A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.     

Growth characteristics of CHO cells in culture

The dependence of the growth characteristics and monolayer formation on the initial cell plating concentration were studied on a permanent CHO cell line. The cells were cultivated under standard conditions on plastic substrate. Initial plating concentrations were varied as 1000, 2000, 3000, 4000, 5000, and 6000 cells/cm². It was shown that the cell growth can be formally described by a standard S-shaped dependence. However, a more detailed analysis revealed inconsistency of the experimental and expected data. Specifically, the cell growth termination produced by monolayer formation does not coincide with the time when the theoretical curves approach a plateau. It is concluded that cell proliferation and monolayer formation are independent processes (at least in CHO cells). Both processes may be considered as analogs of proliferation and morphogenesis in metazoa. In addition, it is shown that the cessation of cell division is induced by reduction in the cell size to some limiting dimension and increasing of the cell polarization rather than contact inhibition of proliferation after the monolayer formation.     

Phospholipase C negatively regulates tyrosine phosphorylation of EGF receptor in A-431 cells

It is known that EGF induces tyrosine phosphorylation and internalization of the EGF receptor in A-431 cells. U73122, an inhibitor of phospholipase C, induces tyrosine phosphorylation of the EGF receptor and its association with phospholipase C still in nonstimulated cells. In U73122 treated cells EGF exerted no effect on these processes. Receptor-mediated endocytosis was not observed in A-431 cells treated with U73122. The reorganization of actin cytoskeleton was detected in U73122 cells.     

EGF receptor degrades via the proteasome-dependent pathway in A-431 cells

It is known that a lot of cell receptors degrade by ubiquitine-proteasome pathway. Here we show that degradation of the epidermal growth factor (EGF) receptor is proteasome-dependent. Treatment of A-431 cells with lactacystine, an inhibitor of proteolytic activities of 26S proteasomes, induces accumulation of the receptor in cells. Incubation of cell lysates with isolated 26S proteasomes leads to diminishing EGF receptor in these cells. Active (tyrosine phosphorylated) EGF receptor is a target of proteolysis by proteasomes.     

Effect of EGF on the intracellular distribution of heat-shock proteins in A431 cells

The intracellular distribution of hsp70 and hdj1 was studied using immunofluorescent method. In nonstimulated cells hsp70 and hdj1 were observed in the cytoplasm of A431 cells. When 100 ng/ml EGF was added for 15 min, both hsp70 and hdj1 were accumulated in the nuclei. Later on (up to 1 h) hsp70 was exported from the nuclei to be observed mainly in the cytoplasm, whereas hdj1 remained in the nuclei. In cells exposed to tyrphostin AG1478, this inhibitor of tyrosine kinase activity of EGF receptor prevented EGF-dependent accumulation of hsp70 and hdj1 in the nuclei. U73122, an inhibitor of phospholipase C activity, induced tyrosine phosphorylation of EGF receptor without EGF stimulation. In cells treated with U73122, both hsp70 and hdj1 were detected in the nuclei of non-stimulated cells. It is concluded that the intracellular distribution of heat shock proteins in A431 cells depends on tyrosine kinase activity of EGF receptor. Here we report for the first time the influence of EGF on the intracellular redistribution of heat shock proteins.     

The position of cleavage furrow in cultured <Emphasis Type="Italic">L-929</Emphasis> and <Emphasis Type="Italic">CHO</Emphasis> cells

The position of the cleavage furrow (random or otherwise) was studied on cultured L-929 (NCTC, clone 929) and CHO cells. CHO cells were seeded uniformly on the surface of Petri dishes; L-929 cells were grown as colonies so that migrating cells could be watched. Cell behavior was registered by time-lapse imaging. Two parameters were analyzed on captured images: the angle between the cell polarization axis and cleavage furrow and the angle between the cell polarization axis or cleavage furrow and the horizontal axis of the image field. It was shown that the position of CHO cells in the dish plane and the value of the angle between the cell polarization axis and the cleavage furrow were random. The L-929 cells migrating from the colony were orientated such that their polarization axis was directed to the colony center and the cleavage furrow was perpendicular to this axis. The nonrandom position of cultivated cells during mitosis and their cleavage furrow during the telophase are discussed.     

Intracellular distribution of proteasomes in A-431 cells

The intracellular proteasome distribution in A-431 cells was shown using methods of cell fractionation and immunofluorescence. In growing cells the distribution of proteasomes was EGF-dependent. In unstimulated cells and within 30 min of EGF treatment, proteasomes were localized in the cytoplasm and nuclei, but not on the plasma membrane. After 30 min of EGF treatment they were observed on the plasma membrane as well. In A-431 cells cultivated for 24 h in the medium with a lowered serum concentration, proteasomes were detected on the plasma membrane already in unstimulated cells. It is suggested that dephosphorylation of the EGF receptor and signalling proteins in unstimulated cells may depend on the proteolytic activity of proteasomes.     

Development and morphofunctional characterization of the osteoblastic phenotype in cell culture in vitro

The objective of this research was to study osteogenic properties of cultured rabbit bone marrow stromal cells, newborn rat cranium bone cells and rat osteocarcoma ROS 17-2/8 cells. For this purpose cytochemical reaction for alkaline phosphatase was performed by the Lowry method, mineral deposition was assessed by staining of the cultures after von Kossa. Cranium bone cells were shown to synthesize alkaline phosphatase (34 +/- 7 nmol/min/10(6) cells), the matrix mineralization being found. Bone marrow stromal cells displayed a lower activity alkaline phosphatase level than did cranium bone cells (4 +/- 0.6 nmol/min/10(6) cells). However, cell cultivation in the presence of dexamethasone in the medium (10(-8) M) induced a higher activity of alkaline phosphatase (9 +/- 1 nmol/min/10(6) cells), mineralization of the extracellular matrix being the case. The highest level of alkaline phosphatase activity was found for ROS 17-2/8 cells (60 +/- 12 nmol/min/10(6) cells) but no matrix mineralization was determined. According to these data, matrix calcification and formation of bone-like nodules are the most important properties of osteoblastic differentiation in vitro.     

Participation of transfused bone marrow cells in reparative osteohistogenesis

The participation of skeletal tissue cell precursors in the repairing regeneration of bone tissue was studied. Bone marrow was taken from donor animals--mice of C57Bl/6-TgN(ACTbGFP) 1 Osb line (The Jackson Laboratory Bar Harbor ME USA line). Nucleated cell fraction was isolated by centrifugation on a density percoll gradient. Recipient mice C57Bl/6 line were irradiated by 7.0-7.5 Gr dose. Intravenous infusion of donor cells and osteoclasts of tibia was done after irradiation of recipient mice. Histological preparations of bone regenerate tissues were studied on 15, 30, and 60 days by confocal microscopy. Donor cells were found as skeletal tissue precursors into periost, endost, bone marrow, and as differentiated cells of newborn tissue of regenerate--osteoblasts, osteocytes, chondrocytes. The data obtained indicate that part of donor bone marrow cells are able to progressive differentiation under recipient bone fractures.