Blood Cell Mitochondrial DNA Content and Premature Ovarian Aging

Primary ovarian insufficiency (POI) is a critical fertility defect characterized by an anticipated and silent impairment of the follicular reserve, but its pathogenesis is largely unexplained. The frequent maternal inheritance of POI together with a remarkable dependence of ovarian folliculogenesis upon mitochondrial biogenesis and bioenergetics suggested the possible involvement of a generalized mitochondrial defect. Here, we verified the existence of a significant correlation between blood and ovarian mitochondrial DNA (mtDNA) content in a group of women undergoing ovarian hyperstimulation (OH), and then aimed to verify whether mtDNA content was significantly altered in the blood cells of POI women. We recruited 101 women with an impaired ovarian reserve: 59 women with premature ovarian failure (POF) and 42 poor responders (PR) to OH. A Taqman copy number assay revealed a significant mtDNA depletion (P<0.001) in both POF and PR women in comparison with 43 women of similar age and intact ovarian reserve, or 53 very old women with a previous physiological menopause. No pathogenic variations in the mitochondrial DNA polymerase γ (POLG) gene were detected in 57 POF or PR women with low blood mtDNA content. In conclusion, blood cell mtDNA depletion is a frequent finding among women with premature ovarian aging, suggesting that a still undetermined but generalized mitochondrial defect may frequently predispose to POI which could then be considered a form of anticipated aging in which the ovarian defect may represent the first manifestation. The determination of mtDNA content in blood may become an useful tool for the POI risk prediction.     

Olfactory functions at the intersection between environmental exposure to manganese and Parkinsonism

The olfactory function can be affected by occupational and environmental exposure to various neurotoxicants that can be transported through the olfactory pathway. Olfactory impairment is a highly recurrent non-motor dysfunction in Parkinson's disease and is considered an early predictive sign of neurodegeneration. Changes in olfactory perception may be caused by a dopaminergic dysregulation, possibly related to changes at the level of dopamine receptors. Manganese is an essential element that can become neurotoxic in various conditions inducing an overload in the organism. Being actively transported through the olfactory tract, manganese can cause impairment of olfactory function and motor coordination in different age groups like children and elderly. Odor and motor changes are interrelated and may be caused by a Mn-induced dopaminergic dysregulation affecting both functions. Given these findings, further research is imperative on the possible role of manganese exposure as a pathogenetic factor for Parkinsonism.     

A balance between Tel1 and Rif2 activities regulates nucleolytic processing and elongation at telomeres

Generation of G-strand overhangs at Saccharomyces cerevisiae yeast telomeres depends primarily on the MRX (Mre11-Rad50-Xrs2) complex, which is also necessary to maintain telomere length by recruiting the Tel1 kinase. MRX physically interacts with Rif2, which inhibits both resection and elongation of telomeres. We provide evidence that regulation of telomere processing and elongation relies on a balance between Tel1 and Rif2 activities. Tel1 regulates telomere nucleolytic processing by promoting MRX activity. In fact, the lack of Tel1 impairs MRX-dependent telomere resection, which is instead enhanced by the Tel1-hy909 mutant variant, which causes telomerase-dependent telomere overelongation. The Tel1-hy909 variant is more robustly associated than wild-type Tel1 to double-strand-break (DSB) ends carrying telomeric repeat sequences. Furthermore, it increases the persistence at a DSB adjacent to telomeric repeats of both MRX and Est1, which in turn likely account for the increased telomere resection and elongation in TEL1-hy909 cells. Strikingly, Rif2 is unable to negatively regulate processing and lengthening at TEL1-hy909 telomeres, indicating that the Tel1-hy909 variant overcomes the inhibitory activity exerted by Rif2 on MRX. Altogether, these findings highlight a primary role of Tel1 in overcoming Rif2-dependent negative regulation of MRX activity in telomere resection and elongation.     

Ultrastructural characterization of calcification onset and progression in subdermally implanted aortic valves. Histochemical and spectrometric data

Detailed characterization of the subdermal model is a significant tool for better understanding of calcification mechanisms occurring in heart valves. In previous ultrastructural investigation on six-week-implantated aortic valve leaflets, modified pre-embedding glutaraldehyde-cuprolinic-blue reactions (GA-CB) enabled sample decalcification with concurrent retention/staining of lipid-containing polyanionic material, which lined cells and cell-derived matrix-vesicle-like bodies (phthalocyanin-positive layers: PPLs) co-localizing with the earliest apatite nucleation sites. Additional post-embedding silver staining (GA-CB-S) revealed PPLs to contain calcium-binding sites. This investigation concerns valve leaflets subjected to shorter implantation times to shed light on the modifications associated with PPLs generation and calcification onset/progression. Spectrometric estimations revealed time-dependent calcium increase, for unreacted samples, and copper modifications indicating an increase in acidic, non-glycanic material, for GA-CB-reacted samples. Two-day-implant thin sections showed emission and subsequent reabsorption of lamellipodium-like protrusions by cells, originating ECM-containing vacuoles, and/or degeneration stages characterized by the appearance of GA-CB-S-reactive, organule-derived dense bodies and progressive dissolution of all cell membranes. In one-week-implants, the first PPL-lined cells were found to co-exist with cells where GA-CB-S-reactive material accumulated, or exudated towards their edges, or outcropped at the ECM milieu, so acquiring PPL features. PPL-derived material was observed increasingly to affect the ECM on thin sections of one-week- to six-week-implants. These results show an endogenous source for PPLs and reveal that a peculiar cascade of cell degenerative steps is associated with valve mineralization in the subdermal model, providing new useful parameters for more reliable comparison of this experimental calcification process versus the physiological and pathological processes.     

Saccharomyces cerevisiae Rif1 cooperates with MRX-Sae2 in promoting DNA-end resection

Diverse roles in DNA metabolism have been envisaged for budding yeast and mammalian Rif1. In particular, yeast Rif1 is involved in telomere homeostasis, while its mammalian counterpart participates in the cellular response to DNA double-strand breaks (DSBs). Here, we show that Saccharomyces cerevisiae Rif1 supports cell survival to DNA lesions in the absence of MRX or Sae2. Furthermore, it contributes to the nucleolytic processing (resection) of DSBs. This Rif1-dependent control of DSB resection becomes important for DSB repair by homologous recombination when resection activities are suboptimal.     

Primary Reactions of the LOV2 Domain of Phototropin Studied with Ultrafast Mid-Infrared Spectroscopy and Quantum Chemistry

Phototropins, major blue-light receptors in plants, are sensitive to blue light through a pair of flavin mononucleotide (FMN)-binding light oxygen and voltage (LOV) domains, LOV1 and LOV2. LOV2 undergoes a photocycle involving light-driven covalent adduct formation between a conserved cysteine and the FMN C(4a) atom. Here, the primary reactions of Avena sativa phototropin 1 LOV2 (AsLOV2) were studied using ultrafast mid-infrared spectroscopy and quantum chemistry. The singlet excited state (S1) evolves into the triplet state (T1) with a lifetime of 1.5 ns at a yield of ∼50%. The infrared signature of S1 is characterized by absorption bands at 1657 cm⁻¹, 1495-1415 cm⁻¹, and 1375 cm⁻¹. The T1 state shows infrared bands at 1657 cm⁻¹, 1645 cm⁻¹, 1491-1438 cm⁻¹, and 1390 cm⁻¹. For both electronic states, these bands are assigned principally to C=O, C=N, C-C, and C-N stretch modes. The overall downshifting of C=O and C=N bond stretch modes is consistent with an overall bond-order decrease of the conjugated isoalloxazine system upon a π-π^∗ transition. The configuration interaction singles (CIS) method was used to calculate the vibrational spectra of the S1 and T1 excited ππ^∗ states, as well as respective electronic energies, structural parameters, electronic dipole moments, and intrinsic force constants. The harmonic frequencies of S1 and T1, as calculated by the CIS method, are in satisfactory agreement with the evident band positions and intensities. On the other hand, CIS calculations of a T1 cation that was protonated at the N(5) site did not reproduce the experimental FMN T1 spectrum. We conclude that the FMN T1 state remains nonprotonated on a nanosecond timescale, which rules out an ionic mechanism for covalent adduct formation involving cysteine-N(5) proton transfer on this timescale. Finally, we observed a heterogeneous population of singly and doubly H-bonded FMN C(4)=O conformers in the dark state, with stretch frequencies at 1714 cm⁻¹ and 1694 cm⁻¹, respectively.     

Effects of a brassinosteroid on growth and electrogenic proton extrusion in Azuki bean epicotyls

Brassinolide, a plant hormone newly isolated from pollen, promotes growth of the stem of a number of plant species. Similar effects are induced by a brassinosteroid (BR), the synthetic 24-epibrassinolide. In this paper the effects of BR on acid secretion and transmembrane electrical potential difference in Azuki bean ( Vigna angularis , Ohwi and Ohashi cv. Takara) epicotyls were determined in short term experiments and compared with the effects on growth. At concentrations between 10^-7 to 10^-5 M , BR stimulates, similarly to indole-3-acetic acid (IAA), growth and H⁺ extrusion and hyperpolarizes the transmembrane electric potential (PD). These effects of BR, as well as those of IAA, are suppressed by inhibitors of RNA and protein synthesis. All these effects of BR and IAA appear roughly additive, even when both hormones are present at their optimal concentrations. The data are interpreted as showing that the action of BR on growth is at least in part mediated by its capability to activate electrogenic proton extrusion. The additivity of the effects of BR and IAA suggests that the primary mechanism of action of the two hormones is different.     

Further studies on oxygen loading in blood from anemic patients

Functional parameters of the whole blood oxygen loading from 66 anemic subjects, compared with those from 66 normal adult of both sexes display the following behaviour: a) Adult males and females differ for p50 values, which are higher in females because of their lower Hb level. This difference is already well known and appears to be statistically significant. b) The same difference is amplified in anemic subjects, where also pH lowering contributes to the right-shift of p50. c) The inverse relationship between p50 and DPG values differs if analysed at the actual pHs, at pH corrected at 7.4, or extrapolated from Bohr effect. In fact, the three relative plots do not parallel, and their most significant intersects drop around 0.8-0.9 value of DPG, and 26-27 torr of p50, i.e. within normal limits.     

Increase of Na+K+ ATPase activity in intact rat brain synaptosomes after their interaction with phosphatidylserine vesicles

Intact synaptosomes prepared from rat brain were incubated with phosphatidylserine vesicles. The synaptosomes incorporated the phospholipid in proportion to its concentration in the preincubation medium. The activity of membrane-bound enzyme $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ ATPase increased proportionally after treatment with phosphatidylserine liposomes.When breaking phosphatidylserine-enriched synaptosomes by osmotic shock or by sonication and when preparing synaptosomal membranes, the expected increase of $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ ATPase activity was not seen. Therefore, cellular integrity was fundamental in order to see the effect of phosphatidylserine on $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ ATPase activity.     

Phylogeography of Eriotheca species complex: insights into the origin and range expansion of apomictic and polyploid trees in Neotropical Savannas

• Polyploidy and whole genome duplication are major evolutionary drivers in plants. Climate variations during the Pleistocene have influenced distribution and range expansion worldwide. Similar trends have been reported for Cerrado plants, but no attempt has been made to link phylogeography with ploidy and breeding changes. Thus, we aimed to (i) assess ploidy and genome size of Eriotheca estevesiae Carv.-Sobr., and compare it with E. pubescens (Mart.) Schott & Endl. (Both included into the Eriotheca Stellate Trichome Species Complex – ESTSC). (ii) Subsequently, we investigated their phylogeography to see whether genetic structure and range expansion trends were similar to those previously described for the Cerrado biome. Finally (iii), we discuss whether ESTSC phylogeographic patterns could be associated with geographic parthenogenesis processes.
• Common cytogenetic techniques and flow cytometry were used to confirm chromosome number and genome size of E. estevesiae. We used three cpDNA regions to analyse 14 ESTSC Cerrado populations, for which we also obtained ploidy level and breeding information. We investigated haplotype diversity, population structure and tested neutrality, aiming to reconstruct phylogeographic scenarios.
• We found three ploidy levels and eight cpDNA haplotypes in ESTSC, one shared by most populations. Haplotype and ploidy distribution corroborated that E. pubescens, the widely distributed polyploid and apomictic species, may have originated from northern diploid and probably sexual E. estevesiae.
• Matrilinear cpDNA links support the idea that apomixis and polyploidy in ESTSC may have allowed range expansion during the Pleistocene, in a process analogous to the geographic parthenogenesis described elsewhere.