Evenly tritium labeled peptides in study of peptide in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50–150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a μg scale of biological samples both in vitro and in vivo.     

The Electromotive Protein Prestin as a Sensitive Core of the Fluorescent Voltage Indicator

Russian Journal of Bioorganic Chemistry - In this study, we first used prestin, an electromotive protein of the mammalian auditory analyzer, as a voltage-sensitive core of a genetically encoded...     

Multimerization of Drosophila sperm protein Mst77F causes a unique condensed chromatin structure

Despite insights on the cellular level, the molecular details of chromatin reorganization in sperm development, which involves replacement of histone proteins by specialized factors to allow ultra most condensation of the genome, are not well understood. Protamines are dispensable for DNA condensation during Drosophila post-meiotic spermatogenesis. Therefore, we analyzed the interaction of Mst77F, another very basic testis-specific protein with chromatin and DNA as well as studied the molecular consequences of such binding. We show that Mst77F on its own causes severe chromatin and DNA aggregation. An intrinsically unstructured domain in the C-terminus of Mst77F binds DNA via electrostatic interaction. This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein. Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation. In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm. Based on these findings we postulate that Mst77F is crucial for sperm development by giving rise to a unique condensed chromatin structure.     

Automated quantification of steatosis: agreement with stereological point counting

Background

Steatosis is routinely assessed histologically in clinical practice and research. Automated image analysis can reduce the effort of quantifying steatosis. Since reproducibility is essential for practical use, we have evaluated different analysis methods in terms of their agreement with stereological point counting (SPC) performed by a hepatologist.

Methods

The evaluation was based on a large and representative data set of 970 histological images from human patients with different liver diseases. Three of the evaluated methods were built on previously published approaches. One method incorporated a new approach to improve the robustness to image variability.

Results

The new method showed the strongest agreement with the expert. At 20× resolution, it reproduced steatosis area fractions with a mean absolute error of 0.011 for absent or mild steatosis and 0.036 for moderate or severe steatosis. At 10× resolution, it was more accurate than and twice as fast as all other methods at 20× resolution. When compared with SPC performed by two additional human observers, its error was substantially lower than one and only slightly above the other observer.

Conclusions

The results suggest that the new method can be a suitable automated replacement for SPC. Before further improvements can be verified, it is necessary to thoroughly assess the variability of SPC between human observers.

     

RISAP Is a TGN-Associated RAC5 Effector Regulating Membrane Traffic during Polar Cell Growth in Tobacco

RAC/ROP GTPases coordinate actin dynamics and membrane traffic during polar plant cell expansion. In tobacco (Nicotiana tabacum), pollen tube tip growth is controlled by the RAC/ROP GTPase RAC5, which specifically accumulates at the apical plasma membrane. Here, we describe the functional characterization of RISAP, a RAC5 effector identified by yeast (Saccharomyces cerevisiae) two-hybrid screening. RISAP belongs to a family of putative myosin receptors containing a domain of unknown function 593 (DUF593) and binds via its DUF593 to the globular tail domain of a tobacco pollen tube myosin XI. It also interacts with F-actin and is associated with a subapical trans-Golgi network (TGN) compartment, whose cytoplasmic position at the pollen tube tip is maintained by the actin cytoskeleton. In this TGN compartment, apical secretion and endocytic membrane recycling pathways required for tip growth appear to converge. RISAP overexpression interferes with apical membrane traffic and blocks tip growth. RAC5 constitutively binds to the N terminus of RISAP and interacts in an activation-dependent manner with the C-terminal half of this protein. In pollen tubes, interaction between RAC5 and RISAP is detectable at the subapical TGN compartment. We present a model of RISAP regulation and function that integrates all these findings.