Assimilatory Nitrate Reduction in Azospirillum Brasilense SP7 : Mutants and Expression of the Genes in Escherichia Coli Recombinant Strains

The intensive study of A. brasilense that has been undertaken is due in part to the desire of microbiologists to understand its nitrogen metabolism. A. brasilense can perform all of the reactions of the nitrogen cycle except for nitrification, however, the details are not yet completely understood on any of the processes. In agriculture, A. brasilense not only supplies fixed nitrogen and nutrient to cereals; but also it significantly contributes to the loss of nitrogen from the soil by denitrification. Enormous amounts of fixed nitrogen are unfortunately lost from the soil to the atmosphere by this denitrification. The elucidation of the interrelationship between nitrogen-fixation and denitrification in this organism will therefore be of great importance. Globally, the No3- metabolism of A. brasilense lessens or adds to human health problems (ozone removal from stratosphere, blue baby syndrome, N03- as carcinogen). Thus, an overall understanding of N03 metabolism in A. brasilense will be of enormous benefit to human kind My study examined N03- and No2- assimilation in A. brasilense and assimilation of purine nitrogen. Various A. brasilense mutants defective in N03- or N02- assimilation were isolated and characterized, and revertants were obtained and studied. In addition, the N03-/N02- assimilation of two recombinant E. coli strains containing a 27 kb segment of A. brasilense DNA (in a cosmid) was also studied

Small molecule-induced simultaneous destabilization of β-catenin and RAS is an effective molecular strategy to suppress stemness of colorectal cancer cells

Background Cancer stem cells (CSCs), the major driver of tumorigenesis, is a sub-population of tumor cells responsible for poor clinical outcomes. However, molecular mechanism to identify targets for controlling CSCs is poorly understood. Methods Gene Set Enrichment Analyses (GSEA) of Wnt/β-catenin and RAS signaling pathways in stem-like subtype of colorectal cancer (CRC) patients were performed using two gene expression data set. The therapeutic effects of destabilization of β-catenin and RAS were tested by treatment of small molecule KYA1797K using CRC patient derived cells. Results Treatment with KYA1797K, a small molecule that destabilizes both β-catenin and RAS via Axin binding, effectively suppresses the stemness of CSCs as shown in CRC spheroids and small intestinal tumors of ApcMin/+/K-RasG12DLA2 mice. Moreover, KYA1797K also suppresses the stemness of cells in CRC patient avatar model systems, such as patient-derived tumor organoids (PDTOs) and patient-derived tumor xenograft (PDTX). Conclusion Our results suggest that destabilization of both β-catenin and RAS is a potential therapeutic strategy for controlling stemness of CRC cells.This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIP) (grants 2016R1A5A1004694, 2019R1A2C3002751, 2018R1D1A1B07050189)

Chromosomal end fusion resulting from telomere erosion increases susceptibility to radiation via multinucleation: Effect of p53

and telomerase activation are frequently found in human cancers. p53 inactivation, however, eliminates or attenuates the biological responses to telomerase inhibition and the eventual telomere erosion. We show that telomere erosion can increase the susceptibility to radiation, irrespective of p53 status. Both telomerase inhibition and critically shortened telomere with significant change of chromosomal end-to-end fusion were essential for the enhancement of radiosensitivity. The enhancement was correlated with greater formation of multinucleated cells. p53 inactivation did not eliminate the observed generation of chromosomal fusion and multinucleation, and the resulting increased susceptibility to radiation, as opposed to the previously proved role of p53 in mediating cellular responses to telomere dysfunction. The present findings suggest the importance of chromosomal end fusion in modulating radiosensitivity rather than p53 DNA damage signaling. Thus, the suggested anticancer radiotherapeutic strategy combined with telomerase inhibition could clinically be applicable to cancers, irrespective of p53 status.We thank Professor Woon Ki Paik for critical reading of the manuscript and Eun-Ju Lee for assistance in the preparation of the manuscript. This work was supported by a grant from National Nuclear R&D program and Human Genome Project (FG-1-1), Korean Ministry of Science and Technology

Valproic Acid Induces Hair Regeneration in Murine Model and Activates Alkaline Phosphatase Activity in Human Dermal Papilla Cells

Alopecia is the common hair loss problem that can affect many people. However, current therapies for treatment of alopecia are limited by low efficacy and potentially undesirable side effects. We have identified a new function for valproic acid (VPA), a GSK3β inhibitor that activates the Wnt/β-catenin pathway, to promote hair re-growth in vitro and in vivo.Topical application of VPA to male C3H mice critically stimulated hair re-growth and induced terminally differentiated epidermal markers such as filaggrin and loricrin, and the dermal papilla marker alkaline phosphatase (ALP). VPA induced ALP in human dermal papilla cells by up-regulating the Wnt/β-catenin pathway, whereas minoxidil (MNX), a drug commonly used to treat alopecia, did not significantly affect the Wnt/β-catenin pathway. VPA analogs and other GSK3β inhibitors that activate the Wnt/β-catenin pathway such as 4-phenyl butyric acid, LiCl, and BeCl(2) also exhibited hair growth-promoting activities in vivo. Importantly, VPA, but not MNX, successfully stimulate hair growth in the wounds of C3H mice.Our findings indicate that small molecules that activate the Wnt/β-catenin pathway, such as VPA, can potentially be developed as drugs to stimulate hair re-growth

Escherichia coli purine repressor: Regulation and structure-function analysis

The Escherichia coli purine repressor (PurR) is a transcriptional regulator for genes involved in purine nucleotide biosynthesis and related pathways. Chemical cross-linking studies show that PurR is a homodimer. Limited proteolysis identified two major structural domains of PurR; an N-terminal 52 amino acid domain for DNA binding and a 284 residue C-terminal domain for corepressor binding and dimerization. These two domains are connected by a short exposed hinge. The 284 amino acid C-terminal domain is homologous to periplasmic sugar binding proteins for arabinose, galactose and ribose. The corepressors, hypoxanthine and guanine, bind cooperatively to each subunit with affinities of 9.3 and 1.5 $\mu$M, respectively. Upon binding of corepressors, PurR undergoes a conformational change that increases its binding affinity for operator DNA. Amino acid residues required for corepressor binding were inferred by sequence comparison of PurR with periplasmic sugar binding proteins of known structure as well as other LacI family repressors, and subsequently replaced by site directed mutagenesis. The analysis of PurR mutants indicates that Asp 146, Trp 147, Arg 196 and Asp275 function in binding the corepressors. Conservation of ligand binding residues between PurR and sugar binding proteins suggests the overall similarity of the PurR corepressor binding domain with sugar binding proteins and leads to the proposal that effector sites must be similar among these proteins. The four mutations constructed perturbed the binding of both hypoxanthine and guanine and thus provides evidence for a single corepressor site per PurR subunit. A significant conformational change of the protease hypersensitive hinge of PurR was detected by binding of operator DNA. To understand the role of the hinge, 16 different mutations in four central hinge residues were produced and characterized. Mutations in the hinge region impaired repressor function in vivo. Several nonfunctional hinge mutants were defective in low affinity binding to operator DNA in the absence of corepressor as well as in high affinity corepressor-dependent binding to operator DNA, although binding of corepressor and general structural characteristics were similar to the wild type PurR. These results establish a role for the hinge region in operator binding and together with the studies of corepressor binding mutants lead to a proposal for two routes to form the holoPurR-operator complex: (i) interaction of apoPurR with operator followed by binding of corepressor to the apoPurR-operator complex and (ii) binding of holoPurR to operator. Homology of PurR and LacI may account for the effector-independent binding to operator DNA. (Abstract shortened by UMI.

β-catenin-mediated signaling: A novel molecular target for chemoprevention with anti-inflammatory substances

Inflammation is thought to play a role in the pathophysiology of cancer. Accumulating evidence from clinical and laboratory-based studies suggests that substances with anti-inflammatory activities are potential candidates for chemoprevention. Recent advances in cellular and molecular biology of cancer shed light on components of intracellular signaling cascades that can be potential molecular targets of chemoprevention with various anti-inflammatory substances. Although cyclooxygenase-2, a primary enzyme that mediates inflammatory responses, has been well recognized as a molecular target for chemoprevention by both synthetic and natural anti-inflammatory agents, the cellular signaling mechanisms that associate inflammation and cancer are not still clearly illustrated. Recent studies suggest that beta-catenin-mediated signaling, which regulates developmental processes, may act as a potential link between inflammation and cancer. This review aims to focus on beta-catenin-mediated signaling pathways, particularly in relation to its contribution to carcinogenesis, and the modulation of inappropriately activated beta-catenin-mediated signaling by nonsteroidal anti-inflammatory drugs and chemopreventive phytochemicals possessing anti-inflammatory properties. (c) 2005 Elsevier B.V All rights reserved

<i>Euodia daniellii</i> Hemsl. Extract and Its Active Component Hesperidin Accelerate Cutaneous Wound Healing via Activation of Wnt/β-Catenin Signaling Pathway

The activation of the Wnt/β-catenin signaling pathway plays a key role in the wound-healing process through tissue regeneration. The extract of Euodia daniellii Hemsl. (E. daniellii), a member of the Rutaceae family, activates the Wnt/β-catenin signaling pathway. However, the function of E. daniellii in wound healing has not yet been elucidated. We performed a migration assay to determine the wound-healing effect of E. daniellii extract in vitro using human keratinocytes and dermal fibroblast. In addition, a mouse acute wound model was used to investigate the cutaneous wound-healing effect of E. daniellii extract in vivo and confirm the potential mechanism. E. daniellii extract enhanced the migration of human keratinocytes and dermal fibroblasts via the activation of the Wnt/β-catenin pathway. Moreover, the E. daniellii extract increased the levels of keratin 14, PCNA, collagen I, and α-SMA, with nuclei accumulation of β-catenin in vitro. E. daniellii extract also efficiently accelerated re-epithelialization and stimulated wound healing in vivo. Furthermore, we confirmed that hesperidin, one of the components of E. daniellii, efficiently accelerated the migration of human keratinocytes and dermal fibroblasts, as well as wound healing in vivo via the activation of the Wnt/β-catenin pathway. Overall, E. daniellii extract and its active component, hesperidin, have potential to be used as therapeutic agents for wound healing