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Mimetics of growth factors and cytokines are promising tools for culturing large numbers of cells and manufacturing regenerative medicine products. In this study, we report single-chain tandem macrocyclic peptides (STaMPtides) as mimetics in a new multivalent peptide format. STaMPtides, which contain two or more macrocyclic peptides with a disulfide-closed backbone and peptide linkers, are successfully secreted into the supernatant by Corynebacterium glutamicum-based secretion technology. Without post-secretion modification steps, such as macrocyclization or enzymatic treatment, bacterially secreted STaMPtides form disulfide bonds, as designed; are biologically active; and show agonistic activities against respective target receptors. We also demonstrate, by cell-based assays, the potential of STaMPtides, which mimic growth factors and cytokines, in cell culture. The STaMPtide technology can be applied to the design, screening, and production of growth factor and cytokine mimetics. Peptides composed of two or more tandemly linked macrocyclic peptides binding to growth factor/cytokine receptors (STaMPtides) are useful growth factor/cytokine mimetics. The peptides can be produced from bacterial cells and bioactivity is demonstrated (HGF, EPO, TPO). [ABSTRACT FROM AUTHOR]

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) 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 EC 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. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer containing 66 kDa p66 and 51 kDa p51 subunits. We previously showed that HIV-1 group M (HIV-1 M) RT and HIV-1 group O (HIV-1 O) RT have higher affinities for dTTP and template-primer (T/P) than Moloney murine leukemia virus RT, which is currently used for cDNA synthesis, suggesting that they might also be useful for cDNA synthesis (Konishi et al. Appl Biochem Biotechnol , 169:77-87). Here, we have increased the thermostability of both HIV-1 M RT and HIV-1 O RT by site-directed mutagenesis. The Asp443 → Ala mutation, which abolishes RNase H activity, was introduced into the p66 subunits of HIV-1 M RT and HIV-1 O RT. The temperatures that reduced the initial activity by 50 % of the resulting mutants, HIV-1 M p66/p51 and HIV-1 O p66/p51, were 44 and 52 °C, respectively, which were higher than those of wild-type HIV-1 M p66/p51 (42 °C) and HIV-1 O p66/p51 (48 °C). The highest temperature at which both HIV-1 M p66/p51 and HIV-1 O p66/p51 exhibited cDNA synthesis activity was 68 °C, which was higher than for the wild-type enzymes (62 and 66 °C, respectively). [ABSTRACT FROM AUTHOR]

Avian myeloblastosis virus reverse transcriptase (AMV RT) is a heterodimer consisting of a 63 kDa α-subunit and a 95 kDa β subunit. Moloney murine leukaemia virus reverse transcriptase (MMLV RT) is a 75 kDa monomer. These two RTs are the most extensively used for conversion of RNA to DNA. We previously developed several mutations that increase the thermostability of MMLV RT and generated a highly stable MMLV RT variant E286R/E302K/L435R/D524A by combining three of them (Glu286→Arg, Glu302→Lys, and Leu435→Arg) and the mutation to abolish RNase H activity (Asp524→Ala) [Yasukawa et al. (2010) J Biotechnol 150:299-306]. To generate a highly stable AMV RT variant, we have introduced the triple mutation of Val238→Arg, Leu388→Arg, and Asp450→Ala into AMV RT α-subunit and the resulted variant V238R/L388R/D450A, was expressed in insect cells and purified. The temperature decreasing the initial activity by 50 %, measured over 10 min, of the variant with or without template primer (T/P), poly(rA)-p(dT), was 50 °C; for the wild-type AMV RT α-subunit (WT) this was 44 °C. The highest temperature at which the variant exhibited cDNA synthesis activity was 64 °C; the WT was 60 °C. A highly stable AMV RT α-subunit is therefore generated by the same mutation strategy as applied to MMLV RT and that positive charges are introduced into RT at positions that have been implicated to interact with T/P by site-directed mutagenesis. [ABSTRACT FROM AUTHOR]