Reduced mannose incorporation into GDP-mannose and dolichol-linked intermediates of N-glycosylation in hamster liver during vitamin A deficiency

Summary The molecular mechanism of reduced incorporation of radioactively labeled mannose into hamster liver glycoconjugates during the progression of vitamin A deficiency was investigated. In particular the in vivo incorporation of [2-³H]mannose into GDP-mannose, dolichyl phosphate mannose (Dol-P-Man), lipid-linked oligosaccharides, and glycopeptides of hamster liver was examined. Hamsters maintained on a vitamin A-free diet showed a reduction in the incorporation of mannose into GDP-mannose about 10 days before clinical signs of vitamin A deficiency could be observed. The decrease in [2-³H]mannose incorporated into GDP-mannose was accompanied by a reduction in label incorporated into Dol-P-Man, lipid linked oligosaccharides and glycopeptides, which became more severe with the progression of vitamin A deficiency. By the time they reached a plateau stage of growth, hamsters fed the vitamin A-free diet showed a 50% reduction in the amount of [2-³H]mannose converted to GDP-mannose, and the radioactivity associated with Dol-P-Man and glycopeptides was reduced by approximately 60% as compared to retinoic acid-supplemented controls. These results strongly indicate that the reduced incorporation of mannose into lipidic intermediates and glycoproteins observed during vitamin A deficiency is due to impaired GDP-mannose synthesis.Abbreviations Dol-P-Man Dolichyl Phosphate Mannose - Dol-P Dolichyl Phosphate     

Ultrastructure of the larval firefly light organ as related to control of light emission

The firefly larva has a pair of light organs consisting of a layer of interdigitating, light emitting cells, covered dorsally with a layer of opaque, white cells. Each light organ is ventilated by one large and several smaller tracheal branches and is innervated by a branch of the segmental nerve containing two axons. These axons branch profusely in the photocyte layer so that several nerve profiles are seen around any photocyte. Nerve terminals contain large dense-core vesicles and small light-core vesicles. Clusters of light-core vesicles surrounding irregularly shaped membrane densifications, presumably the synapses between nerve and photocyte, are common in nerve terminals. Light emitting cells in insects characteristically contain photocyte vesicles. In the larva there are both full and empty photocyte vesicles; the full vesicles contain a matrix with tubular membrane invaginations in contrast to the empty vesicles which contain amorphous membrane invaginations.     

Fuzziness and Heterogeneity of Benthic Metacommunities in a Complex Transitional System

We propose an extension to the metacommunity (MC) concept and a novel operational methodology that has the potential to refine the analysis of MC structure at different hierarchical levels. We show that assemblages of species can also be seen as assemblages of abstract subregional habitat-related metacommunities (habMCs). This intrinsically fuzzy concept recognizes the existence of habMCs that are typically associated with given habitats, while allowing for the mixing and superposition of different habMCs in all sites and for boundaries among subregions that are neither spatially sharp nor temporally constant. The combination of fuzzy clustering and direct gradient analysis permits us to 1) objectively identify the number of habMCs that are present in a region as well as their spatial distributions and relative weights at different sites; 2) associate different subregions with different biological communities; and 3) quantitatively assess the affinities between habMCs and physical, morphological, biogeochemical, and environmental properties, thereby enabling an analysis of the roles and relative importance of various environmental parameters in shaping the spatial structure of a metacommunity. This concept and methodology offer the possibility of integrating the continuum and community unit concepts and of developing the concept of a habMC ecological niche. This approach also facilitates the practical application of the MC concept, which are not currently in common use. Applying these methods to macrophytobenthic and macrozoobenthic hard-substrate assemblages in the Venetian Lagoon, we identified a hierarchical organization of macrobenthic communities that associated different habMCs with different habitats. Our results demonstrate that different reference terms should be applied to different subregions to assess the ecological status of a waterbody and show that a combination of several environmental parameters describes the spatial heterogeneity of benthic communities much better than any single property can. Our results also emphasize the importance of considering heterogeneity and fuzziness when working in natural systems.     

Morphological, biological and molecular characterization of three strains of Trypanosoma cruzi Chagas, 1909 (Kinetoplastida, Trypanosomatidae) isolated from Triatoma sordida (Stal) 1859 (Hemiptera, Reduviidae) and a domestic cat

A study was conducted of the biological, morphological and molecular characters of 3 strains of Trypanosoma cruzi (SI(5), SI(8) and SIGR(3)) isolated from specimens of Triatoma sordida collected in Santo Inácio and a domestic cat. In order to carry out the study, the following parameters were evaluated: pre-patent period, parasitaemia curves, morphology of the parasites, mortality rates, histopathological lesions and molecular typing. The strains presented variable pre-patent periods, low parasitaemia and no animal mortality. The morphological study of trypomastigotes showed a predominance of intermediate-width and short-length forms, as well as low nuclear index. Epimastigotes presented a low nuclear index, intermediate-width forms in strains SI(5) and SI(8), and large-width forms in SIGR(3). A shorter length could be noted in strains SI(8) and SIGR3, whereas SI(5) displayed an intermediate length. The histopathological study did not detect amastigote nests in tissues. The amplification of the divergent domain of 24Sα rRNA, HSP60 and GPI genes of strains SI(5), SI(8) and SIGR(3) classified the 3 strains into Group II. Biological parameters made it possible to classify the strains isolated in Santo Inácio (BA) into Biodeme III, Zymodeme 1 and Group II of T. cruzi.     

Fine characterization of the Iceman's mtDNA haplogroup

Starting from specimens of the intestinal contents of the so-called Tyrolean Iceman or Otzi (5,350-5,100 years before present), it was possible by polymerase chain reaction to amplify fragments of the human mitochondrial DNA (mtDNA) control region that correspond to the sequence found in 1994 at the Munich and Oxford laboratories and which had been attributed to the original DNA of the mummy. The particularly favorable condition of the specimens, showing very low contamination levels, made it easier to extend the analyses to the coding region, which had not previously been considered. The mtDNA of the European population is currently divided into nine (H, T, U, V, W, X, I, J, and K) main groups (haplogroups). The K haplogroup, in particular, is composed of two (K1 and K2) subclusters. The results demonstrate that the Iceman's mtDNA belongs to the K1 subcluster, yet it does not fit any of the three known branches (a, b, and c) into which the K1 subcluster is presently divided. In addition, some other sites, reported to be linked to environmental adaptation or pathologies, were investigated.     

Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-1(26-35) parental or Melan-A/MART-1(26-35(A27L)) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51-73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27-40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25-36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-1(26-35(A27L)) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-1(25-36)/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.     

Relevance of Fatty Acid Covalently Bound to Escherichia coli ��-Hemolysin and Membrane Microdomains in the Oligomerization Process

α-Hemolysin (HlyA) is an exotoxin secreted by some pathogenic strains of Escherichia coli that causes lysis of several mammalian cells, including erythrocytes of different species. HlyA is synthesized as a protoxin, pro-HlyA, which is activated by acylation at two internal lysines Lys-563 and Lys-689. It has been proposed that pore formation is the mechanism of cytolytic activity for this toxin, as shown in experiments with whole cells, planar lipid membranes, and liposomes, but these experiments have yielded conflicting results about the structure of the pore. In this study, HlyA cysteine replacement mutant proteins of amino acids have been labeled with Alexa-488 and Alexa-546. Fluorescence resonance energy transfer measurements, employing labeled toxin bound to sheep ghost erythrocytes, have demonstrated that HlyA oligomerizes on erythrocyte membranes. As the cytotoxic activity is absolutely dependent on acylation, we have studied the role of acylation in the oligomerization, demonstrating that fatty acids are essential in this process. On the other hand, fluorescence resonance energy transfer and the hemolytic activity decrease when the erythrocyte ghosts are cholesterol-depleted, hence indicating the role of membrane microdomains in the clustering of HlyA. Simultaneously, HlyA was found in detergent-resistant membranes. Pro-HlyA has also been found in detergent-resistant membranes, thus demonstrating that the importance of acyl chains in toxin oligomerization is the promotion of protein-protein interaction. These results change the concept of the main role assigned to acyl chain in the targeting of proteins to membrane microdomains.Escherichia coli α-hemolysin, HlyA,⁴ is an exotoxin that elicits a number of responses from mammalian target cells and also alters the membrane permeability of host cells, causing lysis and death (1, 2). Synthesis, maturation, and secretion of E. coli HlyA are determined by the hlyCABD operon (3). The gene A product is a 110-kDa polypeptide corresponding to protoxin (Pro-HlyA), which is matured in bacterial cytosol to the active form (HlyA) by HlyC-directed acylation. This post-translational modification involves a covalent amide linkage of fatty acids at two internal lysine residues (Lys-563 and Lys-689) for activation (4). HlyA activated in vivo consists of a heterogeneous family of up to nine different covalent structures (two acylation sites and three possible modifying groups in each site, C14:0 (68%), C15:0 (26%) and C17:0 (6%) (5)). Although these fatty acids are not required for the binding of the toxin to membranes, they are essential for the hemolytic process, inducing a molten globule conformation and promoting the irreversibility of the binding (6, 7).It has been proposed that pore formation is the mechanism of cytolytic activity for this toxin, as shown in experiments with whole cells, planar lipid membranes, and liposomes. However, these experiments have yielded conflicting results. Although a group of researchers is in favor of a monomer as the active species of the toxin in membranes, other groups postulate that an oligomerization process is involved. Based on experiments with lipid bilayers, Menestrina et al. (8) have suggested that one single HlyA molecule is responsible for the formation of the channel. HlyA has also been recovered from deoxycholate-solubilized erythrocyte membranes as a monomer, indicating either that oligomerization is not required for pore formation or that oligomers are dissociated in the detergent (1).On the other hand, Benz et al. (9) have found that small variations of toxin concentration have had a considerable effect on the specific membrane conductance. An increase in HlyA concentration, by a factor of 5, results in about 40–100-fold higher membrane conductance. This means that several HlyA molecules could be involved in channel formation (9). Besides, they have found that the active channel-forming oligomer and inactive monomer are in an association-dissociation equilibrium (10). In addition, the complementation of inactive deleted mutant proteins of HlyA with the corresponding wild type toxin produces hemolytic activity, suggesting that two or more toxin molecules aggregate before pore formation (11). All of the evidence suggests the formation of an oligomer.Experiments employing erythrocytes and model membranes have shown that the lesion created by HlyA is perhaps a more complicated event than the creation of a simple, static protein-lined pore. We have recently found that addition of nanomolar concentrations of toxin to planar lipid membranes have resulted in a decrease in membrane lifetime up to 3 orders of magnitude in a voltage-dependent manner, a typical behavior of proteolipidic pores (12). Moayeri and Welch (13) have previously demonstrated that osmotic protection of erythrocytes by sugars of different sizes is a function of toxin concentration and assay time. It appears that HlyA induces heterogeneous erythrocyte lesions that increase in size over time and that the rate of the putative growth in the size of HlyA-mediated lesions is temperature-dependent (13).On the other hand, it has been recognized that a variety of pathogens and toxins interacts with microdomains in the plasma membrane. These microdomains are enriched in cholesterol and sphingolipids and probably exist in a liquid-ordered phase, in which lipid acyl chains are extended and ordered (14). Many proteins are targeted to these membrane microdomains by their favorable association with ordered lipids. Interestingly, these proteins are linked to saturated acyl chains, which partition well into these domains (15).In this context, and in view of the fact that acyl chains covalently bound to proteins are determinant of specific protein-protein interactions, this research presents a study of HlyA oligomerization on sheep erythrocytes, as well as the implication of fatty acids and cholesterol-enriched microdomains in this process.