Specific modification at the C-terminal lysine residue of the green fluorescent protein variant, GFPuv, expressed in Escherichia coli

Green fluorescent protein (GFP) is amenable to recombinant expression in various kinds of cells and is widely used in life science research. We found that the recombinant expression of GFPuv, a commonly-used mutant of GFP, in E. coli produced two distinct molecular species as judged by in-gel fluorescence SDS-PAGE. These molecular species, namely form I and II, could be separately purified by anion-exchange chromatography without any remarkable differences in the fluorescence spectra. Mass spectrometric analyses revealed that the molecular mass of form I is almost the same as the calculated value, while that of form II is approximately 1 Da larger than that of form I. Further mass spectrometric top-down sequencing pinpointed the modification in GFPuv form II, where the epsilon-amino group of the C-terminal Lys238 residue is converted into the hydroxyl group. No equivalent modification was observed in the native GFP in jellyfish Aequorea victoria, suggesting that this modification is not physiologically relevant. Crystal structure analysis of the two species verified the structural identity of the backbone and the vicinity of the chromophore. The modification found in this study may also be generated in other GFP variants as well as in other recombinant expression systems

Abnormal spermatogenesis and male infertility in testicular zinc finger protein Zfp318-knockout mice

Zfp318, a mouse gene with a Cys2/His2 zinc finger motif, is mainly expressed in germ cells in the testis. It encodes two alternative transcripts, which regulate androgen receptor-mediated transcriptional activation or repression by overexpression of them. However, the role of Zfp318 is still obscure in vivo, especially in spermatogenesis. To elucidate the role of Zfp318 during gamete production, we established a knockout mouse line. Zfp318-null male mice exhibited infertility, whereas Zfp318-null female mice displayed normal fertility. ZFP318 was expressed during multiple stages of spermatogenesis, from spermatocytes to round spermatids. The nuclei of secondary spermatocytes showed high levels of expression. Histological analysis and quantitative analysis of DNA content showed decreased numbers of both spermatids in the seminiferous tubules and mature spermatozoa in the epididymides of Zfp318-null mice. These results suggest that Zfp318 is expressed as a functional protein in testicular germ cells and plays an important role in meiosis during spermatogenesis

ERRγ enhances cardiac maturation with T-tubule formation in human iPSC-derived cardiomyocytes

ヒトのiPS細胞から新生児レベルまで成熟した心筋細胞を作製する. 京都大学プレスリリース. 2021-06-21.Lowering the cost of heart cell therapies. 京都大学プレスリリース. 2021-06-21.One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human induced pluripotent stem cells (hiPSCs) carrying a TNNI1[EmGFP] and TNNI3[mCherry] double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine

Synthesis of highly conjugated poly(3,9-carbazolyleneethynylenearylene)s emitting variously colored fluorescence

Novel conjugated polymers P1–P7 containing 3, 9-linked carbazole units in the main chain were synthesized by the polycondensation of 3-ethynyl-9-(4-ethynylphenyl)carbazole (EEPCz) and dihaloarenes, and their optical and electrical properties were studied. Polymers with weight-average molecular weights of 4100–48, 000 were obtained in 24–92% yields by the Sonogashira coupling polycondensation in tetrahydrofuran (THF)/Et3N at 30 or 50 °C for 48 h. All the polymers absorbed light around 350 nm. The polymers with electron-accepter units exhibited absorption bands originating from charge transfer. The polymers except the one containing azobenzenes emitted variously colored fluorescence with moderate quantum yields upon excitation at the absorption maxima. P1–P3 were oxidized around 0.6 V, and then reduced around 0.5 V. The conductivity of P3 was 1.1 × 10−14 S/cm at 103 V/cm

Effects of Efflux Transporter Genes on Susceptibility of Escherichia coli to Tigecycline (GAR-936)

The activity of tigecycline, 9-(t-butylglycylamido)-minocycline, against Escherichia coli KAM3 (acrB) strains harboring plasmids encoding various tetracycline-specific efflux transporter genes, tet(B), tet(C), and tet(K), and multidrug transporter genes, acrAB, acrEF, and bcr, was examined. Tigecycline showed potent activity against all three Tet-expressing, tetracycline-resistant strains, with the MICs for the strains being equal to that for the host strain. In the Tet(B)-containing vesicle study, tigecycline did not significantly inhibit tetracycline efflux-coupled proton translocation and at 10 μM did not cause proton translocation. This suggests that tigecycline is not recognized by the Tet efflux transporter at a low concentration; therefore, it exhibits significant antibacterial activity. These properties can explain its potent activity against bacteria with a Tet efflux resistance determinant. Tigecycline induced the Tet(B) protein approximately four times more efficiently than tetracycline, as determined by Western blotting, indicating that it is at least recognized by a TetR repressor. The MICs for multidrug efflux proteins AcrAB and AcrEF were increased fourfold. Tigecycline inhibited active ethidium bromide efflux from intact E. coli cells overproducing AcrAB. Therefore, tigecycline is a possible substrate of AcrAB and its close homolog, AcrEF, which are resistance-modulation-division-type multicomponent efflux transporters

Synthesis of highly conjugated poly(3,9-carbazolyleneethynylenearylene)s emitting variously colored fluorescence

Novel conjugated polymers P1–P7 containing 3, 9-linked carbazole units in the main chain were synthesized by the polycondensation of 3-ethynyl-9-(4-ethynylphenyl)carbazole (EEPCz) and dihaloarenes, and their optical and electrical properties were studied. Polymers with weight-average molecular weights of 4100–48, 000 were obtained in 24–92% yields by the Sonogashira coupling polycondensation in tetrahydrofuran (THF)/Et3N at 30 or 50 °C for 48 h. All the polymers absorbed light around 350 nm. The polymers with electron-accepter units exhibited absorption bands originating from charge transfer. The polymers except the one containing azobenzenes emitted variously colored fluorescence with moderate quantum yields upon excitation at the absorption maxima. P1–P3 were oxidized around 0.6 V, and then reduced around 0.5 V. The conductivity of P3 was 1.1 × 10−14 S/cm at 103 V/cm

Construction of a novel expression vector in Pseudonocardia autotrophica and its application to efficient biotransformation of compactin to pravastatin, a specific HMG-CoA reductase inhibitor

The novel plasmid vector (pTAOR4-Rev) suitable for gene expression in actinomycete strains of Pseudonocardia autotrophica was constructed from 2 P. autotrophica genetic elements, the novel replication origin and the acetone-inducible promoter. The replication origin was isolated from the endogenous plasmid of strain DSM 43082 and the acetone-inducible promoter was determined by analysis of the upstream region of an acetaldehyde dehydrogenase gene homologue in strain NBRC 12743. P. autotrophica strains transformed with pTAOR4-P450, carrying a gene for cytochrome P450 monooxygenase, expressed P450 from the acetone-inducible promoter, as verified by SDS-PAGE and spectral analysis. The biotransformation test of acetone-induced resting cells prepared from a strain of P. autotrophica carrying pTAOR4 that harbors a compactin (CP)-hydroxylating P450 gene revealed 3.3-fold increased production of pravastatin (PV), a drug for hypercholesterolemia. Biotransformation of CP by the same strain in batch culture yielded PV accumulation of 14.3 g/l after 100 hr. The expression vector pTAOR4-Rev and its function-enhancing derivatives provide a versatile approach to industrial biotransformation by Pseudonocardia strains, which can be good hosts for P450 monooxygenase expression