Uridine kinase from Ehrlich ascites carcinoma. Purification and properties of homogeneous enzyme.

Uridine kinase from Ehrlich ascites tumor cells has been purified about 60,000-fold to apparent homogeneity and with an overall recovery of about 40%. This purification was achieved using phosphocellulose and adenosine 5'-triphosphate-agarose affinity chromatography. The subunit molecular mass as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 31,000 daltons. With two-dimensional electrophoresis, only one spot was observed, indicating the absence of isoenzymes. Multiple peaks of activity are routinely observed on ion exchange chromatography or gel filtration, for both crude preparations or homogeneous uridine kinase, in agreement with our earlier results that this enzyme exists as multiple interconvertible oligomeric forms (Payne, R. C., and Traut, T. W. (1982) J. Biol. Chem. 257, 12485-12488). The purified enzyme has a specific activity of 283 mumol/min/mg of protein at 22 degrees C. Initial velocity studies using uridine and ATP are consistent with a sequential mechanism. Km values for uridine, cytidine, and ATP are 40, 57, and 450 microM, respectively. CTP and UTP are competitive inhibitors with respect to ATP, with Ki values for CTP and UTP of 10 and 61 microM, respectively. The enzyme was active with several nucleoside analogs, the Km values being 69 microM (5-fluorouridine), 200 microM (3-deazauridine), and 340 microM (6-azauridine). The pure enzyme is very sensitive to freezing, but can be maintained at O degrees C for 8 weeks with only 20% loss of activity. For long-term storage, enzyme in 50% glycerol can be maintained at -20 degrees C for many months with no detectable loss of activity

Design and Synthesis of a Novel Alpha-Methylene Lactone Chemotherapeutic Agent

Goniothalamin, a natural product isolated from the dried stem bark of Malaysian plants of the genus Goniothalamus, has been shown to induce apoptosis in cancer cells. The bioactivity of this molecule is though to be due to its ability to react with thiols. One mechanism involves its reaction with glutathione, a natural antioxidant found in all cells. Using a four step synthetic sequence, a novel gamma-lactone analogue of goniothalamin has been prepared that replaces the endocylic double bond in goniothalamin\u27s lactone core with an exocyclic double bond. It is anticipated that this alteration will allow the compound to react more rapidly with thiols and therefore increase its cytotoxicity towards cancer cells

Decarbonising the critical sectors of aviation, shipping, road freight and industry to limit warming to 1.5–2°C

Limiting warming to well below 2°C requires rapid and complete decarbonisation of energy systems. We compare economy-wide modelling of 1.5°C and 2°C scenarios with sector-focused analyses of four critical sectors that are difficult to decarbonise: aviation, shipping, road freight transport, and industry. We develop and apply a novel framework to analyse and track mitigation progress in these sectors. We find that emission reductions in the 1.5°C and 2°C scenarios of the IMAGE model come from deep cuts in CO2 intensities and lower energy intensities, with minimal demand reductions in these sectors’ activity. We identify a range of additional measures and policy levers that are not explicitly captured in modelled scenarios but could contribute significant emission reductions. These are demand reduction options, and include less air travel (aviation), reduced transportation of fossil fuels (shipping), more locally produced goods combined with high load factors (road freight), and a shift to a circular economy (industry). We discuss the challenges of reducing demand both for economy-wide modelling and for policy. Based on our sectoral analysis framework, we suggest modelling improvements and policy recommendations, calling on the relevant UN agencies to start tracking mitigation progress through monitoring key elements of the framework (CO2 intensity, energy efficiency, and demand for sectoral activity, as well as the underlying drivers), as a matter of urgency

560 Analysis of 168 primary adult ovarian granulosa cell tumors, its clinicopathological outcome, and risk factors for recurrence

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How does non-nutritive sucking support infant feeding?

Fifty nine premature infants participated in a randomized controlled study to determine the effectiveness of non-nutritive sucking (NNS). It was predicted that NNS would not accelerate the development of full oral feeding or early language skills as sometimes perceived in practice. However, it was predicted that using NNS as a strategy to support parents to identify and respond to early communication and oral readiness signs would increase confidence in infant management and enable quicker discharge home. Infants were aged 26-35 weeks gestation. Infants with no significant difficulties were randomly assigned to one of three groups; Group 1, NNS pre-tube feeding (n=19); Group 2, NNS on onset of tube feeding (n=20) and Group 3, Control (n=20). Follow-up occurred at 6 months. There were no significant differences with number of days to full oral feeding between the groups receiving NNS and the Control group, χ(2)(2, n=59)=4.33, p=.115. A significant difference in number of days in hospital between the Control group and the other two groups was found χ(2) (2, n=59)=7.678, p=.022. Significant changes were noted with the development of more normal sucking patterns in Groups 1-3. At 6 months there were no significant differences in receptive or expressive language skills between all groups. NNS had no significant impact on the transition to full oral feeding or later language development. There was a significant difference in the number of days in hospital between the Control group and the other two groups which involved parents in identification of early communication signs. Possible reasons for this change and future directions are discussed

Insights into the Regulatory Characteristics of the Mycobacterial Dephosphocoenzyme A Kinase: Implications for the Universal CoA Biosynthesis Pathway

Being vastly different from the human counterpart, we suggest that the last enzyme of the Mycobacterium tuberculosis Coenzyme A biosynthetic pathway, dephosphocoenzyme A kinase (CoaE) could be a good anti-tubercular target. Here we describe detailed investigations into the regulatory features of the enzyme, affected via two mechanisms. Enzymatic activity is regulated by CTP which strongly binds the enzyme at a site overlapping that of the leading substrate, dephosphocoenzyme A (DCoA), thereby obscuring the binding site and limiting catalysis. The organism has evolved a second layer of regulation by employing a dynamic equilibrium between the trimeric and monomeric forms of CoaE as a means of regulating the effective concentration of active enzyme. We show that the monomer is the active form of the enzyme and the interplay between the regulator, CTP and the substrate, DCoA, affects enzymatic activity. Detailed kinetic data have been corroborated by size exclusion chromatography, dynamic light scattering, glutaraldehyde crosslinking, limited proteolysis and fluorescence investigations on the enzyme all of which corroborate the effects of the ligands on the enzyme oligomeric status and activity. Cysteine mutagenesis and the effects of reducing agents on mycobacterial CoaE oligomerization further validate that the latter is not cysteine-mediated or reduction-sensitive. These studies thus shed light on the novel regulatory features employed to regulate metabolite flow through the last step of a critical biosynthetic pathway by keeping the latter catalytically dormant till the need arises, the transition to the active form affected by a delicate crosstalk between an essential cellular metabolite (CTP) and the precursor to the pathway end-product (DCoA)

UMP/CMPK Is Not the Critical Enzyme in the Metabolism of Pyrimidine Ribonucleotide and Activation of Deoxycytidine Analogs in Human RKO Cells

Human UMP/CMP kinase was identified based on its enzymatic activity in vitro. The role of this protein is considered critical for the maintenance of pyrimidine nucleotide pool profile and for the metabolism of pyrimidine analogs in cells, based on the in vitro study of partially purified enzyme and recombinant protein. However, no detailed study has yet addressed the role of this protein in nucleotide metabolism in cells.Two stable cell lines in which UMP/CMP kinase (mRNA: AF087865, EC 2.7.4.14) can be either up-regulated or down-regulated were developed using Tet-On Gene Expression Systems. The amount and enzymatic activity of UMP/CMP kinase extracted from these two cell lines can be induced up by 500% or down by 95-98%. The ribonucleotides of endogenous pyrimidine as well as the metabolism of exogenous natural pyrimidine nucleosides and their analogs were not susceptible to the altered amount of UMP/CMP kinase in these two stable RKO cell lines. The level of incorporation of pyrimidine nucleoside analogs, such as gemcitabine (dFdC) and troxacitabine (L-OddC), into cellular DNA and their potency in inhibiting cell growth were not significantly altered by up-regulation or down-regulation of UMP/CMP kinase expression in cells.The UMP/CMP kinase (EC 2.7.4.14) expressed in RKO cells is not critical for the phosphorylation of (d)CMP and the maintenance of natural nucleotide pools. It also does not play an important role in the activation of dFdC and L-OddC. The increase by 500% or decrease by 95-98% in the levels of UMP/CMP kinase do not affect steady state levels of dFdC and L-OddC in RKO cells. Overall, the activity and possible mechanisms of recombinant UMP/CMP kinase expressed in the in vitro system can not be extended to that of UMP/CMP kinase expressed in a cell system or an in vivo system

Sex-specific differences in the synaptonemal complex in the genus Oreochromis (Cichlidae)

Total synaptonemal complex (SC) lengths were estimated from Oreochromis aureus Steindachner (which has a WZ/ZZ sex determination system), O. mossambicus Peters and O. niloticus L. (both of which have XX/XY sex determination systems). The total SC length in oocytes was greater than that in spermatocytes in all three species (194±30 μm and 134±13 μm, 187±22 μm and 127±17 μm, 193±37 μm and 144±19 μm, respectively). These sex-specific differences did not appear to be influenced by the type of sex determination system (the female/male total SC length ratio was 1.45 in O. aureus, 1.47 in O. mossambicus and 1.34 in O. niloticus) and do not correlate with the lack of any overall sex-specific length differences in the current Oreochromis linkage map. Although based on data from relatively few species, there appears to be no consistent relationship between sex-specific SC lengths and linkage map lengths in fish. Neomale (hormonally masculinized genetic female) O. aureus and O. mossambicus had total SC lengths of 138±13 μm and 146±13 μm respectively, more similar to normal males than to normal females. These findings agree with data from other vertebrate species that suggest that phenotypic sex, rather than genotype, determines traits such as total SC length, chiasmata position and recombination pattern, at least for the autosomes

Alpha-Photon Coincidence Spectroscopy Along Element 115 Decay Chains

Produced in the reaction 48Ca+243Am, thirty correlated α-decay chains were observed in an experiment conducted at the GSI Helmholzzentrum für Schwerionenforschung, Darmstadt, Germany. The decay chains are basically consistent with previous findings and are considered to originate from isotopes of element 115 with mass numbers 287, 288, and 289. A set-up aiming specifically for high-resolution charged particle and photon coincidence spectroscopy was placed behind the gas-filled separator TASCA. For the first time, γ rays as well as X-ray candidates were observed in prompt coincidence with the α-decay chains of element 115