Structure and the energy of base pairing in non-natural bases of nucleic acids: the azaguanine-cytosine and azaadenine-thymine base pairs

Watson-Crick optimized geometries and the energies of base pairing for the natural pairs of nucleic bases: adenine-thymine (AT) and guanine-cytosine (GC) have been recalculated by ab initio methods in order to compare results to those found for the non-natural azaadenine-thymine (AAT) and azaguanine-cytosine (AGC) pairs. Geometry optimizations carried out at the HF/6-31G** level and energies obtained at MP2/6-31G**, show that AAT and AGC have hydrogen bonding patterns similar to the natural AT and GC and that the interaction energies (DeltaH0int) for the former are ca. 7 kcal/mol more stable than the latter. Accordingly, the pairs based on azapurines would be favored with respect to the natural pairs. Some possible explanations why nature does not use extensively the azabases in base pairing are given.     

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

Amastigogenesis occurs first when metacyclic trypomastigotes from triatomine urine differentiate into amastigotes inside mammalian host cells and a secondary process when tissue-derived trypomastigotes invade new cells and differentiate newly to amastigotes. Using scanning electron microscopy, we compared the morphological patterns manifested by trypomastigotes and metacyclic forms of Trypanosoma cruzi during their axenic-transformation to amastigotes in acidic medium at 37 C. We show here that in culture MEMTAU medium, secondary and primary axenic amastigogenesis display different morphologies. As already described, we also observed a high differentiation rate of trypomastigotes into amastigotes. Conversely, the transformation rate of in vitro-induced-metacyclic trypomastigotes to amastigotes was significantly slower and displayed distinct patterns of transformation that seem environment-dependent. Morphological comparisons of extracelullar and intracellular amastigotes showed marked similarities, albeit some differences were also detected. SDS-PAGE analyses of protein and glycoprotein from primary and axenic extracelullar amastigotes showed similarities in glycopeptide profiles, but variations between their proteins demonstrated differences in their respective macromolecular constitutions. The data indicate that primary and axenic secondary amastigogenesis of T. cruzi may be the result of different developmental processes and suggest that the respective intracellular mechanisms driving amastigogenesis may not be the same.     

Cultivation of explants of the marine sponge Crambe crambe in closed systems

Explants of the sponge Crambe crambe were cultured in natural seawater, with or without marine microalga (Phaeodactylum tricornutum) in discontinuous flow through systems and in continuous flow-through systems (DFTHS and CFTHS, respectively). Growth was measured as the increase in underwater weight. In the experiment carried out in the CFTHS, the explants average underwater weight increased by up to 1380% of the initial weight in 22-45 days. Growth in DFTHS was much slower producing a gain of up to an average value of 322% of the initial weight in 100-210 days. Growth kinetics varied considerably for different explants. Explants grew fastest in the first 10-days of subculture. The sponges grew better in CFTHS compared with the DFTHS. The high growth rates observed in CFTHS suggest that this technique is a promising method for culturing C. crambe in closed systems.     

Host plant and natural enemy impact on cereal aphid competition in a seasonal environment

Competition between phytophagous insects could be mediated by differential impact of natural enemies or by induced changes in host plant quality. The interaction between the aphids Sipha flava and Rhopalosiphum maidis on the shared host plant, Sorghum halepense , was evaluated during the fall and spring seasons in the presence or absence of natural enemies in a garden experiment. During the fall, S. flava was negatively affected by R. maidis in all treatments. However, during the spring season, S. flava was not affected by R. maidis when natural enemies were excluded, and positively affected by R. maidis with natural enemies present. Rhopalosiphum maidis was negatively affected by S. flava during the fall and spring seasons under all treatments. Laboratory experiments demonstrated that changes in host plant quality induced by S. flava attack and the presence of the parasitoid Lysiphlebus testaceipes negatively affected R. maidis , but not S. flava . Under abiotic conditions simulating fall and spring (lower temperature and daylength in the fall than in the spring), S. flava showed lower performance than R. maidis under "fall" conditions, and both aphid species showed similar life history traits under "spring" conditions. For S. flava , the time to the first reproduction and longevity were longer, and the nymphal production was lower, under "fall" conditions than under "spring" conditions. Our results showed that S. flava is benefited when R. maidis and the parasitoid L. testaceipes are present but the abiotic conditions are not adverse.     

P190-B, a Rho-GTPase-activating protein, is differentially expressed in terminal end buds and breast cancer.

Microdissection and differential display PCR were used to identify genes preferentially expressed in the highly proliferative terminal end buds (TEBs) in the mammary gland of 45-day-old virgin rats. One clone exhibited 87% homology to the human p190-B gene encoding a novel Rho-Gap. Using in situ hybridization, p190-B was detected in both the TEBs and the terminal ducts, with the highest expression observed in the outer layer of TEBs. During normal mammary gland development, p190-B mRNA expression was highest in the virgin mammary gland and decreased during late pregnancy and lactation. Interestingly, increased levels of p190-B mRNA relative to the normal mammary gland were seen in a subset of murine mammary tumors that appeared to be less well differentiated and potentially more aggressive. Transient transfection of a p190-B expression construct into MCF-10A human mammary epithelial cells resulted in disruption of the actin cytoskeleton, which suggests a role for p190-B in regulating the signaling pathways that influence cell migration and invasion. These results suggest that p190-B may be required for virgin mammary gland development, and its aberrant expression may occur in breast cancer.     

Characterization of sialyltransferase mutants using surface plasmon resonance

Sialyltransferases are enzymes responsible for the important sialylation of glycoconjugates. Since crystal structures are not available, other tools are needed to study enzymatic mechanisms. As a model, we used human alpha2,6-sialyltransferase. A putative acceptor-binding domain containing the small and the very small sialyl motifs was randomly mutated. This resulted in enzymes with altered enzymatic activity. Affinity chromatography demonstrated that their binding to donor substrate was maintained. To illustrate the role of the mutated domain in acceptor binding, a method based on surface plasmon resonance was set up. Only at low salt and high acceptor concentration was association of wild-type ST6GalI with asialofetuin demonstrated. As expected, this interaction was affected by cytidine 5'-monophospho-N-acetylneuraminic acid, the donor substrate, which proves the specificity of the interaction. Different types of mutants were found. For some, the drop in activity could be explained by loss in affinity for the acceptor. For others, the catalytic center, but not the acceptor-binding site, was affected. Neither acceptor binding nor catalytic activity were limited to the sialyl motifs. To our knowledge, this is the first example in which surface plasmon resonance is successfully used to demonstrate the binding of a glycosyltransferase to its natural acceptor.     

Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNA-sequencing equipment

The analysis of protein-linked glycans is of increasing importance, both in basic glycobiological research and during the production process of glycoprotein pharmaceuticals. In many cases, the amount of glycoprotein available for typing the glycans is very low. This, combined with the high branching complexity typical for this class of compounds, makes glycan typing a challenging task. We present here methodology allowing the medium-throughput analysis of N-glycans derived from low picomole amounts of glycoproteins using the standard DNA-sequencing equipment available in any life sciences laboratory. The high sensitivity of the overall analytical process (from glycoprotein to results) is obtained using state-of-the-art deglycosylation procedures combined with a highly efficient and reproducible novel postderivatization cleanup step involving Sephadex G10 packed 96-well filterplates. All sample preparation steps (enzymatic deglycosylation with PNGase F, desalting, derivatization with 8-amino-1,3,6-pyrenetrisulfonic acid, and postderivatization cleanup) are performed using 96-well-based plates. This integrated sample preparation scheme is also compatible with capillary electrophoresis and MALDI-TOF-MS platforms already in use in some glycobiology labs and anticipates the higher throughput that will be offered by the capillary-array-based DNA sequencers currently penetrating the market. The described technology should bring high-performance glycosylation analysis within reach of each life sciences lab and thus help expedite the pace of discovery in the field of glycobiology.     

Exocytosis of a 60 kDa protein (calreticulin) from activated hamster oocytes

The sp50 protein localized at the acrosomal region of guinea pig sperm was suggested to participate in acrosome exocytosis, the acrosome reaction (AR). On the other hand, the cortical reaction (CR), also an exocytotic event, occurs during egg activation. The aim of the present work was to identify sp50 and also to define if sp50 is present in hamster eggs, as well as its location before and after CR. Sp50 was identified as calreticulin (CRT), based on: (a) its NH(2)-terminal amino acid (25 aa) sequence, (b) a cross-recognition of pure sp50 and pure CRT with anti-CRT (from Santa Cruz, anti-CRTsc), and anti-sp50 (anti-sp50/CRT) antibodies, respectively, and (c) that both antibodies revealed a 50 kDa protein in a Brij sperm extract. On the other hand, CRT presence in eggs was positively determined by Western blotting (Wb) using anti-sp50/CRT antibody which recognized a 60 kDa protein in the egg extract, and by indirect immunofluorescence (IIF), CRT was located in the cortical granules (CG). It was defined by a granular pattern and co-localization with mannose, a specific carbohydrate of the CG. Additionally, a decrease in CRT concentration occurred in eggs after their activation and, in parallel, the protein was revealed in the egg's incubation medium. In activated eggs with zona pellucida (ZP), CRT remains as a halo in the perivitelline space and around the polar body. From these results we suggest that: (1) CRT is present in the CG of non-activated hamster eggs, (2) CRT is exocytosed during the CR, in response to egg activation, and (3) CRT might participate in the block to polyspermy, together with other CG components.     

An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans

Converting the complete genome sequence of Candida albicans into meaningful biological information will require comprehensive screens for identifying functional classes of genes. Most systems described so far are not applicable to C. albicans because of its difficulty with mating, its diploid nature, and the lack of functional random insertional mutagenesis methods. We examined artificial gene suppression as a means to identify gene products critical for growth of this pathogen; these represent new antifungal drug targets. To achieve gene suppression we combined antisense RNA inhibition and promoter interference. After cloning antisense complementary DNA (cDNA) fragments under control of an inducible GAL1 promoter, we transferred the resulting libraries to C. albicans. Over 2,000 transformant colonies were screened for a promoter-induced diminished-growth phenotype. After recovery of the plasmids, sequence determination of their inserts revealed the messenger RNA (mRNA) they inhibited or the gene they disrupted. Eighty-six genes critical for growth were identified, 45 with unknown function. When used in high-throughput screening for antifungals, the crippled C. albicans strains generated in this study showed enhanced sensitivity to specific drugs.     

In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris

The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an alpha-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and beta-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.