Bioinformatic and partial functional analysis of pEspA and pEspB, two plasmids from Exiguobacterium arabatum sp. nov. RFL1109

The complete nucleotide sequences of two plasmids from Exiguobacterium arabatum sp. nov. RFL1109, pEspA (4563bp) and pEspB (38,945bp), have been determined. Five ORFs were identified in the pEspA plasmid, and putative functions were assigned to two of them. Using deletion mapping approach, the Rep-independent replication region of pEspA, which functions in Bacillus subtilis, was localized within a 0.6kb DNA region. Analysis of the pEspB sequence revealed 42 ORFs. From these, function of two genes encoding enzymes of the Lsp1109I restriction-modification system was confirmed experimentally, while putative functions of another 18 ORFs were suggested based on comparative analysis. Three functional regions have been proposed for the pEspB plasmid: the putative conjugative transfer region, the region involved in plasmid replication and maintenance, and the region responsible for transposition of the IS21 family-like transposable elements.     

Rab14/MACF2 complex regulates endosomal targeting during cytokinesis

Abscission is a complex cellular process that is required for mitotic division. It is well established that coordinated and localized changes in actin and microtubule dynamics are vital for cytokinetic ring formation, as well as establishment of the abscission site. Actin cytoskeleton reorganization during abscission would not be possible without the interplay between Rab11- and Rab35-containing endosomes and their effector proteins, whose roles in regulating endocytic pathways at the cleavage furrow have now been studied extensively. Here, we identified Rab14 as a novel regulator of cytokinesis. We demonstrate that depletion of Rab14 causes either cytokinesis failure or significantly prolongs division time. We show that Rab14 contributes to the efficiency of recruiting Rab11-endosomes to the thin intracellular bridge (ICB) microtubules and that Rab14 knockout leads to inhibition of actin clearance at the abscission site. Finally, we demonstrate that Rab14 binds to microtubule minus-end interacting MACF2/CAMSAP3 complex and that this binding affects targeting of endosomes to the ICB microtubules. Collectively, our data identified Rab14 and MACF2/CAMSAP3 as proteins that regulate actin depolymerization and endosome targeting during cytokinesis.     

Analysis of products of DNA modification by methylases: a procedure for the determination of 5- and N4-methylcytosines in DNA

Although many different methods are used for the identification of methylated heterocyclic bases in DNA not all of them possess the ability to discriminate N4-methylcytosine (m4C) and 5-methylcytosine (m5C). Therefore, some of the methods need additional reexamination. This paper reinvestigates some chromatographic systems (thin-layer chromatography, paper chromatography, electrophoresis) most widely used in the analysis of minor bases occurring in nucleic acids according to their ability to separate m4C and m5C. A simple procedure for the preparation of the sample and a chromatographic system for its analysis was developed. The recommended chromatographic systems may be used for the simultaneous separation of not only of m4C and m5C but also both methylated cytosines together with N6-methyladenine and 7-methylguanine.     

Fiber attenuated total reflection infrared spectroscopy of kidney tissue during live surgery

More than 90% of solid kidney tumors are cancerous and have to be treated by surgical resection where surgical outcomes and patient prognosis are dependent on the tumor discrimination. The development of alternative approaches based on a new generation of fiber attenuated total reflection (ATR) probes could aid tumor identification even under intrasurgical conditions. Herein, fiber ATR IR spectroscopy is employed to distinguish normal and cancerous kidney tissues. Freshly resected tissue samples from 34 patients are investigated under nearly native conditions. Spectral marker bands that allow a reliable discrimination between tumor and normal tissue are identified by a supervised classification algorithm. The absorbance values of the bands at 1025, 1155 and 1240 cm^?1 assigned to glycogen and fructose 1,6‐bisphosphatase are used as the clearest markers for the tissue discrimination. Absorbance threshold values for tumor and normal tissue are determined by discriminant analysis. This new approach allows the surgeon to make a clinical diagnosis.     

Dynamics of indirect symptoms of skeletal muscle damage after stretch-shortening exercise

Healthy untrained men (age 20.4 ± 1.7 years, n = 20) volunteered to participate in an experiment in order to establish dynamics of indirect symptoms of skeletal muscle damage (ISMD) (decrease in maximal isometric voluntary contraction torque (MVCT) and torque evoked by electrostimulation at different frequencies and at different quadriceps muscle length, height (H) of drop jump (DJ), muscle soreness and creatine kinase (CK) activity in the blood) after 100 DJs from 0.75 m height performed with maximal intensity with an interval of 20 s between the jumps (stretch-shortening exercise, SSE). All ISMDs remained even 72 h after SSE (P < 0.01–0.001). The muscle experienced greater decrease (P < 0.01) in torque evoked by electrostimulation (at low stimulation frequencies and at short muscle length in particular) after SSE than neuromuscular performance (MVCT and H of DJ) which demonstrated secondary decrease (P < 0.01) in neuromuscular performance during the first 48 h after SSE. Within 24–72 h after the SSE the subjects felt an acute muscle pain (5–7 points approximately) and the CK activity in the blood was significantly increased up to 1200 IU/L (P < 0.001). A significant correlation between decrease in MVCT and H of DJ 24–48 h after SSE on the one hand and muscle soreness registered within 24–48 h after SSE on the other was observed, whereas correlation between the other indirect symptoms of skeletal muscle damage was not significant.