Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase

Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.     

Magnetic signature of environmental change reflected by Pleistocene lacustrine sediments from the Nihewan Basin, North China

A mineral magnetic study has been conducted on Pleistocene lacustrine sediments from the Xujiayao Paleolithic site within the Nihewan Basin, North China. The magnetic signal is characterized by a dominance of high-coercivity (hematite) minerals that is modulated by low-coercivity magnetite–maghemite assemblages during interglacials. A high-amplitude record of the composition dependent magnetic S_− 0.3 T-parameter varies in accord with composite marine oxygen isotope records, suggesting climate modulation of magnetic composition. The Xujiayao site is situated at the northern outskirts of the Chinese Loess Plateau (CLP) and has neither received significant eolian input nor been exposed to pedogenic processes comparable to those acting on CLP. Mineral magnetic analyses suggest that variations in both magnetite and hematite are primarily controlled by detrital fluxes rather than by authigenic production of magnetic minerals. Here we propose that the fluctuations of magnetic signals at Xujiayao are not derived from varying compositions of eolian flux with glacial/interglacial periods or minute eolian influx during interglacials, but is likely dominated by runoff-related processes, by which some weathered/pedogenized loess and soils with stronger magnetic signals have been eroded and transported to the catchment during interglacials resulting in magnetic enhancement. The inferred continuous deposition from the Brunhes/Matuyama boundary to the paleosol S₁ (ca. 129 ka) gives the age of ca. 500 ka for the sediments carrying the Xujiayao Paleolithic site, suggesting an allogenic origin of the Paleolithic artifacts and mammalian bones dated by U–Th method to ca. 100 ka.     

Lipid production by microalgae <Emphasis Type="Italic">Chlorella protothecoides</Emphasis> with volatile fatty acids (VFAs) as carbon sources in heterotrophic cultivation and its economic assessment

Volatile fatty acids (VFAs) that can be derived from food wastes were used for microbial lipid production by Chlorella protothecoides in heterotrophic cultures. The usage of VFAs as carbon sources for lipid accumulation was investigated in batch cultures. Culture medium, culture temperature, and nitrogen sources were explored for lipid production in the heterotrophic cultivation. The concentration and the ratio of VFAs exhibited significant influence on cell growth and lipid accumulation. The highest lipid yield coefficient and lipid content of C. protothecoides grown on VFAs were 0.187 g/g and 48.7 %, respectively. The lipid content and fatty acids produced using VFAs as carbon sources were similar to those seen on growth and production using glucose. The techno-economic analysis indicates that the biodiesel derived from the lipids produced by heterotrophic C. protothecoides with VFAs as carbon sources is very promising and competitive with other biofuels and fossil fuels.     

Role of an N-Terminal Splice Segment in the Activation of the Cation Channel TRPM2 by ADP-Ribose and Hydrogen Peroxide

In the dysfunctional splice variant TRPM2-ΔN, a stretch of 20 amino acids (aa 537–556) is missing within the N-terminal cytosolic tail of the cation channel TRPM2. The ΔN-stretch overlaps with two IQ-like calmodulin-binding domains. Moreover, it contains two PxxP motifs implicated in protein–protein interactions. Here, we constructed variants to test whether any of these motifs may explain why TRPM2-ΔN does not respond to stimulation with either ADP ribose or hydrogen peroxide. Each of the two IQ-motifs could be removed without loss of channel function. Similarly, deletion of either one or both PxxP motifs had no effect. Moreover, the single point mutation D543E associated with bipolar disorder does not change the activation of TRPM2. We conclude that no functional role can be attributed to any of the structural motifs within the ΔN-stretch that may be a spacer segment for other functional sites in the N terminus.     

Combination of newly developed high quality <Emphasis Type="Italic">Fuqu</Emphasis> with traditional <Emphasis Type="Italic">Daqu</Emphasis> for <Emphasis Type="Italic">Luzhou-flavor</Emphasis> liquor brewing

Application of a compound starter composed of traditional Daqu, high quality Fuqu developed previously in our lab, and alcohol-producing and aroma-producing yeasts in the brewing of Luzhou-flavor liquor was investigated. Compared with traditional Daqu, the compound starter demonstrated apparently higher starch utilization and alcohol productivity both in flasks and in pits. Flavor ingredient analyses showed that the flavor ingredient compositions of liquors produced with the compound starter were similar to those obtained with the traditional process. With appropriate after treatments, the compound starter may be applied in distilleries for elevating the productivity of traditional liquor or producing high quality new-style distilled liquor. As starch utilization can be increased significantly, using this compound starter in liquor brewing is especially meaningful for lessening environmental pollution. These findings may also be helpful for further understanding the mechanisms of Chinese liquor brewing.     

Role of myosin light chain kinase in regulation of basal blood pressure and maintenance of salt-induced hypertension

Vascular tone, an important determinant of systemic vascular resistance and thus blood pressure, is affected by vascular smooth muscle (VSM) contraction. Key signaling pathways for VSM contraction converge on phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin. This phosphorylation is mediated by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) but Ca(2+)-independent kinases may also contribute, particularly in sustained contractions. Signaling through MLCK has been indirectly implicated in maintenance of basal blood pressure, whereas signaling through RhoA has been implicated in salt-induced hypertension. In this report, we analyzed mice with smooth muscle-specific knockout of MLCK. Mesenteric artery segments isolated from smooth muscle-specific MLCK knockout mice (MLCK(SMKO)) had a significantly reduced contractile response to KCl and vasoconstrictors. The kinase knockout also markedly reduced RLC phosphorylation and developed force. We suggest that MLCK and its phosphorylation of RLC are required for tonic VSM contraction. MLCK(SMKO) mice exhibit significantly lower basal blood pressure and weaker responses to vasopressors. The elevated blood pressure in salt-induced hypertension is reduced below normotensive levels after MLCK attenuation. These results suggest that MLCK is necessary for both physiological and pathological blood pressure. MLCK(SMKO) mice may be a useful model of vascular failure and hypotension.     

Purification and Structural Characterization of Dendrobium officinale Polysaccharides and Its Activities

In this study, Dendrobium officinale polysaccharide (named DOPS-1) was isolated from the stems of Dendrobium officinale by hot-water extraction and purified by using Sephadex G-150 column chromatography. The structural characterization, antioxidant and cytotoxic activity were carried out. Based on the results of HPLC, GC, Congo red experiment, together with periodate oxidation, Smith degradation, SEM, FT-IR, and NMR spectral analysis, it expressed that DOPS-1 was largely composed of mannose, glucose and galacturonic acid in a molar ratio of 3.2 : 1.3 : 1. The molecular weight of DOPS-1 was 1530 kDa and the main chain was composed of (1→4)-β-D-Glcp, (1→4)-β-D-Manp and 2-O-acetyl-(1→4)-β-D-Manp. The measurement results of antioxidant activity showed that DOPS-1 had the strong scavenging activities on hydroxyl radicals, DPPH radicals and superoxide radicals and the high reducing ability in vitro. Moreover, DOPS-1 was cytotoxic to all three human cancer cells of MDA-MB-231, A549 and HepG2.