Self-malonylation is an intrinsic property of a chemically synthesized type II polyketide synthase acyl carrier protein

During polyketide biosynthesis, malonyl groups are transferred to the acyl carrier protein (ACP) component of the polyketide synthase (PKS), and it has been shown that a number of type II polyketide ACPs undergo rapid self-acylation from malonyl-CoA in the absence of a malonyl-CoA:holo-acyl carrier protein transacylase (MCAT). More recently, however, the observation of self-malonylation has been ascribed to contamination with Escherichia coli MCAT (FabD) rather than an intrinsic property of the ACP. The wild-type apo-ACP from the actinorhodin (act) PKS of Streptomyces coelicolor (synthetic apo-ACP) has therefore been synthesized using solid-state peptide methods and refolded using the GroEL/ES chaperone system from E. coli. Correct folding of the act ACP has been confirmed by circular dichroism (CD) and 1H NMR. Synthetic apo-ACP was phosphopantetheinylated to 100% by S. coelicolor holo-acyl carrier protein synthase (ACPS), and the resultant holo-ACP underwent self-malonylation in the presence of malonyl-CoA. No malonylation of negative controls was observed, confirming that the use of ACPS and GroEL/ES did not introduce contamination with E. coli MCAT. This result proves unequivocally that self-malonylation is an inherent activity of this PKS ACP in vitro.     

Mutagenic properties of 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, a persistent acetylaminofluorene-derived DNA adduct in mammalian cells

The carcinogen 2-acetylaminofluorene is metabolically activated in cells and reacts with DNA to form N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxyguanosin-N(2)()-yl)-2-acetylaminofluorene (dG-N(2)-AAF) DNA adducts. The dG-N(2)-AAF adduct is the least abundant of the three isomers, but it persists in the tissues of animals treated with this carcinogen. The miscoding and mutagenic properties of dG-C8-AAF and dG-C8-AF have been established; these adducts are readily excised by DNA repair enzymes engaged in nucleotide excision repair. In the present study, oligodeoxynucleotides modified site-specifically with dG-N(2)-AAF were used as DNA templates in primer extension reactions catalyzed by mammalian DNA polymerases. Reactions catalyzed by pol alpha were strongly blocked at a position one base before dG-N(2)-AAF and also opposite this lesion. In contrast, during translesion synthesis catalyzed by pol eta or pol kappa nucleotides were incorporated opposite the lesion. Both pol eta and pol kappa incorporated dCMP, the correct base, opposite dG-N(2)-AAF. In reactions catalyzed by pol eta, small amounts of dAMP misincorporation and one-base deletions were detected at the lesion site. With pol kappa, significant dTMP misincorporation was observed opposite the lesion. Steady-state kinetic analysis confirmed the results obtained from primer extension studies. Single-stranded shuttle vectors containing (5)(')TCCTCCTCXCCTCTC (X = dG-N(2)-AAF, dG-C8-AAF, or dG) were used to establish the frequency and specificity of dG-N(2)-AAF-induced mutations in simian kidney (COS-7) cells. Both lesions promote G --> T transversions overall, with dG-N(2)-AAF being less mutagenic than dG-C8-AAF (3.4% vs 12.5%). We conclude from this study that dG-N(2)-AAF, by virtue of its persistence in tissues, contributes significantly to the mutational spectra observed in AAF-induced mutagenesis and that pol eta, but not pol kappa, may play a role in this process.     

Heterotrimeric type I collagen C-telopeptide conformation as docked to its helix receptor

The amino (N-telo) and carboxyl (C-telo) telopeptides of type I collagen play crucial roles, in vivo and in vitro, in the assembly of collagen fibrils, regulating the axial alignment of the molecules within a fibril, azimuthal orientations of neighboring molecules, and cross-link formation. High-resolution structures of the telopeptides are not available from X-ray diffraction studies, but computational methods permitted prediction of the N-telo structure within a fibril. Here, using a suite of molecular modeling software, the more complex heterotrimeric C-telo of human type I collagen has been built from the correct sequences and energy minimized and the energy minimum confirmed by molecular dynamics. The receptor triple helix was modeled on the basis of the Protein Data Bank coordinates of a collagen-like sequence. Docking of the heterotrimeric C-telopeptide to its receptor showed that hydrophobic interactions involving the short alpha2 C-telopeptide are crucial determinants of its azimuthal orientation within the docked structure. A docked C-telo can interact with only one neighboring helix. The two alpha1(I) C-telo chains in the alpha1(Alpha)-alpha2-alpha1(B) chain stagger do not have identical docked conformations, and one of the alpha1(I) C-telo chains appears to be favored for formation of a cross-link between its K(16C) and a helix K87. Prior studies showed that a docked N-telopeptide can interact with two adjacent collagen monomers, forming a tightly packed region. A recent X-ray analysis showed the N- and C-telo regions pack differently, with the C-telo region being less densely packed than N-telo regions. This difference between N- and C-telopeptide docked structures demonstrates how unique and specific packing can occur in the fibril at each boundary of the type I collagen gap region.     

Role of class A penicillin-binding proteins in PBP5-mediated beta-lactam resistance in Enterococcus faecalis

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of beta-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 DeltaponA DeltapbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of beta-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.     

Repellency and toxicity of mint oil granules to red imported fire ants (Hymenoptera: Formicidae)

Repellency and toxicity of 2% mint oil granules were evaluated against worker red imported fire ants. Solenopsis invicta Buren, in a series of laboratory and field experiments. In continuous exposure experiments, LT50 values ranged from 1.2 h with 164.8 mg/cm2 of 2% mint oil granules to 15.3 h with 1.65 mg/cm2 of granules. LT50 values declined exponentially with increasing rate of mint oil granules. Limited exposure to 164.8 mg/cm2 mint oil granules resulted in > 50% knock down (KD) after 30 min; however, there was no KD at 15 min. Twenty-four hour mortality increased linearly with increasing exposure time. Mean repellency of worker red imported fire ants ranged from 49.2 +/- 5.4% for 0 mg/cm2 (untreated control) of mint oil granules at 30 min to 100% for 147.8 mg/cm2 of mint oil granules at 3 h. Repellency increased with increasing milligrams per square centimeter of mint oil granules and exposure time. In field tests, 100% of mounds opened and treated with mint oil granules were abandoned 5 d after treatment and ants had relocated or formed satellite mounds by 2 d after treatment. Unopened mounds treated topically with mint oil granules were not abandoned, but ants formed satellite mounds 2 d after treatment. Mint oil granules could provide another tool for red imported fire ant integrated pest management, particularly in situations in which conventional insecticides would be inappropriate.     

Comparison of aeration and spinosad for suppressing insects in stored wheat

Field studies were conducted from July 2002 to January 2003 for evaluating the effects of controlled aeration and a commercial biological insecticide, spinosad, in suppressing insect populations in stored wheat. Six cylindrical steel bins were filled with newly harvested (2002 crop year) hard red winter wheat on 9 and 10 July 2002. Each bin contained 30.7 metric tons (1,100 bu) of wheat. Wheat in two bins was left untreated (control), whereas wheat in two bins was treated with spinosad, and in another two bins was subjected to aeration by using aeration controllers. Spinosad was applied to wheat at the time of bin filling to obtain a rate of 1 mg ([AI])/kg. Aeration controllers were set to run the fans when ambient air temperature fell below 23.9, 18.3, and 7.2 degrees C for the first, second, and third cooling cycles, respectively. We added 400 adults each of the rusty grain beetle, Cryptolestes ferrugineus (Stephens); lesser grain borer, Rhyzopertha dominica (F.); and red flour beetle, Tribolium castaneum (Herbst), to the grain at monthly intervals between July and October 2002. Insect density in the bins was estimated monthly by taking 3-kg grain samples from 21 locations within each bin by using a pneumatic grain sampler. No live T. castaneum or C. ferrugineus and very low densities of R. dominica (<0.008 adults per kilogram) were found in wheat treated with spinosad during the 6-mo sampling period. Density of C. ferrugineus and T. castaneum in aerated bins did not exceed two adults per kilogram (the Federal Grain Inspection Service standard for infested wheat), whereas R. dominica increased to 12 adults per kilogram in November 2002, which subsequently decreased to three adults per kilogram in January 2003. In the untreated (control) bins, R. dominica density increased faster than that of C. ferrugineus or T. castaneum. Density of R. dominica peaked at 58 adults per kilogram in October 2002 and decreased subsequently, whereas T. castaneum density was 10 adults per kilogram in October 2002 but increased to 78 adults per kilogram in January 2003. Density of C. ferrugineus increased steadily during the 6-mo study period and was highest (six adults per kilogram) in January 2003. This is the first report comparing the field efficacy of spinosad and aeration in managing insects in farm bins. Our results suggest that spinosad is very effective in suppressing R. dominica, C. ferrugineus, and T. castaneum populations in stored wheat.     

Analysis of autoreactive CD4 T cells in experimental autoimmune encephalomyelitis after primary and secondary challenge using MHC class II tetramers

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is primarily mediated by CD4 T cells specific for Ags in the CNS. Using MHC class II tetramers, we assessed expansion and phenotypic differentiation of polyclonal self-reactive CD4 T cells during EAE after primary and secondary challenge with the specific Ag. After EAE induction in SJL mice with proteolipid protein 139-151, CNS-specific T cells up-regulated activation markers and expanded in the draining lymph nodes and in the spleen. Less than 20% of total autoreactive T cells entered the CNS simultaneously with Th cells of other specificities. Almost all tetramer-positive cells in the CNS were activated and phenotypically distinct from the large peripheral pool. When EAE was induced in Ag-experienced mice, disease symptoms developed earlier and persisted longer; autoreactive T cells were more rapidly activated and invaded the CNS earlier. In striking contrast to specific CTLs that respond after secondary viral challenge, the absolute numbers of autoreactive CD4 T cells were not increased, indicating that the accelerated autoreactivity in Ag-experienced mice is not related to higher frequencies of autoreactive CD4 T cells.     

Regulated expression of the receptor-like tyrosine phosphatase CD148 on hemopoietic cells

CD148 is a receptor-like protein tyrosine phosphatase expressed on a wide variety of cell types. Through the use flow cytometry and immunofluorescence microscopy on tissue sections, we examined the expression of CD148 on multiple murine hemopoietic cell lineages. We found that CD148 is moderately expressed during all stages of B cell development in the bone marrow, as well as peripheral mature B cells. In contrast, CD148 expression on thymocytes and mature T cells is substantially lower. However, stimulation of peripheral T cells through the TCR leads to an increase of CD148 expression. This up-regulation on T cells can be partially inhibited by reagents that block the activity of src family kinases, calcineurin, MEK, or PI3K. Interestingly, CD148 levels are elevated on freshly isolated T cells from MRL lpr/lpr and CTLA-4-deficient mice, two murine models of autoimmunity. Together, these expression data along with previous biochemical data suggest that CD148 may play an important regulatory role to control an immune response.