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1.
Int J Mol Sci ; 24(23)2023 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-38068925

RESUMO

We investigated the tumor immune response in gastric cancer patients receiving third-line nivolumab monotherapy to identify immune-related biomarkers for better patient selection. Nineteen patients (10 males, median age 67 years) who received nivolumab as a third- or later-line therapy were enrolled. We analyzed the tumor immune response in durable clinical benefit (DCB) and non-DCB patients. Pre-treatment and early-on-treatment tumor transcriptomes were examined, and gene expression profiles, immunograms, and T cell receptor (TCR) repertoire were analyzed. DCB was observed in 15.8% of patients, with comparable secondary endpoints (ORR; objective response rate, OS; overall survival, PFS; progression-free survival) to previous trials. The immunograms of individual subjects displayed no significant changes before or early in the treatment, except for the regulatory T cell (Treg) score. Moreover, there were no consistent alterations observed among cases experiencing DCB. The intratumoral immune response was suppressed by previous treatments in most third- or later-line nivolumab recipients. TCR repertoire analysis revealed newly emerged clonotypes in early-on-treatment tumors, but clonal replacement did not impact efficacy. High T cell/Treg ratios and a low UV-radiation-response gene signature were linked to DCB and treatment response. This study emphasizes the tumor immune response's importance in nivolumab efficacy for gastric cancer. High T cell/Treg ratios and specific gene expression signatures show promise as potential biomarkers for treatment response. The tumor-infiltrating immune response was compromised by prior treatments in third-line therapy, implying that, to enhance immunotherapeutic outcomes, commencing treatment at an earlier stage might be preferable. Larger cohort validation is crucial to optimize immune-checkpoint inhibitors in gastric cancer treatment.


Assuntos
Antineoplásicos Imunológicos , Neoplasias Gástricas , Masculino , Humanos , Idoso , Nivolumabe , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/induzido quimicamente , Antineoplásicos Imunológicos/farmacologia , Recidiva Local de Neoplasia/tratamento farmacológico , Receptores de Antígenos de Linfócitos T/genética , Biomarcadores
2.
Biotechnol Lett ; 38(7): 1203-11, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27053084

RESUMO

OBJECTIVE: To characterize Moloney murine leukemia virus (MMLV) reverse transcriptases (RTs) expressed in a cell-free system and in Escherichia coli. RESULTS: We previously expressed MMLV RT using an E. coli expression system and generated a highly thermostable quadruple variant MM4 (E286R/E302K/L435R/D524A) by site-directed mutagenesis. In this study, we expressed the wild-type MMLV RT (WT) and MM4 using a cell-free protein expression system from insect cells. WT exhibited DNA polymerase and RNase H activities, while MM4, in which the catalytic residue for RNase H activity, Asp524 is changed into Ala, exhibited only DNA polymerase activity. MM4, when held at 60 °C for 10 min, retained DNA polymerase activity, while WT, held at 54 °C for 10 min, lost this activity. In the cDNA synthesis reaction (0.5 µl) in which WT or MM4 were exposed to various temperatures and amounts of target RNA in a microarray chip, MM4 exhibited higher thermostability than WT. CONCLUSION: MMLV RT expressed in the cell-free system is indistinguishable from that expressed in E. coli.


Assuntos
Escherichia coli/enzimologia , Escherichia coli/metabolismo , Vírus da Leucemia Murina de Moloney/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Animais , Sistema Livre de Células , Escherichia coli/genética , DNA Polimerase Dirigida por RNA/genética , Temperatura
3.
Protein Expr Purif ; 113: 44-50, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25959458

RESUMO

Moloney murine leukemia virus reverse transcriptase (MMLV RT) contains fingers, palm, thumb, and connection subdomains as well as an RNase H domain. The DNA polymerase active site resides in the palm subdomain, and the RNase H active site is located in the RNase H domain. The RNase H domain contains a positively charged α-helix called the C helix (H(594)GEIYRRR(601)), that is thought to be involved in substrate recognition. In this study, we expressed three versions of the RNase H domain in Escherichia coli, the wild-type domain (WT) (residues Ile498-Leu671) and two variants that lack the regions containing the C helix (Ile593-Leu603 and Gly595-Thr605, which we called ΔC1 and ΔC2, respectively) with a strep-tag at the N-terminus and a deca-histidine tag at the C-terminus. These peptides were purified from the cells by anion-exchange, Ni(2+) affinity, and Strep-Tactin affinity column chromatography, and then the tags were removed by proteolysis. In an RNase H assay using a 25-bp RNA-DNA heteroduplex, WT, ΔC1, and ΔC2 produced RNA fragments ranging from 7 to 16 nucleotides (nt) whereas the full-length MMLV RT (Thr24-Leu671) produced 14-20-nt RNA fragments, suggesting that elimination of the fingers, palm, thumb, and connection subdomains affects the binding of the RNase H domain to the RNA-DNA heteroduplex. The activity levels of WT, ΔC1, and ΔC2 were estimated to be 1%, 0.01%, and 0.01% of full-length MMLV RT activity, indicating that the C helix is important, but not critical, for the activity of the isolated RNase H domain.


Assuntos
Vírus da Leucemia Murina de Moloney/genética , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes/metabolismo , Ribonuclease H/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Escherichia coli/genética , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/enzimologia , RNA/metabolismo , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Ribonuclease H/química , Ribonuclease H/genética , Ribonuclease H/isolamento & purificação , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação
4.
J Nat Med ; 69(3): 432-40, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25663480

RESUMO

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) possesses two distinct enzymatic activities: those of RNA- and DNA-dependent DNA polymerases and RNase H. In the current HIV-1 therapy, all HIV-1 RT inhibitors inhibit the activity of DNA polymerase, but not that of RNase H. We previously reported that ethanol and water extracts of Brasenia schreberi (Junsai) inhibited the DNA polymerase activity of HIV-1 RT [Hisayoshi et al. (2014) J Biol Macromol 14:59-65]. In this study, we screened 43 edible plants and found that ethanol and water extracts of Brasenia schreberi and water extract of Petasites japonicus strongly inhibit not only the activity of DNA polymerase to incorporate dTTP into poly(rA)-p(dT)15 but also the activity of RNase H to hydrolyze the RNA strand of an RNA/DNA hybrid. In addition, these three extracts inhibit HIV-1 replication in human cells, with EC50 values of 1-2 µg/ml. These results suggest that Brasenia schreberi and Petasites japonicus contain substances that block HIV-1 replication by inhibiting the DNA polymerase activity and/or RNase H activity of HIV-1 RT.


Assuntos
Fármacos Anti-HIV/química , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/fisiologia , Petasites/química , Extratos Vegetais/química , Inibidores da Transcriptase Reversa/química , Ribonuclease H/antagonistas & inibidores , Fármacos Anti-HIV/farmacologia , DNA Polimerase Dirigida por DNA/química , Avaliação Pré-Clínica de Medicamentos , Transcriptase Reversa do HIV/química , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Células HeLa , Humanos , Extratos Vegetais/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Replicação Viral/efeitos dos fármacos
5.
Biochem Biophys Res Commun ; 454(2): 269-74, 2014 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-25450388

RESUMO

We have previously used site-directed mutagenesis to introduce basic residues (i.e., Arg; Lys) in the nucleic acid binding cleft of the Moloney murine leukemia virus reverse transcriptase (MMLV RT) in order to increase its template-primer (T/P) binding affinity. Three stabilizing mutations (i.e., E286R, E302K, and L435R) were identified (Yasukawa et al., 2010). Now, we studied the mechanism by which those mutations increase the thermal stability of the RT. The three single-mutants (E286R, E302K, and L435R), an RNase H-deficient MMLV RT (carrying the RNase H-inactivating mutation D524A), a quadruple mutant (E286R/E302K/L435R/D524A, designated as MM4) and the wild-type enzyme (WT) were produced in Escherichia coli. All RTs exhibited similar dissociation constants (Kd) for heteropolymeric DNA/DNA (2.9-6.5 nM) and RNA/DNA complexes (1.2-2.9 nM). Unlike the WT, mutant enzymes (E286R, E302K, L435R, D524A, and MM4) were devoid of RNase H activity, and were not able to degrade RNA in RNA/DNA complexes. These results suggest that the mutations, E286R, E302K, and L435R increase the thermostability of MMLV RT not by increasing its affinity for T/P but by abolishing its RNase H activity.


Assuntos
Substituição de Aminoácidos , Vírus da Leucemia Murina de Moloney/enzimologia , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Ribonuclease H/metabolismo , Sequência de Bases , Domínio Catalítico , DNA/análise , DNA/metabolismo , Escherichia coli/genética , Vírus da Leucemia Murina de Moloney/química , Vírus da Leucemia Murina de Moloney/genética , Mutagênese Sítio-Dirigida , Estabilidade Proteica , RNA/análise , RNA/metabolismo , DNA Polimerase Dirigida por RNA/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura
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