Multiple Propofol-binding Sites in a γ-Aminobutyric Acid Type A Receptor (GABAAR) Identified Using a Photoreactive Propofol Analog

Propofol acts as a positive allosteric modulator of γ-aminobutyric acid type A receptors (GABA_ARs), an interaction necessary for its anesthetic potency in vivo as a general anesthetic. Identifying the location of propofol-binding sites is necessary to understand its mechanism of GABA_AR modulation. [³H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[³H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (etomidate) and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit-binding sites for general anesthetics in the GABA_AR transmembrane domain. Here, we use a photoreactive analog of propofol (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol ([³H]AziPm)) to identify propofol-binding sites in heterologously expressed human α1β3 GABA_ARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [³H]AziPm photoincorporation into GABA_AR subunits maximally by ∼50%. When the amino acids photolabeled by [³H]AziPm were identified by protein microsequencing, we found propofol-inhibitable photolabeling of amino acids in the β3-α1 subunit interface (β3Met-286 in β3M3 and α1Met-236 in α1M1), previously photolabeled by [³H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [³H]AziPm photolabeling of β3Met-227 in βM1, the amino acid in the α1-β3 subunit interface photolabeled by R-[³H]mTFD-MPAB. The propofol-inhibitable [³H]AziPm photolabeling in the GABA_AR β3 subunit in conjunction with the concentration dependence of inhibition of that photolabeling by etomidate or R-mTFD-MPAB also establish that each anesthetic binds to the homologous site at the β3-β3 subunit interface. These results establish that AziPm as well as propofol bind to the homologous intersubunit sites in the GABA_AR transmembrane domain that binds etomidate or R-mTFD-MPAB with high affinity.     

Activity of paraoxonase 1 (PON1) and its relationship to markers of lipoprotein oxidation in healthy Slovaks

Low-density lipoproteins (LDLs), when modified by free radicals derived from artery wall cells, induce atherosclerosis. In contrast to oxidized LDL (ox-LDL), high-density lipoproteins (HDLs) are able to prevent atherosclerosis through a protein with antioxidant properties, paraoxonase 1 (PON1). The purpose of this study was to explore the association between the activity of HDL-associated PON1 and circulating ox-LDL as well as to investigate the relationship between ox-LDL and parameters of lipid profile in thirty Slovaks aged 21-73 years because recent studies have presented controversial results concerning PON1 and its role in LDL oxidation. For determination of circulating ox-LDL sandwich ELISA was used and other lipid parameters were determined by routine laboratory analyses. PON1 activities were assayed by two synthetic substrates - paraoxon and phenyl acetate. Lipid peroxides were determined spectrophotometrically. Of the lipid parameters examined, ox-LDL level correlated positively with total (P < 0.0001) and LDL-cholesterol (P < 0.001). Triacylglycerols (TAG) (P < 0.001), lipid peroxides (P < 0.01) and atherogenic index (AI = total cholesterol/HDL) (P < 0.0001) were also strongly correlated with ox-LDL. No inverse relationships were observed between ox-LDL and HDL-cholesterol or arylesterase/paraoxonase activities of PON1. Furthermore, it was found that ox-LDL (P < 0.01) and lipid peroxides (P < 0.05) were significantly higher in men than in women. PON1 arylesterase activity was marginally affected by sex. The results of this study suggest that the anti-atherogenic properties of HDLs are not directly related to their total concentration and that PON1 activity determined towards synthetic compounds (paraoxon and phenyl acetate) reflects no association with markers of oxidative stress. Furthermore, it follows from our results that men are more susceptible to developing atherosclerosis compared to women.     

MicroRNA‐guided gene expression in prostate cancer: Literature and database overview

Insight into the aberrant expression of microRNAs (miRNAs) and the genes that they regulate during the progression of cancer in general and prostate cancer (PCa) in particular is one of the most important issues in current molecular biomedicine and allows for the discovery of therapeutic or diagnostic miRNA targets. The present study aimed to analyze the available data regarding the direct or indirect effects of miRNAs on the expression of the mRNAs involved in carcinogenesis and to enable updating and optimizing the selection of the corresponding targets. The present review focuses on the data related to the genes with miRNA‐dependent expression during the development of PCa. The data used in this review have been extracted from research papers and the databases STRING, PANTHER and TargetScan, with a special focus on the genes directly associated with cell transformation and the maintenance of the transformed genotype, as well as tumor invasion and spread. The search for miRNA markers of PCa and therapeutically active molecules should rely on bioinformatics resources, such as data from recent experimental studies, as well as meta‐analysis and cross‐analysis of the data on the state of the tumor, patient status, histological/immunohistological data and data on mRNA–miRNA coexpression.     

Coordination conjugates of therapeutic proteins with drug carriers: a new approach for versatile advanced drug delivery

We present here a general system for the coordination attachment of therapeutic proteins to a drug delivery system and its application in combined therapy. Proof of concept is demonstrated by the synthesis and testing of the targeted drug delivery system for cytostatics, which is based on a combination of the drug carrier Zn-porphyrin-cyclodextrin conjugates and their supramolecular coordination complexes with immunoglobulins. This system can be as readily used for a variety of therapeutic and targeting proteins including PAs, MAs, lectins, and HSA. Moreover, it allows combined photodynamic therapy, cell targeted chemotherapy and immunotherapy. When tested in a mouse model with human C32 carcinoma, the therapeutic superiority of the coordination assembly nanosystem was shown in comparison with the efficacy of building blocks used for the construction of the system.     

Glutamatergic plasticity by synaptic delivery of GluR-B(long)-containing AMPA receptors

Activity-driven delivery of AMPA receptors is proposed to mediate glutamatergic synaptic plasticity, both during development and learning. In hippocampal CA1 principal neurons, such trafficking is primarily mediated by the abundant GluR-A subunit. We now report a study of GluR-B(long), a C-terminal splice variant of the GluR-B subunit. GluR-B(long) synaptic delivery is regulated by two forms of activity. Spontaneous synaptic activity-driven GluR-B(long) transport maintains one-third of the steady-state AMPA receptor-mediated responses, while GluR-B(long) delivery following the induction of LTP is responsible for approximately 50% of the resulting potentiation at the hippocampal CA3 to CA1 synapses at the time of GluR-B(long) peak expression-the second postnatal week. Trafficking of GluR-B(long)-containing receptors thus mediates a GluR-A-independent form of glutamatergic synaptic plasticity in the juvenile hippocampus.     

Repeat proliferation and partial endoreplication jointly shape the patterns of genome size evolution in orchids

Although the evolutionary drivers of genome size change are known, the general patterns and mechanisms of plant genome size evolution are yet to be established. Here we aim to assess the relative importance of proliferation of repetitive DNA, chromosomal variation (including polyploidy), and the type of endoreplication for genome size evolution of the Pleurothallidinae, the most species-rich orchid lineage. Phylogenetic relationships between 341 Pleurothallidinae representatives were refined using a target enrichment hybrid capture combined with high-throughput sequencing approach. Genome size and the type of endoreplication were assessed using flow cytometry supplemented with karyological analysis and low-coverage Illumina sequencing for repeatome analysis on a subset of samples. Data were analyzed using phylogeny-based models. Genome size diversity (0.2–5.1 Gbp) was mostly independent of profound chromosome count variation (2n = 12–90) but tightly linked with the overall content of repetitive DNA elements. Species with partial endoreplication (PE) had significantly greater genome sizes, and genomic repeat content was tightly correlated with the size of the non-endoreplicated part of the genome. In PE species, repetitive DNA is preferentially accumulated in the non-endoreplicated parts of their genomes. Our results demonstrate that proliferation of repetitive DNA elements and PE together shape the patterns of genome size diversity in orchids.     

Non-coding keratin variants associate with liver fibrosis progression in patients with hemochromatosis

Background

Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis.

Methods

The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed.

Results

We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury.

Conclusion

In patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development.     

Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels

Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability.