2؉ . Analysis of crz1⌬ cells established Crz1p as the major effector of calcineurin-regulated gene expression in yeast. We identified the Crz1p-binding site as 5-GNG-GC(G/T)CA-3 by in vitro site selection. A similar sequence, 5-GAGGCTG-3, was identified as a common sequence motif in the upstream regions of calcineurin/ Crz1p-dependent genes. This finding is consistent with direct regulation of these genes by Crz1p.

Hyperglycemia is believed to be the major cause of diabetic vascular complications involving both microvessels and arteries as in the retina, renal glomeruli, and aorta. It is unclear by which mechanism hyperglycemia is altering the metabolism and functions of vascular cells, although changes in nonenzymatic protein glycosylation and increases in cellular sorbitol levels have been postulated to be involved. Previously, we have reported that the elevation of extracellular glucose levels with cultured bovine retinal capillary endothelial cells causes an increase in protein kinase C (PKC) activity of the membranous pool with a parallel decrease in the cytosol without alteration of its total activity. Now we demonstrate that the mechanism for the activation of PKC is due to an enhanced de novo synthesis of diacylglycerol as indicated by a 2-fold increase of ['4Cldiacylglycerol labeling from [14C]glucose. The elevated diacylglycerol de novo synthesis is secondarily due to increased formation of precursors derived from glucose metabolism; this formation is enhanced by hyperglycemia as substantiated by elevated [3H1glucose conversion into water. This effect of hyperglycemia on PKC is also observed in cultured aortic smooth muscle and endothelial cells and the retina and kidney of diabetic rats, but not in the brain. Since PKC in vascular cells has been shown to modulate hormone receptor turnover, neovascularization in vitro, and cell growth, we propose that this mechanism of enhancing the membranous PKC activities by hyperglycemia plays an important role in the development of diabetic vascular complications.Hyperglycemia is probably an important etiologic factor in the development of vascular complications in diabetic patients, such as retinopathy (1), nephropathy (2), and accelerated atherosclerosis (3). Nonenzymatic glycosylation (4,5) of protein and accumulation of intracellular sorbitol with reduction of myo-inositol levels (6, 7) have been proposed to be involved in the development of these vascular changes.Protein kinase C (PKC), the Ca2+/phospholipid-dependent protein kinase, appears to be involved in a variety of cellular functions such as signal transduction of cellular responses to hormones, growth factors, neurotransmitters, and drugs (8, 9). In vascular cells, PKC has been shown to modulate growth rate (10), DNA synthesis (11), hormone and growth factor receptor turnover (12), smooth muscle contraction (13-17), and cAMP responses to different hormones (18,19) and to stimulate neovascularization in vitro. Using cultured bovine retinal capillary endothelial cells, a cell type prominently involved in diabetic retinopathy (21), we have previously reported that the membranous pool of PKC activity was increased 100% by elevation of glucose level (22) but not by adding mannitol to the medium. This effect appears to be mostly an increase in the translocation of PKC from cytosol to the membrane since the total PKC activity is not altered and the maximally stimulated membranous PKC activities by phorbol 12...

Heat shock factor (hsf) is the transcriptional activator that governs the transcriptional response of eukaryotic cells to stressful conditions. The structure and regulation of hsf is highly conserved. We describe deletion mutations in hsf+ that alter the ability of Schizosaccharomyces pombe to respond to different stressful conditions. One mutation causes increased sensitivity to cadmium while maintaining near normal sensitivity to heat stress, while another mutation confers increased sensitivity to heat stress but retains normal sensitivity to cadmium. Despite the differential sensitivity of these two strains to cadmium and heat stress, the mutant hsf proteins in each strain were activated by both cadmium and heat. However, we found that these mutations differentially affected the ability of hsf to activate different promoters: one mutated hsf activated the ssp1+ gene better than the wis2+ gene following either stress, while the other mutated hsf activated wis2+ better than ssp1+. We propose that the differential ability of strains that contain these mutant hsfs to survive cadmium and heat stress is not caused by differences in activation of hsf, but is caused instead by differential abilities of the mutant hsfs to activate the appropriate sets of genes needed for survival.

The C‐terminal hydrophobic repeat (CTR) of heat shock transcription factor (HSF) has been proposed to regulate DNA binding by intramolecular interactions with the leucine zipper motifs present in the HSF trimerization domain. Schizosaccharomyces pombe provides a useful model organism for the study of the regulation of HSF DNA binding because, unlike Saccharomyces cerevisiae, S. pombe hsf is highly heat shock inducible for DNA binding and contains a clear homology to the CTR. We examined the role that the CTR plays in the regulation of S. pombe hsf by constructing isogenic strains bearing deletion and point mutations in the chromosomal copy of hsf. Surprisingly, we found that point mutation of key hydrophobic amino acids within the CTR, as well as full deletion of it, yielded factors that show normal binding at normal growth temperatures and full levels of heat‐induced binding. Deletion of the CTR did, however, slightly lower the temperature required for maximal activation. In contrast, a large deletion of the C‐terminus, which removes close to a third of the coding sequence, was deregulated and bound DNA at control temperature. Several of the deletion mutants were significantly reduced in their level of expression, yet they showed wild‐type levels of DNA binding activity following heat shock. These experiments demonstrate that appropriate regulation of the DNA binding activity of S. pombe hsf is not solely dependent upon the CTR, and imply that a feedback mechanism exists that establishes proper levels of DNA binding following heat shock despite mutations that significantly alter levels of total hsf. © 1998 John Wiley & Sons, Ltd.