The production of antibodies against nonnominal immunoglobulin allotypes in rheumatoid arthritis (RA) patients suggests that the immune system of these patients has been exposed to such foreign allotypes. The presence of nonnominal allotypes is, however, a genetic enigma. We searched for nucleotide sequences specific for nonnominal G3m<sup>g</sup> and G3m<sup>b</sup> copies in individuals homozygous for these alleles. Using a sensitive and specific nested polymerase chain reaction (PCR) method with genomic DNA from blood of 18 RA patients and 5 normal controls, we found G3m<sup>g</sup> sequences in 18 of 18 tested G3m<sup>b</sup> homozygous persons. The allele specificity of the PCR fragments was confirmed by sequencing and RFLP analysis. The PCR products contained genomic nonspliced parts of the nonnominal sequences. An analysis of cDNA from inflammatory tissue of 5 RA patients detected nonnominal G3m<sup>b</sup> sequences in 1 of 3 tested G3m<sup>g</sup> homozygotes and G3m<sup>g</sup> sequences in 2 of 2 tested G3m<sup>b</sup> homozygotes. The cDNA-derived PCR products contained sequences from normally spliced nonnominal Ig fragments. The results also showed that the nonnominal Ig sequences were present in very low copy numbers, lower than the Mendelian 1–2 copies per cell. The origin of such a low copy number of Ig gene fragments may be explained by a virus-mediated capture and transfer mechanism of Ig gene fragments generated by the normal Ig switch-associated gene excision process.
AIM: To study the cellular transport of L68Q cystatin C, the cystatin variant causing amyloidosis and brain haemorrhage in patients suffering from hereditary cystatin C amyloid angiopathy (HCCAA). METHODS: Expression vectors for wild-type and L68Q cystatin C were constructed and used to transfect mouse NIH/3T3 cells. Stable cell clones were isolated after cotransfection with pSV2neo. Clones expressing human wild-type and L68Q cystatin C were compared with respect to secreted cystatin C by enzyme linked immunosorbent assay (ELISA), and for intracellular cystatin C by western blotting and immunofluorescence cytochemistry. Colocalisation studies in cells were performed by double staining with antibodies against human cystatin C and marker proteins for lysosomes, the Golgi apparatus, or the endoplasmic reticulum, and evaluated by confocal microscopy. RESULTS: Concentrations of human cystatin C secreted from transfected NIH/3T3 cells were similar to those secreted from human cells in culture. In general, clones expressing the gene encoding L68Q cystatin C secreted slightly lower amounts of the protein than clones expressing wild-type human cystatin C. Both immunofluorescence cytochemistry and western blotting experiments showed an increased accumulation of cystatin C in cells expressing the gene encoding L68Q cystatin C compared with cells expressing the gene for the wild-type protein. The intracellularly accumulating L68Q cystatin C was insoluble and located mainly in the endoplasmic reticulum. CONCLUSIONS: The cellular transport of human cystatin C is impeded by the pathogenic amino acid substitution Leu68-->Gln. The resulting intracellular accumulation and increased localised concentration of L68Q cystatin C might be an important event in the molecular pathophysiology of amyloid formation and brain haemorrhage in patients with HCCAA.
Myocardial infarction and stroke are caused by blood clots forming over a ruptured or denuded atherosclerotic plaque (atherothrombosis). Production of prostaglandin E2 (PGE2) by an inflamed plaque exacerbates atherothrombosis and may limit the effectiveness of current therapeutics. Platelets express multiple G-protein coupled receptors, including receptors for ADP and PGE2. ADP can mobilize Ca2+ and through the P2Y12 receptor can inhibit cAMP production, causing platelet activation and aggregation. Clopidogrel (Plavix), a selective P2Y12 antagonist, prevents platelets from clotting but thereby increases the risk of severe or fatal bleeding. The platelet EP3 receptor for PGE2, like the P2Y12 receptor, also inhibits cAMP synthesis. However, unlike ADP, facilitation of platelet aggregation via the PGE2/EP3 pathway is dependent on co-agonists that can mobilize Ca2+. We used a ligand-based design strategy to develop peri-substituted bicylic acylsulfonamides as potent and selective EP3 antagonists. We show that DG-041, a selective EP3 antagonist, inhibits PGE2 facilitation of platelet aggregation in vitro and ex vivo. PGE2 can resensitize platelets to agonist even when the P2Y12 receptor has been blocked by clopidogrel, and this can be inhibited by DG-041. Unlike clopidogrel, DG-041 does not affect bleeding time in rats, nor is bleeding time further increased when DG-041 is co-administered with clopidogrel. This indicates that EP3 antagonists potentially have a superior safety profile compared to P2Y12 antagonists and represent a novel class of antiplatelet agents.
Cystatin C, an efficient inhibitor of cysteine proteinases, is present in all investigated human extracellular fluids. Dexamethasone caused a significant and dose-dependent increase in the cystatin C secretion of cultivated HeLa cells up to a maximal increase of 80% at 10‐6 mol 1‐1 dexamethasone. Increased production of cystatin C was also observed at lower concentrations, suggesting that glucocorticoids might play a physiological role in the production of cystatin C. The effect of dexamethasone on the cystatin C gene expression was also studied in a transient transfection expression system using chimeric plasmid constructs of the cystatin C gene promoter (positions -2 to -1084) coupled to the structural gene for human growth hormone (hGH). In this system, a small, but statistically significant, increase in hGH secretion was also observed upon dexamethasone treatment, suggesting that the glucocorticoid-induced increase in secretion of cystatin C is due to a promoter-mediated increase in transcription of the cystatin C gene.
Secretory type 2 cystatins, like cystatins C, E/M and F, are thought to be involved in many pathobiological processes, including vascular amyloidosis, rheumatoid arthritis, Alzheimers disease, osteoporosis, viral and bacterial infections, inflammatory disorders and tumour invasion and metastasis. In order to define the levels of cystatins C, E/M, and F in pleural effusions and to investigate whether these cystatins correlate with diagnostic parameters of pleural and lung diseases, we determined their concentrations in 160 pleural effusions. The median concentration of cystatin C in pleural effusions was 1437 ug/l (95.8 nM), ranging between 18-3967 ug/l. Cystatin C did neither correlate with malignant nor with benign diseases. The concentration of cystatin E/M was significantly higher in effusions of primary pleural tumours (mesotheliomas) compared to secondary pleural tumours and benign diseases. Furthermore, there was a significant correlation between the concentration of cystatin E/M of mesotheliomas and the pleural fluid tumour cell count and of cystatin C. The median values of cystatin F were significantly increased in parapneumonic/ empyema thoracis pleural effusions and tuberculous pleurisy compared to malignant pleural effusions, respectively. The concentration of cystatin F in benign effusions correlated significantly with diagnostic parameters and inflammation (total protein; lactate dehydrogenase; C-reactive protein). Finally, only in the group of parapneumonic/empyema thoracis was there a significant correlation between cystatin F and the neutrophil count. In conclusion, pleural effusions of different origin contain high levels of cystatin C, perhaps constituting the major part of an inhibitor reservoir. The level of cystatin E/M appears to be significantly associated with primary pleural tumours and cystatin F correlates with inflammatory processes of lung disorders.
We previously identified Neuregulin1 (NRG1) as a gene contributing to the risk of developing schizophrenia. Furthermore, we showed that NRG1+/- mutant mice display behavioral abnormalities that are reversed by clozapine, an atypical antipsychotic drug used for the treatment of schizophrenia. We now present evidence that ErbB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4), the tyrosine kinase receptor for NRG1 in hippocampal neurons, interacts with two nonreceptor tyrosine kinases, Fyn and Pyk2 (proline-rich tyrosine kinase 2). NRG1 stimulation of cells expressing ErbB4 and Fyn leads to the association of Fyn with ErbB4 and consequent activation. Furthermore, we show that NRG1 signaling, through activation of Fyn and Pyk2 kinases, stimulates phosphorylation of Y1472 on the NR2B subunit of the NMDA receptor (NMDAR), a key regulatory site that modulates channel properties. NR2B Y1472 is hypophosphorylated in NRG1+/- mutant mice, and this defect can be reversed by clozapine at a dose that reverses their behavioral abnormalities. We also demonstrate that short-term synaptic plasticity is altered and theta-burst long-term potentiation is impaired in NRG1+/- mutant mice, and incubation of hippocampal slices from these mice with NRG1 reversed those effects. Attenuated NRG1 signaling through ErbB4 may contribute to the pathophysiology of schizophrenia through dysfunction of NMDAR modulation. Thus, our data support the glutamate hypothesis of schizophrenia.
AbstractA variant of the normal extracellular cysteine protease inhibitor cystatin C (L68Q-cystatin C), is the amyloid precursor in hereditary cystatin C amyloid angiopathy (HCCAA). It has been suggested that the mutation causes cellular entrapment ofL68Q-cystatin C in vivo and that the variant protein is not secreted to extracellular fluids. In order to test this hypothesis, we used matrix-assisted laser desorption ionization time-of-flight mass spectrometry in an effort to demonstrate the presence ofL68Q- along with wildtype cystatin C in plasma and cerebrospinal fluid (CSF) of HCCAA-patients. Plasma from all five investigated HCCAA-patients contained both L68Q- and wildtype cystatin C. The presence of approximately equal amounts of cystatin C dimers and monomers was demonstrated in plasma from HCCAA-patients, whereas only monomers could be found in normal plasma. L68Q-wildtype-cystatin C heterodimers seem to be present in the dimeric cystatin C population. CSF from six HCCAA-patients also contained cystatin C-dimers and monomers, but the dimeric fraction was minute. CSF from control patients did not contain dimeric cystatin C These results suggest that the milieu of L68Q-cystatin C is important for its stability and dimerization status and that certain milieus might hinder its further development into oligomers, amyloid fibrils and other precipiting aggregates.Key Words: amyloidosiscerebral hemorrhagecystatin CHCCAAMALDI-TOFMSmass spectrometry
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