Structure of monoubiquitinated PCNA: Implications for DNA polymerase switching and Okazaki fragment maturation

Ubiquitination of proliferating cell nuclear antigen (PCNA) to ub-PCNA is essential for DNA replication across bulky template lesions caused by UV radiation and alkylating agents, as ub-PCNA orchestrates the recruitment and switching of translesion synthesis (TLS) polymerases with replication polymerases. This allows replication to proceed, leaving the DNA to be repaired subsequently. Defects in a TLS polymerase, Pol η, lead to a form of Xeroderma pigmentosum, a disease characterized by severe skin sensitivity to sunlight damage and an increased incidence of skin cancer. Structurally, however, information on how ub-PCNA orchestrates the switching of these two classes of polymerases is lacking. We have solved the structure of ub-PCNA and demonstrate that the ubiquitin molecules in ub-PCNA are radially extended away from the PCNA without structural contact aside from the isopeptide bond linkage. This unique orientation provides an open platform for the recruitment of TLS polymerases through ubiquitin-interacting domains. However, the ubiquitin moieties, to the side of the equatorial PCNA plane, can place spatial constraints on the conformational flexibility of proteins bound to ub-PCNA. We show that ub-PCNA is impaired in its ability to support the coordinated actions of Fen1 and Pol δ in assays mimicking Okazaki fragment processing. This provides evidence for the novel concept that ub-PCNA may modulate additional DNA transactions other than TLS polymerase recruitment and switching.     

Synthesis and biological evaluation of 2-substituted-4-(3′,4′,5′-trimethoxyphenyl)-5-aryl thiazoles as anticancer agents

Antitumor agents that bind to tubulin and disrupt microtubule dynamics have attracted considerable attention in the last few years. To extend our knowledge of the thiazole ring as a suitable mimic for the cis-olefin present in combretastatin A-4, we fixed the 3,4,5-trimethoxyphenyl at the C4-position of the thiazole core. We found that the substituents at the C2- and C5-positions had a profound effect on antiproliferative activity. Comparing compounds with the same substituents at the C5-position of the thiazole ring, the moiety at the C2-position influenced antiproliferative activities, with the order of potency being NHCH₃ > Me ? N(CH₃)₂. The N-methylamino substituent significantly improved antiproliferative activity on MCF-7 cells with respect to C2-amino counterparts. Increasing steric bulk at the C2-position from N-methylamino to N,N-dimethylamino caused a 1–2 log decrease in activity. The 2-N-methylamino thiazole derivatives 3b, 3d and 3e were the most active compounds as antiproliferative agents, with IC₅₀ values from low micromolar to single digit nanomolar, and, in addition, they are also active on multidrug-resistant cell lines over-expressing P-glycoprotein. Antiproliferative activity was probably caused by the compounds binding to the colchicines site of tubulin polymerization and disrupting microtubule dynamics. Moreover, the most active compound 3e induced apoptosis through the activation of caspase-2, -3 and -8, but 3e did not cause mitochondrial depolarization.     

Systems analysis of eleven rodent disease models reveals an inflammatome signature and key drivers

Common inflammatome gene signatures as well as disease‐specific signatures were identified by analyzing 12 expression profiling data sets derived from 9 different tissues isolated from 11 rodent inflammatory disease models. The inflammatome signature significantly overlaps with known drug targets and co‐expressed gene modules linked to metabolic disorders and cancer. A large proportion of genes in this signature are tightly connected in tissue‐specific Bayesian networks (BNs) built from multiple independent mouse and human cohorts. Both the inflammatome signature and the corresponding consensus BNs are highly enriched for immune response‐related genes supported as causal for adiposity, adipokine, diabetes, aortic lesion, bone, muscle, and cholesterol traits, suggesting the causal nature of the inflammatome for a variety of diseases. Integration of this inflammatome signature with the BNs uncovered 151 key drivers that appeared to be more biologically important than the non‐drivers in terms of their impact on disease phenotypes. The identification of this inflammatome signature, its network architecture, and key drivers not only highlights the shared etiology but also pinpoints potential targets for intervention of various common diseases.     

How Drugs Interact with Transporters: SGLT1 as a Model

Drugs are transported by cotransporters with widely different turnover rates. We have examined the underlying mechanism using, as a model system, glucose and indican (indoxyl-beta-D: -glucopyranoside) transport by human Na(+)/glucose cotransporter (hSGLT1). Indican is transported by hSGLT1 at 10% of the rate for glucose but with a fivefold higher apparent affinity. We expressed wild-type hSGLT1 and mutant G507C in Xenopus oocytes and used electrical and optical methods to measure the kinetics of glucose (using nonmetabolized glucose analogue alpha-methyl-D: -glucopyranoside, alphaMDG) and indican transport, alone and together. Indican behaved as a competitive inhibitor of alphaMDG transport. To examine protein conformations, we recorded SGLT1 capacitive currents (charge movements) and fluorescence changes in response to step jumps in membrane voltage, in the presence and absence of indican and/or alphaMDG. In the absence of sugar, voltage jumps elicited capacitive SGLT currents that decayed to steady state with time constants (tau) of 3-20 ms. These transient currents were abolished in saturating alphaMDG but only slightly reduced (10%) in saturating indican. SGLT1 G507C rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages. Maximal fluorescence increased approximately 150% in saturating indican but decreased approximately 50% in saturating alphaMDG. Modeling indicated that the rate-limiting step for indican transport is sugar translocation, whereas for alphaMDG it is dissociation of Na(+) from the internal binding sites. The inhibitory effects of indican on alphaMDG transport are due to its higher affinity and a 100-fold lower translocation rate. Our results indicate that competition between substrates and drugs should be taken into consideration when targeting transporters as drug delivery systems.     

Phosphorus magnetic resonance spectroscopy 2 h after perinatal cerebral hypoxia-ischemia prognosticates outcome in the newborn piglet

Phosphorus magnetic resonance spectroscopy ((31)P MRS) often reveals apparently normal brain metabolism in the first hours after intrapartum hypoxia-ischemia (HI) at a time when conventional clinical assessment of injury severity is problematic. We aimed to elucidate very-early, injury-severity biomarkers. Twenty-seven newborn piglets underwent cerebral HI: (31)P-MRS measures approximately 2 h after HI were compared between injury groups defined by secondary-energy-failure severity as quantified by the minimum nucleotide triphosphate (NTP) observed after 6 h. For severe and moderate injury versus baseline, [Pi]/[total exchangeable high-energy phosphate pool (EPP)] was increased (p < 0.001 and < 0.02, respectively), and [NTP]/[EPP] decreased (p < 0.03 and < 0.006, respectively): severe-injury [Pi]/[EPP] was also increased versus mild injury (p < 0.04). Mild-injury [phosphocreatine]/[EPP] was increased (p < 0.004). Severe-injury intracellular pH was alkaline versus baseline (p < 0.002). For severe and moderate injury [total Mg]/[ATP] (p < 0.0002 and < 0.02, respectively) and [free Mg] (p < 0.0001 and < 0.02, respectively) were increased versus baseline. [Pi]/[EPP], [phosphocreatine]/[Pi] and [NTP]/[EPP] correlated linearly with injury severity (p < 0.005, < 0.005 and < 0.02, respectively). Increased [Pi]/[EPP], intracellular pH and intracellular Mg approximately 2 h after intrapartum HI may prognosticate severe injury, whereas increased [phosphocreatine]/[EPP] may suggest mild damage. In vivo(31)P MRS may have potential to provide very-early prognosis in neonatal encephalopathy.     

Sex determination of three raptor species using morphology and molecular techniques

ABSTRACT.   Accurate identification of sex is important for many raptor studies, but may be difficult to determine in the field for some species. Because of size differences between males and females, morphological measurements have often been used to sex raptors. However, few investigators have examined the accuracy of using measurements of individuals made at one location to sex individuals of the same species at another location. Our objective was to develop more accurate region-specific methods for determining the sex of Red-tailed Hawks ( Buteo jamaicensis ), Red-shouldered Hawks ( B. lineatus ), and Cooper's Hawks ( Accipiter cooperii ) migrating through and wintering in California. We determined sex using a polymerase chain reaction (PCR)-based genetic test and grouped individuals based on sex, age, and geographic area. We did not combine groups due to differences in measurements between age classes and geographic areas. We then compared a suite of morphological measurements between males and females of each combination, and developed both a discriminant function and a flowchart to determine the sex of Red-tailed and Red-shouldered hawks in the field. The flowcharts were more accurate than the functions for both species. We also confirmed the accuracy of the current flowchart used to determine the sex of Cooper's Hawks migrating along the California coast. These region-specific methods for Red-tailed and Cooper's hawks were generally more accurate than published methods, possibly indicating different populations of these species and highlighting the importance of validating sexing methods when using them in different locations.     

Optimized adeno-associated virus 8 produces hepatocyte-specific Cre-mediated recombination without toxicity or affecting liver regeneration

Engineering viral vectors to produce liver-specific protein expression may help advance understanding of hepatic regeneration and disease states. In addition to introducing genes of interest to the liver, these vectors can be adapted for gene deletion when designed to express Cre recombinase. The ability to use this system requires high, liver-restricted expression, low toxicity, and no effect on the process of interest. We developed an adeno-associated virus 8 (AAV8) with a codon-optimized Cre recombinase under a hepatocyte-specific major urinary protein (MUP) promoter (MUP-iCre-AAV8) that fulfills these requirements. A single intravenous injection of ROSA26R reporter mice, which express lacZ after Cre-mediated recombination, demonstrated homogeneous beta-galactosidase expression limited to hepatocytes after only 7 days. Cre protein expression remained strong for at least 31 days. Serum liver function tests and histology demonstrated minimal liver toxicity. The presence of MUP-iCre-AAV8 did not affect hepatocyte proliferation after partial hepatectomy as measured by Ki67 staining. Conclusion: AAV8 with the MUP promoter, by virtue of its lack of hepatic toxicity or effect on liver regeneration, may be an efficient alternative to complex transgenic methodologies for studies of the mouse liver.     

Dependence of trans-ADP-ribosylation and nuclear glycolysis on the Arg 34-ATP complex of Zn2+ finger I of poly-ADP-ribose polymerase-1

The H-bonded complex of ATP with Arg 34 of Zn2+ finger I of poly-ADP-ribose polymerase-1 (PARP-1) determines trans-oligo-ADP-ribosylation from NAD+ to proteins other than PARP-1. This mechanism was tested in lysolecithin fractions of non-malignant and cancer cells separately and after their recombination. Cellular PARP-1 activity was recovered when the centrifugal sediment was recombined with the supernatant fraction containing cellular ADP-ribose oligomer acceptor proteins. Combination of the matrix fraction (Mx) of cancer cells (lacking OXPHOS) with its supernatant had the same PARP-1 activity as the Mx alone. The supernatant of non-malignant cells was replaced by glycolytic enzymes as ADP-ribose acceptor. The hexokinase activity of the supernatant increased when OXPHOS of intact cells was uncoupled by carbonyl cyanide 4-(trifluoro methoxy) phenylhydrazone. trans-ADP-ribosylation was demonstrated by polyacrylamide gel electrophoresis.