Attenuation of inflammation and cytokine production in rat colitis by a novel selective inhibitor of leukotriene A4 hydrolase.

BACKGROUND AND PURPOSE: Leukotriene B(4) (LTB(4)), formed by the sequential actions of the 5-lipoxygenase (5-LO) and leukotriene A(4) hydrolase (LTA(4)H), is a pro-inflammatory mediator implicated in the pathogenesis of inflammatory bowel disease. However, inhibitors of 5-LO have not proved to be consistent in their therapeutic efficacy in colitis. Another approach to inhibiting LTB(4) synthesis is through the use of inhibitors of LTA(4)H, such as the novel, potent and selective compound, JNJ 26993135. EXPERIMENTAL APPROACH: The effect of oral administration of JNJ 26993135 has been evaluated in a rat model of colitis provoked by colonic instillation of trinitrobenzenesulphonic acid (TNBS). The extent and severity of the macroscopic inflammatory response, the colonic levels of myeloperoxidase (MPO) and LTB(4) and of the pro-inflammatory cytokines, tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were measured. KEY RESULTS: Oral administration of JNJ 26993135 (5, 15 and 30 mg kg(-1), twice a day) dose-dependently reduced both the extent and intensity of the colonic inflammatory damage observed 3 days after TNBS challenge. JNJ 26993135 also dose-dependently reduced the elevated colonic levels of LTB(4), as well as the inflammatory biomarkers, MPO, IL-6 and TNF-alpha. This dosing regimen was supported by the pharmacokinetic profile of JNJ 26993135, along with the demonstration of the inhibition of ex vivo production of LTB(4) in whole blood following oral administration. CONCLUSIONS AND IMPLICATIONS: These results with JNJ 26993135 in the rat TNBS model support the role of LTB(4) in colitis and the potential value of targeting LTA(4)H for the treatment of inflammatory bowel diseases.

HLA class I and class II antigens associated with multiple myeloma in southern Africa.

While the exact aetiology of myeloma is unknown, genetic factors feature among the potential risk factors. The HLA phenotypes in African blacks with myeloma (the commonest haematopoietic malignancy in this group) have not been characterized. The purpose of this study was to determine the HLA class I and class II phenotypes of patients with multiple myeloma and to compare the findings to an ethnically matched control group of 100 individuals. Analysis of the HLA class I and class II phenotypes in 62 myeloma patients revealed: (i) a corresponding statistically significant association with HLA B18 [odds ratio (OR) 6.3; 95% confidence interval (CI) 1.013-39.727; P < 0.005]; (ii) no statistically significant association with HLA B13, Cw2, Cw6 or the DR and DQ antigens; and (iii) a statistically significant negative (protective) association with HLA Cw7 (OR 0.4; 95% CI 0.21-0.87; P < 0.005). This study suggests that although genetic factors may play a role in the multifactorial aetiology of multiple myeloma, with the exception of HLA B18, there is no specific association between HLA types and multiple myeloma in South African blacks.

Characterization and regulation of the major histocompatibility complex-encoded proteins Hsp70-Hom and Hsp70-1/2.

Vertebrate cells contain at least 12 different genes for Hsp70 proteins, 3 of which are encoded in the major histocompatibility complex (MHC) class III region. In the human MHC, these are named Hsp70-1, -2, and -Hom. To characterize these proteins, we have determined their substrate binding specificity, their cellular and tissue distribution, and the regulation of their expression. We show for the first time (1) peptide binding specificity of Hsp70-Hom; (2) endogenous expression of Hsp70-Hom in human cell lines; (3) cytoplasmic location of Hsp70-Hom protein under basal conditions and concentration in the nucleus after heat shock; (4) unique RNA expression profiles in human tissues for each of the MHC-encoded Hsp70s, significantly different from that for the constitutive Hsc70; (5) a relative increase in levels of Hsp70-Hom protein, compared with other Hsp70s, in response to interferon gamma; and (6) a specific increase on lipopolysaccharide (LPS) treatment of in vivo messenger RNA levels for the MHC-encoded Hsp70s and the DnaJ homologue, hdj2, relative to other chaperones. The unique tissue distributions and specific up-regulation by LPS of the MHC-encoded Hsp70s suggest some specialization of functions for these members of the Hsp70 family, possibly in the inflammatory response.

Impaired immunoproteasome assembly and immune responses in PA28-/- mice.

In vitro PA28 binds and activates proteasomes. It is shown here that mice with a disrupted PA28b gene lack PA28a and PA28b polypeptides, demonstrating that PA28 functions as a hetero-oligomer in vivo. Processing of antigenic epitopes derived from exogenous or endogenous antigens is altered in PA28-/- mice. Cytotoxic T lymphocyte responses are impaired, and assembly of immunoproteasomes is greatly inhibited in mice lacking PA28. These results show that PA28 is necessary for immunoproteasome assembly and is required for efficient antigen processing, thus demonstrating the importance of PA28-mediated proteasome function in immune responses.

HSP70 binding sites in the tumor suppressor protein p53.

Mutations within conserved regions of the tumor suppressor protein, p53, result in oncogenic forms of the protein with altered tertiary structures. In most cases, the mutant p53 proteins are selectively recognized and bound by members of the HSP70 family of molecular chaperones, but the binding site(s) in p53 for these chaperones have not been clearly defined. We have screened a library of overlapping biotinylated peptides, spanning the entire human p53 sequence, for binding to the HSP70 proteins, Hsc70 and DnaK. We show that most of the high affinity binding sites for these proteins map to secondary structure elements, particularly beta-strands, in the hydrophobic core of the central DNA binding domain, where the majority of oncogenic p53 mutations are found. Although peptides corresponding to the C-terminal region of p53 also contain potential binding sites, p53 proteins with C-terminal deletions are capable of binding to Hsc70, indicating that this region is not required for complex formation. We propose that mutations in the p53 protein alter the tertiary structure of the central DNA binding domain, thus exposing high affinity HSP70 binding sites that are cryptic in the wild-type molecule.

Common and divergent peptide binding specificities of hsp70 molecular chaperones.

We have studied the binding of synthetic peptides to three hsp70 molecular chaperones, DnaK, BiP, and hsc70, as a model for the interaction of hsp70 proteins with unfolded regions of target polypeptides. We measured the ability of 53 peptides to inhibit the formation of complexes between the hsp70 proteins and denatured lactalbumin. Peptides that bound with highest affinity to all three hsp70 proteins contained stretches of at least 7 residues that included large hydrophobic and basic amino acids, but few or no acidic residues. Amino acid substitutions within one heptameric peptide showed that an important feature for its binding to all three chaperones was a large hydrophobic residue in position 4, while specificity differences between the chaperones were revealed by substitutions at positions 2 and 6. Such specificity differences were frequently observed with other peptides, the most extreme example being a peptide rich in basic residues that bound with high affinity to DnaK, intermediate affinity to hsc70, and negligible affinity to BiP. Substitution of a lysine residue at position 2 in this peptide by tyrosine abolished the specificity difference by increasing the affinities of the DnaK and hsc70 proteins 5- and 20-fold, respectively, and that of BiP by greater than 2 orders of magnitude. Thus, hsp70 proteins can exhibit common or exclusive binding specificities, depending on the peptide sequence.

Peptide-dependent stimulation of the ATPase activity of the molecular chaperone BiP is the result of conversion of oligomers to active monomers.

The molecular chaperone BiP purified from bovine liver (bBiP) exhibits a low basal level of ATPase activity that can be stimulated 3-6-fold by synthetic peptides (Flynn, G. C., Chappell, T. G., and Rothman, J. E. (1989) Science 245, 385-390). By contrast, recombinant murine BiP (rBiP) purified to homogeneity following expression in Escherichia coli exhibits a higher basal level of ATPase activity and is much less stimulated by synthetic peptides. Nondenaturing gel electrophoresis showed that rBiP is predominantly monomeric, while bBiP exists in multiple forms probably corresponding to differentially modified monomeric, dimeric, and higher oligomeric species. Some, but not all, synthetic peptides cause conversion of the oligomeric and modified species of bBiP to a monomeric form. We propose that the peptide-dependent ATPase stimulation observed for BiP reflects the conversion of inactive oligomeric and/or modified species into active monomers.

Low-density lipoprotein receptor point mutation results in expression of both active and inactive surface forms of the same mutant receptor.

LDL receptors, expressed in cultured fibroblasts from patients homozygous for the FH Afrikaner-1 (FH1) mutation (Asp206 to Glu), are transported from the endoplasmic reticulum (ER) to the Golgi apparatus more slowly than in normal cells. In the present study, binding characteristics of FH1 cells for lipoprotein ligands (LDL and beta VLDL) and for receptor-specific monoclonal antibodies pointed to the existence of two surface forms of the same mutant receptor. One of these forms bound lipoproteins with normal high affinity whereas another did not. Binding studies of transfected hamster cells expressing only the mutant human gene confirmed the single-gene origin of the different forms. The existence of functionally distinct forms of the receptor protein was supported by the observation that only lipoprotein-binding receptor molecules were trapped intracellularly and degraded following ammonium chloride treatment of cells in the presence of ligand. The lipoprotein-binding receptor population was indistinguishable from normal receptors with respect to its affinity for LDL and beta VLDL, uptake and degradation of lipoprotein, and receptor recycling. Ligand blotting versus immunoblotting of receptors revealed normal-sized mutant receptors that were not recognized by lipoprotein ligand. Despite these differences, both mutant forms of the receptor were degraded at rates similar to those of normal receptors. We propose that the single amino acid substitution in this receptor interferes with the folding and/or posttranslational processing of precursor molecules in such a way that receptors adopt alternative stable structures.

Deletion of two growth-factor repeats from the low-density-lipoprotein receptor accelerates its degradation.

The region of the low-density-lipoprotein (LDL) receptor showing sequence similarity to the epidermal-growth-factor (EGF) precursor is required for LDL binding and the acid-induced dissociation of ligand and receptor. We describe here a naturally occurring mutant LDL receptor, found in a patient with homozygous familial hypercholesterolaemia, which lacks the first two growth-factor-like repeats of the EGF-precursor-like ('homology') domain. The mutation in the receptor gene is a 2.5 kb deletion including exons 7 and 8. The molecular mass of the mutant receptor (145 kDa) was approx. 15 kDa smaller than the normal LDL receptor. The mutant receptors were derived from precursors (105 kDa) that apparently underwent normal processing. Fibroblasts from the patient had high-affinity binding sites for the the apolipoprotein E-containing ligand, beta VLDL, but did not bind LDL. In the presence of beta VLDL, receptors were rapidly degraded. The mutant receptors also displayed an abnormally rapid turnover, about four times faster than that of normal receptors, in the absence of ligand; this accelerated degradation accounted for the low level of expression of mutant receptors in up-regulated cells. These data support a role for the growth-factor-like repeats in the binding of LDL (but not beta VLDL) and in receptor recycling, and indicate that a normal rate of turnover of unoccupied receptors is dependent on the integrity of these segments of the protein.

Deletion in the first cysteine-rich repeat of low density lipoprotein receptor impairs its transport but not lipoprotein binding in fibroblasts from a subject with familial hypercholesterolemia.

The ligand-binding domain of the low density lipoprotein (LDL) receptor is composed of seven cysteine-rich repeats, each approximately 40 amino acids long. Previous studies by van Driel et al. [van Driel, I. R., Goldstein, J. L., Sudhof, T. C. & Brown, M. S. (1987) J. Biol. Chem. 262, 17443-17449] showed that if the first repeat of the ligand-binding domain (encoded by exon 2) is deleted, the receptor fails to bind an anti-LDL receptor monoclonal antibody (IgG-C7) but continues to bind LDL with high affinity. Cultured fibroblasts from a Black South African Xhosa patient (TT) with the clinical syndrome of homozygous familial hypercholesterolemia demonstrated high-affinity cell-surface binding of 125I-labeled LDL but not 125I-labeled IgG-C7. Previous haplotype analysis, using 10 restriction fragment length polymorphic sites, suggested that the patient inherited two identical LDL receptor alleles. The polymerase chain reaction technique was used to selectively amplify exon 2 of the LDL receptor gene from this patient. Sequence analysis of the amplified fragment disclosed a deletion of six base pairs that removes two amino acids, aspartic acid and glycine, from the first cysteine-rich ligand binding repeat. The mutation creates a new PstI restriction site that can be used to detect the deletion. The existence of this mutant allele confirms that the epitope of IgG-C7 is located in the first cysteine-rich repeat and that this repeat is not necessary for LDL binding. The mutant gene produced a normally sized 120-kilodalton LDL receptor precursor protein that matured to the 160-kilodalton form at less than one-fourth the normal rate. Thus, deletion of two amino acids within the first cysteine-rich repeat retards receptor transport from the endoplasmic reticulum to the cell surface, in contrast to deletion of the entire first repeat, which has no effect on receptor maturation.

Two mutant low-density-lipoprotein receptors in Afrikaners slowly processed to surface forms exhibiting rapid degradation or functional heterogeneity.

Two distinct mutant low-density-lipoprotein receptors in South African Afrikaners exhibit retarded posttranslational processing to mature forms. One mutation gives rise to cell-surface receptors that are subject to abnormally rapid degradation, whereas the other is associated with a functionally heterogeneous surface population degraded at a normal rate.

The effect of cardiotoxin on (Ca2+ + Mg2+)-ATPase of the erythrocyte and sarcoplasmic reticulum.

The effects of cardiotoxin on the ATPase activity and Ca2+-transport of guinea pig erythrocyte and rabbit muscle sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase (E.C.3.6.1.3) were investigated. Erythrocyte (Ca2+ + Mg2+)-ATPase was inhibited by cardiotoxin in a time- and dose-dependent fashion and inhibition appears to be irreversible. Micromolar calcium prevented this inhibitory effect. Specificity for (Ca2+ + Mg2+)-ATPase inhibition by cardiotoxin was indicated since a homologous neurotoxin had no effect. Cardiotoxin did not affect (Ca2+ + Mg2+)-ATPase activity from sarcoplasmic reticulum, but Ca2+-transport was 50% inhibited. This inhibition was not due to an increased Ca2+-efflux and could be the result of an intramolecular uncoupling of ATPase activity from Ca2+-transport. Inhibition of Ca2+-transport by cardiotoxin could not be prevented by millimolar concentrations of Ca2+. It is suggested that the biological effects of cardiotoxin could be a consequence of inhibition of plasma membrane (Ca2+ + Mg2+)-ATPases.