Distribution of mutations in human thymidylate synthase yielding resistance to 5-fluorodeoxyuridine

Thymidylate synthase (TS) catalyzes methylation of dUMP to dTMP and is the target of cancer chemotherapeutic agents (e.g. 5-fluorouracil). Here, we used error-prone PCR to mutagenize the full-length human TS cDNA and then selected mutants resistant to 5-fluorodeoxyuridine in a bacterial complementation system. We found that resistant mutants contained 1-5 amino acid substitutions and that these substitutions were located along the entire length of the polypeptide. Mutations were frequent near the active site Cys(195) and in the catalytically important Arg(50) loop; however, many mutations were also distributed throughout the remainder of the cDNA. Mutants containing a single amino acid replacement identified the following 14 residues as unreported sites of resistance: Glu(23), Thr(51), Thr(53), Val(84), Lys(93), Asp(110), Asp(116), Pro(194), Ser(206), Met(219), His(250), Asp(254), Tyr(258), and Lys(284). Many of these residues are distant from the active site and/or have no documented function in catalysis or resistance. We conclude that mutations distributed throughout the linear sequence and three-dimensional structure of human TS can confer resistance to 5-fluorodeoxyuridine. Our findings imply that long range interactions within proteins affect catalysis at the active site and that mutations at a distance can yield variant proteins with desired properties.     

Formation of carnosine-Cu(II) complexes prevents and reverts the inhibitory action of copper in P2X4 and P2X7 receptors

To further analyze the action of copper on brain synaptic mechanisms, the brain dipeptide carnosine (beta-alanyl-L-histidine) was tested in Xenopus laevis oocytes expressing the rat P2X4 or P2X7 receptors. Ten micromolar copper halved the currents evoked by ATP in both receptors; co-application of carnosine plus copper prevented the metal induced-inhibition with a median effective concentration of 12.1 +/- 3.9 and 12.0 +/- 5.5 microm for P2X4 and P2X7, respectively. Zinc potentiated only the P2X4 ATP-evoked currents; carnosine had no effect over this metal. The relative potency and selectivity of classical metal chelators to prevent the copper inhibition was compared between carnosine and penicillamine (PA), bathophenanthroline (BPh) or L-histidine (His). Their rank order of potency in P2X4 and P2X7 receptors was carnosine = PA = His > BPh > Glycine (Gly) and carnosine = BPh = His > PA > Gly, respectively. The potency to prevent the zinc-induced potentiation in the P2X4 receptor was BPh > PA > His; carnosine, Gly and beta-alanine were inactive. Whereas 1-100 microm carnosine or His alone did not modify the ATP-evoked currents, 10-100 microm PA augmented and 100 microm BPh decreased the ATP-evoked currents. Carnosine was able to revert the copper-induced inhibition restoring the maximal ATP gated current in a concentration-dependent manner. Electronic spectroscopy confirm the formation of carnosine-Cu(II) complexes, mechanism that can account for the prevention and reversal of the copper inhibition, revealing its potential in copper intoxication treatment.     

Evidence consistent with human L1 retrotransposition in maternal meiosis I

We have used a unique polymorphic 3' transduction to show that a human L1, or LINE-1 (long interspersed nucleotide element-1), retrotransposition event most likely occurred in the maternal primary oocyte during meiosis I. We characterized a truncated L1 retrotransposon with a 3' transduction that was inserted, in a Dutch male patient, into the X-linked gene CYBB, thereby causing chronic granulomatous disease. We used the unique flanking sequence to localize the precursor L1 locus, LRE3, to chromosome 2q24.1. In a cell culture assay, the retrotransposition frequency of LRE3 is greater than that for any other element that has been tested to date. The patient's mother had two LRE3 alleles that differed slightly in the 3'-flanking genomic DNA. The patient had a single LRE3 allele that was identical to one of the maternal alleles; however, the patient's insertion matched the maternal LRE3 allele that he did not inherit. Other data indicate that there is only a small chance that the father (unavailable for analysis) carries the precursor LRE3 allele. In addition, paternal origin of the insertion would have required that an LRE3 mRNA transcribed before meiosis II be carried separately from its precursor LRE3 allele in the fertilizing sperm. Since the mother carries a potential precursor allele and the insertion was on the patient's maternal X chromosome, it is highly likely that the insertion originated during maternal meiosis I.     

Elicitor-activated phospholipase A(2) generates lysophosphatidylcholines that mobilize the vacuolar H(+) pool for pH signaling via the activation of Na(+)-dependent proton fluxes

The elicitation of phytoalexin biosynthesis in cultured cells of California poppy involves a shift of cytoplasmic pH via the transient efflux of vacuolar protons. Intracellular effectors of vacuolar proton transport were identified by a novel in situ approach based on the selective permeabilization of the plasma membrane for molecules of < or = 10 kD. Subsequent fluorescence imaging of the vacuolar pH correctly reported experimental changes of activity of the tonoplast proton transporters. Lysophosphatidylcholine (LPC) caused a transient increase of the vacuolar pH by increasing the Na(+) sensitivity of a Na(+)-dependent proton efflux that was inhibited by amiloride. In intact cells, yeast elicitor activated phospholipase A(2), as demonstrated by the formation of LPC from fluorescent substrate analogs, and caused a transient increase of endogenous LPC, as determined by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. It is suggested that LPC generated by phospholipase A(2) at the plasma membrane transduces the elicitor-triggered signal into the activation of a tonoplast H(+)/Na(+) antiporter.     

Broad and complex antifungal activity among environmental isolates of lactic acid bacteria

More than 1200 isolates of lactic acid bacteria isolated from different environments were screened for antifungal activity in a dual-culture agar plate assay. Approximately 10% of the isolates showed inhibitory activity and 4% showed strong activity against the indicator mould Aspergillus fumigatus. The antifungal spectra for 37 isolates with strong activity and five isolates with low or no activity were determined. Several of the strains showed strong inhibitory activity against the moulds A. fumigatus, Aspergillus nidulans, Penicillium commune and Fusarium sporotrichioides, and also against the yeast Rhodotorula mucilaginosa. Penicillium roqueforti and the yeasts Pichia anomala and Kluyveromyces marxianus were not inhibited. Several isolates showed reduced antifungal activity after storage and handling. The majority of the fungal inhibitory isolates were identified by 16S rDNA sequencing as Lactobacillus coryniformis. Lactobacillus plantarum and Pediococcus pentosaceus were also frequently identified among the active isolates. The degree of fungal inhibition was not only related to production of lactic or acetic acid. In addition, antifungal cyclic dipeptides were identified after HPLC separation and several other active fractions were found suggesting a highly complex nature of the antifungal activity.     

Stem cells from midguts of Lepidopteran larvae: clues to the regulation of stem cell fate

Previously, we showed that isolated stem cells from midguts of Heliothis virescens can be induced to multiply in response to a multiplication protein (MP) isolated from pupal fat body, or to differentiate to larval types of mature midgut cells in response to either of 4 differentiation factors (MDFs) isolated from larval midgut cell-conditioned medium or pupal hemolymph. In this work, we show that the responses to MDF-2 and MP in H. virescens stem cells decayed at different time intervals, implying that the receptors or response cascades for stem cell differentiation and multiplication may be different. However, the processes appeared to be linked, since conditioned medium and MDF-2 prevented the action of MP on stem cells; MP by itself appeared to repress stem cell differentiation. Epidermal growth factor, retinoic acid, and platelet-derived growth factor induced isolated midgut stem cells of H. virescens and Lymantria dispar to multiply and to differentiate to mature midgut cells characteristic of prepupal, pupal, and adult lepidopteran midgut epithelium, and to squamous-like cells and scales not characteristic of midgut tissue instead of the larval types of mature midgut epithelium induced by the MDFs. Midgut stem cells appear to be multipotent and their various differentiated fates can be influenced by several growth factors.     

Actin filament polymerization regulates gliding motility by apicomplexan parasites

Host cell entry by Toxoplasma gondii depends critically on actin filaments in the parasite, yet paradoxically, its actin is almost exclusively monomeric. In contrast to the absence of stable filaments in conventional samples, rapid-freeze electron microscopy revealed that actin filaments were formed beneath the plasma membrane of gliding parasites. To investigate the role of actin filaments in motility, we treated parasites with the filament-stabilizing drug jasplakinolide (JAS) and monitored the distribution of actin in live and fixed cells using yellow fluorescent protein (YFP)-actin. JAS treatment caused YFP-actin to redistribute to the apical and posterior ends, where filaments formed a spiral pattern subtending the plasma membrane. Although previous studies have suggested that JAS induces rigor, videomicroscopy demonstrated that JAS treatment increased the rate of parasite gliding by approximately threefold, indicating that filaments are rate limiting for motility. However, JAS also frequently reversed the normal direction of motility, disrupting forward migration and cell entry. Consistent with this alteration, subcortical filaments in JAS-treated parasites occurred in tangled plaques as opposed to the straight, roughly parallel orientation observed in control cells. These studies reveal that precisely controlled polymerization of actin filaments imparts the correct timing, duration, and directionality of gliding motility in the Apicomplexa.     

Synaptojanin is recruited by endophilin to promote synaptic vesicle uncoating

We describe the isolation and characterization of Drosophila synaptojanin (synj) mutants. synj encodes a phosphatidylinositol phosphatase involved in clathrin-mediated endocytosis. We show that Synj is specifically localized to presynaptic terminals and is associated with synaptic vesicles. The electrophysiological and ultrastructural defects observed in synj mutants are strikingly similar to those found in endophilin mutants, and Synj and Endo colocalize and interact biochemically. Moreover, synj; endo double mutant synaptic terminals exhibit properties that are very similar to terminals of each single mutant, and overexpression of Endophilin can partially rescue the functional defects in partial loss-of-function synj mutants. Interestingly, Synj is mislocalized and destabilized at synapses devoid of Endophilin, suggesting that Endophilin recruits and stabilizes Synj on newly formed vesicles to promote vesicle uncoating. Our data also provide further evidence that kiss-and-run is able to maintain neurotransmitter release when synapses are not extensively challenged.