scholarly journals Reverse Transcriptase Activity in Mature Spermatozoa of Mouse

2000 ◽  
Vol 148 (6) ◽  
pp. 1107-1114 ◽  
Author(s):  
Roberto Giordano ◽  
Anna Rosa Magnano ◽  
Germana Zaccagnini ◽  
Carmine Pittoggi ◽  
Nicola Moscufo ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Pcr Amplification ◽  
Reverse Transcriptase Activity ◽  
Pcr Analysis ◽  
Immunogold Electron Microscopy ◽  
Direct Pcr ◽  
Vitro Fertilization ◽  
Rna Molecules ◽  
Exogenous Rna

We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development.

scholarly journals Reverse Transcriptase-PCR Analysis of Bacterial rRNA for Detection and Characterization of Bacterial Species in Arthritis Synovial Tissue

2000 ◽  
Vol 68 (10) ◽  
pp. 6012-6026 ◽  
Author(s):  
Karen E. Kempsell ◽  
Charles J. Cox ◽  
Michael Hurle ◽  
Anthony Wong ◽  
Scott Wilkie ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Synovial Tissue ◽  
Bacterial Species ◽  
Disease Onset ◽  
Pcr Amplification ◽  
Pcr Analysis ◽  
Modified Technique ◽  
Culture Methods ◽  
Reverse Transcriptase Pcr

ABSTRACT Onset of rheumatoid arthritis (RA) is widely believed to be preceded by exposure to some environmental trigger such as bacterial infectious agents. The influence of bacteria on RA disease onset or pathology has to date been controversial, due to inconsistencies between groups in the report of bacterial species isolated from RA disease tissue. Using a modified technique of reverse transcriptase-PCR amplification, we have detected bacterial rRNA in the synovial tissue of late-stage RA and non-RA arthritis controls. This may be suggestive of the presence of live bacteria. Sequencing of cloned complementary rDNA (crDNA) products revealed a number of bacterial sequences in joint tissue from each patient, and from these analyses a comprehensive profile of the organisms present was compiled. This revealed a number of different organisms in each patient, some of which are common to both RA and non-RA controls and are probably opportunistic colonizers of previously diseased tissue and others which are unique species. These latter organisms may be candidates for a specific role in disease pathology and require further investigation to exclude them as causative agents in the complex bacterial millieu. In addition, many of the detected bacterial species have not been identified previously from synovial tissue or fluid from arthritis patients. These may not be easily cultivable, since they were not revealed in previous studies using conventional in vitro bacterial culture methods. In situ hybridization analyses have revealed the joint-associated bacterial rRNA to be both intra- and extracellular. The role of viable bacteria or their nucleic acids as triggers in disease onset or pathology in either RA or non-RA arthritis controls is unclear and requires further investigation.

Functional organization of the retrotransposon Ty from Saccharomyces cerevisiae: Ty protease is required for transposition

1988 ◽  
Vol 8 (4) ◽  
pp. 1421-1431 ◽  
Author(s):  
S D Youngren ◽  
J D Boeke ◽  
N J Sanders ◽  
D J Garfinkel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Reverse Transcriptase ◽  
Functional Organization ◽  
Core Protein ◽  
Reverse Transcriptase Activity ◽  
Protein Domain ◽  
Particle Assembly ◽  
Low Levels ◽  
Viruslike Particles

We used several mutations generated in vitro to further characterize the functions of the products encoded by the TyB gene of the transpositionally active retrotransposon TyH3 from Saccharomyces cerevisiae. Mutations close to a core protein domain of TyB, which is homologous to retroviral proteases, have striking effects on Ty protein processing, the physiology of Ty viruslike particles, and transposition. The Ty protease is required for processing of both TyA and TyB proteins. Mutations in the protease resulted in the synthesis of morphologically and functionally aberrant Ty viruslike particles. The mutant particles displayed reverse transcriptase activity, but did not synthesize Ty DNA in vitro. Ty RNA was present in the mutant particles, but at very low levels. Transposition of a genetically tagged element ceased when the protease domain was mutated, demonstrating that Ty protease is essential for transposition. One of these mutations also defined a segment of TyB encoding an active reverse transcriptase. These results indicate that the Ty protease, like its retroviral counterpart, plays an important role in particle assembly, replication, and transposition of these elements.

scholarly journals In Vitro Interactions between Apricitabine and Other Deoxycytidine Analogues

2007 ◽  
Vol 51 (8) ◽  
pp. 2948-2953 ◽  
Author(s):  
R. Bethell ◽  
J. De Muys ◽  
J. Lippens ◽  
A. Richard ◽  
B. Hamelin ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
High Performance ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Transcriptase Inhibitor ◽  
Reverse Transcriptase Activity ◽  
Peripheral Blood Mononuclear ◽  
Reverse Transcriptase Inhibitor ◽  
Hiv 1

ABSTRACT Apricitabine is a novel deoxycytidine analogue reverse transcriptase inhibitor that is under development for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. Apricitabine is phosphorylated to its active triphosphate by deoxycytidine kinase, which is also responsible for the intracellular phosphorylation of lamivudine (3TC) and emtricitabine (FTC); hence, in vitro studies were performed to investigate possible interactions between apricitabine and these agents. Human peripheral blood mononuclear cells (PBMC) were incubated for 24 h with various concentrations of 3H-labeled or unlabeled apricitabine, 3TC, or FTC. Intracellular concentrations of parent compounds and their phosphorylated derivatives were measured by high-performance liquid chromatography. In other experiments, viral reverse transcriptase activity was measured in PBMC infected with HIV-1 bearing M184V in the presence of various concentrations of apricitabine and 3TC. [3H]apricitabine and [3H]3TC were metabolized intracellularly to form mono-, di-, and triphosphates. 3TC and FTC (1 to 10 μM) produced concentration-dependent decreases in apricitabine phosphorylation; in contrast, apricitabine at concentrations of up to 30 μM had no effect on the phosphorylation of 3TC or FTC. The combination of apricitabine and 3TC reduced the antiviral activity of apricitabine against HIV-1: apricitabine concentrations producing 50% inhibition of viral reverse transcriptase were increased two- to fivefold in the presence of 3TC. These findings suggest that nucleoside reverse transcriptase inhibitors with similar modes of action may show biochemical interactions that affect their antiviral efficacy. It is therefore essential that potential interactions between combinations of new and existing agents be thoroughly investigated before such combinations are introduced into clinical practice.

scholarly journals Functional organization of the retrotransposon Ty from Saccharomyces cerevisiae: Ty protease is required for transposition.

1988 ◽  
Vol 8 (4) ◽  
pp. 1421-1431 ◽  
Author(s):  
S D Youngren ◽  
J D Boeke ◽  
N J Sanders ◽  
D J Garfinkel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Reverse Transcriptase ◽  
Functional Organization ◽  
Core Protein ◽  
Reverse Transcriptase Activity ◽  
Protein Domain ◽  
Particle Assembly ◽  
Low Levels ◽  
Viruslike Particles

We used several mutations generated in vitro to further characterize the functions of the products encoded by the TyB gene of the transpositionally active retrotransposon TyH3 from Saccharomyces cerevisiae. Mutations close to a core protein domain of TyB, which is homologous to retroviral proteases, have striking effects on Ty protein processing, the physiology of Ty viruslike particles, and transposition. The Ty protease is required for processing of both TyA and TyB proteins. Mutations in the protease resulted in the synthesis of morphologically and functionally aberrant Ty viruslike particles. The mutant particles displayed reverse transcriptase activity, but did not synthesize Ty DNA in vitro. Ty RNA was present in the mutant particles, but at very low levels. Transposition of a genetically tagged element ceased when the protease domain was mutated, demonstrating that Ty protease is essential for transposition. One of these mutations also defined a segment of TyB encoding an active reverse transcriptase. These results indicate that the Ty protease, like its retroviral counterpart, plays an important role in particle assembly, replication, and transposition of these elements.

scholarly journals Antimycobacterial and HIV-1 Reverse Transcriptase Activity of Julianaceae and Clusiaceae Plant Species from Mexico

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Rocio Gómez-Cansino ◽  
Clara Inés Espitia-Pinzón ◽  
María Guadalupe Campos-Lara ◽  
Silvia Laura Guzmán-Gutiérrez ◽  
Erika Segura-Salinas ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Antimycobacterial Activity ◽  
Reverse Transcriptase Activity ◽  
Hiv Reverse Transcriptase ◽  
Leaf Extracts ◽  
Calophyllum Brasiliense ◽  
Hplc Profiles ◽  
Low Toxicity ◽  
Hiv Rt

The extracts of 14 Julianaceae and 5 Clusiaceae species growing in Mexico were testedin vitro(50 µg/mL) againstMycobacterium tuberculosisH37Rv and HIV reverse transcriptase (HIV-RT). The Julianaceae bark and leaf extracts inhibitedM. tuberculosis(>84.67%) and HIV-RT (<49.89%). The Clusiaceae leaves extracts also inhibited both targets (>58.3% and >67.6%), respectively. The IC50values for six selected extracts and their cytotoxicity (50 µg/mL) to human macrophages were then determined.Amphipterygium glaucum,A. molle, andA. simplicifoliumfairly inhibitedM. tuberculosiswith IC50of 1.87–2.35 µg/mL; but their IC50against HIV-RT was 59.25–97.83 µg/mL.Calophyllum brasiliense,Vismia baccifera, andVismia mexicanaeffect onM. tuberculosiswas noteworthy (IC503.02–3.64 µg/mL) and also inhibited RT-HIV (IC5026.24–35.17 µg/mL). These 6 extracts (50 µg/mL) presented low toxicity to macrophages (<23.8%). The HPLC profiles ofA. glaucum,A. molle, andA. simplicifoliumindicated that their antimycobacterial activity cannot be related to masticadienonic, 3α, or 3β-hydromasticadienonic acids, suggesting that other compounds may be responsible for the observed activity or this might be a synergy result. The anti-HIV-RT and antimycobacterial activities induced byC. brasiliensecan be attributed to the content of calanolides A, B, as well as soulatrolide.

scholarly journals Genome editing of CCR5 by CRISPR-Cas9 in Mauritian cynomolgus macaque embryos

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jenna Kropp Schmidt ◽  
Nick Strelchenko ◽  
Mi Ae Park ◽  
Yun Hee Kim ◽  
Katherine D. Mean ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hiv Infection ◽  
Genome Editing ◽  
Cynomolgus Macaque ◽  
Pcr Analysis ◽  
Vitro Fertilization ◽  
Cell Therapies ◽  
Cynomolgus Macaques ◽  
Hiv 1

Abstract The discovery that CCR5 serves as an R5-HIV-1 co-receptor, coupled with findings of protection from HIV infection in individuals lacking CCR5, led to the exploration of novel therapeutic strategies for HIV infection based on genome editing of CCR5. Advancing translation of CCR5-mutant-based cellular therapies for HIV requires development of novel physiologically relevant animal models. Mauritian cynomolgus macaques (MCMs), with high degree of MHC allele sharing, are valuable models for HIV-1 research and stem cell therapies. To facilitate the generation of a CCR5-mutant MHC-defined MCM model, we explored editing the CCR5 gene in MCM embryos via CRISPR-Cas9. We refined ovarian stimulation and in vitro fertilization (IVF) methods established for Chinese cynomolgus macaques to generate in vitro MCM embryos. Time-lapse embryo imaging was performed to assess the timing of MCM embryonic developmental events in control and CRISPR-Cas9 microinjected embryos. Using a dual-guide gene targeting approach, biallelic deletions in the CCR5 gene were introduced into ~ 23–37% of MCM embryos. In addition, single blastomere PCR analysis revealed mosaicism in CCR5 editing within the same embryo. Successful development of IVF and CCR5 editing protocols in MCM embryos lays a foundation for the creation of CCR5-mutant MCMs to assess novel stem cell-based HIV therapeutics.

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