Bio-Inspired Fragmentations: Rapid Assembly of Indolones, 2-Quinolinones, and (-)-Goniomitine.

Inspired by the biogenetic origin of goniomitine, new synthetic bio-inspired fragmentation strategies for the synthesis of functionalized 2-quinolinones and indolones have been developed. Remarkable synthetic efficiency was achieved by telescoping several transformations into one-pot reactions, allowing for the direct coupling of 2-alkynyl-anilines and diazo ketones. The synthetic utility was demonstrated by the 5-step asymmetric total synthesis of (-)-goniomitine from 2-ethyl-cyclopentanone.

[The Comparison Between Extraction Methods of Exosomes From Ascites of Colorectal Cancer].

OBJECTIVE: To compare the two different methods to isolate the exosome from the ascites of colorectal cancer (CRC) patient and find the efficient one. METHODS: Exosome from the ascites of CRC patient were isolated by two different methods: density gradient exosome isolation (DG-Exo) and Exo-Quick isolation, and followed by identification with transmission electron microscopy observation and Western blot analysis. And then, Nanodrop was used for protein quantification. RESULTS: Exosome were isolated by both of the two methods. The protein concentration of the exosome isolated by the Exo-Quick isolation were higher than that of DG-Exo. CONCLUSION: Exo-Quick isolation can obtain higher purity and more complete exosome from the ascites.

Metal-Free Ring-Expansion Reaction of Six-membered Sulfonylimines with Diazomethanes: An Approach toward Seven-Membered Enesulfonamides.

A new metal-free, ring-expansion reaction of six-membered N-sulfonylimines with unstable diazomethanes, generated in situ from the N-tosylhydrazones, has been developed. This reaction delivers valuable seven-membered enesulfonamides by a Tiffeneau-Demjanov rearrangement and intramolecular proton transfer tautomerization process. Moreover, this ring-expansion reaction can be carried out in a one-pot fashion and scaled up to the gram scale by using aryl aldehydes, without the need to isolate the N-tosylhydrazone.

FIP200 is involved in murine pseudomonas infection by regulating HMGB1 intracellular translocation.

BACKGROUND: FIP200, a critical autophagy initiating protein, can participate in numerous cellular functions including cancer development; however, its functional role in P. aeruginosa infection of alveolar macrophages is unknown. METHODS: To investigate the role of FIP200 in host defense, we transfected murine alveolar macrophage MH-S cells with FIP200 siRNA. Having confirmed that FIP200 knockdown inhibited PAO1-induced autophagosme formation, we sought to characterize the underlying signaling pathways by immunoblotting. Further, we used fip200 KO mice to study the effects of fip200 deficiency on HMGB1 translocation. RESULTS: We showed that Pseudomonas PAO1 strain infection facilitated autophagosome formation, whereas knockdown of FIP200 inhibited autophagosome formation and HMGB1 expression in MH-S cells. Silencing FIP200 impaired the translocation of HMGB1 to cytosol of MH-S cells and almost abolished acetylation of HMGB1 during PAO1 infection. In contrast, FIP200 overexpression facilitated the cytosol translocation of HMGB1 from nuclei and increased acetylation of HMGB1 in PAO1-infected MH-S cells. Importantly, expression and acetylation of HMGB1 were also significantly down-regulated in fip200 KO mice following PAO1 infection. CONCLUSIONS: Collectively, these findings elucidate that FIP200 may regulate expression and translocation of HMGB1 during PAO1 infection, which may indicate novel therapeutic targets to control pulmonary infection.

Human testis-expressed sequence 101 is limitedly distributed in germinal epithelium of testis and disappears in seminoma.

BACKGROUND: Testis-expressed sequence 101 (TEX101) was found to be highly expressed in testis and involved in acrosome reaction in previous studies. Recently, the metastasis suppressor function of TEX101 in cancer was disclosed, but the comprehensive investigation of its expression has rarely been reported. In this study, the expression features of TEX101 in normal human organs and seminoma were systematically analyzed. RESULTS: Immunohistochemistry demonstrated intense staining of TEX101 in human testis tissues; however, its expression in 27 other types of normal human organs, including the ovary, was negligible. Higher expression of TEX101 was observed in the spermatocytes and spermatids of the testis, but relatively lower staining was detected in spermatogonia. Western blotting showed a single TEX101 band of 38 kDa in human testis, but it did not correspond to the predicted molecular weight of its mature form at 21 KDa. Furthermore, we examined seminoma tissues by immunohistochemistry and found that none of the 36 samples expressed TEX101. CONCLUSIONS: Our data confirmed TEX101 to be a testis protein that could be related to the maturation process of male germ cells. The lack of TEX101 in seminoma indicated its potential role in tumor progression. This characteristic expression of TEX101 could provide a valuable reference for understanding its biological functions.

Expression of CD40 and growth-inhibitory activity of CD40 agonist in ovarian carcinoma cells.

The CD40 receptor is a member of the tumour necrosis factor receptor family and is widely expressed on various cell types. The antitumour activity of CD40 agonist antibody has been observed in B-cell-derived malignancies, but its activity on ovarian cancer remains unclear. However, in this paper, we first confirmed that the anti-CD40 agonist antibody could inhibit the growth of ovarian cancer cells and induce apoptosis. This study investigated the expression of CD40 by ovarian carcinoma tissues and cell lines, at the same time, we evaluated the effect of a recombinant soluble human CD40L (rshCD40L) and an anti-CD40 agonist antibody on cell growth and apoptosis. Flow cytometry and immunohistochemistry assay demonstrated that CD40 was expressed on ovarian carcinoma cell lines and primary ovarian carcinoma cells derived from ascites, as well as on ovarian carcinoma tissues. The growth inhibition of rshCD40L and the anti-CD40 agonist antibody on ovarian carcinoma cells was examined by MTT assay, and the proportion of apoptotic tumour cells was analysed by flow cytometry and Hoechst staining. Our study showed that CD40 was expressed on all ovarian carcinoma cell lines and was examined in 86.2% (162/188) of ovarian cancer tissue samples, but not in normal ovarian tissues (n = 20). Treatment with rshCD40L or anti-CD40 agonist antibody significantly inhibited ovarian carcinoma cell growth and induced apoptosis. Theses results suggest that CD40 is expressed on ovarian carcinoma cells, moreover, that rshCD40L and anti-CD40 agonist antibody have therapeutic potential to inhibit human ovarian cancer growth.

A fully human CD19/CD3 bi-specific antibody triggers potent and specific cytotoxicity by unstimulated T lymphocytes against non-Hodgkin's lymphoma.

The use of a bi-specific antibody (BsAb) is an attractive and specific approach to cancer therapy. We have constructed a fully human recombinant single chain Fv BsAb against CD19 and CD3 that was an effective treatment in an animal model of non-Hodgkin's lymphoma (NHL). The CD19/CD3 BsAb was expressed in CHO cells and purified by Ni-column chromatography. Flow cytometry revealed that the CD19/CD3 BsAb specifically bound to both CD19 and CD3-positive cells. In vitro, the CD19/CD3 BsAb could stimulate T cell proliferation and induce the lysis of cultured Raji cells in the presence of unstimulated T lymphocytes. In vivo, the CD19/CD3 BsAb efficiently inhibited tumour growth in SCID mice of NHL, and the survival time of the mice was significantly prolonged. Therefore, our CD19/CD3 BsAb is a useful tool that could be a suitable candidate for treatment of NHL.

Polyclonal rabbit anti-murine plasmacytoma cell globulins induce myeloma cells apoptosis and inhibit tumour growth in mice.

Multiple myelomas (MMs) are etiologically heterogeneous and there are limited treatment options; indeed, current monoclonal antibody therapies have had limited success, so more effective antibodies are urgently needed. Polyclonal antibodies are a possible alternative because they target multiple antigens simultaneously. In this study, we produced polyclonal rabbit anti-murine plasmacytoma cell immunoglobulin (PAb) by immunizing rabbits with the murine plasmacytoma cell line MPC-11. The isolated PAb bound to plasma surface antigens in several MM cell lines, inhibited their proliferation as revealed by MTT assay, and induce apoptosis as indicated by flow cytometry, microscopic observation of apoptotic changes in morphology, and DNA fragmentation on agarose gels. The cytotoxicity of PAb on MPC-11 cell lines was both dose-dependent and time-dependent; PAb exerted a 50% inhibitory effect on MPC-11 cell viability at a concentration of 200 µg/ml in 48 h. Flow cytometry demonstrated that PAb treatment significantly increased the number of apoptotic cells (48.1%) compared with control IgG (8.3%). Apoptosis triggered by PAb was confirmed by activation of caspase-3, -8, and -9. Serial intravenous or intraperitoneal injections of PAb inhibited tumour growth and prolonged survival in mice bearing murine plasmacytoma, while TUNEL assay demonstrated that PAb induced statistically significant apoptosis (P < 0.05) compared to control treatments. We conclude that PAb is an effective agent for in vitro and in vivo induction of apoptosis in multiple myeloma and that exploratory clinical trials may be warranted.

The effect of miR-7 on behavior and global protein expression in glioma cell lines.

Malignant glioma is a common cancer of the nervous system. Despite recent research efforts in cancer therapy, the prognosis of patients with malignant glioma has remained dismal. MicroRNAs are noncoding RNAs that inhibit the expression of their targets in a sequence-specific manner, and a few have been shown to act as oncogenes or tumor suppressors. Here, we aimed at exploring the precise biological role of microRNA-7 (miR-7) and the global protein changes in glioma cell lines transiently transfected with miR-7. Transfection of miR-7 into glioma cell lines causes inhibition of cell migration and invasion and suppression of tumorigenesis. Moreover, ectopic expression of miR-7 inhibits lung metastases of glioma in vivo. Among 65 protein spots with differential expression separated by 2-DE, 37 proteins were successfully identified by MS/MS analysis. Of those, the 25 downregulated proteins, which include 14-3-3ζ, eukaryotic translation initiation factor 5A (EIF5A), and annexin A4, may be downstream targets of miR-7, a finding that could elucidate some aspects of the behavior of glioma cells at the protein level. In conclusion, the absence of miR-7 function could cause downstream molecules to switch on or off, resulting in glioma development, invasion, and metastases. MiR-7-based gene treatment may be a novel anti-invasion therapeutic strategy for malignant glioma.

Synthesis, characterization, and in vitro and in vivo evaluation of a novel pectin-adriamycin conjugate.

Adriamycin (ADM) has been widely used in the treatment of many types of solid malignant tumor. However, cardiotoxicity, multidrug resistance and a short half-life in vivo are significant problems that limit its clinical application. To resolve these problems, a novel pectin-adriamycin conjugate (PAC) was synthesized by attaching ADM to low-methoxylated pectin via an amide linkage. The ADM content and weight-average molecular weight (Mw) of PAC were greater than 25% (w/w) and 50,360 g/mol, respectively. PAC was highly stable in plasma, but 33.2% of ADM was released from PAC after incubation for 30 h with lysosomes derived from rat liver. PAC was distributed uniformly in the cytoplasm of most A549 cells and accumulated in the nucleus of a few A549 cells after incubation for 30 h. At concentrations equivalent to 0.125-1.000 microg of ADM/mL, PAC did not inhibit the growth of either A594 or B16 cells to the same extent as free ADM or a mixture of ADM and pectin. Interestingly, at all concentrations, PAC inhibited the growth of 2780cp cells in vitro significantly more effectively than ADM or the mixture of ADM and pectin. The anticancer effect of PAC in vivo was evaluated with C57BL/6 mice bearing pulmonary metastases of B16 cells. Compared with ADM and the mixture of ADM and pectin, PAC suppressed tumor growth significantly and prolonged the mean survival time of the B16-inoculated mice. PAC has great potential for development as a tumor targeting polymer-drug.

Preparative isolation and purification of anti-tumor agent ansamitocin P-3 from fermentation broth of Actinosynnema pretiosum using high-performance counter-current chromatography.

Ansamitocin P-3 is a potent anti-tumor maytansinoid found in Actinosynnema pretiosum. However, due to the complexity of the fermentation broth of Actinomycete, how to effectively separate ansamitocin P-3 is still a challenge. In this study, both analytical and preparative high-performance counter-current chromatography were successfully used to separate and purify ansamitocin P-3 from fermentation broth. A total of 28.8 mg ansamitocin P-3 with purity of 98.4% was separated from 160 mg crude sample of fermentation broth in less than 80 min with the two-phase solvent system of hexane-ethyl acetate-methanol-water (0.6:1:0.6:1, v/v/v/v). The purity and structural identification were determined by HPLC, (1)H NMR, (13)C NMR and mass spectroscopy.

Altered protein-expressing profile in hPNAS4-induced apoptosis in A549 human lung adenocarcinoma cells.

Human PNAS4 (hPNAS4) is a recently identified pro-apoptosis gene, which is able to induce apoptosis in A549 human lung adenocarcinoma cells following its overexpression. In this work, we investigated the changes of protein profile in hPNAS4-induced apoptosis in A549 cells through proteomic strategy consisting of two-dimensional electrophoresis (2-DE) coupled with MALDI-Q-TOF mass spectrometry. A total of 20 different proteins with more than 3.0-fold change in expression, including 5 up-regulated and 15 down-regulated proteins were successfully identified by database search. The mRNA transcription levels of the different proteins were further examined by RT-PCT. Functional analyses showed these different proteins are involved in diverse biological processes including metabolism, proteolysis, signal transduction, apoptosis, and redox regulation. Two essential apoptosis-associated protein, annexin A1 and prothymosin alpha, were confirmed by Western blot and showed consistent changes with proteomic detection. Our data provide molecular evidence and possible associated pathway in hPNAS4-induced apoptosis through proteomic strategy, which should be contributed to further investigation on biological function of hPNAS4.

Identification of ATP synthase beta subunit (ATPB) on the cell surface as a non-small cell lung cancer (NSCLC) associated antigen.

BACKGROUND: Antibody-based immunotherapy has achieved some success for cancer. But the main problem is that only a few tumor-associated antigens or therapeutic targets have been known to us so far. It is essential to identify more immunogenic antigens (especially cellular membrane markers) for tumor diagnosis and therapy. METHODS: The membrane proteins of lung adenocarcinoma cell line A549 were used to immunize the BALB/c mice. A monoclonal antibody 4E7 (McAb4E7) was produced with hybridoma technique. MTT cell proliferation assay was carried out to evaluate the inhibitory effect of McAb4E7 on A549 cells. Flow cytometric assay, immunohistochemistry, western blot and proteomic technologies based on 2-DE and mass spectrometry were employed to detect and identify the corresponding antigen of McAb4E7. RESULTS: The monoclonal antibody 4E7 (McAb4E7) specific against A549 cells was produced, which exhibited inhibitory effect on the proliferation of A549 cells. By the proteomic technologies, we identified that ATP synthase beta subunit (ATPB) was the corresponding antigen of McAb4E7. Then, flow cytometric analysis demonstrated the localization of the targeting antigen of McAb4E7 was on the A549 cells surface. Furthermore, immunohistochemistry showed that the antigen of McAb4E7 mainly aberrantly expressed in tumor cellular membrane in non-small cell lung cancer (NSCLC), but not in small cell lung cancer (SCLC). The rate of ectopic expressed ATPB in the cellular membrane in lung adenocarcinoma, squamous carcinoma and their adjacent nontumourous lung tissues was 71.88%, 66.67% and 25.81% respectively. CONCLUSION: In the present study, we identified that the ectopic ATPB in tumor cellular membrane was the non-small cell lung cancer (NSCLC) associated antigen. ATPB may be a potential biomarker and therapeutic target for the immunotherapy of NSCLC.

Immunotherapy of tumors with recombinant adenovirus encoding macrophage inflammatory protein 3beta induces tumor-specific immune response in immunocompetent tumor-bearing mice.

AIM: Tumor immunotherapy aims at activating the body's own immune system to fight an existing tumor. Effective antitumor responses require tumor antigens to be presented to lymphocytes. We aimed to test the hypothesis that intratumoral administration of recombinant adenovirus encoding MIP3beta would induce antitumor immunity by attracting and facilitating the interaction between lymphocytes and dendritic cells. METHODS: A recombinant adenovirus encoding microphage inflammatory protein 3beta (AdMIP3beta) was constructed. The antitumor activity of AdMIP3beta in BALB/c and C57BL/6 mice bearing CT26 colon adenocarcinoma and Lewis lung cancer was evaluated. RESULTS: Immunotherapy with AdMIP3beta resulted in significant inhibition of tumor growth and prolonged survival of tumor-bearing mice. Tumor-specific immune responses elicited by AdMIP3beta include MHC class I-dependent CD8(+) CTL-mediated immune response and IFN-gamma response. Immunohistochemical staining demonstrated numerous CD11c(+) cells and CD3(+) T lymphocytes within tumor tissues of AdMIP3beta-treated mice. These findings suggest that the mechanism of specific antitumor immunity induced by AdMIP3beta may be involved in the chemoattraction of both T lymphocytes and DCs to the tumor site and thus facilitate the process of antigen capture and mature DC to prime naive T cells. CONCLUSION: The present study may be important in the exploration of the potential application of AdMIP3beta in the treatment of a broad spectrum of tumors.

Vascular endothelial cell antigen (VECA), a novel secreted protein: overexpression in Escherichia coli of the hexahistidine- tagged protein and production of polyclonal antibodies.

A novel VECA (vascular endothelial cell antigen) was previously identified by using an antibody pool against antigens in HUVECs (human umbilical-vein endothelial cells). VECA has been evolutionarily conserved in vertebrate species ranging from frog and fish to mouse and human. Bioinformatics analysis indicated that VECA was 10.1 kDa in size, with a predicted signal sequence and transmembrane domain, indicating that VECA may have important biological functions. The present paper describes a procedure for obtaining and purifying human recombinant VECA expressed in Escherichia coli as a fusion protein, via a human VECA cDNA linked pQE30 expression vector to DNA coding for hexahistidine. The purified protein was used to raise anti-(human VECA) polyclonal antibodies, which were suitable for detecting the presence of VECA in cells, cell-culture supernatant and tissues by immunoblotting and immunohistochemistry. To our knowledge, this is the first study on the protein expression and polyclonal-antibody production for human VECA. In addition, we report for the first time the positive identification of VECA in humans at the protein and subcellular level and provide the first experimental verification that VECA was indeed a secreted protein. The anti-(human VECA) polyclonal antibodies prepared may serve as a useful tool for future biological function studies on VECA.

5-(4-Bromo-anilinomethyl-ene)-2,2-dimethyl-1,3-dioxane-4,6-dione.

In the title compound, C(13)H(12)BrNO(4), the dihedral angles between the amino-methyl-ene group and the dioxane ring and between the benzyl ring and the amino-methyl-ene unit are 7.96 (4) and 12.15 (4)°, respectively. The dioxane ring shows a half-boat conformation, in which the C atom between the dioxane ring O atoms is 0.460 (8) Šout of the plane through the remaining ring atoms. An intra-molecular N-H⋯O hydrogen bond may stabilize the planar conformation of the mol-ecule. An inter-molecular C-H⋯O inter-action is also present.

Detection of bovine central nervous system tissue as bovine spongiform encephalopathy risk material by real-time reverse transcriptase-PCR in raw and cooked beef products.

There is increasing evidence of the association of the new variant of Creutzfeldt-Jacob disease (nvCJD) in humans with bovine spongiform encephalopathy (BSE) in cattle. Many countries established legislation of banning central nervous system (CNS) tissues, which are regarded as BSE-specified risk materials (SRM), in human food supply because of the potential transmission of BSE to humans. A real-time reverse transcriptase-PCR assay using the bovine glial fibrillary acidic protein (GFAP) mRNA template for the detection of CNS tissues in raw and cooked beef products was developed in this study. The results showed that (1) this method can detect CNS tissues from bovine and ovine origins, but not from porcine and avian ones; (2) GFAP mRNA can only be detected from brain and spinal cords rather than other tissues; (3) the GFAP mRNA was detectable in CNS tissues even after dilution to 0.001%; and (4) the assay was unaffected by heat treatment at 100 degrees C for 30 min or storage at room temperature for 4 days, and at 4 degrees C for at least 15 days.

[Synthesis of 1,3-dihydro-1,3-dioxo-2H-isoindole derivatives].

OBJECTIVE: To synthesize a series of derivatives of Thalidomide. METHODS: The N-Phthaloyl-L-glutamic anhydride was ammonolyzed with amino acid benzyl esters, followed by hydrogenization. The reaction between the hydogenized products and ammonia gas produced ammonium salt, 1,3-dihydro-1,3-dioxo-2H-isoindole derivatives. RESULTS: Four new derivatives of Thalidomide were obtained and confirmed by spectral detection. CONCLUSION: The derivatives of Thalidomide can be efficiently synthesized under mild conditions.

Betulinic acid impairs metastasis and reduces immunosuppressive cells in breast cancer models.

Breast cancer is the most common female cancer with considerable metastatic potential, explaining the need for new candidates that inhibit tumor metastasis. In our study, betulinic acid (BA), a kind of pentacyclic triterpenoid compound derived from birch trees, was evaluated for its anti-metastasis activity in vitro and in vivo. BA decreased the viability of three breast cancer cell lines and markedly impaired cell migration and invasion. In addition, BA could inhibit the activation of stat3 and FAK which resulted in a reduction of matrix metalloproteinases (MMPs), and increase of the MMPs inhibitor (TIMP-2) expression. Moreover, in our animal experiment, intraperitoneal administration of 10 mg/kg/day BA suppressed 4T1 tumor growth and blocked formation of pulmonary metastases without obvious side effects. Furthermore, histological and immunohistochemical analyses showed a decrease in MMP-9 positive cells, MMP-2 positive cells and Ki-67 positive cells and an increase in cleaved caspase-3 positive cells upon BA administration. Notably, BA reduced the number of myeloid-derived suppressor cells (MDSCs) in the lungs and tumors. Interestingly, in our caudal vein model, BA also obviously suppressed 4T1 tumor pulmonary metastases. These findings suggested that BA might be a potential agent for inhibiting the growth and metastasis of breast cancer.

[Screening proteins related to retinoic acid resistance by proteomic analysis].

OBJECTIVE: To compare the expression profiles of differential proteins between retinoic acid resistant and sensitive cells and screen the proteins related to retinoic acid (RA) resistance by proteomic analysis. METHODS: The total cellular proteins from the RA sensitive cells of the line NB4 and the RA resistant cells of the line MR2 obtained from a patient with acute promyelocytic leukemia (APL) were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and analyzed by PDQuest v7.1 analysis software to screen the differential protein spots. Differentially expressed spots were analyzed by mass spectrometry for peptide mass finger (PMF) data and identified by Mascot software and SWISS-PROT protein database. The differentially expressed proteins were verified by Western blotting assay and semi-quantification RT-PCR. RESULTS: 2-DE patterns of APL cell lines with high-resolution and reproducibility were obtained. The average spots of the RA resistant cell line MR2 and RA sensitive cell line NB4 were 890 +/- 45 and 912 +/- 56 respectively. 57 significantly differentially expressed protein spots were screened, among which 23 protein spots were founded to be upregulated and 34 protein spots down regulated in the RA resistant cell line MR2. 25 differential protein spots were identified by mass spectrometry and 17 proteins were successfully assigned to 13 gene-reading frames, of which one was unknown function protein (FLJ00279) and the others were included in the categories of oncoprotein (DJ-1), transcription factor (MYC promoter-binding protein 1), molecule chaperone (HSP70, HSP60 and protein disulfide isomerase), metabolism protein (prohibitin, triosephosphate isomerase 1, and calreticulin), signal transduction (Rho GDP dissociation inhibitor), and cytoskeleton (ACTG1 protein, Beta 5-tubulin, and keratin 10). The results of Western blotting were similar to those of 2D-PAGE and showed differential expression of DJ-1 and calreticulin in several isoforms. Semi-quantification RT-PCR analysis showed that there was no correlation between the protein expression changes and mRNA levels of HSP70 or HSP60. Those results indicated that post-translational events might modify or shear the protein content of the specific spots. CONCLUSION: The utilization of two-dimensional polyacrylamide gel electrophoresis coupled with mass spectrometry is effective in screening the all-trans RA resistance-associated proteins and could provide novel clue for study of elucidating all-trans RA resistance mechanisms.