PriB stimulates PriA helicase via an interaction with single-stranded DNA

The frequency with which replication forks break down in all organisms requires that specific mechanisms ensure completion of genome duplication. In Escherichia coli a major pathway for reloading of the replicative apparatus at sites of fork breakdown is dependent on PriA helicase. PriA acts in conjunction with PriB and DnaT to effect loading of the replicative helicase DnaB back onto the lagging strand template, either at stalled fork structures or at recombination intermediates. Here we showed that PriB stimulates PriA helicase, acting to increase the apparent processivity of PriA. This stimulation correlates with the ability of PriB to form a ternary complex with PriA and DNA structures containing single-stranded DNA, suggesting that the known single-stranded DNA binding function of PriB facilitates unwinding by PriA helicase. This enhanced apparent processivity of PriA might play an important role in generating single-stranded DNA at stalled replication forks upon which to load DnaB. However, stimulation of PriA by PriB is not DNA structure-specific, demonstrating that targeting of stalled forks and recombination intermediates during replication restart likely resides with PriA alone.     

Synthesis,spectroscopic, and structural characterization of the first aqueous cobalt(II)-citrate complex: toward a potentially bioavailable form of cobalt in biologically relevant fluids

Citric acid represents a class of carboxylic acids present in biological fluids and playing key roles in biochemical processes in bacteria and humans. Its ability to promote diverse coordination chemistries in aqueous media, in the presence of metal ions known to act as trace elements in human metabolism, earmarks its involvement in a number of physiological functions. Cobalt is known to be a central element of metabolically important biomolecules, such as B12, and therefore its biospeciation in biological fluids constitutes a theme worthy of chemical and biological perusal. In an effort to unravel the aqueous chemistry of cobalt in the presence of a physiologically relevant ligand, citrate, the first aqueous, soluble, mononuclear complex has been synthesized and isolated from reaction mixtures containing Co(II) and citrate in a 1:2 molar ratio at pH approximately 8. The crystalline compound (NH4)4[Co(C6H5O7)2] (1) has been characterized spectroscopically (UV/vis, EPR) and crystallographically. Its X-ray structure consists of a distorted octahedral anion with two citrate ligands fulfilling the coordination requirements of the Co(II) ion. The magnetic susceptibility measurements of 1 in the range from 6 to 295 K are consistent with a high-spin complex containing Co(II) with a ground state S=3/2. Corroborating this result is the EPR spectrum of 1, which shows a signal consistent with the presence of a Co(II) system. The spectroscopic and structural properties of the complex signify its potential biological relevance and participation in speciation patterns arising under conditions consistent with those employed for its synthesis and isolation.     

Design,synthesis and anti-melanogenic effect of cinnamamide derivatives

Pigmentation disorders are attributed to excessive melanin which can be produced by tyrosinase. Therefore, tyrosinase is supposed to be a vital target for the treatment of disorders associated with overpigmentation. Based on our previous findings that an (E)-β-phenyl-α,β-unsaturated carbonyl scaffold can play a key role in the inhibition of tyrosinase activity, and the fact that cinnamic acid is a safe natural substance with a scaffolded structure, it was speculated that appropriate cinnamic acid derivatives may exhibit potent tyrosinase inhibitory activity. Thus, ten cinnamamides were designed, and synthesized by using a Horner-Emmons olefination as the key step. Cinnamamides 4 (93.72% inhibition), 9 (78.97% inhibition), and 10 (59.09% inhibition) with either a 2,4-dihydroxyphenyl, or 4-hydroxy-3-methoxyphenyl substituent showed much higher mushroom tyrosinase inhibition at 25?µM than kojic acid (18.81% inhibition), used as a positive control. Especially, the two cinnamamides 4 and 9 having a 2,4-dihydroxyphenyl group showed the strongest inhibition. Docking simulation with tyrosinase revealed that these three cinnamamides, 4, 9, and 10, bind to the active site of tyrosinase more strongly than kojic acid. Cell-based experiments carried out using B16F10 murine skin melanoma cells demonstrated that all three cinnamamides effectively inhibited cellular tyrosinase activity and melanin production in the cells without cytotoxicity. There was a close correlation between cellular tyrosinase activity and melanin content, indicating that the inhibitory effect of the three cinnamamides on melanin production is mainly attributed to their capability for cellular tyrosinase inhibition. These results imply that cinnamamides having the (E)-β-phenyl-α,β-unsaturated carbonyl scaffolds are promising candidates for skin-lighting agents.     

High-fat diet-induced lipidome perturbations in the cortex,hippocampus, hypothalamus,and olfactory bulb of mice

Given their important role in neuronal function, there has been an increasing focus on altered lipid levels in brain disorders. The effect of a high-fat (HF) diet on the lipid profiles of the cortex, hippocampus, hypothalamus, and olfactory bulb of the mouse brain was investigated using nanoflow ultrahigh pressure liquid chromatography-electrospray ionization-tandem mass spectrometry in the current study. For 8?weeks, two groups of 5-week-old mice were fed either an HF or normal diet (6 mice from each group analyzed as the F and N groups, respectively). The remaining mice in both groups then received a 4-week normal diet. Each group was then subdivided into two groups for another 4-week HF or normal diet. Quantitative analysis of 270 of the 359 lipids identified from brain tissue revealed that an HF diet significantly affected the brain lipidome in all brain regions that were analyzed. The HF diet significantly increased diacylglycerols, which play a role in insulin resistance in all regions that were analyzed. Although the HF diet increased most lipid species, the majority of phosphatidylserine species were decreased, while lysophosphatidylserine species, with the same acyl chain, were substantially increased. This result can be attributed to increased oxidative stress due to the HF diet. Further, weight-cycling (yo-yo effect) was found more critical for the perturbation of brain lipid profiles than weight gain without a preliminary experience of an HF diet. The present study reveals systematic alterations in brain lipid levels upon HF diet analyzed either by lipid class and molecular levels.     

Validation of trans-acting elements that promote exon 7 skipping of SMN2 in SMN2-GFP stable cell line

Spinal muscular atrophy is a genetic disease in which the SMN1 gene is deleted. The SMN2 gene exists in all of the patients. Alternative splicing of these two genes are different. More than 90% of exon 7 included form is produced from SMN1 pre-mRNA, whereas only ～20% of exon 7 included form is produced from SMN2 pre-mRNA. Only exon 7 inclusion form produces functional protein. Exon 7 skipped SMN isoform is unstable. Here we constructed a GFP reporter system that recapitulates the alternative splicing of SMN1 and SMN2 pre-mRNA. We designed a system in which GFP protein is expressed only when exon 7 of is included in alternative splicing. The stable cell that expresses SMN1-GFP produces ～4 times more GFP protein than the stable cell line that expresses SMN2-GFP; as demonstrated by microscopy, FACS analysis and immunoblotting. In addition the ratio of exon 7 inclusion and skipping of SMN1-GFP and SMN2-GFP pre-mRNA was similar to endogenous SMN1 and SMN2 pre-mRNA as shown in RT-PCR. Furthermore the knockdown with hnRNP A1 shRNA, a known protein which promotes exon 7 skipping of SMN2, induces exon 7 inclusion of exon 7 in SMN2-GFP pre-mRNA in SMN2-GFP cell line. We conclude that we have established the stable cell lines that recapitulate alternative splicing of the SMN1 and SMN2 genes. The stable cell line can be used to identify the trans-acting elements with siRNA.     

Crystallization,molecular replacement solution,and refinement of tetrameric β-amylase from sweet potato

Sweet potato β-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm × 0.4 mm × 1.0 mm within 2 weeks, belong to the tetragonal space group P4₂2₁2 with unit cell dimensions of a = b = 129.63 Å and c = 68.42 Å. The asymmetric unit contains 1 subunit of β-amylase, with a crystal volume per protein mass (V_M) of 2.57 ų/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric β-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8–2.3 Å (with 2 σ cut-off) with good stereochemistry. The subunit structure of sweet potato β-amylase (crystallized in the absence of α-cyclodextrin) is very similar to that of soybean β-amylase (complexed with α-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent Cα atoms of the two β-amylases is 0.96 Å. Each subunit of sweet potato β-amylase is composed of a large (α/β)₈ core domain, a small one made up of three long loops [L3 (residues 91–150), LA (residues 183–258), and L5 (residues 300–327)], and a long C-terminal loop formed by residues 445–493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (α/β)₈ barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (α/β)₈ barrel. © 1995 Wiley-Liss, Inc.     

Immunostimulating Effects of Extract of Acanthopanax sessiliflorus

As malfunction/absence of immune cells causes a variety of immunosuppressive disorders and chemical synthetic drugs for curing these diseases have many adverse effects, vigorous studies are being conducted. The Acanthopanax family has been used as traditional medicines for gastric ulcer, diabetes, etc. and culinary materials in East-South Asia. In this study, the immunostimulating properties of A. sessiliflorus were evaluated. A. sessiliflorus increased not only the splenocyte number but also immune-related cytokines such as TNF-α. However, it could not upregulate the expressions of IFN-γ and IL-2. A. sessiliflorus increased the swimming time, and comparison of organ weights relative to body weights for immune-related organs such as the spleen and thymus after a forced swim test showed that it could recover the spleen and thymus weights. It also increased the expression of TNF-α and slightly increased the concentration of IFN-γ but not IL-2. From the results, we concluded that as A. sessiliflorus has not only a host defense effect but also a stress-ameliorating property, further study it will be a promising material of immunostimulating material.