Construction and Characterization of a Recombinant Mutant Homolog of the CheW Protein from <Emphasis Type="Italic">Thermotoga petrophila</Emphasis> RKU-1

In this work, a recombinant chemotaxis CheW protein from Thermotoga petrophila RKU-1 (TpeCheW) and its mutant homologue (TpeCheW-mut) have been obtained. Despite the low homology with the CheW protein of the Escherichia coli bacteria, these proteins do not cause metabolic overload and are well expressed by E. coli laboratory strains. A wide range of features and parameters important for isolation of the TpeCheW-mut protein, such as stability over a wide range of temperatures and pH values, high level of expression, solubility, and the possibility of using simple low-stage purification schemes, including heat pretreatment, has been recognized. Possible directions of using this protein in the practice of scientific and applied research have been formulated and justified.     

Validation of portable muscle tone measurement device for quantifying velocity-dependent properties in elbow spasticity.

The objective of this study is to develop a portable device for quantifying the velocity-dependent properties of spastic elbow muscles. Based on a motor-driven system, validation tests of the portable system such as accuracy and response of sensors were first examined. Furthermore, simulated modules (inertia, damper and spring) as well as elbow joints (15 control and 15 hemiplegic subjects) were manually stretched under four different frequencies (1/3, 1/2, 1 and 3/2 Hz) through 60 degrees range of motion. Joint resistance and displacement during sinusoidal stretch were collected for further analysis. Two quantitative parameters (i.e., viscous components under each frequency and averaged viscosity across four frequencies) were derived to estimate the velocity-dependent properties of elbow joint. Tests of simulated modules confirm the manual stretch protocol and data analysis are valid in estimating the velocity-dependent component during a sinusoidal stretch. Compared to normal control, viscous component in each stretch frequency and averaged viscosity were significantly higher in subjects with spasticity (P < 0.001). The viscous component and averaged viscosity were found highly correlated with the modified Ashworth scale. These findings suggest that measurements of viscous component and averaged viscosity during manual sinusoidal stretching using the portable device could be clinically useful in evaluating spasticity.     

Streptomyces-derived actinomycin D inhibits biofilm formation by Staphylococcus aureus and its hemolytic activity

Staphylococcus aureus is a versatile human pathogen that produces diverse virulence factors, and its biofilm cells are difficult to eradicate due to their inherent ability to tolerate antibiotics. The anti-biofilm activities of the spent media of 252 diverse endophytic microorganisms were investigated using three S. aureus strains. An attempt was made to identify anti-biofilm compounds in active spent media and to assess their anti-hemolytic activities and hydrophobicities in order to investigate action mechanisms. Unlike other antibiotics, actinomycin D (0.5 μg ml^?1) from Streptomyces parvulus significantly inhibited biofilm formation by all three S. aureus strains. Actinomycin D inhibited slime production in S. aureus and it inhibited hemolysis by S. aureus and caused S. aureus cells to become less hydrophobic, thus supporting its anti-biofilm effect. In addition, surface coatings containing actinomycin D prevented S. aureus biofilm formation on glass surfaces. Given these results, FDA-approved actinomycin D warrants further attention as a potential antivirulence agent against S. aureus infections.     

Cytosolic PINK1 promotes the targeting of ubiquitinated proteins to the aggresome-autophagy pathway during proteasomal stress

During proteasomal stress, cells can alleviate the accumulation of polyubiquitinated proteins by targeting them to perinuclear aggresomes for autophagic degradation, but the mechanism underlying the activation of this compensatory pathway remains unclear. Here we report that PINK1-s, a short form of Parkinson disease (PD)-related protein kinase PINK1 (PTEN induced putative kinase 1), is a major regulator of aggresome formation. PINK1-s is extremely unstable due to its recognition by the N-end rule pathway, and tends to accumulate in the cytosol during proteasomal stress. Overexpression of PINK1-s induces aggresome formation in cells with normal proteasomal activities, while loss of PINK1-s function leads to a significant decrease in the efficiency of aggresome formation induced by proteasomal inhibition. PINK1-s exerts its effect through phosphorylation of the ubiquitin-binding protein SQSTM1 (sequestosome 1) and increasing its ability to sequester polyubiquitinated proteins into aggresomes. These findings pinpoint PINK1-s as a sensor of proteasomal activities that transduces the proteasomal impairment signal to the aggresome formation machinery.     

The non-coding RNA composition of the mitotic chromosome by 5′-tag sequencing

Mitotic chromosomes are one of the most commonly recognized sub-cellular structures in eukaryotic cells. Yet basic information necessary to understand their structure and assembly, such as their composition, is still lacking. Recent proteomic studies have begun to fill this void, identifying hundreds of RNA-binding proteins bound to mitotic chromosomes. However, by contrast, there are only two RNA species (U3 snRNA and rRNA) that are known to be associated with the mitotic chromosome, suggesting that there are many mitotic chromosome-associated RNAs (mCARs) not yet identified. Here, using a targeted protocol based on 5′-tag sequencing to profile the mammalian mCAR population, we report the identification of 1279 mCARs, the majority of which are ncRNAs, including lncRNAs that exhibit greater conservation across 60 vertebrate species than the entire population of lncRNAs. There is also a significant enrichment of snoRNAs and specific SINE RNAs. Finally, ∼40% of the mCARs are presently unannotated, many of which are as abundant as the annotated mCARs, suggesting that there are also many novel ncRNAs in the mCARs. Overall, the mCARs identified here, together with the previous proteomic and genomic data, constitute the first comprehensive catalogue of the molecular composition of the eukaryotic mitotic chromosomes.     

Seasonal changes in blood cells and biochemical parameters in the Mongolian hamster (<Emphasis Type="Italic">Allocricetulus curtatus</Emphasis>)

It was shown previously that the Mongolian hamster (Allocricetulus curtatus) is a mammalian species with irregular short hibernation. The purpose of the present study was to determine how this status affects seasonal changes in the biochemical and hematological parameters in A. curtatus males under a natural temperature and light regime. It was found that a reduction in circulating white blood cells, specifically lymphocytes, neutrophils, eosinophils, basophils, and monocytes, occurred in winter and bilirubin levels increased in spring. These characteristics make Mongolian hamsters closer to the true hibernating species. At the same time, the character of seasonal changes in the number of red blood cells, glucose, total protein, creatinine, and albumin is closer to species with torpor.