Emmanuel S. Kamberov

Preliminary X-ray diffraction analysis of crystals of the PII protein from Escherichia coli☆

PII protein, which carries metabolic signals regulating the transcription and activity of glutamine synthetase in nitrogen assimilation in Escherichia coli, has been crystallized in space group P2(1) with a = 47.8 A, b = 62.9 A, c = 52.8 A and beta = 100.3 degrees and space group P2(1)2(1)2(1) with a = 52.2 A. b = 64.9 A and c = 100.1 A. Both the monoclinic crystals, which diffract beyond 3.0 A, and the orthorhombic crystals, which diffract beyond 2.5 A, probably have three molecules of 12,400 Da each in the crystallographic asymmetric unit.

Development of a new serum-free cell culture system, McCoy-Plovdiv

Dear Editor: McCoy are synovial cell lines of either human (McCoy A) or murine (McCoy B) origin. These ceils are cultured in Earles minimal essential medium (EMEM), Liverpool-Waymouth medium, or RPMI-1640, supplemented with 5 to 10% of fetal calf serum (Karayiannis et al., 1981; Bose et al., 1986; Meysick et al., 1990; Perotti et al., 1991; Kluytmans et al., 1993; Varanda et al., 1997). McCoy cells are widely used as a model for in vitro cytotoxicity experiments (Vento et al., 1990; Shrivastava et al., 1992; Andersen et al., 1993; Machmoud et al., 1995; Pascual et al., 1997; Varanda et al., 1997; Docheva et al., 1998; Garsia et al., 1998). The presence of serum, however, can lead to interference of serum proteins with the test substances (Stark et al., 1986) and mask the cytotoxic effects. Here we describe the development, stabilization, and the conditions of a continuous culture and preservation of McCoy-Plovdiv clone as a new serum-free cell culture system. McCoy A cell line was obtained from the National Bank of Industrial Microorganisms and Cell Cuhures (NBIMCC), Sofia, Bulgaria, and cultured in MEM+ 10% FCS. In the process of cell adaptation of the new clone to the medium conditions and total absence of serum the effects of three defined growth media were compared: Hams F-12, DMEM, and RPMI-1640. Media were mixed at different ratios as follows: Hams F-12:DMEM (3:1), Hams F-12: DMEM (1:1), Hams F-12:RPMI 1640 (3:1), and Hams F-12:RPM1 1640 (1:1). Cells were evaluated microscopically on their ability for adhesion, spreading, shape, division, monolayer formation, monolayer stability, subconfluent and postconfluent state of the monolayer. These observations determined Hams F-12:DMEM 1:1 (HD) medium as the most suitable for stably maintaining cells in culture. In this growth medium supplemented with DMSO, cells were successfully preserved in liquid nitrogen. We found that 2, 5, 7, or 10% of the cryoprotector in the medium provided 81.7, 83.9, 95.8 and 91.5% cell survival, respectively. Thus for routine storage we use HD + 7% DMSO. Shown in Fig. 1 are microphotographs of McCoy-Plovdiv cells, cultured on cover slips. Inoculation density of 1 • 10 s cell/ml permitted the cells to rapidly adhere to the glass as well as to plastic cell culture flasks. Sixty minutes after inoculation ceils were firmly attached (Fig. 1A). Short and clearly distinct cytoplasmic extensions with various shapes could be observed. Ceils with bipolar fibroblast-like shape were prevailing in the culture 24 h after inoculation (not shown). A confluent culture could be obtained 96 h after inoculation (Fig. 1B). In older cultures cells with epithelial-like shape were predominant. The culture retained its viability as a monolayer 15-17 d after inoculation without change of the medium. Cell death in the culture under these conditions was only moderate. We found that trypsin had to be removed by centrifugation following trypsinization. If this step was omitted, the culture did not develop normally. Cells adhered but did not spread, forming spherical grape-shaped clusters (Fig. 1C).

Biological effects of a novel intestinal peptide--inhibiting enterocytogenin on cultured 3T3 mouse fibroblasts and L5178Y mouse lymphoma cells.

The effects of a new intestinal peptide, inhibiting enterocytogenin (IEG) derived from pig intestinal mucosa were studied in vitro on 3T3 mouse fibroblasts and L5178Y mouse lymphoma cell line. IEG caused considerable growth inhibition together with specific morphological changes, necrotic effects as well as formation of monolayers at the highest concentration applied (1000 micrograms/ml). A biologically active fraction (IEG-BAF) derived by further purification of IEG by gel-filtration, proved to possess most of the described activity. The concentrations of IEG and IEG-BAF inhibiting the growth of L5178Y lymphoma cells by 50% (IC50 values) were calculated to be 759 micrograms/ml and 192 micrograms/ml, respectively. IEG-BAF has a molecular mass of 4450 +/- 180 Da and is most probably a peptidylnucleotidate as revealed by spectral analysis.

Altered bioelectrical and mechanical activities of rat gastric smooth muscle preparations by inhibiting enterocytogenin

Inhibiting enterocytogenin (IEG), a 4.5 kDa nucleopeptide isolated from pig intestinal mucosa induced dose-dependent alterations in the spontaneous contractile and bioelectric activities of rat gastric smooth muscle when applied at 10(-8) to 10(-4) M. Two separate phases were apparent in the effects observed, an initial contractile phase followed by a relaxation phase. The depolarization and the related contraction were reduced by amiloride and to a lesser extent by nifedipine. This reduction resulted in a corresponding decrease in the magnitude of the subsequent relaxation phase. Charybdotoxin and apamin caused a statistically significant decrease in the hyperpolarization and the magnitude of the relaxation phase and increased the duration of the contractile phase. On a caffeine or noradrenaline background the effects induced by IEG were diminished, suggesting that they are mediated through Ca2+ release from the intracellular Ca2+ stores. We hypothesize that the depolarization induced by IEG involves activation of the voltage-dependent Ca2+ channels with subsequent stimulation of the Ca(2+)-dependent K+ channels and late development of hyperpolarization.