Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2

We investigated the control of telomere length by the human telomeric proteins TRF1 and TRF2. To this end, we established telomerase-positive cell lines in which the targeting of these telomeric proteins to specific telomeres could be induced. We demonstrate that their targeting leads to telomere shortening. This indicates that these proteins act in cis to repress telomere elongation. Inhibition of telomerase activity by a modified oligonucleotide did not further increase the pace of telomere erosion caused by TRF1 targeting, suggesting that telomerase itself is the target of TRF1 regulation. In contrast, TRF2 targeting and telomerase inhibition have additive effects. The possibility that TRF2 can activate a telomeric degradation pathway was directly tested in human primary cells that do not express telomerase. In these cells, overexpression of full-length TRF2 leads to an increased rate of telomere shortening.     

Role of the telomeric DNA-binding protein TRF2 in the stability of human chromosome ends

A major issue in telomere research is to understand how the integrity of chromosome ends is preserved. A recent study shows that expression of a dominant-negative form of the human telomeric protein TRF2 increases the number of chromosome fusions in immortalized cells and decreases the quantity of G-rich telomeric DNA 3' overhang, the G tail.⁽¹⁾ Consequently, TRF2 appears to control the structure of the very end of the chromosomal DNA molecule and to prevent recombination between two telomeres. Remarkably, the same study reveals a potential role of TRF2 in cell division control. BioEssays 20:879–883, 1998. © 1998 John Wiley & Sons, Inc.     

Intercalation of proflavine and a platinum derivative of proflavine into double-helical Poly(A)

The equilibria and kinetics of the interactions of proflavine (PR) and its platinum-containing derivative [PtCl(tmen)(2)HNC(13)H(7)(NHCH(2)CH(2))(2)](+) (PRPt) with double-stranded poly(A) have been investigated by spectrophotometry and Joule temperature-jump relaxation at ionic strength 0.1 M, 25 degrees C, and pH 5.2. Spectrophotometric measurements indicate that base-dye interactions are prevailing. T-jump experiments with polarized light showed that effects due to field-induced alignment could be neglected. Both of the investigated systems display two relaxation effects. The kinetic features of the reaction are discussed in terms of a two-step series mechanism in which a precursor complex DS(I) is formed in the fast step, which is then converted to a final complex in the slow step. The rate constants of the fast step are k(1) = (2.5 +/- 0.4) x 10(6) M(-1) s(-1), k(-1) = (2.4 +/- 0.1) x 10(3) s(-1) for poly(A)-PR and k(1) = (2.3 +/- 0.1) x 10(6) M(-1) s(-1), k(-1) = (1.6 +/- 0.2) x 10(3) s(-1) for poly(A)-PRPt. The rate constants for the slow step are k(2) = (4.5 +/- 0.5) x 10(2) s(-1), k(-2) = (1.7 +/- 0.1) x 10(2) s(-1) for poly(A)-PR and k(2) = 9.7 +/- 1.2 s(-1), k(-2) = 10.6 +/- 0.2 s(-1) for poly(A)-PRPt. Spectrophotometric measurements yield for the equilibrium constants and site size the values K = (4.5 +/- 0.1) x 10(3) M(-1), n = 1.3 +/- 0.5 for poly(A)-PR and K = (2.9 +/- 0.1) x 10(3) M(-1), n = 2.3 +/- 0.6 for poly(A)-PRPt. The values of k(1) are similar and lower than expected for diffusion-limited reactions. The values of k(-1) are similar as well. It is suggested that the formation of DS(I) involves only the proflavine residues in both systems. In contrast, the values of k(2) and k(-2) in poly(A)-PRPt are much lower than in poly(A)-PR. The results suggest that in the complex DS(II) of poly(A)-PRPt both proflavine and platinum residues are intercalated. In addition, a very slow process was detected and ascribed to the covalent binding of Pt(II) to the adenine.     

Have we overestimated the benefit of human(ized) antibodies?

The infusion of animal-derived antibodies has been known for some time to trigger the generation of antibodies directed at the foreign protein as well as adverse events including cytokine release syndrome. These immunological phenomena drove the development of humanized and fully human monoclonal antibodies. The ability to generate human(ized) antibodies has been both a blessing and a curse. While incremental gains in the clinical efficacy and safety for some agents have been realized, a positive effect has not been observed for all human(ized) antibodies. Many human(ized) antibodies trigger the development of anti-drug antibody responses and infusion reactions. The current belief that antibodies need to be human(ized) to have enhanced therapeutic utility may slow the development of novel animal-derived monoclonal antibody therapeutics for use in clinical indications. In the case of murine antibodies, greater than 20% induce tolerable/negligible immunogenicity, suggesting that in these cases humanization may not offer significant gains in therapeutic utility. Furthermore, humanization of some murine antibodies may reduce their clinical effectiveness. The available data suggest that the utility of human(ized) antibodies needs to be evaluated on a case-by-case basis, taking a cost-benefit approach, taking both biochemical characteristics and the targeted therapeutic indication into account.Key words: immunogenicity, human anti-mouse antibody, cytokine release syndrome     

Selective elimination of malaria infected erythrocytes by a modified phospholipase A2 in vitro

Pig pancreatic phospholipase A2 does not act on normal erythrocytes, but the membrane penetrating capacity is enhanced by the covalent attachment of one fatty acyl chain to Lys-116 of the enzyme. Taking advantage of the impaired packing of phospholipids in the membrane of Plasmodium infected erythrocytes it was demonstrated that a lauric acid derivative of phospholipase A2 is capable of exclusively attaching the infected erythrocytes in vitro, leaving the uninfected cells undisturbed. The chemically modified phospholipase A2 appeared to cause death of the parasite in cell cultures of infected erythrocytes.     

Animal models of fibromyalgia

Animal models of disease states are valuable tools for developing new treatments and investigating underlying mechanisms. They should mimic the symptoms and pathology of the disease and importantly be predictive of effective treatments. Fibromyalgia is characterized by chronic widespread pain with associated co-morbid symptoms that include fatigue, depression, anxiety and sleep dysfunction. In this review, we present different animal models that mimic the signs and symptoms of fibromyalgia. These models are induced by a wide variety of methods that include repeated muscle insults, depletion of biogenic amines, and stress. All potential models produce widespread and long-lasting hyperalgesia without overt peripheral tissue damage and thus mimic the clinical presentation of fibromyalgia. We describe the methods for induction of the model, pathophysiological mechanisms for each model, and treatment profiles.     

Insomnia,Daytime Sleepiness and Cardio-Cerebrovascular Diseases in the Elderly: A 6-Year Prospective Study

Objective

To examine 1) the associations between history of cardio-cerebrovascular diseases (CVD) and insomnia complaints and excessive daytime sleepiness (EDS), and 2) the relationships between sleep complaints and future CVD in persons over 65.

Methods

CVD was assessed at baseline and during two, four, and six-year follow-up in 5494 non-demented subjects. Self-reported insomnia complaints (poor sleep quality, difficulty in initiating sleep, difficulty in maintening sleep, and early morning awakening), EDS and sleep medication use were evaluated at baseline. Logistic regression models and Cox proportional hazard models, with delayed entry and age of participants as the time scale, were adjusted for socio-demographic, lifestyle and clinical variables.

Results

At baseline, 748 participants had a past-history of CVD. A past-history of CVD was associated with EDS (OR = 1.28 95%CI = [1.05–1.57]) and the number of insomnia complaints (OR = 1.26 95%CI = [1.03–1.55] for 1–2 insomnia complaints; OR = 1.32 95%CI = [1.03–1.71] for ≥3 complaints). In longitudinal analyses, neither the four components of insomnia nor the number of insomnia complaints were significantly associated with first or recurrent CVD events (n = 391 events). EDS was independently associated with future CVD events even after adjusting for prescribed sleep medication and past-history of CVD (HR = 1.35 95%CI = [1.06–1.71]).

Conclusion

Our results suggest that the relationships between sleep complaints and CVD could be complex. Insomnia complaints are more likely a consequence of CVD, whereas EDS appears to be a determinant of CVD independently of past-history of CVD. EDS screening may thus constitute a means of detecting persons at high risk of CVD.     

Chromatin,gene silencing and HIV latency

One of the cellular defenses against virus infection is the silencing of viral gene expression. There is evidence that at least two gene-silencing mechanisms are used against the human immuno-deficiency virus (HIV). Paradoxically, this cellular defense mechanism contributes to viral latency and persistence, and we review here the relationship of viral latency to gene-silencing mechanisms.     

Hormone treatment, estrogen receptor polymorphisms and mortality: a prospective cohort study

Background

The association between hormone treatment (HT) and mortality remains controversial. This study aimed to determine whether the risk of mortality associated with HT use varies depending on the specific characteristics of treatment and genetic variability in terms of the estrogen receptor.

Methodology/Principal Findings

A prospective, population-based study of 5135 women aged 65 years and older who were recruited from three cities in France and followed over six years. Detailed information related to HT use was obtained and five estrogen receptor polymorphisms were genotyped. The total follow-up was 25,436 person-years and during this time 352 women died. Cancer (36.4%) and cardiovascular disease (19.3%) were the major causes of death. Cox proportional hazards models adjusted for age, education, centre, living situation, comorbidity, depression, physical and mental incapacities, indicated no significant association between HT and mortality, regardless of the type or duration of treatment, or the age at initiation. However, the association between HT and all-cause or cancer-related mortality varied across women, with significant interactions identified with three estrogen receptor polymorphisms (p-values = 0.004 to 0.03) in adjusted analyses. Women carrying the C allele of ESR1 rs2234693 had a decreased risk of all-cause mortality with HT (HR: 0.42, 95% CI: 0.18–0.97), while in stark contrast, those homozygous for the T allele had a significantly increased risk of cancer-related mortality (HR: 3.18, 95% CI: 1.23–8.20). The findings were similar for ESR1 rs9340799 and ESR2 rs1271572.

Conclusions/Significance

The risk of mortality was not associated with HT duration, type or age at initiation. It was however not equal across all women, with some women appearing genetically more vulnerable to the effects of HT in terms of their estrogen receptor genotype. These findings, if confirmed in another independent study, may help explain the differential susceptibility of women to the beneficial or adverse effects of HT.