Substrate specificity of the Plasmodium falciparum glycosylphosphatidylinositol biosynthetic pathway and inhibition by species-specific suicide substrates

The substrate specificities of the early glycosylphosphatidylinositol biosynthetic enzymes of Plasmodium were determined using substrate analogues of D-GlcN(alpha)1-6-D-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol (GlcN-PI). Similarities between the Plasmodium and mammalian (HeLa) enzymes were observed. These are as follows: (i) The presence and orientation of the 2'-acetamido/amino and 3'-OH groups are essential for substrate recognition for the de-N-acetylase, inositol acyltransferase, and first mannosyltransferase enzymes. (ii) The 6'-OH group of the GlcN is dispensable for the de-N-acetylase, inositol acyltransferase, all four of the mannosyltransferases, and the ethanolamine phosphate transferase. (iii) The 4'-OH group of GlcNAc is not required for recognition, but substitution interferes with binding to the de-N-acetylase. The 4'-OH group of GlcN is essential for the inositol acyltransferase and first mannosyltransferase. (iv) The carbonyl group of the natural 2-O-hexadecanyl ester of GlcN-(acyl)PI is essential for substrate recognition by the first mannosyltransferase. However, several differences were also discovered: (i) Plasmodium-specific inhibition of the inositol acyltransferase was detected with GlcN-[L]-PI, while GlcN-(2-O-alkyl)PI weakly inhibited the first mannosyltransferase in a competitive manner. (ii) The Plasmodium de-N-acetylase can act on analogues containing N-benzoyl, GalNAc, or betaGlcNAc whereas the human enzyme cannot. Using the parasite specificity of the later two analogues with the known nonspecific de-N-acetylase suicide inhibitor [Smith, T. K., et al. (2001) EMBO J. 20, 3322-3332], GalNCONH(2)-PI and GlcNCONH(2)-beta-PI were designed and found to be potent (IC(50) approximately 0.2 microM), Plasmodium-specific suicide substrate inhibitors. These inhibitors could be potential lead compounds for the development of antimalaria drugs.     

Long-term recovery of macroinvertebrate biota in grossly polluted streams: Re-colonisation as a constraint to ecological quality

To meet targets imposed by the European Water Framework Directive (2000/60/EC) it is vital that measures to improve the status of rivers are both effective and economically viable. Achievement of such aims needs robust understanding of biological responses to changes in water quality vis-à-vis mechanisms of and constraints to the colonization of previously polluted sites. This study therefore examined the long-term chemical and biological changes in historically polluted rivers to elucidate the responses of macroinvertebrate biota to improvements in chemical water quality. For three historically polluted sites in the English Midlands, data from surveys over a period of ca. 50 years were analysed. Ammonia (NH₃) and 5-day biochemical oxygen demand (BOD₅) were used as chemical water quality indicators. Variations in the ecological recovery of the study sites were assessed using an average pollution sensitivity score (Average Score Per Taxon) and the number of taxa present (usually to family level) present in hand-net samples. Ecological recovery varied widely and was influenced by the intensity and spatial extent of the pollution and the proximity of available sources of potential colonisers. At the site most isolated from potential sources of colonizing taxa, no clean-water macroinvertebrate taxa were recorded 30 years after the major sources of pollution ceased. Where clean-water colonisers were more readily available, significant improvements in ecological quality followed within 2–5 years of the improvements in chemical quality. Macroinvertebrate communities and hence monitoring data may thus be indicative of long past conditions or of biological isolation rather than contemporaneous chemical conditions. Combined chemical and biological data were used to explore a generic model for predicting recovery rates and success. Neither BOD₅ nor NH₃ were found to provide a consistent and meaningful prediction of either average pollution tolerance of macroinvertebrate taxa or of the number of taxa present. Long-term relationships between macroinvertebrate variables and chemical water quality variables, however, were non-linear, suggesting that water quality thresholds may have to be exceeded before biological recovery can occur. Even when chemical water quality has been improved substantially, the apparent ecological status of macroinvertebrate communities may not reflect reduced pollution levels attained until adequate time to allow for re-colonisation (possibly decades) has elapsed.     

Field evaluation of a DNA hybridization assay for nuclear polyhedrosis virus in gypsy moth (Lepidoptera: Lymantriidae) larvae.

DNA hybridization assays were used to detect the presence of viral DNA in gypsy moth (Lymantria dispar L.) larvae collected weekly from high density populations or reared from field-collected egg masses. DNA was extracted from larvae, bound to nitrocellulose filters, and hybridized with digoxigenin-labeled L. dispar NPV (LdNPV) DNA probes. The virus incidence determined from DNA hybridization assays was compared with that determined with conventional microscopic examination of larvae for polyhedral inclusion bodies. Among neonates reared from field-collected egg masses, average mortality from LdNPV (15.4%) within 10 d after hatch was not significantly different from the percentage of extracts containing LdNPV DNA (14.8%) found among larvae frozen 5 d after hatch before any mortality occurred. Field-collected larvae were split into two groups: half were frozen immediately and probed for LdNPV DNA and the other half were reared on artificial diet. The proportion containing LdNPV DNA closely approximated the proportion that died within 6 d of collection, but the proportion that died within 13 d of collection was underestimated.     

The extracellular matrix of lip wounds in fetal, neonatal and adult mice.

Wound healing in the fetus occurs rapidly, by a regenerative process and without an inflammatory response, resulting in complete restitution of normal tissue function. By contrast, in the adult, wounds heal with scar formation, which may impair function and inhibit further growth. The cellular mechanisms underlying these differing forms of wound healing are unknown but the extracellular matrix (ECM), through its effects on cell function, may play a key role. We have studied the ECM in upper lip wounds of adult, neonatal and fetal mice at days 14, 16 and 18 of gestation. The spatial and temporal distribution of collagen types I, III, IV, V and VI, fibronectin, tenascin, laminin, chondroitin and heparan sulphates were examined immunohistochemically. Results from the fetal groups were essentially similar whilst there were distinct differences between fetus, neonate and adult. Fibronectin was present at the surface of the wound in all groups at 1 h post-wounding. Tenascin was also present at the wound surface but the time at which it was first present differed between fetus (1 h), neonate (12 h) and adult (24 h). The time of first appearance paralleled the rate of wound healing which was most rapid in the fetus and slowest in the adult. Tenascin inhibits the cell adhesion effect of fibronectin and during development the appearance of tenascin correlates with the initiation of cell migration. During wound healing the appearance of tenascin preceded cell migration and the rapid closure of fetal wounds may be due to the early appearance of tenascin in the wound. Collagen types I, III, IV, V and VI were present in all three wound groups but the timing and pattern of collagen deposition differed, with restoration of the normal collagen pattern in the fetus and a scar pattern in the adult. This confirms that lack of scarring in fetal wounds is due to the organisation of collagen within the wound and not simply lack of collagen formation. The distribution of chondroitin sulphate differed between normal fetal and adult tissues and between fetal and adult wounds. Its presence in the fetal wound may alter collagen fibril formation. No inflammatory response was seen in the fetal wounds. The differences in the ECM of fetal and adult wounds suggests that it may be possible to alter the adult wound so that it heals by a fetal-like process without scar formation, loss of tissue function or restriction of growth.     

Evaluation of the cytoprotective effects of bovine lactoferrin against intestinal toxins using cellular model systems

This study investigated the effects of bovine lactoferrin (BLf) on the growth of different groups of bacteria in vitro. BLf showed a significant inhibitory effect on the growth of selected pathogens but not probiotics. BLf, in combination with probiotics, has the potential to influence the composition of the gut microflora via inhibition of intestinal pathogens with no significant effect on probiotic bacteria.     

The Nuclease Activity of Mre11 Is Required for Meiosis but Not for Mating Type Switching, End Joining, or Telomere Maintenance

The Saccharomyces cerevisiae MRE11 gene is required for the repair of ionizing radiation-induced DNA damage and for the initiation of meiotic recombination. Sequence analysis has revealed homology between Mre11 and SbcD, the catalytic subunit of an Escherichia coli enzyme with endo- and exonuclease activity, SbcCD. In this study, the purified Mre11 protein was found to have single-stranded endonuclease activity. This activity was absent from mutant proteins containing single amino acid substitutions in either one of two sequence motifs that are shared by Mre11 and SbcD. Mutants with allele mre11-D56N or mre11-H125N were partially sensitive to ionizing radiation but lacked the other mitotic phenotypes of poor vegetative growth, hyperrecombination, defective nonhomologous end joining, and shortened telomeres that are characteristic of the mre11 null mutant. Diploids homozygous for the mre11-H125N mutation failed to sporulate and accumulated unresected double-strand breaks (DSB) during meiosis. We propose that in mitotic cells DSBs can be processed by other nucleases that are partially redundant with Mre11, but these activities are unable to process Spo11-bound DSBs in meiotic cells.     

Mechanisms by which resistant starches and non-starch polysaccharide sources affect the metabolism and disposition of the food carcinogen, 2-amino-3-methylimidazo[4,5-f]quinoline

Although both non-starch polysaccharides (NSP) and resistant starches (RS) are included in current definitions of dietary fibre, our previous work has suggested fundamental differences in the way in which these two classes of material affect the disposition and absorption of a dietary carcinogen. The present studies explore whether different effects on carcinogen metabolism could play a role in the contrasting patterns seen previously. Groups of female Wistar rats were pre-fed for 4 weeks one of five types of defined diet (AIN-76). The control diet contained 35% maize starch and no dietary fibre. The RS-containing diets had all the maize starch substituted with either Hi-maize or potato starch. In the NSP-containing diets, 10% of the maize starch was substituted with dietary fibre in the form of either lignified plant cell walls (wheat straw) or soluble dietary fibre (apple pectin). Pre-fed rats were gavaged with the food carcinogen, [2-14C] 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and plasma and urinary metabolites characterized using HPLC at various time intervals after administration. After 4 h gavage, plasma from rats on both RS-containing diets contained significantly higher levels of intact IQ and lower levels of the major metabolites, IQ-5-O-glucuronide and IQ-5-sulfate, as compared with plasma from the negative control group at this time. In contrast, plasma from animals on the NSP-containing wheat straw diet (and to a lesser extent the apple pectin diet) showed significantly lower levels of intact IQ, and significantly higher levels of the two major metabolites, as compared with those from the control rats. These different metabolite profiles were also reflected in different urinary excretion profiles. Urine from rats pre-fed RS-containing diets revealed significantly slower metabolite excretion as compared with urine from rats that had been given the NSP-containing diets. Western blotting methodologies also profiled differences between the effects of these two types of dietary fibre in the expression of xenobiotic metabolizing enzymes. We conclude that changes in activity and expression of xenobiotic metabolising enzymes could play a role in the contrasting effects of these two types of dietary fibre on carcinogen uptake and disposition.     

Comparison of GNRH3 genes across salmonid genera

Genomic sequences of gonadotropin-releasing hormone genes were amplified and examined for sequence divergence among members of three different genera of the subfamily Salmoninae: rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), and Arctic charr (Salvelinus alpinus). Sequences of GNRH3A and GNRH3B (formerly known as sGnRH1 and sGnRH2) were 97-99% similar in coding regions and 94-98% similar in non-coding regions among genera, but comparisons within species between GNRH3A and GNRH3B were only 90-92% similar in coding regions and 83-89% similar in non-coding regions. Polymorphisms in the parents of mapping families for each species allowed for linkage mapping of the GNRH3B gene in all three species and the GNRH3A gene in rainbow trout. GNRH3B maps to linkage group 6 in rainbow trout, linkage group 16 in Atlantic salmon and linkage group 25 in Arctic charr. GNRH3A mapped to linkage group 30 in rainbow trout.     

Cytochrome cd1 Nitrite Reductase NirS Is Involved in Anaerobic Magnetite Biomineralization in Magnetospirillum gryphiswaldense and Requires NirN for Proper d1 Heme Assembly

The alphaproteobacterium Magnetospirillum gryphiswaldense synthesizes magnetosomes, which are membrane-enveloped crystals of magnetite. Here we show that nitrite reduction is involved in redox control during anaerobic biomineralization of the mixed-valence iron oxide magnetite. The cytochrome cd₁-type nitrite reductase NirS shares conspicuous sequence similarity with NirN, which is also encoded within a larger nir cluster. Deletion of any one of these two nir genes resulted in impaired growth and smaller, fewer, and aberrantly shaped magnetite crystals during nitrate reduction. However, whereas nitrite reduction was completely abolished in the ΔnirS mutant, attenuated but significant nitrite reduction occurred in the ΔnirN mutant, indicating that only NirS is a nitrite reductase in M. gryphiswaldense. However, the ΔnirN mutant produced a different form of periplasmic d₁ heme that was not noncovalently bound to NirS, indicating that NirN is required for full reductase activity by maintaining a proper form of d₁ heme for holo-cytochrome cd₁ assembly. In conclusion, we assign for the first time a physiological function to NirN and demonstrate that effective nitrite reduction is required for biomineralization of wild-type crystals, probably by contributing to oxidation of ferrous iron under oxygen-limited conditions.