PSD-95 and SAP97 exhibit distinct mechanisms for regulating K(+) channel surface expression and clustering

Mechanisms of ion channel clustering by cytoplasmic membrane-associated guanylate kinases such as postsynaptic density 95 (PSD-95) and synapse-associated protein 97 (SAP97) are poorly understood. Here, we investigated the interaction of PSD-95 and SAP97 with voltage-gated or Kv K(+) channels. Using Kv channels with different surface expression properties, we found that clustering by PSD-95 depended on channel cell surface expression. Moreover, PSD-95-induced clusters of Kv1 K(+) channels were present on the cell surface. This was most dramatically demonstrated for Kv1.2 K(+) channels, where surface expression and clustering by PSD-95 were coincidentally promoted by coexpression with cytoplasmic Kvbeta subunits. Consistent with a mechanism of plasma membrane channel-PSD-95 binding, coexpression with PSD-95 did not affect the intrinsic surface expression characteristics of the different Kv channels. In contrast, the interaction of Kv1 channels with SAP97 was independent of Kv1 surface expression, occurred intracellularly, and prevented further biosynthetic trafficking of Kv1 channels. As such, SAP97 binding caused an intracellular accumulation of each Kv1 channel tested, through the accretion of SAP97 channel clusters in large (3-5 microm) ER-derived intracellular membrane vesicles. Together, these data show that ion channel clustering by PSD-95 and SAP97 occurs by distinct mechanisms, and suggests that these channel-clustering proteins may play diverse roles in regulating the abundance and distribution of channels at synapses and other neuronal membrane specializations.     

Analysis of variance components in gene expression data

MOTIVATION: A microarray experiment is a multi-step process, and each step is a potential source of variation. There are two major sources of variation: biological variation and technical variation. This study presents a variance-components approach to investigating animal-to-animal, between-array, within-array and day-to-day variations for two data sets. The first data set involved estimation of technical variances for pooled control and pooled treated RNA samples. The variance components included between-array, and two nested within-array variances: between-section (the upper- and lower-sections of the array are replicates) and within-section (two adjacent spots of the same gene are printed within each section). The second experiment was conducted on four different weeks. Each week there were reference and test samples with a dye-flip replicate in two hybridization days. The variance components included week-to-week, animal-to-animal and between-array and within-array variances. RESULTS: We applied the linear mixed-effects model to quantify different sources of variation. In the first data set, we found that the between-array variance is greater than the between-section variance, which, in turn, is greater than the within-section variance. In the second data set, for the reference samples, the week-to-week variance is larger than the between-array variance, which, in turn, is slightly larger than the within-array variance. For the test samples, the week-to-week variance has the largest variation. The animal-to-animal variance is slightly larger than the between-array and within-array variances. However, in a gene-by-gene analysis, the animal-to-animal variance is smaller than the between-array variance in four out of five housekeeping genes. In summary, the largest variation observed is the week-to-week effect. Another important source of variability is the animal-to-animal variation. Finally, we describe the use of variance-component estimates to determine optimal numbers of animals, arrays per animal and sections per array in planning microarray experiments.     

Underphosphorylated BAD interacts with diverse antiapoptotic Bcl-2 family proteins to regulate apoptosis

Survival factors activate kinases which, in turn, phosphorylate the proapoptotic Bcl-xl/Bcl-2-associated death promoter homolog (BAD) protein at key serine residues. Phosphorylated BAD interacts with 14-3-3 proteins, and overexpression of 14-3-3 attenuates BAD-mediated apoptosis. Although BAD is known to interact with Bcl-2, Bcl-w, and Bcl-xL, the exact relationship between BAD and anti- or proapoptotic Bcl-2 proteins has not been analyzed systematically. Using the yeast two-hybrid protein interaction assay, we found that BAD interacted negligibly with proapoptotic Bcl-2 proteins. Even though wild type BAD only interacted with selected numbers of antiapoptotic proteins, underphosphorylated mutant BAD interacted with all antiapoptotic Bcl-2 proteins tested (Bcl-2, Bcl-w, Bcl-xL, Bfl-1/A1, Mcl-1, Ced-9, and BHRF-1). Using nonphosphorylated recombinant BAD expressed in bacteria, direct interactions between BAD and diverse antiapoptotic Bcl-2 members were also observed. Furthermore, apoptosis induced by BAD was blocked by coexpression with Bcl-2, Bcl-w, and Bfl-1. Comparison of BAD orthologs from zebrafish to human indicated the conservation of a 14-3-3 binding site and the BH3 domain during evolution. Thus, highly conserved BAD interacts with diverse antiapoptotic Bcl-2 members to regulate apoptosis.     

Microscale autoradiographic method for the qualitative and quantitative analysis of apoptotic DNA fragmentation

A method combining the advantages of electrophoretic DNA fractionation and autoradiography is described for the qualitative and quantitative analysis of internucleosomal DNA fragmentation that occurs during apoptosis, or “programmed cell death”. This procedure utilizes terminal transferase enzyme to uniformly add one molecule of [α ³²P]-to the 3′-of DNA fragments. Following gel electrophoresis and autoradiographic analysis, the total amount of radiolabel incorporated into the low molecular weight DNA fraction can be quantitated and used to estimate the degree of apoptotic DNA fragmentation in any given sample. This method requires as little as 15 ng of total cellular DNA and increases the sensitivity of apoptotic DNA detection by at least 100-fold over the widely used ethidium bromide staining method. The procedure should prove valuable for the analysis of apoptosis in minute quantities of tissues and cultured cells. © 1993 Wiley-Liss, Inc.     

Antioxidative activity,moisture retention,film formation,and viscosity stability of Auricularia fuscosuccinea,white strain water extract

This study showed that both water extracts (WAF-W) and ethanol extracts (EAF-W) of Auricularia fuscosuccinea (Montagne) Farlow, white strain (AF-W) demonstrated significantly stronger antioxidative effects than did commercially available Tremella fuciformis sporocarp extracts (WSK; with the exception of EAF-W in terms of superoxide radical scavenging activity levels). The moisture retention capacity of WAF-W is as potent as that of sodium hyaluronate (SHA), but less than that of WSK. No corrugation or fissures were observed in WAF-W film; only the SHA and WSK films demonstrated such effects in low-moisture conditions. The WAF-W solution also exhibited stable viscosity at high temperatures, indicating that the WAF-W film was more stable compared with the SHA and WSK films. WAF-W induced no adverse effects when a hen’s egg test was performed on the chorioallantoic membrane (CAM). This study demonstrated that WAF-W exhibits excellent potential as a topical material for skin moisturizing and anti-aging effects.     

Deuterium NMR Study of the Effect of Ergosterol on POPE Membranes

We study the effect of ergosterol on the physical properties of 1-[²H₃₁]palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) multibilayers using deuterium nuclear magnetic resonance. NMR spectra were taken as a function of temperature and ergosterol concentration up to 70 mol %. The spectral first moments show that there is a dramatic difference in the ability of ergosterol to disorder the gel phase and to order the liquid-crystalline phase of POPE membranes, an unusual behavior among lipid/sterol systems studied up to now. Further investigation of the liquid-crystalline phase shows that ergosterol (erg) increases the chain order of POPE-d31, but that this effect saturates at 10 mol % ergosterol. This is in marked contrast to the effect of cholesterol (chol) on POPE membranes: the chain order of POPE increases with cholesterol to at least 45 mol %. Moreover, we found that at higher ergosterol concentrations (>40 mol %) ergosterol decreases the POPE-d31 chain order, which, to our knowledge, has not been directly observed in other lipid/sterol systems. The temperature-composition phase diagram is presented. Finally, at all ergosterol concentrations, the chain order of liquid-crystalline-phase POPE is much smaller than that of comparable POPE/chol membranes. This implies that there is no liquid-ordered phase behavior for POPE/erg membranes.     

Purification and characterization of human truncated prorenin.

Posttranslational processing of enzymatically inactive prorenin to an active form participates in the control of the activity of a key system involved in blood pressure regulation, growth, and other important functions. The issue is complicated because renin can be produced by a number of tissues throughout the body, in addition to the kidney, but the mechanism by which they process prorenin to renin is unknown and difficult to determine because of the small amounts of renin present. In the juxtaglomerular cell of the kidney, a 43 amino acid prosegment is cleaved from the amino terminus of prorenin to generate renin of molecular weight 44,000 [Do, Y. S., Shinagawa, T., Tam, H., Inagami, T., & Hsueh, W. A. (1987) J. Biol. Chem. 262, 1037-1043]. Using human uterine lining or a recombinant human prorenin system, we employed the same approach as that used in kidney, ammonium sulfate precipitation at pH 3.1 followed by pepstatin and H-77 affinity chromatography or gel filtration, to purify to homogeneity a 45,500-MW totally active renin. The specific activity of the active truncated prorenin was 850 Goldblatt units (GU)/mg of protein for chorion-decidua renin and 946 GU/mg of protein for recombinant renin, both similar to that reported for pure human renal renin. Both forms of renin cross-reacted with an antibody generated against 44,00-MW pure human renal renin and with an antibody generated against a peptide identical to the carboxy-terminal one-third of the prosegment.(ABSTRACT TRUNCATED AT 250 WORDS)