Cloning,expression, and isolation from <Emphasis Type="Italic">Escherichia coli</Emphasis> of human protein SURF-6 translationally fused to glutathione-S-transferase

cDNA of human gene Surf-6 (hSutf-6) was amplified and cloned into vector pGEX-2T for the expression in the bacterial system of protein hSURF-6 translationally fused to glutathione-S-transferase. The resulting vector is named as pGEX-2T-GST-hSurf-6. Superproducer of chimeric protein GST-hSURF-6 was obtained on the basis of Escherichia coli strain BL21-CodonPlus(DE3)-RIL. Its purification was performed by the affinity chromatography on L-glatathione-sepharose. The proportion of recombinant protein GST-hSURF-6 in the optimized conditions was not less than 15% of the total bacterial protein, and up to 7 mg of the protein was isolated from 1 liter of culture of the producer strain. The final fraction of eluate contained approximately 80% of GST-hSURF-6. The amount and the purity of the isolated protein were sufficient to immunize animals and obtain antibodies. Protein GST-hSURF-6 can also be used as an affinity ligand for revealing protein partners of hSURF-6 in human cells.     

Photoaction upon adipose tissue cells in vitro

The effect of a change in the optical properties of human adipose tissue cells in vitro after photodynamic action was studied experimentally. The study of kinetics of this process was carried out based on the digital microscopy of thin layers of tissue. The statistical computer processing of the obtained microimages has allowed one to quantitatively estimate the kinetics of the photodynamic after effects on the biotissue. The optical interpretation of images indicates that the observed phenomenon corresponds to the partial lysis of the adipose tissue cells without their complete destruction.     

Temperature effects of interspecies competition between cladoceran species in experimental conditions

In experiments with mono- and polycultures the hypothesis that small-size cladoceran species (Diaphanosoma brachyurum Lievin and Ceriodaphnia reticulata Jurine) are more successful at higher temperatures whereas large-size species (Daphnia magna Straus and Simocephalus vetulus O.F. Müller) under low temperatures was tested. The biomass of Simocephalus vetulus exceeded the biomass of the other species both in mono- and polycultures. An increase of temperature from 18 up to 25°C led to reduction of the biomass of all species. The largest species, D. magna, responded to higher temperature by increased mortality. We assume that the reduction of the biomass of the large species allowed the small C. reticulata to develop successfully in mixed cultures at higher temperature; however it was completely eliminated at lower temperature. Apparently, the survival strategy of large cladocerans involves delayed juvenile development at lower temperatures. However, at higher temperatures, juvenile development of large species accelerates, and their mortality increases. Individual strategies of the studied species to survive at different temperatures are analyzed. On the whole the increase of temperature can result in decrease of the biomass of cladocerans and prevailence of small-size species over large-size ones.     

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification,Distraction Osteogenesis,or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens⁴. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice ^5-7, after application of bioactive molecules ^8,9, after altering physiological parameters of healing ¹⁰, after modifying the amount or time of stabilization ¹¹, after distraction osteogenesis ¹², after creation of a non-union ¹³, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.     

An improved flat embedding technique for immunoelectron microscopy

Immunocytochemical staining has been widely used for localizing various hormonal antigens, protein markers and putative neurotransmitters in tissues. Immunostained sections can be examined light microscopically and specific areas selected for electron microscopic study.     

The Association between Genetic Polymorphism and the Processing Efficiency of miR-149 Affects the Prognosis of Patients with Head and Neck Squamous Cell Carcinoma

MicroRNAs (miRNAs) play important roles in modulating the neoplastic process of cancers including head and neck squamous cell carcinoma (HNSCC). A genetic polymorphism (rs2292832, C>T) has been recently identified in the precursor of miR-149; nevertheless its clinicopathological implications remain obscure. In this study, we showed that miR-149 is down-regulated in HNSCC compared to normal mucosa and this is associated with a poorer patient survival. In addition, HNSCC patients with the T/T genotype have more advanced tumors and a worse prognosis. Multivariate analysis indicated that patients carried the T/T genotype have a 2.81-fold (95% CI: 1.58–4.97) increased risk of nodal metastasis and 1.66-fold (95% CI: 1.05–2.60) increased risk of mortality compared to other groups. T/T genotype also predicted the worse prognosis of buccal mucosa carcinoma subset of HNSCC. In vitro analysis indicated that exogenous miR-149 expression reduces the migration of HNSCC cells. Moreover, HNSCC cell subclones carrying the pri-mir-149 sequence containing the T variant show a low processing efficacy when converting the pre-mir-149 to mature miR-149. These findings suggest that miR-149 suppresses tumor cell mobility, and that the pre-mir-149 polymorphism may affect the processing of miR-149, resulting in a change in the abundance of the mature form miRNA, which, in turn, modulates tumor progression and patient survival.     

Peculiarities of Stone Cockscomb Alectrias alectrolophus (Stichaeidae) Diet in Avacha Bay (Eastern Kamchatka)

Journal of Ichthyology - Information on stone cockscomb Alectrias alectrolophus diet in Avacha Bay (eastern Kamchatka) are presented, and seasonal, local, age, and interannual changes in the...