A designed photoenzyme for enantioselective [2+2] cycloadditions.

The ability to program new modes of catalysis into proteins would allow the development of enzyme families with functions beyond those found in nature. To this end, genetic code expansion methodology holds particular promise, as it allows the site-selective introduction of new functional elements into proteins as noncanonical amino acid side chains1-4. Here we exploit an expanded genetic code to develop a photoenzyme that operates by means of triplet energy transfer (EnT) catalysis, a versatile mode of reactivity in organic synthesis that is not accessible to biocatalysis at present5-12. Installation of a genetically encoded photosensitizer into the beta-propeller scaffold of DA_20_00 (ref. 13) converts a de novo Diels-Alderase into a photoenzyme for [2+2] cycloadditions (EnT1.0). Subsequent development and implementation of a platform for photoenzyme evolution afforded an efficient and enantioselective enzyme (EnT1.3, up to 99% enantiomeric excess (e.e.)) that can promote intramolecular and bimolecular cycloadditions, including transformations that have proved challenging to achieve selectively with small-molecule catalysts. EnT1.3 performs >300 turnovers and, in contrast to small-molecule photocatalysts, can operate effectively under aerobic conditions and at ambient temperatures. An X-ray crystal structure of an EnT1.3-product complex shows how multiple functional components work in synergy to promote efficient and selective photocatalysis. This study opens up a wealth of new excited-state chemistry in protein active sites and establishes the framework for developing a new generation of enantioselective photocatalysts.

Assembly of nuclear dimers of PI3K regulatory subunits is regulated by the Cdc42-activated tyrosine kinase ACK.

Activated Cdc42-associated kinase (ACK) is an oncogenic nonreceptor tyrosine kinase associated with poor prognosis in several human cancers. ACK promotes proliferation, in part by contributing to the activation of Akt, the major effector of class 1A phosphoinositide 3-kinases (PI3Ks), which transduce signals via membrane phosphoinositol lipids. We now show that ACK also interacts with other key components of class 1A PI3K signaling, the PI3K regulatory subunits. We demonstrate ACK binds to all five PI3K regulatory subunit isoforms and directly phosphorylates p85α, p85ß, p50α, and p55α on Tyr607 (or analogous residues). We found that phosphorylation of p85ß promotes cell proliferation in HEK293T cells. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions, where p85α phosphorylated at Tyr607 (pTyr607) also resides, and identify an interaction between pTyr607 and the N-terminal SH2 domain that supports dimerization of the regulatory subunits. We infer from this that ACK targets p110-independent p85 and further postulate that these regulatory subunit dimers undertake novel nuclear functions underpinning ACK activity. We conclude that these dimers represent a previously undescribed mode of regulation for the class1A PI3K regulatory subunits and potentially reveal additional avenues for therapeutic intervention.

Synthesis and Conformational Analysis of Fluorinated Uridine Analogues Provide Insight into a Neighbouring-Group Participation Mechanism.

Fluorinated nucleoside analogues have attracted much attention as anticancer and antiviral agents and as probes for enzymatic function. However, the lack of direct synthetic methods, especially for 2',3'-dideoxy-2',3'-difluoro nucleosides, hamper their practical utility. In order to design more efficient synthetic methods, a better understanding of the conformation and mechanism of formation of these molecules is important. Herein, we report the synthesis and conformational analysis of a 2',3'-dideoxy-2',3'-difluoro and a 2'-deoxy-2'-fluoro uridine derivative and provide an insight into the reaction mechanism. We suggest that the transformation most likely diverges from the SN1 or SN2 pathway, but instead operates via a neighbouring-group participation mechanism.

Pac13 is a Small, Monomeric Dehydratase that Mediates the Formation of the 3'-Deoxy Nucleoside of Pacidamycins.

The uridyl peptide antibiotics (UPAs), of which pacidamycin is a member, have a clinically unexploited mode of action and an unusual assembly. Perhaps the most striking feature of these molecules is the biosynthetically unique 3'-deoxyuridine that they share. This moiety is generated by an unusual, small and monomeric dehydratase, Pac13, which catalyses the dehydration of uridine-5'-aldehyde. Here we report the structural characterisation of Pac13 with a series of ligands, and gain insight into the enzyme's mechanism demonstrating that H42 is critical to the enzyme's activity and that the reaction is likely to proceed via an E1cB mechanism. The resemblance of the 3'-deoxy pacidamycin moiety with the synthetic anti-retrovirals, presents a potential opportunity for the utilisation of Pac13 in the biocatalytic generation of antiviral compounds.

Recent and episodic volcanic and glacial activity on Mars revealed by the High Resolution Stereo Camera.

The large-area coverage at a resolution of 10-20 metres per pixel in colour and three dimensions with the High Resolution Stereo Camera Experiment on the European Space Agency Mars Express Mission has made it possible to study the time-stratigraphic relationships of volcanic and glacial structures in unprecedented detail and give insight into the geological evolution of Mars. Here we show that calderas on five major volcanoes on Mars have undergone repeated activation and resurfacing during the last 20 per cent of martian history, with phases of activity as young as two million years, suggesting that the volcanoes are potentially still active today. Glacial deposits at the base of the Olympus Mons escarpment show evidence for repeated phases of activity as recently as about four million years ago. Morphological evidence is found that snow and ice deposition on the Olympus construct at elevations of more than 7,000 metres led to episodes of glacial activity at this height. Even now, water ice protected by an insulating layer of dust may be present at high altitudes on Olympus Mons.

Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme.

The crystal structure of an unmodified hammerhead RNA in the absence of divalent metal ions has been solved, and it was shown that this ribozyme can cleave itself in the crystal when divalent metal ions are added. This biologically active RNA fold is the same as that found previously for two modified hammerhead ribozymes. Addition of divalent cations at low pH makes it possible to capture the uncleaved RNA in metal-bound form. A conformational intermediate, having an additional Mg(II) bound to the cleavage-site phosphate, was captured by freeze-trapping the RNA at an active pH prior to cleavage. The most significant conformational changes were limited to the active site of the ribozyme, and the changed conformation requires only small additional movements to reach a proposed transition-state.

1-Aryl-3,4-dihydroisoquinoline inhibitors of JNK3.

A series of 1-aryl-3,4-dihydroisoquinoline inhibitors of JNK3 are described. Compounds 20 and 24 are the most potent inhibitors (pIC50 7.3 and 6.9, respectively in a radiometric filter binding assay), with 10- and 1000-fold selectivity over JNK2 and JNK1, respectively, and selectivity within the wider mitogen-activated protein kinase (MAPK) family against p38alpha and ERK2. X-ray crystallography of 16 reveals a highly unusual binding mode where an H-bond acceptor interaction with the hinge region is made by a chloro substituent.

Does a single metal ion bridge the A-9 and scissile phosphate groups in the catalytically active hammerhead ribozyme structure?

We have constructed a model structure that we believe represents the strongest possible physically and chemically reasonable representation of a hypothesized catalytically active hammerhead ribozyme structure in which a single divalent metal ion bridges the A9 and scissile phosphate groups. It has been proposed that such a structure arises from a conformational change in which the so-called ground-state structure (as observed by X-ray crystallography) rearranges in such a way that the pro-R oxygen atoms of both the A9 and scissile phosphate groups are directly coordinated by a single divalent metal ion in the transition-state of the hammerhead ribozyme cleavage reaction. We show that even the small subset of possible model structures that are consistent with these requirements, and that are stereochemically and sterically reasonable, are contradicted by experimental evidence. We also demonstrate that even a minimal subset of assumptions, i.e. that stems I and II are helical and that the two phosphate groups are coordinated by a divalent metal ion in the standard octahedral geometry, are sufficient to lead to this contradiction. We therefore conclude that such a mechanism of hammerhead ribozyme catalysis is untenable, at least in its present formulation.

Synthesis and characterisation of oligodeoxynucleotides containing thio analogues of (6-4) pyrimidine-pyrimidinone photo-dimers.

A method for the preparation of an oligodeoxynucleotide, 20 bases in length, containing centrally located thio analogues of (6-4) pyrimidine-pyrimidinone thymine photo-dimers is reported. The approach is based on the selective irradiation, at 350 nm, of a Tp4ST (4ST = 4-thiothymidine) step within a 20-mer having the sequence: d(ACTCGGACCT(4sT)CGCTGTGAT). Conversion of the S5-(6-4)/S5-thietane pyrimidine-pyrimidinone, initially formed, to its S5-Dewar isomer is by a subsequent irradiation at 300 nm. Both of the photo-dimer-containing oligonucleotides were purified by HPLC (ion exchange and reverse phase) and characterised by base composition analysis. The S5-(6-4)/S5-thietane pyrimidine-pyrimidinone containing 20-mer has a characteristic UV absorbance at 320 nm and exhibits strong fluorescence when excited at this wavelength. As expected, conversion to the S5-Dewar isomer abolished both the 320 nm absorbance and the fluorescence emission. The lengths of the oligonucleotides produced allowed the formation of stable double-stranded DNA, by hybridisation to a complementary sequence. Examination of these duplexes by circular dichroism spectroscopy showed that they formed B-DNA, with little changes to their gross structure as compared to the parent duplex. However, local structural perturbations in the region of the photo-dimer cannot be excluded. The S5-(6-4)/S5-thietane photoproduct lowered the tm by 10.5 deg. C and the Dewar isomer by 12 deg. C. The degree of curvature induced in the DNA sequence by the introduction of the photo-dimers was assessed by analysing the migration of modified and unmodified multimer ladders on polyacrylamide gels. Both photoproducts induced considerable bending into the DNA. A comparison with a six-base-pair T tract, a bending standard that has a known bend angle of 19 degrees, gave values of around 47 degrees for the S5-(6-4)/S5-thietane product and about 28 degrees for the S5-Dewar isomer.

The three-dimensional structures of two complexes between recombinant MS2 capsids and RNA operator fragments reveal sequence-specific protein-RNA interactions.

Crystal structures of two complexes between recombinant MS2 capsids and RNA operator fragments have been determined at 2.7 A resolution. The coat protein of the RNA bacteriophage MS2 is bifunctional; it forms the icosahedral virus shell to protect the viral nucleic acid and it acts as a translational repressor by binding with high specificity to a unique site on the RNA, a single stem-loop structure, containing the initiation codon of the gene for the viral replicase. In order to determine the structure of these protein-RNA complexes, we have used chemically synthesized variants of the stem-loop fragment and soaked them into crystals of recombinant capsids. The RNA stem-loop, as bound to the protein, forms a crescent-like structure and interacts with the surface of the beta-sheet of a coat protein dimer. It makes protein contacts with seven phosphate groups on the 5' side of the stem-loop, with a pyrimidine base at position -5, which stacks onto a tyrosine, and with two exposed adenine bases, one in the loop and one at a bulge in the stem. Replacement of the wild-type uridine with a cytosine at position -5 increases the affinity of the RNA to the dimer significantly. The complex with RNA stem-loop having cytosine at this position differs from that of the wild-type complex mainly by having one extra intramolecular RNA interaction and one extra water-mediated hydrogen bond.

The hammerhead, hairpin and VS ribozymes are catalytically proficient in monovalent cations alone.

BACKGROUND: The catalytic activity of RNA enzymes is thought to require divalent metal ions, which are believed to facilitate RNA folding and to play a direct chemical role in the reaction. RESULTS: We have found that the hammerhead, hairpin and VS ribozymes do not require divalent metal ions, their mimics such as [Co(NH3)6]3+, or even monovalent metal ions for efficient self-cleavage. The HDV ribozyme, however, does appear to require divalent metal ions for self-cleavage. For the hammerhead, hairpin and VS ribozymes, very high concentrations of monovalent cations support RNA-cleavage rates similar to or exceeding those observed in standard concentrations of Mg2+. Analysis of all reaction components by inductively coupled plasma-optical emission spectrophotometry (ICPOES) and the use of a variety of chelating agents effectively eliminate the possibility of contaminating divalent and trivalent metal ions in the reactions. For the hairpin ribozyme, fluorescence resonance energy transfer experiments demonstrate that high concentrations of monovalent cations support folding into the catalytically proficient tertiary structure. CONCLUSIONS: These results directly demonstrate that metal ions are not obligatory chemical participants in the reactions catalysed by the hammerhead, hairpin, and VS ribozymes. They permit us to suggest that the folded structure of the RNA itself contributes more to the catalytic function than was previously recognised, and that the presence of a relatively dense positive charge, rather than divalent metal ions, is the general fundamental requirement. Whether this charge is required for catalysis per se or simply for RNA folding remains to be determined.

Outcomes of 807 Thompson hip hemiarthroplasty procedures and the effect of surgical approach on dislocation rates.

The majority of displaced intracapsular fractures in our unit are managed with a Thompson hip hemiarthroplasty. Recent UK guidance from the National Institute for Health and Care Excellence has, however, advised against the continued used of the Thompson implant in patients with hip fracture. The aim of this study was to review the outcomes and complications after Thompson hip hemiarthroplasty, including the impact of modern surgical approaches and cementing, whilst controlling for confounding factors. We reviewed the outcomes following Thompson hip hemiarthroplasty from a series of 807 cases performed between April 2008 and November 2013. Of these, 721 (89.3%) were cemented and 86 (10.7%) uncemented. A total of 575 (71.3%) procedures were performed in female patients. The anterolateral approach was performed in 753 (93.3%) and the posterior approach with enhanced soft tissue repair in 54 (6.7%). Overall, there were 23 dislocations (2.9%). Dislocation following the posterior approach occurred in 13.0% (seven of 54) in comparison to 2.1% (16 of 753) with the anterolateral approach (odds ratio (OR) 8.5 (95% confidence interval (CI) 2.8-26.3), p < 0.001). Patients were discharged home in 459 cases (56.9%), to a care home or other hospital in 273 cases (33.8%). Of the total number of patients, 75 died during their admission (9.3%), and 51.8% (338 of 653) returned home within 30 days. The 30-day mortality was 7.1% (57 cases) and the 1-year mortality was 16.6% (116 of 699). We recommend against the continued use of the posterior approach in hip hemiarthroplasty, as enhanced soft tissue repair did not reduce the dislocation rates to an acceptable level in this series utilising the Thompson implant. Our findings, however, demonstrate satisfactory results for patients treated with the Thompson hip hemiarthroplasty performed through an anterolateral approach. We suggest that the continued use of this implant in a carefully selected patient cohort is justifiable.

Auricular microelectrostimulation: naloxone-reversible attenuation of opiate abstinence syndrome.

This study evaluated the effects in rats of very low amplitude (10 mu amp) charge-balanced 10-Hz stimulation delivered bilaterally to low impedance points on the outer ear. This microelectrostimulation markedly and significantly reduced the number of opiate abstinence signs observed following a week of continuous morphine infusion. This effect was prevented by subcutaneous injection of 3 mg/kg naloxone, suggesting that stimulation of endogenous opioid activity plays a major role in the actions of auricular microelectrostimulation.

Ability analysis of gender relevance and sex differences in cardiovascular response to behavioral challenge.

Cardiovascular effects of gender-specific ability perceptions (ability perceptions linked to one's identity as being female or male) were examined under different task conditions. In Study I, participants were led to believe that either men (masculine task) or women (feminine task) tend to do well on a memory task and then were provided the chance to avoid noise by attaining a low or high performance standard. As expected, sex differences in systolic blood pressure response during performance depended not only on task type but also on the degree of challenge. In Study 2, high standard effects were strengthened and extended through the use of an appetitive procedure and the inclusion of conditions in which the performance standard was extreme. Findings are discussed in terms of an interactional analysis of ability percepts, task demand, and cardiovascular responsivity.

Difficulty as a determinant of cardiovascular response: moderating effect of instrumentality in an alleviation paradigm.

Female subjects performed 40 trials of an easy or difficult digit-recognition task while being exposed to intermittent presentations of a noxious noise. Half (high instrumentality) were told that, if they performed well, there was a high chance that they would work without noise in a subsequent trial period. The rest (low instrumentality) were told that, if they performed well, there was a low chance that they would work without noise in the subsequent trial period. Thus, a good performance insured either a high or low probability of alleviating the aversive stimulus. Results indicated that systolic responsiveness was proportional to task demand when the instrumentality of alleviative behavior was high, but low under both task conditions when the instrumentality of alleviative behavior was low. Reaction time and performance data were generally consistent with the view that these effects were due to group differences in task engagement, or 'active coping'. The main findings conceptually replicate and extend results from two previous studies that crossed difficulty and instrumentality manipulations. They also call further into question familiar theoretical conceptions that intimate direct effects of incentive value and perceived control on cardiovascular reactivity.

Psychoendocrinological aspects of affective disorders.

Psychoendocrinological studies have opened a new approach to understanding affective disorders. In this study, the links of affective illnesses to changes in endocrine secretions--particularly adrenal, gonadal, growth, pineal, thyroidal, and prolactin--were reviewed with the object of adding to the number of depressed whose symptoms can be relieved.

Psychopharmacological therapy of deviant sexual behavior.

Psychopharmacological approaches to controlling male deviant sexual behavior, especially sexual recidivism and sexual deviants on probation, have been reported in psychiatric literature. In Europe, the drug cyproterone acetate, and in the United States, medroxyprogesterone acetate, Provera, and in the long-acting form, Depo-Provera, have all benefitted exhibitionists and pedophiliacs, and reduced sex drive in sexual deviants. The combination of pharmacotherapy and either psychotherapy or behavioral therapy has been the most effective approach to reducing the sex drive of sexual deviants.

Marijuana's effects on human cognitive functions, psychomotor functions, and personality.

Marijuana is complex chemically and not yet fully understood, but it is not a narcotic. Like alcohol, marijuana acts as both stimulant and depressant, but it lingers in body organs longer than alcohol. Smoking marijuana can injure mucosal tissue and may have more carcinogenic potential than tobacco. Research has indicated that marijuana intoxication definitely hinders attention, long-term memory storage, and psychomotor skills involved in driving a car or flying a plane. Expectations and past experience with marijuana have often influenced results more than pharmacological aspects have. Marijuana has triggered psychotic episodes in those more vulnerable. Psychological and some instances of physiological dependence on marijuana have been demonstrated. As a psychoactive drug, marijuana surely alters mental functioning. Although it is possible that chronic use of marijuana produces irreversible damage to mind or brain areas, this has not been determined by research.

Psychopharmacological investigation of panic disorder by means of lactate infusion.

The contribution of research with lactate infusion to an understanding of agoraphobia, generalized anxiety disorder, and panic disorder (PD) was reviewed. Lactate-induced panic seems to differentiate panic disorder from generalized anxiety disorder. Panic disorder seems to have important links to depression; both respond to tricyclic antidepressant drugs and neither responds well to the benzodiazepine antianxiety drugs. Response to pharmacotherapy, epidemiological surveys, and familial studies support the distinction between panic disorder and generalized anxiety disorder and the overlap between major depression and panic disorder. Understanding the mechanism of lactate-induced attacks may provide a better understanding of the pathophysiology of naturally occurring panic.