Phagocytosis is coupled to the formation of phagosome-associated podosomes and a transient disruption of podosomes in human macrophages

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.     

Simple and selective high-performance liquid chromatographic method for estimating plasma quinidine levels

A reversed-phase, high-performance liquid chromatographic method employing fluorescence detection is described for the rapid quantification of plasma levels of quinidine, dihydroquinidine and 3-hydroxyquinidine. It involves protein precipitation with acetonitrile followed by direct injection of the supernatant into the chromatograph. For the preparation of plasma standards, pure 3-hydroxyquinidine was isolated from human urine by a simplified thin-layer chromatographic procedure. The mobile phase for the chromatography was a mixture of 1.5 mM aqueous phosphoric acid and acetonitrile (90:10) at a flow-rate of 2 ml/min. The intra-assay coefficient of variation for the assay of quinidine and 3-hydroxyquinidine over the concentration range 2.5–20 μmole/l was < 1% for both. Interassay coefficients of variation for quinidine (10 μmole/l) and 3-hydroxyquinidine (5 μmole/l) were 3.5% and 4.0% with detection limits of 50 and 25 μmole/l respectively. The method correlated well (r² = 0.96) with an independently developed gas—liquid chromatographic—nitrogen detection assay for quinidine which also possessed a high degree of precision. (Intra-assay coefficient of variation 3.6% at 20 μmole/l). As expected, comparison of the high-performance liquid chromatographic assay with a published protein precipitation—fluorescence assay showed poor correlation (r² = 0.78).     

Identification of HLA-B44 subtypes associated with extended MHC haplotypes

The HLA class I antigen B44 is found in each of two different extended major histocompatibility haplotypes (allele combinations of HLA-B, HLA-DR, and complement genes BF, C2, C4A, and C4B in linkage disequilibrium). Using isoelectric focusing, two variants of HLA-B44 were identified. The basic variant was found in all cell lines with the extended haplotype HLA-B44, DR7, FC31, and the acidic variant in all cell lines with the extended haplotype HLA-B44, DR4, SC30. The occurrence of each antigen variant with a unique extended haplotype explains previous observations concerning the nonrandom association of B44 variants with DR antigens.     

Coupling between phosphoinositide breakdown and early mitogenic events in fibroblasts. Studies with fluoroaluminate, vanadate, and pertussis toxin

In the preceding paper (Paris, S., and Pouysségur J. (1987) J. Biol. Chem. 262, 1970-1976), AlF4- and vanadate have been shown to induce inositol phosphate formation in resting hamster fibroblasts (CCL39). In this study, we show that these two phosphate analogs are good tools to explore the causal relationship between phosphoinositide breakdown and early mitogenic events. AlF4- can activate, very similarly to the mitogen alpha-thrombin: the amiloride-sensitive Na+/H+ antiport, the bumetanide-sensitive Na+/K+/Cl- co-transport, and the expression of c-myc mRNA. The link between phospholipase C activation and these early events of the mitogenic response is demonstrated by the similarity of all dose-response curves for NaF and AlCl3 and by the common sensitivity of the four events to pertussis toxin. Vanadate likewise stimulates the Na+/H+ antiport through a pertussis toxin-sensitive pathway. On longer incubations, both fluoride and vanadate were found to be toxic and failed to induce DNA synthesis. Therefore, we have used pertussis toxin to investigate the link between phospholipase C activation and commitment to DNA synthesis. We show that pertussis toxin strikingly inhibits thrombin-induced reinitiation of DNA synthesis but does not affect the stimulation by the epidermal or fibroblast growth factors, two mitogens that do not stimulate phosphoinositide breakdown in CCL39 cells. In conclusion, these studies demonstrate that activation of phospholipase C, if not an obligatory step in the action of all growth factors, plays a crucial role in the mitogenic signaling pathway of alpha-thrombin.     

The study of a French family with two duplicated C4A haplotypes

Summary The finding of two duplicated C4A haplotypes in a normal French family led to a detailed study of their C4 polymorphism. The father had an extremely rare A^*6A^*11, B^* QO haplotype inherited by all of his children and the mother had the more common A^*3A^*2, B^*QO haplotype. Two HLA identical daughters only have four C4A alleles. The father's A11 allotype expresses Ch: 1 (Chido) rather than Rg:1 (Rodgers) and represents a new Ch phenotype Ch: 1,-2,-3,-4,-5,-6. In order to clarify the genetic background in this unusual family, DNA studies of restriction fragment length polymorphisms (RFLPs) were undertake. The father's rare haplotype, which expresses two C4A allotypes, results from a long and a short C4 gene normally associated with the A^*6, B^*1 that also exhibits the BglII RFLP. As it travels in an extended MHC haplotype HLA A2, B57 (17), C2^*C, BF^*S, DR7 that is most frequently associated with A^*6, B^*1, we postulate that the short C4B has been converted in the chain region to a C4A gene which produces a C4A protein. This report of a short C4A gene is the first example in the complex polymorphism of C4.     

Mammalian cell processing of a unique uracil residue in simian virus 40 DNA.

The processing of a unique uracil in DNA has been studied in mammalian cells. A synthetic oligodeoxyribonucleotide carrying a potential Bgl II restriction site, where one base has been substituted with a uracil, was inserted in the early intron of SV40 genome. Various heteroduplexes were constructed in such a manner that the restitution of an active Bgl II restriction site corresponds in each case to the specific substitution of the uracil by one of the four bases normally present in the DNA. DNA cuts by this restriction enzyme in one or several constructed heteroduplexes immediately determine the type of base pair substitution produced at the site of the U residue. When the uracil is inserted opposite a purine it is fully repaired; when facing a guanine it is replaced by a cytosine and opposite an adenine it is replaced by a thymine. These results indicate the error-free repair of uracil when it appears in the cell with the usual mechanisms such as cytosine deamination or incorporation of dUTP in place of dTTP during replication. When the uracil is inserted opposite a pyrimidine no error free repair at all is detected for U:C or U:T mismatches. It appears, moreover, that in approximately 18% of the cases U:T mismatch leads to a C:G base pairing. In the majority of the U:pyrimidine mismatches, mutations occur in the vicinity of the uracil, including base substitutions and frameshifts by addition of one or several bases.     

Nasal allergy to fungi in guinea pigs

Sensitized guinea pigs produced specific IgG and IgE antibodies toward Cladosporium and Alternaria. In presence of fungal extracts, nasal mast cells degranulate. Ultrastructural modifications of the cells during degranulation have been established. The ciliary epithelium and the ciliary beating are not affected by fungal allergens.