Correlation of structure to antitumor activities of five derivatives of a beta-glucan from Poria cocos sclerotium

A water-insoluble (1-->3)-beta-D-glucan isolated from fresh sclerotium of Poria cocos was, respectively, sulfated, carboxymethylated, methylated, hydroxyethylated, and hydroxypropylated, to afford five water-soluble derivatives. Their weight-average molecular masses (Mw) and intrinsic viscosities ([eta]) were determined by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS, and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The antitumor activities, against Sarcoma 180 tumor cell (S-180) and gastric carcinoma cell strain (MKN-45 and SGC-7901) of the native beta-glucan and the five derivatives, were tested in vitro and in vivo. The Mw values of the five derivatives in PBS were determined to be 3.8 x 10(4), 18.9 x 10(4), 16.0 x 10(4), 76.8 x 10(4), and 224.3 x 10(4), respectively. The high Mw values of the hydroxyethylated and hydroxypropylated derivatives in aqueous solution resulted from aggregation, and their true Mw values obtained in dimethyl sulfoxide were 20.1 x 10(4) and 19.1 x 10(4). The sulfated and carboxymethylated derivatives having DS of 1.0-1.3 show good water solubility, and exist as relatively expanded chains in aqueous solution. Interestingly, the native beta-glucan did not show antitumor activity, whereas the sulfated and carboxymethylated derivatives exhibit significant antitumor activities against S-180 and gastric carcinoma tumor cells. This work showed that good water solubility, relatively high chain stiffness, and moderate molecular mass of the derivatives in aqueous solution contribute beneficial to enhancement of antitumor activity.     

J蛋白研究进展

J蛋白（J-domain protein）是一类分子中含有J结构域的蛋白质大家族,大部分J蛋白具有分子伴侣的功能。J蛋白作为热休克蛋白70（HSP70）的同伴蛋白与HSP70组成分子伴侣机器,参与蛋白质分子折叠、组装、转运以及信号转导等多种细胞过程。此外,J蛋白在植物对环境胁迫的反应及其他生理过程中起重要作用。     

A cell-based screen for inhibitors of protein folding and degradation

Cancer cells are exposed to external and internal stresses by virtue of their unrestrained growth, hostile microenvironment, and increased mutation rate. These stresses impose a burden on protein folding and degradation pathways and suggest a route for therapeutic intervention in cancer. Proteasome and Hsp90 inhibitors are in clinical trials and a 20S proteasome inhibitor, Velcade, is an approved drug. Other points of intervention in the folding and degradation pathway may therefore be of interest. We describe a simple screen for inhibitors of protein synthesis, folding, and proteasomal degradation pathways in this paper. The molecular chaperone-dependent client v-Src was fused to firefly luciferase and expressed in HCT-116 colorectal tumor cells. Both luciferase and protein tyrosine kinase activity were preserved in cells expressing this fusion construct. Exposing these cells to the Hsp90 inhibitor geldanamycin caused a rapid reduction of luciferase and kinase activities and depletion of detergent-soluble v-Src::luciferase fusion protein. Hsp70 knockdown reduced v-Src::luciferase activity and, when combined with geldanamycin, caused a buildup of v-Src::luciferase and ubiquitinated proteins in a detergent-insoluble fraction. Proteasome inhibitors also decreased luciferase activity and caused a buildup of phosphotyrosine-containing proteins in a detergent-insoluble fraction. Protein synthesis inhibitors also reduced luciferase activity, but had less of an effect on phosphotyrosine levels. In contrast, certain histone deacetylase inhibitors increased luciferase and phosphotyrosine activity. A mass screen led to the identification of Hsp90 inhibitors, ubiquitin pathway inhibitors, inhibitors of Hsp70/Hsp40-mediated refolding, and protein synthesis inhibitors. The largest group of compounds identified in the screen increased luciferase activity, and some of these increase v-Src levels and activity. When used in conjunction with appropriate secondary assays, this screen is a powerful cell-based tool for studying compounds that affect protein synthesis, folding, and degradation.     

Elongated mouse chromosomes suitable for enhanced molecular cytogenetics

Characterization of genetic disorders in humans and animal models requires identification of chromosomal aberrations. However, identifying fine deletions or insertion in metaphase chromosomes has been always a challenge due to limitations of resolution. In this study we developed a rapid method for chromosome elongation using two different intercalating agents: ethidium bromide and 5-bromo-2′-deoxyuridine (BrdU), together with a short-term mitotic block using colcemid. About 70% of the chromosomes from cells that underwent this elongation procedure reached three times longer than those prepared from control cells. FISH experiments using elongated chromosomes revealed a duplicated region of chromosome 11 that was not visible in cells prepared with conventional methods.     

旱麦草属种质资源的随机扩增多态性DNA（RAPDs）分析

利用３１个１０ｂｐ随机引物对来自我国新疆和中东地区的１１份旱麦草属种质资源材料以及两个普通小麦亲本和１个普通小麦与东方旱麦草的远缘杂种后代进行了ＲＡＰＤ分析。对扩增形成的３２１条谱带进行的研究发现,该属植物在新疆地区具有较丰富的遗传多样性,同时还将１个根据形态性状定名为光穗旱麦草的样品修正为东方旱麦草。研究还揭示出旱麦草属与普通小麦之间有明显的遗传分化,并发现四部体光穗旱麦草的两个基因组分别来自二     

Extended N6 substitution of rigid C2-arylethynyl nucleosides for exploring the role of extracellular loops in ligand recognition at the A3 adenosine receptor

2-Arylethynyl-(N)-methanocarba adenosine 5′-methyluronamides containing rigid N⁶-(trans-2-phenylcyclopropyl) and 2-phenylethynyl groups were synthesized as agonists for probing structural features of the A₃ adenosine receptor (AR). Radioligand binding confirmed A₃AR selectivity and N⁶-1S,2R stereoselectivity for one diastereomeric pair. The environment of receptor-bound, conformationally constrained N⁶ groups was explored by docking to an A₃AR homology model, indicating specific hydrophobic interactions with the second extracellular loop able to modulate the affinity profile. 2-Pyridylethynyl derivative 18 was administered orally in mice to reduce chronic neuropathic pain in the chronic constriction injury model.     

Isolation of a <Emphasis Type="Italic">Latimeria menadoensis</Emphasis><Emphasis Type="Italic">heat shock protein 70</Emphasis> (<Emphasis Type="Italic">Lmhsp70</Emphasis>) that has all the features of an inducible gene and encodes a functional molecular chaperone

Molecular chaperones facilitate the correct folding of other proteins, and heat shock proteins form one of the major classes of molecular chaperones. Heat shock protein 70 (Hsp70) has been extensively studied, and shown to be critically important for cellular protein homeostasis in almost all prokaryotic and eukaryotic systems studied to date. Since there have been very limited studies conducted on coelacanth chaperones, the main objective of this study was to genetically and biochemically characterize a coelacanth Hsp70. We have successfully isolated an Indonesian coelacanth (L. menadoensis) hsp70 gene, Lmhsp70, and found that it contained an intronless coding region and a potential upstream regulatory region. Lmhsp70 encoded a typical Hsp70 based on conserved structural and functional features, and the predicted upstream regulatory region was found to contain six potential promoter elements, and three potential heat shock elements (HSEs). The intronless nature of the coding region and the presence of HSEs suggested that Lmhsp70 was stress-inducible. Phylogenetic analyses provided further evidence that Lmhsp70 was probably inducible, and that it branched as a clade intermediate between bony fish and tetrapods. Recombinant LmHsp70 was successfully overproduced, purified and found to be functional using ATPase activity assays. Taken together, these data provide evidence for the first time that the coelacanth encodes a functional molecular chaperone system. K. W. Modisakeng and M. Jiwaji contributed equally to this study.     

Spectroscopic characterization and molecular weight distribution of dissolved organic matter in sediment porewaters from Lake Erhai, Southwest China

Dissolved organic matter (DOM) in sediment porewaters from Lake Erhai, Southwest China was investigated using dissolved organic carbon (DOC) concentration, UV absorbance, fluorescence and molecular weight distribution. DOC exhibited a high concentration at the sediment–water interface with a rapid decrease to the oxic–anoxic interface at approximately 7 cm, and then increased with depth. Similar trends were also found for the UV absorption coefficients at 254 and 280 nm in the porewaters. DNA in the sediment was also measured, which confirmed the high abundance of aerobic bacteria in the upper layer of the sediment. Both humic-like (peaks A and C) and protein-like (peaks B and D) fluorescence were observed in the porewater DOM, and their fluorescence intensities exhibited a similar porewater profile as DOC concentration. A strong correlation was found between the peak fluorescence intensity ratio r(A, C) and r(D, B). Both the fluorescence index and UV absorption coefficient at 254 nm suggested a dramatic increase in aromaticity of porewater DOM across the oxic–anoxic interface. Porewater DOM exhibited a multimodal distribution of molecular weight with a relatively low polydispersity. The results of this study offer significant insight into the nature and properties of DOM in freshwater ecosystems.     

Three-dimensional solution structure of an archaeal FKBP with a dual function of peptidyl prolyl cis-trans isomerase and chaperone-like activities

Here we report the solution structure of an archaeal FK506-binding protein (FKBP) from a thermophilic archaeum, Methanococcus thermolithotrophicus (MtFKBP17), which has peptidyl prolyl cis-trans isomerase (PPIase) and chaperone-like activities, to reveal the structural basis for the dual function. In addition to a typical PPIase domain, a newly identified domain is formed in the flap loop by a 48-residue insert that is required for the chaperone-like activity. The new domain, called IF domain (the Insert in the Flap), is a novel-folding motif and exposes a hydrophobic surface, which we consider to play an important role in the chaperone-like activity.     

Mapping Hsp47 binding site(s) using CNBr peptides derived from type I and type II collagen

As a crucial molecular chaperone in collagen biosynthesis, Hsp47 interacts with the nascent form as well as the mature triple-helical form of procollagen. The location(s) of Hsp47 binding sites on the collagen molecule are, as yet, unknown. We have examined the substrate specificity of Hsp47 in vitro using well-characterized CNBr peptide fragments of type I and type II collagen along with radiolabeled, recombinant Hsp47. Interaction of these peptides with Hsp47 bound to collagen-coated microtiter wells showed several binding sites for Hsp47 along the length of the alpha1 and alpha2 chains of type I collagen and the alpha1 chain of type II collagen, with the N-terminal regions showing the strongest affinities. The latter observation was also supported by the results of a ligand-blot assay. Except for two peptides in the alpha2(I) chain, peptides that showed substantial binding to Hsp47 did so in their triple-helical and not random-coil form. Unlike earlier studies that used peptide models for collagen, the results obtained here on fragments of type I and type II collagen identify, for the first time, binding of Hsp47 to specific regions of the collagen molecule. They also point to additional structural requirements for Hsp47 binding besides the known preference for third-position Arg residues and the triple-helical conformation.