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Biomedical subjects

M A Ferguson

Publications and source records attributed to M A Ferguson.

At least 37 records · Page 2Linked to original sources
PubMed2000

Cloning, expression, and characterization of the acyl-CoA-binding protein in African trypanosomes.

African trypanosomes are shielded from their hosts' defenses by a coat of variant surface glycoprotein molecules, each of which is attached to the plasma membrane by a glycosylphosphatidylinositol anchor. During the later stages of glycosylphosphatidylinositol biosynthesis, myristic acid is incorporated into the anchor from the donor myristoyl-CoA by a series of unique fatty acid remodeling and exchange reactions. We have cloned and expressed a recombinant trypanosome acyl-CoA-binding protein that has a preference for binding relatively short chain acyl-CoAs and that has a high affinity for binding myristoyl-CoA (K(d) = 3.5 x 10(-10) M). This protein enhances fatty acid remodeling of glycosylphosphatidylinositol precursors in the trypanosome cell-free system. We speculate that the trypanosome acyl-CoA-binding protein plays an active role in supplying myristoyl-CoA to the fatty acid remodeling machinery in the parasite.

Acyl Coenzyme A↗
PubMed2000

Highly purified glycosylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents.

Intracellular protozoan parasites are potent stimulators of cell-mediated immunity. The induction of macrophage proinflammatory cytokines by Trypanosoma cruzi is considered to be important in controlling the infection and the outcome of Chagas' disease. Here we show that the potent tumour necrosis factor-alpha-, interleukin-12- and nitric oxide-inducing activities of T.cruzi trypomastigote mucins were recovered quantitatively in a highly purified and characterized glycosylphosphatidylinositol (GPI) anchor fraction of this material. The bioactive trypomastigote GPI fraction was compared with a relatively inactive GPI fraction prepared from T. cruzi epimastigote mucins. The trypomastigote GPI structures were found to contain additional galactose residues and unsaturated, instead of saturated, fatty acids in the sn-2 position of the alkylacylglycerolipid component. The latter feature is essential for the extreme potency of the trypomastigote GPI fraction, which is at least as active as bacterial endotoxin and Mycoplasma lipopeptide and, therefore, one of the most potent microbial proinflammatory agents known.

Amino Acid Sequence↗
PubMed2000

Isolation and characterization of glycosylphosphatidylinositol-anchored, mucin-like surface glycoproteins from bloodstream forms of the freshwater-fish parasite Trypanosoma carassii.

Wild and farmed freshwater fishes are widely and heavily parasitized by the haemoflagellate Trypanosoma carassii. In contrast, common carp, a natural host, can effectively control experimental infections by the production of specific anti-parasite antibodies. In this study we have identified and partially characterized mucin-like glycoproteins which are expressed in high abundance [(6. 0+/-1.7)x10(6) molecules.cell(-1)] at the surface of the bloodstream trypomastigote stage of the parasite. The polypeptide backbone of these glycoproteins is dominated by threonine, glycine, serine, alanine, valine and proline residues, and is modified at its C-terminus by a glycosylphosphatidylinositol membrane anchor. On average, each polypeptide carries carbohydrate chains composed of about 200 monosaccharide units (galactose, N-acetylglucosamine, xylose, sialic acid, fucose, mannose and arabinose), which are most probably O-linked to hydroxy amino acids. The mucin-like molecules are the target of the fish's humoral immune response, but do not undergo antigenic variation akin to that observed for the variant surface glycoprotein in salivarian trypanosomes. The results are discussed with reference to the differences between natural and experimental infections, and in relation to the recently delineated molecular phylogeny of trypanosomes.

Amino Acids↗
PubMed2000

Effect of previous surgery on abdominal opening time.

PURPOSE: The purpose of this study was to document prospectively the time required to gain access to the abdomen to perform a planned procedure in patients with and without previous surgery. METHODS: Patients were obtained from the consecutive cases of 11 surgeons at three colorectal surgery centers. Opening time (skin incision to retractor placement) was measured and recorded in the operating room by the circulating nurse or by an independent researcher. Demographic data including the number and type of previous operations and the presence and severity of adhesions were recorded by the staff surgeon. A comparison of opening times between patients with and without previous abdominal operations was conducted. RESULTS: One hundred ninety-eight patients had abdominal operations. Fifty-five percent had previous abdominal procedures. Patients with prior surgery required a mean of 21 minutes to open their abdomens, whereas patients without prior surgery required a mean of 6 minutes (P < 0.01). The median times were 17 and 6 minutes, respectively. Eighty-three percent of patients with prior surgery had adhesions, whereas only 7 percent of patients had adhesions on their initial operation. Patients with prior surgery also had higher grade adhesions (P < 0.001). Irrespective of previous surgery, comparing patients with adhesions with those without, patients with adhesions required a mean of 22 minutes to open, whereas the lack of adhesions resulted in a mean opening time of 6 minutes. CONCLUSIONS: Previous surgery and the presence of adhesions add significant time to opening the abdomen.

Abdomen↗
PubMed2000

Delayed effects of exercise on the plasma leptin concentration.

Recent studies have concluded that a single exercise session has no immediate effect on the plasma concentration of leptin, a putative satiety factor. We tested the hypothesis that an increase in energy expenditure would decrease the leptin concentration but the effects would be manifest in a 48-hour period following exercise. Eleven active males completed two treadmill exercise sessions with different energy expenditure (800 or 1,500 kcal) at 70% maximal O2 consumption (Vo2max). Subjects maintained constant energy intake on the day before, the day of, and 2 days after exercise, as verified by dietary recall. Compared with preexercise in either exercise session, there were no differences in plasma leptin concentrations following exercise (0 and 24 hours postexercise) except at 48 hours postexercise, where an approximately 30% decrease (P < .05) was observed. With either duration of exercise, plasma glucose increased about 10% (P < .05), insulin decreased 35% to 46% (P < .05), and cortisol increased 41% to 50% (P < .05, 1,500 kcal only) immediately following exercise, but returned to preexercise values at 24 and 48 hours postexercise. A statistically significant correlation was observed between the changes in leptin and insulin (r = .49, P < .0001). Single exercise sessions of varying energy expenditure decreased the plasma leptin concentration after 48 hours in association with a preceding decrease in insulin.

Adult↗
PubMed2000

Cloning, expression and functional characterisation of a peroxiredoxin from the potato cyst nematode Globodera rostochiensis.

We report the cloning, expression and functional characterisation of a peroxidase belonging to the peroxiredoxin family from the potato cyst nematode Globodera rostochiensis, the first molecule of this type from any nematode parasitic on plants. The G. rostochiensis peroxiredoxin catalyses the breakdown of hydrogen peroxide, but not cumene or t-butyl hydroperoxide, in a trypanosomatid reducing system comprising trypanothione reductase, trypanothione and tryparedoxin. In common with its homologues from Onchocerca volvulus and Brugia malayi, the G. rostochiensis enzyme is present on the surface of invasive and post-infective juveniles despite the apparent lack of a cleavable N-terminal signal peptide. The possibility that the G. rostochiensis peroxiredoxin plays a role in protection of the parasite from plant defence responses is discussed.

Amino Acid Sequence↗
PubMed2000

C-reactive protein binds to phosphorylated carbohydrates.

C-reactive protein (CRP) is a major acute phase protein in man. In order to more fully understand the physiological role of this serum protein, we have demonstrated high avidity binding for a defined chemically synthesized carbo-hydrate ligand which represents the repeating disaccharide of lipophosphoglycan, the major surface glycoconjugate of the unicellular parasite Leishmania donovani. Increasing the number of phosphorylated disaccharides in a molecule from one up to seven did not increase the avidity for CRP, however increasing this to 10 potential CRP binding sites did. In order to define the important features of this complex and variable structure for CRP binding we competed CRP binding to whole Leishmania parasites with amino, sulfated, phosphorylated, and unsubstituted monosaccharides, of which only phosphorylated monosaccharides were able to inhibit. Both the carbohydrate and the position of phosphorylation influenced the avidity for CRP. Synthetic oligosaccharides and phospho-oligosaccharides of various lengths and conformations were used to define the structural requirements for CRP recognition. The optimum structure for recognition of a single phosphate group was between two monosaccharide pyranose rings, and within a linear rather than a cyclic molecule. This stresses the importance of the interaction of the CRP binding site with both the carbohydrate and the phosphate group. CRP function may be mediated via the recognition of large arrays of phosphorylated carbohydrates as are characteristic of the surface of microorganisms.

C-Reactive Protein↗
PubMed2000

Protein structure controls the processing of the N-linked oligosaccharides and glycosylphosphatidylinositol glycans of variant surface glycoproteins expressed in bloodstream form Trypanosoma brucei.

The variant surface glycoproteins (VSGs) of Trypanosoma brucei are a family of homodimeric glycoproteins that adopt similar shapes. An individual trypanosome expresses one VSG at a time in the form of a dense protective mono-layer on the plasma membrane. VSG genes are expressed from one of several polycistronic transcription units (expression sites) that contain several expression site associated genes. We used a transformed trypanosome clone expressing two different VSGs (VSG121 and VSG221) from the same expression site (that of VSG221) to establish whether the genotype of the trypanosome clone or the VSG structure itself controls VSG N-linked oligosaccharide and GPI anchor glycan processing. In-gel release and fluorescent labeling of N-linked oligosaccharides and on-blot fluorescent labeling and release of GPI anchor glycans were employed to compare the carbohydrate structures of VSG121 and VSG221 when expressed individually in wild-type trypanosome clones and when expressed together in the transformed trypanosome clone. The data indicate that the genotype of the trypanosome clone has no effect on the N-linked oligosaccharide structures present on a given VSG variant and only a minor effect on the GPI anchor glycans. The latter is most likely an effect of changes in inter-VSG packing when two VGSs are expressed simultaneously. Thus, N-linked oligosaccharide and GPI anchor processing enzymes appear to be constitutively expressed in bloodstream form African trypanosomes and the tertiary and quaternary structures of the VSG homodimers appear to dictate the processing and glycoform microheterogeneity of surface-expressed VSGs.

Animals↗
PubMed2000

Fractionation of membrane components from tachyzoite forms of Toxoplasma gondii: differential recognition by immunoglobulin M (IgM) and IgG present in sera from patients with acute or chronic toxoplasmosis.

Tachyzoite forms of Toxoplasma gondii were subjected to a sequential organic solvent extraction, which allows fractionation of membrane components according to their degrees of hydrophobicity, yielding three fractions named F1 (most hydrophobic) to F3 (least hydrophobic). Fractions F2 (80.85% specificity and 86.95% sensitivity) and F3 (89.36% specificity and 93.61% sensitivity) gave the best results, being preferentially recognized by immunoglobulin M (IgM) and IgG in sera from patients with acute and chronic toxoplasmosis, respectively. Improved scores of specificity (100%) and sensitivity (100%) were achieved when a secondary antibody against human IgG1 instead of total IgG was employed to measure the reactivity of IgG antibodies with the F3 fraction. To purify tachyzoite antigens recognized by human IgM or IgG antibodies, the F2 or F3 fraction was loaded onto an octyl-Sepharose column and eluted with a propan-1-ol gradient. The main antigen(s) recognized by IgM or IgG eluted in a single peak from the octyl-Sepharose resin loaded with either F2 (30 to 50% propan-1-ol) or F3 (15 to 35% propan-1-ol), respectively. These semipurified fractions gave improved scores when used to detect T. gondii-specific IgM (95.7% specificity and 81.8% sensitivity) or IgG (100% specificity and 93. 75% sensitivity) in an enzyme-linked immunosorbent assay. Further biochemical and immunological analyses of antigens partially purified from F2 and F3 indicate that glycoinositolphospholipids are preferentially recognized by IgM, whereas proteins of approximately 30 to 40 kDa are recognized by IgG, elicited during T. gondii infection in humans.

Acute Disease↗
PubMed2000

Mucin-like molecules form a negatively charged coat that protects Trypanosoma cruzi trypomastigotes from killing by human anti-alpha-galactosyl antibodies.

In the presence of sialic acid donors Trypanosoma cruzi acquires up to 10(7) sialic acid residues on its surface, in a reaction catalyzed by its unique trans-sialidase. Most of these sialic acid residues are incorporated into mucin-like glycoproteins. To further understand the biological role of parasite sialylation, we have measured the amount of mucin in this parasite. We found that both epimastigote and trypomastigote forms have the same number of mucin molecules per surface area, although trypomastigotes have less than 10% of the amount of glycoinositol phospholipids, the other major surface glycoconjugate of T. cruzi. Based on the estimated surface area of each mucin, we calculated that these molecules form a coat covering the entire trypomastigote cell. The presence of the surface coat is shown by transmission electron microscopy of Ruthenium Red-stained parasites. The coat was revealed by binding of antibodies isolated from Chagasic patients that react with high affinity to alpha-galactosyl epitopes present in the mucin molecule. When added to the trypomastigote, these antibodies cause an extensive structural perturbation of the parasite coat with formation of large blebs, ultimately leading to parasite lysis. Interestingly, lysis is decreased if the mucin coat is heavily sialylated. Furthermore, addition of MgCl2 reverses the protective effect of sialylation, suggesting that the sialic acid negative charges stabilize the surface coat. Inhibition of sialylation by anti-trans-sialidase antibodies, found in immunized animals, or human Chagasic sera, also increase killing by anti-alpha-galactosyl antibodies. Therefore, the large amounts of sialylated mucins, forming a surface coat on infective trypomastigote forms, have an important structural and protective role.

Animals↗
PubMed1999

Selective inhibitors of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei.

Synthetic analogues of D-GlcNalpha1-6D-myo-inositol-1-HPO(4)-3(sn-1, 2-diacylglycerol) (GlcN-PI), with the 2-position of the inositol residue substituted with an O-octyl ether [D-GlcNalpha1-6D-(2-O-octyl)myo-inositol-1-HPO(4)-3-sn-1, 2-dipalmitoylglycerol; GlcN-(2-O-octyl) PI] or O-hexadecyl ether [D-GlcNalpha1-6D-(2-O-hexadecyl)myo-inositol-1-HPO(4)-3-sn-1, 2-dipalmitoylglycerol; GlcN-(2-O-hexadecyl)PI], were tested as substrates or inhibitors of glycosylphosphatidylinositol (GPI) biosynthetic pathways using cell-free systems of the protozoan parasite Trypanosoma brucei (the causative agent of human African sleeping sickness) and human HeLa cells. Neither these compounds nor their N-acetyl derivatives are substrates or inhibitors of GPI biosynthetic enzymes in the HeLa cell-free system but are potent inhibitors of GPI biosynthesis in the T.brucei cell-free system. GlcN-(2-O-hexadecyl)PI was shown to inhibit the first alpha-mannosyltransferase of the trypanosomal GPI pathway. The N-acetylated derivative GlcNAc-(2-O-octyl)PI is a substrate for the trypanosomal GlcNAc-PI de-N-acetylase and this compound, like GlcN-(2-O-octyl)PI, is processed predominantly to Man(2)GlcN-(2-O-octyl)PI by the T.brucei cell-free system. Both GlcN-(2-O-octyl)PI and GlcNAc(2-O-octyl)PI also inhibit inositol acylation of Man(1-3)GlcN-PI and, consequently, the addition of the ethanolamine phosphate bridge in the T.brucei cell-free system. The data establish these substrate analogues as the first generation of in vitro parasite GPI pathway-specific inhibitors.

Acylation↗
PubMed1999

The procyclin repertoire of Trypanosoma brucei. Identification and structural characterization of the Glu-Pro-rich polypeptides.

The surface of the insect stages of the protozoan parasite Trypanosoma brucei is covered by abundant glycosyl phosphatidylinositol (GPI)-anchored glycoproteins known as procyclins. One type of procyclin, the EP isoform, is predicted to have 22-30 Glu-Pro (EP) repeats in its C-terminal domain and is encoded by multiple genes. Because of the similarity of the EP isoform sequences and the heterogeneity of their GPI anchors, it has been impossible to separate and characterize these polypeptides by standard protein fractionation techniques. To facilitate their structural and functional characterization, we used a combination of matrix-assisted laser desorption ionization and electrospray mass spectrometry to analyze the entire procyclin repertoire expressed on the trypanosome cell. This analysis, which required removal of the GPI anchors by aqueous hydrofluoric acid treatment and cleavage at aspartate-proline bonds by mild acid hydrolysis, provided precise information about the glycosylation state and the number of Glu-Pro repeats in these proteins. Using this methodology we detected in a T. brucei clone the glycosylated products of the EP3 gene and two different products of the EP1 gene (EP1-1 and EP1-2). Furthermore, only low amounts of the nonglycosylated products of the GPEET and EP2 genes were detected. Because all procyclin genes are transcribed polycistronically, the latter finding indicates that the expression of the GPEET and EP2 genes is post-transcriptionaly regulated. This is the first time that the whole procyclin repertoire from procyclic trypanosomes has been characterized at the protein level.

Amino Acid Sequence↗
PubMed1999

The GPI biosynthetic pathway as a therapeutic target for African sleeping sickness.

African sleeping sickness is a debilitating and often fatal disease caused by tsetse fly transmitted African trypanosomes. These extracellular protozoan parasites survive in the human bloodstream by virtue of a dense cell surface coat made of variant surface glycoprotein. The parasites have a repertoire of several hundred immunologically distinct variant surface glycoproteins and they evade the host immune response by antigenic variation. All variant surface glycoproteins are anchored to the plasma membrane via glycosylphosphatidylinositol membrane anchors and compounds that inhibit the assembly or transfer of these anchors could have trypanocidal potential. This article compares glycosylphosphatidylinositol biosynthesis in African trypanosomes and mammalian cells and identifies several steps that could be targets for the development of parasite-specific therapeutic agents.

Aminoacyltransferases↗
PubMed1999

Synthesis of some second-generation substrate analogues of early intermediates in the biosynthetic pathway of glycosylphosphatidylinositol membrane anchors.

1-D-6-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl)-2-O-octyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (23) and the corresponding 2-O-hexadecyl-D-myo-inositol compound 24 have been prepared as substrate analogues of an early intermediate in the biosynthetic pathway of glycosylphosphatidylinositol (GPI) membrane anchors. 1-D-6-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol 1-(1,2-di-O-octyl-sn-glycerol 3-phosphate) has also been prepared as a substrate analogue. Biological evaluation of the analogues 23 and 24 revealed that they are neither substrates nor inhibitors of GPI biosynthetic enzymes in the human (HeLa) cell-free system but are potent inhibitors at different stages of GPI biosynthesis in the Trypanosoma brucei cell-free system.

Animals↗
PubMed1999

Structural studies on the polar glycoinositol phospholipids of Trypanosoma (Schizotrypanum) dionisii from bats.

The polar glycoinositol phospholipids (GIPLs) of a Trypanosoma species that belongs to the Schizotrypanum subgenus were purified by reversed-phase and normal-phase liquid chromatography and analysed by negative-ion mode electrospray-mass spectrometry (ES-MS). The phosphatidylinositol moieties were released by nitrous acid deamination and identified as ceramide- and alkylacylglycerol-containing species. The structures of the GIPLs were determined using chemical treatments, sequential exoglycosidase digestions and positive-ion mode ES-MS-MS. All of the GIPLs were based on the same Man alpha1-2Man alpha1-2Man alpha1-6Man alpha1-4(NH2-CH2CH2-HPO3-)GlcN-PI core with single terminal Galf residue substitutions either on the terminal nonreducing Man or on the second alphaMan residue from the inositol and with either ethanolamine phosphate or 2-aminoethylphosphonate on the third alphaMan residue from the inositol. The T. (S.) dionisii GIPLs are compared with those of T. (S.) cruzi, a closely related species of the Schizotrypanum subgenus.

Animals↗
PubMed1999

Typing of Leishmania lipophosphoglycans by electrospray mass spectrometry.

A method has been developed to identify the repeating phosphosaccharide units of Leishmania lipophosphoglycans using electrospray mass-spectrometry (ES-MS). Cone voltage-induced fragmentation of intact lipophosphoglycan was found to be as effective as analysis of mild acid hydrolysates in identifying the degree of modification of the repeating units of lipophosphoglycans derived from Leishmania mexicana and Leishmania major. This finding was exploited in a 'rapid-analysis' method in which a crude organic extract of approximately 2 x 10(9) L. major promastigote cells was loaded onto a reverse-phase cartridge for immediate elution into the mass-spectrometer. Using this approach, it was possible to identify the repeating units by total ion scanning and scanning for parents of the m/z 79 (PO3-) fragment ion. This approach is suitable for quick-typing of lipophosphoglycan repeats and was shown to detect alterations in repeat side chains caused by: (1) culturing L. major promastigotes in the presence of L-fucose; and (2) in vitro metacyclogenesis of L. major promastigotes. It is anticipated that the method will be applicable to small samples of cultured field isolates or genetically-manipulated strains.

Animals↗