Silver-mediated radical oxytrifluoromethylation of unsaturated carboxylic acids for the synthesis of γ-trifluoromethylthio lactones.

An efficient silver-mediated oxidative trifluoromethylthiolation of unsaturated carboxylic acids to construct trifluoromethylthiol-containing lactones has been disclosed. In this protocol no metal-catalysts was added, and preliminary mechanism investigations suggested that a free-radical pathway should be involved in the process. High functional group tolerance and excellent yields were demonstrated by the efficient preparation of a wide range of γ-trifluoromethylthiolated phthalides.

Penfluridol targets acid sphingomyelinase to inhibit TNF signaling and is therapeutic against inflammatory autoimmune diseases.

BACKGROUND: Penfluridol, isolated from an FDA-approved small-molecule drug library as an inhibitor of tumor necrosis factor α (TNFα)-stimulated NF-κB activation, is clinically used to treat chronic schizophrenia and related disorders. This study is aimed to investigate the therapeutic effect of penfluridol on TNFα-stimulated inflammatory autoimmune diseases, particularly inflammatory arthritis. METHODS: Various in vitro studies to confirm the inhibitory effect of penfluridol on TNFα-induced NF-κB activity in bone marrow-derived macrophages or Raw 264.7 macrophage cell line. In vivo studies assessed the therapeutic effects of penfluridol in various disease models, including TNFα transgenic mice, collagen-induced arthritis, DSS-induced colitis, and TNBS-induced colitis. Identification and characterization of the binding of penfluridol to acid sphingomyelinase using bioinformatics and drug affinity responsive target stability assay. Acid sphingomyelinase activity assays to reveal penfluridol-mediated inhibition of acid sphingomyelinase activity. siRNA knockdown experiments to illustrate the dependence of penfluridol's anti-TNF activity on acid sphingomyelinase. RESULTS: Penfluridol effectively inhibited TNFα-induced NF-κB activation in vitro and alleviated the severity of arthritis and colitis in vivo. Mechanistic studies revealed that penfluridol bound to acid sphingomyelinase and inhibited its activation. In addition, knockdown of acid sphingomyelinase largely abolished the inhibitory effects of penfluridol on TNFα-induced inflammatory cytokine production. Furthermore, penfluridol suppressed the differentiation of spleen naive CD4+T cells to TH1 and TH17 and inhibited M1 macrophage polarization. CONCLUSION: This study provides the rationale for the possible innovative use of penfluridol as a newly identified small-molecule drug for TNFα-driven diseases, such as inflammatory arthritis and colitis.

[Identification of human small supernumerary marker chromosomes and discussion of its research value].

OBJECTIVE: To identify the origin of human small supernumerary marker chromosomes (sSMCs) using fluorescent in situ hybridization (FISH) combined with G-banding karyotype analysis, and to discuss their mechanisms of formation and research value. METHODS: Cep-FISH and SubcenM-FISH were used to analyze sSMCs in 3 patients for whom the result of G-banding was 47,XN,+mar. RESULTS: The FISH result of case 1 was 47,XY,+mar.ish inv dup(22)(q11.1)(D22Z4++,D14/22Z1+, RP11-172D7-). The marker has formed exclusively by heterochromatin. A boy was delivered later with no apparent clinical abnormalities. The FISH result of case 2 was 47,XX,+mar.ish r(10)(p11.2q11.2) (cep10+, RP11-232C13+, RP11-178A10+)[25]/46,XX[10]. The marker has formed by heterochromatin and nearby centromere. A girl was delivered later with no clinical abnormalities. The FISH result of case 3 was 47,XY,+mar.ish inv dup(22)(q11.1)(D22Z4+,D14/22Z1+). The marker has also formed exclusively by euchromatin. Fetal abnormalities were detected by type B ultrasonography, but were not necessarily related with the marker. CONCLUSION: The diversity of sSMCs has posed a great challenge for prenatal diagnosis. Identification of sSMCs will require combined karyotype analysis and FISH or other molecular techniques such as microarray based comparative genomic hybridization or sequencing. For its specific structure, the sSMCs may also provide a valuable tool for gene mapping, heterochromatin research and gene therapy.

Lead induces oxidative stress, DNA damage and alteration of p53, Bax and Bcl-2 expressions in mice.

Oxidative stress is considered as a possible molecular mechanism involved in lead toxicity. This study was carried out to investigate whether lead acetate could induce oxidative stress in mice, and the following damages as well. Lead acetate was given orally to mice for 4 weeks at doses of 0, 10, 50, 100mg/kg body weight every other day, respectively. Production of reactive oxygen species (ROS) and malondialdehyde (MDA) were measured as indicators of oxidative stress. DNA damage in peripheral blood lymphocytes was determined by comet assay. Ultrastructure alteration was detected using transmission electron microscopy. The alterations of p53, Bax, and Bcl-2 expression were determined by western blotting. The results showed that lead acetate significantly increased the levels of ROS and MDA in mice. Meanwhile, severe DNA damage and ultrastructure alterations were obviously observed. In addition, p53 and Bax expressions increased and the imbalance of Bax/Bcl-2 occurred. Therefore, it strongly suggests that lead may induce oxidative stress and change the expressions of apoptosis-related proteins in mouse liver.

[Progress in mechanism of microcystin toxicity].

OBJECTIVE: Microcystin is one of the monocyclic heptapeptides produced primarily by microcystis aeruginosa. Recent studies suggest that microcystin can induce cell apoptosis, as well as oxidative stress and mitochondrial alteration. Studies also indicate that Bcl-2 family and p53 may play an important role in the apoptosis induced by microcystin.

Proteomic analysis of cellular response to microcystin in human amnion FL cells.

Microcystins (MC), the potent inhibitor of protein phosphatase 1 and 2A, are hepatotoxins of increasing importance due to its high acute toxicity and potent tumor promoting activity. So far, the exact mechanisms of MC-induced hepatotoxicity and tumor promoting activity have not been fully elucidated. To better understand the mechanisms underlying microcystin-RR (MC-RR) induced toxicity as well as provide the possibility for the establishment of biomarkers for MC-RR exposure, differential proteome analysis on human amnion FL cells treated by MC-RR was carried out using two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Image analysis of silver-stained 2-dimensional gels revealed that 89 proteins showed significant differential expression in MC-RR treated cells compared with control, and 8 proteins were unique to MC-RR treated cells and 8 proteins were only detected in control cells. Sixty-six proteins were further identified with high confidence by peptide mass fingerprinting. Some of the identified differentially expressed proteins have clearly relationship with the process of apoptosis, signal transduction, and cytoskeleton alteration which are consistent with the literature. The functional implications of alterations in the levels of these proteins were discussed. However, most of which have not been reported previously to be involved in cellular processes responded to MC-RR. Therefore, this work will provide new insight into the mechanism of MC-RR toxicity.

Altered expression of p53, Bcl-2 and Bax induced by microcystin-LR in vivo and in vitro.

It has been reported that MC-LR could induce apoptosis in a variety of cell types. Although the induction of oxidative stress and mitochondrial alteration played critical role in MC-LR induced apoptosis, but the exact mechanisms of MC-LR induced apoptosis are still unknown. In spite of extensive studies on MC-LR mediated cell damages, there is little information on the protein expression of p53 and Bcl-2, Bax in vivo and in vitro, which are vital regulator of apoptosis in response to a variety of stimuli. The present study was undertaken to determine the expression level of p53 and Bcl-2, Bax in cultured hepatocytes and rat liver tissues. The results show that MC-LR can increase the expression of p53 and Bax significantly both in vivo and in vitro, however, MC-LR can only decrease the expression of Bcl-2 significantly in vitro and there is no difference observed in vivo. It can be concluded that the expression of p53, Bcl-2 and Bax are involved in the regulation of MC-LR induced apoptosis.

[Expression of P53 and Bax in rat liver induced by microcystin LR in vivo].

OBJECTIVE: To examine the expression of P53, Bcl-2 and Bax proteins in rat liver after exposed to microcystin LR. METHODS: SD rats received microsystin LR by gastric perfusion. The expression of P53, Bax and Bcl-2 in liver was detected by Western blot. RESULTS: The expression of P53 and Bax in each treatment group increased significantly compared with that in control group(P<0.05), with the exception of 0.1 microg/kg LR exposure group. Moreover, with exposure levels increasing the expression of P53 and Bax increased gradually; while no changes of the expression of Bcl-2 were observed. CONCLUSION: P53 and Bax may play important roles in microcystin LR induced apoptosis, but Bcl-2 seems not be involved in this process.

[Cloning and sequence analysis of the preprotein cDNA of human bone morphogenetic protein 12].

OBJECTIVE: To clone the cDNA of the preprotein cDNA of human bone morphogenetic protein 12 (hBMP12). METHODS: Two primers were designed according to hBMP12 sequence reported in GenBank. The hBMP12 preprotein cDNA was obtained by reverse transcriptional (RT)-PCR from the mRNA extracted from human placenta, followed by cloning into pTARGETTM plasmid and sequence analysis of the plasmid pT(ARGE)T(TM)-BMP12. RESULTS: DNA agarose gel electrophoresis showed that the product of RT-PCR was about 920 bp, as was consistent with the result of PCR detection of the recombinant plasmid. The result of sequence analysis was in agreement with the reported hBMP12 sequence in GenBank. CONCLUSION: The preprotein of hBMP12 cDNA has been successfully cloned with correct sequence.

[Morphological changes of collagen fibrils during the formation of autogenous tendon induced by human hair keratin artificial tendon].

OBJECTIVE: To observe the morphological changes of collagen fibrils during the formation of autogenous tendon induced by human hair keratin (HHK) artificial tendon. METHODS: Rabbit models of injured tendon were established in which implantation of HHK artificial tendon was performed to observe the formation of autogenous tendon under light microscope and electron microscope at 3, 6, 9, 12 and 16 weeks after HHK implantation. RESULTS AND CONCLUSION: During autogenous tendon formation induced by HHK artificial tendon, the tendon cells of the impaired end of the tendon and beneath the tendon membrane dedifferentiate and are capable of collagen secretion, followed by the formation of typesI,II and III collagen fibrils, and eventually, the majority of the tendon cells disappear with the collagenization of the tendon.

Degradation of human hair keratin scaffold implanted for repairing injured skeletal muscles.

OBJECTIVE: To observe the in vivo degradation process of human hair keratin (HHK) scaffold after implantation in rabbits. METHODS: Seven New Zealand rabbits were divided into 4 groups including a control group and 3 operation groups. HHK scaffold was implanted, after partial resection of the skeletal muscles, in rabbits of the 3 operation groups, followed by observation 1, 3, and 6 weeks later respectively. Routine morphological observation, histochemistry with ubiquitin and electron microscopy were performed. HHK scaffold incorporated 3 types of HHK with different degradation speeds, respectively designated types F, B, and Z. RESULTS: Light microscopic observation revealed that human hair cuticles began to strip off at the first postoperative week, and the material was homogeneous on the surface of which macrophagocytes and multinuclear giant cells adhered. At the third week HHK scaffold was degraded into particles as seen under electron microscope and was phagocytosed by macrophagocytes and multinuclear giant cells. Ubiquitin enzymatic histochemistry demonstrated that macrophagocytes, multinuclear giant cells were positive at the first week. At sixth week, further degradation of HHK scaffold occurred when newly generated muscles were seen beside the HHK. CONCLUSIONS: HHK scaffold is initially degraded extracellularly by ubiquitin system into particles, which are phagocytosed by the cells and degraded by the cooperation of lysosome and ubiquitin; meanwhile the satellite cells are activated, beginning to proliferate and eventually fused into newly generated muscle fibers.

Differentiation and telomerase activity of human mesenchymal stem cells.

OBJECTIVE: To study the differentiation of human mesenchymal stem cells (MSCs) derived from human fetal bone marrow and observe its telomerase activity. METHODS: MSCs were isolated from fetal bone marrow of the femur followed by cell culture and amplification. In situ hybridization was employed to detect telomerase activity in the MSCs. Subcutaneous implantation of MSCs into nude mice was performed to observe their differentiation potentials 4 weeks after the implantation. RESULTS: Human MSCs were found positive for telomerase activity and they showed the potential to differentiate into such tissue cells as of the bone, cartilage, adipose, skeletal muscle, tendon-like tissue and unmyelinated nerve fiber-like bundles. CONCLUSION: Human MSCs are multipotent stem cells that may differentiate into many types of tissue cells with high levels of telomerase activity.