Site-specific phosphorylation of synapsin I by Ca2+/calmodulin-dependent protein kinase II in pancreatic betaTC3 cells: synapsin I is not associated with insulin secretory granules.
(25/9318)
Increasing evidence supports a physiological role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in the secretion of insulin from the pancreatic beta-cell, but the precise sites of action are not known. A role of this enzyme in neuroexocytosis is implicated by its phosphorylation of a vesicle-associated protein, synapsin I. Because of emerging similarities to the neuron with respect to exocytotic mechanisms, the expression and phosphorylation of synapsin I in the beta-cell have been studied. Synapsin I expression in clonal mouse beta-cells (betaTC3) and primary rat islet beta-cells was initially confirmed by immunoblot analysis. By immunoprecipitation, in situ phosphorylation of synapsin I was induced in permeabilized betaTC3 cells within a Ca2+ concentration range shown to activate endogenous CaM kinase II under identical conditions. Proteolytic digests of these immunoprecipitates revealed that calcium primarily induced the increased phosphorylation of sites identified as CaM kinase II-specific and distinct from protein kinase A-specific sites. Immunofluorescence and immunogold electron microscopy verified synapsin I expression in betaTC3 cells and pancreatic slices but demonstrated little if any colocalization of synapsin I with insulin-containing dense core granules. Thus, although this study establishes that synapsin I is a substrate for CaM kinase II in the pancreatic beta-cell, this event appears not to be important for the mobilization of insulin granules. (+info)
Control of replicative life span in human cells: barriers to clonal expansion intermediate between M1 senescence and M2 crisis.
(26/9318)
The accumulation of genetic abnormalities in a developing tumor is driven, at least in part, by the need to overcome inherent restraints on the replicative life span of human cells, two of which-senescence (M1) and crisis (M2)-have been well characterized. Here we describe additional barriers to clonal expansion (Mint) intermediate between M1 and M2, revealed by abrogation of tumor-suppressor gene (TSG) pathways by individual human papillomavirus type 16 (HPV16) proteins. In human fibroblasts, abrogation of p53 function by HPVE6 allowed escape from M1, followed up to 20 population doublings (PD) later by a second viable proliferation arrest state, MintE6, closely resembling M1. This occurred despite abrogation of p21(WAF1) induction but was associated with and potentially mediated by a further approximately 3-fold increase in p16(INK4a) expression compared to its level at M1. Expression of HPVE7, which targets pRb (and p21(WAF1)), also permitted clonal expansion, but this was limited predominantly by increasing cell death, resulting in a MintE7 phenotype similar to M2 but occurring after fewer PD. This was associated with, and at least partly due to, an increase in nuclear p53 content and activity, not seen in younger cells expressing E7. In a different cell type, thyroid epithelium, E7 also allowed clonal expansion terminating in a similar state to MintE7 in fibroblasts. In contrast, however, there was no evidence for a p53-regulated pathway; E6 was without effect, and the increases in p21(WAF1) expression at M1 and MintE7 were p53 independent. These data provide a model for clonal evolution by successive TSG inactivation and suggest that cell type diversity in life span regulation may determine the pattern of gene mutation in the corresponding tumors. (+info)
Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source.
(27/9318)
Myoblasts, the precursors of skeletal muscle fibers, can be induced to withdraw from the cell cycle and differentiate in vitro. Recent studies have also identified undifferentiated subpopulations that can self-renew and generate myogenic cells (Baroffio, A., M. Hamann, L. Bernheim, M.-L. Bochaton-Pillat, G. Gabbiani, and C.R. Bader. 1996. Differentiation. 60:47-57; Yoshida, N., S. Yoshida, K. Koishi, K. Masuda, and Y. Nabeshima. 1998. J. Cell Sci. 111:769-779). Cultured myoblasts can also differentiate and contribute to repair and new muscle formation in vivo, a capacity exploited in attempts to develop myoblast transplantation (MT) for genetic modification of adult muscle. Our studies of the dynamics of MT demonstrate that cultures of myoblasts contain distinct subpopulations defined by their behavior in vitro and divergent responses to grafting. By comparing a genomic and a semiconserved marker, we have followed the fate of myoblasts transplanted into muscles of dystrophic mice, finding that the majority of the grafted cells quickly die and only a minority are responsible for new muscle formation. This minority is behaviorally distinct, slowly dividing in tissue culture, but rapidly proliferative after grafting, suggesting a subpopulation with stem cell-like characteristics. (+info)
Hypermutation in Ig V genes from mice deficient in the MLH1 mismatch repair protein.
(28/9318)
During somatic hypermutation of Ig V genes, mismatched nucleotide substitutions become candidates for removal by the DNA mismatch repair pathway. Previous studies have shown that V genes from mice deficient for the MSH2 and PMS2 mismatch repair proteins have frequencies of mutation that are comparable with those from wild-type (wt) mice; however, the pattern of mutation is altered. Because the absence of MSH2 and PMS2 produced different mutational spectra, we examined the role of another protein involved in mismatch repair, MLH1, on the frequency and pattern of hypermutation. MLH1-deficient mice were immunized with oxazolone Ag, and splenic B cells were analyzed for mutations in their V kappa Ox1 light chain genes. Although the frequency of mutation in MLH1-deficient mice was twofold lower than in wt mice, the pattern of mutation in Mlh1-/- clones was similar to wt clones. These findings suggest that the MLH1 protein has no direct effect on the mutational spectrum. (+info)
In vivo and in vitro activation of T cells after administration of Ag-negative heat shock proteins.
(29/9318)
Heat shock proteins (HSP) Hsp70 and gp96 prime class I-restricted cytotoxic T cells against Ags present in the cells from which they were isolated. The immunization capacity of HSPs is believed to rely on their ability to bind antigenic peptides. In this study, we employed the well-established OVA and beta-galactosidase (beta-gal) antigenic model systems. We show that in vitro long-term established OVA and beta-gal-specific CTL clones release TNF-alpha and IFN-gamma when incubated with Ag-negative Hsp70 and gp96. In the absence of antigenic peptides, HSP-mediated secretion of TNF-alpha and IFN-gamma requires cell contact of the APC with the T cell but is not MHC-I restricted. Moreover, Hsp70 molecules purified from Ag-negative tissue, e.g., negative for antigenic peptide, are able to activate T cells in vivo, leading to significant higher frequencies in OVA-specific CD8+ T cells. In unprimed animals, these T cells lyse OVA-transfected cell lines and produce TNF-alpha and IFN-gamma after Ag stimulus. Taken together our data show that, besides the well-established HSP/peptide-specific CTL induction and activation, a second mechanism exists by which Hsp70 and gp96 molecules activate T cells in vivo and in vitro. (+info)
Inflammatory cytokines provide a third signal for activation of naive CD4+ and CD8+ T cells.
(30/9318)
The effects of inflammatory cytokines on naive T cells have been studied using MHC protein/peptide complexes on microspheres, thus avoiding the use of APCs whose functions may be affected by the cytokines. IL-1, but not IL-12, increased proliferation of CD4+ T cells in response to Ag and IL-2, which is consistent with effects on in vivo priming of CD4+ cells. In contrast, proliferation of CD8+ T cells to Ag and IL-2 required IL-12, and IL-12 replaced adjuvant in stimulating an in vivo response to peptide. These results support a model in which distinct inflammatory cytokines act directly on naive CD4+ and CD8+ T cells to provide a third signal, along with Ag and IL-2, to optimally activate differentiation and clonal expansion. (+info)
Fine specificity and MHC restriction of trinitrophenyl-specific CTL.
(31/9318)
In this study, the fine specificity and MHC restriction of a CTL response specific to the trinitrophenyl (TNP) hapten was analyzed. Based on the structure of peptide/Kb complexes and ternary TCR/Ag/MHC complexes, four TNP peptides, two octamers, and two nonamers were chosen for eliciting anti-TNP CTL responses. Hapten was conjugated at position 4 in the octamers and at position 5 in the nonamers, positions which should allow engagement of the hapten by TCRs. Potent CTL activity for each of the TNP peptides was obtained that was highly hapten-specific; however, there were considerable differences in the extent of cross-reactivity with other TNP peptides, with the octamers generating more cross-reactive CTL than the nonamers. MHC restriction analysis suggested that anti-hapten responses were less dependent on MHC recognition than anti-peptide responses. This was evidenced by the relative ease of detecting cross-reactivity to haptenated peptides presented by allo-MHC and by the relative insensitivity of anti-hapten vs anti-peptide CTL to mutations in the Kb molecule at potential TCR interaction sites. One potential explanation for this insensitivity to MHC mutation was the finding that the anti-hapten response appeared to be of higher avidity, since a > 100-fold difference in the amount of Ag required to sensitize target cells was found between these two types of Ags. (+info)
Normal development in porcine thymus grafts and specific tolerance of human T cells to porcine donor MHC.
(32/9318)
The induction of T cell tolerance is likely to play an essential role in successful xenotransplantation in humans. In this study, we show that porcine thymus grafts in immunodeficient mice support normal development of polyclonal, functional human T cells. These T cells were specifically tolerant to MHC Ags of the porcine thymus donor and responded to nondonor porcine xenoantigens and alloantigens. Exogenous IL-2 did not abolish tolerance, suggesting central clonal deletion rather than anergy as the likely tolerance mechanism. Our study suggests that the thymic transplantation approach to achieving tolerance with restoration of immunocompetence may be applicable to xenotransplantation of pig tissues to humans. (+info)