scholarly journals Sequence-Directed Base Mispairing in Human Oncogenes

1998 ◽  
Vol 18 (8) ◽  
pp. 4659-4669 ◽  
Author(s):  
Lavanya Lall ◽  
Richard L. Davidson
Keyword(s):  
Mammalian Cells ◽  
Point Mutations ◽  
Inhibitory Effect ◽  
Relative Difference ◽  
Human Tumors ◽  
Sequence Specificity ◽  
Klenow Polymerase ◽  
Free Environment ◽  
Base Mispairing

ABSTRACT The most frequently observed mutations in ras oncogenes in solid human tumors are GC→AT transitions at the 3′ G residue of the GG doublet in codon 12 of these oncogenes. We had shown previously that mutagenesis by thymidine occurred with the same sequence specificity in mammalian cells, in that mutagenesis occurred preferentially at the 3′ G of GG doublets. In this study, in vitro DNA synthesis experiments were carried out to assess the effect of local DNA sequence on base mispairing in order to determine the mechanism of sequence-directed mutagenesis by thymidine and its possible relationship to activating point mutations in N-, Ki- and Ha-ras oncogenes in solid human tumors. To avoid complicating the interpretation of the results because of the occurrence of mismatch repair as well as base misincorporation, the experiments were carried out in a repair-free environment with exonuclease-free Klenow polymerase. The results of these experiments showed that misincorporation of deoxyribosylthymine (dT) occurred with several-fold-greater efficiency opposite the 3′ G compared to the 5′ G of the GG doublet in codon 12 of human ras oncogenes. These results further demonstrated that the relative difference in the extent of dT misincorporation opposite the 3′ G and the 5′ G of GG doublets in codon 12 in the various ras oncogenes was affected by the base immediately upstream of the doublet. Within the GG doublet, it was seen that the 5′ G and 3′ G residues had an effect on the extent of dT misincorporation opposite each other. The 5′ G was shown to have a stimulatory effect on dT misincorporation opposite the 3′ G, while the 3′ G was shown to have an inhibitory effect on dT misincorporation opposite the 5′ G. Presumably, these mutual interactions within GG doublets are additive, such that the large differential in dT misincorporation observed between the 3′ G and 5′ G residues in GG doublets is the end result of the combined stimulatory and inhibitory effects within these doublets. Since the observed pattern of dT misincorporation within GG doublets corresponds to the most frequent mode of activation of ras oncogenes in solid human tumors, the results of these experiments suggest that sequence-directed dT misincorporation may be involved in the pattern of activation of humanras oncogenes, by causing GC→AT transitions preferentially at the 3′ G of the GG doublet in codon 12 of these oncogenes.

scholarly journals Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease

2001 ◽  
Vol 82 (4) ◽  
pp. 765-773 ◽  
Author(s):  
Andres Merits ◽  
Lidia Vasiljeva ◽  
Tero Ahola ◽  
Leevi Kääriäinen ◽  
Petri Auvinen
Keyword(s):  
Mammalian Cells ◽  
Active Sites ◽  
Insect Cells ◽  
Semliki Forest Virus ◽  
Point Mutations ◽  
Proteolytic Processing ◽  
Wild Type ◽  
In Trans ◽  
Polyprotein Precursor

The RNA replicase proteins of Semliki Forest virus (SFV) are translated as a P1234 polyprotein precursor that contains two putative autoproteases. Point mutations introduced into the predicted active sites of both proteases nsP2 (P2) and nsP4 (P4), separately or in combination, completely abolished virus replication in mammalian cells. The effects of these mutations on polyprotein processing were studied by in vitro translation and by expression of wild-type polyproteins P1234, P123, P23, P34 and their mutated counterparts in insect cells using recombinant baculoviruses. A mutation in the catalytic site of the P2 protease, C478A, (P2CA) completely abolished the processing of P12CA34, P12CA3 and P2CA3. Co-expression of P23 and P12CA34 in insect cells resulted in in trans cleavages at the P2/3 and P3/4 sites. Co-expression of P23 and P34 resulted in cleavage at the P3/4 site. In contrast, a construct with a mutation in the active site of the putative P4 protease, D6A, (P1234DA) was processed like the wild-type protein. P34 or its truncated forms were not processed when expressed alone. In insect cells, P4 was rapidly destroyed unless an inhibitor of proteosomal degradation was used. It is concluded that P2 is the only protease needed for the processing of SFV polyprotein P1234. Analysis of the cleavage products revealed that P23 or P2 could not cleave the P1/2 site in trans.

scholarly journals Simple Silica Column–Based Method to Quantify Inorganic Polyphosphates in Cartilage and Other Tissues

Cartilage ◽  
2017 ◽  
Vol 9 (4) ◽  
pp. 417-427 ◽  
Author(s):  
Whitaik David Lee ◽  
Rahul Gawri ◽  
Toshikazu Shiba ◽  
Ae-Ri Ji ◽  
William L. Stanford ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Inhibitory Effect ◽  
Proteinase K ◽  
Inorganic Polyphosphates ◽  
Column Method ◽  
Spin Column ◽  
Mammalian Tissues ◽  
Biological Sources ◽  
Low Levels

Objective. Inorganic polyphosphates (polyP) play a multitude of roles in mammalian biology. PolyP research is hindered by the lack of a simple and sensitive quantification method. The aim of this study was to develop a robust method for quantifying the low levels of polyP in mammalian tissue such as cartilage, which is rich in macromolecules that interfere with its determination. Design. Native and in vitro formed tissues were digested with proteinase K to release sequestrated polyP. The tissue digest was loaded on to silica spin columns, followed by elution of bound polyP and various treatments were assessed to minimize non-polyP fluorescence. The eluent was then quantified for polyP content using fluorometry based on DAPI (4′,6-diamidino-2-phenylindole) fluorescence shift occurring with polyP. Results. Proteinase K pretreatment reduced the inhibitory effect of proteins on polyP recovery. The eluent was contaminated with nucleic acids and glycosaminoglycans, which cause extraneous fluorescence signals. These were then effectively eliminated by nucleases treatment and addition of concentrated Tris buffer. PolyP levels were quantified and recovery ratio determined using samples spiked with a known amount of polyP. This silica spin column method was able to recover at least 80% of initially loaded polyP, and detect as little as 10−10 mol. Conclusions. This sensitive, reproducible, easy to do method of quantifying polyP will be a useful tool for investigation of polyP biology in mammalian cells and tissues. Although the protocol was developed for mammalian tissues, this method should be able to quantify polyP in most biological sources, including fluid samples such as blood and serum.

scholarly journals Reversal of Mefloquine and Quinine Resistance in Plasmodium falciparum with NP30

2003 ◽  
Vol 47 (8) ◽  
pp. 2393-2396 ◽  
Author(s):  
Michelle Ciach ◽  
Kathleen Zong ◽  
Kevin C. Kain ◽  
Ian Crandall
Keyword(s):  
Plasmodium Falciparum ◽  
Multidrug Resistance ◽  
Genetic Analysis ◽  
Resistance Genes ◽  
Mammalian Cells ◽  
Point Mutations ◽  
In Vitro Assays ◽  
Drug Efflux ◽  
P Glycoprotein

ABSTRACT Quinoline resistance in malaria is frequently compared with P-glycoprotein-mediated multidrug resistance (mdr) in mammalian cells. We have previously reported that nonylphenolethoxylates, such as NP30, are potential Plasmodium falciparum P-glycoprotein substrates and drug efflux inhibitors. We used in vitro assays to compare the ability of verapamil and NP30 to sensitize two parasite isolates to four quinolines: chloroquine (CQ), mefloquine (MF), quinine (QN), and quinidine (QD). NP30 was able to sensitize (reversal, >80%) P. falciparum to MF, QN, QD, and, to a lesser extent, CQ. The presence of 2 μM verapamil had no effect on mefloquine resistance; however, the presence of verapamil modulated the activities of QN and QD in a manner parallel to that observed for CQ. Genetic analysis of putative quinoline resistance genes did not suggest an association between known point mutations in pfcrt and pfmdr1 and NP30 sensitization activity. We conclude that the sensitization action of NP30 is distinct both phenotypically and genotypically from that of verapamil.

scholarly journals Cyclosporin Analogs Inhibit In Vitro Growth of Cryptosporidium parvum

1998 ◽  
Vol 42 (4) ◽  
pp. 843-848 ◽  
Author(s):  
Margaret E. Perkins ◽  
Teresa W. Wu ◽  
Sylvie M. Le Blancq
Keyword(s):  
Cryptosporidium Parvum ◽  
Mammalian Cells ◽  
Inhibitory Effect ◽  
Positive Control ◽  
Short Term ◽  
Apicomplexan Parasites ◽  
Psc 833 ◽  
Drug Assays ◽  
Sdz Psc 833

ABSTRACT Cyclosporine and nonimmunosuppressive cyclosporin (CS) analogs were demonstrated to be potent inhibitors of the growth of the intracellular parasite Cryptosporidium parvum in short-term (48-h) in vitro cultures. Fifty-percent inhibitory concentrations (IC50s) were 0.4 μM for SDZ 033-243, 1.0 μM for SDZ PSC-833, and 1.5 μM for cyclosporine. Two other analogs were less effective than cyclosporine: the IC50 of SDZ 205-549 was 5 μM, and that of SDZ 209-313 was 7 μM. These were much lower than the IC50 of 85 μM of paromomycin, a standard positive control for in vitro drug assays for this parasite. In addition, intracellular growth of excysted sporozoites that had been incubated for 1 h in cyclosporine was significantly reduced, suggesting that the drug can inhibit sporozoite invasion. The cellular activities of the CS analogs used have been characterized for mammalian cells and protozoa. The two analogs that were most active in inhibiting C. parvum, SDZ PSC-833 and SDZ 033-243, bind weakly to cyclophilin, a peptidyl proline isomerase which is the primary target of cyclosporine and CS analogs. However, they are potent modifiers of the activity of the P glycoproteins/multidrug resistance (MDR) transporters, members of the ATP-binding cassette (ABC) superfamily. Hence, both cyclophilin and some ABC transporters may be targets for this class of drugs, although drugs that preferentially interact with the latter are more potent. Cyclosporine (0.5 μM) had no significant chemosensitizing activity. That is, it did not significantly increase sensitivity to paromomycin, suggesting that an ABC transporter is not critical in the efflux of this drug. Cyclosporine at concentrations up to 50 μM was not toxic to host Caco-2 cells in the CellTiter 96 assay. The results of this study complement those of studies of the inhibitory effect of cyclosporine and CS analogs on other apicomplexan parasites, Plasmodium falciparum, Plasmodium vivax, and Toxoplasma gondii.

Intracellular redistribution of Ku immunoreactivity in response to cell-cell contact and growth modulating components in the medium

1996 ◽  
Vol 109 (7) ◽  
pp. 1937-1946 ◽  
Author(s):  
J.W. Fewell ◽  
E.L. Kuff
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Primary Cultures ◽  
Human Keratinocytes ◽  
Inhibitory Effect ◽  
Cell Contact ◽  
Nuclear Component ◽  
Confluent Cell ◽  
Cell Cell

Ku is a heterodimeric protein first recognized as a human autoantigen but now known to be widely distributed in mammalian cells. Analysis of repair-deficient mutant cells has shown that Ku is required for DNA repair, and roles in DNA replication and transcription have also been suggested on the basis of in vitro observations. Ku is generally regarded as a nuclear component. However, in the present paper, we show that a quantitatively significant fraction (half or more) of Ku is located in the cytoplasm of cultured primate cells, and that major changes in epitope accessibility of both nuclear and cytoplasmic Ku components are associated with the transition from sparse to confluent cell densities. The same changes in immunoreactivity were seen in HeLa, 293, CV-1 (monkey) and HPV-transformed keratinocyte cell lines, and in primary cultures of human keratinocytes. The immunostaining pattern of sparsely grown cells could be converted to the ‘confluent’ configuration by re-plating them at the same low density on a monolayer of mouse 3T3 cells. The confluent antigen pattern could also be induced in sparse cells within 15–30 minutes by exposure of the cells to serum- or Ca(2+)-free medium or overnight with 2 mM hydroxyurea. Somatostatin at 0.12 mM blocked the effects of serum/Ca2+ deprivation of Ku p70 antigen distribution in sparse CV-1 cells, and in confluent cultures reversed the usual nuclear concentration of p70 immunoreactivity. However, somatostatin did not alter the expected immunostaining patterns of p86. Preliminary studies indicate that sparse CV-1 cells, but not HeLa cells, respond to as little as 1 pM of TGF-beta 1 in the culture medium by the rapid appearance of nuclear immunoreactivity. TGF-alpha had no apparent effect. These findings are consistent with the participation of Ku in a signal transduction system responsive to the inhibitory effect of cell-cell contact on the one hand and to cytokines and growth-supportive components of the culture medium on the other.

Munc18b regulates core SNARE complex assembly and constitutive exocytosis by interacting with the N-peptide and the closed-conformation C-terminus of syntaxin 3

2010 ◽  
Vol 431 (3) ◽  
pp. 353-361 ◽  
Author(s):  
Ren-Wang Peng ◽  
Claudio Guetg ◽  
Eric Abellan ◽  
Martin Fussenegger
Keyword(s):  
Mammalian Cells ◽  
Ectopic Expression ◽  
Inhibitory Effect ◽  
Snare Complex ◽  
Closed Conformation ◽  
The Core ◽  
C Terminus ◽  
Physiological Impact ◽  
Syntaxin 3

The interaction between SM (Sec1/Munc18) and SNARE (soluble N-ethylmaleimide-sensitive factor-attachment receptor) proteins constitutes the core eukaryotic membrane fusion machinery which manages exocytosis by mediating fusion of constitutively exocytic vesicles with the plasma membrane. However, mechanistic details on the nature and the physiological impact of SM–SNARE interactions remain largely elusive. Detailed characterization of the interaction profiles between Munc18b and its cognate SNAREs, Stx3 (syntaxin 3), SNAP-23 (soluble N-ethylmaleimide-attachment protein 23) and VAMP8 (vesicle-associated membrane protein 8), revealed that Munc18b binds Stx3, VAMP8 and the assembled core SNARE complex consisting of Stx3, SNAP-23 and VAMP8. Dissection of the Munc18b–Stx3 heterodimer suggested that Munc18b interacts with Stx3's conserved N-peptide as well as with its closed-conformation C-terminus encompassing the Habc domain, a linker and the SNARE (H3) motif. Deletion of the Habc domain or mutations interrupting the intramolecular binding of the Habc and H3 domains abrogated the Munc18b–Stx3 interaction. Although only the N-peptide deletion mutant, but not the soluble wild-type Stx3, is assembled into the core SNARE complex in the presence of Munc18b in vitro, ectopic expression of this SM protein increases constitutive exocytosis in mammalian cells. Our results suggest that Munc18b is functionally coupled to the assembly of exocytic SNARE complexes and increases exocytosis by interacting with the N-peptide and closed-conformation C-terminus of Stx3, thereby neutralizing the secretion-inhibitory effect of this SNARE.

scholarly journals In Vitro Antiplasmodium and Chloroquine Resistance Reversal Effects of Andrographolide

2019 ◽  
Vol 2019 ◽  
pp. 1-16
Author(s):  
Zaid O. Ibraheem ◽  
Roslaini Abd Majid ◽  
Hasidah Mohd Sidek ◽  
Sabariah Md Noor ◽  
Mun Fei Yam ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Drug Sensitivity ◽  
Vero Cells ◽  
Inhibitory Effect ◽  
Chloroquine Resistance ◽  
Safety Level ◽  
Reversal Effect ◽  
Resistance Reversal

The emergence of drug-resistant strains of Plasmodium falciparum is the worst catastrophe that has ever confronted the dedicated efforts to eradicate malaria. This urged for searching other alternatives or sensitizers that reverse chloroquine resistance. In this experiment, the potential of andrographolide to inhibit plasmodial growth and reverse CQ resistance was tested in vitro using the SYBRE green-1-based drug sensitivity assay and isobologram technique, respectively. Its safety level toward mammalian cells was screened as well against Vero cells and RBCs using MTT-based drug sensitivity and RBC hemolysis assays, respectively. Its effect against hemozoin formation was screened using β-hematin formation and heme fractionation assays. Its molecular characters were determined using the conventional tests for the antioxidant effect measurement and the in silico molecular characterization using the online free chemi-informatic Molinspiration software. Results showed that andrographolide has a moderate antiplasmodium effect that does not entitle it to be a substituent for chloroquine. Furthermore, andrographolide ameliorated the sensitivity of the parasite to chloroquine. Besides, it showed an indirect inhibitory effect against hemozoin formation within the parasite and augmented the chloroquine-induced inhibition of hemozoin formation. The study suggests that its chloroquine resistance reversal effect may be due to inhibition of chloroquine accumulation or due to its impact on the biological activity of the parasite. Overall, this in vitro study is a clue for the reliability of andrographolide to be added with chloroquine for reversal of chloroquine resistance and tolerance, but further in vivo studies are recommended to confirm this notion. In spite of its prominent and safe in vitro and in vivo growth inhibitory effect and its in vitro chloroquine resistance reversing effect, it is inapplicable to implement it in malaria chemotherapy to substitute chloroquine or to reverse its resistance.

scholarly journals Inhibitory Effect of Aureobasidin A on Toxoplasma gondii

2005 ◽  
Vol 49 (5) ◽  
pp. 1794-1801 ◽  
Author(s):  
Sabrina Sonda ◽  
Giusy Sala ◽  
Riccardo Ghidoni ◽  
Andrew Hemphill ◽  
Jean Pieters
Keyword(s):  
Toxoplasma Gondii ◽  
Mammalian Cells ◽  
Cell Metabolism ◽  
Opportunistic Pathogen ◽  
Inhibitory Effect ◽  
Stage Conversion ◽  
Congenital Birth Defects ◽  
Aureobasidin A ◽  
Sphingolipid Biosynthesis

ABSTRACT The apicomplexan parasite Toxoplasma gondii is a leading opportunistic pathogen associated with AIDS and congenital birth defects. Due to the need for identifying new parasite-specific treatments, the possibility of targeting sphingolipid biosynthesis in the parasite was investigated. Aureobasidin A, an inhibitor of the enzyme synthesizing the sphingolipid inositol phosphorylceramide, which is present in fungi, plants, and some protozoa but absent in mammalian cells, was found to block in vitro T. gondii replication without affecting host cell metabolism. Aureobasidin A treatment did not induce tachyzoite to bradyzoite stage conversion in T. gondii but resulted in a loss of intracellular structures and vacuolization within the parasite. In addition, aureobasidin A inhibited sphingolipid synthesis in T. gondii. Sphingolipid biosynthetic pathways may therefore be considered targets for the development of anti-T. gondii agents.

Investigation of the mechanism of action of a potent pateamine A analog, des-methyl, des-amino pateamine A (DMDAPatA)

2020 ◽  
Vol 98 (4) ◽  
pp. 502-510
Author(s):  
Sai Shilpa Kommaraju ◽  
Julieta Aulicino ◽  
Shruthi Gobbooru ◽  
Jing Li ◽  
Mingzhao Zhu ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Inhibitory Effect ◽  
Initiation Factor ◽  
Cellular Activity ◽  
Proliferating Cells ◽  
Eif4f Complex ◽  
Antiproliferative Agent ◽  
Pateamine A ◽  
The Impact

The natural product pateamineA (PatA) is a highly potent antiproliferative agent. PatA and the simplified analog desmethyl, desamino pateamineA (DMDAPatA) have exhibited cytotoxicity selective for rapidly proliferating cells, and have been shown to inhibit cap-dependent translation initiation through binding to eIF4A (eukaryotic initiation factor 4A) of the eIF4F complex. PatA and DMDAPatA are both known to stimulate the RNA-dependent ATPase, and ATP-dependent RNA helicase activities of eIF4A. The impact of other eIF4F components, eIF4E and eIF4G, on DMDAPatA action were investigated in vitro and in cultured mammalian cells. The perturbation of the eIF4A–eIF4G association was found to be eIF4E- and mRNA cap-dependent. An inhibitory effect on helicase activity of eIF4A was observed when it was part of a complex that mimicked the eIF4F complex. We propose a model of action for DMDAPatA (and by supposition PatA) where the cellular activity of the compound is dependent on an “active” eIF4F complex.

scholarly journals Genetic dissection of the signaling domain of a mammalian steroid receptor in yeast.

1992 ◽  
Vol 3 (11) ◽  
pp. 1245-1257 ◽  
Author(s):  
M J Garabedian ◽  
K R Yamamoto
Keyword(s):  
Signal Transduction ◽  
Glucocorticoid Receptor ◽  
Mammalian Cells ◽  
Rank Order ◽  
Point Mutations ◽  
Steroid Receptor ◽  
Genetic Dissection ◽  
Signaling Domain

The mechanism of signal transduction by steroid receptor proteins is complex and not yet understood. We describe here a facile genetic strategy for dissection of the rat glucocorticoid receptor "signaling domain," a region of the protein that binds and transduces the hormonal signal. We found that the characteristics of signal transduction by the receptor expressed in yeast were similar to those of endogenous receptors in mammalian cells. Interestingly, the rank order of particular ligands differed between species with respect to receptor binding and biological efficacy. This suggests that factors in addition to the receptor alone must determine or influence ligand efficacy in vivo. To obtain a collection of receptors with distinct defects in signal transduction, we screened in yeast an extensive series of random point mutations introduced in that region in vitro. Three phenotypic classes were obtained: one group failed to bind hormone, a second displayed altered ligand specificity, and a third bound hormone but lacked regulatory activity. Our results demonstrate that analysis of glucocorticoid receptor action in yeast provides a general approach for analyzing the mechanism of signaling by the nuclear receptor family and may facilitate identification of non-receptor factors that participate in this process.

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