Differential inhibition of inducible T cell cytokine secretion by potent iron chelators

Effector functions and proliferation of T helper (Th) cells are influenced by cytokines in the environment. Th1 cells respond to a synergistic effect of interleukin-12 (IL-12) and interleukin-18 (IL-18) to secrete interferon-gamma (IFN-gamma). In contrast, Th2 cells respond to interleukin-4 (IL-4) to secrete IL-4, interleukin-13 (IL-13), interleukin-5 (IL-5), and interleukin-10 (IL-10). The authors were interested in identifying nonpeptide inhibitors of the Th1 response selective for the IL-12/IL-18-mediated secretion of IFN-gamma while leaving the IL-4-mediated Th2 cytokine secretion relatively intact. The authors established a screening protocol using human peripheral blood mononuclear cells (PBMCs) and identified the hydrazino anthranilate compound 1 as a potent inhibitor of IL-12/IL-18-mediated IFN-gamma secretion from CD3(+) cells with an IC(50) around 200 nM. The inhibitor was specific because it had virtually no effect on IL-4-mediated IL-13 release from the same population of cells. Further work established that compound 1 was a potent intracellular iron chelator that inhibited both IL-12/IL-18- and IL-4-mediated T cell proliferation. Iron chelation affects multiple cellular pathways in T cells. Thus, the IL-12/IL-18-mediated proliferation and IFN-gamma secretion are very sensitive to intracellular iron concentration. However, the IL-4-mediated IL-13 secretion does not correlate with proliferation and is partially resistant to potent iron chelation.     

Polyamines are absorbed through a y+ amino acid carrier in rat intestinal epithelial cells

Summary. Due to the similarity in transport characteristics of polyamines and the y⁺ basic amino acid system, we hypothesized that both substrates could be moving through a common carrier site. Competitive and cross inhibition experiments in intestinal epithelial cells revealed the possibility of a common transport site. N-ethylmalemide (NEM) inhibited both lysine and putrescine transport, confirming that both were carried by a y⁺ transporter. Overexpressing the y⁺ transporter CAT-1 in a polyamine transport-deficient cell line, CHO-MG, did not reconstitute polyamine-transport. Thus, polyamines are not traveling through CAT-1. To determine if lysine is carried by a polyamine transport site, an antizyme-overexpressing cell line was used. Antizyme overexpression decreased polyamine uptake by 50%; in contrast, lysine transport was unaffected. Therefore, lysine is not traveling through a polyamine transport site. It appears that polyamines and lysine are likely traveling through a common unknown y⁺ transport site.     

Quantitative interdependence of coeffectors, CcpA and cre in carbon catabolite regulation of Bacillus subtilis

The phosphoproteins HPrSerP and CrhP are the main effectors for CcpA-mediated carbon catabolite regulation (CCR) in Bacillus subtilis. Complexes of CcpA with HPrSerP or CrhP regulate genes by binding to the catabolite responsive elements (cre). We present a quantitative analysis of HPrSerP and CrhP interaction with CcpA by surface plasmon resonance (SPR) revealing small and similar equilibrium constants of 4.8 +/- 0.4 microm for HPrSerP-CcpA and 19.1 +/- 2.5 microm for CrhP-CcpA complex dissociation. Forty millimolar fructose-1,6-bisphosphate (FBP) or glucose-6-phosphate (Glc6-P) increases the affinity of HPrSerP to CcpA at least twofold, but have no effect on CrhP-CcpA binding. Saturation of binding of CcpA to cre as studied by fluorescence and SPR is dependent on 50 microm of HPrSerP or > 200 microm CrhP. The rate constants of HPrSerP-CcpA-cre complex formation are k(a) = 3 +/- 1 x 10(6) m(-1).s(-1) and k(d) = 2.0 +/- 0.4 x 10(-3).s(-1), resulting in a K(D) of 0.6 +/- 0.3 nm. FBP and Glc6-P stimulate CcpA-HPrSerP but not CcpA-CrhP binding to cre. Maximal HPrSerP-CcpA-cre complex formation in the presence of 10 mm FBP requires about 10-fold less HPrSerP. These data suggest a specific role for FBP and Glc6-P in enhancing only HPrSerP-mediated CCR.     

Structural analysis of hyperperiodic DNA from Caenorhabditis elegans

Several bioinformatics studies have identified an unexpected but remarkably prevalent ~10 bp periodicity of AA/TT dinucleotides (hyperperiodicity) in certain regions of the Caenorhabditis elegans genome. Although the relevant C.elegans DNA segments share certain sequence characteristics with bent DNAs from other sources (e.g. trypanosome mitochondria), the nematode sequences exhibit a much more extensive and defined hyperperiodicity. Given the presence of hyperperiodic structures in a number of critical C.elegans genes, the physical characteristics of hyperperiodic DNA are of considerable interest. In this work, we demonstrate that several hyperperiodic DNA segments from C.elegans exhibit structural anomalies using high-resolution atomic force microscopy (AFM) and gel electrophoresis. Our quantitative analysis of AFM images reveals that hyperperiodic DNA adopts a significantly smaller mean square end-to-end distance, hence a more compact coil structure, compared with non-periodic DNA of similar length. While molecules remain capable of adopting both bent and straight (rod-like) configurations, indicating that their flexibility is still retained, examination of the local curvatures along the DNA contour length reveals that the decreased mean square end-to-end distance can be attributed to the presence of long-scale intrinsic bending in hyperperiodic DNA. Such bending is not detected in non-periodic DNA. Similar studies of shorter, nucleosome-length DNAs that survived micrococcal nuclease digestion show that sequence hyperperiodicity in short segments can likewise induce strong intrinsic bending. It appears, therefore, that regions of the C.elegans genome display a significant correlation between DNA sequence and unusual mechanical properties.     

Modifying oocytes and embryos to improve their cryopreservation

Numerous studies indicate that in vitro-produced bovine embryos do not survive cryopreservation as well as those produced in vivo. Furthermore, embryos cultured in vitro in the absence of blood serum are more cryotolerant than embryos cultured in media containing serum. Although in vivo-produced embryos are more cryotolerant, there appear to be breed differences. Most if not all of these observations are correlated with cytoplasmic lipid content of embryos; more and larger lipid droplets are associated with reduced cryotolerance. This review concerns strategies for modifying oocytes and embryos to increase cryosurvival. Reduction of cytoplasmic lipid content of embryos with phenazine ethosulfate (PES), a compound that oxidizes NADPH, even improved cryotolerance of bovine embryos cultured in the absence of serum. Whether cytoplasmic lipid content per se or associated changes in lipid composition of cell membranes are responsible for differences in cryotolerance is unknown. Increasing cholesterol content of membranes of sperm and oocytes via cholesterol-loaded cyclodextrin also appears to improve cryotolerance. While lipids have been emphasized and appear to be important, non-lipid aspects of cell composition also likely affect cryotolerance, and might be modified to improve cryotolerance. Additional research on mechanisms of variation in cryotolerance will be applicable to circumvent cryo-intolerance attributable to variation associated with the individual animal, breed, species, cell type, and factors such as nutrition and season of the year.     

Vitrification of bovine oocytes after treatment with cholesterol-loaded methyl-beta-cyclodextrin

A major site of cryoinjury during cryopreservation of mammalian oocytes is the plasma membrane. Chilling can irreversibly damage plasma membrane integrity during the lipid phase transition that occurs upon cooling. Membranes containing higher cholesterol concentrations are more fluid at lower temperatures and therefore less sensitive to cooling. The purpose of this study was to determine if cryosurvival of vitrified oocytes could be improved by incubation with cholesterol-loaded methyl-beta-cyclodextrin (CLC) prior to vitrification in the presence or absence of fetal calf serum (FCS), and if cholesterol could enter oocytes through cumulus cells and the zona pellucida. Cumulus-enclosed oocytes incubated with various concentrations (0, 0.75 or 1.5 mg/mL) of CLC in the presence of FCS for 25-45 min prior to vitrification did not result in different rates of development after warming of vitrified oocytes, followed by in vitro fertilization. However, there was an increase (P<0.05) in cleavage and number of eight-cell embryos from oocytes preincubated for 1h with 2mg/mL CLC in a chemically defined system and then handled and vitrified in chemically defined media, in comparison to those not exposed to CLC prior to vitrification or to those handled and vitrified in the presence of FCS (55, 41 and 38% eight-cell embryos, respectively). Fluorescence was seen in cumulus-oocyte complexes (COCs) previously exposed to CLC containing cholesterol labeled with a fluorescent dye; fluorescence was also seen in oocytes after removal of the cumulus cells. Oocytes not exposed to the labeled cholesterol did not fluoresce. Cholesterol from CLC readily entered cumulus cells and oocytes and improved survival in chemically defined vitrification systems.     

Embryo production from superovulated cattle following insemination of sexed sperm

Two trials were conducted to ascertain fertilization rate, embryo quality and numbers of transferable embryos in superovulated heifers and cows inseminated with sexed sperm. Inseminates contained 2 x 10(6), 10 x 10(6) or 20 x 10(6) total sperm enriched for the X- or Y-chromosome ( approximately 90%) by flow cytometry/cell sorting. Non-sexed inseminates contained 40 x 10(6) total sperm. Donors in each trial were allocated to one of each of the bulls included in that study. Each donor was inseminated with frozen/thawed sperm from the same bull for each treatment in successive courses of superstimulation with twice daily i.m. injections of FSH for 4 d. Heifers and cows were inseminated 12 and 24 h after visually observed standing estrus in Trial 1. In Trial 2, a single timed inseminate was used 70-72 h following PGF(2alpha). Ova/embryos were collected non-surgically 7-7.5 d after insemination. In both trials, fewer ova were fertilized with sexed versus non-sexed treatments and with 2 x 10(6) sexed sperm compared to higher doses (P < 0.05). However, insemination of 20 x 10(6) total sexed sperm of >or=90% purity resulted in similar numbers of transferable embryos of the desired sex compared to that for non-sexed sperm.     

Identification of a novel arabinofuranosyltransferase (AftA) involved in cell wall arabinan biosynthesis in Mycobacterium tuberculosis

The cell wall mycolyl-arabinogalactan-peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis, and is the target of several anti-tubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB. Following a detailed bioinformatics analysis of genes surrounding the conserved emb locus, we present the identification and characterization of a novel arabinofuranosyltransferase AftA (Rv3792). The enzyme catalyzes the addition of the first key arabinofuranosyl residue from the sugar donor beta-D-arabinofuranosyl-1-monophosphoryldecaprenol to the galactan domain of the cell wall, thus "priming" the galactan for further elaboration by the arabinofuranosyltransferases. Because aftA is an essential gene in M. tuberculosis, we deleted its orthologue in Corynebacterium glutamicum to produce a slow growing but viable mutant. Analysis of its cell wall revealed the complete absence of arabinose resulting in a truncated cell wall structure possessing only a galactan core with a concomitant loss of cell wall-bound mycolates. Complementation of the mutant was fully restored to the wild type phenotype by Cg-aftA. In addition, by developing an in vitro assay using recombinant Escherichia coli expressing Mt-aftA and use of cell wall galactan as an acceptor, we demonstrated the transfer of arabinose from beta-D-arabinofuranosyl-1-monophosphoryldecaprenol to galactan, and unlike the Mt-Emb proteins, Mt-AftA was not inhibited by ethambutol. This newly discovered glycosyltransferase represents an attractive drug target for further exploitation by chemotherapeutic intervention.