A bistable gene switch for antibiotic biosynthesis: the butyrolactone regulon in Streptomyces coelicolor

Many microorganisms, including bacteria of the class Streptomycetes, produce various secondary metabolites including antibiotics to gain a competitive advantage in their natural habitat. The production of these compounds is highly coordinated in a population to expedite accumulation to an effective concentration. Furthermore, as antibiotics are often toxic even to their producers, a coordinated production allows microbes to first arm themselves with a defense mechanism to resist their own antibiotics before production commences. One possible mechanism of coordination among individuals is through the production of signaling molecules. The gamma-butyrolactone system in Streptomyces coelicolor is a model of such a signaling system for secondary metabolite production. The accumulation of these signaling molecules triggers antibiotic production in the population. A pair of repressor-amplifier proteins encoded by scbA and scbR mediates the production and action of one particular gamma-butyrolactone, SCB1. Based on the proposed interactions of scbA and scbR, a mathematical model was constructed and used to explore the ability of this system to act as a robust genetic switch. Stability analysis shows that the butyrolactone system exhibits bistability and, in response to a threshold SCB1 concentration, can switch from an OFF state to an ON state corresponding to the activation of genes in the cryptic type I polyketide synthase gene cluster, which are responsible for production of the hypothetical polyketide. The switching time is inversely related to the inducer concentration above the threshold, such that short pulses of low inducer concentration cannot switch on the system, suggesting its possible role in noise filtering. In contrast, secondary metabolite production can be triggered rapidly in a population of cells producing the butyrolactone signal due to the presence of an amplification loop in the system. S. coelicolor was perturbed experimentally by varying concentrations of SCB1, and the model simulations match the experimental data well. Deciphering the complexity of this butyrolactone switch will provide valuable insights into how robust and efficient systems can be designed using "simple" two-protein networks.     

Purified Human Synovium Mesenchymal Stem Cells as a Good Resource for Cartilage Regeneration

Mesenchymal stem cells (MSCs) have the ability to differentiate into a variety of lineages and to renew themselves without malignant changes, and thus hold potential for many clinical applications. However, it has not been well characterized how different the properties of MSCs are depending on the tissue source in which they resided. We previously reported a novel technique for the prospective MSC isolation from bone marrow, and revealed that a combination of cell surface markers (LNGFR and THY-1) allows the isolation of highly enriched MSC populations. In this study, we isolated LNGFR⁺ THY-1 ⁺ MSCs from synovium using flow cytometry. The results show that the synovium tissue contained a significantly larger percentage of LNGFR ⁺ THY-1 ⁺ MSCs. We examined the colony formation and differentiation abilities of bone marrow-derived MSCs (BM-MSCs) and synovium-derived MSCs (SYN-MSCs) isolated from the same patients. Both types of MSCs exhibited a marked propensity to differentiate into specific lineages. BM-MSCs were preferentially differentiated into bone, while in the SYN-MSC culture, enhanced adipogenic and chondrogenic differentiation was observed. These data suggest that the tissue from which MSCs are isolated should be tailored according to their intended clinical therapeutic application.     

Establishment of a detection system for demethylating agents using an endogenous promoter CpG island

Disturbances of epigenetic information that result in changes in DNA methylation patterns are involved in carcinogenesis and other human disorders. Detection of agents that can cause epigenetic alterations--i.e. epimutagens--is therefore an important objective. We have developed and now describe the first detection system for demethylating agents that involves an endogenous promoter CpG island (CGI). After screening 10 promoter CGIs of genes silenced in human cancers, a CGI of the FLJ32130 gene was found to respond sensitively to a known demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), by abundantly re-expressing its mRNA. After introducing the Hyg(r)-EGFP fusion gene into exon 3 of the FLJ32130 gene by homologous recombination, we isolated one clone that had the expected recombination outcomes and designated it F117. Two subclones (F117-47 and F117-123) of this original clone that did not share its propensity for leaky expression of the Hyg(r)-EGFP mRNA were then isolated, and methylation of their 5' CGI was confirmed. The addition of 5-aza-dC at doses of 0.1 microM or higher led to their 5' CGI being demethylated, and to Hyg(r)-EGFP being expressed; the anticipated fluorescence was readily confirmed by fluorescence microscopy. We believe that this is the first assay system that detects agents that disturb the methylated status of a CGI that regulates an endogenous promoter.     

Discovery of novel scaffolds for γ-secretase modulators without an arylimidazole moiety

Gamma-secretase modulators (GSMs) selectively inhibit the production of amyloid-β 42 (Aβ42) and may therefore be useful in the management of Alzheimer’s disease. Most heterocyclic GSMs that are not derived from nonsteroidal anti-inflammatory drugs contain an arylimidazole moiety that potentially inhibits cytochrome P450 (CYP) activity. Here, we discovered imidazopyridine derivatives that represent a new class of scaffold for GSMs, which do not have a strongly basic end group such as arylimidazole. High-throughput screening identified 2-methyl-8-[(2-methylbenzyl)oxy]-3-(pyridin-4-yl)imidazo[1,2-a]pyridine (3a), which inhibited the cellular production of Aβ42 (IC₅₀?=?7.1?µM) without changing total production of Aβ. Structural optimization of this series of compounds identified 5-[8-(benzyloxy)-2-methylimidazo[1,2-a]pyridin-3-yl]-2-ethylisoindolin-1-one (3m) as a potent inhibitor of Aβ42 (IC₅₀?=?0.39?µM) but not CYP3A4. Further, 3m demonstrated a sustained pharmacokinetic profile in mice and sufficiently penetrated the brain.     

Functional evolution of duplicated odorant-binding protein genes, Obp57d and Obp57e, in Drosophila

Odorant-binding proteins (OBPs) are extracellular proteins found in insect chemosensilla, where they participate in the sensing of odors, tastes, and pheromones. Although a large number of OBP genes have been identified in insect genomes, their molecular functions and biological roles have been clarified in limited cases. Two OBP genes, Obp57d and Obp57e, were involved in the evolution of host-plant preference in Drosophila sechellia. Comparative analyses of the Obp57d/e genomic sequences from 27 closely related species suggested that the two genes arose by tandem gene duplication and functionally diverged from each other. In this study, the functional evolution of Obp57d and Obp57e was examined by in vitro binding assays using recombinant proteins synthesized in a bacterial system. Compared to the ancestral Dpse\OBP57de, Dmel\OBP57d was more specialized to tridecanoic acid while Dmel\OBP57e was generalized regarding their binding affinity, suggesting that the two OBP genes underwent subfunctionalization and neofunctionalization. A behavioral analysis using knockout flies supported that the biological role is different between OBP57d and OBP57e in vivo. Site-directed mutagenesis of the evolutionarily conserved amino acids revealed that these residues play an important role in protein folding. These findings provide a clue to understanding how the repertoire of OBP genes is maintained in a genome under natural selection.     

An amino acid substitution in PBP-3 in Haemophilus influenzae associate with the invasion to bronchial epithelial cells

Haemophilus influenzae is a common pathogen of respiratory infections. We examined whether beta-lactamase-negative ampicillin-resistant (BLNAR) strains that are known to have ampicillin resistance due to a substitution of amino acid of penicillin binding protein (PBP)-3, differ from beta-lactamase-negative ampicillin-susceptible strains with regard to invasion of bronchial epithelium. After 3h incubation of each of 34 beta-lactamase-negative ampicillin-susceptible and 57 BLNAR strains in the presence of BEAS-2B cells, a human bronchial epithelium cell line, extracellular bacteria were killed using gentamicin and intracellular bacteria numbered. All nine strains in which the efficiency of invasion was 1% or higher were BLNAR strains. The rate of invasion was significantly greater in strains with PBP-3 amino acid substitution (Met377 to Ile, Ser385 to Thr, Leu389 to Phe, and Asn526 to Lys) (n=34) than in those with no amino acid substitution. Electron microscopy showed that high invasive BLNAR strains were observed in cytoplasm of BEAS-2B cell layer. The injured cells were 9.44+/-1.76% among attaching cells examined by trypan blue staining after 6h. These data may suggest that the amino acid substitution of the PBP in BLNAR strains may at least partly play roles in macropinocytosis, leading to the invasion and injury to epithelial cells.     

A Src family inhibitor (PP1) potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells

Connexin (Cx) genes exert negative growth effects on tumor cells with certain cell specificity. We have recently reported that Cx32 acts as a tumor suppressor gene in renal cancer cells due to the inhibition of Src-dependent signaling. In line with the previous study, here we examined if a Src family inhibitor (PP1) could potentiate tumor-suppressive effect of Cx32 in Caki-2 cell from human renal cell carcinoma. In order to clarify the potentialization of PP1, using Cx32-transfected Caki-2 cells and mock-transfected Caki-2 cells, we estimated difference in cytotoxic effect of PP1 on the two cell clones in vitro as well as in vivo. PP1 showed more cytotoxic effect on Caki-2 cells having Cx32 positive expression than that of Cx32 negative expression at lower doses. This potentialization was also observed in xenograft model of nude mice. The potentialization of the effect mainly depended on the induction of apoptosis but not the control of cell growth. In conjugation with this event, the reduction of anti-apoptotic molecules (Bcl-2 and Bcl-xL) was caused by the combination of Cx32 expression and PP1 treatment in Caki-2 cells. These results suggest that PP1 potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells through the reduction of anti-apoptotic molecules.     

Epigenetic mechanisms regulate the prostaglandin E receptor 2 in breast cancer

The increase in local oestrogen production seen in oestrogen receptor positive (ER+) breast cancers is driven by increased activity of the aromatase enzyme. CYP19A1, the encoding gene for aromatase, is often overexpressed in the oestrogen-producing cells of the breast adipose fibroblasts (BAFs) surrounding an ER+ tumour, and the molecular processes underlying this upregulation is important in the development of breast-specific aromatase inhibitors for breast cancer therapy. Prostaglandin E2 (PGE2), a factor secreted by tumours, is known to stimulate CYP19A1 expression in human BAFs. The hormonal regulation of this process has been examined; however, what is less well understood is the emerging role of epigenetic mechanisms and how they modulate PGE2 signalling. This present study characterises the epigenetic processes underlying expression of the prostanoid receptor EP2 in the context of ER+ breast cancer. Sodium bisulphite sequencing of CpG methylation within the promoter region of EP2 revealed that an inverse correlation existed between methylation levels and relative EP2 expression in breast cancer cell lines MDA-MB-231, MCF7 and MCF10A but not in HS578t and T47D. Inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5aza) and histone deacetylation with Trichostatin A (TSA) resulted in upregulation of EP2 mRNA in all cell lines with varying influences of each epigenetic process observed. Expression of EP2 was detected in human BAFs despite a natively methylated promoter, and this expression was further increased upon 5aza treatment. An examination of 3 triple negative, 3 ductal carcinoma in situ and 3 invasive ductal carcinoma samples revealed that there was no change in EP2 promoter methylation status between normal and cancer associated stroma, despite observed differences in relative mRNA levels. Although EP2 methylation status is inversely correlated to expression levels in established breast cancer cell lines, we could not identify that such a correlation existed in tumour-associated stroma cells.     

Effects of U0126 and fibroblast growth factor on gene expression profile in Ciona intestinalis embryos as revealed by microarray analysis

Fibroblast growth factor (FGF) induces the notochord and mesenchyme in ascidian embryos, via extracellular signal-regulated kinase (ERK) that belongs to the mitogen-activated protein kinase (MAPK) family. A cDNA microarray analysis was carried out to identify genes affected by an inhibitor of MAPK/ERK kinase (MEK), U0126, in embryos of the ascidian Ciona intestinalis. Data obtained from the microarray and in situ hybridization suggest that the majority of genes are downregulated by U0126 treatment. Genes that were downregulated in U0126-treated embryos included Ci-Bra and Ci-Twist-like1 that are master regulatory genes of notochord and mesenchyme differentiation, respectively. The plasminogen mRNA was downregulated by U0126 in presumptive endoderm cells. This suggests that a MEK-mediated extracellular signal is necessary for gene expression in tissues whose specification does not depend on cell-to-cell interaction. Among 85 cDNA clusters that were not affected by U0126, 30 showed mitochondria-like mRNA localization in the nerve cord/muscle lineage blastomeres in the equatorial region. The expression level and asymmetric distribution of these mRNA were independent of MEK signaling.     

Discovery of {1-[4-(2-{hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl}-1H-benzimidazol-1-yl)piperidin-1-yl]cyclooctyl}methanol,systemically potent novel non-peptide agonist of nociceptin/orphanin FQ receptor as analgesic for the treatment of neuropathic pain: Design,synthesis, and structure–activity relationships

Neuropathic pain is a serious chronic disorder caused by lesion or dysfunction in the nervous systems. Endogenous nociceptin/orphanin FQ (N/OFQ) peptide and N/OFQ peptide (NOP) receptor [or opioid-receptor-like-1 (ORL1) receptor] are located in the central and peripheral nervous systems, the immune systems, and peripheral organs, and have a crucial role in the pain sensory system. Indeed, peripheral or intrathecal N/OFQ has displayed antinociceptive activities in neuropathic pain models, and inhibitory effects on pain-related neurotransmitter releases and on synaptic transmissions of C- and Aδ-fibers. In this study, design, synthesis, and structure–activity relationships of peripheral/spinal cord-targeting non-peptide NOP receptor agonist were investigated for the treatment of neuropathic pain, which resulted in the discovery of highly selective and potent novel NOP receptor full agonist {1-[4-(2-{hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl}-1H-benzimidazol-1-yl)piperidin-1-yl]cyclooctyl}methanol 1 (HPCOM) as systemically (subcutaneously) potent new-class analgesic. Thus, 1 demonstrates dose-dependent inhibitory effect against mechanical allodynia in chronic constriction injury-induced neuropathic pain model rats, robust metabolic stability and little hERG potassium ion channel binding affinity, with its unique and potentially safe profiles and mechanisms, which were distinctive from those of N/OFQ in terms of site-differential effects.