The murine chaperonin 10 gene family contains an intronless, putative gene for early pregnancy factor, Cpn10-rs1

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. Human platelet-derived EPF shares amino acid sequence identity with chaperonin 10 (Cpn10), a mitochondrial matrix protein which functions as a molecular chaperone. The striking differences in cellular localization and function of the two proteins suggest differential regulation of production reflecting either alternative transcription of the same gene or transcription from different genes. In mammals and more distantly related genera, there is a large gene family with homology to CPN 10 cDNA, which includes intronless copies of the coding sequence. To determine whether this could represent the gene for EPF, we have screened a mouse genomic library and sequenced representative Cpn10 family members, looking for a functional gene distinct from that of Cpn 10, which could encode EPF. Eight distinct genes were identified. Cpn10 contains introns, while other members are intronless. Six of these appear to be pseudogenes, and the remaining member, Cpn10-rs1, would encode a full-length protein. The 309-bp open reading frame (ORF) is identical to that of mouse Cpn10 cDNA with the exception of three single-base changes, two resulting in amino acid changes. Only one further single nucleotide difference between the Cpn10-rs1 and Cpn10 cDNAs is observed, located in the 3' UTR. Single nucleotide primer extension was applied to discriminate between Cpn10-rs1 and Cpn10 expression. Cpn10, which is ubiquitous, was detected in all tissue samples tested, whereas Cpn10-rs1 was expressed selectively. The pattern was completely coincident with known patterns of EPF activity, strongly suggesting that Cpn10-rs1 does encode EPF. The complete ORF of Cpn10-rs1 was expressed in E. coli. The purified recombinant protein was found to be equipotent with native human platelet-derived EPF in the bioassay for EPF, the rosette inhibition test.

The effects of pregnancy on myelin basic protein-induced experimental autoimmune encephalomyelitis in Lewis rats: Suppression of clinical disease, modulation of cytokine expression in the spinal cord inflammatory infiltrate and suppression of lymphocyte p

Problem: The present study was performed to explore the effects of pregnancy on experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats by inoculation with myelin basic protein (MBP) (MBP-EAE). Method of study: MBP-EAE was induced in pregnant and non-pregnant rats and severity of disease evaluated. Serum from pregnant and non-pregnant rats was used in standard lymphocyte proliferation assays. Real-time polymerase chain reaction (PCR) was used to investigate the expression of cytokine mRNA in the inflammatory cells obtained from the spinal cord of rats on day 15 after inoculation. Results: Pregnant rats developed less severe disease than non-pregnant rats. Serum from pregnant rats suppressed the proliferation of T lymphocytes in response to MBP. There was significantly increased expression of IL-4. IL-10 and TNF-alpha mRNA in the spinal cord infiltrate of pregnant rats. Conclusion: Circulating humoral factors and alteration in cytokine production by inflammatory cells may contribute to the suppression of EAE in pregnant rats.

Chronic hypoxia induced down-regulation of angiotensinogen expression in rat epididymis

The presence of an intrinsic renin-angiotensin system (RAS) in the rat epididymis has been previously established by showing the expression of several key RAS components, and in particular angiotensinogen, the indispensable element for the intracellular generation of angiotensin II. In this study, the possible involvement of this local epididymal RAS in the testicular effects of chronic hypoxia was investigated. Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR), Western blotting and by in situ hybridization histochemistry of the rat epididymis were used to show changes in localization and expression of angiotensinogen. Results from RT-PCR analysis demonstrated that chronic hypoxia caused a marked decrease (60%) in the expression of angiotensinogen mRNA, when compared with that in the normoxic epididymis. Western blot analysis demonstrated a less decrease (35%) in the expression of angiotensinogen protein. In situ hybridization histochemistry showed that the reduced angiotensinogen mRNA in chronic hypoxia was specifically localized to the epididymal epithelium from the cauda, corpus and caput regions of the epididymis; a distribution similar to that of normoxic rats. It was concluded that chronic hypoxia decreases the transcriptional and translational expression of angiotensinogen, and thus local formation of angiotensin II, in the rat epididymis. (C) 2001 Elsevier Science B.V. All rights reserved.

Inhibition of lipid raft-dependent signaling by a dystrophy-associated mutant of caveolin-3

Specific point mutations in caveolin-3, a predominantly muscle-specific member of the caveolin family, have been implicated in limb-girdle muscular dystrophy and in rippling muscle disease. We examined the effect of these mutations on caveolin-3 localization and function. Using two independent assay systems, Raf activation in fibroblasts and neurite extension in PC12 cells, we show that one of the caveolin-3 point mutants, caveolin-3-C71W, specifically inhibits signaling by activated H-Ras but not by K-Ras. To gain insights into the effect of the mutant protein on H-Ras signaling, we examined the localization of the mutant proteins in fibroblastic cells and in differentiating myotubes. Unlike the previously characterized caveolin-3-DGV mutant, the inhibitory caveolin-3-C71W mutant reached the plasma membrane and colocalized with wild type caveolins. In BHK cells, caveolin-3-C71W associated with caveolae and in differentiating muscle cells with the developing T-tubule system. In contrast, the caveolin-3-P104L mutant accumulated in the Golgi complex and had no effect on H-Ras-mediated Raf activation. Inhibition by caveolin-3-C71W was rescued by cholesterol addition, suggesting that the mutant protein perturbs cholesterol-rich raft domains. Thus, we have demonstrated that a naturally occurring caveolin-3 mutation can inhibit signaling involving cholesterol-sensitive raft domains.

RelB nuclear translocation mediated by C-terminal activator reigons of Epstein Barr virus-encoded latent membrane protein 1 and its effect on antigen-presenting function in B cells

Previous studies have shown that Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) is uniquely able to up-regulate the expression of the peptide transporters (referred to as TAP-1 and TAP-2) and major histocompatibility complex (MHC) class I in Burkitt's lymphoma (BL) cell lines. This up-regulation is often accompanied by a restoration of antigen-presenting function as measured by the ability of these cells to present endogenously expressed viral antigen to cytotoxic T lymphocytes. Here we show that the expression of LMP1 resulted in up-regulation and nuclear translocation of RelB that were coincident with increased expression of MHC class I in BL cells. Deletion of the C-terminal activator regions (CTARs) of LMP1 significantly impaired the abilities of LMP1 to translocate RelB into the nucleus and to up-regulate the expression of antigen-processing genes. Further analysis with single-point mutations within the CTARs confirmed that the residues critical for NF-kappaB activation directly contribute to antigen-processing function regulation in BL cells. This LMP1-mediated effect was blocked following expression of either dominant negative IkappaBalpha S32/36A, an NF-kappaB inhibitor, or antisense RelB. These observations indicate that upregulation of antigen-presenting function in B cells mediated by LMP1 is signaled through the NF-kappaB subunit RelB. The data provide a mechanism by which LMP1 modulates immunogenicity of Epstein-Barr virus-infected normal and malignant cells.

Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection

Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV Proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles. At times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.

Limitations of HLA-transgenic mice in presentation of HLA-restricted cytotoxic T-cell epitopes from endogenously processed human papillomavirus type 16 E7 protein

We investigated the use of mice transgenic for human leucocyte antigen (HLA) A*0201 antigen-binding domains to test vaccines composed of defined HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitopes of human papillomavirus (HPV) type 16 E7 oncoprotein. HPV is detected in >90% of cervical carcinomas. HPV16 E7 oncoprotein transforms cells of the uterine cervix and functions as a tumour-associated antigen to which immunotherapeutic strategies may be directed. We report that although the HLA A*0201 E7 epitope peptides function both to prime for E7 CTL responses, and to sensitize target cells for E7-directed CTL killing in situations where antigen processing is not required, the epitopes are not processed out of either endogenously expressed or immunization-introduced E7, by the mouse antigen-processing and presentation machinery. Thus (1) CTL induced by HLA A*0201 peptide immunization killed E7 peptide-pulsed target cells, but did not kill target cells expressing whole E7; (2) immunization with whole E7 protein did not elicit CTL directed to HLA A*0201-restricted E7 CTL epitopes; (3) HLA A*0201-restricted CTL epitopes expressed in the context of a DNA polytope vaccine did not activate E7-specific T cells either in 'conventional' HLA A*0201 transgenic (A2.1K(b) ) mice, or in HHD transgenic mice in which expression of endogenous H-2 class 1 is precluded; and (4) HLA A*0201 E7 peptide epitope immunization was incapable of preventing the growth of an HLA A*0201- and E7-expressing tumour. There are generic implications for the universal applicability of HLA-class 1 transgenic mice for studies of human CTL epitope presentation in murine models of human infectious disease where recognition of endogenously processed antigen is necessary. There are also specific implications for the use of HLA A2 transgenic mice for the development of E7-based therapeutic vaccines for cervical cancer.

Accumulation of human heat shock protein 60-reactive T cells in the gingival tissues of periodontitis patients

Heat shock protein 60s (hsp60) are remarkably immunogenic, and both T-cell and antibody responses to hsp60 have been reported in various inflammatory conditions. To clarify the role of hsp60 in T-cell responses in periodontitis, we examined the proliferative response of peripheral blood mononuclear cells (PBMC), as well as the cytokine profile and T-cell clonality, for periodontitis patients and controls following stimulation with recombinant human hsp60 and Porphyromonas gingivalis GroEL. To confirm the infiltration of hsp60-reactive T-cell clones into periodontitis lesions, nucleotide sequences within complementarity-determining region 3 of the T-cell receptor (TCR) beta-chain were compared between hsp60-reactive peripheral blood T cells and periodontitis lesion-infiltrating T cells. Periodontitis patients demonstrated significantly higher proliferative responses of PBMC to human hsp60, but not to P. gingivalis GroEL, than control subjects. The response was inhibited by anti-major histocompatibility complex class 11 antibodies. Analysis of the nucleotide sequences of the TCR demonstrated that human hsp60-reactive T-cell clones and periodontitis lesion-infiltrating T cells have the same receptors, suggesting that hsp60-reactive T cells accumulate in periodontitis lesions. Analysis of the cytokine profile demonstrated that hsp60-reactive PBMC produced significant levels of gamma interferon (IFN-gamma) in periodontitis patients, whereas P. gingivalis GroEL did not induce any, skewing toward a type1 or type2 cytokine profile. In control subjects no significant expression of IFN-gamma or interleukin 4 was induced. These results suggest that periodontitis patients have human hsp60-reactive T cells with a type I cytokine profile in their peripheral blood T-cell pools.

Expression of a truncated Brca1 protein delays lactational mammary development in transgenic mice

To address the hypothesis that certain disease-associated mutants of the breast-ovarian cancer susceptibility gene BRCA1 have biological activity in vivo, we have expressed a truncated Brca1 protein (trBrca1) in cell-lines and in the mammary gland of transgenic mice. Immunofluorescent analysis of transfected cell-lines indicates that trBRCA1 is a stable protein and that it is localized in the cell cytoplasm. Functional analysis of these cell-lines indicates that expression of trBRCA1 confers an increased radiosensitivity phenotype on mammary epithelial cells, consistent with abrogation of the BRCA1 pathway. MMTV-trBrca1 transgenic mice from two independent lines displayed a delay in lactational mammary gland development, as demonstrated by altered histological profiles of lobuloalveolar structures. Cellular and molecular analyses indicate that this phenotype results from a defect in differentiation, rather than altered rates of proliferation or apoptosis. The results presented in this paper are consistent with trBrca1 possessing dominant-negative activity and playing an important role in regulating normal mammary development. They may also have implications for germline carriers of BRCA1 mutations.

Myelin proteolipid protein - the first 50 years

Myelin proteolipid protein (PLP), the most abundant protein of central nervous system (CNS) myelin, is a hydrophobic integral membrane protein. Because of its physical properties, which make it difficult to work with, progress towards determining the exact function(s) and disease associations of myelin PLP has been slow. However, recent molecular biology advances have given new life to investigations of PLP, and suggest that it has multiple functions within myelin and is of importance in several neurological disorders. (C) 2002 Elsevier Science Ltd. All rights reserved.