14-3-3 connects glycogen synthase kinase-3 beta to tau within a brain microtubule-associated tau phosphorylation complex

In a recent study, we reported that in bovine brain extract, glycogen synthase kinase-3beta and tau are parts of an approximately 400-500 kDa microtubule-associated tau phosphorylation complex (Sun, W., Qureshi, H. Y., Cafferty, P. W., Sobue, K., Agarwal-Mawal, A., Neufield, K. D., and Paudel, H. K. (2002) J. Biol. Chem. 277, 11933-11940). In this study, we find that when purified brain microtubules are subjected to Superose 12 gel filtration column chromatography, the dimeric scaffold protein 14-3-3 zeta co-elutes with the tau phosphorylation complex components tau and GSK3 beta. From gel filtration fractions containing the tau phosphorylation complex, 14-3-3 zeta, GSK3 beta, and tau co-immunoprecipitate with each other. From extracts of bovine brain, COS-7 cells, and HEK-293 cells transfected with GSK3 beta, 14-3-3 zeta co-precipitates with GSK3 beta, indicating that GSK3 beta binds to 14-3-3 zeta. From HEK-293 cells transfected with tau, GSK3 beta, and 14-3-3 zeta in different combinations, tau co-immunoprecipitates with GSK3 beta only in the presence of 14-3-3 zeta. In vitro, approximately 10-fold more tau binds to GSK3 beta in the presence of than in the absence of 14-3-3 zeta. In transfected HEK-293 cells, 14-3-3 zeta stimulates GSK3 beta-catalyzed tau phosphorylation in a dose-dependent manner. These data indicate that in brain, the 14-3-3 zeta dimer simultaneously binds and bridges tau and GSK3 beta and stimulates GSK3 beta-catalyzed tau phosphorylation.     

Interacting genetic loci on chromosomes 20 and 10 influence extreme human obesity

Obesity is a multigenic trait that has a substantial genetic component. Animal models confirm a role for gene-gene interactions, and human studies suggest that as much as one-third of the heritable variance may be due to nonadditive gene effects. To evaluate potential epistatic interactions among five regions, on chromosomes 7, 10, and 20, that have previously been linked to obesity phenotypes, we conducted pairwise correlation analyses based on alleles shared identical by descent (IBD) for independent obese affected sibling pairs (ASPs), and we determined family-specific nonparametric linkage (NPL) scores in 244 families. The correlation analyses were also conducted separately, by race, through use of race-specific allele frequencies. Conditional analyses for a qualitative trait (body mass index [BMI] >/=27) and hierarchical models for quantitative traits were used to further refine evidence of gene interaction. Both the ASP-specific IBD-sharing probability and the family-specific NPL score revealed that there were strong positive correlations between 10q (88-97 cM) and 20q (65-83 cM), through single-point and multipoint analyses with three obesity thresholds (BMI >/=27, >/=30, and >/=35) across African American and European American samples. Conditional analyses for BMI >/=27 found that the LOD score at 20q rises from 1.53 in the baseline analysis to 2.80 (empirical P=.012) when families were weighted by evidence for linkage at 10q (D10S1646) through use of zero-one weights (weight(0-1)) and to 3.32 (empirical P<.001) when proportional weights (weight(prop)) were used. For percentage fat mass, variance-component analysis based on a two-locus epistatic model yielded significant evidence for interaction between 20q (75 cM) and the chromosome 10 centromere (LOD = 1.74; P=.024), compared with a two-locus additive model (LOD = 0.90). The results from multiple methods and correlated phenotypes are consistent in suggesting that epistatic interactions between loci in these regions play a role in extreme human obesity.     

Induction of cytochrome P450 1A is required for circulation failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish

The mechanism of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is thought to result from changes in gene expression via the aryl hydrocarbon receptor (AHR). The induction of cytochrome P450 1A (CYP1A) in various organs is a cardinal effect of TCDD. However, whether CYP1A is involved in endpoints of TCDD toxicity is controversial. We investigated the role of CYP1A in TCDD-induced developmental toxicities using gene knock-down with morpholino antisense oligos. Exposure of zebrafish embryos to TCDD, at concentrations eliciting the hallmark endpoints of developmental toxicity, induced CYP1A in the heart and vascular endothelium throughout the body. This induction by TCDD was markedly inhibited by morpholinos to zebrafish arylhydrocarbon receptor 2 (zfAHR2-MO) and to zebrafish CYP1A (zfCYP1A-MO). The zfAHR2-MO but not the zfCYP1A-MO inhibited zfCYP1A mRNA expression, indicating the specificities of these morpholinos. Injection of either zfAHR2-MO or zfCYP1A-MO blocked the representative signs of TCDD developmental toxicity in zebrafish, pericardial edema and trunk circulation failure. The morpholinos appeared do not affect normal development in TCDD-untreated embryos. These results suggest a mediatory role of zfCYP1A induction through zfAHR2 activation in causing circulation failure by TCDD in zebrafish. This is the first molecular evidence demonstrating an essential requirement for CYP1A induction in TCDD-evoked developmental toxicities in any vertebrate species.     

Tight-binding inhibition by alpha-naphthoflavone of human cytochrome P450 1A2

Human cytochrome P450 (P450) enzymes exhibit remarkable diversity in their substrate specificities, participating in oxidation reactions of a wide range of xenobiotic drugs. Previously, we reported that alpha-naphthoflavone (ANF) is bound to the recombinant P450 1A2 tightly and stabilizes an overall enzyme conformation. The present study is designed to determine the type of P450 1A2 inhibition exerted by ANF, using two different substrates of P450 1A2, 7-ethoxycoumarin (EOC) and 7-ethoxyresorufin (EOR). ANF is generally known as a competitive inhibitor of the enzyme. However, in our tight-binding enzyme kinetics study, ANF acts as noncompetitive inhibitor in 7-ethoxycoumarin O-deethylation (ECOD) (K(i)=55.0 nM), but as competitive inhibitor in 7-ethoxyresorufin O-deethylation (EROD) (K(i)=1.4 nM). Based on homology modeling studies, ANF is positioned to bind to a hydrophobic cavity next to the active site where it may cause a direct effect on substrate binding. It is agreed with the predicted binding site of ANF in P450 3A4, in which ANF is rather known as a stimulating modulator. Our results suggest that ANF binds near the active site of P450 1A2 and exhibits differential inhibition mechanisms, possibly depending on the molecular structure of the substrate.     

Increased Ca2+ storage capacity in the sarcoplasmic reticulum by overexpression of HRC (histidine-rich Ca2+ binding protein)

The histidine-rich Ca(2+) binding protein (HRC) is a high capacity Ca(2+) binding protein in the sarcoplasmic reticulum (SR). Because HRC appears to interact directly with triadin, HRC may play a role in the regulation of Ca(2+) release during excitation-contraction coupling. In this study, we examined the physiological effects of HRC overexpression in rat neonatal cardiomyocytes. Both caffeine-induced and depolarization-induced Ca(2+) release from the SR were increased significantly in the HRC overexpressing cardiomyocytes. Consistently, the Ca(2+) content, normally depleted from the SR in the presence of cyclopiazonic acid (CPA), remained elevated in these cells. In contrast, the density and the ryanodine-binding kinetics of the ryanodine receptor (RyR)/Ca(2+) release channel were slightly reduced or not significantly altered in the HRC overexpressing cardiomyocytes. We suggest that HRC is involved in the regulation of releasable Ca(2+) content into the SR.     

A naturally enhanced green fluorescent protein from magnificent sea anemone (Heteractis magnifica) and its functional analysis

A novel fluorescent protein termed hmGFP homologous to the green fluorescent protein (GFP) from Aequorea victoria was cloned from the tentacles of sea anemone Heteractis magnifica by EST sequencing and analysis of cDNA library and followed by using RT-PCR. The sequence analysis suggested that the chromophore, consensus amino acids, and secondary structure of 11 beta-strands of hmGFP were similar to those of GFP from other species. The recombinant hmGFP protein with high purity was obtained by the fusion expression of pETTRX-hmGFP in Escherichia coli and subsequent purification. The pH sensitivity and fluorescence spectroscopy of recombinant hmGFP were characterized. The excitation spectrum of recombinant hmGFP has a rather wide major peak with a maximum at 490 nm and a shoulder at 420 nm, and its emission spectrum at 510 nm. The expression of hmGFP and the chimera IPL through hmGFP in CHO cells has shown that the fusion protein IPL through hmGFP has retained the normal membrane targeting of the IPL from Dasyatis akajei, as well as maintaining fluorescent properties similar to those of native hmGFP, suggesting a promising prospect of the application in biotechnology research for the new protein.     

Self-stabilized CpG DNAs optimally activate human B cells and plasmacytoid dendritic cells

We recently showed that 5'-terminal secondary structures in CpG DNA affect activity significantly more than those at the 3'-end [Biochem. Biophys. Res. Commun. 306 (2003) 948]. The need for an accessible 5'-end of CpG DNA for activity suggested that the receptor reads the DNA sequence from this end. In continuation of these studies, we have designed immunomodulatory oligonucleotides (IMOs), consisting of a nine-mer stimulatory domain, containing a CpG motif and a hairpin-loop structure at the 3'-end, referred to as self-stabilized CpG DNAs. We studied the ability of self-stabilized CpG DNAs to stimulate human B-cell proliferation and interferon-alpha (IFN-alpha) secretion in plasmacytoid dendritic cell (pDC) culture assays. Self-stabilized CpG DNAs activated human B cells and induced plasmacytoid dendritic cells to secrete high levels of IFN-alpha. While both stimulatory and secondary structures in CpG DNAs were required for pDC activation, CpG motifs were sufficient to activate B cells. Interestingly, CpG motifs were not required for activity in the hairpin duplex region. Further modifications of the hairpin duplex region with a mixture of oligodeoxynucleotides and oligo-2'-O-methylribonucleotides in a heteroduplex formation permitted activation of both human B cells and pDCs.     

Electrostatic contributions to the stability of a thermophilic cold shock protein

The thermophilic Bacillus caldolyticus cold shock protein (Bc-Csp) differs from the mesophilic Bacillus subtilis cold shock protein B (Bs-CspB) in 11 of the 66 residues. Stability measurements of Schmid and co-workers have implicated contributions of electrostatic interactions to the thermostability. To further elucidate the physical basis of the difference in stability, previously developed theoretical methods that treat electrostatic effects in both the folded and the unfolded states were used in this paper to study the effects of mutations, ionic strength, and temperature. For 27 mutations that narrow the difference in sequence between Bc-Csp and Bs-CspB, calculated changes in unfolding free energy (Delta G) and experimental results have a correlation coefficient of 0.98. Bc-Csp appears to use destabilization of the unfolded state by unfavorable charge-charge interactions as a mechanism for increasing stability. Accounting for the effects of ionic strength and temperature on the electrostatic free energies in both the folded and the unfolded states, explanations for two important experimental observations are presented. The disparate ionic strength dependences of Delta G for Bc-Csp and Bs-CspB were attributed to the difference in the total charges (-2e and -6e, respectively). A main contribution to the much higher unfolding entropy of Bs-CspB was found to come from the less favorable electrostatic interactions in the folded state. These results should provide insight for understanding the thermostability of other thermophilic proteins.