http://academic.oup.com/milmed/article-pdf/146/7/506/24126715/milmed-146-7-506.pdf

Goodman and Gilman's The Pharmacological Basis of Therapeutics

Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.

Erythromycin inhibits protein synthesis by its effect on ribosome function (14, 118, 119) The metabolic modifications that enable cells to cope with the inhibitory action of erythromycin fall under major headings that include (i) target site alteration, (ii) antibiotic modification, and (iii) altered antibiotic transport This minireview concentrates on target site alteration, which for erythromycin is the 50S subunit of the ribosome The first clinical isolates of macrolide-resistant staphylococci were described in reports from France, England, Japan, and the United States shortly after the introduction of erythromycin into clinical practice in 1953 On the basis of current understanding of the biochemistry of erythromycin’s action, resistance in most of the strains that were described in early reports can be ascribed to a posttranscriptional modification of the 23S rRNA by an adenine-specific N-methyltransferase (methylase) specified by a class of genes bearing the name erm (erythromycin ribosome methylation) The last decade has seen the isolation and characterization of approximately 30 erm genes from diverse sources, ranging from clinical pathogens to actinomycetes that produce antibiotics; for many of these genes, both the respective nucleotide sequences that encode the methylases as well as the flanking sequences that control their expression have been determined A tabulation of the erm genes that have been described is presented in Table 1 Any discussion of mechanisms of resistance to macrolide antibiotics must include the chemically distinct, but functionally overlapping, lincosamide and streptogramin B families as well This type of resistance has therefore also been referred to as MLS resistance Members of the MLS antibiotic superfamily include, among the macrolides, carbomycin, clarithromycin, erythromycin, josamycin, midecamycin, mycinamicin, niddamycin, rosaramicin, roxithromycin, spiramycin, and tylosin; among the lincosamides, celesticetin, clindamycin, and lincomycin; and among the streptogramins, staphylomycin S, streptogramin B, and vernamycin B The streptogramin family is subdivided into A and B groups or alternatively into M and S groups, respectively Methylation of A2058 confers resistance to the Band S-group streptogramins but not to the Aand M-group streptogramins The reason for this grouping was originally based on empirical observations from clinical bacteriology that resistance to one class often involved resistance to the other two classes (11, 16, 35, 39, 41, 135); however, (i) the three classes of antibiotics interact competitively when binding to the 50S subunit, and only one antibiotic molecule can bind per 50S subunit (129, 130); this suggests that the binding sites for these antibiotics overlap or at least functionally interact (ii) Nucleotide alterations in 23S rRNA, both mutational and posttranscriptional, that confer coresistance to MLS antibiotics appear to cluster in the peptidyltransferase region in 23S rRNA domain V, providing a physical basis and a common location for their sites of action (50, 101–104, 109, 110, 128) (Fig 1 and Table 2), and (iii) footprinting experiments show that the nucleotides in 23S rRNA domain V are protected by bound MLS antibiotics against modification by agents such as dimethyl sulfate (DMS) and kethoxal that can derivatize purine and pyrimidine bases in single-stranded DNA or RNA (26, 76) (Table 3) The erm family of genes is not alone in conferring clinical resistance to macrolide antibiotics A notable early exception to the established MLS resistance pattern was the MS pattern reported by Janosy and coworkers (58, 59), who described clinical isolates that were coresistant to erythromycin and streptogramin B but that remained susceptible to lincosamide antibiotics The molecular basis for resistance in these strains was subsequently shown by Ross et al (94) to involve the active efflux of erythromycin and streptogramin B but not clindamycin Additional mechanisms of macrolide resistance, all associated with the acquisition of new genetic information, including structural modification of erythromycin by phosphorylation (82), glycosylation (60), and lactone ring cleavage by erythromycin esterase (2, 83), have been added to the list Mechanisms involving mutational alteration of genes that normally reside in the host and that encode either ribosomal protein or rRNA have also been described and will be discussed below in detail Reviews of erythromycin resistance that relate to material covered in the present work have been presented previously (4, 18, 20, 21, 28, 29, 133) Recent developments in the synthetic chemistry of semisynthetic macrolides, including the biological and clinical aspects of their actions, have been reviewed by Kirst (65, 66) A forthcoming review covers the inducible nature of MLS resistance and its implications for the mechanism of action of erythromycin (134)

Erythromycin resistance by ribosome modification.

The gut hormone and neuropeptide ghrelin affects energy balance and growth hormone release through hypothalamic action that involves synaptic plasticity in the melanocortin system. Ghrelin binding is also present in other brain areas, including the telencephalon, where its function remains elusive. Here we report that circulating ghrelin enters the hippocampus and binds to neurons of the hippocampal formation, where it promotes dendritic spine synapse formation and generation of long-term potentiation. These ghrelin-induced synaptic changes are paralleled by enhanced spatial learning and memory. Targeted disruption of the gene that encodes ghrelin resulted in decreased numbers of spine synapses in the CA1 region and impaired performance of mice in behavioral memory testing, both of which were rapidly reversed by ghrelin administration. Our observations reveal an endogenous function of ghrelin that links metabolic control with higher brain functions and suggest novel therapeutic strategies to enhance learning and memory processes.

Ghrelin controls hippocampal spine synapse density and memory performance

Multiplex bead array assays: performance evaluation and comparison of sensitivity to elisa

In this article, the authors compare the assay performance measures, signal window, Z' factor, and assay variability ratio. They examine their mathematical formulae for similarities and differences, describe their statistical sampling properties using the results of a computer simulation, and illustrate their use with example data. Based on these results, the authors recommend the Z' factor as a preferred measure of assay performance for screening assays and point out that none of these measures are adequate for characterizing concentration-response assays.

https://journals.sagepub.com/doi/pdf/10.1177/1087057105285610

A comparison of assay performance measures in screening assays: signal window, Z' factor, and assay variability ratio.

Tumor angiogenesis is essential for tumor growth and metastasis formation. Luminex methodology was used to measure the levels of four angiogenic cytokines in cell culture medium and in the plasma of mice bearing human tumors. We obtained plasma and conditioned culture medium from 12 different human tumor cell lines. Tumor necrosis factor-alpha (TNF-α), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGF-β) were determined by the Luminex FlowMetrix assay. VEGF, TNF-α, and bFGF were undetectable in non-tumor-bearing animals. HS746T gastric cancer and Caki-1 renal cell cancer cells in culture produced high levels of VEGF (1000 and 450 pg/106 cells, respectively). High levels of TGF-β were produced by HS746T gastric carcinoma and Calu-6 non-small-cell lung carcinoma (3000 and 1000 pg/106 cells, respectively). Caki-1 renal cell carcinoma and Calu-6 non-small-cell lung carcinoma cells in culture produced high levels of bFGF (42 and 10 pg/106 cells, respectively). Caki-1, SW2 SCLC, HCT-116 and HT-29 colon tumors produced high plasma levels of VEGF (200, 220, 42, and 151 pg/ml, respectively) and TGF-β (31, 36, 45, 32 pg/ml, respectively). A positive linear correlation was seen between tumor volume and VEGF in SW2 (r=0.87) and Caki-1 (r=0.47) tumors, and a moderate correlation in HCT116 tumors (r=0.3). Angiogenic profiles in the plasma of nude mice bearing human tumors may be useful to identify appropriate biomarkers for antiangiogenic therapy, as diagnostic and prognostic tools, and to monitor the responses of individual tumors to antiangiogenic therapy.

Circulating angiogenic growth factor levels in mice bearing human tumors using Luminex Multiplex technology.

A gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tylr) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tylr phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyls) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyls mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyls mutant obtained by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage lambda Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tylr gene is not closely linked to tylosin biosynthetic genes.

Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae.

Spiramycin antibiotic biosynthetic genes of Streptomyces ambofaciens are provided by the present invention, in addition to a variety of recombinant DNA vectors. The genes also function in other macrolide producing organisms. The genes can be used to increase or otherwise alter the macrolide antibiotic-producing ability of an organism. The present invention also provides host strains comprising mutant spiramycin biosynthetic genes which can be used to generate novel antibiotics. Also provided is a method for preparing the mutant gene comprising mutating cloned spiramycin biosynthetic DNA by transposon mutagenesis with subsequent transformation into a macrolide-antibiotic producing host and homologous recombination into its genome, to generate stable mutant cell lines.

Macrolide biosynthetic genes for use in streptomyces and other organisms

Phenotypic lead generation strategies seek to identify compounds that modulate complex, physiologically relevant systems, an approach that is complementary to traditional, target-directed strategies. Unlike gene-specific assays, phenotypic assays interrogate multiple molecular targets and signaling pathways in a target "agnostic" fashion, which may reveal novel functions for well-studied proteins and discover new pathways of therapeutic value. Significantly, existing compound libraries may not have sufficient chemical diversity to fully leverage a phenotypic strategy. To address this issue, Eli Lilly and Company launched the Phenotypic Drug Discovery Initiative (PD(2)), a model of open innovation whereby external research groups can submit compounds for testing in a panel of Lilly phenotypic assays. This communication describes the statistical validation, operations, and initial screening results from the first PD(2) assay panel. Analysis of PD(2) submissions indicates that chemical diversity from open source collaborations complements internal sources. Screening results for the first 4691 compounds submitted to PD(2) have confirmed hit rates from 1.6% to 10%, with the majority of active compounds exhibiting acceptable potency and selectivity. Phenotypic lead generation strategies, in conjunction with novel chemical diversity obtained via open-source initiatives such as PD(2), may provide a means to identify compounds that modulate biology by novel mechanisms and expand the innovation potential of drug discovery.

https://journals.sagepub.com/doi/pdf/10.1177/1087057111405379

Karen Leigh Cox

Papers

A comparison of assay performance measures in screening assays: signal window, Z' factor, and assay variability ratio.

Circulating angiogenic growth factor levels in mice bearing human tumors using Luminex Multiplex technology.

Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae.

Macrolide biosynthetic genes for use in streptomyces and other organisms

Open Innovation for Phenotypic Drug Discovery The PD2 Assay Panel