Macular Microcysts in Mitochondrial Optic Neuropathies: Prevalence and Retinal Layer Thickness Measurements

Purpose

To investigate the thickness of the retinal layers and to assess the prevalence of macular microcysts (MM) in the inner nuclear layer (INL) of patients with mitochondrial optic neuropathies (MON).

Methods

All patients with molecularly confirmed MON, i.e. Leber’s Hereditary Optic Neuropathy (LHON) and Dominant Optic Atrophy (DOA), referred between 2010 and 2012 were enrolled. Eight patients with MM were compared with two control groups: MON patients without MM matched by age, peripapillary retinal nerve fiber layer (RNFL) thickness, and visual acuity, as well as age-matched controls. Retinal segmentation was performed using specific Optical coherence tomography (OCT) software (Carl Zeiss Meditec). Macular segmentation thickness values of the three groups were compared by one-way analysis of variance with Bonferroni post hoc corrections.

Results

MM were identified in 5/90 (5.6%) patients with LHON and 3/58 (5.2%) with DOA. The INL was thicker in patients with MON compared to controls regardless of the presence of MM [133.1±7μm vs 122.3±9μm in MM patients (p<0.01) and 128.5±8μm vs. 122.3±9μm in no-MM patients (p<0.05)], however the outer nuclear layer (ONL) was thicker in patients with MM (101.4±1mμ) compared to patients without MM [77.5±8mμ (p<0.001)] and controls [78.4±7mμ (p<0.001)]. ONL thickness did not significantly differ between patients without MM and controls.

Conclusion

The prevalence of MM in MON is low (5-6%), but associated with ONL thickening. We speculate that in MON patients with MM, vitreo-retinal traction contributes to the thickening of ONL as well as to the production of cystic spaces.     

Copper(II) complexes with chicken prion repeats: influence of proline and tyrosine residues on the coordination features

The prion protein (PrP^c) is a copper-binding glycoprotein that can misfold into a β-sheet-rich and pathogenic isoform (PrP^sc) leading to prion diseases. The first non-mammalian PrP^c was identified in chicken and it was found to keep many structural motifs present in mammalian PrP^c, despite the low sequence identity (approximately 40%) between the two primary structures. The present paper describes the synthesis and the coordination properties of some hexapeptide fragments (namely, PHNPGY , HNPGYP and NPGYPH) as well as a bishexapeptide (PHNPGYPHNPGY), which encompasses two hexarepeats. The copper(II) complexes were characterized by means of potentiometric, UV–vis, circular dichroism and electron paramagnetic resonance techniques. We also report the synthesis of three hexapeptides (PHNPGF, HNPGFP and NPGFPH), in which one tyrosine was replaced by phenylalanine as well as two bishexapeptides in which either one (PHNPGFPHNPGY and PHNPGYPHNPGF), or two tyrosines were replaced by phenylalanine, in order to check whether tyrosine was involved in copper(II) binding. Overall, the results indicate that the major copper(II) species formed by the chicken PrP dodecapeptides are stabler than the analogous species reported for the peptide fragments containing two octarepeat peptides from the mammalian prion protein. It is concluded that the presence of four prolyl residues, that are break points in copper coordination, induces the metal-assisted formation of macrochelates as well as the formation of binuclear species. Furthermore, it has been shown that the phenolic group is directly involved in the formation of copper binuclear species.Electronic Supplementary Material Supplementary material is available for this article at .This revised version was published online in June 2005 with corrections to the text. The author name LaMendola has been corrected to La Mendola.     

Ticks (Acari: Ixodidae) of the state of Amazonas,Brazil

The tick fauna of Brazil is currently composed by 72 species. The state of Amazonas is the largest of Brazil, with an area of ≈ 19% of the Brazilian land. Besides its vast geographic area, only 19 tick species have been reported for Amazonas. Herein, lots containing ticks from the state of Amazonas were examined in three major tick collections from Brazil. A total of 5933 tick specimens were examined and recorded, comprising 2693 males, 1247 females, 1509 nymphs, and 484 larvae. These ticks were identified into the following 22 species: Amblyomma cajennense sensu lato, Amblyomma calcaratum, Amblyomma coelebs, Amblyomma dissimile, Amblyomma dubitatum, Amblyomma geayi, Amblyomma goeldii, Amblyomma humerale, Amblyomma latepunctatun, Amblyomma longirostre, Amblyomma naponense, Amblyomma oblongoguttatum, Amblyomma ovale, Amblyomma rotundatum, Amblyomma scalpturatum, Amblyomma varium, Dermacentor nitens, Haemaphysalis juxtakochi, Ixodes cf. Ixodes fuscipes, Ixodes luciae, Rhipicephalus microplus, Rhipicephalus sanguineus sensu lato. Ticks were collected from 17 (27.4%) out of the 62 municipalities that currently compose the state of Amazonas. The following four species are reported for the first time in the state of Amazonas: A. coelebs, A. dubitatum, H. juxtakochi, and Ixodes cf. I. fuscipes. The only tick species previously reported for Amazonas and not found in the present study is Amblyomma parvum. This study provides a great expansion of geographical and host records of ticks for the state of Amazonas, which is now considered to have a tick fauna composed by 23 species. It is noteworthy that we report 1391 Amblyomma nymphs that were identified to 13 different species.     

Tetracycline‐controlled expression of glycosylated porcine interferon‐gamma in mammalian cells

Abstract

Tetracycline‐controlled expression plasmids that allow inducible expression of proteins in mammalian cells (Gossen & Bujard, 1992), have been used to express porcine interferon‐γ in the RK‐13 rabbit kidney cell line. Following neomycin selection, stable clones produced recombinant, glycosylated porcine interferon‐γ (rGPoIFN‐γ) only after removal of tetracycline (Tc). Southern blot analysis of one clone showed that approximately 50 copies of IFN‐γ cDNA were present in the cell genome. In the absence of Tc, stable clones secreted large amounts of rGPoIFN‐γ (up to 16 μg/ml) into the medium supplemented with 10% FCS and high glucose concentration. Molecular weight comparison of ³⁵S‐Methionine, labelled rGPoIFN‐γ with natural leukocytic IFN‐γ after immunoprecipitation, revealed 4 major glycoforms with apparent Mr of 27,000; 25,000; 20,000 and 18,500, that are almost identical in both IFN‐γ species. In both cases, all 4 glycoforms resolved into 2 polypeptide monomers with apparent Mr of 16,500 and 14,500 upon deglycosylation with N‐glycosydase F. The biological activity of rGPoIFN‐γ was in the same range as that of natural leukocytic PoIFN‐γ (2 × 10⁶ U/mg). Eventually, this recombinant mammalian IFN‐γ should constitute a very useful substitute for leukocyte PoIFN‐γ in in vitro or in vivo experiments.     

CXC chemokine receptor 4 is expressed in uveal malignant melanoma and correlates with the epithelioid-mixed cell type

PURPOSE: Although relatively rare, uveal melanoma is the most common ocular tumor of adults. Up to half of uveal melanoma patients die of metastatic disease. CXCR4, a chemokine receptor, is a prognostic factor in cutaneous melanoma involved in angiogenesis and metastasis formation. The aim of this study was to evaluate the expression of CXCR4 in uveal melanoma. METHODS: CXCR4 was detected by immunohistochemistry in 44 samples of uveal melanoma. Staining was categorized into three semiquantitative classes based on the rate of stained (positive) tumor cells: absence of staining, <50% of cell (+) and >50% (++). Correlations between CXCR4 expression, data on patient and tumor features were studied by contingency tables and the chi2 test. Time-to-event curves were studied using the Kaplan-Meier method. Univariate analysis was performed using the log-rank test. Ninety-five percent confidence intervals (95% CI) of hazard ratios were also reported. RESULTS: Staining for CXCR4 protein was absent in 18 tumors (40.9%), present in <50% of cells in 19 (43.2%) and in >50% of cells in 7 (15.9%) tumors. CXCR4 expression correlated to the epithelioid-mixed cell type (P=0.030). No statistically significant relation emerged between CXCR4 expression, largest tumor diameter (LTD) and extracellular matrix patterns as evaluated through histological patterns stained with periodic acid-Schiff (PAS). Events occurred in 2 out of 18 patients (11.1%) with negative tumors (2 deaths), in 3 out of 19 patients (15.8%) with <50% of positive tumor cells (2 deaths and 1 occurrence of metastases) and in 1 out of 7 patients (14.3%) with >50% of positive tumor cells (1 occurrence of metastases). The cell type (P=0.0457) but not CXCR4 showed prognostic value at univariate analysis. CONCLUSION: This study shows that CXCR4 is commonly expressed in uveal melanoma and correlates with cell type a well-established prognostic factor.     

Binding of aflatoxins to the 20S proteasome: effects on enzyme functionality and implications for oxidative stress and apoptosis

Aflatoxins (AF) are contaminants of improperly stored foods; they are potent genotoxic and carcinogenic compounds, exerting their effects through damage to DNA. They can also induce mutations that increase oxidative damage. The goal of this study was to evaluate the possibility that a third mechanism could be involved in the carcinogenic action of aflatoxins, namely, direct binding to key enzymes involved in the regulatory pathways of the cell cycle, thereby modulating enzyme functionality. The 20S constitutive and immunoproteasome peptidase and proteolytic activities were assayed in the presence of aflatoxins B1, G1 and M1. All three toxins activated multiple peptidase activities of the proteasome. Aflatoxin (AF) M1 was the most potent activator of proteasome activity, while the constitutive 20S proteasome was specifically stimulated by AFG1. Furthermore, the effects of AFB1 on cultured hepatoma cells were investigated and the various proteasomal activities determined with cell lysates were differently affected. Taking into account the key role of the proteasome in cellular defense against oxidative stress, the carbonyl group content and the activities of antioxidant enzymes in cell lysates were analyzed. The proapoptotic effect of AFB1 was also investigated by measuring caspase-3 activity and cellular levels of p27 and IkappaBalpha.     

Genetic Barriers to Historical Gene Flow between Cryptic Species of Alpine Bumblebees Revealed by Comparative Population Genomics

Evidence is accumulating that gene flow commonly occurs between recently diverged species, despite the existence of barriers to gene flow in their genomes. However, we still know little about what regions of the genome become barriers to gene flow and how such barriers form. Here, we compare genetic differentiation across the genomes of bumblebee species living in sympatry and allopatry to reveal the potential impact of gene flow during species divergence and uncover genetic barrier loci. We first compared the genomes of the alpine bumblebee Bombus sylvicola and a previously unidentified sister species living in sympatry in the Rocky Mountains, revealing prominent islands of elevated genetic divergence in the genome that colocalize with centromeres and regions of low recombination. This same pattern is observed between the genomes of another pair of closely related species living in allopatry (B. bifarius and B. vancouverensis). Strikingly however, the genomic islands exhibit significantly elevated absolute divergence (d_XY) in the sympatric, but not the allopatric, comparison indicating that they contain loci that have acted as barriers to historical gene flow in sympatry. Our results suggest that intrinsic barriers to gene flow between species may often accumulate in regions of low recombination and near centromeres through processes such as genetic hitchhiking, and that divergence in these regions is accentuated in the presence of gene flow.     

Polygyny in the tree swallow Tachycineta bicolor: a result of the cost of searching for an unmated male

The costs imposed by parental care duties on an individual's future survival and reproduction generate conflicts because parents should attempt to minimize their investment in the present brood, and exploit the parental care of the other parent. This conflict is likely to contribute to cases of both polygamy and desertion. Here, we study the costs of polygyny in the tree swallow Tachycineta bicolor , using observations on 52 nests that were attended by polygynous males over 14 y. Both females mated to polygynous males paid reproductive costs at several stages of the nesting cycle. Clutches laid by social mates of polygynous versus monogamous males did not differ in size. However, initial brood sizes for polygynously mated females were lower because a higher proportion of their eggs failed to hatch. Likewise, fledging success was lower and nest predation rates were higher, perhaps reflecting the direct or indirect effects of reduced male attention. These results demonstrate that females pay a productivity cost when breeding with reduced male parental care. In contrast, polygynous males fledge on average more young than monogamous ones and clearly benefit from the association. We suggest that a mate-search cost is leading to the few cases of polygamous males: although females are likely evaluating males for their prospective dedication to the breeding attempt, in a short-lived bird with a short breeding season, the cost to females of searching for a more dedicated male is the risk of not breeding at all.     

Micronuclei, genetic polymorphisms and cardiovascular disease mortality in a nested case-control study in Italy

AIM: To validate the predictive value of micronuclei (MN) in peripheral blood lymphocytes (PBL) and glutathione-S-transferases (GSTs) polymorphisms (GSTM1 and GSTT1) for mortality risk (MR) of cardiovascular diseases (CVD). METHODS: Blood samples from 1650 healthy subjects selected from the general population were collected between June 1991 and November 1993, and slides were immediately prepared for MN assessment. The vital status, or the cause of death, was monitored for all subjects until January 2005. At the end of the follow-up, 111 deaths were recorded and 39 CVD cases were observed (age range=42-88 years). Two thousand binucleated (BN) cells/subject were scored for the MN assay and GSTs genotypes were assessed on the DNA extracted from the blood or serum samples. RESULTS: A significantly higher MN frequency was recorded for the case group in comparison with the control group (n=67, Kruskall-Wallis test, p=0.006) and GSTT1 null genotype was significantly less frequent in CVD patients (chi(2)-test, p=0.036). The influence of other factors were evaluated using a unconditional logistic regression that confirmed a significant association of GSTT1 positive genotype with an increased OR for CVD (OR=6.29, 95% CI 1.32-29.95) beside a significant effect of age (OR=1.13, 95% CI 1.03-1.26 year(-1)). Finally, subjects with an higher MN frequency showed a higher MR for CVD (Log-rank test, p=0.001). CONCLUSIONS: MN confirmed to be a suitable cytogenetic biomarker for early prediction of CVD death. The GSTT1 positive genotype is associated with an increased MR for CVD.     

Regulation of microRNA-145 by growth arrest and differentiation

MicroRNA145 (miR145), a tumor suppressor miR, has been reported to inhibit growth of human cancer cells, to induce differentiation and to cause apoptosis, all conditions that result in growth arrest. In order to clarify the functional effects of miR145, we have investigated its expression in diverse conditions and different cell lines. Our results show that miR145 levels definitely increase in differentiating cells and also in growth-arrested cells, even in the absence of differentiation. Increased expression during differentiation sometimes occurs as a late event, suggesting that miR145 could be required either early or late during the differentiation process.