The HLA class II genotypes were determined in the B-lymphoblastoid cell lines selected for the Tenth International Histocompatibility Workshop. The HLA class II genes were determined by the PCR-SSOP method using the reagents provided by the Eleventh Histocompatibility Workshop. Additional studies have been performed for further characterization of HLA class II polymorphism on these cell lines. It is observed that several cell lines have HLA class II haplotypes with the same DRB1, DQA1 and DQB1 alleles on both haplotypes but different alleles at the other class II loci, confirming that these cell lines are not truly HLA class II-homozygous. Other cell lines carried HLA class II haplotypes which were only different at the DRB1 gene. These results suggest double recombination events or gene conversion-like events in generation of HLA DR, DQ haplotypes. These cell lines provide an important tool as references for HLA DNA typing.
DNA fragmentation factor (DFF) functions downstream of caspase-3 and directly triggers DNA fragmentation during apoptosis. Here we described the identification and characterization of DFF35, an isoform of DFF45 comprised of 268 amino acids. Functional assays have shown that only DFF45, not DFF35, can assist in the synthesis of highly active DFF40. Using the deletion mutants, we mapped the function domains of DFF35/45 and demonstrated that the intact structure/conformation of DFF45 is essential for it to function as a chaperone and assist in the synthesis of active DFF40. Whereas the amino acid residues 101-180 of DFF35/45 mediate its binding to DFF40, the amino acid residues 23-100, which is homologous between DFF35/45 and DFF40, may function to inhibit the activity of DFF40. In contrast to DFF45, DFF35 cannot work as a chaperone, but it can bind to DFF40 more strongly than DFF45 and can inhibit its nuclease activity. These findings suggest that DFF35 may function in vivo as an important alternative mechanism to inhibit the activity of DFF40 and further, that the inhibitory effects of both DFF35 and DFF45 on DFF40 can put the death machinery under strict control.Apoptosis is fundamentally important in a variety of physiological and pathological processes. Apoptotic cells undergo an orchestrated cascade of events characterized by distinct morphological changes including membrane blebbing, cytoplasmic and nuclear condensation, chromatin aggregation, and formation of apoptotic bodies (1, 2). Activation of the caspase cascade is a key molecular event in the process of apoptosis (3, 4). Apoptotic signals, including growth factor and interleukin deprivation, activation of Fas, ionizing radiation, and a series of chemicals acting as upstream signals, can convert the precursors of caspases into the active enzymes (2). Several important downstream substrates of caspase, such as gelsolin (5), p21-activated kinase-2 (PAK-2) (6), and DNA fragmentation factor 45 (DFF45) 1 (7), whose cleavages clearly induce specific well characterized steps in apoptosis, have been recently identified. The cleavage of chromatin into the nucleosomal fragments, which distinguishes apoptosis from oncosis and necrosis, is a key element in the cell death process and is believed to be mediated by Mg 2ϩ /Ca 2ϩ required and Zn 2ϩ -sensitive nuclease (8 -12).We have previously identified a triplet of nuclease proteins named NP 42-50 that causes DNA degradation in vitro when cells undergo apoptosis (13). The similarity in molecular weight and biochemical characteristics between NP 42-50 and the recently identified DFF40 led us to further investigate these molecules. DFF is a heterodimeric protein composed of DFF45 and DFF40 subunits. DFF45 has been found to be the substrate of caspase-3, and DFF40 has also been cloned and found to be a DNA fragmentation nuclease (7,14,15). Cleavage of the DFF45 by caspase-3 during apoptosis releases DFF40 that degrades chromosomal DNA into nucleosomal fragments. Similar findings have also been described recently in the...
SUMMARYCD26, a T-cell activation antigen that has dipeptidyl peptidase IV activity in its extracellular domain and has also been shown to play an important role in T-cell activation. The earliest biochemical events seen in stimulated T lymphocytes activated through the engagement of the T-cell receptor (TCR) is the tyrosine phosphorylation of a panel of cellular proteins. In this study we demonstrate that antibody-induced cross-linking of CD26 in CD26-transfected Jurkat cells induced tyrosine phosphorylation of several intracellular proteins with a similar pattern to that seen after TCR/CD3 stimulation. Herbimycin A, an inhibitor of the src family protein tyrosine kinases dramatically inhibited this CD26-mediated effect on tyrosine phosphorylation. Major tyrosine phosphorylated proteins were identified by immunoblotting, and included p56lck, p59fyn, zeta associated protein-tyrosine kinase of 70 000 MW (ZAP-70), mitogen-activated protein ( MAP) kinase, c-Cbl, and phospholipase Cc. CD26-induced tyrosine phosphorylation of MAP kinase correlated with increased MAP kinase activity. In addition, CD26 was costimulatory to CD3 signal transduction since co-cross-linking of CD26 and CD3 antigens induced prolonged and increased tyrosine phosphorylation in comparison with CD3 activation alone. We therefore conclude that CD26 is a true costimulatory entity that can up-regulate the signal transducing properties of the TCR.
We have typed 64 Japanese patients with mixed connective tissue disease (MCTD) and 53 Japanese patients with systemic lupus erythematosus (SLE) for HLA-DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, and DPB1 genes by the HLA-DNA typing method using the PCR-SSOP technique. Frequencies of HLA-DRB1*0401, DRB1*0901, DRB4*0101, and DQA1*03 were increased and those of HLA-DRB1*0405 and DQB1*0401 were decreased in the patients with MCTD, while the frequencies of HLA-DRB1*1501, DRB5*0101, and DQB1*0602 were increased in the patients with SLE. The typing results suggest that susceptibility to MCTD is strongly associated with the HLA-DRB1*0401-DRB4*0101-DQA1*03-DQB1*0301 haplotype, and that to SLE is associated with the HLA-DRB1*1501-DRB5*0101-DQA1*0102-DQB1*0602 haplotype. The observation that the MCTD-associated HLA alleles are distinct from the SLE-associated ones may support the clinical entity of MCTD different from SLE.
Multiple myeloma is a disease in which angiogenesis is postulated to be a target for therapy. Based on this hypothesis, we conducted a phase II trial of ZD6474 (Zactima; a VEGFR inhibitor) 100 mg p.o. daily in patients with relapsed multiple myeloma. The primary efficacy endpoint was objective response as assessed by reduction in M protein. There were 18 patients with a mean age of 64 years. One patient was ineligible and one was not evaluable. Overall, ZD6474 was well tolerated and pharmacokinetic testing demonstrated that adequate drug levels were achieved. The most common drug-related adverse events were nausea, vomiting, fatigue, rash, pruritus, headache, diarrhea, dizziness, and sensory neuropathy, all of which were Grade I-II in severity. There were no drug-related serious adverse events. Laboratory adverse events were infrequent: one patient had Grade III anemia, and there were no Grade III changes in biochemistry. No significant QTc interval changes were seen. There were no responses in M protein levels. In conclusion, ZD6474 was well tolerated at a dose of 100 mg per day and achieved plasma levels predicted to inhibit VEGF signaling. However, this was not reflected in clinical benefit since none of the patients had a reduction in M protein.
These results indicate that terfenadine has a specific inhibitory effect on TH2-type cytokine production induced by several ways of costimulatory activation.
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