Evidence of lipid peroxidation and protein phosphorylation in cells upon oxidative stress photo-generated by fullerols

An oxidative stress (OS) state is characterized by the generation of Reactive Oxygen Species (ROS) in a biological system above its capacity to counterbalance them [1]. Exposure to OS induces the accumulation of intracellular ROS, which in turn causes cell damage in the form of protein, lipid, and/or DNA oxidations. Such conditions are believed to be linked to numerous diseases or simply to the ageing of tissues. However, the controlled generation of ROS via photosensitizing drugs or photosensitizers (PS) is now widely used to treat various tumors and other infections [2,3]. Here we present a method to track the chemical changes in a cell after exposure to oxidative stress. OS is induced via fullerols, a custom made water soluble derivative of fullerene (C(60)), under visible light illumination. Synchrotron-based Fourier Transform InfraRed Microspectroscopy (S-FTIRM) was used to assess the chemical makeup of single cells after OS exposure. Consequently, a chemical fingerprint of oxidative stress was probed in this study through an increase in the bands linked with lipid peroxidation (carbonyl ester group at 1740 cm(-1)) and protein phosphorylation (asymmetric phosphate stretching at 1240 cm(-1)).     

Susceptibility of the common hamster (Cricetus cricetus) to Francisella tularensis and its effect on the epizootiology of tularemia in an area where both are endemic

Francisella tularensis is a highly infectious zoonotic agent causing the disease tularemia. The common hamster (Cricetus cricetus) is considered a pest in eastern Europe, and believed to be a source of human tularemia infections. We examined the role of the common hamster in the natural cycle of tularemia using serologic methods on 900 hamsters and real-time polymerase chain reaction (PCR) on 100 hamsters in an endemic agricultural area. We collected 374 Ixodes acuminatus ticks from the hamsters and tested them by real-time PCR. All tests were negative. To examine clinical signs, pathology, and histopathology of acute tularemia infection similar to the natural infection, two hamsters were infected with a large dose of a wild strain of F. tularensis ssp. holarctica. After a short period of apathy, the animals died on the eighth and ninth days postinfection. The pathologic, histopathologic, and immunohistochemical examination contributed to the diagnosis of septicemia in both cases. Our results confirmed previous findings that common hamsters are highly sensitive to F. tularensis. We conclude that although septicemic hamsters may pose substantial risk to humans during tularemia outbreaks, hamsters in interepizootic periods do not act as a main reservoir of F. tularensis.     

New microsatellite loci for Narcissus papyraceus (Amarillydaceae) and cross-amplification in other congeneric species

? Premise of the study: Microsatellite loci from a genomic library of the species Narcissus papyraceus were optimized and characterized for studies of population genetics. ? Methods and Results: Eleven markers that were successfully amplified showed polymorphism when tested on 50 individuals from two populations in southern Spain and northern Morocco. Overall, the number of alleles per locus ranged between 4 and 15. Between 8 and 11 loci successfully amplified in other eight Narcissus species. ? Conclusions: These markers will enable genetic diversity studies of N. papyraceus across its distribution range and conduct paternity analyses among individuals differing in flower morphology.     

An investigation of the occurrence and properties of the mitochondrial intermediate-conductance Ca2+-activated K+ channel mtKCa3.1

The mitochondrial intermediate-conductance Ca²⁺-activated K⁺ channel mtK_Ca3.1 has recently been discovered in the HCT116 colon tumor-derived cell line, which expresses relatively high levels of this protein also in the plasma membrane. Electrophysiological recordings revealed that the channel can exhibit different conductance states and kinetic modes, which we tentatively ascribe to post-translational modifications. To verify whether the localization of this channel in mitochondria might be a peculiarity of these cells or a more widespread feature we have checked for the presence of mtK_Ca3.1 in a few other cell lines using biochemical and electrophysiological approaches. It turned out to be present at least in some of the cells investigated. Functional assays explored the possibility that mtK_Ca3.1 might be involved in cell proliferation or play a role similar to that of the Shaker-type K_V1.3 channel in lymphocytes, which interacts with outer mitochondrial membrane-inserted Bax thereby promoting apoptosis (Szabò, I. et al., Proc. Natl. Acad Sci. USA 105 (2008) 14861–14866). A specific K_Ca3.1 inhibitor however did not have any detectable effect on cell proliferation or death.     

The sensitivity of microscopy and PCR-based detection methods affecting estimates of prevalence of blood parasites in birds

Inferences about the evolution of host-parasitic relationships are often made based on the prevalence of avian malaria, which is usually estimated in a large sample of birds using either microscopic or molecular screening of blood samples. However, different techniques often have variable accuracy; thus, screening methodology can raise issues about statistical bias if method sensitivity varies systematically across parasites or hosts. To examine this possibility, published information was collected on the prevalence of species in 4 genera of avian blood parasites ( Plasmodium, Haemoproteus, Leucocytozoon, and Trypanosoma) from various sources that used different tools. The data were tested to determine if the application of different methods provided different estimates for the same hosts. In these comparisons between the main methodologies, the PCR-based molecular methods were generally found to provide higher estimates for Plasmodium spp. prevalence than microscopic tools, while there was no significant tendency for such a trend in species of Haemoproteus and Leucocytozoon. When analyzing intraspecific variance of prevalence within molecular studies, some studies provided consistently higher estimates for Haemoproteus spp. prevalence than others, indicating that differences between studies can affect detected estimates. Within microscopic studies, surveys that examined more microscopic fields were more likely to report higher prevalence for Plasmodium spp. than those relying on fewer microscopic fields. Consequently, studies making comparisons across parasite genera and/or host species from different sources need to consider several types of bias originating from variation in method sensitivity.     

Histopathology of <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> (Acari: Ixodidae) ticks fed on resistant hosts

Histological features of Rhipicephalus sanguineus ticks fed on dog, a non resistant host, and on guinea pig, a resistant host, were compared. Unfed ticks and ticks from each host species were collected during first and third infestation and processed for histology. Many ticks from guinea pigs, especially during third infestation, were unattached, dehydrated and small. Only the midgut of ticks fed on guinea pigs had host leukocytes. Vacuolization of midgut cells was observed in all ticks, with exception of those fed on dogs for more than 96 h. Ticks of guinea pigs, particularly from third infestation, had vacuolated tracheae and swelling of malpighian tubules. Solely ticks from third infestation of guinea pigs displayed vacuolization of oocytes. Ticks fed on guinea pigs also had an increased number of guanine spherules. Observed alterations in ticks from guinea pigs are discussed.     

Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA

Small regulatory RNA repertoires in biological samples are heterogeneous mixtures that may include species arising from varied biosynthetic pathways and modification events. Small RNA profiling and discovery approaches ought to capture molecules in a way that is representative of expression level. It follows that the effects of RNA modifications on representation should be minimized. The collection of high-quality, representative data, therefore, will be highly dependent on bias-free sample manipulation in advance of quantification. We examined the impact of 2'-O-methylation of the 3'-terminal nucleotide of small RNA on key enzymatic reactions of standard front-end manipulation schemes. Here we report that this common modification negatively influences the representation of these small RNA species. Deficits occurred at multiple steps as determined by gel analysis of synthetic input RNA and by quantification and sequencing of derived cDNA pools. We describe methods to minimize the effects of 2'-O-methyl modification of small RNA 3'-termini using T4 RNA ligase 2 truncated, and other optimized reaction conditions, demonstrating their use by quantifying representation of miRNAs and piRNAs in cDNA pools prepared from biological samples.     

The structural plasticity of SCA7 domains defines their differential nucleosome‐binding properties

SAGA (Spt–Ada–Gcn5 acetyltransferase), a coactivator complex involved in chromatin remodelling, harbours both histone acetylation and deubiquitination activities. ATXN7/Sgf73 and ATXN7L3, two subunits of the SAGA deubiquitination module, contain an SCA7 domain characterized by an atypical zinc‐finger. We show that the yeast Sgf73–SCA7 domain is not required to recruit Sgf73 into SAGA. Instead, it binds to nucleosomes, a property that is conserved in the human ATXN7–SCA7 domain but is lost in the ATXN7L3 domain. The solution structures of the SCA7 domain of both ATXN7 and ATXN7L3 reveal a new, common zinc‐finger motif at the heart of two distinct folds, providing a molecular basis for the observed functional differences.