Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light

Green fluorescent protein (GFP) and GFP-like proteins represent invaluable genetically encoded fluorescent probes. In the last few years a new class of photoactivatable fluorescent proteins (PAFPs) capable of pronounced light-induced spectral changes have been developed. Except for tetrameric KFP1 (ref. 4), all known PAFPs, including PA-GFP, Kaede, EosFP, PS-CFP, Dronpa, PA-mRFP1 and KikGR require light in the UV-violet spectral region for activation through one-photon excitation--such light can be phototoxic to some biological systems. Here, we report a monomeric PAFP, Dendra, derived from octocoral Dendronephthya sp. and capable of 1,000- to 4,500-fold photoconversion from green to red fluorescent states in response to either visible blue or UV-violet light. Dendra represents the first PAFP, which is simultaneously monomeric, efficiently matures at 37 degrees C, demonstrates high photostability of the activated state, and can be photoactivated by a common, marginally phototoxic, 488-nm laser line. We demonstrate the suitability of Dendra for protein labeling and tracking to quantitatively study dynamics of fibrillarin and vimentin in mammalian cells.     

A new synthesis of 2'-C-methylnucleosides starting from D-ribose

A general method have been developed for the synthesis of 2'-C-methylnucleosides from 2,3-O-isopropylidene-2-hydroxymethyl-5-O-trityl-D-ribofuranose (2).     

Comparison of methods for clustering independent EEG components in healthy subjects and patients with postconcussion syndrome after traumatic brain injury

The efficiencies of three clustering methods for independent components of 19-channel baseline EEG in location of pathological cerebral activity sources were compared. The samples comprised 518 healthy subjects and 87 patients with postconcussion syndrome after traumatic brain injury (TBI). Clustering of independent component topographies, the spatial coordinates of equivalent dipole sources corresponding to independent component topographies, and locations of the maximums of the equivalent source current density calculated by standardized low resolution electromagnetic tomography (sLORETA) were compared. A comparison of the power spectra of independent components showed a significant increase in the EEG power in the Δ, θ, and α bands for sources located in the frontal and temporal lobes of TBI patients compared to healthy subjects. The method of clustering of independent component topographies proved to be the most sensitive of the methods compared.     

The coding region of far-red fluorescent protein Katushka contains a strong donor splice site

Computer analysis of the sequence encoding the red fluorescent protein Katushka revealed a strong donor splice site in the 3′-terminal region. A model vector encoding protein Katushka and green fluorescent protein TagGFP2 separated by a fragment of the gene tafazzin, has been constructed for experimental verification of the functional activity of this site. Splicing of this pre-mRNA should lead to the frameshift between the Katushka and TagGFP2 proteins in the case of normal splicing of the tafazzin sequence. In the case of the use of the donor splice site within a katushka sequence, it should lead to the appearance of the fusion protein Katushka-TagGFP2. Flow cytometry showed that the expression of this construct in mammalian cells led to bright red and green fluorescence. Therefore, the splice site within the gene katushka is really functional. Disruption of this splice site using site-directed mutagenesis, without changing the amino acid sequence of the Katushka protein, led to the disappearance of the green signal that corresponds to the normal splicing of tafazzin. Mutant variant of the coding sequence of the Katushka protein can be used for the analysis of pre-mRNA splicing in individual cells using fluorescence microscopy and flow cytofluorimetry.     

Temperature conditions of the formation of frost damages in conifer trees in the high latitudes of Western Siberia

Frost damage to the bottom of the stem at a height of 0.2 m and at the height of the position of the thermometer in the weather station (2 m) and higher in the Siberian spruce (Picea obovata Ledeb.) and Siberian larch (Larix sibirica Ledeb.) growing at the northern limits of their natural habitat were studied in order to reveal the upper threshold temperature conditions of their formation. Possible causes of differences in the distribution of frost damage in the stem of the spruce and larch are discussed.     

A Family of Genes of Multidomain Free Lectins from a Planarian: Structure,Expression, and the Use as Markers for Monitoring Regeneration

A family of genes of the asexual race of planarian Girardia tigrina were described that encode proteins that belong to the superfamily of C-type lectins and were demonstrated to have a unique domain organization. The genes are differentially expressed in the planarian body. The protein products of at least two genes (scarf2 and gtlec1) are expressed in specifically differentiated gland cells of the planarian and secreted into the environment through long cell necks. A comparison of the results obtained by electron microscopy and immunohistochemistry with literature data allows the assignment of these cells to the group of adhesion glands. The observation of the regeneration of the cell necks in normal and artificial two-headed planaria indicated that the dorsoventral contact at the edge of the head part of the planarian body directs and maintains the growth of the gtLec1-producing cell necks during regeneration.     

Impaired step-tracking control in patients with cerebellar and pyramidal lesions

Kinematic and electromyographic characteristics of step-tracking movements performed with and without visual guidance were investigated in patients with prevalently intermediary and lateral cerebellar and pyramidal tract lesions. Parameters of the phasic programmed component of motion were shown to be little affected by damage to the intermediary cerebellar system, whereas the maintenance phase was severely disrupted and performed as low-amplitude fluctuations around a target level. Lateral cerebellar lesions led to impaired control over the accuracy of the phasic programmed component of motion, a rapid shift into a new position, executed by patients belonging to this group with a constant hypermetric error. Their ability to sustain the position was maintained, however. The phasic component lost its programmatic pattern in patients with pyramidal lesions; the transition to a new position took the form of a slow, fragmented approximation of the limb towards its goal (approximating tracking) in this group, and the maintenance phase was unsteady. Findings are discussed in the light of the theory that the lateral and intermediary systems of the cerebellum are connected with the processes of programming and sustained control of motion, respectively.Institute for Research into Information Transmission, Academy of Sciences of the USSR, Moscow; Institute of Neurology, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 291–299, May–June, 1987.