The immunomodulatory activity of the betulonic acid based compound.

The immunomodulatory activity of a betulonic acid-based compound with furocoumarin (BABCF; 2-azido, 9-N-methylpiperazinomethyl oreozelone) has been investigated. Male C57BL/6 mice (aged 3 months) treated with the cytostatic agent cyclophosphamide (CP) and intact individuals served as experimental models. The expression of genes was studied in bone marrow (IL-12, IL-10, IL-1ß, TNF-α, TGF-ß, M-CSF, GM-CSF) or in the suspension of peritoneal cells (IL-12, IL-10; as the injection site). The surface markers of T-lymphocytes (CD3, CD4, and CD8) in fractions of venous blood mononuclear cells (MNCs) were determined by means of flow cytometry using antibodies. Histological and morphometric studies were performed to assess the impact of CP and BABCF on the thymus. BABCF caused a pronounced (about 3-fold) increase in relative expression of the GM-KSF gene. BABCF caused a local increase in the expression of IL-12 in the peritoneal cavity cells and restored the relative content of T-lymphocytes in the blood of CP-treated mice treated affecting mainly CD3⁺CD4⁺ lymphocytes. This substance reduced the tissue density of the thymic cortex and thymic medulla in CP-treated mice. Thus, results of this study suggest that BABCF exhibits a stimulating effect on the cellular link of immunity and promotes maintenance of the number of T-lymphocytes in the blood due to their migration from the central organs of the immune system.

Effect of Melatonin on the Content of CD3low and CD3hi T Cells in the Thymus of Mice with Functional Pinealectomy.

Continuous lighting for 14 days (functional pinealectomy model) leads to a decrease in the relative number of CD3low and CD3hi T lymphocytes and the CD3low/CD3hi ratio in the thymus of C57BL/6 mice. Intragastric administration of melatonin in physiological doses (1 mg/kg body weight, 14 days) against the background of functional pinealectomy restores the percentage of CD3low and CD3hi thymocytes and CD3low/CD3hi ratio to the control values. Hence, prolonged continuous illumination inhibits the differentiation and maturation of young thymocytes into mature forms, while melatonin treatment helps to compensate the effects of functional pinealectomy triggering cell proliferation in the thymus from the earliest stages of proliferation and differentiation of T cells. Thus, melatonin has immunotropic properties and can be used for correction of the consequences of functional pinealectomy.

Light-Induced Functional Pinealectomy. Effect on the Thymus of C57BL/6 Mice.

Light-induced functional pinealectomy was simulated in C57BL/6 mice by 14-day exposure to constant lighting. Immunophenotyping of CD3hi and CD3low thymocytes was performed by staining with CD3-APC antibodies followed by flow cytofluorometry. To study the cell cycle distribution of thymus cells, the content of intracellular DNA was measured by the level PI inclusion. In animals with light-induced functional pinealectomy, blood leukocyte content, the relative number of CD3low and CD3hi T cells in the thymus, and the ratio of CD3low/CD3hi thymocytes decreased. The number of G0/G1-phase thymus cells (non-dividing cells) increased and the content of S-phase cells (division phase) decreased. Continuous lighting stimulated the development of thymocyte apoptosis. The results obtained indicate that prolonged 24-h illumination inhibits differentiation and maturation of young CD3low thymocytes into mature CD3hi forms and leads to the development of T-cell apoptosis in the thymus and, as a consequence, to leukopenia.

In Vitro Effects of Lithium Carbonate on Cell Cycle, Apoptosis, and Autophagy in Hepatocellular Carcinoma-29 Cells.

We studied the effects of lithium carbonate on the cell cycle, apoptosis, and autophagy in hepatocellular carcinoma-29 cells (HCC-29) in vitro. Flow cytofluorometry analysis revealed accumulation of G2/M-phase HCC-29 cells and increase in the number of apoptotic cells in 48 h after administration of 5 mM lithium carbonate. Induction of autophagy in HCC-29 cells was detected by transmission electron microscopy and immunofluorescence staining. Thus, lithium carbonate produces an antitumor effect by arresting cell cycle in the G2/M-phase and induction of apoptosis and autophagy in HCC-29 cells, which confirms the lithium potential as a promising drug for the treatment of hepatocellular carcinoma.

Correction to: Construction of a Pichia pastoris strain efficiently producing recombinant human granulocyte-colony stimulating factor (rhG-CSF) and study of its biological activity on bone marrow cells.

The original publication has been updated. The acknowledgment was omitted from the original article and is published below.

Construction of a Pichia pastoris strain efficiently producing recombinant human granulocyte-colony stimulating factor (rhG-CSF) and study of its biological activity on bone marrow cells.

Non-glycosylated, recombinant human granulocyte colony-stimulating factor (rhG-CSF), produced by Escherichia coli (filgrastim, leukostim) is widely used to treat a number of serious human diseases and aids in the recovery post bone marrow transplantation. Although glycosylation is not required for the manifestation of the biological activity of G-CSF, a number of studies have shown that the carbohydrate residue significantly increases the physicochemical stability of the G-CSF molecule. Therefore, the aim of the present study was to design a Pichia pastoris strain capable of producing glycosylated rhG-CSF, and to study its effects on rat bone marrow cells. The nucleotide sequence of the rhG-CSF gene has been optimized for expression in P. pastoris, synthesized, cloned into the pPICZαA vector and expressed under the control of the AOX promoter in P. pastoris X33. One of the selected clones secreting rhG-CSF, produced 100-120 mg/l of rhG-CSF three days post-induction with methanol. The recombinant cytokine was purified using two-step, ion-exchange chromatography. The final yield of purified G-CSF was 35 mg/L of culture medium. The biological activity of rhG-CSF was examined in rat bone marrow cells. The P. pastoris strain was designed to produce relatively high levels of rhG-CSF. The rhG-CSF protein had a strong stimulating effect on the growth of rat bone marrow cells, which was comparable to that of the commercial drug leukostim, but showed a more persistent effect on granulocyte cells and monocyte sprouts, enabling the enhanced maintenance of the viability of the cells into the 4th day of incubation.

Apolipoprotein A-I Stimulates Cell Proliferation in Bone Marrow Cell Culture.

Culturing of bone marrow cells in serum-free RPMI-1640 medium led to a decrease in the rate of DNA biosynthesis. Addition of HDL or their main protein component apolipoprotein A-I to the culture medium dose-dependently increased the rate of [3H]-thymidine incorporation into DNA. The maximum stimulation was achieved at HDL concentration of 80 µg/ml and apolipoprotein A-I concentration of 20 µg/ml. To identify the target-cells of apolipoprotein A-I, we used thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU) that incorporates into cell DNA at the stage of replicative DNA synthesis (S phase) and can be detected by fluorescence microscopy. In bone marrow cell culture, apolipoprotein A-I stimulates the proliferation of monocyte (monoblasts, promonocytes) and granulocyte (myeloblasts, promyelocytes) progenitor cells, as well as bone marrow stromal cells.

Effect of Native and Modified Apolipoprotein A-I on DNA Synthesis in Cultures of Different Cells.

Culturing of bone marrow cells in serum-free RPMI-1640 medium for 24 h was accompanied by a decrease in the rate of [3H]-thymidine incorporation into DNA. Addition of native apolipoprotein A-I (apoA-I) or plasma LDL and HDL to the culture medium increased this parameter. In contrast to native apoA-I, its modified form decelerated DNA synthesis in bone marrow cells. A similar inhibitory effect of modified protein was observed in cultures of human embryonic kidney cells (HEK293) and in rapidly proliferating mouse macrophage cell line ANA-1. The only exclusion was human myeloid cell line U937: neither native nor modified apoA-I affected DNA synthesis in these cells. Thus, the regulatory effects of apoA-I are tissue-specific; this protein can produce either stimulatory or inhibitory effect on DNA biosynthesis in cells depending on its conformation.

[Comparative analysis of morphofunctional indicators of peritoneal macrophages in mice of the cancer line A/SN and white outbred mice].

A study of the functional state of peritoneal macrophages was conducted of white outbred mice (W/o) which were resistant to cancer pathology as well as the mice of the A/sn line prone to development of cancer diseases. It was reveald that due to induction of aseptic inflammation ( induced by starch administration), chemotaxis of macrophages of the oncological line was 2.5-3 times reduced and capacity to adhesion was 1.5-2 times lower than capacity of macrophages of the control group (W/o). The paint on F-actin of cytoskeleton in cells showed that cells of the control group form filopodia as a result of adhesion to the substrate, during intercellular contacts as well as during macrophage incubation with retinoic acid. Macrophages A/sn can form pseudopodia when exposed to retinoic acid. Macrophages of both groups of mice were shown to be producer of endonucleases. The same activity of these enzymes was provided by the number of cells which was 1.5-2 times fewer in mice of the A/sn line.

[Genetic variability of antigenic structure of the Lpm-protein and alpha2-macroglobulin among non-mustelid mammals]. / Geneticheskaia izmenchivost' antigennoi struktury Lpm-proteina i alpha2-makroglobulina u nekunikh mlekopitaiushchikh.

The data on interspecific distribution and antigenic variability of homologues of mink Lpm-protein (Lpm) and alpha 2M-globulin (alpha 2M) among 43 species of 6 orders of mammals are presented. Strong variability of antigenic structure of Lpm and alpha 2M of Rodentia and Artiodactyla orders was established. This fact allows to classify the macroglobulins studied as belonging to the group of evolutionary high-speed plasma globulins. In the plasma of human and green marmoset (Cercopithecus aephiops) the only homologue of alpha 2M was found. The lack of reaction to Lpm in above-mentioned species allows to postulate significant differences between Carnivora and Primates in the organization of this gene cluster. Unique features of antigenic variability of Lpm and alpha 2M were also fixed among other systematical groups tested. Taken together, the data obtained stimulate scientific interest to the study of molecular organization of the Lpm and alpha 2M gene families.

[Genetics and evolution of the mink Lpm-system. VIII. Characteristic changes in the antigenic structure of Lpm-protein and alpha2- macroglobulin in Mustelidae family]. / Genetika i évoliutsiia Lpm-sistemy amerikanskoi norki. Soobshchenie VIII. Osobennosti izmenenii antigennoi struktury Lpm-proteina i alpha2-makroglobulina v semeistve kun'ikh.

Interspecific variability of the isotypic structure of two macroglobulins (Lpm and alpha 2M) in Mustelidae family was investigated using monospecific rabbit antisera against Lpm and alpha 2M Mustela vison. It was established that all the species studied may be divided into four groups, according to the degree of their homology with Lpm: 1) M. vison; 2) M. altaica, M. erminea, Kolonocus sibirica, Putorius eversmany, P. putorius, Luteola lutreola; 3) Vormela peregusna; 4) Martes martes, M. foina, M. zibellina. The greatest divergence of Martes gene species from M. vison and other species studied is determined by two different immunogenetic approaches, the first based on the whole isotype of Lpm and the other using allotypic markers of individual Lpm genes. The variability of the isotypic structure of alpha 2M was lower than that revealed in Lpm. V. peregusna is the only species which significantly differs from all above-mentioned species, including M. vison. The differences established for degree of variability between Lpm and alpha 2M in Mustelidae family may be connected with the evolutionary peculiarities of these two genetic system of serum alpha-macroglobulins, which being phylogenetically related, are still structurally divergent.

[Localization of the multigene Lpm locus in chromosome 9 of the American mink]. / Lokalizatsiia mul'tigennogo Lpm-lokusa v khromosome 9 amerikanskoi norki.

The results of genetic study on linkage of Lpm locus with peptidase B gene are presented. Investigation of 111 offspring back-crosses shows that Lpm allotypes and allelic variants of peptidase B are inherited in concert. The frequency of recombination between the Lpm locus and peptidase B gene is 11 +/- 3% in male. Since it was earlier established that peptidase B gene is a marker of chromosome 9, our data indicate that the Lpm loci family is situated in the chromosome 9 of domestic mink.