Telomerase synthesizes telomeric DNA repeats at the ends of eukaryotic chromosomes. The RNA component of the enzyme (hTR) provides the template for telomere synthesis, which is catalyzed by telomerase reverse transcriptase (hTERT). Little is known regarding the subcellular localization of hTR and hTERT and the pathway by which telomerase is assembled. Here we report the first glimpse of the detailed subcellular localization of endogenous hTR in human cells, which we obtained by fluorescence in situ hybridization (FISH). Our studies have revealed a distinctive hTR localization pattern in cancer cells. We have found that hTR accumulates within intranuclear foci called Cajal bodies in all typical tumor-derived cell lines examined (in which telomerase is active), but not in primary or ALT cells (where little or no hTERT is present). Accumulation of hTR in the Cajal bodies of primary cells is induced when hTERT is ectopically expressed. Moreover, we report that hTERT is also found in Cajal bodies. Our data suggest that Cajal bodies are involved in the assembly and/or function of human telomerase.

Background: CYP2C9 polymorphisms are associated with decreased S-warfarin clearance and lower maintenance dosage. Decreased expression of VKORC1 resulting from the ؊1639G>A substitution has also been implicated in lower warfarin dose requirements. We investigated the additional contribution of this polymorphism to the variance in warfarin dose. Methods: Sixty-five patients with stable anticoagulation were genotyped for CYP2C9 and VKORC1 with Tag-It™ allele-specific primer extension technology. Plasma Swarfarin concentrations and warfarin maintenance dose were compared among patients on the basis of the VKORC1 ؊1639G>A genotype. Results: Eighty percent of CYP2C9*1/*1 patients stabilized on <4.0 mg/day warfarin had at least 1 VKORC1 ؊1639A allele. Mean warfarin doses (SD) were 6.7 (3.3), 4.3 (2.2), and 2.7 (1.2) mg/day for patients with the VKORC1 ؊1639GG, GA, and AA genotypes, respectively. Steady-state plasma concentrations of S-warfarin were lowest in patients with the VKORC1 ؊1639AA genotype and demonstrated a positive association with the VKORC1 ؊1639G allele copy number (trend P ‫؍‬ 0.012). A model including VKORC1 and CYP2C9 geno-

Telomerase is the enzyme essential to complete the replication of the terminal DNA of most eukaryotic chromosomes. In humans, this enzyme is composed of the telomerase reverse transcriptase (hTERT) and telomerase RNA (hTR) subunits. hTR has been found in the nucleolus, a site of assembly of ribosomes as well as other ribonucleoproteins (RNPs). We therefore tested whether the hTERT component is also found in the nucleolus, where it could complex with the hTR RNA to form a functional enzyme. We report here that hTERT does indeed localize to the nucleolus, and we mapped the domain responsible for this localization to the hTRbinding region of the protein by deletion analysis. Substitution mutations in two of the three conserved hTRbinding domains in this nucleolar localization domain (NoLD) abolished nucleolar localization. However, another mutation that impeded hTR binding did not alter this subcellular localization. Additionally, wild type hTERT was detected in the nucleolus of cells that failed to express hTR. Taken together, we propose that the nucleolar localization of hTERT involves more than just the association with the hTR subunit. Furthermore, the coincidental targeting of both the hTR and hTERT subunits to the nucleolus supports the premise that the assembly of telomerase occurs in the nucleolus.Telomerase is a reverse transcriptase ribonucleoprotein (RNP) 1 complex composed of a reverse transcriptase catalytic protein subunit (TERT) that copies a template region of an accompanying RNA subunit (TR) onto telomeres as DNA (1). In humans, this enzyme is of great medical importance because of its pivotal role in unlimited cellular proliferation, a hallmark of cancer cells (2). The union of the RNA and protein subunits to form an RNP is essential for telomerase activity (3).Based on a comparison of the amino acid sequence of TERT from organisms of many different kingdoms and on mutational analysis, it has been possible to identify a number of discrete domains in TERT. The central region of the catalytic subunit contains seven motifs found in reverse transcriptases, which define the catalytic core (4 -17). The C terminus of TERT is highly divergent, both at the sequence and the functional level (18,19). The N-terminal region is more conserved, containing domain I and the DAT (dissociates activities of telomerase) domain (18, 20) followed by domains II and III (18,20,21) and the T motif (1, 5, 6), which are essential for telomere elongation. Substitution mutations in domains II and III or the T motif decrease TERT binding to the telomerase RNA in yeast (18), ciliate (22, 23), or human cells (20,24) and correspondingly result in a dysfunctional enzyme. Deletion analysis has also defined the region extending from amino acids 326 to 613, which harbors all three of the aforementioned domains, as the minimum region required for hTR binding (22), although mutations in domain I can also have some effect on hTR binding (24).The site of assembly of the telomerase RNP has not been determined; however, there is growing e...

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A BS TRACT: Background: Circulating cholesterol levels have been linked to PD, but not directly to brain physiology. Objective: To assess whether brain cholesterol metabolism is related to PD. Methods: Sixty PD patients and 64 controls were recruited from an academic movement disorder clinic (2009)(2010)(2011)(2012). Thirty-five PD patients and 33 controls returned approximately 36 months later. Fasting plasma (S)24-OH-cholesterol (brain-derived cholesterol metabolite) and 27-OH-cholesterol (peripheral cholesterol metabolite) were quantified. Odds ratios for PD were derived from logistic regression models, adjusting for potential confounders. Relationships between the oxysterols and clinical measurements were explored using Spearman correlation coefficients. Results: Mean age of PD subjects was 63.8 AE 8.3 years and disease duration was 5.0 AE 5.4 years. Plasma (S)24-OHcholesterol levels were inversely associated with the odds of having PD, with an odds ratio of 0.92 (95% confidence interval: 0.87-0.97) for each 1-ng/mL increase (P = 0.004).Compared to the lowest tertile, the odds ratio was 0.34 (0.12-0.98) for the second tertile (P = 0.045) and 0.08 (0.02-0.31) for the highest tertile (P < 0.001). Higher (S)24-OHcholesterol levels also were correlated with better sense of smell (r = 0.35; P = 0.01). No significant associations were found between clinical measures and 27-OH-cholesterol, a peripheral cholesterol metabolite. Furthermore, (S)24-OHcholesterol levels were stable over time, whereas 27-OHcholesterol decreased with time in both cases and controls. Conclusions: Results indicate that plasma (S)24-OHcholesterol (possibly reflecting brain cholesterol metabolism) is inversely linked to PD, is relatively stable over time, and may serve as a new biomarker for PD. Further investigation is necessary to determine the mechanistic and clinical implications.