High Density Lipoprotein Stimulated Migration of Macrophages Depends on the Scavenger Receptor Class B,Type I,PDZK1 and Akt1 and Is Blocked by Sphingosine 1 Phosphate Receptor Antagonists

HDL carries biologically active lipids such as sphingosine-1-phosphate (S1P) and stimulates a variety of cell signaling pathways in diverse cell types, which may contribute to its ability to protect against atherosclerosis. HDL and sphingosine-1-phosphate receptor agonists, FTY720 and SEW2871 triggered macrophage migration. HDL-, but not FTY720-stimulated migration was inhibited by an antibody against the HDL receptor, SR-BI, and an inhibitor of SR-BI mediated lipid transfer. HDL and FTY720-stimulated migration was also inhibited in macrophages lacking either SR-BI or PDZK1, an adaptor protein that binds to SR-BI''s C-terminal cytoplasmic tail. Migration in response to HDL and S1P receptor agonists was inhibited by treatment of macrophages with sphingosine-1-phosphate receptor type 1 (S1PR1) antagonists and by pertussis toxin. S1PR1 activates signaling pathways including PI3K-Akt, PKC, p38 MAPK, ERK1/2 and Rho kinases. Using selective inhibitors or macrophages from gene targeted mice, we demonstrated the involvement of each of these pathways in HDL-dependent macrophage migration. These data suggest that HDL stimulates the migration of macrophages in a manner that requires the activities of the HDL receptor SR-BI as well as S1PR1 activity.     

Striking phenotypic and functional differences in lamina propria lymphocytes from the large and small intestine of mice

Although intraepithelial T lymphocytes of the large intestine (LI) are known to differ from those of the small intestine (SI) in phenotype and function, differences in LI and SI lamina propria (LP) lymphocyte populations have not been clearly established. In this work we found striking phenotypic differences between SI and LI LP lymphocyte populations from Balb/c mice analyzed by flow cytometry. In the LI most lymphocytes were B cells and the predominant T cells were TCR-alpha beta+, CD8+. In contrast, in the SI most T lymphocytes were CD4+ expressing TCR-alpha beta+, although a higher proportion expressed TCR-gamma delta+ than in the LI. In T cells the expression of adhesion molecules and cytokines was also different between SI and LI. The proportion of LP T cells expressing alpha4beta7 and L-selectin was higher in the LI than in the SI; whereas a greater proportion of cells expressing alpha(E)beta7 were detected in the SI than in LI. Higher proportions of T cells expressing L-selectin and alpha4beta1 were detected in the intraepithelial compartment of the LI than that of the SI, whereas the number of T cells expressing alpha(E)beta7 was much higher in the SI than in the LI. The proportion of T cells spontaneously producing IL-2, IFN gamma, and IL-4 at the intraepithelial and lamina propria, in the small and large intestine, was different indicating that distinctive functional features exist in the lymphocyte populations residing at the different intestinal compartments.     

Identification of new protein-protein interactions involving the products of the chromosome- and plasmid-encoded type IV secretion loci of the phytopathogen Xanthomonas axonopodis pv. citri

The recently sequenced genome of the bacterial plant pathogen Xanthomonas axonopodis pv. citri contains two virB gene clusters, one on the chromosome and one on a 64-kb plasmid, each of which codes for a previously uncharacterized type IV secretion system (T4SS). Here we used a yeast two-hybrid assay to identify protein-protein interactions in these two systems. Our results revealed interactions between known T4SS components as well as previously uncharacterized interactions involving hypothetical proteins coded by open reading frames in the two X. axonopodis pv. citri virB loci. Our results indicate that both loci may code for previously unidentified VirB7 proteins, which we show interact with either VirB6 or VirB9 or with a hypothetical protein coded by the same locus. Furthermore, a set of previously uncharacterized Xanthomonas proteins have been found to interact with VirD4, whose gene is adjacent to the chromosomal virB locus. The gene for one member of this family is found within the chromosomal virB locus. All these uncharacterized proteins possess a conserved 120-amino-acid domain in their C termini and may represent a family of cofactors or substrates of the Xanthomonas T4SS.     

Pyruvate kinase revisited: the activating effect of K+

For more than 50 years, it has been known that K(+) is an essential activator of pyruvate kinase (Kachmar, J. F., and Boyer, P. D. (1953) J. Biol. Chem. 200, 669-683). However, the role of K(+) in the catalysis by pyruvate kinase has not been totally understood. Previous studies without K(+) showed that the affinity of ADP-Mg(2+) depends on the concentration of phosphoenolpyruvate, although the kinetics of the enzyme at saturating K(+) concentrations show independence in the binding of substrates (Reynard, A. M., Hass, L. F., Jacobsen, D. D. & Boyer, P. D. (1961) J. Biol. Chem. 236, 2277-2283). Here, we explored the kinetics of the enzyme with and without K(+). The results show that without K(+), the kinetic mechanism of pyruvate kinase changes from random to ordered with phosphoenol-pyruvate as first substrate. V(max) with K(+) was about 400 higher than without K(+). In the presence of K(+), the affinities for phosphoenol-pyruvate, ADP-Mg(2+), oxalate, and ADP-Cr(2+) were 2-6-fold higher than in the absence of K(+). This as well as fluorescence data also indicate that K(+) is involved in the acquisition of the active conformation of the enzyme, allowing either phosphoenolpyruvate or ADP to bind independently (random mechanism). In the absence of K(+), ADP cannot bind to the enzyme until phosphoenolpyruvate forms a competent active site (ordered mechanism). We propose that K(+) induces the closure of the active site and the arrangement of the residues involved in the binding of the nucleotide.     

Cytogenetic Analysis of Experimental Hybrids in Species of Triatominae (Hemiptera-Reduviidae)

A cytogenetic analysis was performed in experimental hybrids between species of Chagas disease transmitting bugs with remarkable differences in the amount and distribution of heterochromatin. Using C-banding technique, we identified the parental species chromosomes and analysed the meiotic behaviour in the male hybrids between Triatoma platensis and T. infestans, T. platensis and T. delpontei, and T. infestans and T. rubrovaria. The two former hybrids have an entirely normal meiotic behaviour despite the extensive differences in C-banded karyotypes observed in the parental species, indicating that heterochromatin differences between homeologous chromosomes are not a barrier that influences meiotic synapsis and recombination. On the contrary, the experimental hybrids between T. infestans and T. rubrovaria show failures in pairing of homeologous chromosomes that lead to the production of abnormal spermatids and hybrid sterility. Our data suggest that karyotypic repatterning within triatomines has involved at least two different pathways. Among closely related species, chromosomal changes have largely involved addition or deletion of heterochromatic regions. In more distant species, chromosomal rearrangements (i.e. inversions and translocations) have also arisen. Hybridisation data also allow to hypothesize about the origin and divergence of this taxonomic group, as well as the mechanisms that maintain species isolation.     

Amino-terminally truncated Abeta peptide species are the main component of cotton wool plaques

Cotton wool plaques (CWPs) are round lesions that lack a central amyloid core. CWPs have been observed in individuals affected by early-onset familial Alzheimer disease (FAD) associated with mutations in the presenilin 1 (PSEN1) gene. Here we present the characterization of the amyloid-beta (Abeta) peptides deposited in the brain of an individual affected by FAD carrying the novel missense (V261I) mutation in the PSEN1 gene. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to determine the Abeta peptide species present in the cerebral and cerebellar cortices, in leptomeningeal vessels, and in CWPs isolated by laser microdissection (LMD). Our results indicate that amino-terminally truncated Abeta peptide species ending at residues 42 and 43 are the main Abeta peptides deposited in brain parenchyma and LMD-CWPs in association with the PSEN1 V261I mutation. Full-length Abeta1-42 and Abeta1-43 peptide species were underrepresented. CWPs were not found to be associated with vessels and did not contain Abeta1-40 peptides, the main component of the vascular deposits. Although Abeta deposits were present mostly in the form of CWPs in the cerebral cortex and as diffuse deposits in the cerebellar cortex, a similar array of amino-terminally truncated Abeta peptide species was seen in both cases. The biochemical data support the concept that parenchymal and vascular amyloid deposits are associated with a different array of Abeta peptide species. The generation and parenchymal deposition of highly insoluble amino-terminally truncated Abeta peptides may play an important role in the pathogenesis of AD and must be taken into consideration in developing new diagnostic and therapeutic strategies.     

Entamoeba histolytica: Soluble and membrane-associated neutral sphingomyelinase-C and other unidentified esterase activity

Sphingomyelinase (SMase) activity was measured in Entamoeba histolytica particulate and soluble subcellular fractions. The effects on SMase of incubation time, total protein concentration, pH, and several divalent cations were determined. SMase-C and other unidentified esterase activity were detected in soluble and particulate fractions. SMase-C was 94.5-96.0% higher than the unidentified esterase activity. Soluble and insoluble SMase-C specific activities increased with protein dose and incubation time. Soluble and insoluble SMase-C activities were maximum at pH 7.5 and were dependent on Mg²⁺, Mn²⁺, or Co²⁺, and inhibited by Zn²⁺, Hg²⁺, Ca²⁺, and EDTA. SMase-C was active in the pH range of 3-10 and its maximum activity was at pH 7.5. The soluble and insoluble SMases have remarkably similar physicochemical properties, strongly suggesting that E. histolytica has just one isoform of neutral SMase-C that had not been described before and might be essential for E. histolytica metabolism or virulence.     

Role of the septin Cdc10 in the virulence of Candida albicans

The relationship between the morphology and virulence of Candida albicans has aroused interest in the study of the proteins involved in its morphogenesis. We present virulence data for one important element in fungal morphogenesis-septins. We disrupted CaCDC10 and studied the virulence in a mouse infection model and the different steps followed by the fungus during the infection: adherence to epithelial cells, organ colonisation, macrophage phagocytosis, and host survival. We found the altered subcellular localisation of Int1--a C. albicans adhesin- in the septin null mutants. The Int1 mislocalisation and the defects in the cell wall of defective CaCdc10 strains permit us to propose a model for explaining the biological meaning of the absence of virulence presented by these septin mutants.     

Bcl-2 protects against oxidative stress while inducing premature senescence

Replicative senescence is a cellular response to stress that has been postulated to serve as a tumor suppression mechanism and a contributor to aging. Numerous experimental studies have demonstrated that an apparently identical senescent state can also be prematurely induced in vitro in different cell types following sublethal oxidative stress stimuli. The former suggests a molecular link between cell cycle regulation and cell survival that could involve regulatory proteins such as Bcl-2. There is strong evidence that, in addition to its well-known effects on apoptosis, Bcl-2 is involved in antioxidant protection and regulation of cell cycle progression. The aim of this work was to determine if the protection against oxidative stress mediated by Bcl-2 could prevent or delay oxidative stress-induced senescence. Using a retroviral infection system, Bcl-2 was overexpressed in primary, nonembryonic mice fibroblasts obtained from lungs derived from 2-month-old CD1 mice. Fibroblasts overexpressing Bcl-2 were exposed to 75 microM H2O2 for 2 h to induce SIPS. The rate of proliferation and the increment of senescent cells were then determined. Our results indicate that overexpression of Bcl-2 protected primary fibroblasts against oxidative stress-mediated reduction in cell proliferation, but did not prevent premature senescence.     

Biological and biochemical quality of the Artemia (Anostraca: Artemiidae) population from Real de Salinas saltworks, Calkiní, Campeche, Mexico

Cysts of Artemia spp. collected from February 1997 to February 2000 in the Real de Salinas solar saltworks, Campeche, Mexico, were compared with Artemia franciscana (batch number 8,131 Microfeast Artemia Cysts, Texas, USA). The variables determined in these two populations were: number of cysts per gram, hatching percentage, hatching efficiency, hatching rate, hatching synchrony and hatching biomass, as well as diameter of the cysts and length of the nauplii (instar I). For Salinas, the average diameters of the encapsulated and decapsulated cysts were 230.5 +/- 4.14 and 221.8 +/- 3.39 microm, respectively. The thickness of the cyst shell was 4.35 +/- 0.68 microm and the length of the nauplii was 388.11 +/- 4.39 microm, this last value is among the smallest reported in the literature. For the commercial population of A. franciscana, the average diameters of the encapsulated and decapsulated cysts were 230.21 +/- 12.49 and 216.96 +/- 13.71, respectively. With respect to the corion thickness and length of the nauplii the values were 6.62 +/- 2.72 and 424.70 +/- 30.08, respectively. The protein value of the cysts (47.91 %) and nauplii (50.5 %) of Artemia population from Real de Salinas, are considered adequate to be used as food in aquaculture. The results indicate that the population from Real de Salinas presents positive features for its use in aquaculture in the region.