Selecting for and Checking Cells with HGPRT Deficiency for Hybridoma Production

For drug-selective media to work for hybridoma selection, myeloma cells expressing a mutation abrogating the function of their HGPRT gene (and subsequently unable to produce purines for DNA biosynthesis) are used. HGPRT will recognize 8-AG as a substrate and convert it to the monophosphate nucleotide. The 8-AG-containing nucleotide is then processed further and incorporated into DNA and RNA, where it is toxic. Therefore, cells with a functional HGPRT enzyme grown in the presence of 8-AG will die. Cells that are deficient in HGPRTase cannot incorporate 8-AG in vivo and thus continue to grow. Cells that have been selected for resistance to 8-AG should be checked periodically to ensure that they maintain sensitivity to drugs that block the de novo synthesis of DNA. In addition, all myeloma cell lines should be checked periodically for reversion of their drug selection markers. Any line that is not killed completely by drug selection should either be reselected or replaced with a new line.

How ATP and dATP act as molecular switches to regulate enzymatic activity in the prototypic bacterial class Ia ribonucleotide reductase

Class Ia ribonucleotide reductases (RNRs) are subject to allosteric regulation to maintain the appropriate deoxyribonucleotide levels for accurate DNA biosynthesis and repair. RNR activity requires a precise alignment of its α2 and β2 subunits such that a catalytically-essential radical species is transferred from β2 to α2. In E. coli, when too many deoxyribonucleotides are produced, dATP binding to RNR generates an inactive α4β4 state in which α2 and β2 are separated, preventing radical transfer. ATP binding breaks the α−β interface, freeing β2 and restoring activity. Here we investigate the molecular basis for allosteric activity regulation in the prototypic E. coli class Ia RNR. Through the determination of six crystal structures we are able to establish how dATP binding creates a binding pocket for β on α that traps β2 in the inactive α4β4 state. These structural snapshots also reveal the numerous ATP-induced conformational rearrangements that are responsible for freeing β2. We further discover, and validate through binding and mutagenesis studies, a previously unknown nucleotide binding site on the α subunit that is crucial for the ability of ATP to dismantle the inactive α4β4 state. These findings have implications for the design of allosteric inhibitors for bacterial RNRs.

Protein and DNA Biosynthesis Demonstrated in Host Cell-Free Phagosomes Containing Anaplasma phagocytophilum or Ehrlichia chaffeensis in Axenic Media

ABSTRACT Rickettsiae belong to the Anaplasmataceae family, which includes mostly tick-transmitted pathogens causing human, canine, and ruminant diseases. Biochemical characterization of the pathogens remains a major challenge because of their obligate parasitism. We investigated the use of an axenic medium for growth of two important pathogens—Anaplasma phagocytophilum and Ehrlichia chaffeensis—in host cell-free phagosomes. We recently reported that the axenic medium promotes protein and DNA biosynthesis in host cell-free replicating form of E. chaffeensis, although the bacterial replication is limited. We now tested the hypothesis that growth on axenic medium can be improved if host cell-free rickettsia-containing phagosomes are used. Purification of phagosomes from A. phagocytophilum- and E. chaffeensis-infected host cells was accomplished by density gradient centrifugation combined with magnet-assisted cell sorting. Protein and DNA synthesis was observed for both organisms in cell-free phagosomes with glucose-6-phosphate and/or ATP. The levels of protein and DNA synthesis were the highest for a medium pH of 7. The data demonstrate bacterial DNA and protein synthesis for the first time in host cell-free phagosomes for two rickettsial pathogens. The host cell support-free axenic growth of obligate pathogenic rickettsiae will be critical in advancing research goals in many important tick-borne diseases impacting human and animal health.

Detection of a G-Quadruplex as a Regulatory Element in Thymidylate synthase for Gene Silencing Using Polypurine Reverse Hoogsteen Hairpins

Thymidylate synthase (TYMS) enzyme is an anti-cancer target given its role in DNA biosynthesis. TYMS inhibitors (e.g., 5-Fluorouracil) can lead to drug resistance through an autoregulatory mechanism of TYMS that causes its overexpression. Since G-quadruplexes (G4) can modulate gene expression, we searched for putative G4 forming sequences (G4FS) in the TYMS gene that could be targeted using polypurine reverse Hoogsteen hairpins (PPRH). G4 structures in the TYMS gene were detected using the quadruplex forming G-rich sequences mapper and confirmed through spectroscopic approaches such as circular dichroism and NMR using synthetic oligonucleotides. Interactions between G4FS and TYMS protein or G4FS and a PPRH targeting this sequence (HpTYMS-G4-T) were studied by EMSA and thioflavin T staining. We identified a G4FS in the 5’UTR of the TYMS gene in both DNA and RNA capable of interacting with TYMS protein. The PPRH binds to its corresponding target dsDNA, promoting G4 formation. In cancer cells, HpTYMG-G4-T decreased TYMS mRNA and protein levels, leading to cell death, and showed a synergic effect when combined with 5-fluorouracil. These results reveal the presence of a G4 motif in the TYMS gene, probably involved in the autoregulation of TYMS expression, and the therapeutic potential of a PPRH targeted to the G4FS.

Tetrahydrocarbazoles as Novel Class of DNA Biosynthesis Inhibitors in Bacteria

Background: High-throughput screening (HTS) of large-compound libraries is a convenient and cost-effective tool for novel drug discovery. However, in many cases, HTS platforms are not well adapted to perform cell-based screening assay with simultaneous identification of the mechanism of action for active compounds. Methods: Previously, we have described a unique double-reporter system that can be used to reveal compounds having antibacterial activity. This construction is based on two genes of Katushka2S and RFP that encode proteins with different imaging signature. The upregulated expression of the first gene attributes directly to translation inhibition in prokaryotes while the high expression of the second one is unambiguously associated with DNA biosynthesis restriction (SOS-response). Results: In the current work, we have applied this system for the identification of novel smallmolecule compounds with antibacterial activity in HTS scale. Compounds were selected from the ChemDiv in-house collection (of total 47K molecules) following the maximum diversity in structure. Based on the obtained results, we have revealed a small series of tetrahydrocarbazole derivatives with antibacterial activity. The hit compound inhibited bacterial growth with a MIC value of 21 μg/ml and blocked DNA biosynthesis. No activity towards translation has been observed. In spite of a relatively moderate potency, as compared to the control sample (erythromycin, MIC=3 μg/ml), this molecule provides good selectivity index (SI>333, HEK239). Conclusion: Tetrahydrocarbazole derivatives can be properly regarded as an attractive starting point for further development and optimization.

Structure of a trapped radical transfer pathway within a ribonucleotide reductase holocomplex

Ribonucleotide reductases (RNRs) are a diverse family of enzymes that are alone capable of generating 2′-deoxynucleotides de novo and are thus critical in DNA biosynthesis and repair. The nucleotide reduction reaction in all RNRs requires the generation of a transient active site thiyl radical, and in class I RNRs, this process involves a long-range radical transfer between two subunits, α and β. Because of the transient subunit association, an atomic resolution structure of an active α2β2 RNR complex has been elusive. We used a doubly substituted β2, E52Q/(2,3,5)-trifluorotyrosine122-β2, to trap wild-type α2 in a long-lived α2β2 complex. We report the structure of this complex by means of cryo–electron microscopy to 3.6-angstrom resolution, allowing for structural visualization of a 32-angstrom-long radical transfer pathway that affords RNR activity.

The Effects of a Novel Series of KTTKS Analogues on Cytotoxicity and Proteolytic Activity

KTTKS is a matrikine that originates from the proteolytic hydrolysis of collagen. This peptide stimulates ECM production and types I and III collagen expression in vitro. A more stable form of KTTKS is pal-KTTKS, known as Matrixyl® or palmitoyl pentapeptide-3. A series of novel pentapeptides, analogues of KTTKS with the general formula X-KTTKS-OH(NH2), where X = acetyl, lipoyl, palmitoyl residues, was designed and synthesized. Their effect on amidolytic activity of urokinase, thrombin, trypsin, plasmin, t-PA, and kallikrein were tested. Cytotoxic tests on fibroblasts, as well as collagen and DNA biosynthesis tests for selected peptides, were also carried out. The test results showed that the most active plasmin inhibitors were palmitoyl peptides, whether in acid or amide form. No biological effects of lysine modification to arginine in the synthesized peptides were found. None of the synthesized peptides was not cytotoxic on fibroblasts, and three of them showed cell growth. These three compounds showed no concentration-activity relationship in the collagen and DNA biosynthesis assays.

Beneficial Effects of Vitamins K and D3 on Redox Balance of Human Osteoblasts Cultured with Hydroxyapatite-Based Biomaterials

Hydroxyapatite-based biomaterials are commonly used in surgery to repair bone damage. However, the introduction of biomaterials into the body can cause metabolic alterations, including redox imbalance. Because vitamins D3 and K (K1, MK-4, MK-7) have pronounced osteoinductive, anti-inflammatory, and antioxidant properties, it is suggested that they may reduce the adverse effects of biomaterials. The aim of this study was to investigate the effects of vitamins D3 and K, used alone and in combination, on the redox metabolism of human osteoblasts (hFOB 1.19 cell line) cultured in the presence of hydroxyapatite-based biomaterials (Maxgraft, Cerabone, Apatos, and Gen-Os). Culturing of the osteoblasts in the presence of hydroxyapatite-based biomaterials resulted in oxidative stress manifested by increased production of reactive oxygen species and decrease of glutathione level and glutathione peroxidase activity. Such redox imbalance leads to lipid peroxidation manifested by an increase of 4-hydroxynonenal level, which is known to influence the growth of bone cells. Vitamins D3 and K were shown to help maintain redox balance and prevent lipid peroxidation in osteoblasts cultured with hydroxyapatite-based biomaterials. The strongest effect was observed for the combination of vitamin D3 and MK-7. Moreover, vitamins promoted growth of the osteoblasts, manifested by increased DNA biosynthesis. Therefore, it is suggested that the use of vitamins D3 and K may protect redox balance and support the growth of osteoblasts affected by hydroxyapatite-based biomaterials.