Circular dichroism and structure of the complex of acridine orange with poly(L-glutamic acid)

Circular dichroism and absorption spectra have been measured on solutions of acridine orange and poly(L -glutamic acid) mixed at two molar ratios of carboxyl group-to-dye, P/D 25 and 0.8, and at different pH's. Characteristic circular dichroism is induced at the absorption bands of acridine orange when the P/D is 25 and the solution is acidic. Another type of circular dichroism is manifest at neutral and alkaline pH when P/D is 0.8. For the induction of the former type of circular dichroism, a helical array of acridine-orange dimers bound to the α-helix is postulated, in which the dye molecular planes are almost perpendicular to the helical axis. Assuming the helical geometry and optical parameters, combined with the observed magnitude of transition electric moment, the rotatory strength of the complex is calculated to the zeroth order approximation, and the observed circular dichroism spectra have been reproduced.     

A single highly mutable catalytic site amino acid is critical for DNA polymerase fidelity

DNA polymerases contain active sites that are structurally superimposable and conserved in amino acid sequence. To probe the biochemical and structure-function relationship of DNA polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (Motif A; amino acids 605-617), and a fraction of all selected active Taq pol I (291 out of 8000) was tested for base pairing fidelity; seven unique mutants that efficiently misincorporate bases and/or extend mismatched bases were identified and sequenced. These mutants all contain substitutions of one specific amino acid, Ile-614, which forms part of the hydrophobic pocket that binds the base and ribose portions of the incoming nucleotide. Mutant Taq pol Is containing hydrophilic substitution I614K exhibit 10-fold lower base misincorporation fidelity, as well as a high propensity to extend mispairs. In addition, these low fidelity mutants containing hydrophilic substitution for Ile-614 can bypass damaged templates that include an abasic site and vinyl chloride adduct ethenoA. During polymerase chain reaction, Taq pol I mutant I614K exhibits an error rate that is >20-fold higher relative to the wild-type enzyme and efficiently catalyzes both transition and transversion errors. These studies have generated polymerase chain reaction-proficient mutant polymerases containing substitutions within the active site that confers low base pairing fidelity and a high error rate. Considering the structural and sequence conservation of Motif A, it is likely that a similar substitution will yield active low fidelity DNA polymerases that are mutagenic.     

Induction of LYVE-1/stabilin-2-positive liver sinusoidal endothelial-like cells from embryoid bodies by modulation of adrenomedullin-RAMP2 signaling

Embryonic stem cells (ESCs) are a useful source for various cell lineages. So far, however, progress toward reconstitution of mature liver morphology and function has been limited. We have shown that knockout mice deficient in adrenomedullin (AM), a multifunctional endogenous peptide, or its receptor-activity modifying protein (RAMP2) die in utero due to poor vascular development and hemorrhage within the liver. In this study, using embryoid bodies (EBs)-culture system, we successfully induced liver sinusoidal endothelial-like cells by modulation of AM-RAMP2. In an EB differentiation system, we found that co-administration of AM and SB431542, an inhibitor of transforming growth factor β (TGFβ) receptor type 1, markedly enhanced differentiation of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)/stabilin-2-positive endothelial cells. These cells showed robust endocytosis of acetylated low-density lipoprotein (Ac-LDL) and upregulated expression of liver sinusoidal endothelial cells (LSECs)-specific markers, including factor 8 (F8), Fc-γ receptor 2b (Fcgr2b), and mannose receptor C type 1 (Mrc1), and also possessed fenestrae-like structure, a key morphological feature of LSECs. In RAMP2-null liver, by contrast, LYVE-1 was downregulated in LSECs, and the sinusoidal structure was disrupted. Our findings highlight the importance of AM-RAMP2 signaling for development of LSECs.     

Identification of Novel Inhibitors of M. tuberculosis Growth Using Whole Cell Based High-Throughput Screening

Despite the urgent need for new antitubercular drugs, few are on the horizon. To combat the problem of emerging drug resistance, structurally unique chemical entities that inhibit new targets will be required. Here we describe our investigations using whole cell screening of a diverse collection of small molecules as a methodology for identifying novel inhibitors that target new pathways for Mycobacterium tuberculosis drug discovery. We find that conducting primary screens using model mycobacterial species may limit the potential for identifying new inhibitors with efficacy against M. tuberculosis. In addition, we confirm the importance of developing in vitro assay conditions that are reflective of in vivo biology for maximizing the proportion of hits from whole cell screening that are likely to have activity in vivo. Finally, we describe the identification and characterization of two novel inhibitors that target steps in M. tuberculosis cell wall biosynthesis. The first is a novel benzimidazole that targets mycobacterial membrane protein large 3 (MmpL3), a proposed transporter for cell wall mycolic acids. The second is a nitro-triazole that inhibits decaprenylphosphoryl-β-d-ribose 2'-epimerase (DprE1), an epimerase required for cell wall biosynthesis. These proteins are both among the small number of new targets that have been identified by forward chemical genetics using resistance generation coupled with genome sequencing. This suggests that methodologies currently employed for screening and target identification may lead to a bias in target discovery and that alternative methods should be explored.     

Morphological Effects,Rate of Incorporation,and the Enzymatic Action of Botulinum ADP-Ribosyltransferase,Known as C3 Exoenzyme,on Human Neuroblastoma GOTO Cells

The susceptibility of various lines of cultured cells to botulinum ADP-ribosyltransferase, known as C3 exoenzyme, was examined. Human neuroblastoma GOTO cells were most sensitive. The C3 exoenzyme caused a change in cell shape that involved extension of neurites. The exoenzyme evoked the outgrowth of neurites from chick ganglion as effectively as nerve growth factor, suggesting that C3 exoenzyme possesses neurotropic activity. Experiments with¹²⁵I-labeled enzyme revealed that C3 exoenzyme was rapidly incorporated into cells but the number of incorporated enzyme molecules was small. Once C3 exoenzyme had been incorporated, ADP-ribosylation of the substrate (Rho protein) in GOTO cells occurred immediately and rapidly reached a maximum level. However, some of Rho proteins remained unmodified even after induction of the change in morphology. These findings suggest that ADP-ribosylation by C3 exoenzyme is directly associated with the differentiation of GOTO cells but that other events may also participate in this process.     

Ion access pathway to the transmembrane pore in P2X receptor channels

P2X receptors are trimeric cation channels that open in response to the binding of adenosine triphosphate (ATP) to a large extracellular domain. The x-ray structure of the P2X4 receptor from zebrafish (zfP2X4) receptor reveals that the extracellular vestibule above the gate opens to the outside through lateral fenestrations, providing a potential pathway for ions to enter and exit the pore. The extracellular region also contains a void at the central axis, providing a second potential pathway. To investigate the energetics of each potential ion permeation pathway, we calculated the electrostatic free energy by solving the Poisson-Boltzmann equation along each of these pathways in the zfP2X4 crystal structure and a homology model of rat P2X2 (rP2X2). We found that the lateral fenestrations are energetically favorable for monovalent cations even in the closed-state structure, whereas the central pathway presents strong electrostatic barriers that would require structural rearrangements to allow for ion accessibility. To probe ion accessibility along these pathways in the rP2X2 receptor, we investigated the modification of introduced Cys residues by methanethiosulfonate (MTS) reagents and constrained structural changes by introducing disulfide bridges. Our results show that MTS reagents can permeate the lateral fenestrations, and that these become larger after ATP binding. Although relatively small MTS reagents can access residues in one of the vestibules within the central pathway, no reactive positions were identified in the upper region of this pathway, and disulfide bridges that constrain movements in that region do not prevent ion conduction. Collectively, these results suggest that ions access the pore using the lateral fenestrations, and that these breathe as the channel opens. The accessibility of ions to one of the chambers in the central pathway likely serves a regulatory function.