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M A Ferguson

Publications and source records attributed to M A Ferguson.

At least 55 records · Page 3Linked to original sources
PubMed1999

A novel glycosylphosphatidylinositol in African trypanosomes. A possible catabolic intermediate.

The major glycosylphosphatidylinositols (GPIs) in African trypanosomes are glycolipid A, the precursor of the variant surface glycoprotein membrane anchor, and glycolipid C, a species identical to glycolipid A except that it contains an acylated inositol. Both glycolipids A and C contain dimyristoyl glycerol and are efficiently labeled with [3H]myristate in a cell-free system. We now report a novel GPI known as lipid X. This GPI is radiolabeled strongly with [3H]palmitate (and very poorly with [3H]myristate or [3H]stearate) in digitonin-permeabilized cells. The structure of lipid X is Man1GlcNAc-(2O-palmitoyl)-D-myo-inositol-1-HPO4-3(lyso-pa lmitoylglyce rol). Metabolically, lipid X exists as an intermediate, and can be detected only under conditions in which its formation is stimulated (e.g. by EDTA) or its breakdown is inhibited (e.g. by Co2+). Lipid X has not been observed previously because these conditions do not support GPI biosynthesis. We speculate that lipid X is an intermediate in the catabolism of conventional trypanosome GPIs, possibly deriving from breakdown of glycolipid C.

Acyl Coenzyme A↗
PubMed1999

Regulation of macrophage IL-12 synthesis by Leishmania phosphoglycans.

It is now generally accepted that IFN-gamma, secreted by Th1 cells, is the most potent cytokine leading to macrophage activation and host resistance against infection with the intracellular protozoan parasite Leishmania. It is also established that IL-12 is a critical cytokine involved in the differentiation and expansion of Th1 cells. Therefore, the ability of Leishmania parasites to actively suppress IL-12 production by host macrophages may be an important strategy for parasite survival. Here we report that a major parasite cell surface molecule, phosphoglycan (PG), of Leishmania could selectively inhibit the synthesis of IL-12(p40, p70) by activated murine macrophages. Furthermore, synthetic PG (sPG) was able to inhibit IL-12 release in a dose-dependent manner. Inhibition was dependent on the galactose(beta1-4)mannose(alpha1)-PO4 repeating units and not the glycophosphoinositol lipid anchor of lipophosphoglycan. At the concentration used, sPG had no effect on the release of TNF-alpha or IL-6 in activated macrophages. The inhibition of IL-12(p40) production was at the transcriptional level, but was not mediated through NF kappaB inhibition. These data demonstrate that PG may be an important molecule for the establishment and survival of the parasite in permissive hosts.

Animals↗
PubMed1999

Life-cycle and biology of Sychnocotyle kholo n.g., n. sp. (Trematoda : Aspidogastrea) in Emydura macquarii (Pleurodira : Chelidae) from southern Queensland, Australia.

Sychnocotyle kholo n. g., n. sp. (Aspidogastrea: Aspidogastridae) is described from the small intestine of the freshwater turtle Emydura macquarii (Pleurodira: Chelidae). The new genus is distinguished from other aspidogastrids by the possession of the following suite of characters: no cirrus-sac; no hermaphroditic duct; four rows of alveoli on the ventral disc but no prominent papillae; Laurer's canal opening to the exterior. Eggs hatched to cotylocidia within 37-41 days at 17-24 degrees C. The life-cycle is obligate two-host, involving a mollusc and a freshwater turtle. Juvenile forms, almost entirely single worm infections, were found in the molluscs Corbiculina sp. (Bivalvia: Corbiculidae) and Thiara balonnensis (Prosobranchia: Thiaridae). Sychnocotyle is most likely to have evolved in Australia after the separation of Gondwanaland from Laurasia 120- 100 mya.

Animals↗
PubMed1999

The preparation of neoglycoconjugates containing inter-saccharide phosphodiester linkages as potential anti-Leishmania vaccines.

The Leishmania express complex glycoconjugates containing phosphosaccharide repeat units at all stages of their life-cycle. One of these molecules, lipophosphoglycan (LPG) has been suggested to be a vaccine candidate. To assess the immunological properties of Leishmania phosphosaccharides, we have prepared neoglycoproteins and neoglycolipids containing synthetic Leishmania phosphosaccharide repeats. The coupling procedure uses the dec-9-enyl spacer of previously synthesised phosphosaccharides for linkage to protein and phospholipid. This alkene moiety is converted by ozonolysis to an aldehyde which is then attached to protein and phospholipid amino groups by reductive amination. The procedure produces neoglycoconjugates in good yield and without compromising the labile phosphodiester linkages within the phosphosaccharide chains.

Animals↗
PubMed1999

Differential inhibitory mechanism of cyclic AMP on TNF-alpha and IL-12 synthesis by macrophages exposed to microbial stimuli.

Microbial stimuli such as bacterial lipopolysaccharide (LPS) or glycosylphosphatidylinositol-mucins derived from Trypanosoma cruzi trypomastigotes (tGPI-mucins) are effective stimulators of the synthesis of cytokines by macrophages. Here, we evaluated the ability of cyclic AMP mimetic or elevating agents to modulate TNF-alpha and IL-12 synthesis by murine inflammatory macrophages. Cholera Toxin (ChTx) inhibited tGPI-mucins (2.5 nM) or LPS (100 ng ml(-1)) induced TNF-alpha and IL-12(p40) synthesis in a concentration-dependent manner. Similarly, the cyclic AMP mimetics, 8-bromo cyclic AMP or dibutyryl cyclic AMP, or prostaglandin (PG) E2 inhibited the synthesis of both cytokines by macrophages exposed to microbial stimuli. The protein kinase A inhibitor H-89 partially reversed the inhibitory effects of dibutyryl cyclic AMP and PGE2 on both IL-12(p40) and TNF-alpha synthesis. Pretreatment of macrophages with dibutyryl cyclic AMP or ChTx augmented the synthesis of IL-10 triggered by microbial products. Elevation of cyclic AMP inhibited the synthesis of TNF-alpha, but not IL-12(p40), by inflammatory macrophages from IL-10 knockout mice. Kinetic studies showed that synthesis of both TNF-alpha and IL-10 peaked at 8 h and IL-12 at 24 h after stimulation with microbial stimuli. Together, our findings favour the hypothesis that the cyclic AMP inhibitory activity on IL-12(p40) but not on TNF-alpha synthesis is dependent on de novo protein synthesis, most likely involving IL-10, by macrophages stimulated with microbial products. Accordingly, dibutyryl cyclic AMP inhibited IL-12(p40) synthesis only when added before or at the same time of the stimuli. In contrast, the effect of this cyclic AMP analogue on TNF-alpha synthesis was protracted and observed even 2 h after the addition of the stimuli.

Animals↗
PubMed1999

Effects of exercise training and its cessation on components of the insulin resistance syndrome in obese children.

OBJECTIVE: To determine the effect of exercise training (ET) on components of the insulin resistance syndrome (IRS) in obese children. DESIGN: Randomized, modified cross-over study, with subjects assigned to one of two conditions: (1) 4 months of ET followed by 4 months of no-ET; or (2) 4 months of no-ET followed by 4 months of ET. Measurements were made at three time points: 0, 4 and 8 months. SUBJECTS: 79 obese, but otherwise healthy children (age: 7-11 y, percent fat (%fat) 27-61%). MEASUREMENTS: Plasma lipid and lipoprotein concentrations, plasma insulin and glucose concentrations; %fat; submaximal heart rate (HR) as an index of fitness. EXERCISE TRAINING: ET was offered 5 d/week 40 min/d. For the 73 children who completed 4 months of ET, the mean attendance was 80% (that is, 4 d/week) and the average HR during ET was 157 bpm. RESULTS: Significant (P < 0.05) group x time interactions were found for plasma triglyceride (TG) and insulin concentrations and %fat. The average change for both groups, from just before ET to just after the 4 month ET was -0.24 mmol.l-1 for TG, -25.4 pmol.l-1 for insulin and -1.6 units for %fat. When Group 1 ceased ET, over the following 4 month period the average change for insulin was +26.6 pmol.l-1 and for %fat +1.3 units. CONCLUSION: Some components (plasma TG, insulin, %fat) of the IRS are improved as a result of 4 months of ET in obese children. However, the benefits of ET are lost when obese children become less active.

Analysis of Variance↗
PubMed1999

Effects of physical training and its cessation on the hemostatic system of obese children.

BACKGROUND: Physical training can improve hemostatic function in adults, thereby reducing heart disease risk, but no information is available in children on whether physical training can enhance hemostatic function. OBJECTIVE: The purpose of this investigation was to examine the effects of a physical training program on hemostatic variables in a biethnic group of obese children. DESIGN: Children were randomly assigned to 2 groups. Group 1 participated in physical training for 4 mo and then ceased physical training for 4 mo, whereas group 2 did no physical training for the first 4 mo and then participated in physical training for 4 mo. Plasma hemostatic variables [fibrinogen, plasminogen activator inhibitor 1 (PAI-1), and D-dimer) were measured at months 0, 4, and 8. RESULTS: Analyses of variance revealed no significant group-by-time interactions for the hemostatic variables. When data from both groups were combined there was a significant decrease in D-dimer after 4 mo of physical training (P < 0.05). Factors explaining individual differences in responsiveness to the physical training revealed that individuals with greater percentage fat before physical training showed greater reductions in fibrinogen and D-dimer, and that blacks showed greater reductions in D-dimer than whites (P < 0.05). Stepwise multiple linear regression showed that only higher prephysical training concentrations of fibrinogen, PAI-1, and D-dimer explained significant proportions of the variation in changes in these variables. CONCLUSIONS: In obese children, 4-mo periods of physical training did not lead to significant changes in hemostatic variables. Children with greater adiposity and concentrations of hemostatic factors before physical training showed greater reductions in hemostatic variables after physical training than did children with lesser values.

Analysis of Variance↗
PubMed1999

Differences between the trypanosomal and human GlcNAc-PI de-N-acetylases of glycosylphosphatidylinositol membrane anchor biosynthesis.

De-N-acetylation of N-acetylglucosaminyl-phosphatidylino-sitol (GlcNAc-PI) is the second step of glycosylphosphatidylino-sitol (GPI) membrane anchor biosynthesis in eukaryotes. This step is a prerequisite for the subsequent processing of glucosaminyl-phosphatidylinositol (GlcN-PI) that leads to mature GPI membrane anchor precursors, which are transferred to certain proteins in the endoplasmic reticulum. In this article, we used a direct de-N-acetylase assay, based on the release of [14C]acetate from synthetic GlcN[14C]Ac-PI and analogues thereof, and an indirect assay, based on the mannosylation of GlcNAc-PI analogues, to study the substrate specificities of the GlcNAc-PI de-N-acetylase activities of African trypanosomes and human (HeLa) cells. The HeLa enzyme was found to be more fastidious than the trypanosomal enzyme such that, unlike the trypanosomal enzyme, it was unable to act on a GlcNAc-PI analogue containing 2-O-octyl-d- myo -inositol or on the GlcNAc-PI diastereoisomer containing l- myo -inositol (GlcNAc-P(l)I). These results suggest thatselective inhibition of the trypanosomal de-N-acetylase may be possible and that this enzyme should be considered as a possible therapeutic target. The lack of strict stereospecificity of the trypanosomal de-N-acetylase for the d- myo -inositol component was also seen for the trypanosomal GPI alpha-manno-syltransferases when GlcNAc-P(l)I was added to the trypanosome cell-free system, but not when GlcN-P(l)I was used. In an attempt to rationalize these data, we modeled the structure and dynamics of d-GlcNAcalpha1-6d- myo -inositol-1-HPO4-( sn )-3-glycerol and its diastereoisomer d-GlcNAcalpha1-6l- myo -inositol-1-HPO4-( sn )-3-glycerol. These studies indicate that the latter compound visits two energy minima, one of which resembles the low-energy conformer of former compound. Thus, it is conceivable that the trypanosomal de-N-acetylase acts on GlcNAc-P(l)I when it occupies a GlcNAc-PI-likeconformation and that GlcN-P(l)I emerging from the de-N-acetylase may be channeled to the alpha-mannosyltransferases in this conformation.

Amidohydrolases↗
PubMed1999

The structure, biosynthesis and functions of glycosylphosphatidylinositol anchors, and the contributions of trypanosome research.

The discovery of glycosylphosphatidylinositol (GPI) membrane anchors has had a significant impact on several areas of eukaryote cell biology. Studies of the African trypanosome, which expresses a dense surface coat of GPI-anchored variant surface glycoprotein, have played important roles in establishing the general structure of GPI membrane anchors and in delineating the pathway of GPI biosynthesis. The major cell-surface molecules of related parasites are also rich in GPI-anchored glycoproteins and/or GPI-related glycophospholipids, and differences in substrate specificity between enzymes of trypanosomal and mammalian GPI biosynthesis may have potential for the development of anti-parasite therapies. Apart from providing stable membrane anchorage, GPI anchors have been implicated in the sequestration of GPI-anchored proteins into specialised membrane microdomains, known as lipid rafts, and in signal transduction events.

Animals↗
PubMed1998

A synthetic acceptor substrate for Trypanosoma brucei UDP-Gal: GPI anchor side-chain alpha-galactosyltransferases.

A synthetic analogue of a trisaccharide fragment of the Trypanosoma brucei Variant Surface Glycoprotein (VSG) GlycosylPhosphatidyInositiol (GPI) anchor, Gal-alpha-1,3(Man-alpha-1,6)-Man-alpha-O-octyl (1), serves as a substrate for two T. brucei alpha-galactosyltransferases. The principle tetrasaccharide product derived from (1) contains a Gal-alpha-1,2-Gal linkage.

Animals↗
PubMed1998

Transformation of monomorphic Trypanosoma brucei bloodstream form trypomastigotes into procyclic forms at 37 degrees C by removing glucose from the culture medium.

African trypanosomes have been shown previously to undergo efficient transformation from bloodstream forms to procyclic (insect dwelling) forms in vitro by adding citrate and/or cis-aconitate to the culture medium and lowering incubation temperature to 27 degrees C. In this paper, it is shown that strain 427 monomorphic bloodstream forms of Trypanosoma brucei grown in axenic culture at 37 degrees C can be transformed to procyclic forms by simply replacing the glucose carbon source in the culture medium with glycerol. The removal of glucose from the medium results in the loss of the variant surface glycoprotein, the acquisition of cell surface procyclic acidic repetitive protein, the synthesis of procyclic-specific glycosylphosphatidylinositol precursors and the acquisition of substantial resistance to salicyl hydroxamic acid and glycerol within 72 h. A procyclic-specific cytoskeletal protein, known to be a marker of the late stage of transformation, is fully expressed by 96 h but full trans-sialidase activity appears only after 18-30 days. The transformation process described here is slower and less efficient than that previously described for monomorphic trypanosomes, using citrate and/or cis-aconitate and temperature shift as triggers. However, the separation of the transformation process from these stimuli is significant and the effects of glucose deprivation described here may reflect some of the events that occur in vivo in the tsetse fly midgut, where glucose levels are known to be very low.

Animals↗
PubMed1998

Structure of the glycosylphosphatidylinositol membrane anchor glycan of a class-2 variant surface glycoprotein from Trypanosoma brucei.

The neutral glycan fraction of the glycosylphosphatidylinositol (GPI) membrane anchor of a class-2 variant surface glycoprotein (VSG) from Trypanosoma brucei was isolated following aqueous hydrogen fluoride dephosphorylation and nitrous acid deamination of the purified glycoprotein. The neutral glycans were fractionated by high-pH anion exchange chromatography and gel-filtration and six major glycan structures were solved by a combination of one and two-dimensional NMR, composition analysis, methylation linkage analysis and electrospray-mass spectrometry. The glycans were similar to those previously described for class-1 VSGs, in that they contained the linear trimannosyl sequence Manalpha1-2Manalpha1-6Man and a complex alpha-galactose branch of up to Galalpha1-2Galalpha1-6(Galalpha1-2)Gal, but most also contained an additional galactose residue attached alpha1-2 to the non-reducing terminal mannose residue and about one-third contained an additional galactose residue attached beta1-3 to the middle mannose residue. The additional complexity of the class-2 VSG GPI glycans is discussed in terms of a biosynthetic model that explains the full range of mature GPI structures that can be expressed on different VSG classes by the same trypanosome clone.

Animals↗
PubMed1998

The glycosylation of the variant surface glycoproteins and procyclic acidic repetitive proteins of Trypanosoma brucei.

Trypanosoma brucei, in common with the other African trypanosomes, exhibits unusual cell-surface molecular architecture. The bloodstream form of the parasite is coated with a continuous layer of approximately five million variant surface glycoprotein (VSG) dimers that provide the parasite with a macromolecular diffusion barrier to guard against lysis by the alternative complement pathway. The procyclic form of the parasite has a more diffuse cell-surface coat made up of approximately 2.5 million copies of procyclic acidic repetitive protein (PARP). Within the VSG and PARP coats exist lower-abundance surface glycoproteins such as receptors and nutrient transporters. Both the VSG molecules and the PARP molecules are attached to the membrane via glycosylphosphatidylinositol (GPI) membrane anchors and the VSGs and one form of PARP are N-glycosylated. In this article, the structures of the N-glycans and the GPI anchors of T. brucei VSGs and PARPs are reviewed and simple models of the surfaces of bloodstream and procyclic trypomastigotes are presented.

Animals↗
PubMed1998

The glycosyl-inositol-phosphate and dimyristoylglycerol moieties of the glycosylphosphatidylinositol anchor of the trypanosome variant-specific surface glycoprotein are distinct macrophage-activating factors.

The TNF-alpha-inducing capacity of different trypanosome components was analyzed in vitro, using as indicator cells a macrophage cell line (2C11/12) or peritoneal exudate cells from LPS-resistant C3H/HeJ mice and LPS-sensitive C3H/HeN mice. The variant-specific surface glycoprotein (VSG) was identified as the major TNF-alpha-inducing component present in trypanosome-soluble extracts. Both soluble (sVSG) and membrane-bound VSG (mfVSG) were shown to manifest similar TNF-alpha-inducing capacities, indicating that the dimyristoylglycerol (DMG) compound of the mfVSG anchor was not required for TNF-alpha triggering. Detailed analysis indicated that the glycosyl-inositol-phosphate (GIP) moiety was responsible for the TNF-alpha-inducing activity of VSG and that the presence of the GIP-associated galactose side chain was essential for optimal TNF-alpha production. Furthermore, the results showed that the responsiveness of macrophages toward the TNF-alpha-inducing activity of VSG was strictly dependent on the activation state of the macrophages, since resident macrophages required IFN-gamma preactivation to become responsive. Comparative analysis of the ability of both forms of VSG to activate macrophages revealed that mfVSG but not sVSG stimulates macrophages toward IL-1alpha secretion and acquisition of LPS responsiveness. The priming activity of mfVSG toward LPS responsiveness was also demonstrated in vivo and may be relevant during trypanosome infections, since Trypanosoma brucei-infected mice became gradually LPS-hypersensitive during the course of infection. Collectively, the VSG of trypanosomes encompasses two distinct macrophage-activating components: while the GIP moiety of sVSG mediates TNF-alpha induction, the DMG compound of the mfVSG anchor contributes to IL-1 alpha induction and LPS sensitization.

Animals↗