RET/GFRα Signals Are Dispensable for Thymic T Cell Development In Vivo

Identification of thymocyte regulators is a central issue in T cell biology. Interestingly, growing evidence indicates that common key molecules control neuronal and immune cell functions. The neurotrophic factor receptor RET mediates critical functions in foetal hematopoietic subsets, thus raising the possibility that RET-related molecules may also control T cell development. We show that Ret, Gfra1 and Gfra2 are abundantly expressed by foetal and adult immature DN thymocytes. Despite the developmentally regulated expression of these genes, analysis of foetal thymi from Gfra1, Gfra2 or Ret deficient embryos revealed that these molecules are dispensable for foetal T cell development. Furthermore, analysis of RET gain of function and Ret conditional knockout mice showed that RET is also unnecessary for adult thymopoiesis. Finally, competitive thymic reconstitution assays indicated that Ret deficient thymocytes maintained their differentiation fitness even in stringent developmental conditions. Thus, our data demonstrate that RET/GFRα signals are dispensable for thymic T cell development in vivo, indicating that pharmacological targeting of RET signalling in tumours is not likely to result in T cell production failure.     

Single cell studies of the cell cycle and some models

Analysis of growth and division often involves measurements made on cell populations, which tend to average data. The value of single cell analysis needs to be appreciated, and models based on findings from single cells should be taken into greater consideration in our understanding of the way in which cell size and division are co-ordinated. Examples are given of some single cell analyses in mammalian cells, yeast and other microorganisms. There is also a short discussion on how far the results are in accord with simple models.     

Evidence that P36, a human sperm acrosomal antigen involved in the fertilization process is triosephosphate isomerase

P36 is one of the immunodominant sperm antigens identified by antibodies eluted from the spermatozoa of infertile men. In a previous study, we isolated and characterized this auto-antigen as a glycoprotein with several isoforms. Specific rabbit antibodies were produced to investigate sperm topography and the role of P36 in the fertilization process and we showed that P36 is present on the equatorial segment of acrosome-reacted spermatozoa and is involved in sperm-binding and the penetration of zona-free hamster oocytes. In the present study, we demonstrated, by means of immunofluorescence and electron microscopy, that P36 is present all over the acrosomal membranes of non-reacted spermatozoa. We also investigated the role of P36 in the acrosome reaction and sperm binding to the zona pellucida (ZP). The exposure of capacitated spermatozoa to rabbit anti-P36 antibodies had no effect on primary fixation to the ZP, but inhibited secondary binding to the ZP and the Ca2+ ionophore-induced acrosome reaction. These results suggest that P36, an acrosomal antigen, is involved in several steps of the fertilization process. On two-dimensional Western blots, human anti-sperm antibodies (ASA) and rabbit anti-P36 antibodies recognized five to six isoforms of P36, all 36/37 kDa in size, with a pI between 5.1 and 5.7. Two major spots were identified as human triosephosphate isomerase (TPI) by MALDI-TOF mass spectrometry. Anti-TPI antibodies were shown to react with the isoforms recognized by human and rabbit anti-P36 antibodies. We also demonstrated the presence of TPI in human sperm heads. Further studies are underway to establish whether there is a sperm-specific isoform of TPI and its role in sperm function.     

Apoptosis of phagocytic cells induced by Candida albicans and production of IL-10

Macrophages co-incubated with Candida albicans strain CR1 in vitro showed early signs of apoptosis, but evolved to necrosis after 2 h. In this study, we investigated whether strain CR1 caused apoptosis or necrosis of macrophages after its inoculation into mice peritoneal cavity, and whether this correlated with the secretion of IL-10. Peritoneal macrophages from mice that received an inoculum of C. albicans CR1 showed signs of apoptosis and necrosis from 30 min to 2 h afterwards, whereas heat-killed C. albicans did not cause those effects. IL-10 production was low during the first 6 h post-infection, when macrophages predominated in the peritoneal exudate, whereas its higher production after 24 h correlated with an increase of neutrophils in the exudate. Treatment of CR1 with pepstatin (an inhibitor of proteinases) prevented the process of apoptosis and significantly reduced IL-10 production, suggesting that the increased production of IL-10 was caused by processes occurring during the initial phase of infection, such as apoptosis, necrosis and uptake of death cells.     

A Suitable <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> CVC Strain for Post-Genome Studies

The genome sequence of the pathogen Xylella fastidiosa Citrus Variegated Chlorosis (CVC) strain 9a5c has revealed many genes related to pathogenicity mechanisms and virulence determinants. However, strain 9a5c is resistant to genetic transformation, impairing mutant production for the analysis of pathogenicity mechanisms and virulence determinants of this fastidious phytopathogen. By screening different strains, we found out that cloned strains J1a12, B111, and S11400, all isolated from citrus trees affected by CVC, are amenable to transformation, and J1a12 has been used as a model strain in a functional genomics program supported by FAPESP (São Paulo State Research Foundation). However, we have found that strain J1a12, unlike strains 9a5c and B111, was incapable of inducing CVC symptoms when inoculated in citrus plants. We have now determined that strain B111 is an appropriate candidate for post-genome studies of the CVC strain of X. fastidiosa.     

Cadmium tolerance plasticity in Rhizobium leguminosarum bv. viciae: glutathione as a detoxifying agent

Rhizobium leguminosarum bv. viciae strains expressing different degrees of tolerance to metal stress were used in this work to study the basic mechanisms underlying heavy metal tolerance. We used various parameters to evaluate this response. The strains' growth responses under different Cd2+ concentrations were determined and we reported variation in Cd2+ tolerance. Total soluble protein content decreased drastically, revealing the toxic effects that intracellular Cd2+ imposes on cellular metabolism, but this decrease in protein content was particularly evident in sensitive and moderately tolerant strains. Tolerant strains presented the highest intracellular and wall-bound Cd2+ concentrations. Cd2+ induced increases in the expression of some specific proteins, which were identical in all tolerant strains. Glutathione levels remained unaltered in the sensitive strain and increased significantly in tolerant and moderately tolerant strains, suggesting the importance of glutathione in coping with metal stress. This work suggests that efflux mechanisms may not be the only system responsible for dealing with heavy metal tolerance. A clear correlation between glutathione levels and Cd2+ tolerance is reported, thus adding a novel aspect in bacteria protection against heavy metal deleterious effects.     

Thermodynamics of membrane domains

The concept of lipid rafts and the intense work toward their characterization in biological membranes has spurred a renewed interest in the understanding of domain formation, particularly in the case of cholesterol-containing membranes. The thermodynamic principles underlying formation of domains, rafts, or cholesterol/phospholipid complexes are reviewed here, along with recent work in model and biological membranes. A major motivation for this review was to present those concepts in a way appropriate for the broad readership that has been drawn to the field. Evidence from a number of different techniques points to the conclusion that lipid-lipid interactions are generally weak; therefore, in most cases, massive phase separations are not to be expected in membranes. On the contrary, small, dynamic lipid domains, possibly stabilized by proteins are the most likely outcome. The results on mixed lipid bilayers are used to discuss recent experiments in biological membranes. The clear indication is that proteins partition preferentially into fluid, disordered lipid domains, which is contrary to their localization in ordered, cholesterol/sphingomyelin rafts inferred from detergent extraction experiments on cell membranes. Globally, the evidence appears most consistent with a membrane model in which the majority of the lipid is in a liquid-ordered phase, with dispersed, small, liquid-disordered domains, where most proteins reside. Co-clustering of proteins and their concentration in some membrane areas may occur because of similar preferences for a particular domain but also because of simultaneous exclusion from other lipid phases. Specialized structures, such as caveolae, which contain high concentrations of cholesterol and caveolin are not necessarily similar to bulk liquid-ordered phase.     

Diversity of Cyanobacterial Hydrogenases, a Molecular Approach

In an effort to elucidate the diversity of cyanobacterial hydrogenases, we used a molecular approach. Filamentous strains from a broad range of sources were screened for the presence of hup (uptake hydrogenase), xisC (rearrangement within hupL), and hox (bidirectional hydrogenase) genes. As expected, an uptake hydrogenase seems to be present in all N₂-fixing cyanobacteria. On the other hand, no evidence was found for the presence of a conventional bidirectional enzyme in several strains. Similarly, the presence of xisC is not a characteristic shared by all the heterocyst-forming cyanobacteria. Although tempting, it is not possible to establish a correlation between the presence/absence of the bidirectional hydrogenase and the occurrence of xisC. The natural molecular variation of hydrogenases in cyanobacteria is certainly a field to explore, both to understand the physiological functions of the respective enzymes and to identify a genetic background to be used when constructing a strain for photobiological H₂ production in a bioreactor. Received: 3 November 1999 / Accepted: 8 December 1999     

Hydrolysis by plasma kallikrein of fluorogenic peptides derived from prorenin processing site

Human plasma kallikrein (HPK) activates plasma prorenin to renin, and the physiological significance of this activation is still unknown. In this paper we investigated the efficiency and the cleavage pattern of the hydrolysis by HPK of the internally quenched fluorescent peptides (qf-peptides) derived from the amino acid sequence of human prorenin cleavage site. The peptide Abz-F-S-Q-P-M-K-R-L-T-L-G-N-T-T-Q-EDDnp (Abz=ortho-aminobenzoic acid, and EDDnp=N-[2,4-dinitrophenyl]-ethylene diamine), that corresponds to the amino acid sequence P(7) to P(7)' of human prorenin cleavage site, is hydrolyzed at the correct processing site (R-L bond) with k(cat)/K(m)=85 mM(-1) s(-1). Alanine was scanned in all positions from P(5) to P(5)' in order to investigate the substrate specificity requirements of HPK.The qf-peptides derived from the equivalent segment of rat prorenin, that has Lys-Lys as basic amino acid pair, and the peptide Abz-NVTSPVQ-EDDnp that contains the proposed cleavage site of rat prorenin have very low susceptibility to hydrolysis by rat plasma kallikrein. These data are according to the previously reported absence of rat plasma prorenin activation by rat plasma kallikrein (RPK), and with the view that prorenin activation in rat requires alternative enzymes and/or mechanism.All the obtained peptides described in this paper were also assayed with bovine trypsin that was taken as a reference protease because it is commonly used to activate prorenin.