Replication checkpoint protein Mrc1 is regulated by Rad3 and Tel1 in fission yeast

Fission yeast Mrc1 (mediator of replication checkpoint 1) is an adaptor checkpoint protein required for Rad3-dependent activation of the checkpoint kinase Cds1 in response to arrest of replication forks. Here we report studies on the regulation of Mrc1 by phosphorylation. Replication arrest induced by hydroxyurea (HU) induces Mrc1 phosphorylation that is detected by a change in Mrc1 electrophoretic mobility. Phosphorylation is maintained in cds1Delta, rad3Delta, and tel1Delta single mutants but eliminated in a rad3Delta tel1Delta double mutant. Mrc1 has two clusters of S/TQ motifs that are potential Rad3/Tel1 phosphorylation sites. Mutation of six S/TQ motifs in these two clusters strongly impairs Mrc1 phosphorylation. Two motifs located at S604 and T645 are vital for HU resistance. The T645A mutation strongly impairs a Cds1-Mrc1 yeast two-hybrid interaction that is dependent on a functional forkhead-associated (FHA) domain in Cds1, indicating that phosphorylation of T645 mediates Mrc1's association with Cds1. Consistent with this model, the T645 region of Mrc1 effectively substitutes for the T11 region of Cds1 that is thought to be phosphorylated by Rad3 and to mediate FHA-dependent oligomerization of Cds1. The S/TQ cluster that includes S604 is needed for Mrc1's increased association with chromatin in replication-arrested cells. These data indicate that Rad3 and Tel1 regulate Mrc1 through differential phosphorylation to control Cds1.     

Extracellular Matrix Lumican Promotes Bacterial Phagocytosis,and Lum?/? Mice Show Increased Pseudomonas aeruginosa Lung Infection Severity

Phagocytosis is central to bacterial clearance, but the exact mechanism is incompletely understood. Here, we show a novel and critical role for lumican, the connective tissue extracellular matrix small leucine-rich repeat proteoglycan, in CD14-mediated bacterial phagocytosis. In Psuedomonas aeruginosa lung infections, lumican-deficient (Lum^−/−) mice failed to clear the bacterium from lungs, tissues, and showed a dramatic increase in mortality. In vitro, phagocytosis of nonopsonized Gram-negative Escherichia coli and P. aeruginosa was inhibited in Lum^−/− peritoneal macrophages (MΦs). Lumican co-localized with CD14, CD18, and bacteria on Lum^+/+ MΦ surfaces. Using two different P. aeruginosa strains that require host CD14 (808) or CD18/CR3 (P1) for phagocytosis, we showed that lumican has a larger role in CD14-mediated phagocytosis. Recombinant lumican (rLum) restored phagocytosis in Lum^−/− MΦs. Surface plasmon resonance showed specific binding of rLum to CD14 (K_A = 2.15 × 10⁶ m⁻¹), whereas rLumY20A, and not rLumY21A, where a tyrosine in each was replaced with an alanine, showed 60-fold decreased binding. The rLumY20A variant also failed to restore phagocytosis in Lum^−/− MΦs, indicating Tyr-20 to be functionally important. Thus, in addition to a structural role in connective tissues, lumican has a major protective role in Gram-negative bacterial infections, a novel function for small leucine-rich repeat proteoglycans.     

8-Methyladenosine-substituted analogues of 2-5A: synthesis and their biological activities.

8-Methyladenosine-substituted analogues of 2-5A, p5'A2'p5'A2'p5'(me8A), p5'A2'p5'(me8A)2'p5'(me8A), p5'(me8A)2'p5'(me8A)2'p5'(me8A), and p5'(me8A) 2'p5'A2'p5'A, were prepared via a modification of a lead ion-catalyzed ligation reaction. These 2-5A monophosphates were converted into the corresponding 5'-triphosphates. Substitution of an 8-methyladenosine residue at the third position (2'-terminus) of the oligonucleotides increased the stability to snake venom phosphodiesterase digestion. Both binding and activation of mouse liver 2-5A dependent ribonuclease (RNase L) by the various 8-methyladenosine-substituted 2-5A analogues were examined. Among the 8-methyladenosine-substituted trimer analogues, the analogues with 8-methyladenosine residing in the 2'-terminal position showed the strongest binding affinity and were several times more effective than 2-5A itself as an inhibitor of translation.     

Prediction of splice junctions in mRNA sequences.

A general method based on the statistical technique of discriminant analysis is developed to distinguish boundaries of coding and non-coding regions in nucleic acid sequences. In particular, the method is applied to the prediction of splicing sites in messenger RNA precursors. Information used for discrimination includes consensus sequence patterns around splice junctions, free energy of snRNA and mRNA base pairing, and statistical differences between coding and non-coding regions such as periodic appearance of specific bases in coding regions reflecting the non-random usage of degenerate codons. Given the reading frame of an exon (but not the exon/intron boundaries), the method will predict the following exon, namely, the intron to be excised out. When applied to human sequences in the GenBank database, the method correctly identified 80% of true splice junctions.     

Immature Pacific bluefin tuna, <Emphasis Type="Italic">Thunnus orientalis</Emphasis>, utilizes cold waters in the Subarctic Frontal Zone for trans-Pacific migration

The habitat and movements of a Pacific bluefin tuna were investigated by reanalyzing archival tag data with sea surface temperature data. During its trans-Pacific migration to the eastern Pacific, the fish took a direct path and primarily utilized waters, in the Subarctic Frontal Zone (SFZ). Mean ambient temperature during the trans-Pacific migration was 14.5 ± 2.9 (°C ± SD), which is significantly colder than the waters typically inhabited by bluefin tuna in their primary feeding grounds in the western and eastern Pacific (17.6 ± 2.1). The fish moved rapidly through the colder water, and the heat produced during swimming and the thermoconservation ability of bluefin tuna likely enabled it to migrate through the cold waters of the SFZ.     

Low copy number and limited variability of proviral DNA in alveolar macrophages from HIV-1-infected patients: evidence for genetic differences in HIV-1 between lung and blood macrophage populations.

BACKGROUND: We investigated the human immunodeficiency virus (HIV) proviral DNA sequence and copy number in alveolar macrophages (AM) and peripheral blood monocytes (PBM) from 10 HIV-positive patients without any active concurrent pulmonary disease to understand the nature of HIV-1 infection in vivo in the lung microenvironment. MATERIALS AND METHODS: The 10 seropositive patients without active pulmonary disease were selected based on chest roentegenography and pathological/cytological test of bronchoalveolar (BAL) fluid. In order to determine accurate proviral copy numbers, AM and PBM were isolated to 99 and 94% purity, respectively, and quantitative polymerase chain reaction (PCR), with a sensitivity to detect three copies of HIV proviral DNA per 10(5) cells, was applied. For analysis of genetic variation in HIV-1, PCR-amplified HIV-1 DNA from AM and PBM of five patients were subcloned and 2-12 clones from each sample underwent DNA sequence analysis of HIV-1 gp120 V3-V5. Heteroduplex mobility assays were performed to confirm the results of the sequence analysis. RESULTS: The proviral copy number in AM or PBM were less than 20 copies/10(5) cells in all patients, and five patients had less than the detection limit. There was no significant difference in HIV copy number between AM and PBM. No correlation was found between PBM/AM HIV copy number and CD4+ lymphocyte count in the peripheral blood. Sequence analysis revealed that the mean intrapatient genetic similarity in AM was 97.5 +/- 0.18% (n = 107), which was significantly higher than that in PBM (96.2 +/- 0.26% (n = 94), p < 0.001), suggesting that variability of HIV-1 DNA in AM was relatively limited. Divergence occurred when AM derived HIV-1 sequence was compared with PBM derived sequence from the same patient (95.8 +/- 0.17% (n = 223) p < 0.001). Phylogenetic analysis of DNA sequence demonstrated complete separation of HIV lineages from lung and blood in four of five patients. CONCLUSIONS: The results suggest the HIV-1 infection in AM is restricted in vivo with low viral burden and homogenous genotype. We propose that the pulmonary microenvironment may limit the extent of HIV-1 infection.     

Improvement of the glutaryl-7-aminocephalosporanic acid acylase activity of a bacterial gamma-glutamyltranspeptidase

7-Aminocephalosporanic acid (7-ACA) is an important material in the production of semisynthetic cephalosporins, which are the best-selling antibiotics worldwide. 7-ACA is produced from cephalosporin C via glutaryl-7-ACA (GL-7-ACA) by a bioconversion process using d-amino acid oxidase and cephalosporin acylase (or GL-7-ACA acylase). Previous studies demonstrated that a single amino acid substitution, D433N, provided GL-7-ACA acylase activity for gamma-glutamyltranspeptidase (GGT) of Escherichia coli K-12. In this study, based on its three-dimensional structure, residues involved in substrate recognition of E. coli GGT were rationally mutagenized, and effective mutations were then combined. A novel screening method, activity staining followed by a GL-7-ACA acylase assay with whole cells, was developed, and it enabled us to obtain mutant enzymes with enhanced GL-7-ACA acylase activity. The best mutant enzyme for catalytic efficiency, with a k(cat)/K(m) value for GL-7-ACA almost 50-fold higher than that of the D433N enzyme, has three amino acid substitutions: D433N, Y444A, and G484A. We also suggest that GGT from Bacillus subtilis 168 can be another source of GL-7-ACA acylase for industrial applications.     

Artesunate: developmental toxicity and toxicokinetics in monkeys

BACKGROUND: The developmental toxicity, toxicokinetics, and hematological effects of the antimalarial drug, artesunate, were previously studied in rats and rabbits and have now been studied in cynomolgus monkeys. METHODS: Groups of up to 15 pregnant females were dosed on Gestation Days (GD) 20–50 or for 3–7‐day intervals. RESULTS: At 30 mg/kg/day, 6 embryos died between GD30 and GD40. Histologic examination of 3 live embryos (GD26–GD36) revealed a marked reduction in embryonic erythroblasts and cardiomyopathy. At 12 mg/kg/day, 6 embryos died between GD30 and GD45. Four surviving fetuses examined on GD100 had no malformations, but long bone lengths were slightly decreased. At the developmental no‐adverse‐effect‐level (4 mg/kg/day), maternal plasma AUC was 3.68 ng.h/mL for artesunate and 6.93 ng.h/ml for its active metabolite, dihydroartemisinin (DHA). No developmental toxicity occurred with administration of 12 mg/kg/day for 3 or 7 days, GD29–31 or GD27–33 (maternal plasma AUC of 9.84 ng.h/mL artesunate and 16.4 ng.h/mL DHA). Exposures at embryotoxic doses were substantially lower than human therapeutic exposures. However, differences in monkey and human Vss for artesunate (0.5 L/kg vs. 0.18 L/kg) confound relying solely on AUC for assessing human risk. Decreases in reticulocyte count occur at therapeutic doses in humans. Changes to reticulocyte counts at embryotoxic doses in monkeys (≥12 mg/kg/day) were variable and generally minor. CONCLUSIONS: Artesunate was embryolethal at ≥12 mg/kg/day when dosed for at least 12 days at the beginning of organogenesis, but not when dosed for 3 or 7 days, indicating that developmental toxicity of artesunate is dependent upon duration of dosing in cynomologus monkeys. Birth Defects Res (Part B) 83:418–434, 2008. © 2008 Wiley‐Liss, Inc.