Dynamic control of yeast MAP kinase network by induced association and dissociation between the Ste50 scaffold and the Opy2 membrane anchor

Membrane localization of the Ste11 MAPKKK is essential for activation of both the filamentous growth/invasive growth (FG/IG) MAP kinase (MAPK) pathway and the SHO1 branch of the osmoregulatory HOG MAPK pathway, and is mediated by binding of the Ste50 scaffold protein to the Opy2 membrane anchor. We found that Opy2 has two major (CR-A and CR-B), and one minor (CR-D), binding sites for Ste50. CR-A binds Ste50 constitutively and can transmit signals to both the Hog1 and Fus3/Kss1 MAPKs. CR-B, in contrast, binds Ste50 only when Opy2 is phosphorylated by Yck1/Yck2 under glucose-rich conditions and transmits the signal preferentially to the Hog1 MAPK. Ste50 phosphorylation by activated Hog1/Fus3/Kss1 MAPKs downregulates the HOG MAPK pathway by dissociating Ste50 from Opy2. Furthermore, Ste50 phosphorylation, together with MAPK-specific protein phosphatases, reduces the basal activity of the HOG and the mating MAPK pathways. Thus, dynamic regulation of Ste50-Opy2 interaction fine-tunes the MAPK signaling network.     

Structural insights into the hot spot amino acid residues of mushroom tyrosinase for the bindings of thujaplicins

Tyrosinase inhibitors are important agents for cosmetic products. We examined here the inhibitory effects of three isomers of thujaplicins (α, β and γ) on mushroom tyrosinase and analyzed their binding modes using a homology model from the crystal structure of Streptomyces castaneoglobisporus tyrosinase (PDB ID: 1wx2). All the thujaplicins were found to be competitive inhibitors and γ-thujaplicin has the most potent inhibitory activity (IC(50)=0.07μM). It is noted that there are good correlations between their observed IC(50) values and their binding free energies calculated by MM-GB/SA. The binding modes of thujaplicins were predicted to be similar to that of Tyr98 of caddie protein (ORF378), which was co-crystallized with S. castaneoglobisporus tyrosinase. Furthermore, free energy decomposition analysis indicated that the potent inhibitory activity of γ-thujaplicin is due to the interactions with His242, Val243 and Pro257 (hot spot amino acid residues) at the active site of tyrosinase. These results provide a novel structural insight into the hot spot of mushroom tyrosinase for the specific binding of γ-thujaplicin.     

Adaptor functions of Cdc42, Ste50, and Sho1 in the yeast osmoregulatory HOG MAPK pathway

The yeast high osmolarity glycerol (HOG) signaling pathway can be activated by either of the two upstream pathways, termed the SHO1 and SLN1 branches. When stimulated by high osmolarity, the SHO1 branch activates an MAP kinase module composed of the Ste11 MAPKKK, the Pbs2 MAPKK, and the Hog1 MAPK. To investigate how osmostress activates this MAPK module, we isolated both gain-of-function and loss-of-function alleles in four key genes involved in the SHO1 branch, namely SHO1, CDC42, STE50, and STE11. These mutants were characterized using an HOG-dependent reporter gene, 8xCRE-lacZ. We found that Cdc42, in addition to binding and activating the PAK-like kinases Ste20 and Cla4, binds to the Ste11-Ste50 complex to bring activated Ste20/Cla4 to their substrate Ste11. Activated Ste11 and its HOG pathway-specific substrate, Pbs2, are brought together by Sho1; the Ste11-Ste50 complex binds to the cytoplasmic domain of Sho1, to which Pbs2 also binds. Thus, Cdc42, Ste50, and Sho1 act as adaptor proteins that control the flow of the osmostress signal from Ste20/Cla4 to Ste11, then to Pbs2.     

Synergistic interaction of xyloglucan and xanthan investigated by rheology, differential scanning calorimetry, and NMR

A new synergistic interaction between tamarind seed xyloglucan and xanthan was found and investigated by rheology, differential scanning calorimetry (DSC), and NMR. The effect of the acetyl and pyruvate groups in the side chain in xanthan on the synergistic interaction was also examined. The shear moduli G' and G' ' of the mixture solution of xyloglucan and native (or acetate-free) xanthan increased steeply at around 22 degrees C upon cooling. An exothermic DSC peak appeared at the same temperature. A drastic decrease in the of the acetyl and pyruvate groups of the xanthan side chain was observed from 1H NMR spectra only in the mixture at low temperatures (<25 degrees C). It was found that the pyruvate group is more restricted in the mixture solution compared with the acetyl group. The mixture of xyloglucan and pyruvate-free xanthan showed no synergistic interaction. We concluded that this synergistic interaction is caused by the intermolecular binding between xyloglucan and xanthan, and, in the heterotypic junction zones, the xanthan side chain becomes a new state that is different from both the coil and helix states.     

The mouse embryo autonomously acquires anterior-posterior polarity at implantation

The earliest recognizable sign of patterning of the mouse embryo along the anteroposterior (A-P) axis is the migration of the distal visceral endoderm (DVE) toward the future anterior side. Here we report an asymmetry in the mouse embryo at an unexpectedly early stage. The gene for Lefty1, a Nodal antagonist that influences the direction of DVE migration, was found to be asymmetrically expressed in the primitive endoderm of the implanting blastocyst. Lefty1 expression begins randomly in the inner cell mass (ICM) of the blastocyst but is regionalized to one side of the tilted ICM shortly after implantation. Asymmetric expression of Lefty1 can be established by in vitro culture, indicating that it does not require interaction with the uterus. The asymmetric Lefty1 expression is induced by Nodal signaling, although Nodal and genes for its effectors are expressed symmetrically. This asymmetry in molecular patterning of the mouse embryo pushes back the origin of the A-P body axis to the peri-implantation stage.     

Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions

Adiponectin plays a central role as an antidiabetic and antiatherogenic adipokine. AdipoR1 and AdipoR2 serve as receptors for adiponectin in vitro, and their reduction in obesity seems to be correlated with reduced adiponectin sensitivity. Here we show that adenovirus-mediated expression of AdipoR1 and R2 in the liver of Lepr(-/-) mice increased AMP-activated protein kinase (AMPK) activation and peroxisome proliferator-activated receptor (PPAR)-alpha signaling pathways, respectively. Activation of AMPK reduced gluconeogenesis, whereas expression of the receptors in both cases increased fatty acid oxidation and lead to an amelioration of diabetes. Alternatively, targeted disruption of AdipoR1 resulted in the abrogation of adiponectin-induced AMPK activation, whereas that of AdipoR2 resulted in decreased activity of PPAR-alpha signaling pathways. Simultaneous disruption of both AdipoR1 and R2 abolished adiponectin binding and actions, resulting in increased tissue triglyceride content, inflammation and oxidative stress, and thus leading to insulin resistance and marked glucose intolerance. Therefore, AdipoR1 and R2 serve as the predominant receptors for adiponectin in vivo and play important roles in the regulation of glucose and lipid metabolism, inflammation and oxidative stress in vivo.     

Interactions between Serum Vitamin D Levels and Vitamin D Receptor Gene FokI Polymorphisms for Renal Function in Patients with Type 2 Diabetes

Background

We aimed to examine associations among serum 25-hydroxyvitamin D (25OHD) levels, 1,25-dihyroxyvitamin D (1,25OHD) levels, vitamin D receptor (VDR) polymorphisms, and renal function based on estimated glomerular filtration rate (eGFR) in patients with type 2 diabetes.

Methods

In a cross-sectional study of 410 patients, chronic kidney disease (CKD) stage assessed by eGFR was compared with 25OHD, 1,25OHD, and VDR FokI (rs10735810) polymorphisms by an ordered logistic regression model adjusted for the following confounders: disease duration, calendar month, use of angiotensin converting enzyme inhibitors/angiotensin receptor blockers or statins, and serum calcium, phosphate, and intact parathyroid hormone levels.

Results

1,25OHD levels, rather than 25OHD levels, showed seasonal oscillations; peak levels were seen from May to October and the lowest levels were seen from December to February. These findings were evident in patients with CKD stage 3∼5 but not stage 1∼2. eGFR was in direct proportion to both 25OHD and 1,25OHD levels (P<0.0001), but it had stronger linearity with 1,25OHD (r = 0.73) than 25OHD (r = 0.22) levels. Using multivariate analysis, 1,25OHD levels (P<0.001), but not 25OHD levels, were negatively associated with CKD stage. Although FokI polymorphisms by themselves showed no significant associations with CKD stage, a significant interaction between 1,25OHD and FokITT was observed (P = 0.008). The positive association between 1,25OHD and eGFR was steeper in FokICT and CC polymorphisms (r = 0.74) than FokITT polymorphisms (r = 0.65).

Conclusions

These results suggest that higher 1,25OHD levels may be associated with better CKD stages in patients with type 2 diabetes and that this association was modified by FokI polymorphisms.     

华北二月兰-春玉米轮作体系中土壤硝态氮的时空变化特征

二月兰-春玉米轮作生产体系是近年来为解决华北地区出现的大面积冬闲田而提出的冬绿肥-春玉米生产新模式.本文依托定位试验,研究了该体系从二月兰翻压到玉米收获期间的土壤硝态氮时空变化特征.结果表明：土壤硝态氮含量呈玉米生育前期高后期低的时间变化特征和硝态氮含量峰值随着生育期的推移逐渐下移的空间变化特征,且土壤硝态氮含量随施肥量的增加而显著增加.翻压二月兰对土壤硝态氮含量的时空变化有一定影响,冬春季种植二月兰可降低0～180 cm土壤硝态氮累积量;二月兰翻压后,春玉米苗期与喇叭口期土壤硝态氮规律基本一致,主要集中在0～20 cm土层,0～100 cm土壤剖面为有二月兰处理高于无二月兰处理,100～180 cm土壤剖面则为有二月兰处理低于无二月兰处理;抽雄期以后,土壤硝态氮含量普遍较低,100～180 cm土层土壤硝态氮含量为有二月兰处理略高于无二月兰处理.总体上,翻压二月兰可以增加0～180 cm土层土壤硝态氮保蓄量.     

Mutational deglycosylation of the Fc portion of immunoglobulin G causes O-sulfation of tyrosine adjacently preceding the originally glycosylated site

Mutagenesis directed to a specific glycosylation site has been widely used to examine biological roles of individual glycans. However, occurrence of any post-translational modification on such deglycosylated mutants has not yet been well characterized. Here we performed mass spectrometric analyses of the Fc fragment of an unglycosylated mutant of mouse immunoglobulin G2b, whose conserved N-glycosylation site, i.e. Asn297, was substituted with alanine. We found that a major part of this mutant is sulfated at Tyr296, which adjacently precedes the originally glycosylated site. Our findings demonstrate that mutational deglycosylation can induce an unexpected post-translational modification in the protein.     

Detection of sulfur-containing compounds in control and cadmium-exposed rat organs by high-performance liquid chromatography--vacuum-ultraviolet inductively coupled plasma-atomic emission spectrometry (HPLC-ICP)

Sulfur-containing compounds in biological samples were separated by high-performance gel permeation chromatography and detected with a vacuum-ultraviolet inductively coupled plasma-atomic emission spectrometer. Distribution profiles of sulfur in the supernatants of liver, kidney, spleen, lung, and pancreas of control and cadmium-exposed rats were determined along with cadmium, copper, iron, phosphorus, and zinc profiles. Changes in sulfur distribution were induced by cadmium exposure not only in the metallothionein fraction, but also in the high-molecular-weight protein fraction, indicating the effect of cadmium exposure on diverse endogenous sulfur-containing compounds. Glutathione and taurine also were detected simultaneously as distinct peaks.