Quantification of Uncoupling Protein 2 Reveals Its Main Expression in Immune Cells and Selective Up-Regulation during T-Cell Proliferation

Uncoupling protein 2 (UCP2) is an inner mitochondrial membrane protein. Although the protein was discovered in 1997, its function and even its tissue distribution are still under debate. Here we present a quantitative analysis of mRNA and protein expression in various mice tissues, revealing that UCP2 is mainly expressed in organs and cells associated with the immune system. Although the UCP2 gene is present in the brain, as demonstrated using quantitative RT-PCR, the protein was not detectable in neurons under physiological conditions. Instead, we could detect UCP2 in microglia, which act in the immune defense of the central nervous system. In lymphocytes, activation led to a ten-fold increase of UCP2 protein expression simultaneously to the increase in levels of other mitochondrial proteins, whereas lymphocyte re-stimulation resulted in the selective increase of UCP2. The highest detected level of UCP2 expression in stimulated T-cells (0.54 ng/(μg total cellular protein)) was approximately 200 times lower than the level of UCP1 in brown adipose tissue from room temperature acclimated mice. Both the UCP2 expression pattern and the time course of up-regulation in stimulated T-cells imply UCP2's involvement in the immune response, probably by controlling the metabolism during cell proliferation.     

Advantages and Limitations of Different p62-Based Assays for Estimating Autophagic Activity in Drosophila

Levels of the selective autophagy substrate p62 have been established in recent years as a specific readout for basal autophagic activity. Here we compared different experimental approaches for using this assay in Drosophila larvae. Similar to the more commonly used western blots, quantifying p62 dots in immunostained fat body cells of L3 stage larvae detected a strong accumulation of endogenous p62 aggregates in null mutants for Atg genes and S6K. Importantly, genes whose mutation or silencing results in early stage lethality can only be analyzed by microscopy using clonal analysis. The loss of numerous general housekeeping genes show a phenotype in large-scale screens including autophagy, and the p62 assay was potentially suitable for distinguishing bona fide autophagy regulators from silencing of a DNA polymerase subunit or a ribosomal gene that likely has a non-specific effect on autophagy. p62 accumulation upon RNAi silencing of known autophagy regulators was dependent on the duration of the knockdown effect, unlike in the case of starvation-induced autophagy. The endogenous p62 assay was more sensitive than a constitutively overexpressed p62-GFP reporter, which showed self-aggregation and large-scale accumulation even in control cells. We recommend western blots for following the conversion of overexpressed p62-GFP reporters to estimate autophagic activity if sample collection from mutant larvae or adults is possible. In addition, we also showed that overexpressed p62 or Atg8 reporters can strongly influence the phenotypes of each other, potentially giving rise to false or contradicting results. Overexpressed p62 aggregates also incorporated Atg8 reporter molecules that might lead to a wrong conclusion of strongly enhanced autophagy, whereas expression of an Atg8 reporter transgene rescued the inhibitory effect of a dominant-negative Atg4 mutant on basal and starvation-induced autophagy.     

The TT genotype of methylenetetrahydrofolate reductase 677C>T polymorphism increases the susceptibility to pediatric ischemic stroke: meta-analysis of the 822 cases and 1,552 controls

The 677C>T polymorphism within methylenetetrahydrofolate reductase (MTHFR) gene is related to an elevated level of homocysteine. Thus it may be considered as a genetic risk factor in ischemic stroke. Apparently studies of this type of polymorphism in childhood stroke have shown conflicting results. We performed meta-analysis of all the data that are available in relation with MTHFR polymorphism and the risk of ischemic stroke in children. We searched PubMed (last search dated December 2010) using "MTHFR polymorphism", "ischemic stroke" "child", "children", "pediatric stroke" as keywords and reference lists of studies and reviews on the topic. Finally, 15 case-control studies corresponded to the inclusion criteria for meta-analysis. These studies involved the total number of 822 children and adolescents after ischemic stroke and 1,552 control subjects. Fixed or random effects models were used depending on the heterogeneity between the studies. The association between ischemic stroke and 677C>T polymorphism within MTHFR gene was observed in three of the studies. The pooled analysis showed that TT genotype of MTHFR gene is more common in stroke patients than in controls (p = 0.0402, odds ratio = 1.57, 95 % confidence interval 1.02-2.41). The Egger's test did not reveal presence of a publication bias. The results based on a sizeable group of cases and controls have proved that the 677C>T polymorphism in MTHFR gene is associated with the development of ischemic stroke in children.     

The C/A polymorphism in intron 11 of the XPC gene plays a crucial role in the modulation of an individual’s susceptibility to sporadic colorectal cancer

Epidemiological data show that colorectal cancer (CRC) is the second most frequent malignancy worldwide. The involvement of “minor impact genes” such as XME and DNA-repair genes in the etiology of sporadic cancer has been postulated by other authors. We focused on analyzing polymorphisms in DNA-repair genes in CRC. We considered the following genes involved in DNA-repair pathways: base excision repair (OGG1 Ser326Cys, XRCC1 Trp194Arg and Arg399Gln); nucleotide excision repair [XPA (−4)G/A, XPC C/A (i11) and A33512C (Lys939Gln), XPD Asp312Asn and A18911C (Lys751Gln), XPF Arg415Gln, XPG Asp1104His, ERCC1 C118T]; homologous recombination repair [NBS1 Glu185Gln, Rad51 135G/C, XRCC3 C18067 (Thr241Met)]. The study group consisted of 133 patients diagnosed with sporadic CRC, while the control group was composed of 100 age-matched non-cancer volunteers. Genotyping was performed by PCR and PCR-RFLP. Fisher’s exact test with a Bonferroni correction for multiple testing was used. We found that: (i) XPC C/A (i11) heterozygous variant is associated with increased risk of CRC [OR is 2.07 (95% CI 1.1391, 3.7782) P = 0.038], (ii) XPD A18911C (Lys751Gln) is associated with decreased risk of CRC [OR = 0.4497, (95% CI 0.2215, 0.9131) P = 0.031] for an individual with at least one A allele at this locus. (1) The XPC C/A (i11) genotype is associated with an increased risk of sporadic colorectal cancer. (2) The NER pathway has been highlighted in our study, as a most important in modulation of individual susceptibility to sCRC.     

Secondary Cell Wall Polymers of Enterococcus faecalis Are Critical for Resistance to Complement Activation via Mannose-binding Lectin

The complement system is part of our first line of defense against invading pathogens. The strategies used by Enterococcus faecalis to evade recognition by human complement are incompletely understood. In this study, we identified an insertional mutant of the wall teichoic acid (WTA) synthesis gene tagB in E. faecalis V583 that exhibited an increased susceptibility to complement-mediated killing by neutrophils. Further analysis revealed that increased killing of the mutant was due to a higher rate of phagocytosis by neutrophils, which correlated with higher C3b deposition on the bacterial surface. Our studies indicated that complement activation via the lectin pathway was much stronger on the tagB mutant compared with wild type. In concordance, we found an increased binding of the key lectin pathway components mannose-binding lectin and mannose-binding lectin-associated serine protease-2 (MASP-2) on the mutant. To understand the mechanism of lectin pathway inhibition by E. faecalis, we purified and characterized cell wall carbohydrates of E. faecalis wild type and V583ΔtagB. NMR analysis revealed that the mutant strain lacked two WTAs with a repeating unit of →6)[α-l-Rhap-(1→3)]β-d-GalpNAc-(1→5)-Rbo-1-P and →6) β-d-Glcp-(1→3) [α-d-Glcp-(1→4)]-β-d-GalpNAc-(1→5)-Rbo-1-P→, respectively (Rbo, ribitol). In addition, compositional changes in the enterococcal rhamnopolysaccharide were noticed. Our study indicates that in E. faecalis, modification of peptidoglycan by secondary cell wall polymers is critical to evade recognition by the complement system.     

Multiple factors correlating with wing malformations in the population of Parnassius apollo (Lepidoptera: Papilionidae) restituted from a low number of individuals: A mini review

The Apollo butterfly, Parnassius apollo (Linnaeus), was common in Europe over 100 years ago, but currently it is considered as near threatened. Different conservation programs have promoted the persistence of this species;however, it is still endangered. An example of such programs was the action devoted to reestablish the Apollo butterfly population in Pieniny National Park (Poland) from only 20-30 individuals which had survived till the last decade of the 20th century. This reintroduction has been successful;however, unexpected developmental problems appeared. Butterflies with deformed or reduced wings became frequent in the population living in the natural habitat, and particularly among those reared under seminatural conditions (in the same environment, but fenced by a net). Until recently, reasons for these malformations remained unknown. However, reports published during last months indicated that there are genetic, biochemical, and microbiological factors contributing to this phenomenon. In the malformed individuals, lesions in the wingless gene and dysfunctions of laccase 1 and 2 were found to be significantly more frequent than in normal insects. A large fraction of butterflies with deformed or reduced wings was devoid of the prokaryotic symbiont Wolbachia, which was present in most normal individuals. Moreover, Yersinia pseudotuberculosis (Pfeiffer) Smith and Thai, and Serratia sp., bacteria pathogenic to insects, were detected in the biological material from both normal and malformed butterflies from this population. These findings are summarized and discussed in this review, in the light of conservation of insects and restitution of their populations from a low number of individuals.     

Bacteriophages engineered to display foreign peptides may become short-circulating phages

Bacteriophages draw scientific attention in medicine and biotechnology, including phage engineering, widely used to shape biological properties of bacteriophages. We developed engineered T4-derived bacteriophages presenting seven types of tissue-homing peptides. We evaluated phage accumulation in targeted tissues, spleen, liver and phage circulation in blood (in mice). Contrary to expectations, accumulation of engineered bacteriophages in targeted organs was not observed, but instead, three engineered phages achieved tissue titres up to 2 orders of magnitude lower than unmodified T4. This correlated with impaired survival of these phages in the circulation. Thus, engineering of T4 phage resulted in the short-circulating phage phenotype. We found that the complement system inactivated engineered phages significantly more strongly than unmodified T4, while no significant differences in phages’ susceptibility to phagocytosis or immunogenicity were found. The short-circulating phage phenotype of the engineered phages suggests that natural phages, at least those propagating on commensal bacteria of animals and humans, are naturally optimized to escape rapid neutralization by the immune system. In this way, phages remain active for longer when inside mammalian bodies, thus increasing their chance of propagating on commensal bacteria. The effect of phage engineering on phage pharmacokinetics should be considered in phage design for medical purposes.     

Comparison of Chemical Composition of the Essential Oils from Different Botanical Organs of Pinus mugo Growing in Poland

The essential oils from needles, twigs, bark, wood, cones and young shoots of Pinus mugo were analyzed by GC, GC/MS, and ¹H‐NMR spectroscopy. More than 130 compounds were identified. The oils differed in the quantitative composition. The principal components of the oil from twigs with needles were 3‐carene (23.8 %), myrcene (22.3 %), and α‐pinene (10.3 %). The needle oil contained mainly α‐pinene (18.6 %), 3‐carene (11.3 %), and bornyl acetate (8.3 %). The oils from twigs without needles, young shoots, bark, and wood were dominated by 3‐carene (28.6 %, 15.0 %, 18.5 %, and 34.6 %, respectively) and myrcene (23.4 %, 24.0 %, 24.6 %, and 9.4 %, respectively). In the cone oil (E)‐β‐caryophyllene was the main constituent (24.0 %).