Okadaic acid induces hyperphosphorylated forms of tau protein in human brain slices.

Hyperphosphorylated forms of the microtubule-associated protein tau are components of the paired helical filaments (PHFs) seen in patients with Alzheimer's disease. Slices of human lateral temporal cortex were obtained from tissues removed incidental to resections for intractable hippocampal epilepsy. Tau phosphorylation in temporal lobe slices was determined using mobility shifts after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection with the monoclonal antibodies Alz-50, 5E2, and Tau-1. The results indicate that tau phosphorylation was altered in a dose-dependent manner by the phosphatase inhibitor okadaic acid, but not by N-methyl-D-aspartate, quisqualate, or kainate. The slowest mobility forms of tau, termed "PHF-like tau," produced by okadaic acid treatment were dephosphorylated by purified protein phosphatase 2B (calcineurin). Formation of PHF-like tau peptides was blocked by KN-62, 1[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, an inhibitor of Ca2+/calmodulin-dependent protein kinase II. The protein kinase inhibitor staurosporine also prevented formation of PHF-like tau. These data suggest that phosphorylation of tau is regulated by Ca(2+)-dependent protein kinases and okadaic acid-sensitive protein phosphatases, alterations of which may be implicated in the pathogenesis of Alzheimer's disease.

The properties of a new polymerase III transcription factor reveal that transcription complexes can assemble by more than one pathway.

We have resolved a previously unidentified factor (TFIIID) that is required for in vitro transcription of polymerase III templates. Our ability to resolve factor D from each of the other components of the transcription machinery (polymerase and transcription factors IIIB and IIIC) allowed us to test the capacity of these separated components to form stable complexes with tRNA genes. We find that none of the individual components binds detectably to tRNA genes, but that certain combinations of transcription factors do bind. Our results show that TFIIID is essential for binding and that formation of a full transcription complex can proceed by either of two different pathways.

Cycloheximide sensitivity in regulation of acyl coenzyme A:cholesterol acyltransferase activity in Chinese hamster ovary cells. 1. Effect of exogenous sterols.

Chinese hamster ovary cells grown in medium containing low-density lipoprotein (LDL) express high acyl coenzyme A:cholesterol acyltransferase (ACAT) activity as measured by an [3H]oleate pulse. Removal of LDL from the medium causes rapid inactivation of ACAT activity; the t1/2 for the initial inactivation rate is 0.8 h. Preincubation with protein synthesis inhibitors (cycloheximide or emetine) for 2 h or longer lengthens the t1/2 for the initial inactivation rate to approximately 2.1 h. When LDL is removed for more than 10 h, the cells contain only 3% of the original ACAT activity. Cycloheximide under this condition causes an 8-fold increase in ACAT activity; the increase approaches a maximum in 6-8 h. The extent of ACAT activation by cycloheximide inversely depends on exogenous sterol present in the medium; LDL diminishes the activation, while cationized LDL or 25-hydroxycholesterol completely abolishes the activation. Adding LDL back to the sterol-free medium causes a 40-70-fold increase in ACAT activity; however, the activation of LDL is not further augmented if the cells are pretreated with cycloheximide. The above observations are qualitatively confirmed by ACAT assays in vitro with cell homogenates. LDL or cycloheximide has no effect on the rates of 3H-labeled triglyceride and 3H-labeled polar lipid synthesis. Efflux of prelabeled cholesterol from cells is cycloheximide-insensitive. Rates of degradation of [3H]-leucine-pulse-labeled total protein in cells grown with or without LDL are identical. The above results imply the existence of at least one specific short-lived factor that directly or indirectly inhibits ACAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl-CoA:cholesterol acyltransferase in Chinese hamster ovary cells. Enzyme activity determined after reconstitution in phospholipid/cholesterol liposomes.

Acyl-CoA:cholesterol acyltransferase from Chinese hamster ovary (CHO) cells was solubilized by deoxycholate, and then reconstituted in phosphatidylcholine/cholesterol liposomes. This reconstituted activity was totally dependent upon the cholesterol content of the mixture and showed saturation for cholesterol. Analysis of the reconstituted enzyme on linear Ficoll gradients shows that the enzyme has been incorporated into phospholipid/cholesterol liposomes. The CHO cell enzyme activity as measured by conventional assay (using cellular cholesterol as the substrate) was activated approximately 20-fold by low density lipoprotein. This activation process was independent of protein synthesis. When the above cell homogenates were assayed after optimal reconstitution, the activation produced by low density lipoprotein was essentially completely abolished. There was also no change in enzyme activity measured after reconstitution when cells were switched from sterol-containing medium to sterol-free medium, in contrast to a more than 7-fold drop in enzyme activity when assayed without reconstitution. These results suggest that the enzyme activity in intact cells is controlled by the content and composition of cellular lipids associated with the enzyme molecule. Since the intracellular messenger of low density lipoprotein is known to be cholesterol, it is likely that this enzyme activity in intact cells is primarily controlled by the cholesterol content in the vicinity of the enzyme molecule.

Solubilization, partial purification, and reconstitution in phosphatidylcholine-cholesterol liposomes of acyl-CoA:cholesterol acyltransferase.

Acyl-CoA:cholesterol acyltransferase (ACAT) was solubilized from pig liver microsomes with a combination of 1:1% deoxycholate and 1 M potassium chloride. This solubilized activity was then reconstituted in lipid vesicles by diluting the extract into a solution of phosphatidylcholine, cholesterol, and sodium cholate, followed by dialysis. The reconstituted activity was shown to be dependent upon cholesterol in the reconstitution mixture and also shown to vary with changes in the phospholipid headgroup: phosphatidylethanolamine was most active, phosphatidylcholine was next, and phosphatidylserine or phosphatidylinositol was inhibitory. The reconstituted activity showed a migration pattern of ficoll gradients that was distinct from that of the unreconstituted enzyme and similar to that of phospholipid-cholesterol liposomes. These method provide a technique to assay the ACAT activity in defined lipid environment. The solubilized ACAT fraction was further purified by ammonium acetate fractionation and Sepharose 4B column chromatography. The entire purification procedure yielded a 150-fold increase in ACAT specific activity with 40% of the original activity recovered.