Purification and characterization of insulin-mimetic inositol phosphoglycan-like molecules from grass pea (Lathyrus sativus) seeds.

BACKGROUND: Signal transduction through the hydrolysis of glycosyl-phosphatidylinositol (GPI) leading to the release of the water-soluble inositol phosphoglycan (IPG) molecules has been demonstrated to be important for mediating some of the actions of insulin and insulin-like growth factor-I (IGF-I). MATERIALS AND METHODS: In the present study, GPI from grass pea (Lathyrus sativus) seeds has been purified and partially characterized on the basis of its chromatographic properties and its compositional analysis. RESULTS: The results indicate that it shows similarities to GPI previously isolated from other sources such as rat liver. IPG was generated from L. sativus seed GPI by hydrolysis with a GPI-specific phospholipase D (GPI-PLD). This IPG inhibited protein kinase A (PKA) in an in vitro assay, caused cell proliferation in explanted cochleovestibular ganglia (CVG), and decreased 8-Br-cAMP-induced phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression in cultured hepatoma cells. CONCLUSIONS: Our data indicate that L. sativus seed IPG possess insulin-mimetic activities. This may explain why L. sativus seeds have been used in some traditional medicines to ameliorate diabetic symptoms.

Towards the in vitro reconstitution of caveolae. Asymmetric incorporation of glycosylphosphatidylinositol (GPI) and gangliosides into liposomal membranes.

Large unilamellar vesicles consisting of phospholipids with or without cholesterol have been prepared containing GPI and/or gangliosides asymmetrically located in the outer leaflet of the bilayer. Such asymmetric distribution of GPI and gangliosides is found in 'rafts' and caveolae. Using these vesicles, GPI can be readily hydrolysed by phospholipases. Both cholesterol and ganglioside are seen to inhibit, in an additive way, the hydrolytic activity of GPI-specific phospholipase D.

[Diagnosis of thalassemias and hemoglobinopathies by HPLC (high performance liquid chromatography): study of 627 patients]. / Diagnose der Thalassämien und Hämoglobinopathien mittels HPLC (high performance liquid chromatography): eine Studie von 627 Patienten.

Red cell haemolysates of 627 patients with mainly microcytic anaemia were subjected to HPLC for diagnosis of thalassaemia (thal) or haemoglobinopathy during 1998. Thalassaemia was diagnosed in 16.3% (95 beta-thal minor, 1 beta-thal major, 2 delta beta-thal heterozygote, 4 alpha-thal1), haemoglobinopathies in 3.5% (10 Hb S including 3 Hb S-alpha-thal, 1 homozygote, 1 Hb SC and 1 Hb SE; 6 Hb E including 3 homozygotes; 3 Hb Lepore heterozygotes; 1 Hb K; 1 Hb O-Arab*; 1 Hb K-Ibadan* [* = confirmed by DNA sequencing]). In 10.7% of patients severe iron-deficiency (ferritin < 7 micrograms/l) was the cause of microcytosis (MCV 72.1 +/- 2.6 fl) and anaemia (Hb 97.2 +/- 9.8 g/l). The beta-thal minor group showed prominent microcytosis (MCV 66.9 +/- 2.6 fl) but only mild anaemia (Hb 114.1 +/- 12.9 g/l). Variant Hb K-Ibadan und Hb O-Arab were found during quantification of HbA1c. Patients with beta-thal minor or severe iron-deficiency anaemia were identified with equal frequency in adult females, children and adolescents of both sexes; however, in adult males beta-thal minor was the most frequent aetiology (> 90%) of microcytic anaemia. Our results demonstrate the diagnostic value of red cell lysate HPLC and ferritin determination when evaluating unclear microcytic anaemia. This approach, together with die HbA1c-quantification by HPLC, will render possible detailed diagnosis of thalassaemia and haemoglobinopathies.

In vitro phosphorylation of purified glycosylphosphatidylinositol-specific phospholipase D.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was phosphorylated in vitro by cAMP-dependent protein kinase (PKA) and by tyrosine kinase. Phosphorylation by PKA occurred in the 110 kDa native form of GPI-PLD as well as in multiple proteolytic degradation products and caused a significant decrease in enzyme activity. Dephosphorylation by treatment with alkaline phosphatase completely restored GPI-PLD activity. In addition, incubation of GPI-PLD with trypsin, which results in the generation of distinct peptide fragments, resulted in complete dephosphorylation of radiolabeled GPI-PLD. The site of phosphorylation by PKA was assigned to Thr-286. Tyrosine phosphorylation was only observed in a proteolytically processed fragment of GPI-PLD but not in the 110 kDa native form and had no effect on GPI-PLD activity.

Signalling properties of glycosylphosphatidylinositols and their regulated release from membranes in the turnover of glycosylphosphatidylinositol-anchored proteins.

Glycosylphosphatidylinositols are involved in transmembrane signalling, and their signal mediated release from the cell membrane has been demonstrated for several hormones and growth factors. Presently, however, only indirect evidence exists for the enzyme responsible for the signal mediated release of glycosylphosphatidylinositols. Indirect evidence from product identification led to the conclusion that in mammals the hormone sensitive activity is that of a glycosylphosphatidylinositol anchor hydrolyzing phospholipase C. On the other hand a mammalian glycosylphosphatidylinositol anchor hydrolyzing phospholipase D is well-established. This enzyme most likely functions in the intracellular turnover of glycosylphosphatidylinositols, however, its possible relation to the signalling properties of glycosylphosphatidylinositols remains unclear.

In vitro incorporation of GPI-anchored proteins into human erythrocytes and their fate in the membrane.

In many different cells, glycosylphosphatidylinositol (GPI)-anchored molecules are clustered in membrane microdomains that resist extraction by detergents at 4 degrees C. In this report, we identified the presence of such domains in human erythrocytes and examined the ability of exogenously-added GPI-anchored molecules to colocalize with the endogenous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three purified GPI-anchored proteins having different GPI lipid moieties resulted in their efficient and correct incorporation into the membrane. The extent of membrane insertion was dependent on the intactness of the GPI lipid moiety. However, unlike the endogenous GPI-anchored proteins, the in vitro incorporated GPI molecules were not resistant to membrane extraction by Triton X-100 at 4 degrees C. In addition, in contrast to the endogenous GPI-anchored proteins, they were not preferentially released from erythrocytes during vesiculation induced by calcium loading of the cells. These results suggest that in vitro incorporated GPI-linked molecules are excluded from pre-existing GPI-enriched membrane areas in human erythrocytes and that these microdomains may represent the sites of membrane vesicle formation.

Expression of intracellular and GPI-anchored forms of GPI-specific phospholipase D in COS-1 cells.

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is a secretory protein present in high amounts in mammalian body fluids. Its cDNA has been isolated and encodes a signal peptide of 23 amino acids and the mature protein of 816 amino acids. We generated cDNAs encoding a signal peptide-deficient and a GPI-anchored form of GPI-PLD and transiently transfected these constructs into COS-1 cells. The signal peptide-deficient form of GPI-PLD was expressed as a 90-kDa protein that was catalytically active and was localized intracellularly. Cells transfected with cDNA encoding the GPI-anchored form of GPI-PLD expressed a catalytically active enzyme of 100 kDa that could be labelled with [3H]ethanolamine demonstrating its modification by a GPI structure. Expression of the GPI-anchored form of GPI-PLD resulted in the release of endogenous GPI-anchored alkaline phosphatase from COS-1 cells, whereas expression of the intracellular form of GPI-PLD had no effect on membrane attachment of endogenous alkaline phosphatase. Similarly, in cells cotransfected with GPI-anchored placental alkaline phosphatase (PLAP) and the GPI-anchored form of GPI-PLD, PLAP was released into the cell culture supernatant while expression of the signal peptide-deficient form of GPI-PLD did not affect the amount of cell-associated PLAP.

Addition of G418 and other aminoglycoside antibiotics to mammalian cells results in the release of GPI-anchored proteins.

Resistance to the neomycin analogue G418 forms the basis of a dominant marker selection system for mammalian cells transfected with the bacterial neomycin gene. We found that COS-1 cells stably transfected with the neomycin resistance gene had a greater than 50% reduction in cell-associated glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (AP). A similarly reduced amount of AP was also observed in wild-type COS-1 cells incubated in the presence of G418 or other aminoglycoside antibiotics. The AP was released from cells into the culture supernatant in its GPI-anchored form. Our data suggest that the G418-induced reduction of AP involves a vesiculation process of COS-1 cells.

Short-term results of IPS-Empress full-porcelain crowns.

PURPOSE: A leucite-reinforced, glass-ceramic material was recently introduced for clinical use. In this clinical trial, IPS-Empress material was tested in the form of full-porcelain crowns. MATERIALS AND METHODS: Thirty-four patients were restored with 78 full-porcelain crowns. After etching the crowns with hydrofluoric acid, they were silanized and luted using dentin bonding agents and resin composite cement, which was primarily a dual-cured type. The 41 anterior and 37 posterior crowns were evaluated clinically with a mirror and probe, radiographically, and using clinical photographs, according to the modified United States Public Health Service criteria. Restorations having neither charlie nor delta criteria were defined as successful, and their survival rate was calculated according to Kaplan-Meier analysis. RESULTS: The mean observation period for the 78 restorations was 19.7 +/- 8.5 months. Seventy-four crowns were defined as successful. Four restorations failed because of fractures. Three of the four failures occurred in the first 2 months after cementation. The survival rate was estimated to be 95% successful after 2 years in service. Eighty percent of the crowns demonstrated an excellent esthetic result. CONCLUSIONS: The initial clinical results of this esthetic full-porcelain crown system are encouraging. However, because of fatigue phenomena for all ceramic materials, a longer observation period is needed to provide a definitive prognosis about the long-term clinical behavior.

Uptake and intracellular stability of glycosylphosphatidylinositol-specific phospholipase D in neuroblastoma cells.

Glycosylphosphatidylinositol-specific phospholipase D from mammalian serum has been described to be relatively stable towards the action of proteases in vitro, and it has been speculated that the enzyme may only be active on glycosylphosphatidylinositol-anchored substrates after its proteolytic processing in an intracellular compartment following uptake from body fluids. To test this hypothesis, we studied the possible uptake and intracellular processing of purified glycosylphosphatidylinositol-specific phospholipase D into the mouse neuroblastoma cell line N2A. We found that after incubation of neuroblastoma cells with glycosylphosphatidylinositol-specific phospholipase D at 37 degrees C the amount of cell-associated glycosylphosphatidylinositol-specific phospholipase D activity increased in a concentration- and time-dependent way. A similar uptake was also observed with 125I-labeled intact and trypsin-treated form of glycosylphosphatidylinositol-specific phospholipase D. We found that the incorporated radiolabeled proteins were processed intracellularly to distinct low molecular mass products, and that this process was in part inhibited by the presence of chloroquine during incubation.

The C-terminus of glycosylphosphatidylinositol-specific phospholipase D is essential for biological activity.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) (EC 3.1.4.50) from mammalian serum is a 115 kDa glycoprotein consisting of 816 amino acids. We found that C-terminal deletions of only two to five amino acids reduced GPI-PLD enzymatic activity by roughly 70% as compared to wild-type protein. C-terminal deletions of more than five amino acids resulted in a complete loss of GPI-PLD enzymatic activity. Point mutations at position 811 indicate that Tyr-811 may play a major role in maintaining the biological activity of GPI-PLD.

The neuronal cell-adhesion molecule axonin-1 is specifically released by an endogenous glycosylphosphatidylinositol-specific phospholipase.

Axonin-1, a member of the immunoglobulin/fibronectin type-III family of cell-adhesion molecules, occurs both as a glycosylphosphatidylinositol-(glycosylPtdIns)-anchored membrane-bound and a soluble form. In vivo observations show that the major part of axonin-1 is found in the soluble fraction and that soluble axonin-1 perturbs neurite fasciculation and pathfinding in the developing chicken embryo. This has prompted further investigations into the mechanism of the axonin-1 release. We demonstrate here that axonin-1 released from dorsal root ganglion neurons contains ethanolamine and inositol, components of the glycosylPtdIns anchor. Secreted axonin-1 does not exhibit the cross-reacting determinant epitope, an indication that the cleavage of the anchor is not mediated by a phosphatidylinositol-specific phospholipase C. Treatment of dorsal root ganglion neurons with 1,10-phenanthroline, an inhibitor of glycosylPtdIns-specific phospholipase D, reduces the release of axonin-1 by 56%. Moreover, glycosylPtdIns-specific phospholipase D activity was detected in dorsal root ganglion neurons and brain. These results suggest that axonin-1 is released from the membrane by an endogenously expressed glycosylPtdIns-specific phospholipase D in vivo. With domain-swaping experiments between axonin-1 and its non-released relative F11, deletion mutants and monoclonal antibodies, we demonstrate that the fourth fibronectin type-III-like domain of axonin-1 is required for the generation of the soluble form of axonin-1.

Six years of clinical experience with an all-ceramic system.

The search for tooth-colored and metal-free restorations is one of the major challenges in dental research. For several decades, ceramic has been used as a restorative material because of its aesthetics and stability. Unfortunately, the survival rate of most all-ceramic systems seems unsatisfactory; due to the natural brittleness of ceramic, fractures have been the primary reason for the high failure rate. Since 1988, the University of Zurich Dental School, Switzerland, has been working with the IPS Empress all-ceramic system (Ivoclar/Williams, Amherst, NY). This article reports the clinical and research data from approximately 3,000 all-ceramic restorations.

Differential interaction of the monoclonal antibody AE-1 with acetylcholinesterase oligomers and monomers from rabbit muscle microsomes, human brain and fetal bovine serum.

The monoclonal antibody AE-1 raised against acetylcholinesterase (AChE) from human erythrocytes (HE) is shown to react with active asymmetric and tetrameric AChE components from rabbit muscle microsomes (RMM), and with tetrameric forms from human brain (HB) or fetal bovine serum (FBS). However, it failed to bind to AChE monomers from RMM or HB. The results of Western blot revealed that the determinant for AE-1 consisted of a conformational domain, not a primary sequence region, in the AChE subunit. The antibody recognized HE monomers and FBS dimers, but not FBS monomers. The formation of labile immunocomplexes between AE-1 and AChE subunits may explain the lack of interaction between the antibody and the monomers from non-erythrocyte sources.

Short-term results of IPS-Empress inlays and onlays.

PURPOSE: A leucite-reinforced, glass-ceramic material was recently introduced for clinical use. In this clinical trial, IPS-Empress inlays and onlays were evaluated using the modified United States Public Health Service (USPHS) criteria. MATERIALS AND METHODS: The teeth of 36 patients were restored with 105 posterior inlays and 25 onlays, fabricated by an indirect technique. After etching the restorations with hydrofluoric acid, they were silanized and luted using composite cements. The restorations were evaluated visually, clinically with a mirror and probe, and by bitewing radiographs and clinical photographs, using modified USPHS criteria. Restorations having neither charlie nor delta criteria were defined as successful, and their survival rate was calculated according to Kaplan-Meier analysis. RESULTS: The mean observation period for the 130 restorations was 23.4 +/- 6.1 months. After 2 years, 127 restorations were successful with an estimated survival rate of 97.5%. Three restorations failed because of fractures. The esthetic results were excellent. CONCLUSIONS: The initial clinical results of this esthetic restorative material are encouraging. However, because of fatigue phenomena for all ceramic materials, a longer observation period is needed to provide a definitive prognosis of the long-term clinical behavior.

Subcellular distribution of glycosylphosphatidylinositol-specific phospholipase D in rat liver.

Glycosylphosphatidylinositol (GPI)-hydrolysing enzymes have been described in many mammalian tissues and body fluids; however, their site(s) of action and in vivo functions have remained unclear. In order to identify a possible intracellular site of GPI hydrolysis, we studied the subcellular distribution of GPI-hydrolysing activity in rat liver. We found that purified fractions from rat liver hydrolysed the GPI moieties of two GPI-anchored proteins with the specificity of a phospholipase D. This GPI-specific phospholipase D (GPI-PLD) activity was found to be highly enriched in a lysosomal fraction and showed a similar intracellular distribution to that of typical lysosomal enzymes. Our results indicate that lysosomes may represent a possible intracellular site of GPI-PLD action.

Phosphatidylinositol hydrolysis by Trypanosoma brucei glycosylphosphatidylinositol phospholipase C.

Detergent-solubilized glycosylphosphatidylinositol (GPI)-anchored structures can be cleaved by C-type phospholipases isolated from peanuts and bloodstream cells of the African trypanosome, Trypanosoma brucei. The two enzymes differ in their reported ability to hydrolyze phosphatidylinositol (PI); while the peanut enzyme readily hydrolyzes PI in vitro, the T. brucei enzyme was reported to be virtually inactive against PI and consequently named GPI-specific phospholipase C (GPI-PLC). In this paper, we describe experiments in which we reinvestigated the substrate specificity of T. brucei GPI-PLC by incubating the purified enzyme with Triton X-100/PI-mixed micelles and by studying PI hydrolysis. We found that PI hydrolysis occurred in a detergent-dependent fashion over the range of concentrations tested (5 microM to 1 mM PI). At 5 microM PI, hydrolysis was maximal at 0.005% Triton X-100, whereas at 1 mM PI, maximal hydrolysis required 0.05% Triton X-100. Hydrolysis of both PI and GPI was strongly affected by the presence of phospholipids. Endogenous PI was hydrolyzed during osmotic and detergent lysis of trypanosomes under conditions used to obtain quantitative hydrolysis of the GPI-anchored trypanosome variant surface glycoprotein. PI hydrolysis in the lysates was inhibited by sodium p-chloromercuriphenylsulfonate but unaffected by EGTA, consistent with the proposal that hydrolysis is due to GPI-PLC. These results suggest that the function of T. brucei GPI-PLC may be to regulate PI as well as (or instead of) GPI levels.

The membrane anchor of mammalian brain acetylcholinesterase consists of a single glycosylated protein of 22 kDa.

Mammalian brain acetylcholinesterase (AChE; EC 3.1.1.7) is membrane-bound through a structural subunit of about 20 kDa. So far little is known about this anchor because it is only detectable after hydrophobic labelling. In the present study we demonstrate that the two bands migrating around 20 kDa on SDS-PAGE derive from the same protein containing the same N-terminal amino acid sequence. The difference in their mobility is due to different N-glycosidation. Radioalkylation of cysteine residues reveals that the anchor contains just the two cysteine residues involved in binding the catalytic subunits.

Monoclonal antibodies against a C-terminal peptide of human brain acetylcholinesterase distinguish between erythrocyte and brain acetylcholinesterases.

Monoclonal antibodies (mAbs) were raised against a peptide of the 10 C-terminal amino acids of human brain acetylcholinesterase (AChE): H-Tyr-Ser-Lys-Gln-Asp-Arg-Cys-Ser-Asp-Leu-OH. Two positive clones (mAbs 190-1 and 190-2) were selected and tested for their ability to distinguish between mammalian brain and erythrocyte AChEs. In a solid-phase enzyme antigen immunoassay as well as by Western- and dot-blot analysis, both antibodies showed clear binding to AChE from human and bovine brain but not to AChE from erythrocytes. MAbs 190-1 and 190-2 reacted with neither AChE from electric eel nor butyrylcholinesterase from human serum. Both antibodies were used in a quantitative assay for AChE in amniotic fluids, where AChE activity could be found only in samples from open neural tube-defect pregnancies, but not in fluids from normal pregnancies or in artificially blood-contaminated samples.

Six years of clinical experience with an all-ceramic system.

The search for tooth-colored and metal-free restorations is one of the major challenges in dental research. For several decades, ceramic has been used as a restorative material because of its aesthetics and stability. Unfortunately, the survival rate of most all-ceramic systems seems unsatisfactory; due to the natural brittleness of ceramic, fractures have been the primary reason for the high failure rate. Since 1988, the University of Zurich Dental School, Switzerland, has been working with the IPS Empress all-ceramic system (Ivoclar/Williams, Amherst, NY). This article reports the clinical and research data from approximately 3,000 all-ceramic restorations.