Studies on NK cell purification by negative selection in human peripheral blood

We have attempted to improve negative selection procedures for the large scale purification of human CD _in3 ^– CD56⁺ NK cells. In a series of experiments, purifications of NK cells from 10⁸ PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3⁺ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 10⁸ cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 10⁹ PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior toin vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads.     

d-Hydantoinase from anaerobic microorganisms

Summary Of 373 anaerobic microbial isolates screened for the enzymatic conversion of dihydrouracil to N-carbamyl--alanine, several strains of Clostridium spp., C. glycolicum, C. subterminale and Peptococcus anaerobius were positive. These Clostridium and Peptococcus strains produced also N-carbamyl-d-amino acids from the respective 5-monosubstituted hydantoins. The d-hydantoinase activity from whole cell suspensions of P. anaerobius strain CRDA 303 was characterized with regard to pH and temperature stability and activity by using dihydrouracil (DHU) and isopropylhydantoin (IPH) as substrates. The d-hydantoinase from P. anaerobius was optimal at 60°C and at pH 6.5–9.5 for the substrate DHU. It was stable up to 55°C and at pH 5.0–9.5 and could be stored at 4°C under an aerobic atmosphere for at least 14 days. Offprint requests to: A. Morin     

A protein of Halobacterium halobium immunologically related to the v-myc gene product

A 70 kDa protein of Halobacterium halobium cross-reacts with an antiserum directed against the v-myc gene product of the avian myelocytomatosis virus (MC29). This cross-reaction is in agreement with hybridization studies which indicate that H. halobium possesses DNA and RNA sequences homologous to the v-myc gene.     

Evidence for bidirectional transverse diffusion of spin-labeled phospholipids in the plasma membrane of guinea pig blood cells

The distribution and transverse diffusion kinetics of four spin-labeled phospholipid analogues (two with choline heads: phosphatidylcholine (PC) and sphingomyelin (SM); two with amino heads: phosphatidylserine (PS) and phosphatidylethanolamine (PE) were studied in the plasma membrane of guinea pig blood cells: erythrocytes, reticulocytes, and leukemic lymphocytes. Nitroxide reduction by the internal content of the cells was used as an indicator to determine the phospholipids that penetrated the cells. The reduction rates were in the order, PS greater than PE greater than PC greater than SM in all cells. Reoxidation of phospholipids extracted by serum albumin revealed the distribution of the phospholipids at a given time. In all cells, the distribution equilibrium was reached in less than 2 h and the amounts left in the external leaflet were in the following proportional order: PS less than PE less than PC less than SM. In the erythrocytes and especially in the reticulocytes, the shape change induced by adding phospholipids relaxed partially or completely at a lower speed but kept the same proportional order as at equilibrium. All the results were analyzed quantitatively with a simple kinetic model including the rates of transverse diffusion (flip and flop), the exchange between plasma membrane and internal membranes, and the reduction rate of free radicals (determined in either the internal or external membrane leaflet). The calculated rate constants of transverse diffusion varied from 2 x 10(-3) to 1.2 x 10(-1) min-1 for the flip and from 4 x 10(-3) to 1.2 x 10(-1) for the flop, depending on the polar head and the cell type. Possible interpretations of the external phospholipid reduction mechanism and cell deformation are discussed.     

Patterns in the development of the ultrastructure of elements of the microcirculatory system in the early stages of human embryogenesis

By means of transmissive electron microscopy methods, general regularities in development of the microcirculatory system have been studied at early stages of the human prenatal ontogenesis in functionally different organs. Ultrastructure of two cell types has been described in the mesenchyme of human embryos. Formation mechanisms of the primary blood vessels belonging to the protocapillary type are revealed. Structural peculiarities of the primary protocapillary network differentiating into various links of the secondary organospecific hemomicrocirculatory bed are distinguished. Certain stageness in development of the microcirculatory system is stated, its blood circulatory compartment including. Two stages are determined in development of the microcirculatory system: prevascular and vascular microcirculation. The latter includes the precirculatory and circulatory phases.     

Relationships of the Col plasmids E2, E3, E4, E5, E6, and E7: Restriction mapping and colicin gene fusions

Thirteen ColE plasmids representing the E2-E7 types have been compared by restriction mapping. Over 80% of their restriction sites were found to be similarly positioned, indicating that these plasmids share a common structure. Three variants are ColE2-CA42 and ColE7-K317, both of which contain 1.8-kb DNA segments in place of a 2.5-kb segment common to the other plasmids, and ColE6-CT14, which has an additional 5.0-kb DNA segment compared to the other plasmids. The colicin (col), immunity (imm), and colicin release (hic) genes of these plasmids have been localized to regions corresponding to those known for ColE3-CA38 and ColE2-P9, with the imm and hic genes adjacent to the 3' end of the col gene. Active colicin is produced from hybrid col genes containing 5' and 3' ends from different E-type plasmids. The 3'-termini of the fused col genes specify the colicin type.     

Bufonid nucleocytoplasmic hybrids arrested at the early gastrula stage lack a fibronectin-containing fibrillar extracellular matrix

Summary Most hybrids betweenBufo bufo andB. calamita obtained by nuclear transplantation become arrested at the early gastrula stage. In both parental controls and the hybrid embryos, the presence and distribution of extracellular matrix was analysed with fluorescent wheat germ agglutinin and by immunolabelling with antibodies directed against fibronectin. InB. bufo andB. calamita gastrulae and in the few hybrids that complete gastrulation, the inner surface of the blastocoel roof is covered by a fibronectin-rich fibrillar matrix. In nucleocytoplasmic hybrids whose development was arrested at the gastrula stage, the fibronectin-containing extracellular matrix was either totally absent or poorly developed and disorganized.