Myosin light chain kinase colocalizes with nonmuscle myosin IIB in myofibril precursors and sarcomeric Z-lines of cardiomyocytes

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility involving actin and myosin II. MLCK is widely present in vertebrate tissues including the myocardium. However, the role of MLCK in cardiomyocyte function is not known. Previous attempts to gain insight into possible roles and identify potential molecular partners were disappointing and equivocal due to cross reactivity of early antibodies with striated muscle MLCK, which has a different genetic locus and a divergent amino acid sequence from the above mentioned enzyme. Using an immunofluorescence approach and a panel of antibodies directed against MLCK, cytoskeletal, and sarcomeric proteins, we localized MLCK to myofibril precursors and Z-lines of sarcomeres in embryonic and adult cardiomyocytes. The same structures contained nonmuscle myosin IIB implicating this protein as a possible target of MLCK. Our results suggest a role for MLCK in cardiomyocyte differentiation and contraction through regulation of nonmuscle myosin IIB.     

Mammalian CLASP1 and CLASP2 cooperate to ensure mitotic fidelity by regulating spindle and kinetochore function

CLASPs are widely conserved microtubule plus-end-tracking proteins with essential roles in the local regulation of microtubule dynamics. In yeast, Drosophila, and Xenopus, a single CLASP orthologue is present, which is required for mitotic spindle assembly by regulating microtubule dynamics at the kinetochore. In mammals, however, only CLASP1 has been directly implicated in cell division, despite the existence of a second paralogue, CLASP2, whose mitotic roles remain unknown. Here, we show that CLASP2 localization at kinetochores, centrosomes, and spindle throughout mitosis is remarkably similar to CLASP1, both showing fast microtubule-independent turnover rates. Strikingly, primary fibroblasts from Clasp2 knockout mice show numerous spindle and chromosome segregation defects that can be partially rescued by ectopic expression of Clasp1 or Clasp2. Moreover, chromosome segregation rates during anaphase A and B are slower in Clasp2 knockout cells, which is consistent with a role of CLASP2 in the regulation of kinetochore and spindle function. Noteworthy, cell viability/proliferation and spindle checkpoint function were not impaired in Clasp2 knockout cells, but the fidelity of mitosis was strongly compromised, leading to severe chromosomal instability in adult cells. Together, our data support that the partial redundancy of CLASPs during mitosis acts as a possible mechanism to prevent aneuploidy in mammals.     

Effect of <Emphasis Type="Italic">NtDCN1</Emphasis> gene activated during embyogenesis on organogenesis in tobacco tissue culture

The role of NtDCN1 gene in organogenesis in the culture of tobacco (Nicotiana tabacum L.) somatic tissues was studied. This gene is specifically expressed in tobacco microspores induced for somatic embryogenesis. This gene knockout resulted in a disturbance of formation and development of embyoids from microspores. In leaf disks and calli derived from tobacco lines with active and inactivated NtDCN1 gene, we studied induction of shoots and roots. A comparative analysis of tobacco line morphogenetic responses in vitro showed that NtDCN1 gene inactivation enhanced shoot formation and suppressed rhizogenesis, whereas this gene reactivation returned organogenesis processes to control level. Difference between lines was manifested only at a definite ratio between exogenous hormones supplied. The involvement of NtDCN1 gene in line responses to exogenous auxin is discussed. The results obtained permit a supposition that the NtDCN1 gene is critical for regulation not only somatic embryogenesis but also organogenesis.     

A new yeast strain for brewery: Properties and advantages

Beer is a natural product and is a multicomponent system that has both positive and negative consumer properties. Organoleptical off-flavors of beer are difficult to eliminate. Yeasts are the main active component of the system. The relationship between beer quality and yeast usage is well known. New industrial strains for brewery are continuously developed. An industrial yeast Saccharomyces cerevisiae strain was obtained and showed high technological properties, including efficient fermentation, a reduced production of sulfur hydrate, and a high diacetyl reduction rate. The advantages made it possible to develop new brands of beer and nonalcoholic products. The commercial use of the strain was patented. The strain was deposited in the Russian Collection of Industrial Microorganisms.     

Laccase of the lignolytic fungus Trametes hirsuta: Purification and characterization of the enzyme, and cloning and primary structure of the gene

The main physicochemical characteristics of the major isoform of the laccase secreted by the fungus Trametes hirsuta 072 were studied. The enzyme belongs to the group of high redox potential laccases (E _T1 ⁰ 790 ± 5), and it oxidizes with high efficiency various substrates of phenolic nature. The gene of this isoform was cloned, and its nucleotide sequence was determined. The length of the complete gene is 2134 bp. It comprises 11 exons and 10 introns. Analysis of the amino acid sequence of T. hirsuta 072 laccase demonstrated a high homology to the other laccases secreted by fungi of the genus Trametes.     

Plant-produced recombinant influenza vaccine based on virus-like HBc particles carrying an extracellular domain of M2 protein

Conventional influenza vaccines are based on a virus obtained in chicken embryos or its components. The high variability of the surface proteins of influenza virus, hemagglutinin and neuraminidase, requires strain-specific vaccines matching the antigenic specificity of newly emerging virus strains to be developed. A recombinant vaccine based on a highly conservative influenza virus protein M2 fused to a nanosized carrier particle can be an attractive alternative to traditional vaccines. We have constructed a recombinant viral vector based on potato X virus that provides for expression in the Nicotiana benthamiana plants of a hybrid protein M2eHBc consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen (HBc). This vector was introduced into plant cells by infiltrating leaves with agrobacteria carrying the viral vector. The hybrid protein M2eHBc was synthesized in the infected N. benthamiana plants in an amount reaching 1–2% of the total soluble protein and formed virus-like particles with the M2e peptide presented on the surface. Methods of isolation and purification of M2eHBc particles from plant producers were elaborated. Experiments on mice have shown a high immunogenicity of the plant-produced M2eHBc particles and their protective effect against lethal influenza challenge. The developed transient expression system can be used for production of M2e-based candidate influenza vaccine in plants.     

Glucose metabolism and template synthesis in the mitotic cycle of human diploid fibroblasts

The correlation between the rates of protein and nucleic acid synthesis and the activity of the key enzymes of glycolysis (hexokinase, phosphofructokinase) and pentose phosphate cycle (glucose-6-phosphate dehydrogenase) in the mitotic cycle of human diploid fibroblasts synchronized by double thymidine block was studied. It was found that the removal of the thymidine block is followed by short-term (presumably, non-specific) simultaneous stimulation of matrix syntheses, as well as by glycolytic and pentose phosphate cycle enzyme syntheses. By the beginning of the S-phase, all the processes appear to be inhibited, followed by gradual activation of glycolysis and pentose phosphate cycle reactions. The implementation of the cell cycle is concomitant with stepwise transitions of protein and hexokinase synthesis rates and ATP content to one of the following levels--basal, intermediate or maximal. Changes in the activity of glucose-6-phosphate dehydrogenase in the course of the cell cycle appear as oscillations, those in phosphofructokinase as alternative states. At stage M, the oscillatory processes are temporarily quenched, whereas the ATP content occupies an intermediate level. In contrast with diploid fibroblasts, in transformed T9 cells the enzyme activity is much higher, and the fluctuations in activity throughout the cell cycle are less noticeable. Presumably, in transformed cells the enzyme activity is at the maximum level and is not prone to effector regulation.     

Effect of alkylating derivatives of cyclic adenosine-3' ,5'-monophosphate on proliferation of mouse bone marrow stem cells

The effect of the physiological concentration of cyclic adenosine-3' ,5'-monophosphate (cAMP) analogues on the proliferation of mouse bone marrow stem hemopoietic cells (CFUs) was examined. The stimulating effect was estimated from the decrease in CFUs expressed in the percentage derived from comparing the number of spleen colonies in the control and experimental groups treated with hydroxyurea 10(-3) M (incubation with hydroxyurea resuted in the cell death in S-phase). Cyclic AMP stimulted the proliferation of CFUs by 60%, while its analogues such as 8-(N-chloroacetylaminoethylamino)-cAMP, 1-(N-chloroacetylaminoethyoxy)-cAMP and 1-(N-(p-fluorosulfonyl)-benzoylaminoethoxy)-cAMP stimulated the proliferation by 39.2%, 32.4% and 21.9%, respectively. Therefore, the synthetic analogues of cAMP were not only far from inhibiting the proliferation of CFUs but, on the contrary, exerted a stimualting effect unlike most antineoplastic alkylating drugs that depress hemopoiesis up to its total aplasia.