Selective laser trabeculoplasty in the treatment of pseudoexfoliation glaucoma in patients allergic to all anti-glaucoma drops

Secondary chronic open-angle glaucoma associated with pseudoexfoliation (PEX) syndrome accounts for approximately 25% of all glaucomas and represents the most common identifiable cause of glaucoma overall. Selective laser trabeculoplasty (SLT) is effective in reducing intraocular pressure (IOP) in glaucomatous patients and has the advantage of preserving surrounding structures. We report here SLT treatment of a 82 year old female with a secondary developed open-angle pseudoexfoliation glaucoma allergic to all anti glaucoma eye drops especially those which contain bensalconium chloridum as preservative. Since patient was allergic also to methyl-cellulose, we performed SLT with water as a mediator. Patient had PEX syndrome for 10 years, immature cataracts on both eyes, and best corrected visual acuity (BCVA) 0.7 on the right and 0.2 on the left eye. We have monitored intraocular pressure (IOP), the changes in the visual field and optic nerve. Preoperative IOP was 28 mmHg on the right and 30 mmHg on the left eye. The follow up period was 24 months with time points for measured parameters every 3 months. After 18 months IOP remained in the normal values (average 17 mmHg) on the right eye, but on the left eye it increased up to 28 mmHg. SLT re-treatment was carried out on the left eye and the IOP stabilized again on the values between 16-18mmHg. There were no significant change in the visual field and optic nerve configuration before and after SLT (C/D value for right eye: 0.3-0.4; C/D left eye: 0.5). Based on this case report, SLT seems to be very effective treatment for maintaining regular IOP in patient with PEX who is allergic to all types of medications.     

Evidence that cytochrome b559 is involved in superoxide production in photosystem II: effect of synthetic short-chain plastoquinones in a cytochrome b559 tobacco mutant

Light-induced production of superoxide (O2*-) in spinach PSII (photosystem II) membrane particles was studied using EPR spin-trapping spectroscopy. The presence of exogenous PQs (plastoquinones) with a different side-chain length (PQ-n, n isoprenoid units in the side-chain) enhanced O2*- production in the following order: PQ-1>PQ-2>PQ-9. In PSII membrane particles isolated from the tobacco cyt (cytochrome) b559 mutant which carries a single-point mutation in the beta-subunit and also has a decreased amount of the alpha-subunit, the effect of PQ-1 was less than in the wild-type. The increase in LP (low-potential) cyt b559 content, induced by the incubation of spinach PSII membrane particles at low pH, resulted in a significant increase in O2*- formation in the presence of PQ-1, whereas it had little effect on O2*- production in the absence of PQ-1. The enhancement of O2*- formation induced by PQ-1 was not abolished by DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Under anaerobic conditions, dark oxidation of LP cyt b559 increased, as pH was decreased. The presence of molecular oxygen significantly enhanced dark oxidation of LP cyt b559. Based on these findings it is suggested that short-chain PQs stimulate O2*- production via a mechanism that involves electron transfer from Pheo- (pheophytin) to LP cyt b559 and subsequent auto-oxidation of LP cyt b559.     

Detecting hydrogen peroxide in leaves in vivo – a comparison of methods

Four hydrogen peroxide detecting probes, 3,3′‐diaminobenzidine (DAB), Amplex Red (AR), Amplex Ultra Red (AUR) and a europium–tetracycline complex (Eu₃Tc) were infiltrated into tobacco leaves and tested for sensitivity to light, toxicity, subcellular localization and capacity to detect H₂O₂ in vivo. In the absence of leaves, in water solutions, AUR was very much sensitive to strong light, AR showed slight light sensitivity, while DAB and Eu₃Tc were insensitive to irradiation. When infiltrated into the leaves, the probes decreased the photochemical yield (Φ_PSII) in the following order of effect AR > DAB > AUR > Eu₃Tc. With the exception of Eu₃Tc, all probes stimulated the build‐up of non‐photochemical quenching either temporally (DAB, AUR) or permanently (AR), showing that their presence may already limit the photosynthetic capacity of leaves, even in the absence of additional stress. This should be taken into account when using these probes in plant stress experiments. Confocal laser scanning microscopy studies with the three fluorescent H₂O₂ probes showed that the localizations of Eu₃Tc and AUR were mainly intercellular. AR partly penetrated into leaf chloroplasts but probably not into the thylakoid membranes. Photosynthesis‐related stress applications of AR seem to be limited by the low availability of internal leaf peroxidases. Applications of AR for kinetic H₂O₂ measurements would require a co‐infiltration of external peroxidase, imposing another artificial modifying factor and thus taking experiments further from ideal, in vivo conditions. Our results suggest that the studied H₂O₂ probes should be used in leaf studies with caution, carefully balancing benefits and artifacts.     

In Depth Characterization of Repetitive DNA in 23 Plant Genomes Reveals Sources of Genome Size Variation in the Legume Tribe Fabeae

The differential accumulation and elimination of repetitive DNA are key drivers of genome size variation in flowering plants, yet there have been few studies which have analysed how different types of repeats in related species contribute to genome size evolution within a phylogenetic context. This question is addressed here by conducting large-scale comparative analysis of repeats in 23 species from four genera of the monophyletic legume tribe Fabeae, representing a 7.6-fold variation in genome size. Phylogenetic analysis and genome size reconstruction revealed that this diversity arose from genome size expansions and contractions in different lineages during the evolution of Fabeae. Employing a combination of low-pass genome sequencing with novel bioinformatic approaches resulted in identification and quantification of repeats making up 55–83% of the investigated genomes. In turn, this enabled an analysis of how each major repeat type contributed to the genome size variation encountered. Differential accumulation of repetitive DNA was found to account for 85% of the genome size differences between the species, and most (57%) of this variation was found to be driven by a single lineage of Ty3/gypsy LTR-retrotransposons, the Ogre elements. Although the amounts of several other lineages of LTR-retrotransposons and the total amount of satellite DNA were also positively correlated with genome size, their contributions to genome size variation were much smaller (up to 6%). Repeat analysis within a phylogenetic framework also revealed profound differences in the extent of sequence conservation between different repeat types across Fabeae. In addition to these findings, the study has provided a proof of concept for the approach combining recent developments in sequencing and bioinformatics to perform comparative analyses of repetitive DNAs in a large number of non-model species without the need to assemble their genomes.     

ATP and MO25α Regulate the Conformational State of the STRADα Pseudokinase and Activation of the LKB1 Tumour Suppressor

Pseudokinases lack essential residues for kinase activity, yet are emerging as important regulators of signal transduction networks. The pseudokinase STRAD activates the LKB1 tumour suppressor by forming a heterotrimeric complex with LKB1 and the scaffolding protein MO25. Here, we describe the structure of STRADα in complex with MO25α. The structure reveals an intricate web of interactions between STRADα and MO25α involving the αC-helix of STRADα, reminiscent of the mechanism by which CDK2 interacts with cyclin A. Surprisingly, STRADα binds ATP and displays a closed conformation and an ordered activation loop, typical of active protein kinases. Inactivity is accounted for by nonconservative substitution of almost all essential catalytic residues. We demonstrate that binding of ATP enhances the affinity of STRADα for MO25α, and conversely, binding of MO25α promotes interaction of STRADα with ATP. Mutagenesis studies reveal that association of STRADα with either ATP or MO25α is essential for LKB1 activation. We conclude that ATP and MO25α cooperate to maintain STRADα in an “active” closed conformation required for LKB1 activation. It has recently been demonstrated that a mutation in human STRADα that truncates a C-terminal region of the pseudokinase domain leads to the polyhydramnios, megalencephaly, symptomatic epilepsy (PMSE) syndrome. We demonstrate this mutation destabilizes STRADα and prevents association with LKB1. In summary, our findings describe one of the first structures of a genuinely inactive pseudokinase. The ability of STRADα to activate LKB1 is dependent on a closed “active” conformation, aided by ATP and MO25α binding. Thus, the function of STRADα is mediated through an active kinase conformation rather than kinase activity. It is possible that other pseudokinases exert their function through nucleotide binding and active conformations.     

Expansion of Microsatellites on Evolutionary Young Y Chromosome

Sex chromosomes are an ideal system to study processes connected with suppressed recombination. We found evidence of microsatellite expansion, on the relatively young Y chromosome of the dioecious plant sorrel (Rumex acetosa, XY1Y2 system), but no such expansion on the more ancient Y chromosomes of liverwort (Marchantia polymorpha) and human. The most expanding motifs were AC and AAC, which also showed periodicity of array length, indicating the importance of beginnings and ends of arrays. Our data indicate that abundance of microsatellites in genomes depends on the inherent expansion potential of specific motifs, which could be related to their stability and ability to adopt unusual DNA conformations. We also found that the abundance of microsatellites is higher in the neighborhood of transposable elements (TEs) suggesting that microsatellites are probably targets for TE insertions. This evidence suggests that microsatellite expansion is an early event shaping the Y chromosome where this process is not opposed by recombination, while accumulation of TEs and chromosome shrinkage predominate later.     

Comparison of two approaches for quantitative O-linked glycan analysis used in characterization of recombinant proteins

The principal aim of this study was to demonstrate the optimization and fine-tuning of quantitative and nonselective analysis of O-linked glycans released from therapeutic glycoproteins. Two approaches for quantitative release of O-linked glycans were examined: ammonia-based β-elimination and hydrazinolysis deglycosylation strategies. A significant discrepancy in deglycosylation activity was observed between the ammonia-based and hydrazinolysis procedures. Specifically, the release of O-glycans from glycoproteins was approximately 20 to 30 times more efficient with hydrazine compared with ammonia-based β-elimination reagent. In addition, the ammonia-based reagent demonstrated bias in the release of particular glycan species. A robust quantitative hydrazinolysis procedure was developed for characterization of O-glycans. The method performance parameters were evaluated. It was shown that this procedure is superior for quantitative nonselective release of O-glycans. Identity confirmation and structure elucidation of O-glycans from hydrophilic interaction chromatography (HILIC) fractions was also demonstrated using linear ion trap Fourier transform mass spectrometry (LTQ FT MS) with mass accuracy below 1 ppm.     

ACTH-induced hypertension is dependent on the ouabain-binding site of the alpha2-Na+-K+-ATPase subunit

ACTH-induced-hypertension is commonly employed as a model of stress-related hypertension, and despite extensive investigation, the mechanisms underlying elevated blood pressure (BP) are not well understood. We have reported that ACTH treatment increases tail-cuff systolic pressure in wild-type mice but not in mutant mice expressing ouabain-resistant alpha(2)-Na(+)-K(+)-ATPase subunits (alpha2(R/R) mice). Since tail-cuff measurements involve restraint stress, the present study used telemetry to distinguish between an effect of ACTH on resting BP vs. an ACTH-enhanced stress response. We also sought to explore the mechanisms underlying ACTH-induced BP changes in mutant alpha2(R/R) mice vs. wild-type mice (ouabain-sensitive alpha(2)-Na(+)-K(+)-ATPase, alpha2(S/S) mice). Baseline BP was not different between the two genotypes, but after 5 days of ACTH treatment, BP increased in alpha2(S/S) (104.0 +/- 2.6 to 117.7 +/- 3.0 mmHg) but not in alpha2(R/R) mice (108.2 +/- 3.2 to 111.5 +/- 4.0 mmHg). To test the hypothesis that ACTH hypertension is related to inhibition of alpha(2)-Na(+)-K(+)-ATPase on vascular smooth muscle by endogenous cardiotonic steroids, we measured BP and regional blood flow. Results suggest a differential sensitivity of renal, mesenteric, and cerebral circulations to ACTH and that the response depends on the ouabain sensitivity of the alpha(2)-Na(+)-K(+)-ATPase. Baseline cardiac performance was elevated in alpha2(S/S) but not alpha2(R/R) mice. Overall, the data establish that the alpha(2)-Na(+)-K(+)-ATPase ouabain-binding site is of central importance in the development of ACTH-induced hypertension. The mechanism appears to be related to alterations in cardiac performance, and perhaps vascular tone in specific circulations, presumably caused by elevated levels of circulating cardiotonic steroids.