Ultrastructure of the spermatozoa of the flatworms Phaenocelis peleca and Boninia divae (Platyhelminthes, Polycladida)

The ultrastructure of spermatozoa of the acotylean Phaenocelis peleca and the cotylean Boninia divae is described. All spermatozoa are filiform and biflagellate with a 9+"1" microtubular pattern in the axoneme. Sperm characters in P. peleca follow the morphologies described for other acotyleans, with axonemes exiting the sperm shaft at the distal end and remaining in close contact with the sperm membrane. The nucleus occupies the proximal region of the shaft, and two types of dense bodies and mitochondria are located at the distal end. Unlike other members of the Cotylea, the axonemes of B. divae spermatozoa are incorporated into the sperm shaft, leaving the shaft at some distance from the distal end and then remaining free. This type of morphology is characteristic for acotyleans. Additionally, the spermatozoa of B. divae contain only one type of dense bodies plus a unique structure, which we call a central core. The nucleus in this species is unique as well; it shows periodic constrictions and rings of electron-dense granules, characters that further contribute to the distinct status of Boniniidae.     

A point mutation in the ectodomain-transmembrane 2 interface eliminates the inhibitory effects of ethanol in P2X4 receptors

ATP-gated P2X4 receptors (P2X4R) are abundantly expressed in the CNS. However, little is known about the molecular targets for ethanol action in P2X4Rs. The current investigation tested the hypothesis that the ectodomain-transmembrane (TM) interface contains residues that are important for the action of ethanol in P2X4Rs. Wild type (WT) and mutant P2X4R were expressed in Xenopus oocytes. ATP concentration–response curves and ethanol (10–200 mM)-induced changes in ATP EC₁₀-gated currents were determined using two-electrode voltage clamp (−70 mV). Alanine substitution at the ectodomain-TM1 interface (positions 50–61) resulted in minimal changes in ethanol response. On the other hand, alanine substitution at the ectodomain-TM2 interface (positions 321–337) identified two key residues (D331 and M336) that significantly reduced ethanol inhibition of ATP-gated currents without causing marked changes in ATP I _max, EC₅₀, or Hill's slope. Other amino acid substitutions at positions 331 and 336 significantly altered or eliminated the modulatory effects of ethanol. Linear regression analyses revealed a significant relationship between hydropathy and polarity, but not molecular volume/molecular weight of the residues at these two positions. The results support the proposed hypothesis and represent an important step toward developing ethanol-insensitive receptors for investigating the role of P2X4Rs in mediating behavioral effects of ethanol.     

Mycobacterium szulgai identification by hsp65 gene sequencing in an HIV-positive patient with non-Hodgkin's lymphoma

Mycobacterium szulgai, described for the first time in 1972, is a rare human pathogen that mainly causes pulmonary non-tubercular mycobacteriosis. We report its isolation and identification from a bronchoalveolar lavage specimen by hsp65 gene sequencing analysis in an HIV-positive patient with non-Hodgkin's lymphoma.     

Extension of G-quadruplex DNA by ciliate telomerase

Telomeric DNA can fold into four-stranded structures known as G-quadruplexes. Here we investigate the ability of G-quadruplex DNA to serve as a substrate for recombinant Tetrahymena and native Euplotes telomerase. Inter- and intramolecular G-quadruplexes were gel-purified and their stability examined using native gel electrophoresis, circular dichroism (CD) and thermal denaturation. While intermolecular G-quadruplexes were highly stable, they were excellent substrates for both ciliate telomerases in primer extension assays. In contrast, intramolecular G-quadruplexes formed in K+ exhibited biphasic unfolding and were not extended by ciliate telomerases. Na+-stabilised intramolecular G-quadruplexes were extended by telomerase owing to their rapid rate of dissociation. The Tetrahymena telomerase protein component bound to inter- but not intramolecular K+-stabilised G-quadruplexes. This study provides evidence that parallel intermolecular G-quadruplexes can serve as substrates for telomerase in vitro, their extension being mediated through direct interactions between this higher-order structure and telomerase.     

Biomphalaria tenagophila: dynamics of populations of resistant and susceptible strains to Schistosoma mansoni, with or without pressure of the parasite

Resistant (Taim, RS) and susceptible albino (Joinville, SC) Biomphalaria tenagophila populations were kept together, at different proportions, throughout a 18-month-period. Some of the snail groups were submitted to Schistosoma mansoni infection. The targets of this study were (a) to analyze the populational dynamics among resistant and susceptible individuals to S. mansoni; (b) to study the resistance phenotype in descendants of cross-breeding; (c) to observe whether the parasite could exert any kind of selection in those snail populations. Throughout the experiment it could be observed that the susceptible B. tenagophila strain (Joinville) underwent a selective pressure of the parasite that was negative, since the individuals showed a high mortality rate. Although B. tenagophila (Taim) population presented a higher mortality rate without pressure of the parasite, this event was compensated by a reproductive capacity. B. tenagophila Taim was more fecund than B. tenagophila Joinville and was able to transmit the resistance character to their descendants. F1 generation obtained by cross-breeding between resistant and susceptible lineages was completely resistant to S. mansoni infection, irrespective of the Taim proportion. Moreover, less than 5% of F2 progeny were susceptible to S. mansoni infection.     

Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes

During apoptosis, cytochrome c (cyt c) is released from intermembrane space of mitochondria into the cytosol where it triggers the caspase-dependent machinery. We discovered that cyt c plays another critical role in early apoptosis as a cardiolipin (CL)-specific oxygenase to produce CL hydroperoxides required for release of pro-apoptotic factors [Kagan, V. E., et al. (2005) Nat. Chem. Biol. 1, 223-232]. We quantitatively characterized the activation of peroxidase activity of cyt c by CL and hydrogen peroxide. At low ionic strength and high CL/cyt c ratios, peroxidase activity of the CL/cyt c complex was increased >50 times. This catalytic activity correlated with partial unfolding of cyt c monitored by Trp(59) fluorescence and absorbance at 695 nm (Fe-S(Met(80)) band). The peroxidase activity increase preceded the loss of protein tertiary structure. Monounsaturated tetraoleoyl-CL (TOCL) induced peroxidase activity and unfolding of cyt c more effectively than saturated tetramyristoyl-CL (TMCL). TOCL/cyt c complex was found more resistant to dissociation by high salt concentration. These findings suggest that electrostatic CL/cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost. In the presence of CL, cyt c peroxidase activity is activated at lower H(2)O(2) concentrations than for isolated cyt c molecules. This suggests that redistribution of CL in the mitochondrial membranes combined with increased production of H(2)O(2) can switch on the peroxidase activity of cyt c and CL oxidation in mitochondria-a required step in execution of apoptosis.     

Neisseria meningitidis expressing lgtB lipopolysaccharide targets DC-SIGN and modulates dendritic cell function

Neisseria meningitidis lipopolysaccharide (LPS) has been identified as a major determinant of dendritic cell (DC) function. Here we report that one of a series of meningococcal mutants with defined truncations in the lacto-N-neotetraose outer core of the LPS exhibited unique strong adhesion and internalization properties towards DC. These properties were mediated by interaction of the GlcNAc(beta1-3)-Gal(beta1-4)-Glc-R oligosaccharide outer core of lgtB LPS with the dendritic-cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) lectin receptor. Activation of DC-SIGN with this novel oligosaccharide ligand skewed T-cell responses driven by DC towards T helper type 1 activity. Thus, the use of lgtB LPS may provide a powerful instrument to selectively induce the desired arm of the immune response and potentially increase vaccine efficacy.     

Magic-angle spinning NMR studies of cell wall bound aromatic-aliphatic biopolyesters associated with strengthening of intercellular adhesion in potato (Solanum tuberosum L.) tuber parenchyma

Intercellular adhesion strengthening, a phenomenon that compromises the texture and the edible quality of potatoes (Solanum tuberosum L.), has been induced reproducibly by exposure to low-pH acetic acid solutions under tissue culture conditions. The resulting parenchyma tissues have been examined by solid-state nuclear magnetic resonance (NMR) in order to characterize the biopolymer(s) thought to be associated with this syndrome. Cross polarization-magic angle spinning (CPMAS) (13)C NMR has been used to establish the presence of a polyphenol-suberin-like aromatic-aliphatic polyester within an abundant cell wall polysaccharide matrix in potato tubers that exhibit hardening due to strengthened intercellular adhesion. Dipolar dephasing and CP chemical shift anisotropy experiments suggest that the aromatic domain is composed primarily of guaiacyl and sinapyl groups. Two-dimensional wide-line separation experiments show that the biopolymer associated with parenchyma hardening contains rigid polysaccharide cell walls and mobile aliphatic long-chain fatty acids; (1)H spin diffusion experiments show that these flexible aliphatic chains are proximal to both the phenolics and a subpopulation of the cell wall polysaccharides. Finally, high-resolution MAS NMR of parenchyma samples swelled in DMSO in conjunction with two-dimensional through-bond and through-space NMR spectroscopy provides evidence for covalent linkages among the polysaccharide, phenolic, and aliphatic domains of the intercellular adhesion-strengthening biopolymer in potato parenchyma tissue.