Genetically encoded calcium indicator with NTnC-like design and enhanced fluorescence contrast and kinetics.

BACKGROUND: The recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca2+-binding moiety inserted into the mNeonGreen fluorescent protein. NTnC binds two times less Ca2+ ions while maintaining a higher fluorescence brightness at the basal level of Ca2+ in neurons as compared with the calmodulin-based GECIs, such as GCaMPs. In spite of NTnC's high brightness, pH-stability, and high sensitivity to single action potentials, it has a limited fluorescence contrast (F-Ca2+/F+Ca2+) and slow Ca2+ dissociation kinetics. RESULTS: Herein, we developed a new NTnC-like GECI with enhanced fluorescence contrast and kinetics by replacing the mNeonGreen fluorescent subunit of the NTnC indicator with EYFP. Similar to NTnC, the developed indicator, named iYTnC2, has an inverted fluorescence response to Ca2+ (i.e. becoming dimmer with an increase of Ca2+ concentration). In the presence of Mg2+ ions, iYTnC2 demonstrated a 2.8-fold improved fluorescence contrast in vitro as compared with NTnC. The iYTnC2 indicator has lower brightness and pH-stability, but similar photostability as compared with NTnC in vitro. Stopped-flow fluorimetry studies revealed that iYTnC2 has 5-fold faster Ca2+ dissociation kinetics than NTnC. When compared with GCaMP6f GECI, iYTnC2 has up to 5.6-fold faster Ca2+ association kinetics and 1.7-fold slower dissociation kinetics. During calcium transients in cultured mammalian cells, iYTnC2 demonstrated a 2.7-fold higher fluorescence contrast as compared with that for the NTnC. iYTnC2 demonstrated a 4-fold larger response to Ca2+ transients in neuronal cultures than responses of NTnC. iYTnC2 response in neurons was additionally characterized using whole-cell patch clamp. Finally, we demonstrated that iYTnC2 can visualize neuronal activity in vivo in the hippocampus of freely moving mice using a nVista miniscope. CONCLUSIONS: We demonstrate that expanding the family of NTnC-like calcium indicators is a promising strategy for the development of the next generation of GECIs with smaller molecule size and lower Ca2+ ions buffering capacity as compared with commonly used GECIs.

[Compartmentalization of ROS-mediated signal transduction].

The localization of signaling molecules close to their targets is the central principle of cell signaling. The colocalization of multicomponent signaling complexes is realized through protein scaffolds that provide better specificity than undirected diffusion ofthe same components. ROS-generating complexes have been suggested to follow this principle by specific intracellular localization of ROS production and the limitation of ROS diffusion distances. However, the lack of adequate methods did not allow direct detection of local ROS production to confirm the model ofredox signaling compartmentalization. Nevertheless, evidences of local ROS production and restriction of diffusion were provided by kinetic modeling and data on the subcellular localization of NADPH-oxidase isoforms, their adapter proteins and local restriction of ROS diffusion. Here we shall discuss the properties of antioxidant system which prevents uncontrolled ROS diffusion from the sites of generation to the adjacent subcellular compartments; the current data of the specific localization NADPH-oxidases activity and its influence on intracellular processes; the recent evidences of the ROS diffusion restriction.

Regulation of organelle movement in melanophores by protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2A (PP2A).

We used melanophores, cells specialized for regulated organelle transport, to study signaling pathways involved in the regulation of transport. We transfected immortalized Xenopus melanophores with plasmids encoding epitope-tagged inhibitors of protein phosphatases and protein kinases or control plasmids encoding inactive analogues of these inhibitors. Expression of a recombinant inhibitor of protein kinase A (PKA) results in spontaneous pigment aggregation. alpha-Melanocyte-stimulating hormone (MSH), a stimulus which increases intracellular cAMP, cannot disperse pigment in these cells. However, melanosomes in these cells can be partially dispersed by PMA, an activator of protein kinase C (PKC). When a recombinant inhibitor of PKC is expressed in melanophores, PMA-induced pigment dispersion is inhibited, but not dispersion induced by MSH. We conclude that PKA and PKC activate two different pathways for melanosome dispersion. When melanophores express the small t antigen of SV-40 virus, a specific inhibitor of protein phosphatase 2A (PP2A), aggregation is completely prevented. Conversely, overexpression of PP2A inhibits pigment dispersion by MSH. Inhibitors of protein phosphatase 1 and protein phosphatase 2B (PP2B) do not affect pigment movement. Therefore, melanosome aggregation is mediated by PP2A.

[NO synthetase in the cells of different tissues and organs of the mouse]. / Issledovanie NO-sintetazy v kletkakh razlichnykh tkanei i organov myshi.

Cellular NO synthase was assayed in bone marrow, spleen, thymus, lymph nodes, epidermis, hypodermic connective tissue, small gut, peritoneal exudate, liver, kidney, and heart by cytochemical and immunofluorescent methods. As a rule the enzymatic activity was found in differentiated cells that finished proliferation (mature hemopoietic cells, epithelium of gut villi, etc.). The enzyme activity was undetectable in actively reproducing low-differentiated cells (basal layer of the epidermis, crypt cells of the small gut, or blast hemopoietic forms). NO is proposed to participate in regulation of tissue-specific terminal differentiation (maturation) of the cells.

[In vitro transcription of esterase S gene in Drosophila virilis]. / Transkriptsiia in vitro gena ésterazy S Drosophila virilis.

In vitro transcription of D. virilis esterase S gene and it's dependence on reaction conditions and template configuration was studied. Localization of major in vivo transcription initiation points were proved. In vitro transcription can start from alternative points, localised at positions -125 and -342. Dependence of the transcription efficiency of the presence of the 5'-flanking region is shown.

[Primary structure of the esterase s gene from Drosophila virilis]. / Pervichnaia struktura gena ésterazy S Drosophila virilis.

Complete nucleotide sequence of the esterase S gene of Drosophila virilis has been determined. The length of the gene from the transcription initiation site down to the polyadenylation signal is about 1850 bp. Conceptual translation of the DNA sequence reveals a protein 549 amino acid residues long, its presumptive active site being highly similar to active sites of the known esterases of insects and mammals.

[Integration and expression of the human somatotropic hormone gene in Teleostei]. / Integratsiia i ékspressiia gena somatotropnogo gormona cheloveka u kostistykh ryb.

Plasmid DNA containing human growth hormone gene was microinjected into cytoplasm of loach (Misgurnus fossilis L.) fertilized eggs. After plasmid injection, more than 50% of embryos reached the hatching stage. In control experiments embryogenesis was completed giving 72.5% of injected and 90% of intact larvae. Southern blot hybridization analysis revealed integration of injected recombinant DNA constructions into fish chromosome's DNA (2-30 copies per genome), without any significant rearrangements. Significant increase in length and weight of transgenic fish was observed in experiments (P less than 0.01). S1-analysis of RNA demonstrated correlation of the amount of specific RNA molecules and the accelerated growth of the individual specimens and proper utilization of the transcriptional start points in transgenic fish.

[Primary structure of the transcription initiation region of the esterase S gene of Drosophila virilis]. / Pervichnaia struktura oblasti initsiatsii transkriptsii gena ésterazy S Drosophila virilis.

The transcription initiation region of the gene for tissue-specific esterase S of D. virilis has been analysed. By means of high-resolution S1-mapping we located a set of transcription initiation points, two of them being major ones. The position of the major start points, but not the efficiency of their usage, does not depend on age, sex or strain of the flies. The transcription initiation region is sequenced. The region contains various motifs characteristic for eukaryotic promoters.

[Transfer of the gene of the BTSh 70 heat-shock protein from Drosophila into the mouse genome]. / Perenos gena belka teplovogo shoka drozofily BTSh 70 v genom myshei.

The integration of the gene of the Drosophila heat shock protein BTSh 70 into the mouse genome has been described. Approximately 20 copies of the gene were integrated.

Structure of nuclear pre-mRNA. VIII. Isolation and characterization of complementary sequences.

After the annealing of pre-mRNA at high Cot values, up to 20% of the material becomes resistant to the action of ribonuclease. This has been attributed to the presence of self-complementary sequences (scRNA) in the pre-mRNA. The most important properties of scRNA, which was isolated in preparative amounts, have been studied. The approximate dimensions of the complementary segments are 45-50 nucleotides. Hybridization experiments have shown that scRNA is transcribed from repeated segments of the genome. It may be postulated that the rapidly hybridizing pre-mRNA fraction consists mainly of self-complementary sequences.