Isolation of plasmid DNA from Bifidobacterium

Conditions for accumulation of the biomass and a procedure for isolation of plasmid DNA from bifidobacteria in microquantities were developed. It was shown that all the strains tested had 1 to 3 plasmids of different molecular weights electrophoretically detected. Relation between the detected plasmid DNA and bifidobacteria resistance to tetracycline and fusidin as well as utilization of some carbohydrates is discussed.     

Common antigens of stable group A streptococcal L forms and muscle fibers of human myocardium]

The immunofluorescence method was used to demonstrate that the antigens of stable streptococcus L-forms and of the cytoplasmic membrane of human myocardium muscle fibers were common. The common antigen was included into the composition of the surface membrane of the muscle cell adjacent to the sarcolemma, and of the transverse tubule membranes of the sarcoplasma reticulum passing in the Z-band region of the muscle fiber sarcomeres. The reactions is completely prevented by the exhaustion of the anti-serum to the antigen of the L-forms with the homogenate of human myocardium tissues or a suspension of the L-form cultures grown both on meat and on casein media. Exhaustion with tissue homogenate of other organs (the liver) or with concentrated nutrient medium practically failed to influence the extent of this reaction. The authors believe that the commonnes of the antigens of stable L-forms of streptococcus, group A, cultures and of the myocardium could serve as one of the causes of prolonged persistence of L-forms in human and animal organism.     

Comprehensive comparative analysis of kinesins in photosynthetic eukaryotes

Background

Kinesins, a superfamily of molecular motors, use microtubules as tracks and transport diverse cellular cargoes. All kinesins contain a highly conserved ~350 amino acid motor domain. Previous analysis of the completed genome sequence of one flowering plant (Arabidopsis) has resulted in identification of 61 kinesins. The recent completion of genome sequencing of several photosynthetic and non-photosynthetic eukaryotes that belong to divergent lineages offers a unique opportunity to conduct a comprehensive comparative analysis of kinesins in plant and non-plant systems and infer their evolutionary relationships.

Results

We used the kinesin motor domain to identify kinesins in the completed genome sequences of 19 species, including 13 newly sequenced genomes. Among the newly analyzed genomes, six represent photosynthetic eukaryotes. A total of 529 kinesins was used to perform comprehensive analysis of kinesins and to construct gene trees using the Bayesian and parsimony approaches. The previously recognized 14 families of kinesins are resolved as distinct lineages in our inferred gene tree. At least three of the 14 kinesin families are not represented in flowering plants. Chlamydomonas, a green alga that is part of the lineage that includes land plants, has at least nine of the 14 known kinesin families. Seven of ten families present in flowering plants are represented in Chlamydomonas, indicating that these families were retained in both the flowering-plant and green algae lineages.

Conclusion

The increase in the number of kinesins in flowering plants is due to vast expansion of the Kinesin-14 and Kinesin-7 families. The Kinesin-14 family, which typically contains a C-terminal motor, has many plant kinesins that have the motor domain at the N terminus, in the middle, or the C terminus. Several domains in kinesins are present exclusively either in plant or animal lineages. Addition of novel domains to kinesins in lineage-specific groups contributed to the functional diversification of kinesins. Results from our gene-tree analyses indicate that there was tremendous lineage-specific duplication and diversification of kinesins in eukaryotes. Since the functions of only a few plant kinesins are reported in the literature, this comprehensive comparative analysis will be useful in designing functional studies with photosynthetic eukaryotes.     

Assays for in vitro monitoring of human airway smooth muscle (ASM) and human pulmonary arterial vascular smooth muscle (VSM) cell migration

Migration of human pulmonary vascular smooth muscle (VSM) cells contributes to vascular remodeling in pulmonary arterial hypertension and atherosclerosis. Evidence also indicates that, in part, migration of airway smooth muscle (ASM) cells may contribute to airway remodeling associated with asthma. Here we describe migration of VSM and ASM cells in vitro using Transwell or Boyden chamber assays. Because dissecting signaling mechanisms regulating cell migration requires molecular approaches, our protocol also describes how to assess migration of transfected VSM and ASM cells. Transwell or Boyden chamber assays can be completed in approximately 8 h and include plating of serum-deprived VSM or ASM cell suspension on membrane precoated with collagen, migration of cells toward chemotactic gradient and visual (Transwell) or digital (Boyden chamber) analysis of membrane. Although the Transwell assay is easy, the Boyden chamber assay requires hands-on experience; however, both assays are reliable cell-based approaches providing valuable information on how chemotactic and inflammatory factors modulate VSM and ASM migration.     

Comparison of in vitro and in vivo methods of evaluation of the efficacy of influenza virus hemagglutinin inhibitors

One of the problem in the selection of the most effective antiviral preparations with a broad spectrum of antiviral protective activity, is the "continuity" of assays of different level of complexity so, that the most effective antiviral therapeutic, selected by in vitro assays would be the most effective in vivo. Comparative study of the efficacy of the influenza virus inhibitor in the assays of inhibition of virus binding with fetuin, inhibition of infectious focus forming units in MDCK cells, inhibition of virus yield in infected MDCK cells, and inhibition of influenza virus infectivity in mice infected by viral aerosol are presented. The value of 50% inhibiting concentration IC50 for the pare "influenza virus strain A/NIB/23/89-MA-inhibitor tetra-Aca6-6'SLN" corresponded to 6-10 microM and was invariant for three different tests--in vitro assay of inhibition of virus binding with fetuin, inhibition of yield in infected MDCK cell culture, and inhibition of virus infectivity in mice, but not for the assay of inhibition of infectious focus forming units in cell culture.     

Association of immune system gene polymorphisms with quantitative features which are pathogenetically important in chronic viral hepatitis

Association study was performed for genetic polymorphisms IL4 C(-590)T, IL4RA Ile50Val, TNF G(-308)A, to estimate their effect on quantitative features which are pathogenetically important for chronic viral hepatitis course, i.e. levels of IL4, IL10, IL12, tumor necrosis factor alpha, fibronectin, collagenase, protease inhibitors, macroglobulines, elastases, free and protein-bound hydroxyproline. It has been shown that A allele of TNF G(-308)A polymorphism is associated with decreased TNF-alpha, increased IL4 and IL12, as well as with low level of protein-bound hydroxyproline. In addition, association of CT genotype of IL4 C(-590)T polymorphism and high level of protein-bound hydroxyproline has been identified.     

The Effect of Seasonal Shifts in Temperature on the Lipid Composition of Marine Macrophytes

The comparative study of lipid composition was carried out in four species of marine algae, Ahnfeltia tobuchiensis, Laminaria japonica, Sargassum pallidum, and Ulva fenestrata, as well as a higher plant grass wrack (Zostera marina). Plants were collected in the Japan Sea in spring at 2.9 and 5.5°C and in summer at 23°C. The main lipid components of membranes were determined, and the general patterns of the ratio of phospholipids (PL), glycolipids (GL), betaine (BL), and neutral (NL) lipids were discerned. The relative content of NL in all species (except A. tobuchiensis) was higher in summer. The level of triacylglycerols was as high as 18–37%. The content of individual classes of PL and GL varied between the spring and summer samples, the relative content of PL being higher in spring. In most species, the ratio of PL to GL decreased in summer. The content of free sterols did not depend on the season. The molar ratios of phosphatidylcholine and diacylglycerol-o-(hydroxymethyl)-(N,N,N-trimethyl)homoserine to free sterols varied from 0.9 to 1.7. The seasonal changes of lipid composition were apparently related to macrophyte adaptation to water temperature and to biology of their development.     

Role of Mitogen-Activated Protein Kinases in Stimulation of Smooth Muscle Cell Migration by Urokinase

The urokinase-type plasminogen activator, or urokinase, stimulates proliferation, adhesion, and migration of different type cells both due to its proteolytic activity and by activation of intracellular transduction pathways after interaction with the external cell surface. It is suggested that activation of p42/p44^erk1,2 MAP-kinases in response to specific receptor binding to the urokinase N-terminal domain is the key event in initiation of cell migration. However, we have found that the central kringle-domain of urokinase has its own target on the cell surface, and that its binding causes a migration response of human smooth muscle cells (SMCs). In the present study, we have shown that the urokinase kringle-domain is required to activate the p38 MAP kinase cascade, and that its inhibition leads to suppression of the migration response of SMCs. On the contrary, stimulation of the p42/p44^erk1,2 MAP-kinase cascade is determined only by proteolytic activity of urokinase and does not depend on its binding to SMCs. Selective inhibition of the p42/p44^erk1,2 MAP-kinase cascade produced a depression of the SMC migration induced by catalytically active urokinase, but did not affect the migration induced by non-active urokinase. It is concluded that binding of the urokinase kringle-domain to a yet unidentified target at the SMC surface is required for activation of the p38 MAP-kinase cascade and of the cell migration. Urokinase was shown to stimulate phosphorylation and activation of regulatory light myosin chains that are required to increase the cytoskeleton dynamics and SMC motility. The participation of p42/p44^erk1,2 and p38 MAP-kinase cascade in the realization of this effect is discussed.