Production of recombinant human lactoferrin in the allantoic fluid of embryonated chicken eggs and its characteristics.

The human iron-binding protein lactoferrin (hLf) has been implicated in a number of important physiological pathways, including those regulating immune function and tumor growth. In an effort to develop an efficient system for production of recombinant hLf (rhLf) that is structurally and functionally equivalent to the natural protein, we generated a recombinant CELO (chicken embryo lethal orphan) avian adenovirus containing an expression cassette for hLf. Embryonated chicken eggs were infected with the generated CELO-Lf virus.

Recombinant avian adenovirus CELO expressing the human interleukin-2: characterization in vitro, in ovo and in vivo.

In our study, a recombinant adenovirus based on the avian adenovirus CELO genome, has been constructed that contains the human interleukin-2 gene. We have shown the production of biologically active recombinant interleukin-2 in vitro (LMH and 293 cells) and in ovo (chicken embryos) infected with recombinant virus CELO-IL2. An increase in the median survival time of C57BL/6 mice carrying B16 melanoma tumors has been demonstrated after multiple intra-tumors injections of the recombinant adenovirus CELO-IL2.

[The induction of neovascularization in chorioallantoic membrane of chicken embryos transfected by a recombinant plasmid containing human angiogenin gene].

A method was elaborated to evaluate the biological activity of expression products of gene in the plasmid vectors, which are crucial for the synthesis of growth factor of blood vessels. It was proven as possible that the chrioallantonic membrane (CAM) of chicken's embryos could be transfected by recombinant plasmids containing both the reporter and target genes. The efficiency of CAM transfection was assessed by a plasmid carrying the reporter gene of green fluorescent protein (GFP).

[Eukaryotic vectors of Celo avian adenovirus genome, carrying GFP and human IL-2 genes].

Recombinant CELO avian adenoviruses carrying green fluorescent protein (GFP) and and human interleukin-2 (IL-2) genes were obtained by homologous recombination in cell culture. The resultant recombinant CELO viruses are reproduced in chick embryos in the renal tubular and chorionic allantoic membrane cells. The ability of CELO vectors to transduce human and animal cells was studied in vitro (in cell cultures) and in vivo (in laboratory animals).

[Transcription of anti-apoptotic proteins from CELO and EDS avian adenoviruses at early stages of infection].

Transcription of E1b region of avian CELO and EDS adenoviruses was investigated in infection of primary chicken kidney cell culture with these viruses. Transcription of Gam-1 gene of CELO virus and predicted large T antigen of EDS virus was observed 4 and 24 h after cell infection.

[Early transcription of FAV-1 (CELO) avian adenovirus genome].

The patterns of RNA from cells infected with avian adenovirus chicken embryo lethal orphan (CELO) virus were analyzed by Northern hybridization method. Early RNAs are specifically hybridized with several fragments of the Eco-RI restriction map: A (49-81%), B (0.2-20.

[Analysis of core virion polypeptides from the pathogen causing chicken egg-drop syndrome].

The cores of egg-drop syndrome virus (EDS-76) were isolated by the pyridine technique. EDS-76 proved to be much more resistant to pyridine disruption than other adenoviruses and treatment with 10% pyridine did not lead to complete dissociation of capsid and cores; only increase of pyridine concentration to 20% produced satisfactory results. At least three polypeptides (24, 10.

Receptor-mediated transfection of murine and ovine mammary glands in vivo.

Transfection of HC-11 murine epithelial mammary cells as well as murine and sheep mammary glands were carried out using insulin-containing constructs that deliver DNA by receptor-mediated endocytosis to receptor-expressing cells. In vivo transfection of mammary gland tissue with the luciferase gene was carried out by introducing the DNA constructs into the mammary ducts of both mice and sheep. The successful transfection of ewe mammary glands was demonstrated by the detection of luciferase activity in mammary gland biopsy material up to a month after a single administration of the construct.

Thiophilic adsorption: rapid purification of F(ab)2 and Fc fragments of IgG1 antibodies from murine ascitic fluid.

A thiophilic adsorption method has been developed for rapid purification and separation of mouse F(ab)2 and Fc fragments obtained after proteolytic digestion of IgG1 monoclonal antibodies. Partially purified Mabs were digested with papain. Thiophilic chromatography was performed using stepwise elution with decreasing concentrations of ammonium sulphate.

[Detection of surface immunoglobulins on peripheral lymphocytes in patients with infectious allergic bronchial asthma].

During the examination of 45 patients with infectious allergic asthma and 21 healthy donors the relative number of B-lymphocytes, determined by direct immunofluorescence, was found to be normal in 88% of the patients. In 80% of the patients the relative number of lymphocytes, carrying surface immunoglobulins, exceeded the upper limit of the norm immediately after their isolation, which was due to the presence of exogenous immunoglobulins, partially dissociating from the cell surface after 1-hour incubation at 37 degrees C. The results obtained in this study are of importance in the evaluation of the function of the immune system.

[Determination of phospholipase A2 activity in liver mitochondria using fluorescent dye 1,8-anilinonaphthalene sulfonate].

Quantitative estimation of free fatty acids was carried out in liver mitochondria of normal and thyroidectomized rabbits by means of two procedures--spectrophotometry using rhodamine 6Zh and a fluorescent method with 1,8-aniline naphthalene sulfonate /ANS/ as a dye. The use of ANS enabled to estimate quantitatively the free fatty acids beginning from 25 nmol in a sample /with eggor of about 17%/ by visual evaluation of the spot areas. As compared with the rhodamine procedure, use of ANS permitted to increase 5-fold the sensitivity of free fatty acid estimation, to elevate 6-fold the accuracy of the analysis, to overcome the step of fatty acid elution from chromatogramms and to decrease the time required for analysis.

[Role of phospholipase A2 in anoxic injury to the energy-dependent functions of the mitochondria].

An increase in concentration of free fatty acids was observed simultaneously with a decrease in energy dependent functions of mitochondria (oxidative phosphorylation and Ca2+-accumulating ability) after their preincubation under anoxic conditions in vitro with Ca2+ ions (11-14 nmol/mg of mitochondrial protein). Impairment of mitochondria and accumulation of fatty acids were prevented by inhibitors of phospholipase A2 from mitochondria. The anoxic impairment of mitochondria appears to be caused by Ca2+-activated phospholipase A2.