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.     

Adverse reactions to acetylcysteine and effects of overdose.

Since the introduction in 1979 of intravenous acetylcysteine (Parvolex) as an antidote for overdosage of paracetamol the National Poisons Information Service and the manufacturer have been notified of 38 adverse reactions that were anaphylactoid in nature and 19 accidental overdoses. The most common feature of the anaphylactoid reaction to normal dosage was rash; other features reported included angioedema, hypotension, and bronchospasm; all the patients recovered. The features associated with an overdose of acetylcysteine were similar but more severe; two patients died, but the extent to which the overdose of acetylcysteine may have been implicated was not clear in either case.     

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.     

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.     

Purification and characterisation of PQQ-dependent glucose dehydrogenase from Erwinia sp. 34-1

A pyrroloquinoline quinone-dependent glucose dehydrogenase from an isolate of Erwinia sp. has been purified to homogeneity and characterised. SDS-PAGE showed a single band of 88.4 kDa. The enzyme activity was optimal at 47°C and pH 7.5–8.5. The Michaelis constants for d-glucose and PMS were 3.2 mM and 132 M, respectively (50 mM glycine–NaOH, at pH 8.0).     

The role of unstructured highly charged regions on the stability and specificity of dimerization of two-stranded alpha-helical coiled-coils: analysis of the neck-hinge region of the kinesin-like motor protein Kif3A

We investigated the folding, stability, and specificity of dimerization of the neck-hinge region (residues 356-416) of the kinesin-like protein Kif3A. We showed that the predicted coiled-coil on its own (residues 356-377) will fold autonomously in solution. We then explored the ability of oppositely charged regions to specify heterodimer formation in coiled-coils by synthesizing analogs of the neck coiled-coil region with and without various negatively and positively charged extensions to the C-terminus of the neck coiled-coil and characterizing these analogs by circular dichroism spectroscopy. The charged region alone (residues 378-416) adopted a random-coil structure and this region remained unfolded in the presence of the coiled-coil. Redox experiments demonstrated that oppositely charged regions specified the formation of a hetero-two-stranded coiled-coil. Denaturation studies with urea demonstrated a decrease in coiled-coil stability with the addition of negatively charged residues in the homostranded coiled-coil; conversely, the addition of the positively charged region (residues 403-416) of Kif3A C-terminally to the neck coiled-coil did not affect coiled-coil stability. Overall, our results suggest that electrostatic attractions drive the specificity of heterodimerization of the coiled-coil, not the removal of positive or negative charge-charge repulsions, while maintaining the stability of the heterodimer compared to that of the stablest homodimer.     

H-Y antigen expression in different tissues from transsexuals

Summary H-Y-antigen expression was analyzed in patients with transsexuality. Peripheral blood lymphocytes and various tissues were examined using the cytotoxicity assay of Goldberg et al. (1971). Peripheral blood lymphocytes from healthy male and female subjects were used as controls as well as tissues from nontranssexual individuals and from male and female C57Bl/6J mice. In three female-to-male transsexuals the peripheral blood lymphocytes were H-Y antigen positive. In these patients also their ovaries, uterus, and mammae were found to be H-Y antigen positive. Three male-to-female transsexuals were examined. The peripheral blood lymphocytes in two of these patients were found to be H-Y antigen negative. Their testes were also H-Y antigen negative, as well as the epididymus, the corpus cavernosum penis, and the cremaster muscle which was analyzed in one of them. One male-to-female transsexual had peripheral blood lymphocytes which were H-Y antigen positive; this patient had testis and corpus cavernosum penis which were also H-Y-antigen positive.