Interaction between cells and elastin fibers: An ultrastructural and immunocytochemical study

Mesenchymal cells (fibroblasts, smooth muscle cells) and endothelial cells were shown to interact with elastin fibers. The strong adhesion of elastin fibers to these cells is mediated by a cell membrane complex with a major glycoprotein component of 120 kDa designated as elastonectin. This interaction was studied by transmission electron microscopy (TEM) and immunocytochemical techniques using antibodies raised against the elastin adhesive proteins. When fibroblasts and smooth muscle cells were cultured in presence of elastin fibers, TEM showed an adhesion mechanism that takes place over several sites along the plasma membrane of these cells. Endothelial cells showed a very close association with elastin, emitting “pseudopodia” that embody the fibers. TEM, indirect immunofluorescence, immunoperoxidase, and confocal microscopy showed the presence and localization of cell membrane components synthesized in large quantities when cells were incubated in presence of elastin. Cells without elastin fibers barely revealed the adhesive membrane complex. These results confirm and extend previous findings concerning the presence of an inducible cell membrane complex that mediates the adhesion of elastin fibers to these cell types. © 1994 Wiley-Liss, Inc.     

Tyrosine phosphorylation signaling regulates Ca2+ entry by affecting intracellular pH during human sperm capacitation

Capacitation is a mandatory process for the acquisition of mammalian sperm fertilization competence and involves the activation of a complex and still not fully understood system of signaling pathways. Under in vitro conditions, there is an increase in both protein tyrosine phosphorylation (pTyr) and intracellular Ca²⁺ levels in several species. In human sperm, results from our group revealed that pTyr signaling can be blocked by inhibiting proline-rich tyrosine kinase 2 (PYK2). Based on the role of PYK2 in other cell types, we investigated whether the PYK2-dependent pTyr cascade serves as a sensor for Ca ²⁺ signaling during human sperm capacitation. Flow cytometry studies showed that exposure of sperm to the PYK2 inhibitor N-[2-[[[2-[(2,3-dihydro-2-oxo-1 H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]- N-methyl-methanesulfonamide hydrate (PF431396) produced a significant and concentration-dependent reduction in intracellular Ca ²⁺ levels during capacitation. Further studies revealed that PF431396-treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca ²⁺ channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca ²⁺ and pH levels after 2 hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca ²⁺ levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH ₄Cl, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca ²⁺ entry in human sperm through regulation of cytoplasmic pH. These results contribute to a better understanding of the modulation of the polymodal CatSper and signaling pathways involved in human sperm capacitation.     

Plasmolysis induced by toluene in a cyoB mutant of Pseudomonas putida

The cyoABCDE gene cluster of Pseudomonas putida DOT-T1E encodes a terminal cytochrome oxidase. A 500-bp 'cyoB' DNA fragment was cloned in pCHESI Omega Km and used to generate a cyoB knock-out mutant in vivo. The mutant strain was not limited in the generation of proton-motif force, although when grown on minimal medium with glucose or citrate, the CyoB mutant exhibited a slight increase in duplication time with respect to the wild-type strain. This effect was even more pronounced when toluene was supplied in the gas phase. In consonance with the negative effect of toluene on the growth was the finding that the CyoB mutant was hypersensitive to sudden 0.3% (v/v) toluene shocks, in contrast with the wild-type strain. This effect was particularly exacerbated in cells that reached the stationary phase. The increased sensitivity to solvents of the CyoB mutant did not appear to be related to the inability of the cells to strengthen the membrane package or to induce the efflux pumps in response to the solvent, but rather to solvent-induced plasmolysis that may be triggered by wrinkles in the cytoplasmic membrane at the poles of the mutant cells, and invagination of the outer membranes, which eventually lead to cell death.     

An ACTH-like peptide immunocortin: effect on the activity of cells from the rat adrenal cortex

The effect of immunocortin, an ACTH-like decapeptide VKKPGSSVKV corresponding to the 11-20 sequence of the variable part of the human IgG1 heavy chain on the content of 11-hydroxycorticosteroids (CS) in rat adrenal glands and blood serum in vivo was studied. An intramuscular injection of immunocortin at a dose of 10 microg/kg was found in an hour to induce a twofold decrease in CS content in the adrenal glands and a 1.8-fold increase in the blood serum CS content. At the same time, an immunocortin dose of 100 microg/kg exerted practically no effect on the CS content and its dose of 1000 microg/kg increased the CS content both in adrenal glands and in blood serum by 1.6 and 2.2 times, respectively. Four hours after the injection of any of the three doses of immunocortin, the CS content in adrenal glands did not differ from the control value, and after 24 h the content decreased threefold. Immunocortin was shown to be bound by the ACTH receptors in the membranes of the rat adrenal cortex with a high affinity and specificity (inhibiting the specific binding of 125I-labeled ACTH-(11-24) peptide with Ki of 1.2 nM).     

DNA immunization with the ribosomal P2beta gene of Trypanosoma cruzi fails to induce pathogenic antibodies

Patients with chronic Chagas' heart disease (cChHD) develop a strong IgG response against the C-terminal region of the Trypanosoma cruzi ribosomal P2beta protein (TcP2beta). These antibodies have been shown to exert an in vitro chronotropic effect on cardiocytes through stimulation of the beta1-adrenergic receptor (beta1-AR). Moreover, the presence of antibodies recognizing the TcP2beta C-terminus was associated with cardiac alterations in mice immunized with the corresponding recombinant protein. Here, we demonstrate that DNA immunization could be used to modulate the specificity of the anti-TcP2beta humoral response in order to avoid the production of pathogenic antibodies. After DNA injection, we detected IgG antibodies that were directed only to internal epitopes of the TcP2beta molecule and that did not exert anti-beta1-AR functional activity, measured as an increase in intracellular cAMP levels of transfected COS-7 cells. Accordingly, DNA-immunized mice did not present electrocardiographic alterations. These data demonstrate that anti-TcP2beta antibodies elicited by DNA immunization are completely different in their specificity and functional activity from those produced during T. cruzi infection.     

Expression in insect cells of active mature cruzipain from Trypanosoma cruzi,containing its C-terminal domain

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi, is a member of the papain family that contains a C-terminal domain in the mature enzyme, in addition to a catalytic moiety homologous to papain and some mammalian cathepsins. The native enzyme is expressed as a complex mixture of isoforms and has not been crystallized. Previous attempts to express recombinant mature cruzipain containing the C-terminal domain have failed. For this reason, the three-dimensional structure of the complete mature enzyme is not known, although the structure of a recombinant truncated molecule lacking the C-terminal domain (cruzaindeltac) has been determined. We report here the expression of active, N-glycosylated, complete mature cruzipain in an insect cell/baculovirus system. The purified recombinant enzyme, obtained with a yield of about 0.2 mg/100 ml of culture supernatant, has an apparent molecular mass similar, and an identical N-terminal sequence, compared with the native enzyme. The expressed protein is able to process itself by self-proteolysis, leaving the isolated C-terminal domain, and has kinetic properties similar to those of native cruzipain, although some differences in substrate specificity were found. These results open up the possibility of obtaining recombinant intact mature cruzipain of a quality and in quantity suitable for X-ray crystallography.     

Wealth, status, and fitness: a historical study of Norwegians in variable environments

Wealth and status covary with lifetime reproductive success in preindustrial human populations. Local ecology is likely to modify this association, but details of this presumed relationship are not yet known. We sought to determine whether local ecology modifies the relationship between status and fitness (number of grandchildren). Our approach to the problem was to measure variation in fitness relative to status (landless or with land) and to local ecology (inland versus coastal communities). We also analyzed life history traits that might explain observed variations in fitness. Our results confirm previous findings that both status (landless=9.9 vs. with land=16.5) and ecology (inland=12.3 vs. coast=14.1) affect the number of grandchildren produced by a female in pre-industrial society. We also found that the differences in number of children between the status groups were less pronounced on the coast (landless=12.0 vs. with land=16.1) than inland (landless=7.8 vs. with land=16.8). Our findings are novel because they suggest that the fitness consequences of human status may depend on details of local ecology. We discuss four different mechanisms that could account for these fitness differences: (1) differential reproductive rate of mothers, (2) differential marriage rate of children (3) differential survival rate of children, and (4) different social practices (breastfeeding, inheritance of property and diet).     

Role of mitochondrial electron transport complex I in coenzyme Q1 reduction by intact pulmonary arterial endothelial cells and the effect of hyperoxia

The objective was to determine the impact of intact normoxic and hyperoxia-exposed (95% O(2) for 48 h) bovine pulmonary arterial endothelial cells in culture on the redox status of the coenzyme Q(10) homolog coenzyme Q(1) (CoQ(1)). When CoQ(1) (50 microM) was incubated with the cells for 30 min, its concentration in the medium decreased over time, reaching a lower level for normoxic than hyperoxia-exposed cells. The decreases in CoQ(1) concentration were associated with generation of CoQ(1) hydroquinone (CoQ(1)H(2)), wherein 3.4 times more CoQ(1)H(2) was produced in the normoxic than hyperoxia-exposed cell medium (8.2 +/- 0.3 and 2.4 +/- 0.4 microM, means +/- SE, respectively) after 30 min. The maximum CoQ(1) reduction rate for the hyperoxia-exposed cells, measured using the cell membrane-impermeant redox indicator potassium ferricyanide, was about one-half that of normoxic cells (11.4 and 24.1 nmol x min(-1) x mg(-1) cell protein, respectively). The mitochondrial electron transport complex I inhibitor rotenone decreased the CoQ(1) reduction rate by 85% in the normoxic cells and 44% in the hyperoxia-exposed cells. There was little or no inhibitory effect of NAD(P)H:quinone oxidoreductase 1 (NQO1) inhibitors on CoQ(1) reduction. Intact cell oxygen consumption rates and complex I activities in mitochondria-enriched fractions were also lower for hyperoxia-exposed than normoxic cells. The implication is that intact pulmonary endothelial cells influence the redox status of CoQ(1) via complex I-mediated reduction to CoQ(1)H(2), which appears in the extracellular medium, and that the hyperoxic exposure decreases the overall CoQ(1) reduction capacity via a depression in complex I activity.     

Increased expression of the dyslexia candidate gene DCDC2 affects length and signaling of primary cilia in neurons

DCDC2 is one of the candidate susceptibility genes for dyslexia. It belongs to the superfamily of doublecortin domain containing proteins that bind to microtubules, and it has been shown to be involved in neuronal migration. We show that the Dcdc2 protein localizes to the primary cilium in primary rat hippocampal neurons and that it can be found within close proximity to the ciliary kinesin-2 subunit Kif3a. Overexpression of DCDC2 increases ciliary length and activates Shh signaling, whereas downregulation of Dcdc2 expression enhances Wnt signaling, consistent with a functional role in ciliary signaling. Moreover, DCDC2 overexpression in C. elegans causes an abnormal neuronal phenotype that can only be seen in ciliated neurons. Together our results suggest a potential role for DCDC2 in the structure and function of primary cilia.