Molecular characterization of the interleukin-1 receptor (IL-1R) on monocytes and polymorphonuclear cells.

Primary human monocytes and monocytic cells express an interleukin 1 receptor (IL-1R) which is similar in molecular weight and IL-1 binding characteristics to the IL-1R expressed on B lymphocytes (type II). Northern blot analysis of monocytic cells using a cDNA probe from the recently isolated type II IL-1R indicates that this mRNA is detectable by 4 h and accumulates for at least 24 h following treatment with IL-1R inducing drugs. The time course of induction of this mRNA is slower than that of the type I IL-1R mRNA which is also transcribed in monocytic cells but does not appear to be translated. Sequence analysis of a monocyte-derived cDNA corresponding to the type II IL-1R mRNA shows that the monocyte and B-cell mRNAs are identical. Comparison of monocyte IL-1R peptide maps with those of the type II IL-1R suggests that the two surface IL-1R are identical. This was confirmed serologically using a polyclonal antiserum raised against the type II IL-1R. Data are presented which indicate that primary human neutrophils can also be induced to express abundant type II IL-1R.     

Segregation Analyses and Gene-Centromere Distances in Zebrafish

The gol-1, gol-2, alb-1 and spa-1 mutations affect pigment pattern in the zebrafish. We show here that these loci are unlinked to each other. In addition, gene-centromere distances were determined for these loci by analysis of half-tetrads obtained by the inhibition of the second meiotic division. The fractions of tetratype (second-division segregation) tetrads range from 0.24 (spa-1) to 0.89 (gol-1). The observation of greater than 0.67 second-division segregation indicates that the zebrafish has high chiasma interference.     

Affinity purification and chemical analysis of the interleukin-1 receptor

Interleukins-1 alpha and -1 beta regulate the metabolism of cells through a common plasma membrane receptor protein. In this study, it is demonstrated that the interleukin-1 (IL-1) receptor from detergent solutions of EL-4 cells can be stably adsorbed to nitrocellulose with full retention of IL-1 binding activity. This assay system was used to monitor the purification of the IL-1 receptor and to investigate the effects of several chemical modifications on receptor binding activity. IL-1 receptors extracted from EL-4 6.1 C10 cells can be bound to and specifically eluted from IL-1 alpha coupled to Sepharose. The affinity chromatography method resulted in the identification by polyacrylamide gel electrophoresis and silver staining of a protein of Mr 82,000 that was present in fractions exhibiting IL-1 binding activity. Experiments in which the cell-surface proteins of EL-4 cells were radiolabeled and 125I-labeled receptor was purified by affinity chromatography suggested that the Mr 82,000 protein was expressed on the plasma membrane. N-Glycanase treatment of this material showed that 23-35% of the total Mr (82,000) of the receptor is N-linked carbohydrate.     

Retention of ligand binding activity by the extracellular domain of the IL-1 receptor

The IL-1R on murine T cells is an 80-kDa cell surface glycoprotein which binds both IL-1 alpha and IL-1 beta. We have recently isolated a cDNA clone encoding this molecule. From the primary sequence mature receptor is predicted to be a 557 residue integral membrane protein with a 319 residue carbohydrate-rich extracellular region. We have constructed a cDNA clone encoding this region of the protein (residues 1 to 316). Expression of this cDNA in HeLa cells leads to secretion of a soluble IL-1 alpha binding protein into the culture medium. Quantitative binding experiments with the truncated receptor show that it possesses IL-1 binding properties which are indistinguishable from those of full length IL-1R. Gel filtration chromatography experiments show that a complex can be formed between a single truncated receptor molecule and a single IL-1 alpha molecule.     

Human beta-casein. Amino acid sequence and identification of phosphorylation sites

The primary structure of human beta-casein has been determined by automated Edman degradation of the intact protein and of peptides derived therefrom by hydrolysis with trypsin and by chemical cleavage with cyanogen bromide. For each form of this multiphosphorylated protein (0-5 P/molecule), phosphorylated sites at specific seryl and threonyl residues have been identified. These are located near the amino terminus, within the first 10 residues of this 212-amino acid molecule. Sequence comparison of human beta-casein with the bovine and ovine proteins reveals 50% identity and a 10-residue shifted alignment relationship. Locations of prolyl and charged residues are generally conserved for the three homologues. The sequence data indicate the existence of genetic polymorphism involving uncharged residues in human beta-casein.     

On the PAM matrix model of protein evolution

The internal consistency of the PAM matrix model of protein evolution is here investigated. The 1 PAM matrix has been constructed from amino acid replacements observed in closely related sequences. Such replacements are of two types, those that do not require an intermediate amino acid replacement and those that do. The second type of replacement must generally be produced by a repetition of the first. This allows data on the first type to be used in predicting data on the second type so that some elements of the 1 PAM matrix may be used to predict others. A discrepancy of more than two orders of magnitude is found between the predictions and the data when this is carried out. This is partly accounted for by an error in constructing the matrix. However, it also seems necessary that the basic model be modified. Several possibilities are considered. One of these is to incorporate a site-dependent spectrum of mutabilities associated with each amino acid.      

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.     

Feedback loops in intracellular signal processing and their potential for identifying novel signalling proteins

Mapping of intracellular signalling networks has attracted a lot of attention during recent years. Approaches which have been used to characterise functional and physical relationships within signalling systems and to identify novel components have included bioinformatics, genomics, proteomics and genetics. However, the importance of feedback loops in the regulation of signal processing systems has often been ignored. Here we report a novel focus for our previous screening platform, which evaluates the potential of autocatalytic feedback for functional screening of cDNA expression libraries. We confirm that the use of such systems is feasible. We propose that the use of feedback loops in functional screens may introduce a bias towards the detection of signalling molecules, which are likely to be of key regulatory importance due to the sensitivity of the system towards their expression levels.