Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila,Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90°C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70°C) and Escherichia coli (max. growth temp., 47°C) with respect to (a) bihelical content of rRNA; (b) G·C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. 1. Subunits of C. acidophila ribosomes (T_m = 90–93°C) exhibit considerable thermal tolerance over their B. acidocaldarius (T_m = 77°C) and E. coli counterparts (T_m = 72°C). 2. Based on the ‘melting’ hyperchromicities of the intact ribosomal subunits a 51–55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67–70%, appears to be involved in hydrogen bonding in the naked rRNA species. 3. The G·C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63–64% G·C, compared to 58.5% G·C for B. acidocaldarius and 55% G·C for E. coli. 4. The increment in ribosome T_m values with increasing thermophily is greater than the increase in T_m for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). 5. The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. 6. Compared to E. coli, the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.     

The evolution and maintenance of virulence in Staphylococcus aureus: a role for host-to-host transmission?

Despite progress in our understanding of infectious disease biology and prevention, the conditions that select for the establishment and maintenance of microbial virulence remain enigmatic. To address this aspect of pathogen biology, we focus on two members of the Staphylococcus genus - Staphylococcus aureus and Staphylococcus epidermidis - and consider why S. aureus has evolved to become more virulent than S. epidermidis. Several hypotheses to explain this phenomenon are discussed and a mathematical model is used to argue that a complex transmission pathway is the key factor in explaining the evolution and maintenance of virulence in S. aureus. In the case of S. epidermidis, where skin contact affords easier transmission between hosts, high levels of virulence do not offer an advantage to this pathogen.     

Infectious molecular clones from a simian immunodeficiency virus-infected rapid-progressor (RP) macaque: evidence of differential selection of RP-specific envelope mutations in vitro and in vivo

A minor fraction of simian immunodeficiency virus (SIV)-infected macaques progress rapidly to AIDS in the absence of SIV-specific immune responses. Common mutations in conserved residues of env in three SIVsmE543-3-infected rapid-progressor (RP) macaques suggest the evolution of a common viral variant in RP macaques. The goal of the present study was to analyze the biological properties of these variants in vitro and in vivo through the derivation of infectious molecular clones. Virus isolated from a SIVsmE543-3-infected RP macaque, H445 was used to inoculate six naive rhesus macaques. Although RP-specific mutations dominated in H445 tissues, they represented only 10% of the population of the virus stock, suggesting a selective disadvantage in vitro. Only one of these macaques (H635) progressed rapidly to AIDS. Plasma virus during primary infection of H635 was similar to the inoculum. However, RP-specific mutations were apparently rapidly reselected by 4 to 9 weeks postinfection. Terminal plasma from H635 was used as a source of viral RNA to generate seven full-length, infectious molecular clones. With the exception of one clone, which was similar to SIVsmE543-3, clones with RP-specific mutations replicated with delayed kinetics in rhesus peripheral blood mononuclear cells and human T-cell lines. None of the clones replicated in monocyte-derived or alveolar macrophages, and all used CCR5 as their major coreceptor. RP variants appear to be well adapted to replicate in vivo in RP macaques but are at a disadvantage in tissue culture compared to their parent, SIVsmE543-3. Therefore, tissue culture may not provide a good surrogate for replication of RP variants in macaques. These infectious clones will provide a valuable reagent to study the roles of specific viral variants in rapid progression in vivo.     

Activation of the lutropin/choriogonadotropin receptor in MA-10 cells leads to the tyrosine phosphorylation of the focal adhesion kinase by a pathway that involves Src family kinases

We show that activation of the endogenous or recombinant lutropin/choriogonadotropin receptor (LHR) in mouse Leydig tumor cells (MA-10 cells) leads to the tyrosine phosphorylation of the focal adhesion kinase (FAK) and one of its substrates (paxillin). Using specific antibodies to the five tyrosine residues of FAK that become phosphorylated, we show that activation of the LHR increases the phosphorylation of Tyr576 and Tyr577, but it does not affect the phosphorylation of Tyr397, Tyr861, or Tyr925. Because FAK is a prominent substrate for the Src family of tyrosine kinases (SFKs) we tested for their involvement in the LHR-mediated phosphorylation of FAK-Tyr576. Src is not detectable in MA-10 cells, but two other prominent members of this family (Fyn and Yes) are present. The LHR-mediated phosphorylation of FAK-Tyr576 is readily inhibited by PP2 (a pharmacological inhibitor of SFKs) and by dominant-negative mutants of SKFs. Moreover, activation of the LHR in MA-10 cells results in the stimulation of the activity of Fyn and Yes, and overexpression of either of these two tyrosine kinases enhances the LHR-mediated phosphorylation of FAK-Tyr576. Studies involving activation of other G protein-coupled receptors, overexpression of the different Galpha-subunits, and the use of second messenger analogs suggest that the LHR-induced phosphorylation of FAK-Tyr576 in MA-10 cells is mediated by SFKs, and that this family of kinases is, in turn, independently or cooperatively activated by the LHR-induced stimulation of Gs and Gq/11-mediated pathways.     

OX40 ligation of CD4+ T cells enhances virus-specific CD8+ T cell memory responses independently of IL-2 and CD4+ T regulatory cell inhibition

We have previously shown that CD4(+) T cells are required to optimally expand viral-specific memory CD8(+) CTL responses using a human dendritic cell-T cell-based coculture system. OX40 (CD134), a 50-kDa transmembrane protein of the TNFR family, is expressed primarily on activated CD4(+) T cells. In murine models, the OX40/OX40L pathway has been shown to play a critical costimulatory role in dendritic cell/T cell interactions that may be important in promoting long-lived CD4(+) T cells, which subsequently can help CD8(+) T cell responses. The current study examined whether OX40 ligation on ex vivo CD4(+) T cells can enhance their ability to "help" virus-specific CTL responses in HIV-1-infected and -uninfected individuals. OX40 ligation of CD4(+) T cells by human OX40L-IgG1 enhanced the ex vivo expansion of HIV-1-specific and EBV-specific CTL from HIV-1-infected and -uninfected individuals, respectively. The mechanism whereby OX40 ligation enhanced help of CTL was independent of the induction of cytokines such as IL-2 or any inhibitory effect on CD4(+) T regulatory cells, but was associated with a direct effect on proliferation of CD4(+) T cells. Thus, OX40 ligation on CD4(+) T cells represents a potentially novel immunotherapeutic strategy that should be investigated to treat and prevent persistent virus infections, such as HIV-1 infection.     

Oxidative and nitrative modifications of enkephalins by reactive nitrogen species

The interaction of Leucine-enkephalin (Leu-enkephalin) with reactive nitrogen species has been investigated. Reactive nitrogen species are capable of nitrating and oxidizing Leu-enkephalin. HPLC analysis shows the formation of two major enkephalin derivatives by peroxynitrite. The tyrosine amino-terminal residue of Leu-enkephalin is converted either to 3-nitrotyrosine thus producing nitroenkephalin and to dityrosine by dimerization with the production of an enkephalin dimer. The evidence of the formation of the nitroenkephalin and of the enkephalin dimer—dienkephalin—was achieved by electrospray ionisation mass spectrometry. In addition to peroxynitrite, the methylene blue photosensitized oxidation of enkephalin in the presence of nitrite leads to the formation of the nitrated peptide. Moreover, the nitropeptide can be also obtained by peroxidase-generated nitrogen reactive species.     

Oxidative stress,erythrocyte ageing and plasma non-protein-bound iron in diabetic patients

Increased oxidative stress and decreased life span of erythrocytes (RBCs) are repeatedly reported in diabetes. In the aim to elucidate the mechanism of the latter, i.e. the events leading to erythrocyte ageing, this study determined in RBCs from diabetic patients iron release in a free desferrioxamine-chelatable form (DCI), methemoglobin (MetHb) formation, binding of autologous IgG to membrane proteins and in plasma non-protein-bound iron (NPBI), F₂-Isoprostanes (F₂-IsoPs) and advanced oxidation protein products (AOPP). DCI and MetHb were higher in diabetic RBCs than in controls and autologous IgG binding occurred in a much higher percentage of diabetic patients than controls. A significant correlation between DCI and IgG binding was found in diabetic RBCs. Plasma NPBI, esterified F₂-IsoPs and AOPP were higher in diabetic patients and a significant correlation was found between plasma NPBI and intra-erythrocyte DCI. The increased DCI and autologous IgG binding appear to be important factors in the accelerated removal of RBCs from the blood stream in diabetes and the increase in plasma NPBI could play an important role in the increased oxidative stress.     

Chaperonin 60: a paradoxical,evolutionarily conserved protein family with multiple moonlighting functions

Chaperonin 60 is the prototypic molecular chaperone, an essential protein in eukaryotes and prokaryotes, whose sequence conservation provides an excellent basis for phylogenetic analysis. Escherichia coli chaperonin 60 (GroEL), the prototype of this family of proteins, has an established oligomeric‐structure‐based folding mechanism and a defined population of folding partners. However, there is a growing number of examples of chaperonin 60 proteins whose crystal structures and oligomeric composition are at variance with GroEL, suggesting that additional complexities in the protein‐folding function of this protein should be expected. In addition, many organisms have multiple chaperonin 60 proteins, some of which have lost their protein‐folding ability. It is emerging that this highly conserved protein has evolved a bewildering variety of additional biological functions – known as moonlighting functions – both within the cell and in the extracellular milieu. Indeed, in some organisms, it is these moonlighting functions that have been left after the loss of the protein‐folding activity. This highlights the major paradox in the biology of chaperonin 60. This article reviews the relationship between the folding and non‐folding (moonlighting) activities of the chaperonin 60 family and discusses current knowledge on their molecular evolution focusing on protein domains involved in the non‐folding chaperonin functions in an attempt to understand the emerging biology of this evolutionarily ancient protein family.     

Systematics, biogeography, and evolution of the Neotropical peacock basses Cichla (Perciformes: Cichlidae)

To investigate forces influencing diversification in Neotropical fishes, the phylogenetic relationships among species and populations of the cichlid genus Cichla were examined. Mitochondrial DNA was sequenced for 454 individuals of the 5 nominal Cichla species and several putative undescribed species. Phylogenetic analyses support the distinction of two major clades of Cichla. Clade A includes C. temensis and two undescribed species from the lower Amazonas and Xingu Rivers. Clade B includes C. orinocensis, C. monoculus, C. ocellaris. C, intermedia, and an undescribed species from the upper Madeira River. Species boundaries were relatively well-circumscribed for clade B, while incomplete lineage sorting was inferred for clade A. Three probable instances of introgression were observed, including a regional population of C. orinocensis from the Negro River that shows a history of introgression. Biogeographic patterns from Cichla are partially congruent with those seen in several other Neotropical fish clades, and the diversification of Cichla species is inferred to result from both vicariance and sympatric divergence.     

Can the introduction of Xenopus laevis affect native amphibian populations? Reduction of reproductive occurrence in presence of the invasive species

Biological invasions are regarded as a form of global change and potential cause of biodiversity loss. Xenopus laevis is an anuran amphibian native to sub-Saharan Africa with strong invasive capacity, especially in geographic regions with a Mediterranean climate. In spite of the worldwide diffusion of X. laevis, the effective impact on local ecosystems and native amphibian populations is poorly quantified. A large population of X. laevis occurs in Sicily and our main aim of this work was to assess the consequences of introduction of this alien species on local amphibian populations. In this study we compare the occurrence of reproduction of native amphibians in ponds with and without X. laevis, and before and after the alien colonization. The results of our study shows that, when X. laevis establishes a conspicuous population in a pond system, the populations of Discoglossus pictus, Hyla intermedia and Pelophylax synklepton esculentus show clear signs of distress and the occurrence of reproduction of these native amphibians collapses. In contrast, the populations of Bufo bufo do not appear to be affected by the alien species. Since the Sicilian population of X. laevis shows a strong dispersal capacity, proportionate and quick interventions become necessary to bound the detriment to the Sicilian amphibians populations.