The bottleneck in AZT activation.

Nucleoside-based inhibitors of reverse transcriptase were the first drugs to be used in the chemotherapy of AIDS. After entering the cell, these substances are activated to their triphosphate form by cellular kinases, after which they are potent chain terminators for the growing viral DNA. The two main factors limiting their efficacy are probably interrelated. These are the insufficient degree of reduction of viral load at the commencement of treatment and the emergence of resistant variants of the virus. The reason for the relatively poor suppression of viral replication appears to be inefficient metabolic activation. Thus, for the most extensively used drug, 3'-azido-3'-deoxythymidine (AZT), whereas phosphorylation to the monophosphate is facile, the product is a very poor substrate for the next kinase in the cascade, thymidylate kinase. Because of this, although high concentrations of the monophosphate can be reached in the cell, the achievable concentration of the active triphosphate is several orders of magnitude lower. Determination of the structure of thymidylate kinase as a complex with AZT monophosphate (AZTMP) together with studies on the kinetics of its phosphorylation have now led to a detailed understanding of the reasons for and consequences of the poor substrate properties.

Outcome of Weekly Carboplatin-Paclitaxel-based Definitive Chemoradiation in Oesophageal Cancer in Patients Not Considered to be Suitable for Platinum-Fluoropyrimidine-based Treatment: A Multicentre, Retrospective Review.

AIMS: Although cisplatin-fluoropyrimidine-based definitive chemoradiotherapy (dCRT) is a standard of care for oesophageal cancer, toxicity is significant and limits its use in elderly and frail patients. Weekly carboplatin-paclitaxel-based dCRT provides a viable alternative, although prospective data are lacking in the dCRT setting. Here we report the results of a national, multicentre retrospective review of outcome in patients treated with weekly carboplatin-paclitaxel-based dCRT. MATERIALS AND METHODS: In this multicentre retrospective study of nine radiotherapy centres across the UK we evaluated the outcome of patients who had non-metastatic, histologically confirmed carcinoma of the oesophagus (adenocarcinoma, squamous cell or undifferentiated; World Health Organization performance status 0-2; stage I-III disease) and had been selected to receive weekly carboplatin-paclitaxel-based dCRT as they were considered not suitable for cisplatin-fluoropyrimidine-based dCRT. dCRT consisted of carboplatin AUC 2 and paclitaxel 50 mg/m2 (days 1, 8, 15, 22, 29) and the recommended radiation dose was 50 Gy in 25 daily fractions. We assessed overall survival, progression-free survival (PFS; overall, local and distant), proportion of patients who were failure free at the response assessment (12 weeks after dCRT), treatment compliance and toxicity. RESULTS: In total, 214 patients from nine UK centres were treated between 15 February 2013 and 19 March 2019: 39.7% of patients were ≥75 years; 18.7% ≥ 80 years. Indications for weekly carboplatin-paclitaxel-based dCRT were comorbidities (47.2%), clinician choice (36.4%) and poor tolerance/progression on cisplatin-fluoropyrimidine induction chemotherapy (15.8%). The median overall survival was 24.28 months (95% confidence interval 20.07-30.09) and the median PFS was 16.33 months (95% confidence interval 14.29-20.96). Following treatment, 69.1% (96/139) had a combined complete response on endoscopy with non-progression (complete response/partial response/stable disease) on imaging. The 1- and 2-year overall survival rates for this patient group were 81.9% (95% confidence interval 75.6-86.8%) and 50.6% (95% confidence interval 40.5-60.0%), respectively. Thirty-three per cent (n = 70) of patients experienced at least one grade 3 + acute toxicity (grade 3/4 haematological: 10%; grade 3/4 non-haematological: 32%) and there were no treatment-related deaths. 86.9% of patients completed at least four cycles of concomitant weekly carboplatin-paclitaxel-based chemotherapy and planned radiotherapy was completed in 97.7% (209/214). CONCLUSION: Weekly carboplatin-paclitaxel-based CRT seems to be well tolerated in elderly patients and in those with comorbidities, where cisplatin-fluoropyrimidine-based dCRT is contraindicated. Survival outcomes are comparable with cisplatin-fluoropyrimidine-based dCRT.

Infrared image of venus at the time of pioneer venus probe encounter.

An image of the infrared emission from the Earth-facing hemisphere of Venus was obtained at the time the Pioneer Venus probes penetrated the atmosphere. The thermal structure of the atmosphere at the 85-millibar level included regions of rapidly varying polar features, a solar-related postdawn warm area, and a nonsolar-fixed nighttime warm area. The probes succeeded in entering each of these three thermal regions.

Formation of a transition-state analog of the Ras GTPase reaction by Ras-GDP, tetrafluoroaluminate, and GTPase-activating proteins.

Unlike the alpha subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins, Ras-related GTP-binding proteins have hitherto been considered not to bind or become activated by tetrafluoroaluminate (AIF4-). However, the product of the proto-oncogene ras in its guanosine diphosphate (GDP)-bound form interacted with AIF4 - in the presence of stoichiometric amounts of either of the guanosine triphosphatase (GTPase)-activating proteins (GAPs) p120GAP and neurofibromin. Neither oncogenic Ras nor a GAP mutant without catalytic activity produced such a complex. Together with the finding that the Ras-binding domain of the protein kinase c-Raf, whose binding site on Ras overlaps that of the GAPs, did not induce formation of such a complex, this result suggests that GAP and neurofibromin stabilize the transition state of the GTPase reaction of Ras.

Extreme ultraviolet observations from voyager 2 encounter with jupiter.

Extreme ultraviolet spectral observations of the Jovian planetary system made during the Voyager 2 encounter have extended our knowledge of many of the phenomena and physical processes discovered by the Voyager 1 ultraviolet spectrometer. In the 4 months between encounters, the radiation from Io's plasma torus has increased in intensity by a factor of about 2. This change was accompanied by a decrease in plasma temperature of about 30 percent. The high-latitude auroral zones have been positively associated with the magnetic projection of the plasma torus onto the planet. Emission in molecular hydrogen bands has been detected from the equatorial regions of Jupiter, indicating planetwide electron precipitation. Hydrogen Lyman alpha from the dark side of the planet has been measured at an intensity of about 1 kilorayleigh. An observation of the occultation of alpha Leonis by Jupiter was carried out successfully and the data are being analyzed in detail.

Extreme ultraviolet observations from voyager 1 encounter with jupiter.

Observations of the optical extreme ultraviolet spectrum of the Jupiter planetary system during the Voyager 1 encounter have revealed previously undetected physical processes of significant proportions. Bright emission lines of S III, S IV, and O III indicating an electron temperature of 10(5) K have been identified in preliminary analyses of the Io plasma torus spectrum. Strong auroral atomic and molecular hydrogen emissions have been observed in the polar regions of Jupiter near magnetic field lines that map the torus into the atmosphere of Jupiter. The observed resonance scattering of solar hydrogen Lyman alpha by the atmosphere of Jupiter and the solar occultation experiment suggest a hot thermosphere (>/= 1000 K) wvith a large atomic hydrogen abundance. A stellar occultation by Ganymede indicates that its atmosphere is at most an exosphere.

Hypofractionated Radiotherapy in Oesophageal Cancer for Patients Unfit for Systemic Therapy: A Retrospective Single-Centre Analysis.

AIMS: Chemoradiotherapy (CRT) is established as a superior treatment option to definitive radiotherapy in the non-surgical management of oesophageal cancer. For patients precluded from CRT through choice or comorbidity there is little evidence to guide delivery of single-modality radiotherapy. In this study we outline outcomes for patients unfit for CRT who received a hypofractionated radiotherapy (HRT) regimen. MATERIALS AND METHODS: A retrospective UK single-centre analysis of 61 consecutive patients with lower- or middle-third adenocarcinoma (OAC; 61%) or squamous cell carcinoma of the oesophagus managed using HRT with radical intent between April 2009 and 2014. Treatment consisted of 50 Gy in 16 fractions (n = 49, 80.3%) or 50-52.5 Gy in 20 fractions (n = 12, 19.7%). Outcomes were referenced against a contemporaneous comparator cohort of 80 (54% OAC) consecutive patients managed with conventionally fractionated CRT within the same centre. RESULTS: Three-year and median overall survival were, respectively, 56.9% and 29 months with HRT compared with 55.5% and 26 months for CRT; adjusted hazard ratio 0.79 (95% confidence interval 0.48-1.28). Grade 3 and 4 toxicity rates were low at 16.4% (n = 10) for those receiving HRT and 40.2% (n = 32) for the CRT group. In patients with OAC, CRT delivered superior overall survival (hazard ratio 0.46; 95% confidence interval 0.25-0.85) and progression-free survival (hazard ratio 0.45; 95% confidence interval 0.23-0.88) when compared with HRT. CONCLUSIONS: The HRT regimen described here was safe and tolerable in patients unable to receive CRT, and delivered promising survival outcomes. The use of HRT for the treatment of oesophageal cancer, both alone and as a sequential or concurrent treatment with chemotherapy, requires further study. New precision radiotherapy technologies may provide additional scope for improving outcomes in oesophageal cancer using HRT-based approaches and should be evaluated.

The pre-hydrolysis state of p21(ras) in complex with GTP: new insights into the role of water molecules in the GTP hydrolysis reaction of ras-like proteins.

BACKGROUND: In numerous biological events the hydrolysis of guanine triphosphate (GTP) is a trigger to switch from the active to the inactive protein form. In spite of the availability of several high-resolution crystal structures, the details of the mechanism of nucleotide hydrolysis by GTPases are still unclear. This is partly because the structures of the proteins in their active states had to be determined in the presence of non-hydrolyzable GTP analogues (e.g. GppNHp). Knowledge of the structure of the true Michaelis complex might provide additional insights into the intrinsic protein hydrolysis mechanism of GTP and related nucleotides. RESULTS: The structure of the complex formed between p21(ras) and GTP has been determined by X-ray diffraction at 1.6 A using a combination of photolysis of an inactive GTP precursor (caged GTP) and rapid freezing (100K). The structure of this complex differs from that of p21(ras)-GppNHp (determined at 277K) with respect to the degree of order and conformation of the catalytic loop (loop 4 of the switch II region) and the positioning of water molecules around the gamma-phosphate group. The changes in the arrangement of water molecules were induced by the cryo-temperature technique. CONCLUSIONS: The results shed light on the function of Gln61 in the intrinsic GTP hydrolysis reaction. Furthermore, the possibility of a proton shuffling mechanism between two attacking water molecules and an oxygen of the gamma-phosphate group can be proposed for the basal GTPase mechanism, but arguments are presented that render this protonation mechanism unlikely for the GTPase activating protein (GAP)-activated GTPase.

Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate.

BACKGROUND: Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes the reversible phosphoryltransfer between ATP and TMP to yield ADP and TDP. In addition to its vital role in supplying precursors for DNA synthesis, human TMPK has an important medical role participating in the activation of a number of anti-HIV prodrugs. RESULTS: Crystal structures of human TMPK in complex with TMP and ADP, TMP and the ATP analog AppNHp, TMP with ADP and the phosphoryl analog AlF(3), TDP and ADP, and the bisubstrate analog TP(5)A were determined. The conformations of the P-loop, the LID region, and the adenine-binding loop vary according to the nature of the complex. Substitution of ADP by AppNHp results in partial closure of the P-loop and the rotation of the TMP phosphate group to a catalytically unfavorable position, which rotates back in the AlF(3) complex to a position suitable for in-line attack. In the fully closed state observed in the TP(5)A and the TDP-ADP complexes, Asp15 interacts strongly with the 3'-hydroxyl group of TMP. CONCLUSIONS: The observed changes of nucleotide state and conformation and the corresponding protein structural changes are correlated with intermediates occurring along the reaction coordinate and show the sequence of events occurring during phosphate transfer. The low catalytic activity of human TMPK appears to be determined by structural changes required to achieve catalytic competence and it is suggested that a mechanism might exist to accelerate the activity.

Biochemical and biological consequences of changing the specificity of p21ras from guanosine to xanthosine nucleotides.

The D119N mutation of p21ras was prepared by site-directed mutagenesis. Its nucleotide binding properties were investigated using fluorescently labelled guanosine and xanthosine nucleotides. Its affinity for guanosine nucleotides is severely reduced, with a concomitant increase in the affinity for xanthosine nucleotides, which leads to an almost complete reversal of base specificity. The protein is a GTPase as well as a XTPase and the hydrolysis reaction can be efficiently stimulated by GAP. Dissociation of XDP from the mutant is stimulated by the guanine nucleotide exchange factor Cdc25Mm in a similar manner to that of GDP from wildtype. The interaction of the mutant with the effector domain of c-Raf kinase or Ral-GEF is normal. In microinjection experiments in PC12 and NIH3T3 cells the protein behaves as an oncogenic mutant due to its high dissociation rate for GDP. However, when the protein is loaded with XDP before microinjection the onset of the oncogenic signal can be efficiently retarded. Thus, the protein behaves initially as wildtype and later as an oncogenic protein.

Rapid determination of the affinity of 28- and 14-mer phosphorothioate oligonucleotides for HIV-1 reverse transcriptase by fluorescence spectroscopy.

Intrinsic fluorescence of human immunodeficiency virus type 1 reverse transcriptase (E.C. 2.7.7.49) and displacement experiments of a fluorescent template.primer probe were used to study the interaction of the enzyme with several types of 28- and 14-mer normal or phosphorothioate oligodeoxycytidinylates and their duplexes with poly(rI). The two methods gave convergent results and allowed in each case fast determinations of ligand affinities for the enzyme. The dissociation constants (Kd) obtained from intrinsic fluorescence changes were slightly lower than those determined from the less direct competitive displacement experiments. In all cases, the enzyme displayed better recognition of the hybrid than of the unannealed oligonucleotide. The Kd values of phosphorothioate oligomers and their hybrids were lower than those of the corresponding normal oligomers and hybrids, but the difference was not as significant as in the case of the Ki constants for (dC)28 and S(dC)28 (Majumdar et al. (1989) Biochemistry 28, 1340). The affinities of the annealed phosphorothioate oligodeoxycytidinylates for the enzyme were found to be larger than for any other compounds in this series (Kd of poly(rI).S(dC)28: 0.28 nM at 25 degrees C). Changing the beta stereochemistry of the oligomer bases to alpha did not alter the affinity of the oligodeoxycytidinylate and its hybrids for the enzyme.

Dimerization kinetics of HIV-1 and HIV-2 reverse transcriptase: a two step process.

The dimerization processes of the human immunodeficiency virus (HIV) types 1 and 2 reverse transcriptase (RTs) from their subunits have been investigated using a number of complementary approaches (fluorescence spectroscopy, size exclusion-HPLC and polymerase activity assay). The formation of the native heterodimeric form of HIV-1 and HIV-2 RT occurs in a two step process. The first step is a concentration-dependent association of the two subunits (p66 and p51) to give a heterodimeric intermediate, which slowly isomerizes to the "mature" heterodimeric form of the enzyme. For both RTs, the first step behaves as a second order reaction with similar association rate constants (in the range of 2 x 10(4) to 4 x 10(4) M-1 s-1). This initial dimerization results in a 25% quenching of the intrinsic fluorescence and a 30% decrease in the accessibility of the tryptophan hydrophobic cluster to solvent as revealed by iodide quenching experiments and by monitoring the binding of 1-anilino-8-naphthalenesulphonate. The formation of the intermediate-RT form appears to involve hydrophobic regions of the subunits containing tryptophan residues. This intermediate form is devoid of polymerase activity, but is able to bind primer/template with high affinity. The final stage of the mature RT-heterodimer formation occurs in a slow first order reaction, which is 12-fold faster for HIV-2 (1.2 h-1) than HIV-1 RT (0.1 h-1). At micromolar concentrations, this slow isomerization constitutes the rate limiting step of the RT maturation and the structural change involved appears to be partly associated with the catalytic site, as shown using fluorescent labelled primer/template. On the basis of both the presently available X-ray structure of the HIV-1 RT and the predicted structure of HIV-2 RT, the thumb subdomain of the p51 subunit seems to be involved in this maturation step, which is probably the interaction of this domain with the RNAse H domain of the large subunit. The placement of the fingers subdomain of p51 in the palm subdomain of the p66 subunit may also be associated with formation of mature heterodimeric RTs.

Refined model for primer/template binding by HIV-1 reverse transcriptase: pre-steady-state kinetic analyses of primer/template binding and nucleotide incorporation events distinguish between different binding modes depending on the nature of the nucleic acid substrate.

The kinetic mechanism of nucleic acid substrate binding and nucleotide incorporation by human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) was analysed using synthetic DNA/DNA and DNA/RNA primer/templates (p/t) without predicted secondary structures in the single-stranded region. Determination of the pre-steady-state kinetics of p/t binding by a combination of stopped-flow and quench flow methods indicate a branched binding mechanism for the HIV-1 RT/nucleic acid interaction. Analysis of p/t-RT association by stopped-flow measurements suggest a three-step binding mode with an initial second-order step followed by two isomerisation steps with rates of about 6 s(-1)and 0.5 s(-1), respectively. Determination of the rate-limiting step of the association process via single turnover, single nucleotide incorporation analysis by quench flow measurements revealed two binding events (the initial second-order step cannot be detected with this experimental set-up) with rates of 4 - 7 s(-1)and 0.4 - 0. 7 s(-1), respectively, indicating that both binding events exist in parallel. Thorough pre-steady-state analysis of single turnover, single nucleotide incorporation kinetics showed that dNTP incorporation occurs with a biphasic exponential burst followed by a linear phase. The exponential burst consists of a fast phase with rates of 20 - 60 s(-1) and a slow phase with rates of 0.5 - 2 s(-1), respectively. The relative distribution of these two burst amplitudes differs significantly depending upon which substrate is used. The DNA/RNA-RT complex shows primarily fast incorporation (>80 %) whereas less than 45 % of the DNA/DNA-RT complex incorporate dNTP rapidly. The same relative distribution of amplitudes concerning the two substrates is also found for the association process of RT and p/t. Analysis of dNTP incorporation of the preformed RT-p/t complex in the presence of a nucleic acid competitor shows no effect on the biphasic burst amplitude, however the linear phase disappears. Here, a refined model of the mechanism of RT-p/t binding is presented which is based on the suggestion that two different RT-p/t complexes are formed, i.e. a productive enzyme/substrate complex which is capable of nucleotide incorporation and a non-productive complex which has to undergo an isomerisation before dNTP incorporation can occur. In addition, binding of RT to its substrate can lead to a dead end complex that is not capable of dNTP incorporation.

Time-resolved cryo-electron microscopic study of the dissociation of actomyosin induced by photolysis of photolabile nucleotides.

The rapid release of a substrate or other ligand from photolabile precursors in a thin layer suspension of biological specimens followed by rapid freezing provides a method of trapping and visualizing short-lived states in a dynamic system. We demonstrate here the first successful application of this method to study the interaction of actin filaments with myosin subfragment 1 (S1) after release of nucleotides. The results obtained suggest that structural changes in actin filaments occur as a result of interaction with S1.

High-resolution crystal structure of S. cerevisiae Ypt51(DeltaC15)-GppNHp, a small GTP-binding protein involved in regulation of endocytosis.

Ypt/Rab proteins are membrane-associated small GTP-binding proteins which play a central role in the coordination, activation and regulation of vesicle-mediated transport in eukaryotic cells. We present the 1.5 A high-resolution crystal structure of Ypt51 in its active, GppNHp-bound conformation. Ypt51 is an important regulator involved in the endocytic membrane traffic of Saccharomyces cerevisiae. The structure reveals small but significant structural differences compared with H-Ras p21. The effector loop and the catalytic loop are well defined and stabilized by extensive hydrophobic interactions. The switch I and switch II regions form a well-defined epitope for hypothetical effector protein binding. Sequence comparisons between the different isoforms Ypt51, Ypt52 and Ypt53 provide the first insights into determinants for specific effector binding and for fine-tuning of the intrinsic GTP-hydrolysis rate.

Temperature-dependent equilibrium between the open and closed conformation of the p66 subunit of HIV-1 reverse transcriptase revealed by site-directed spin labelling.

X-ray crystallographic studies of human immunodeficiency virus type 1 reverse transcriptase complexed with or without substrates or inhibitors show that the heterodimeric enzyme adopts distinct conformations that differ in the orientation of the so-called thumb subdomain in the large subunit. Site-directed spin labelling of mutated residue positions W24C and K287C is applied here to determine the distances between the fingers and thumb subdomains of liganded and unliganded RT in solution. The inter-spin distances of a DNA/DNA and a pseudoknot RNA complexed reverse transcriptase in solution was found to agree with the respective crystal data of the open and closed conformations. For the unliganded reverse transcriptase a temperature-dependent equilibrium between these two states was observed. The fraction of the closed conformation decreased from 95% at 313 K to 65% at 273 K. The spectral separation between the two structures was facilitated by the use of a perdeuterated ([15)N]nitroxide methane-thiosulfonate spin label.

Crystallization and preliminary X-ray analysis of the human c-H-ras-oncogene product p21 complexed with GTP analogues.

The catalytic domain (amino acid residues 1 to 166) of the human ras-oncogene product p21 complexed with the GTP analogues beta,gamma-imido-GTP (GMPPNP), beta,gamma-methylene-GTP (GMPPCP), and guanosine-5'-(gamma-thiotriphosphate) (GTP gamma S) have been been crystallized. Crystals of the GMPPNP and GMPPCP complexes are well suited for high resolution X-ray crystallography. They belong to space group P3(1)21 (or its enantiomorph P3(2)21) with unit cell axes a=b=40.3 A and c = 162.2 A.

Potentiating AZT activation: structures of wild-type and mutant human thymidylate kinase suggest reasons for the mutants' improved kinetics with the HIV prodrug metabolite AZTMP.

The 60-fold reduced phosphorylation rate of azidothymidine (AZT) monophosphate (AZTMP), the partially activated AZT metabolite, by human thymidylate kinase (TMPK) severely limits the efficacy of this anti-HIV prodrug. Crystal structures of different TMPK nucleotide complexes indicate that steric hindrance by the azido group of AZTMP prevents formation of the catalytically active closed conformation of the P-loop of TMPK. The F105Y mutant and a chimeric mutant that contains sequences of the human and Escherichia coli enzyme phosphorylate AZTMP 20-fold faster than the wild-type enzyme. The structural basis of the increased activity is assigned to stabilization of the closed P-loop conformation.

Equine infectious anemia virus transactivator is a homeodomain-type protein.

Lentiviral transactivator (Tat) proteins are essential for viral replication. Tat proteins of human immunodeficiency virus type 1 and bovine immunodeficiency virus form complexes with their respective RNA targets (Tat responsive element, TAR), and specific binding of the equine anemia virus (EIAV) Tat protein to a target TAR RNA is suggested by mutational analysis of the TAR RNA. Structural data on equine infectious anemia virus Tat protein reveal a helix-loop-helix-turn-helix limit structure very similar to homeobox domains that are known to bind specifically to DNA. Here we report results of gel-shift and footprinting analysis as well as fluorescence and nuclear magnetic resonance spectroscopy experiments that clearly show that EIAV Tat protein binds to DNA specifically at the long terminal repeat Pu.1 (GTTCCTGTTTT) and AP-1 (TGACGCG) sites, and thus suggest a common mechanism for the action of some of the known lentiviral Tat proteins via the AP-1 initiator site. Complex formation with DNA induces specific shifts of the proton NMR resonances originating from amino acids in the core and basic domains of the protein.