Functional proteomics identifies miRNAs to target a p27/Myc/phospho-Rb signature in breast and ovarian cancer.

The myc oncogene is overexpressed in almost half of all breast and ovarian cancers, but attempts at therapeutic interventions against myc have proven to be challenging. Myc regulates multiple biological processes, including the cell cycle, and as such is associated with cell proliferation and tumor progression. We identified a protein signature of high myc, low p27 and high phospho-Rb significantly correlated with poor patient survival in breast and ovarian cancers. Screening of a miRNA library by functional proteomics in multiple cell lines and integration of data from patient tumors revealed a panel of five microRNAs (miRNAs) (miR-124, miR-365, miR-34b*, miR-18a and miR-506) as potential tumor suppressors capable of reversing the p27/myc/phospho-Rb protein signature. Mechanistic studies revealed an RNA-activation function of miR-124 resulting in direct induction of p27 protein levels by binding to and inducing transcription on the p27 promoter region leading to a subsequent G1 arrest. Additionally, in vivo studies utilizing a xenograft model demonstrated that nanoparticle-mediated delivery of miR-124 could reduce tumor growth and sensitize cells to etoposide, suggesting a clinical application of miRNAs as therapeutics to target the functional effect of myc on tumor growth.

Recruitment of beta-2-glycoprotein 1 to cell surfaces in extrinsic and intrinsic apoptosis.

Apoptotic cells and phagocytes have developed a diverse array of distinct ligand-receptor systems that drive the recognition and uptake of dying cells. Phagocytes recognize apoptotic cells either directly, by binding to specific ligands at their cell surface, or indirectly, by binding to bridging proteins that bind these ligands. Previous observations showed that the plasma bridging protein beta2GP1, binds PS containing vesicles, and enhances their binding and engulfment by phagocytes in vitro. In this study we show that apoptotic cells injected intravenously and intraperitonealy into syngeneic mice recruited the PS binding protein, beta2GP1. Examination of peritoneal exudates and spleen thin sections showed that only the injected apoptotic cells picked up endogenous beta2GP1. Recovery of cells from the peritoneum showed that apoptotic cells bearing beta2GP1 were clustered around host peritoneal phagocytes. In addition, tissue sections from mice injected with Fas antibody showed colocalization of beta2GP1 with TUNEL-positive apoptotic cells. These results provide evidence that endogenous beta2GP1 binds apoptotic cells in vivo, suggesting that the protein plays an important physiologic role in the recognition of dying cells.

In vitro and in vivo activities of Syn2190, a novel beta-lactamase inhibitor.

Syn2190, a monobactam derivative containing 1,5-dihydroxy-4-pyridone as the C-3 side chain, is a potent inhibitor of group 1 beta-lactamase. The concentrations of inhibitor needed to reduce the initial rate of hydrolysis of substrate by 50% for Syn2190 against these enzymes were in the range of 0.002 to 0.01 microM. These values were 220- to 850-fold lower than those of tazobactam. Syn2190 showed in vitro synergy with ceftazidime and cefpirome. This synergy was dependent on the concentration of the inhibitor against group 1 beta-lactamase-producing strains, such as Pseudomonas aeruginosa, Enterobacter cloacae, Citrobacter freundii, and Morganella morganii. However, against beta-lactamase-derepressed mutants of P. aeruginosa, the MICs of ceftazidime plus Syn2190 were not affected by the amount of beta-lactamase, and the values were the same for the parent strains. The MICs at which 50% of isolates are inhibited (MIC(50)s) of ceftazidime plus Syn2190 were 2- to 16-fold lower than those of ceftazidime alone for ceftazidime-resistant, clinically isolated gram-negative bacteria. Similarly, the MIC(50)s of cefpirome plus Syn2190 were two- to eightfold lower for cefpirome-resistant clinical isolates. The synergies of Syn2190 plus ceftazidime or cefpirome observed in vitro were also reflected in vivo. Syn2190 improved the efficacies of both cephalosporins in both a murine systemic infection model with cephalosporin-resistant rods and urinary tract infection models with cephalosporin-resistant P. aeruginosa.

Beta-lactamase inhibitors: agents to overcome bacterial resistance.

The extensive use of beta-lactam antibiotics in hospitals and community has created major resistance problems leading to increased morbidity, mortality and health-care costs. Resistance is most often mediated by -lactamases, which have emerged in both gram-positive and gram-negative bacteria. A novel approach to countering bacterial beta-lactamases is the delivery of a beta-lactam antibiotic in combination with a beta-lactamase inhibitor. Several such combinations are currently available, containing inhibitors clavulanic acid, sulbactam and tazobactam. These inhibitors are not, however, active against all beta-lactamases and the AmpC chromosomal enzymes that are hyperproduced by some enterobacteria and pseudomonas are a particular gap. Moreover, genes for these AmpC enzymes have begun to escape to plasmids. Consequently, there is a growing need for new broad-spectrum beta-lactamase inhibitors. This review offers an overview of synthetic beta-lactamase inhibitors, emphasizing information on their structures, and highlighting their activity against various beta-lactamases, particularly AmpC enzymes. Effective inhibition of AmpC enzymes are to be found among the penems and monobactams, but none of these has yet proved suitable for pharmaceutical development.

SYN-1012: a new beta-lactamase inhibitor of penem skeleton.

A new beta-lactamase inhibitor, SYN-1012, with a penem skeleton was synthesized and its biological activity compared with clavulanic acid, sulbactam, tazobactam and BRL-42715. The beta-lactamase inhibitory activity of SYN-1012 was comparable to BRL-42715. Clavulanate and penam sulphones (sulbactam and tazobactam) were more active against TEM-1 and OXA-1, but were less active against TEM-3 and cephalosporinase (Case) than SYN-1012. In combination with piperacillin, SYN-1012 exhibited comparable or slightly lower synergistic effects than BRL-42715 against all the Gram-positive and Gram-negative isolates tested with only exception of Pseudomonas aeruginosa. The separate combinations of SYN-1012 and BRL-42715 with ceftazidime and cefotaxime provided comparable results against Gram-negatives, but not against Gram-positive isolates. Tazobactam was inferior to SYN-1012 in all cases. In comparison to tazobactam, SYN-1012 and BRL-42715 were relatively unstable in human and mouse plasma, and in mouse liver and kidney homogenates. Serum level of SYN-1012 and BRL-42715 after an intravenous administration of 20 mg/kg in rabbit was undetectable after 1 hour.

Chemical modification of tazobactam. Synthesis of 2 beta-[(4-substituted)-1,2,3-triazol-1-yl]methyl penicillanic acid sulfone derivatives.

A series of 2 beta-[(4-substituted)-1,2,3-triazol-1-yl] methyl penicillanic acid sulfones was synthesized as beta-lactamase inhibitors. Many of these compounds showed good in vitro inhibitory activity against penicillinase, cefotaximase and plasmid-mediated class III TEM enzymes, but exhibited weaker cephalosporinase inhibition. One member in this series--2 beta-[(4-pyridiniummethyl)-1,2,3-triazol- 1-yl]-6,6-dihydropenicillanate 1,1-dioxide (12a), when tested in combination with piperacillin, showed excellent synergistic activity against microorganisms producing plasmid-mediated enzymes, but had insufficient activity against microorganisms producing chromosomally mediated class I enzymes.

Synthesis and beta-lactamase inhibitory activity of 6-[(1-heteroarylthioethyl-1,2,3-triazol-4-yl)-methylene]penam sulfones.

The synthesis of beta-lactamase inhibitory activity of a series of sodium 6-[(1-heteroarylthioethyl-1,2,3-triazol-4-yl)methylene]pe nicillanate, 1,1-dioxides are described. Their activity was compared with tazobactam and sulbactam. The Z-isomers were more active than the E-isomers. The in vitro activity of the Z-isomers of the phenylthiadiazole derivatives (13a and 15a) was better than sulbactam against the tested beta-lactamases and comparable to tazobactam especially against TEM-2 and cephalosporinase. But their synergistic activity with five antibiotics was inferior to tazobactam.

Collagen induced MMP-2 activation in human breast cancer.

Matrix metalloproteinase-2 (MMP-2), a zymogen requiring proteolytic activation for catalytic activity, has been implicated broadly in the invasion and metastasis of many cancer model systems, including human breast cancer (HBC). MMP-2 has been immunolocalized to carcinomatous human breast, where the degree of activation of MMP-2 correlates well with tumor grade and patient prognosis. Using Matrigel assays, we have stratified HBC cell lines for invasiveness in vitro, and compared this to their potential for metastatic spread in nude mice. HBC cell lines expressing the mesenchymal marker protein vimentin were found to be highly invasive in vitro, and tended to form metastases in nude mice. We have further discovered that culture on collagen-I gels (Vitrogen; Vg) induces MMP-2-activator in highly invasive but not poorly invasive HBC cell lines. As seen for other MMP-2-activator inducing regimens, this induction requires protein synthesis and an intact MMP-2 hemopexin-like domain, appears to be mediated by a cell surface activity, and can be inhibited by metalloproteinase inhibitors. The induction is highly specific to collagen I, and is not seen with thin coatings of collagen I, collagen IV, laminin, or fibronectin, or with 3-dimensional gels of laminin, Matrigel, or gelatin. This review focuses on collagen I and MMP-2, their localization and source in HBC, and their relationship(s) to MMP-2 activation and HBC metastasis. The relevance of collagen I in activation of MMP-2 in vivo is discussed in terms of stromal cell: tumor cell interaction for collagen I deposition, MMP-2 production, and MMP-2-activation. Such cooperativity may exist in vivo for MMP-2 participation in HBC dissemination. A more complete understanding of the regulation of MMP-2-activator by type I collagen may provide new avenues for improved diagnosis and prognosis of human breast cancer.

Analysis of genes coding for the major and minor fimbrial subunits of the Prs-like fimbriae F165(1) of porcine septicemic Escherichia coli strain 4787.

Septicemic Escherichia coli 4787 of porcine origin produces a Prs-like fimbrial antigen, F165(1) which agglutinates sheep erythrocytes in a similar manner to Prs fimbriae, but unlike the latter, also agglutinates pig erythrocytes. In a previous study, we reported the cloning of the f165(1) operon and showed that it encodes a Prs-like adhesin. Here, we report the sequence of the f165(1)A, and f165(1)EFG genes. f165(1)A encodes a protein of 161 amino acids preceded by a signal peptide of 21 amino acids. A size of 19.3 kDa was calculated for the processed F165(1)A protein. The E, F, and G open reading frames potentially give rise to mature proteins of 149, 148 and 313 amino acids respectively. The F165(1)A protein showed significant similarity with the major subunit protein of P-fimbriae of F11 serotype, differing only in four positions. F165(1)E and F165(1)G were found to be closely related to the PrsE and PrsG proteins of Prs-fimbriae variants, whereas F165(1)F was found to be almost completely identical to the F protein of various P and Prs fimbriae. Our results indicated that the f165(1) is a mosaic operon consisting of sequences related to both the pap operon and the prs operon.

Structure and copy number analyses of pap-, sfa-, and afa-related gene clusters in F165-positive bovine and porcine Escherichia coli isolates.

Pathogenic F165-positive Escherichia coli isolates of porcine and bovine origin possess gene clusters related to extraintestinal E. coli fimbrial operons pap, sfa, and afaI. Probes from different segments of the pap, sfa, and afaI operons were used in Southern hybridization to analyze 18 F165-positive, mannose-resistant hemagglutinating E. coli isolates possessing pap- and sfa-, pap- and afa-, or pap-related sequences. Only single copies of the pap-, sfa-, or afa-related sequences were found among the isolates, and the position of each sequence with respect to those of adjacent sequences was variable. Expression of the P and F adhesins individually or concurrently was associated with the presence of a single copy of pap-related sequences. Gene clusters related to pap were structurally similar to the pap operon in certain isolates or the prs operon in others. sfa-related sequences showed few internal structural polymorphisms and were structurally similar to the foc rather than the sfa operon. afa-related sequences showed many internal structural polymorphisms compared with the afa-related sequences from prototype strain KS52. These results demonstrate that the pap- and sfa-related sequences in F165-positive isolates are closely related to the prototype pap operon and foc operon of the P family and Sfa family, respectively. afa-related sequences, on the other hand, display heterogeneity and differ from the prototype afaI operon.

Preparations of liposomal fluconazole and their in vitro antifungal activity.

Fluconazole was successfully incorporated into multilamellar (MLV) and large unilamellar liposomes (LUV). Both MLV and LUV were stable up to 72 h in saline but were less stable in the high-resolution medium. The MLV-entrapped fluconazole was found to be four-fold more active than LUV-entrapped fluconazole against Candida pseudotropicalis and over six-fold more active against C. albicans. The MLV-fluconazole was one-fold less active than free fluconazole in terms of its endpoints (MIC value). However, when compared with free fluconazole, MLV-fluconazole was one-fold more active against two strains of C. albicans and equally active against C. kefyr and C. parapsilosis. In an incubation time-dependent assay against C. tropicalis, MLV-Fluconazole was one-fold more active after 16 h incubation and two-fold less active than fluconazole after 24 or 36 h post-incubation. Our results demonstrate the usefulness of liposomal formulation of the water-soluble azole, fluconazole, in the limited in vitro assay method used.

Effect of valine and the herbicide sulfometuron methyl on acetolactate synthase activity in nuclear and plasmid-borne sulphometuron methyl resistant Saccharomyces cerevisiae strains.

Acetolactate synthase (ALS) specific activity was evaluated in isogenic lines of Saccharomyces cerevisiae carrying the wild-type ILV2 gene or mutations in this gene for resistance to the herbicide sulfometuron methyl (SM). Statistical comparisons were made between two nuclear alleles and among five alleles borne on a YE chimaeric plasmid transformed into a strain carrying a 1.5-kilobase deletion in the nuclear ILV2 gene. Decreased ALS activity of plasmid-borne SM-resistant mutations was shown not to be caused by copy number effects. ALS-specific activity in strains carrying the wild-type ILV2 allele exhibited strong feedback inhibition by valine and was sensitive to SM. All nuclear and plasmid-borne SM-resistance alleles resulted in ALS-specific activity highly resistant to SM and resistant to valine feedback inhibition.

Synthesis and beta-lactamase inhibitory properties of 2 beta-[(1,2,3-triazol-1-yl)methyl]-2 alpha-methylpenam-3 alpha-carboxylic acid 1,1-dioxide and related triazolyl derivatives.

Benzhydryl 2 beta-[(1,2,3-triazol-1-yl)methyl]-2 alpha-methylpenam- 3 alpha-carboxylate 1,1-dioxide was prepared by heating benzhydryl 2 beta-(azidomethyl)-2 alpha-methylpenam-3 alpha-carboxylate 1,1-dioxide with (trimethylsilyl)acetylene. The ester group was removed by hydrogenolysis to give sodium 2 beta-[(1,2,3-triazol-1-yl)methyl]-2 alpha-methylpenam-3 alpha-carboxylate 1,1-dioxide (3i, YTR-830), which was found to be a potent inhibitor of various bacterial beta-lactamases. A series of related compounds was prepared in a similar way, and all of these compounds show excellent beta-lactamase inhibitory properties.

Syntheses of pure (9Z, 11Z), (9E, 11E), (9E, 11Z), and (9Z,11E)-9,11-hexadecadienals-possible candidate pheromones.

The title compounds were prepared by six different routes, and recommendations are given for the more convenient procedures in laboratory-scale syntheses. Modifications in the literature preparations of the 9E,11E and 9E,11Z isomers are described. Baseline separation of a prepared mixture of all four isomers of the (9Z, 11Z), (9E, 11E), (9E, 11Z), and (9Z, 11E)-9,11-hexadecadienals was achieved using GC methods with standard capillary columns. [(13)C]NMR spectroscopy of the alkene carbon atoms clearly differentiates between theZ,Z, E,E and eitherE,Z orZ,E isomers of the precursor dienols and thus of the dienals.