Multidrug-resistance (MDR) phenotype of human osteosarcoma cells evaluated by quantitative morphological and electron microscopy analyses

Summary— Multidrug-resistant (MDR) variants of a human osteosarcoma cell line (U-2 OS) have been recently obtained by continuous exposure to doxorubicin (DX). The growth and phenotypic characteristics of these cell lines have been demonstrated to be related to the level of expression of P-glycoprotein. In this work, the morphological changes associated with MDR have been evaluated by quantitative image analysis and transmission electron microscopy. Resistant cells present morphological changes with respect to sensitive cells at both cytoplasmic and nuclear level. Some of these changes appear to be related to the degree of resistance but not to the direct presence of DX, since deprived cells maintain some modified characters, while others are partly lost. These findings suggest that DX exposure affects cell metabolism causing progressive changes of the cell morphotype.     

Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana

The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.     

n-3脂肪酸代谢产物抗炎作用的研究进展

炎症反应是机体正常组织对感染和损伤的应答,然而过度的炎症反应往往会引起急性和慢性疾病的发生.最近研究发现,由n-3多不饱和脂肪酸二十碳五烯酸和二十二碳六烯酸代谢产生的resolvins和protectins两类化合物,具有很强的抗炎和炎症修复活性.综述了resolvins和protectin D1的来源、抗炎作用和抗炎机制,为进一步开展n-3多不饱和脂肪酸及其代谢产物的抗炎作用研究、为炎症的防治提供新的思路.     

DX16 is a novel SR protein phosphorylated by DOA

The serine-arginine-rich (SR) proteins belong to a conserved splicing factor family that not only is essential for constitutive pre-mRNA splicing, but also plays important roles in regulation of alternative splicing. Dx16 is a member of SR protein family in Drosophila. In order to get more insight of dx16 function, we identified the proteins interacting with DX16 through yeast two-hybrid and GST-pull down assays. DX16 interacts with the U1 snRNP subunit CG7564, the SR protein RBP1 and the SR protein kinase DOA. The first and second serine-and arginine-rich regions of DOA are required for the interaction between DOA and DX16. DX16 could be phosphorylated by DOA in vitro and DX16 is highly phosphorylated in vivo. Immunofluorescence microscopy results reveal that doa and dx16 are both highly expressed in embryonic central nervous system. These results suggest that DX16 could be a novel SR protein phosphorylated by DOA and it may participate in the formation of splicing complex through its interactions with other splicing related proteins.     

Separation methods for camptothecin and related compounds

This paper reviews working procedures for the analytical determination of camptothecin and analogues. We give an overview of aspects such as the chemistry, structure–activity relationships, stability and mechanism of action of these antitumor compounds. The main body of the review describes separation techniques. Sample treatment and factors influencing high-performance liquid chromatography development are delineated. Published high-performance liquid chromatographic methods are summarized to demonstrate the variability and versatility of separation techniques and a critical evaluation of separation efficiency, detection sensitivity and specificity of these methods is reported.     

Functional and pathological roles of the 12- and 15-lipoxygenases

The 12/15-lipoxygenase enzymes react with fatty acids producing active lipid metabolites that are involved in a number of significant disease states. The latter include type 1 and type 2 diabetes (and associated complications), cardiovascular disease, hypertension, renal disease, and the neurological conditions Alzheimer’s disease and Parkinson’s disease. A number of elegant studies over the last thirty years have contributed to unraveling the role that lipoxygenases play in chronic inflammation. The development of animal models with targeted gene deletions has led to a better understanding of the role that lipoxygenases play in various conditions. Selective inhibitors of the different lipoxygenase isoforms are an active area of investigation, and will be both an important research tool and a promising therapeutic target for treating a wide spectrum of human diseases.     

促炎症消退新介质：消退素与保护素

炎症启动（initiation）、发展之后的消退（resolution）是受到体内促消退介质（pro-resolving mediator）调控的主动过程。继发现由花生四烯酸衍生的脂氧素（lipoxin）后,新近又从炎症消退阶段的炎性渗出物中分离出由ω-3多不饱和脂肪酸转化而来的消退素（resolvin）与保护素（protectin）,它们也具有强效的抗炎促消退效应,成为促炎症消退介质的新成员。     

Bcl-2 acts subsequent to and independent of Ca fluxes to inhibit apoptosis in thapsigargin- and glucocorticoidmtreated mouse lymphoma cells

The mechanism by which Bcl-2 inhibits apoptosis is unknown. One proposal is that Bcl-2 regulates intracellular Ca²⁺ fluxes thought to mediate apoptosis. In the present study, we investigated Bcl-2's mechanism of action by determining the effect of Bcl-2 on intracellular Ca²⁺ fluxes in the WEH17.2 mouse lymphoma cell line, which does not express Bcl-2, and its stable transfectant, which expresses a high level of Bcl-2. Treatment with the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin produced marked alterations in intracellular Ca²⁺ homeostasis in both WEH17.2 and W.Hb12 cells, including elevation of free cytosolic Ca²⁺, endoplasmic reticulum Ca²⁺ pool depletion, capacitative entry of extracellular Ca²⁺, and increased loading of Ca²⁺ into mitochondria. Similar changes in intracellular Ca²⁺ occurred spontaneously in both cell lines following exponential growth. In both situations, W.Hb12 cells maintained optimal viability despite marked alterations in intracellular Ca ²⁺' whereas WEH17.2 cells underwent apoptosis. Treatment with the glucocorticoid hormone, dexamethasone, induced apoptosis in WEH17.2 cells, but not in W.HB12 cells, even though dexamethasone treatment did not alter intracellular Ca²⁺ homeostasis in either cell line. These findings indicate that Bcl-2 acts downstream from intracellular Ca ²⁺ fluxes in a pathway where Ca²⁺-dependent and Ca²⁺-independent death signals converge.     

Toxoplasma gondii: an evaluation of diagnostic value of recombinant antigens in a murine model

Laboratory diagnostics of toxoplasmosis depends primarily on serological methods detecting specific antibodies. Since these methods do not always enable specific and sensitive recognition of the infection and phase of toxoplasmosis, the search for new diagnostic tools continues. Recombinant antigens promise a new alternative in diagnostics of Toxoplasma gondii infections. In this work the usefulness of six recombinant T. gondii antigens: GRA1, GRA6, GRA7, p35, SAG1, and SAG2 in the detection of primary murine toxoplasmosis was evaluated. Sera obtained from infected mice differing in their natural susceptibility to T. gondii infection, BALB/c (relatively resistant) and C57BL/6 (relatively susceptible), were tested using ELISA. During acute infection high response to GRA7, GRA6, and p35 antigens was noticed, whereas a strong reactivity with surface antigens SAG1 and SAG2 was characteristic for chronic toxoplasmosis. Our results show that the recombinant antigens are useful in distinguishing between acute and chronic toxoplasmosis regardless of the genetically determined susceptibility of the host.