Knowledge, Attitudes and Practices of Drivers towards Road Traffic Regulations in Trinidad, West Indies

This study aims to test the knowledge, practices & attitudes of drivers in Trinidad through issuing a questionnaire based on driving practices in relation to seat belt use, alcohol consumption, fatigue & distraction, and a quiz, which focuses on road regulations. The information obtained from this study can be used to prevent accidents by identifying: poor knowledge, driving practices and attitudes in the driving population of Trinidad.

Description of site-specific morphology of keloid phenotypes in an Afrocaribbean population.

By examining the keloid scars of 211 Afrocaribbean patients presenting to the Plastic Surgery unit in Kingston, Jamaica, we have described site-specific morphologies of scarring; keloid disease is not a homogenous biological entity. All cases conformed to clinical criteria for diagnosis of keloid scarring: 369 keloid scars were present in 137 females (2-83 years; mean 29.6 years; SD+/-14.9 years) and 74 males (5-90 years, mean 29.5 years; SD+/-15.0 years). Morphologies were specific to each anatomical site: trunk scars (n=45,12.1%) were geometrically shaped with clear margins or irregular in outline, surface and margin; back single scars were well-demarcated botryoid but multiple scars were butterfly-shaped, spheroidal and irregular; chest scars (n=72,20.1%) were butterfly or nonbutterfly shaped found most commonly in the midsternal line; upper limb scars (n=57,15.3%) mostly in the deltoid region (propeller shaped) or elsewhere nodular, linear to irregular; ear (n=85,23%) commonest site being the lobe, having reniform to bulbous shape; face and neck (n=60,16.2%) scars were firm nodular to hard; posterior auricular scars were either horizontal and oblong-shaped or vertical and reniform in outline; scalp scars (n=11,2.8%) were commonest in the occipital area varying from small papules to large plaques; lower limb scars (n=39,10.5%) varied from propeller, butterfly, petalloid to dum-bell-shaped. Three plantar and eight pubic keloids were rare findings. Recognition of different morphological phenotypes is necessary in understanding genotypic predisposition and aiding diagnosis, treatment and prognosis of keloid scars.

Karyotype relationships between distantly related marsupials from South America and Australia.

Reciprocal chromosome painting and G-banding were used to compare the karyotypes of three Australian marsupials (Sminthopsis crassicaudata, Macropus eugenii, Trichosurus vulpecula) and one South American marsupial (Monodelphis domestica). The results revealed only a limited number of rearrangements between these species and that the four karyotypes can be described as different combinations of fifteen conserved segments. Five chromosomes are totally conserved between M. domestica (pairs 1, 2, 5, 8 and the X) and the presumed 2n = 14 Australian ancestral karyotype, while M. domestica pairs 3 and 6 and 4 and 7 would have been involved in fusion/fission rearrangements. Chromosome comparisons are presented in a chromosome homology map. Although the species studied diverged 70 million years ago, the karyotype of Monodelphis domestica is highly conserved in relation to those of Australian marsupials.

Requirement of mitogen-activated protein kinases and I kappa B phosphorylation for induction of proinflammatory cytokines synthesis by macrophages indicates functional similarity of receptors triggered by glycosylphosphatidylinositol anchors from parasitic protozoa and bacterial lipopolysaccharide.

In the present study, we evaluated the ability of GPI-anchored mucin-like glycoproteins purified from Trypanosoma cruzi trypomastigotes (tGPI-mucin) to trigger phosphorylation of different mitogen-activated protein kinases (MAPKs) and related transcription factors in inflammatory macrophages. Kinetic experiments show that the peak of extracellular signal-related kinase (ERK)-1/ERK-2, stress-activated protein kinase (SAPK) kinase-1/mitogen-activated protein kinase (MAPK) kinase-4, and p38/SAPK-2, phosphorylation occurs between 15 and 30 min after macrophage stimulation with tGPI-mucin or GPI anchors highly purified from tGPI-mucins (tGPI). The use of the specific inhibitors of ERK-1/ERK-2 (PD 98059) and p38/SAPK-2 (SB 203580) phosphorylation also indicates the role of MAPKs, with possible involvement of cAMP response element binding protein, in triggering TNF-alpha and IL-12 synthesis by IFN-gamma-primed-macrophages exposed to tGPI or tGPI-mucin. In addition, tGPI-mucin and tGPI were able to induce phosphorylation of I kappa B, and the use of SN50 peptide, an inhibitor of NF-kappa B translocation, resulted in 70% of TNF-alpha synthesis by macrophages exposed to tGPI-mucin. Finally, the similarity of patterns of MAPK and I kappa B phosphorylation, the concentration of drugs required to inhibit cytokine synthesis, as well as cross-tolerization exhibited by macrophages exposed to tGPI, tGPI-mucin, or bacterial LPS, suggest that receptors with the same functional properties are triggered by these different microbial glycoconjugates.

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.

Payment deferred: strychnine poisoning in Nicaragua 65 years ago.

BACKGROUND: In 1933 in Leon, Nicaragua, a 22-year-old woman died after an acute convulsive illness in which she experienced trismus, opisthotonos, and hyperpyrexia. Three years later her husband, Oliverio Castaneda, was convicted of her murder and that of 2 other people in the same city. METHODS: We went to Nicaragua to investigate documents involved with that case and evaluate whether the verdict of murder by strychnine was substantiated by the data. We present the results of the investigation and provide information about the practice of medicine, pharmacy, and toxicology early in this century. RESULTS: The clinical picture in all 3 cases suggests strychnine poisoning. The clinical, toxicological, and circumstantial evidence is strong and implicates Castaneda as a murderer and strychnine as the weapon. CONCLUSION: We conclude that Oliverio Castaneda was the probable perpetrator of three 1933 strychnine murders in Leon and that he may have previously used strychnine to kill others in Nicaragua and neighboring countries.

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.

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.

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.

Visceral adipose tissue and cardiovascular risk factors in obese children.

OBJECTIVE: In adults visceral adipose tissue (VAT) has been shown to be more highly correlated with cardiovascular (CV) risk factors than are other measures of adiposity such as subcutaneous abdominal adipose tissue (SAAT), percent body fat (%BF), or total body fat mass (TFM). We examined the relations between these measures of fatness and CV risk factors in obese children. STUDY DESIGN: Subjects were 64 obese (27% to 61% BF) children (24 black girls, 19 white girls, 11 black boys, 10 white boys) aged 7 to 11 years. VAT and SAAT were measured with magnetic resonance imaging. TFM and %BF were determined with dual x-ray absorptiometry. Hierarchical stepwise multiple regression analyses were used to determine the proportions of variance in CV risk factors explained by the demographic and adiposity measures. RESULTS: VAT but not SAAT, %BF, or TFM explained a significant proportion of the variance (r2 range = 0.10 to 0.21) in several lipid/lipoprotein risk factors including triacylglycerols, high-density lipoprotein cholesterol, the ratio of total cholesterol to high-density lipoprotein cholesterol, and low-density lipoprotein particle size. CONCLUSION: Many of the deleterious relations between VAT and lipid/lipoprotein risk factors seen in adults were already present in this sample of obese children.

Glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated from Trypanosoma cruzi trypomastigotes initiate the synthesis of proinflammatory cytokines by macrophages.

Components of Trypanosoma cruzi able to induce the production of IL-12 and other proinflammatory cytokines by macrophages were identified. Murine inflammatory macrophages were cultured with live parasites or with cellular components from different developmental forms of T. cruzi (i.e., trypomastigotes, amastigotes, metacyclic trypomastigotes, and epimastigotes), and the cytokine levels were measured after 24 and 48 h. Our results indicate that live trypomastigotes or live amastigotes (but not live epimastigotes or live metacyclic trypomastigotes) as well as trypomastigote extracts (but not extracts derived from epimastigotes) induce IL-12 and TNF-alpha synthesis by macrophages. Such biological activity is enhanced in membrane preparations from trypomastigotes. Further enrichment of the trypomastigote-derived monokine-inducing factor was obtained by solvent extraction and hydrophobic-interaction chromatography. The resultant purified molecules are a family of closely related glycoconjugates with predominant species at 70 to 80 and 120 to 200 kDa. These molecules are composed of carbohydrate chains O-linked to a polypeptide backbone that is anchored to the trypomastigote membrane via a glycosylphosphatidylinositol structure. The trypomastigote-derived glycoconjugates are active in inducing cytokine synthesis by macrophages at concentrations of 100 ng/ml. These effects are highly potentiated by IFN-gamma. Mapping of the glycoconjugate molecules to characterize the structural requirements for macrophage activation suggested that nonsaturated acyl fatty acid chains and periodate-sensitive units from the glycosylphosphatidylinositol anchor are important elements for the infective trypomastigote form to initiate cytokine synthesis by macrophages.

Differences in the distribution of versican, decorin, and biglycan in atherosclerotic human coronary arteries.

The distributions of versican, biglycan, and decorin have been examined in segments of normal and atherosclerotic human coronary arteries using antibodies directed against the core proteins of these macromolecules. Versican immunostaining was prominent throughout the extracellular matrix (ECM) in regions of the vessels that contained abundant smooth-muscle cells, such as in diffuse intimal thickenings, fibrous caps, and in zones of loose, myxoid connective tissue. Versican also was present in smooth-muscle-rich thrombi and at borders of the lipid-rich cores of advanced atherosclerotic lesions. Biglycan immunostaining was observed in diffuse intimal thickenings, fibrous caps, and myxoid areas, but, unlike versican, it was abundant in the lipid-rich core of advanced plaques. However, biglycan immunostaining was absent in smooth-muscle cell-enriched thrombi. Decorin immunostaining paralleled biglycan immunostaining except that it was conspicuously absent in the myxoid areas of the plaque and markedly reduced in diffuse intimal thickenings. Both biglycan and decorin immunostaining were consistently associated with some of the microvessels in the thrombi and in advanced atherosclerotic plaques. Taken together, these results indicate that specific proteoglycans distribute to topographically defined regions of normal and atherosclerotic human coronary arteries and that these different distributions may indicate a diversity of functions in normal and pathologic processes of the arterial wall.

Structure of the N-linked oligosaccharide of the main diagnostic antigen of the pathogenic fungus Paracoccidioides brasiliensis.

The major diagnostic antigen of Paracoccidioides brasiliensis is the exocellularly secreted 43,000 Da glycoprotein (gp43) which contains a single N-linked oligosaccharide chain. This oligosaccharide, although poorly immunogenic in man, is responsible for the cross-reactivity of the gp43 with sera from patients with histoplasmosis, and may have a role in fungal virulence. It contains a neutral high-mannose core (Man7GlcNAc2) to which a (1-->6)-linked alpha-D-Manp chain of variable length, substituted at the 2-O positions by single alpha-D-Manp residues, is attached. A terminal unit of beta-D-galactofuranose is (1-->6)-linked to one of the (1-->2)-linked mannosyl residues, either in the C or in the A arm of the oligosaccharide. The heterogeneity of the oligosaccharide is determined by the different sizes of the A arm and the sites of insertion of the beta-galactofuranosyl unit. The complete structure was determined by methylation analysis, 1H-NMR, mass spectrometry, acetolysis and mannosidase degradation. Electrospray mass spectrometry showed that the oligosaccharide comprises several subtypes ranging from Hex18GlcNAc2 to Hex10GlcNAc2 which accounts for the diffuse migration of the gp43 in polyacrylamide gels. The average size of the most frequent subtype is Hex13.6GlcNAc2. Dilute acid treatment to remove beta-D-Galf reduced the molecular masses of the majority of the subtypes by a single sugar unit.

The lipid structure of the glycosylphosphatidylinositol-anchored mucin-like sialic acid acceptors of Trypanosoma cruzi changes during parasite differentiation from epimastigotes to infective metacyclic trypomastigote forms.

The major acceptors of sialic acid on the surface of metacyclic trypomastigotes, which are the infective forms of Trypanosoma cruzi found in the insect vector, are mucin-like glycoproteins linked to the parasite membrane via glycosylphosphatidylinositol anchors. Here we have compared the lipid and the carbohydrate structure of the glycosylphosphatidylinositol anchors and the O-linked oligosaccharides of the mucins isolated from metacyclic trypomastigotes and noninfective epimastigote forms obtained in culture. The single difference found was in the lipid structure. While the phosphatidylinositol moiety of the epimastigote mucins contains mainly 1-O-hexadecyl-2-O-hexadecanoylphosphatidylinositol, the phosphatidylinositol moiety of the metacyclic trypomastigote mucins contains mostly (approximately 70%) inositol phosphoceramides, consisting of a C18:0 sphinganine long chain base and mainly C24:0 and C16:0 fatty acids. The remaining 30% of the metacyclic phosphatidylinositol moieties are the same alkylacylphosphatidylinositol species found in epimastigotes. In contrast, the glycosylphosphatidylinositol glycan cores of both molecules are very similar, mainly Man alpha 1-2Man alpha 1-2Man alpha 1- 6Man alpha 1-4GlcN. The glycans are substituted at the GlcN residue and at the third alpha Man distal to the GlcN residue by ethanolamine phosphate or 2-aminoethylphosphonate groups. The structures of the desialylated O-linked oligosaccharides of the metacyclic trypomastigote mucin-like molecules, released by beta-elimination with concomitant reduction, are identical to the structures reported for the epimastigote mucins (Previato, J. O., Jones, C., Gonçalves, L. P. B., Wait, R., Travassos, L. R., and Mendoça-Previato, L. (1994) Biochem. J. 301, 151-159). In addition, a significant amount of nonsubstituted N-acetylglucosaminitol was released from the mucins of both forms of the parasite. Taken together, these results indicate that when epimastigotes transform into infective metacyclic trypomastigotes, the phosphatidylinositol moiety of the glycosylphosphatidylinositol anchor of the major acceptor of sialic acid is modified, while the glycosylphosphatidylinositol anchor and O-linked sugar chains remain essentially unchanged.

Characterization of the lipid moiety of the glycosylphosphatidylinositol anchor of Trypanosoma cruzi 1G7-antigen.

The 90-kDa stage-specific 1G7-antigen has been implicated in the invasion of host cells by the metacyclic forms of Trypanosoma cruzi. The antigen is attached to the plasma membrane via glycosylphosphatidylinositol, the partial structure of which was the first to be determined for a protein of this parasite. In this study, the complete structure of the lipid component of the anchor was determined by electrospray mass spectrometry, gas chromatography mass spectrometry, phospholipase sensitivity and high-performance thin-layer chromatography of the diaradylglycerol components after benzoylation. These analyses showed that the lipid moiety of 1G7-antigen is composed essentially of 1-O-hexadecyl-2-O-hexadecanoyl-phosphatidylinositol and 1-O-hexadecyl-2-O-octadecanoyl-phosphatidylinositol. The high sensitivity of the electrospray mass spectrometric analysis unexpectedly revealed the presence of a small proportion of putative inositol-phosphoceramide structures, and confirmed the absence of inositol-acylated species. An interesting finding was that the biosynthetic incorporation of [3H]palmitate labelled solely the acyl position, and not the 1-O-alkyl chain in the 1G7-antigen anchor.

Lytic anti-alpha-galactosyl antibodies from patients with chronic Chagas' disease recognize novel O-linked oligosaccharides on mucin-like glycosyl-phosphatidylinositol-anchored glycoproteins of Trypanosoma cruzi.

Sera of patients with chronic Chagas' disease (American trypanosomiasis) contain elevated levels of anti-alpha-galactosyl antibodies that are lytic to Trypanosoma cruzi. The T. cruzi trypomastigote F2/3 antigen complex recognized by these antibodies runs as a broad smear on SDS/PAGE [Almeida, Krautz, Krettli and Travassos (1993) J. Clin. Lab. Anal. 7, 307-316]. Treatment of T. cruzi trypomastigote cells with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) abolished most of their reactivity to chronic Chagas'-disease ((Chagasic, Ch) anti-alpha-galactosyl antibodies (anti-Gal). The F2/3 antigen complex, purified by solvent extraction and hydrophobic-interaction chromatography, contained 60% carbohydrate by weight and substantial amounts of Thr, Ser, Glx, Asx, Gly, Ala and Pro, but relatively few hydrophobic amino acids. The presence of myoinositol, ethanolamine and 1-O-hexadecylglycerol suggested the presence of glycosyl-phosphatidylinositol membrane anchors. This was confirmed by PI-PLC treatment, which rendered the F2/3 molecules hydrophilic and reactive to anti-(cross-reacting determinant) antibodies. The majority of the GlcNAc content of the F2/3 antigens was found at the reducing termini of oligosaccharides in O-glycosidic linkage to Thr residues. These O-linked oligosaccharides could be released by beta-elimination and by mild hydrazinolysis. The smallest released oligosaccharitol that was reactive with the Ch anti-Gal was Gal alpha 1-3Gal beta 1-4GlcNAcol (where GlcNAcol is N-acetyl-glucosaminitol). Several other Gal-containing oligosaccharitols were observed, most of which were branched and contained 4,6-di-O-substituted GlcNAcol at their reducing termini. About half of the total released oligosaccharitols could bind to immobilized Ch anti-Gal, but none of them bound to the anti-Gal isolated from normal human sera. These data suggest that the specificities of the Ch anti-Gal are quite different from the natural anti-Gal isolated from normal human sera. Therefore, these novel T. cruzi O-linked oligosaccharides are highly immunogenic under the conditions of natural infection and are the targets for lytic Ch anti-Gal.

O- and N-glycosylation of the Leishmania mexicana-secreted acid phosphatase. Characterization of a new class of phosphoserine-linked glycans.

The protozoan parasite Leishmania mexicana secretes a heavily glycosylated 100-kDa acid phosphatase (sAP) which is associated with one or more polydisperse proteophosphoglycans. Most of the glycans in this complex were released using mild acid hydrolysis conditions that preferentially cleave phosphodiester linkages. The released saccharides were shown to consist of monomeric mannose and a series of neutral and phosphorylated glycans by Dionex high performance liquid chromatography, methylation analysis, exoglycosidase digestions, and one-dimensional 1H NMR spectroscopy. The neutral species comprised a linear series of oligosaccharides with the structures [Man alpha 1-2]1-5Man. The phosphorylated oligosaccharides were characterized as PO4-6Gal beta 1-4Man and PO4-6[Glc beta 1-3]Gal beta 1-4Man. The attachment of these glycans to the polypeptide backbone via the linkage, Man alpha 1-PO4-Ser, is suggested by: 1) the finding that more than 60% of the serine residues in the polypeptide are phosphorylated and 2) the resistance of the phosphoserine residues to alkaline phosphatase digestion unless the sAP was first treated with either mild acid (to release all glycans) or jack bean alpha-mannosidase (to release neutral mannose glycans). Analysis of the partially resolved components of the complex indicated that the most of the O-linked glycans on the 100-kDa phosphoglycoprotein comprised mannose and the mannose-oligosaccharides. In contrast the major O-linked glycans on the proteophosphoglycan were short phosphoglycan chains, containing on average two repeat units per chain. In addition to the O-linked glycans, both components in the sAP complex contained N-linked glycans. The N-glycanase F-released glycans were characterized by Bio-Gel P4 chromatography and exoglycosidase digestions to be the biantennary oligomannose type with the structures Glc1Man6GlcNAc2 and Man6GlcNAc2. The O-linked glycans of the sAP complex are similar to those found in the phosphoglycan chains of the abundant surface lipophosphoglycan, but differ in having much shorter phosphoglycan chains and a more diverse series of mannose cap oligosaccharides. These data suggest that there are marked differences in the ability of different glycosyltransferases to utilize peptide-linked versus glycolipid-linked acceptors.

The detection of phospholipase-resistant and -sensitive glycosyl-phosphatidylinositol membrane anchors by western blotting.

Glycosyl-phosphatidylinositol (GPI) membrane anchors are present on a large number of eukaryotic plasma membrane proteins. Some of these anchors can be cleaved with bacterial phosphatidylinositol-specific phospholipases C, and a glycosyl-phosphatidylinositol-specific phospholipase C from Trypanosoma brucei, to reveal an epitope called the cross-reacting determinant. Other glycosyl-phosphatidylinositol anchors are resistant to the action of these enzymes prior to treatment with mild base. A simple method is described for identifying both phospholipase-sensitive and -resistant anchors using anti-cross-reacting determinant antibodies on Western blots. This procedure represents a high-sensitivity general method for the identification of GPI-anchored proteins.