the cytoskeleton, was used as a control and this receptor did not cosediment with actin filaments. Determination of the actin-binding domain of the EGFR was done by measuring competition of either a polyclonal antibody or synthetic peptides on EGFR cosedimentation with F-actin. A synthetic peptide was made homologous to amino acid residues 984-996 (HL-33) of the EGFR which shows high homology with the actin-binding domain of Acanthamoeba profilin. A polyclonal antibody raised against HL-33 was found to prevent cosedimentation of EGFR with F-actin. This peptide HL-33 was shown to bind directly to actin in contrast with a synthetic peptide homologous to residues 1001-1013 (HL-34). During cosedimentation, HL-33 competed for actin binding of the EGFR and HL-34 did not, indicating that the EGFR contains one actin-binding site. These results demonstrate that the EGFR is an actin-binding protein which binds to actin via a domain containing amino acids residues 984-996.     

Transforming growth factor-beta inhibits the production of IgG, IgM, and IgA in human lymphocyte cultures.

Transforming growth factor beta (TGF beta) has potent immunoregulatory effects acting on both T and B cells. It strongly inhibits secretion of IgG and IgM in human and murine B cell cultures, but has been shown to have an enhancing effect on IgA production in the mouse. We have studied the effect of TGF beta on the production of IgA in human lymphocyte cultures. The addition of TGF beta to pokeweed-stimulated peripheral blood lymphocytes resulted in a suppression of IgA production of both subclasses, similar in magnitude to the suppression of IgG and IgM production. Membrane IgA expression was not increased by culturing tonsillar lymphocytes with TGF beta. In conclusion, we find no evidence for a selective enhancing effect of TGF beta on IgA synthesis in humans, in contrast to the findings reported in mice.