Tel2p, a regulator of yeast telomeric length in vivo, binds to single-stranded telomeric DNA in vitro

The telomeres of the yeast Saccharomyces cerevisiae consist of a duplex region of TG_1–3 repeats that acquire a single-stranded 3’ extension of the TG_1–3 strand at the end of S-phase. The length of these repeats is kept within a defined range by regulators such as the TEL2-encoded protein (Tel2p). Here we show that Tel2p can specifically bind to single-stranded TG_1–3. Tel2p binding produced several shifted bands; however, only the slowest migrating band contained Tel2p. Methylation protection and interference experiments as well as gel shift experiments using inosine-containing probes indicated that the faster migrating bands resulted from Tel2p-mediated formation of DNA secondary structures held together by G-G interactions. Tel2p bound to single-stranded substrates that were at least 19 bases in length and contained 14 bases of TG_1–3, and also to double-stranded/single-stranded hybrid substrates with a 3’ TG_1–3 overhang. Tel2p binding to a hybrid substrate with a 24 base single-stranded TG_1–3 extension also produced a band characteristic of G-G-mediated secondary structures. These data suggest that Tel2p could regulate telomeric length by binding to the 3’ single-stranded TG_1–3 extension present at yeast telomeres. Received: 12 November 1998; in revised form: 6 April 1999 / Accepted: 13 April 1999     

Balance training and exercise in geriatric patients

Objective measures of gait and balance which meet the criteria of reliability and validity are required as a basis for exercise regimens. We established reference values of clinically relevant locomotor and balance performances for geriatric patients. We are using these data for evaluating the effects of different therapeutic approaches to locomotor and balance disorders. Reference values for chair rising. We administered a battery of five tests concerning neuromuscular function, locomotion and balance to a sample of 212 participants without apparent locomotor deficits (139 women, 73 men, mean age 70,5 years, SD 6,78 , median 70 years, range 60 to 90 years, recruited by public announcements). The test battery comprised the 'chair rising test' for measuring lower extremity neuromuscular function (five repetitions of rising from a chair as quickly as possible with arms crossed over the chest). The test has been proven reliable, valid, sensible and predictive for falls and future locomotor status and ADL-status. Chair rising [sec/5x], Range: 5.4-19.4, Mean: 9.1 (women:9.2, men:9.0), SD: 1.97, Median: 8.9. Training of balance and muscle power with Galileo 2000 - preliminary results. Galileo is a device for whole body vibration/oscillatory muscle stimulation. The subject stands with bended knees and hips on a rocking platform with a sagittal axle, which thrusts alternatively the right and left leg 7-14 mm upwards with a frequency of 27 Hz, thereby lengthening the extensor muscles of the lower extremities. The reflexive reaction of the neuromuscular system is a chain of rapid muscle contractions. We conducted a randomized controlled trial, n=34 (age: mean 67y, range 61-85, 11 female), cross-over design, intervention group 2 months training program three times a week (each session 3x2 minutes), performance tests of all participants every two weeks). The first 19 subjects have finished the intervention period. They reached mean performance gains in chair rising of 18%, strikingly different to the constant values of the controls! We interpret the findings as improvements in muscle power by the oscillative muscle stimulation.     

Constitutive apical membrane recycling in Aplysia enterocytes

In Aplysia californica enterocytes, alanine-stimulated Na+ absorption increases both apical membrane exocytosis and fractional capacitance (fCa; a measure of relative apical membrane surface area). These increases are thought to reduce membrane tension during periods of nutrient absorption that cause the enterocytes to swell osmotically. In the absence of alanine, exocytosis and fCa are constant. These findings imply equal rates of constitutive endocytosis and exocytosis and constitutive recycling of the apical plasma membrane. Thus, the purpose of this study was to confirm and determine the relative extent of constitutive apical membrane recycling in Aplysia enterocytes. Biotinylated lectins are commonly used to label plasma membranes and to investigate plasma membrane recycling. Of fourteen biotinylated lectins tested, biotinylated wheat germ agglutinin (bWGA) bound preferentially to the enterocytes apical surface. Therefore, we used bWGA, avidin D (which binds tightly to biotin), and the UV fluorophore 7-amino-4-methylcoumarin-3-acetic acid (AMCA)-conjugated avidin D to assess the extent of constitutive apical membrane recycling. A temperature-dependent (20 vs. 4 degrees C) experimental protocol employed the use of two tissues from each of five snails and resulted in a approximately 60% difference in apical surface fluorescence intensity. Because the extent of membrane recycling is proportional to the difference in surface fluorescence intensity, this difference reveals a relatively high rate of constitutive apical membrane recycling in Aplysia enterocytes.     

Aging, oxidative responses, and proliferative capacity in cultured mouse aortic smooth muscle cells

The cellular mechanisms that contribute to the acceleration of atherosclerosis in aging populations are poorly understood, although it is hypothesized that changes in the proliferative capacity of vascular smooth muscle cells is contributory. We addressed the relationship among aging, generation of reactive oxygen species (ROS), and proliferation in primary culture smooth muscle cells (SMC) derived from the aortas of young (4 mo old) and aged (16 mo old) mice to understand the phenotypic modulation of these cells as aging occurs. SMC from aged mice had decreased proliferative capacity in response to alpha-thrombin stimulation, yet generated higher levels of ROS and had constitutively increased mitogen-activated protein kinase activity, in comparison with cells from younger mice. These effects may be explained by dysregulation of cell cycle-associated proteins such as cyclin D1 and p27Kip1 in SMC from aged mice. Increased ROS generation was associated with decreased endogenous antioxidant activity, increased lipid peroxidation, and mitochondrial DNA damage. Accrual of oxidant-induced damage and decreased proliferative capacity in SMC may explain, in part, the age-associated transition to plaque instability in humans with atherosclerosis.