Overexpression of Arabidopsis ATX1 retards plant growth under severe copper deficiency

In a previous study, we demonstrated that Arabidopsis Antioxidant Protein1 (ATX1) plays an essential role in copper (Cu) homeostasis, conferring tolerance to both excess and subclinically deficient Cu. The Cu-binding motif MXCXXC was required for the physiological function of ATX1. In this study, we found that overexpression of ATX1 resulted in hypersensitivity to severe Cu deficiency despite enhancing tolerance to subclinical Cu deficiency. However, overexpression of mutated ATX1, replacing the Cu-binding motif MXCXXC with MXGXXG, abolished the hypersensitivity, for no differences from the wild type under the same conditions. Thus, the expression of ATX1 must be cautiously regulated to avoid homeostatic imbalance with the over-chelation of Cu.     

Real time visualization of protein kinase activity in living cells.

A library of fluorescently labeled protein kinase C (PKC) peptide substrates was prepared to identify a phosphorylation-induced reporter of protein kinase activity. The lead PKC substrate displays a 2.5-fold change in fluorescence intensity upon phosphorylation. PKC activity is readily sampled in cell lysates containing the activated PKCs. Immunodepletion of conventional PKCs from the cell lysate eliminates the fluorescence response, suggesting that this peptide substrate is selectively phosphorylated by PKCalpha, beta, and gamma. Finally, living cells microinjected with the peptide substrate exhibit a 2-fold increase in fluorescence intensity upon exposure to a PKC activator. These results suggest that peptide-based protein kinase biosensors may be useful in monitoring the temporal and spatial dynamics of PKC activity in living cells.     

XLID CUL4B mutants are defective in promoting TSC2 degradation and positively regulating mTOR signaling in neocortical neurons

Truncating or missense mutation of cullin 4B (CUL4B) is one of the most prevalent causes underlying X-linked intellectual disability (XLID). CUL4B-RING E3 ubiquitin ligase promotes ubiquitination and degradation of various proteins. Consistent with previous studies, overexpression of wild-type CUL4B in 293 cells enhanced ubiquitylation and degradation of TSC2 or cyclin E. The present study shows that XLID mutant (R388X), (R572C) or (V745A) CULB failed to promote ubiquitination and degradation of TSC2 or cyclin E. Adenoviruses-mediated expression of wild-type CUL4B decreased protein level of TSC2 or cyclin E in cultured neocortical neurons of frontal lobe. Furthermore, shRNA-mediated CUL4B knockdown caused an upregulation of TSC2 or cyclin E. XLID mutant (R388X), (R572C) or (V745A) CUL4B did not downregulate protein expression of TSC2 or cyclin E in neocortical neurons. By promoting TSC2 degradation, CUL4B could positively regulate mTOR activity in neocortical neurons of frontal cortex. Consistent with this hypothesis, CUL4B knockdown-induced upregulation of TSC2 in neocortical neurons resulted in a decreased protein level of active phospho-mTOR^Ser2448 and a reduced expression of active phospho-p70S6K^Thr389 and phospho-4E-BP1^Thr37/46, two main substrates of mTOR-mediated phosphorylation. Wild-type CUL4B also increased protein level of active phospho-mTOR^Ser2448, phospho-p70S6K^Thr389 or phospho-4E-BP1^Thr37/46. XLID CUL4B mutants did not affect protein level of active phospho-mTOR^Ser2448, phospho-p70S6K^Thr389 or phospho-4E-BP1^Thr37/46. Our results suggest that XLID CUL4B mutants are defective in promoting TSC2 degradation and positively regulating mTOR signaling in neocortical neurons.     

Raman spectra of a solid antifreeze glycoprotein and its liquid and frozen aqueous solutions.

Raman spectroscopy was used to study the anomalous decrease in the freezing temperature of water produced by an antifreeze glycoprotein obtained from the sera of an Antarctic fish. An active fraction of this glycoprotein has a molecular weight of approximately 18,000 by equilibrium sedimentation compared to an apparent weight of 20 by freezing temperature depression. The Raman spectra of water present in a 1% antifreeze glycoprotein solution and of ice frozen from this solution were indistinguishable from the spectra of pure water and ice, respectively. These results indicate that the bulk properties of water and ice are unaffected by the presence of the antifreeze glycoprotein. Raman measurements on ice grown slowly, using as seed an oriented single crystal of ice in contact with 1% glycoprotein solutions, showed that the active glycoprotein was not excluded from the ice phase. On the other hand, we found that a smaller, inactive glycoprotein was excluded. Comparison of the Raman spectra of active and inactive glycoprotein components as solids, in 5% solutions, and rapidly frozen 5% solutions, showed that the two components differ in conformation and possibly in the environment of their carbohydrate hydroxyls. These observations suggest that hydrogen bonding of the carbohydrate hydroxyls of the active glycoprotein at the ice-solution interface may physically prevent growth of the ice lattice.     

Identification of the probable coding region for exon 2 of cytochrome oxidase polypeptide I from Aspergillus nidulans mitochondrion

Hypothetical protein URFe of Aspergillus nidulans mitochondrion is homologous with the amino end of cytochrome oxidase (EC 1.9.3.1) polypeptide I. Unidentified reading frame URFe does not contain a suitable initiation codon and codes for a protein with a length of only 91 residues, corresponding to about 20% of cytochrome oxidase polypeptide I. It is proposed that this region codes for the second exon of the cox 1 gene of Aspergillus mitochondrion. Possible candidates for the 2- to 3-residue amino-terminal exon 1 are discussed.     

The Xanthine Derivative KMUP-1 Attenuates Serotonin-Induced Vasoconstriction and K+-Channel Inhibitory Activity via the PKC Pathway in Pulmonary Arteries

Serotonin (5-hydroxytryptamine, 5-HT) is a potent pulmonary vasoconstrictor that promotes pulmonary artery smooth muscle cell (PASMC) proliferation. 5-HT-induced K⁺ channel inhibition increases [Ca²⁺]_i in PASMCs, which is a major trigger for pulmonary vasoconstriction and development of pulmonary arterial hypertension (PAH). This study investigated whether KMUP-1 reduces pulmonary vasoconstriction in isolated pulmonary arteries (PAs) and attenuates 5-HT-inhibited K⁺ channel activities in PASMCs. In endothelium-denuded PA rings, KMUP-1 (1 μM) dose-dependently reduced 5-HT (100 μM) mediated contractile responses. Responses to KMUP-1 were reversed by K⁺ channel inhibitors (TEA, 10 mM, 4-aminopyridine, 5 mM, and paxilline, 10 μM). In primary PASMCs, KMUP-1 also dose-dependently restored 5-HT-inhibited voltage-gated K⁺-channel (Kv1.5 and Kv2.1) and large-conductance Ca²⁺-activated K⁺-channel (BK_Ca) proteins, as confirmed by immunofluorescent staining. Furthermore, 5-HT (10 μM)-inhibited Kv1.5 protein was unaffected by the PKA inhibitor KT5720 (1 μM) and the PKC activator PMA (1 μM), but these effects were reversed by KMUP-1 (1 μM), 8-Br-cAMP (100 μM), chelerythrine (1 μM), and KMUP-1 combined with a PKA/PKC activator or inhibitor. Notably, KMUP-1 reversed 5-HT-inhibited Kv1.5 protein and this response was significantly attenuated by co-incubation with the PKC activator PMA, suggesting that 5-HT-mediated PKC signaling can be modulated by KMUP-1. In conclusion, KMUP-1 ameliorates 5-HT-induced vasoconstriction and K⁺-channel inhibition through the PKC pathway, which could be valuable to prevent the development of PAH.     

Obesity and Hepatic Steatosis Are Associated with Elevated Serum Amyloid Beta in Metabolically Stressed APPswe/PS1dE9 Mice

Diabesity-associated metabolic stresses modulate the development of Alzheimer’s disease (AD). For further insights into the underlying mechanisms, we examine whether the genetic background of APPswe/PS1dE9 at the prodromal stage of AD affects peripheral metabolism in the context of diabesity. We characterized APPswe/PS1dE9 transgenic mice treated with a combination of high-fat diet with streptozotocin (HFSTZ) in the early stage of AD. HFSTZ-treated APPswe/PS1dE9 transgenic mice exhibited worse metabolic stresses related to diabesity, while serum β-amyloid levels were elevated and hepatic steatosis became apparent. Importantly, two-way analysis of variance shows a significant interaction between HFSTZ and genetic background of AD, indicating that APPswe/PS1dE9 transgenic mice are more vulnerable to HFSTZ treatment. In addition, body weight gain, high hepatic triglyceride, and hyperglycemia were positively associated with serum β-amyloid, as validated by Pearson’s correlation analysis. Our data suggests that the interplay between genetic background of AD and HFSTZ-induced metabolic stresses contributes to the development of obesity and hepatic steatosis. Alleviating metabolic stresses including dysglycemia, obesity, and hepatic steatosis could be critical to prevent peripheral β-amyloid accumulation at the early stage of AD.