The Effect of Sample Handling on Cross Sectional HIV Incidence Testing Results

To determine if mishandling prior to testing would make a sample from a chronically infected subject appear recently infected when tested by cross-sectional HIV incidence assays.Serum samples from 31 subjects with chronic HIV infection were tested. Samples were subjected to different handling conditions, including incubation at 4 °C, 25 °C and 37 °C, for 1, 3, 7 or 15 days prior to testing. Samples were also subjected to 1,3, 7 and 15 freeze-thaw cycles prior to testing. Samples were tested using the BED capture enzyme immuno assay (BED-CEIA), Vironostika-less sensitive (V-LS), and an avidity assay using the Genetic Systems HIV-1/HIV-2 plus O EIA (avidity assay).Compared to the sample that was not subjected to any mishandling conditions, for the BED-CEIA, V-LS and avidity assay, there was no significant change in test results for samples incubated at 4 °C or 25 °C prior to testing. No impact on test results occurred after 15 freeze-thaw cycles. A decrease in assay results was observed when samples were held for 3 days or longer at 37 °C prior to testing.Samples can be subjected up to 15 freeze-thaw cycles without affecting the results the BED-CEIA, Vironostika-LS, or avidity assays. Storing samples at 4 °C or 25 °C for up to fifteen days prior to testing had no impact on test results. However, storing samples at 37°C for three or more days did affect results obtained with these assays

Down-regulation of ATM protein sensitizes human prostate cancer cells to radiation-induced apoptosis

Treatment with the protein kinase C activator 12-O-tetradecanoylphorbol 12-acetate (TPA) enables radiation-resistant LNCaP human prostate cancer cells to undergo radiation-induced apoptosis, mediated via activation of the enzyme ceramide synthase ( CS) and de novo synthesis of the sphingolipid ceramide (Garzotto, M., Haimovitz-Friedman, A., Liao, W. C., White-Jones, M., Huryk, R., Heston, D. W. W., Cardon-Cardo, C., Kolesnick, R., and Fuks, Z. ( 1999) Cancer Res. 59, 5194-5201). Here, we show that TPA functions to decrease the cellular level of the ATM ( ataxia telangiectasia mutated) protein, known to repress CS activation ( Liao, W.-C., Haimovitz-Friedman, A., Persaud, R., McLoughlin, M., Ehleiter, D., Zhang, N., Gatei, M., Lavin, M., Kolesnick, R., and Fuks, Z. ( 1999) J. Biol. Chem. 274, 17908 - 17917). Gel shift analysis in LNCaP and CWR22-Rv1 cells demonstrated a significant reduction in DNA binding of the Sp1 transcription factor to the ATM promoter, and quantitative reverse transcription-PCR showed a 50% reduction of ATM mRNA between 8 and 16 h of TPA treatment, indicating that TPA inhibits ATM transcription. Furthermore, treatment of LNCaP, CWR22-Rv1, PC-3, and DU-145 human prostate cells with antisense-ATM oligonucleotides, which markedly reduced cellular ATM levels, significantly enhanced radiation-induced CS activation and apoptosis, leading to apoptosis at doses as a low as 1 gray. These data suggest that the CS pathway initiates a generic mode of radiation- induced apoptosis in human prostate cancer cells, regulated by a suppressive function of ATM, and that ATM might represent a potential target for pharmacologic inactivation with potential clinical applications in human prostate cancer

Common Hydrogen Bond Interactions in Diverse Phosphoryl Transfer Active Sites

<div><p>Phosphoryl transfer reactions figure prominently in energy metabolism, signaling, transport and motility. Prior detailed studies of selected systems have highlighted mechanistic features that distinguish different phosphoryl transfer enzymes. Here, a top-down approach is developed for comparing statistically the active site configurations between populations of diverse structures in the Protein Data Bank, and it reveals patterns of hydrogen bonding that transcend enzyme families. Through analysis of large samples of structures, insights are drawn at a level of detail exceeding the experimental precision of an individual structure. In phosphagen kinases, for example, hydrogen bonds with the O_3β of the nucleotide substrate are revealed as analogous to those in unrelated G proteins. In G proteins and other enzymes, interactions with O_3β have been understood in terms of electrostatic favoring of the transition state. Ground state quantum mechanical calculations on model compounds show that the active site interactions highlighted in our database analysis can affect substrate phosphate charge and bond length, in ways that are consistent with prior experimental observations, by modulating hyperconjugative orbital interactions that weaken the scissile bond. Testing experimentally the inference about the importance of O_3β interactions in phosphagen kinases, mutation of arginine kinase Arg₂₈₀ decreases k_cat, as predicted, with little impact upon K_M.</p></div

Synthesis and reactivity of a cyclopentadienyl-indenyl ligand ring-coupled by a chiral bridge derived from ethyl (S)-(-) lactate

1477-9226In order to prepare a new unsymmetrical chiral ligand, C9H7CH(CH3)CH2C5H5, with an indenyl moiety connected to a cyclopentadienyl unit by a chiral ethylene bridge, we reacted the optically active tosylate 4, derived from ethyl (S)-(-) lactate, with LiCp. The only product resulting from this reaction was an optically active spirocyclopropane 6 obtained with a high diastereoselectivity (81.8% de). We also observed that, when the reduction of the ethyl indene lactate 2 was realized with an excess of LiAlH4 in Et2O under reflux, spirocyclopropane 6 was obtained in high yield with the same diastereoselectivity. When tosylate 4 was reacted with MgCp2, in place of LiCp, ligand 5 was obtained in good yield as a mixture of two double-bond isomers of the Cp unit. Ligand 5 was fully structurally characterized after conversion into transition monometallic complexes such as C9H7CH(CH3)CH2[small eta]5-C5H4Mo(CO)3Me 7 and C9H7CH(CH3)CH2[small eta]5-C5H4Rh(COD) 9, in which the indene moiety was kept intact due to the difference in reactivity of the indenyl moiety with respect to the cyclopentadienyl unit. An attempt to prepare a heterobimetallic complex from the sodium indenide salt of 7 and [RhCl(COD)]2, afforded a rhodium cyclopentadienyl complex 9, resulting from a metal exchange reaction with loss of the molybdenum part initially coordinated to the Cp unit, and conservation of the optical purity. An homobimetallic rhodium complex 10 could be prepared by deprotonation of ligand 5 with TlOEt, followed by quenching with [RhCl(COD)]2. The bis-rhodium complex 10 is obtained as a mixture of two diastereoisomers 10a and 10b with respect to the planar chirality of the indenyl ring system, with a good diastereoisomeric excess de = 70%. The structures of both complexes 9 and major (pS)-diastereoisomer 10a were determined by single crystal X-ray diffraction

Enzyme structures can be categorized according to the fate of the bound nucleoside triphosphate (NTP).

<p>A) Phosphoryl transfer in which the O_3β―P_γ bond is cleaved. B) Reactions in which the P_α―O_3α bond is cleaved and C) Structures where the bound NTP does not undergo a chemical reaction. Red lettering indicates the atoms in the scissile bond and red arrows depict the transfer of electrons in going from reactants to products.</p

Impact of (secondary) interactions with γ-oxygens on O_3β—P_γ bond elongation induced by (primary) O_3β interactions.

<p>Secondary interactions with γ-oxygens have modest impact (much smaller than the direct effects characterized by Summerton et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108310#pone.0108310-Summerton1" target="_blank">[16]</a>) and are substantial only for charged donors.</p

Arginine kinase active site mutation.

<p>Selected interactions of the nucleotide in the crystal structure of the transition state analog complex of Horseshoe Crab Arginine Kinase (AK, PDB ID 1M15) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108310#pone.0108310-Yousef2" target="_blank">[48]</a>. Carbon = green, nitrogen = dark blue, oxygen = red and phosphorus = orange. In AK, Arg₂₈₀ contacts the O_3β oxygen of ADP, an α-oxygen and the Asp₃₂₄ side chain. In ATP, the O_3β oxygen bridges to the γ-phosphate which is mimicked by nitrate in this transition state analog complex. Hydrogen bonds are shown with red dotted lines.</p

Effects of a hydrogen bond at O_3β on orbital and interaction energies in Structure1.

<p>Shortening the hydrogen bond between N-methylacetamide and methyl triphosphate: A) decreases the orbital energy of the σ*(O_3β—P_γ) anti-bonding orbital; while (B) leaving unchanged both the n(O_γ) donor orbital energies and (C) F_i,j, a measure of the overlap between the n(O_γ) lone pair orbitals and σ*(O_3β—P_γ). D denotes hydrogen bond donor. σ* denotes σ*(O_3β—P_γ).</p

Non-catalytic (blue) active sites have a preference positively charged hydrogen bond donors (a–b) whereas in catalytic (red) active sites neutral interactions are favored (c–d).

<p>Mean numbers of hydrogen bonds were measured for non-bridging (a & c) and bridging (b & d) oxygens. The catalytic group is composed of both O_3β—P_γ and P_α—O_3α structure sets. Positive donors include Lys, Arg, and His side chains and neutral donors include Asn, Gln, Trp, Ser Thr,Tyr, Cys side chains, the nucleotide O2′ and O3′ oxygens and all backbone nitrogens (except Pro).</p

Models used to test the dependence of hyperconjugation and O_3β―P_γ bond length on enzyme-ligand interactions.

<p>N-methylacetamide (A) was used to model a (neutral) protein backbone amide hydrogen bond to O_3β, using methyl triphosphate to model an NTP nucleotide. Structures 2 (B) and 3 (C) were used to investigate the effects of protonation at the γ-oxygens. Additional active site hydrogen bonds, represented in Structures 4 through 6 (D–F), were used to assess the secondary effects of different types of O_γ hydrogen bonds. Acetamide (E) 1-propylaminium (F) were used to model asparagine and lysine side chains respectively. Structure 7 (G) was used to investigate the impact of hydrogen bonding at a nonbridging β-oxygen on hyperconjugation and O_3β―P_γ bond length. Hydrogen bonds are shown with dashed red lines. White = hydrogen, gray = carbon, red = oxygen, blue = nitrogen, orange = phosphorus.</p