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Human fibroblasts expressing the catalytic component of human telomerase (hTERT) have been followed for 250-400 population doublings. As expected, telomerase activity declined in long term culture of stable transfectants. Surprisingly, however, clones with average telomere lengths several kilobases shorter than those of senescent parental cells continued to proliferate. Although the longest telomeres shortened, the size of the shortest telomeres was maintained. Cells with subsenescent telomere lengths proliferated for an additional 20 doublings after inhibiting telomerase activity with a dominant-negative hTERT mutant. These results indicate that, under conditions of limiting telomerase activity, cis-acting signals may recruit telomerase to act on the shortest telomeres, argue against the hypothesis that the mortality stage 1 mechanism of cellular senescence is regulated by telomere positional effects (in which subtelomeric loci silenced by long telomeres are expressed when telomeres become short), and suggest that catalytically active telomerase is not required to provide a protein-capping role at the end of very short telomeres.

The lipid bilayer technique was adapted to the functional reconstitution of ion channels from the endoplasmic reticulum of a higher plant. This was obtained at high purity from touch‐sensitive tendrils of Bryonia dioica. In this preparation, a calcium‐selective strongly rectifying channel is prevailing whose single‐channel properties have been characterized. The single‐channel conductance is 29 pS in 50 mM CaCl2. The Ca2+: K+ selectivity was determined to be approximately 6.6. The channel is voltage‐gated and, more importantly, the gating voltage is strongly shifted towards more negative voltages when a transmembrane Ca2+ gradient is applied. Thus, at physiological voltages across the endoplasmic reticulum membrane, the channel's open probability will be governed largely by the chemical potential gradient of Ca2+, generated by the Ca(2+)‐ATPase in that same membrane. The calcium release channel described here is effectively blocked by Gd3+ which also completely suppresses a tendril's reaction to touch, suggesting that this channel could be a key element of calcium signaling in higher plant mechanotransduction. Its molecular characteristics and inhibitor data show it to be the first known member of a hitherto unrecognized class of calcium channels.

Procedures have been developed which allow the preparation of highly pure endoplasmic reticulum and plasma membrane from tendrils ofBryonia dioica. These and further membrane fractions were used to study vanadate-sensitive ATPase activity as well as Mg2+ATP-driven transport of45Ca2+. Calcium-translocating ATPases were detected in the endoplasmic reticulum, the plasma membrane and the mitochondrial fraction and characterized kinetically and with respect to the effects of various inhibitors. The endoplasmic-reticulum Ca2+-translocating ATPase was stimulated by KCl and was calmodulin-dependent. The plasma-membrane enzyme was not affected by these agents. These, as well as the inhibitor data, show that the Ca2+-translocating ATPases of the endoplasmic reticulum and the plasma membrane are distinctly different enzymes. Upon mechanical stimulation, the activities of the vanadate-sensitive K+, Mg2+-ATPase and the Ca2+-translocating ATPase(s) increased rapidly and transiently, indicating that increasing transmembrane proton and calcium fluxes are involved in the early stages of tendril coiling.

A catalytically active, vanadate-sensitive P-type ATPase with enzymatic, inhibitor and immunological properties similar to the plasmalemma enzyme is associated with the rough, but not smooth, endoplasmic reticulum obtained from tendril tissue of Bryonia dioicaJacq. using a density shift discontinuous two-step gradient purification procedure. The enzyme is present in this fraction in similar abundance as in the plasmalemma and constitutes approximately 32% of the total vanadate-sensitive ATPase activity of the microsomal fraction. ATP-dependent proton pumping activity was shown for this membrane using the fluorescent dye 9-amino-6-chloro-2-methoxyacridine. Proton potentials may be of relevance for rough endoplasmic reticulum functions, at least in tendrils.