Two domains are critical for the nuclear localization of soluble adenylyl cyclase

Soluble adenylyl cyclase (sAC) is a newly identified source of cyclic adenosine 3',5'-monophosphate (cAMP). Unlike the well-known transmembrane adenylyl cyclases (tmACs), sAC locates to the nucleus, mitochondria and microtubules. For most cAMP-signaling microdomains, there is always an AC nearby, for example tmAC. But it was until the discovery of sAC that there was not known cAMP resource in the nucleus. sAC associates with nuclear cAMP-signaling microdomains, which were once considered to depend on the diffusion of cAMP produced by tmAC. In this report, we focus on the truncated soluble adenylyl cyclase (tsAC), the most common existence form of sAC in tissues. Two domains (145-200 aa and 257-318 aa) related with sAC nuclear localization were present here. The findings provide evidence that these two domains are critical for the nuclear localization of sAC and they collocated with the catalytic domains.     

海马位置细胞对空间信息的处理

海马位置细胞接收各种来源的空间信息后,可对这些信息进行加工处理,在海马内形成认知地图或加强联合皮层内细胞集群的突触联系以形成对空间位置的永久记忆。海马内的空间信息输出后,在伏核（nucleus accumbens,NAC）。内与其它来源的信息进行整合,最终通过运动环路形成目标指向性行为。     

Levels of selenium, zinc, copper, and antioxidant enzyme activity in patients with leukemia

Essential elements, mainly selenium and zinc, were involved in protection against oxidative stress in cells. Oxidation could lead to the formation of free radicals that have been implicated in the pathogenesis of many diseases, including leukemia. Leukemia is a neoplastic disease that is susceptible to antioxidant enzyme and essential elements alterations. This study was undertaken to examine the levels of essential elements, antioxidant enzymes activities, and their relationships with different types of leukemia. Serum selenium, zinc, and copper concentrations, red blood cell glutathione peroxidase (GPx) activities, plasma Cu−Zn superoxide dismutase (Cu−Zn SOD) activities and lipid peroxidation (LPO) levels were determined in 49 patients with different types of leukemia before initial treatment. Serum selenium and zinc concentrations were lower in leukemia patients than those of controls (p<0.01). Serum copper concentration was higher in leukemia patients than that of controls (p<0.01). The activities GPx and Cu−Zn SOD were significantly increased in leukemia patients, especially with acute leukemia (AL), acute lymphoid leukemia (ALL), and acute nonlymphoid leukemia (ANLL) (p<0.05), whereas no difference was found between those of chronic myelogeneous leukemia and the controls. The levels of LPO were normal as controls. Serum selenium concentration was not correlated with GPx, and serum levels of zinc and copper were not related to Cu−Zn SOD. Serum zinc levels had a negative correlation with the absolute peripheral blast cells, whereas serum copper had a positive correlation with the absolute peripheral blast cells. Increased GPx and Cu−Zn SOD activities and normal levels of LPO, which were a protective responses, were an indicator of mild oxidative stress; it mights indicate that the essentials elements alterations in leukemia patients were mostly dependent on tumor activity. Changes of their levels demonstrated that there are low selenium, zinc, and high copper status in leukemia patients. The decrease of plasma zinc and increase of the Cu/Zn ratio could be the index that showed an unfavorable prognosis of acute leukemia.     

Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO

Despite the availability of numerous gene fusion systems, recombinant protein expression in Escherichia coli remains difficult. Establishing the best fusion partner for difficult-to-express proteins remains empirical. To determine which fusion tags are best suited for difficult-to-express proteins, a comparative analysis of the newly described SUMO fusion system with a variety of commonly used fusion systems was completed. For this study, three model proteins, enhanced green fluorescent protein (eGFP), matrix metalloprotease-13 (MMP13), and myostatin (growth differentiating factor-8, GDF8), were fused to the C termini of maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and SUMO tags. These constructs were expressed in E. coli and evaluated for expression and solubility. As expected, the fusion tags varied in their ability to produce tractable quantities of soluble eGFP, MMP13, and GDF8. SUMO and NUS A fusions enhanced expression and solubility of recombinant proteins most dramatically. The ease at which SUMO and NUS A fusion tags were removed from their partner proteins was then determined. SUMO fusions are cleaved by the natural SUMO protease, while an AcTEV protease site had to be engineered between NUS A and its partner protein. A kinetic analysis showed that the SUMO and AcTEV proteases had similar KM values, but SUMO protease had a 25-fold higher kcat than AcTEV protease, indicating a more catalytically efficient enzyme. Taken together, these results demonstrate that SUMO is superior to commonly used fusion tags in enhancing expression and solubility with the distinction of generating recombinant protein with native sequences.     

Synthesis and immunosuppressive activity of new artemisinin derivatives. Part 2: 2-[12(beta or alpha)-dihydroartemisinoxymethyl(or 1'-ethyl)]phenoxyl propionic acids and esters

Another series of novel dihydroartemisinin derivatives were synthesized and assessed for their cytotoxicity of lymphocyte, inhibitory activity on mitogen-induced spleen lymphocyte proliferation in vitro. Some of the compounds exhibited inhibitory effects on ConA-induced T cell and LPS-induced B cell proliferation comparable to or more potent than parent artemisinin.     

Protein kinase Czeta mediates insulin-induced glucose transport through actin remodeling in L6 muscle cells

Protein kinase C (PKC) zeta has been implicated in insulin-induced glucose uptake in skeletal muscle cell, although the underlying mechanism remains unknown. In this study, we investigated the effect of PKCzeta on actin remodeling and glucose transport in differentiated rat L6 muscle cells expressing myc-tagged glucose transporter 4 (GLUT4). On insulin stimulation, PKCzeta translocated from low-density microsomes to plasma membrane accompanied by increase in GLUT4 translocation and glucose uptake. Z-scan confocal microscopy revealed a spatial colocalization of relocated PKCzeta with the small GTPase Rac-1, actin, and GLUT4 after insulin stimulation. The insulin-mediated colocalization, PKCzeta distribution, GLUT4 translocation, and glucose uptake were inhibited by wortmannin and cell-permeable PKCzeta pseudosubstrate peptide. In stable transfected cells, overexpression of PKCzeta caused an insulin-like effect on actin remodeling accompanied by a 2.1-fold increase in GLUT4 translocation and 1.7-fold increase in glucose uptake in the absence of insulin. The effects of PKCzeta overexpression were abolished by cell-permeable PKCzeta pseudosubstrate peptide, but not wortmannin. Transient transfection of constitutively active Rac-1 recruited PKCzeta to new structures resembling actin remodeling, whereas dominant negative Rac-1 prevented the insulin-mediated PKCzeta translocation. Together, these results suggest that PKCzeta mediates insulin effect on glucose transport through actin remodeling in muscle cells.     

Syntheses, structure and properties of chiral copper(II) complexes with end-on azide bridge and chiral-bipyridine ligand

A novel one-dimensional chiral copper(II) complex with single end-on (EO) azide bridge and chiral 2,2-bipyridine ligand, [Cu(N₃)₂(L)]_n (1), and a mononuclear chiral copper(II) complex, [Cu(N₃)₂(L)] (2) (L = (1R)-6,6-dimethyl-5,7-methano-2-(2-pyridinyl)-4,5,6,7-tetrahydroquinoline), have been synthesized and characterized. The crystal structure determination shows that complex 1 is a one-dimensional chiral coordination polymer with non-equivalent Cu-N(azide) bonds, in which the central Cu(II) ion is penta-coordinated in the form of a slightly distorted square-based pyramid. Compound 2 is a four-coordinated mononuclear complex where the Cu(II) ion has a highly distorted tetrahedronal environment. Both complexes 1 and 2 crystallize in the chiral space group: P2₁2₁2₁ and P₁, respectively. The magnetic studies show that there exists antiferromagnetic interaction between the copper(II) ions in complex 1.     

Wnt信号通路对成纤维细胞Rat-1生长及表型的调控

构建Wnt-3a的真核表达拽体并稳定转染人鼠成纤维细胞Rat-1,建立Wnt信号通路持续激活的细胞模型,以探讨Wnt信号通路激活对咳细胞的牛K以及某些表型特征的影响。结果表明：Wnt信号通路持续激活时,Rat-1细胞形态表现为细胞长度的增加,其折光性以及呈绳索状的成束密集排布：MTT以及流式细胞仪检测表明稳定转染后细胞增殖率明显高于正常对照组,进入G2期的细胞增多,细胞增殖分裂能力增强：Transwell小窄迁移实验证实转染组的细胞迁移率略高于对照组,但无显著性差异;体外划痕实验表明,稳定转染后的Rat-1细胞在划痕后伤口愈合时问显著缩短。结果提示：体外Wnt信号通路的激沂能够引起成纤维细胞某些表型改变,并促进细胞增殖,加速体外伤口的修复。     

Interactions Between Vacuolar H<Superscript>+</Superscript>-ATPases and Microfilaments in Osteoclasts

Vacuolar H⁺-ATPases (V-ATPases) are transported from cytosolic compartments to the ruffled plasma membrane of osteoclasts as they activate to resorb bone. Transport of V-ATPases is essential for bone resorption, and is associated with binding interactions between V-ATPases and microfilaments that are mediated by an actin-binding site in subunit B. This site is contained within 44 amino acids in the amino terminal domain, and requires a sequence motif that resembles an actin-binding motif found in mammalian profilin 1. Small alterations in the profilin-like sequence disrupt the actin-binding activity of subunit B. The interaction between V-ATPases and microfilaments in osteoclasts is regulated in response to changes in phosphatidylinositol-3 kinase activity. During internalization of V-ATPases from the plasma membrane of osteoclasts after a cycle of resorption, V-ATPases bind microfilaments that are in podosomes, dynamic actin-based structures, also present in metastatic cancer cells. Studies are ongoing to establish the physiological role of the microfilament-binding activity of subunit B in osteoclasts and in other cells.     

The comparison of the estimation of enzyme kinetic parameters by fitting reaction curve to the integrated Michaelis-Menten rate equations of different predictor variables

The estimation of enzyme kinetic parameters by nonlinear fitting reaction curve to the integrated Michaelis-Menten rate equation ln(S(0)/S)+(S(0)-S)/K(m)=(V(m)/K(m))xt was investigated and compared to that by fitting to (S(0)-S)/t=V(m)-K(m)x[ln(S(0)/S)/t] (Atkins GL, Nimmo IA. The reliability of Michaelis-Menten constants and maximum velocities estimated by using the integrated Michaelis-Menten equation. Biochem J 1973;135:779-84) with uricase as the model. Uricase reaction curve was simulated with random absorbance error of 0.001 at 0.075 mmol/l uric acid. Experimental reaction curve was monitored by absorbance at 293 nm. For both CV and deviation <20% by simulation, K(m) from 5 to 100 micromol/l was estimated with Eq. (1) while K(m) from 5 to 50 micromol/l was estimated with Eq. (2). The background absorbance and the error in the lag time of steady-state reaction resulted in negative K(m) with Eq. (2), but did not affect K(m) estimated with Eq. (1). Both equations gave better estimation of V(m). The computation time and the goodness of fit with Eq. (1) were 40-fold greater than those with Eq. (2). By experimentation, Eq. (1) yielded K(m) consistent with the Lineweaver-Burk plot analysis, but Eq. (2) gave many negative parameters. Apparent K(m) by Eq. (1) linearly increased, while V(m) were constant, vs. xanthine concentrations, and the inhibition constant was consistent with the Lineweaver-Burk plot analysis. These results suggested that the integrated rate equation that uses the predictor variable of reaction time was reliable for the estimation of enzyme kinetic parameters and applicable for the characterization of enzyme inhibitors.