Ligand-binding specificity of an invertebrate (Manduca sexta) putative cellular retinoic acid binding protein

Intracellular lipid-binding proteins (iLBPs) are small cytoplasmic proteins that specifically interact with hydrophobic ligands. Fatty acid-binding proteins (FABPs), cellular retinoic acid-binding proteins (CRABPs) and cellular retinol-binding proteins (CRBPs) belong to the iLBP family. A recently identified insect (Manduca sexta) iLBP has been reported to possibly represent an invertebrate CRABP mimicking the role of CRABPs in vertebrate organisms. The presence in this protein of the characteristic binding triad residues involved in the interaction with ligand carboxylate head groups, a feature pertaining to several FABPs and to CRABPs, and the close phylogenetic relationships with both groups of vertebrate heart-type FABPs and CRBPs/CRABPs, makes it difficult to assign it to either FABPs or CRABPs. However, its negligible interaction with retinoic acid and high affinity (K(d) values in the 10(-8) M range) for fatty acids have been established by means of direct and competitive binding assays. As shown by phylogenetic analysis, the M. sexta iLBP belongs to a wide group of invertebrate iLBPs, which, besides being closely related phylogenetically, share distinctive features, such as the conservation of chemically distinct residues in their amino acid sequences and the ability to bind fatty acids. Our results are in keeping with the lack of cellular retinoid-binding proteins in invertebrates and with their later appearance during the course of chordate evolution.     

Flux of sterol intermediates in a yeast strain deleted of the lanosterol C-14 demethylase Erg11p

Lanosterol C-14 demethylase Erg11p of the yeast Saccharomyces cerevisiae catalyzes the enzymatic step following formation of lanosterol by the lanosterol synthase Erg7p in lipid particles (LP). Localization experiments employing microscopic inspection and cell fractionation revealed that Erg11p in contrast to Erg7p is associated with the endoplasmic reticulum (ER). An erg11Delta mutation in erg3Delta background, which is required to circumvent lethality of the erg11 defect, did not only change the sterol pattern but also the sterol distribution within the cell. Whereas in wild type the plasma membrane was highly enriched in ergosterol and LP harbored large amounts of sterol precursors in the form of steryl esters, sterol intermediates were more or less evenly distributed among organelles of erg11Delta erg3Delta. This distribution is not result of the erg3Delta background, because in the erg3Delta strain the major intermediate formed, ergosta-7,22-dienol, is also highly enriched in the plasma membrane similar to ergosterol in wild type. These results indicate that (i) exit of lanosterol from LP occurs independently of functional Erg11p, (ii) random supply of sterol intermediates to all organelles of erg11Delta erg3Delta appears to compensate for the lack of ergosterol in this mutant, and (iii) preferential sorting of ergosterol in wild type, but also of ergosta-7,22-dienol in erg3Delta, supplies sterol to the plasma membrane.     

Mutations in the "lid" region affect chain length specificity and thermostability of a Pseudomonas fragi lipase

The cold-adapted Pseudomonas fragi lipase (PFL) displays highest activity on short-chain triglyceride substrates and is rapidly inactivated at moderate temperature. Sequence and structure comparison with homologous lipases endowed with different substrate specificity and stability, pointed to three polar residues in the lid region, that were replaced with the amino acids conserved at equivalent positions in the reference lipases. Substitutions at residues T137 and T138 modified the lipase chain-length preference profile, increasing the relative activity towards C8 substrates. Moreover, mutations conferred to PFL higher temperature stability. On the other hand, replacement of the serine at position 141 by glycine destabilized the protein.     

2.45 GHz radiofrequency fields alter gene expression in cultured human cells

The biological effect of radiofrequency (RF) fields remains controversial. We address this issue by examining whether RF fields can cause changes in gene expression. We used the pulsed RF fields at a frequency of 2.45 GHz that is commonly used in telecommunication to expose cultured human HL-60 cells. We used the serial analysis of gene expression (SAGE) method to measure the RF effect on gene expression at the genome level. We observed that 221 genes altered their expression after a 2-h exposure. The number of affected genes increased to 759 after a 6-h exposure. Functional classification of the affected genes reveals that apoptosis-related genes were among the upregulated ones and the cell cycle genes among the downregulated ones. We observed no significant increase in the expression of heat shock genes. These results indicate that the RF fields at 2.45 GHz can alter gene expression in cultured human cells through non-thermal mechanism.     

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.     

Structure-function studies of PANDER, an islet specific cytokine inducing cell death of insulin-secreting beta cells

PANDER (pancreatic derived factor, FAM3B) is a novel cytokine, present in insulin secretory granules, that induces apoptosis of alpha and beta cells of mouse, rat, and human islets in a dose- and time-dependent manner, and may be implicated in diabetes. PANDER has the predicted secondary structure of 4 alpha-helical bundles with an up-up-down-down topology, and two disulfide bonds. Eleven mutated PANDERs were constructed and expressed in beta-TC3 cells to identify the essential region of PANDER involved in beta-cell death. Beta-cell function was assessed by assays of cell viability and insulin secretion. Based on quantitative real-time RT-PCR all mutant PANDERs had similar mRNA expression levels in beta-TC3 cells. Immunoblotting showed that ten of eleven mutant PANDER proteins were synthesized and detected in beta-TC3 cells. A mutant PANDER with no signal peptide, however, was not expressed. Truncation of helix D alone caused a 40-50% decrease in PANDER's activity, while truncation of both helices C and D resulted in a 75% loss of activity. In contrast, truncation of the N-terminus of PANDER (helix A, the loop between helices A and B, and the first two cysteines) had no effect on PANDER-induced beta-cell death. The third and fourth cysteines of PANDER, C91 and C229, were shown to form one disulfide bond and be functionally important. Finally, the region between Cys91 and Phe152 constitutes the active part of PANDER, based on the demonstration that mutants with truncation of helix B or C caused decreased beta-cell death and did not inhibit insulin secretion, as compared to wild-type PANDER. Hence, helices B and C and the second disulfide bond of PANDER are essential for PANDER-induced beta-cell death.     

Calcium and magnesium binding to human centrin 3 and interaction with target peptides

There are four isoforms of centrin in mammals, with variable sequence, tissue expression, and functional properties. We have recently characterized a number of structural, ion, and target binding properties of human centrin isoform HsCen2. This paper reports a similar characterization of HsCen3, overexpressed in Escherichia coli and purified by phase-reversed chromatography. Equilibrium and dynamic binding studies revealed that HsCen3 has one mixed Ca(2+)/Mg(2+) binding site of high affinity (K(d) = 3 and 10 microM for Ca(2+) and Mg(2+), respectively) and two Ca(2+)-specific sites of low affinity (K(d) = 140 microM). The metal-free protein is fragmented by an unidentified protease into a polypeptide segment of 11 kDa, which was purified by HPLC, and identified by mass spectrometry as the segment of residues 21-112. Similarly, controlled trypsinolysis on Ca(2+)-bound HsCen3 yielded a mixture of segments of residues 1-124 and 1-125. The Ca(2+)/Mg(2+) site could be assigned to this segment and thus resides in the N-terminal half of HsCen3. Temperature denaturation experiments, circular dichroism, and utilization of fluorescence hydrophobic probes allowed us to propose that the metal-free protein has molten globule characteristics and that the dication-bound forms are compact with a polar surface for the Mg(2+) form and a hydrophobic exposed surface for the Ca(2+) form. Thus, HsCen3 could be classified as a Ca(2+) sensor protein. In addition, it is able to bind strongly to a model target peptide (melittin), as well as to peptides derived from the protein XPC and Kar1p, with a moderate Ca(2+) dependence.     

Enhanced tolerance to autoimmune uveitis in CD200-deficient mice correlates with a pronounced Th2 switch in response to antigen challenge

A single exposure to inhaled Ag 10 days before immunization leads to long term, Ag-specific tolerance. Respiratory tract myeloid APCs are implicated, but how regulation is invoked, and how tolerance is sustained are unclear. This study examines the in vivo function of the myeloid regulatory molecule CD200 in the process of tolerance induction. Despite earlier onset of experimental autoimmune uveitis in sham-tolerized, CD200-deficient mice, disease incidence and subsequent severity were actually reduced compared with those in wild-type mice. Protection was more effective and long term, lasting at least 28 days. Halting disease progression and tolerance in CD200(-/-) mice correlated with a marked increase in Th2-associated cytokine production by Ag-challenged splenocytes. Reduced overall disease and enhanced tolerance in the CD200-deficient mice in this model system were unexpected and may be related to altered populations of MHC class II(low) APC in the respiratory tract compared with wild-type mice together with associated activation of STAT6 in draining lymph nodes of tolerized mice. These data indicate that in the absence of default inhibitory CD200 receptor signaling, alternative, powerful regulatory mechanisms are invoked. This may represent either permissive dominant Th2 activation or an altered hierarchy of negative signaling by other myeloid cell-expressed regulatory molecules.     

Cryptococcus neoformans capsular glucuronoxylomannan induces expression of fas ligand in macrophages

The major component of capsular material of Cryptococcus neoformans is glucuronoxylomannnan (GXM), a polysaccharide that exhibits potent immunosuppressive properties in vitro and in vivo. The results reported here show that 1) soluble purified GXM induces a prompt, long-lasting, and potent up-regulation of Fas ligand (FasL) on macrophages, 2) the up-regulation of FasL is related to induced synthesis and increased mobilization to the cellular surface, 3) this effect is largely mediated by interaction between GXM and TLR4, 4) FasL up-regulation occurs exclusively in GXM-loaded macrophages, 5) macrophages that show up-regulation of FasL induce apoptosis of activated T cells expressing Fas and Jurkat cells that constitutively express Fas, and 6) anti-Fas Abs rescue T cells from apoptosis induced by GXM. Collectively our results reveal novel aspects of the immunoregulatory properties of GXM and suggest that this nontoxic soluble compound could be used to dampen the immune response, to promote or accelerate the death receptor, and to fix FasL expression in a TLR/ligand-dependent manner. In the present study, we delineate potential new therapeutic applications for GXM that exploit death receptors as key molecular targets in regulating cell-mediated cytotoxicity, immune homeostasis, and the immunopathology of diseases.