Cryoradiolysis of oxygenated cytochrome P450 17A1 with lyase substrates generates expected products.

When subjected to γ-irradiation at cryogenic temperatures the oxygenated complexes of Cytochrome P450 CYP17A1 (CYP17A1) bound with either of the lyase substrates, 17α-Hydroxypregnenolone (17-OH PREG) or 17α-Hydroxyprogesterone (17-OH PROG) are shown to generate the corresponding lyase products, dehydroepiandrosterone (DHEA) and androstenedione (AD) respectively. The current study uses gas chromatography-mass spectrometry (GC/MS) to document the presence of the initial substrates and products in extracts of the processed samples. A rapid and efficient method for the simultaneous determination of residual substrate and products by GC/MS is described without derivatization of the products. It is also shown that no lyase products were detected for similarly treated control samples containing no nanodisc associated CYP17 enzyme, demonstrating that the product is formed during the enzymatic reaction and not by GC/MS conditions, nor the conditions produced by the cryoradiolysis process.

Nanodiscs for the study of membrane proteins.

Nanodiscs represent a versatile tool for studies of membrane proteins and protein-membrane interactions under native-like conditions. Multiple variations of the Nanodisc platform, as well as new experimental methods, have been recently developed to understand various aspects of structure, dynamics and functional properties of systems involved in signaling, transport, blood coagulation and many other critically important processes. In this mini-review, we focus on some of these exciting recent developments that utilize the Nanodisc platform.

Edge-Terminated AlGaN/GaN/AlGaN Multi-Quantum Well Impact Avalanche Transit Time Sources for Terahertz Wave Generation.

In our pursuit of high-power terahertz (THz) wave generation, we propose innovative edge-terminated single-drift region (SDR) multi-quantum well (MQW) impact avalanche transit time (IMPATT) structures based on the AlxGa1-xN/GaN/AlxGa1-xN material system, with a fixed aluminum mole fraction of x = 0.3. Two distinct MQW diode configurations, namely p+-n junction-based and Schottky barrier diode structures, were investigated for their THz potential. To enhance reverse breakdown characteristics, we propose employing mesa etching and nitrogen ion implantation for edge termination, mitigating issues related to premature and soft breakdown. The THz performance is comprehensively evaluated through steady-state and high-frequency characterizations using a self-consistent quantum drift-diffusion (SCQDD) model. Our proposed Al0.3Ga0.7N/GaN/Al0.3Ga0.7N MQW diodes, as well as GaN-based single-drift region (SDR) and 3C-SiC/Si/3C-SiC MQW-based double-drift region (DDR) IMPATT diodes, are simulated. The Schottky barrier in the proposed diodes significantly reduces device series resistance, enhancing peak continuous wave power output to approximately 300 mW and DC to THz conversion efficiency to nearly 13% at 1.0 THz. Noise performance analysis reveals that MQW structures within the avalanche zone mitigate noise and improve overall performance. Benchmarking against state-of-the-art THz sources establishes the superiority of our proposed THz sources, highlighting their potential for advancing THz technology and its applications.

SINEs as Potential Expression Cassettes: Impact of Deletions and Insertions on Polyadenylation and Lifetime of B2 and Ves SINE Transcripts Generated by RNA Polymerase III.

Short Interspersed Elements (SINEs) are common in the genomes of most multicellular organisms. They are transcribed by RNA polymerase III from an internal promoter comprising boxes A and B. As transcripts of certain SINEs from mammalian genomes can be polyadenylated, such transcripts should contain the AATAAA sequence as well as those called ß- and τ-signals. One of the goals of this work was to evaluate how autonomous and independent other SINE parts are ß- and τ-signals. Extended regions outside of ß- and τ-signals were deleted from SINEs B2 and Ves and the derived constructs were used to transfect HeLa cells in order to evaluate the relative levels of their transcripts as well as their polyadenylation efficiency. If the deleted regions affected boxes A and B, the 5'-flanking region of the U6 RNA gene with the external promoter was inserted upstream. Such substitution of the internal promoter in B2 completely restored its transcription. Almost all tested deletions/substitutions did not reduce the polyadenylation capacity of the transcripts, indicating a weak dependence of the function of ß- and τ-signals on the neighboring sequences. A similar analysis of B2 and Ves constructs containing a 55-bp foreign sequence inserted between ß- and τ-signals showed an equal polyadenylation efficiency of their transcripts compared to those of constructs without the insertion. The acquired poly(A)-tails significantly increased the lifetime and thus the cellular level of such transcripts. The data obtained highlight the potential of B2 and Ves SINEs as cassettes for the expression of relatively short sequences for various applications.

Solvent isotope effects in the catalytic cycle of P450 CYP17A1: Computational modeling of the hydroxylation and lyase reactions.

The catalytic cycle of the cytochromes P450 (CYP) requires two electrons from a protein redox partner and two protons from water to generate the main catalytic intermediate, a ferryl-oxo complex with π-cation on the heme porphyrin ring, termed Compound 1. The protonation steps are at least partially rate-limiting, therefore the steady-state rates of P450 catalysis are usually slower in deuterated solvent (D2O) by a factor of 1.5-3. However, in several P450 systems a pronounced inverse kinetic solvent isotope effect (KSIE ∼0.4-0.7) is observed, where the reaction is faster in D2O. This raises an important mechanistic question: Is this inverse solvent isotope effect compatible with Compound 1 catalyzed reactions, or is it indicative of another catalytic intermediate being involved? In this communication we use exhaustive numerical modeling of the P450 steady-state kinetics to demonstrate that a significant inverse KSIE cannot be obtained for a pure Compound 1 driven catalytic cycle of P450. Rather, an alternative, protonation independent, catalytic intermediate needs to be introduced. This result is applicable to the broad spectrum of P450s in nature, but as an example we use the extensively documented inverse isotope effect in the human steroid biosynthetic P450 CYP17A1 where the involvement of a heme peroxo anion intermediate has been characterized. Based on this analysis, we show that the observation of an inverse KSIE can be used as a general mechanistic probe for reaction cycle intermediates in the cytochromes P450.

Tail Wags Dog's SINE: Retropositional Mechanisms of Can SINE Depend on Its A-Tail Structure.

SINEs, non-autonomous short retrotransposons, are widespread in mammalian genomes. Their transcripts are generated by RNA polymerase III (pol III). Transcripts of certain SINEs can be polyadenylated, which requires polyadenylation and pol III termination signals in their sequences. Our sequence analysis divided Can SINEs in canids into four subfamilies, older a1 and a2 and younger b1 and b2. Can_b2 and to a lesser extent Can_b1 remained retrotranspositionally active, while the amplification of Can_a1 and Can_a2 ceased long ago. An extraordinarily high Can amplification was revealed in different dog breeds. Functional polyadenylation signals were analyzed in Can subfamilies, particularly in fractions of recently amplified, i.e., active copies. The transcription of various Can constructs transfected into HeLa cells proposed AATAAA and (TC)n as functional polyadenylation signals. Our analysis indicates that older Can subfamilies (a1, a2, and b1) with an active transcription terminator were amplified by the T+ mechanism (with polyadenylation of pol III transcripts). In the currently active Can_b2 subfamily, the amplification mechanisms with (T+) and without the polyadenylation of pol III transcripts (T-) irregularly alternate. The active transcription terminator tends to shorten, which renders it nonfunctional and favors a switch to the T- retrotransposition. The activity of a truncated terminator is occasionally restored by its elongation, which rehabilitates the T+ retrotransposition for a particular SINE copy.

Midazolam as a Probe for Heterotropic Drug-Drug Interactions Mediated by CYP3A4.

Human cytochrome P450 CYP3A4 is involved in the processing of more than 35% of current pharmaceuticals and therefore is responsible for multiple drug-drug interactions (DDI). In order to develop a method for the detection and prediction of the possible involvement of new drug candidates in CYP3A4-mediated DDI, we evaluated the application of midazolam (MDZ) as a probe substrate. MDZ is hydroxylated by CYP3A4 in two positions: 1-hydroxy MDZ formed at lower substrate concentrations, and up to 35% of 4-hydroxy MDZ at high concentrations. The ratio of the formation rates of these two products (the site of metabolism ratio, SOM) was used as a measure of allosteric heterotropic interactions caused by effector molecules using CYP3A4 incorporated in lipid nanodiscs. The extent of the changes in the SOM in the presence of effectors is determined by chemical structure and is concentration-dependent. MD simulations of CYP3A4 in the lipid bilayer suggest that experimental results can be explained by the movement of the F-F' loop and concomitant changes in the shape and volume of the substrate-binding pocket. As a result of PGS binding at the allosteric site, several residues directly contacting MDZ move away from the substrate molecule, enabling the repositioning of the latter for minor product formation.

Brain Trauma, Glucocorticoids and Neuroinflammation: Dangerous Liaisons for the Hippocampus.

Glucocorticoid-dependent mechanisms of inflammation-mediated distant hippocampal damage are discussed with a focus on the consequences of traumatic brain injury. The effects of glucocorticoids on specific neuronal populations in the hippocampus depend on their concentration, duration of exposure and cell type. Previous stress and elevated level of glucocorticoids prior to pro-inflammatory impact, as well as long-term though moderate elevation of glucocorticoids, may inflate pro-inflammatory effects. Glucocorticoid-mediated long-lasting neuronal circuit changes in the hippocampus after brain trauma are involved in late post-traumatic pathology development, such as epilepsy, depression and cognitive impairment. Complex and diverse actions of the hypothalamic-pituitary-adrenal axis on neuroinflammation may be essential for late post-traumatic pathology. These mechanisms are applicable to remote hippocampal damage occurring after other types of focal brain damage (stroke, epilepsy) or central nervous system diseases without obvious focal injury. Thus, the liaisons of excessive glucocorticoids/dysfunctional hypothalamic-pituitary-adrenal axis with neuroinflammation, dangerous to the hippocampus, may be crucial to distant hippocampal damage in many brain diseases. Taking into account that the hippocampus controls both the cognitive functions and the emotional state, further research on potential links between glucocorticoid signaling and inflammatory processes in the brain and respective mechanisms is vital.

Memantine Disrupts Motor Coordination through Anxiety-like Behavior in CD1 Mice.

Memantine is an FDA approved drug for the treatment of Alzheimer's disease. It reduces neurodegeneration in the hippocampus and cerebral cortex through the inhibition of extrasynaptic NMDA receptors in patients and mouse models. Potentially, it could prevent neurodegeneration in other brain areas and caused by other diseases. We previously used memantine to prevent functional damage and to retain morphology of cerebellar neurons and Bergmann glia in an optogenetic mouse model of spinocerebellar ataxia type-1 (SCA1). However, before suggesting wider use of memantine in clinics, its side effects must be carefully evaluated. Blockers of NMDA receptors are controversial in terms of their effects on anxiety. Here, we investigated the effects of chronic application of memantine over 9 weeks to CD1 mice and examined rotarod performance and anxiety-related behaviors. Memantine-treated mice exhibited an inability to adapt to anxiety-causing conditions which strongly affected their rotarod performance. A tail suspension test revealed increased signs of behavioral despair. These data provide further insights into the potential deleterious effects of memantine which may result from the lack of adaptation to novel, stressful conditions. This effect of memantine may affect the results of tests used to assess motor performance and should be considered during clinical trials of memantine in patients.

XMCD and ab initio study of interface-engineered ultrathin Ru/Co/W/Ru films with perpendicular magnetic anisotropy and strong Dzyaloshinskii-Moriya interaction.

Understanding the nature of recently discovered spin-orbital induced phenomena and a definition of a general approach for "ferromagnet/heavy-metal" layered systems to enhance and manipulate spin-orbit coupling, spin-orbit torque, and the Dzyaloshinskii-Moriya interaction (DMI) assisted by atomic-scale interface engineering are essential for developing spintronics and spin-orbitronics. Here, we exploit X-ray magnetic circular dichroism (XMCD) spectroscopy at the L2,3-edges of 5d and 4d non-magnetic heavy metals (W and Ru, respectively) in ultrathin Ru/Co/W/Ru films to determine their induced magnetic moments due to the proximity to the ferromagnetic layer of Co. The deduced orbital and spin magnetic moments agree well with the theoretically predicted values, highlighting the drastic effect of constituting layers on the system's magnetic properties and the strong interfacial DMI in Ru/Co/W/Ru films. As a result, we demonstrate the ability to simultaneously control the strength of magnetic anisotropy and intermixing-enhanced DMI through the interface engineered inversion asymmetry in thin-film chiral ferromagnets, which are a potential host for stable magnetic skyrmions.

Neuroinflammatory Cytokine Response, Neuronal Death, and Microglial Proliferation in the Hippocampus of Rats During the Early Period After Lateral Fluid Percussion-Induced Traumatic Injury of the Neocortex.

Time course of changes in neuroinflammatory processes in the dorsal and ventral hippocampus was studied during the early period after lateral fluid percussion-induced neocortical traumatic brain injury (TBI) in the ipsilateral and contralateral hemispheres. In the ipsilateral hippocampus, neuroinflammation (increase in expression of pro-inflammatory cytokines) was evident from day 1 after TBI and ceased by day 14, while in the contralateral hippocampus, it was mainly limited to the dorsal part on day 1. TBI induced an increase in hippocampal corticosterone level on day 3 bilaterally and an accumulation of Il1b on day 1 in the ipsilateral hippocampus. Activation of microglia was observed from day 7 in different hippocampal areas of both hemispheres. Neuronal cell loss was detected in the ipsilateral dentate gyrus on day 3 and extended to the contralateral hippocampus by day 7 after TBI. The data suggest that TBI results in distant hippocampal damage (delayed neurodegeneration in the dentate gyrus and microglia proliferation in both the ipsilateral and contralateral hippocampus), the time course of this damage being different from that of the neuroinflammatory response.

Mechanism of the Clinically Relevant E305G Mutation in Human P450 CYP17A1.

Steroid metabolism in humans originates from cholesterol and involves several enzyme reactions including dehydrogenation, hydroxylation, and carbon-carbon bond cleavage that occur at regio- and stereo-specific points in the four-membered ring structure. Cytochrome P450s occur at critical junctions that control the production of the male sex hormones (androgens), the female hormones (estrogens) as well as the mineralocorticoids and glucocorticoids. An important branch point in human androgen production is catalyzed by cytochrome P450 CYP17A1 and involves an initial Compound I-mediated hydroxylation at the 17-position of either progesterone (PROG) or pregnenolone (PREG) to form 17-hydroxy derivatives, 17OH-PROG and 17OH-PREG, with approximately similar efficiencies. Subsequent processing of the 17-hydroxy substrates involves a C17-C20 bond scission (lyase) activity that is heavily favored for 17OH-PREG in humans. The mechanism for this lyase reaction has been debated for several decades, some workers favoring a Compound I-mediated process, with others arguing that a ferric peroxo- is the active oxidant. Mutations in CYP17A1 can have profound clinical manifestations. For example, the replacement of the glutamic acid side with a glycine chain at position 305 in the CYP17A1 structure causes a clinically relevant steroidopathy; E305G CYP17A1 displays a dramatic decrease in the production of dehydroepiandrosterone from pregnenolone but surprisingly increases the activity of the enzyme toward the formation of androstenedione from progesterone. To better understand the functional consequences of this mutation, we self-assembled wild-type and the E305G mutant of CYP17A1 into nanodiscs and examined the detailed catalytic mechanism. We measured substrate binding, spin state conversion, and solvent isotope effects in the hydroxylation and lyase pathways for these substrates. Given that, following electron transfer, the ferric peroxo- species is the common intermediate for both mechanisms, we used resonance Raman spectroscopy to monitor the positioning of important hydrogen-bonding interactions of the 17-OH group with the heme-bound peroxide. We discovered that the E305G mutation changes the orientation of the lyase substrate in the active site, which alters a critical hydrogen bonding of the 17-alcohol to the iron-bound peroxide. The observed switch in substrate specificity of the enzyme is consistent with this result if the hydrogen bonding to the proximal peroxo oxygen is necessary for a proposed nucleophilic peroxoanion-mediated mechanism for CYP17A1 in carbon-carbon bond scission.

Analysis of SINE Families B2, Dip, and Ves with Special Reference to Polyadenylation Signals and Transcription Terminators.

Short Interspersed Elements (SINEs) are eukaryotic non-autonomous retrotransposons transcribed by RNA polymerase III (pol III). The 3'-terminus of many mammalian SINEs has a polyadenylation signal (AATAAA), pol III transcription terminator, and A-rich tail. The RNAs of such SINEs can be polyadenylated, which is unique for pol III transcripts. Here, B2 (mice and related rodents), Dip (jerboas), and Ves (vespertilionid bats) SINE families were thoroughly studied. They were divided into subfamilies reliably distinguished by relatively long indels. The age of SINE subfamilies can be estimated, which allows us to reconstruct their evolution. The youngest and most active variants of SINE subfamilies were given special attention. The shortest pol III transcription terminators are TCTTT (B2), TATTT (Ves and Dip), and the rarer TTTT. The last nucleotide of the terminator is often not transcribed; accordingly, the truncated terminator of its descendant becomes nonfunctional. The incidence of complete transcription of the TCTTT terminator is twice higher compared to TTTT and thus functional terminators are more likely preserved in daughter SINE copies. Young copies have long poly(A) tails; however, they gradually shorten in host generations. Unexpectedly, the tail shortening below A10 increases the incidence of terminator elongation by Ts thus restoring its efficiency. This process can be critical for the maintenance of SINE activity in the genome.

Inpatient versus outpatient intravenous diuresis for the acute exacerbation of chronic heart failure.

BACKGROUND: We established an IV outpatient diuresis (IVOiD) clinic and conducted a quality improvement project to evaluate safety, effectiveness and costs associated with outpatient versus inpatient diuresis for patients presenting with acute decompensated heart failure (ADHF) to the emergency department (ED). METHODS: Patients who were clinically diagnosed with ADHF in the ED, but did not have high-risk features, were either diuresed in the hospital or in the outpatient IVOiD clinic. The dose of IV diuretic was based on their home maintenance diuretic dose. The outcomes measured were the effects of diuresis (urine output, weight, hemodynamic and laboratory abnormalities), 30-90 day readmissions, 30-90 day death and costs. RESULTS: In total, 36 patients (22 inpatients and 14 outpatients) were studied. There were no significant differences in the baseline demographics between groups. The average inpatient stay was six days and the average IVOiD clinic days were 1.2. There was no significant difference in diuresis per day of treatment (1159 vs. 944 ml, p = 0.46). There was no significant difference in adverse outcomes, 30-90 day readmissions or 30-90 day deaths. There was a significantly lower cost in the IVOiD group compared to the inpatient group ($839.4 vs. $9895.7, p=<0.001). CONCLUSIONS: Outpatient IVOiD clinic diuresis may be a viable alternative to accepted clinical practice of inpatient diuresis for ADHF. Further studies are needed to validate this in a larger cohort and in different sites.

Neuroinflammation and Neuronal Loss in the Hippocampus Are Associated with Immediate Posttraumatic Seizures and Corticosterone Elevation in Rats.

Hippocampal damage after traumatic brain injury (TBI) is associated with late posttraumatic conditions, such as depression, cognitive decline and epilepsy. Mechanisms of selective hippocampal damage after TBI are not well understood. In this study, using rat TBI model (lateral fluid percussion cortical injury), we assessed potential association of immediate posttraumatic seizures and changes in corticosterone (CS) levels with neuroinflammation and neuronal cell loss in the hippocampus. Indices of distant hippocampal damage (neurodegeneration and neuroinflammation) were assessed using histological analysis (Nissl staining, Iba-1 immunohistochemical staining) and ELISA (IL-1ß and CS) 1, 3, 7 and 14 days after TBI or sham operation in male Wistar rats (n = 146). IL-1ß was elevated only in the ipsilateral hippocampus on day 1 after trauma. CS peak was detected on day 3 in blood, the ipsilateral and contralateral hippocampus. Neuronal cell loss in the hippocampus was demonstrated bilaterally; in the ipsilateral hippocampus it started earlier than in the contralateral. Microglial activation was evident in the hippocampus bilaterally on day 7 after TBI. The duration of immediate seizures correlated with CS elevation, levels of IL-1ß and neuronal loss in the hippocampus. The data suggest potential association of immediate post-traumatic seizures with CS-dependent neuroinflammation-mediated distant hippocampal damage.

Midazolam as a Probe for Drug-Drug Interactions Mediated by CYP3A4: Homotropic Allosteric Mechanism of Site-Specific Hydroxylation.

We developed an efficient and sensitive probe for drug-drug interactions mediated by human CYP3A4 by using midazolam (MDZ) as a probe substrate. Using global analysis of four parameters over several experimental data sets, we demonstrate that the first MDZ molecule (MDZ1) binds with high affinity at the productive site near the heme iron and gives only hydroxylation at the 1 position (1OH). The second midazolam molecule (MDZ2) binds at an allosteric site at the membrane surface and perturbs the position and mobility of MDZ1 such that the minor hydroxylation product at the 4 position (4OH) is formed in a 1:2 ratio (35%). No increase in catalytic rate is observed after the second MDZ binding. Hence, the site of the 1OH:4OH metabolism ratio is a sensitive probe for drugs, such as progesterone, that bind with high affinity to the allosteric site and serve as effectors. We observe similar changes in the MDZ 1OH:4OH ratio in the presence of progesterone (PGS), suggesting a direct communication between the active and allosteric sites. Mutations introduced into the F-F' loop indicate that residues F213 and D214 are directly involved in allosteric interactions leading to MDZ homotropic cooperativity, and these same residues, together with L211, are involved in heterotropic allosteric interactions in which PGS is the effector and MDZ the substrate. Molecular dynamics simulations provide a mechanistic picture of the origin of this cooperativity. These results show that the midazolam can be used as a sensitive probe for drug-drug interactions in human P450 CYP3A4.

Substrate-Specific Allosteric Effects on the Enhancement of CYP17A1 Lyase Efficiency by Cytochrome b5.

CYP17A1 is an essential human steroidogenic enzyme, which catalyzes two sequential reactions leading to the formation of androstenedione from progesterone and dehydroepiandrosterone from pregnenolone. The second reaction is the C17-C20 bond scission, which is strongly dependent on the presence of cytochrome b5 and displays a heretofore unexplained more pronounced acceleration when 17OH-progesteone (17OH-PROG) is a substrate. The origin of the stimulating effect of cytochrome b5 on C-C bond scission catalyzed by CYP17A1 is still debated as mostly due to either the acceleration of the electron transfer to the P450 oxy complex or allosteric effects of cytochrome b5 favoring active site conformations that promote lyase activity. Using resonance Raman spectroscopy, we compared the effect of Mn-substituted cytochrome b5 (Mn-Cytb5) on the oxy complex of CYP17A1 with both proteins co-incorporated in lipid nanodiscs. For CYP17A1 with 17OH-PROG, a characteristic shift of the Fe-O mode is observed in the presence of Mn-b5, indicating reorientation of a hydrogen bond between the 17OH group of the substrate from the terminal to the proximal oxygen atom of the Fe-O-O moiety, a configuration favorable for the lyase catalysis. For 17OH-pregnenolone, no such shift is observed, the favorable H-bonding orientation being present even without Mn-Cytb5. These new data provide a precise allosteric interpretation for the more pronounced acceleration seen for the 17OH-PROG substrate.

Differential early effects of traumatic brain injury on spike-wave discharges in Sprague-Dawley rats.

Unprovoked seizures in the late period of traumatic brain injury (TBI) occur in almost 20% of humans and experimental animals, psychiatric comorbidities being common in both situations. The aim of the study was to evaluate epileptiform activity in the early period of TBI induced by lateral fluid percussion brain injury in adult male Srague-Dawley rats and to reveal potential behavioral and pathomorphological correlates of early electrophysiological alterations. One week after TBI the group of animals was remarkably heterogeneous regarding the incidence of bifrontal 7-Hz spikes and spike-wave discharges (SWDs). It consisted of 3 typical groups: a) rats with low baseline and high post-craniotomy SWD level; b)with constantly low both baseline and post-craniotomy SWD levels; c) constantly high both baseline and post-craniotomy SWD levels. Rats with augmented SWD occurrence after TBI demonstrated freezing episodes accompanying SWDs as well as increased anxiety-like behavior (difficulty of choosing). The discharges were definitely associated with sleep phases. The incidence of SWDs positively correlated with the area of glial activation in the neocortex but not in the hippocampus.The translational potential of the data is revealing new pathophysiological links between epileptiform activity appearance, direct cortical and distant hippocampal damage and anxiety-like behavior, putative early predictors of late posttraumatic pathology.

Nanodiscs: A toolkit for membrane protein science.

Membrane proteins are involved in numerous vital biological processes, including transport, signal transduction and the enzymes in a variety of metabolic pathways. Integral membrane proteins account for up to 30% of the human proteome and they make up more than half of all currently marketed therapeutic targets. Unfortunately, membrane proteins are inherently recalcitrant to study using the normal toolkit available to scientists, and one is most often left with the challenge of finding inhibitors, activators and specific antibodies using a denatured or detergent solubilized aggregate. The Nanodisc platform circumvents these challenges by providing a self-assembled system that renders typically insoluble, yet biologically and pharmacologically significant, targets such as receptors, transporters, enzymes, and viral antigens soluble in aqueous media in a native-like bilayer environment that maintain a target's functional activity. By providing a bilayer surface of defined composition and structure, Nanodiscs have found great utility in the study of cellular signaling complexes that assemble on a membrane surface. Nanodiscs provide a nanometer scale vehicle for the in vivo delivery of amphipathic drugs, therapeutic lipids, tethered nucleic acids, imaging agents and active protein complexes. This means for generating nanoscale lipid bilayers has spawned the successful use of numerous other polymer and peptide amphipathic systems. This review, in celebration of the Anfinsen Award, summarizes some recent results and provides an inroad into the current and historical literature.

Identification of nucleotide sequences and some proteins involved in polyadenylation of RNA transcribed by Pol III from SINEs.

We have previously reported that not only transcripts of RNA polymerase II (pol II), but also one type of RNA transcribed by RNA polymerase III (pol III), undergo AAUAAA-dependent polyadenylation. Such an unusual feature is inherent in Short Interspersed Elements (SINEs) from genomes of certain mammals. For polyadenylation of its transcript, SINE should contain, besides an AATAAA hexamer and a transcription terminator, two specific regions: ß, located downstream of box B of a promoter, and τ, preceding AATAAA. Here, using nucleotide substitutions in SINEs B2 (mouse) and Ves (bat), we identified nucleotides of ß regions necessary for polyadenylation of their transcripts. These sequences (ß signals) are the following: ACCACATgg in B2 and GGGCATGT in Ves. Using this approach, we identified τ signal of SINE B2 (GCTACagTGTACTTACAT), where TGTA tetramer is most important for polyadenylation. In Ves, τ region is a long polypyrimidine motif which is able to interact with PTB protein in Ves transcripts. We demonstrated by knockdown that B2 and Ves transcript polyadenylation is performed by canonical poly(A) polymerase with the participation of proteins CSPF-160 and Fip1, the known factors of mRNA polyadenylation. We also showed that a factor CFIm partaking in polyadenylation of many mRNAs, is involved only in polyadenylation of B2 transcripts. CFIm seems to interact with τ signal of В2 RNA and thereby facilitates the recruiting of other proteins engaged in polyadenylation. Thus, SINEs utilize at least some proteins involved in polyadenylation of pol II transcripts to polyadenylate their pol III transcripts.