In-cell Catalysis by Tethered Organo-Osmium Complexes Generates Selectivity for Breast Cancer Cells.

Anticancer agents that exhibit catalytic mechanisms of action offer a unique multi-targeting strategy to overcome drug resistance. Nonetheless, many in-cell catalysts in development are hindered by deactivation by endogenous nucleophiles. We have synthesised a highly potent, stable Os-based 16-electron half-sandwich ('piano stool') catalyst by introducing a permanent covalent tether between the arene and chelated diamine ligand. This catalyst exhibits antiproliferative activity comparable to the clinical drug cisplatin towards triple-negative breast cancer cells and can overcome tamoxifen resistance. Speciation experiments revealed Os to be almost exclusively albumin-bound in the extracellular medium, while cellular accumulation studies identified an energy-dependent, protein-mediated Os accumulation pathway, consistent with albumin-mediated uptake. Importantly, the tethered Os complex was active for in-cell transfer hydrogenation catalysis, initiated by co-administration of a non-toxic dose of sodium formate as a source of hydride, indicating that the Os catalyst is delivered to the cytosol of cancer cells intact. The mechanism of action involves the generation of reactive oxygen species (ROS), thus exploiting the inherent redox vulnerability of cancer cells, accompanied by selectivity for cancerous cells over non-tumorigenic cells.

Using TLC-MALDI-TOF to Interrogate In Vitro Peptidyl Proximal Preferences of PARP14 and Glycohydrolase Specificity.

The transfer of ADP-ribose (ADPr) from nicotinamide adenine dinucleotide (NAD+) to target proteins is mediated by a class of human diphtheria toxin-like ADP-ribosyltransferases (ARTDs; previously referred to as poly-ADP-ribose polymerases or PARPs) and the removal of ADPr is catalyzed by a family of glycohydrolases. Although thousands of potential ADPr modification sites have been identified using high-throughput mass-spectrometry, relatively little is known about the sequence specificity encoded near the modification site. Herein, we present a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method that facilitates the in vitro analysis of proximal factors that guide ARTD target selection. We identify a minimal 5-mer peptide sequence that is necessary and sufficient to drive glutamate/aspartate targeting using PARP14 while highlighting the importance of the adjacent residues in PARP14 targeting. We measure the stability of the resultant ester bond and show that non-enzymatic removal is pH and temperature dependent, sequence independent, and occurs within hours. Finally, we use the ADPr-peptides to highlight differential activities within the glycohydrolase family and their sequence preferences. Our results highlight (1) the utility of MALDI-TOF in analyzing proximal ARTD-substrate interactions and (2) the importance of peptide sequences in governing ADPr transfer and removal.

Identification of a Novel PARP14 Site Motif and Glycohydrolase Specificity Using TLC-MALDI-TOF.

Transfer of ADP-ribose (ADPr) from nicotinamide adenine dinucleotide (NAD+) to target proteins is mediated by a class of human poly-ADP-ribose polymerases, PARPs, and removal of ADPr is catalyzed by a family of glycohydrolases. Although thousands of potential ADPr modification sites have been identified using high-throughput mass-spectrometry, relatively little is known about sequence specificity encoded near the modification site. Herein, we present a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method that facilitates the discovery and validation of ADPr site motifs. We identify a minimal 5-mer peptide sequence that is sufficient to drive PARP14 specific activity while highlighting the importance of the adjacent residues in PARP14 targeting. We measure the stability of the resultant ester bond and show that non-enzymatic removal is sequence independent and occurs within hours. Finally, we use the ADPr-peptide to highlight differential activities within the glycohydrolase family and their sequence specificities. Our results highlight: 1) the utility of MALDI-TOF in motif discovery and 2) the importance of peptide sequence in governing ADPr transfer and removal.

Rapid Analysis of ADP-Ribosylation Dynamics and Site-Specificity Using TLC-MALDI.

Poly(ADP-ribose) polymerases, PARPs, transfer ADP-ribose onto target proteins from nicotinamide adenine dinucleotide (NAD+). Current mass spectrometric analytical methods require proteolysis of target proteins, limiting the study of dynamic ADP-ribosylation on contiguous proteins. Herein, we present a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method that facilitates multisite analysis of ADP-ribosylation. We observe divergent ADP-ribosylation dynamics for the catalytic domains of PARPs 14 and 15, with PARP15 modifying more sites on itself (+3-4 ADP-ribose) than the closely related PARP14 protein (+1-2 ADP-ribose)─despite similar numbers of potential modification sites. We identify, for the first time, a minimal peptide fragment (18 amino-acids) that is preferentially modified by PARP14. Finally, we demonstrate through mutagenesis and chemical treatment with hydroxylamine that PARPs 14/15 prefer acidic residues. Our results highlight the utility of MALDI-TOF in the analysis of PARP target modifications and in elucidating the biochemical mechanism governing PARP target selection.

Combining Chemical Genetics with Proximity-Dependent Labeling Reveals Cellular Targets of Poly(ADP-ribose) Polymerase 14 (PARP14).

Poly(ADP-ribose) polymerase 14 (PARP14) is a member of the PARP family of enzymes that transfer ADP-ribose from NAD+ to nucleophilic amino acids on target proteins, a process known as mono-ADP-ribosylation (MARylation). PARP14 is involved in normal immune function through the IL-4 signaling pathway and is a prosurvival factor in multiple myeloma and hepatocellular carcinoma. A mechanistic understanding of the physiological and pathophysiological roles of PARP14 has been limited by the dearth of PARP14-specific MARylation targets. Herein we engineered a PARP14 variant that uses an NAD+ analog that is orthogonal to wild-type PARPs for identifying PARP14-specific MARylation targets. Combining this chemical genetics approach with a BioID approach for proximity-dependent labeling of PARP14 interactors, we identified 114 PARP14-specific protein substrates, several of which are RNA regulatory proteins. One of these targets is PARP13, a protein known to play a role in regulating RNA stability. PARP14 MARylates PARP13 on several acidic amino acids. This study not only reveals crosstalk among PARP family members but also highlights the advantage of using disparate approaches for identifying the direct targets of individual PARP family members.

Sensory integration deficits support a dimensional view of psychosis and are not limited to schizophrenia.

Visual dysfunction is commonplace in schizophrenia and occurs alongside cognitive, psychotic and affective symptoms of the disorder. Psychophysical evidence suggests that this dysfunction results from impairments in the integration of low-level neural signals into complex cortical representations, which may also be associated with symptom formation. Despite the symptoms of schizophrenia occurring in a range of disorders, the integration deficit has not been tested in broader patient populations. Moreover, it remains unclear whether such deficits generalize across other sensory modalities. The present study assessed patients with a range of psychotic and nonpsychotic disorders and healthy controls on visual contrast detection, visual motion integration, auditory tone detection and auditory tone integration. The sample comprised a total of 249 participants (schizophrenia spectrum disorder n=98; bipolar affective disorder n=35; major depression n=31; other psychiatric conditions n=31; and healthy controls n=54), of whom 178 completed one or more visual task and 71 completed auditory tasks. Compared with healthy controls and nonpsychotic patients, psychotic patients trans-diagnostically were impaired on both visual and auditory integration, but unimpaired in simple visual or auditory detection. Impairment in visual motion integration was correlated with the severity of positive symptoms, and could not be accounted for by a reduction in processing speed, inattention or medication effects. Our results demonstrate that impaired sensory integration is not specific to schizophrenia, as has previously been assumed. Instead, sensory deficits are closely related to the presence of positive symptoms independent of diagnosis. The finding that equivalent integrative sensory processing is impaired in audition is consistent with hypotheses that propose a generalized deficit of neural integration in psychotic disorders.

Phase I metabolites of mephedrone display biological activity as substrates at monoamine transporters.

BACKGROUND AND PURPOSE: 4-Methyl-N-methylcathinone (mephedrone) is a synthetic stimulant that acts as a substrate-type releaser at transporters for dopamine (DAT), noradrenaline (NET) and 5-HT (SERT). Upon systemic administration, mephedrone is metabolized to several phase I compounds: the N-demethylated metabolite, 4-methylcathinone (nor-mephedrone); the ring-hydroxylated metabolite, 4-hydroxytolylmephedrone (4-OH-mephedrone); and the reduced keto-metabolite, dihydromephedrone. EXPERIMENTAL APPROACH: We used in vitro assays to compare the effects of mephedrone and synthetically prepared metabolites on transporter-mediated uptake and release in HEK293 cells expressing human monoamine transporters and in rat brain synaptosomes. In vivo microdialysis was employed to examine the effects of i.v. metabolite injection (1 and 3 mg·kg(-1) ) on extracellular dopamine and 5-HT levels in rat nucleus accumbens. KEY RESULTS: In cells expressing transporters, mephedrone and its metabolites inhibited uptake, although dihydromephedrone was weak overall. In cells and synaptosomes, nor-mephedrone and 4-OH-mephedrone served as transportable substrates, inducing release via monoamine transporters. When administered to rats, mephedrone and nor-mephedrone produced elevations in extracellular dopamine and 5-HT, whereas 4-OH-mephedrone did not. Mephedrone and nor-mephedrone, but not 4-OH-mephedrone, induced locomotor activity. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that phase I metabolites of mephedrone are transporter substrates (i.e. releasers) at DAT, NET and SERT, but dihydromephedrone is weak in this regard. When administered in vivo, nor-mephedrone increases extracellular dopamine and 5-HT in the brain whereas 4-OH-mephedrone does not, suggesting the latter metabolite does not penetrate the blood-brain barrier. Future studies should examine the pharmacokinetics of nor-mephedrone to determine its possible contribution to the in vivo effects produced by mephedrone.

Identifying Family-Member-Specific Targets of Mono-ARTDs by Using a Chemical Genetics Approach.

ADP-ribosyltransferases (ARTD1-16) have emerged as major downstream effectors of NAD(+) signaling in the cell. Most ARTDs (ARTD7 and 8, 10-12, and 14-17) catalyze the transfer of a single unit of ADP-ribose from NAD(+) to target proteins, a process known as mono-ADP-ribosylation (MARylation). Progress in understanding the cellular functions of MARylation has been limited by the inability to identify the direct targets for individual mono-ARTDs. Here, we engineered mono-ARTDs to use an NAD(+) analog that is orthogonal to wild-type ARTDs. We profiled the MARylomes of ARTD10 and ARTD11 in vitro, identifying isoform-specific targets and revealing a potential role for ARTD11 in nuclear pore complex biology. We found that ARTD11 targeting is dependent on both its regulatory and catalytic domains, which has important implications for how ARTDs recognize their targets. We anticipate that our chemical genetic strategy will be generalizable to all mono-ARTD family members based on the similarity of the mono-ARTD catalytic domains.

Identifying Direct Protein Targets of Poly-ADP-Ribose Polymerases (PARPs) Using Engineered PARP Variants-Orthogonal Nicotinamide Adenine Dinucleotide (NAD+) Analog Pairs.

Poly-ADP-ribose polymerases (PARPs) comprise a family of 17 distinct enzymes that catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to acceptor sites on protein targets. PARPs have been implicated in a number of essential signaling pathways regulating both normal cell function and pathophysiology. To understand the physiological role of each PARP family member in the cell we need to identify the direct targets for each unique PARP in a cellular context. PARP-family member-specific target identification is challenging because of their shared catalytic mechanism and functional redundancy. To address this challenge, we have engineered a PARP variant that efficiently uses an orthogonal NAD+ analog, an analog that endogenous PARPs cannot use, as a substrate for ADP-ribosylation. The protocols in this unit describe a general procedure for using engineered PARP variants-orthogonal NAD+ analog pairs for labeling and identifying the direct targets of the poly-subfamily of PARPs (PARPs 1-3, 5, and 6).

Selective inhibition of PARP10 using a chemical genetics strategy.

The lack of inhibitors that are selective for individual poly-ADP-ribose polymerase (PARP) family members has limited our understanding of their roles in cells. Here, we describe a chemical genetics approach for generating selective inhibitors of an engineered variant of PARP10. We synthesized a series of C-7 substituted 3,4-dihydroisoquinolin-1(2H)-one (dq) analogues designed to selectively inhibit a mutant of PARP10 (LG-PARP10) that contains a unique pocket in its active site. A dq analogue containing a bromo at the C-7 position demonstrated a 10-fold selectivity for LG-PARP10 compared to its WT counterpart. This study provides a platform for the development of selective inhibitors of individual PARP family members that will be useful for decoding their cellular functions.

Private land manager capacity to conserve threatened communities under climate change.

Major global changes in vegetation community distributions and ecosystem processes are expected as a result of climate change. In agricultural regions with a predominance of private land, biodiversity outcomes will depend on the adaptive capacity of individual land managers, as well as their willingness to engage with conservation programs and actions. Understanding adaptive capacity of landholders is critical for assessing future prospects for biodiversity conservation in privately owned agricultural landscapes globally, given projected climate change. This paper is the first to develop and apply a set of statistical methods (correlation and bionomial regression analyses) for combining social data on land manager adaptive capacity and factors associated with conservation program participation with biophysical data describing the current and projected-future distribution of climate suitable for vegetation communities. We apply these methods to the Tasmanian Midlands region of Tasmania, Australia and discuss the implications of the modelled results on conservation program strategy design in other contexts. We find that the integrated results can be used by environmental management organisations to design community engagement programs, and to tailor their messages to land managers with different capacity types and information behaviours. We encourage environmental agencies to target high capacity land managers by diffusing climate change and grassland management information through well respected conservation NGOs and farm system groups, and engage low capacity land managers via formalized mentoring programs.

Engineering the substrate specificity of ADP-ribosyltransferases for identifying direct protein targets.

Adenosine diphosphate ribosyltransferases (ARTDs; ARTD1-17 in humans) are emerging as critical regulators of cell function in both normal physiology and disease. These enzymes transfer the ADP-ribose moiety from its substrate, nicotinamide adenine dinucleotide (NAD(+)), to amino acids of target proteins. The functional redundancy and overlapping target specificities among the 17 ARTDs in humans make the identification of direct targets of individual ARTD family members in a cellular context a formidable challenge. Here we describe the rational design of orthogonal NAD(+) analogue-engineered ARTD pairs for the identification of direct protein targets of individual ARTDs. Guided by initial inhibitor studies with nicotinamide analogues containing substituents at the C-5 position, we synthesized an orthogonal NAD(+) variant and found that it is used as a substrate for several engineered ARTDs (ARTD1, -2, and -6) but not their wild-type counterparts. Comparing the target profiles of ARTD1 (PARP1) and ARTD2 (PARP2) in nuclear extracts highlighted the semi-complementary, yet distinct, protein targeting. Using affinity purification followed by tandem mass spectrometry, we identified 42 direct ARTD1 targets and 301 direct ARTD2 targets. This represents a powerful new technique for identifying direct protein targets of individual ARTD family members, which will facilitate studies delineating the pathway from ARTD activation to a given cellular response.

Total intravenous anesthesia affecting spike sources of magnetoencephalography in pediatric epilepsy patients: focal seizures vs. non-focal seizures.

PURPOSE: Magnetoencephalography (MEG) provides source localization of interictal spikes. This study evaluated the inhibitory effects of propofol on MEG spike sources (MEGSSs) among different types of seizures in patients who underwent two separate MEG studies with and without total intravenous anesthesia (TIVA) using propofol. METHODS: We studied 19 children (1-14 years; mean, 6.2 years) who had MEG with and without TIVA. TIVA was administered using propofol (0.03-0.06 mg/kg/min) to record MEG with simultaneous EEG. We analyzed number of spikes of MEG and MEGSSs comparing MEG studies done with and without TIVA. RESULTS: Seizures were divided into nine focal seizure (FS) with/without secondary generalization, five epileptic spasm (ES), and five generalized seizure (GS). TIVA significantly decreased the number of MEG spikes/min (from 4.5 to 2.0) in five FS without secondary generalization (p<0.05). The number of MEG spikes/min was significantly lower (1.9) in FS than that in non-FS (ES+GS, 6.1) (p<0.01). MEGSSs without TIVA were clustered in 15 patients (6FS; 4ES; 5GS), scattered in four (3FS; 1ES). MEG under TIVA showed clusters in 10 patients (1FS; 4ES; 5GS), scatters in three (2FS; 1ES) and no MEGSS in six patients with FS. Under TIVA, nine (90%) of ten patients with non-FS showed MEGSSs clusters compared to one (11%) of nine patients with FS (p<0.01). CONCLUSIONS: Reduction of MEGSSs occurred in patients with FS under TIVA. Diffuse/generalized spikes in non-FS are not affected by TIVA. Propofol may decrease focal spikes in the epileptic cortex in FS. Cortical hyperexcitability in non-FS group would be stronger or more extensive than that in the FS group of patients.

Muscle ciliary neurotrophic factor receptor α promotes axonal regeneration and functional recovery following peripheral nerve lesion.

Ciliary neurotrophic factor (CNTF) administration maintains, protects, and promotes the regeneration of both motor neurons (MNs) and skeletal muscle in a wide variety of models. Expression of CNTF receptor α (CNTFRα), an essential CNTF receptor component, is greatly increased in skeletal muscle following neuromuscular insult. Together the data suggest that muscle CNTFRα may contribute to neuromuscular maintenance, protection, and/or regeneration in vivo. To directly address the role of muscle CNTFRα, we selectively-depleted it in vivo by using a "floxed" CNTFRα mouse line and a gene construct (mlc1f-Cre) that drives the expression of Cre specifically in skeletal muscle. The resulting mice were challenged with sciatic nerve crush. Counting of nerve axons and retrograde tracing of MNs indicated that muscle CNTFRα contributes to MN axonal regeneration across the lesion site. Walking track analysis indicated that muscle CNTFRα is also required for normal recovery of motor function. However, the same muscle CNTFRα depletion unexpectedly had no detected effect on the maintenance or regeneration of the muscle itself, even though exogenous CNTF has been shown to affect these functions. Similarly, MN survival and lesion-induced terminal sprouting were unaffected. Therefore, muscle CNTFRα is an interesting new example of a muscle growth factor receptor that, in vivo under physiological conditions, contributes much more to neuronal regeneration than to the maintenance or regeneration of the muscle itself. This novel form of muscle-neuron interaction also has implications in the therapeutic targeting of the neuromuscular system in MN disorders and following nerve injury. J. Comp. Neurol. 521: 2947-2965, 2013. © 2013 Wiley Periodicals, Inc.

Ciliary neurotrophic factor receptor regulation of adult forebrain neurogenesis.

Appropriately targeted manipulation of endogenous neural stem progenitor (NSP) cells may contribute to therapies for trauma, stroke, and neurodegenerative disease. A prerequisite to such therapies is a better understanding of the mechanisms regulating adult NSP cells in vivo. Indirect data suggest that endogenous ciliary neurotrophic factor (CNTF) receptor signaling may inhibit neuronal differentiation of NSP cells. We challenged subventricular zone (SVZ) cells in vivo with low concentrations of CNTF to anatomically characterize cells containing functional CNTF receptors. We found that type B "stem" cells are highly responsive, whereas type C "transit-amplifying" cells and type A neuroblasts are remarkably unresponsive, as are GFAP(+) astrocytes found outside the SVZ. CNTF was identified in a subset of type B cells that label with acute BrdU administration. Disruption of in vivo CNTF receptor signaling in SVZ NSP cells, with a "floxed" CNTF receptor α (CNTFRα) mouse line and a gene construct driving Cre recombinase (Cre) expression in NSP cells, led to increases in SVZ-associated neuroblasts and new olfactory bulb neurons, as well as a neuron subtype-specific, adult-onset increase in olfactory bulb neuron populations. Adult-onset receptor disruption in SVZ NSP cells with a recombinant adeno-associated virus (AAV-Cre) also led to increased neurogenesis. However, the maintenance of type B cell populations was apparently unaffected by the receptor disruption. Together, the data suggest that endogenous CNTF receptor signaling in type B stem cells inhibits adult neurogenesis, and further suggest that the regulation may occur in a neuron subtype-specific manner.

An independent audit of the Australian food industry's voluntary front-of-pack nutrition labelling scheme for energy-dense nutrition-poor foods.

BACKGROUND/OBJECTIVE: Since 2006, the Australian food industry has promoted its front-of-pack (FOP) food labelling system-the Daily Intake Guide (DIG)-as a success story of industry self-regulation. With over 4000 products already voluntary featuring the DIG, the industry argues that government regulation of FOP nutrition labelling is simply unnecessary. However, no independent audit of the industry's self-regulation has ever been undertaken and we present the first such Australian data. SUBJECTS/METHODS: Energy-dense nutrient-poor (EDNP) snacks were audited at nine Australian supermarkets, including biscuits, candy, ice creams, chocolates, crisps, sports drinks, energy drinks, flavoured milks, sweetened juices and soft drinks. In these categories nutrition labels were recorded for 728 EDNP products in various packaging sizes. RESULTS: The DIG was displayed on 66% of audited EDNP products but most of these (75%) did not report saturated fat and sugar content. Only generic supermarket EDNP products were likely to display saturated fat and sugar content, compared with very few branded products (48% vs 4%, P<0.001). Branded products not displaying fat and sugar content contained on average 10-times more saturated fat than those displaying such (10% vs 1% DI, P<0.001) and nearly twice as much sugar (21 vs 13% DI, P<0.05). CONCLUSIONS: Most Australian manufacturers of EDNP products have adopted the DIG; consistent with industry claims of widespread adoption, but almost all still avoid displaying the high saturated fat and sugar content of their products by opting for the 'energy alone' option, violating the industry's own voluntarily guidelines and highlighting serious weaknesses with the industry's self-regulation.

Genome-wide characterization of the phosphate starvation response in Schizosaccharomyces pombe.

BACKGROUND: Inorganic phosphate is an essential nutrient required by organisms for growth. During phosphate starvation, Saccharomyces cerevisiae activates the phosphate signal transduction (PHO) pathway, leading to expression of the secreted acid phosphatase, PHO5. The fission yeast, Schizosaccharomyces pombe, regulates expression of the ScPHO5 homolog (pho1+) via a non-orthologous PHO pathway involving genetically identified positive (pho7+) and negative (csk1+) regulators. The genes induced by phosphate limitation and the molecular mechanism by which pho7+ and csk1+ function are unknown. Here we use a combination of molecular biology, expression microarrays, and chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to characterize the role of pho7+ and csk1+ in the PHO response. RESULTS: We define the set of genes that comprise the initial response to phosphate starvation in S. pombe. We identify a conserved PHO response that contains the ScPHO5 (pho1+), ScPHO84 (SPBC8E4.01c), and ScGIT1 (SPBC1271.09) orthologs. We identify members of the Pho7 regulon and characterize Pho7 binding in response to phosphate-limitation and Csk1 activity. We demonstrate that activation of pho1+ requires Pho7 binding to a UAS in the pho1+ promoter and that Csk1 repression does not regulate Pho7 enrichment. Further, we find that Pho7-dependent activation is not limited to phosphate-starvation, as additional environmental stress response pathways require pho7+ for maximal induction. CONCLUSIONS: We provide a global analysis of the transcriptional response to phosphate limitation in S. pombe. Our results elucidate the conserved core regulon induced in response to phosphate starvation in this ascomycete distantly related to S. cerevisiae and provide a better understanding of flexibility in environmental stress response networks.

The effect of retail cigarette pack displays on unplanned purchases: results from immediate postpurchase interviews.

OBJECTIVE: To assess the influence of point-of-sale (POS) cigarette displays on unplanned purchases. METHODS: Intercept interviews were conducted with customers observed purchasing cigarettes from retail outlets featuring POS cigarette displays. Measures included intention to purchase cigarettes prior to entering the store, unprompted and prompted salience of POS tobacco displays, urge to buy cigarettes as a result of seeing the POS display, brand switching and support for a ban on POS cigarette displays. RESULTS: In total, 206 daily smokers aged 18-76 years (90 male, 116 female) were interviewed. Unplanned cigarette purchases were made by 22% of participants. POS displays influenced nearly four times as many unplanned purchases as planned purchases (47% vs 12%, p<0.01). Brand switching was reported among 5% of participants, half of whom were influenced by POS displays. Four times as many smokers were supportive of a ban on POS tobacco displays than unsupportive (49% vs 12%), and 28% agreed that such a ban would make it easier to quit. CONCLUSIONS: POS tobacco displays act as a form of advertising even in the absence of advertising materials. They stimulate unplanned cigarette purchases, play an important role in brand selection and tempt smokers trying to quit. This justifies removing POS tobacco displays from line of sight-something that very few smokers in our sample would object to.

Thermodynamic examination of trinucleotide bulged RNA in the context of HIV-1 TAR RNA.

RNA structures contain many bulges and loops that are expected to be sites for inter- and intra-molecular interactions. Nucleotides in the bulge are expected to influence the structure and recognition of RNA. The same stability is assigned to all trinucleotide bulged RNA in the current secondary structure prediction models. In this study thermal denaturation experiments were performed on four trinucleotide bulged RNA, in the context of HIV-1 TAR RNA, to determine whether the bulge sequence affects RNA stability and its divalent ion interactions. Cytosine-rich bulged RNA were more stable than uracil-rich bulged RNA in 1 M KCl. Interactions of divalent ions were more favorable with uracil-rich bulged RNA by approximately 2 kcal/mol over cytosine-rich bulged RNA. The UCU-TAR RNA (wild type) is stabilized by 1.7 kcal/mol in 9.5 mM Ca(2+) as compared with 1 M KCl, whereas no additional gain in stability is measured for CCC-TAR RNA. These results have implications for base substitution experiments traditionally employed to identify metal ion binding sites. To our knowledge, this is the first systematic study to quantify the effect of small sequence changes on RNA stability upon interactions with divalent ions.

An immortalized rat ventral mesencephalic cell line, RTC4, is protective in a rodent model of stroke.

One therapeutic approach to stroke is the transplantation of cells capable of trophic support, reinnervation, and/or regeneration. Previously, we have described the use of novel truncated isoforms of SV40 large T antigen to generate unique cell lines from several primary rodent tissue types. Here we describe the generation of two cell lines, RTC3 and RTC4, derived from primary mesencephalic tissue using a fragment of mutant T antigen, T155c (cDNA) expressed from the RSV promoter. Both lines expressed the glial markers vimentin and S100beta, but not the neuronal markers NeuN, MAP2, or beta-III-tubulin. A screen for secreted trophic factors revealed substantially elevated levels of platelet-derived growth factor (PDGF) in RTC4, but not RTC3 cells. When transplanted into rat cortex, RTC4 cells survived for at least 22 days and expressed PDGF. Because PDGF has been reported to reduce ischemic injury, we examined the protective functions of RTC4 cells in an animal model of stroke. RTC4 or RTC3 cells, or vehicle, were injected into rat cortex 15-20 min prior to a 60-min middle cerebral artery ligation. Forty-eight hours later, animals were sacrificed and the stroke volume was assessed by triphenyl-tetrazolium chloride (TTC) staining. Compared to vehicle or RTC3 cells, transplanted RTC4 cells significantly reduced stroke volume. Overall, we generated a cell line with glial properties that produces PDGF and reduces ischemic injury in a rat model of stroke.