Regulation by phagic T-lymphocytes of a (pluripotent?) organ stem cell present in adult human blood. A beneficial exception to self-tolerance.

Stem cells isolated from adult human blood are able to give rise to several different kinds of cell types such as mesenchymal cells, including striated muscle cells, hepatocytes, and endothelial-cells. Because independently studied by authors whose interests focused on particular tissue types, these stem cells have been described as different. However, they might well represent one unique population of pluripotent stem cells in homeostatic equilibrium with the 'reserve' stem cells buried in organs. In the blood, these stem cells have a monocytic phenotype. In in vitro culture, once they have adhered, they spontaneously differentiate into diverse types of cells reminiscent of embryonic stem cells in culture. Normally, they are almost quiescent cells. But under precise circumstances such as wound-healing, they may proliferate and migrate to the right organ to give rise there to the right type of cells, in order to participate in the repair process. Indeed, such a powerful stem cell needs to be tightly controlled. We illustrate here, by time-lapse videocinematography, how a special subpopulation of T-lymphocytes, for which we coined the name 'phagic T-lymphocytes' (PTLs), destroys these stem cells as soon as they differentiate in vitro, i.e., without the purpose of a repair. These stem cells express constitutively HLA-DR molecules and therefore can act as antigen-presenting cells able to activate phagic T-lymphocytes. The targets of these activated phagic T-lymphocytes are the differentiated stem cell themselves. Phagic T-lymphocytes are attracted by the stem cells, circulate around them, then penetrate and circulate inside them until the latter 'explode'. This mechanism of destruction by phagic T-lymphocytes is unique and seems to be normally restricted to stem cells. It represents a beneficial exception in self-tolerance since it avoids the accumulation of these stem cells out of healing purposes. Interestingly, in disorders such as fibrosis and/or some malignant proliferations, these stem cells proliferate, escape destruction by phagic T-lymphocytes and, as a consequence, accumulate, giving rise to a 'tissue' when cultured in vitro.

Fusion of Leishmania amazonensis parasitophorous vacuoles with phagosomes containing zymosan particles: cinemicrographic and ultrastructural observations.

Studies on fixed preparations have shown that vacuoles containing zymosan (Z) particles internalized by infected macrophages can selectively fuse with the large parasitophorous vacuoles (PVs) that shelter Leishmania amazonensis. To examine the kinetics of vacuolar fusion in individual cells, particles were followed by time-lapse cinemicrography from their uptake to their entry in a PV. Newly formed Z-containing vacuoles moved centripetally and, if they contacted a PV, the two vacuoles remained closely apposed for variable, often extended, periods of time before they eventually fused. Transmission electron microscopy confirmed that the cytoplasm separating the partner vacuoles could be reduced to a very thin layer. Initiation of fusion was indicated by reduced refractility of the boundary between Z vacuoles and target PVs. Within a few minutes the PV enlarged and encompassed the Z particles, which remained immobile throughout. The interval between phagocytosis and fusion, 50 +/- 7.4 min (N = 17; range, 4 to 108 min), suggests that most but not all Z vacuoles underwent significant maturation by the time of fusion. Some particles were transferred singly, others entered PVs in groups of 2 or more, and additional clustered transfers to the same vacuole were also observed. These observations provide a baseline for studies of the biochemical mechanisms and the pharmacological control of the fusion of Leishmania PVs, and for the comparison of the fusion behavior of the PVs with that of other phagocytically derived vacuoles.

Cloning and analysis of calbindin-D28K cDNA and its expression in the central nervous system.

The vitamin D-dependent calcium-binding protein (CaBP), calbindin-D28K (CaBP28K), is present in the central nervous system (CNS), the sensory system, and kidneys of mammals and birds. Recent studies have indicated that several other CaBPs of very similar Mrs are also present in the CNS. This study was carried out to establish the relationship between CaBP28K and other CaBP, particularly spot 35, to provide a basis for further studies on the tissue-specific regulation and distribution of CaBP28K. A cloned pC28 cDNA was isolated from a rat brain expression library using synthetic oligodeoxyribonucleotides (oligos) complementary to rat spot-35 mRNA. This pC28 cDNA had an open reading frame (ORF) of 783 nucleotides (nt) coding for a 261-aa, 30-kDa protein. There was 100% homology between the pC28 sequence and that of the CaBP28K isolated from rat brain cDNA library using a chicken intestinal CaBP28K probe (Hunziker and Schrickel, 1988). Thus the aa and nt sequences of rat CaBP28K and spot 35 are identical. Primer extension studies and Northern analyses show that the major species of CaBP28K mRNA contains a 5'-untranslated region of 132 nt, a coding region of 261 codons and a 3'-untranslated region of 804 nt without the poly(A) tail. The rat CaBP28K probe hybridizes to one major RNA species (1.9 kb) and two minor ones (2.8 and 3.2 kb) in the cerebellum, hippocampus, retina and kidney. This distribution correlates well with the distribution of CaBP28K itself in these organs. Comparison of the genomic organization of the CaBP28K gene with that of other members of the 'EF-hand' CaBP family emphasizes that the CaBP28K gene diverged from the others at the first duplication of the gene encoding one CaBP domain. All the members of the 'EF-hand' gene CaBP family evolved by exon shuffling and specific genomic rearrangements.

Immobilized colipase affinities for lipases B, A, C and their terminal peptide (336-449): the lipase recognition site lysine residues are located in the C-terminal region.

Zonal high-performance affinity chromatography has been used in order to study the interactions between pig isolipases A, B and C and the terminal peptide chain fragment 336-449 of the pig lipase on the one hand, and the homolog colipase bound to the inert LiChrosorb diol support on the other. A mathematical treatment led the to assessment of the dissociation constant of the lipase-colipase complex using isolipases or the terminal peptide as eluted acceptors and colipase as silica-bound ligand (Mahé, N., Léger, C.L., Linard, A. and Alessandri, J.-M. (1987) J. Chromatogr. 395, 511-521). A higher affinity of isolipase B as compared to isolipases A and C towards colipase was observed (KD, respectively, of 0.68, 11 and 12 microM) at pH 6.5. Under the same chromatographic conditions, the terminal peptide chain interacted with the bound colipase (KD 0.70 microM, close to that of isolipase B). The chromatographic behaviors of both native and chemically modified lipase and terminal peptide were very similar. In particular, guanidination of lysine residues of both peptide and isolipase B led to the loss of interactions with colipase. The same result was observed with the peptide preincubated in the presence of increasing amounts of free colipase. Accordingly, it is suggested that, firstly, a preferential association of isolipase B to colipase could take place and, secondly, the colipase recognition site of lipase could be located in the C-terminal region, the conformational structure of the terminal peptide not being affected by the enzymic cleavage and, therefore, being largely independent of the rest of the polypeptide molecule. On the other hand, a lower colipase affinity for isolipases A or C than for isolipase B or the C-terminal peptide could tentatively be attributed to a non-local (distant) disturbing effect of the negatively charged glycan chain, as sialic acid is present in both isoforms A and C. Finally, the present paper confirms and extends earlier studies on lipase-colipase interactions.

The rat vitamin-D-dependent calcium-binding protein (9-kDa CaBP) gene. Complete nucleotide sequence and structural organization.

The structural organization of the entire rat vitamin-D-dependent calcium-binding protein (9-kDa CaBP) gene was determined by analysis of overlapping genomic clones isolated from a rat genomic library using the rat 9-kDa CaBP cDNA [Desplan C., Heidmann O., Lillie J., Auffray C. and Thomasset M. (1983) J. Biol. Chem. 258, 13502-13505]. These clones together span 30 kbp of rat genomic DNA, with the rat 9-kDa CaBP gene lying in the middle. The 9-kDa CaBP gene is 2.5 kbp long and contains three exons interrupted by two introns. The first exon contains almost the entire 5' untranslated region. The second exon codes for the calcium-binding site I, the third exon codes for site II and the 3' untranslated region. Therefore each of the calcium-binding domains is encoded by single, separate exons. The transcription initiation site was identified by S1 nuclease mapping and primer extension. A consensus sequence TATAAA is localized 31 bp upstream from the cap site and the 'CCAAT-box' lies upstream from the transcription start. Single (AC)25 and (AG)23 repeats are present in the second intron together with an Alu-like sequence. Repetitive elements are present 5 kbp upstream from the cap site and in the 3' flanking region. Comparison of the known rat CaBP sequences (9-kDa CaBP, 28-kDa CaBP, S100 protein) shows that the 9-kDa CaBP is more closely related to the S100 protein than to the 28-kDa CaBP. There is no evidence to indicate that 9-kDa CaBP has arisen from the 28-kDa CaBP.