Epigenetic regulation of amniotic fluid mesenchymal stem cell differentiation to the mesodermal lineages at normal and fetus-diseased gestation

Human mesenchymal stem cells isolated from amniotic fluid (AF-MSCs) demonstrate the potency for self-renewal and multidifferentiation, and can, therefore, be a potential alternative source of stem cells adapted for therapeutic purposes. The object of this study is to evaluate the efficacy of MSCs from AF when the pregnancy is normal or when the fetus is affected during pregnancy to differentiate into mesodermal lineage tissues and to elucidate epigenetic states responsible for terminal adipogenic and osteogenic differentiation. The morphology of AF-MSCs from two cell sources and the expression of the cell surface-specific (CD44, CD90, and CD105) markers and pluripotency (Oct4, Nanog, Sox2, and Rex1) genes were quite similar and underwent mesodermal lineage differentiation because this is shown by the typical cell morphology and of genes’ expression specific for adipogenic (peroxisome proliferator-activated receptor-ɣ, adiponectin) and osteoblastic (alkaline phosphatase, osteopontin, and osteocalcin) differentiation. Terminal lineage-specific differentiation was related to differential expression of miR-17, miR-21, miR-34a, and miR-146a, decreased levels of acetylated H4 and H3K9, trimethylated H3K4 and H3K9, and the retention of H3K27me3 along with a reduction in the levels of HDAC1, DNMT1, and PRC1/2 proteins (BMI1/SUZ12). No significant distinction could be identified in the levels of expression of all epigenetic or pluripotency markers between undifferentiated MSCs isolated from AF of normal gestation and pregnancy where the fetus was damaged and between those differentiated toward adipocytes or osteoblasts. The expressional changes of those marks and microRNAs that occurred during terminal differentiation to mesodermal tissues indicate subtle epigenetic regulation in AF-MSCs when the condition of the fetus is healthy normal or diseased. More detailed studies of epigenetic mechanisms may offer a better understanding of AF-MSCs differentiation in fetus-diseased conditions and their usage in an autologous therapeutic application and prenatal disease research.     

Pyrrolidinone-bearing methylated and halogenated benzenesulfonamides as inhibitors of carbonic anhydrases

Two series of benzenesulfonamides bearing methyl groups at ortho/ortho or meta/ortho positions and a pyrrolidinone moiety at para position were synthesized and tested as inhibitors of the twelve catalytically active human carbonic anhydrase (CA) isoforms. Observed binding affinities were determined by fluorescent thermal shift assay and intrinsic binding affinities representing the binding of benzenesulfonamide anion to the Zn(II)-bound water form of CA were calculated. Introduction of dimethyl groups into benzenesulfonamide ring decreased the binding affinity to almost all CA isoforms, but gained in selectivity towards one CA isoform. A chloro group at the meta position of 2,6-dimethylbenzenesulfonamide derivatives did not influence the binding to CA I, but it increased the affinity to all other CAs, especially, CA VII and CA XIII (up to 500 fold). The compounds may be used for further development of CA inhibitors with higher selectivity to particular CA isoforms.     

Neonatal mice genetically modified to express the elastase inhibitor elafin are protected against the adverse effects of mechanical ventilation on lung growth

Mechanical ventilation (MV) with O(2)-rich gas (MV-O(2)) offers life-saving treatment for newborn infants with respiratory failure, but it also can promote lung injury, which in neonates translates to defective alveolar formation and disordered lung elastin, a key determinant of lung growth and repair. Prior studies in preterm sheep and neonatal mice showed that MV-O(2) stimulated lung elastase activity, causing degradation and remodeling of matrix elastin. These changes yielded an inflammatory response, with TGF-β activation, scattered elastic fibers, and increased apoptosis, culminating in defective alveolar septation and arrested lung growth. To see whether sustained inhibition of elastase activity would prevent these adverse pulmonary effects of MV-O(2), we did studies comparing wild-type (WT) and mutant neonatal mice genetically modified to express in their vascular endothelium the human serine elastase inhibitor elafin (Eexp). Five-day-old WT and Eexp mice received MV with 40% O(2) (MV-O(2)) for 24-36 h. WT and Eexp controls breathed 40% O(2) without MV. MV-O(2) increased lung elastase and MMP-9 activity, resulting in elastin degradation (urine desmosine doubled), TGF-β activation (pSmad-2 increased 6-fold), apoptosis (cleaved-caspase-3 increased 10-fold), and inflammation (NF-κB activation, influx of neutrophils and monocytes) in lungs of WT vs. unventilated controls. These changes were blocked or blunted during MV-O(2) of Eexp mice. Scattered lung elastin and emphysematous alveoli observed in WT mice after 36 h of MV-O(2) were attenuated in Eexp mice. Both WT and Eexp mice showed defective VEGF signaling (decreased lung VEGF-R2 protein) and loss of pulmonary microvessels after lengthy MV-O(2), suggesting that elafin's beneficial effects during MV-O(2) derived primarily from preserving matrix elastin and suppressing lung inflammation, thereby enabling alveolar formation during MV-O(2). These results suggest that degradation and remodeling of lung elastin can contribute to defective lung growth in response to MV-O(2) and might be targeted therapeutically to prevent ventilator-induced neonatal lung injury.     

Chloroplasts of Arabidopsis are the source and a primary target of a plant-specific programmed cell death signaling pathway

Enhanced levels of singlet oxygen ((1)O(2)) in chloroplasts trigger programmed cell death. The impact of (1)O(2) production in chloroplasts was monitored first in the conditional fluorescent (flu) mutant of Arabidopsis thaliana that accumulates (1)O(2) upon a dark/light shift. The onset of (1)O(2) production is rapidly followed by a loss of chloroplast integrity that precedes the rupture of the central vacuole and the final collapse of the cell. Inactivation of the two plastid proteins EXECUTER (EX1) and EX2 in the flu mutant abrogates these responses, indicating that disintegration of chloroplasts is due to EX-dependent signaling rather than (1)O(2) directly. In flu seedlings, (1)O(2)-mediated cell death signaling operates as a default pathway that results in seedlings committing suicide. By contrast, EX-dependent signaling in the wild type induces the formation of microlesions without decreasing the viability of seedlings. (1)O(2)-mediated and EX-dependent loss of plastid integrity and cell death in these plants occurs only in cells containing fully developed chloroplasts. Our findings support an as yet unreported signaling role of (1)O(2) in the wild type exposed to mild light stress that invokes photoinhibition of photosystem II without causing photooxidative damage of the plant.     

An arc of unpaired "hinge bases" facilitates information exchange among functional centers of the ribosome

Information must be shared and functions coordinated among the spatially distinct functional centers of the ribosome. To address these issues, a yeast-based genetic system enabling generation of stable strains expressing only mutant forms of rRNA was devised. The B1a bridge (helix 38) has been implicated in the subtle modulation of numerous ribosomal functions. Base-specific mutations were introduced into helix 38 at sites affecting the B1a bridge and where it contacts the aminoacyl-tRNA (aa-tRNA) D-loop. Both sets of mutants promoted increased affinities for aa-tRNA but had different effects in their responses to two A-site-specific drugs and on suppression nonsense codons. Structural analyses revealed an arc of nucleotides in 25S rRNA that link the B1a bridge, the peptidyltransferase center, the GTPase-associated center, and the sarcin/ricin loop. We propose that a series of regularly spaced "hinge bases" provide fulcrums around which rigid helices can reorient themselves depending on the occupancy status of the A-site.     

Indapamide-like benzenesulfonamides as inhibitors of carbonic anhydrases I, II, VII, and XIII

A series of novel 2-chloro-5-[(1-benzimidazolyl- and 2-benzimidazolylsulfanyl)acetyl]benzene-sulfonamides were designed and synthesized. Their binding to recombinant human carbonic anhydrase (hCA) isozymes I, II, VII, and XIII was determined by isothermal titration calorimetry and thermal shift assay. The designed S-alkylated benzimidazole derivatives exhibited stronger binding than the indapamide-like N-alkylated benzimidazoles, with the K(d) reaching about 50-100 nM with drug-targeted hCAs VII and XIII. The cocrystal structures of selected compounds with hCA II were determined by X-ray crystallography, and structural features of the binding event were revealed.     

Testosterone and Visceral Fat in Midlife Women: The Study of Women's Health Across the Nation (SWAN) Fat Patterning Study

Visceral fat (VF) increases with the menopause and is an independent predictor of the metabolic syndrome, diabetes, and cardiovascular disease (CVD) in women. Little is known about how hormonal changes during the menopausal transition are related to the increase in VF. We aimed to determine the relationship between bioavailable testosterone and VF in middle‐aged women at various stages of the menopausal transition and whether this relationship is independent of age and other CVD risk factors. The Study of Women's Health Across the Nation (SWAN) is a longitudinal, community‐based study. This report uses baseline data from a population‐based longitudinal ancillary study at the Chicago site to examine the cross‐sectional relationship between testosterone and computed tomography (CT)–assessed VF in women at different stages of the menopausal transition. Included are 359 women (47.2% black), aged 42–60 years, who were randomly selected from a complete community census in which a 72% participation rate was achieved. In multivariate models, bioavailable testosterone was associated with VF independent of age, race, percent total body fat, and other cardiovascular risk factors. Bioavailable testosterone was a stronger predictor than estradiol and was interchangeable in its strength of association with sex hormone–binding globulin (SHBG). As bioavailable testosterone was associated with VF even after adjusting for insulin resistance, this suggests that it plays an important role in regional fat distribution. Our findings may have direct implications in explaining the effect of menopause‐related testosterone predominance on VF accumulation and subsequent cardiovascular risk.