TANK2, a new TRF1-associated poly(ADP-ribose) polymerase, causes rapid induction of cell death upon overexpression.

Tankyrase (TANK1) is a human telomere-associated poly(ADP-ribose) polymerase (PARP) that binds the telomere-binding protein TRF1 and increases telomere length when overexpressed. Here we report characterization of a second human tankyrase, tankyrase 2 (TANK2), which can also interact with TRF1 but has properties distinct from those of TANK1. TANK2 is encoded by a 66-kilobase pair gene (TNKS2) containing 28 exons, which express a 6.7-kilobase pair mRNA and a 1166-amino acid protein. The protein shares 85% amino acid identity with TANK1 in the ankyrin repeat, sterile alpha-motif, and PARP catalytic domains but has a unique N-terminal domain, which is conserved in the murine TNKS2 gene. TANK2 interacted with TRF1 in yeast and in vitro and localized predominantly to a perinuclear region, similar to the properties of TANK1. In contrast to TANK1, however, TANK2 caused rapid cell death when highly overexpressed. TANK2-induced death featured loss of mitochondrial membrane potential, but not PARP1 cleavage, suggesting that TANK2 kills cells by necrosis. The cell death was prevented by the PARP inhibitor 3-aminobenzamide. In vivo, TANK2 may differ from TANK1 in its intrinsic or regulated PARP activity or its substrate specificity.

Expression of alpha 1D subunit mRNA is correlated with L-type Ca2+ channel activity in single neurons of hippocampal "zipper" slices.

L-type voltage-sensitive Ca(2+) channels (L-VSCCs) play an important role in developmental and aging processes, as well as during normal function of brain neurons. Here, we tested a prediction of the hypothesis that membrane density of functional L-VSCCs is regulated by the level of gene expression for its alpha(1D) pore-forming subunit. If so, alpha(1D) mRNA and L-VSCC activity should be positively correlated within individual neurons. Conventional methods of aspiration and/or acute cell dissociation used in prior single-cell studies have generally yielded variable and incomplete recovery of intracellular mRNA. Thus, quantitative relationships between channel function and expression have been difficult to define. In this study, we used the partially dissociated ("zipper") hippocampal slice preparation as a method for collecting a single neuron's mRNA complement. This preparation, developed to expose neuronal somata for recording, also enables the extraction of a neuron with major processes largely intact. Thus, single-cell measures of gene/mRNA expression can be based on approximately the cell's full set of mRNA transcripts. In adult and aged rat hippocampal zipper slices, L-VSCC activity was first recorded in CA1 neurons in cell-attached patch mode. The same neurons were then extracted and collected for semiquantitative reverse transcriptase-PCR analysis of alpha(1D) and calmodulin A (CaM) mRNA content. Across multiple single neurons, a significant, positive correlation was found between the rank orders of L-VSCC activity and of alpha(1D), but not CaM, mRNA expression. Thus, these studies support the possibility that the level of alpha(1D) gene expression regulates the density of functional L-VSCCs.

TIN2, a new regulator of telomere length in human cells.

Telomeres are DNA-protein structures that cap linear chromosomes and are essential for maintaining genomic stability and cell phenotype. We identified a novel human telomere-associated protein, TIN2, by interaction cloning using the telomeric DNA-binding-protein TRF1 as a bait. TIN2 interacted with TRF1 in vitro and in cells, and co-localized with TRF1 in nuclei and metaphase chromosomes. A mutant TIN2 that lacks amino-terminal sequences effects elongated human telomeres in a telomerase-dependent manner. Our findings suggest that TRF1 is insufficient for control of telomere length in human cells, and that TIN2 is an essential mediator of TRF1 function.

The influence of age on collagen and non-collagen protein production by human gingival epithelial cells.

The effects of donor age on the proliferation and secretory phenotype of cultured human gingival epithelial cells were investigated. Pure cultures of epithelial cells were isolated from human gingiva of old (61-75 yr) and young (18-30 yr) adults and serially cultivated in a serum-free medium at 37 degrees C in humidified air containing 5% CO2. For each experiment, cells were seeded at 150/mm2 and the medium changed every other day. Cell number, collagen and non-collagen protein production and relative collagen synthesis (percentage collagen synthesized) were determined at days 2, 4, 6 and 8. Epithelial strains from old and young adults became confluent by day 8 and there were no differences in their rates of proliferation. Likewise there was no difference in collagen production between the two groups; however, cells from elderly individuals produced significantly less non-collagen protein. Over time the decrease in non-collagen protein production ranged from 56% below the non-collagen protein levels of epithelium from young adults at day 2 to 24% below at day 8. The reduction of non-collagen protein coupled with the unchanged secretion of collagen resulted in a statistically significant increase in relative collagen synthesis by epithelial cells from elderly individuals. These differences in non-collagen protein production and relative collagen synthesis by cultured gingival epithelium of old adults suggest a selective conversion in protein secretion.