Deficiency of the Deubiquitinase UCHL1 Attenuates Pulmonary Arterial Hypertension.

BACKGROUND: The ubiquitin-proteasome system regulates protein degradation and the development of pulmonary arterial hypertension (PAH), but knowledge about the role of deubiquitinating enzymes in this process is limited. UCHL1 (ubiquitin carboxyl-terminal hydrolase 1), a deubiquitinase, has been shown to reduce AKT1 (AKT serine/threonine kinase 1) degradation, resulting in higher levels. Given that AKT1 is pathological in pulmonary hypertension, we hypothesized that UCHL1 deficiency attenuates PAH development by means of reductions in AKT1. METHODS: Tissues from animal pulmonary hypertension models as well as human pulmonary artery endothelial cells from patients with PAH exhibited increased vascular UCHL1 staining and protein expression. Exposure to LDN57444, a UCHL1-specific inhibitor, reduced human pulmonary artery endothelial cell and smooth muscle cell proliferation. Across 3 preclinical PAH models, LDN57444-exposed animals, Uchl1 knockout rats (Uchl1-/-), and conditional Uchl1 knockout mice (Tie2Cre-Uchl1fl/fl) demonstrated reduced right ventricular hypertrophy, right ventricular systolic pressures, and obliterative vascular remodeling. Lungs and pulmonary artery endothelial cells isolated from Uchl1-/- animals exhibited reduced total and activated Akt with increased ubiquitinated Akt levels. UCHL1-silenced human pulmonary artery endothelial cells displayed reduced lysine(K)63-linked and increased K48-linked AKT1 levels. RESULTS: Supporting experimental data, we found that rs9321, a variant in a GC-enriched region of the UCHL1 gene, is associated with reduced methylation (n=5133), increased UCHL1 gene expression in lungs (n=815), and reduced cardiac index in patients (n=796). In addition, Gadd45α (an established demethylating gene) knockout mice (Gadd45α-/-) exhibited reduced lung vascular UCHL1 and AKT1 expression along with attenuated hypoxic pulmonary hypertension. CONCLUSIONS: Our findings suggest that UCHL1 deficiency results in PAH attenuation by means of reduced AKT1, highlighting a novel therapeutic pathway in PAH.

New molecular evidence on the members of the genus Ortholinea (Cnidaria, Myxozoa) and the description of Ortholinea hamsiensis n. sp. infecting the urinary bladder of European anchovy Engraulis engrasicolus in the Black Sea.

Members of the genus Ortholinea are among the worldwide distributed myxozoan parasites that mainly infect marine fish. In this study, a new myxosporean species, Ortholinea hamsiensis n. sp., was isolated from the urinary bladder of European anchovy Engraulis engrasicolus collected from the Sinop coasts of the Black Sea. The prevalence and density values of infection were 1.4% and 1­5 individuals in the field of view (1 + ), respectively. Mature myxospores are subspherical with slight tapering down to the less pronounced tip in the frontal view and subspherical in the sutural view. Myxospores measured 9.1 ± 0.25 (8.8­9.9) µm in length, 9.2 ± 0.11 (8.9­9.4) µm in thickness, and 8.4 ± 0.33 (8.2-9.1) µm in width. Two polar capsules equal in size measured 3.1 ± 0.11 (3.0­3.3) µm in length and 2.7 ± 0.11 (2.6­2.9) µm in width. The polar tubule had 3­4 coils. Along with morphological peculiarities, the results of the 18S rDNA also revealed it to be a new species for science compared to the other species of the genus. In this study, another myxosporean species O. gobiusi was also detected in round goby Neogobius melanostomus with a prevalence of infection value of 4.8% and a density of 1­5 individuals in the field of view (1 + ). The present study also provided the first data of 18S rDNA of O. gobiusi from N. melanostomus and type species of the genus O. divergens from Gobius niger and the phylogenetic relationships of these species with other Ortholinea species have been revealed.

Molecular and morphological description of Ceratomyxa scophthalmi sp. nov. (Myxozoa) infecting Scophthalmus maeoticus and first report of Myxidium finnmarchicum in the Black Sea.

In the present study, 2 species of myxozoan parasites, the novel Ceratomyxa scophthalmi sp. nov. and Myxidium finnmarchicum, were documented from the gallbladder of Scophthalmus maeoticus inhabiting the Turkish Black Sea coast at Sinop. C. scophthalmi sp. nov. had mostly spherical, clavate, and fine-grained polymorphic plasmodia that ranged in diameter from 16.0 to 29.0 µm. Spores were arcuate with a convex anterior margin and concave posterior. Valves of spores were highly elongated and unequal in thickness with one valve 1.1-1.3 times thicker than the other. Valves narrowed gradually toward slightly truncated ends. The mean (range) dimensions of spores were 9.4 (8.5-10.0) µm in length and 49.6 (44.5-55.0) µm in thickness, with polar capsules that were 3.1 (2.7-3.5) µm in length and 3.2 (2.7-3.5) µm in width. Polar filaments were coiled with 4-5 turns. Large numbers of immature forms of C. scophthalmi sp. nov., which were often 'crumpled', and mature forms were found together in the gallbladder bile. Along with morphological differences, phylogenetic analysis based on 18S rDNA, including pairwise nucleotide similarities with other related Ceratomyxa species, suggests C. scophthalmi as a novel species. Moreover, this report provides the first morphological and molecular descriptions of M. finnmarchicum outside of its original geographical location and type host species.

Genetic structure and demographic history of endangered Alburnus tarichi (Güldenstädt, 1814) populations from Lake Van basin in Turkey inferred from mtDNA analyses.

Genetic diversity, genetic structure, and demographic history of the endemic and endangered cyprinid species Alburnus tarichi based on samples from 17 populations consisting of resident and potamodromous specimens from the Lake Van basin in eastern Turkey were analyzed using two mitochondrial DNA markers. A. tarichi populations in the Lake Van basin are genetically heterogeneous, as indicated by the high haplotype and low nucleotide diversity of 1233 bp of the 16S rRNA marker (44 haplotypes; 70 polymorphic sites, haploid diversity (Hd) = 0.9130, π = 0.0032) and 1140 bp of the cyt b marker (47 haplotypes; 82 polymorphic sites, Hd = 0.9339, π = 0.0057). Clades were separated by average sequence divergences of 1.94% (II vs. III), 1.80% (I vs. III), and 0.66% (I vs. II). Based on these clades, AMOVA analysis revealed that 80.76% of the total variation occurred among populations, 10.74% occurred within populations, and only 8.50% occurred between populations within groups for the concatenated 16S rRNA-cyt b sequences. Pairwise FST values varied from 0.0167 to 0.9705 for the concatenated 16S rRNA-cyt b dataset, emphasizing the high genetic variation among populations. The time since the endemic tarek populations split from their last common ancestor has been dated to 5.647 Ma (95% highest posterior density: 4.183-7.011 Ma) in the Messinian Stage. Recent population expansion for tarek populations has been determined by neutrality tests and mismatch distribution analyses. The results of this study provide valuable information on the genetic population structure, conservation, and management of this species.

IL-18 mediates sickle cell cardiomyopathy and ventricular arrhythmias.

Previous reports indicate that IL18 is a novel candidate gene for diastolic dysfunction in sickle cell disease (SCD)-related cardiomyopathy. We hypothesize that interleukin-18 (IL-18) mediates the development of cardiomyopathy and ventricular tachycardia (VT) in SCD. Compared with control mice, a humanized mouse model of SCD exhibited increased cardiac fibrosis, prolonged duration of action potential, higher VT inducibility in vivo, higher cardiac NF-κB phosphorylation, and higher circulating IL-18 levels, as well as reduced voltage-gated potassium channel expression, which translates to reduced transient outward potassium current (Ito) in isolated cardiomyocytes. Administering IL-18 to isolated mouse hearts resulted in VT originating from the right ventricle and further reduced Ito in SCD mouse cardiomyocytes. Sustained IL-18 inhibition via IL-18-binding protein resulted in decreased cardiac fibrosis and NF-κB phosphorylation, improved diastolic function, normalized electrical remodeling, and attenuated IL-18-mediated VT in SCD mice. Patients with SCD and either myocardial fibrosis or increased QTc displayed greater IL18 gene expression in peripheral blood mononuclear cells (PBMCs), and QTc was strongly correlated with plasma IL-18 levels. PBMC-derived IL18 gene expression was increased in patients who did not survive compared with those who did. IL-18 is a mediator of sickle cell cardiomyopathy and VT in mice and a novel therapeutic target in patients at risk for sudden death.

Protective effects of melatonin on lung damage associated with one-lung ventilation: An experimental study.

BACKGROUND: This study aims to investigate the protective effect of melatonin on lung damage induced by one-lung ventilation in a rat model. METHODS: A total of 20 healthy, Sprague-Dawley male rats were randomized into two equal groups as control (n=10) and melatonin groups (n=10). The control group underwent 60 min of one-lung ventilation, followed by 30 min of two-lung ventilation. In the melatonin group, the rats were administered 10 mg/kg melatonin intraperitoneally 10 min before the start of the experiment. At the end of both ventilation periods, tissue samples were obtained from the lungs of the control and melatonin groups for biochemical analysis and histopathological examinations. Tissue superoxide dismutase, malondialdehyde, and tumor necrosis factor-alpha levels were measured. Lung tissue samples were examined based on the presence and amount of alveolar congestion, intra-alveolar bleeding, and leukocyte and lymphocyte infiltration. RESULTS: At the end of the study, lung tissue malondialdehyde (3.8±0.9 vs. 1.8±0.8 µM; p<0.001) and tumor necrosis factor-alpha levels (47.2±15.0 vs. 21.8±7.2 pg/mL; p<0.001) of the melatonin group were found to significantly decrease, compared to the control group. Superoxide dismutase levels of the melatonin group increased at the end of both ventilation periods, and the increase at the end of one-lung ventilation was found to be statistically significant (0.6±0.2 vs. 1.3±0.7 U/mL; p<0.05). Histopathological examination demonstrated that the tissue damage was less in the melatonin group. There was a significant decrease in the alveolar congestion in this group (p=0.0401). Although other histopathological parameters decreased in the melatonin group, no significant difference was found. CONCLUSION: Our study results demonstrate that melatonin has protective effects on the lung damage induced by one-lung ventilation both at biochemical and histopathological levels in rats.

A Genome-Wide Screen Reveals a Role for the HIR Histone Chaperone Complex in Preventing Mislocalization of Budding Yeast CENP-A.

Centromeric localization of the evolutionarily conserved centromere-specific histone H3 variant CENP-A (Cse4 in yeast) is essential for faithful chromosome segregation. Overexpression and mislocalization of CENP-A lead to chromosome segregation defects in yeast, flies, and human cells. Overexpression of CENP-A has been observed in human cancers; however, the molecular mechanisms preventing CENP-A mislocalization are not fully understood. Here, we used a genome-wide synthetic genetic array (SGA) to identify gene deletions that exhibit synthetic dosage lethality (SDL) when Cse4 is overexpressed. Deletion for genes encoding the replication-independent histone chaperone HIR complex (HIR1, HIR2, HIR3, HPC2) and a Cse4-specific E3 ubiquitin ligase, PSH1, showed highest SDL. We defined a role for Hir2 in proteolysis of Cse4 that prevents mislocalization of Cse4 to noncentromeric regions for genome stability. Hir2 interacts with Cse4 in vivo, and hir2∆ strains exhibit defects in Cse4 proteolysis and stabilization of chromatin-bound Cse4 Mislocalization of Cse4 to noncentromeric regions with a preferential enrichment at promoter regions was observed in hir2∆ strains. We determined that Hir2 facilitates the interaction of Cse4 with Psh1, and that defects in Psh1-mediated proteolysis contribute to increased Cse4 stability and mislocalization of Cse4 in the hir2∆ strain. In summary, our genome-wide screen provides insights into pathways that regulate proteolysis of Cse4 and defines a novel role for the HIR complex in preventing mislocalization of Cse4 by facilitating proteolysis of Cse4, thereby promoting genome stability.

Polo kinase Cdc5 associates with centromeres to facilitate the removal of centromeric cohesin during mitosis.

Sister chromatid cohesion is essential for tension-sensing mechanisms that monitor bipolar attachment of replicated chromatids in metaphase. Cohesion is mediated by the association of cohesins along the length of sister chromatid arms. In contrast, centromeric cohesin generates intrastrand cohesion and sister centromeres, while highly cohesin enriched, are separated by >800 nm at metaphase in yeast. Removal of cohesin is necessary for sister chromatid separation during anaphase, and this is regulated by evolutionarily conserved polo-like kinase (Cdc5 in yeast, Plk1 in humans). Here we address how high levels of cohesins at centromeric chromatin are removed. Cdc5 associates with centromeric chromatin and cohesin-associated regions. Maximum enrichment of Cdc5 in centromeric chromatin occurs during the metaphase-to-anaphase transition and coincides with the removal of chromosome-associated cohesin. Cdc5 interacts with cohesin in vivo, and cohesin is required for association of Cdc5 at centromeric chromatin. Cohesin removal from centromeric chromatin requires Cdc5 but removal at distal chromosomal arm sites does not. Our results define a novel role for Cdc5 in regulating removal of centromeric cohesins and faithful chromosome segregation.

A new Pseudophoxinus (Teleostei, Cyprinidae) species from Southwestern Anatolia, with remarks on the distribution of the genus in western Anatolia.

Pseudophoxinus burduricus sp. n. is described from drainages of Salda and Burdur lakes, southwestern Turkey. It is distinguished from other Anatolian Pseudophoxinus by a combination of characters: lateral line incomplete, with 21-39 (commonly 26-37) perforated scales and 47-57+1-2 scales in lateral series; 10½-12½ scale rows between lateral line and dorsal fin origin, 3-4(5) scale rows between lateral line and the pelvic fin origin; dorsal fin commonly with 7½ branched rays; anal fin commonly with 6½ branched rays; 7-8(9) gill rakers on the first branchial arch; a faint and diffuse epidermal black stripe from eye to caudal fin base in alive and preserved individuals; mouth slightly subterminal, tip of mouth cleft on about level of lower margin of eye; snout rounded, its length greater than eye diameter. Comparison is given with all Pseudophoxinus species from western Anatolia.

Reactive oxygen species homeostasis and signalling during drought and salinity stresses.

Water deficit and salinity, especially under high light intensity or in combination with other stresses, disrupt photosynthesis and increase photorespiration, altering the normal homeostasis of cells and cause an increased production of reactive oxygen species (ROS). ROS play a dual role in the response of plants to abiotic stresses functioning as toxic by-products of stress metabolism, as well as important signal transduction molecules. In this review, we provide an overview of ROS homeostasis and signalling in response to drought and salt stresses and discuss the current understanding of ROS involvement in stress sensing, stress signalling and regulation of acclimation responses.

Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants expressing proteins of unknown function.

Over one-quarter of all plant genes encode proteins of unknown function that can be further classified as proteins with obscure features (POFs), which lack currently defined motifs or domains, or proteins with defined features, which contain at least one previously defined domain or motif. Although empirical data in the form of transcriptome and proteome profiling suggest that many of these proteins play important roles in plants, their functional characterization remains one of the main challenges in modern biology. To begin the functional annotation of proteins with unknown function, which are involved in the oxidative stress response of Arabidopsis (Arabidopsis thaliana), we generated transgenic Arabidopsis plants that constitutively expressed 23 different POFs (four of which were specific to Arabidopsis) and 18 different proteins with defined features. All were previously found to be expressed in response to oxidative stress in Arabidopsis. Transgenic plants were tested for their tolerance to oxidative stress imposed by paraquat or t-butyl hydroperoxide, or were subjected to osmotic, salinity, cold, and heat stresses. More than 70% of all expressed proteins conferred tolerance to oxidative stress. In contrast, >90% of the expressed proteins did not confer enhanced tolerance to the other abiotic stresses tested, and approximately 50% rendered plants more susceptible to osmotic or salinity stress. Two Arabidopsis-specific POFs, and an Arabidopsis and Brassica-specific protein of unknown function, conferred enhanced tolerance to oxidative stress. Our findings suggest that tolerance to oxidative stress involves mechanisms and pathways that are unknown at present, including some that are specific to Arabidopsis or the Brassicaceae.

The zinc finger network of plants.

The zinc finger domain enables different proteins to interact with or bind DNA, RNA, or other proteins, and is present in the proteomes of many different organisms. Proteins containing zinc finger domain(s) were found to play important roles in eukaryotic cells regulating different signal transduction pathways and controlling processes, such as development and programmed cell death. There are many types of zinc finger proteins, classified according to the number and order of the Cys and His residues that bind the Zinc ion. Among these, the C2H2-type zinc finger proteins, with 176 members in Arabidopsis thaliana, constitute one of the largest families of transcriptional regulators in plants. They are mostly plant-specific and contain a conserved QALGGH sequence within their zinc finger domain. Recent studies revealed that C2H2 zinc finger proteins could function as key transcriptional repressors involved in the defense and acclimation response of plants to different environmental stress conditions. Here we highlight recent functional characterization studies of different C2H2 proteins in Arabidopsis, and suggest that many of these proteins function as part of a large regulatory network that senses and responds to different environmental stimuli.

The EAR-motif of the Cys2/His2-type zinc finger protein Zat7 plays a key role in the defense response of Arabidopsis to salinity stress.

Cys2/His2-type zinc finger proteins, which contain the EAR transcriptional repressor domain, are thought to play a key role in regulating the defense response of plants to biotic and abiotic stress conditions. Although constitutive expression of several of these proteins was shown to enhance the tolerance of transgenic plants to abiotic stress, it is not clear whether the EAR-motif of these proteins is involved in this function. In addition, it is not clear whether suppression of plant growth, induced in transgenic plants by different Cys2/His2 EAR-containing proteins, is mediated by the EAR-domain. Here we report that transgenic Arabidopsis plants constitutively expressing the Cys2/His2 zinc finger protein Zat7 have suppressed growth and are more tolerant to salinity stress. A deletion or a mutation of the EAR-motif of Zat7 abolishes salinity tolerance without affecting growth suppression. These results demonstrate that the EAR-motif of Zat7 is directly involved in enhancing the tolerance of transgenic plants to salinity stress. In contrast, the EAR-motif appears not to be involved in suppressing the growth of transgenic plants. Further analysis of Zat7 using RNAi lines suggests that Zat7 functions in Arabidopsis to suppress a repressor of defense responses. A yeast two-hybrid analysis identified putative interactors of Zat7 and the EAR-domain, including WRKY70 and HASTY, a protein involved in miRNA transport. Our findings demonstrate that the EAR-domain of Cys2/His2-type zinc finger proteins plays a key role in the defense response of Arabidopsis to abiotic stresses.

Gain- and loss-of-function mutations in Zat10 enhance the tolerance of plants to abiotic stress.

C(2)H(2)-zinc finger proteins that contain the EAR repressor domain are thought to play a key role in modulating the defense response of plants to abiotic stress. Constitutive expression of the C(2)H(2)-EAR zinc finger protein Zat10 in Arabidopsis was found to elevate the expression of reactive oxygen-defense transcripts and to enhance the tolerance of plants to salinity, heat and osmotic stress. Surprisingly, knockout and RNAi mutants of Zat10 were also more tolerant to osmotic and salinity stress. Our results suggest that Zat10 plays a key role as both a positive and a negative regulator of plant defenses.