Frataxin from Psychromonas ingrahamii as a model to study stability modulation within the CyaY protein family

Adaptation of life to low temperatures influences both protein stability and flexibility. Thus, proteins from psychrophilic organisms are excellent models to study relations between these properties. Here we focused on frataxin from Psychromonas ingrahamii (pFXN), an extreme psychrophilic sea ice bacterium that can grow at temperatures as low as − 12 °C. This α/β protein is highly conserved and plays a key role in iron homeostasis as an iron chaperone. In contrast to other frataxin homologs, chemical and temperature unfolding experiments showed that the thermodynamic stability of pFXN is strongly modulated by pHs: ranging from 5.5 ± 0.9 (pH 6.0) to 0.9 ± 0.3 kcal mol^− 1 (pH 8.0). This protein was crystallized and its X-ray structure solved at 1.45 Å. Comparison of B-factor profiles between Escherichia coli and P. ingrahamii frataxin variants (51% of identity) suggests that, although both proteins share the same structural features, their flexibility distribution is different. Molecular dynamics simulations showed that protonation of His44 or His67 in pFXN lowers the mobility of regions encompassing residues 20–30 and the C-terminal end, probably through favorable electrostatic interactions with residues Asp27, Glu42 and Glu99. Since the C-terminal end of the protein is critical for the stabilization of the frataxin fold, the predictions presented may be reporting on the microscopic origin of the decrease in global stability produced near neutral pH in the psychrophilic variant. We propose that suboptimal electrostatic interactions may have been an evolutionary strategy for the adaptation of frataxin flexibility and function to cold environments.     

The Role of Vesicle Trafficking and Release in Oligodendrocyte Biology

Oligodendrocytes are a subtype of glial cells found within the central nervous system (CNS), responsible for the formation and maintenance of specialized myelin membranes which wrap neuronal axons. The development of myelin requires tight coordination for the cell to deliver lipid and protein building blocks to specific myelin segments at the right time. Both internal and external cues control myelination, thus the reception of these signals also requires precise regulation. In late years, a growing body of evidence indicates that oligodendrocytes, like many other cell types, may use extracellular vesicles (EVs) as a medium for transferring information. The field of EV research has expanded rapidly over the past decade, with new contributions that suggest EVs might have direct involvement in communications with neurons and other glial cells to fine tune oligodendroglial function. This functional role of EVs might also be maladaptive, as it has likewise been implicated in the spreading of toxic molecules within the brain during disease. In this review we will discuss the field’s current understanding of extracellular vesicle biology within oligodendrocytes, and their contribution to physiologic and pathologic conditions.

     

A retrospective study of DNA prevalence of porcine parvoviruses in Mexico and its relationship with porcine circovirus associated disease

Worldwide, many emerging porcine parvoviruses (PPVs) have been linked to porcine circovirus-2 (PCV2) associated disease (PCVAD), which includes post-weaning multi-systemic wasting syndrome (PMWS), PCV2-related reproductive failure (PCV2-RF), as well as other syndromes. To determine the DNA prevalence of PPVs and their relationship with PMWS and PCV2-RF in Mexico, 170 formalin-fixed paraffin-embedded tissues were selected from archival collections to detect PPVs using a nested polymerase chain reaction. The tissues were composed of 50 PMWS cases, 20 age-matched tissues from healthy pigs, 56 PCV2-related reproductive failure (PCV2⁺-RF) cases, and 44 PCV2^--RF cases. Overall, PPV2 and PPV6 were the most prevalent species (90.0% and 74.7%, respectively). In 8–11 week old pigs, the highest prevalence was for PPV6 and PPV3. Concerning reproductive failure, the PCV2-affected farms had a significantly higher prevalence for PPV6 (61.6%) and PPV5 (36.4%) than the PCV2-unaffected farms (35.0% and 5.0%, respectively). The concurrent infection rate was high, being significant for PPV2/PPV4 and PPV1/PPV5 within the PMWS cases and for PPV6/PPV5 among the PCV2⁺-RF tissues. PPV5 showed a significant relationship with PMWS, whereas PPV5 and PPV6 were significant for PCVAD. The prevalence and coinfection rate of PPVs in Mexico were markedly higher than that described in other countries, denoting that PPV5 and PPV6 might have a potential role in PCVAD in Mexico. It is concluded that it is likely that the density population of pigs in Mexico is contributing to high PPV inter-species and PCV2 coinfections which might lead to a different pathogenic outcome.     

Pseudomembranous Candidiasis in HIV/AIDS Patients in Cali, Colombia

Candida albicans is the most frequently isolated yeast from the oral cavity of HIV/AIDS individuals. The use of fluconazole has increased the number of resistant or less-sensitive Candida species different than C. albicans. The purpose of this study was to identify the Candida species producing pseudomembranous candidiasis in patients suffering from AIDS, their relationship with CD4⁺ counts and their sensitivity to fluconazole and itraconazole. We studied 71 patients at a hospital in the city of Cali. Samples of white plaque were seeded on CHROMagar Candida, yeast identification was done with API 20C Aux, and susceptibility testing was determined by E test. Ninety-three yeast isolates were obtained, 52 single and 41 mixed. C. albicans was the most isolated, followed by C. glabrata. An increased frequency of isolates and variety of Candida species occurred in patients with a CD4⁺ cell count ≤100 cells/mm³ without significant differences (p = 0.29). The susceptibility study showed that 8 (8.6 %) isolates were resistant to fluconazole and 11 (11.8 %) to itraconazole, while 6 (8.8 %) C. albicans were simultaneously resistant. No association was found between the isolates of C. albicans or Candida species different than C. albicans and the use of fluconazole (p = 0.21). The results of this study indicate that in the tested population, fluconazole continues to be the best treatment option for oropharyngeal candidiasis in patients suffering from AIDS (HIV/AIDS); however, susceptibility tests are necessary in patients who present therapeutic failure.     

DNA Topological Context Affects Access to Eukaryotic DNA Topoisomerase I

Abstract

We have analyzed the reactivity of a 217 base pair segment of the intrinsically curved Crithidia fasciculata kinetoplast DNA towards eukaryotic DNA topoisomerase I. The substrates were open [linear fragment and nicked circle] and closed minidomains [closed relaxed circle and circles with linking differences of ?1 and ?2], We interpreted the results with the aid of a model that was used to predict the structures of the topoisomers. The modelling shows that the ΔLk(?l) form is unusually compact because of the curvature in the DNA. To determine the role of sequence-directed curvature in both the experimental and modeling studies, controls were examined in which the curved Crithidia sequence was replaced by an uncurved sequence obtained from the plasmid pBR322.

Reactivity of the Crithidia DNA [as analyzed both by the cleavage and the topoisomerization reactions] markedly varied among the DNA forms: (i) the hierarchy of overall reactivity observed is: linear fragment > nicked circular, closed circular [ΔLk(O)], interwound [ΔLk(?2)] > bent interwound [ΔLk(?l)]; (ii) the intensity of several cleavage positions differs among DNA forms.

The results show that eukaryotic DNA topoisomerase I is very sensitive to the conformation of the substrates and that its reactivity is modulated by the variation of the compactness of the DNA molecule. The C. fasciculata sequence contains a highly curved segment that determines the conformation of the closed circle in a complex way.     

Attenuated and Replication-Competent Vaccinia Virus Strains M65 and M101 with Distinct Biology and Immunogenicity as Potential Vaccine Candidates against Pathogens

Replication-competent poxvirus vectors with an attenuation phenotype and with a high immunogenic capacity of the foreign expressed antigen are being pursued as novel vaccine vectors against different pathogens. In this investigation, we have examined the replication and immunogenic characteristics of two vaccinia virus (VACV) mutants, M65 and M101. These mutants were generated after 65 and 101 serial passages of persistently infected Friend erythroleukemia (FEL) cells. In cultured cells of different origins, the mutants are replication competent and have growth kinetics similar to or slightly reduced in comparison with those of the parental Western Reserve (WR) virus strain. In normal and immune-suppressed infected mice, the mutants showed different levels of attenuation and pathogenicity in comparison with WR and modified vaccinia Ankara (MVA) strains. Wide genome analysis after deep sequencing revealed selected genomic deletions and mutations in a number of viral open reading frames (ORFs). Mice immunized in a DNA prime/mutant boost regimen with viral vectors expressing the LACK (Leishmania homologue for receptors of activated C kinase) antigen of Leishmania infantum showed protection or a delay in the onset of cutaneous leishmaniasis. Protection was similar to that triggered by MVA-LACK. In immunized mice, both polyfunctional CD4⁺ and CD8⁺ T cells with an effector memory phenotype were activated by the two mutants, but the DNA-LACK/M65-LACK protocol preferentially induced CD4⁺ whereas DNA-LACK/M101-LACK preferentially induced CD8⁺ T cell responses. Altogether, our findings showed the adaptive changes of the WR genome during long-term virus-host cell interaction and how the replication competency of M65 and M101 mutants confers distinct biological properties and immunogenicity in mice compared to those of the MVA strain. These mutants could have applicability for understanding VACV biology and as potential vaccine vectors against pathogens and tumors.     

Morphometric and molecular variation in concert: taxonomy and genetics of the reticulate Pyrenean and Iberian alpine spiny fescues (Festuca eskia complex,Poaceae)

The Iberian mountain spiny fescues are a reticulate group of five diploid grass taxa consisting of three parental species and two putative hybrids: F. × souliei (F. eskia × F. quadriflora) and F. × picoeuropeana (F. eskia × F. gautieri). Phenotypic and molecular studies were conducted with the aim of determining the taxonomic boundaries and genetic relationships of the five taxa and disentangling the origins of the two hybrids. Statistical analyses of 31 selected phenotypic traits were conducted on individuals from 159 populations and on nine type specimens. Molecular analyses of random amplified polymorphic DNA (RAPD) markers were performed on 29 populations. The phenotypic analyses detected significant differences between the five taxa and demonstrated the overall intermediacy of the F. × picoeuropeana and F. × souliei between their respective parents. The RAPD analysis corroborated the genetic differentiation of F. eskia, F. gautieri and F. quadriflora and the intermediate nature of the two hybrids; however, they also detected genetic variation within F. × picoeuropeana. These results suggest distinct origins for F. × picoeuropeana in the Cantabrian and Pyrenean mountains, with the sporadic Pyrenean populations having potentially resulted from recent hybridizations and the stabilized Cantabrian ones from older events followed by potential displacements of the parents. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 676–706.     

Amarcord: I Remember

I have tried to offer a historical account of a success story, as I saw it develop from the early times when it interested only a few aficionados to the present times when it has pervaded most of cell biochemistry and physiology. It is of course the story of calcium signaling. It became my topic of work when I was a young postdoctoral fellow at The Johns Hopkins University. I entered it through a side door, that of mitochondria, which had been my area of work during my earlier days in Italy. The 1960s and 1970s were glorious times for mitochondrial calcium signaling, but the golden period was not going to last. As I have discussed below, mitochondrial calcium gradually lost appeal, entering a long period of oblivion. Its fading happened as the general area of calcium signaling was instead experiencing a phase of explosive growth, with landmark discoveries at the molecular and cellular levels. These discoveries established that calcium signaling was one of the most important areas of cell biology. However, mitochondria as calcium partners were not dead; they were only dormant. In the 1990s, they were rescued from their state of neglect to the central position of the regulation of cellular calcium signaling, which they had once rightly occupied. Meanwhile, it had also become clear that calcium is an ambivalent messenger. Hardly anything important occurs in cells without the participation of the calcium message, but calcium must be controlled with absolute precision. This is an imperative necessity, which becomes unfortunately impaired in a number of disease conditions that transform calcium into a messenger of death.     

Coarse Architecture of the Transient Receptor Potential Vanilloid 1 (TRPV1) Ion Channel Determined by Fluorescence Resonance Energy Transfer

The transient receptor potential vanilloid 1 ion channel is responsible for the perception of high temperatures and low extracellular pH, and it is also involved in the response to some pungent compounds. Importantly, it is also associated with the perception of pain and noxious stimuli. Here, we attempt to discern the molecular organization and location of the N and C termini of the transient receptor potential vanilloid 1 ion channel by measuring FRET between genetically attached enhanced yellow and cyan fluorescent protein to the N or C terminus of the channel protein, expressed in transfected HEK 293 cells or Xenopus laevis oocytes. The static measurements of the domain organization were mapped into an available cryo-electron microscopy density of the channel with good agreement. These measurements also provide novel insights into the organization of terminal domains and their proximity to the plasma membrane.